Method for detecting content of chemical components in cotton flower medicinal material

Abstract

The invention relates to the field of traditional Chinese medicine quality detection, in particular to a method for detecting the content of chemical components in cotton flower medicinal materials. The detection method comprises the following steps: providing a reference substance solution, wherein a reference substance comprises one or more of hyperoside, isoquercitrin, astragalus smicus glycoside, quercetin, quercetin-7-O-glucoside and quercetin-3 '-glucoside; preparing a test solution by using a cotton flower medicinal material sample to be detected; and detecting the reference substance solution and the test solution by high performance liquid chromatography, and determining the content of chemical components corresponding to the reference substance in the to-be-detected cotton flower medicinal material sample, the high performance liquid chromatography meets one or more of the following conditions: a stationary phase is a C18 chromatographic column; the mobile phase comprises a mobile phase A, a mobile phase B and a mobile phase C, the mobile phase A comprises methanol, the mobile phase B comprises acetonitrile, and the mobile phase C comprises a phosphoric acid aqueous solution. The whole method is high in sensitivity, simple and convenient to operate, relatively low in cost and short in detection time.

Description

Technical Field

[0001] The present application relates to the field of quality testing of traditional Chinese medicine, and in particular to a method for testing the content of chemical components in cotton flower medicinal materials. Background Art

[0002] Cotton flowers are the dried flowers of Gossypium herbaceum L. or G. hirsutum L., plants of the Malvaceae family, and are a commonly used medicinal material in Uyghur medicine.

[0003] In Uyghur medicine, cotton flowers are used to treat various mental illnesses, brain diseases, immune disorders, and hepatitis. According to literature, cotton flowers have a damp-heat nature and a slightly bitter taste. Their effects include: removing dampness and heat, calming the mind and inducing sleep, aromatically invigorating the orifices, nourishing the nerves, and tonifying the heart and mind. Indications: Various dry-cold neurological disorders, such as decreased mental capacity and neurasthenia. The 2020 edition of the "Xinjiang Uyghur Autonomous Region Traditional Chinese Medicine and Uyghur Medicinal Pieces Preparation Specifications" describes their functions and indications as removing dampness and heat, calming the mind and inducing sleep, aromatically invigorating the orifices, nourishing the nerves, and tonifying the heart and mind. They are used to treat dry-cold or black bile diseases, dry-cold brain neurological disorders, palpitations, restlessness, insomnia, depression, decreased mental capacity, and neurasthenia.

[0004] The regulations cover cotton flower characteristics, thin-layer identification, microscopic identification, impurities, moisture, total ash, and extract determination. Under the content determination section, hyperoside content is used to evaluate cotton flower quality. This single component, hyperoside, is used as a quality evaluation indicator, making it inaccurate and incomplete for quality control of cotton flower medicinal materials. A quality testing method that can comprehensively reflect the quality of cotton flower medicinal materials is urgently needed. Summary of the Invention

[0005] Based on this, one or more embodiments of the present application provide a method for detecting the content of chemical components in cotton flower medicinal materials, including the following technical solutions:

[0006] One or more embodiments of the present application provide a method for detecting the content of chemical components in cotton flower medicinal materials, the detection method comprising the following steps:

[0007] Providing a reference solution, wherein the reference substance in the reference solution includes one or more of hyperoside, isoquercetin, astragalin, quercetin, quercetin-7-O-glucoside, and quercetin-3'-glucoside;

[0008] Prepare a test solution using the cotton flower medicinal material sample to be tested; and,

[0009] The reference solution and the test solution are tested by high performance liquid chromatography to determine the content of the chemical components corresponding to the reference solution in the cotton flower medicinal material sample to be tested;

[0010] The high performance liquid chromatography method satisfies one or more of the following conditions:

[0011] (a) The stationary phase is a C18 chromatographic column;

[0012] (b) The mobile phase includes mobile phase A, mobile phase B, and mobile phase C, wherein the mobile phase A includes methanol, the mobile phase B includes acetonitrile, and the mobile phase C includes a phosphoric acid aqueous solution.

[0013] In some embodiments of the present application, the C18 chromatographic column includes a Hypersil Gold C18 chromatographic column, 250 mm×4.6 mm, 5 μm.

[0014] In some embodiments of the present application, the mobile phase C comprises a phosphoric acid aqueous solution having a phosphoric acid concentration of 0.08 wt % to 0.12 wt %.

[0015] In some embodiments of the present application, the high performance liquid chromatography method further satisfies the following condition: a gradient elution program is used.

[0016] In some embodiments of the present application, the gradient elution procedure comprises:

[0017] From 0 min to 5 min, the volume proportion of the mobile phase A increased from 10% to 40%, the volume proportion of the mobile phase B was 0%, and the volume proportion of the mobile phase C decreased from 90% to 60%;

[0018] From 5 min to 6 min, the volume proportion of the mobile phase A decreased from 40% to 0%, the volume proportion of the mobile phase B increased from 0% to 13%, and the volume proportion of the mobile phase C increased from 60% to 87%;

[0019] 6 min-27 min, the volume proportion of the mobile phase A is 0%, the volume proportion of the mobile phase B is 13%, and the volume proportion of the mobile phase C is 87%;

[0020] From 27 min to 32 min, the volume proportion of the mobile phase A was 0%, the volume proportion of the mobile phase B increased from 13% to 18%, and the volume proportion of the mobile phase C decreased from 87% to 82%;

[0021] 32 min-37 min, the volume proportion of the mobile phase A is 0%, the volume proportion of the mobile phase B is 18%, and the volume proportion of the mobile phase C is 82%;

