Construction method and application of fingerprint spectrum of Hexishi chrysanthemum

Abstract

The invention provides a construction method and application of a fingerprint spectrum of Hexishi chrysanthemum. The construction method comprises the following steps: a) preparation of reference substance solutions: taking aesculetin, scutellarin, apigenin and luteolin as reference substances, respectively adding solutions for dissolving to obtain the reference substance solutions, and the reference substance solutions comprise an aesculetin solution, a scutellarin solution, an apigenin solution and a luteolin solution; b) preparation of a test solution: mixing the powder of the Hexishi chrysanthemum with a methanol solution, carrying out heating reflux treatment, cooling, and filtering to obtain the test solution; and c) placing the test solution and the plurality of reference substance solutions in a high performance liquid chromatograph, and measuring to obtain a high performance liquid chromatogram of the test solution and high performance liquid chromatograms of the reference substance solutions. The method can solve the problem of lack of a method for controlling the quality of the Hexishi chrysanthemum in the prior art, and is suitable for the field of traditional Chinese medicine analysis.

Description

Technical Field

[0001] The present invention relates to the field of traditional Chinese medicine analysis, and in particular to a method for constructing a fingerprint spectrum of Chrysanthemum indica and an application thereof. Background Art

[0002] Hexinia polydichotoma (Ostenf.) HLYang, belongs to the Asteraceae family, is generally about 15-40 cm high, and is a perennial herb. It grows in sandy land, sandy land edges, lowlands between dunes, Gobi gullies and sandy land fields, at an altitude of -42-1800 meters. Hexinia polydichotoma is a xerophyte with small yellow flowers and degenerate leaves. Because its branches resemble deer horns, it is also known as deer horn grass. It has extremely high ornamental value and is a superior ground cover plant and bonsai plant for urban greening in arid areas. Because of its drought resistance and salt-alkali resistance, it is widely distributed in the desert areas of the Tarim Basin in Xinjiang. It is an important windbreak and sand-fixing plant and has important ecological value in the desert edge. Modern research reports on Hexinia polydichotoma mainly focus on reports on desert plants in ecology, landscape, etc. Chinese patent CN114984075B discloses a preparation method and preparation and use of Hexinia polydichotoma extract, which is mainly used for medicinal value. However, in the prior art, there is no report on the quality control method for Hexi Chrysanthemum as a medicinal material. Summary of the invention

[0003] The main purpose of the present invention is to provide a method for constructing a fingerprint of Chrysanthemum chinense and its application, so as to solve the problem that there is a lack of a method for quality control of Chrysanthemum chinense in the prior art.

[0004] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, a method for constructing a fingerprint of Chrysanthemum officinale is provided, and the construction method comprises: a) preparing a reference solution: taking scutellaria baicalin, apigenin and luteolin as reference substances, respectively adding solutions to dissolve them to obtain reference solutions, and the reference solutions comprise scutellaria baicalin solution, apigenin solution and luteolin solution; b) preparing a test solution: mixing the powder of Chrysanthemum officinale with a methanol solution and heating and refluxing the mixture, cooling the mixture and filtering the mixture to obtain a test solution; c) placing the test solution and a plurality of reference solution solutions in a high performance liquid chromatograph, and measuring and obtaining a high performance liquid chromatogram of the test solution and a high performance liquid chromatogram of the reference solution.

[0005] Furthermore, the concentration of scutellarin in the scutellarin solution is 55.00-75.00 μg / mL; preferably, the concentration of scutellarin in the scutellarin solution is 50.00-125.00 μg / mL; preferably, the concentration of apigenin in the apigenin solution is 50.00-90.00 μg / mL; preferably, the concentration of luteolin in the luteolin solution is 80.00-100.00 μg / mL.

[0006] Furthermore, the heating reflux treatment time is 30-60 min; preferably, the heating reflux treatment temperature is 80-85° C.; preferably, the volume fraction of methanol in the methanol solution is 50%-80%.

[0007] Furthermore, the filtering includes: filtering using a 0.22 μm microporous filter membrane.

[0008] Furthermore, the chromatographic conditions of the high performance liquid chromatograph include: the chromatographic column uses octadecylsilane as a filler, acetonitrile as a mobile phase A, and a 0.1-0.4% (v / v) aqueous solution of an inorganic acid as a mobile phase B, a flow rate of 0.8-1.2 mL / min, a column temperature of 25-35° C., a detection wavelength of 240-255 nm, and a gradient elution; the elution conditions are: 0-10 min, the volume fraction of the mobile phase A is 8%, and the volume fraction of the mobile phase B is 92%; 10-17 min, the volume fraction of the mobile phase A increases from 8% to 15%, and the volume fraction of the mobile phase B decreases from 92% to 85%; 17-32 min, the volume fraction of the mobile phase A increases from 15% to 17%, and the volume fraction of the mobile phase B decreases from 85% to 83%; 32-6 2min, the volume fraction of mobile phase A increases from 17% to 27%, and the volume fraction of mobile phase B decreases from 83% to 73%; 62-82min, the volume fraction of mobile phase A increases from 27% to 28%, and the volume fraction of mobile phase B decreases from 73% to 72%; 82-92min, the volume fraction of mobile phase A increases from 28% to 31%, and the volume fraction of mobile phase B decreases from 72% to 69%; 92-100min, the volume fraction of mobile phase A increases from 31% to 45%, and the volume fraction of mobile phase B decreases from 69% to 55%; 100-110min, the volume fraction of mobile phase A decreases from 45% to 8%, and the volume fraction of mobile phase B increases from 55% to 92%; preferably, the chromatographic column is an Eternity XT-5-C18 column.

