Construction method and detection method of characteristic chromatogram of xuchangqing standard decoction and dispensing granules

Abstract

The application relates to the technical field of traditional Chinese medicine detection and analytical chemistry, and particularly relates to a characteristic spectrum construction method and detection method of Xuchangqing standard decoction and formula granules. The specific steps comprise mixing a test sample and an extraction solvent, performing ultrasonic extraction or hot reflux extraction, and preparing a test sample solution; performing high performance liquid chromatography analysis on the test sample solution; the high performance liquid chromatography analysis conditions comprise: gradient elution is performed with acetonitrile as mobile phase A and acid aqueous solution with a volume concentration of 0.1%-0.2% as mobile phase B, and the gradient elution program comprises: 0-30 min, the volume percentage of the mobile phase A is increased from 7% to 14%. The characteristic spectrum of the application has better specificity, can be applied to Xuchangqing standard decoction and formula granules, and can accurately determine the characteristic components.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine detection and analytical chemistry technology, specifically to the method for constructing characteristic spectra and detection methods for Xu Changqing standard decoction and formula granules. Background Technology

[0002] Cynanchum paniculatum (Bge.) Kitag., a plant in the Asclepiadaceae family, is a dried root and rhizome. It is harvested in autumn, impurities are removed, and it is dried in the shade. It has a pungent taste and warm properties. It enters the liver and stomach meridians. It has the effects of dispelling wind, resolving dampness, relieving pain, and relieving itching. It is used for rheumatic pain, stomach pain and bloating, toothache, back pain, injuries from falls, urticaria, and eczema.

[0003] The 2025 edition of the Chinese Pharmacopoeia only addresses the quality control of *Cynanchum paniculatum* (Xu Changqing) based on its physical properties, microscopic and thin-layer chromatography identification, and the specified content limit for paeonol. Due to the diversity of components in traditional Chinese medicinal materials, measuring only a single indicator cannot reflect the complexity of these materials or accurately indicate their quality. Paeonol is a fat-soluble component, while *Cynanchum paniculatum* is traditionally used in decoctions. Therefore, selecting water-soluble components as the detection indicator is more conducive to reflecting the pharmacodynamic material basis of *Cynanchum paniculatum* under the decoction system. The original properties of traditional Chinese medicinal materials are destroyed after decoction, making it impossible to identify the authenticity of the decoction through physical identification. Furthermore, traditional Chinese medicine preparations such as granules are based on standard decoctions, which also presents the problem of difficulty in identifying whether the raw material is *Cynanchum paniculatum*. Therefore, to ensure the effectiveness and safety of *Cynanchum paniculatum* decoction-related products, it is necessary to establish detection methods applicable to *Cynanchum paniculatum* decoction-related products (standard decoctions and granules).

[0004] Currently, most quality testing research on products related to the decoction of Cynanchum paniculatum focuses on paeonol. Existing studies have shown that vanillic acid possesses anti-inflammatory and antioxidant activities; vanillic acid is highly water-soluble and is a representative chemical component in products related to the decoction of Cynanchum paniculatum, possessing research value in the study of the material basis of Cynanchum paniculatum in treating rheumatic pain, traumatic injuries, urticaria, eczema, and other diseases. Currently reported methods for determining vanillic acid content are only applicable to Cynanchum paniculatum raw materials; there are currently no reports on methods for determining vanillic acid content in its decoction.

[0005] In addition, Cynanchum paniculatum is rich in a large number of active compounds with acetophenone as the parent ring. Among them, vanillinone (4'-hydroxy-3'-methoxyacetophenone) and isovanillinone (4-hydroxy-3-methoxyacetophenone) are also key active components in Cynanchum paniculatum. The two compounds are isomers of each other and have similar chemical properties. Existing detection methods have failed to separate the two substances. Summary of the Invention

[0006] To address the problems existing in the prior art, one or more embodiments of this application provide a method for constructing characteristic spectra and a method for detecting Xu Changqing standard decoction and formula granules.

[0007] The specific technical solution is as follows:

[0008] The first aspect of this application provides a method for constructing the characteristic spectrum of Xu Changqing standard decoction or formula granules, including the following steps:

[0009] The test sample and extraction solvent are mixed and subjected to ultrasonic extraction or hot reflux extraction to obtain the test sample solution.

[0010] The test solution was analyzed by high performance liquid chromatography.

[0011] The conditions for the high performance liquid chromatography analysis include: gradient elution using acetonitrile as mobile phase A and an aqueous solution of acid with a volume concentration of 0.1%-0.2% as mobile phase B. The gradient elution program includes: 0-30 min, during which the volume percentage of mobile phase A increases from 7% to 14%.

[0012] In some embodiments, the conditions for the high-performance liquid chromatography analysis further include at least one of the following features:

[0013] (1) The chromatographic column is packed with octadecylsilane-bonded silica gel. (2) The column temperature is 25℃-40℃. (3) The detection wavelength is 260nm-290nm. (4) The flow rate of the mobile phase is 0.3mL / min-0.35mL / min. (5) The injection volume is 1μL-2μL. (6) The acid includes one or more of phosphoric acid, formic acid and acetic acid.

[0014] In some embodiments, the chromatographic column has the following specifications: a length of 150 mm, an inner diameter of 2.1 mm, and a packing particle size of 1.6 μm-2.2 μm.

[0015] In some embodiments, the extraction solvent comprises an aqueous solution of alcohol with a volume concentration of 50%-70%;

[0016] Optionally, the alcohol includes one or more of methanol and ethanol.

[0017] In some embodiments, the conditions for ultrasonic extraction include: ultrasonic power of 200W-500W, frequency of 30kHz-50kHz, and ultrasonic time of 15min-45min; and / or,

[0018] The conditions for hot reflux extraction include: a heating temperature of 80℃-90℃ and a heating time of 15min-45min.

[0019] In some embodiments, the weight-to-volume ratio of the test sample to the extraction solvent is 0.4-0.6 g: 25 mL.

[0020] In some embodiments, the preparation method of the Xu Changqing standard decoction includes the following steps:

[0021] Take the medicinal herb *Cynanchum paniculatum*, soak it in water, add the first water for the first decoction, the weight ratio of the first water to the medicinal herb *Cynanchum paniculatum* is (10~12):1, filter to obtain the first filtrate and the first residue, add the second water to the first residue for the second decoction, the weight ratio of the second water to the medicinal herb *Cynanchum paniculatum* is (8~10):1, filter to obtain the second filtrate, and combine the first filtrate and the second filtrate.

[0022] In some embodiments, the preparation method of the Xu Changqing formula granules includes the following steps:

[0023] Take the medicinal material *Cynanchum paniculatum*, add water and perform steam distillation to collect paeonol; add a third water to the residue after steam distillation for a third decoction, the mass ratio of the third water to the residue after steam distillation is (8-10):1, filter to obtain a third filtrate and a third filter residue, add a fourth water to the third filter residue for a fourth decoction, the mass ratio of the fourth water to the third residue is (6-8):1, filter to obtain a fourth filtrate, combine the third filtrate and the fourth filtrate, concentrate to obtain a clear extract; add a first excipient to the clear extract and mix, dry to obtain a dry extract powder; mix the paeonol, the dry extract powder and the second excipient, and granulate.

