Method for determining contents of phenylpropanoid and flavonoid components in folium artemisiae argyi

Abstract

The invention relates to a method for determining the content of phenylpropanoid and flavonoid components in folium artemisiae argyi, which is characterized in that folium artemisiae argyi is taken as a research object, isochlorogenic acid C is selected as an internal reference substance to determine the content of phenylpropanoid and flavonoid components in folium artemisiae argyi by a quantitative analysis of multi-components by single marker, and the method comprises the following steps: extracting folium artemisiae argyi by using an extraction solvent to prepare a test solution; dissolving the reference substance by using a dissolving reagent to prepare a reference substance mixed solution; respectively injecting the test solution and the reference substance mixed solution, and carrying out ultra-high performance liquid chromatography detection; isochlorogenic acid C is used as an internal reference substance, the content of the to-be-detected component in the folium artemisiae argyi is calculated according to a formula, and finally it is proved that the quantitative analysis of multi-components by single marker can be applied to synchronous quantitative determination of characteristic phenylpropanoid and flavonoid components of the folium artemisiae argyi. The established method is rapid, stable, stable in base line and high in specificity, more comprehensively reflects the quality condition of the folium artemisiae argyi medicinal material compared with a single determination index, is high in practicability, simple and convenient to operate and high in accuracy, and saves the detection cost.

Description

Technical Field

[0001] The present application relates to the technical field of content detection of traditional Chinese medicine, and in particular to a method for determining the content of phenylpropanoids and flavonoids in wormwood. Background Art

[0002] Artemisia argyi is the dried leaves of Artemisia argyi Levl.et Vant., a plant of the Asteraceae family. It is pungent, bitter, warm and slightly toxic. It enters the liver, spleen and kidney meridians. It is picked in summer before the flowers bloom, and then removed of impurities and dried in the sun. It can be taken internally to warm the meridians and stop bleeding, dispel cold and relieve pain; it can be used externally to remove dampness and relieve itching. The clinical application of Artemisia argyi has a long history. It was first recorded as a medicine in the "Famous Doctors' Records" of the Wei and Jin Dynasties. Since then, it has been recorded in many documents of successive dynasties. It is still widely used in clinical practice as a commonly used medicine in TCM gynecology.

[0003] The main chemical components of wormwood are volatile oils, flavonoids, phenylpropanoids, etc., which have multiple pharmacological effects such as antibacterial, antiviral, hemostatic, anti-tumor, liver protection and choleretic, antioxidant, antitussive and antiasthmatic, analgesic and anti-inflammatory, hypoglycemic, and immunomodulatory. The 2020 edition of the Chinese Pharmacopoeia only controls the content of volatile oil components eucalyptol and borneol in wormwood, and lacks content determination indicators that can fully reflect the intrinsic quality of medicinal materials. Summary of the invention

[0004] Based on this, it is necessary to provide a method for determining the content of phenylpropanoids and flavonoids in Artemisia argyi, which can accurately evaluate and control the quality of Artemisia argyi medicinal materials, thereby ensuring the safety and effectiveness of its clinical use.

[0005] The technical solution is as follows:

[0006] In one aspect of the present invention, a method for determining the content of phenylpropanoids and flavonoids in wormwood is provided, comprising the following steps:

[0007] Extracting Artemisia argyi with an extraction solvent to prepare a test solution;

[0008] The reference substance is dissolved using a dissolving reagent to prepare a reference substance mixed solution; the reference substance includes isochlorogenic acid C, and one or more of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, cyanidin and isoeupretinoic acid reference substances;

[0009] The test solution and the reference solution are injected separately for ultra-high performance liquid chromatography detection;

[0010] Taking isochlorogenic acid C as the internal reference, the content of the component to be tested in the test solution was calculated according to the following formula:

[0011] Ci = f*Cs*(Ai / As);

[0012] Where f is the relative correction factor of the component to be tested in Artemisia argyi, As is the peak area of ​​the internal reference substance isochlorogenic acid C in the reference substance mixed solution, Cs is the concentration of the internal reference substance isochlorogenic acid C, Ai is the peak area of ​​the component to be tested in the test solution, and Ci is the concentration of the component to be tested in Artemisia argyi;

[0013] The components to be tested in the wormwood leaf include phenylpropanoids and flavonoids.

[0014] In one embodiment, the phenylpropanoid and flavonoid components include one or more of isochlorogenic acid C, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, cyanidin and isoeupretinoic acid.

[0015] In one embodiment, the method for determining the content of phenylpropanoids and flavonoids in wormwood also includes the following steps: using isochlorogenic acid C as an internal reference, injecting a reference solution of different concentrations, performing ultra-high performance liquid chromatography detection, recording the peak area corresponding to the component i to be tested in the reference solution of different concentrations, and calculating according to the following formula to take the average value as the relative correction factor:

[0016] f = (As / Cs) / (Ai / Ci);

[0017] In the formula, As is the peak area of ​​the internal reference isochlorogenic acid C, Cs is the concentration of the internal reference isochlorogenic acid C, Ai is the peak area of ​​the reference substance i of the component to be tested, and Ci is the concentration of the reference substance i of the component to be tested; the component i to be tested includes neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, cyanidin or isoeupretinoic acid.

[0018] In one embodiment, the ultra-high performance liquid chromatography detection conditions include: mobile phase A is acetonitrile, mobile phase B is formic acid aqueous solution, and gradient elution is used.

[0019] In one embodiment, the gradient elution procedure comprises:

[0020] From 0 to 10 min, the volume percentage of the mobile phase A increases from 8% to 10%;

[0021] 10min-18min, the volume percentage of the mobile phase A increases from 10% to 14%;

[0022] From 18min to 27min, the volume percentage of the mobile phase A increases from 14% to 20%;

[0023] From 27min to 32min, the volume percentage of the mobile phase A increases from 20% to 21%;

[0024] From 32min to 50min, the volume percentage of the mobile phase A increases from 21% to 40%;

[0025] From 50 min to 62 min, the volume percentage of the mobile phase A increases from 40% to 70%.

