Method for constructing characteristic spectrum of yinong tea and method for determining content of to-be-tested component of yinong tea

Abstract

The application relates to the technical field of health-care food detection, in particular to a construction method of a characteristic spectrum of Yuni Hong tea and a content determination method of a to-be-detected component of the Yuni Hong tea. Chromatographic detection conditions of the application include: a mobile phase: acetonitrile (A), water (B), gradient elution (0-6 min, 4-12% A; 6-12 min, 12-15% A; 12-22 min, 15-19% A; 22-23 min, 19-21% A; 23-26 min, 21-26% A; 26-28 min, 26% A; 28-29 min, 26-29% A; 29-38 min, 29-33% A; 38-38.5 min, 33-4% A; 38.5-40 min, 4% A). Through adoption of suitable sample treatment and chromatographic detection conditions, the application realizes relatively comprehensive detection of the Yuni Hong tea and constructs the characteristic spectrum of the Yuni Hong tea, and provides a more scientific judgment basis for quality control of the Yuni Hong tea.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of health food detection, in particular to a construction method of a characteristic spectrum of Yuni Hongcha and a content determination method of a to-be-tested component thereof. BACKGROUND

[0002] Yuni Hongcha is a health food with the effect of relieving physical fatigue, which is composed of black tea, Cistanche, jujube, Herba Pykagliae and Acanthopanax; its approval number is Guo Shi Jian Zhu Zhi G20240286, and the specified effective components are 6.5 g of tea polyphenols, 0.45 g of total saponins and 0.05 g of icariin per 100 g. Since Yuni Hongcha contains prescription medicinal materials and has complex chemical components, it is difficult to comprehensively reflect the internal chemical information of Yuni Hongcha by using single compound for identification or content determination, which is not conducive to the overall quality control.

[0003] Traditional Chinese medicine (TCM) fingerprint spectrum can describe the overall characteristics of TCM and achieve the control of the overall quality of TCM by using appropriate fuzzy processing means, thanks to its advantages of comprehensiveness, macroscopicness and fuzzy analysis. This spectrum technology can visualize the overall characteristics of multiple chemical components in TCM and reveal the quality defects that may be ignored by traditional detection methods. Based on the comprehensive action characteristics of multiple components and multiple action points of TCM, the fingerprint spectrum provides a comprehensive method for modern TCM quality control from the perspective of “all components”, which is especially suitable for the quality control of non-single-component drugs.

[0004] At present, there is still a lack of systematic qualitative research on Yuni Hongcha. The specified effective components in Yuni Hongcha are all chromatographically detectable components, and the development of the characteristic spectrum and the content determination method of Yuni Hongcha according to the principle of TCM fingerprint spectrum will help to more comprehensively and accurately ensure the quality of Yuni Hongcha. SUMMARY

[0005] Therefore, one or more embodiments of the present application provide a construction method of a characteristic spectrum of Yuni Hongcha and a content determination method of a to-be-tested component thereof. The method of the present application can more comprehensively evaluate the internal chemical components of Yuni Hongcha and accurately determine the content of the to-be-tested component.

[0006] The technical solution of the present application comprises the following steps:

[0007] A construction method of a characteristic spectrum of Yuni Hongcha, wherein the composition of the Yuni Hongcha comprises black tea, Cistanche, jujube, Herba Pykagliae and Acanthopanax;

[0008] The construction method of the characteristic spectrum of the Yuni Hongcha comprises the following steps:

[0009] A test Yuni Hongcha is taken and an extraction solvent is added for extraction to obtain a test solution;

[0010] The test product solution is subjected to liquid chromatography detection to obtain the characteristic spectrum of the Yinger tea;

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

[0012] (1) acetonitrile is used as mobile phase A and water is used as mobile phase B;

[0013] (2) gradient elution is adopted, and the program of the gradient elution includes:

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

[0015] 6 min ~ 12 min, the volume percentage of the mobile phase A is increased from 12% to 15%;

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

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

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

[0019] 26 min ~ 28 min, the volume percentage of the mobile phase A is maintained at 26%;

[0020] 28 min ~ 29 min, the volume percentage of the mobile phase A is increased from 26% to 29%;

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

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

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

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

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

[0026] (2) the column temperature is 24 ℃ ~ 26 ℃;

[0027] (3) the injection amount is 8 μL ~ 12 μL;

[0028] (4) the flow rate is 0.8 mL / min to 1.2 mL / min; and

[0029] (5) the detection wavelength is 205 nm to 215 nm.

