Identification method and application of rhizoma dryopteris crassirhizomae decoction piece standard decoction and counterfeit product thereof

Abstract

The invention relates to a method for identifying standard decoction of rhizoma dryopteris crassirhizomae decoction pieces and counterfeit products thereof and application, and the method comprises the following steps: respectively extracting the standard decoction of rhizoma dryopteris crassirhizomae decoction pieces and counterfeit products thereof by using an extraction solvent to prepare a test solution; dissolving a flavaspidic acid AB reference substance by using a dissolving reagent to prepare a reference substance solution; and performing high performance liquid chromatography detection on the test solution and the reference solution to obtain the content of flavaspidic acid AB in the standard decoction of the rhizoma dryopteris crassirhizomae decoction pieces. The content determination method is established for the characteristic component flavaspidic acid AB of the rhizoma dryopteris crassirhizomae decoction piece standard decoction, so that the rhizoma dryopteris crassirhizomae decoction piece standard decoction can be qualitatively and quantitatively identified, the medication safety of the rhizoma dryopteris crassirhizomae decoction piece standard decoction is ensured, and the method is used for quality control of the rhizoma dryopteris crassirhizomae decoction piece The establishment of the method not only provides a basis for the establishment of the quality standard of the rhizoma dryopteris crassirhizomae decoction piece standard decoction, but also provides an important reference for the establishment of the quality standard of rhizoma dryopteris crassirhizomae formula granules and related traditional Chinese medicine preparations thereof.

Description

Technical Field

[0001] The present application relates to the technical field of drug analysis, and in particular to a method and application for identifying a standard decoction of Mianma Guanzhong slices and counterfeits thereof. Background Art

[0002] Standard decoctions of Chinese herbal medicines are single-flavor decoctions of Chinese herbal medicines prepared by standardized processes under the guidance of traditional Chinese medicine theory and based on clinical application, with reference to modern extraction methods. As a standard substance and standard system, they can be used to standardize clinical medication, regulate new forms of decoctions including formula granules that are currently widely used in clinical practice, and ensure the accuracy of medication and consistency of dosage. Therefore, the use of modern analytical technology to establish the quality standards of standard decoctions of Chinese herbal medicines can provide an important reference for the establishment of quality standards for modern Chinese medicine preparations such as corresponding formula granules and classic prescriptions.

[0003] Mianma Guanzhong is the dried rhizome and petiole residue of Dryopteris crassirhizoma Nakai, a plant of the Dryopteris family. Mianma Guanzhong contains ingredients such as phloroglucinols, flavonoids, terpenes and phenylpropanoids, which have the effects of clearing heat and detoxifying, and expelling insects. Modern studies have shown that phloroglucinols are the main components of the antiviral, antibacterial, antitumor and anthelmintic pharmacological activities of Mianma Guanzhong. As a commonly used antiviral drug in clinical practice, Mianma Guanzhong has played an important role in fighting against the new coronavirus infection. In recent years, due to the increase in demand for Mianma Guanzhong, counterfeit products have frequently appeared in the market, such as Zifan Guanzhong, Xiao Guanzhong, Dryopteris Guanzhong, and Globose rhizomes. The different efficacy will affect the safety of Mianma Guanzhong. However, there are few studies on the quality standards of Mianma Guanzhong, and its clinical safety and effectiveness cannot be guaranteed.

[0004] The 2020 edition of the Chinese Pharmacopoeia only records the properties, microscopic, and thin-layer identification methods of the medicinal material Corydalis guanzhong. The medicinal material Corydalis guanzhong is not included in the provincial Chinese medicinal material standards, and is only included in the processing specifications of some Chinese herbal medicines. Its identification methods include property identification and microscopic identification methods, and there are no content determination indicators. In the research on the quality standards of Chinese medicine formula granules in various provinces, total flavonoids and chlorogenic acid were selected as content determination indicators, which are not specific. In the current literature research, the quality control methods for Corydalis guanzhong include the determination of total flavonoids content by ultraviolet method and the determination of total phloroglucinol content by ultraviolet method. However, the use of total flavonoids and total phloroglucinols as content determination indicators lacks specificity and accuracy. There is no literature report on the content determination method of chlorogenic acid AB, the active ingredient in Corydalis guanzhong.

[0005] There are abundant resources of Guanzhong varieties, and there are many Guanzhong varieties commonly used in different regions. In recent years, Mianma Guanzhong has played a significant role in antiviral (anti-new coronavirus, influenza virus and respiratory tract-related viruses), which has increased its demand, and counterfeits have frequently appeared in the market. According to investigations, common counterfeits such as Guanzhong purpurogenum, Guanzhong scutellariae, Guanzhong dactyliferae, and rhizomes of Pteridaceae affect the safety of Mianma Guanzhong based on their different efficacy. It is even more difficult to identify the authenticity of standard decoctions of medicinal pieces that have lost their appearance characteristics. Therefore, it is necessary to establish a method for determining the content of characteristic components of standard decoctions of Mianma Guanzhong medicinal pieces, so as to qualitatively and quantitatively identify standard decoctions of Mianma Guanzhong medicinal pieces and ensure their safety. Summary of the invention

[0006] Based on this, it is necessary to provide a method and application for identifying the standard decoction of Mianma Guanzhong slices and its counterfeits. This method establishes a content determination method for the characteristic ingredient malonic acid AB in the standard decoction of Mianma Guanzhong slices, so as to qualitatively and quantitatively identify the standard decoction of Mianma Guanzhong slices and ensure its safety.

