Methods and applications for identifying and counterfeiting of standard decoctions of Dryopteris crassirhizoma and its products.

Abstract

This invention relates to a method and application for identifying standard decoctions of Dryopteris crassirhizoma and its adulterants. The method includes the following steps: extracting standard decoctions of Dryopteris crassirhizoma and its adulterants using extraction solvents to prepare test solutions; dissolving a reference standard of oxaliplatin AB using a dissolving reagent to prepare a reference solution; and performing high-performance liquid chromatography (HPLC) on the test solution and the reference solution to obtain the content of oxaliplatin AB in the standard decoction of Dryopteris crassirhizoma. This invention establishes a method for determining the content of oxaliplatin AB, a characteristic component of the standard decoction of Dryopteris crassirhizoma, thereby enabling qualitative and quantitative identification of the standard decoction, ensuring its safe use, and for quality control of the standard decoction. The establishment of this method not only provides a basis for establishing quality standards for the standard decoction of Dryopteris crassirhizoma, but also provides an important reference for establishing quality standards for Dryopteris crassirhizoma granules and related traditional Chinese medicine preparations.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of drug analysis, in particular to a standard decoction of dryopteris crassirhizoma and a method for identifying counterfeit products thereof and application. BACKGROUND

[0002] The standard decoction of traditional Chinese medicine slices is prepared by referring to modern extraction methods and using a standard process under the guidance of traditional Chinese medicine theory and based on clinical application. As a standard substance and a standard system, the standard decoction of traditional Chinese medicine slices can be used for standardizing clinical medication, standardizing the new slice form including formula granules, and guaranteeing the accuracy and consistency of the dose. Therefore, by using modern analysis techniques, the quality standard of the standard decoction of traditional Chinese medicine slices can provide an important reference for the establishment of the quality standard of corresponding formula granules and classical famous prescriptions and other modern traditional Chinese medicine preparations.

[0003] Dryopteris crassirhizoma is the dry rhizome and petiole residue of Dryopteris crassirhizoma Nakai. Dryopteris crassirhizoma contains phloroglucinols, flavonoids, terpenes and phenylpropanoids, and has the effects of clearing heat and resolving toxins and expelling parasites. Modern studies have shown that phloroglucinols are the main active ingredients of dryopteris crassirhizoma for antiviral, antibacterial, antitumor and anthelmintic pharmacological activities. As a commonly used antiviral drug in clinical practice, dryopteris crassirhizoma has played an important role in fighting against the new coronavirus infection. In recent years, due to the increasing demand for dryopteris crassirhizoma, counterfeit products have frequently appeared in the market, such as Osmunda japonica, small dryopteris crassirhizoma, Dryopteris crassirhizoma, and rhizome of Bolbitis. Different efficacy will affect the safety of dryopteris crassirhizoma. However, there are few studies on the quality standard of dryopteris crassirhizoma, which cannot guarantee the safety and effectiveness of its clinical use.

[0004] The 2020 edition of Chinese Pharmacopoeia only records the appearance, microscopic and thin-layer identification methods of dryopteris crassirhizoma. The provincial standard of traditional Chinese medicine does not collect dryopteris crassirhizoma, and only collects its identification methods including appearance identification and microscopic identification in the standard of some traditional Chinese medicine slices. There is no content determination index. In the quality standard research of provincial formula granules of traditional Chinese medicine, total flavonoids and chlorogenic acid are selected as content determination indexes, which lack specificity. In the current literature research, the quality control methods of dryopteris crassirhizoma include determination of total flavonoids content by ultraviolet method and determination of total phloroglucinol content by ultraviolet method. However, the determination of total flavonoids and total phloroglucinols as content determination indexes lacks specificity and accuracy. There is no literature report on the content determination method of the effective component of dryopteris crassirhizoma, i.e. yellow dryopteris acid AB.

[0005] Pteridium aquilinum has rich resources, and there are many Pteridium aquilinum varieties in different regions. In recent years, Pteridium aquilinum has played a significant role in resisting viruses (such as new coronavirus, influenza virus and respiratory tract related viruses), which has increased the demand for Pteridium aquilinum. However, counterfeit products frequently appear in the market. After investigation, it is found that common counterfeit products such as Pteridium aquilinum, Pteridium aquilinum, Pteridium aquilinum, Pteridium aquilinum and rhizome of Pteridium aquilinum have different effects on the efficacy of Pteridium aquilinum, which affects the safety of Pteridium aquilinum. It is difficult to identify the authenticity of the standard decoction pieces of Pteridium aquilinum based on the appearance of the medicinal materials. Therefore, it is necessary to establish a content determination method for the characteristic components of the standard decoction pieces of Pteridium aquilinum, so as to qualitatively and quantitatively identify the standard decoction pieces of Pteridium aquilinum and ensure the safety of Pteridium aquilinum. SUMMARY

[0006] Therefore, it is necessary to provide a method for identifying Pteridium aquilinum and its counterfeit products and application, which establishes a content determination method for the characteristic component of Pteridium aquilinum, i.e., yellow Pteridium acid AB, so as to qualitatively and quantitatively identify Pteridium aquilinum and ensure the safety of Pteridium aquilinum.

[0007] The technical solution of the present application is as follows:

[0008] In one aspect of the present application, a method for identifying Pteridium aquilinum and its counterfeit products is provided, which comprises the following steps:

[0009] Providing standard decoctions of Pteridium aquilinum and its counterfeit products, respectively;

[0010] Extracting the standard decoctions of Pteridium aquilinum and its counterfeit products using extraction solvents to prepare sample solutions;

[0011] Dissolving the yellow Pteridium acid AB reference substance using a dissolving reagent to prepare a reference substance solution;

[0012] Performing high performance liquid chromatography detection on the sample solution and the reference substance solution to obtain the content of yellow Pteridium acid AB in the standard decoction pieces of Pteridium aquilinum;

[0013] The conditions for high performance liquid chromatography detection include: the mobile phase A is methanol, the mobile phase B is phosphoric acid aqueous solution, and isocratic elution is adopted; the volume ratio of the mobile phase A and the mobile phase B is (70-85):(30-15).

