Establishment of characteristic chromatogram and content determination of phloroglucinolic components in Aspidium hearing and its adulterants

Abstract

The present application relates to a kind of phloroglucinol characteristic map of Pteroxygonum giraldii plant, content determination construction method and confused product identification application, the present application constructs a kind of Pteroxygonum giraldii plant phloroglucinol component HPLC characteristic map method, and combining high performance liquid chromatography-mass spectrometry, first realize the characterization of Pteroxygonum giraldii plant phloroglucinol component, the characteristic peak of this characteristic map method is well separated, method precision, repeatability and durability are good, can be more comprehensive to reflect the main chemical component characteristics of Pteroxygonum giraldii plant, and can be used for Pteroxygonum giraldii confused product identification, provide important reference for the establishment of Pteroxygonum giraldii plant quality standard.In addition, the characteristic map method constructed by the present application can simultaneously determine the content of active ingredient of Pteroxygonum giraldii plant yellow Pteroxygonum acid AB, first realize the determination of the content of yellow Pteroxygonum acid AB in Pteroxygonum giraldii plant, and the method is validated, specificity, precision, repeatability, accuracy.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical analysis technology, and in particular to a characteristic spectrum of phloroglucinol components in the medicinal herb Dryopteris crassirhizoma, a method for constructing its content determination, and its application in identifying adulterants. Background Technology

[0002] Traditional Chinese medicine (TCM) chemical components constitute a complex, multi-component system. Traditional chemical drug quality control methods are insufficient for evaluating the intrinsic quality of TCM. Fingerprint / characteristic chromatograms, due to their holistic control approach, can comprehensively reflect the types and quantities of chemical components contained in TCM and its preparations, thus providing an overall description and evaluation of drug quality. Their application in the research field of TCM chemical components and quality control is increasing. While there are numerous studies applying fingerprint / characteristic chromatograms to the quality control of TCM materials, existing research on *Dryopteris crassirhizoma* fingerprint / characteristic chromatograms mainly focuses on water-soluble components, primarily phenylpropanoids such as chlorogenic acid, caffeic acid, and protocatechuic acid, followed by a few flavonoid components. For example, some literature has established HPLC characteristic chromatograms for processed slices, standard decoctions, and formulated granules, showing 12 common peaks and identifying 7 chromatographic peak components, mainly phenylpropanoids such as chlorogenic acid, neochlorogenic acid, and caffeic acid. However, research indicates that the main chemical components of Dryopteris crassirhizoma are phloroglucinols, including phloroglucinol AB, phloroglucinol ABA, and phloroglucinol ABBA, which are also the material basis for the various pharmacological activities exhibited by Dryopteris crassirhizoma, such as antiviral and antibacterial effects. Therefore, existing fingerprint / characteristic chromatograms of Dryopteris crassirhizoma cannot fully reflect its intrinsic quality.

[0003] Dryopteris crassirhizoma Nakai, a plant belonging to the Dryopteris family, is a dried rhizome and petiole remnants. It contains phloroglucinols, flavonoids, terpenoids, and phenylpropanoids, and possesses heat-clearing, detoxifying, and anthelmintic properties. Modern research indicates that phloroglucinols are the main components responsible for its antiviral, antibacterial, antitumor, and anthelmintic pharmacological activities. In recent years, due to increased demand for Dryopteris crassirhizoma, counterfeit products have frequently appeared in the market, such as Dryopteris crassirhizoma var. crassirhizoma, Dryopteris crassirhizoma var. crassirhizoma, and rhizomes of other fern species. These differences in efficacy affect the safety of using Dryopteris crassirhizoma. However, existing methods for distinguishing Dryopteris crassirhizoma from its counterfeits are limited, relying mainly on traditional morphological and microscopic identification methods and cross-sectional microscopic identification techniques. However, judging appearance is subjective and inapplicable to powdered medicinal materials that have lost their morphological appearance, presenting significant limitations. Therefore, it is necessary to establish fingerprint / characteristic spectra of Dryopteris crassirhizoma that can characterize its main chemical component, phloroglucinol, and the detection index of its effective component content as a standard for its qualitative identification, so as to ensure its medication safety. Summary of the Invention

[0004] Therefore, it is necessary to provide a characteristic spectrum of resorcinol components in Dryopteris crassirhizoma, a method for constructing content determination, and an application for identifying adulterants, which can provide a more comprehensive and effective evaluation for the quality control of Dryopteris crassirhizoma.

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

[0006] In a first aspect, the present invention provides a method for constructing a characteristic spectrum of phloroglucinol components from the medicinal herb Dryopteris crassirhizoma, comprising the following steps:

[0007] The medicinal material Dryopteris crassirhizoma was extracted using an extraction solvent to prepare a test solution;

[0008] A reference solution was prepared by dissolving the phloroglucinol components of Dryopteris crassirhizoma using a dissolving agent.

[0009] The test solution and the reference solution were analyzed by high performance liquid chromatography to construct a characteristic spectrum of phloroglucinol components in Dryopteris crassirhizoma.