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

[0008] One aspect of the present invention provides a method for identifying a standard decoction of Mianma Guanzhong slices and its counterfeits, comprising the following steps:

[0009] Provide standard decoctions of Mianma Guanzhong slices and their counterfeits respectively;

[0010] The standard decoctions of Mianma Guanzhong pieces and their counterfeits were extracted using extraction solvents to prepare test sample solutions;

[0011] Dissolve the scutellaria baicalenic acid AB reference substance using a dissolving reagent to prepare a reference substance solution;

[0012] The test solution and the reference solution are subjected to high performance liquid chromatography to obtain the content of scutellaria baicalensis acid AB in the standard decoction of the scutellaria baicalensis slices;

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

[0014] In one embodiment, the HPLC detection satisfies at least one of the following conditions:

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

[0016] (2) Column temperature is 20℃~30℃:

[0017] (3) Flow rate: 0.5 mL min-1 ~1.5mL·min -1 ;

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

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

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

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

[0022] (2) The extraction method is ultrasonic extraction, reflux extraction or extraction extraction;

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

[0024] (4) The mass volume ratio of the standard decoction of the Mianma Guanzhong slices and their counterfeits to the extraction solvent is 0.5 g: (15-50) mL.

[0025] In one embodiment, obtaining the content of cypermethrin acid AB in the standard decoction of the Cyperus rotundus slices comprises the following steps:

[0026] Dissolve the reference substance using a dissolving reagent to prepare reference substance solutions of different concentrations;

[0027] Injecting the reference substance solutions of different concentrations respectively, and performing high performance liquid chromatography detection;

[0028] Obtain the peak areas corresponding to schizonepenic acid AB in the high performance liquid chromatography detection spectrum of the reference solution of different concentrations, take the concentration of the reference solution as the abscissa, take the peak areas corresponding to schizonepenic acid AB in the reference solution of different concentrations as the ordinate, draw a standard curve, and calculate the linear regression equation;

[0029] Substituting the peak area corresponding to scutellaria AB in the HPLC detection spectrum obtained by performing HPLC detection on the test solution into the linear regression equation, the content of scutellaria AB in the test solution is calculated;

[0030] Wherein, the reference substance includes scutellaria baicalenic acid AB.

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

[0032] In one embodiment, the dissolving agent is methanol.

[0033] In one embodiment, the content of scutellaria baicalensis acid AB in the standard decoction of the Rhizoma Cynomorii Scutellariae decoction pieces is 0.100 mg / g to 0.750 mg / g.

[0034] In one embodiment, the counterfeit products include one or more of the following: single-bud dog spine fern, Japanese dog spine, small dog spine, cycad dog spine, brown ear fern, dog spine, octopus fern, terminal dog spine and bulbil dog spine.

[0035] Another aspect of the present invention provides the use of the above-mentioned standard decoction of Mianma Guanzhong slices and the method for identifying counterfeits thereof in the quality control of Mianma Guanzhong slices.

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

[0037] This application establishes a content determination method for the characteristic ingredient of the standard decoction of Mianma Guanzhong slices, phloroglucinol compound phloroglucinol AB, that is, by adopting specific high-performance liquid chromatography detection conditions for the standard decoction of Mianma Guanzhong slices, a HPLC content determination method for the active ingredient phloroglucinol compound phloroglucinol AB in the standard decoction of Mianma Guanzhong slices is established, so that the standard decoction of Mianma Guanzhong slices can be qualitatively and quantitatively identified to ensure its medication safety, and used for the quality control of the standard decoction of Mianma Guanzhong slices. At the same time, the method has good specificity, peak purity, linear relationship, precision, repeatability, stability, accuracy, and durability through methodological verification. The establishment of this method not only provides a basis for the establishment of the quality standard of the standard decoction of Mianma Guanzhong slices, but also provides an important reference for the establishment of the quality standard of Mianma Guanzhong formula granules and related Chinese medicine preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0039] Figure 1 This is the ultraviolet absorption curve of the standard product of AB flavone acid in Example 1.

[0040] Figure 2 This is a comparative chromatogram of different organic phase elutions for the determination of the content of AB in the standard decoction of Mianma Guanzhong slices in Example 1.

[0041] Figure 3This is a comparative chromatogram of different types of acid elution for the determination of the content of AB in the standard decoction of Mianma Guanzhong slices in Example 1.

[0042] Figure 4 This is a comparative chromatogram of different acid concentrations for the determination of the content of AB in the standard decoction of Mianma Guanzhong slices in Example 1.

[0043] Figure 5 This is a comparative chromatogram of isocratic elution with different proportions of mobile phases for the determination of the content of AB in the standard decoction of Mianma Guanzhong slices in Example 1.

[0044] Figure 6 This is a comparative chromatogram of different column temperatures for determining the content of scutellaria baicalensis acid AB in the standard decoction of the Mianma Guanzhong slices in Example 1.

[0045] Figure 7 This is a graph showing the specificity of the determination of the AB content of the Mianma acid in the standard decoction of the Mianma Guanzhong slices in Example 1.

[0046] Figure 8 This is a peak purity investigation diagram for the determination of the content of AB content of mianmaic acid in the standard decoction of Mianma Guanzhong slices in Example 1.

[0047] Fig. 9 This is the standard curve of AB of scutellaria baicalenic acid in Example 1. DETAILED DESCRIPTION

[0048] The present application will be further described in detail below in conjunction with specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thoroughly understood.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0050] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0051] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0052] Herein, the optional scope of "and / or", "or / and", and "and / or" includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, wherein said any and all combinations include any two relevant listed items, any more relevant listed items, or a combination of all relevant listed items. (It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in the present application, the technical solution undoubtedly includes technical solutions that are all connected by "logical AND", and also undoubtedly includes technical solutions that are all 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 (that is, technical solutions that are all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, a combination of any two or any three of A, B, C, and D, and also includes a combination of four items of A, B, C, and D (that is, a technical solution that is all connected by "logical AND").)