[0014] In one embodiment, the high performance liquid chromatography detection meets at least one of the following conditions:

[0015] (1) The detection wavelength is 210 nm-400 nm:

[0016] (2) The column temperature is 20℃-30℃:

[0017] (3) The flow rate is 0.5 mL·min-1 ~1.5 mL·min -1 ;

[0018] (4) The injection volume is 5 μL to 15 μL.

[0019] In one embodiment, the volume percentage of phosphoric acid in the aqueous phosphoric acid solution is 0.1% to 0.5%.

[0020] In one embodiment, the extraction step satisfies at least one of the following conditions:

[0021] (1) The solvent for extraction is an alcohol or an aqueous solution of an alcohol; optionally, the alcohol is methanol or ethanol; the volume percentage of the alcohol in the aqueous solution is 50% to 99%.

[0022] (2) The extraction methods are ultrasonic extraction, reflux extraction or extraction.

[0023] (3) The extraction time is 15 min to 60 min;

[0024] (4) The mass-volume ratio of the standard decoction and extraction solvent of the *Dryopteris crassirhizoma* slices and their adulterants is 0.5 g: (15-50) mL.

[0025] In one embodiment, obtaining the content of oxalis acid AB in the standard decoction of *Dryopteris crassirhizoma* slices includes the following steps:

[0026] The reference standard was dissolved using a dissolving reagent to prepare reference standard solutions of different concentrations;

[0027] The reference standard solutions of different concentrations were injected separately and detected by high performance liquid chromatography.

[0028] The peak areas corresponding to oxaliplatin A and B in the high performance liquid chromatography (HPLC) spectra of the reference solutions at different concentrations were obtained. A standard curve was plotted with the concentration of the reference solution as the x-axis and the peak areas corresponding to oxaliplatin A and B in the reference solutions at different concentrations as the y-axis. The linear regression equation was then calculated.

[0029] The content of oxalic acid AB in the test sample solution is calculated by substituting the peak area corresponding to oxalic acid AB in the high performance liquid chromatography (HPLC) chromatogram obtained by HPLC detection of the test sample solution into the linear regression equation.

[0030] The reference standard includes oxaliplatin AB.

[0031] In one embodiment, the volume ratio of mobile phase A to mobile phase B is 80:20.

[0032] In one of the embodiments, the dissolving reagent is methanol.

[0033] In one of the embodiments, the content of the flavonoid AB in the standard decoction of the dry piece of Drynariae Rhizoma is 0.100 mg / g-0.750 mg / g.

[0034] In one of the embodiments, the counterfeit products include one or more of Drynaria propinquua, Drynaria japonica, Drynaria fortunei, Drynaria suffruticosa, Drynaria brownii, Drynaria barbata, Drynaria fortunei var. purpurea, Drynaria barbata var. apiculata and Drynaria barbata var. gemmifera.

[0035] In another aspect of the present application, the application provides the use of the method for identifying the standard decoction of the dry piece of Drynariae Rhizoma and its counterfeit products in the quality control of the dry piece of Drynariae Rhizoma.

[0036] Compared with the prior art, the present application has the following beneficial effects:

[0037] The present application establishes a content determination method for the characteristic component flavonoid AB in the standard decoction of the dry piece of Drynariae Rhizoma, i.e. a HPLC content determination method for the active component flavonoid AB in the standard decoction of the dry piece of Drynariae Rhizoma is established by using specific high performance liquid chromatography detection conditions for the standard decoction of the dry piece of Drynariae Rhizoma, so that the standard decoction of the dry piece of Drynariae Rhizoma can be identified qualitatively and quantitatively, the safety of the medication is ensured, and the quality control of the standard decoction of the dry piece of Drynariae Rhizoma is used. At the same time, the method is verified, and the specificity, peak purity, linear relationship, precision, repeatability, stability, accuracy and durability are good. The establishment of the method not only provides a basis for the establishment of the quality standard of the standard decoction of the dry piece of Drynariae Rhizoma, but also provides an important reference for the establishment of the quality standard of Drynariae Rhizoma Granules and related traditional Chinese medicine preparations. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the specific embodiments or the prior art, the drawings needed in the following specific embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creating labor.

[0039] Figure 1 It is the UV absorption curve graph of the standard product of flavonoid AB in Example 1.

[0040] Figure 2 It is the comparative chromatogram of the content determination of flavonoid AB in the standard decoction of the dry piece of Drynariae Rhizoma in Example 1 in different organic phase elution.

[0041] Figure 3Comparative chromatogram of different kinds of acid elution for determination of content of Huangma acid AB in standard decoction of Radix Sieboldi in Example 1.

[0042] Figure 4 Comparative chromatogram of different concentrations of acid elution for determination of content of Huangma acid AB in standard decoction of Radix Sieboldi in Example 1.

[0043] Figure 5 Comparative chromatogram of different proportions of mobile phase isocratic elution for determination of content of Huangma acid AB in standard decoction of Radix Sieboldi in Example 1.

[0044] Figure 6 Comparative chromatogram of different column temperatures for determination of content of Huangma acid AB in standard decoction of Radix Sieboldi in Example 1.

[0045] Figure 7 Specificity investigation chromatogram for determination of content of Huangma acid AB in standard decoction of Radix Sieboldi in Example 1.

[0046] Figure 8 Peak purity investigation chromatogram for determination of content of Huangma acid AB in standard decoction of Radix Sieboldi in Example 1.

[0047] Figure 9 Standard curve of Huangma acid AB in Example 1. DETAILED DESCRIPTION

[0048] The present application can be implemented in numerous ways, and is not limited to the embodiments described herein. Rather, the embodiments are provided as examples of implementing the application to enable those skilled in the art to practice the application without undue experimentation.

[0049] 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 in the description herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0050] Unless otherwise indicated, or unless the context clearly indicates otherwise, the terms or phrases used in this document have the following meanings:

[0051] As used herein, "one or more" means any one, any two, or any two or more of the recited items.

[0052] In this document, the optional scope of "and / or", "or / and", "and / or" includes any one of two or more related listed items, and also includes any and all combinations of related listed items, including any two related listed items, any more related listed items, or a combination of all related listed items. (It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it is understood that in this application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0053] In this document, "further", "furthermore", "in particular" and the like are used to describe purposes, indicating differences in content, but should not be understood as limiting the scope of protection of the application.