[0022] From 37 min to 65 min, the volume proportion of the mobile phase A was 0%, the volume proportion of the mobile phase B increased from 18% to 30%, and the volume proportion of the mobile phase C decreased from 82% to 70%;

[0023] From 65 min to 70 min, the volume proportion of the mobile phase A was 0%, the volume proportion of the mobile phase B increased from 30% to 85%, and the volume proportion of the mobile phase C decreased from 70% to 15%;

[0024] 70 min-75 min, the volume proportion of the mobile phase A is 0%, the volume proportion of the mobile phase B is 85%, and the volume proportion of the mobile phase C is 15%;

[0025] From 75 min to 80 min, the volume proportion of the mobile phase A increased from 0% to 10%, the volume proportion of the mobile phase B decreased from 85% to 0%, and the volume proportion of the mobile phase C increased from 15% to 90%;

[0026] From 80 min to 87 min, the volume proportion of the mobile phase A was 10%, the volume proportion of the mobile phase B was 0%, and the volume proportion of the mobile phase C was 90%.

[0027] In some embodiments of the present application, the high performance liquid chromatography method further satisfies one or more of the following conditions:

[0028] (1) Column temperature is 20℃-35℃;

[0029] (2) The injection volume is 5 μL-15 μL;

[0030] (3) The detection wavelength is 350nm-360nm; and

[0031] (4) The flow rate is 0.8 mL / min-1 mL / min.

[0032] In some embodiments of the present application, the reference solution satisfies one or more of the following conditions:

[0033] (A) the solvent in the reference solution includes methanol; and

[0034] (B) The concentration of each reference substance in the reference substance solution is independently 80 μg / mL to 120 μg / mL.

[0035] In some embodiments of the present application, the steps of preparing the test solution include:

[0036] The cotton flower medicinal material sample to be tested is mixed with an extraction solvent for extraction, and the extract is collected to prepare the test solution.

[0037] In some embodiments of the present application, the extraction method includes heating reflux extraction.

[0038] In some embodiments of the present application, the heating reflux extraction satisfies one or more of the following conditions:

[0039] (I) The extraction temperature is 70°C to 80°C;

[0040] (II) The extraction time of heating and reflux is 15min-120min;

[0041] (III) the extraction solvent for the extraction under heating and reflux comprises methanol; and

[0042] (IV) The dosage ratio of the cotton flower medicinal material sample to be tested and the extraction solvent is 0.5 g: (25-100) mL.

[0043] Compared with traditional technologies, the advantages of this application include:

[0044] This application discovered active ingredients that can characterize the quality of cotton flower medicinal materials, such as hyperoside, isoquercetin, astragalin, quercetin, quercetin-7-O-glucoside, and quercetin-3'-glucoside. Based on this, this application specifically established a detection method for detecting the content of this active ingredient. This detection method is based on a suitable stationary phase and mobile phase, and is further combined with a suitable gradient elution procedure. It can well separate the various components and ultimately achieve analytical detection, providing an overall quality control basis for the quality standards of authentic cotton flower medicinal materials. The entire method has high sensitivity, simple operation, low cost, and short detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0046] Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 The chromatograms obtained corresponding to Schemes 1 to 5 in Example 1 are shown;

[0047] Figure 6 、 Figure 7 、 Figure 8 The chromatograms obtained at different flow rates in Example 1 are as follows;

[0048] Figure 9 、 Figure 10 、 Figure 11 The chromatograms obtained under different injection amounts in Example 1 are as follows;

[0049] Figure 12 、 Figure 13 The chromatograms obtained by different extraction methods in Example 1 are shown below:

[0050] Figure 14 、 Figure 15 、 Figure 16 The chromatograms obtained under different extraction solvents in Example 1 are shown;

[0051] Figure 17 、 Figure 18 、 Figure 19 The chromatograms obtained under different material ratios in Example 1 are shown below:

[0052] Figure 20 、 Figure 21 、 Figure 22 、 Figure 23 The chromatograms obtained at different extraction times in Example 1 are shown;

[0053] Figure 24 、 Figure 25 、 Figure 26 、 Figure 27 The chromatograms obtained at different column temperatures in Example 1 are shown. DETAILED DESCRIPTION

[0054] Below in conjunction with accompanying drawing, embodiment and example, the application is described in further detail.It should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms and is not limited to the embodiment and example described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the application.It should be understood that the application can be implemented without one or more of these details.

[0055] 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 pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only and are not intended to limit this application.

[0056] the term

[0057] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0058] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of 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 connected by "logical and" and also undoubtedly includes technical solutions 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 of all being 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 four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0059] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0060] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0061] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0062] Herein, "preferred", "better", "more preferred" and "suitable" are merely 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.

[0063] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0064] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0065] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0066] In this 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.

[0067] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0068] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0069] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0070] 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 in their entirety and for all purposes. When 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 cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement 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 adaptively amended according to the description in this application.

[0071] Cotton flower chemical composition determination utilizes modern analytical techniques, such as high-performance liquid chromatography (HPLC) and mass spectrometry (MS), to qualitatively and quantitatively analyze the complex chemical components in cotton flowers. By establishing specific chromatographic profiles, it accurately reflects the types and contents of various components in cotton flowers, providing a scientific basis for cotton flower quality control and standardization.

[0072] Tao et al. used a C18 column with gradient elution using methanol, acetonitrile, and 0.2% aqueous phosphoric acid to establish a method for the simultaneous determination of seven flavonoids in cotton flowers, including quercetin, hyperoside, isoquercetin, astragaloside IV, kaempferol, and quercetin-3-O-glucoside. In 2009, they isolated and purified three flavonoids, quercetin, and kaempferol, from cotton flowers using high-speed countercurrent chromatography.