[0009] Further, the inorganic acid includes phosphoric acid.

[0010] Furthermore, the construction method also includes: importing the high performance liquid chromatogram of the test solution and the high performance liquid chromatogram of the reference solution into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for analysis, thereby obtaining the fingerprint of Hexi chrysanthemum.

[0011] In order to achieve the above-mentioned purpose, according to the second aspect of the present invention, a quality detection method for Hexi chrysanthemum is provided, and the quality detection method comprises: using the above-mentioned Hexi chrysanthemum fingerprint map construction method to construct a fingerprint map of the Hexi chrysanthemum sample to be tested, and obtaining the fingerprint map of the Hexi chrysanthemum sample to be tested; comparing the obtained fingerprint map of the Hexi chrysanthemum sample to be tested with the standard fingerprint map obtained under the same fingerprint map detection conditions for similarity, so as to realize the quality detection of the Hexi chrysanthemum sample to be tested.

[0012] Furthermore, there are 12 peaks in the standard fingerprint; among them, peak No. 1 is the peak of scutellariae, peak No. 2 is the peak of baicalin, peak No. 9 is the peak of apigenin, and peak No. 11 is the peak of luteolin.

[0013] Furthermore, taking peak 11 as the positioning peak, the relative retention time is 1.000; the average relative retention times of the other 11 common peaks are: the average relative retention time of peak 1 is 0.2346±0.0023; the average relative retention time of peak 2 is 0.4851±0.0030; the average relative retention time of peak 3 is 0.5432±0.0020; the average relative retention time of peak 4 is 0.6017±0.0036; the average relative retention time of peak 5 is 0.61 42±0.0023; the average relative retention time of peak 6 was 0.6443±0.0026; the average relative retention time of peak 7 was 0.7253±0.0021; the average relative retention time of peak 8 was 0.7570±0.0023; the average relative retention time of peak 9 was 0.8008±0.0016; the average relative retention time of peak 10 was 0.8315±0.0016; the average relative retention time of peak 12 was 1.1340±0.0028.

[0014] By applying the technical solution of the present invention, a high-performance liquid phase fingerprint of Hexi chrysanthemum is established, which can detect multiple chemical components in Hexi chrysanthemum. The fingerprint construction method is simple, and the obtained fingerprint is stable and reliable, with high accuracy and good repeatability, which can realize rapid, accurate and comprehensive quality control of Hexi chrysanthemum medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0016] Figure 1 The fingerprint of the precision test in Example 1 of the present invention is shown.

[0017] Figure 2 The fingerprint of the stability test in Example 1 of the present invention is shown.

[0018] Figure 3 The fingerprint of the repeatability test in Example 1 of the present invention is shown.

[0019] Figure 4 The chromatogram of aesculetin in Example 1 according to the present invention is shown.

[0020] Figure 5 The chromatogram of scutellariae in Example 1 of the present invention is shown.

[0021] Figure 6 A chromatogram of apigenin in Example 1 according to the present invention is shown.

[0022] Figure 7 The chromatogram of luteolin in Example 1 according to the present invention is shown.

[0023] Figure 8 The standard fingerprint of Hexi Chrysanthemum medicinal material in Example 1 of the present invention is shown. DETAILED DESCRIPTION

[0024] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0025] As mentioned in the background technology, there is a lack of methods for component analysis and quality control of Hexi chrysanthemum in the prior art. In the previous research of the inventor, it was found that the plant contains a large number of chemical components, mainly phenolic acids, flavonoids, polysaccharides, saponins, phenylpropanoids, etc.; it has a variety of biological activities. The fingerprint of Chinese medicinal materials is obtained by using certain analytical methods and detection instruments. It can identify the characteristics of the Chinese medicinal materials and is one of the methods that can more comprehensively reflect the intrinsic quality of Chinese medicinal materials.

[0026] Therefore, in this application, the inventor attempts to use the HPLC method to establish a fingerprint of Hexi chrysanthemum to provide a basis for the development and utilization, scientific identification and quality control of Hexi chrysanthemum, and on this basis proposes a series of protection schemes of this application.