[0054] In this application, if no special instructions are given, the numerical range is considered to be continuous and includes the minimum value and the maximum value of the range, and every value between the minimum value and the maximum value. Further, when the range refers to an integer, every integer between the minimum value and the maximum value of the range is included. In addition, when multiple ranges are provided to describe a feature or a characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0055] In this application, the percentage content involved, if no special instructions are given, refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0056] In this application, the percentage concentration involved, if no special instructions are given, refers to the final concentration. The final concentration refers to the proportion of the added ingredient in the system after the ingredient is added.

[0057] The temperature parameters in the present application, if not particularly limited, allow for constant temperature treatment, and also allow for treatment within a certain temperature range. The constant temperature treatment allows for fluctuations within the accuracy range controlled by the instrument. Fluctuations within a range such as ±5℃, ±2℃, ±1℃, ±0.5℃, ±0.4℃, ±0.3℃, ±0.2℃, ±0.1℃ are allowed. Normal temperature or room temperature in the present application refers to no temperature control operation, and generally refers to 4-35℃, preferably 20±5℃.

[0058] In the present application, the technical features described in an open manner include both closed technical solutions consisting of listed features, and open technical solutions containing listed features.

[0059] The quantitative analysis of certain active ingredient or active part in traditional Chinese medicinal materials by using appropriate chemical methods or instrumental analysis methods, and the determination of whether the test results meet the provisions of the drug standard to judge the quality of the drug, is an important method for controlling and evaluating the quality of the drug. According to the investigation, the current standard of Aspidium hearing in the Pharmacopoeia does not regulate the content index representing the quality, only the appearance, microscopic identification and thin layer identification are regulated, and the quality standard is still not perfect. There are few reports on the research of the standard decoction quality standard of Aspidium hearing slices. Since the main active ingredient of Aspidium hearing, resorcylic acid, has small polarity, it is difficult to establish a chromatographic separation method, so most of the quality standard research of Aspidium hearing medicinal materials and formula granules selects total flavonoids and chlorogenic acid as the content determination index component, which lacks specificity.

[0060] Huangma acid AB is a resorcylic acid active ingredient in Aspidium hearing slices, and its chemical formula is C 22 H 26 O8, and the structure is as follows:

[0061]

[0062] In view of the deficiencies of the existing quality control index components of Aspidium hearing, such as lack of specificity, inability to identify counterfeit products, and lack of accuracy, the technical personnel of the present application have established an HPLC content determination method of resorcylic acid active ingredient Huangma acid AB in Aspidium hearing slice standard decoction, which is used for quality control of Aspidium hearing slice standard decoction. The establishment of the content determination method not only provides a basis for the establishment of the quality standard of Aspidium hearing slice standard decoction, but also provides an important reference for the establishment of the quality standard of Aspidium hearing formula granules and related traditional Chinese medicine preparations.

[0063] In one aspect of the present application, a method for determining the content of Huangma acid AB in Aspidium hearing slice standard decoction is provided, comprising the following steps:

[0064] Standard decoctions of Aspidium hearing slices and their counterfeit products are provided respectively;

[0065] The standard decoction of the Pteroxygonum giraldii drinking pieces and its counterfeit is extracted by using the extraction solvent respectively to prepare the test solution;

[0066] The reference substance solution is prepared by dissolving the Pterygospermin AB reference substance by using the dissolving reagent;

[0067] The test solution and the reference substance solution are detected by high performance liquid chromatography to obtain the content of Pterygospermin AB in the Pteroxygonum giraldii drinking pieces standard decoction;

[0068] The conditions of the high performance liquid chromatography detection include: the mobile phase A is methanol, the mobile phase B is phosphoric acid aqueous solution, and isocratic elution is adopted; the volume ratio of the mobile phase A and the mobile phase B is (70-85):(30-15). Understandably, the volume ratio of the mobile phase A and the mobile phase B includes but is not limited to 70:30, 75:25, 80:20, 85:15.

[0069] In some examples, the conditions of the high performance liquid chromatography detection include: the detection wavelength is 210nm-400nm. Understandably, the detection wavelength includes but is not limited to 210nm, 250nm, 300nm, 350nm, 400nm.

[0070] In some examples, the conditions of the high performance liquid chromatography detection include: the column temperature is 20℃-30℃. Understandably, the column temperature includes but is not limited to 20℃, 22℃, 24℃, 26℃, 28℃, 30℃.

[0071] In some examples, the conditions of the high performance liquid chromatography detection include: the flow rate is 0.5mL·min -1 -1.5mL·min -1 . Understandably, the flow rate includes but is not limited to 0.5mL·min -1 , 0.75mL·min -1 , 1.0mL·min -1 , 1.25mL·min -1 , 1.5mL·min -1 .

[0072] In some examples, the conditions of the high performance liquid chromatography detection include: the injection volume is 5μL-15μL. Understandably, the injection volume includes but is not limited to 5μL, 7μL, 9μL, 11μL, 13μL, 15μL.

[0073] In some examples, in the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.1%-0.5%. Understandably, in the phosphoric acid aqueous solution, the volume percentage of phosphoric acid includes but is not limited to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%.

[0074] In some examples, the conditions of the extracting step include that the solvent for extraction is an alcohol or an aqueous alcohol solution.

[0075] In some examples, the conditions of the extracting step include that the alcohol is methanol or ethanol.

[0076] In some examples, the aqueous alcohol solution has a volume percentage of alcohol ranging from 50% to 99%. It is understood that the volume percentage of alcohol in the aqueous alcohol solution includes but is not limited to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%.

[0077] In some examples, the conditions of the extracting step include that the extraction method is ultrasonic extraction, reflux extraction, or extraction extraction.

[0078] In some examples, the conditions of the extracting step include that the extraction time ranges from 15 minutes to 60 minutes. It is understood that the extraction time includes but is not limited to 15 minutes, 30 minutes, 45 minutes, and 60 minutes.

[0079] In some examples, the conditions of the extracting step include that the mass-to-volume ratio of the standard decoction of the dried rhizome of Aspidium and its counterfeit to the extraction solvent ranges from 0.5g:(15-50)mL. It is understood that the mass-to-volume ratio of the standard decoction of the dried rhizome of Aspidium and its counterfeit to the extraction solvent includes but is not limited to 0.5g:15mL, 0.5g:20mL, 0.5g:30mL, 0.5g:40mL, and 0.5g:50mL.