[0073] Zhao Yongxin et al. studied the extraction and purification processes of cotton flower extract and identified 20 flavonoids using column chromatography and HPLC-MS. In 2013, they used high-speed countercurrent chromatography (HSCC) to separate and purify isoquercetin from cotton flower extract in a one-step process. The solvent system consisted of ethyl acetate and water (1:1), with the upper phase as the stationary phase and the lower phase as the mobile phase. The main engine speed was 800 r / min, the volume flow rate was 2.0 mL / min, and the detection wavelength was 254 nm. The structure of the obtained compound was identified by spectrometry, and the purity of the product was determined by thin-layer chromatography and high-performance liquid chromatography. Further separation under these conditions yielded an isoquercetin reference substance with a mass fraction of 99%, establishing a method for preparing an isoquercetin standard from cotton flower extract.

[0074] Wu Tao et al. established an HPLC method for the determination of hyperoside and isoquercetin in tablets. The chromatographic column was Phenomenex Synergi Fusion-RP80A (250 mm × 4.60 mm, 4 μm), the mobile phase was methanol-acetonitrile-0.1% phosphoric acid aqueous solution (19:6:75), the column temperature was 30 ° C, the volume flow rate was 1.0 mL / min, the detection wavelength was 360 nm, and the injection volume was 10 μL.

[0075] Elliger et al. isolated and identified 3,5,7,4,-tetrahydroxy-8-methoxyflavone from the immature flower buds of cotton plants.

[0076] In 2008, Yi Yang et al. used high-speed countercurrent chromatography to separate and identify quercetin, quercetin-3,-glucoside, isoquercetin, and quercetin-7-O-glucoside from cotton flowers. In 2010, mass spectrometry was used to further separate and identify four secondary flavonols in cotton extracts using high-speed countercurrent chromatography, namely astragaloside, hyperoside, 8-methoxy-kaempferol-7-rhamnoside, and 4,-methoxyquercetin-7-O-β-D-glucoside. Zhao Yongxin et al. [6-7] Column chromatography and HPLC-MS^n technology were used to identify 20 flavonoid compounds. In 2013, cotton flower extract was separated and purified by high-speed countercurrent chromatography to prepare isoquercetin. The structure of the obtained compound was identified by spectroscopy, and the purity of the product was determined by thin-layer chromatography and high-performance liquid chromatography. Wu Tao et al. [8] A HPLC method for the determination of hyperoside and isoquercetin in cotton flower total flavonoids tablets was established.

[0077] Sai Liman Hader used high-performance liquid chromatography-electrospray ionization tandem mass spectrometry to determine the histidine content in cotton flower petal extracts. This elution procedure, used for determining the active ingredients in cotton flower medicinal materials, does not ensure the separation and identification of all active ingredients. It is not possible to simultaneously determine hyperoside, isoquercitrin, astragalin, quercetin, quercetin-7-O-glucoside, and quercetin-3'-glucoside.

[0078] However, the above-mentioned traditional method for determining the content of active ingredients in cotton flowers by HPLC cannot fully characterize the quality of cotton flower medicinal materials.

[0079] Based on this, an embodiment of the present application provides a method for detecting the content of chemical components in cotton flower medicinal materials, the detection method comprising the following steps:

[0080] Providing a reference solution, wherein the reference substance in the reference solution includes one or more of hyperoside, isoquercetin, astragalin, quercetin, quercetin-7-O-glucoside, and quercetin-3'-glucoside;

[0081] Prepare a test solution using the cotton flower medicinal material sample to be tested; and,

[0082] The reference solution and the test solution are tested by high performance liquid chromatography to determine the content of the chemical components corresponding to the reference solution in the cotton flower medicinal material sample to be tested;

[0083] The high performance liquid chromatography method satisfies one or more of the following conditions:

[0084] (a) The stationary phase is a C18 chromatographic column;

[0085] (b) The mobile phase includes mobile phase A, mobile phase B, and mobile phase C, wherein the mobile phase A includes methanol, the mobile phase B includes acetonitrile, and the mobile phase C includes a phosphoric acid aqueous solution.

[0086] In some embodiments of the present application, the C18 chromatographic column includes a Hypersil Gold C18 chromatographic column, 250 mm×4.6 mm, 5 μm.

[0087] In some embodiments of the present application, the mobile phase C comprises a phosphoric acid aqueous solution having a phosphoric acid concentration of 0.08 wt%-0.12 wt% (eg, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.11 wt%, 0.12 wt%).

[0088] In some embodiments of the present application, the high performance liquid chromatography method further satisfies the following condition: a gradient elution program is used.

[0089] In some embodiments of the present application, the gradient elution procedure comprises:

[0090] From 0 min to 5 min, the volume proportion of the mobile phase A increased from 10% to 40%, the volume proportion of the mobile phase B was 0%, and the volume proportion of the mobile phase C decreased from 90% to 60%;

[0091] From 5 min to 6 min, the volume proportion of the mobile phase A decreased from 40% to 0%, the volume proportion of the mobile phase B increased from 0% to 13%, and the volume proportion of the mobile phase C increased from 60% to 87%;

[0092] 6 min-27 min, the volume proportion of the mobile phase A is 0%, the volume proportion of the mobile phase B is 13%, and the volume proportion of the mobile phase C is 87%;

[0093] From 27 min to 32 min, the volume proportion of the mobile phase A was 0%, the volume proportion of the mobile phase B increased from 13% to 18%, and the volume proportion of the mobile phase C decreased from 87% to 82%;

[0094] 32 min-37 min, the volume proportion of the mobile phase A is 0%, the volume proportion of the mobile phase B is 18%, and the volume proportion of the mobile phase C is 82%;