[0026] In one embodiment, the volume percentage of formic acid in the formic acid aqueous solution is 0.1%-0.3%.

[0027] In one embodiment, the extraction solvent includes methanol-water solution;

[0028] Optionally, in the methanol aqueous solution, the volume percentage of methanol is 60%-80%.

[0029] In one embodiment, the ultra-high performance liquid chromatography detection also satisfies at least one of the following conditions:

[0030] (1) The chromatographic column is a C18 chromatographic column;

[0031] (2) Column temperature is 20°C-30°C;

[0032] (3) Flow rate: 0.3 mL / min-0.5 mL / min;

[0033] (4) The injection volume is 1 μL-3 μL;

[0034] (5) The wavelength is 315nm-335nm.

[0035] In one embodiment, the dissolving agent is methanol.

[0036] Another aspect of the present invention provides the use of the above-mentioned method for determining the content of phenylpropanoids and flavonoids in Artemisia argyi in the quality control of Artemisia argyi medicinal materials.

[0037] Compared with the prior art, this application has the following beneficial effects:

[0038] 1. This application uses isochlorogenic acid C as an internal reference to carry out a one-test-multiple-evaluation method to determine the content of phenylpropanoids and flavonoids in Artemisia argyi. Isochlorogenic acid C is inexpensive, has clear efficacy and is easy to obtain. It can save research costs and improve analysis efficiency to the greatest extent, and can reduce solvent consumption and is beneficial to environmental protection.

[0039] 2. In view of the shortcomings of the existing single component and multi-index content determination methods, this application has established for the first time a one-measurement and multi-evaluation content determination method for the eight main phenylpropanoids and flavonoids in Artemisia argyi. Only one reference substance is needed to simultaneously determine the contents of six phenylpropanoids and two flavonoids, which can more comprehensively reflect the quality of Artemisia argyi medicinal materials. According to methodological investigation, the method has good specificity, good peak purity, good precision, good linear relationship between the six phenylpropanoids and two flavonoids, good reproducibility, the test sample is stable within 24 hours, the method has good intermediate precision, can be stably reproduced in different laboratories and equipment, high sample recovery rate, and high accuracy.

[0040] 3. There was no significant difference in the content determination results between the external standard method and the one-measurement-multiple-evaluations method, and the relative correction factors and relative retention time positioning between the components had good reproducibility and durability, indicating that the one-measurement-multiple-evaluations method can be applied to the quality control of the simultaneous determination of the contents of multiple medicinal components in Artemisia argyi.

[0041] In summary, this application uses Artemisia argyi as the research object, and through chromatographic condition development, methodological verification, determination of relative correction factors and relative retention time positioning, and verification of measurement results, it is confirmed that the one-measurement-multiple-evaluation method can be applied to the simultaneous quantitative determination of characteristic phenylpropanoid and flavonoid components of Artemisia argyi. The established method is fast, stable, has a stable baseline and strong specificity, and reflects the quality status of Artemisia argyi medicinal materials more comprehensively than a single measurement indicator. The detection method of this application is highly practical, easy to operate, highly accurate, and saves detection costs, which is more conducive to environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0043] Figure 1 This is a graph showing the specificity of the content determination method in Example 1.

[0044] Figure 2 It is the UV absorption curve and peak purity diagram of the reference substance in Example 1, wherein, from left to right: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and caffeic acid.

[0045] Figure 3 The ultraviolet absorption curve and peak purity diagram of the reference substance in Example 1, wherein, from left to right: isochlorogenic acid B, isochlorogenic acid C, cyanidin and isoeupretinoic acid.

[0046] Figure 4This is the ultraviolet absorption curve and peak purity diagram of the mugwort sample in Example 1, where, from left to right: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, and caffeic acid.

[0047] Figure 5 This is the ultraviolet absorption curve and peak purity diagram of the mugwort sample in Example 1, where, from left to right: isochlorogenic acid B, isochlorogenic acid C, cyanidin and isoeupretinoic acid.

[0048] Figure 6 This is a graph showing the results of the linear relationship investigation of the content determination method in Example 1.

[0049] Figure 7 This is a graph showing the results of the intermediate precision investigation (reference substance) of the HIMADZU LC-40D xs ultra-high performance liquid chromatograph in Example 1. DETAILED DESCRIPTION

[0050] The present application will be further described in detail below in conjunction with specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thoroughly understood.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0052] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0053] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0054] Herein, the optional scope of "and / or", "or / and", and "and / or" includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, wherein said any and all combinations include any two relevant listed items, any more relevant listed items, or a combination of all relevant listed items.

[0055] Herein, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0056] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0057] The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0058] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0059] The temperature parameters in this application, unless otherwise specified, allow both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±2°C, ±1°C, ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, ±0.1°C are allowed. Normal temperature or room temperature in this application refers to no temperature control operation, generally 4°C to 35°C, preferably 20±5°C.

[0060] For conventional quantitative analysis, when using the external standard method (ESM) for multi-component quantitative analysis, there are problems such as the difficulty in obtaining standard reference materials and their high prices. The quantitative analysis of multi-components by single marker (QAMS) method is a quality evaluation method for Chinese medicine based on the possible internal relationship and regularity between the same type of active ingredients in Chinese medicine. It uses the internal functional relationship and proportional relationship of the active ingredients to obtain the simultaneous quantitative results of multiple components through a single component. It can simultaneously monitor multiple analytes by measuring cheap, stable and easily available standard reference materials, and has the advantages of being economical, efficient and fast. In recent years, QAMS has been widely used in the simultaneous determination of the content of multiple components in Chinese herbal medicines and has been widely recognized in the quality control research of Chinese herbal medicines. This method uses the relationship between the active ingredients in the sample, selects only one chemical component (internal reference, usually a typical component and the reference material is cheap and easy to obtain), and establishes the relative correction factor between the internal reference and other multiple components through strict methodological investigation to achieve the determination of the content of multiple similar components.