[0053] Herein, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0054] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0055] The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0056] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0057] The temperature parameters in this application, unless otherwise specified, allow both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±2°C, ±1°C, ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, and ±0.1°C are allowed. Normal temperature or room temperature in this application refers to no temperature control operation, generally 4°C to 35°C, preferably 20±5°C.

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

[0059] It is an important method to control and evaluate the quality of drugs by using appropriate chemical methods or instrumental analysis methods to quantitatively analyze certain (some) effective ingredients or effective parts in Chinese medicinal materials, and to judge the quality of drugs by whether the measurement results meet the requirements of drug standards. According to the survey, the current pharmacopoeia standard for Mianma Guanzhong medicinal materials does not stipulate the content indicators that characterize the quality quality, but only stipulates its properties, microscopic identification and thin layer identification items, and the quality standards are still imperfect. There are also few reports on the quality standards of Mianma Guanzhong decoction pieces. Since the main active ingredients of Mianma Guanzhong, the phloroglucinol compounds, have low polarity and it is difficult to establish a chromatographic separation method, most of the quality standard studies of Mianma Guanzhong medicinal materials and formula granules choose total flavonoids and chlorogenic acid as content determination indicator components, which lack specificity.

[0060] Phloroglucinol compound, the active ingredient in Limianma Guanzhong decoction pieces, has a chemical formula of C 22 H 26 O8, the structure is as follows:

[0061]

[0062] In view of the shortcomings of the existing quality control index components of Mianma Guanzhong, such as lack of specificity, inability to identify counterfeits, and lack of accuracy, the technical personnel of this application have established an HPLC content determination method for the active ingredient phloroglucinol compound phloroglucinic acid AB in the standard decoction of Mianma Guanzhong slices after extensive research, which is used for the quality control of the standard decoction of Mianma Guanzhong slices. The establishment of this content determination method not only provides a basis for the establishment of the quality standard of the standard decoction of Mianma Guanzhong slices, but also provides an important reference for the establishment of the quality standard of Mianma Guanzhong formula granules and related Chinese medicine preparations.

[0063] One aspect of the present invention provides a method for determining the content of cypermethrin acid AB in a standard decoction of Rhizoma Cynomorii Scutellariae, comprising the following steps:

[0064] Provide standard decoctions of Mianma Guanzhong slices and their counterfeits respectively;

[0065] The standard decoctions of Mianma Guanzhong pieces and their counterfeits were extracted using extraction solvents to prepare test sample solutions;

[0066] Dissolve the scutellaria baicalenic acid AB reference substance using a dissolving reagent to prepare a reference substance solution;

[0067] The test solution and the reference solution are subjected to high performance liquid chromatography to obtain the content of scutellaria baicalensis acid AB in the standard decoction of the scutellaria baicalensis slices;

[0068] The conditions for the HPLC detection include: mobile phase A is methanol, mobile phase B is phosphoric acid aqueous solution, and isocratic elution is used; the volume ratio of mobile phase A to mobile phase B is (70-85): (30-15). It can be understood that the volume ratio of mobile phase A to mobile phase B includes but is not limited to 70:30, 75:25, 80:20, 85:15.

[0069] In some examples, the conditions for the HPLC detection include: the detection wavelength is 210nm to 400nm. It is understandable that the detection wavelength includes but is not limited to 210nm, 250nm, 300nm, 350nm, and 400nm.

[0070] In some examples, the conditions for the HPLC detection include: a column temperature of 20° C. to 30° C. It is understood that the column temperature includes but is not limited to 20° C., 22° C., 24° C., 26° C., 28° C., and 30° C.

[0071] In some of the examples, the conditions for the HPLC detection include: a flow rate of 0.5 mL min -1 ~1.5mL·min -1 It is understood that the flow rate includes but is not limited to 0.5 mL min -1 、0.75mL·min -1 、1.0mL·min -1 、1.25mL·min -1 、1.5mL·min -1 .

[0072] In some examples, the conditions for the HPLC detection include: injection volume of 5 μL to 15 μL. It is understandable that the injection volume includes but is not limited to 5 μL, 7 μL, 9 μL, 11 μL, 13 μL, and 15 μL.

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

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

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

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

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

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

[0079] In some of the examples, the conditions of the extraction step include: the mass volume ratio of the standard decoction of the Mianma Guanzhong slices and their counterfeits to the extraction solvent is 0.5g: (15-50)mL. It is understandable that the mass volume ratio of the standard decoction of the Mianma Guanzhong slices and their counterfeits to the extraction solvent includes but is not limited to 0.5g: 15mL, 0.5g: 20mL, 0.5g: 30mL, 0.5g: 40mL, 0.5g: 50mL.

[0080] In some examples, obtaining the content of cypermethrin acid AB in the standard decoction of the Cyperus rotundus slices comprises the following steps:

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

[0082] Injecting the reference substance solutions of different concentrations respectively, and performing high performance liquid chromatography detection;

[0083] Obtain the peak areas corresponding to schizonepenic acid AB in the high performance liquid chromatography detection spectrum of the reference solution of different concentrations, take the concentration of the reference solution as the abscissa, take the peak areas corresponding to schizonepenic acid AB in the reference solution of different concentrations as the ordinate, draw a standard curve, and calculate the linear regression equation;

[0084] Substituting the peak area corresponding to scutellaria AB in the HPLC detection spectrum obtained by performing HPLC detection on the test solution into the linear regression equation, the content of scutellaria AB in the test solution is calculated;

[0085] Wherein, the reference substance includes scutellaria baicalenic acid AB.