[0010] The conditions for high-performance liquid chromatography detection include: mobile phase A is a mixture of acetonitrile, methanol and phosphoric acid aqueous solution, mobile phase B is phosphoric acid aqueous solution, and gradient elution is used;

[0011] The gradient elution procedure is as follows:

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

[0013] The volume percentage of mobile phase A was maintained at 64% for 60-65 minutes.

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

[0015] In some embodiments, the high-performance liquid chromatography detection satisfies at least one of the following conditions:

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

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

[0018] (3) The flow rate is 0.5 mL·min -1 ~1.5 mL·min -1 ;

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

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

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

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

[0023] The phosphoric acid aqueous solution contains 0.05% to 0.4% phosphoric acid by volume; and / or

[0024] The dissolving agent is methanol.

[0025] In some embodiments, the method for constructing the characteristic spectrum of resorcinols in the Dryopteris crassirhizoma herb involves using high performance liquid chromatography-mass spectrometry to identify the characteristic peaks of the characteristic spectrum of the Dryopteris crassirhizoma herb.

[0026] The conditions for the high performance liquid chromatography-mass spectrometry (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 gradient elution procedure is as follows:

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

[0029] The volume percentage of mobile phase A was maintained at 64% for 60-65 minutes.

[0030] The characteristic peaks of the characteristic spectrum of the herbal medicine Dryopteris crassirhizoma include: peak 1 is oxalic acid AP, peak 2 is noroxalic acid PB, and peak 8 is oxalic acid AB.

[0031] In some embodiments, the conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) further include: the volume percentage of acetonitrile and methanol in mobile phase A is (1-8):(1-2); and / or

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

[0033] The formic acid aqueous solution contains formic acid at a volume percentage of 0.05% to 0.3%.

[0034] In some embodiments, the step of extracting the Dryopteris crassirhizoma herb using an extraction solvent satisfies at least one of the following conditions:

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

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

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

[0038] (4) The mass-volume ratio of the medicinal material *Dryopteris crassirhizoma* and the extraction solvent is 1 g: (15-50) mL.

[0039] A second aspect of the present invention provides a method for determining the content of phloroglucinol components in the medicinal herb Dryopteris crassirhizoma, comprising the following steps:

[0040] The medicinal material Dryopteris crassirhizoma was extracted using an extraction solvent to prepare a test solution;

[0041] The reference standard was dissolved using a dissolving reagent to prepare reference standard solutions of different concentrations;

[0042] The reference standard solutions of different concentrations were injected separately and detected by high performance liquid chromatography.

[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 the linear regression equation.

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

[0045] The resorcinol component includes phloroglucinol AB; the reference standard 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 Dryopteris crassirhizoma and its adulterants, comprising the following steps:

[0047] Extraction solvents were used to extract the medicinal material Dryopteris crassirhizoma and its adulterants to prepare test solutions;

[0048] The test solution was subjected to high performance liquid chromatography to detect the characteristic spectrum of Dryopteris crassirhizoma and its adulterants, and to observe whether the characteristic spectrum of each adulterant had the characteristic peaks of phloroglucinol components corresponding to the characteristic spectrum of Dryopteris crassirhizoma.

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

[0050] A fourth aspect of the present invention provides a method for preparing oxaliplatin AB, comprising the following steps:

[0051] The powder of Dryopteris crassirhizoma was extracted using an extraction solvent. The resulting extract was then concentrated and dried to prepare an extract of Dryopteris crassirhizoma.

[0052] The extract of Dryopteris crassirhizoma was mixed with water, and the resulting mixture was extracted using an extraction solvent.

[0053] The obtained extract was separated by column chromatography, and the chromatographic solution containing the phloroglucinol component of Dryopteris crassirhizoma was collected.

[0054] The chromatography solution was purified to prepare oxaliplatin AB.

[0055] In some embodiments, the extraction solvent is an aqueous ethanol solution; optionally, the volume percentage of ethanol in the aqueous ethanol 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] This application constructs a characteristic spectrum of resorcinol components in Dryopteris crassirhizoma using specific high-performance liquid chromatography (HPLC) conditions. This method has good separation of the characteristic peaks, good precision, repeatability, and robustness, and can comprehensively reflect the main chemical component characteristics of Dryopteris crassirhizoma, providing an overall quality control basis for the quality standards of Dryopteris crassirhizoma.

[0060] Meanwhile, this application combines high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) to characterize the phloroglucinol components of Dryopteris crassirhizoma, and can be used to identify adulterants of Dryopteris crassirhizoma. It can provide an important reference for the establishment of quality standards for Dryopteris crassirhizoma and provide a more comprehensive and effective evaluation for the quality control of Dryopteris crassirhizoma.

[0061] Furthermore, the characteristic spectral method constructed in this application can simultaneously determine the content of oxalic acid AB, an active ingredient in Dryopteris crassirhizoma. It first achieves the determination of the content of oxalic acid AB in Dryopteris crassirhizoma. After methodological verification, the method has good specificity, precision, repeatability, accuracy, and robustness, providing a reference for the selection of content indicators when establishing quality standards for Dryopteris crassirhizoma. Attached Figure Description

[0062] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0063] Figure 1 This is a full-wavelength scan 3D image of the characteristic spectrum of the medicinal herb Dryopteris crassirhizoma in Example 1.