[0061] At present, the 2020 edition of the Chinese Pharmacopoeia only stipulates the content of volatile oil components eucalyptol and borneol under the wormwood leaf, lacking a comprehensive and scientific quality standard. After consulting the literature, the quantitative analysis research on wormwood leaves focuses on the determination of the main component content by the conventional external standard method, but there are problems such as large consumption of reference substances, high cost and low efficiency; the one-side multi-evaluation method established by a small number of studies can simultaneously determine fewer components, and can only determine one type of component in the phenylpropanoid or flavonoid components, which has certain limitations as an overall quality control method for wormwood leaves. Based on this, the technical personnel of this application found through a large number of studies that the establishment of a QAMS that can simultaneously determine the main various phenylpropanoid and flavonoid components in wormwood leaves can be accurately applied to the overall quality control and evaluation of wormwood leaves. The detection method is simple, stable, and has strong specificity, which can shorten the analysis time, save research costs and improve analysis efficiency. The method can achieve the simultaneous determination of the contents of eight characteristic phenylpropanoid and flavonoid components in wormwood leaves, and can accurately evaluate and control the quality of wormwood medicinal materials, thereby ensuring the safety and effectiveness of its clinical medication.

[0062] In one aspect of the present invention, a method for determining the content of phenylpropanoids and flavonoids in wormwood is provided, comprising the following steps:

[0063] Extracting Artemisia argyi with an extraction solvent to prepare a test solution;

[0064] The reference substance is dissolved using a dissolving reagent to prepare a reference substance mixed solution; the reference substance includes isochlorogenic acid C, and one or more of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, cyanidin and isoeupretinoic acid reference substances;

[0065] The test solution and the reference solution are injected separately for ultra-high performance liquid chromatography detection;

[0066] Taking isochlorogenic acid C as the internal reference, the content of the component to be tested in the test solution was calculated according to the following formula:

[0067] Ci = f*Cs*(Ai / As);

[0068] Where f is the relative correction factor of the component to be tested in Artemisia argyi, As is the peak area of ​​the internal reference isochlorogenic acid C, Cs is the concentration of the internal reference isochlorogenic acid C in the reference solution, Ai is the peak area of ​​the component to be tested in Artemisia argyi, and Ci is the concentration of the component to be tested in the test solution;

[0069] The components to be tested in the wormwood leaf include phenylpropanoids and flavonoids.

[0070] In some examples, the phenylpropanoid and flavonoid components include one or more of isochlorogenic acid C, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, cyanidin and isoeupretinoic acid.

[0071] In some of the examples, the method for determining the content of phenylpropanoids and flavonoids in wormwood also includes the following steps: using isochlorogenic acid C as an internal reference, injecting a reference solution of different concentrations, performing ultra-high performance liquid chromatography detection, recording the peak area corresponding to the component i to be tested in the reference solution of different concentrations, and calculating according to the following formula to take the average value as the relative correction factor:

[0072] f = (As / Cs) / (Ai / Ci);

[0073] In the formula, As is the peak area of ​​the internal reference isochlorogenic acid C, Cs is the concentration of the internal reference isochlorogenic acid C, Ai is the peak area of ​​the reference substance i of the component to be tested, and Ci is the concentration of the reference substance i of the component to be tested; the component i to be tested includes neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, cyanidin or isoeupretinoic acid.

[0074] In some of the examples, the relative retention time method is used to locate the chromatographic peaks to be measured, including the following steps: accurately locate the positions of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, brown cyanidin and isoeupretinoic acid in Artemisia argyi by the relative retention time method, and calculate the relative retention time according to the following formula: T = ti / ts;

[0075] Where ti is the retention time of the reference substance of the component to be tested in Artemisia argyi, and ts is the retention time of the internal reference substance isochlorogenic acid C.

[0076] In some of the examples, the method for determining the content of phenylpropanoids and flavonoids in wormwood also includes the following steps: using isochlorogenic acid C as an internal reference, recording the peak areas of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, cyanidin and isoeupretinoic acid, and calculating the relative correction factor according to the following formula:

[0077] f = (As / Cs) / (Ai / Ci);

[0078] In the formula, As is the peak area of ​​the internal reference isochlorogenic acid C reference substance s, Cs is the concentration of the internal reference isochlorogenic acid C reference substance s, Ai is the peak area of ​​the reference substance i of the component to be tested, and Ci is the concentration of the reference substance i of the component to be tested.

[0079] In some of the examples, the conditions for ultra-high performance liquid chromatography detection include: mobile phase A is acetonitrile, mobile phase B is formic acid aqueous solution, and gradient elution is used.

[0080] In some examples, the gradient elution procedure includes:

[0081] From 0 to 10 min, the volume percentage of the mobile phase A increases from 8% to 10%;

[0082] 10min-18min, the volume percentage of the mobile phase A increases from 10% to 14%;

[0083] From 18min to 27min, the volume percentage of the mobile phase A increases from 14% to 20%;

[0084] From 27min to 32min, the volume percentage of the mobile phase A increases from 20% to 21%;

[0085] From 32min to 50min, the volume percentage of the mobile phase A increases from 21% to 40%;

[0086] From 50 min to 62 min, the volume percentage of the mobile phase A increases from 40% to 70%.