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

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

[0088] In some of the examples, the content of scutellaria baicalensis acid AB in the standard decoction of the Mianma Guanzhong slices is 0.100 mg / g to 0.750 mg / g.

[0089] In some of the examples, the counterfeits include one or more of the following: single-bud dogwood fern, Japanese dogwood fern, small dogwood fern, cycad dogwood fern, brown ear fern, dogwood fern, octopus fern, terminal dogwood fern, and bulbil dogwood fern.

[0090] Another aspect of the present invention provides the use of the above-mentioned standard decoction of Mianma Guanzhong slices and the method for identifying counterfeits thereof in the quality control of Mianma Guanzhong slices.

[0091] The following is further described in conjunction with specific embodiments. The raw materials involved in the following specific embodiments, unless otherwise specified, can all be commercially available; the instruments used, unless otherwise specified, can all be commercially available; the processes involved, unless otherwise specified, are all routinely selected by those skilled in the art.

[0092] The following are specific embodiments.

[0093] Example 1

[0094] This embodiment provides a method for constructing an HPLC determination method for the content of scutellaria baicalensis AB in a standard decoction of Rhizoma Cynomorii Scutellariae and its application in identifying counterfeits, as follows:

[0095] 1. Instruments, reagents and test drugs

[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 × 250 mm, 5 μm), Kromasil 100-5-C18 chromatographic column (4.6 mm × 250 mm, 5 μm), Agilent ZORBAX Extend-C18 chromatographic column (4.6 mm × 250 mm, 5 μm), digitally controlled ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), one-tenth balance (ME204E, Mettler-Toledo), one-millionth electronic analytical balance (XP26, Mettler-Toledo), rotary evaporator (RE-3000, Shanghai Yarong Biochemical Instrument Factory), ultrapure water system (Milli-Q Direct, Merck & Co., Ltd.). Ethanol, methanol (Tianjin Fuyu Fine Chemical Co., Ltd.), and petroleum ether 60-90°C (Tianjin Best Chemical Co., Ltd.) were all analytical grade; acetonitrile, methanol (Merck Co., Ltd.), and phosphoric acid (Tianjin Komiou Chemical Reagent Co., Ltd.) were all chromatographic grade; ultrapure water (self-made in the laboratory); and scutellaria baicalensis AB reference substance (self-made in the laboratory).

[0097] 2. Preparation of Mianma Guanzhong Decoction Pieces

[0098] The decoction pieces of Mianma Guanzhong are prepared according to the processing regulations of the Mianma Guanzhong item in the 2020 edition of the Chinese Pharmacopoeia. The specific processing method is: remove impurities, spray with clean water, wash, moisten thoroughly, cut into thick slices, dry, and sieve out ash to obtain the product.

[0099] 3. Preparation of standard decoction of Mianma Guanzhong slices

[0100] Take 100g of Mianma Guanzhong slices, add water and decoct twice. For the first decoction, add 9 times the amount of water, soak for 30 minutes, boil over high heat (500W), then keep it slightly boiling over low heat (200W) for 30 minutes, filter it while hot with a 350-mesh sieve, and quickly cool the filtrate with cold water; add 7 times the amount of water for the second decoction, boil over high heat (500W), then keep it slightly boiling over low heat (200W) for 25 minutes, filter it while hot with a 350-mesh sieve, and quickly cool the filtrate with cold water, and combine the two filtrates. The decoction was transferred to a 2000mL round-bottom flask and concentrated to 100mL under reduced pressure and low temperature using a rotary evaporator (temperature: 65°C; vacuum degree: -0.08 to -0.1MPa); under magnetic stirring, it was dispensed into 10mL vials, with a dispensing volume of 2mL per bottle. After dispensing, it was transferred to a vacuum freeze dryer for freeze drying, taken out, and covered with an aluminum cap to obtain a standard decoction freeze-dried powder. The batch numbers of standard decoction freeze-dried powder prepared from 15 batches of Mianma Guanzhong decoction pieces from different origins are shown in Table 1.

[0101] Table 115 Batch of Mianma Guanzhong Standard Decoction Freeze-dried Powder and Medicinal Materials Origin Information

[0102]

[0103] 4. Establishment of the determination method of AB content of scutellaria baicalensis

[0104] 4.1 Preparation of reference solution

[0105] Accurately weigh 2.353 mg of the AB standard substance of scutellariae acid, place it in a 50 mL volumetric flask, and add methanol to make a standard solution containing 44.547 μg per 1 mL.

[0106] 4.2 Optimization of chromatographic conditions

[0107] 4.2.1 Selection of detection wavelength

[0108] The full-wave scanning of the AB reference solution of scutellaria baicalensis was performed, and the absorption spectrum of the reference solution in the range of 210nm to 400nm was recorded. The results are shown in Figure 1 .

[0109] The results showed that the maximum absorption wavelength of scutellaria baicalensis AB was at 298nm.

[0110] 4.2.2 Choice of mobile phase

[0111] The effects of different organic phases (acetonitrile, methanol, acetonitrile: methanol -1:1) on chromatographic peak separation and baseline were investigated. Figure 2 .

[0112] The results showed that when methanol was used as the organic phase for elution, the baseline of the chromatogram was stable and the separation effect of the chromatographic peaks of scutellaria baicalenic acid AB was good, so methanol was selected as the final elution organic phase.

[0113] 4.2.3 Choice of acid type

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

[0115] The results showed that when phosphoric acid aqueous solution was used for elution of the aqueous phase, the baseline of the chromatogram was stable and the separation effect of the chromatographic peaks of scutellaria baicalenic acid AB was better, so phosphoric acid was selected as the acid added to the final aqueous phase.

