Construction method of chrysanthemum-tang compound fingerprint, fingerprint and application thereof

Abstract

The application discloses a kind of construction methods of Jujing compound fingerprint, comprising: (1) Jujing compound is made into test solution;(2) using chlorogenic acid, 6'-O-β-D-glucosyl gentiopicroside, swertiamarin, gentiopicroside, swertisin, 3-O-coffeicoyl shikimic acid, astilbin, astilbinin, rutin, isofraxin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside and zephyranthine preparation mixed reference solution;(3) respectively test solution and mixed reference solution are injected into high performance liquid chromatograph, the similarity analysis is carried out to the obtained chromatogram, and Jujing compound fingerprint is obtained.The application also provides Jujing compound fingerprint obtained by the construction method and its application in identifying and / or evaluating the quality of Jujing compound.The Jujing compound fingerprint of the application can comprehensively, objectively and accurately detect and evaluate the quality of Jujing compound, so as to ensure the safety and effectiveness of patient medication.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method of a Jujing compound fingerprint, the fingerprint and application thereof. BACKGROUND

[0002] The Jujing compound is composed of chicory, smilax glabra, gentiana, poria cocos and sophora fruit, wherein the chicory and smilax glabra invigorate the spleen, expel dampness and turbidity, dredge the joints, regulate uric acid metabolism, and are the monarch drug; the poria cocos and gentiana expel wind and dampness, remove arthralgia, and intervene in gouty inflammatory swelling and pain, and are the ministerial drug; the sophora fruit clears heat, cools blood, eliminates inflammation and relieves pain, and is the auxiliary drug. The whole prescription has the functions of invigorating the spleen, expelling dampness and turbidity, reducing uric acid, eliminating swelling and relieving pain. Literature research and network pharmacology research show that the prescription contains various chemical components that can act on uric acid generation, excretion and inflammation pathways, has the advantages of multiple components, multiple targets and multiple effects, and is a comprehensive anti-gout drug that is consistent with the modern pathological understanding of gout.

[0003] At present, the research on the Jujing compound is mainly focused on clinical application and pharmacology and efficacy, but there is a lack of effective methods for analyzing the influence of the differences in internal efficacy components on the quality of the Jujing compound.

[0004] The traditional Chinese medicine fingerprint is an effective means for identifying traditional Chinese medicine products and evaluating the quality of the products. For example, the construction method of the HPLC fingerprint of chicory and hairy chicory and the application of the obtained HPLC fingerprint are studied in “Construction of HPLC Fingerprint of Chicory and Hairy Chicory and Pattern Recognition Research” (Chinese Journal of Chinese Materia Medica, Vol. 38, No. 9, pp. 1401-1405). However, the components of the traditional Chinese medicine compound are complex, and the fingerprint construction method for single traditional Chinese medicine material in the document cannot be applied to the traditional Chinese medicine compound. In addition, the chicory and hairy chicory are extracted by ultrasonic extraction with an organic solvent methanol, which is inconsistent with the clinic.

[0005] Therefore, it is an urgent problem to be solved to establish the ultra-high performance liquid chromatography fingerprint characteristic spectrum of the Jujing compound, analyze the Jujing compound by a traditional Chinese medicine chromatography characteristic spectrum similarity evaluation system, establish a control fingerprint, and provide a basis for the quality control of the Jujing compound. SUMMARY

[0006] The present application aims to provide a construction method of a Jujing compound fingerprint, the fingerprint and application thereof, so as to realize comprehensive, objective and accurate evaluation of the quality of the Jujing compound.

[0007] The present application provides a construction method of a Jujing compound fingerprint, which comprises the following steps:

[0008] (1) preparing a test solution of the Jujing compound;

[0009] (2) at least two reference substances selected from the group consisting of chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swertiamarin, gentiopicroside, swertisin, 3-O-caffeoylshikimic acid, neoreviresin, reviresin, rutin, isoreviresin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside and zephyrol are used to prepare a mixed reference substance solution;

[0010] (3) the test substance solution and the mixed reference substance solution are injected into a high performance liquid chromatograph, similarity analysis is performed on the obtained chromatogram, and the jujing compound fingerprint is obtained.

