Characteristic chromatogram and content determination construction method of phloroglucinol components in rhizoma dryopteris crassirhizomae medicinal material and application of identification of adulterants

Abstract

The invention relates to a male fern rhizome medicinal material phloroglucinol characteristic chromatogram, a content determination construction method and adulterant identification application, and discloses an HPLC characteristic chromatogram method of phloroglucinol components in a male fern rhizome medicinal material, which is combined with a high performance liquid chromatography-mass spectrometry method. Firstly, the characterization of phloroglucinol components in the rhizoma dryopteris crassirhizomae medicinal material is realized, the specific chromatogram method is relatively good in separation degree of each characteristic peak, relatively good in precision, repeatability and durability, capable of relatively comprehensively reflecting the characteristics of main chemical components of the rhizoma dryopteris crassirhizomae medicinal material, and capable of being used for identifying rhizoma dryopteris crassirhizomae adulterants; the important reference is provided for establishing the quality standard of the rhizoma dryopteris crassirhizomae medicinal material. Besides, the content of the active ingredient flavaspidic acid AB in the rhizoma dryopteris crassirhizomae medicinal material can be simultaneously measured by the specific chromatogram method constructed by the invention, the content of the flavaspidic acid AB in the rhizoma dryopteris crassirhizomae medicinal material is firstly measured, and methodology verifies that the method has specificity, precision, repeatability and accuracy.

Description

Technical Field

[0001] The present application relates to the field of drug analysis technology, and in particular to a characteristic spectrum of phloroglucinol components of the medicinal material Guanzhong, a method for constructing a content determination method, and an application for identifying adulterants. Background Art

[0002] The chemical composition of traditional Chinese medicine is a complex multi-component system. The traditional chemical drug quality control model is difficult to evaluate the intrinsic quality of traditional Chinese medicine. Fingerprint / characteristic spectrum, due to its overall control concept, can more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine and its preparations, and then comprehensively describe and evaluate the quality of drugs. It is increasingly used in the field of chemical composition and quality control research of traditional Chinese medicine. There are many studies on the application of fingerprint / characteristic spectrum to the quality control of traditional Chinese medicine, but the existing research on fingerprint / characteristic spectrum of Mianma Guanzhong mainly focuses on components with good water solubility, mainly phenylpropanoids such as chlorogenic acid, caffeic acid, and protocatechuic acid, followed by a few flavonoid components. For example, the HPLC characteristic spectrum of decoction pieces, standard decoctions, and formula granules established in the literature shows 12 common peaks, and 7 chromatographic peak components are identified, mainly phenylpropanoid components such as chlorogenic acid, neochlorogenic acid, and caffeic acid. However, studies have shown that the main chemical components of Rhizoma Cyrthospermi are phloroglucinols, including phloroglucinol AB, phloroglucinol ABA, Rhizoma Cyrthospermi ABBA, etc., which are also the material basis for Rhizoma Cyrthospermi to exhibit antiviral, antibacterial and other pharmacological activities. Therefore, the existing Rhizoma Cyrthospermi fingerprints / characteristic maps cannot fully reflect the intrinsic quality of Rhizoma Cyrthospermi.

[0003] Mianma Guanzhong is the dried rhizome and petiole residue of Dryopteris crassirhizoma Nakai, a plant of Dryopteris crassirhizoma Nakai. Mianma Guanzhong contains phloroglucinols, flavonoids, terpenes and phenylpropanoids, and has the effects of clearing heat, detoxifying and expelling insects. Modern studies have shown that phloroglucinol compounds are the main components of Mianma Guanzhong that exert antiviral, antibacterial, antitumor and expelling insects. In recent years, due to the increase in demand for Mianma Guanzhong, counterfeits have frequently appeared in the market, such as Osmanthus fernus, small Guanzhong, Dryopteris crassirhizoma Nakai, and rhizomes of Pteridaceae, which affect the safety of Mianma Guanzhong based on different efficacy. However, there are few existing means to distinguish Mianma Guanzhong from its counterfeits. Only traditional properties, micro-properties identification methods, and cross-section microscopic identification techniques are used for differentiation and research, but the judgment of appearance properties is subjective, and it is not applicable to medicinal powder that has lost its appearance properties, which has great limitations. Therefore, it is necessary to establish a fingerprint spectrum / characteristic spectrum of the main chemical components of the phloroglucinol class and an effective ingredient content detection index as a standard for its qualitative identification, thereby ensuring its safety in use. Summary of the invention

[0004] Based on this, it is necessary to provide a characteristic spectrum of the phloroglucinol components of the medicinal material Corydalis guanyin, a content determination construction method and an application for identification of adulterants, which can provide a more comprehensive and effective evaluation for the quality control of the medicinal material Corydalis guanyin.

[0005] The technical solution of this application is as follows:

[0006] In a first aspect of the present invention, a method for constructing a characteristic spectrum of phloroglucinol components of the medicinal material Rhizoma Cynomorii is provided, comprising the following steps:

[0007] Extract the Mianma Guanzhong medicinal material using an extraction solvent to prepare a test solution;

[0008] The phloroglucinol components of the medicinal material of Mianma Guanzhong were dissolved by using a dissolving reagent to prepare a reference solution;

[0009] The test solution and the reference solution are subjected to high performance liquid chromatography to construct a characteristic spectrum of phloroglucinol components of the medicinal material of Mianma Guanzhong;

[0010] The conditions for the HPLC detection include: mobile phase A is a mixture of acetonitrile, methanol and aqueous phosphoric acid, mobile phase B is aqueous phosphoric acid, and gradient elution is used;

[0011] The procedure of the gradient elution is:

[0012] From 0 to 60 min, the volume percentage of mobile phase A increased from 60% to 64%;

[0013] From 60 to 65 minutes, the volume percentage of mobile phase A was maintained at 64%;

[0014] The phloroglucinol components include phloroglucinol AP, norphloroglucinol PB and phloroglucinol AB.

[0015] In some embodiments, the HPLC detection satisfies at least one of the following conditions:

[0016] (1) The detection wavelength is 210nm~400nm;

[0017] (2) Column temperature is 20℃~40℃;

[0018] (3) Flow rate: 0.5 mL min -1 ~1.5mL·min -1 ;

[0019] (4) The injection volume is 1 μL to 10 μL;

[0020] (5) The chromatographic column is a C18 chromatographic column.

[0021] In some embodiments, the volume percentages of acetonitrile and methanol in the mobile phase A are (1-8): (1-2); and / or

[0022] The volume ratio of the total volume of acetonitrile and methanol to the phosphoric acid aqueous solution is 1:(0.1% to 0.3%); and / or

[0023] The volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.4%; and / or

[0024] The dissolving agent is methanol.

[0025] In some of the embodiments, the method for constructing the characteristic spectrum of phloroglucinols of the Mianma Guanzhong medicinal material adopts high performance liquid chromatography-mass spectrometry to identify the characteristic peaks of the characteristic spectrum of the Mianma Guanzhong medicinal material;

[0026] The conditions of the HPLC-MS method include: mobile phase A is a mixture of acetonitrile, methanol and formic acid aqueous solution, mobile phase B is formic acid aqueous solution, and gradient elution is used;

[0027] The procedure of the gradient elution is:

[0028] From 0 to 60 min, the volume percentage of mobile phase A increased from 60% to 64%;

[0029] From 60 to 65 minutes, the volume percentage of mobile phase A was maintained at 64%;

[0030] The characteristic peaks of the characteristic spectrum of the medicinal material of Rhizoma Cyrthospermi include: Peak 1 is cyrthospermic acid AP, Peak 2 is norcyrthospermic acid PB, and Peak 8 is cyrthospermic acid AB.

[0031] In some embodiments, the conditions of the HPLC-MS method further include: in the mobile phase A, the volume percentages of acetonitrile and methanol are (1-8): (1-2); and / or

[0032] The volume ratio of the total volume of acetonitrile and methanol to the formic acid aqueous solution is 0.1% to 0.3%; and / or

[0033] In the formic acid aqueous solution, the volume percentage of formic acid is 0.05% to 0.3%.

[0034] In some embodiments, the step of extracting the medicinal material of Cynomorium mianmai using an extraction solvent satisfies at least one of the following conditions:

[0035] (1) The extraction solvent is methanol or methanol-water solution; optionally, the volume percentage of methanol in the methanol-water solution is 30% to 70%;

[0036] (2) The extraction method is ultrasonic extraction or reflux extraction;

[0037] (3) The extraction time is 15 to 60 minutes;

[0038] (4) The mass volume ratio of the Mian Ma Guan Zhong medicinal material and the extraction solvent is 1 g: (15-50) mL.

[0039] The second aspect of the present invention provides a method for determining the content of phloroglucinol components in the medicinal material of Rhizoma Cynomorii, comprising the following steps:

[0040] Extract the Mianma Guanzhong medicinal material using an extraction solvent to prepare a test solution;

[0041] Dissolve the reference substance using a dissolving reagent to prepare reference substance solutions of different concentrations;

[0042] Injecting the reference substance solutions of different concentrations respectively, and performing high performance liquid chromatography detection;

[0043] Obtain the peak areas corresponding to the active ingredients in the reference solution at different concentrations, plot a standard curve with the concentration of the reference solution as the abscissa and the peak areas corresponding to the active ingredients in the reference solution at different concentrations as the ordinate, and calculate a linear regression equation;

[0044] Obtaining the peak area corresponding to the active ingredient in the test solution, and substituting it into the linear regression equation to calculate the content of the active ingredient in the test solution;

[0045] Wherein, the phloroglucinol component includes phloroglucinol AB; the reference substance includes phloroglucinol AB; the preparation method of the test solution and the conditions for high performance liquid chromatography detection are the same as above.

[0046] A third aspect of the present invention provides a method for identifying the medicinal material of Rhizoma Cynomorii and its mixed products, comprising the following steps:

[0047] Extracting the Mianma Guanzhong medicinal material and its adulterants with extraction solvents respectively to prepare test sample solutions;

[0048] The test solution is subjected to high performance liquid chromatography to construct characteristic spectra of the Mianma Guanzhong medicinal material and its adulterants, and observe whether the characteristic spectra of each adulterant have corresponding characteristic peaks of phloroglucinol components with the characteristic spectra of the Mianma Guanzhong medicinal material;

[0049] Wherein; the preparation method of the test solution and the conditions for high performance liquid chromatography detection are the same as above.

[0050] The fourth aspect of the present invention provides a method for preparing scutellaria baicalenic acid AB, comprising the following steps:

[0051] Extracting the powder of the medicinal material of Mienma cyrtomium by using an extraction solvent, concentrating and drying the obtained extract, and preparing a medicinal material of Mienma cyrtomium extract;

[0052] Mixing the extract of the Chinese cyrtomium sibiricum and water, and extracting the resulting mixture using an extraction solvent;

[0053] The obtained extract is separated by column chromatography, and the chromatographic liquid containing the phloroglucinol component of the mianma guanzhong, flavocal acid AB, is collected;

[0054] The chromatographic solution is purified to prepare scutellariae acid AB.

