Construction method of characteristic chromatogram of Yunnan black tea and method for determining content of components to be detected of Yunnan black tea

Abstract

The invention relates to the technical field of health-care product food detection, in particular to a construction method of a characteristic spectrum of remote black tea and a content determination method of a to-be-detected component of the remote black tea. The chromatographic detection conditions of the invention comprise: a mobile phase comprising acetonitrile (A) and water (B), gradient elution (0-6 min, 4-12% A); 6-12 min, 12%-15% of A is obtained; 15%-19% of A is selected from 12 min to 22 min; 22-23 min, 19-21% of A is taken; 23-26 min, 21-26% of A is obtained; 26% A is determined after 26-28 min; 28-29 min, 26-29% of A is obtained; when the time is 29-38 minutes, 29-33% of A is obtained; 38-38.5 min, 33-4% of A is added; 38.5-40 min, 4% of A). According to the method, by adopting proper sample treatment and chromatographic detection conditions, comprehensive detection of the remote black tea is realized, the characteristic chromatogram of the remote black tea is constructed, and a more scientific judgment basis is provided for quality control of the remote black tea.

Description

Technical Field

[0001] The present application relates to the technical field of health care food detection, and in particular to a method for constructing a characteristic spectrum of Yunhong tea and a method for determining the content of its components to be detected. Background Art

[0002] Yunhong tea is a health food that can relieve physical fatigue. It is composed of black tea, Cistanche deserticola, jujube, Flos ophiopogonis and Acanthopanax senticosus. The approval number is National Food and Health Note G20240286, and the effective ingredients are 6.5g of tea polyphenols, 0.45g of total saponins and 0.05g of icariin per 100g. Since Yunhong tea contains prescription medicinal materials and has complex chemical components, identification or content determination of a single compound alone cannot fully reflect the internal chemical information of Yunhong tea, which is not conducive to overall quality control.

[0003] With its comprehensive, macroscopic and fuzzy analysis advantages, the fingerprint of traditional Chinese medicine can achieve overall quality control of traditional Chinese medicine by describing the overall characteristics of traditional Chinese medicine and using appropriate fuzzy processing methods. This mapping technology can visualize the overall characteristics of various chemical components in traditional Chinese medicine, revealing quality defects that may be overlooked by traditional detection methods. Based on the comprehensive action characteristics of traditional Chinese medicine with multiple components and multiple action points, the fingerprint spectrum provides a comprehensive method for modern traditional Chinese medicine quality control from the perspective of "all ingredients", which is especially suitable for quality monitoring of non-single-ingredient drugs.

[0004] There is currently a lack of systematic qualitative research on Yunhong tea. The specified effective ingredients in Yunhong tea are all chromatographically detectable ingredients. Developing a characteristic spectrum and content determination method for Yunhong tea based on the principle of traditional Chinese medicine fingerprint will help to ensure the quality of Yunhong tea more comprehensively and accurately. Summary of the invention

[0005] Based on this, one or more embodiments of the present application provide a method for constructing a characteristic spectrum of Yunhong tea and a method for determining the content of its components to be tested. The method of the present application can comprehensively evaluate the internal chemical composition of Yunhong tea and accurately determine the content of the components to be tested.

[0006] The technical solution of this application includes the following steps:

[0007] A method for constructing a characteristic spectrum of Yunhong tea, wherein the composition of the Yunhong tea includes black tea, Cistanche deserticola, jujube, epimedium and Acanthopanax senticosus;

[0008] The method for constructing the characteristic spectrum of Yun black tea comprises the following steps:

[0009] Take the Yun black tea to be tested, add the extraction solvent to extract, and obtain the test solution;

[0010] The test solution is subjected to liquid chromatography to obtain a characteristic spectrum of the Yunhong tea;

[0011] The conditions for the liquid chromatography detection include:

[0012] (1) Acetonitrile was used as mobile phase A and water was used as mobile phase B;

[0013] (2) Gradient elution is used, wherein the procedure of the gradient elution comprises:

[0014] From 0min to 6min, the volume percentage of the mobile phase A increases from 4% to 12%;

[0015] 6min-12min, the volume percentage of the mobile phase A increases from 12% to 15%;

[0016] From 12min to 22min, the volume percentage of the mobile phase A increases from 15% to 19%;

[0017] From 22min to 23min, the volume percentage of the mobile phase A increased from 19% to 21%;

[0018] From 23min to 26min, the volume percentage of the mobile phase A increased from 21% to 26%;

[0019] 26min-28min, maintaining the volume percentage of the mobile phase A at 26%;

[0020] From 28 to 29 minutes, the volume percentage of the mobile phase A increased from 26% to 29%;

[0021] From 29min to 38min, the volume percentage of the mobile phase A increased from 29% to 33%;

[0022] 38min-38.5min, the volume percentage of the mobile phase A decreases from 33% to 4%;

[0023] 38.5min-40min, the volume percentage of the mobile phase A is maintained at 4%.