[0027] In a first typical embodiment of the present application, a method for constructing a fingerprint of Chrysanthemum officinale is provided, and the method comprises: a) preparing a reference solution: taking scutellaria baicalin, apigenin and luteolin as reference substances, respectively adding solutions to dissolve them to obtain reference solutions, and the reference solutions comprise scutellaria baicalin solution, apigenin solution and luteolin solution; b) preparing a test solution: mixing the powder of Chrysanthemum officinale with a methanol solution and heating and refluxing the mixture, cooling the mixture and filtering the mixture to obtain a test solution; c) placing the test solution and a plurality of reference solution solutions in a high performance liquid chromatograph, and measuring and obtaining a high performance liquid chromatogram of the test solution and a high performance liquid chromatogram of the reference solution.

[0028] The fingerprint of traditional Chinese medicine refers to a chromatogram that can indicate the chemical characteristics of certain Chinese medicinal materials after they have been properly processed and analyzed using certain analytical methods. Through this method, the fingerprint of Hexi chrysanthemum can be constructed to achieve accurate qualitative and quantitative analysis of the main active ingredients in Hexi chrysanthemum, providing a powerful tool for quality control and efficacy evaluation of Hexi chrysanthemum. In the production process of traditional Chinese medicine preparations, this method can be used for the screening and quality control of raw materials to ensure the effectiveness and consistency of the final product.

[0029] In a preferred embodiment, the concentration of scutellarin in the scutellarin solution is 55.00-75.00 μg / mL; preferably, the concentration of scutellarin in the scutellarin solution is 50.00-125.00 μg / mL; preferably, the concentration of apigenin in the apigenin solution is 50.00-90.00 μg / mL; preferably, the concentration of luteolin in the luteolin solution is 80.00-100.00 μg / mL.

[0030] By optimizing the concentration of the reference solution, the sensitivity and accuracy of the detection can be improved, making the fingerprint of Hexi chrysanthemum clearer and facilitating subsequent analysis and comparison.

[0031] The concentration of aesculetin in the aesculetin solution includes but is not limited to 55, 60, 65, 70 or 75 μg / mL.

[0032] The concentration of scutellarin in the above scutellarin solution includes but is not limited to 50, 75, 100 or 125 μg / mL.

[0033] The concentration of apigenin in the above-mentioned esculetin solution includes but is not limited to 50, 60, 70, 80, 90 or 100 μg / mL.

[0034] The concentration of luteolin in the above-mentioned esculetin solution includes but is not limited to 80, 85, 90, 95 or 100 μg / mL.

[0035] In a preferred embodiment, the heating reflux treatment time is 30-60 minutes; preferably, the heating reflux treatment temperature is 80-85°C.

[0036] In this application, the chemical components in the Hexi chrysanthemum sample are extracted by heating reflux. Appropriate reflux time can ensure that the chemical components in the Hexi chrysanthemum are fully dissolved, improve the extraction efficiency, and avoid the degradation of components caused by long-term reflux. In the optimization of traditional Chinese medicine extraction technology, the setting of this parameter is of great significance for improving the utilization efficiency of medicinal materials and reducing production costs.

[0037] In a preferred embodiment, the volume fraction of methanol in the methanol solution is 50%-80%.

[0038] By adjusting the volume fraction of methanol, the extraction conditions can be optimized, so that the specific components in Hexi chrysanthemum can be extracted more effectively, while reducing the interference of impurities. In the quality control of traditional Chinese medicine, this optimization can improve the accuracy and reliability of detection and ensure the safety and effectiveness of traditional Chinese medicine preparations.

[0039] In a preferred embodiment, the filtering includes: filtering using a 0.22 μm microporous filter membrane.

[0040] The use of microporous filter membrane can effectively remove impurities and particles in the extract, ensuring that subsequent high performance liquid chromatography analysis can be carried out normally.

[0041] In a preferred embodiment, the chromatographic conditions of the high performance liquid chromatograph include: a chromatographic column with octadecylsilane (C18) as a filler, acetonitrile as a mobile phase A, a 0.1-0.4% (v / v) aqueous solution of an inorganic acid as a mobile phase B, a flow rate of 1 mL / min, a column temperature of 25-35° C., a detection wavelength of 240-255 nm, and a gradient elution.

[0042] The elution conditions are as follows: 0-10 min, the volume fraction of mobile phase A is 8%, and the volume fraction of mobile phase B is 92%; 10-17 min, the volume fraction of mobile phase A increases from 8% to 15%, and the volume fraction of mobile phase B decreases from 92% to 85%; 17-32 min, the volume fraction of mobile phase A increases from 15% to 17%, and the volume fraction of mobile phase B decreases from 85% to 83%; 32-62 min, the volume fraction of mobile phase A increases from 17% to 27%, and the volume fraction of mobile phase B decreases from 83% to 73%; 62-82 min, the volume fraction of mobile phase A increased from 27% to 28%, and the volume fraction of mobile phase B decreased from 73% to 72%; from 82 to 92 min, the volume fraction of mobile phase A increased from 28% to 31%, and the volume fraction of mobile phase B decreased from 72% to 69%; from 92 to 100 min, the volume fraction of mobile phase A increased from 31% to 45%, and the volume fraction of mobile phase B decreased from 69% to 55%; from 100 to 110 min, the volume fraction of mobile phase A decreased from 45% to 8%, and the volume fraction of mobile phase B increased from 55% to 92%.

