CN112649543A

CN112649543A - Construction method of fingerprint of Guajilin Ganhe tea and standard fingerprint thereof

Info

Publication number: CN112649543A
Application number: CN202110066555.6A
Authority: CN
Inventors: 招嘉文; 杜蔼媚; 陈铠怡; 张淑雯; 周奕姝
Original assignee: Sinopharm Group Dezhong Foshan Pharmaceutical Co Ltd
Current assignee: Sinopharm Group Dezhong Foshan Pharmaceutical Co Ltd
Priority date: 2021-01-19
Filing date: 2021-01-19
Publication date: 2021-04-13
Anticipated expiration: 2041-01-19
Also published as: CN112649543B

Abstract

The application discloses a construction method of a fingerprint of Gujilin Ganhe tea and a standard fingerprint thereof, which comprises the following steps: s1, preparing a reference substance solution; s2, preparing a test solution; s3, determination: and respectively sucking the reference substance solution and the test solution to be injected into a liquid chromatograph to obtain the fingerprint of the original Jilin Ganjia tea. The fingerprint spectrum of the source Jilin Ganhe tea can comprehensively reflect the overall appearance of various components in the formula of the source Jilin Ganhe tea, can qualitatively and quantitatively analyze the quality of the source Jilin Ganhe tea, has the advantages of rapidness, stability, high precision, strong reproducibility and the like, and provides a new method for controlling the quality of the source Jilin Ganhe tea.

Description

Construction method of fingerprint of Guajilin Ganhe tea and standard fingerprint thereof

Technical Field

The application relates to the technical field of construction of traditional Chinese medicine fingerprint spectrums, in particular to a construction method of a fingerprint spectrum of virginine ganhe tea and a standard fingerprint spectrum thereof.

Background

The Chinese patent medicine has the characteristics of complex system, multiple components and multiple action targets. At present, the clinical curative effect of the Chinese patent medicine is more understood, but the action mechanism of the Chinese patent medicine is not completely understood, and the quality of the Chinese patent medicine is not comprehensive when the Chinese patent medicine is evaluated by independently measuring one or two index compounds. Therefore, most Chinese patent medicines begin to be characterized by adopting a fingerprint pattern mode, the traditional Chinese medicine fingerprint pattern is a comprehensive and quantifiable quality control means, the basic attributes of the traditional Chinese medicine fingerprint pattern are integrity and fuzziness, which correspond to the integrity principle of the traditional Chinese medicine theory and the fuzziness of the action mechanism of the traditional Chinese medicine, so that the quality of the traditional Chinese medicine can be comprehensively evaluated by comprehensively utilizing the fingerprint pattern and combining with the intelligent information processing of a computer. The traditional Chinese medicine fingerprint spectrum is a chromatogram or a spectrogram which can represent the chemical characteristics of certain traditional Chinese medicines or traditional Chinese medicine preparations and is obtained by adopting a certain analysis means after the traditional Chinese medicines or the traditional Chinese medicine preparations are properly processed, can comprehensively reflect the types and the quantities of the chemical components contained in the medicinal materials, effectively reflects the integrity and the comprehensive action of the traditional Chinese medicine components, and has been widely applied to the aspects of analysis and identification, quality control and the like of the traditional Chinese medicines due to the characteristics of rapidness, accuracy and the like.

The Yuan Jilin Ganhe tea as a Chinese patent medicine mainly comprises 35 traditional Chinese medicinal materials such as climbing groundsel herb, vitex negundo leaf, gardenia, kudzuvine root, baical skullcap root and the like, has the effects of dispelling wind and clearing heat, relieving summer heat and helping digestion, and promoting the production of body fluid to quench thirst, and is used for treating cold and fever, headache, joint pain, dyspepsia and distention, abdominal pain and vomiting and diarrhea. The quality standard of the product only comprises qualitative identification of sweet wormwood herb thin-layer chromatography, but no content determination method or other qualitative identification items are established, the original Jilin Ganhe tea is used as a Chinese herbal compound, the contained chemical components are very complex, the standard cannot completely reflect the overall quality characteristics of the product, and the quality of the product cannot be controlled integrally.

Disclosure of Invention

The application mainly aims to provide a construction method of a fingerprint of original Jilin Ganhe tea, and aims to solve the technical problem that the quality characteristics of products cannot be comprehensively reflected by the existing qualitative identification method of the sweet wormwood herb thin-layer chromatography of the original Jilin Ganhe tea.

In order to achieve the purpose, the construction method of the fingerprint of the source Jilin Ganjia tea, which is provided by the application, comprises the following steps:

s1, preparing a reference substance solution;

s2, preparing a test solution;

s3, determination: respectively sucking the reference substance solution and the test solution to be injected into a liquid chromatograph to obtain fingerprint spectra of the original Jilin Ganjia tea;

wherein, the conditions of the high performance liquid chromatography in the step S3 comprise: using octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A, and glacial acetic acid solution as mobile phase B to perform gradient elution at flow rate of 1ml/min and column temperature of 20-30 deg.C.

Optionally, in the step S3, the high performance liquid chromatography conditions further include: the chromatographic column is Welch Ultimate AQ C184.6X 250mm, 5 μm, and the detection wavelength is 250-290 nm.

Optionally, in the step S3, the gradient elution condition is: 0-15min, 5-15% of mobile phase A and 95-85% of mobile phase B; 15-40min, 15-19% of mobile phase A and 85-81% of mobile phase B; 40-65min, 19-60% of mobile phase A and 81-40% of mobile phase B; 65-75min, 60-95% of mobile phase A and 40-5% of mobile phase B; 75-76min, 95% -5% of mobile phase A and 5% -95% of mobile phase B; 76-83min, mobile phase A5%, mobile phase B95%.