[0024] In one embodiment, the conditions for liquid chromatography detection also include at least one of the following:

[0025] (1) The filler of the chromatographic column is octadecylsilane bonded silica gel;

[0026] (2) Column temperature is 24°C to 26°C;

[0027] (3) The injection volume is 8 μL to 12 μL;

[0028] (4) a flow rate of 0.8 mL / min to 1.2 mL / min; and,

[0029] (5) The detection wavelength is 205nm~215nm.

[0030] In one embodiment, the length of the chromatographic column is 240 mm to 260 mm, the diameter is 4.4 mm to 4.8 mm, and the particle size of the filler is 4.8 μm to 5.2 μm.

[0031] In one embodiment, the extraction solvent is an organic alcohol, and optionally, the organic alcohol includes methanol; and / or,

[0032] The extraction method includes ultrasound, and optionally, the ultrasound frequency is 40 Hz to 60 Hz, and the time is 45 min to 55 min; and / or,

[0033] The mass volume ratio of the Yunhong tea to the extraction solvent is 1-2 g:100 mL.

[0034] In one embodiment, the characteristic spectrum of the Yunhong tea includes 7 characteristic peaks, namely peak 1, peak 2, peak 3, peak 4, peak 5, peak 6 and peak 7, wherein peak 1 is syringin, peak 2 is echinacoside, peak 3 is verbascoside, peak 4 is epimedin A, peak 5 is epimedin B, peak 6 is epimedin C, and peak 7 is icariin;

[0035] Taking peak 1 as the control, the characteristic peaks and their relative retention times are as follows: peak 2: 1.56±10%; peak 3: 2.18±10%; peak 4: 2.82±10%; peak 5: 2.87±10%; peak 6: 2.91±10%; peak 7: 2.99±10%.

[0036] A method for determining the content of a component to be tested in Yunhong tea, wherein the composition of the Yunhong tea includes black tea, Cistanche deserticola, jujube, epimedium and Acanthopanax senticosus;

[0037] The method for determining the content of the index components of the Yun black tea comprises the following steps:

[0038] Take a reference substance of the component to be tested, add a dissolving solvent to dissolve it, and prepare reference substance solutions of different concentrations;

[0039] Taking solutions of the reference substance of different concentrations for liquid chromatography detection to obtain a standard curve of concentration and peak area;

[0040] Take the Yunhong tea to be tested, add the extraction solvent to extract, and obtain the test solution;

[0041] Taking the test solution for liquid chromatography detection, obtaining the peak area of ​​the test component and substituting it into the standard curve of concentration and peak area to calculate the concentration;

[0042] The conditions for the liquid chromatography detection include:

[0043] (1) Acetonitrile was used as mobile phase A and water was used as mobile phase B;

[0044] (2) Gradient elution is used, wherein the procedure of the gradient elution comprises:

[0045] From 0min to 6min, the volume percentage of the mobile phase A increases from 4% to 12%;

[0046] 6min-12min, the volume percentage of the mobile phase A increases from 12% to 15%;

[0047] From 12min to 22min, the volume percentage of the mobile phase A increases from 15% to 19%;

[0048] From 22min to 23min, the volume percentage of the mobile phase A increased from 19% to 21%;

[0049] From 23min to 26min, the volume percentage of the mobile phase A increased from 21% to 26%;

[0050] 26min-28min, maintaining the volume percentage of the mobile phase A at 26%;

[0051] From 28 to 29 minutes, the volume percentage of the mobile phase A increased from 26% to 29%;

[0052] From 29min to 38min, the volume percentage of the mobile phase A increased from 29% to 33%;

[0053] 38min-38.5min, the volume percentage of the mobile phase A decreases from 33% to 4%;

[0054] 38.5min-40min, the volume percentage of the mobile phase A is maintained at 4%.

[0055] In one embodiment, the conditions for liquid chromatography detection also include at least one of the following:

[0056] (1) The filler of the chromatographic column is octadecylsilane bonded silica gel;

[0057] (2) Column temperature is 24°C to 26°C;

[0058] (3) The injection volume is 8 μL to 12 μL;

[0059] (4) a flow rate of 0.8 mL / min to 1.2 mL / min; and,

[0060] (5) The detection wavelength is 205nm~215nm.

[0061] In one embodiment, the length of the chromatographic column is 240 mm to 260 mm, the diameter is 4.4 mm to 4.8 mm, and the particle size of the filler is 4.8 μm to 5.2 μm.

[0062] In one embodiment, the extraction solvent is an organic alcohol, and optionally, the organic alcohol includes methanol; and / or,

[0063] The extraction method includes ultrasound, and optionally, the ultrasound frequency is 40 Hz to 60 Hz, and the time is 45 min to 55 min; and / or,

[0064] The mass volume ratio of the Yunhong tea to the extraction solvent is 1-2 g:100 mL.

[0065] In one embodiment, the dissolving solvent is an organic alcohol. Optionally, the organic alcohol includes methanol.