[0064] Figure 2 This is a comparative chromatogram of different detection wavelengths for the characteristic chromatograms of Dryopteris crassirhizoma in Example 1.

[0065] Figure 3 This is a comparative chromatogram of different elution gradients for the characteristic chromatograms of Dryopteris crassirhizoma in Example 1.

[0066] Figure 4 This is a comparative chromatogram of different organic phases eluted from the characteristic chromatogram of Dryopteris crassirhizoma in Example 1.

[0067] Figure 5 This is a comparative chromatogram of the characteristic chromatograms of Dryopteris crassirhizoma in Example 1 at different column temperatures.

[0068] Figure 6 The chromatograms of the characteristic chromatograms of Dryopteris crassirhizoma in Example 1 are investigated using different extraction solvents.

[0069] Figure 7 The chromatograms are for different extraction methods of the medicinal herb Dryopteris crassirhizoma in Example 1.

[0070] Figure 8 The chromatograms are for investigating the different amounts of extraction solvents used in the characteristic chromatograms of Dryopteris crassirhizoma in Example 1.

[0071] Figure 9 The chromatograms are for different extraction times of the herbal medicine Dryopteris crassirhizoma in Example 1.

[0072] Figure 10 The chromatograms are for different extraction times of the herbal medicine Dryopteris crassirhizoma in Example 1.

[0073] Figure 11 The chromatograms are for investigating the different amounts of extraction solvents used in the characteristic chromatograms of Dryopteris crassirhizoma in Example 1.

[0074] Figure 12 The common pattern of the characteristic chromatograms of 16 batches of Dryopteris crassirhizoma in Example 1.

[0075] Figure 13The images show the total ion chromatogram and ultraviolet absorption chromatogram of the *Dryopteris crassirhizoma* medicinal material test solution in Example 1.

[0076] Figure 14 This is the first-order mass spectrometry scan (negative ion mode) of peak 1 in Example 1.

[0077] Figure 15 This is the first-order mass spectrum (positive ion mode) of peak 1 in Example 1.

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

[0079] Figure 17 This is a secondary mass spectrometry scan (positive ion mode) of the peak at m / z = 403.1393 in Example 1.

[0080] Figure 18 The structure of AP in Example 1 is shown.

[0081] Figure 19 This represents a possible fragmentation mode (negative ion mode) for the peak at m / z = 403.1393 in Example 1.

[0082] Figure 20 This is the first-order mass spectrometry scan (negative ion mode) of peak 2 in Example 1.

[0083] Figure 21 This is the first-order mass spectrum (positive ion mode) of peak 2 in Example 1.

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

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

[0086] Figure 24 The PB structure of norflavomaric acid in Example 1 is shown.

[0087] Figure 25 This represents a possible fragmentation mode (negative ion mode) for the peak at m / z = 417.1550 in Example 1.

[0088] Figure 26 This is the first-order mass spectrometry scan (negative ion mode) of peak 8 in Example 1.

[0089] Figure 27 This is the first-order mass spectrum (positive ion mode) of peak 8 in Example 1.

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

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

[0092] Figure 30 The structure of AB-type oxaliplatin in Example 1 is shown.

[0093] Figure 31 This represents a possible fragmentation mode (negative ion mode) for the peak at m / z = 417.1550 in Example 1.

[0094] Figure 32 The image shows the 1H NMR spectrum of the AB monomer of ramie acid prepared in Example 1.

[0095] Figure 33 The image shows the carbon NMR spectrum of the AB monomer of ramie acid prepared in Example 1.

[0096] Figure 34 The characteristic chromatograms of the AB monomer of ramie acid prepared in Example 1 are compared with those of the test sample.

[0097] Figure 35 This is a comparative characteristic spectrum of the medicinal material Dryopteris crassirhizoma in Example 1.

[0098] Figure 36 This is a specificity-investigating chromatogram of the medicinal herb Dryopteris crassirhizoma in Example 1.

[0099] Figure 37 The graphs for different column temperatures are the characteristic graphs of the medicinal herb Dryopteris crassirhizoma in Example 1.

[0100] Figure 38 The image shows the characteristic spectra of Dryopteris crassirhizoma in Example 1 under different flow rates.

[0101] Figure 39 This is a comparative chromatogram of different chromatographic columns for the characteristic chromatogram of Dryopteris crassirhizoma in Example 1.

[0102] Figure 40 This is a comparison of the characteristic spectra of the *Dryopteris crassirhizoma* medicinal material and its adulterants in Example 1.

[0103] Figure 41 This is a specificity test diagram for the determination of AB content of ylang-ylang acid in Dryopteris crassirhizoma in Example 1.

[0104] Figure 42 This is a graph showing the purity of the peak of AB content in the *Dryopteris crassirhizoma* herb from Example 1.

[0105] Figure 43This is the AB standard curve of ramie acid in Example 1.

[0106] Figure 44 The characteristic spectrum of the medicinal material *Dryopteris crassirhizoma* (S1) in Example 2 was determined. Detailed Implementation

[0107] The present application will be further described in detail below with reference to specific embodiments. The present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application.