[0024] In some embodiments, the method for constructing the feature map further includes the following steps:

[0025] A reference solution is prepared by mixing a reference substance with an organic solvent; the reference substance includes one or more of vanillic acid, p-hydroxyacetophenone, vanillin, isovillin, and 2,4-dihydroxyacetophenone.

[0026] The reference solution was analyzed by high-performance liquid chromatography.

[0027] Optionally, the mass-to-volume ratio of vanillic acid, p-hydroxyacetophenone, vanillinone, isovanillinone, 2,4-dihydroxyacetophenone, and the organic solvent is independently selected from 8-12 μg:1 mL.

[0028] In some embodiments, the feature spectrum obtained by constructing the feature spectrum includes 7 common peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, and peak 7; wherein, peak 1 is vanillic acid, peak 2 is p-hydroxyacetophenone, peak 3 is vanillyl acetophenone, peak 4 is isovanillyl acetophenone, and peak 6 is 2,4-dihydroxyacetophenone.

[0029] Furthermore, taking peak 2 as a reference, the characteristic peaks and their relative retention times are as follows: peak 1: 0.65±10%, peak 3: 1.40±10%, peak 4: 1.45±10%, peak 5: 1.67±10%, peak 6: 1.79±10%, peak 7: 1.94±10%.

[0030] The second aspect of this application provides a method for detecting Xu Changqing standard decoction or formula granules, including the following steps:

[0031] A reference solution is prepared by mixing a reference substance with an organic solvent; the reference substance includes one or more of vanillic acid, p-hydroxyacetophenone, vanillin, isovillin, and 2,4-dihydroxyacetophenone.

[0032] The sample to be tested and the extraction solvent are mixed and subjected to ultrasonic extraction or hot reflux extraction to obtain the sample solution.

[0033] The reference solution and the test solution were analyzed by high performance liquid chromatography.

[0034] The conditions for the high performance liquid chromatography analysis include: gradient elution using acetonitrile as mobile phase A and an aqueous solution of acid with a volume concentration of 0.1%-0.2% as mobile phase B. The gradient elution program includes: 0-30 min, during which the volume percentage of mobile phase A increases from 7% to 14%.

[0035] The method for constructing the characteristic spectrum of the standard decoction or formula granules of Cynanchum paniculatum in this application can construct the characteristic spectrum of the water extract of Cynanchum paniculatum-related products—standard decoction and formula granules. The method is simple, stable, highly specific, and has a short analysis time, and can realize the overall quality stability evaluation of the water extract.

[0036] The detection method for the standard decoction or formula granules of Cynanchum paniculatum in this application can accurately determine one or more of vanillic acid, p-hydroxyacetophenone, vanillinone, isovanillinone, and 2,4-dihydroxyacetophenone in the above-mentioned Cynanchum paniculatum water extract-related products, filling the gap in the existing technology for the lack of detection and analysis of the above-mentioned key pharmacologically active substances. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0038] Figure 1This is a comparison of the characteristic spectra of Xu Changqing standard decoction under the wavelength range of 210nm-260nm in this application.

[0039] Figure 2 This is a comparison of the characteristic spectra of Xu Changqing standard decoction under the wavelength range of 265nm-285nm in this application.

[0040] Figure 3 This is a comparison of the characteristic spectra of Xu Changqing standard decoction under the wavelength range of 290nm-330nm in this application.

[0041] Figure 4 This is a comparison of the characteristic spectra of Xu Changqing standard decoction under different mobile phase conditions in this application.

[0042] Figure 5 This is a comparison of the characteristic spectra of Xu Changqing standard decoction under different column temperatures in this application.

[0043] Figure 6 This is a comparison of the characteristic spectra of Xu Changqing standard decoction under different flow rate conditions in this application.

[0044] Figure 7 This is a comparison of the characteristic spectra of the Xu Changqing standard decoction under different elution gradients in this application.

[0045] Figure 8 Comparison of characteristic chromatograms of Xu Changqing standard decoction under different brands and models of chromatographic columns in this application.

[0046] Figure 9 This is a comparison of the characteristic spectra of the standard decoction of Cynanchum paniculatum under different extraction solvent conditions in this application.

[0047] Figure 10 The chromatograms are for the specificity investigation of the test solution of Xu Changqing standard decoction, the reference solution of p-hydroxyacetophenone and the blank solvent of this application.

[0048] Figure 11 This is a superimposed image of the characteristic spectra of 10 batches of Xu Changqing standard decoction in this application.

[0049] Figure 12 This is a superimposed image of the characteristic spectra of 10 batches of Cynanchum paniculatum formula granules in this application.

[0050] Figure 13 This is the comparative characteristic spectrum (BT-R) of the Xu Changqing standard decoction in this application.

[0051] Figure 14 This is the KL-R (Knowledge and Limestone Species) characteristic spectrum of the Xu Changqing formula granules in this application.

[0052] Figure 15The total ion chromatogram and ultraviolet absorption chromatogram of the Xu Changqing test sample solution are shown in the application.

[0053] Figure 16 A comparison image of the Xu Changqing test sample and the control sample submitted for the application.

[0054] Figure 17 This is a standard curve for the determination of vanillic acid content in this application. Detailed Implementation

[0055] The present application is further described below with reference to the embodiments, examples, and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Furthermore, it should be understood that after reading the teachings of this application, those skilled in the art can make various alterations or modifications to the application, and these equivalent forms also fall within the protection scope of the appended claims.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0057] the term:

[0058] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0059] The standard decoctions in this application are single-herb Chinese medicinal decoctions prepared using standardized processes, guided by traditional Chinese medicine theory and based on clinical application, and referencing modern extraction methods. The Chinese herbal formula granules in this application are a series of pure Chinese medicine products made from traditional Chinese medicinal herbs that conform to processing standards, refined through modern pharmaceutical technology including extraction, concentration, separation, drying, granulation, and packaging. Understandably, the aforementioned standard decoctions and Chinese herbal formula granules can essentially preserve all the characteristics of the original Chinese medicinal herbs.

[0060] The standard materials of Chinese medicinal materials referred to in this application are reference medicinal materials sold by the China National Institutes for Food and Drug Control or working reference medicinal materials tested by drug testing institutes in various provinces and cities.

[0061] The term "and / or" as used herein includes any one of two or more related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations encompass any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," it should be understood that, in this application, the technical solution undoubtedly includes solutions connected using "logical AND," and also undoubtedly includes solutions connected using "logical OR." For example, "A and / or B" includes three parallel solutions: 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, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0062] In this document, terms such as "preferred," "better," and "more preferred" are merely descriptions of implementation methods or examples that achieve better results, and should be understood as not constituting a limitation on the scope of protection of this application.

[0063] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0064] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0065] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, optional numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. Furthermore, 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 to which they are included.

[0066] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, or ±0.1°C.

[0067] In this application, weight can be a well-known unit of mass in the field of traditional Chinese medicine, such as μg, mg, g, or kg.

[0068] The first aspect of this application provides a method for constructing the characteristic spectrum of Xu Changqing standard decoction or formula granules, including the following steps:

[0069] The test sample and extraction solvent are mixed and subjected to ultrasonic extraction or hot reflux extraction to obtain the test sample solution.

[0070] The test solution was analyzed by high performance liquid chromatography.

[0071] The conditions for high performance liquid chromatography analysis include: gradient elution with acetonitrile as mobile phase A and an aqueous solution of acid with a volume concentration of 0.1%-0.2% as mobile phase B. The gradient elution program includes: 0-30 min, with the volume percentage of mobile phase A increasing from 7% to 14%.