[0043] The above chromatographic conditions can ensure that the various components in Hexi chrysanthemum are well separated on the chromatogram, making the fingerprint clearer and facilitating subsequent analysis and comparison. In the quality control and efficacy research of traditional Chinese medicine, this optimization can improve the accuracy and reliability of detection and provide a scientific basis for the standardized production and clinical application of traditional Chinese medicine.

[0044] In a preferred embodiment, the chromatographic column is an Eternity XT-5-C18 column.

[0045] In a preferred embodiment, the inorganic acid includes phosphoric acid.

[0046] Using phosphoric acid as an inorganic acid can adjust the pH value of the mobile phase, optimize chromatographic separation conditions, and improve the sensitivity and selectivity of detection. In the analysis of the chemical components of traditional Chinese medicine, this choice can improve the accuracy and reliability of detection and provide a scientific basis for the standardized production and clinical application of traditional Chinese medicine.

[0047] In a preferred embodiment, the above construction method also includes: importing the high performance liquid chromatogram of the test solution and the high performance liquid chromatogram of the reference solution into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for analysis, thereby obtaining the fingerprint of Hexi chrysanthemum.

[0048] In this application, for the similarity evaluation of fingerprints, the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) is used. This software is only used for similarity evaluation. The high-performance liquid chromatogram of the test solution and the high-performance liquid chromatogram of the reference solution are introduced into the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System for analysis, which can objectively and comprehensively reflect the similarities between fingerprints and provide an effective means for fingerprint evaluation.

[0049] In a second typical embodiment of the present application, a quality detection method for Hexi chrysanthemum is provided, and the above-mentioned quality detection method comprises: using the above-mentioned Hexi chrysanthemum fingerprint map construction method to construct a fingerprint map of the Hexi chrysanthemum sample to be tested, and obtaining the fingerprint map of the Hexi chrysanthemum sample to be tested; comparing the obtained fingerprint map of the Hexi chrysanthemum sample to be tested with the standard fingerprint map obtained under the same fingerprint map detection conditions for similarity, so as to realize the quality detection of the Hexi chrysanthemum sample to be tested.

[0050] By comparing the fingerprints of the sample to be tested and the standard sample, the quality of Hexi chrysanthemum can be quickly and accurately judged, the types and contents of multiple compounds in the sample to be tested can be identified, and the quality of the sample to be tested and the single medicinal material in the sample can be determined by the attribution of the characteristic peaks of the single medicinal material. This avoids the tediousness and time-consuming nature of traditional detection methods and improves detection efficiency and accuracy. In the production process of traditional Chinese medicine preparations, this method can be used for the screening and quality control of raw materials to ensure the effectiveness and consistency of the final product.

[0051] In a preferred embodiment, there are 12 peaks in the standard fingerprint, among which Peak 1 is the peak of scutellariae, Peak 2 is the peak of baicalin, Peak 9 is the peak of apigenin, and Peak 11 is the peak of luteolin.

[0052] By marking the peaks of the main components of Hexi chrysanthemum, the fingerprints of different samples can be more intuitively identified and compared, improving the accuracy and reliability of detection. In the quality control of traditional Chinese medicine, this optimization can improve the efficiency of detection and provide a scientific basis for the standardized production and clinical application of traditional Chinese medicine.

[0053] In a preferred embodiment, peak 11 is used as the positioning peak, and the relative retention time is 1.000; the average relative retention times of the other 11 common peaks are: the average relative retention time of peak 1 is 0.2346±0.0023; the average relative retention time of peak 2 is 0.4851±0.0030; the average relative retention time of peak 3 is 0.5432±0.0020; the average relative retention time of peak 4 is 0.6017±0.0036; the average relative retention time of peak 5 is 0 .6142±0.0023; the average relative retention time of peak 6 was 0.6443±0.0026; the average relative retention time of peak 7 was 0.7253±0.0021; the average relative retention time of peak 8 was 0.7570±0.0023; the average relative retention time of peak 9 was 0.8008±0.0016; the average relative retention time of peak 10 was 0.8315±0.0016; the average relative retention time of peak 12 was 1.1340±0.0028.

[0054] By setting the range of relative retention time, it is possible to ensure good consistency in the detection of different batches of Hexi chrysanthemum samples, avoiding deviations in test results caused by slight differences in experimental conditions. In the production process of traditional Chinese medicine preparations, this optimization can improve the accuracy and reliability of detection and ensure the quality stability and safety of traditional Chinese medicine preparations.

[0055] The following sources of instruments and reagents are for illustrative purposes: Agilent 1260 high performance liquid chromatograph (Agilent), ME-55 1 / 10000 electronic analytical balance (Mettler), ME-204T 1 / 10000 electronic analytical balance (Mettler), acetonitrile (chromatographic grade, Fisher), methanol (analytical grade, Sigma), phosphoric acid (analytical grade, Tianjin Zhiyuan Fine Chemical Co., Ltd.), aesculetin (supplied by China Food and Drug Administration), Content determination 110741-202310, purity 98.3%), scutellariae baicalin (China Food and Drug Inspection Institutes, for content determination 110842-202411, purity 91.0%), apigenin (China Food and Drug Inspection Institutes, for content determination 111901-202205, purity 98.4%), luteolin (China Food and Drug Inspection Institutes, for content determination 111520-202107, purity 96.3%).