[0080] In some examples, obtaining the content of xanthohumol AB in the standard decoction of the dried rhizome of Aspidium includes the following steps:

[0081] Dissolve the control sample using a dissolving reagent to prepare control sample solutions of different concentrations;

[0082] Respectively sample the control sample solutions of different concentrations for high-performance liquid chromatography detection;

[0083] Obtain the peak areas of xanthohumol AB in the high-performance liquid chromatography detection spectrum of the control sample solutions of different concentrations, draw a standard curve with the concentrations of the control sample solutions as the abscissa and the peak areas of xanthohumol AB in the control sample solutions of different concentrations as the ordinate, and calculate the linear regression equation;

[0084] The peak area corresponding to the yellow Ma acid AB in the high performance liquid chromatography detection spectrum obtained by detecting the test product solution by high performance liquid chromatography is substituted into the linear regression equation to calculate the content of the yellow Ma acid AB in the test product solution;

[0085] The control product includes the yellow Ma acid AB.

[0086] In some examples, the volume ratio of the mobile phase A and the mobile phase B is 80:20.

[0087] In some examples, the dissolving reagent is methanol.

[0088] In some examples, the content of the yellow Ma acid AB in the standard decoction of the Pteridium aquilinum L. var. latiusculum (Desv.) Underw. is 0.100 mg / g-0.750 mg / g.

[0089] In some examples, the pseudo-product includes one or more of the following: Monachosorum pubescens, Drynaria fortunei, Pteridium latiusculum, Cycas revoluta, Leptoleonia brownii, Pteridium aquilinum, Osmunda japonica, Pteridium aquilinum var. pubescens and Pteridium aquilinum var. esculentum.

[0090] In another aspect of the present application, the application provides the use of the standard decoction of the Pteridium aquilinum L. var. latiusculum (Desv.) Underw. and the identification method of the pseudo-product thereof in the quality control of the Pteridium aquilinum L. var. latiusculum (Desv.) Underw.

[0091] The following will be further described in combination with specific examples, and the raw materials involved in the following specific examples, if not specifically stated, can be sourced from the market; the instruments used, if not specifically stated, can be sourced from the market; the processes involved, if not specifically stated, are conventionally selected by those skilled in the art.

[0092] The following are specific examples.

[0093] Example 1

[0094] The present example provides a construction of a HPLC determination method of the content of the yellow Ma acid AB in the standard decoction of the Pteridium aquilinum L. var. latiusculum (Desv.) Underw. and the identification application of the pseudo-product thereof, which is specifically as follows:

[0095] 1. Instruments, reagents and reagents

[0096] Waters high performance liquid chromatograph (Arc, Waters Corporation), Agilent high performance liquid chromatograph (1260, Agilent), Waters XSelect HSS T3 chromatographic column (4.6 mm x 250 mm, 5 μm), Kromasil 100-5-C18 chromatographic column (4.6 mm x 250 mm, 5 μm), Agilent ZORBAX Extend-C18 chromatographic column (4.6 mm x 250 mm, 5 μm), numerical control ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instruments Co., Ltd.), one-hundredth scale (ME204E, Mettler-Toledo), one-millionth electronic analytical balance (XP26, Mettler-Toledo), rotary evaporator (RE-3000, Shanghai Yalong Biochemical Instrument Factory), ultrapure water system (Milli-Q Direct, Merck), ethanol, methanol (Tianjin Fuyu Fine Chemical Co., Ltd.), petroleum ether 60-90℃ (Tianjin Baishi Chemical Co., Ltd.), all of which are analytical pure; acetonitrile, methanol (Merck), phosphoric acid (Tianjin Kemio Chemical Reagent Co., Ltd.), all of which are chromatographic pure; ultrapure water (laboratory self-made); Picroside AB reference substance (laboratory self-made).

[0097] 2. Preparation of Pteroxygonum graveolens slice

[0098] Pteroxygonum graveolens slice was prepared according to the processing method in Chinese Pharmacopoeia 2020 edition. The specific processing method is as follows: remove impurities, spray with clean water, wash, soak, cut into thick slices, dry, and sieve off the ash, to obtain the product.

[0099] 3. Preparation of standard decoction of Pteroxygonum graveolens slice

[0100] Take 100 g of Pteroxygonum graveolens slice, add water and decoct twice. For the first decoction, add 9 times the amount of water, soak for 30 minutes, then boil with strong fire (500W) and keep the temperature at a low boil with gentle fire (200W) for 30 minutes. Filter the decoction with a 350-mesh sieve while it is hot, and quickly cool the filtrate with cold water. For the second decoction, add 7 times the amount of water, boil with strong fire (500W), then keep the temperature at a low boil with gentle fire (200W) for 25 minutes. Filter the decoction with a 350-mesh sieve while it is hot, and quickly cool the filtrate with cold water. Combine the filtrates of the two decoctions. Transfer the decoction to a 2000-mL round-bottom flask, and concentrate it under reduced pressure and low temperature (temperature: 65℃; vacuum degree: -0.08 to -0.1 MPa) to 100 mL. Under magnetic stirring, divide the decoction into 10-mL vials, with each vial containing 2 mL of the decoction. After the division is complete, transfer the vials to a vacuum freeze-drying machine for freeze-drying. Remove the vials, and replace the caps with aluminum caps. The standard decoction freeze-dried powder is obtained. The batch numbers of the standard decoction freeze-dried powder prepared from Pteroxygonum graveolens slices from 15 different production areas are shown in Table 1.

[0101] Table 1 Batch numbers of standard decoction freeze-dried powder of Pteroxygonum graveolens slice from 15 different production areas

[0102]

[0103] 4. Establishment of determination method of flavonoids in Huangsimacordata

[0104] 4.1 Preparation of reference solution

[0105] Accurately weigh 2.353 mg of Huangsimacordata AB reference substance into a 50 mL volumetric flask, add methanol to make a reference solution containing 44.547 μg per 1 mL.