Optionally, in the step S3, the volume fraction of the glacial acetic acid solution is 0.5% -1%.

Optionally, in step S1, respectively weighing chlorogenic acid reference, geniposide reference, puerarin reference, baicalin reference and ammonium glycyrrhizinate reference, and respectively adding 50% ethanol to obtain mixed solution;

each 1ml of the mixed solution contains chlorogenic acid, geniposide, puerarin, baicalin and ammonium glycyrrhizinate 30 μ g.

Optionally, in step S2, pulverizing and sieving source Jilin Ganjia tea, collecting 1.5-3.5g, adding 40-60ml of water, heating and refluxing, cooling for 40-60min, adding water to make up for the loss, mixing well, filtering to obtain a filtrate, collecting 5-15ml of the filtrate, extracting the filtrate with water saturated n-butanol to obtain 5-15ml of an extract, extracting for 3-5 times, mixing the extracts, evaporating to dryness to obtain a residue, adding 50% ethanol to dissolve the residue, adding 50% ethanol to dilute, mixing well, and filtering.

The application also provides a standard fingerprint of the original Jilin Ganhe tea, which is measured by the construction method of any one of the original Jilin Ganhe tea fingerprints: the standard fingerprint spectrum comprises 23 characteristic peaks, wherein the peak 5 is chlorogenic acid, the peak 7 is geniposide, the peak 9 is puerarin, the peak 22 is baicalin, and the peak 23 is glycyrrhizic acid.

Optionally, the average relative retention time of the No. 1 peak is 0.34-0.38, and the RSD is 0.12%; the average relative retention time of the No. 2 peak is 0.56-0.60, and the RSD is 0.06%; the average relative retention time of the No. 3 peak is 0.59-0.63, and the RSD is 0.04%; the average relative retention time of the No. 4 peak is 0.80-0.84, and the RSD is 0.05%; the average relative retention time of the No. 5 peak is 0.83-0.87, and the RSD is 0.06%; the average relative retention time of the No. 6 peak is 0.92-0.96, and the RSD is 0.03%; the average relative retention time of No. 7 peak is 0.94-0.98, and RSD is 0.02%; peak 8 has an average relative retention time of 0.98 and an RSD of 0.04%; peak 9 has an average relative retention time of 1 and an RSD of 0%; the average relative retention time of the No. 10 peak is 1.03-1.07, and the RSD is 0.04%; the average relative retention time of No. 11 peak is 1.04-1.08, and RSD is 0.05%; the average relative retention time of the No. 12 peak is 1.08-1.12, and the RSD is 0.05%; the average relative retention time of the No. 13 peak is 1.26-1.30, and the RSD is 0.06%; the average relative retention time of No. 14 peak is 1.29-1.31, and the RSD is 0.07%; the average relative retention time of the No. 15 peak is 1.33-1.37, and the RSD is 0.09%; the average relative retention time of the No. 16 peak is 1.52-1.56, and the RSD is 0.08%; the average relative retention time of the No. 17 peak is 1.56-1.60, and the RSD is 0.08%; the average relative retention time of the No. 18 peak is 1.68-1.72, and the RSD is 0.08%; the average relative retention time of the 19 th peak is 1.72-1.74, and the RSD is 0.08%; the average relative retention time of the No. 20 peak is 1.77-1.81, and the RSD is 0.07%; the average relative retention time of the No. 21 peak is 2.46-2.50, and the RSD is 0.21%; the average relative retention time of No. 22 peak is 2.55-2.59, and RSD is 0.22%; the average relative retention time of the No. 23 peak is 3.05-3.09, and the RSD is 0.27%.

Alternatively, peak No. 1 has an average relative retention time of 0.36; peak 2 average relative retention time 0.58; peak 3 has an average relative retention time of 0.61; peak No. 4 had an average relative retention time of 0.82; peak 5 has an average relative retention time of 0.85; peak No. 6 has an average relative retention time of 0.94; peak No. 7 average relative retention time 0.96; peak 8 average relative retention time 0.98; peak 9 has an average relative retention time of 1; peak 10 has an average relative retention time of 1.05; peak 11 average relative retention time 1.06; peak 12 average relative retention time 1.10; peak 13 has an average relative retention time of 1.28; peak 14 average relative retention time 1.31; peak 15 has an average relative retention time of 1.35; peak 16 has an average relative retention time of 1.54; peak 17 average relative retention time 1.58; peak 18 average relative retention time 1.70; peak 19 has an average relative retention time of 1.74; peak 20 has an average relative retention time of 1.79; peak 21 has an average relative retention time of 2.48; peak No. 22 has an average relative retention time of 2.57; peak 23 had an average relative retention time of 3.07.

Optionally, the

peaks

1, 3 and 20 are attributed to senecio scandens; 4. the peak attributes of No. 6, 12, 14, 15, 17, 18 and 21 are the medicinal material of folium Viticis Cannabifoliae; the attribution of No. 7 peak is gardenia medicinal material; 9. the attribution of No. 10, No. 11 and No. 13 peaks is a kudzu root medicinal material; 19, the attribution of the peak is sophora flower medicinal material; the 22 # peak belongs to scutellaria baicalensis; 23, the attribution of the peak is liquorice; 2. peak 5 is the common peak of Senecio scandens, Vitex negundo L.leaf and Murraya koenigii; 8. peak 16 is the common peak of Qianlieguang and Vitex negundo L.leaf medicinal materials.