[0055] In some embodiments, the extraction solvent is an ethanol aqueous solution; optionally, the volume percentage of ethanol in the ethanol aqueous solution is 80% to 99%;

[0056] The extraction solvent is one or more of petroleum ether, ethyl acetate and n-butanol;

[0057] The elution solution used in the column chromatography method includes a mixed solution of petroleum ether, ethyl acetate and formic acid.

[0058] Compared with the prior art, this application has the following beneficial effects:

[0059] The present application constructs a characteristic spectrum of the phloroglucinol components of the Mianma Guanzhong medicinal material containing multiple characteristic peaks by using specific high performance liquid chromatography conditions to detect and analyze the Mianma Guanzhong medicinal material. The characteristic spectrum method has good separation of the characteristic peaks, and the method has good precision, repeatability and durability. It can more comprehensively reflect the main chemical composition characteristics of the Mianma Guanzhong medicinal material, and provide an overall quality control basis for the quality standards of the Mianma Guanzhong medicinal material.

[0060] At the same time, this application combines high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to achieve the characterization of the phloroglucinol components of the Mianma Guanzhong medicinal material, and can be used to identify Mianma Guanzhong adulterated products. It can provide an important reference for the establishment of quality standards for Mianma Guanzhong medicinal materials, and provide a more comprehensive and effective evaluation for the quality control of Mianma Guanzhong medicinal materials.

[0061] In addition, the characteristic spectrum method constructed in this application can simultaneously determine the content of the active ingredient scutellaria baicalensis AB in the medicinal material of Coptis chinensis, and first realize the determination of the content of scutellaria baicalensis AB in the medicinal material of Coptis chinensis. After methodological verification, this method has good specificity, precision, repeatability, accuracy and durability, which provides a reference for the selection of content indicators when establishing the quality standard of Coptis chinensis medicinal material. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] 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.

[0063] Figure 1 This is a full-wavelength scanning 3D spectrum of the characteristic spectrum of the medicinal material Corydalis cyrtomium in Example 1.

[0064] Figure 2 It is a comparative chromatogram of different detection wavelengths of the characteristic spectrum of the medicinal material of Mianma Guanzhong in Example 1.

[0065] Figure 3 It is a comparative chromatogram of different elution gradients of the characteristic spectrum of the medicinal material of Mianma Guanzhong in Example 1.

[0066] Figure 4 This is a comparative chromatogram of the characteristic spectrum of the medicinal material Rhizoma Cynomorii in Example 1 eluted with different organic phases.

[0067] Figure 5 It is a comparative chromatogram of the characteristic spectrum of the medicinal material of Mianma Guanzhong at different column temperatures in Example 1.

[0068] Figure 6 The chromatograms of different extraction solvents for the characteristic spectrum of the medicinal material of Mianma Guanzhong in Example 1 are shown.

[0069] Figure 7 This is a chromatogram showing different extraction methods for the characteristic spectrum of the medicinal material Rhizoma Cynomorii in Example 1.

[0070] Figure 8 This is a chromatogram showing the characteristic spectrum of the medicinal material Rhizoma Cynomorii in Example 1 with different extraction solvent dosages.

[0071] Fig. 9 The chromatograms of the characteristic spectrum of the medicinal material of Mianma Guanzhong at different extraction times in Example 1 are shown.

[0072] Fig.10 This is a chromatogram showing the different extraction times of the characteristic spectrum of the medicinal material of Mianma Guanzhong in Example 1.

[0073] Fig.11 This is a chromatogram of the characteristic spectrum of the medicinal material Rhizoma Cynomorii in Example 1, which was examined using different amounts of extraction solvents.

[0074] Fig.12 This is the common pattern of the characteristic spectra of 16 batches of Rhizoma Cyperi in Example 1.

[0075] Fig.13The total ion flow diagram and ultraviolet absorption chromatogram of the test solution of the Mianma Guanzhong medicinal material in Example 1 are shown.

[0076] Fig.14 This is the primary mass spectrometry scan of peak No. 1 in Example 1 (negative ion mode).

[0077] Fig.15 This is the primary mass spectrometry scan of peak No. 1 in Example 1 (positive ion mode).

[0078] Fig.16 This is the secondary mass spectrometry scan of the peak m / z=403.1393 in Example 1 (negative ion mode).

[0079] Fig.17 It is the secondary mass spectrometry scan diagram (positive ion mode) of the peak m / z=403.1393 in Example 1.

[0080] Fig.18 It is the structural formula of flavone acid AP in Example 1.

[0081] Fig.19 This is the possible fragmentation mode of the peak m / z=403.1393 in Example 1 (negative ion mode).

[0082] Fig. 20 This is the primary mass spectrometry scan of peak No. 2 in Example 1 (negative ion mode).

[0083] Fig.21 This is the primary mass spectrometry scan of Peak 2 in Example 1 (positive ion mode).

[0084] Fig. 22 This is the secondary mass spectrometry scan of the peak m / z=417.1550 in Example 1 (negative ion mode).

[0085] Fig.23 This is the secondary mass spectrometry scan of the peak m / z=417.1550 in Example 1 (positive ion mode).

[0086] Fig.24 It is the structural formula of norflavonoids acid PB in Example 1.

[0087] Fig.25 This is the possible fragmentation mode of the peak m / z=417.1550 in Example 1 (negative ion mode).

[0088] Fig.26 This is the primary mass spectrometry scan of peak No. 8 in Example 1 (negative ion mode).

[0089] Fig. 27 This is the primary mass spectrometry scan of peak No. 8 in Example 1 (positive ion mode).

[0090] Fig.28 This is the secondary mass spectrometry scan of the peak m / z=417.1555 in Example 1 (negative ion mode).

[0091] Fig.29 This is the secondary mass spectrometry scan of the peak m / z=417.1555 in Example 1 (positive ion mode).

[0092] Fig.30 It is the structural formula of scutellariae acid AB in Example 1.

[0093] Fig.31 This is the possible fragmentation mode of the peak m / z=417.1550 in Example 1 (negative ion mode).

[0094] Fig.32 This is the H NMR spectrum of the scutellaria baicalenic acid AB monomer prepared in Example 1.

[0095] Fig.33 This is the NMR carbon spectrum of the scutellaria baicalenic acid AB monomer prepared in Example 1.

[0096] Fig.34 The characteristic spectrum of the AB monomer of scutellaria baicalensis prepared in Example 1 is compared with that of the test sample.

[0097] Fig.35 This is the reference characteristic spectrum of the medicinal material Rhizoma Cynomorii in Example 1.

[0098] Fig.36 This is a graph showing the specificity of the characteristic graph of the medicinal material Rhizoma Cynomorii in Example 1.

[0099] Fig.37 This is the characteristic spectrum of the medicinal material Rhizoma Cynomorii in Example 1 observed at different column temperatures.

[0100] Fig.38 This is the characteristic spectrum of the medicinal material Mianma Guanzhong in Example 1, and the spectrum is investigated at different flow rates.

[0101] Fig.39 This is a comparative chromatogram of the characteristic spectrum of the medicinal material Rhizoma Cynomorii in Example 1 using different chromatographic columns.

[0102] Fig.40 This is a comparison chart of the characteristic spectra of the Mianma Guanzhong medicinal material and its counterfeit product in Example 1.

[0103] Fig.41 This is a graph showing the specificity of the determination of the AB content of scutellaria baicalensis in the medicinal material Cyperus rotundus in Example 1.

[0104] Fig.42 This is a peak purity investigation diagram for the determination of the AB content of scutellaria baicalensis in the medicinal material of Example 1.

[0105] Fig.43This is the standard curve of AB of scutellaria baicalenic acid in Example 1.

[0106] Fig.44 This is the determination of the characteristic spectrum of the Mianma Guanzhong medicinal material (S1) in Example 2. DETAILED DESCRIPTION

[0107] 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.

[0108] 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.

[0109] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0110] As used herein, "one or more" refers to any one, any two, or any two or more of the listed items.

[0111] 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.

[0112] 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.

[0113] Herein, "first aspect", "second aspect", "third aspect", "fourth aspect", etc. are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. In addition, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. In the description of the present application, the meaning of "several" is at least one, such as one, two, etc., unless otherwise clearly and specifically defined.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] According to the investigation, the 2020 edition of the Chinese Pharmacopoeia lacks fingerprint / characteristic spectrum and active ingredient content determination items for the Mianma Guanzhong medicinal material, making it difficult to control its quality as a whole. Therefore, the quality standards of Mianma Guanzhong medicinal materials need to be further improved. Fingerprint / characteristic spectrum can characterize the main representative chemical components, and quantitative analysis of the effective ingredients of Chinese medicinal materials can judge the quality of the medicine. It is an important method to control and evaluate the quality of medicinal materials and identify related counterfeits. Studies have shown that the chemical components of Mianma Guanzhong are mainly phloroglucinols, which are also the main material basis for the antiviral and antibacterial pharmacological activities of Mianma Guanzhong. Therefore, it is necessary to establish a characteristic spectrum of phloroglucinols in Mianma Guanzhong to further comprehensively control its quality. The current reported research on fingerprint / characteristic spectrum of Mianma Guanzhong medicinal materials mainly focuses on non-main active ingredients such as phenylpropanoids and flavonoids. The content detection indicators are mainly total flavonoids, total phloroglucinols or chlorogenic acids, which are not accurate or specific. At present, there is a lack of reports on the content determination of phloroglucinol components with good pharmacological activity in Rhizoma Cynomorii, such as phloroglucinic acid AB[12,13].

[0120] In view of the shortcomings of the existing quality control methods for Corydalis guanyin, such as the inability to distinguish counterfeits, the non-specificity of quality control ingredients, and the poor accuracy, the technical personnel of the present application unexpectedly discovered after extensive research that a method for constructing a characteristic spectrum and content determination of Corydalis guanyin phloroglucinols that can identify counterfeits, which is efficient and specific, can be provided. That is, by establishing a characteristic spectrum of small-polar phloroglucinols for Corydalis guanyin, its main active ingredients, phloroglucinols, are simultaneously characterized as much as possible, and the content of the exclusive effective ingredient, scutellaria baicalensis AB, thereby effectively and qualitatively identifying related confusing products, comprehensively evaluating and controlling them, ensuring their clinical safety, and providing an important reference for establishing comprehensive quality standards for medicinal materials.