[0095] From 37 min to 65 min, the volume proportion of the mobile phase A was 0%, the volume proportion of the mobile phase B increased from 18% to 30%, and the volume proportion of the mobile phase C decreased from 82% to 70%;

[0096] From 65 min to 70 min, the volume proportion of the mobile phase A was 0%, the volume proportion of the mobile phase B increased from 30% to 85%, and the volume proportion of the mobile phase C decreased from 70% to 15%;

[0097] 70 min-75 min, the volume proportion of the mobile phase A is 0%, the volume proportion of the mobile phase B is 85%, and the volume proportion of the mobile phase C is 15%;

[0098] From 75 min to 80 min, the volume proportion of the mobile phase A increased from 0% to 10%, the volume proportion of the mobile phase B decreased from 85% to 0%, and the volume proportion of the mobile phase C increased from 15% to 90%;

[0099] From 80 min to 87 min, the volume proportion of the mobile phase A was 10%, the volume proportion of the mobile phase B was 0%, and the volume proportion of the mobile phase C was 90%.

[0100] In some embodiments of the present application, the high performance liquid chromatography method further satisfies one or more of the following conditions:

[0101] (1) The column temperature is 20°C-35°C (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35°C);

[0102] (2) The injection volume is 5 μL-15 μL (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 μL);

[0103] (3) The detection wavelength is 350 nm to 360 nm (e.g., 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360 nm); and

[0104] (4) The flow rate is 0.8 mL / min-1 mL / min (e.g., 0.8, 0.9, 0.95, 1 mL / min).

[0105] In some embodiments of the present application, the reference solution satisfies one or more of the following conditions:

[0106] (A) the solvent in the reference solution includes methanol; and

[0107] (B) The concentration of each reference substance in the reference substance solution is independently 80 μg / mL to 120 μg / mL (e.g., 80, 85, 90, 95, 100, 105, 110, 115, 120 μg / mL).

[0108] In some embodiments of the present application, the steps of preparing the test solution include:

[0109] The cotton flower medicinal material sample to be tested is mixed with an extraction solvent for extraction, and the extract is collected to prepare the test solution.

[0110] In some embodiments of the present application, the extraction method includes heating reflux extraction.

[0111] In some embodiments of the present application, the heating reflux extraction satisfies one or more of the following conditions:

[0112] (I) the temperature of the extraction by heating under reflux is 70° C. to 80° C. (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80° C.);

[0113] (II) the extraction time under heating and reflux is 15 min to 120 min (e.g., 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 min);

[0114] (III) the extraction solvent for the extraction under heating and reflux comprises methanol; and

[0115] (IV) The dosage ratio of the cotton flower medicinal material sample to be tested and the extraction solvent is 0.5g:(25-100)mL, for example, 0.5g:25mL, 0.5g:30mL, 0.5g:35mL, 0.5g:40mL, 0.5g:45mL, 0.5g:50mL, 0.5g:55mL, 0.5g:60mL, 0.5g:65mL, 0.5g:70mL, 0.5g:75mL, 0.5g:80mL, 0.5g:85mL, 0.5g:90mL, 0.5g:95mL, and 0.5g:100mL.

[0116] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0117] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0118] The mobile phase selected in this application is methanol, acetonitrile and phosphoric acid gradient elution. By using specific HPLC chromatographic conditions to detect and analyze cotton flower medicinal materials, a method for simultaneously determining the content of 6 chemical components in cotton flower medicinal materials was constructed. This method is simple, reliable, objective, and has good precision, repeatability, specificity and stability. Further, the obtained liquid phase spectrum of cotton flower medicinal materials was analyzed, and 6 index components including hyperoside, isoquercetin, astragalin, quercetin, quercetin-7-O-glucoside, and quercetin-3'-glucoside were identified. These six components have high response peak types in cotton flower medicinal materials and are the main peak types that can be detected under this liquid phase method of cotton flower medicinal materials, providing an overall quality control basis for the quality standard of authentic cotton flower medicinal materials. The details are as follows:

[0119] 1. Medicinal materials, reagents, and equipment

[0120] 1.1 Experimental herbs

[0121] Cotton flower medicinal material (provided by Xinjiang Xinlvbao Pharmaceutical Co., Ltd., batch number MHH-YP-230404).

[0122] 1.2 Experimental reagents and materials

[0123] Methanol, acetonitrile, ethanol, phosphoric acid, ultrapure water; quercetin-7-O-glucoside (Batch No.: B20531, content ≥98%, Shanghai Yuanye Biotechnology Co., Ltd.), hyperoside (Batch No.: 11521-202310, content 94.7%, China Food and Drug Administration), isoquercetin (Batch No.: WP24041012, content ≥98%, Sichuan Weikeqi Biotechnology Co., Ltd.), astragaloside (Batch No.: WP24012505, content ≥98%, Sichuan Weikeqi Biotechnology Co., Ltd.), quercetin-3'-glucoside (Batch No.: WP24051012, content ≥98%, Sichuan Weikeqi Biotechnology Co., Ltd.), quercetin (100081-201610, content 99.8%, China Food and Drug Administration).

[0124] 1.3 Instruments and Equipment

[0125] A Vanquish Core high-performance liquid chromatograph (Thermo Fisher Scientific Inc.) was used; a Hypersil Gold C18 chromatographic column (250 mm × 4.6 mm, 5 μm) was used; an MS205DU / A analytical balance was used (Mettler Toli Technology Co., Ltd.); a PX423ZH / E electronic balance was used (Ohaus Instruments (Shanghai) Co., Ltd.); an A510200BT ultrasonic cleaner was used (Tianjin Aotesense Ultrasonic Instrument Co., Ltd.); and an UPL-II-40RZ ultrapure water machine was used (Sichuan Youpu Ultrapure Technology Co., Ltd.).