[0108] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0109] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0110] In this article, "one or more" refers to any one, two or more of the listed items.

[0111] In this document, the optional range of "and / or", "or / and", and "and / or" includes any one of two or more of the related listed items, as well as any and all combinations of the related listed items, including any two related listed items, any more related listed items, or a combination of all the related listed items.

[0112] In this document, terms such as “further,” “even further,” and “especially” are used to describe the purpose and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0113] In this document, terms such as "first aspect," "second aspect," "third aspect," and "fourth aspect" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly specifying the importance or quantity of the indicated technical features. Furthermore, terms such as "first," "second," "third," and "fourth" serve only as a non-exhaustive enumeration and should be understood as not constituting a closed limitation on quantity. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified. In the description of this application, "several" means at least one, such as one, two, etc., unless otherwise explicitly specified.

[0114] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0115] Unless otherwise specified, the percentage content mentioned in this application refers to mass percentage for solid-liquid mixtures and solid-phase-solid mixtures, and volume percentage for liquid-phase-liquid mixtures.

[0116] Unless otherwise specified, all percentage concentrations mentioned in this application refer to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of that component.

[0117] Unless otherwise specified, the temperature parameters in this application allow for both isothermal processing and processing within a certain temperature range. The isothermal processing allows temperature fluctuations within the precision range of instrument control, such as ±5℃, ±2℃, ±1℃, ±0.5℃, ±0.4℃, ±0.3℃, ±0.2℃, and ±0.1℃. In this application, room temperature or ambient temperature refers to the absence of temperature control operations, generally meaning 4℃ to 35℃, and preferably 20±5℃.

[0118] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0119] Research indicates that the 2020 edition of the Chinese Pharmacopoeia lacks fingerprint / characteristic chromatograms and active ingredient content determination items for Dryopteris crassirhizoma, making it difficult to control its overall quality. Therefore, the quality standards for Dryopteris crassirhizoma need further improvement. Fingerprint / characteristic chromatograms can characterize major representative chemical components, and quantitative analysis of the active ingredients in traditional Chinese medicine can determine the quality of the medicine, making them important methods for controlling and evaluating the quality of medicinal materials and identifying related counterfeit products. Studies have shown that the main chemical component of Dryopteris crassirhizoma is phloroglucinol, which is also the main material basis for its various pharmacological activities such as antiviral and antibacterial effects. Therefore, it is necessary to establish a characteristic chromatogram of phloroglucinol in Dryopteris crassirhizoma to further comprehensively control its quality. Currently reported studies on fingerprint / characteristic chromatograms of Dryopteris crassirhizoma mainly focus on non-major active ingredients such as phenylpropanoids and flavonoids. The content detection indicators mainly selected are total flavonoids, total phloroglucinol, or chlorogenic acid, which lack accuracy or specificity. Currently, there are no reports on the content determination of phloroglucinol components with good pharmacological activity, such as phloroglucinol AB [12,13], in Dryopteris crassirhizoma.

[0120] To address the shortcomings of existing quality control methods for Dryopteris crassirhizoma, such as the inability to distinguish counterfeit products, lack of specificity of quality control components, and poor accuracy, the researchers of this application unexpectedly discovered, through extensive research, a highly efficient and specific method for constructing a characteristic spectrum of resorcinols in Dryopteris crassirhizoma and determining its content. Specifically, by establishing a characteristic spectrum of low-polarity resorcinols in Dryopteris crassirhizoma, the main active ingredient, resorcinols, can be characterized simultaneously as much as possible, and the content of the specific active ingredient, phloroglucinol AB, can be determined. This allows for effective and qualitative identification of related adulterants, comprehensive evaluation and quality control, ensuring clinical medication safety, and providing an important reference for establishing comprehensive medicinal material quality standards.

[0121] In a first aspect, the present invention provides a method for constructing a characteristic spectrum of phloroglucinol components from the medicinal herb Dryopteris crassirhizoma, comprising the following steps:

[0122] The medicinal material Dryopteris crassirhizoma was extracted using an extraction solvent to prepare a test solution;

[0123] A reference solution was prepared by dissolving the phloroglucinol components of Dryopteris crassirhizoma using a dissolving agent.

[0124] The test solution and the reference solution were analyzed by high performance liquid chromatography to construct a characteristic spectrum of phloroglucinol components in Dryopteris crassirhizoma.

[0125] The conditions for high-performance liquid chromatography detection include: mobile phase A is a mixture of acetonitrile, methanol and phosphoric acid aqueous solution, mobile phase B is phosphoric acid aqueous solution, and gradient elution is used;

[0126] The gradient elution procedure is as follows:

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

[0128] The volume percentage of mobile phase A was maintained at 64% for 60-65 minutes.

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

[0130] In some examples, the conditions for high-performance liquid chromatography (HPLC) detection include a detection wavelength of 210 nm to 400 nm. Further, the detection wavelength is 254 nm to 330 nm. It is understood that the detection wavelengths of HPLC include, but are not limited to, 254 nm, 265 nm, 295 nm, 315 nm, and 330 nm.