[0066] The present application achieves a relatively comprehensive detection of Yunhong tea and constructs a characteristic spectrum of Yunhong tea by adopting appropriate sample processing and chromatographic detection conditions. The characteristic spectrum prepared by this method is characteristic, reproducible and operable, and can provide relatively rich characteristic peak information, providing a more comprehensive and scientific judgment basis for the quality control of Yunhong tea.

[0067] The construction method of the present application fully considers the material benchmark of Yunhong tea. Based on the interaction between the chemical components contained in Yunhong tea, the retention time of each characteristic peak is fixed within a certain range, and will produce differences when counterfeit and inferior medicinal materials are used or the product is deteriorated. Therefore, the quality of Yunhong tea products can be monitored more intuitively, accurately and quickly. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0069] Figure 1a and Figure 1b This is a comparison chart of the chromatographic detection results of different extraction solvents in Example 1 of the present application, wherein: Figure 1a A, B, and C are chromatograms of water, 8% acetonitrile, and 65% ethanol as extraction solvents, respectively; Figure 1b D, E, and F are chromatograms with 85% ethanol, ethanol, and methanol as extraction solvents, respectively;

[0070] Figure 2 This is a comparison chart of the chromatographic detection results of different extraction conditions in Example 1 of the present application, wherein: Figure 2 A and B are chromatograms of ultrasonic extraction and heating reflux extraction as extraction methods, respectively;

[0071] Figure 3 This is a comparison chart of the chromatographic detection results of different mobile phases in Example 1 of the present application, wherein: Figure 3 A, B, and C are chromatograms of acetonitrile-0.3% phosphoric acid, acetonitrile-water, and acetonitrile-0.1% formic acid as mobile phases, respectively;

[0072] Figure 4 This is a comparison chart of the chromatographic detection results at different flow rates in Example 1 of the present application, wherein: Figure 4 A, B, and C are chromatograms with flow rates of 0.8 mL / min, 1 mL / min, and 1.2 mL / min, respectively;

[0073] Figure 5 This is a comparison chart of the chromatographic detection results of different injection amounts in Example 1 of the present application, wherein: Figure 5 A, B, and C are chromatograms with 5 μL, 10 μL, and 15 μL injection volumes, respectively;

[0074] Figure 6 It is a comparison chart of the chromatographic test results of the blank solvent, the reference solution and the test solution in Example 1 of the present application, wherein: Figure 6 A is the chromatogram of blank solvent. Figure 6 B is the chromatogram of the reference solution. Figure 6 C is the chromatogram of the test solution. DETAILED DESCRIPTION

[0075] The present application will be further described below in conjunction with the embodiments and examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. In addition, it should be understood that after reading the content taught in the present application, those skilled in the art may make various changes or modifications to the present application, and these equivalent forms also fall within the protection scope of the claims attached to the present application.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0077] the term

[0078] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0079] The term "and / or" used in this article includes any one of two or more related listed items, and also includes any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0080] Herein, "preferred", "preferable", "better", etc. are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0081] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0082] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0083] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0084] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range of the instrument control. Fluctuations within the range of ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, and ±0.1°C are allowed.

[0085] In the present application, weight may be μg, mg, g, kg or other mass units known in the chemical industry.

[0086] In the chromatogram obtained by liquid chromatography detection in the present application, the horizontal axis is minutes (min) and the vertical axis is the response value (mAU).

[0087] In one aspect of the present application, a method for constructing a characteristic spectrum of Yunhong tea is provided, wherein the composition of Yunhong tea includes black tea, Cistanche deserticola, jujube, epimedium and Acanthopanax senticosus;

[0088] The method for constructing the characteristic spectrum of Yun black tea comprises the following steps:

[0089] Take the Yun black tea to be tested, add the extraction solvent to extract, and obtain the test solution;

[0090] The test solution is subjected to liquid chromatography to obtain a characteristic spectrum of Yunhong tea;

[0091] The conditions for liquid chromatography detection include:

[0092] (1) Acetonitrile was used as mobile phase A and water was used as mobile phase B;

[0093] (2) Gradient elution is used. The procedure of gradient elution includes:

[0094] From 0 to 6 minutes, the volume percentage of mobile phase A increased from 4% to 12%;

[0095] From 6 to 12 minutes, the volume percentage of mobile phase A increased from 12% to 15%;

[0096] From 12 to 22 minutes, the volume percentage of mobile phase A increased from 15% to 19%;

[0097] From 22 to 23 minutes, the volume percentage of mobile phase A increased from 19% to 21%;

[0098] From 23 to 26 minutes, the volume percentage of mobile phase A increased from 21% to 26%;

[0099] 26min-28min, maintain the volume percentage of mobile phase A at 26%;

[0100] From 28 to 29 minutes, the volume percentage of mobile phase A increased from 26% to 29%;

[0101] From 29 to 38 min, the volume percentage of mobile phase A increased from 29% to 33%;

[0102] From 38 to 38.5 min, the volume percentage of mobile phase A decreased from 33% to 4%;

[0103] From 38.5 min to 40 min, the volume percentage of mobile phase A was maintained at 4%.