[0126] 2. Experimental Procedure

[0127] 2.1 Preparation of reference substance stock solution

[0128] Accurately weigh 10 mg each of hyperoside reference substance, isoquercetin reference substance, astragaloside reference substance, quercetin reference substance, quercetin-7-O-glucoside reference substance, and quercetin-3'-glucoside reference substance, and place them in 10 mL volumetric flasks. Dissolve them in methanol and dilute to the mark to prepare solutions containing 1 mg per mL. These solutions are used as stock solutions ①, ②, ③, ④, ⑤, and ⑥, respectively.

[0129] 2.2 Preparation of mixed reference solution

[0130] Accurately measure 0.1 mL each of the stock solutions ①, ②, ③, ④, ⑤, and ⑥ under 2.1, add 4 mL of methanol to prepare a solution containing 100 μg / mL each of hyperoside, isoquercetin, astragalin, quercetin, quercetin-7-O-glucoside, and quercetin-3'-glucoside as the mixed reference solution.

[0131] 2.3 Preparation of test solution

[0132] Weigh about 0.5 g of cotton flower powder (passed through No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of methanol, stopper it tightly, weigh it, heat it in a 75°C water bath, reflux it for 60 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0133] 2.4 Blank solution

[0134] Accurately add 50 mL of methanol to a stoppered conical flask, seal it tightly, weigh it, heat it in a 75°C water bath, reflux and extract it for 60 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0135] 2.5 Chromatographic conditions

[0136] A Hypersil Gold C18 column (250 mm × 4.6 mm, 5 μm) was used for gradient elution according to the requirements of 2.6. The column temperature was 25°C, the injection volume was 10 μL, and the detection wavelength was 352 nm.

[0137] 2.6 Elution procedure

[0138] Flow was investigated using five gradient elution schemes: methanol-water, acetonitrile-water, methanol-0.1wt% phosphoric acid, acetonitrile-0.1wt% phosphoric acid, and methanol-acetonitrile-0.1wt% phosphoric acid. The optimal elution procedure was determined based on experimental results such as the theoretical plate number, peak separation, and asymmetry.

[0139] Scheme 1: The mobile phase is methanol-water, the flow rate is 1.0 mL / min, the column temperature is 30°C, the injection volume is 10 μL, and the detection wavelength is 352 nm. The specific experimental results are shown in Figure 1 , Figure 1 In the above figure, all chromatographic peaks were not effectively separated. The results in the figure show that the separation degree of substances was poor in the methanol-water mobile phase system.

[0140] Table 1 Elution gradient of Scheme 1 (%, volume percentage)

[0141] time Mobile phase A: methanol Mobile phase C: water 0min-30min 10％ 90％ 30min-31min 10％→20％ 90％→80％ 31min-45min 20％ 80％ 45min-46min 20％→40％ 80％→60％ 46min-60min 40％ 60％ 60min-61min 40％→85％ 60％→15％ 61min-70min 85％ 15％ 70min-80min 85％→10％ 15％→90％ 80min-90min 10％ 90％

[0142] Scheme 2: The mobile phase is acetonitrile-water, the flow rate is 1.0 mL / min, the column temperature is 30 °C, the injection volume is 10 μL, and the detection wavelength is 352 nm. The specific experimental results are shown in Figure 2 , Figure 2 In the figure, 1, 2, and 3 represent the peaks of quercetin-7-O-glucoside, isoquercetin, and quercetin-3'-glucoside, respectively. The results in the figure show that the baseline noise is large and the separation is poor in the acetonitrile-water mobile phase system.

[0143] Table 2 Elution gradient of scheme 2 (%, volume percentage)

[0144] time Mobile phase B: acetonitrile Mobile phase C: water 0min-30min 10％ 90％ 30min-31min 10％→20％ 90％→80％ 31min-60min 20％ 80％ 60min-61min 20％→85％ 80％→15％ 61min-70min 85％ 15％ 70min-75min 85％→10％ 15％→90％ 75min-85min 10％ 90％

[0145] Scheme 3: The mobile phase is methanol-0.1% phosphoric acid, the flow rate is 1.0 mL / min, the column temperature is 30°C, the injection volume is 10 μL, and the detection wavelength is 352 nm. The specific experimental results are shown in Figure 3 , Figure 3 In the figure, 1, 2, 3, and 4 represent the peaks of quercetin-7-O-glucoside, isoquercetin, astragaloside, and quercetin-3'-glucoside, respectively. The results in the figure show that under the methanol-0.1% phosphoric acid mobile phase system, the running time is long and the substance separation is poor.

[0146] Table 3 Elution gradient of scheme 3 (%, volume percentage)

[0147] time Mobile phase A: methanol Mobile phase D: 0.1% phosphoric acid 0min-30min 10％ 90％ 30min-31min 10％→20％ 90％→80％ 31min-45min 20％ 80％ 45min-46min 20％→40％ 80％→60％ 46min-60min 40％ 60％ 60min-61min 40％→85％ 60％→15％ 61min-70min 85％ 15％ 70min-75min 85％→10％ 15％→90％ 75min-85min 10％ 90％

[0148] Scheme 4: The mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, the flow rate is 1.0 mL / min, the column temperature is 30°C, the injection volume is 10 μL, and the detection wavelength is 352 nm. The specific experimental results are shown in Figure 4 , Figure 4 In the figure, 1, 2, 3, and 4 represent the peaks of quercetin-7-O-glucoside, isoquercetin, astragaloside, and quercetin-3'-glucoside, respectively. The results in the figure show that under the acetonitrile-0.1% phosphoric acid mobile phase system, the running time is long and the substance separation is poor.