[0131] In some examples, the conditions for the high-performance liquid chromatography (HPLC) detection include a column temperature of 20°C to 40°C. It is understood 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 these examples, the conditions for the high-performance liquid chromatography detection include a flow rate of 0.5 mL / min. -1 ~1.5 mL·min -1 Understandably, the flow rate for the high-performance liquid chromatography detection includes, but is not limited to, 0.5 mL / min. -1 0.75 mL·min -1 1.0 mL·min -1 1.25 mL·min -1 1.5 mL·min -1 .

[0133] In some of these examples, the conditions for the high-performance liquid chromatography (HPLC) detection include an injection volume of 1 μL to 10 μL. It is understood 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 these examples, the conditions for the high-performance liquid chromatography detection include: the column being a C18 column.

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

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

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

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

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

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

[0141] In some of these examples, the extraction time ranged from 15 to 60 minutes.

[0142] In some of these examples, the mass-to-volume ratio of the herb *Dryopteris crassirhizoma* to the extraction solvent is 1 g: (15–50) mL.

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

[0144] In some of these 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 these examples, the volume ratio of the extraction solvent to the extraction solvent is (15–40) mL: 25 mL.

[0146] In some examples, the method for constructing the characteristic spectrum of resorcinols in the Dryopteris crassirhizoma herb uses high performance liquid chromatography-mass spectrometry to identify the characteristic peaks of the characteristic spectrum of the Dryopteris crassirhizoma herb.

[0147] The conditions for the high performance liquid chromatography-mass spectrometry (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 gradient elution procedure is as follows:

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

[0150] The volume percentage of mobile phase A was maintained at 64% for 60-65 minutes.

[0151] The characteristic peaks of the characteristic spectrum of the herbal medicine Dryopteris crassirhizoma include: peak 1 is oxalic acid AP, peak 2 is noroxalic acid PB, and peak 8 is oxalic acid AB.

[0152] In some examples, the conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) further include: the volume percentage of acetonitrile and methanol in mobile phase A is (1-8):(1-2). Understandably, the volume percentage of acetonitrile and methanol in mobile phase A includes, but is not limited to, 1:1, 5:1, 8:1, 3:2, 5:2, and 7:2.

[0153] In some examples, the conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) further include: the volume ratio of the total volume of acetonitrile and methanol to the aqueous formic acid solution is 0.1% to 0.3%. It is understood that the volume ratio of the total volume of acetonitrile and methanol to the aqueous formic acid solution includes, but is not limited to, 0.1%, 0.2%, and 0.3%.

[0154] In some examples, the conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) further include: the volume percentage of formic acid in the formic acid aqueous solution is 0.05% to 0.3%. Understandably, 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 of these examples, the high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) method also satisfies at least one of the following conditions:

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

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

[0158] (3) The flow rate is 0.5 mL·min -1 ~1.5 mL·min -1 ;

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

[0160] In some of these examples, the chromatographic peaks of the *Dryopteris crassirhizoma* herb characteristic chromatogram contain eight characteristic peaks; among which the three identified characteristic peaks are: peak 1 is oxaliplatin AP, peak 2 is noroxaliplatin PB, and peak 8 is oxaliplatin AB.

[0161] A second aspect of the present invention provides a method for determining the content of phloroglucinol components in the medicinal herb Dryopteris crassirhizoma, comprising the following steps:

[0162] The medicinal material Dryopteris crassirhizoma was extracted using an extraction solvent to prepare a test solution;

[0163] The reference standard was dissolved using a dissolving reagent to prepare reference standard solutions of different concentrations;

[0164] The reference standard solutions of different concentrations were injected separately and detected by high performance liquid chromatography.

[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 the linear regression equation.

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

[0167] The resorcinol component includes phloroglucinol AB; the reference standard 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 these examples, the extraction step in the method for determining the content of phloroglucinol components in the medicinal herb Dryopteris crassirhizoma satisfies at least one of the following conditions:

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

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

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

[0172] (4) The mass-volume ratio of the medicinal material *Dryopteris crassirhizoma* 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 Dryopteris crassirhizoma and its adulterants, comprising the following steps:

[0174] Extraction solvents were used to extract the medicinal material Dryopteris crassirhizoma and its adulterants to prepare test solutions;

[0175] The test solution was subjected to high performance liquid chromatography to detect the characteristic spectrum of Dryopteris crassirhizoma and its adulterants, and to observe whether the characteristic spectrum of each adulterant had the characteristic peaks of phloroglucinol components corresponding to the characteristic spectrum of Dryopteris crassirhizoma.

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

[0177] Quantitative analysis of certain effective components or effective parts in traditional Chinese medicinal materials using appropriate chemical or instrumental analytical methods, and judging the quality of the medicine based on whether the test results meet the requirements of the drug standard, is an important method for controlling and evaluating drug quality. According to research, the current pharmacopoeia standard for *Dryopteris crassirhizoma* does not specify content indicators to characterize quality; it only specifies its appearance, microscopic identification, and thin-layer chromatography identification, indicating that the quality standard is still incomplete. *Dryopteris crassirhizoma* has abundant resources, and many varieties are commonly used in different regions. In recent years, *Dryopteris crassirhizoma* has played a significant role in antiviral activity (against COVID-19, influenza, and respiratory-associated viruses), leading to increased demand and the frequent appearance of counterfeit products in the market. Research shows that common counterfeit products include *Dryopteris crassirhizoma* var. *sinensis*, *Dryopteris crassirhizoma var. *sinensis*, *Dryopteris crassirhizoma var. *sinensis*, and rhizomes of *Dryopteris crassirhizoma*, which affect the safety of *Dryopteris crassirhizoma* due to differences in efficacy. Therefore, it is necessary to establish fingerprint / characteristic spectra of *Dryopteris crassirhizoma* that can characterize its main chemical components and effective component content detection indicators as standards for qualitative identification, thereby ensuring its safe use.