[0116] 4.2.4 Selection of acid concentration

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

[0118] The results showed that when 0.2% and 0.5% phosphoric acid aqueous solutions were used for elution, the baseline of the chromatogram was stable and the separation effect of the chromatographic peaks of scutellaria baicalensis AB was better. Since the pH value of 0.5% phosphoric acid aqueous solution was low and it had a greater impact on the performance of the chromatographic column, 0.2% was selected as the acid concentration in the final aqueous phase.

[0119] 4.2.5 Selection of mobile phase ratio

[0120] The effect of isocratic elution with different mobile phase ratios (organic phase: aqueous phase-70:30, organic phase: aqueous phase-75:25, organic phase: aqueous phase-80:20, organic phase: aqueous phase-85:15) on the chromatographic peak separation and baseline was investigated. The organic phase was methanol and the aqueous phase was 0.2% phosphoric acid aqueous solution. The results are shown in Figure 5 .

[0121] The results showed that when the organic phase: aqueous phase ratio of 80:20 was used for elution, the baseline of the chromatogram was stable and the separation effect of the chromatographic peaks of scutellaria baicalenic acid AB was better, so the organic phase: aqueous phase ratio of 80:20 was selected for isocratic elution.

[0122] 4.2.6 Selection of column temperature

[0123] The effects of different column temperatures (20°C, 25°C, 30°C, 35°C, 40°C) on chromatographic peak separation and baseline were investigated. The results are shown in Figure 6 .

[0124] The results showed that when the column temperature was 20℃~30℃ for elution, the peak shape of the chromatographic peak of scutellaria baicalensis AB was better and the separation effect was better. Considering all factors, 30℃ was selected as the final column temperature.

[0125] 4.2.7 Determination of chromatographic conditions

[0126] According to the above experimental results, the HPLC conditions for determining the content of AB in the standard decoction of Mianma Guanzhong slices are determined as follows:

[0127] Chromatographic column: Octadecylsilane bonded silica gel as filler (4.6 mm × 250 mm, 5 μm); methanol-0.2% phosphoric acid aqueous solution (80:20) as mobile phase; volume flow rate of 1.0 mL min -1 ; Detection wavelength was 298nm; column temperature was 30℃; injection volume was 10μL.

[0128] 4.3 Investigation of test sample treatment conditions

[0129] 4.3.1 Investigation of extraction solvent types

[0130] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, and use methanol solution, 70% methanol aqueous solution, 50% methanol aqueous solution, 95% ethanol aqueous solution, 70% ethanol aqueous solution, 50% ethanol aqueous solution as extraction solvents, respectively, to investigate the types of extraction solvents for the same batch of test samples. The results showed that when methanol was used as the extraction solvent, the content of scutellaria baicalensis AB was the highest, and methanol was finally selected as the extraction solvent for the determination of scutellaria baicalensis AB content in the standard decoction of Mianma Guanzhong slices.

[0131] 4.3.2 Investigation of extraction methods

[0132] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, use methanol solution as the extraction solvent, and investigate the extraction methods of the same batch of test samples by ultrasonic treatment and heating reflux. The results show that there is no significant difference between the content of scutellaria baicalensis AB measured in the heating reflux extraction and the ultrasonic treatment extraction. Considering the simplicity of operation, ultrasonic treatment was finally selected as the extraction method for the determination of scutellaria baicalensis AB content in the standard decoction of Mianma Guanzhong slices.

[0133] 4.3.3 Investigation of extraction time

[0134] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, use methanol solution as the extraction solvent, use ultrasonic treatment as the extraction method, and investigate the extraction time of the same batch of test samples at 15min, 30min, 45min, and 60min. The results show that when the extraction time is 30min, the scutellaria acid AB can be completely extracted, and finally 30min is selected as the extraction time for the determination of the scutellaria acid AB content in the standard decoction of Mianma Guanzhong slices.

[0135] 4.3.4 Investigation of the amount of extraction solvent

[0136] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, use methanol solution as the extraction solvent, ultrasonic treatment as the extraction method, 30min as the extraction time, and use 15mL, 25mL, and 50mL to investigate the amount of extraction solvent for the same batch of test samples. The results show that when the amount of extraction solvent is 25mL, the scutellaria acid AB can be completely extracted, and finally 25mL is selected as the extraction solvent amount for the determination of the scutellaria acid AB content in the standard decoction of Mianma Guanzhong slices.

[0137] 4.3.5 Investigation of extraction times

[0138] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, use methanol solution as extraction solvent, ultrasonic treatment as extraction method, 30min as extraction time, 25mL as extraction solvent dosage, and investigate the extraction times of 2, 3, 4, and 5 for the same batch of test samples. The results show that when the extraction times are 3, the scutellaria acid AB can be completely extracted, and finally 3 times is selected as the extraction times for the determination of the scutellaria acid AB content in the standard decoction of Mianma Guanzhong slices.

[0139] 4.3.6 Investigation of the amount of extraction solvent

[0140] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, use methanol solution as extraction solvent, ultrasonic treatment as extraction method, 30min as extraction time, 25mL as extraction solvent dosage, 3 times as extraction times, and 15, 25, 40mL petroleum ether (60℃~90℃) for the same batch of test samples to investigate the amount of extraction solvent. The results show that when the amount of extraction solvent is 25mL, the scutellaria baicalensis AB can be completely extracted, and finally 25mL is selected as the amount of extraction solvent for the determination of the content of scutellaria baicalensis AB in the standard decoction of Mianma Guanzhong slices.