[0149] Table 4 Elution gradient of scheme 4 (%, volume percentage)

[0150] time Mobile phase B: acetonitrile Mobile phase D: 0.1% phosphoric acid 0min-30min 10％ 90％ 30min-31min 10％→20％ 90％→80％ 31min-60min 20％ 80％ 60min-61min 20％→85％ 80％→15％ 61min-70min 85％ 15％ 70min-75min 85％→10％ 15％→90％ 75min-85min 10％ 90％

[0151] Scheme 5: The mobile phase is methanol-acetonitrile-0.1% phosphoric acid, the flow rate is 1.0 mL / min, the column temperature is 30°C, the injection volume is 10 μL, and the detection wavelength is 352 nm. The specific experimental results are shown in Figure 5 In the mobile phase system of methanol-acetonitrile-0.1% phosphoric acid, all components can be detected with good separation and peak shape, with high detection capability and resolution.

[0152] Table 5

[0153] time Mobile phase A: methanol Mobile phase B: acetonitrile Mobile phase D: 0.1% phosphoric acid 0min-5min 10％→40％ 0％ 90％→60％ 5min-6min 40％→0％ 0％→13％ 60％→87％ 6min-27min 0％ 13％ 87％ 27min-32min 0％ 13％→18％ 87％→82％ 32min-37min 0％ 18％ 82％ 37min-65min 0％ 18％→30％ 82％→70％ 65min-70min 0％ 30→85 70％→15％ 70min-75min 0％ 85％ 15％ 75min-80min 0％→10％ 85％→0％ 15％→90％ 80min-87min 10％ 0％ 90％

[0154] Based on the above content, it was decided to adopt Plan 5 for subsequent testing.

[0155] 2.7 Flow rate investigation

[0156] Referring to Scheme 5, a C18 column was used with methanol-acetonitrile-0.1% phosphoric acid water as the mobile phase. Keeping other conditions unchanged, the flow rates were set to 0.8 mL / min, 0.9 mL / min, and 1.0 mL / min, respectively, to investigate the effects of different flow rates on the separation of the components of the cotton flower sample. The specific experimental results are shown in Figure 6 、 Figure 7 and Figure 8 At a flow rate of 0.8 mL / min, the separation degree was low due to the similar elution time of quercetin-7-O-glucoside and hyperoside. At flow rates of 0.9 mL / min and 1.0 mL / min, the differences in separation degree and peak shape between the peaks were small. The separation degree of astragaloside was slightly lower at a flow rate of 1.0 mL / min, so a flow rate of 0.9 mL / min was selected.

[0157] 2.8 Sample injection volume investigation

[0158] Referring to Scheme 5, a C18 column was used with methanol-acetonitrile-0.1% phosphoric acid water as the mobile phase. Keeping other conditions unchanged, the injection volumes were set to 5 μL, 10 μL, and 15 μL respectively to investigate the effects of different injection volumes on the separation of the components of the cotton flower sample. The specific experimental results are shown in Figure 9 、 Figure 10 and Figure 11 The peak shape of the 15μL injection volume was poor, while the peak shape and separation of the 5μL and 10μL injection volumes were ideal, so the instrument standard injection volume of 10μL was selected as the method injection volume.

[0159] 2.9 Investigation of different extraction methods

[0160] Referring to Scheme 5, a C18 chromatographic column was used with methanol-acetonitrile-0.1% phosphoric acid water as the mobile phase. Keeping other conditions unchanged, the cotton flower samples were prepared by ultrasonic extraction and reflux extraction, respectively, to investigate the effects of different extraction methods on the separation of cotton flower samples.

[0161] (1) Ultrasonic extraction: Weigh about 0.5 g of cotton flower powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of methanol, seal it tightly, weigh it, place it in an ultrasonic machine and extract it at 50 Hz for 60 min, let it stand at room temperature, weigh it again, make up the lost weight with methanol, shake it well, filter it, take the filtrate, and prepare the test solution.

[0162] (2) Reflux extraction: Weigh about 0.5 g of cotton flower powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of methanol, stopper it tightly, weigh it, heat it in a 75°C water bath, reflux it for 60 min, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, take the filtrate, and prepare the test solution.

[0163] Specific experimental results can be found in Figure 12 、 Figure 13 The content of each component in the reflux extraction method is higher than that in the ultrasonic extraction method, so the reflux extraction method is selected.

[0164] 2.10 Screening of different extraction solvents

[0165] Referring to Scheme 5, a C18 column was used with methanol-acetonitrile-0.1% phosphoric acid water as the mobile phase. Keeping other conditions unchanged, the cotton flower samples were extracted with methanol, ethanol, and water respectively. The effects of different extraction solvents on the separation of the components of the cotton flower samples were investigated. The specific experimental results are shown in Figure 14 、 Figure 15 、 Figure 16 Comparing the peak shapes and peak areas of the test samples extracted with different solvents, methanol extraction produced more components and had the highest content, so methanol was selected as the extraction solvent.

[0166] 2.11 Material Ratio Investigation

[0167] Referring to Scheme 5, a C18 column was used with methanol-acetonitrile-0.1% phosphoric acid water as the mobile phase. Keeping other conditions unchanged, the mass volume ratio of cotton flower sample to extraction solvent was 0.5g:25mL, 0.5g:50mL, and 0.5g:100mL, respectively. The effects of different material ratios on the separation of the components of cotton flower samples were investigated. The specific experimental results are shown in Figure 17 、 Figure 18 、 Figure 19 The extraction rate was lower when the material ratio was 0.5 g:25 mL, and the extraction rates were similar when the material ratios were 0.5 g:50 mL and 0.5 g:100 mL. For energy saving considerations, the material ratio of 0.5 g:50 mL was selected.