[0106] 4.2 Optimization of chromatographic conditions

[0107] 4.2.1 Selection of detection wavelength

[0108] Full-wave scanning was performed on the Huangsimacordata AB reference solution, and the absorption spectrum of the reference substance in the range of 210 nm to 400 nm was recorded, and the results are shown in Figure 1 .

[0109] The results showed that Huangsimacordata AB had a maximum absorption wavelength at 298 nm.

[0110] 4.2.2 Selection of mobile phase

[0111] The effects of different organic phases (acetonitrile, methanol, acetonitrile:methanol-1:1) on the separation degree of chromatographic peaks and baseline were investigated, and the results are shown in Figure 2 .

[0112] The results showed that when methanol was selected as the elution organic phase, the baseline of the chromatogram was smooth and the separation effect of Huangsimacordata AB chromatographic peaks was better, so methanol was selected as the final elution organic phase.

[0113] 4.2.3 Selection of acid type

[0114] The effects of different acid aqueous solutions (phosphoric acid, formic acid, acetic acid) on the separation degree of chromatographic peaks and baseline were investigated, and the results are shown in Figure 3 .

[0115] The results showed that when phosphoric acid aqueous solution was selected as the elution water phase, the baseline of the chromatogram was smooth and the separation effect of Huangsimacordata AB chromatographic peaks was better, so phosphoric acid was selected as the acid added in the final water phase.

[0116] 4.2.4 Selection of acid concentration

[0117] The effects of different concentrations of phosphoric acid aqueous solution (0.1%, 0.2%, 0.5%) on the separation degree of chromatographic peaks and baseline were investigated, and the results are shown in Figure 4 .

[0118] The results show that when the water phase is eluted by 0.2% and 0.5% phosphoric acid aqueous solution, the baseline of the chromatogram is stable and the separation effect of the chromatographic peak of Huangma Acid AB is better. Since the pH value of 0.5% phosphoric acid aqueous solution is lower, it has a greater impact on the performance of the chromatographic column, therefore, 0.2% is selected as the final acid concentration in the water phase.

[0119] 4.2.5 Selection of the proportion of the mobile phase

[0120] The effects of isocratic elution of different proportions of the mobile phase (organic phase: water phase-70:30, organic phase: water phase-75:25, organic phase: water phase-80:20, organic phase: water phase-85:15) on the separation degree of the chromatographic peak and the baseline are investigated, wherein the organic phase is methanol and the water phase is 0.2% phosphoric acid aqueous solution, and the results are shown in Table 4.2.5. Figure 5 .

[0121] The results show that when the organic phase: water phase-80:20 is selected for elution, the baseline of the chromatogram is stable and the separation effect of the chromatographic peak of Huangma Acid AB is better, therefore, isocratic elution of the organic phase: water phase 80:20 is selected.

[0122] 4.2.6 Selection of the column temperature

[0123] The effects of different column temperatures (20℃, 25℃, 30℃, 35℃, 40℃) on the separation degree of the chromatographic peak and the baseline are investigated, and the results are shown in Table 4.2.6. Figure 6 .

[0124] The results show that when the column temperature is 20℃-30℃, the peak shape of the chromatographic peak of Huangma Acid AB is better and the separation effect is better, therefore, 30℃ is selected as the final column temperature.

[0125] 4.2.7 Determination of the chromatographic conditions

[0126] According to the above experimental results, the high performance liquid chromatography conditions for determining the content of Huangma Acid AB in the standard decoction of Radix Wilsoniae Pteridis are determined as follows:

[0127] The chromatographic column is filled with octadecylsilane-bonded silica gel (4.6mm x 250mm, 5μm), the mobile phase is methanol-0.2% phosphoric acid aqueous solution (80:20), the volume flow rate is 1.0mL·min -1 , the detection wavelength is 298nm, the column temperature is 30℃, and the injection volume is 10μL.

[0128] 4.3 Investigation of the processing conditions of the test sample

[0129] 4.3.1 Investigation of the types of extraction solvents

[0130] Take the standard decoction of Radix Asarum, grind it finely, take about 0.5g, and use methanol solution, 70% methanol aqueous solution, 50% methanol aqueous solution, 95% ethanol aqueous solution, 70% ethanol aqueous solution, and 50% ethanol aqueous solution as the extraction solvent, respectively, to investigate the type of extraction solvent for the same batch of test samples. The results show that the content of xanthohumol AB measured by methanol as the extraction solvent is the highest, and methanol is finally selected as the extraction solvent for the determination of the content of xanthohumol AB in the standard decoction of Radix Asarum.

[0131] 4.3.2 Investigation of extraction method

[0132] Take the standard decoction of Radix Asarum, grind it finely, take about 0.5g, and use methanol solution as the extraction solvent, respectively, to investigate the type of extraction solvent for the same batch of test samples. The results show that there is no significant difference in the content of xanthohumol AB measured by heating reflux extraction and ultrasonic treatment extraction, and ultrasonic treatment is finally selected as the extraction method for the determination of the content of xanthohumol AB in the standard decoction of Radix Asarum.

[0133] 4.3.3 Investigation of extraction time

[0134] Take the standard decoction of Radix Asarum, grind it finely, take about 0.5g, and use methanol solution as the extraction solvent, respectively, to investigate the type of extraction solvent for the same batch of test samples. The results show that there is no significant difference in the content of xanthohumol AB measured by heating reflux extraction and ultrasonic treatment extraction, and ultrasonic treatment is finally selected as the extraction method for the determination of the content of xanthohumol AB in the standard decoction of Radix Asarum.

[0135] 4.3.4 Investigation of extraction solvent dosage

[0136] Take the standard decoction of Radix Asarum, grind it finely, take about 0.5g, and use methanol solution as the extraction solvent, respectively, to investigate the type of extraction solvent for the same batch of test samples. The results show that there is no significant difference in the content of xanthohumol AB measured by heating reflux extraction and ultrasonic treatment extraction, and ultrasonic treatment is finally selected as the extraction method for the determination of the content of xanthohumol AB in the standard decoction of Radix Asarum.