[0011] Optionally, in step (1), the extract powder or extract powder of the jujing compound is extracted with methanol, the supernatant is filtered to obtain a filtrate as the test substance solution;

[0012] Preferably, in step (1), the extract powder or extract powder of the jujing compound is extracted with 70% methanol, the supernatant is filtered with a 0.22 μm microporous filter to obtain a filtrate as the test substance solution.

[0013] Optionally, in step (2), chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swertiamarin, gentiopicroside, swertisin, 3-O-caffeoylshikimic acid, neoreviresin, reviresin, rutin, isoreviresin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside and zephyrol are used to prepare a mixed reference substance solution.

[0014] Optionally, in step (2), the reference substances are mixed and then dissolved in methanol to obtain the mixed reference substance solution; or the reference substances are respectively prepared into reference substance solutions with methanol, and then mixed to obtain the mixed reference substance solution.

[0015] Preferably, in step (2), the reference substances are mixed in equal proportions and then dissolved in 70% methanol to obtain the mixed reference substance solution, in which the content of each reference substance is 1 μg / ml; or the reference substances are respectively prepared into reference substance solutions with 70% methanol, and then mixed to obtain the mixed reference substance solution, in which the content of each reference substance is 1 μg / ml.

[0016] Optionally, in step (3), the chromatographic column of the high performance liquid chromatograph is an ACCQUITY UPLC HSS T3 chromatographic column, methanol is used as mobile phase A, 0.1% formic acid aqueous solution is used as mobile phase B, the flow rate is 0.3 mL / min, the column temperature is 30 ℃, the injection amount is 2 μL, and the detection wavelength is 300 nm.

[0017] Preferably, the chromatographic column of the high performance liquid chromatograph adopts an ACCQUITY UPLC HSS T3 chromatographic column with a specification of 2.1*100 mm, 1.8 μm.

[0018] Optionally, in step (3), the elution condition of the high performance liquid chromatograph is:

[0019] 0~2 minutes, the volume fraction of mobile phase A is increased from 5% to 10%, and the volume fraction of mobile phase B is decreased from 95% to 90%;

[0020] 2~16 minutes, the volume fraction of mobile phase A is increased from 10% to 40%, and the volume fraction of mobile phase B is decreased from 90% to 60%;

[0021] 16~26 minutes, the volume fraction of mobile phase A is increased from 40% to 54%, and the volume fraction of mobile phase B is decreased from 60% to 46%;

[0022] 26~26.5 minutes, the volume fraction of mobile phase A is increased from 54% to 95%, and the volume fraction of mobile phase B is decreased from 46% to 5%;

[0023] 26.5~27.5 minutes, the volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%;

[0024] 27.5~28 minutes, the volume fraction of mobile phase A is decreased from 95% to 5%, and the volume fraction of mobile phase B is increased from 5% to 95%.

[0025] The application further provides a Jujing compound fingerprint obtained by the construction method, and the Jujing compound fingerprint comprises 15 fingerprint peaks.

[0026] The application further provides application of the Jujing compound fingerprint in identification and / or evaluation of the quality of the Jujing compound.

[0027] The application further provides a method for identifying and / or evaluating the quality of the Jujing compound, comprising: comparing the high performance liquid chromatogram of the Jujing compound to be tested with the above-mentioned Jujing compound fingerprint, and identifying the similarity greater than 0.9 as qualified.

[0028] From the above technical solution, the method for constructing the Jujing compound fingerprint, the Jujing compound fingerprint and the application thereof have at least the following beneficial effects:

[0029] According to the structural properties of the active ingredients contained in the Jujing compound, including flavonoids, organic acids, iridoid glycosides and the like, the chromatographic analysis conditions such as the composition of the mobile phase, the gradient elution program, the detection wavelength, the chromatographic column and the column temperature are determined through experiments. It is proved through multiple experiments that the Jujing compound fingerprint obtained by using the construction method of the application can comprehensively, objectively and accurately detect and evaluate the quality of the Jujing compound, which has important significance for ensuring the safety and effectiveness of the patient's medication.

[0030] For the Jujing compound with complex components, the application more comprehensively reveals the fingerprint thereof through the ultra-high performance liquid chromatography (UHPLC), which is a further supplement to the research on the fingerprint of the single medicinal material Jujing and Maojujing. In addition, the Jujing compound is extracted by the method of water decoction, which is closer to the clinical medication. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 The retention time of the common fingerprint peaks of the Jujing compound in 10 batches in Example 1 of the application is shown.