[0104] In one embodiment, the conditions for liquid chromatography detection also include at least one of the following:

[0105] (1) The filler of the chromatographic column is octadecylsilane bonded silica gel;

[0106] (2) Column temperature is 24°C to 26°C;

[0107] (3) The injection volume is 8 μL to 12 μL;

[0108] (4) a flow rate of 0.8 mL / min to 1.2 mL / min; and,

[0109] (5) The detection wavelength is 205nm~215nm.

[0110] In one embodiment, the filler of the chromatographic column is octadecylsilane bonded silica gel.

[0111] In one embodiment, the length of the chromatographic column is 240 mm to 260 mm, the diameter is 4.4 mm to 4.8 mm, and the particle size of the filler is 4.8 μm to 5.2 μm.

[0112] In one embodiment, the model of the chromatographic column is a ZORBAX Eclipse Plus C18 chromatographic column.

[0113] In one embodiment, the column temperature is 24°C to 26°C, for example 24°C, 25°C, 26°C, etc.

[0114] In one embodiment, the injection volume is 8 μL to 12 μL, for example 8 μL, 10 μL, 12 μL, etc.

[0115] In one embodiment, the flow rate is 0.8 mL / min to 1.2 mL / min, for example 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, etc.

[0116] In one embodiment, the detection wavelength is 205 nm to 215 nm, for example 205 nm, 206 nm, 207 nm, 208 nm, 209 nm, 210 nm, 211 nm, 212 nm, 213 nm, 214 nm, 215 nm, etc.

[0117] In one embodiment, the extraction solvent is an organic alcohol. Optionally, the organic alcohol includes methanol.

[0118] In one embodiment, the extraction method includes ultrasound. Optionally, the frequency of ultrasound is 40Hz to 60Hz, for example 40Hz, 45Hz, 50Hz, 55Hz, 60Hz, etc., and the time is 45min to 55min, for example 45min, 48min, 50min, 52min, 55min, etc.

[0119] In one embodiment, the mass volume ratio of Yunhong tea to the extraction solvent is 1-2 g:100 mL, for example 1 g:100 mL, 1.5 g:100 mL, 2 g:100 mL, etc.

[0120] In one embodiment, the characteristic spectrum of Yunhong tea includes 7 characteristic peaks, namely peak 1, peak 2, peak 3, peak 4, peak 5, peak 6 and peak 7, wherein peak 1 is syringin, peak 2 is echinacoside, peak 3 is verbascoside, peak 4 is epimedin A, peak 5 is epimedin B, peak 6 is epimedin C, and peak 7 is icariin;

[0121] Taking peak 1 as the control, the characteristic peaks and their relative retention times are as follows: peak 2: 1.56±10%; peak 3: 2.18±10%; peak 4: 2.82±10%; peak 5: 2.87±10%; peak 6: 2.91±10%; peak 7: 2.99±10%.

[0122] In another aspect of the present application, a method for determining the content of a component to be tested in Yunhong tea is provided, wherein the composition of Yunhong tea includes black tea, Cistanche deserticola, jujube, Rhizoma Epimedii and Acanthopanax senticosus.

[0123] The method for determining the content of the index components of Yun black tea comprises the following steps:

[0124] Take the reference substance of the component to be tested, add a solvent to dissolve it, and prepare reference substance solutions of different concentrations;

[0125] Take reference substance solutions of different concentrations for liquid chromatography detection to obtain a standard curve of concentration and peak area;

[0126] Take the Yunhong tea to be tested, add the extraction solvent to extract, and obtain the test solution;

[0127] Take the solution of the test product for liquid chromatography detection, obtain the peak area of ​​the test component and substitute it into the standard curve of concentration and peak area to calculate the concentration;

[0128] The conditions for liquid chromatography detection include:

[0129] (1) Acetonitrile was used as mobile phase A and water was used as mobile phase B;

[0130] (2) Gradient elution is used. The procedure of gradient elution includes:

[0131] From 0 to 6 minutes, the volume percentage of mobile phase A increased from 4% to 12%;

[0132] From 6 to 12 minutes, the volume percentage of mobile phase A increased from 12% to 15%;

[0133] From 12 to 22 minutes, the volume percentage of mobile phase A increased from 15% to 19%;

[0134] From 22 to 23 minutes, the volume percentage of mobile phase A increased from 19% to 21%;

[0135] From 23 to 26 minutes, the volume percentage of mobile phase A increased from 21% to 26%;

[0136] 26min-28min, maintain the volume percentage of mobile phase A at 26%;

[0137] From 28 to 29 minutes, the volume percentage of mobile phase A increased from 26% to 29%;

[0138] From 29 to 38 min, the volume percentage of mobile phase A increased from 29% to 33%;

[0139] From 38 to 38.5 min, the volume percentage of mobile phase A decreased from 33% to 4%;

[0140] From 38.5 min to 40 min, the volume percentage of mobile phase A was maintained at 4%.