[0087] In some examples, the volume percentage of formic acid in the formic acid aqueous solution is 0.1%-0.3%. It can be understood that the volume percentage of formic acid in the formic acid aqueous solution includes but is not limited to 0.1%, 0.15%, 0.2%, 0.25%, and 0.3%.

[0088] In some examples, the extraction solvent includes aqueous methanol.

[0089] In some examples, the volume percentage of methanol in the methanol aqueous solution is 60%-80%. It can be understood that the volume percentage of methanol in the methanol aqueous solution includes but is not limited to 60%, 65%, 70%, 75%, and 80%.

[0090] In some of the examples, the conditions for the ultra-high performance liquid chromatography detection include: the chromatographic column is a C18 chromatographic column.

[0091] In some examples, the conditions for the ultra-high performance liquid chromatography detection include: a column temperature of 20°C-30°C. It is understandable that the column temperature for the ultra-high performance liquid chromatography detection includes but is not limited to 20°C, 22°C, 24°C, 26°C, 28°C, and 30°C. Further, the column temperature for the ultra-high performance liquid chromatography detection is 23°C-27°C.

[0092] In some of the examples, the conditions for the ultra-high performance liquid chromatography detection include: a flow rate of 0.3 mL / min-0.5 mL / min. It is understandable that the flow rate for the ultra-high performance liquid chromatography detection includes but is not limited to 0.3 mL / min, 0.35 mL / min, 0.4 mL / min, 0.45 mL / min, and 0.5 mL / min. Further, the flow rate for the ultra-high performance liquid chromatography detection is 0.35 mL / min-0.45 mL / min.

[0093] In some examples, the conditions for the ultra-high performance liquid chromatography detection include: the injection volume is 1 μL-3 μL. It can be understood that the injection volume of the ultra-high performance liquid chromatography detection includes but is not limited to 1 μL, 1.5 μL, 2 μL, 2.5 μL, and 3 μL.

[0094] In some examples, the ultra-high performance liquid chromatography detection conditions include: a wavelength of 315nm-335nm. It can be understood that the wavelength of the ultra-high performance liquid chromatography detection includes but is not limited to 315nm, 320nm, 325nm, 330nm, and 335nm.

[0095] In some of the examples, the conditions for the ultra-high performance liquid chromatography detection include: the filler of the chromatographic column is octadecyl bonded silica gel.

[0096] In some examples, the dissolving agent is methanol.

[0097] Another aspect of the present invention provides the use of the above-mentioned method for determining the content of phenylpropanoids and flavonoids in Artemisia argyi in the quality control of Artemisia argyi medicinal materials.

[0098] The one-test-multiple-evaluation quality control method for characteristic phenylpropanoids and flavonoids in wormwood established in this application is reported for the first time. This application uses isochlorogenic acid C as an internal reference to carry out a one-test-multiple-evaluation method to determine the content of phenylpropanoids and flavonoids in wormwood. Only one reference substance is needed to simultaneously determine the content of six phenylpropanoids and two flavonoids, which can more comprehensively reflect the quality of wormwood medicinal materials. Studies have reported that isochlorogenic acid C has multiple active effects such as anti-inflammatory, antibacterial, and antiviral. The present invention found that all batches of wormwood are rich in isochlorogenic acid C, and this component is the main potential quality difference marker, with moderate retention time, good peak shape, stable properties and low price, and is selected as the internal reference in this method. It shows that the established f system has good stability and applicability, and can be applied to the simultaneous quantitative determination of multiple characteristic components of wormwood (Table 8). The stability and applicability of relative retention time positioning are good, and the chromatographic peak to be measured can be accurately located (Table 9). The relative correction factor and relative retention time positioning determined in this study have been verified by the system and standards. They are relatively fixed under the same analytical conditions and without background negative interference, and can be directly applied to the simultaneous quantitative analysis of multiple components of mugwort raw materials and their products without repeated investigation and verification. The effects of Waters CORTECS C18 (4.6mm×150mm, 2.7μm) chromatographic column batches, ultra-high performance liquid chromatography models (Waters H-Class, SHIMADZU LC-40D xs and Thermo Vanquish), column temperature (23℃, 25℃ and 27℃) and flow rate (0.38mL / min, 0.40mL / min and 0.42mL / min) on the system durability of relative correction factor and relative retention time positioning were systematically investigated. The established method can be stably reproduced between different batches of chromatographic columns and ultra-high performance liquid chromatography, and the small fluctuations in column temperature and flow rate can meet the system suitability requirements of the analysis, with good durability and practicality.

[0099] The chromatographic conditions and one-measurement-multiple-evaluation method established by the present invention were used to determine the characteristic phenylpropanoid and flavonoid components, namely, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid C, cyanidin and isoeupretinoic acid in 18 batches of mugwort leaves from the main producing areas. The relative deviation (Relative deviation, RD / %) was used as the standard, and the external standard sampling method (ESM) was used to compare and verify the QAMS results. There was no significant difference in the content determination results of the QAMS and ESM methods, and RD<1%, indicating that the QAMS content determination method is accurate and can be applied to the quality control of the simultaneous determination of the content of multiple pharmacological components of mugwort leaves.

[0100] The following is further described in conjunction with specific embodiments. The raw materials involved in the following specific embodiments, unless otherwise specified, can all be commercially available; the instruments used, unless otherwise specified, can all be commercially available; the processes involved, unless otherwise specified, are all routinely selected by those skilled in the art.

[0101] The following are specific embodiments.