[0137] 4.3.5 Investigation of extraction times

[0138] Take the standard decoction of Radix Asarum, grind it finely, take about 0.5 g, use methanol solution as the extraction solvent, use ultrasonic treatment as the extraction method, take 30 min as the extraction time, take 25 mL as the extraction solvent dosage, take 3 times as the extraction times, and take 15, 25, 40 mL of petroleum ether (60-90°C) to investigate the extraction solvent dosage for the same batch of test samples. The results show that when the extraction solvent dosage is 25 mL, the yellow asarum acid AB can be completely extracted, and finally 25 mL is selected as the extraction solvent dosage for the determination of the content of yellow asarum acid AB in the standard decoction of Radix Asarum.

[0139] 4.3.6 Investigation of extraction solvent dosage

[0140] Take the standard decoction of Radix Asarum, grind it finely, take about 0.5 g, use methanol solution as the extraction solvent, use ultrasonic treatment as the extraction method, take 30 min as the extraction time, take 25 mL as the extraction solvent dosage, take 3 times as the extraction times, and take 15, 25, 40 mL of petroleum ether (60-90°C) to investigate the extraction solvent dosage for the same batch of test samples. The results show that when the extraction solvent dosage is 25 mL, the yellow asarum acid AB can be completely extracted, and finally 25 mL is selected as the extraction solvent dosage for the determination of the content of yellow asarum acid AB in the standard decoction of Radix Asarum.

[0141] 4.3.7 Investigation of drying method

[0142] Take the standard decoction of Radix Asarum, grind it finely, take about 0.5 g, use methanol solution as the extraction solvent, use ultrasonic treatment as the extraction method, take 30 min as the extraction time, take 25 mL as the extraction solvent dosage, take 3 times as the extraction times, and take 15, 25, 40 mL of petroleum ether (60-90°C) to investigate the extraction solvent dosage for the same batch of test samples. The results show that when the extraction solvent dosage is 25 mL, the yellow asarum acid AB can be completely extracted, and finally 25 mL is selected as the extraction solvent dosage for the determination of the content of yellow asarum acid AB in the standard decoction of Radix Asarum.

[0143] 4.3.8 Determination of test sample solution preparation method

[0144] According to the above experimental results, the sample pretreatment method for the determination of the content of yellow asarum acid AB in the standard decoction of Radix Asarum is determined as follows:

[0145] Take the standard decoction of Radix Asarum, grind it finely, take about 0.5 g, place it in a conical flask with a stopper, accurately add 25 mL of methanol solution, weigh it, ultrasonically treat it for 30 min, cool it, weigh it again, make up the weight loss with methanol, shake it well, filter it, evaporate it to dryness under reduced pressure with a rotary evaporator, dissolve the residue in 25 mL of water, extract it 3 times with 25 mL of petroleum ether (60-90°C), combine the solvents, evaporate them to dryness under reduced pressure at low temperature (40°C) with a rotary evaporator, dissolve the residue in methanol to 5 mL, shake it well, filter it, and take the filtrate, which is obtained.

[0146] 4.4 Methodology validation

[0147] 4.4.1 Specificity investigation

[0148] Take the standard decoction of Guan Zhong of Radix Viburni, grind it finely, take about 0.5 g, accurately weigh and prepare the test solution according to the preparation method of the test solution under item 4.3. Take 10 μL of the AB control solution of Radix Viburni acid and the blank solvent, respectively, and inject them into the liquid chromatograph. Determine them according to the chromatographic conditions under item 4.2. The results are shown in Table 3. Figure 7 .

[0149] The results show that the test sample chromatogram has the same chromatographic peak at the corresponding retention time as the control sample chromatogram, and the blank solvent does not interfere, indicating that the method has good specificity.

[0150] 4.4.2 Peak purity investigation

[0151] Take the standard decoction of Guan Zhong of Radix Viburni, grind it finely, take about 0.5 g, accurately weigh and prepare the test solution according to the preparation method of the test solution under item 4.3. Take 10 μL of the AB control solution of Radix Viburni acid and the blank solvent, respectively, and inject them into the liquid chromatograph. Determine them according to the chromatographic conditions under item 4.2. The results are shown in Table 3. Figure 8 .

[0152] The results show that the AB peak of Radix Viburni acid in the sample does not detect impurity peaks, and the peak purity meets the requirements, indicating that under the chromatographic conditions, the peak purity of AB of Radix Viburni acid meets the requirements.

[0153] 4.4.3 Investigation of linear relationship

[0154] Accurately weigh 25.392 mg of the AB control of Radix Viburni acid, and place it in a 10 mL volumetric flask. Add methanol to make a control stock solution containing 2403.630 μg per 1 mL. Accurately pipette 0.2 mL, 0.2 mL, 0.5 mL, 1.0 mL, 2.0 mL, 1.5 mL, and 2.0 mL of the above stock solution into 25 mL, 10 mL, and 5 mL volumetric flasks, respectively, and add methanol to constant volume. Prepare control solutions containing 19.229 μg, 48.073 μg, 120.182 μg, 240.363 μg, 480.726 μg, 721.089 μg, and 961.452 μg per 1 mL, respectively. Accurately pipette the above control solution linear solutions, and determine them by sample injection under the chromatographic conditions under item 4.1. Record the chromatographic peak area. Take the peak area as the vertical coordinate (y) and the control concentration as the horizontal coordinate (x), and draw the standard curve, as shown in Table 2. Figure 9 .

[0155] Table 2 Investigation results of AB of Radix Viburni acid

[0156]

[0157] The results showed that the regression equation for oxalis AB was y = 14978x - 161849, with a correlation coefficient r = 0.9999, indicating that oxalis AB at a concentration of 19.229 μg·mL⁻¹ was effective. -1 ~961.452 μg·mL -1 Within the specified range, the injection concentration and peak area showed a good linear relationship.

[0158] 4.4.4 Precision Examination

[0159] Accurately pipette the reference solution from section "4.1" (C = 218.097 μg·mL). -1 The sample was injected six times under the chromatographic conditions described in section "4.2", and the RSD value was calculated based on the peak area of ​​AB oxaliplatin. The results showed that the RSD value of the peak area of ​​AB oxaliplatin was 2.23% after six consecutive injections of the same reference solution, which is less than 3.0%, indicating good instrument precision.