[0032] Figure 2 The control fingerprint obtained by using the Jujing compound in 10 batches in Example 1 of the application is shown. DETAILED DESCRIPTION

[0033] In order to fully understand the purpose, features and effects of the application, the application is described in detail through the following specific embodiments. The method of the application adopts the conventional method or device in the art except the following content. Unless otherwise specified, the technical and scientific terms involved in the application have the meanings commonly understood by the person skilled in the art.

[0034] In the present application, the so-called "Juling compound" refers to a traditional Chinese medicine composition prepared from radix taraxaci, poria cocos, smilax glabra, sophora fruit and gentiana. Preferably, the traditional Chinese medicine composition is prepared from radix taraxaci 6-18 parts by weight, poria cocos 5-15 parts by weight, smilax glabra 10-60 parts by weight, sophora fruit 5-10 parts by weight and gentiana 3-10 parts by weight; further preferably, the traditional Chinese medicine composition is prepared from radix taraxaci 6-10 parts by weight, poria cocos 10-15 parts by weight, smilax glabra 20-40 parts by weight, sophora fruit 5-10 parts by weight and gentiana 3-10 parts by weight; most preferably, the traditional Chinese medicine composition is prepared from radix taraxaci 9 parts by weight, poria cocos 10 parts by weight, smilax glabra 30 parts by weight, sophora fruit 6 parts by weight and gentiana 9 parts by weight. Specifically, reference can be made to the traditional Chinese medicine composition described in the Chinese invention patent application with the application number 202210821181.9, which is incorporated herein by reference in its entirety.

[0035] Juling compound contains a variety of chemical components that can act on uric acid generation, excretion and inflammatory pathways, and has the advantages of multiple components, multiple targets and multiple effects in prevention and treatment. However, at present, the content control of traditional Chinese medicine compound preparations basically has only one or two index components, and the quality control is single and the index is few, so the existing evaluation method is difficult to fully reflect the quality status of Juling compound composition with multiple herbs, multiple components and multiple targets.

[0036] In view of these problems, the inventors of the present application apply traditional Chinese medicine HPLC fingerprint technology to the quality evaluation of Juling compound, optimize and improve the chromatographic analysis conditions of HPLC fingerprint technology such as mobile phase composition, gradient elution program, detection wavelength, chromatographic column and column temperature according to the active components and structural properties of Juling compound, and screen matching reference substances, thereby proposing a method for constructing the fingerprint of Juling compound.

[0037] In the present application, the so-called "Juling compound" refers to a traditional Chinese medicine composition prepared from radix taraxaci, poria cocos, smilax glabra, sophora fruit and gentiana. Preferably, the traditional Chinese medicine composition is prepared from radix taraxaci 6-18 parts by weight, poria cocos 5-15 parts by weight, smilax glabra 10-60 parts by weight, sophora fruit 5-10 parts by weight and gentiana 3-10 parts by weight; further preferably, the traditional Chinese medicine composition is prepared from radix taraxaci 6-10 parts by weight, poria cocos 10-15 parts by weight, smilax glabra 20-40 parts by weight, sophora fruit 5-10 parts by weight and gentiana 3-10 parts by weight; most preferably, the traditional Chinese medicine composition is prepared from radix taraxaci 9 parts by weight, poria cocos 10 parts by weight, smilax glabra 30 parts by weight, sophora fruit 6 parts by weight and gentiana 9 parts by weight. Specifically, reference can be made to the traditional Chinese medicine composition described in the Chinese invention patent application with the application number 202210821181.9, which is incorporated herein by reference in its entirety.

[0038] In the present application, the so-called "Juling compound" refers to a traditional Chinese medicine composition prepared from radix taraxaci, poria cocos, smilax glabra, sophora fruit and gentiana. Preferably, the traditional Chinese medicine composition is prepared from radix taraxaci 6-18 parts by weight, poria cocos 5-15 parts by weight, smilax glabra 10-60 parts by weight, sophora fruit 5-10 parts by weight and gentiana 3-10 parts by weight; further preferably, the traditional Chinese medicine composition is prepared from radix taraxaci 6-10 parts by weight, poria cocos 10-15 parts by weight, smilax glabra 20-40 parts by weight, sophora fruit 5-10 parts by weight and gentiana 3-10 parts by weight; most preferably, the traditional Chinese medicine composition is prepared from radix taraxaci 9 parts by weight, poria cocos 10 parts by weight, smilax glabra 30 parts by weight, sophora fruit 6 parts by weight and gentiana 9 parts by weight. Specifically, reference can be made to the traditional Chinese medicine composition described in the Chinese invention patent application with the application number 202210821181.9, which is incorporated herein by reference in its entirety.