The construction method of the fingerprint of the Yuan Jilin Ganhe tea and the standard fingerprint thereof have the following beneficial effects: the fingerprint characteristics of the large-polarity components in the original Jilin Ganhe tea are inspected mainly by using a high performance liquid chromatography technology so as to better reflect the correlation of the pharmacological and pharmacodynamic bases of the original Jilin Ganhe tea, the method has good specificity, and the characteristic peak is not interfered by impurity peaks; the precision, the repeatability and the stability all meet the requirements, and the result shows that the fingerprint of the original Jilin Ganhe tea obtained by the method can comprehensively reflect the overall appearance of various components in the original Jilin Ganhe tea formula, can qualitatively and quantitatively analyze the quality of the original Jilin Ganhe tea and provides a new method for controlling the quality of the original Jilin Ganhe tea.

Drawings

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a fingerprint of the source Jilin Ganhe tea provided by the present application;

FIG. 2 is a fingerprint of the source Jilin Ganhu tea provided by the present application at different detection wavelengths;

FIG. 3 is a fingerprint of the source Jilin Ganhu tea provided by the present application on different chromatographic columns;

FIG. 4 is a fingerprint of the source Jilin Ganjin tea provided by the present application under different mobile phases;

FIG. 5 is a fingerprint of glacial acetic acid of the original Jilin Ganhu tea provided by the present application at different volume fractions;

FIG. 6 is a fingerprint of the source Jilin Ganhu tea provided by the present application at different column temperatures;

FIG. 7 is a fingerprint of the source Jilin Ganhu tea provided by the present application under different extraction solvents;

FIG. 8 is a fingerprint of the source Jilin Ganhe tea provided by the present application under different extraction methods;

FIG. 9 is a fingerprint of the source Jilin Ganjin provided by the present application at different reflux times;

FIG. 10 is a specificity chromatogram of chlorogenic acid;

FIG. 11 is a chromatogram of Gardenia jasminoides Ellis speciality;

FIG. 12 is a chromatogram of a specificity of Pueraria lobata;

FIG. 13 is a chromatogram of Scutellaria baicalensis Georgi speciality;

FIG. 14 is a chromatogram of a specificity of Glycyrrhiza glabra;

FIG. 15 is a fingerprint of a repeat study of Yuan Jilin Ganhe tea;

FIG. 16 is a fingerprint of source Jilin Ganhe tea precision investigation;

FIG. 17 is a fingerprint of the source Jilin Ganjin tea measured by different operators respectively;

FIG. 18 is a finger print of Yuan Jilin Ganhe tea detected by different liquid chromatographs;

FIG. 19 is a fingerprint of source Jilin Ganhe tea stability study;

figure 20 is a fingerprint of 15 batches of source Jilin Ganhe tea.

The implementation, functional features and advantages of the objectives of the present application will be further explained with reference to the accompanying drawings.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The application provides a construction method of a fingerprint of a souji forest ganhe tea. In the embodiment of the application, the construction method of the fingerprint of the sourgine ganhe tea comprises the following steps:

s1, preparing a reference substance solution;

s2, preparing a test solution;

The original Jilin Ganhe tea is a traditional tea, the main active ingredients in the product are water-soluble substances with larger polarity, and the properties of the active ingredients are combined, so that the scheme mainly applies a high performance liquid chromatography technology to investigate the fingerprint characteristics of the large-polarity ingredients in the original Jilin Ganhe tea so as to better reflect the correlation of the pharmacological and pharmacodynamic bases of the original Jilin Ganhe tea, and the method has good specificity and no impurity peak interference in characteristic peaks; the precision, the repeatability and the stability all meet the requirements, the method is stable and reliable, the adaptability is good, the constructed fingerprint spectrum can comprehensively reflect the quality information of the original Jilin Ganju tea, and the method has the advantages of quickness, stability, high precision, strong repeatability and the like, and can be used for quality control of the original Jilin Ganju tea.

In order to determine the optimal mobile phase, in this embodiment, the influence of acetonitrile-water, acetonitrile-phosphoric acid solution with volume fraction of 0.1%, acetonitrile-glacial acetic acid solution with volume fraction of 0.5%, methanol-phosphoric acid solution with volume fraction of 0.1%, and methanol-glacial acetic acid solution with volume fraction of 0.5% on the chromatographic peak is respectively considered, and the specific detection result is shown in fig. 4, which shows that when the mobile phase system is acetonitrile-glacial acetic acid solution with volume fraction of 0.5%, the overall baseline is relatively stable, the chromatographic peak separation degree is relatively good, the number of chromatographic peaks is relatively large, the number of miscellaneous peaks is relatively small, and the peak emergence time is relatively fast, so in this embodiment, the acetonitrile + glacial acetic acid solution with volume fraction of 0.5% is selected as the final mobile phase. The scheme measures the fingerprint spectra at different column temperatures, specifically the influence on the chromatographic peak at 20 ℃, 25 ℃, 28 ℃, 30 ℃ and 35 ℃, the specific detection result is shown in fig. 6, and the separation effect at 35 ℃ is poor, so the preferred column temperature in the embodiment is 20-30 ℃, and the chromatographic peak can achieve the best separation effect at 28 ℃.

In an embodiment of the present application, in the step S3, the high performance liquid chromatography conditions further include: the chromatographic column is Welch Ultimate AQ C184.6X 250mm, 5 μm, and the detection wavelength is 250-290 nm.