[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 the organic alcohol comprises methanol optionally; and / or,

[0032] The extraction method comprises ultrasonic, and the frequency of the ultrasonic is 40 Hz to 60 Hz, and the time is 45 min to 55 min; and / or,

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

[0034] In one embodiment, the characteristic spectrum of the Yohong tea comprises seven 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 ducnohaidin A, peak 5 is ducnohaidin B, peak 6 is ducnohaidin C, and peak 7 is icariin.

[0035] Taking peak 1 as a control, the relative retention time of each characteristic peak is 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%; and peak 7: 2.99±10%.

[0036] A content determination method of a to-be-tested component of a Yohong tea, wherein the Yohong tea comprises black tea, cistanche, jujube, epimedium and acanthopanax;

[0037] The content determination method of the index component of the Yohong tea comprises the following steps:

[0038] A control sample of the to-be-tested component is taken, dissolved with a dissolving solvent, and different concentrations of the control sample solution are prepared;

[0039] The control sample solution of different concentrations is taken for liquid chromatography detection, and a standard curve of concentration and peak area is obtained;

[0040] The Yohong tea to be tested is taken, and an extraction solvent is used for extraction, and a to-be-tested product solution is obtained;

[0041] The to-be-tested product solution is taken for liquid chromatography detection, and the peak area of the to-be-tested component is obtained and substituted into the standard curve of concentration and peak area to calculate the concentration;

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

[0043] (1) acetonitrile as mobile phase A and water as mobile phase B;

[0044] (2) gradient elution, the program of the gradient elution includes:

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

[0046] 6 min ~ 12 min, the volume percentage of the mobile phase A is increased from 12% to 15%;

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

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

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

[0050] 26 min ~ 28 min, the volume percentage of the mobile phase A is maintained at 26%;

[0051] 28 min ~ 29 min, the volume percentage of the mobile phase A is increased from 26% to 29%;

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

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

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

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

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

[0057] (2) the column temperature is 24 ~ 26℃;

[0058] (3) the injection volume is 8 ~ 12 μL;

[0059] (4) the flow rate is 0.8 ~ 1.2 mL / min; and,

[0060] (5) the detection wavelength is 205nm-215nm.

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

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

[0063] The extraction method comprises ultrasonic, and optionally, the frequency of the ultrasonic is 40Hz-60Hz, and the time is 45min-55min; and / or,

[0064] The mass-volume ratio of the Goutte de Rosée and the extraction solvent is 1-2g:100mL.

[0065] In one embodiment, the dissolving solvent is an organic alcohol, and optionally, the organic alcohol comprises methanol.

[0066] The present application realizes the comprehensive detection of Goutte de Rosée and the construction of the characteristic spectrum of Goutte de Rosée by using suitable sample processing and chromatographic detection conditions. The characteristic spectrum prepared by the method has characteristics, reproducibility and operability, and can provide rich characteristic peak information, thereby providing a more comprehensive and scientific basis for the quality control of Goutte de Rosée.

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

[0068] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0069] Figure 1a and Figure 1b The comparison chart of the chromatographic detection results of different extraction solvents in Example 1 of the present application is shown in the following table. Figure 1a In the table, A, B and C are the chromatograms of water, 8% acetonitrile and 65% ethanol as the extraction solvent, respectively. Figure 1b In the table, D, E and F are the chromatograms of 85% ethanol, ethanol and methanol as the extraction solvent, respectively.

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

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

[0072] Figure 4 The comparative chart of chromatographic detection results of different flow rates of Example 1 of the present application, wherein, Figure 4 A, B, C in the chart are chromatograms of 0.8 mL / min, 1 mL / min, 1.2 mL / min as flow rates, respectively;

[0073] Figure 5 The comparative chart of chromatographic detection results of different injection amounts of Example 1 of the present application, wherein, Figure 5 A, B, C in the chart are chromatograms of 5 μL, 10 μL, 15 μL as injection amounts, respectively;

[0074] Figure 6 The comparative chart of chromatographic detection results of blank solvent, reference solution and sample solution of Example 1 of the present application, wherein, Figure 6 A in the chart is the chromatogram of blank solvent, Figure 6 B in the chart is the chromatogram of reference solution, Figure 6 C in the chart is the chromatogram of sample 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 not intended to limit the scope of the present application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the content taught in the present application, and these equivalent forms also fall within the protection scope of the claims of the present application.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the description of the present application herein only for the purpose of describing specific embodiments of the present application and is not intended to limit the present application.