[0168] 2.12 Extraction time investigation

[0169] Referring to Scheme 5, a C18 column was used with methanol-acetonitrile-0.1% phosphoric acid water as the mobile phase. Keeping other conditions unchanged, the reflux extraction time of the cotton flower sample was set at 15 min, 30 min, 60 min, and 120 min, respectively. The effects of different extraction times on the separation of the components of the cotton flower sample were investigated. The specific experimental results are shown in Figure 20 、 Figure 21 、 Figure 22 、 Figure 23 The content was slightly lower when the extraction time was 15 min than when it was 30 min. The content of each substance was not much different when it was 60 min than when it was 120 min. Therefore, 60 min was selected as the extraction time.

[0170] 2.13 Column temperature investigation

[0171] Referring to Scheme 5, a C18 column was used with methanol-acetonitrile-0.1% phosphoric acid water as the mobile phase. Keeping other conditions unchanged, the column temperature was set at 20°C, 25°C, 30°C, and 35°C respectively to investigate the effect of different column temperatures on the separation of cotton flower samples. The specific experimental results are shown in Figure 24 、 Figure 25 、 Figure 26 、 Figure 27 The method was stable at different temperatures. The peak shape and separation were best when the column temperature was 25°C, so 25°C was selected as the final column temperature.

[0172] 2.14 Determined detection methods

[0173] According to the above content, in the method of this application: the preparation of the test solution is as described in Item 2.3. The chromatographic conditions are as described in Item 2.5. The elution procedure is as described in Scheme 5 in Item 2.6, with a flow rate of 0.9 mL / min.

[0174] 2.15 Methodological Validation

[0175] After the detection method is determined, the reliability of the method is verified from the aspects of precision, repeatability, durability, and quantitative limit.

[0176] (1) Precision

[0177] For the same cotton flower test solution, inject the sample six times continuously according to the chromatographic conditions determined in 2.14 for detection and analysis. Calculate the sample content and relative standard deviation (RSD). The results are shown in the following table.

[0178] Table 6 Precision

[0179]

[0180] The test results show that the precision of the six index components are all qualified.

[0181] (2) Repeatability

[0182] Take the same batch of samples and prepare 6 test solutions in parallel. Determine according to the chromatographic conditions under 2.14 and calculate the sample content and relative standard deviation (RSD). The results are shown in the following table.

[0183] Table 7 Repeatability

[0184]

[0185] The test results show that the repeatability of the six index components are all qualified.

[0186] (3) Durability

[0187] Take the same test solution, place it at room temperature, and inject 10 μL at 0, 2, 4, 6, 8, 10, 12, 18, 24, and 36 h according to the chromatographic conditions in 2.14. Perform detection and analysis and record the peak areas of the six component peaks. Calculate the sample content and relative standard deviation (RSD) within 36 h. The results are shown in the following table.

[0188] Table 8 Durability

[0189]

[0190]

[0191] The test results show that the durability of the six index components are all qualified.

[0192] (4) Linear relationship

[0193] Accurately pipette 1 μL, 2 μL, 4 μL, 6 μL, 8 μL, 10 μL, and 12 μL of the mixed reference solution into a liquid chromatograph for analysis according to the chromatographic conditions in 2.14. Plot a standard curve using peak area (Y) as the ordinate and injection volume (X (μg)) as the abscissa. The results are shown in the table below. Within the corresponding linear ranges, the correlation coefficients (r) for all six chemical components were greater than 0.999, indicating good linearity.

[0194] Table 9 Linear relationship of 6 chemical components

[0195] chemical composition Linear range (ug / ml) Linear equations r-value Quercetin-7-O-glucoside 12.179-146.151 Y=0.396X-0.060 0.9997 Hyperoside 3.215-38.579 Y=0.434X-0.085 0.9999 Isoquercetin 21.732-260.782 Y=0.550X+0.903 0.9995 Astragaloside 1.304-15.647 Y=0.539X+0.061 0.9995 Quercetin-3'-glucoside 15.374-184.491 Y=0.464X+0.443 0.9995 Quercetin 1.04-12.476 Y=0.748X-0.165 0.9997

[0196] (5) Sample recovery rate

[0197] Take 6 portions of the known sample, take about 0.5 g of each portion, accurately weigh, and place them in conical flasks respectively. Accurately add appropriate amounts of reference stock solutions of hyperoside, isoquercetin, astragalin, quercetin, quercetin-7-O-glucoside, and quercetin-3'-glucoside. Prepare the test solution as described above, and inject 10 μL of each sample for determination under the chromatographic conditions under 2.14. Calculate the recovery rate. The results are shown in the following table.

[0198] Table 10 Sample recovery

[0199]

[0200] (6) Limit of quantification and detection line

[0201] Following the chromatographic conditions in 2.14, a blank baseline was collected and the baseline noise was measured. Separately, the reference solution in 2.2 was diluted incrementally and the peak heights of each peak were measured according to the chromatographic conditions in 2.14. The injection volumes at signal-to-noise ratios of (S / N ≥ 10) and (S / N ≥ 3) were defined as the limit of quantification and limit of detection, respectively. The results, shown in Table 6 below, demonstrate the high sensitivity of this method.

[0202] Table 11 Limit of quantification and detection limit

[0203]

[0204] 3. Application Examples

[0205] Prepare the test solution as described in 2.3. Prepare three aliquots. Follow the chromatographic conditions described in 2.5 and the elution procedure described in 2.6, Scheme 5, for gradient elution at a flow rate of 0.9 mL / min.