The method for constructing the fingerprint of the source Jilin Ganhe tea in the scheme preferably selects the following parameters and conditions, and S3. determination: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, measuring, and recording the chromatographic peak within 65 min. Wherein, chromatographic conditions and system applicability tests are carried out, and octadecylsilane chemically bonded silica is used as a filling agent; performing gradient elution by using acetonitrile as a mobile phase A and using a 0.5% glacial acetic acid solution as a mobile phase B; the flow rate was 1 ml/min. The detection wavelength in the high performance liquid chromatography condition is 250-290 nm. When the mobile phase A is acetonitrile and the mobile phase B is 0.5 percent glacial acetic acid (V/V) solution, linear gradient elution is carried out according to the parameters, PDA monitoring is carried out, the wavelength scanning range is 190nm-400nm, and a specific fingerprint is shown in figure 2, so that the ultraviolet absorption of many characteristic chromatographic peaks (such as glycyrrhizic acid, geniposide and puerarin) of the source Jilin Ganjin and tea is near 250nm, therefore, the detection wavelength of 250-290nm is selected, the baseline is stable in the wavelength range of 250nm, the impurity interference is less, and 250nm is further preferably used as the detection wavelength of the fingerprint of the source Jilin Ganjin and tea in the actual detection. The column in the HPLC conditions was Welch Ultimate AQ C184.6X 250mm, 5 μm. In order to determine the best chromatographic column, the scheme measures the fingerprint of a plurality of chromatographic columns: as shown in FIG. 3, it was found that the effect on the chromatogram was the best when a Welch Ultrate AQ C18 (4.6X 250mm, 5 μm) column was used as a Waters XSelect HSS T3 (4.6X 250mm, 5 μm), Ecosil C18 (4.6X 250mm, 5 μm), Welch Ultrate XB C18 (4.6X 250mm, 5 μm), respectively, under the same gradient, and it was found that the whole separation effect of the chromatographic peaks was the best when a Welch Ultrate AQ C18 (4.6X 250mm, 5 μm) column was used, and thus a liquid chromatography column in which Welch Ultrate AQ C18 is a Jilin sweet and tea fingerprint spectrum was preferable.

In an embodiment of the present application, in the step S3, the gradient elution condition is: 0-15min, 5-15% of mobile phase A and 95-85% of mobile phase B; 15-40min, 15-19% of mobile phase A and 85-81% of mobile phase B; 40-65min, 19-60% of mobile phase A and 81-40% of mobile phase B; 65-75min, 60-95% of mobile phase A and 40-5% of mobile phase B; 75-76min, 95-5% of mobile phase A and 78-95% of mobile phase B5; 76-83min, mobile phase A5%, mobile phase B95%. The above is the optimum gradient elution conditions obtained in this example, and the chromatogram has good peak shape and resolution within the above range.

In an embodiment of the present application, in the step S3, the volume fraction of the glacial acetic acid solution is 0.5% to 1%. In order to obtain the optimal glacial acetic acid volume fraction, the fingerprint of each volume fraction of glacial acetic acid is measured in this embodiment, and the influence of glacial acetic acid solutions with different volume fractions on a chromatographic peak is examined, wherein the glacial acetic acid solutions mainly include acetonitrile-0.2% glacial acetic acid, acetonitrile-0.5% glacial acetic acid, and acetonitrile-1% glacial acetic acid, and other chromatographic conditions such as an elution gradient and a column temperature are kept consistent, and the measurement result is shown in fig. 5, and the result shows that when the glacial acetic acid volume fraction is 0.5% to 1%, the separation is better, the peak emergence time is faster, and the glacial acetic acid solution with the volume fraction of 0.5% is preferred.

In an embodiment of the present application, in step S1, a chlorogenic acid reference substance, a geniposide reference substance, a puerarin reference substance, a baicalin reference substance, and an ammonium glycyrrhizinate reference substance are respectively weighed, and then 50% ethanol is respectively added to prepare mixed solutions; each 1ml of the mixed solution contains chlorogenic acid, geniposide, puerarin, baicalin and ammonium glycyrrhizinate 30 μ g.

In the scheme, the above parameters and conditions are preferably selected for preparing the reference substance solution, and appropriate amounts of chlorogenic acid reference substance, geniposide reference substance, puerarin reference substance, baicalin reference substance and ammonium glycyrrhizinate reference substance are taken, and 50% ethanol solvent is added to prepare a mixed reference substance solution. According to the pharmacodynamic ingredients and the main chemical properties of each medicine, corresponding reference substance solution is prepared for positioning comparison, so that the constructed standard fingerprint spectrum can comprehensively reflect the quality information of the Ganjing tea, the defects of single quality control standard and one side are avoided, and the method can be used for comprehensive quality evaluation and control of the Ganjing tea.

In an embodiment of the application, in step S2, the source Jilin Ganjia tea is pulverized and sieved to obtain 1.5-3.5g, 40-60ml of water is added, the mixture is heated and refluxed for 40-60min and then cooled, the water is added to make up the loss, the filtrate is obtained by mixing and filtering, 5-15ml of the filtrate is obtained, the filtrate is extracted with water-saturated n-butanol to obtain 5-15ml of extracting solution, the extracting solution is extracted for 3-5 times, the extracting solution is combined and evaporated to dryness to obtain evaporation residue, 50% ethanol is added to dissolve the evaporation residue, 50% ethanol is added to dilute the evaporation residue, and the mixture is filtered after mixing.