[0121] In a first aspect of the present invention, a method for constructing a characteristic spectrum of phloroglucinol components of the medicinal material Rhizoma Cynomorii is provided, comprising the following steps:

[0122] Extract the Mianma Guanzhong medicinal material using an extraction solvent to prepare a test solution;

[0123] The phloroglucinol components of the medicinal material of Mianma Guanzhong were dissolved by using a dissolving reagent to prepare a reference solution;

[0124] The test solution and the reference solution are subjected to high performance liquid chromatography to construct a characteristic spectrum of phloroglucinol components of the medicinal material of Mianma Guanzhong;

[0125] The conditions for the HPLC detection include: mobile phase A is a mixture of acetonitrile, methanol and aqueous phosphoric acid, mobile phase B is aqueous phosphoric acid, and gradient elution is used;

[0126] The procedure of the gradient elution is:

[0127] From 0 to 60 min, the volume percentage of mobile phase A increased from 60% to 64%;

[0128] From 60 to 65 minutes, the volume percentage of mobile phase A was maintained at 64%;

[0129] The phloroglucinol components include phloroglucinol AP, norphloroglucinol PB and phloroglucinol AB.

[0130] In some examples, the conditions for the HPLC detection include: a detection wavelength of 210nm to 400nm. Further, the wavelength of the HPLC detection is 254nm to 330nm. It is understandable that the wavelength of the HPLC detection includes but is not limited to 254nm, 265nm, 295nm, 315nm, and 330nm.

[0131] In some examples, the conditions for the HPLC detection include: a column temperature of 20° C. to 40° C. It is understandable that the column temperature for the HPLC detection includes but is not limited to 20° C., 24° C., 28° C., 30° C., 36° C., and 40° C.

[0132] 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 of the HPLC detection 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 .

[0133] In some examples, the conditions for the HPLC detection include: the injection volume is 1 μL to 10 μL. It is understandable that the injection volume includes but is not limited to 1 μL, 3 μL, 5 μL, 7 μL, and 10 μL.

[0134] In some of the examples, the conditions for the HPLC detection include: the chromatographic column is a C18 chromatographic column.

[0135] In some examples, the volume percentage of acetonitrile and methanol in the mobile phase A is (1-8):(1-2). It can be understood that the volume percentage of acetonitrile and methanol in the mobile phase A includes but is not limited to 1:1, 5:1, 8:1, 3:2, 5:2, 7:2.

[0136] In some examples, the volume ratio of the total volume of acetonitrile and methanol to the phosphoric acid aqueous solution is 1:(0.1% to 0.3%). It is understood that the volume ratio of the total volume of acetonitrile and methanol to the phosphoric acid aqueous solution includes but is not limited to 1:0.1%, 1:0.2%, and 1:0.3%.

[0137] In some examples, the volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.4%. It is understandable that the volume percentage of phosphoric acid in the phosphoric acid aqueous solution includes but is not limited to 0.05%, 0.1%, 0.2%, 0.3%, and 0.4%.

[0138] In some examples, the dissolving agent is methanol.

[0139] In some of the examples, the extraction solvent is methanol or a methanol-water solution; optionally, in the methanol-water solution, the volume percentage of methanol is 30% to 70%.

[0140] In some of these examples, the extraction method is ultrasonic extraction or reflux extraction.

[0141] In some of the examples, the extraction time was 15 min to 60 min.

[0142] In some of the examples, the mass-to-volume ratio of the Glehnia littoralis medicinal material and the extraction solvent is 1 g: (15-50) mL.

[0143] In some of the examples, the preparation of the test solution further includes the step of extracting the extracted solution using an extraction solvent.

[0144] In some of the examples, the extraction solvent is a mixture of water and petroleum ether; further, the volume percentage of water and petroleum ether is (1-3): (1-3).

[0145] In some of the examples, the volume ratio of the extraction solvent to the extraction solvent is (15-40) mL:25 mL.

[0146] In some of the examples, the method for constructing the characteristic spectrum of phloroglucinols of the Mianma Guanzhong medicinal material adopts high performance liquid chromatography-mass spectrometry to identify the characteristic peaks of the characteristic spectrum of the Mianma Guanzhong medicinal material;

[0147] The conditions of the HPLC-MS method include: mobile phase A is a mixture of acetonitrile, methanol and formic acid aqueous solution, mobile phase B is formic acid aqueous solution, and gradient elution is used;

[0148] The procedure of the gradient elution is:

[0149] From 0 to 60 min, the volume percentage of mobile phase A increased from 60% to 64%;

[0150] From 60 to 65 minutes, the volume percentage of mobile phase A was maintained at 64%;

[0151] The characteristic peaks of the characteristic spectrum of the medicinal material of Rhizoma Cyrthospermi include: Peak 1 is cyrthospermic acid AP, Peak 2 is norcyrthospermic acid PB, and Peak 8 is cyrthospermic acid AB.

[0152] In some examples, the conditions of the HPLC-MS method also include: the volume percentage of acetonitrile and methanol in the mobile phase A is (1-8): (1-2). It can be understood that the volume percentage of acetonitrile and methanol in the mobile phase A includes but is not limited to 1:1, 5:1, 8:1, 3:2, 5:2, 7:2.

[0153] In some examples, the conditions of the HPLC-MS method also include: the volume ratio of the total volume of the acetonitrile and methanol to the formic acid aqueous solution is 0.1% to 0.3%. It can be understood that the volume ratio of the total volume of the acetonitrile and methanol to the formic acid aqueous solution includes but is not limited to 0.1%, 0.2%, and 0.3%.

[0154] In some examples, the conditions of the HPLC-MS method also include: the volume percentage of formic acid in the formic acid aqueous solution is 0.05% to 0.3%. It can be understood that the volume percentage of formic acid in the formic acid aqueous solution includes but is not limited to 0.05%, 0.1%, 0.2%, and 0.3%.

[0155] In some examples, the HPLC-MS method further satisfies at least one of the following conditions:

[0156] (1) The detection wavelength is 210nm~400nm;

[0157] (2) Column temperature is 20℃~40℃;

[0158] (3) Flow rate: 0.5 mL min -1 ~1.5mL·min -1 ;

[0159] (4) Injection volume: 1 μL to 10 μL.

[0160] In some of the examples, the chromatographic peaks of the characteristic spectrum of the medicinal material Corydalis guanyin contain 8 characteristic peaks, among which the 3 identified characteristic peaks include: Peak 1 is guanylic acid AP, Peak 2 is norguanylic acid PB, and Peak 8 is guanylic acid AB.

[0161] The second aspect of the present invention provides a method for determining the content of phloroglucinol components in the medicinal material of Rhizoma Cynomorii, comprising the following steps:

[0162] Extract the Mianma Guanzhong medicinal material using an extraction solvent to prepare a test solution;

[0163] Dissolve the reference substance using a dissolving reagent to prepare reference substance solutions of different concentrations;

[0164] Injecting the reference substance solutions of different concentrations respectively, and performing high performance liquid chromatography detection;

[0165] Obtain the peak areas corresponding to the active ingredients in the reference solution at different concentrations, plot a standard curve with the concentration of the reference solution as the abscissa and the peak areas corresponding to the active ingredients in the reference solution at different concentrations as the ordinate, and calculate a linear regression equation;

[0166] Obtaining the peak area corresponding to the active ingredient in the test solution, and substituting it into the linear regression equation to calculate the content of the active ingredient in the test solution;

[0167] Wherein, the phloroglucinol component includes phloroglucinol AB; the reference substance includes phloroglucinol AB; the preparation method of the test solution and the conditions for high performance liquid chromatography detection are the same as above.

[0168] In some of the examples, in the method for determining the content of phloroglucinol components in the Mianma Guanzhong medicinal material, the extraction step satisfies at least one of the following conditions:

[0169] (1) The extraction solvent is methanol or a methanol aqueous solution; optionally, the volume percentage of methanol in the methanol aqueous solution is 50%-95%;

[0170] (2) The extraction method is ultrasonic extraction or reflux extraction;

[0171] (3) The extraction time is 15 to 45 minutes;

[0172] (4) The mass volume ratio of the Mian Ma Guan Zhong medicinal material and the extraction solvent is 1.0 g: (15-50) mL.

[0173] A third aspect of the present invention provides a method for identifying the medicinal material of Rhizoma Cynomorii and its mixed products, comprising the following steps:

[0174] Extracting the Mianma Guanzhong medicinal material and its adulterants with extraction solvents respectively to prepare test sample solutions;

[0175] The test solution is subjected to high performance liquid chromatography to construct characteristic spectra of the Mianma Guanzhong medicinal material and its adulterants, and observe whether the characteristic spectra of each adulterant have corresponding characteristic peaks of phloroglucinol components with the characteristic spectra of the Mianma Guanzhong medicinal material;

[0176] Wherein; the preparation method of the test solution and the conditions for high performance liquid chromatography detection are the same as above.

[0177] 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 the medicinal materials of Mianma Guanzhong 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. Guanzhong varieties are rich in resources, and there are many varieties of Guanzhong used in different regions. In recent years, Mianma Guanzhong has played a significant role in antiviral (anti-new coronavirus, influenza virus and respiratory related viruses), which has increased its demand, and counterfeit products have frequently appeared in the market. According to the survey, common counterfeit products such as Ziqi Guanzhong, Xiao Guanzhong, Dryopteris Guanzhong, and Globose Pteridaceae rhizomes affect the safety of Mianma Guanzhong based on different efficacy. Therefore, it is necessary to establish a fingerprint spectrum / characteristic spectrum of Mianma Guanzhong that can characterize its main chemical components and an effective ingredient content detection index as a standard for its qualitative identification, thereby ensuring its drug safety.

[0178] The fourth aspect of the present invention provides a method for preparing scutellaria baicalenic acid AB, comprising the following steps:

[0179] Extracting the powder of the medicinal material of Mienma cyrtomium by using an extraction solvent, concentrating and drying the obtained extract, and preparing a medicinal material of Mienma cyrtomium extract;

[0180] Mixing the extract of the Chinese cyrtomium sibiricum and water, and extracting the resulting mixture using an extraction solvent;

[0181] The obtained extract is separated by column chromatography, and the chromatographic liquid containing the phloroglucinol component of the mianma guanzhong, flavocal acid AB, is collected;

[0182] The chromatographic solution is purified to prepare scutellariae acid AB.

[0183] In some of the examples, in the step of extracting the powder of the medicinal material Glehnia littoralis using an extraction solvent, the extraction solvent is an ethanol-water solution; optionally, the volume percentage of ethanol in the ethanol-water solution is 80%-99%.

[0184] In some of the examples, in the step of extracting the Corydalis cyrtomium powder using an extraction solvent, the mass ratio of the extraction solvent to the Corydalis cyrtomium powder is (8-15):1.

[0185] In some examples, the extraction solvent is one or more of petroleum ether, ethyl acetate and n-butanol.

[0186] In some examples, the elution solution used in the column chromatography method includes a mixed solution of petroleum ether, ethyl acetate and formic acid.

[0187] In some of the examples, in the elution solution, the volume ratio of petroleum ether, ethyl acetate and formic acid is (20-10):1:0.2.