[0077] Terminology

[0078] Unless otherwise indicated or contradictory, the terms or phrases used herein have the following meanings:

[0079] The selection range of the term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, which includes any two related listed items, any more related listed items, or a combination of 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 the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B, and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (i.e., the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C, and D, i.e., includes a combination of any two or any three of A, B, C, and D, and also includes a four-item combination of A, B, C, and D (i.e., the technical solution connected by "logical and").

[0080] Herein, "preferably", "more preferably", "even more preferably", and the like only describe embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present application.

[0081] In the present application, "further", "even further", "in particular", and the like are used for description purposes, and represent differences in content, but should not be understood as a limitation on the protection scope of the present application.

[0082] In the present application, in the technical features described in an open manner, a closed technical solution composed of listed features is included, and an open technical solution containing the listed features is also included.

[0083] In the present application, in relation to a numerical interval (i.e., a numerical range), if no special description is provided, it is considered that the optional numerical distribution is continuous within the numerical interval, and includes both numerical end points (i.e., the minimum value and the maximum value) of the numerical range, and each numerical value between the two numerical end points. If no special description is provided, when the numerical interval only points to integers within the numerical interval, the two end point integers of the numerical range, and each integer between the two end points are included. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise indicated, the ranges disclosed herein should be understood to include any and all sub-ranges therein.

[0084] The temperature parameters in the present application, if not particularly limited, allow for constant temperature treatment as well as for variations within a certain temperature interval. It is to be understood that the constant temperature treatment allows for fluctuations within the accuracy of the instrument control. Fluctuations within a range of, for example, ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, ±0.1°C are allowed.

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

[0086] In the present application, the chromatogram obtained by liquid chromatography detection has a horizontal coordinate of minutes (min) and a vertical coordinate of response value (mAU).

[0087] In one aspect of the present application, a method for constructing a characteristic map of Yuni Hongcha is provided, the composition of Yuni Hongcha comprising Hongcha, Cistanche, Jujube, Herba Epimedii, and Acanthopanax;

[0088] The method for constructing a characteristic map of Yuni Hongcha comprises the following steps:

[0089] A test Yuni Hongcha is taken, and an extraction solvent is added for extraction to obtain a test solution;

[0090] The test solution is subjected to liquid chromatography detection to obtain a characteristic map of Yuni Hongcha;

[0091] The conditions for liquid chromatography detection include:

[0092] (1) acetonitrile is used as mobile phase A, and water is used as mobile phase B;

[0093] (2) gradient elution is adopted, and the program for gradient elution comprises:

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

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

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

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

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

[0099] 26 min to 28 min, the volume percentage of mobile phase A is maintained at 26%;

[0100] 28min~29min, the volume percentage of mobile phase A is increased from 26% to 29%;

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

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

[0103] 38.5min~40min, the volume percentage of mobile phase A is maintained at 4%.

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

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

[0106] (2) the column temperature is 24~26℃;

[0107] (3) the injection volume is 8~12μL;

[0108] (4) the flow rate is 0.8~1.2mL / min; and,

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

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

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

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

[0113] In one embodiment, the column temperature is 24~26℃, for example, 24℃, 25℃, 26℃, etc.

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

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

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

[0117] In one embodiment, the extraction solvent is an organic alcohol, which can be methanol.

[0118] In one embodiment, the extraction method comprises ultrasonic, which can be at a frequency of 40Hz-60Hz, for example, 40Hz, 45Hz, 50Hz, 55Hz, 60Hz, etc., for a time of 45min-55min, for example, 45min, 48min, 50min, 52min, 55min, etc.

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

[0120] In one embodiment, the characteristic spectrum of the Yuhong tea comprises 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 damianol A, peak 5 is damianol B, peak 6 is damianol C, and peak 7 is icariin.