[0072] The test samples mentioned above were selected from Xu Changqing standard decoction or Xu Changqing formula granules.

[0073] In some embodiments, the chromatographic column is packed with octadecylsilane-bonded silica gel.

[0074] In some embodiments, the chromatographic column has the following specifications: a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.6 μm-2.2 μm, such as 1.6 μm, 1.8 μm, 2 μm, 2.2 μm, etc.

[0075] In some embodiments, the column temperature is 25°C-40°C, for example 25°C, 30°C, 35°C, 40°C, etc.

[0076] In some embodiments, the detection wavelength is 260nm-290nm, for example 260nm, 265nm, 270nm, 275nm, 280nm, 285nm, 290nm, etc.

[0077] In some embodiments, the flow rate of the mobile phase is 0.3 mL / min to 0.35 mL / min, for example 0.3 mL / min, 0.31 mL / min, 0.32 mL / min, 0.33 mL / min, 0.34 mL / min, 0.35 mL / min, etc.

[0078] In some embodiments, the injection volume is 1 μL-2 μL, for example 1 μL, 1.5 μL, 2 μL, etc.

[0079] In some embodiments, the acid in mobile phase B includes one or more of phosphoric acid, formic acid, and acetic acid.

[0080] In some embodiments, the extraction solvent comprises an aqueous solution of alcohol with a volume concentration of 50%-70%; the volume concentration of the aqueous solution of alcohol is, for example, 50%, 60%, 70%, etc.

[0081] Alternatively, the alcohol includes one or more of methanol and ethanol.

[0082] In some embodiments, the conditions for ultrasonic extraction include: ultrasonic power of 200W-500W, frequency of 30kHz-50kHz, and ultrasonic time of 15min-45min. Examples of ultrasonic power include 200W, 300W, 400W, and 500W; examples of frequency include 30kHz, 40kHz, and 50kHz; and examples of ultrasonic time include 15min, 20min, 25min, 30min, 35min, 40min, and 45min.

[0083] In some embodiments, the conditions for hot reflux extraction include: a heating temperature of 80°C-90°C (e.g., 80°C, 85°C, 90°C, etc.) and a heating time of 15 min-45 min (e.g., 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, etc.).

[0084] In some embodiments, the weight-to-volume ratio of the sample to the extraction solvent is 0.4-0.6 g: 25 mL, for example, 0.4 g: 25 mL, 0.5 g: 25 mL, 0.6 g: 25 mL, etc.

[0085] In some embodiments, the preparation method of Xu Changqing standard decoction includes the following steps:

[0086] Take the medicinal herb *Cynanchum paniculatum*, soak it in water, add the first water for the first decoction, the weight ratio of the first water to the medicinal herb *Cynanchum paniculatum* is (10~12):1, filter to obtain the first filtrate and the first residue, add the second water to the first residue for the second decoction, the weight ratio of the second water to the medicinal herb *Cynanchum paniculatum* is (8~10):1, filter to obtain the second filtrate, and combine the first filtrate and the second filtrate.

[0087] The weight ratio of the first water to Xu Changqing medicinal material can be selected from (10~12):1, for example, 10:1, 11:1, 12:1, etc., and the weight ratio of the second water to Xu Changqing medicinal material can be selected from (8~10):1, for example, 8:1, 9:1, 10:1, etc.

[0088] Optionally, the first simmering time is 10 min to 40 min, for example, 10 min, 20 min, 30 min, 40 min, etc., and more preferably 20 min to 30 min; the second simmering time is 10 min to 40 min, for example, 10 min, 20 min, 30 min, 40 min, etc., and more preferably 10 min to 20 min.

[0089] In some embodiments, the preparation method of Xu Changqing formula granules includes the following steps:

[0090] Take the medicinal material *Cynanchum paniculatum*, add water and perform steam distillation to collect paeonol; add a third water to the residue after steam distillation for a third decoction, the mass ratio of the third water to the residue after steam distillation is (8-10):1, filter to obtain a third filtrate and a third filter residue, add a fourth water to the third filter residue for a fourth decoction, the mass ratio of the fourth water to the third residue is (6-8):1, filter to obtain a fourth filtrate, combine the third filtrate and the fourth filtrate, concentrate to obtain a clear extract; add a first excipient to the clear extract and mix, dry to obtain a dry extract powder; mix the paeonol, the dry extract powder and the second excipient, and granulate.

[0091] The mass ratio of the third water to the residue after steam distillation is (8-10):1, for example, 8:1, 9:1, 10:1, etc., and the mass ratio of the fourth water to the third residue is (6-8):1, for example, 6:1, 7:1, 8:1, etc.

[0092] Optionally, in the step of taking Xu Changqing medicinal material and adding water for steam distillation, the steam distillation time is 2-4 hours, and the weight of water added is 4-6 times that of Xu Changqing medicinal material.

[0093] Optionally, the third decoction time is 10 min to 40 min, for example, 10 min, 20 min, 30 min, 40 min, etc., and more preferably 20 min to 40 min; the fourth decoction time is 10 min to 40 min, for example, 10 min, 20 min, 30 min, 40 min, etc., and more preferably 20 min to 40 min.

[0094] Optionally, the relative density of the extract is 1.08 to 1.10. Relative density refers to the ratio of the density of a substance to the density of water.

[0095] Alternatively, the drying method may include spray drying.

[0096] Optionally, the first excipient includes maltodextrin, and the second excipient includes maltodextrin and silicon dioxide.

[0097] In some embodiments, the preparation method of Xu Changqing standard formula granules includes the following steps: Take Xu Changqing medicinal material, remove impurities, wash quickly, and cut into sections. Take 3500g of Xu Changqing slices, add 5 times the amount of water, steam distill for 3 hours, and collect paeonol; after distillation, add water to the residue and decoct twice, adding 9 times the amount of water for the first time and decoct for 0.5 hours, and adding 7 times the amount of water for the second time and decoct for 0.5 hours. Filter the decoction, concentrate under reduced pressure to a clear extract with a relative density of 1.08-1.10, filter, add an appropriate amount of maltodextrin, mix well, spray dry to obtain dry extract powder; pulverize paeonol, mix it with the dry extract powder and an appropriate amount of silicon dioxide and maltodextrin, dry granulate to make 1000g, and package to obtain the product.

[0098] In some embodiments, the method for constructing feature maps further includes the following steps:

[0099] A reference solution is prepared by mixing a reference substance with an organic solvent; the reference substance includes one or more of vanillic acid, p-hydroxyacetophenone, vanillin, isovillin, and 2,4-dihydroxyacetophenone.

[0100] The reference solution was analyzed by high performance liquid chromatography.

[0101] Optionally, the mass-to-volume ratio of vanillic acid, p-hydroxyacetophenone, vanillin, isovillin, 2,4-dihydroxyacetophenone, and the organic solvent is independently selected from 8-12 μg: 1 mL.

[0102] In some embodiments, the characteristic spectrum obtained by constructing the characteristic spectrum includes 7 common peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, and peak 7; wherein, peak 1 is vanillic acid, peak 2 is p-hydroxyacetophenone, peak 3 is vanillic acid, peak 4 is isovanillic acid, and peak 6 is 2,4-dihydroxyacetophenone.