[0188] In some of the examples, during the separation process, a mixed solution of petroleum ether, ethyl acetate and formic acid in a volume ratio is sequentially used for gradient elution, and the chromatographic solution containing the scutellaria baicalenic acid AB is collected.

[0189] In some of these examples, the solvent used in the purification step was a mixture of petroleum ether and ethyl acetate.

[0190] 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.

[0191] Example 1

[0192] This embodiment provides a method for constructing a characteristic spectrum of phloroglucinols in the medicinal material of Mianma Guanzhong and a method for determining the content of effective ingredients and an application for identifying adulterants, as follows:

[0193] 1. Instruments and Materials

[0194] 1.1 Medicinal Materials

[0195] 16 batches of Mianma Guanzhong medicinal material samples were collected from different regions in Northeast China, as shown in Table 1. They were identified by the Quality Center of Guangdong Yifang Pharmaceutical Co., Ltd. as the dried rhizomes and petiole residues of Dryopteris crassirhizoma, a plant of the Dryopteris family.

[0196] Table 1 Origin information of Mianma Guanzhong medicinal materials

[0197]

[0198] 1.2 Instruments and reagents

[0199] Waters high performance liquid chromatograph (Arc, Waters Corporation), Agilent high performance liquid chromatograph (1260, Agilent Corporation), Thermo (ultra) high performance liquid chromatograph (Vanquish, Thermo Fisher), Thermo Vanquish Flex (ultra) high performance liquid-Thermo Fisher QE high resolution mass spectrometer (Thermo Fisher), Waters XSelect HSSC18 column (4.6 mm × 250 mm, 5 μm), Kromasil 100-5-C18 column (4.6 mm × 250 mm, 5 μm), Agilent ZORBAX Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm), Thermo Scientific™ Acclaim™ 120C18 column (4.6 mm × 250 mm, 5 μm), Agilent ZORBAX Extend-C18 chromatographic column (4.6 mm × 250 mm, 5 μm), CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), 1 / 10,000 balance (ME204E, Mettler-Toledo), 1 / 1000 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.), petroleum ether 60-90 ° C (Tianjin Best Chemical Co., Ltd.) were all analytical grade; acetonitrile, methanol (Merck Co., Ltd.), phosphoric acid (Tianjin Komiou Chemical Reagent Co., Ltd.) were all chromatographic grade; formic acid (Shanghai Anpu Experimental Technology Co., Ltd.) was UPLC / LC-MS grade; ultrapure water (laboratory-made); and scutellaria baicalensis AB reference substance (laboratory-made).

[0200] 2. Experimental methods

[0201] 2.1 Establishment of HPLC characteristic spectrum of Rhizoma Cynomorii

[0202] 2.1.1 Optimization of chromatographic conditions

[0203] (1) Selection of detection wavelength

[0204] By consulting the literature, 254 nm, 280 nm, and 330 nm were selected as the detection wavelengths of the Mianma Guanzhong medicinal material samples. The chromatographic conditions were as follows: chromatographic column: Agilent ZORBAX RRHD StableBond C18 chromatographic column (2.1×150 mm, 1.8 μm); mobile phase: A was methanol, and B was a 0.1% mass concentration phosphoric acid aqueous solution; the gradient elution order was: 0-30 min, 10%→60% A; 30-50 min, 60%→80% A; 50-60 min, 80%→95% A; 60-65 min, 95%→100% A; volume flow rate 0.30 mL·min -1 ; Column temperature 30℃; injection volume 1μL, and full wave scanning, record the absorption spectrum of the sample in the range of 210nm~400nm, the results are shown in Figure 1 and Figure 2 .

[0205] The results show that the chromatographic peak information is more at the detection wavelength of 280nm, so 280nm was selected as the detection wavelength of the characteristic spectrum.

[0206] (2) Selection of mobile phase gradient

[0207] As shown in Table 2-A, Table 2-B, Table 2-C and Table 2-D below, the chromatographic conditions are: chromatographic column: Waters XSelect HSSC18 chromatographic column (4.6 mm×250 mm, 5 μm); mobile phase: A is acetonitrile-methanol (7:2) (0.2% phosphoric acid), B is 0.2% phosphoric acid aqueous solution; volume flow rate 1.0 mL·min -1 ; detection wavelength 280nm; column temperature 25℃; injection volume 5μL, the effect of different elution gradients on chromatographic peak separation and baseline was investigated, the results are shown in Figure 3 .

[0208] Table 2-A Gradient 1

[0209]

[0210] Table 2-B Gradient 2

[0211]

[0212] Table 2-C Gradient 3

[0213]

[0214] Table 2-D Gradient 4

[0215]

[0216] The results showed that when gradient 4 was selected as the elution gradient, the baseline of the characteristic spectrum was stable and the separation effect of each chromatographic peak was good, so gradient 4 was used as the final elution gradient.

[0217] (3) Selection of mobile phase

[0218] The effects of different organic phase ratios (acetonitrile:methanol-8:1, acetonitrile:methanol-5:1, acetonitrile:methanol-7:2, acetonitrile:methanol-2:1, acetonitrile:methanol-1:1) (0.2% phosphoric acid) on the separation and baseline of each chromatographic peak of the characteristic spectrum were investigated. The chromatographic conditions were as follows: chromatographic column: Waters XSelect HSS C18 column (4.6mm×250mm, 5μm); gradient elution order: 0-60min, 60%→64%A, 40%→36%B; 60-65min, 64%A, 36%B; volume flow rate 1.0mL·min -1 ; Detection wavelength 280nm; Column temperature 25℃; Injection volume 5μL, Results see Figure 4 .

[0219] The results showed that when acetonitrile: methanol -7:2 (0.2% phosphoric acid) was used as the organic phase ratio for elution, the baseline of the characteristic spectrum was stable and the separation effect of each chromatographic peak was good, so acetonitrile: methanol -7:2 (0.2% phosphoric acid) was used as the final elution organic phase ratio.

[0220] (4) Selection of column temperature

[0221] The effects of different column temperatures (20°C, 25°C, 30°C, 40°C) on the separation of each chromatographic peak and baseline of the characteristic spectrum were investigated. The chromatographic conditions were as follows: chromatographic column: Waters XSelect HSS C18 chromatographic column (4.6mm×250mm, 5μm); mobile phase: A was acetonitrile-methanol (7:2) (0.2% phosphoric acid), and B was a 0.2% phosphoric acid aqueous solution; gradient elution order: 0-60min, 60%→64%A, 40%→36%B; 60-65min, 64%A, 36%B; volume flow rate 1.0mL·min -1 ; Detection wavelength 280nm; injection volume 5μL, results see Figure 5 .

[0222] The results showed that when the column temperature was 25℃ for elution, the peak shapes of the chromatographic peaks in the characteristic spectrum were better and the separation was higher, so 25℃ was used as the final column temperature.

[0223] (5) Determination of chromatographic conditions

[0224] According to the above experimental results, the HPLC conditions for the characteristic spectrum of Mianma Guanzhong medicinal material are determined as follows:

[0225] Chromatographic column: Waters XSelect HSS C18 chromatographic column (4.6 mm × 250 mm, 5 μm); mobile phase A was acetonitrile-methanol (7:2) (0.2% phosphoric acid), and mobile phase B was 0.2% phosphoric acid aqueous solution; gradient elution order: 0-60 min, 60% → 64% A, 40% → 36% B; 60-65 min, 64% A, 36% B; volume flow rate 1.0 mL min -1 ; Detection wavelength 280nm; column temperature 25℃; injection volume 5μL.

[0226] 2.1.2 Investigation of test sample treatment conditions

[0227] (1) Investigation of extraction solvent types

[0228] The sample size of the medicinal material was 1.0 g, and 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, and methanol solution were used as extraction solvents. The types of extraction solvents were investigated for the same batch of test samples. The results are shown in Figure 6 The results showed that the peak area of ​​the methanol solution was the largest, the peak areas of the 70% methanol aqueous solution and the 50% methanol aqueous solution were significantly reduced, and the 30% methanol aqueous solution had fewer peaks, so methanol solution was selected as the extraction solvent for the test sample.

[0229] (2) Investigation of extraction methods

[0230] The sample size of the medicinal material was 1.0 g, and methanol solution was used as the extraction solvent. Ultrasonic extraction and reflux extraction were used to investigate the extraction methods of the same batch of test samples. The results are shown in Figure 7 The results showed that there was no significant difference in the chromatographic peak area between ultrasonic extraction and reflux extraction, but the chromatogram baseline of ultrasonic extraction was relatively stable and the ultrasonic extraction operation was simpler, so ultrasonic extraction was selected as the extraction method for the test sample.

[0231] (3) Investigation of the amount of extraction solvent

[0232] The sample size of the medicinal material was 1.0 g, methanol solution was used as the extraction solvent, and ultrasonic extraction was performed. The extraction solvent dosage was selected as 15 mL, 25 mL, and 50 mL respectively. The same batch of test samples was investigated for the extraction solvent dosage. The results are shown in Figure 8 The results showed that when the extraction solvent dosage was 15mL, 25mL, and 50mL, there was no significant difference in the peak areas of the samples, and the peaks were obvious. Considering energy conservation and ensuring complete extraction, 25mL was selected as the extraction solvent dosage.

[0233] (4) Investigation of extraction time

[0234] The sample size of the medicinal material was 1.0 g, and 25 mL of methanol solution was used as the extraction solvent. The ultrasonic extraction method was used, and the ultrasonic time was selected to be 15 min, 30 min, 45 min, and 60 min respectively. The ultrasonic time of the same batch of test samples was investigated, and the results are shown in Fig. 9 The results showed that there was no significant difference in the peak areas of the samples after ultrasonic treatment for 15min, 30min, 45min and 60min, and the peaks were obvious. Considering energy saving and ensuring complete extraction, 30min was selected as the ultrasonic time.

[0235] (5) Investigation of extraction times

[0236] The sample size of the medicinal material was 1.0 g, and 25 mL of methanol solution was used as the extraction solvent. The ultrasonic extraction method was used, the ultrasonic time was 30 min, and the extraction times were 2, 3, and 4 times. The extraction times of the same batch of test samples were investigated. The results are shown in Fig.10 The results showed that after extraction for 2, 3, and 4 times, there was no significant difference in the peak areas of the samples, and the peaks were obvious. Considering energy conservation and ensuring complete extraction, 3 times was selected as the number of extractions.

[0237] (6) Investigation of the amount of extraction solvent used

[0238] The sample size of the medicinal material was 1.0 g, 25 mL of methanol solution was used as the extraction solvent, ultrasonic extraction method was used, ultrasonic time was 30 min, extraction times were 3 times, and the amount of extraction solvent used was 15 mL, 25 mL, and 40 mL. The results are shown in Fig.11 The results showed that when the extraction solvent dosage was 15mL, 25mL, and 40mL, there was no significant difference in the peak areas of the samples, and the peaks were obvious. Considering energy conservation and ensuring complete extraction, 25mL was selected as the extraction solvent dosage.