[0178] A fourth aspect of the present invention provides a method for preparing oxaliplatin AB, comprising the following steps:

[0179] The powder of Dryopteris crassirhizoma was extracted using an extraction solvent. The resulting extract was then concentrated and dried to prepare an extract of Dryopteris crassirhizoma.

[0180] The extract of Dryopteris crassirhizoma was mixed with water, and the resulting mixture was extracted using an extraction solvent.

[0181] The obtained extract was separated by column chromatography, and the chromatographic solution containing the phloroglucinol component of Dryopteris crassirhizoma was collected.

[0182] The chromatography solution was purified to prepare oxaliplatin AB.

[0183] In some examples, in the step of extracting the powder of Dryopteris crassirhizoma using an extraction solvent, the extraction solvent is an aqueous ethanol solution; optionally, the volume percentage of ethanol in the aqueous ethanol solution is 80%-99%.

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

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

[0186] In some of these examples, the column chromatography method uses an elution solution comprising a mixture of petroleum ether, ethyl acetate, and formic acid.

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

[0188] In some examples, the separation process involves gradient elution with a mixture of petroleum ether, ethyl acetate, and formic acid in a volume ratio, and collection of the chromatographic solution containing the said flavomaric acid AB.

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

[0190] The following detailed description is provided in conjunction with specific embodiments. Unless otherwise specified, all raw materials used in the following embodiments are commercially available; all instruments used are commercially available; and all processes involved are conventionally selected by those skilled in the art unless otherwise specified.

[0191] Example 1

[0192] This embodiment provides a method for constructing a characteristic spectrum of resorcinols in Dryopteris crassirhizoma and determining the content of active ingredients, as well as its application in identifying adulterants, as detailed below:

[0193] 1. Instruments and Materials

[0194] 1.1 Medicinal Materials

[0195] Sixteen batches of samples of Dryopteris crassirhizoma were collected from different regions in Northeast China, as detailed in Table 1. They were identified by the Quality Center of Guangdong Yifang Pharmaceutical Co., Ltd. as dried rhizomes and petiole remnants of Dryopteris crassirhizoma, a plant of the Dryopteris family.

[0196] Table 1. Origin information of *Dryopteris crassirhizoma* (a type of medicinal herb).

[0197]

[0198] 1.2 Instruments and Reagents

[0199] Waters HPLC system (Arc, Waters Corporation), Agilent HPLC system (1260, Agilent Technologies), Thermo (Ultra-High Performance Liquid Chromatography) system (Vanquish, Thermo Fisher Scientific), Thermo Vanquish Flex (Ultra-High Performance Liquid Chromatography-Thermo Fisher QE High Resolution Mass Spectrometry System (Thermo Fisher Scientific), Waters XSelect HSSC18 column (4.6mm × 250mm, 5μm), Kromasil 100-5-C18 column (4.6mm × 250mm, 5μm), Agilent ZORBAX Eclipse XDB-C18 column (4.6mm × 250mm, 5μm), Thermo Scientific™ Acclaim™ 120C18 column (4.6mm × 250mm, 5μm), Agilent ZORBAX Extend-C18 column (4.6mm×250mm, 5μm), CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), 0.01% balance (ME204E, Mettler Toledo), 0.1% electronic analytical balance (XP26, Mettler Toledo), rotary evaporator (RE-3000, Shanghai Yarong Biochemical Instrument Factory), ultrapure water system (Milli-Q Direct, Merck KGaA). Ethanol, methanol (Tianjin Fuyu Fine Chemical Co., Ltd.), and petroleum ether (60–90℃, Tianjin Baishi Chemical Co., Ltd.) were all analytical grade; acetonitrile, methanol (Merck KGaA), and phosphoric acid (Tianjin Kemeio Chemical Reagent Co., Ltd.) were chromatographic grade; formic acid (Shanghai Anpu Experimental Technology Co., Ltd.) was UPLC / LC-MS grade; ultrapure water (laboratory-made); ramie acid AB reference standard (laboratory-made).

[0200] 2. Experimental Methods

[0201] 2.1 Establishment of HPLC characteristic chromatograms of Dryopteris crassirhizoma.