[0103] Furthermore, taking peak 2 as a reference, the characteristic peaks and their relative retention times are as follows: peak 1: 0.65±10%, peak 3: 1.40±10%, peak 4: 1.45±10%, peak 5: 1.67±10%, peak 6: 1.79±10%, peak 7: 1.94±10%.

[0104] The second aspect of this application provides a method for detecting Xu Changqing standard decoction or formula granules, including the following steps:

[0105] A reference solution is prepared by mixing a reference substance with an organic solvent; the reference substance includes one or more of vanillic acid, p-hydroxyacetophenone, vanillin, isovillin, and 2,4-dihydroxyacetophenone.

[0106] The sample to be tested and the extraction solvent are mixed and subjected to ultrasonic extraction or hot reflux extraction to obtain the sample solution.

[0107] The reference solution and the test solution were analyzed by high performance liquid chromatography.

[0108] The conditions for high performance liquid chromatography analysis include: gradient elution with acetonitrile as mobile phase A and an aqueous solution of acid with a volume concentration of 0.1%-0.2% as mobile phase B. The gradient elution program includes: 0-30 min, with the volume percentage of mobile phase A increasing from 7% to 14%.

[0109] The following are some specific examples.

[0110] Example 1

[0111] This embodiment explores and establishes the construction method of the characteristic spectrum of Xu Changqing standard decoction or formula granules. The specific steps are as follows:

[0112] 1. Instruments and Reagents

[0113] 1.1. Instruments

[0114] Waters UHPLC (Waters H-Class, Waters Corporation), Thermo UHPLC (Vanquish, Thermo Fisher Scientific (China) Co., Ltd.), Agilent UHPLC (Model 1290, Agilent); Waters ACQUITY UPLC BEH C18 column (2.1mm × 150mm, 1.7μm), Waters ACQUITY UPLC HSS T3 column (2.1mm × 150mm, 1.8μm), Thermo Acclaim 120 C18 column (2.1mm × 150mm, 2.2μm), Waters CORTECS T3 column (2.1mm × 150mm, 1.6μm), Waters BEH C18 column (2.1 × 100mm, 1.7μm), YMC Triart C18 column (2.1 × 100mm, 1.9μm), Thermo... Syncronis aQ column (2.1×100mm, 1.7μm); 0.0001g balance (ME204E, Mettler Toledo), 0.00000g balance (XP26, Mettler Toledo), CNC ultrasonic cleaner (KQ500D, Kunshan Ultrasonic Instrument Co., Ltd.), constant temperature water bath (HWS28, Shanghai Yiheng Technology Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck KGaA).

[0115] 1.2. Reagents

[0116] Ethanol (Xilong Scientific Co., Ltd.) and methanol (Xilong Scientific Co., Ltd.) were analytical grade; phosphoric acid (Tianjin Kemeio Chemical Reagent Co., Ltd.), formic acid (Tianjin Kemeio Chemical Reagent Co., Ltd.), and acetonitrile (Thermo Fisher Scientific (China) Co., Ltd.) were chromatographic grade; and water was ultrapure water (prepared in the laboratory).

[0117] 1.3. Drug Trial

[0118] Vanillic acid (batch number: 110776-201503, content: 99.8%, China National Institutes for Food and Drug Control); Vanillin ethyl ketone (batch number: ST79120120-12683, content: 98%, Shanghai Shidander Biotechnology Co., Ltd.); Isovanillin ethyl ketone (batch number: ST783101-14738, content: 98%, Shanghai Shidander Biotechnology Co., Ltd.); 2,4-Dihydroxyacetophenone (batch number: B29F7H10229, content: 99%, Shanghai Yuanye Biotechnology Co., Ltd.); p-Hydroxyacetophenone (batch number: 111897-201602, content: 99.9%, China National Institutes for Food and Drug Control);

[0119] Table 1 details the batch numbers of the 10 batches of Xu Changqing standard decoction and their corresponding medicinal material origins, as well as the 10 batches of Xu Changqing formula granules and their corresponding medicinal material origins.

[0120] Table 1. Source Information of 10 Batches of Cynanchum paniculatum Medicinal Materials

[0121]

[0122] 1.4. Preparation of Xu Changqing Standard Decoction and Formula Granules

[0123] Preparation of Xu Changqing Standard Decoction: Take Xu Changqing medicinal material, soak it in water, add the first water for the first decoction, the weight ratio of the first water to Xu Changqing medicinal material is 11:1, the decoction time is 20 minutes, filter to obtain the first filtrate and the first residue, add the second water to the first residue for the second decoction, the weight ratio of the second water to Xu Changqing medicinal material is 9:1, the decoction time is 15 minutes, filter to obtain the second filtrate, combine the first filtrate and the second filtrate, concentrate, freeze dry to obtain the freeze-dried powder of the standard decoction.

[0124] Preparation of Xu Changqing Formula Granules: Take Xu Changqing medicinal material, add 5 times the amount of water and steam distill for 3 hours to collect paeonol; add third water to the residue after steam distillation for a third decoction, the mass ratio of third water to the residue after steam distillation is 9:1, the decoction time is 30 minutes, filter to obtain third filtrate and third filter residue, add fourth water to the third filter residue for a fourth decoction, the mass ratio of fourth water to the third residue is 7:1, the decoction time is 30 minutes, filter to obtain fourth filtrate, combine the third filtrate and fourth filtrate, concentrate to a relative density of 1.08-1.10 to obtain clear extract, add 4% by weight of the first excipient maltodextrin to the clear extract and mix, spray dry to obtain dry extract powder; pulverize paeonol, mix with dry extract powder and 4% by weight of the second excipient (a mixture of silicon dioxide and maltodextrin in a 1:1 weight ratio), dry granulate to obtain formula granules.

[0125] 2. Chromatographic conditions and sample preparation

[0126] 2.1 Chromatographic conditions for characteristic spectra

[0127] A Waters ACQUITY UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 μm) was used; acetonitrile was used as mobile phase A and 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the specifications in Table 2; the flow rate was 0.32 mL per minute; the column temperature was 30 °C; the detection wavelength was 274 nm; and the injection volume was 2 μL.

[0128] Table 2 Gradient Elution Table

[0129]

[0130] 2.2 Chromatographic conditions for vanillic acid content determination

[0131] A Waters BEH C18 column (2.1×100 mm, 1.7 μm) was used; the mobile phase was methanol:acetonitrile (1:1)-0.2% phosphoric acid solution (3:97); the flow rate was 0.3 mL / min; the column temperature was 30℃; the detection wavelength was 217 nm; and the injection volume was 1 μL.

[0132] 2.3 Preparation of reference solution

[0133] Accurately weigh appropriate amounts of vanillic acid and p-hydroxyacetophenone reference standards, add methanol to prepare solutions containing 10 μg per 1 mL, shake well, and the solution is obtained.