[0102] Example 1

[0103] This embodiment provides a method for determining the contents of eight phenylpropanoids and flavonoid components in Artemisia argyi, as follows:

[0104] 1. Instruments, reagents and test drugs

[0105] Instruments: Waters ultra-high performance liquid chromatograph (H-Class, Waters Corporation, USA), SHIMADZU ultra-high performance liquid chromatograph (LC 40Dxs, Shimadzu Corporation, Japan), Thermo ultra-high performance liquid chromatograph (Vanquish, Thermo Fisher Scientific Inc., USA), Waters Cortecs C18 chromatographic column (4.6 mm × 150 mm, 2.7 μm), one-ten-thousandth balance (ME204E, Mettler-Toledo), one-millionth balance (XP26, Mettler-Toledo), electric constant temperature water bath (HWS-28, Shanghai Yiheng Technology Co., Ltd.), ultrapure water machine (Milli-Q Direct 8 / 16 system, Merck & Co., Ltd.).

[0106] Reagents: Methanol (Xilong Scientific Co., Ltd.) was of analytical grade; formic acid (Tianjin Komiou Chemical Reagent Co., Ltd., chromatographic grade), acetonitrile (Merck Co., Ltd., chromatographic grade), and water was ultrapure water (self-made in the laboratory).

[0107] Test drugs: neochlorogenic acid (batch number: ST06230120, content 98%, Shanghai Shidande Standard Technology Service Co., Ltd.); chlorogenic acid (batch number: 110753-202119, content 96.3%, China Food and Drug Inspection Institute); cryptochlorogenic acid (batch number: ST07850120, content 98%, Shanghai Shidande Standard Technology Service Co., Ltd.); caffeic acid (batch number: 110885-201703, content ≥99.7%, China Food and Drug Inspection Institute); isochlorogenic acid B (batch number: 110885-201703, content ≥99.7%, China Food and Drug Inspection Institute); No.: ST06590120, content 98%, Shanghai Shidande Standard Technical Service Co., Ltd.); isochlorogenic acid C (wkq23011207, content 98%, Sichuan Weikeqi Biotechnology Co., Ltd.); brown cyanidin (batch number: Z05801909018; content ≥98%, Chengdu Ruifensidedan Biotechnology Co., Ltd.); isoeupretinoic acid (batch number: wkq22062813, content 98%, Sichuan Weikeqi Biotechnology Co., Ltd.); the source information of the medicinal materials used in the research is shown in Table 1.

[0108] Table 1 Information of medicinal materials and decoction pieces

[0109]

[0110] 2. Experimental Procedure

[0111] 2.1 Chromatographic conditions and system suitability test

[0112] Octadecyl bonded silica gel was used as filler (Waters Cortecs C18 column, column length 150 mm, inner diameter 4.6 mm, particle size 2.7 μm), acetonitrile was used as mobile phase A, 0.2% formic acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in Table 2; the flow rate was 0.4 mL per minute, the column temperature was 25 ° C, and the detection wavelength was 325 nm. The injection volume of the reference solution and the test solution was 2 μL. The theoretical plate number calculated based on the isochlorogenic acid C peak shall not be less than 5000.

[0113] Table 2 Mobile phase gradient table

[0114]

[0115] 2.2 Preparation of reference solution

[0116] Artemisia argyi medicinal material reference solution: accurately weigh 4.136 mg of neochlorogenic acid reference, 15.988 mg of chlorogenic acid reference, 4.08 mg of cryptochlorogenic acid reference, 2.12 mg of caffeic acid reference, 11.976 mg of isochlorogenic acid B reference, 24.944 mg of isochlorogenic acid C reference, 4.332 mg of cyanidin reference and 12.236 mg of isoeuphenin reference, place in a 100 mL volumetric flask, and dilute to 60 mL with methanol. The mixed reference substance stock solution was prepared by adjusting the scale to prepare a mixed reference substance stock solution containing 40.9248 μg of neochlorogenic acid reference substance, 153.964 μg of chlorogenic acid reference substance, 39.984 μg of cryptochlorogenic acid reference substance, 21.1364 μg of caffeic acid reference substance, 117.3648 μg of isochlorogenic acid B reference substance, 244.4512 μg of isochlorogenic acid C reference substance, 42.4536 μg of cyanidin reference substance and 119.9128 μg of isoeuphenin reference substance per 1 mL.

[0117] 2.3 Preparation of test solution

[0118] Weigh about 0.2g of mugwort powder (pass through No. 3 sieve), add 25mL of 70% methanol, ultrasonically treat (power 300W, frequency 40kHZ) for 30min, let it cool to room temperature, weigh it again, and make up the lost mass with 70% methanol. Filter it with a 0.22μm filter membrane, and take the filtrate as the test solution.

[0119] 2.4 Determination of relative correction factor and relative retention time positioning

[0120] 0.05mL, 0.1mL, 0.3mL, 0.5mL, 1mL and 3mL of the mixed reference solution prepared in the above steps were taken respectively, placed in a 5.0mL volumetric flask, and methanol was added to the scale, shaken well, and a series of mixed reference solutions of different concentrations were prepared, and determined according to the above chromatographic conditions. With isochlorogenic acid C as the internal reference, the peak areas of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, brown cyanidin and isoeupretinoin were recorded, according to the relative correction factor f = (As / Cs) / (Ai / Ci), where As is the peak area of ​​the internal reference isochlorogenic acid C reference s, Cs is the concentration of the internal reference isochlorogenic acid C reference s, Ai is the peak area of ​​the reference i of the component to be tested, and Ci is the concentration of the reference i of the component to be tested. The relative retention time method is used to locate the chromatographic peak to be measured. The relative retention time T = ti / ts, where ti is the retention time of the reference substance of the component to be measured, and ts is the retention time of the internal reference substance isochlorogenic acid C.