[0141] The conditions for liquid chromatography detection in the method for determining the content of the components to be tested in Yunhong tea in the embodiment of the present application are the same as the conditions for liquid chromatography detection in the method for constructing the fingerprint of Yunhong tea in any of the above technical solutions.

[0142] The extraction solvent and extraction method used in the method for determining the content of the test component of Yunhong tea in the embodiment of the present application shall preferably refer to the extraction solvent and extraction method in the method for constructing the fingerprint of Yunhong tea in any of the above technical solutions.

[0143] In one embodiment, the mass volume ratio of the tested Yunhong tea to the extraction solvent is 1-2 g:100 mL.

[0144] In one embodiment, the concentration of the syringoside reference solution can be 1.0 μg / mL to 8.9 μg / mL, the concentration of the echinacoside reference solution can be 75.8 μg / mL to 682.3 μg / mL, the concentration of the verbascoside reference solution can be 7.8 μg / mL to 69.8 μg / mL, the concentration of the epimedin A reference solution can be 0.9 μg / mL to 8.5 μg / mL, the concentration of the epimedin B reference solution can be 3.3 μg / mL to 29.3 μg / mL, the concentration of the epimedin C reference solution can be 3.2 μg / mL to 28.6 μg / mL, and the concentration of the icariin reference solution can be 3.7 μg / mL to 33.0 μg / mL.

[0145] The following are some specific embodiments.

[0146] For experimental parameters not specified in the following specific embodiments, reference is made to the instructions given in the present application document, and reference may also be made to experimental manuals in the art or other experimental methods known in the art, or to experimental conditions recommended by manufacturers.

[0147] The raw materials and reagents involved in the following specific examples can be obtained from commercial sources, or can be prepared by those skilled in the art according to known methods.

[0148] Example 1

[0149] 1. Instruments and Materials

[0150] Instruments: High performance liquid chromatograph (model Vanquish Core, Thermo Fisher Scientific), analytical balance (model MS205DU, Mettler), ultrasonic cleaner (model AS10200BT, Tianjin Aotesense Instrument Co., Ltd.), circulating water multi-purpose vacuum pump (model SHZ-DII, Beijing Yongguangming Medical Instrument Co., Ltd.), electric constant temperature water bath (model DZKW-S-6, Beijing Yongguangming Medical Instrument Co., Ltd.), water purifier (model UPL-II-40RZ, Sichuan Youpu Ultrapure Technology Co., Ltd.).

[0151] Reference substances: syringoside (batch number 111574-202106, purity 94.3%), echinacoside (batch number 111670-201907, purity 91.8%), verbascoside (batch number 111530-201914, purity 95.2%), epimedin C (batch number 111780-201905, purity 94.3%), and icariin (batch number 110737-202017, purity 98.1%) were purchased from China Food and Drug Inspection Institutes; epimedin A (batch number WP23042711, purity 98.0%) and epimedin B (batch number WP23062006, purity 98.0%) were purchased from Sichuan Weikeqi Biotechnology Co., Ltd.

[0152] Reagents: Methanol and acetonitrile were of chromatographic grade; water was purified water.

[0153] Yunhong tea samples: 4 batches of Yunhong tea bags were all produced by Zhengzhou Linnuo Pharmaceutical Co., Ltd. and were numbered according to different production batches, namely 20220321, 20220322, 20220323 and 20231008.

[0154] 2. Methods and Results

[0155] 2.1 Reference solution

[0156] Accurately weigh appropriate amounts of each reference substance of syringin, echinacoside, verbascoside, epimedin A, epimedin B, epimedin C and icariin into a 10 mL volumetric flask, and use methanol to prepare reference substance stock solutions with mass concentrations of 0.988 mg / mL, 0.936 mg / mL, 0.970 mg / mL, 0.939 mg / mL, 0.984 mg / mL, 0.962 mg / mL and 0.989 mg / mL, respectively; pipette appropriate amounts of each reference substance stock solution into a 10 mL volumetric flask, make up to volume with methanol, shake well, and prepare each series of reference substance solutions of different concentrations.

[0157] Among them: the concentrations of the syringoside series reference solution are 1.0μg / mL, 3.0μg / mL, 5.0μg / mL, 7.0μg / mL and 8.9μg / mL; the concentrations of the echinacoside series reference solution are 75.8μg / mL, 227.4μg / mL, 379.0μg / mL, 530.7μg / mL and 682.3μg / mL; the concentrations of the verbascoside series reference solution are 7.8μg / mL, 23.3μg / mL, 38.8μg / mL, 54.3μg / mL and 69.8μg / mL; the concentrations of the epimedin A series reference solution are 0.9μg / mL, 2.8 μg / mL, 4.7μg / mL, 6.6μg / mL and 8.5μg / mL; the concentrations of epimedin B series reference solution were 3.3μg / mL, 9.8μg / mL, 16.3μg / mL, 22.8μg / mL and 29.3μg / mL; the concentrations of epimedin C series reference solution were 3.2μg / mL, 9.5μg / mL, 15.9μg / mL, 22.2μg / mL and 28.6μg / mL; the concentrations of icariin series reference solution were 3.7μg / mL, 11.0μg / mL, 18.3μg / mL, 25.6μg / mL and 33.0μg / mL.