The following parameters and conditions are preferred for the preparation of the test article in the scheme: taking the original Jilin Ganjin and the tea content, crushing, sieving by a third sieve, taking 2.5g, precisely weighing, placing in a conical flask with a plug, precisely adding 50ml of water, sealing the plug, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the weight loss by water, shaking uniformly, and filtering. Precisely measuring 10ml of the subsequent filtrate, placing in a separating funnel, extracting with water saturated n-butanol under shaking for 4 times, 10ml each time, mixing n-butanol extractive solutions, evaporating, dissolving the residue with 50% ethanol, transferring to a 10ml measuring flask, adding 50% ethanol to dilute to the scale, shaking, filtering, and collecting the subsequent filtrate. The theoretical plate number should not be as low as 80000 calculated as puerarin peak. The traditional Chinese medicine chromatogram fingerprint similarity evaluation system is adopted to identify the fingerprint, the operation is simple, convenient and quick, the similarity result obtained by the method is used for evaluating the fingerprint of the Yuan Jilin Ganju tea, and the conclusion is objective and accurate. The particle size of the screened source Jilin Ganjin and tea is 342-368 mu m. When the source Jilin Ganju tea is crushed and sieved to the fineness, the whole body is dispersed, and the separation effect in the subsequent detection process is better.

In order to further improve the detection effect of the fingerprint, two extraction modes are selected for determination, 3 backflow times are selected for determination, the specific fingerprint test result is shown in fig. 8 and 9, when water backflow and water ultrasound are selected for respective extraction, due to the fact that the source Jilin sweet tea is light in weight, the ultrasonic effect is not ideal, backflow extraction is complete, and backflow extraction is preferred in the embodiment. When the mixture is heated and refluxed for 30 minutes, 60 minutes and 90 minutes respectively, the extraction effect after heating for 60 minutes is obviously better than that after heating for 30 minutes, and meanwhile, the extraction effect is not obviously different from that after heating for 90 minutes. Therefore, in order to save the detection cost, the heating reflux time in the present embodiment is 40 to 60min, and the extraction (heating reflux) time is preferably 60 min.

The original Jilin Ganhe tea prescription has many medicinal tastes and complex traditional Chinese medicine components, the ratio of three main tea medicinal materials (namely, folium Viticis negundo, senecio scandens and Mussaenda yunnanensis) in the prescription is more than 58%, and the tea contains a large amount of water-soluble pigments and other impurities, so that the color of a sample solution of a fingerprint is dark brown, the background interference is large, the separation and identification of chromatographic peaks are influenced, and the damage to chromatographic columns is large, so the pigments and impurity interference of the tea medicinal materials are required to be removed firstly in the establishment process of the fingerprint, and the characteristic peaks of the medicinal tastes of the prescription can be kept to the maximum extent. According to the scheme, water is used as an extraction solvent for heating, and a water-saturated n-butyl alcohol liquid-liquid extraction technology is adopted, so that the medicinal components in the prescription medicine can be enriched in the water-saturated n-butyl alcohol solution, and the purified test solution is obtained through separation, so that the background interference is greatly reduced, the damage to chromatographic columns is reduced, the characteristic peaks of the prescription medicine can be embodied in the purified test solution, and the quality of the whole fingerprint characteristic spectrum is greatly improved. In the scheme, water is preferably used as an extraction solvent, mainly because when the extraction solvent is 50% ethanol and 70% ethanol, chlorophyll impurities are more, the solution is dark green, as shown in fig. 7, when the extraction solvent is 50% ethanol and 70% ethanol, chromatographic impurities are more, background interference is larger, when water is used as the extraction solvent, chromatographic impurity peaks are less, background interference is relatively less, and chromatographic peak areas corresponding to all reference substances are relatively consistent, so that effective ingredients extracted by water are relatively complete, and original Jilin Ganhe tea is a traditional tea preparation, and water is selected as the extraction solvent, so that the correlation of the pharmacological and pharmacodynamic bases of the tea preparation is better reflected.

By optimizing the preparation method of the sample solution and optimizing chromatographic conditions in the embodiments, the fingerprint baseline of the original Jilin Ganhu tea is more stable, the chromatographic peak separation degree is better, the number of chromatographic peaks is more, the number of impure peaks is less, the peak emergence time is faster, and the information amount is large.

In order to verify the accuracy of the construction method of the fingerprint of the source Jilin Ganhe tea, the scheme comprises the following experiments:

1. specificity test

Precisely sucking corresponding negative control solution (lacking corresponding medicinal materials), reference solution and sample solution each 10 μ L, and determining according to the same chromatographic conditions. The specific determination results are shown in fig. 10 to fig. 14, the negative control solution has no corresponding chromatographic peak at the characteristic peak position of the reference medicinal material and the sample solution, which indicates that the sample solution fingerprint determination is non-interference, and the specificity of the construction method of the Yuan Jilin Ganju tea fingerprint is good.

2. Repeatability test

The same laboratory technician A takes the same batch of test articles (batch number: 20107, provided by Dezhong (Foshan) pharmaceutical industry Co., Ltd., national drug group), prepares 6 test article solutions according to the preparation of the test article solutions, precisely absorbs 10 mu L of the test article solutions, injects the test article solutions into a liquid chromatograph, performs sample injection measurement according to the high performance liquid chromatography condition, records fingerprint spectrums for 6 times, compares the fingerprint spectrums with generated comparison fingerprint spectrums, calculates the similarity, as shown in figure 15, the result similarity is 1, the total peak retention time RSD is less than 1%, and the method is proved to have good repeatability.