[0039] (1) Prepare a test sample solution from Juling compound.

[0040] In the present application, the extract powder or extract powder of Jiling compound can be used to prepare the test solution.

[0041] In a preferred embodiment, the extract powder of Jiling compound is used to prepare the test solution. Specifically, the extract powder of Jiling compound is accurately weighed and placed in a conical flask with a plug, extracted with 70% methanol, weighed again after the extract is cooled to room temperature, and the missing weight is made up with 70% methanol solution. The supernatant is filtered with a filter membrane, and the filtrate is obtained as the test solution of Jiling compound. The preparation method of the extract powder of Jiling compound can refer to the related schemes in the prior art, and the present application is not limited in particular.

[0042] In another preferred embodiment, the extract powder of Jiling compound is used to prepare the test solution. Specifically, the extract powder is accurately weighed and placed in a conical flask with a plug, added with 70% methanol (v / v) to 10 mL, diluted to 6 mg / mL, shaken, ultrasonically filtered for 30 min, and the filtrate is obtained as the test solution.

[0043] The preparation method of the extract powder of Jiling compound can refer to the related schemes in the prior art. For example, the extract powder of Jiling compound can be prepared by the following method: the Jiling compound proportionally accurate Jiling, Yunnan rhizoma gentianae, sophora fruit, tuckahoe, and smilax rhizome decoction pieces are weighed and added with 12 times the amount of water to soak for 30 min, and then heated and decocted. After boiling, decoct for 1.5 h, filter. Repeat the extraction for 3 times, combine the water decoction, and reduce the pressure to concentrate to contain 1 g / mL of crude drug in a rotary evaporator. The compound concentrated liquid is placed in a water bath at 80 ℃ to evaporate until no flow is obtained, and then placed in a vacuum drying oven for drying. The drying temperature is kept at 80 ℃, and the drying is continued for 7 days. After taking out, it is placed at room temperature and crushed to obtain the compound extract powder.

[0044] (2) Preparation of mixed reference solution.

[0045] According to the characteristics of the active ingredients of Jiling compound, at least 2 substances are selected from the group consisting of chlorogenic acid, 6'-O-β-D-glucosylgentialoside, swertiamarin, gentiopicroside, swertisin, 3-O-coffeoylshikimic acid, neoreinvin, reinvirin, rutin, isoreinvin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and zephyranthine as the reference, and preferably, chlorogenic acid, 6'-O-β-D-glucosylgentialoside, swertiamarin, gentiopicroside, swertisin, 3-O-coffeoylshikimic acid, neoreinvin, reinvirin, rutin, isoreinvin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and zephyranthine are used as the reference to prepare the mixed reference solution.

[0046] In the present application, the mixed control solution can be obtained by mixing the control samples and then dissolving them in methanol, or the control samples can be prepared into control sample solutions respectively using methanol, and then mixed to obtain the mixed control solution.

[0047] In a preferred embodiment, chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swertiamarin, gentiopicroside, sweroside, 3-O-caffeoylshikimic acid, reichardtianoside, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, zephyranthine reference samples are precisely weighed respectively, placed in a volumetric flask, dissolved and diluted with 70% methanol, shaken uniformly, filtered with a microporous filter membrane to obtain a mixed control solution.

[0048] In another preferred embodiment, chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swertiamarin, gentiopicroside, sweroside, 3-O-caffeoylshikimic acid, reichardtianoside, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, zephyranthine reference samples are precisely weighed respectively, dissolved in 70% methanol to prepare a solution containing about 100 μg per 1 ml (i.e. 100 μg / ml of control sample solution). 1 ml of each of the above control sample solutions is precisely measured and placed in the same 100 ml volumetric flask, 70% methanol is added to the mark, shaken uniformly to obtain a mixed control solution, which contains 1 μg of each control sample per 1 ml of mixed control solution, i.e. the content of each control sample in the mixed control solution is 1 μg / ml.