[0239] (7) Determination of the preparation method of the test solution

[0240] According to the above experimental results, the sample pretreatment method of the characteristic spectrum of Mianma Guanzhong medicinal material is determined as follows:

[0241] Take about 1.0g of the powder of the medicinal material of Corydalis cyrtonema (passed through a No. 3 sieve), weigh it accurately, put it in a stoppered conical flask, add 25mL of methanol solution accurately, weigh it, treat it with ultrasound 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, make up the volume of the residue to 10mL with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0242] (8) Preparation of reference solution:

[0243] Accurately weigh 2.304 mg of the AB standard substance of scutellariae flavescent acid, place it in a 10 mL volumetric flask, and add methanol to make a standard solution containing 218.097 μg per 1 mL.

[0244] 2.1.3 Determination of characteristic peaks

[0245] Take 16 batches of Mianma Guanzhong medicinal materials samples, prepare the test solution according to the test solution preparation method determined in "2.1.2", accurately pipette 5 μL of the above test solution and reference solution respectively, inject and measure according to the chromatographic conditions in "2.1.1", use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" to identify the common peaks of the characteristic spectra of 16 batches of Mianma Guanzhong medicinal materials, and select 8 common peaks with known components, good peak shape and separation, and high purity as the characteristic peaks of Mianma Guanzhong medicinal materials, such as Fig.12 As shown, the chromatographic peak AB of the reference substance obtained from the Rhizoma Cynomorii is taken as the reference peak S, the relative retention time of each characteristic peak and the S peak is calculated, and the average relative retention time of each characteristic peak of the characteristic spectra of 16 batches of Rhizoma Cynomorii medicinal materials is calculated, and the characteristic peak is located using the average relative retention time of each characteristic peak.

[0246] The results showed that the characteristic spectra of 16 batches of Corydalis guanyin were analyzed, and the peak corresponding to the AB reference peak of corydalis guanyin acid was taken as S peak. The relative retention times of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7 and S peak were calculated. The relative retention times should be within ±10% of the specified values, and the specified values ​​are: 0.49 (peak 1), 0.60 (peak 2), 0.72 (peak 3), 0.77 (peak 4), 0.82 (peak 5), 0.86 (peak 6), and 0.96 (peak 7).

[0247] 2.1.4 High-resolution mass spectrometry identification of characteristic peaks

[0248] (1) HPLC-MS conditions

[0249] Liquid chromatography conditions: Column: Waters XSelect HSS C18 column (4.6 mm × 250 mm, 5 μm); mobile phase A was acetonitrile-methanol (7:2) (0.2% formic acid), mobile phase B was 0.2% formic acid aqueous solution; gradient elution order: 0-60 min, 60% → 64% A, 40% → 36% B; 60-65 min, 64% A, 36% B; volume flow rate 1.0 mL min -1 ; Detection wavelength 280nm; column temperature 25℃; injection volume 5μL.

[0250] Mass spectrometry conditions: see Table 3.

[0251] Table 3 Mass spectrometry parameters

[0252]

[0253] (2) Preparation of test solution

[0254] Take about 1.0g of the powder of the medicinal material of Corydalis cyrtonema (passed through a No. 3 sieve), weigh it accurately, put it in a stoppered conical flask, add 25mL of methanol solution accurately, weigh it, treat it with ultrasound 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, make up the volume of the residue to 10mL with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0255] (3) Sample measurement

[0256] Accurately pipette 5 μL of the test solution and inject it into the liquid chromatography-mass spectrometer. Use the above liquid chromatography conditions and mass spectrometry conditions to detect the test solution. The total ion flow diagram and ultraviolet absorption chromatogram of the test solution are shown in Fig.13 .

[0257] (4) Results analysis

[0258] Through mass spectrometry accurate molecular weight and fragment ion comparison analysis, and matching with literature data (Wang Juan. Screening and metabolic study of anti-H5N1 influenza virus activity of pyrrolidone compounds in Mianma Guanzhong [D]. Beijing University of Chinese Medicine, 2022.; Ren Qiang. Research on active ingredients of traditional Chinese medicine against acute lymphoblastic leukemia [D]. Peking Union Medical College, 2010.; Ren Qiang, Xia Tianrui, Wang Yunlong, et al. Mass spectrometry fragmentation and spectral rules of pyrrolidone chemical components in Dryopteris plants [J]. Chinese Herbal Medicine, 2015, 46(06): 932-937.), three components of pyrrolidone AP (peak 1), norpyrrolidone PB (peak 2), and pyrrolidone AB (peak 8) were confirmed in the characteristic spectrum of Mianma Guanzhong medicinal material. The compound information is shown in Table 4, and the specific analysis process is shown in Table 4. Figure 14 to Figure 34 .

[0259] Table 4 Mass spectrometry identification results of compounds in the medicinal material of Mianma Guanzhong

[0260]

[0261] ① Peak 1

[0262] Extract the primary mass spectrum of peak 1, the time period is 26.48min-27.11min. Fig.14 , Fig.15 .

[0263] In the primary mass spectrum extraction diagram of peak 1, the peak with m / z 403.1393 has the highest response value in negative ion mode, which is [MH] - Peak; m / z 405.1538 peak in positive ion mode should be [M+H] + peak; from its precise molecular weight, it is inferred that its molecular formula may be C 21 H 24 O 8 ; Extract the secondary graph of the m / z 403.1393 peak with a collision energy of 40. The results show that the main ion fragments are 195.0653, 151.0752, 207.0654, etc. For detailed results, see Fig.16 ; Extract the secondary graph of the m / z 405.1538 peak with a collision energy of 40. The results show that the main ion fragments are 197.0809, 179.0702, 209.0809, etc. For detailed results, see Fig.17 .

[0264] The precise molecular weight and secondary ion fragment information of the signal peak were matched with the compounds in the literature database. The results showed that the compound with the highest matching degree of m / z 403.1393 peak was scutellariae AP, with the structural formula shown in Fig.18 For possible cleavage modes, see Fig.19 According to the fragment ion comparison results and the structural formula fragmentation inference analysis, it is suggested that the compound may be scutellaria baicalenic acid AP.

[0265] ② Peak 2

[0266] Extract the primary mass spectrum of peak 2, the time period is 36.63min-38.10min. Fig. 20 , Fig.21 .

[0267] In the primary mass spectrum extraction diagram of peak 2, the peak with m / z 417.1550 has the highest response value in negative ion mode, which is [MH] - Peak; m / z 419.1693 peak in positive ion mode should be [M+H] + peak; from its precise molecular weight, it is inferred that its molecular formula may be C 22 H 26 O 8 ; Extract the secondary graph of the peak m / z 417.1550 with a collision energy of 40. The results show that the main ion fragments are 209.0813, 195.0655, 165.0910, etc. For detailed results, see Fig. 22 ; Extract the secondary graph of the peak m / z 419.1693 with a collision energy of 40. The results show that the main ion fragments are 211.0963, 193.0860, 137.0596, etc. For detailed results, see Fig.23 .

[0268] The precise molecular weight and secondary ion fragment information of the signal peak were matched with the compounds in the literature database. The results showed that the compound with the highest matching degree of m / z 417.1550 peak was nor-methylenetetracycline PB, with the structural formula shown in Fig.24 For possible cleavage modes, see Fig.25 According to the fragment ion comparison results and the fragmentation inference analysis of the structural formula, it is suggested that the compound may be norflavonoids PB.

[0269] ③ Peak 8

[0270] Extract the primary mass spectrum of peak 8, the time period is 56.78min-58.19min. Fig.26 , Fig. 27 .

[0271] In the primary mass spectrum extraction diagram of peak 8, the peak with m / z 417.1555 has the highest response value in negative ion mode, which is [MH] - Peak; m / z 419.1693 peak in positive ion mode should be [M+H] + peak; from its precise molecular weight, it is inferred that its molecular formula may be C 22 H 26 O 8 ; Extract the secondary graph of the peak m / z 417.1550 with a collision energy of 40. The results show that the main ion fragments are 209.0813, 195.0655, 165.0910, etc. For detailed results, see Fig.28 ; Extract the secondary map of the peak m / z 419.1693 with a collision energy of 40. The results show that the main ion fragments are 211.0962, 193.0859, 137.0596, etc. For detailed results, see Fig.29 .

[0272] The precise molecular weight and secondary ion fragment information of the signal peak were matched with the compounds in the literature database. The results showed that the compound with the highest matching degree of m / z 417.1550 peak was scutellariae AB, with the structural formula shown in Fig.30 For possible cleavage modes, see Fig.31 According to the fragment ion comparison results and the structural formula fragmentation inference analysis, it is suggested that the compound may be scutellaria baicalenic acid AB.

[0273] 2.1.5 Confirmation of characteristic peaks

[0274] In view of the fact that the standard of pyrogallol compounds in Rhizoma Cynomorii is difficult to purchase on the market, the present application also provides a method for preparing Rhizoma Cynomorii acid AB reference substance, which can obtain high-purity Rhizoma Cynomorii acid AB with high efficiency and yield. The specific steps are as follows:

[0275] A) 25 kg of dried powder of Rhizoma Cynomorii (passed through a No. 3 sieve) was percolated with 10 times the amount of 95% ethanol solution, filtered, the filtrates were combined, and then concentrated and dried under reduced pressure to obtain 7.8 kg of a total extract;

[0276] B) the 95% ethanol extract was dispersed in water, and extracted with petroleum ether, ethyl acetate, and n-butanol in sequence to obtain 824 g of petroleum ether fraction, 1447 g of ethyl acetate fraction, and 1088 g of n-butanol fraction;

[0277] C) subjecting the petroleum ether fraction to chromatography separation and purification to obtain the compound.

[0278] Step C) The step of chromatographic separation and purification of the petroleum ether portion is as follows:

[0279] The petroleum ether part was subjected to column chromatography on silica gel, and a gradient elution of petroleum ether-ethyl acetate-formic acid with a volume ratio of (20:1:0.2) → (10:1:0.2) was used. After merging, 15 fractions were obtained; after removing the solvent from fraction 10, an extract was obtained, and petroleum ether (60°C-90°C) was added to wash until the washing liquid was clear, and then ethyl acetate was added to dissolve, and the extract was allowed to stand for crystallization. The bottom solid was removed and continued to be washed with petroleum ether (60°C-90°C) until the washing liquid was clear, and the solvent was removed to obtain 415 mg of a bright yellow powder solid; after removing the solvent from fraction 11, an extract was obtained, and petroleum ether (60°C-90°C) was added to wash until the washing liquid was clear, and then ethyl acetate was added to wash until the washing liquid was clear, and the solvent was removed to obtain 1646 mg of a bright yellow powder solid. The obtained solids were detected by high performance liquid chromatography and were the same substance, and were combined. Its structure was analyzed by nuclear magnetic resonance technology, and the result was identified as scutellaria baicalensis AB, such as Fig.32 , Fig.33 . And through nuclear magnetic quantitative detection, its purity reached 94.66%.