3. Precision test

The test solution is taken and continuously measured for 6 times according to the formulated test conditions. The fingerprint is recorded and compared with the generated comparison fingerprint, and the similarity is calculated, as shown in figure 16, the result similarity is 1, the retention time RSD of the common peak is less than 1 percent, and the precision of the method is good.

4. Intermediate precision test

Another test person B prepared 6 parts of the test solution under the above conditions (test person A) and measured under the predetermined chromatographic conditions. And recording the fingerprint, bringing the fingerprint into similarity software, and respectively calculating the similarity of the fingerprint and the comparison fingerprint. As shown in FIG. 17, the results show that the similarity between each fingerprint and the control fingerprint is 1 and the retention time RSD of the common peak is less than 1% under the preparation of different people, which indicates that the method has good intermediate precision.

Taking the same sample solution, respectively testing the same sample solution on a Waters e2695 liquid chromatograph, a Thermo Ultimate3000 liquid chromatograph and a Waters AQUITY Arc liquid chromatograph according to specified chromatographic conditions, wherein the specific test results are shown in FIG. 18, and the fingerprint spectrums obtained by respectively adopting the Waters e2695 liquid chromatograph, the Thermo Ultimate3000 liquid chromatograph and the Waters AQUITY Arc liquid chromatograph are analyzed by a similarity evaluation system, the similarities are 1, 0.997 and 1 in sequence, so that the results of the liquid chromatographs of different brands or models have no obvious difference, and the intermediate precision of the method is good.

5. Stability test

A portion of the test solution was taken and assayed at 0, 3, 7, 13, 25 and 45 hours, respectively. Recording each fingerprint, bringing the fingerprint into similarity software, wherein each fingerprint has 23 characteristic peaks, as shown in fig. 19, in the measurement results of different time periods, the similarity between each fingerprint and the fingerprint in 0 hour is 1, and the retention time RSD is less than 1%. The sample is shown to be stable in the measurement result within 45 hours.

The same determination method is adopted to respectively perform (single needle) fingerprint determination on 15 batches of samples of the original Jilin Ganjin and tea, record each fingerprint, bring the fingerprint into similarity software to obtain a graph 20, generate a reference spectrum, and calculate the similarity between the 15 batches of samples to be tested and the reference fingerprint, wherein the specific similarity result is shown in the following table 1, and the result shows that the fingerprint similarity of the 15 batches of samples of the original Jilin Ganjin and tea is more than 0.99, which shows that the quality consistency degree of the 15 batches of samples of the original Jilin Ganjin and tea is high, and the production process is stable and controllable.

TABLE 1.15 lots of fingerprint similarity summary table for Yuan Jilin Ganju tea

Serial number	Batch number	Similarity to control map
			1	18035	1.000
2	18057	0.995
			3	18096	0.997
4	18125	0.997
			5	18155	0.999
6	19005	0.998
			7	19040	0.998
8	19069	0.995
			9	19100	0.994
10	19135	0.996
			11	20023	0.994
12	20045	0.992
			13	20066	0.993
14	20089	0.999
			15	20107	0.994

The application also provides a source Jilin Ganhe tea standard fingerprint, which is measured by any one of the above construction methods of the source Jilin Ganhe tea fingerprint, the construction method of the source Jilin Ganhe tea fingerprint refers to the above embodiments, and as the source Jilin Ganhe tea fingerprint adopts all the technical schemes of all the above embodiments, all the effects brought by the technical schemes of the above embodiments are at least achieved, and the details are not repeated here.

As shown in fig. 1, a high performance liquid chromatography is adopted to perform full-wavelength scanning, and the retention time and the ultraviolet absorption spectrum of a reference substance are compared to confirm a characteristic peak compound, wherein the standard fingerprint spectrum of the original Jilin Ganhu tea comprises 23 characteristic peaks, wherein the peak 5 is chlorogenic acid, the peak 7 is geniposide, the peak 9 is puerarin, the peak 22 is baicalin, and the peak 23 is glycyrrhizic acid. The scheme determines that 23 common characteristic peaks exist in the fingerprint of the original Jilin Ganjin tea, constructs the standard fingerprint of the original Jilin Ganjin tea, and utilizes the mixed reference substance solution to identify the common peaks of chlorogenic acid, geniposide, puerarin, baicalin and glycyrrhizic acid, the constructed standard fingerprint can comprehensively reflect the quality information of the Ganjin tea, and the method has the advantages of rapidness, stability, high precision, strong reproducibility and the like, avoids the defects of single quality control standard and one-sidedness in the prior art, and can be used for comprehensive quality evaluation and control of the Ganjin tea.

Compared with the contrast fingerprint, the similarity is not lower than 0.90 through the calculation of the traditional Chinese medicine chromatogram fingerprint similarity evaluation system software. And the newly established fingerprint spectrum is used for carrying out comprehensive quality comparison on 15 batches of original Jilin Ganjin tea samples, so that the quality consistency of the product is proved, and the conclusion is objective and accurate.