[0121] With peak 1 as a control, the relative retention times of the characteristic peaks 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%; and peak 7: 2.99±10%.

[0122] In another aspect of the present application, a content determination method of a to-be-tested component of Yuhong tea is provided, wherein the composition of the Yuhong tea comprises black tea, cistanche, jujube, epimedium and acanthopanax.

[0123] The content determination method of the index component of the Yuhong tea comprises the following steps:

[0124] A control sample of the to-be-tested component is taken, dissolved with a solvent, and different concentrations of the control sample solution are prepared;

[0125] The control sample solution of different concentrations is taken for liquid chromatography detection, and a standard curve of concentration and peak area is obtained;

[0126] The Yuhong tea to be tested is taken, and an extraction solvent is used for extraction, and a to-be-tested product solution is obtained;

[0127] The peak area of the to-be-tested component is obtained by liquid chromatography detection, and the concentration is calculated by substituting the peak area into the standard curve of the concentration and the peak area.

[0128] The conditions of liquid chromatography detection include:

[0129] (1) acetonitrile as mobile phase A and water as mobile phase B;

[0130] (2) gradient elution, and the gradient elution program includes:

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

[0132] 6 min~12 min, the volume percentage of mobile phase A is increased from 12% to 15%;

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

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

[0135] 23 min~26 min, the volume percentage of mobile phase A is increased from 21% to 26%;

[0136] 26 min~28 min, the volume percentage of mobile phase A is maintained at 26%;

[0137] 28 min~29 min, the volume percentage of mobile phase A is increased from 26% to 29%;

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

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

[0140] 38.5 min~40 min, the volume percentage of mobile phase A is maintained at 4%.

[0141] The conditions of liquid chromatography detection in the content determination method of the to-be-tested component of the Camellia japonica of the embodiments of the present application are preferably the same as the conditions of liquid chromatography detection in the construction method of the fingerprint of the Camellia japonica of any one of the technical solutions above.

[0142] The extraction solvent and the extraction method used in the content determination method of the to-be-tested component of the Camellia japonica of the embodiments of the present application are preferably the same as the extraction solvent and the extraction method in the construction method of the fingerprint of the Camellia japonica of any one of the technical solutions above.

[0143] In one embodiment, the mass-volume ratio of the sample and the extraction solvent is 1-2 g: 100 mL.

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

[0145] The following are some specific embodiments.

[0146] In the following specific embodiments, the experimental parameters not specified can be preferably referred to the guidance given in the present application, and can also be referred to the experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturers.

[0147] In the following specific embodiments, the raw materials and reagents involved can be obtained commercially or prepared by those skilled in the art according to known means.

[0148] Embodiment 1

[0149] 1. Instruments and materials

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

[0151] Reference substances: Syringin (Batch No. 111574-202106, purity 94.3%), Echinacoside (Batch No. 111670-201907, purity 91.8%), Verbascoside (Batch No. 111530-201914, purity 95.2%), Daurisoline C (Batch No. 111780-201905, purity 94.3%), Icariin (Batch No. 110737-202017, purity 98.1%) were purchased from China Institute for Food and Drug Control; Daurisoline A (Batch No. WP23042711, purity 98.0%), Daurisoline B (Batch No. WP23062006, purity 98.0%) were purchased from Sichuan Weikexi Biological Technology Co., Ltd.

[0152] Reagents: Methanol and acetonitrile were chromatographically pure; water was purified water.

[0153] Yunhong tea samples: Four batches of Yunhong tea bags were produced by Zhengzhou Linuo Pharmaceutical Co., Ltd. and were numbered according to the production batch, in the order of 20220321, 20220322, 20220323 and 20231008.

[0154] 2. Method and results

[0155] 2.1 Reference substance solution

[0156] An appropriate amount of each reference substance of Syringin, Echinacoside, Verbascoside, Daurisoline A, Daurisoline B, Daurisoline C and Icariin was precisely weighed into a 10 mL volumetric flask, and methanol was used to prepare reference 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. An appropriate amount of each reference stock solution was transferred into a 10 mL volumetric flask, and methanol was used to make up to the volume, shake well, and prepare different concentrations of each series of reference substance solutions, which were obtained.