[0049] (3) The test sample solution and the mixed control sample solution are injected into the high performance liquid chromatograph, and similarity analysis is performed on the obtained chromatogram to obtain the Jilong compound fingerprint.

[0050] In the present application, the chromatographic conditions are as follows: the chromatographic column is ACCQUITY UPLC HSS T3 chromatographic column, the specification of which is preferably an inner diameter of 2.1 mm, a column length of 100 mm, a particle size of 1.8 μm, methanol is used as mobile phase A, 0.1% formic acid aqueous solution is used as mobile phase B, the flow rate is 0.3 mL / min, the column temperature is 30 ℃, the injection amount is 2 μL, and the detection wavelength is 300 nm.

[0051] In the present application, the gradient elution is performed according to the following table:

[0052]

[0053] The chromatogram obtained by high performance liquid chromatography is imported into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system (version 2012.130723) to obtain the Jilong compound fingerprint.

[0054] It should be noted that the various substances involved in the construction method of the present application, such as traditional Chinese medicinal materials, reference substances, etc., can be obtained by market purchase.

[0055] In a second aspect, the present application provides a Jujing compound fingerprint spectrum, comprising 15 fingerprint peaks.

[0056] Reference Figure 2 In the numbering in the present application, the key peaks of the Jujing compound fingerprint spectrum of the present application include fingerprint peaks 2, 3, 5, 8, 9, 10, 11, etc. Among them, the relative retention time of peak 2 is 9.64 min, and the corresponding chemical substance is chlorogenic acid; the relative retention time of peak 3 is 10.57 min, and the corresponding chemical substance is 6'-O-β-D-glucosylgentiopicroside; the relative retention time of peak 5 is 11.51 min, and the corresponding chemical substance is gentiopicroside; the relative retention time of peak 8 is 16.73 min, and the corresponding chemical substance is neorendering new wife glycoside; the relative retention time of peak 9 is 17.16 min, and the corresponding chemical substance is astilbin; the relative retention time of peak 10 is 18.50 min, and the corresponding chemical substance is rutin; the relative retention time of peak 11 is 18.90 min, and the corresponding chemical substance is isorendering new wife glycoside.

[0057] The Jujing compound fingerprint spectrum of the present application can be used for comprehensive, objective and accurate detection and evaluation of the quality of Jujing compound, so in a third aspect, the present application provides a method for identifying and / or evaluating the quality of Jujing compound, comprising: obtaining the high performance liquid chromatogram of the Jujing compound to be tested by using the above-mentioned chromatographic conditions and gradient elution program, comparing the high performance liquid chromatogram with the Jujing compound fingerprint spectrum of the present application, and if the similarity is greater than 0.9, it is considered that the Jujing compound to be tested is a qualified product.

[0058] Example

[0059] The present application will be further described by way of examples below, but the present application is not limited in the scope of the examples. The experimental methods not specified in the following examples are selected according to conventional methods and conditions, or according to the instructions of the commodity.

[0060] Example 1: Establishment and application of Jujing compound reference fingerprint spectrum

[0061] 1. Instruments and reagents, medicinal materials

[0062] Waters ACQUITY UPLC H-CLASS ultra-high performance liquid chromatograph, ACCQUITY UPLC HSS T3 chromatographic column; KQ-5200E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., power 250 W, frequency 40 kHz), MTW120 electronic balance (Shenzhen Meters & Instruments Co., Ltd.); pulverizer (Xiaoxiong Electrical Appliance Co., Ltd.).

[0063] Methanol, formic acid (chromatographically pure, Fisher Company, USA); distilled water (Watsons Industrial Co., Ltd.); pure water was provided by the laboratory.

[0064] Five medicinal materials of Chong Lou compound were collected from all over the country, 10 batches of each medicinal material, and the origin and batch were shown in Table 1 and Table 2.