In one embodiment of the present application, the average relative retention time of peak 1 is 0.34-0.38, and the RSD is 0.12%; the average relative retention time of the No. 2 peak is 0.56-0.60, and the RSD is 0.06%; the average relative retention time of the No. 3 peak is 0.59-0.63, and the RSD is 0.04%; the average relative retention time of the No. 4 peak is 0.80-0.84, and the RSD is 0.05%; the average relative retention time of the No. 5 peak is 0.83-0.87, and the RSD is 0.06%; the average relative retention time of the No. 6 peak is 0.92-0.96, and the RSD is 0.03%; the average relative retention time of No. 7 peak is 0.94-0.98, and RSD is 0.02%; peak 8 has an average relative retention time of 0.98 and an RSD of 0.04%; peak 9 has an average relative retention time of 1 and an RSD of 0%; the average relative retention time of the No. 10 peak is 1.03-1.07, and the RSD is 0.04%; the average relative retention time of No. 11 peak is 1.04-1.08, and RSD is 0.05%; the average relative retention time of the No. 12 peak is 1.08-1.12, and the RSD is 0.05%; the average relative retention time of the No. 13 peak is 1.26-1.30, and the RSD is 0.06%; the average relative retention time of No. 14 peak is 1.29-1.31, and the RSD is 0.07%; the average relative retention time of the No. 15 peak is 1.33-1.37, and the RSD is 0.09%; the average relative retention time of the No. 16 peak is 1.52-1.56, and the RSD is 0.08%; the average relative retention time of the No. 17 peak is 1.56-1.60, and the RSD is 0.08%; the average relative retention time of the No. 18 peak is 1.68-1.72, and the RSD is 0.08%; the average relative retention time of the 19 th peak is 1.72-1.74, and the RSD is 0.08%; the average relative retention time of the No. 20 peak is 1.77-1.81, and the RSD is 0.07%; the average relative retention time of the No. 21 peak is 2.46-2.50, and the RSD is 0.21%; the average relative retention time of No. 22 peak is 2.55-2.59, and RSD is 0.22%; the average relative retention time of the No. 23 peak is 3.05-3.09, and the RSD is 0.27%. According to the scheme, 5 common peaks can be identified from the common peaks through the parameters such as the retention time and the like, wherein the 5 peak is chlorogenic acid, the 7 peak is geniposide, the 9 peak is puerarin, the 22 peak is baicalin, and the 23 peak is glycyrrhizic acid.

In one embodiment of the present application, peak 1 has an average relative retention time of 0.36; peak 2 average relative retention time 0.58; peak 3 has an average relative retention time of 0.61; peak No. 4 had an average relative retention time of 0.82; peak 5 has an average relative retention time of 0.85; peak No. 6 has an average relative retention time of 0.94; peak No. 7 average relative retention time 0.96; peak 8 average relative retention time 0.98; peak 9 has an average relative retention time of 1; peak 10 has an average relative retention time of 1.05; peak 11 average relative retention time 1.06; peak 12 average relative retention time 1.10; peak 13 has an average relative retention time of 1.28; peak 14 average relative retention time 1.31; peak 15 has an average relative retention time of 1.35; peak 16 has an average relative retention time of 1.54; peak 17 average relative retention time 1.58; peak 18 average relative retention time 1.70; peak 19 has an average relative retention time of 1.74; peak 20 has an average relative retention time of 1.79; peak 21 has an average relative retention time of 2.48; peak No. 22 has an average relative retention time of 2.57; peak 23 had an average relative retention time of 3.07.

In an embodiment of the present application, the

peaks

1, 3 and 20 are attributed to senecio scandens; 4. the peak attributes of No. 6, 12, 14, 15, 17, 18 and 21 are the medicinal material of folium Viticis Cannabifoliae; the attribution of No. 7 peak is gardenia medicinal material; 9. the attribution of No. 10, No. 11 and No. 13 peaks is a kudzu root medicinal material; 19, the attribution of the peak is sophora flower medicinal material; the 22 # peak belongs to scutellaria baicalensis; 23, the attribution of the peak is liquorice; 2. peak 5 is the common peak of Senecio scandens, Vitex negundo L.leaf and Murraya koenigii; 8. peak 16 is the common peak of Qianlieguang and Vitex negundo L.leaf medicinal materials. The characteristic peaks can be detected in specific medicinal materials, and the main components are derived from the medicinal materials.

The above description is only a preferred embodiment of the present application, and not intended to limit the scope of the present application, and all modifications and equivalents of the technical solutions that can be directly or indirectly applied to other related fields without departing from the spirit of the present application are intended to be included in the scope of the present application.

Claims

1. A construction method of fingerprint of Guijilin Ganhe tea is characterized by comprising the following steps:

s1, preparing a reference substance solution;

s2, preparing a test solution;

s3, determination: respectively sucking the reference substance solution and the test solution, injecting the reference substance solution and the test solution into a liquid chromatograph for high performance liquid chromatography analysis, and recording chromatographic peaks to obtain fingerprint spectra of the source Jilin Ganhe tea;

2. The method for constructing fingerprint of guilinggan and cha as claimed in claim 1, wherein in step S3, the hplc conditions further include: the chromatographic column is Welch Ultimate AQC 184.6X 250mm, 5 μm, and the detection wavelength is 250-290 nm.

3. The method for constructing fingerprint of Guerin and tea of claim 2, wherein in step S3, the gradient elution condition is: 0-15min, 5-15% of mobile phase A and 95-85% of mobile phase B; 15-40min, 15-19% of mobile phase A and 85-81% of mobile phase B; 40-65min, 19-60% of mobile phase A and 81-40% of mobile phase B; 65-75min, 60-95% of mobile phase A and 40-5% of mobile phase B; 75-76min, 95% -5% of mobile phase A and 5% -95% of mobile phase B; 76-83min, mobile phase A5%, mobile phase B95%.

4. The method for constructing fingerprint of sougilin ganhe tea as claimed in claim 2, wherein in the step S3, the volume fraction of the glacial acetic acid solution is 0.5% -1%.