[0202] 2.1.1 Optimization of chromatographic conditions

[0203] (1) Selection of detection wavelength

[0204] Based on literature review, 254 nm, 280 nm, and 330 nm were selected as the detection wavelengths for *Dryopteris crassirhizoma* samples. The chromatographic conditions were as follows: column: Agilent ZORBAX RRHD StableBond C18 column (2.1 × 150 mm, 1.8 μm); mobile phase: A was methanol, B was 0.1% phosphoric acid aqueous solution; gradient elution sequence: 0–30 min, 10% → 60% A; 30–50 min, 60% → 80% A; 50–60 min, 80% → 95% A; 60–65 min, 95% → 100% A; flow rate: 0.30 mL / min. -1 The column temperature was 30℃; the injection volume was 1 μL, and a full-wavelength scan was performed to record the absorption spectrum of the sample in the range of 210 nm to 400 nm. The results are shown in [Figure number missing]. Figure 1 and Figure 2 .

[0205] The results showed that there was more chromatographic peak information at a detection wavelength of 280 nm. Therefore, 280 nm was selected as the detection wavelength for the characteristic spectrum.

[0206] (2) Selection of mobile phase gradient

[0207] As shown in Tables 2-A, 2-B, 2-C, and 2-D below, the chromatographic conditions were as follows: Column: Waters XSelect HSSC18 column (4.6 mm × 250 mm, 5 μm); Mobile phase: A was acetonitrile-methanol (7:2) (0.2% phosphoric acid), B was 0.2% phosphoric acid aqueous solution; Flow rate: 1.0 mL / min -1 The detection wavelength was 280 nm; the column temperature was 25 ℃; and the injection volume was 5 μL. The effects of different elution gradients on peak resolution and baseline were investigated. The results are shown in [Figure number missing]. Figure 3 .

[0208] Table 2-A Gradient 1

[0209]

[0210] Table 2-B Gradient 2

[0211]

[0212] Table 2 - Gradient 3 of C

[0213]

[0214] Table 2-D Gradient 4

[0215]

[0216] The results showed that when gradient 4 was used as the elution gradient, the baseline of the characteristic spectrum was stable and the separation of each chromatographic peak was good. Therefore, 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 resolution and baseline of each chromatographic peak in the characteristic chromatogram were investigated. The chromatographic conditions were as follows: column: Waters XSelect HSS C18 column (4.6 mm × 250 mm, 5 μm); gradient elution sequence: 0–60 min, 60% → 64% A, 40% → 36% B; 60–65 min, 64% A, 36% B; flow rate: 1.0 mL / min. -1 Detection wavelength: 280 nm; column temperature: 25 °C; injection volume: 5 μL. Results are shown below. 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 chromatogram was stable and the separation of each chromatographic peak was good. Therefore, 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℃, 25℃, 30℃, 40℃) on the resolution and baseline of each chromatographic peak in the characteristic chromatogram were investigated. The chromatographic conditions were as follows: column: Waters XSelect HSS C18 column (4.6mm × 250mm, 5μm); mobile phase: A was acetonitrile-methanol (7:2) (0.2% phosphoric acid), B was 0.2% aqueous phosphoric acid solution; gradient elution sequence: 0–60 min, 60% → 64% A, 40% → 36% B; 60–65 min, 64% A, 36% B; flow rate: 1.0 mL / min. -1 Detection wavelength: 280 nm; injection volume: 5 μL; results are shown below. Figure 5 .

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

[0223] (5) Determination of chromatographic conditions

[0224] Based on the above experimental results, the high-performance liquid chromatography conditions for the characteristic chromatogram of Dryopteris crassirhizoma were determined as follows:

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

[0226] 2.1.2 Investigation of sample treatment conditions

[0227] (1) Investigation of extraction solvent types

[0228] The sample size of the medicinal material was 1.0 g. Extraction solvents were applied to the same batch of test samples using 30% methanol aqueous solution, 50% methanol aqueous solution, 70% methanol aqueous solution, and methanol solution, respectively. The results are shown below. Figure 6 The results showed that the peak area of ​​the methanol solution was the largest, while 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. Therefore, methanol solution was selected as the extraction solvent for the test sample.

[0229] (2) Examination of extraction methods

[0230] The sample size of the medicinal material was 1.0 g. Methanol solution was used as the extraction solvent. The extraction methods were investigated using both ultrasonic extraction and reflux extraction on the same batch of test samples. The results are shown below. Figure 7 The results showed that there was no significant difference in the peak area between ultrasonic extraction and reflux extraction, but the baseline of the chromatogram obtained by ultrasonic extraction was more stable, and the ultrasonic extraction operation was simpler. Therefore, ultrasonic extraction was selected as the extraction method for the test sample.

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

[0232] The sample size of the medicinal material was 1.0 g. Methanol solution was used as the extraction solvent, and ultrasonic extraction was employed. The solvent volumes were selected as 15 mL, 25 mL, and 50 mL for different batches of the same sample. The results are shown below. Figure 8 The results showed that when the extraction solvent volume was 15 mL, 25 mL, and 50 mL, there was no significant difference in the peak area of ​​each sample, and the peaks were clearly eluted. Considering energy conservation and ensuring complete extraction, 25 mL was selected as the extraction solvent volume.

[0233] (4) Examination 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. Ultrasonic extraction was performed at different times: 15 min, 30 min, 45 min, and 60 min. The ultrasonic extraction time was investigated for the same batch of samples, and the results are shown below. Figure 9 The results showed that there was no significant difference in the peak area of ​​the sample after sonication for 15 min, 30 min, 45 min, and 60 min, and the peaks were clearly eluted. Considering energy conservation and ensuring complete extraction, 30 min was selected as the sonication time.