[0056] The above-mentioned method for constructing the fingerprint of Hexi chrysanthemum and the method for detecting the quality of Hexi chrysanthemum can use any instruments and reagents in the prior art, and are not limited to the specific instruments and reagents used in this application.

[0057] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0058] Example 1

[0059] [Source] The dried aerial parts of Hexinia polydichotoma (Ostenf.) HLYang.

[0060] 1. Sample collection: A total of 8 batches of Hexi chrysanthemum samples were received, and detailed information is shown in Table 1.

[0061] Table 1 Hexi Chrysanthemum Sample Information

[0062] serial number name Medicinal Parts Origin Collection time HXJ-1 Hexi Chrysanthemum Dry above ground parts Xinjiang 2019.11 HXJ-2 Hexi Chrysanthemum Dry above ground parts Xinjiang 2020.10 HXJ-3 Hexi Chrysanthemum Dry above ground parts Xinjiang 2022.06 HXJ-4 Hexi Chrysanthemum Dry above ground parts Xinjiang 2022.06 HXJ-5 Hexi Chrysanthemum Dry above ground parts Xinjiang 2023.05 HXJ-6 Hexi Chrysanthemum Dry above ground parts Xinjiang 2023.05 HXJ-7 Hexi Chrysanthemum Dry above ground parts Xinjiang 2023.10 HXJ-8 Hexi Chrysanthemum Dry above ground parts Xinjiang 2023.10

[0063] The Hexinia polydichotoma sample HXJ-5 was made into a herbarium specimen, which was identified as the above-ground part of the Hexinia polydichotoma (Ostenf.) HLYang. After the samples were collected, all 8 samples were crushed, sieved, and set aside.

[0064] 2. Instruments and Reagents

[0065] 2.1 Experimental equipment

[0066] Agilent 1260 high performance liquid chromatograph, ME-55 1 / 100,000 electronic analytical balance, ME-204T 1 / 10,000 electronic analytical balance.

[0067] 2.2 Experimental reagents

[0068] Acetonitrile, methanol, purified water, phosphoric acid, scutellariae, baicalin, apigenin, and luteolin.

[0069] 3. Experimental Methods

[0070] 3.1 Chromatographic conditions

[0071] Kromasil Eternity XT-5-C18 column (250×4.6 mm, 5 μm); mobile phase: acetonitrile (A), 0.2% volume fraction phosphoric acid solution (B), gradient program as shown in Table 2; detection wavelength 248 nm; column temperature 30° C.; injection volume 10 μL.

[0072] Table 2

[0073] Time (min) Acetonitrile (%) Phosphoric acid aqueous solution (%) 0 8 92 6 8 92 17 15 85 32 17 83 62 27 73 82 28 72 92 31 69 100 45 55 110 8 92

[0074] 3.2 Preparation of reference solution and test solution

[0075] Accurately weigh the above four reference substances, place them in four brown volumetric flasks respectively, add methanol solution to dissolve to volume, shake well, and prepare reference substance solutions of 62.08 μg / mL of fraxinus ethyl, 110.05 μg / mL of scutellarin, 57.05 μg / mL of apigenin, and 85.02 μg / mL of luteolin respectively.

[0076] Preparation of the test solution: Weigh 1 g of Hexi Chrysanthemum medicinal material powder into a conical flask, accurately add 25 mL of 60% volume fraction methanol solution, weigh the weight, reflux for 40 minutes and weigh the weight, make up the lost weight with 60% volume fraction methanol solution, shake well, filter, and filter through a microporous filter membrane (0.22μm) to obtain the test solution.

[0077] 4. Methodological investigation

[0078] 4.1 Precision test

[0079] Take the above test solution, repeat the injection 6 times under the above liquid chromatography conditions, each injection of 10 μL, and examine the consistency of the relative retention time and relative peak area of ​​the main chromatographic peaks. The results are shown in Table 3, Table 4 (with luteolin as the reference peak) and Figure 1 .