[0280] Take about 1.0g of the medicinal material of Rhizoma Cynomorii, weigh it accurately, prepare the test solution according to the test solution preparation method determined in "2.1.2", take 5μL of the Rhizoma Cynomorii acid AB reference solution and the test solution, inject them into the liquid chromatograph, and determine them according to the chromatographic conditions in "2.1.1". The results are shown in Fig.34 .

[0281] The results showed that the retention time of the monomer component of scutellaria baicalensis AB was consistent with that of chromatographic peak 8, and the mass spectrometry fragment ions were the same, which confirmed that chromatographic peak 8 was scutellaria baicalensis AB.

[0282] 2.1.6 Preparation of feature maps

[0283] The HPLC characteristic spectra of 16 batches of Mianma Guanzhong medicinal materials were matched using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and a reference spectrum was generated by the average method to establish a reference characteristic spectrum of Mianma Guanzhong medicinal materials, such as Fig.35The characteristic spectrum has 8 characteristic peaks. The peak corresponding to the AB reference peak of scutellaria baicalensis is taken as the S peak, and the relative retention time of peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7 and S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is: 0.49 (peak 1), 0.60 (peak 2), 0.72 (peak 3), 0.77 (peak 4), 0.82 (peak 5), 0.86 (peak 6), 0.96 (peak 7).

[0284] 2.1.7 Methodological Validation

[0285] (1) Specificity inspection

[0286] Take about 1.0g of the medicinal material of Rhizoma Cynomorii, weigh it accurately, and prepare the test solution according to the test solution preparation method determined in "2.1.2". Take 5μL of the Rhizoma Cynomorii acid AB reference solution and blank solvent respectively, inject them into the liquid chromatograph, and determine them according to the chromatographic conditions in "2.1.1". The results are shown in Fig.36 .

[0287] 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.

[0288] (2) Precision inspection

[0289] Take about 1.0g of the medicinal material of Corydalis cyrtonema, weigh it accurately, prepare the test solution according to the test sample preparation method determined under "2.1.2", repeat the injection 6 times according to the chromatographic conditions under "2.1.1", and analyze the injection. Take the AB peak of Corydalis cyrtonema as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The results show that the same test solution was injected 6 times in a row, taking the AB peak of Corydalis cyrtonema as the reference peak S, the relative retention time RSD value of each characteristic peak and the S peak was in the range of 0.05% to 0.08%, and the relative peak area RSD value was in the range of 1.79% to 3.39%, all less than 5.0%, indicating that the instrument has good precision.

[0290] (3) Repeatability study

[0291] Take about 1.0g of the medicinal material of Corydalis cyrtonema, weigh it accurately, prepare 6 test sample solutions according to the test sample preparation method determined under "2.1.2", and analyze them according to the chromatographic conditions under "2.1.1". Take the AB peak of Corydalis cyrtonema as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The results showed that the same batch of samples was measured 6 times, taking the AB peak of Corydalis cyrtonema as the reference peak S, the relative retention time RSD value of each characteristic peak and the S peak was in the range of 0.07% to 0.11%, and the relative peak area RSD value was in the range of 1.41% to 3.62%, all less than 5.0%, indicating that the method has good repeatability.

[0292] (4) Stability test

[0293] Take about 1.0g of the medicinal material of Corydalis cyrtonema, weigh it accurately, and analyze it according to the preparation method of the test sample determined under "2.1.2" and the chromatographic conditions under "2.1.1" at 0, 4 hours, 8 hours, 12 hours, 18 hours, and 24 hours, respectively. Take the AB peak of Corydalis cyrtonema as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The results show that the same test solution is analyzed at 0, 4 hours, 8 hours, 12 hours, 18 hours, and 24 hours, take the AB peak of Corydalis cyrtonema as the reference peak S, the relative retention time RSD value of each characteristic peak and the S peak is in the range of 0.05% to 0.08%, and the relative peak area RSD value is in the range of 1.15% to 4.45%, all less than 5.0%, indicating that the test solution has good relative stability within 24 hours.

[0294] (5) Intermediate precision study

[0295] Other analysts of this project team operated on different dates and different chromatographs, took about 1.0g of the same batch of Corydalis cyrtonema medicinal materials, accurately weighed them, and analyzed them according to the test sample preparation method determined under "2.1.2" and the chromatographic conditions under "2.1.1". The relative retention time and relative peak area of ​​each characteristic peak and the S peak were calculated with the AB peak of Corydalis cyrtonema as the reference peak S, and the RSD value was calculated. The results showed that the same batch of samples was repeated 6 times by different analysts operating on different instruments at different times. The relative retention time RSD values ​​of each characteristic peak and the S peak were in the range of 0.12% to 0.23%, and the relative peak area RSD values ​​were in the range of 1.32% to 3.11%, all less than 5.0%. The RSD values ​​of the six data of relative retention time and repeatability test were in the range of 0.10% to 0.75%, and the RSD values ​​of the six data of relative peak area and repeatability test were in the range of 2.06% to 4.34%, both of which were less than 5.0%, indicating that the relative retention time and relative peak area of ​​each characteristic peak had good intermediate precision on different instruments.

[0296] (6) Durability test

[0297] ① Investigation of different column temperatures

[0298] Take about 1.0g of the same batch of Mianma Guanzhong medicinal materials, weigh accurately, and prepare the test solution according to the test sample preparation method specified in "2.1.2". Except for the column temperature of 23℃, 25℃, and 27℃, the other chromatographic conditions are the same as those specified in "2.1.1". Sample injection and determination are performed. Taking smilax acid AB as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The results are shown in Fig.37 .

[0299] The results showed that at different column temperatures, the RSD values ​​of the relative retention time of each characteristic peak and the S peak were in the range of 0.19% to 1.61%, and the RSD values ​​of the relative peak area were in the range of 1.38% to 4.60%, all less than 5.0%, indicating that the column temperature of ±2°C had little effect on the relative retention time and relative peak area of ​​each characteristic peak, indicating that different column temperatures have good durability.

[0300] ② Investigation of different flow rates

[0301] Take about 1.0 g of the same batch of Mianma Guanzhong medicinal materials, weigh them accurately, and prepare the test solution according to the test sample preparation method specified in "2.1.2", except that the flow rate is 0.9 mL min -1 、1.0mL·min -1 and 1.1 mL·min -1 , other chromatographic conditions are the same as those specified in "2.1.1", sample injection and determination, with scutellariae acid AB as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak and S peak, and calculate the RSD value. Fig.38 .

[0302] The results showed that at different flow rates, the relative retention time RSD values ​​of each characteristic peak and S peak were in the range of 0.19% to 1.15%, and the relative peak area RSD values ​​were in the range of 0.38% to 1.86%, both of which were less than 3.0%, indicating that when the flow rate was ±0.1 mL·min -1 When the flow rate is changed, the change of relative retention time and relative peak area has little effect.

[0303] ③Investigation of different chromatographic columns

[0304] Take about 1.0g of the same batch of Rhizoma Cynomorium officinale, weigh accurately, and prepare the test solution according to the test sample preparation method specified in "2.1.2". Except that the chromatographic columns are Kromasil 100-5-C18 (4.6mm×250mm, 5μm), AgilentZORBAX Eclipse XDB-C18 (4.6mm×250mm, 5μm), Thermo ScientificTM AcclaimTM 120C18 (4.6mm×250mm, 5μm), Agilent ZORBAX Extend-C18 (4.6mm×250mm, 5μm), and Waters XSelectHSS C18 (4.6mm×250mm, 5μm), other chromatographic conditions are the same as those specified in "2.1.1". Samples were injected and measured. The results are shown in Table 2. Fig.39 .

[0305] The results showed that compared with other columns with different fillers, the peak shape of each chromatographic peak in the chromatogram was better and the separation was higher when using Waters XSelect HSS C18 (4.6mm×250mm, 5μm) column. Therefore, it is recommended to fix WatersXSelect HSS C18 (4.6mm×250mm, 5μm) as the chromatographic column for further research.

[0306] 2.1.8 Comparison of characteristic spectra of different adulterants

[0307] Take about 1.0g of each of the medicinal materials of single-bud dog fern, Japanese dog fern, small dog fern, cycad, brown ear fern, cycad, cycad, aster, terminal bud dog fern, pearl bud dog fern, and mianma dog fern, accurately weigh, prepare the test sample solution according to the test sample preparation method determined in "2.1.2", and inject and analyze according to the chromatographic conditions in "2.1.1". The results are as follows Fig.40 shown.

[0308] The results showed that there were differences in the characteristic spectra of medicinal materials such as Osmanthus fern, Brown ear fern, Pearly buds of Cyperus rotundus, Cibotium wilfordii, Japanese Cibotium wilfordii, Cycas revoluta, Single buds of Cibotium wilfordii, Small buds of Cyperus rotundus, Cyperus rotundus and Mianma rotundus. Osmanthus fern only contained characteristic peak 3, Brown ear fern contained characteristic peaks 3, peak 4, peak 5, peak 7, and peak 8, Pearly buds of Cyperus rotundus contained peaks 4 and peak 6, Terminal buds of Cibotium wilfordii only contained characteristic peak 4, Japanese Cibotium wilfordii contained characteristic peaks 4 and peak 6, Cycas revoluta only contained characteristic peak 6, Single buds of Cibotium wilfordii contained characteristic peaks 4, peak 5, peak 6, and peak 7, Small buds of Cyperus rotundus only contained characteristic peak 4, and Cyperus rotundus contained characteristic peaks 4 and peak 5. The peak area ratios of the counterfeit and the medicinal material Cyperus rotundus were calculated. Only Cyperus rotundus contained characteristic peaks 1 and 2; the peak area of ​​characteristic peak 3 in Cyperus rotundus and Blang'erjue was close to that of Cyperus rotundus; except for the peak area of ​​characteristic peak 4 in Pteris uniflorus, which was close to that of Cyperus rotundus, the peak areas of characteristic peak 4 in Blang'erfern, Cyperus rotundus, Cyperus rotundus, Cyperus rotundus, Cyperus rotundus, and Cyperus rotundus were significantly smaller than those in Pteris rotundus; the peak area of ​​characteristic peak 5 in Pteris uniflorus was close to that of Cyperus rotundus, while the peak areas of characteristic peak 5 in Blang'erfern and Cyperus rotundus were significantly smaller than those in Pteris rotundus; the peak area of ​​characteristic peak 6 in Pteris uniflorus was close to that of Cyperus rotundus, while the peak areas of characteristic peak 6 in Pteris uniflorus, Cyperus rotundus, Cyperus rotundus, and Cycas rotundus were significantly smaller than those in Pteris rotundus; the peak area of ​​characteristic peak 7 in Pteris uniflorus was close to that of Cyperus rotundus, while the peak area of ​​characteristic peak 7 in Blang'erfern was significantly smaller than that in Pteris rotundus; the peak area of ​​characteristic peak 8 in Blang'erfern was about twice that of Cyperus rotundus. In summary, the established characteristic spectrum method can effectively distinguish Mianma Guanzhong from various confusing products.