[0134] 2.4 Preparation of the test solution

[0135] Take an appropriate amount of freeze-dried powder or granules of Xu Changqing standard decoction, grind it into a fine powder, take about 0.5g, place it in a stoppered conical flask, add 10mL of 70% methanol, weigh it, sonicate it (power 300W, frequency 40kHz) for 30 minutes, cool it, make up the weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0136] 3. Comparison of chromatographic conditions for characteristic spectra

[0137] 3.1 Comparison of detection wavelengths

[0138] Accurately pipette 2 μL of the test solution and insert a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 μm). Use acetonitrile-0.1% phosphoric acid solution as the mobile phase and perform gradient elution according to the specifications in Table 2. The flow rate is 0.32 mL / min, the column temperature is 30℃, and a full wavelength scan of 210-400 nm is performed. Compare the chromatograms of Xu Changqing standard decoction at different wavelengths to select the optimal detection wavelength. The results are as follows: Figure 1-3 As shown in the figure. The results indicate that when the wavelength is less than 230 nm, the chromatographic peaks are chaotic and the baseline is unstable; when the wavelength is 230 nm to 260 nm, there is less chromatographic peak information; when the wavelength is 260 nm to 290 nm, the overall baseline is stable, the chromatographic peak response is moderate, and the resolution and peak shape are both good, with 274 nm being the best; when the wavelength is greater than 290 nm, the chromatographic peak response weakens. Therefore, the detection wavelength for the characteristic chromatogram of this application can be 260 nm to 290 nm. Since the chromatographic peak response is highest at a wavelength of 274 nm, the detection wavelength of 274 nm was selected for subsequent studies.

[0139] 3.2 Comparison of different mobile phases

[0140] Accurately pipette 2 μL of the test solution and use a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 μm) with acetonitrile as the organic phase and different types and concentrations of acid as the aqueous phase, eluting according to the gradient specified in Table 2. The flow rate is 0.32 mL / min, the column temperature is 30℃, and the detection wavelength is 274 nm. The effect of different aqueous phases on the characteristic chromatogram of *Cynanchum paniculatum* is investigated. The results are as follows: Figure 4 As shown, when phosphoric acid, acetic acid, and formic acid were used as the aqueous phase, the number of chromatographic peaks and the resolution did not differ significantly. The effect of 0.1%–0.2% phosphoric acid on the chromatogram was investigated; the results showed that different concentrations of phosphoric acid had little effect on the peak shape and resolution. Therefore, different concentrations of phosphoric acid, acetic acid, and formic acid can be used as the aqueous phase.

[0141] 3.3 Comparison of different column temperatures

[0142] Accurately pipette 2 μL of the test solution and insert it into a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 μm). Use acetonitrile-0.1% phosphoric acid solution as the mobile phase and perform gradient elution according to the specifications in Table 2. The flow rate is 0.32 mL / min, and the column temperature is set to 25℃, 30℃, 35℃, and 40℃. The detection wavelength is 274 nm. Compare the similarities and differences in the characteristic chromatograms. The results show ( Figure 5 When the column temperature is between 25℃ and 40℃, the resolution, peak shape and response of each characteristic peak are not significantly different. In order to extend the life of the chromatographic column and improve the temperature control effect of the chromatograph at room temperature, this application selects a column temperature of 30℃ for subsequent research.

[0143] 3.4 Comparison of different flow rates

[0144] Accurately pipette 2 μL of the test solution and insert a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 μm). Use acetonitrile-0.1% phosphoric acid solution as the mobile phase and perform gradient elution according to the specifications in Table 2. The column temperature is 30 °C, the detection wavelength is 274 nm, and the flow rates are set to 0.25 mL / min, 0.28 mL / min, 0.30 mL / min, 0.32 mL / min, and 0.35 mL / min, respectively. Compare the similarities and differences in the characteristic chromatograms. The results are as follows: Figure 6 As shown, when the flow rate is 0.25 mL / min to 0.28 mL / min, the separation of some characteristic peaks is poor. When the flow rate is 0.30 mL / min to 0.35 mL / min, the peak shape and separation of each characteristic peak are good and the differences are not significant. Therefore, this method has good robustness when the flow rate is 0.30 mL / min to 0.35 mL / min.

[0145] 3.5 Comparison of elution gradients

[0146] Accurately pipette 2 μL of the test solution and insert a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 μm). Use acetonitrile-0.1% phosphoric acid solution as the mobile phase and perform gradient elution according to the specifications in Table 3. The flow rate is 0.32 mL / min, the column temperature is 30 °C, and the detection wavelength is 274 nm. Comparison of chromatograms for different gradient elutions is shown below. Figure 7 As shown, when the elution gradient is gradient 3, the separation effect of each chromatographic peak is good and the analysis time is moderate.

[0147] Table 3 Gradient elution table

[0148]

[0149] 3.6 Comparison of chromatographic columns

[0150] The test solution of Xu Changqing standard decoction was analyzed using chromatographic columns of different brands, models, and particle sizes to compare the effects of different chromatographic columns on the characteristic chromatograms. This application selected four chromatographic columns for comparison: Waters ACQUITY UPLC BEH C18 column (2.1mm × 150mm, 1.7μm), Waters ACQUITY UPLC HSS T3 column (2.1mm × 150mm, 1.8μm, numbered), Thermo Acclaim 120 C18 column (2.1mm × 150mm, 2.2μm), and Waters CORTECS T3 column (2.1mm × 150mm, 1.6μm) to analyze the effects of different chromatographic columns on the characteristic chromatograms of Xu Changqing standard decoction. The results showed ( Figure 8 Different brands of chromatographic columns have a significant impact on the separation of each characteristic peak. Among them, the Waters ACQUITY UPLC BEH C18 column (2.1mm×150mm, 1.7μm) has the best separation effect on each chromatographic peak.

[0151] 4. Investigation on the preparation of the test solution

[0152] 4.1 Investigation of Extraction Solvents

[0153] Take approximately 0.5 g of lyophilized powder of Xu Changqing standard decoction (BT01), prepare 6 parallel groups (2 portions per group), accurately weigh them, and place them in stoppered conical flasks. Accurately add 10 mL each of methanol, 70% methanol, 50% methanol, ethanol, 70% ethanol, and 50% ethanol, respectively, weigh them, sonicate for 30 minutes, cool, weigh them again, replenish the lost weight with the appropriate solvent, shake well, filter, and collect the filtrate to obtain the test solution. Inject and determine the chromatographic conditions under section "2.1", record the peak areas of the 7 characteristic peaks, and calculate the "total peak area / sample weight" value; calculate the vanillic acid content by injection and determination under the chromatographic conditions under section "2.2". The results are shown in Table 4. Figure 9 As shown in the figure. The results show that when methanol and ethanol are used as extraction solvents, the peak shapes of each characteristic peak are poor. There is no significant difference in the peak shape and separation effect of each characteristic peak when other extraction solvents are used. Among them, when 70% methanol is used as extraction solvent, the "total peak area / sample weight" value of the seven characteristic peaks is the largest, and the extraction efficiency of vanillic acid is the highest. Therefore, 70% methanol will be selected as the extraction solvent in subsequent studies.

[0154] Table 4 Results of the Extraction Solvent Study

[0155]

[0156] 4.2 Examination of Extraction Methods

[0157] Take approximately 0.5 g of the lyophilized powder of Xu Changqing standard decoction (BT01), make two parallel groups, two portions per group, accurately weigh them, place them in a stoppered conical flask, accurately add 25 mL of 70% methanol, weigh them, and extract by reflux in a water bath and by ultrasonic extraction for 30 minutes respectively. After cooling, weigh them again, make up the weight loss with 70% methanol, shake well, filter, and collect the filtrate to obtain the test solution. Inject and determine the chromatographic conditions under section "2.1", record the peak areas of the seven characteristic peaks, and calculate the "total peak area / sample weight" value; inject and determine the vanillic acid content under the chromatographic conditions under section "2.2", and the results are shown in Table 5. The results show that there is no significant difference in the peak shape, separation effect, and "total peak area / sample weight" value of each characteristic peak when using different extraction methods, and there is also no significant difference in vanillic acid content. Considering the simplicity of operation, ultrasonic extraction was selected subsequently.