[0080] Table 3 Precision test table (relative retention time of common peaks)

[0081]

[0082]

[0083] Table 4 Precision test table (common peak relative peak area)

[0084] 1 2 3 4 5 6 average value RSD Peak 1 0.3201 0.3187 0.3198 0.3222 0.3245 0.3198 0.3209 0.662 Peak 2 2.3105 2.3198 2.3178 2.3102 2.3201 2.3199 2.3164 0.205 Peak 3 1.4756 1.4732 1.4685 1.4705 1.4763 1.4698 1.4723 0.218 Peak 4 10.0258 10.0098 10.0256 10.0895 10.0003 10.1589 10.0517 0.608 Peak 5 1.3865 1.3725 1.3798 1.3802 1.3895 1.3715 1.3800 0.524 Peak 6 2.1698 2.1321 2.0998 2.1057 2.1698 2.1035 2.1301 1.540 Peak 7 20.4587 20.3995 20.3987 20.4089 20.4546 20.4106 20.4218 0.134 Peak 8 6.7775 6.6574 6.6721 6.7546 6.7999 6.8002 6.7436 0.942 Peak 9 0.3274 0.3279 0.3265 0.3397 0.3359 0.3287 0.3310 1.643 Peak 10 0.6745 0.6666 0.6589 0.6702 0.6678 0.6798 0.6696 1.067 Peak 11 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.000 Peak 12 1.6861 1.6952 1.6865 1.6905 1.6894 1.6729 1.6868 0.447

[0085] 4.2 Stability test

[0086] The same test solution was taken and tested at 0, 12, and 24 hours under the above-mentioned liquid chromatography conditions, with 10 μL injected each time, to examine the consistency of the relative retention time and relative peak area of ​​the main chromatographic peaks. The results are shown in Tables 5 and 6 (with luteolin as the reference peak) and Figure 2 .

[0087] Table 5 Stability test table (relative retention time of common peaks)

[0088]

[0089]

[0090] Table 6 Stability test table (common peak relative peak area)

[0091] Peak 0h 12h 24h average value RSD(%) Peak 1 0.3199 0.3156 0.3179 0.3178 0.677 Peak 2 2.3175 2.3105 2.3146 2.3142 0.152 Peak 3 1.4795 1.4789 1.4687 1.4757 0.411 Peak 4 10.0016 10.0178 10.0297 10.0164 0.141 Peak 5 1.3749 1.3763 1.3687 1.3733 0.295 Peak 6 2.1302 2.1378 2.1278 2.1319 0.245 Peak 7 20.3967 20.3978 20.3899 20.3948 0.021 Peak 8 6.6589 6.6514 6.6715 6.6606 0.152 Peak 9 0.3218 0.3201 0.3156 0.3192 1.004 Peak 10 0.6588 0.6612 0.6503 0.6568 0.872 Peak 11 1.0000 1.0000 1.0000 1.0000 0.000 Peak 12 1.6845 1.6955 1.6904 1.6901 0.326

[0092] 4.3 Repeatability test

[0093] Take the same batch of samples and prepare 6 test sample solutions according to the test sample preparation method. Under the above liquid chromatography conditions, the samples were injected and analyzed. The results are shown in Table 7 and Table 8 (with luteolin as the reference peak) and Figure 3 .

[0094] Table 7 Repeatability test table (relative retention time of common peaks)

[0095]

[0096]

[0097] Table 8 Repeatability test table (common peak relative peak area)

[0098] Peak 1 2 3 4 5 6 average value RSD(%) Peak 1 0.3050 0.3214 0.3185 0.3210 0.3211 0.3052 0.3051 2.55 Peak 2 2.3158 2.3241 2.2564 2.2927 2.3015 2.4152 2.3655 2.30 Peak 3 1.4895 1.4765 1.4364 1.5558 1.4766 1.4526 1.4711 2.78 Peak 4 10.0424 10.2148 10.0562 10.2348 10.1547 10.2546 10.1485 0.90 Peak 5 1.3576 1.3678 1.3584 1.4533 1.4097 1.3825 1.3701 2.69 Peak 6 2.1546 2.1587 2.0818 2.1800 2.0978 2.1784 2.1665 1.96 Peak 7 20.1806 21.0546 20.3556 21.0874 20.4241 20.3985 20.2896 1.88 Peak 8 6.8832 6.5431 6.6191 6.8467 6.8098 6.5123 6.6977 2.44 Peak 9 0.3231 0.3358 0.3264 0.3331 0.3378 0.3457 0.3344 2.43 Peak 10 0.6622 0.6321 0.6524 0.6651 0.6874 0.6732 0.6677 2.84 Peak 11 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 Peak 12 1.6761 1.7423 1.6562 1.7064 1.6458 1.7514 1.7137 2.61

[0099] 5. Establishment of the standard fingerprint of Hexi Chrysanthemum medicinal materials

[0100] Accurately pipette 10 μL of each of the reference solution and the test solution, inject them into the HPLC instrument, perform HPLC determination, and record the chromatogram. Figure 4 As shown in the chromatogram of scutellariae scutellariae Figure 5 As shown in the chromatogram of apigenin Figure 6 The chromatogram of luteolin is shown in Figure 7 shown.

[0101] Based on the fingerprints of 8 batches of Hexi chrysanthemum medicinal materials, standard fingerprints were formulated, such as Figure 8 shown. Figure 8 Peak 1 is aesculetin, peak 2 is scutellarin, peak 9 is apigenin, and peak 11 is luteolin, a total of 4 peaks. S1 to S8 correspond to the source information of Chinese medicinal materials HXJ-1 to HXJ-8 in Table 1, and R represents the reference spectrum.