[0158] 2.2 Test solution

[0159] 2.2.1 Selection of extraction solvent

[0160] The effects of six different extraction solvents, water, 8% acetonitrile, 65% ethanol, 85% ethanol, ethanol, and methanol, on the extraction effect were investigated. Figure 1a A, B, and C are chromatograms of water, 8% acetonitrile, and 65% ethanol as extraction solvents, respectively; Figure 1b D, E, and F are chromatograms of 85% ethanol, ethanol, and methanol as extraction solvents, respectively; Figure 1a and Figure 1b The comparison results show that some chromatographic peaks are missing when the extraction solvents are water, 8% acetonitrile, 65% ethanol, and 85% ethanol. Compared with the chromatographic peaks under the conditions of methanol and ethanol, methanol has better peak shape and separation, and methanol has higher extraction efficiency, so methanol is selected as the extraction solvent.

[0161] 2.2.2 Selection of extraction conditions

[0162] The effects of two different extraction methods, ultrasonic extraction and heating reflux extraction, on the extraction effect were investigated. Figure 2 A and B are chromatograms of ultrasonic extraction and heating reflux extraction as extraction methods, respectively. Figure 2The comparison results show that the chromatographic peak shapes and separations obtained by the two extraction methods are good, and the extraction efficiency is basically the same. Considering the simplicity of experimental operation, ultrasonic extraction is selected as the extraction method.

[0163] 2.2.3 Determine the preparation method of the test product

[0164] Based on the above investigation results, the determined preparation method is as follows: accurately weigh 0.75 g of Yun black tea, place it in a stoppered conical flask, accurately add 50 mL of methanol, ultrasonically extract for 50 minutes, filter the extract solution with quantitative filter paper, dry the filtrate in a constant temperature water bath at 75°C for 2 hours, make up to volume with methanol in a 25 mL volumetric flask, shake well, and obtain.

[0165] 2.3 Chromatographic conditions

[0166] 2.3.1 Mobile phase investigation

[0167] The chromatograms of acetonitrile-0.3% phosphoric acid, acetonitrile-water, and acetonitrile-0.1% formic acid were compared. Figure 3 A, B and C are chromatograms of acetonitrile-0.3% phosphoric acid, acetonitrile-water and acetonitrile-0.1% formic acid as mobile phases, respectively; Figure 3 The comparison results show that the peak shape, separation and baseline of the chromatographic peak when the mobile phase is acetonitrile-water are better than those of other mobile phases, so acetonitrile-water is determined to be the mobile phase.

[0168] 2.3.2 Flow rate investigation

[0169] The chromatograms of 0.8mL / min, 1mL / min, and 1.2mL / min were compared. Figure 4 A, B and C are chromatograms with flow rates of 0.8 mL / min, 1 mL / min and 1.2 mL / min respectively; Figure 4 The comparison results show that the separation of peak 1 and peak 3 is better than that of other flow rates when the flow rate is 1 mL / min, so the flow rate is determined to be 1 mL / min.

[0170] 2.3.3 Sample volume investigation

[0171] The chromatograms of 5μL, 10μL, and 15μL were compared. Figure 5 A, B and C are chromatograms with injection volumes of 5 μL, 10 μL and 15 μL respectively; Figure 5 The comparison results show that the chromatographic peak shape and separation are better when the injection volume is 10μL and 15μL. Considering the chromatographic peak stability and sample recovery rate, the injection volume is determined to be 10μL.

[0172] 2.3.4 Determine the chromatographic conditions

[0173] Based on the above investigation results, the chromatographic conditions determined are as follows: using a ZORBAX Eclipse Plus C18 chromatographic column (250mm×4.6mm, 5mm); mobile phase acetonitrile (A)-water (B), gradient elution (0-6min, 4-12% A; 6-12min, 12-15% A; 12-22min, 15-19% A; 22-23min, 19-21% A; 23-26min, 21-26% A; 26-28min, 26% A; 28-29min, 26-29% A; 29-38min, 29-33% A; 38-38.5min, 33-4% A; 38.5-40min, 4% A); volume flow rate 1.0mL / min; column temperature 25℃; detection wavelength 210nm; injection volume 10mL.

[0174] 2.4 Methodological Review

[0175] 2.4.1 Specificity Investigation

[0176] Accurately pipette 10 mL of blank solvent (methanol), solution under "2.1" and solution under "2.2", and perform injection measurement under the chromatographic conditions of "2.3". Figure 6 A, B, and C are the chromatograms of blank solvent, solution under "2.1" and solution under "2.2" as injection solution respectively; Figure 6 The results showed that the chromatograms of the test solution and the reference solution had chromatographic peaks with the same retention time at corresponding positions, while the blank solvent did not. In addition, under this condition, the separation degree of the chromatographic peaks of each component was greater than 1.5, and other components had no interference with the determination of the test components. The theoretical plate number based on syringin was not less than 5000, indicating that the method had good specificity.