[0121] 2.5 Content determination and verification

[0122] The chromatographic peaks of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, cyanidin and isochlorogenic acid C were determined by Ts, relative retention time Tneochlorogenic acid / isochlorogenic acid C, Tchlorogenic acid / isochlorogenic acid C, Tcryptochlorogenic acid / isochlorogenic acid C, Tcaffeic acid / isochlorogenic acid C, Tisochlorogenic acid B / isochlorogenic acid C, Tcyanidin / isochlorogenic acid C and Tisozeupetin / isochlorogenic acid C, and the peak areas Ai1 of neochlorogenic acid, Ai2 of chlorogenic acid, Ai3 of cryptochlorogenic acid, Ai4 of caffeic acid, Ai5 of isochlorogenic acid B, Ai6 of cyanidin and Ai7 of isozeupetin were obtained;

[0123] The concentration Ci1 of neochlorogenic acid was determined by As, Ai1, Cs and the relative correction factor fneochlorogenic acid / isochlorogenic acid C; the concentration Ci2 of chlorogenic acid was determined by As, Ai2, Cs and the relative correction factor fchlorogenic acid / isochlorogenic acid C; the concentration Ci3 of cryptochlorogenic acid was determined by As, Ai3, Cs and the relative correction factor fcryptochlorogenic acid / isochlorogenic acid C; the concentration Ci4 of caffeic acid was determined by As, Ai4, Cs and the relative correction factor fcaffeic acid / isochlorogenic acid C; the concentration Ci5 of isochlorogenic acid B was determined by As, Ai5, Cs and the relative correction factor fisochlorogenic acid B / isochlorogenic acid C; the concentration Ci6 of cyanidin was determined by As, Ai6, Cs and the relative correction factor fcyanidin / isochlorogenic acid C; the concentration Ci7 of isocyanidin was determined by As, Ai7, Cs and the relative correction factor fisocyanidin / isochlorogenic acid C.

[0124] Under the above optimized chromatographic conditions, the chromatogram of the wormwood test solution showed baseline-separated characteristic peaks of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid C, cyanidin and isocyanidin. Using isochlorogenic acid C as the internal reference, the positions of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, cyanidin and isocyanidin were accurately located by relative retention time, and their accurate contents were further calculated by the above relative correction factors. The results were compared by external standard method (ESM) and QAMS method, and the accuracy of QAMS results was evaluated by relative deviation (RD).

[0125] 2.6 Methodological Review

[0126] 2.6.1 Specificity inspection

[0127] Take the reference solution under "2.2", 70% methanol blank solvent and the test solution under "2.3" and analyze them according to the chromatographic conditions under "2.1". The results show (see Figure 1), the test sample chromatogram has the same chromatographic peak at the retention time corresponding to the reference sample chromatogram, and there is no interference from the blank solvent, indicating that the method has good specificity.

[0128] 2.6.2 Peak purity investigation

[0129] Take the powder of mugwort leaves (pass through No. 3 sieve), prepare the test solution according to the method under "2.3", scan the reference and test samples at 190-400nm according to the chromatographic conditions of "2.1", and compare the characteristic wavelengths of the components to be tested. The results show that the chromatographic peak capacity of the test and reference samples at 325nm is large, the response is high, the peak shape is symmetrical (the asymmetry is between 0.995-1.05) and the peak purity is good ( Figure 2-5 ), which is significantly better than other absorption wavelengths. Therefore, the optimal absorption wavelength was determined to be 325nm, and the components to be measured achieved baseline separation at this wavelength, and no impurity peaks were detected, which met the requirements.

[0130] 2.6.3 Precision investigation

[0131] Take the powder of mugwort leaves (pass through No. 3 sieve), prepare the test solution according to the method under "2.3", repeat the injection and determination 6 times according to the chromatographic conditions of "2.1", record the peak areas of chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid C, cyanidin and isoeupretinoic acid, and calculate their RSD values ​​(Table 3). The results show that the peak area RSD values ​​of the six characteristic phenylpropanoid components and the two flavonoid components are ≤3.0%, indicating that the method has good precision.

[0132] Table 3 Results of precision test of reference substances

[0133]

[0134] 2.6.4 Linear Investigation

[0135] Take 0.05mL, 0.1mL, 0.3mL, 0.5mL, 1mL and 3mL of the mixed reference solution prepared under "2.2" respectively, put them into 5.0mL volumetric flasks, add methanol to the scale, shake well, and prepare a series of mixed reference solutions of different concentrations, and measure them under the above chromatographic conditions. Linear regression is performed with peak area as the ordinate and concentration as the abscissa ( Figure 6 The results showed that the six phenylpropanoids and two flavonoid components in Artemisia argyi had a good linear relationship.

[0136] 2.6.5 Repeatability study

[0137] Take the powder of mugwort leaves (pass through No. 3 sieve), prepare 6 test solutions in parallel according to the method under "2.3", repeat the injection and determination 6 times according to the chromatographic conditions of "2.1", record the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid C, cyanidin and isoeupretinoic acid, and calculate their RSD values ​​(Table 4). The results show that the RSD values ​​of the contents of six phenylpropanoids and two flavonoids are ≤3.0%, indicating that the established method has good reproducibility.

[0138] Table 4 Repeatability test results of the test products

[0139]

[0140] 2.6.6 Stability study

[0141] Take the powder of mugwort leaves (pass through No. 3 sieve), prepare the test solution according to the method under "2.3", and analyze the samples at 0, 7, 14, 17, 20, and 24 hours, record the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid C, brown cyanidin and isoeupretinoic acid, and calculate their RSD values ​​(Table 5). The results show that the RSD values ​​of the contents of six phenylpropanoids and two flavonoids are all ≤3.0%, indicating that the test sample is stable within 24 hours.