[0065] Table 1

[0066]

[0067] Table 2

[0068]

[0069] 2. Construction of Chong Lou compound fingerprint

[0070] (1) Preparation of test solution

[0071] According to Chong Lou 9 g, Fuling 10 g, Tufuling 30 g, Huai Mi 6 g and Qinjia 9 g, accurately weigh each decoction piece of S1 to S10 batch in Table 1 and Table 2 to obtain 10 batches of test samples. For the 10 batches of test samples, the following operations were carried out respectively:

[0072] After adding 12 times water and soaking for 30 min, heating and decocting. After boiling, decocting for 1.5 h, filtering. Repeating extraction for 3 times, combining water decoction, reducing pressure concentration to contain 1 g / mL of crude drug in rotary evaporator, to obtain compound water decoction concentrate. After the compound concentrate was placed in 80 ℃ water bath and evaporated to no flow, it was placed in a vacuum drying oven, the drying temperature was kept at 80 ℃, and the drying was continued for 7 days. After taking out and placing at room temperature, it was pulverized to obtain compound extract powder;

[0073] Precisely weigh the extract powder, place it in a stoppered conical flask, add 70% methanol to 10 mL, dilute to 6 mg / mL, shake, ultrasonic for 30 min, filter, take the filtrate, filter through 0.22 μm microporous filter membrane, to obtain the test solution.

[0074] (2) Preparation of mixed reference solution

[0075] A certain amount of chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swertiamarin, gentiopicroside, sweroside, 3-O-caffeoylshikimic acid, reichardtin, astilbin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, zephyranthine reference substance were precisely weighed, dissolved in 70% methanol to prepare a solution containing about 100 μg per 1 ml. 1 ml of the above reference solution was precisely measured and placed in the same 100 ml volumetric flask, 70% methanol was added to the mark, and shaken to obtain a mixed reference solution (1 μg of each reference substance per 1 ml);

[0076] (3) Determination of Jilong Compound Fingerprint by Ultra-high Performance Liquid Chromatography

[0077] The 10 batches of Jilong Compound test sample solutions prepared in step (1) and the mixed reference solution prepared in step (2) were injected into a high performance liquid chromatograph.

[0078] Chromatographic conditions: ACCQUITY UPLC HSS T3 chromatographic column (inner diameter 2.1 mm, column length 100 mm, particle size 1.8 μm) was used, methanol was used as mobile phase A, 0.1% formic acid aqueous solution was used as mobile phase B, the flow rate was 0.3 mL / min, the column temperature was 30 ℃, the injection volume was 2 μL, the detection wavelength was 300 nm, and gradient elution was performed according to the following table.

[0079]

[0080] The obtained fingerprint chromatograms of the 10 batches of Jilong Compound test sample solutions were exported and imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (version 2012.130723): the chromatographic peaks that existed in the chromatograms of the 10 batches of Jilong Compound and had high content were selected as common peaks; the median calculation method was used to generate the control fingerprint of Jilong Compound, the relative retention time and relative peak area of each common peak were calculated, and there were 15 common fingerprint peaks in the control fingerprint, and the similarity of the fingerprint chromatograms of the 10 batches of test sample solutions was greater than 0.90.

[0081] The retention time of the 15 common fingerprint peaks is shown in Table Figure 1 The control fingerprint obtained in this example is shown in Figure Figure 2

[0082] Example 2: Methodology Investigation

[0083] 1. Precision experiment

[0084] ​The same batch of Jilingsuifang powder was precisely taken, and a test sample solution was prepared according to the method of step (1) in Example 1. The sample was injected 6 times according to the chromatographic conditions of step (3) in Example 1, and the RSD values of the peak areas of common peaks 1, 2, 3, 5, 6, 7, 8 and 10 were all less than 4.0%, and the RSD values of the retention times were all less than 1.0%, proving that the instrument precision was good.

[0085] 2. Reproducibility test

[0086] The same batch of Jilingsuifang powder was precisely taken, and 6 test sample solutions were prepared in parallel according to the method of step (1) in Example 1. The sample was injected 6 times according to the chromatographic conditions of step (3) in Example 1, and the RSD values of the peak areas of common peaks 1, 2, 3, 5, 6, 7, 8 and 10 were all less than 3.5%, and the RSD values of the retention times were all less than 1.0%, proving that the method had good reproducibility.

[0087] 3. Stability test

[0088] The same batch of Jilingsuifang powder was precisely taken, and a test sample solution was prepared according to the method of step (1) in Example 1. The sample was injected and analyzed at 0 h, 1 h, 2 h, 5 h, 12 h and 24 h after sample preparation according to the chromatographic conditions of step (3) in Example 1, and the RSD values of the peak areas of common peaks 1, 2, 3, 5, 6, 7, 8 and 10 were all less than 3.5%, and the RSD values of the retention times were all less than 1.0%, proving that the test sample solution had good stability within 24 h.