[0158] Table 5. Results of the Extraction Method Examination

[0159]

[0160] 4.3 Examination of extraction time

[0161] Take approximately 0.5 g of lyophilized powder of Xu Changqing standard decoction (BT01), in triplicate (two portions per group), accurately weigh each portion, place them in a stoppered conical flask, accurately add 25 mL of 70% methanol, weigh again, and sonicate for 15 minutes, 30 minutes, and 45 minutes respectively. After cooling, weigh again, replenish the lost weight with 70% methanol, shake well, filter, and collect the filtrate to obtain the test solution. Inject and determine the sample according to the chromatographic conditions in section "2.1", record the peak areas of the seven characteristic peaks, and calculate the "total peak area / sample weight" value. Calculate the vanillic acid content according to the chromatographic conditions in section "2.2", and the results are shown in Table 6. The results show that the "total peak area / sample weight" values ​​do not differ significantly at different extraction times. To ensure complete extraction in subsequent experiments, an extraction time of 30 minutes was selected.

[0162] Table 6 Results of the Extraction Time Examination

[0163]

[0164] 4.4 Investigation on the amount of extraction solvent used

[0165] Take approximately 0.5g of lyophilized powder of Xu Changqing standard decoction (BT01), make 3 parallel groups, 2 portions per group, accurately weigh them, place them in stoppered conical flasks, accurately add 15mL, 25mL, and 50mL of 70% methanol respectively, weigh them, sonicate for 30 minutes, cool, weigh them again, make up the weight loss with 70% methanol, shake well, filter, and collect the filtrate. Inject and determine according to the chromatographic conditions under section "2.1", record the peak area of ​​7 characteristic peaks respectively, and calculate the value of "(total peak area × solvent volume) / sample volume", the results are shown in Table 7. The results show that there is no significant difference in the peak shape and resolution of each characteristic peak when using different extraction solvent volumes, and there is no significant difference in the value of "(total peak area × solvent volume) / sample volume", and the measured vanillic acid content is also not significantly different, indicating that 15mL has been completely extracted. In order to ensure complete extraction in subsequent experiments, the extraction solvent volume is selected as 25mL.

[0166] Table 7 Results of the Investigation on Extraction Solvent Dosage

[0167]

[0168] 5. Examination of Feature Mapping Methodology

[0169] 5.1 Specificity Examination

[0170] Accurately pipette 2 μL each of the Xu Changqing standard decoction test solution (BT01), p-hydroxyacetophenone reference solution, and blank solvent (70% methanol), and inject them for analysis according to the chromatographic conditions described in section "2.1". The results show ( Figure 10The test sample and the reference sample showed the same chromatographic peaks at the corresponding retention times, and the blank solvent did not interfere, indicating that the established method has good specificity.

[0171] 5.2 Precision Test

[0172] Accurately pipette the same sample solution of Xu Changqing standard decoction (BT01) and repeat the injection and determination 6 times under the chromatographic conditions in section "2.1". Using the p-hydroxyacetophenone chromatographic peak as the reference peak S, calculate the relative retention time of each characteristic peak and peak S, and calculate the RSD value. The results show (Table 8) that the RSD value of the relative retention time of each characteristic peak is less than 1.0%, indicating that the instrument precision is good.

[0173] Table 8 Precision test results

[0174]

[0175] 5.3 Stability Test

[0176] Accurately pipette the same sample of Xu Changqing standard decoction (BT01) and inject it at 0, 2, 4, 6, 8, 14, 18, 20, 24, and 30 hours according to the chromatographic conditions in section "2.1". Using the p-hydroxyacetophenone chromatographic peak as the reference peak S, calculate the relative retention time of each characteristic peak and peak S, and calculate the RSD value. The results show (Table 9) that the RSD value of the relative retention time of each characteristic peak is less than 1.0%, indicating that the sample solution has good stability within 30 hours.

[0177] Table 9 Stability Results

[0178]

[0179] 5.4 Reproducibility Test

[0180] Six test solutions were prepared from the same batch of Xu Changqing standard decoction lyophilized powder (BT01) according to the method in section "2.4", and injected for determination under the chromatographic conditions in section "2.1". Using the p-hydroxyacetophenone chromatographic peak as the reference peak S, the relative retention times of each characteristic peak and peak S were calculated, and the RSD values ​​were also calculated. The results (Table 10) show that the RSD values ​​of the relative retention times of each characteristic peak are all less than 1.0%, indicating that the established method has good reproducibility.

[0181] Table 10 Reproducibility Results

[0182]

[0183] 6. Determination of characteristic spectra of Xu Changqing standard decoction and formula granules and establishment of common patterns

[0184] Ten batches of lyophilized powder of Xu Changqing standard decoction and ten batches of Xu Changqing formulation granules were used to prepare test solutions according to the method in section "2.4". Characteristic chromatographic analysis was performed according to section "2.1", using the p-hydroxyacetophenone chromatographic peak as the reference peak S. The relative retention times of each characteristic peak and peak S were calculated. The experimental results are shown in Tables 11-12. The overlay chromatograms of the ten batches of Xu Changqing standard decoction are shown below. Figure 11 As shown, the superimposed image of the granules from 10 batches of the formula is as follows. Figure 12 As shown. Seven common peaks with good peak shape and resolution were selected as characteristic peaks of the Xu Changqing characteristic chromatogram. The characteristic chromatograms of 10 batches of Xu Changqing standard decoction and 10 batches of formula granules were matched using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System". A control chromatogram was generated based on the average. The control characteristic chromatograms of Xu Changqing standard decoction and formula granules (BT-R, KL-R) are shown below. Figure 13-14 As shown.

[0185] Based on the determination results of the characteristic chromatograms of 10 batches of Xu Changqing standard decoction, it is tentatively determined that the characteristic chromatogram of Xu Changqing standard decoction should present 7 characteristic peaks, among which peak 2 should be consistent with the retention time of the reference peak. The peak corresponding to the p-hydroxyacetophenone reference peak is the S peak. The relative retention times of each characteristic peak and the S peak are calculated, and their relative retention times should be within ±10% of the specified values, which are: 0.65 (peak 1), 1.40 (peak 3), 1.45 (peak 4), 1.67 (peak 5), 1.79 (peak 6), and 1.94 (peak 7). Based on the relative retention time data of 10 batches of Xu Changqing standard decoction, it is tentatively determined that the relative retention times of each characteristic peak in the characteristic chromatogram of Xu Changqing formula granules are consistent with those of the standard decoction, and the relative retention times of all 10 batches of formula granules are within the specified range.

[0186] Table 11 Results of Characteristic Spectrum Determination of 10 Batches of Standard Decoctions

[0187]

[0188] Table 12 Results of Characteristic Spectrum Determination of 10 Batches of Formulation Granules

[0189]

[0190] 7. Identification of characteristic peaks in the characteristic spectrum of Xu Changqing

[0191] Based on the liquid chromatography conditions of the characteristic spectrum of Xu Changqing standard decoction, the compounds in the test solution of Xu Changqing standard decoction were analyzed by mass spectrometry. The unknown compounds were identified by matching the spectral data with the spectral data in the local mass spectrometry database and referring to relevant literature.