[0309] 2.2 Establishment of the determination method of AB content of scutellaria baicalensis

[0310] 2.2.1 Preparation of reference solution

[0311] Accurately weigh 2.304 mg of the AB standard substance of scutellariae flavescent acid, place it in a 10 mL volumetric flask, and add methanol to make a standard solution containing 218.097 μg per 1 mL.

[0312] 2.2.2 Optimization of chromatographic conditions

[0313] Same as the characteristic spectrum chromatographic conditions under "2.1.1".

[0314] 2.2.3 Investigation of test sample treatment conditions

[0315] (1) Investigation of extraction solvent types

[0316] The sample size of the medicinal material was 1.0 g, and methanol solution, 70% methanol aqueous solution, 50% methanol aqueous solution, 95% ethanol aqueous solution, 70% ethanol aqueous solution, and 50% ethanol aqueous solution were used as extraction solvents, respectively. The types of extraction solvents were investigated 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 medicinal material of scutellaria baicalensis.

[0317] (2) Investigation of extraction methods

[0318] The sample size of the medicinal material was 1.0 g, and methanol solution was used as the extraction solvent. The same batch of test samples were extracted by ultrasonic treatment and heating reflux. The results showed that there was no significant difference between the content of scutellaria baicalensis AB measured in the heating reflux extraction and the content measured by ultrasonic treatment extraction. Combined with the characteristic spectrum pretreatment conditions, ultrasonic treatment was finally selected as the extraction method for the determination of scutellaria baicalensis AB content in the medicinal material.

[0319] (3) Investigation of extraction time

[0320] The sample size of the medicinal material was 1.0g, methanol solution was used as the extraction solvent, ultrasonic treatment was used as the extraction method, and the extraction time of the same batch of test samples was investigated at 15min, 30min, and 45min. The results showed that the extraction time of 30min could completely extract the scutellaria baicalensis acid AB, and 30min was finally selected as the extraction time for the determination of the scutellaria baicalensis acid AB content in the medicinal material of scutellaria baicalensis.

[0321] (4) Investigation of the amount of extraction solvent

[0322] The sample size of the medicinal material was 1.0g, methanol solution was used as the extraction solvent, ultrasonic treatment was used as the extraction method, 30min was used as the extraction time, and 15mL, 25mL, and 50mL were used to investigate the amount of extraction solvent used for the same batch of test samples. The results showed that when the amount of extraction solvent was 25mL, the scutellaria baicalensis AB could be completely extracted, and 25mL was finally selected as the amount of extraction solvent used for the determination of the content of scutellaria baicalensis AB in the medicinal material.

[0323] (5) Investigation of extraction times

[0324] The sample size of the medicinal material was 1.0 g, methanol solution was used as the extraction solvent, ultrasonic treatment was used as the extraction method, 30 min was used as the extraction time, 25 mL was used as the extraction solvent, and the number of extractions for the same batch of test samples was investigated 2, 3, 4, and 5 times. The results showed that when the number of extractions was 3, the scutellaria baicalensis acid AB could be completely extracted, and 3 times was finally selected as the extraction number for the determination of the scutellaria baicalensis acid AB content in the medicinal material of Scutellaria baicalensis.

[0325] (6) Investigation of the amount of extraction solvent used

[0326] The sample size of the medicinal material was 1.0 g, methanol solution was used as the extraction solvent, ultrasonic treatment was used as the extraction method, 30 min was used as the extraction time, 25 mL was used as the extraction solvent dosage, 3 times was used as the extraction number, and 15 mL, 25 mL, and 40 mL were used to investigate the extraction solvent dosage of the same batch of test samples. The results showed that when the extraction solvent dosage was 25 mL, the scutellaria baicalensis acid AB could be completely extracted, and 25 mL was finally selected as the extraction solvent dosage for the determination of the scutellaria baicalensis acid AB content in the medicinal material of Scutellaria baicalensis.

[0327] (7) Determination of the preparation method of the test solution

[0328] According to the above experimental results, the sample pretreatment method for the determination of AB content of scutellaria baicalensis is determined as follows:

[0329] Take about 1.0g of the powder of the medicinal material of Corydalis cyrtonema (passed through a No. 3 sieve), weigh it accurately, put it in a stoppered conical flask, add 25mL of methanol solution accurately, weigh it, treat it with ultrasound 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, make up the volume of the residue to 10mL with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0330] 2.2.4 Methodological validation

[0331] (1) Specificity inspection

[0332] Take about 1.0g of the medicinal material of Rhizoma Cynomorii, weigh it accurately, and prepare the test solution according to the test solution preparation method determined in "2.2.3". Take 5μL of the Rhizoma Cynomorii acid AB reference solution and blank solvent respectively, inject them into the liquid chromatograph, and determine them according to the chromatographic conditions in "2.2.2". The results are shown in Fig.41 .

[0333] 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.

[0334] (2) Peak purity investigation

[0335] Take about 1.0g of the medicinal material of Rhizoma Cynomorii, weigh it accurately, prepare the test solution according to the test solution preparation method determined in "2.2.3", accurately pipette the test solution and Rhizoma Cynomorii acid AB reference solution, inject them into the liquid chromatograph, perform scanning detection at 210nm~400nm with the DAD detector according to the chromatographic conditions in "2.2.2", calculate the peak purity, and the results are shown in Fig.42 .

[0336] 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.

[0337] (3) Linear relationship investigation

[0338] 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 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, 961.452 μg, and 2403.630 μg per 1 mL, respectively; accurately pipette the above reference substance solution linear solution, inject 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 5. Fig.43 .

[0339] Table 5 Results of linear investigation of AB of yellow cotton acid

[0340]

[0341] The results showed that the regression equation of scutellaria scutellariae AB ​​was: y = 14,991.1720x-166,093.8476, and its correlation coefficient r = 1.0000, indicating that scutellaria scutellariae AB ​​had a concentration of 19.229 μg·mL -1 ~2403.630μg·mL -1 There is a good linear relationship between the injection concentration and the peak area in the range of 1.

[0342] (4) Precision inspection

[0343] Accurately pipette the reference solution (C = 218.097 μg mL) under "2.2.1" -1 ), repeat the injection 6 times according to the chromatographic conditions under "2.2.2", 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 0.77% after 6 injections of the same reference solution, which is less than 3.0%, indicating that the instrument has good precision.

[0344] (5) Repeatability study

[0345] Take about 1.0g of the medicinal material of Mianma Guanzhong, weigh it accurately, and make 6 parallel copies. According to the preparation method of the test solution determined under "2.2.3", 6 test solutions were prepared, and the samples were injected and analyzed according to the chromatographic conditions under "2.2.2", 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 0.81%. 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 0.1% to 1%, the repeatability RSD limit is <2%, indicating that the repeatability of this analytical method is good.

[0346] (6) Stability investigation

[0347] Take about 1.0g of the medicinal material of Corydalis cyrtonema, weigh it accurately, and make 6 parallel portions. Prepare the test solution according to the test solution preparation method determined under "2.2.3". According to the chromatographic conditions under "2.2.2", the sample was injected and analyzed at 0, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, and 24 hours, and the peak area of ​​​​corydalis acid AB in the test solution was determined, and the peak area RSD value was calculated. The results show that the same sample solution was injected and determined at 0, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, and 24 hours, and the RSD value of the peak area of ​​​​corydalis acid AB was 2.75%, indicating that the test solution under this method has good stability within 24 hours.

[0348] (7) Intermediate precision study

[0349] Different experimenters were selected to measure at different times and with different instruments. About 1.0g of the medicinal material of Mianma Guanzhong was taken, accurately weighed, and 6 parallel portions were prepared according to the test solution preparation method determined under "2.2.3". The test was carried out according to the chromatographic conditions under "2.2.2". 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 1.42%, which was 2.51% 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 is 0.1% to 1%, the intermediate precision RSD limit is <4%. Therefore, different analysts operate on different dates and different chromatographs, and the intermediate precision of this method is good.

[0350] (8) Accuracy test

[0351] 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 three replicates 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 0.5g of the powder of the determined content of the scutellaria baicalensis in a conical flask, accurately weigh it, and prepare the test solution according to the method under "2.2.3". Samples were injected and determined according to the chromatographic conditions under "2.2.2", and the results are shown in Table 6.

[0352] Table 6. Results of sample recovery of AB content determination of maltose acid in Mianma Guanzhong medicinal material

[0353]

[0354]

[0355] The results showed that the recovery rate of scutellaria AB was 96.15%. 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 0.1% to 1%, the recovery limit is 92% to 105%. The sample recovery rate of scutellaria AB ranged from 92.79% to 100.85%, the average sample recovery rate was 96.15%, and the RSD value was 2.91%, indicating that the method has good accuracy.

[0356] (9) Durability test

[0357] ① Investigation of different column temperatures

[0358] Take about 1.0g of the medicinal material of Mianma Guanzhong, weigh it accurately, and prepare the test solution according to the test solution preparation method determined under "2.2.3". Except that the column temperature is 23℃, 25℃ and 27℃ respectively, other conditions are the chromatographic conditions under "2.2.2". Sample injection analysis is performed to determine the content of AB and RSD value of scutellaria baicalensis in the test solution. The results show that the RSD value of AB content of scutellaria baicalensis in the medicinal material of Mianma Guanzhong measured at different column temperatures (±2℃) is 1.48%, indicating that the analytical method has good durability within the range of column temperature ±2℃.

[0359] ② Investigation of different flow rates

[0360] Take about 1.0g of Mianma Guanzhong medicinal material, weigh it accurately, and prepare the test solution according to the test solution preparation method specified in "2.2.3", except that the flow rate is 0.9mL·min -1 、1.0mL·min -1 and 1.1 mL·min -1 Except for the above conditions, all other conditions were in accordance with the chromatographic conditions under "2.2.2". 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 (±0.1 mL·min-1 The RSD value of the AB content of scutellaria baicalensis in the herbal medicine of Rhizoma Cynomorium was 2.07% under the flow rate of ±0.1 mL·min -1 Durability is good within the range.

[0361] (10) Sample measurement

[0362] Take 16 batches of Corydalis cyrtonema medicinal materials, prepare 16 test solutions according to the test solution preparation method under "2.2.3", inject and measure according to the chromatographic conditions under "2.2.2", record the peak area, and calculate the content of Corydalis cyrtonema acid AB by external standard method. The results are shown in Table 7.