[0142] Table 5 Stability test results of the test products

[0143]

[0144] 2.6.7 Intermediate precision study

[0145] The analysis was performed by different analysts in different laboratories and with different brands of instruments (Waters H-Class and ThermoVanquish). Take the powder of Artemisia argyi (passed through No. 3 sieve), prepare 6 test solutions in parallel according to the method in item "2.3", repeat the injection and determination 6 times according to the chromatographic conditions in item "2.1", record the contents of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid C, cyanidin and isoeupretinoic acid, and calculate their RSD values ​​( Figure 7 and Table 6). The results showed that the RSD values ​​of the contents of the six phenylpropanoids and two flavonoids were all ≤3.0%, indicating that the established method had good intermediate precision and could be stably reproduced in different laboratories and equipment.

[0146] Table 6 Investigation on the intermediate precision of the determination of eight phenylpropanoids and flavonoids in Artemisia argyi

[0147]

[0148] 2.6.8 Sample recovery rate investigation

[0149] Accurately weigh 9 portions of wormwood powder (passed through No. 3 sieve) with known content, each portion is about 0.1g. Accurately add corresponding reference solids at 50%, 100%, and 150% of the content of the four components to be tested, and each gradient is repeated in 3 groups. Samples are injected and analyzed according to the preparation method under "2.3" and the chromatographic conditions under "2.1", and the sample recovery rates and RSD values ​​of the four components to be tested are calculated (Table 7). The results showed that the recoveries of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid C, cyanidin and isoeupretinoic acid were in the range of 95.30%-103.68% (RSD, 2.798%), 98.93%-104.46% (RSD, 2.566%), 93.35%-100.18% (RSD, 2.426%), 94.28%-100.04% (RSD, 2.844%), respectively. (RSD, 2.264%), 94.04%-102.41% (RSD, 2.944%), 93.76%-98.75% (RSD, 1.391%), 94.19%-102.29% (RSD, 2.746%), 95.40%-102.49% (RSD, 2.316%) met the recovery limit standards of AOAC, indicating that the established method has high accuracy and high sample recovery rate.

[0150] Table 7 Recovery results of eight phenylpropanoids and flavonoids in Artemisia argyi

[0151]

[0152] 2.7 Determination of relative correction factors and relative retention time positioning of the measured indicators

[0153] The present application found that all batches of Artemisia argyi were rich in isochlorogenic acid C, and this component was the main potential quality difference marker with moderate retention time, good peak shape, stable properties and low price, and was selected as the internal reference in this method.

[0154] The results showed that the relative correction factors f of isochlorogenic acid C, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, cyanidin and isoeupretinoic acid were 新绿原酸 / 异绿原酸C 、f 绿原酸 / 异绿原酸C 、f 隐绿原酸 / 异绿原酸C 、f 咖啡酸 / 异绿原酸C 、f 异绿原酸B / 异绿原酸C 、f 棕矢车菊素 / 异绿原酸C and f 异泽兰黄素 / 异绿原酸C The relative retention time was 1.213, 1.186, 1.277, 0.671, 1.009, 1.157 and 1.048, respectively, and the RSDs were all <3%, indicating that the established f system had good stability and applicability and could be applied to the simultaneous quantitative determination of multiple characteristic components of Artemisia argyi (Table 8). 新绿原酸 / 异绿原酸C、T 绿原酸 / 异绿原酸C 、T 隐绿原酸 / 异绿原酸C 、T 咖啡酸 / 异绿原酸C 、T 异绿原酸B / 异绿原酸C 、T 棕矢车菊素 / 异绿原酸C and T 异泽兰黄素 / 异绿原酸C They are 0.220, 0.350, 0.398, 0.455, 0.890, 1.389 and 1.552 respectively, with RSD < 3%, indicating that they have good stability and applicability, and can accurately locate the chromatographic peak to be measured (Table 9). The relative correction factor and relative retention time positioning determined in this application have been verified by the system and the standard, and are relatively fixed under the same analytical conditions and without background negative interference, and can be directly applied to the simultaneous quantitative analysis of multiple components of wormwood raw materials and their products without repeated inspection and verification.

[0155] Table 8 Relative correction factor positioning of eight phenylpropanoids and flavonoids in Artemisia argyi by one-test-multiple-evaluation method

[0156]

[0157] Table 9 Relative retention time positioning of eight phenylpropanoids and flavonoids in Artemisia argyi by one-test-multiple-evaluation method

[0158]

[0159] 2.8 Durability study of relative correction factors and relative retention times of the measured indicators

[0160] The determination was carried out under the chromatographic conditions specified in this application, and the serial dilution mixed reference solution under "2.6.4 Linearity Study" was accurately aspirated to further examine the system durability of relative correction factors and relative retention time positioning, and to verify the accuracy of the one-measurement-multiple-evaluation method for determining the characteristic phenylpropanoid and flavonoid components of mugwort.

[0161] This application systematically investigated the effects of Waters CORTECS C18 (4.6mm×150mm, 2.7μm) chromatographic column batches, ultra-high performance liquid chromatograph models (Waters H-Class, SHIMADZU LC-40D xs and Thermo Vanquish), column temperature (23℃, 25℃ and 27℃) and flow rate (0.38mL / min, 0.40mL / min and 0.42mL / min) on the system durability of relative correction factor and relative retention time positioning (Tables 10 and 11). The results showed that the RSD of relative correction factor and relative retention time positioning was <3%, indicating that the established method can be stably reproduced between different batches of chromatographic columns and ultra-high performance liquid chromatographs, and the small fluctuations in column temperature and flow rate can meet the system suitability requirements of the analysis, and both durability and practicality are good.