[0141] 4.3.7 Investigation of drying methods

[0142] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, use methanol solution as extraction solvent, ultrasonic treatment as extraction method, 30min as extraction time, 25mL as extraction solvent dosage, 3 times as extraction times, 25mL as extraction solvent dosage, and low temperature (40℃) reduced pressure drying and high temperature (80℃) normal pressure drying to investigate the drying methods of the same batch of test samples. The results showed that the content of scutellaria baicalensis AB measured in low temperature (40℃) reduced pressure drying was significantly higher than that in high temperature (80℃) normal pressure drying, and finally low temperature (40℃) reduced pressure drying was selected as the drying method for the determination of scutellaria baicalensis AB content in the standard decoction of Mianma Guanzhong slices.

[0143] 4.3.8 Determination of the preparation method of the test solution

[0144] According to the above experimental results, the sample pretreatment method for the determination of AB content of scutellaria baicalensis in the standard decoction of scutellaria baicalensis slices is determined as follows:

[0145] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, put it in a stoppered conical flask, accurately add 25mL of methanol solution, weigh it, ultrasonicate it for 30min, let it cool, weigh it again, make up the loss with methanol, shake it well, filter it, evaporate it to dryness under reduced pressure on a rotary evaporator, dissolve the residue with 25mL of water, add 25mL of petroleum ether (60℃~90℃) and extract it 3 times, combine the solvents, evaporate it to dryness under reduced pressure on a rotary evaporator at low temperature (40℃), make up the volume of the residue to 5mL with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0146] 4.4 Methodological validation

[0147] 4.4.1 Specificity Investigation

[0148] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, weigh it accurately, prepare the test solution according to the test solution preparation method determined in "4.3", take 10μL of the AB reference solution of scutellaria baicalensis and blank solvent respectively, inject them into the liquid chromatograph, and determine them according to the chromatographic conditions in "4.2". The results are shown in Figure 7 .

[0149] The results showed that the test sample chromatogram had the same chromatographic peak at the corresponding retention time as the reference sample chromatogram, and there was no interference from the blank solvent, indicating that the method had good specificity.

[0150] 4.4.2 Peak purity investigation

[0151] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, weigh it accurately, prepare the test solution according to the test solution preparation method determined in "4.3", accurately pipette the test solution and the Mianma acid AB reference solution, inject them into the liquid chromatograph, and perform scanning detection at 210nm~400nm with the DAD detector according to the chromatographic conditions in "4.2", calculate the peak purity, and the results are shown in Figure 8 .

[0152] The results showed that no impurity peaks were detected in the AB peak of scutellaria baicalensis in the sample, and the peak purity met the requirements, indicating that under this chromatographic condition, the purity of the AB peak of scutellaria baicalensis met the requirements.

[0153] 4.4.3 Investigation of linear relationship

[0154] Accurately weigh 25.392 mg of scutellaria baicalensis AB reference substance, place it in a 10 mL volumetric flask, and add methanol to prepare a reference substance 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-mentioned stock solution into 25 mL, 10 mL, and 5 mL volumetric flasks, respectively, add methanol to make up to the scale, and prepare reference substance 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-mentioned reference substance solution linear solution, and measure according to the chromatographic conditions under "4.1", and record the chromatographic peak area. The peak area is used as the ordinate (y) and the concentration of the reference substance is used as the abscissa (x), and a standard curve is drawn, as shown in Table 2. Fig. 9 .

[0155] Table 2 Results of linear investigation of AB of yellow cotton acid

[0156]

[0157] The results showed that the regression equation of scutellaria AB was: y = 14978x-161849, and its correlation coefficient r = 0.9999, indicating that scutellaria AB had a concentration of 19.229 μg·mL -1 ~961.452μg·mL -1 There is a good linear relationship between the injection concentration and the peak area in the range of 1.

[0158] 4.4.4 Precision investigation

[0159] Accurately pipette the reference solution (C = 218.097 μg mL) under item "4.1" -1 ), repeat the injection 6 times according to the chromatographic conditions under "4.2", and calculate the RSD value based on the peak area of ​​scutellaria AB. The results show that the RSD value of the peak area of ​​scutellaria AB was 2.23% after 6 injections of the same reference solution, which is less than 3.0%, indicating that the instrument has good precision.

[0160] 4.4.5 Repeatability study

[0161] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, weigh it accurately, and make 6 parallel portions. According to the preparation method of the test solution determined under "4.3", 6 test solutions were prepared, and the sample was injected and analyzed according to the chromatographic conditions under "4.2", and the content of AB and RSD value of scutellariae acid were calculated. The results showed that the same batch of samples was measured 6 times, and the RSD value of AB content of scutellariae acid was 2.49%. According to the "Guidelines for Validation of Analytical Methods for Drug Quality Standards" of the 2020 edition of the Chinese Pharmacopoeia, when the content of the tested component in the sample is 100μg / g~1mg / g, the repeatability RSD limit is <3%, indicating that the repeatability of this analytical method is good.

[0162] 4.4.6 Stability investigation

[0163] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, weigh it accurately, and make 6 parallel portions. Prepare the test solution according to the test solution preparation method determined under "4.3". According to the chromatographic conditions under "4.2", the peak areas of scutellaria acid AB in the test solution were measured at 0, 3 hours, 6 hours, 9 hours, 12 hours, 16 hours, 20 hours, and 24 hours, and the peak area RSD value was calculated. The results showed that the same test solution was sampled and measured at 0, 3 hours, 6 hours, 9 hours, 12 hours, 16 hours, 20 hours, and 24 hours, and the RSD value of the peak area of ​​scutellaria acid AB was 1.64%, indicating that the test solution under this method had good stability within 24 hours.