[0177] 2.4.2 Investigation of linear relationship

[0178] Accurately measure each series of reference solution of different concentrations prepared under item "2.1" and inject them separately, and measure them under the chromatographic conditions of item "2.3". Regression is performed with the mass concentration of each component as the abscissa (x) and the peak area as the ordinate (y). The results are shown in Table 1. The results show that the 7 components in the reference solution have good linearity within their respective ranges, and the mass concentrations at signal-to-noise ratios of 3:1 and 10:1 are taken as the detection limit and quantification limit, respectively. The results are shown in Table 1.

[0179] Table 1 Linear relationship of each component

[0180]

[0181] 2.4.3 Precision test

[0182] Accurately pipette 10 mL of the reference solution under "2.1" and continuously inject and measure 6 times under the chromatographic conditions of "2.3". The RSDs of the peak areas of syringin, echinacoside, verbascoside, epimedin A, epimedin B, epimedin C and icariin were 1.13%, 1.90%, 0.32%, 0.92%, 1.15%, 1.07% and 0.23%, respectively, indicating that the instrument has good precision.

[0183] 2.4.4 Stability test

[0184] Accurately weigh 0.75 g of Yunhong tea (20231008), prepare the test solution according to the method under "2.2", and inject and measure the samples under the chromatographic conditions of "2.3" at 0h, 2h, 4h, 8h, 16h and 24h. The RSDs of the peak areas of syringin, echinacoside, verbascoside, epimedin A, epimedin B, epimedin C and icariin were 2.04%, 2.07%, 1.92%, 2.89%, 1.93%, 3.02% and 2.21%, respectively, indicating that the test solution has good stability within 24h.

[0185] 2.4.5 Repeatability test

[0186] Accurately weigh 6 portions of the same batch of Yunhong tea samples (20231008), each about 0.75 g, and prepare the test solution in parallel according to the method under "2.2". The samples were injected and determined under the chromatographic conditions of "2.3". The average contents of syringin, echinacoside, verbascoside, epimedin A, epimedin B, epimedin C and icariin were 0.119 mg / g, 18.115 mg / g, 1.262 mg / g, 0.200 mg / g, 0.852 mg / g, 0.694 mg / g and 0.598 mg / g, respectively, and the RSDs were 2.54%, 1.75%, 2.87%, 3.95%, 3.64%, 4.86% and 2.03%, respectively.

[0187] 2.4.6 Sample recovery test

[0188] Accurately weigh 9 portions of Yun black tea sample (20231008), each portion is about 0.375g, and appropriate amount of reference solution is accurately added at the ratio of 50%, 100% and 150%, respectively. Prepare the test solution in parallel as in "2.2", inject and measure under the chromatographic conditions in "2.3", record the peak area, calculate the average recovery rate and RSD. The results are shown in Table 2.

[0189] Table 2 Results of sample recovery test for each component (n=9)

[0190]

[0191]

[0192] Example 2

[0193] Take another 4 batches of Yunhong tea samples to be tested for content determination. Accurately weigh 0.75g of samples from different batches of Yunhong tea, take 3 parallel portions from each batch, prepare the test solution according to the conditions under "2.2.3", inject and determine according to the chromatographic conditions established under "2.3.4", record the chromatogram, record the peak areas of the 7 components, calculate the concentration according to the linear relationship equation in Table 1, and the results are shown in Table 3.

[0194] Table 3 Determination results of the content of each component in different batches (mg / g, n=3)

[0195]

[0196] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0197] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0198] The above-described embodiments only express several implementation methods of the present application, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can also be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solutions provided in the present application through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic spectrum of Yun black tea, characterized in that: The composition of the Yun black tea includes black tea, Cistanche deserticola, jujube, epimedium and Acanthopanax senticosus; The method for constructing the characteristic spectrum of Yun black tea comprises the following steps: Take the Yun black tea to be tested, add the extraction solvent to extract, and obtain the test solution; The test solution is subjected to liquid chromatography to obtain a characteristic spectrum of the Yunhong tea; The conditions for the liquid chromatography detection include: (1) Acetonitrile was used as mobile phase A and water was used as mobile phase B; (2) Gradient elution is used, wherein the procedure of the gradient elution comprises: From 0min to 6min, the volume percentage of the mobile phase A increases from 4% to 12%; 6min-12min, the volume percentage of the mobile phase A increases from 12% to 15%; From 12min to 22min, the volume percentage of the mobile phase A increases from 15% to 19%; From 22min to 23min, the volume percentage of the mobile phase A increased from 19% to 21%; From 23min to 26min, the volume percentage of the mobile phase A increased from 21% to 26%; 26min-28min, maintaining the volume percentage of the mobile phase A at 26%; From 28min to 29min, the volume percentage of the mobile phase A increased from 26% to 29%; From 29min to 38min, the volume percentage of the mobile phase A increased from 29% to 33%; 38min-38.5min, the volume percentage of the mobile phase A decreases from 33% to 4%; 38.5min-40min, the volume percentage of the mobile phase A is maintained at 4%.