[0102] By comparing the chromatograms of Hexi chrysanthemum medicinal materials, 12 peaks were determined to be common peaks, among which peak No. 11 was luteolin. The luteolin peak was used as the reference peak to calculate the relative retention time and relative standard deviation of each common peak. The results are shown in Tables 9 and 10.

[0103] Table 9 Relative retention time of common peaks of 8 batches of samples

[0104]

[0105]

[0106] Table 10 Relative peak areas of common peaks of 8 batches of samples

[0107] Peak S1 S2 S3 S4 S5 S6 S7 S8 average value RSD(%) Peak 1 0.3089 0.3159 0.3254 0.3145 0.3327 0.3578 0.3257 0.3785 0.3324 7.20 Peak 2 2.3248 2.3756 2.5424 2.0056 2.2378 2.3545 2.4789 2.0489 2.2961 8.29 Peak 3 1.4578 1.4875 1.4021 1.4736 1.4569 1.4987 1.5239 1.4856 1.4733 2.45 Peak 4 10.2569 10.0056 10.6863 10.0008 10.2856 10.5897 10.6720 10.5897 10.3858 2.76 Peak 5 1.3026 1.3659 1.3278 1.3478 1.3521 1.3698 1.3920 1.2859 1.3430 2.65 Peak 6 2.1879 2.0018 2.5896 2.4587 2.1045 2.0587 2.1874 2.2225 2.2264 9.02 Peak 7 20.5846 20.4875 20.2147 20.9871 20.3564 19.9997 20.8745 20.0054 20.4387 1.80 Peak 8 6.2348 6.5879 6.2341 6.8974 6.8745 6.0871 6.0247 6.5874 6.4410 5.32 Peak 9 0.3158 0.3589 0.3784 0.3248 0.3147 0.3987 0.3546 0.3248 0.3463 9.04 Peak 10 0.6289 0.6201 0.6874 0.6178 0.6421 0.6352 0.6874 0.6547 0.6467 4.29 Peak 11 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.00 Peak 12 1.6574 1.6874 1.7498 1.6387 1.7841 1.5896 1.6487 1.7421 1.6872 3.92

[0108] 6. Similarity evaluation

[0109] The similarity of the fingerprints of the components of 8 batches of samples was calculated using the 2012 edition of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine". The results are shown in Table 11.

[0110] Table 11 Similarity evaluation results of HPLC fingerprints of 8 batches of Hexi chrysanthemum medicinal materials

[0111] Sample No. S1 S2 S3 S4 S5 S6 S7 S8 R S1 1 S2 0.901 1 S3 0.895 0.889 1 S4 0.912 0.875 0.897 1 S5 0.878 0.865 0.882 0.879 1 S6 0.876 0.854 0.856 0.886 0.869 1 S7 0.867 0.836 0.869 0.875 0.896 0.873 1 S8 0.893 0.847 0.879 0.896 0.924 0.851 0.922 1 R 0.856 0.863 0.855 0.846 0.873 0.845 0.872 0.843 1

[0112] The present invention adopts HPLC method to establish a research method for fingerprint spectrum of 8 batches of Hexi chrysanthemum medicinal materials, and identifies 4 components, establishes characteristic spectrum, and obtains good precision, stability and repeatability of the method through methodological verification. From the similarity result analysis of Hexi chrysanthemum fingerprint spectrum, the similarity of 8 batches of Hexi chrysanthemum medicinal materials fingerprint spectrum is greater than 0.8, and the overall picture of fingerprint spectrum is basically consistent, indicating the consistency of chemical composition of different batches of Hexi chrysanthemum.

[0113] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0114] (1) The present invention establishes a method for constructing a fingerprint spectrum of Hexi chrysanthemum medicinal materials. Through the construction method, a standard fingerprint spectrum of Hexi chrysanthemum medicinal materials can be obtained. The similarity between the HPLC fingerprint spectrum of Hexi chrysanthemum medicinal materials established by the method provided by the present invention and the reference spectrum is greater than 0.8, which can effectively characterize its quality, better evaluate the relative stability of the control medicinal materials, improve the quality evaluation system of Hexi chrysanthemum medicinal materials, and provide a theoretical and practical basis for the comprehensive and effective control of its quality.

[0115] (2) The HPLC fingerprint of Hexi Chrysanthemum established in the present invention calibrated 12 common peaks, identified four common peaks of fraxinoside, scutellarin, apigenin, and luteolin, thus avoiding the singleness of the quality control of Hexi Chrysanthemum medicinal materials.

[0116] (3) The sample pretreatment method of the present invention is simple, the characteristic components of the established fingerprint spectrum are preserved intact, the repeatability and stability are good, the precision is high, and it has a certain specificity.