[0363] Table 7 Determination results of AB content of 16 batches of cyperus rotundus medicinal materials

[0364]

[0365] The results showed that the content of 16 batches of cyperus rotundus medicinal materials ranged from 1.1846mg / g to 2.7330mg / g, with a mean of 1.6884mg / g, SD of 0.3874mg / g, a range of ±30% of the mean of 1.1819mg / g to 2.1949mg / g, and a range of ±3 times the mean of SD of 0.5261mg / g to 2.8507mg / g. Considering the representativeness of the 16 batches of samples, the value of -3 times the mean of SD was taken as the limit to determine the content of 16 batches of cyperus rotundus medicinal materials containing 1.1846mg / g to 2.7330mg / g. The content of 16 batches of cyperus rotundus medicinal materials containing 1.1819mg / g to 2.1949mg / g was ±3 times the mean of SD, and the content of ... 22 H 26 O 8 ) shall not be less than 0.050%.

[0366] 2.2.5 Determination of counterfeit medicinal material samples

[0367] Take about 1.0 g of each of the medicinal materials of single-bud dog fern, Japanese dog fern, small dog fern, cycad dog fern, brown ear fern, dog fern, cycad dog fern, aster fern, terminal dog fern, pearl bud dog fern, and mianma dog fern, and weigh them accurately. Prepare the test solution according to the test solution preparation method under "2.2.3". Inject and measure according to the chromatographic conditions under "2.2.2", record the peak area, and calculate the content of scutellaria baicalensis AB by external standard method. The results are shown in Table 8.

[0368] Table 8 Determination results of AB content of scutellaria baicalensis in Chinese medicine and counterfeit Chinese medicine

[0369]

[0370] The results showed that the content of scutellaria baicalensis AB in the medicinal material Brown Ear Fern was significantly lower than that in the medicinal material Corydalis chuanxiong, and the other counterfeit medicinal materials did not contain scutellaria baicalensis AB, indicating that using scutellaria baicalensis AB as an indicator for determining the content of Corydalis chuanxiong medicinal material has good specificity.

[0371] Example 2

[0372] This embodiment provides a method for constructing a characteristic spectrum of phloroglucinols in the medicinal material of Mianma Guanzhong and a method for determining the content of effective ingredients and an application for identifying adulterants, as follows:

[0373] Among them, the batch number of Mianma Guanzhong medicinal material is S1;

[0374] Chromatographic conditions: Column: Waters XSelect HSS C18 column (4.6 mm × 250 mm, 5 μm); Mobile phase A is acetonitrile-methanol (7:2) (0.2% phosphoric acid), Mobile phase B is 0.2% phosphoric acid aqueous solution; Gradient elution order: 0-60 min, 60% → 64% A, 40% → 36% B; 60-65 min, 64% A, 36% B; Volume flow rate 1.0 mL min -1 ; Detection wavelength 280nm; column temperature 25℃; injection volume 5μL.

[0375] Preparation of reference solution: Accurately weigh 2.304 mg of scutellariae AB ​​reference substance, place it in a 10 mL volumetric flask, and add methanol to make a reference substance solution containing 218.097 μg per 1 mL.

[0376] Preparation of test solution: Take about 1.0g of Corydalis cyrtonema powder (passed through No. 3 sieve), weigh it accurately, put it in a stoppered conical flask, add 25mL of methanol solution accurately, weigh it, treat it with ultrasound 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, make up the volume to 10mL with methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0377] Determination method: Accurately pipette 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0378] Determination results: The medicinal material of Corydalis cyrtonema with batch number S1 has 8 characteristic peaks. Peak 8 corresponding to the reference peak of scutellaria baicalensis AB is taken as S peak. The relative retention times of peaks 1, 2, 3, 4, 5, 6, 7 and S peak are calculated. The relative retention times are 0.49 (peak 1), 0.60 (peak 2), 0.72 (peak 3), 0.77 (peak 4), 0.82 (peak 5), 0.86 (peak 6), and 0.96 (peak 7). The content of scutellaria baicalensis AB in the medicinal material of Corydalis cyrtonema with batch number S1 is 0.1637%, which is within the specified range of medicinal material content. The results are shown in Tables 9, 10, Fig.44 .

[0379] Table 9 Calculation results of relative retention time of each characteristic peak of characteristic spectrum of Mianma Guanzhong medicinal material (batch number: S1)

[0380]

[0381]

[0382] Table 10 Determination results of the content of scutellaria baicalensis AB in the medicinal material of Mianma Guanzhong (batch number: S1)

[0383]

[0384] 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.

[0385] 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 constructing a characteristic spectrum of phloroglucinol components of the medicinal material Rhizoma Cynomorii, characterized in that: The following steps are involved: Extract the Mianma Guanzhong medicinal material using an extraction solvent to prepare a test solution; The phloroglucinol components of the medicinal material of Mianma Guanzhong were dissolved by using a dissolving reagent to prepare a reference solution; The test solution and the reference solution are subjected to high performance liquid chromatography to construct a characteristic spectrum of phloroglucinol components of the medicinal material of Mianma Guanzhong; The conditions for the HPLC detection include: mobile phase A is a mixture of acetonitrile, methanol and aqueous phosphoric acid, mobile phase B is aqueous phosphoric acid, and gradient elution is used; The procedure of the gradient elution is: From 0 to 60 min, the volume percentage of mobile phase A increased from 60% to 64%; From 60 to 65 minutes, the volume percentage of mobile phase A was maintained at 64%; The phloroglucinol components include phloroglucinol AP, norphloroglucinol PB and phloroglucinol AB.

2. The method for constructing the characteristic spectrum of phloroglucinol components of the medicinal material of Rhizoma Cynomorii 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℃~40℃; (3) Flow rate: 0.5 mL min -1 ~1.5mL·min -1 ; (4) The injection volume is 1 μL to 10 μL; (5) The chromatographic column is a C18 chromatographic column.

3. The method for constructing the characteristic spectrum of phloroglucinol components of the medicinal material of Rhizoma Cynomorii according to claim 1, characterized in that: In the mobile phase A, the volume percentages of acetonitrile and methanol are (1-8): (1-2); and / or The volume ratio of the total volume of acetonitrile and methanol to the phosphoric acid aqueous solution is 1:(0.1% to 0.3%); and / or The volume percentage of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.4%; and / or The dissolving agent is methanol.

4. The method for constructing the characteristic spectrum of phloroglucinol components of the medicinal material of Rhizoma Cynomorii according to claim 1, characterized in that: High performance liquid chromatography-mass spectrometry is used to identify characteristic peaks of the characteristic spectrum of the medicinal material Rhizoma Cynomorii. The conditions of the HPLC-MS method include: mobile phase A is a mixture of acetonitrile, methanol and formic acid aqueous solution, mobile phase B is formic acid aqueous solution, and gradient elution is used; The procedure of the gradient elution is: From 0 to 60 min, the volume percentage of mobile phase A increased from 60% to 64%; From 60 to 65 minutes, the volume percentage of mobile phase A was maintained at 64%; The characteristic peaks of the characteristic spectrum of the medicinal material of Rhizoma Cyrthospermi include: Peak 1 is cyrthospermic acid AP, Peak 2 is norcyrthospermic acid PB, and Peak 8 is cyrthospermic acid AB.

5. The method for constructing the characteristic spectrum of phloroglucinol components of the medicinal material of Rhizoma Cyrthospermi according to claim 4, characterized in that: The conditions of the high performance liquid chromatography-mass spectrometry method also include: in the mobile phase A, the volume percentage of acetonitrile and methanol is (1-8): (1-2); and / or The volume ratio of the total volume of acetonitrile and methanol to the formic acid aqueous solution is 0.1% to 0.3%; and / or In the formic acid aqueous solution, the volume percentage of formic acid is 0.05% to 0.3%.

6. The method for constructing the characteristic spectrum of phloroglucinol components of the medicinal material of Rhizoma Cynomorii according to claim 1, characterized in that: The step of extracting the medicinal material of Mianma Guanzhong using an extraction solvent satisfies at least one of the following conditions: (1) The extraction solvent is methanol or methanol-water solution; optionally, the volume percentage of methanol in the methanol-water solution is 30% to 70%; (2) The extraction method is ultrasonic extraction or reflux extraction; (3) The extraction time is 15 to 60 minutes; (4) The mass volume ratio of the Mian Ma Guan Zhong medicinal material and the extraction solvent is 1 g: (15-50) mL.

7. A method for determining the content of phloroglucinol components in the medicinal material Rhizoma Cynomorii, characterized in that: The following steps are involved: Extract the Mianma Guanzhong medicinal material using an extraction solvent to prepare a test solution; 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 the active ingredients in the reference solution at different concentrations, plot a standard curve with the concentration of the reference solution as the abscissa and the peak areas corresponding to the active ingredients in the reference solution at different concentrations as the ordinate, and calculate a linear regression equation; Obtaining the peak area corresponding to the active ingredient in the test solution, and substituting it into the linear regression equation to calculate the content of the active ingredient in the test solution; Wherein, the phloroglucinol component includes phloroglucinol AB; the reference substance includes phloroglucinol AB; the preparation method of the test solution and the conditions for high performance liquid chromatography detection are the same as any one of claims 1 to 4.

8. A method for identifying the medicinal material of Rhizoma Cynomorii and its adulterants, characterized in that: The following steps are involved: Extracting the Mianma Guanzhong medicinal material and its adulterants with extraction solvents respectively to prepare test sample solutions; The test solution is subjected to high performance liquid chromatography to construct characteristic spectra of the Mianma Guanzhong medicinal material and its adulterants, and observe whether the characteristic spectra of each adulterant have corresponding characteristic peaks of phloroglucinol components with the characteristic spectra of the Mianma Guanzhong medicinal material; in ； The preparation method of the test solution and the conditions for high performance liquid chromatography detection are the same as any one of claims 1 to 4.

9. A method for preparing scutellaria serrata AB, characterized in that: The following steps are involved: Extracting the powder of the medicinal material of Mienma cyrtomium by using an extraction solvent, concentrating and drying the obtained extract, and preparing a medicinal material of Mienma cyrtomium extract; Mixing the extract of the Chinese cyrtomium sibiricum and water, and extracting the resulting mixture using an extraction solvent; The obtained extract is separated by column chromatography, and the chromatographic liquid containing the phloroglucinol component of the mianma guanzhong, flavocal acid AB, is collected; The chromatographic solution is purified to prepare scutellariae acid AB.

10. The method for preparing scutellaria baicalenic acid AB according to claim 9, characterized in that: The extraction solvent is an ethanol aqueous solution; optionally, the volume percentage of ethanol in the ethanol aqueous solution is 80% to 99%; The extraction solvent is one or more of petroleum ether, ethyl acetate and n-butanol; The elution solution used in the column chromatography method includes a mixed solution of petroleum ether, ethyl acetate and formic acid.

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