[0089] Through the above research, it can be determined that the UHPLC fingerprint of Jilingsuifang is established by using the fingerprint technology, which can quickly and intuitively identify the effective material basis in a complex traditional Chinese medicine decoction system. Meanwhile, combined with the gray correlation analysis and the partial least squares analysis, the quality differences between samples of different batches are evaluated, which provides a reference for the key quality attribute research of Jilingsuifang, and provides a beneficial reference for the preparation process, quality control and product safety and stability evaluation of subsequent related preparations.

[0090] Finally, it should be noted that: the above-mentioned is only a specific embodiment of the present application, of course, the person skilled in the art can modify and change the present application, if these modifications and changes belong to the scope of the claims of the present application and its equivalent technology, all should be considered as the protection scope of the present application.

Claims

1. A method for constructing a compound fingerprint of Jujing, characterized in that, The method comprises the following steps: (1) extracting the extract powder of the Chukang compound with methanol to obtain a supernatant, filtering the supernatant to obtain a filtrate as a test solution; The Chukang compound is composed of chicory, smilax glabra, gentiana, poria cocos, and sophora fruit; (2) preparing a mixed control solution with chlorogenic acid, 6'-O-β-D-glucosylgentiopicroside, swertiamarin, gentiopicroside, swertisin, 3-O-caffeoylshikimic acid, astilbin, astralin, rutin, isoastilbin, isochlorogenic acid C, quercitrin, kaempferol-3-O-rutinoside, and zephyranthine; (3) injecting the test solution and the mixed control solution into a high-performance liquid chromatograph, and performing similarity analysis on the obtained chromatogram to obtain a fingerprint of the Chukang compound; The chromatographic column of the high-performance liquid chromatograph is an ACCQUITY UPLC HSS T3 chromatographic column, methanol is used as mobile phase A, 0.1% formic acid aqueous solution is used as mobile phase B, and the detection wavelength is 300 nm; The elution conditions of the high-performance liquid chromatograph are as follows: 0-2 minutes, the volume fraction of mobile phase A is increased from 5% to 10%, and the volume fraction of mobile phase B is decreased from 95% to 90%; 2-16 minutes, the volume fraction of mobile phase A is increased from 10% to 40%, and the volume fraction of mobile phase B is decreased from 90% to 60%; 16-26 minutes, the volume fraction of mobile phase A is increased from 40% to 54%, and the volume fraction of mobile phase B is decreased from 60% to 46%; 26-26.5 minutes, the volume fraction of mobile phase A is increased from 54% to 95%, and the volume fraction of mobile phase B is decreased from 46% to 5%; 26.5-27.5 minutes, the volume fraction of mobile phase A is 95%, and the volume fraction of mobile phase B is 5%; 27.5-28 minutes, the volume fraction of mobile phase A is decreased from 95% to 5%, and the volume fraction of mobile phase B is increased from 5% to 95%.

2. The construction method according to claim 1, characterized in that, In step (1), the extract powder of the Chukang compound is extracted with 70% methanol, the obtained supernatant is filtered with a 0.22 μm microporous filter to obtain a filtrate as the test solution.

3. The construction method of claim 1, wherein, In step (2), the mixed control solution is obtained by dissolving the control substances with methanol; or, the control substances are respectively prepared into control substance solutions with methanol, and then mixed to obtain the mixed control solution.

4. The construction method according to claim 3, characterized in that, In step (2), the control substances are mixed in equal proportions and then dissolved with 70% methanol to obtain the mixed control solution with a content of 1 μg / ml of each control substance; or, the control substances are respectively prepared into control substance solutions with 70% methanol, and then mixed to obtain the mixed control solution with a content of 1 μg / ml of each control substance.

5. The construction method of claim 1, wherein In step (3), the flow rate of the mobile phase is 0.3 mL / min, the column temperature of the chromatographic column is 30 ℃, and the injection amount is 2 μL.

6. The construction method of claim 1, wherein, The chromatographic column of the high performance liquid chromatograph in step (3) is an ACCQUITY UPLC HSS T3 chromatographic column with a specification of 2.1 mm x 100 mm, 1.8 μm.

7. The use of the Jujing compound fingerprint spectrum of any one of claims 1-6 in identifying and / or evaluating the quality of the Jujing compound.

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