2. The construction method according to claim 1, characterized in that: The conditions for the liquid chromatography detection also include at least one of the following: (1) The filler of the chromatographic column is octadecylsilane bonded silica gel; (2) Column temperature is 24°C to 26°C; (3) The injection volume is 8 μL to 12 μL; (4) a flow rate of 0.8 mL / min to 1.2 mL / min; and, (5) The detection wavelength is 205nm~215nm.

3. The construction method according to claim 2, characterized in that: The length of the chromatographic column is 240 mm to 260 mm, the diameter is 4.4 mm to 4.8 mm, and the particle size of the filler is 4.8 μm to 5.2 μm.

4. The construction method according to any one of claims 1 to 3, characterized in that: The extraction solvent is an organic alcohol, and optionally, the organic alcohol includes methanol; and / or, The extraction method includes ultrasound, and optionally, the frequency of ultrasound is 40 Hz to 60 Hz, and the time is 45 min to 55 min; and / or, The mass volume ratio of the Yunhong tea to the extraction solvent is 1-2 g:100 mL.

5. The construction method according to any one of claims 1 to 3, characterized in that: The characteristic spectrum of the Yunhong tea includes 7 characteristic peaks, namely peak 1, peak 2, peak 3, peak 4, peak 5, peak 6 and peak 7, wherein peak 1 is syringin, peak 2 is echinacoside, peak 3 is verbascoside, peak 4 is epimedin A, peak 5 is epimedin B, peak 6 is epimedin C, and peak 7 is icariin; Taking peak 1 as the control, the characteristic peaks and their relative retention times are as follows: peak 2: 1.56±10%; peak 3: 2.18±10%; peak 4: 2.82±10%; peak 5: 2.87±10%; peak 6: 2.91±10%; peak 7: 2.99±10%.

6. A method for determining the content of a component to be tested in Yunhong tea, characterized in that: The composition of the Yun black tea includes black tea, Cistanche deserticola, jujube, epimedium and Acanthopanax senticosus; The method for determining the content of the index components of the Yun black tea comprises the following steps: Take a reference substance of the component to be tested, add a dissolving solvent to dissolve it, and prepare reference substance solutions of different concentrations; Taking solutions of the reference substance of different concentrations for liquid chromatography detection to obtain a standard curve of concentration and peak area; Take the Yunhong tea to be tested, add the extraction solvent to extract, and obtain the test solution; Taking the test solution for liquid chromatography detection, obtaining the peak area of ​​the test component and substituting it into the standard curve of concentration and peak area to calculate the concentration; The conditions for the liquid chromatography detection include: (1) Acetonitrile was used as mobile phase A and water was used as mobile phase B; (2) Gradient elution is used, wherein the procedure of the gradient elution comprises: From 0min to 6min, the volume percentage of the mobile phase A increases from 4% to 12%; 6min-12min, the volume percentage of the mobile phase A increases from 12% to 15%; From 12min to 22min, the volume percentage of the mobile phase A increases from 15% to 19%; From 22min to 23min, the volume percentage of the mobile phase A increased from 19% to 21%; From 23min to 26min, the volume percentage of the mobile phase A increased from 21% to 26%; 26min-28min, maintaining the volume percentage of the mobile phase A at 26%; From 28min to 29min, the volume percentage of the mobile phase A increased from 26% to 29%; From 29min to 38min, the volume percentage of the mobile phase A increased from 29% to 33%; 38min-38.5min, the volume percentage of the mobile phase A decreases from 33% to 4%; 38.5min-40min, the volume percentage of the mobile phase A is maintained at 4%.

7. The content determination method according to claim 6, characterized in that: The conditions for the liquid chromatography detection also include at least one of the following: (1) The filler of the chromatographic column is octadecylsilane bonded silica gel; (2) Column temperature is 24°C to 26°C; (3) The injection volume is 8 μL to 12 μL; (4) a flow rate of 0.8 mL / min to 1.2 mL / min; and, (5) The detection wavelength is 205nm~215nm.

8. The content determination method according to claim 7, characterized in that: The length of the chromatographic column is 240 mm to 260 mm, the diameter is 4.4 mm to 4.8 mm, and the particle size of the filler is 4.8 μm to 5.2 μm.

9. The content determination method according to any one of claims 6 to 8, characterized in that: The extraction solvent is an organic alcohol, and optionally, the organic alcohol includes methanol; and / or, The extraction method includes ultrasound, and optionally, the frequency of ultrasound is 40 Hz to 60 Hz, and the time is 45 min to 55 min; and / or, The mass volume ratio of the Yunhong tea to the extraction solvent is 1-2 g:100 mL.

10. The content determination method according to any one of claims 6 to 8, characterized in that: The dissolving solvent is an organic alcohol, and optionally, the organic alcohol includes methanol.

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