[0160] 4.4.5 Repeatability Test

[0161] The standard decoction of *Dryopteris crassirhizoma* slices was finely ground. Approximately 0.5g was accurately weighed and prepared in six parallel portions. Six test solutions were prepared according to the method specified in section "4.3". The solutions were then analyzed under the chromatographic conditions specified in section "4.2". The content of anaerobic acid AB and the RSD value were calculated. The results showed that, after six repeated determinations of the same batch of samples, the RSD value of anaerobic acid AB was 2.49%. According to the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is between 100 μg / g and 1 mg / g, the repeatability RSD limit is <3%, indicating that the repeatability of this analytical method is good.

[0162] 4.4.6 Stability Test

[0163] A standard decoction of *Dryopteris crassirhizoma* slices was finely ground. Approximately 0.5g was accurately weighed and prepared in six parallel portions. The test solution was prepared according to the method specified in section "4.3". Chromatographic analysis was performed under the conditions described in section "4.2" at 0, 3, 6, 9, 12, 16, 20, and 24 hours. The peak area of ​​*Dryopteris crassirhizoma* AB in the test solution was determined, and the peak area RSD was calculated. The results showed that for the same test solution, the RSD of the peak area of ​​*Dryopteris crassirhizoma* AB was 1.64% at 0, 3, 6, 9, 12, 16, 20, and 24 hours, indicating good stability of the test solution within 24 hours under this method.

[0164] 4.4.7 Intermediate precision investigation

[0165] Select different experimental personnel at different times, different instruments, take the standard decoction of Radix Aristolochiae Contortae, grind finely, take about 0.5 g, accurately weigh, parallel 6 times, prepare the test solution according to the preparation method of the test solution under "4.3", measure under the chromatographic conditions under "4.2", sample respectively, measure the content of aristolochic acid AB in the test solution, calculate the content of aristolochic acid AB and RSD value, and compare with the results of repeatability test. The results show that the same batch of samples is repeatedly measured 6 times by different personnel at different times on different instruments, the RSD value of aristolochic acid AB content is 1.99%, and the RSD value of 6 data of repeatability test is 2.32%. According to the "Guiding Principles for Analysis Method Validation of Drug Quality Standards" in Chinese Pharmacopoeia 2020 edition, when the content of the measured component in the sample is 100 μg / g-1 mg / g, the intermediate precision RSD limit is <6%, therefore, the intermediate precision of this method is good under the operation of different analysts on different dates and different chromatographs.

[0166] 4.4.8 Accuracy investigation

[0167] The sample recovery method was used, and the ratio of the amount of the reference substance to the content of the test component in the test sample was controlled at about 0.5:1, 1:1 and 1.5:1 to design 3 groups of experiments, each with 3 parallel samples. An appropriate amount of aristolochic acid AB reference substance was accurately weighed into a conical flask with a stopper, and about 0.25 g of Radix Aristolochiae Contortae standard decoction powder with a measured content was accurately weighed into a conical flask. The test solution was prepared according to the method under "4.3". The sample was injected and measured under the chromatographic conditions under "4.2", and the results are shown in Table 3.

[0168] Table 3 Determination of aristolochic acid AB content in Radix Aristolochiae Contortae standard decoction sample recovery rate results

[0169]

[0170] The results show that the recovery rate of aristolochic acid AB is 101.01%. According to the "Guiding Principles for Analysis Method Validation of Drug Quality Standards" in Chinese Pharmacopoeia 2020 edition, when the content of the measured component in the sample is 100 μg / g-1 mg / g, the recovery limit is 90%-108%. The sample recovery rate of aristolochic acid AB is in the range of 96.81%-104.92%, the average sample recovery rate is 101.01%, and the RSD value is 2.81%. It is proved that the accuracy of this method is good.

[0171] 4.4.9 Durability investigation

[0172] ① Different chromatographic column investigation

[0173] Take the standard decoction of Radix Aspidii, grind it finely, take about 0.5 g, accurately weigh and prepare the test solution according to the preparation method of test solution under item 4.3. Except that the column is Kromasil 100-5-C18(4.6mm x 250mm, 5μm), Waters Xselect HSS T3(4.6mm x 250mm, 5μm), Agilent ZORBAX Extend-C18(4.6mm x 250mm, 5μm) respectively, other conditions are according to the chromatographic conditions under item 4.2, sample analysis, determination of the content of Radix Aspidii in the test solution and RSD value. The results show that the separation effect of different brands of column is good, the RSD value of Radix Aspidii is 0.76%, which indicates that the method has good durability to different brands of column.

[0174] ②Investigation of different column temperatures

[0175] Take the standard decoction of Radix Aspidii, grind it finely, take about 0.5 g, accurately weigh and prepare the test solution according to the preparation method of test solution under item 4.3. Except that the column temperature is 28℃, 30℃ and 32℃ respectively, other conditions are according to the chromatographic conditions under item 4.2, sample analysis, determination of the content of Radix Aspidii in the test solution and RSD value. The results show that the RSD value of Radix Aspidii in the standard decoction of Radix Aspidii is 1.42% under different column temperatures(±2℃), which indicates that the analysis method has good durability within the range of column temperature ±2℃.

[0176] ③Investigation of different flow rates

[0177] Take the standard decoction of Radix Aspidii, grind it finely, take about 0.5 g, accurately weigh and prepare the test solution according to the preparation method of test solution under item 4.3. Except that the flow rate is 0.8mL·min -1 , 1.0mL·min -1 and 1.2mL·min -1 respectively, other conditions are according to the chromatographic conditions under item 4.2, sample analysis, determination of the content of Radix Aspidii in the test solution and RSD value. The results show that the RSD value of Radix Aspidii in the standard decoction of Radix Aspidii is 1.25% under different flow rates(±0.2mL·min -1 ), which indicates that the analysis method has good durability within the range of flow rate ±0.2mL·min -1 .

[0178] 4.4.10 Sample determination

[0179] Take 15 batches of standard decoction of Tsunma Guanchou, prepare 15 test solution according to the method of test solution preparation in 4.3, inject sample according to the chromatographic conditions in 4.2, record peak area, calculate the content of yellow Tsunma acid AB by external standard method, the results are shown in Table 4.