5. The method of claim 1, wherein in step S1, a chlorogenic acid reference, a geniposide reference, a puerarin reference, a baicalin reference, and an ammonium glycyrrhizinate reference are weighed, respectively, and 50% ethanol is added to prepare a mixed solution;

6. The method for constructing fingerprint of yijilin ganhe tea as claimed in claim 1, wherein in step S2, the yijilin ganhe tea is pulverized, sieved, 1.5-3.5g is taken, 40-60ml of water is added, the mixture is heated and refluxed for 40-60min and then cooled, the water is added to make up the loss, the mixture is uniformly mixed and filtered to obtain filtrate, 5-15ml of the filtrate is taken, water saturated n-butanol is used for extracting the filtrate to obtain 5-15ml of extract, the extract is extracted for 3-5 times, the extract is combined and then evaporated to dryness to obtain evaporation slag, 50% of ethanol is added to dissolve the evaporation slag, 50% of ethanol is added for dilution, and the mixture is uniformly mixed and filtered.

7. The standard fingerprint of Yuanjilin gan and cha as determined by the method for constructing the fingerprint of Yuanjilin gan and cha as claimed in any one of claims 1-6, wherein the standard fingerprint comprises 23 characteristic peaks, wherein the peak 5 is chlorogenic acid, the peak 7 is jasminoidin, the peak 9 is puerarin, the peak 22 is baicalin, and the peak 23 is glycyrrhizic acid.

8. The standard fingerprint of Guerin and tea of claim 7, wherein the average relative retention time of peak 1 is 0.34-0.38, and the RSD is 0.12%;

the average relative retention time of the No. 2 peak is 0.56-0.60, and the RSD is 0.06%;

the average relative retention time of the No. 3 peak is 0.59-0.63, and the RSD is 0.04%;

the average relative retention time of the No. 4 peak is 0.80-0.84, and the RSD is 0.05%;

the average relative retention time of the No. 5 peak is 0.83-0.87, and the RSD is 0.06%;

the average relative retention time of the No. 6 peak is 0.92-0.96, and the RSD is 0.03%;

the average relative retention time of No. 7 peak is 0.94-0.98, and RSD is 0.02%;

peak 8 has an average relative retention time of 0.98 and an RSD of 0.04%;

peak 9 has an average relative retention time of 1 and an RSD of 0%;

the average relative retention time of the No. 10 peak is 1.03-1.07, and the RSD is 0.04%;

the average relative retention time of No. 11 peak is 1.04-1.08, and RSD is 0.05%;

the average relative retention time of the No. 12 peak is 1.08-1.12, and the RSD is 0.05%;

the average relative retention time of the No. 13 peak is 1.26-1.30, and the RSD is 0.06%;

the average relative retention time of No. 14 peak is 1.29-1.31, and the RSD is 0.07%;

the average relative retention time of the No. 15 peak is 1.33-1.37, and the RSD is 0.09%;

the average relative retention time of the No. 16 peak is 1.52-1.56, and the RSD is 0.08%;

the average relative retention time of the No. 17 peak is 1.56-1.60, and the RSD is 0.08%;

the average relative retention time of the No. 18 peak is 1.68-1.72, and the RSD is 0.08%;

the average relative retention time of the 19 th peak is 1.72-1.74, and the RSD is 0.08%;

the average relative retention time of the No. 20 peak is 1.77-1.81, and the RSD is 0.07%;

the average relative retention time of the No. 21 peak is 2.46-2.50, and the RSD is 0.21%;

the average relative retention time of No. 22 peak is 2.55-2.59, and RSD is 0.22%;

the average relative retention time of the No. 23 peak is 3.05-3.09, and the RSD is 0.27%.

9. The standard fingerprint of Guerin and tea of claim 7, wherein the average relative retention time of peak 1 is 0.36;

peak 2 average relative retention time 0.58;

peak 3 has an average relative retention time of 0.61;

peak No. 4 had an average relative retention time of 0.82;

peak 5 has an average relative retention time of 0.85;

peak No. 6 has an average relative retention time of 0.94;

peak No. 7 average relative retention time 0.96;

peak 8 average relative retention time 0.98;

peak 9 has an average relative retention time of 1;

peak 10 has an average relative retention time of 1.05;

peak 11 average relative retention time 1.06;

peak 12 average relative retention time 1.10;

peak 13 has an average relative retention time of 1.28;

peak 14 average relative retention time 1.31;

peak 15 has an average relative retention time of 1.35;

peak 16 has an average relative retention time of 1.54;

peak 17 average relative retention time 1.58;

peak 18 average relative retention time 1.70;

peak 19 has an average relative retention time of 1.74;

peak 20 has an average relative retention time of 1.79;

peak 21 has an average relative retention time of 2.48;

peak No. 22 has an average relative retention time of 2.57;

peak 23 had an average relative retention time of 3.07.

10. The standard fingerprint of Yuan Jilin gan and cha as claimed in claim 7, wherein the 1, 3, 20 peak attributes are senecio scandens; 4. the peak attributes of No. 6, 12, 14, 15, 17, 18 and 21 are the medicinal material of folium Viticis Cannabifoliae; the attribution of No. 7 peak is gardenia medicinal material; 9. the attribution of No. 10, No. 11 and No. 13 peaks is a kudzu root medicinal material; 19, the attribution of the peak is sophora flower medicinal material; the 22 # peak belongs to scutellaria baicalensis; 23, the attribution of the peak is liquorice; 2. peak 5 is the common peak of Senecio scandens, Vitex negundo L.leaf and Murraya koenigii; 8. peak 16 is the common peak of Qianlieguang and Vitex negundo L.leaf medicinal materials.