[0157] The concentrations of the following reference solutions were as follows: syringin series: 1.0 μg / mL, 3.0 μg / mL, 5.0 μg / mL, 7.0 μg / mL, and 8.9 μg / mL; echinacoside series: 75.8 μg / mL, 227.4 μg / mL, 379.0 μg / mL, 530.7 μg / mL, and 682.3 μg / mL; verbascoside series: 7.8 μg / mL, 23.3 μg / mL, 38.8 μg / mL, 54.3 μg / mL, and 69.8 μg / mL; and astragaloside A series: 0.9 μg / mL, 2.8 μg / mL, and 8.9 μg / mL. The concentrations of the reference solutions for the icariin B series were 3.3 μg / mL, 9.8 μg / mL, 16.3 μg / mL, 22.8 μg / mL, and 29.3 μg / mL; the concentrations of the reference solutions for the icariin C series were 3.2 μg / mL, 9.5 μg / mL, 15.9 μg / mL, 22.2 μg / mL, and 28.6 μg / mL; and the concentrations of the reference solutions for the icariin series were 3.7 μg / mL, 11.0 μg / mL, 18.3 μg / mL, 25.6 μg / mL, and 33.0 μg / 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 efficiency were investigated. Figure 1a Chromatograms A, B, and C are for water, 8% acetonitrile, and 65% ethanol as extraction solvents, respectively. Figure 1b Chromatograms D, E, and F are for extraction with 85% ethanol, ethanol, and methanol, respectively; according to Figure 1a and Figure 1b The comparison results showed that some chromatographic peaks were missing when the extraction solvents were water, 8% acetonitrile, 65% ethanol, and 85% ethanol. Compared with the chromatographic peaks under the conditions of methanol and ethanol, methanol had better peak shape and resolution, and methanol had higher extraction efficiency. Therefore, methanol was chosen as the extraction solvent.

[0161] 2.2.2 Selection of Extraction Conditions

[0162] The effects of two different extraction methods, ultrasonic extraction and reflux extraction, on the extraction efficiency were investigated. Figure 2 Chromatograms A and B in the image show ultrasonic extraction and reflux extraction, respectively, as extraction methods. Figure 2The comparison results show that the peak shape and separation degree of the chromatographic peaks obtained by the two extraction methods are good, and the extraction efficiency is basically consistent. Considering the simplicity of the experimental operation, ultrasonic extraction is selected as the extraction method.

[0163] 2.2.3 Determination of test sample preparation method

[0164] Based on the above investigation results, the preparation method is determined as follows: accurately weigh 0.75 g of Yuhong tea, place it in a conical flask with a stopper, accurately add 50 mL of methanol, ultrasonic extract for 50 min, filter the extraction solution with a quantitative filter paper, dry the filtrate in a constant temperature water bath at 75°C for 2 h, and then dilute to 25 mL with methanol in a volumetric flask, shake well, and obtain the sample.

[0165] 2.3 Chromatographic conditions

[0166] 2.3.1 Investigation of mobile phase

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

[0168] 2.3.2 Investigation of flow rate

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

[0170] 2.3.3 Investigation of injection volume

[0171] The chromatograms of 5 μL, 10 μL and 15 μL are compared, Figure 5 wherein A, B and C are the chromatograms of 5 μL, 10 μL and 15 μL as the injection volume; according to the comparison results, Figure 5 the peak shape and separation degree when the injection volume is 10 μL and 15 μL are good, considering the chromatographic peak stability and sample recovery rate, so the injection volume is determined to be 10 μL.

[0172] 2.3.4 Determination of chromatographic conditions

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

[0174] 2.4 Methodology investigation

[0175] 2.4.1 Specificity investigation

[0176] Precisely take 10 mL of the blank solvent (methanol), the solution under item "2.1" and the solution under item "2.2", and inject them under the chromatographic conditions of item "2.3" for determination, Figure 6 Figures A, B and C are chromatograms of the blank solvent, the solution under item "2.1" and the solution under item "2.2" as injection liquids, respectively; according to Figure 6 The results show that the chromatogram of the test sample solution and the chromatogram of the reference substance solution have chromatographic peaks with the same retention time at the corresponding positions, while the blank solvent does not, and the separation degree of each component chromatographic peak under the condition is greater than 1.5, other components do not interfere with the determination of the tested components, and the theoretical plate number is not less than 5000 with syringin as the reference, indicating that the method has good specificity.