[0164] 4.4.7 Intermediate precision study

[0165] Different experimenters were selected to measure at different times and with different instruments. The standard decoction of Mianma Guanzhong slices was taken, ground into powder, and about 0.5g was taken, accurately weighed, and 6 parallel portions were prepared. The test solution was prepared according to the test solution preparation method determined under "4.3". The test was carried out according to the chromatographic conditions under "4.2". The samples were injected separately to determine the content of scutellaria baicalensis AB in the test solution, and the content and RSD value of scutellaria baicalensis AB were calculated and compared with the results of the repeatability test. The results showed that the same batch of samples were operated by different personnel on different instruments at different times, and the determination was repeated 6 times. The RSD value of scutellaria baicalensis AB content was 1.99%, which was 2.32% compared with the RSD value of 6 data in the repeatability test. According to the "Guidelines for Validation of Analytical Methods for Drug Quality Standards" of the 2020 edition of the Chinese Pharmacopoeia, when the content of the tested component in the sample was 100μg / g~1mg / g, the intermediate precision RSD limit was <6%. Therefore, different analysts operated on different dates and different chromatographs, and the intermediate precision of this method was good.

[0166] 4.4.8 Accuracy test

[0167] The sample recovery method was used to design three groups of experiments according to the ratio of the amount of reference substance added to the content of the test substance to be tested at about 0.5:1, 1:1, and 1.5:1, with 3 parallels in each group. Accurately weigh an appropriate amount of the AB reference substance of scutellaria baicalensis in a stoppered conical flask, and then take about 0.25g of the powder of the standard decoction of scutellaria baicalensis slices with the content determined in a conical flask, accurately weigh it, and prepare the test solution according to the method under "4.3". Sample injection and determination were carried out according to the chromatographic conditions under "4.2", and the results are shown in Table 3.

[0168] Table 3. Results of sample recovery of AB content of scutellaria baicalensis in standard decoction of scutellaria baicalensis slices

[0169]

[0170] The results showed that the recovery rate of scutellaria AB was 101.01%. According to the "Guidelines for Validation of Analytical Methods for Drug Quality Standards" of the 2020 edition of the Chinese Pharmacopoeia, when the content of the test component in the sample is between 100 μg / g and 1 mg / g, the recovery limit is 90% to 108%. The sample recovery rate of scutellaria AB ranged from 96.81% to 104.92%, the average sample recovery rate was 101.01%, and the RSD value was 2.81%, indicating that the method has good accuracy.

[0171] 4.4.9 Durability test

[0172] ① Investigation of different chromatographic columns

[0173] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, weigh it accurately, and prepare the test solution according to the test solution preparation method determined under "4.3". Except that the chromatographic columns are Kromasil 100-5-C18 (4.6mm×250mm, 5μm), Waters Xselect HSS T3 (4.6mm×250mm, 5μm), and Agilent ZORBAX Extend-C18 (4.6mm×250mm, 5μm), the other conditions are the chromatographic conditions under "4.2". Samples are injected and analyzed to determine the content of scutellaria baicalensis AB and the RSD value in the test solution. The results show that the separation effect of different brands of chromatographic columns is good, and the RSD value of scutellaria baicalensis AB content is 0.76%, indicating that the method has good durability for different brands of chromatographic columns.

[0174] ② Investigation of different column temperatures

[0175] Take the standard decoction of Mianma Guanzhong slices, grind it into powder, take about 0.5g, weigh it accurately, prepare the test solution according to the test solution preparation method determined under "4.3", except that the column temperature is 28℃, 30℃ and 32℃ respectively, other conditions are the chromatographic conditions under "4.2", sample analysis, and determine the content of AB and RSD value of scutellaria baicalensis in the test solution. The results showed that the RSD value of AB content of scutellaria baicalensis in the standard decoction of Mianma Guanzhong slices measured at different column temperatures (±2℃) was 1.42%, indicating that the analytical method has good durability within the range of column temperature ±2℃.

[0176] ③ Investigation of different flow rates

[0177] Take the standard decoction of Mian Ma Guan Zhong slices, grind it into powder, take about 0.5 g, weigh it accurately, and prepare the test solution according to the test solution preparation method specified in "4.3", except that the flow rate is 0.8 mL·min -1 、1.0mL·min -1 and 1.2 mL·min -1 Except for the above conditions, all other conditions were in accordance with the chromatographic conditions under "4.2". Samples were injected and analyzed to determine the content of AB and RSD value of scutellariae acid in the test solution. The results showed that at different flow rates (±0.2 mL·min -1 ) measured at a flow rate of ±0.2 mL min -1 Durability is good within the range.

[0178] 4.4.10 Sample determination

[0179] Take 15 batches of standard decoction of Rhizoma Cynomorii Scutellariae, prepare 15 test solutions according to the test solution preparation method under "4.3", inject and measure according to the chromatographic conditions under "4.2", record the peak area, and calculate the content of Rhizoma Cynomorii acid AB by external standard method. The results are shown in Table 4.

[0180] Table 415 Batch of Mianma Guanzhong Decoction Standard Decoction Results of Mianma Acid AB Content Determination

[0181]

[0182]

[0183] The results showed that the content of scutellaria baicalensis acid AB in 15 batches of standard decoctions of Mianma Guanzhong slices ranged from 0.0895 mg / g to 0.5914 mg / g, with a mean of 0.3006 mg / g, SD of 0.1556 mg / g, a range of ±30% of the mean of 0.2104 mg / g to 0.3908 mg / g, and a range of ±3 times the mean of SD of -0.1662 mg / g to 0.7674 mg / g. Considering the representativeness of the 15 batches of samples, the minimum value was taken as the lower limit, and the mean + 3 times the SD was taken as the upper limit, that is, the range was: 0.0895 mg / g to 0.7674 mg / g, and the adjusted data was: 0.100 mg / g to 0.750 mg / g. Therefore, it was finally determined that the standard decoction of Mianma Guanzhong slices contained scutellaria baicalensis acid AB (C 22 H 26 O8) should be 0.100mg / g~0.750mg / g.