[0180] Table 4 Determination results of the content of yellow Tsunma acid AB in 15 batches of standard decoction of Tsunma Guanchou

[0181]

[0182]

[0183] The results show that the content of yellow Tsunma acid AB in 15 batches of standard decoction of Tsunma Guanchou is in the range of 0.0895mg / g-0.5914mg / g, the average content is 0.3006mg / g, SD is 0.1556mg / g, the range of average content±30% is 0.2104mg / g-0.3908mg / g, the range of average content±3SD is-0.1662mg / g-0.7674mg / g. Considering the representativeness of 15 batches of samples, take the minimum value as the lower limit, the average content+3SD as the upper limit, i.e. the range is 0.0895mg / g-0.7674mg / g, adjust the data to 0.100mg / g-0.750mg / g. Therefore, the final determination of the content of yellow Tsunma acid AB in standard decoction of Tsunma Guanchou (C 22 H 26 O8) should be 0.100mg / g-0.750mg / g.

[0184] 4.5 Determination of standard decoction sample of pseudo-drug

[0185] Take standard decoction of single bud dog spine fern, Japanese dog spine Tsunma, small Tsunma, Tsunma of Cycas revoluta, Brown ear fern, Tsunma, purple Tsunma, top bud dog spine Tsunma, bead bud Tsunma, Tsunma Guanchou, grind finely, take about 0.5g each, accurately weigh, prepare test solution according to the method of test solution preparation in 4.3, inject sample according to the chromatographic conditions in 4.2, record peak area, calculate the content of yellow Tsunma acid AB by external standard method, the results are shown in Table 5.

[0186] Table 5 Determination results of the content of yellow Tsunma acid AB in standard decoction of Tsunma Guanchou and pseudo-drug

[0187]

[0188] The results show that yellow Tsunma acid AB is not contained or lower than the specified content range value (0.100mg / g-0.750mg / g) of standard decoction of Tsunma Guanchou in each pseudo-drug standard decoction, which indicates that yellow Tsunma acid AB has good specificity as the determination index of the content of standard decoction of Tsunma Guanchou.

[0189] Each technical feature of the above-described embodiments can be combined with any other technical feature, and for the sake of brevity, not all possible combinations are described, but it is understood that the scope of the present disclosure encompasses all such possible combinations.

[0190] The above-described embodiments are merely illustrative of several embodiments of the present application, and the description is relatively specific and detailed, but should not be construed as limiting the scope of the patent application. It should be noted that for those skilled in the art, some modifications and improvements can be made without departing from the concept of the present application, and these are all within the scope of the present application. Therefore, the scope of the patent of the present application should be based on the appended claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for identifying standard decoction of Aspidium and its counterfeit, characterized in that, The method comprises the following steps: respectively providing standard decoctions of Qianma Guanzong and its counterfeit; respectively extracting the standard decoctions of Qianma Guanzong and its counterfeit using extraction solvents to prepare test sample solutions; the extraction solvents are alcohol or aqueous alcohol, the alcohol is methanol or ethanol, and the extraction methods are ultrasonic extraction, reflux extraction or extraction extraction; dissolving the Xanthohumol AB control sample using a dissolving reagent to prepare a control sample solution; performing high performance liquid chromatography detection on the test sample solution and the control sample solution, observing whether there is a chromatographic peak response in the chromatogram of the test sample solution at a position corresponding to the retention time of the chromatographic peak of the control sample solution, and obtaining the content of Xanthohumol AB in the standard decoction of Qianma Guanzong; The conditions for the high performance liquid chromatography detection include: the mobile phase A is methanol, the mobile phase B is phosphoric acid aqueous solution, and isocratic elution is adopted; the volume ratio of the mobile phase A and the mobile phase B is (70-85):(30-15); and the chromatographic column uses octadecylsilane bonded silica gel as the filler.

2. The method according to claim 1, wherein the method is characterized by, The high performance liquid chromatography detection meets at least one of the following conditions: (1) the detection wavelength is 210nm-400nm; (2) the column temperature is 20℃-30℃; (3) flow rate of 0.5 mL • min -1 1.5 mL • min -1 ; (4) the injection volume is 5μL-15μL.

3. The method according to claim 1, wherein the method is characterized by, In the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.1%-0.5%.

4. The method according to claim 1, wherein the method is characterized by, The extraction step meets at least one of the following conditions: (1) in the aqueous alcohol, the volume percentage of alcohol is 50%-99%; (2) the extraction time is 15min-60min; (3) the mass-volume ratio of the standard decoctions of Qianma Guanzong and its counterfeit and the extraction solvent is 0.5g:(15-50)mL.

5. The method according to claim 1, wherein the method is characterized by, Obtaining the content of Xanthohumol AB in the standard decoction of Qianma Guanzong comprises the following steps: dissolving the control sample using a dissolving reagent to prepare control sample solutions of different concentrations; respectively injecting the control sample solutions of different concentrations to perform high performance liquid chromatography detection; obtaining the peak areas of Xanthohumol AB in the high performance liquid chromatography detection maps of the control sample solutions of different concentrations, taking the concentrations of the control sample solutions as the abscissa and the peak areas of Xanthohumol AB in the control sample solutions of different concentrations as the ordinate, drawing a standard curve, and calculating a linear regression equation; substituting the peak area of Xanthohumol AB in the high performance liquid chromatography detection map of the test sample solution obtained by high performance liquid chromatography detection into the linear regression equation to calculate the content of Xanthohumol AB in the test sample solution; The control sample comprises Xanthohumol AB.

6. The method for identifying standard decoction of Pteroxygonum hupehense Diels and its counterfeit according to any one of claims 1-5, characterized in that, The volume ratio of the mobile phase A and the mobile phase B is 80:

20.

7. The method according to any one of claims 1-5, wherein the method is characterized by, The dissolving reagent is methanol.

8. The method according to any one of claims 1-5, wherein the method is characterized by, The content of Xanthohumol AB in the standard decoction of Qianma Guanzong is 0.100mg / g-0.750mg / g.

9. The method according to any one of claims 1-5, wherein the method is characterized by, The counterfeit includes one or more of the following: Shandou Goujizhong, Riken Guanzhong, Xiaoguanzhong, Suotie Guanzhong, Brown Ear Fern, Guanzhong, Zixi Guanzhong, Dingya Goujizhong and Zhuyan Guanzhong.

10. The use of the method for identifying the standard decoction of Pteroxygonum heterophllum and its counterfeit in the quality control of Pteroxygonum heterophllum.

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