[0192] 7.1 Liquid chromatography-mass spectrometry conditions

[0193] Except for mobile phase B being changed to 0.1% formic acid, the detection conditions for liquid chromatography are as detailed in "2.1 Characteristic Chromatography" for standard injection detection, and the detection conditions for mass spectrometry are as detailed in Table 13.

[0194] Table 13 Mass Spectrometry Parameters

[0195]

[0196] 7.2 Compound Identification

[0197] The test solution was analyzed using the above-mentioned liquid chromatography and mass spectrometry conditions. By comparing the retention times of the reference standard in liquid chromatography and analyzing the precise molecular weight and fragment ion composition in mass spectrometry, five components were identified in the characteristic chromatogram of Xu Changqing decoction: vanillic acid (peak 1), p-hydroxyacetophenone (peak 2), vanillinone (peak 3), isovillinone (peak 4), and 2,4-dihydroxyacetophenone (peak 6). The total ion chromatogram and ultraviolet absorption chromatogram of the test solution are shown below. Figure 15 The comparison chart between the test sample and the reference sample is shown below. Figure 16 Compound information is shown in Table 14.

[0198] Table 14 Compound Identification Results

[0199]

[0200] 8. Determination of vanillin and isovillin content

[0201] Take appropriate amounts of vanillin and isovillin reference standards, and prepare a reference solution containing 10 μg per mL with 70% methanol. Prepare test solutions using the method described in section "2.4" for 10 batches of Xu Changqing standard decoction lyophilized powder and 10 batches of Xu Changqing formula granules. Analyze the reference solutions and test solutions separately according to section "2.1". Calculate the contents of vanillin and isovillin in the 20 batches of samples using the external standard method. The results are shown in Table 15. The vanillin content range of the 10 batches of Xu Changqing standard decoction is 0.049 mg / g to 0.078 mg / g, and the isovillin content range is 0.161 mg / g to 0.233 mg / g. Combined with the extract obtained from the preparation of the standard decoction... Based on the data from 10 batches of standard decoctions and the production volume of prepared formulation granules, the provisional limits for vanillin content in Xu Changqing standard decoctions are set at 0.030 mg / g to 0.085 mg / g and isovanillin content at 0.12 mg / g to 0.27 mg / g. For Xu Changqing formulation granules, the provisional limits for vanillin content are set at 0.020 mg / g to 0.085 mg / g and isovanillin content at 0.08 mg / g to 0.30 mg / g. The vanillin content range of the 10 batches of formulation granules is 0.021 mg / g to 0.074 mg / g, and the isovanillin content range is 0.091 mg / g to 0.293 mg / g, all within the provisional ranges.

[0202] Table 15 Results of determination of vanillin and isovillin content

[0203]

[0204] Example 2

[0205] This embodiment detects the content of relevant substances in the standard solvent and formulation granules of Cynanchum paniculatum.

[0206] 1. Methodological investigation of vanillic acid content determination

[0207] 1.1 Specificity Examination

[0208] Accurately pipette 2 μL each of the Xu Changqing standard decoction test solution (BT01), reference solution, and blank solvent, and inject them for analysis under the chromatographic conditions described in section "2.2". The results show ( Figure 16 The test sample and the reference sample showed the same chromatographic peaks at the corresponding retention times, and the blank solvent did not interfere, indicating that the established method has good specificity.

[0209] 1.2 Linear Examination

[0210] Accurately weigh 2.633 mg of vanillic acid reference standard, place it in a 25 mL volumetric flask, and add methanol to the mark to obtain a vanillic acid reference standard stock solution with a concentration of 105.109 μg / mL.

[0211] Accurately measure 10 mL of the above vanillic acid reference stock solution into a 20 mL volumetric flask, add methanol to the mark, and shake well to prepare a reference solution containing 52.555 μg of vanillic acid per mL. Accurately measure 5 mL, 2 mL, and 1 mL of the above 52.555 μg / mL reference solution into 10 mL volumetric flasks, add methanol to the mark, and shake well to prepare reference solutions containing 26.277 μg, 10.511 μg, and 5.255 μg of vanillic acid per mL, respectively. Accurately measure 1 mL of the above 10.511 μg / mL reference solution into a 10 mL volumetric flask, add methanol to the mark, and shake well to prepare a reference solution containing 1.051 μg of vanillic acid per mL. Accurately pipette 2 μL of each of the above reference solutions and reference stock solutions, and inject them sequentially under the chromatographic conditions described in section "2.2". Record the peak areas. Plot the peak area on the ordinate (y) and the standard concentration on the abscissa (x), as shown in Table 16, and then plot the standard curve. Figure 17 The results showed that the linear regression equation was y = 17950.91x + 9703.32, with a correlation coefficient r = 0.9997, indicating a good linear relationship between vanillic acid concentration and peak area within the concentration range of 1.051 μg / mL to 105.109 μg / mL.

[0212] Table 16 Results of Linearity Study

[0213]

[0214] 1.3 Accuracy Assessment

[0215] Accurately weigh 2.633 mg of vanillic acid reference standard, place it in a 100 mL volumetric flask, add methanol to prepare a solution containing 26.277 μg per mL, shake well, and the vanillic acid reference standard stock solution is obtained for later use.

[0216] Accurately pipette 1 mL of the above stock solution into six parallel portions, evaporate the solvent to dryness, and then add approximately 0.25 g of the pre-determined content of Xu Changqing standard decoction (BT01) to each portion, accurately weighing the total amount. Prepare the test solution according to the method specified in section "2.4". Determine the recovery rate under the chromatographic conditions in section "2.2", and calculate the recovery rate. The results are shown in Table 17. The experimental results show that the vanillic acid recovery rate ranges from 91.34% to 97.49%, with an average recovery rate of 95.11% and an RSD of 2.64%, indicating good recovery.

[0217] Table 17 Results of Vanillic Acid Recovery Test

[0218]

[0219] 1.4 Column robustness test

[0220] To compare the effects of different types of chromatographic columns on the determination of vanillic acid content in Xu Changqing standard decoction, three columns were investigated: Waters BEH C18 (2.1×100mm, 1.7μm), YMC Triart C18 (2.1×100mm, 1.9μm), and Thermo Syncronis aQ (2.1×100mm, 1.7μm). The test solution of Xu Changqing standard decoction and the vanillic acid reference solution were accurately pipetted, and the vanillic acid content was determined according to the chromatographic conditions in section "2.2". The results (Table 18) show that the resolution and purity of vanillic acid met the quantitative requirements for all brands of columns. The RSD value of vanillic acid content determined using the three columns was 1.48%, indicating that the analytical method has good robustness with different brands of columns.

[0221] Table 18 Results of Column Robustness Study

[0222]

[0223] 1.5 Flow Rate Durability Test

[0224] The effects of different flow rates (0.28 mL / min, 0.30 mL / min, and 0.32 mL / min) on the determination of vanillic acid content in Xu Changqing standard decoction were compared. The test solution and vanillic acid reference solution of Xu Changqing standard decoction were accurately pipetted, and the vanillic acid content was determined according to the chromatographic conditions in section "2.2". The results (Table 19) show that the RSD value of vanillic acid content was 0.46% when the flow rate was between 0.28 mL / min and 0.32 mL / min, indicating that the analytical method has good robustness within the flow rate range of 0.28 mL / min to 0.32 mL / min.