[0162] Table 10 Durability investigation of relative correction factor positioning

[0163]

[0164] Table 11 Durability study of relative retention time positioning

[0165]

[0166] 2.9 Verification of content determination results by one-test-multiple-evaluation method and external standard method

[0167] After methodological validation and durability investigation, the chromatographic conditions established in this application and the one-measurement-multiple-evaluation method were used to determine the characteristic phenylpropanoid and flavonoid components, namely, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid C, cyanidin and isoeupretinoic acid in 18 batches of mugwort leaves from the main producing areas. The relative deviation (RD / %) was used as the standard, and the external standard sampling method (ESM) was used to compare and verify the QAMS results. There was no significant difference in the content determination results of QAMS and ESM methods, and RD<1%, indicating that the content determination method of QAMS is accurate and can be applied to the quality control of the simultaneous determination of the content of multiple pharmacological components of mugwort leaves (Table 12).

[0168] Table 12 ESM and QAMS determination of 8 characteristic phenylpropanoids and flavonoids in Artemisia argyi (n=8)

[0169]

[0170] (Continued Table 12)

[0171]

[0172] 3. Conclusion

[0173] This application uses Artemisia argyi as the research object, and through the development of chromatographic conditions, methodological verification, determination of relative correction factors and relative retention time positioning, and verification of measurement results, it is confirmed that the one-measurement-multiple-evaluation method can be applied to the simultaneous quantitative determination of characteristic phenylpropanoid and flavonoid components of Artemisia argyi. The established method is fast, stable, has a stable baseline and strong specificity, and reflects the quality status of Artemisia argyi medicinal materials more comprehensively than a single measurement indicator. The detection method of this application is highly practical, easy to operate, highly accurate, saves detection costs, and is more conducive to environmental protection.

[0174] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0175] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A method for determining the content of phenylpropanoids and flavonoids in wormwood, characterized in that: The following steps are involved: Extracting Artemisia argyi with an extraction solvent to prepare a test solution; The reference substance is dissolved using a dissolving reagent to prepare a reference substance mixed solution; the reference substance includes isochlorogenic acid C, and one or more of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, cyanidin and isoeupretinoic acid reference substances; The test solution and the reference solution are injected separately for ultra-high performance liquid chromatography detection; Taking isochlorogenic acid C as the internal reference, the content of the component to be tested in the test solution was calculated according to the following formula: Ci = f*Cs*(Ai / As); Where f is the relative correction factor of the component to be tested, As is the peak area of ​​the internal reference isochlorogenic acid C, Cs is the concentration of the internal reference isochlorogenic acid C in the reference solution, Ai is the peak area of ​​the component to be tested in Artemisia argyi, and Ci is the concentration of the component to be tested in the test solution; The components to be tested include phenylpropanoids and flavonoids.

2. The method for determining the content of phenylpropanoids and flavonoids in wormwood according to claim 1, characterized in that: The phenylpropanoid and flavonoid components include one or more of isochlorogenic acid C, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, cyanidin and isoeupretinoic acid.

3. The method for determining the content of phenylpropanoids and flavonoids in wormwood according to claim 1, characterized in that: The following steps are also included: Using isochlorogenic acid C as the internal reference, inject mixed solutions of reference substances of different concentrations, perform ultra-high performance liquid chromatography detection, record the peak areas corresponding to the component i to be tested in the mixed solutions of reference substances of different concentrations, and calculate the average value as the relative correction factor according to the following formula: f = (As / Cs) / (Ai / Ci); In the formula, As is the peak area of ​​the internal reference isochlorogenic acid C, Cs is the concentration of the internal reference isochlorogenic acid C, Ai is the peak area of ​​the reference substance i of the component to be tested, and Ci is the concentration of the reference substance i of the component to be tested; the component i to be tested includes neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, cyanidin or isoeupretinoic acid.

4. The method for determining the content of phenylpropanoids and flavonoids in wormwood according to claim 1, characterized in that: The ultra-high performance liquid chromatography detection conditions include: mobile phase A is acetonitrile, mobile phase B is formic acid aqueous solution, and gradient elution is adopted.

5. The method for determining the content of phenylpropanoids and flavonoids in wormwood according to claim 4, characterized in that: The procedure of the gradient elution includes: From 0 to 10 min, the volume percentage of the mobile phase A increases from 8% to 10%; 10min-18min, the volume percentage of the mobile phase A increases from 10% to 14%; From 18min to 27min, the volume percentage of the mobile phase A increases from 14% to 20%; From 27min to 32min, the volume percentage of the mobile phase A increases from 20% to 21%; From 32min to 50min, the volume percentage of the mobile phase A increases from 21% to 40%; From 50 min to 62 min, the volume percentage of the mobile phase A increases from 40% to 70%.

6. The method for determining the content of phenylpropanoids and flavonoids in wormwood according to claim 4, characterized in that: In the formic acid aqueous solution, the volume percentage of formic acid is 0.1%-0.3%.

7. The method for determining the content of phenylpropanoids and flavonoids in wormwood according to claim 1, characterized in that: The extraction solvent includes methanol-water solution; Optionally, in the methanol aqueous solution, the volume percentage of methanol is 60%-80%.

8. The method for determining the content of phenylpropanoids and flavonoids in wormwood according to claim 1, characterized in that: The ultra-high performance liquid chromatography detection also satisfies at least one of the following conditions: (1) The chromatographic column is a C18 chromatographic column; (2) Column temperature is 20°C-30°C; (3) Flow rate: 0.3 mL / min-0.5 mL / min; (4) The injection volume is 1 μL-3 μL; (5) The wavelength is 315nm-335nm.

9. The method for determining the content of phenylpropanoids and flavonoids in wormwood according to claim 1, characterized in that: The dissolving agent is methanol.

10. Use of the method for determining the content of phenylpropanoids and flavonoids in Artemisia argyi according to any one of claims 1 to 9 in the quality control of Artemisia argyi medicinal materials.

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