[0177] 2.4.2 Investigation of linear relationship

[0178] Precisely take different concentrations of each series of reference substance solutions prepared under item "2.1" and inject them, and determine them under the chromatographic conditions of item "2.3", and regress 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, which shows that the 7 components in the reference substance solution have good linearity in their respective ranges, and the mass concentrations at the signal-to-noise ratios of 3:1 and 10:1 are taken as the detection limit and the quantification limit, respectively, and the results are shown in Table 1.

[0179] Table 1 Linear relationship of each component

[0180]

[0181] 2.4.3 Precision test

[0182] The precision of 10 mL of the reference solution under item "2.1" was measured under the chromatographic conditions of item "2.3" for 6 times in succession, and the peak area RSDs 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 precision was good.

[0183] 2.4.4 Stability test

[0184] 0.75 g of Yuni Hongcha (20231008) was precisely weighed, and the test sample solution was prepared according to the method under item "2.2". The sample was injected and determined under the chromatographic conditions of item "2.3" at 0 h, 2 h, 4 h, 8 h, 16 h and 24 h, and the peak area RSDs 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 sample solution had good stability within 24 h.

[0185] 2.4.5 Reproducibility test

[0186] Six portions of the same batch of Yuni Hongcha sample (20231008) were precisely weighed, each about 0.75 g, and the test sample solutions were prepared in parallel according to the method under item "2.2". The sample was injected and determined under the chromatographic conditions of item "2.3", and 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 Spiking recovery test

[0188] Nine portions of Yuni Hongcha sample (20231008) were precisely weighed, each about 0.375 g, and appropriate amounts of reference solution were precisely added at a ratio of 50%, 100% and 150% respectively. The test sample solutions were prepared in parallel according to the method under item "2.2", and the sample was injected and determined under the chromatographic conditions of item "2.3". The peak area was recorded, and the average spiking recovery rate and RSD were calculated. The results are shown in Table 2.

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

[0190]

[0191]

[0192] Example 2

[0193] The content of the 4 batches of Yuhong tea samples to be tested was determined. 0.75 g of different batches of Yuhong tea samples was accurately weighed, 3 samples were taken in parallel for each batch, the sample solution was prepared according to the conditions in item 2.2.3, the sample was injected for determination according to the chromatographic conditions established in item 2.3.4, the chromatogram was recorded, the peak areas of 7 components were recorded, the concentration was calculated 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 the documents mentioned in the present application are cited as references in the present application, as if each document is cited as a reference individually. Unless and to the extent that the documents cited in the present application conflict with the application purpose and / or technical scheme of the present application, the cited documents are cited in the present application in their entirety, in their entirety purpose. When the present application refers to the cited documents, the definition of the relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present application refers to the cited documents, the examples, preferred modes of the relevant technical features cited are also incorporated into the present application as references, but are limited to the implementation of the present application. It should be understood that when the cited content conflicts with the description in the present application, the present application is used as the reference or is modified according to the description in the present application.

[0197] Each technical feature of the above-mentioned embodiments and examples can be combined in any suitable manner. In order to make the description simple, each technical feature in the above-mentioned embodiments and examples is not described in all possible combinations, however, as long as the combination of the technical features does not exist, it should be considered that it is within the scope of the present description.

[0198] The above-mentioned embodiments only express several embodiments of the present application, but should not be construed as limiting the scope of the patent application. It should be noted that for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, which are within the scope of the present application. In addition, it should be understood that after reading the above description of the present application, those skilled in the art can make various modifications or modifications to the present application, and the equivalent forms obtained are also within the scope of the present application. It should also be understood that the technical solutions provided by the present application can be obtained by logical analysis, reasoning or limited experiments, which are within the scope of the claims of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description can be used to explain the content of the claims.