[0206] Table 12 Contents of six chemical components in cotton flower medicinal materials (%)

[0207] name Quercetin-7-O-glucoside Hyperoside Isoquercetin Astragaloside Quercetin-3'-glucoside Quercetin Sample 1 0.493％ 0.139％ 0.860％ 0.051％ 0.581％ 0.041％ Sample 2 0.488％ 0.138％ 0.853％ 0.050％ 0.580％ 0.040％ Sample 3 0.486％ 0.138％ 0.850％ 0.051％ 0.584％ 0.040％

[0208] According to the above table, the detection method is stable and effective.

[0209] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various 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 the description in this specification.

[0210] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that, for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection 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 scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A method for detecting the content of chemical components in cotton flower medicinal materials, characterized in that: The detection method comprises the following steps: Providing a reference solution, wherein the reference substance in the reference solution includes one or more of hyperoside, isoquercetin, astragalin, quercetin, quercetin-7-O-glucoside, and quercetin-3'-glucoside; Prepare a test solution using the cotton flower medicinal material sample to be tested; and, The reference solution and the test solution are tested by high performance liquid chromatography to determine the content of the chemical components corresponding to the reference solution in the cotton flower medicinal material sample to be tested; The high performance liquid chromatography method satisfies one or more of the following conditions: (a) The stationary phase is a C18 chromatographic column; (b) The mobile phase includes mobile phase A, mobile phase B, and mobile phase C, wherein the mobile phase A includes methanol, the mobile phase B includes acetonitrile, and the mobile phase C includes a phosphoric acid aqueous solution.

2. The method for detecting the content of chemical components in cotton flower medicinal materials according to claim 1, characterized in that: The C18 chromatographic column includes a Hypersil Gold C18 chromatographic column, 250 mm×4.6 mm, 5 μm.

3. The method for detecting the content of chemical components in cotton flower medicinal materials according to claim 1, characterized in that: The mobile phase C includes a phosphoric acid aqueous solution having a phosphoric acid concentration of 0.08 wt % to 0.12 wt %.

4. The method for detecting the content of chemical components in cotton flower medicinal materials according to claim 1, characterized in that: The high performance liquid chromatography method further satisfies the following conditions: a gradient elution program is adopted.

5. The method for detecting the content of chemical components in cotton flower medicinal materials according to claim 4, characterized in that: The gradient elution procedure includes: From 0 min to 5 min, the volume proportion of the mobile phase A increased from 10% to 40%, the volume proportion of the mobile phase B was 0%, and the volume proportion of the mobile phase C decreased from 90% to 60%; From 5 min to 6 min, the volume proportion of the mobile phase A decreased from 40% to 0%, the volume proportion of the mobile phase B increased from 0% to 13%, and the volume proportion of the mobile phase C increased from 60% to 87%; 6 min-27 min, the volume proportion of the mobile phase A is 0%, the volume proportion of the mobile phase B is 13%, and the volume proportion of the mobile phase C is 87%; From 27 min to 32 min, the volume proportion of the mobile phase A was 0%, the volume proportion of the mobile phase B increased from 13% to 18%, and the volume proportion of the mobile phase C decreased from 87% to 82%; 32 min-37 min, the volume proportion of the mobile phase A is 0%, the volume proportion of the mobile phase B is 18%, and the volume proportion of the mobile phase C is 82%; From 37 min to 65 min, the volume proportion of the mobile phase A was 0%, the volume proportion of the mobile phase B increased from 18% to 30%, and the volume proportion of the mobile phase C decreased from 82% to 70%; From 65 min to 70 min, the volume proportion of the mobile phase A was 0%, the volume proportion of the mobile phase B increased from 30% to 85%, and the volume proportion of the mobile phase C decreased from 70% to 15%; 70 min-75 min, the volume proportion of the mobile phase A is 0%, the volume proportion of the mobile phase B is 85%, and the volume proportion of the mobile phase C is 15%; From 75 min to 80 min, the volume proportion of the mobile phase A increased from 0% to 10%, the volume proportion of the mobile phase B decreased from 85% to 0%, and the volume proportion of the mobile phase C increased from 15% to 90%; From 80 min to 87 min, the volume proportion of the mobile phase A was 10%, the volume proportion of the mobile phase B was 0%, and the volume proportion of the mobile phase C was 90%.

6. The method for detecting the content of chemical components in cotton flower medicinal materials according to any one of claims 1 to 5, characterized in that: The high performance liquid chromatography method further satisfies one or more of the following conditions: (1) Column temperature is 20℃-35℃; (2) The injection volume is 5 μL-15 μL; (3) The detection wavelength is 350nm-360nm; and (4) The flow rate is 0.8 mL / min-1 mL / min.

7. The method for detecting the content of chemical components in cotton flower medicinal materials according to any one of claims 1 to 5, characterized in that: The reference solution meets one or more of the following conditions: (A) the solvent in the reference solution includes methanol; and (B) The concentration of each reference substance in the reference substance solution is independently 80 μg / mL to 120 μg / mL.

8. The method for detecting the content of chemical components in cotton flower medicinal materials according to any one of claims 1 to 5, characterized in that: The preparation steps of the test solution include: The cotton flower medicinal material sample to be tested is mixed with an extraction solvent for extraction, and the extract is collected to prepare the test solution.

9. The method for detecting the content of chemical components in cotton flower medicinal materials according to claim 8, characterized in that: The extraction method includes heating reflux extraction.

10. The method for detecting the content of chemical components in cotton flower medicinal materials according to claim 9, characterized in that: Heating reflux extraction meets one or more of the following conditions: (I) The extraction temperature is 70°C to 80°C; (II) The extraction time of heating and reflux is 15min-120min; (III) the extraction solvent for the extraction under heating and reflux comprises methanol; and (IV) The dosage ratio of the cotton flower medicinal material sample to be tested and the extraction solvent is 0.5 g: (25-100) mL.

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