[0117] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing a fingerprint of Chrysanthemum chinense, characterized in that: The construction method comprises: a) Preparation of reference solution: taking aesculetin, scutellarin, apigenin and luteolin as reference substances, respectively adding solutions to dissolve them to obtain the reference solution, wherein the reference solution includes aesculetin solution, scutellarin solution, apigenin solution and luteolin solution; b) Preparation of the test solution: mixing the powder of the chrysanthemum hupensis with a methanol solution and heating and refluxing the mixture, cooling the mixture and filtering the mixture to obtain the test solution; c) placing the test solution and a plurality of reference solution in a high performance liquid chromatograph, and obtaining a high performance liquid chromatogram of the test solution and a high performance liquid chromatogram of the reference solution.

2. The construction method according to claim 1, characterized in that: The concentration of the aesculetin in the aesculetin solution is 55.00-75.00 μg / mL; Preferably, the concentration of scutellarin in the scutellarin solution is 50.00-125.00 μg / mL; Preferably, the concentration of the apigenin in the apigenin solution is 50.00-90.00 μg / mL; Preferably, the concentration of luteolin in the luteolin solution is 80.00-100.00 μg / mL.

3. The construction method according to claim 1, characterized in that: The heating reflux treatment time is 30-60min; Preferably, the temperature of the heating reflux treatment is 80-85°C; Preferably, the volume fraction of methanol in the methanol solution is 50%-80%.

4. The construction method according to claim 1, characterized in that: The filtering comprises: filtering using a 0.22 μm microporous filter membrane.

5. The construction method according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatograph include: The chromatographic column uses octadecylsilane as filler, acetonitrile as mobile phase A, 0.1-0.4% (v / v) aqueous solution of inorganic acid as mobile phase B, flow rate of 1 mL / min, column temperature of 25-35° C., detection wavelength of 240-255 nm, and gradient elution; The elution conditions are: 0-10min, the volume fraction of mobile phase A was 8%, and the volume fraction of mobile phase B was 92%; From 10 to 17 min, the volume fraction of mobile phase A increased from 8% to 15%, and the volume fraction of mobile phase B decreased from 92% to 85%; From 17 to 32 min, the volume fraction of mobile phase A increased from 15% to 17%, and the volume fraction of mobile phase B decreased from 85% to 83%; From 32 to 62 min, the volume fraction of mobile phase A increased from 17% to 27%, and the volume fraction of mobile phase B decreased from 83% to 73%; From 62 to 82 min, the volume fraction of mobile phase A increased from 27% to 28%, and the volume fraction of mobile phase B decreased from 73% to 72%; From 82 to 92 min, the volume fraction of mobile phase A increased from 28% to 31%, and the volume fraction of mobile phase B decreased from 72% to 69%; From 92 to 100 min, the volume fraction of mobile phase A increased from 31% to 45%, and the volume fraction of mobile phase B decreased from 69% to 55%; From 100 to 110 min, the volume fraction of mobile phase A decreased from 45% to 8%, and the volume fraction of mobile phase B increased from 55% to 92%; Preferably, the chromatographic column is an Eternity XT-5-C18 column.

6. The construction method according to claim 5, characterized in that: The inorganic acid includes phosphoric acid.

7. The construction method according to any one of claims 1 to 6, characterized in that: The construction method also includes: importing the high performance liquid chromatogram of the test solution and the high performance liquid chromatogram of the reference solution into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for analysis, thereby obtaining the fingerprint of the Hexi chrysanthemum.

8. A method for detecting the quality of Hexi chrysanthemum, characterized in that: include: The fingerprint spectrum of the Hexi chrysanthemum sample to be tested is constructed using the method for constructing the fingerprint spectrum of Hexi chrysanthemum described in any one of claims 1 to 7 to obtain the fingerprint spectrum of the Hexi chrysanthemum sample to be tested; the similarity of the obtained fingerprint spectrum of the Hexi chrysanthemum sample to be tested is compared with the standard fingerprint spectrum obtained under the same fingerprint spectrum detection conditions to achieve quality detection of the Hexi chrysanthemum sample to be tested.

9. The quality inspection method according to claim 8, characterized in that: There are 12 peaks in the standard fingerprint. Among them, peak No. 1 is the peak of the scutellarin, peak No. 2 is the peak of the baicalin, peak No. 9 is the peak of the apigenin, and peak No. 11 is the peak of the luteolin.

10. The quality inspection method according to claim 9, characterized in that: The peak No. 11 is taken as the positioning peak, and the relative retention time is 1.000; The average relative retention times of the other 11 common peaks are: The average relative retention time of peak 1 was 0.2346 ± 0.0023; The average relative retention time of peak 2 was 0.4851 ± 0.0030; The average relative retention time of peak 3 was 0.5432 ± 0.0020; The average relative retention time of peak 4 was 0.6017 ± 0.0036; The average relative retention time of peak 5 was 0.6142 ± 0.0023; The average relative retention time of peak 6 was 0.6443 ± 0.0026; The average relative retention time of peak 7 was 0.7253 ± 0.0021; The average relative retention time of peak 8 was 0.7570 ± 0.0023; The average relative retention time of peak 9 was 0.8008 ± 0.0016; The average relative retention time of peak 10 was 0.8315 ± 0.0016; The average relative retention time of peak 12 was 1.1340±0.0028.

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