[0184] 4.5 Determination of counterfeit decoction pieces and standard decoction samples

[0185] Take standard decoctions of single-bud dog fern, Japanese dog fern, small dog fern, cycad dog fern, brown ear fern, dog fern, osmanthus fern, terminal bud dog fern, pearl bud dog fern, and mianma dog fern, grind them into powder, take about 0.5g of each, weigh them accurately, prepare the test solution according to the test solution preparation method determined under "4.3", inject and measure according to the chromatographic conditions under "4.2", record the peak area, and calculate the content of scutellaria baicalensis AB by external standard method. The results are shown in Table 5.

[0186] Table 5 Determination results of AB content of cyperus rotundus and its counterfeit decoction pieces

[0187]

[0188] The results showed that the content of malachite acid AB was not contained in the standard decoctions of all counterfeit medicinal pieces or was lower than the specified range of content of the standard decoction of Mianma Guanzhong medicinal pieces (0.100 mg / g~0.750 mg / g), indicating that using malachite acid AB as an indicator for content determination of the standard decoction of Mianma Guanzhong medicinal pieces has good specificity.

[0189] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0190] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A method for identifying a standard decoction of Mianma Guanzhong slices and its counterfeits, characterized in that: The following steps are involved: Provide standard decoctions of Mianma Guanzhong slices and their counterfeits respectively; The standard decoctions of Mianma Guanzhong pieces and their counterfeits were extracted using extraction solvents to prepare test sample solutions; Dissolve the scutellaria baicalenic acid AB reference substance using a dissolving reagent to prepare a reference substance solution; Performing high performance liquid chromatography on the test solution and the reference solution, observing whether the chromatogram of the test solution has a chromatographic peak response at a position corresponding to the retention time of the chromatographic peak of the reference solution, and obtaining the content of scutellaria baicalensis acid AB in the standard decoction of the Mianma Guanzhong decoction pieces; The conditions for the high performance liquid chromatography detection include: mobile phase A is methanol, mobile phase B is phosphoric acid aqueous solution, and isocratic elution is adopted; the volume ratio of the mobile phase A to the mobile phase B is (70-85): (30-15).

2. The method for identifying the standard decoction of Mianma Guanzhong slices and its counterfeits according to claim 1, characterized in that: The high performance liquid chromatography detection satisfies at least one of the following conditions: (1) The detection wavelength is 210nm~400nm: (2) Column temperature is 20℃~30℃: (3) Flow rate: 0.5 mL min -1 ~1.5mL·min -1 ; (4) The injection volume is 5 μL to 15 μL.

3. The method for identifying the standard decoction of Mianma Guanzhong slices and its counterfeits according to claim 1, characterized in that: In the phosphoric acid aqueous solution, the volume percentage of phosphoric acid is 0.1% to 0.5%.

4. The method for identifying the standard decoction of Mianma Guanzhong slices and its counterfeits according to claim 1, characterized in that: The extraction step satisfies at least one of the following conditions: (1) The extraction solvent is alcohol or an aqueous solution of alcohol; optionally, the alcohol is methanol or ethanol; the volume percentage of alcohol in the aqueous solution of alcohol is 50% to 99%; (2) The extraction method is ultrasonic extraction, reflux extraction or extraction extraction; (3) The extraction time is 15 to 60 minutes; (4) The mass volume ratio of the standard decoction of the Mianma Guanzhong slices and their counterfeits to the extraction solvent is 0.5 g: (15-50) mL.

5. The method for identifying the standard decoction of Mianma Guanzhong slices and its counterfeits according to claim 1, characterized in that: Obtaining the content of cypermethrin acid AB in the standard decoction of the Cyperus rotundus slices comprises the following steps: Dissolve the reference substance using a dissolving reagent to prepare reference substance solutions of different concentrations; Injecting the reference substance solutions of different concentrations respectively, and performing high performance liquid chromatography detection; Obtain the peak areas corresponding to schizonepenic acid AB in the high performance liquid chromatography detection spectrum of the reference solution of different concentrations, take the concentration of the reference solution as the abscissa, take the peak areas corresponding to schizonepenic acid AB in the reference solution of different concentrations as the ordinate, draw a standard curve, and calculate the linear regression equation; Substituting the peak area corresponding to scutellaria AB in the HPLC detection spectrum obtained by performing HPLC detection on the test solution into the linear regression equation, the content of scutellaria AB in the test solution is calculated; Wherein, the reference substance includes scutellaria baicalenic acid AB.

6. The method for identifying the standard decoction of Mianma Guanzhong slices and its counterfeits according to any one of claims 1 to 5, characterized in that: The volume ratio of the mobile phase A to the mobile phase B is 80:

20.

7. The method for identifying the standard decoction of Mianma Guanzhong slices and its counterfeit according to any one of claims 1 to 5, characterized in that: The dissolving agent is methanol.

8. The method for identifying the standard decoction of Mianma Guanzhong slices and its counterfeits according to any one of claims 1 to 5, characterized in that: The content of scutellaria baicalensis acid AB in the standard decoction of the Rhizoma Cynomorii Scutellariae decoction pieces is 0.100 mg / g to 0.750 mg / g.

9. The method for identifying the standard decoction of Mianma Guanzhong slices and its counterfeit according to any one of claims 1 to 5, characterized in that: The counterfeits include one or more of single-bud dog spine fern, Japanese dog spine, small dog spine, cycad dog spine, brown ear fern, dog spine, octopus fern, terminal dog spine and bulbil dog spine.

10. Use of the standard decoction of Mianma Guanzhong slices and the method for identifying counterfeits thereof as described in any one of claims 1 to 9 in the quality control of Mianma Guanzhong slices.

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