[0225] Table 19 Results of Flow Velocity Durability Test

[0226]

[0227] 1.6 Column Temperature Durability Test

[0228] The effects of different column temperatures (28℃, 30℃, and 32℃) on the determination of vanillic acid content in Xu Changqing standard decoction were compared. The test solution and vanillic acid reference solution of Xu Changqing standard decoction were accurately pipetted, and the vanillic acid content was determined according to the chromatographic conditions in section "2.2". The results (Table 20) show that the RSD value of vanillic acid content was 0.35% when the column temperature was between 28℃ and 32℃, indicating that the analytical method has good robustness within the flow rate range of 28℃ to 32℃.

[0229] Table 20 Results of Column Temperature Durability Test

[0230]

[0231] 1.7 Vanillic acid content determination in samples

[0232] Ten batches of freeze-dried powder of Xu Changqing standard decoction and ten batches of Xu Changqing formula granules were prepared into test solutions according to the method in section "2.4". The content was determined and analyzed according to section "2.2". The vanillic acid content of the 20 batches of samples was calculated. The results are shown in Table 21. The vanillic acid content of the ten batches of Xu Changqing standard decoction ranged from 0.075 mg / g to 0.125 mg / g. Based on the yield of the prepared standard decoction, the content limit of Xu Changqing standard decoction was tentatively set at 0.06 mg / g to 0.15 mg / g. Based on the data of the ten batches of standard decoction and the yield of the prepared formula granules, the content limit of Xu Changqing formula granules was tentatively set at 0.04 mg / g to 0.13 mg / g. The vanillic acid content of the ten batches of formula granules ranged from 0.060 mg / g to 0.108 mg / g, all of which were within the tentative range.

[0233] Table 21 Results of Vanillic Acid Content Determination

[0234]

[0235] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0236] The embodiments described above merely illustrate several implementation methods of this application and should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Furthermore, it should be understood that after reading the above teachings, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for constructing the characteristic spectrum of Xu Changqing standard decoction or formula granules, characterized in that, Includes the following steps: The test sample and extraction solvent are mixed and subjected to ultrasonic extraction or hot reflux extraction to obtain the test sample solution. The test solution was analyzed by high performance liquid chromatography. The conditions for the high performance liquid chromatography analysis include: gradient elution using acetonitrile as mobile phase A and an aqueous solution of acid with a volume concentration of 0.1%-0.2% as mobile phase B. The gradient elution program includes: 0-30 min, during which the volume percentage of mobile phase A increases from 7% to 14%.

2. The method for constructing the feature map according to claim 1, characterized in that, The conditions for the high-performance liquid chromatography analysis also include at least one of the following characteristics: (1) The chromatographic column is packed with octadecylsilane-bonded silica gel. (2) The column temperature is 25℃-40℃. (3) The detection wavelength is 260nm-290nm. (4) The flow rate of the mobile phase is 0.3mL / min-0.35mL / min. (5) The injection volume is 1μL-2μL. (6) The acid includes one or more of phosphoric acid, formic acid and acetic acid.

3. The method for constructing a feature map according to claim 2, characterized in that, The chromatographic column has the following specifications: length 150 mm, inner diameter 2.1 mm, and packing material particle size 1.6 μm-2.2 μm.

4. The method for constructing a feature map according to any one of claims 1-3, characterized in that, The extraction solvent includes an aqueous solution of alcohol with a volume concentration of 50%-70%; Optionally, the alcohol includes one or more of methanol and ethanol.

5. The method for constructing a feature map according to any one of claims 1-3, characterized in that, The conditions for ultrasonic extraction include: ultrasonic power of 200W-500W, frequency of 30kHz-50kHz, and ultrasonic time of 15min-45min; and / or, The conditions for hot reflux extraction include: a heating temperature of 80℃-90℃ and a heating time of 15min-45min.

6. The method for constructing a feature map according to any one of claims 1-3, characterized in that, The weight-to-volume ratio of the test sample to the extraction solvent is 0.4-0.6 g: 25 mL.

7. The method for constructing a feature map according to any one of claims 1-3, characterized in that, The preparation method of the Xu Changqing standard decoction includes the following steps: taking Xu Changqing medicinal material, soaking it in water, adding first water for a first decoction, the weight ratio of the first water to the Xu Changqing medicinal material being (10~12):1, filtering to obtain a first filtrate and a first residue, adding second water to the first residue for a second decoction, the weight ratio of the second water to the Xu Changqing medicinal material being (8~10):1, filtering to obtain a second filtrate, and combining the first filtrate and the second filtrate; and / or, The preparation method of the Xu Changqing formula granules includes the following steps: taking Xu Changqing medicinal material, adding water for steam distillation, and collecting paeonol; adding a third water to the residue after steam distillation for a third decoction, the mass ratio of the third water to the residue after steam distillation being (8-10):1, filtering to obtain a third filtrate and a third filter residue, adding a fourth water to the third filter residue for a fourth decoction, the mass ratio of the fourth water to the third residue being (6-8):1, filtering to obtain a fourth filtrate, combining the third filtrate and the fourth filtrate, concentrating to obtain a clear extract; adding a first excipient to the clear extract and mixing, drying to obtain a dry extract powder; mixing the paeonol, the dry extract powder, and the second excipient, and granulating.

8. The method for constructing a feature map according to any one of claims 1-3, characterized in that, It also includes the following steps: A reference solution is prepared by mixing a reference substance with an organic solvent; the reference substance includes one or more of vanillic acid, p-hydroxyacetophenone, vanillin, isovillin, and 2,4-dihydroxyacetophenone. The reference solution was analyzed by high-performance liquid chromatography. Optionally, the mass-to-volume ratio of vanillic acid, p-hydroxyacetophenone, vanillinone, isovanillinone, 2,4-dihydroxyacetophenone, and the organic solvent is independently selected from 8-12 μg:1 mL.

9. The method for constructing a feature map according to any one of claims 1-3, characterized in that, The constructed characteristic spectrum includes 7 common peaks: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, and peak 7; among which, peak 1 is vanillic acid, peak 2 is p-hydroxyacetophenone, peak 3 is vanillin, peak 4 is isovanillin, and peak 6 is 2,4-dihydroxyacetophenone. Furthermore, taking peak 2 as a reference, the characteristic peaks and their relative retention times are as follows: peak 1: 0.65±10%, peak 3: 1.40±10%, peak 4: 1.45±10%, peak 5: 1.67±10%, peak 6: 1.79±10%, peak 7: 1.94±10%.

10. A method for detecting Xu Changqing standard decoction or formula granules, characterized in that, Includes the following steps: A reference solution is prepared by mixing a reference substance with an organic solvent; the reference substance includes one or more of vanillic acid, p-hydroxyacetophenone, vanillin, isovillin, and 2,4-dihydroxyacetophenone. The sample to be tested and the extraction solvent are mixed and subjected to ultrasonic extraction or hot reflux extraction to obtain the sample solution. The reference solution and the test solution were analyzed by high performance liquid chromatography. The conditions for the high performance liquid chromatography analysis include: gradient elution using acetonitrile as mobile phase A and an aqueous solution of acid with a volume concentration of 0.1%-0.2% as mobile phase B. The gradient elution program includes: 0-30 min, during which the volume percentage of mobile phase A increases from 7% to 14%.

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