Claims

1. A method for constructing a characteristic map of a Gynostemma pentaphyllum, characterized in that, The composition of the Yuni Hongcha tea comprises Hongcha tea, Cistanche, jujube, Epimedium and Acanthopanax; The method for constructing the characteristic map of the Yuni Hongcha tea comprises the following steps: 0.75 g of the Yuni Hongcha tea is taken, 50 mL of methanol is added, and ultrasonic extraction is performed for 50 min, the extraction solution is filtered, the filtrate is dried in a water bath at 75℃ for 2 h, the dried product is made up to 25 mL with methanol to obtain a test solution; The test solution is subjected to liquid chromatography detection to obtain the characteristic map of the Yuni Hongcha tea; The liquid chromatography detection conditions comprise: a ZORBAX Eclipse Plus C18 chromatographic column is used, the column length is 250 mm, the inner diameter is 4.6 mm, and the particle size of the filler is 5 mm; acetonitrile is used as the mobile phase A, and water is used as the mobile phase B; gradient elution is adopted, and the gradient elution program comprises: 0 min~6 min, the volume percentage of the mobile phase A is increased from 4% to 12%; 6 min~12 min, the volume percentage of the mobile phase A is increased from 12% to 15%; 12 min~22 min, the volume percentage of the mobile phase A is increased from 15% to 19%; 22 min~23 min, the volume percentage of the mobile phase A is increased from 19% to 21%; 23 min~26 min, the volume percentage of the mobile phase A is increased from 21% to 26%; 26 min~28 min, the volume percentage of the mobile phase A is maintained at 26%; 28 min~29 min, the volume percentage of the mobile phase A is increased from 26% to 29%; 29 min~38 min, the volume percentage of the mobile phase A is increased from 29% to 33%; 38 min~38.5 min, the volume percentage of the mobile phase A is decreased from 33% to 4%; 38.5 min~40 min, the volume percentage of the mobile phase A is maintained at 4%; The volume flow rate is 1.0 mL / min; the column temperature is 25℃; the detection wavelength is 210 nm; and the injection volume is 10 μL.

2. The construction method of claim 1, wherein, The characteristic map of the Yuni Hongcha tea comprises seven 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 epimedioid A, peak 5 is epimedioid B, peak 6 is epimedioid C, and peak 7 is icariin; With peak 1 as the control, the relative retention times of the characteristic peaks 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%; and peak 7: 2.99±10%.

3. A method for determining the content of a component to be measured in a rooibos tea, characterized in that The composition of the Yuni Hongcha tea comprises Hongcha tea, Cistanche, jujube, Epimedium and Acanthopanax; The method for determining the content of the index components of the Yuni Hongcha tea comprises the following steps: The control sample of the component to be determined is taken, dissolved with a dissolving solvent, and different concentrations of the control sample solution are prepared; The control sample solutions of different concentrations are subjected to liquid chromatography detection to obtain a standard curve of concentration and peak area; The control sample of the component to be determined is taken, dissolved with a dissolving solvent, and different concentrations of the control sample solution are prepared; Take 0.75 g of the to-be-tested Yinger tea and add 50 mL of methanol to ultrasonically extract for 50 min. Filter the extraction solution, dry the filtrate in a water bath at 75 ℃ for 2 h, and then use methanol to fix the dried product to 25 mL to obtain a to-be-tested solution; Take the to-be-tested solution for liquid chromatography detection to obtain the peak area of the to-be-tested component and then substitute it into the standard curve of the concentration and the peak area to calculate the concentration; The liquid chromatography detection conditions include: using a ZORBAX Eclipse Plus C18 chromatographic column with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 mm; Using acetonitrile as mobile phase A and water as mobile phase B; Gradient elution is adopted, and the gradient elution program includes: 0 min~6 min, the volume percentage of the mobile phase A is increased from 4% to 12%; 6 min~12 min, the volume percentage of the mobile phase A is increased from 12% to 15%; 12 min~22 min, the volume percentage of the mobile phase A is increased from 15% to 19%; 22 min~23 min, the volume percentage of the mobile phase A is increased from 19% to 21%; 23 min~26 min, the volume percentage of the mobile phase A is increased from 21% to 26%; 26 min~28 min, the volume percentage of the mobile phase A is maintained at 26%; 28 min~29 min, the volume percentage of the mobile phase A is increased from 26% to 29%; 29 min~38 min, the volume percentage of the mobile phase A is increased from 29% to 33%; 38 min~38.5 min, the volume percentage of the mobile phase A is decreased from 33% to 4%; 38.5 min~40 min, the volume percentage of the mobile phase A is maintained at 4%; The volume flow rate is 1.0 mL / min; the column temperature is 25 ℃; the detection wavelength is 210 nm; and the injection amount is 10 μL.

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