[0235] (5) Examination of the number of extractions

[0236] The sample size of the medicinal material was 1.0 g. 25 mL of methanol solution was used as the extraction solvent. Ultrasonic extraction was performed for 30 min, with extractions repeated 2, 3, and 4 times for the same batch of sample. The results are shown below. Figure 10 The results showed that there was no significant difference in the peak area of ​​the sample after 2, 3, and 4 extractions, and the peaks were clearly eluted. Considering energy conservation and ensuring complete extraction, 3 extractions were selected as the number of extractions.

[0237] (6) Investigation on 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 was performed for 30 min, and the extraction was repeated three times. The solvent volumes were 15 mL, 25 mL, and 40 mL for the same batch of samples. The results are shown below. Figure 11 The results showed that when the extraction solvent volume was 15 mL, 25 mL, and 40 mL, there was no significant difference in the peak area of ​​each sample, and the peaks were clearly defined. Considering energy conservation and ensuring complete extraction, 25 mL was selected as the extraction solvent volume.

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

[0240] Based on the above experimental results, the sample pretreatment method for the characteristic spectrum of Dryopteris crassirhizoma was determined as follows:

[0241] Accurately weigh approximately 1.0g of powdered Dryopteris crassirhizoma (passed through a No. 3 sieve), place it in a stoppered conical flask, accurately add 25mL of methanol solution, weigh, sonicate for 30min, cool, weigh again, replenish the lost weight with methanol, shake well, filter, evaporate to dryness under reduced pressure using a rotary evaporator, dissolve the residue in 25mL of water, add 25mL of petroleum ether (60℃~90℃) and extract 3 times, combine the solvents, evaporate to dryness under reduced pressure using a rotary evaporator, dilute the residue to 10mL with methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0242] (8) Preparation of reference solution:

[0243] Accurately weigh 2.304 mg of oxalic acid AB reference standard, place it in a 10 mL volumetric flask, and add methanol to prepare a reference standard solution containing 218.097 μg per mL.

[0244] 2.1.3 Determination of characteristic peaks

[0245] Sixteen batches of *Dryopteris crassirhizoma* samples were collected. Test solutions were prepared according to the method specified in section "2.1.2". 5 μL of each of the test solution and reference solution were precisely pipetted and injected according to the chromatographic conditions specified in section "2.1.1". The common peaks of the characteristic chromatograms of the 16 batches of *Dryopteris crassirhizoma* were identified using the *Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software*. Eight common peaks with known components, good peak shape and resolution, and high purity were selected as the characteristic peaks of *Dryopteris crassirhizoma*. Figure 12 As shown, the AB chromatographic peak of the reference standard was taken as the reference peak S. The relative retention time of each characteristic peak and peak S was calculated. The average relative retention time of each characteristic peak in the characteristic chromatograms of 16 batches of Dryopteris crassirhizoma was calculated. The characteristic peaks were located using the average relative retention time of each characteristic peak.

[0246] The results showed that the characteristic spectra of 16 batches of Dryopteris crassirhizoma were analyzed. The peak corresponding to the reference peak of AB oxalis acid was taken as the S peak. The relative retention times of peaks 1, 2, 3, 4, 5, 6, and 7 with the S peak were calculated. The relative retention times should be within ±10% of the specified values. 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) High performance liquid chromatography-mass spectrometry conditions

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

[0250] Mass spectrometry conditions: see Table 3.

[0251] Table 3 Mass Spectrometry Parameters

[0252]

[0253] (2) Preparation of the test solution

[0254] Accurately weigh approximately 1.0g of powdered Dryopteris crassirhizoma (passed through a No. 3 sieve), place it in a stoppered conical flask, accurately add 25mL of methanol solution, weigh, sonicate for 30min, cool, weigh again, replenish the lost weight with methanol, shake well, filter, evaporate to dryness under reduced pressure using a rotary evaporator, dissolve the residue in 25mL of water, add 25mL of petroleum ether (60℃~90℃) and extract 3 times, combine the solvents, evaporate to dryness under reduced pressure using a rotary evaporator, dilute the residue to 10mL with methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0255] (3) Sample determination

[0256] Accurately pipette 5 μL of the test solution and inject it into the liquid chromatography-mass spectrometry (LC-MS) instrument. Analyze the test solution under the above-described LC and MS conditions. The total ion chromatogram and UV absorption chromatogram of the test solution are shown below. Figure 13 .

[0257] (4) Results Analysis

[0258] By comparing the precise molecular weight and fragment ion analysis using mass spectrometry, and matching the data with literature data (Wang Juan. Screening and metabolic study of the activity of phloroglucinol compounds against H5N1 influenza virus in Dryopteris crassirhizoma [D]. Beijing University of Chinese Medicine, 2022.; Ren Qiang. Study on the active components 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 spectroscopic rules of phloroglucinol chemical components in Dryopteris crassirhizoma [J]. Chinese Traditional and Herbal Drugs, 2015, 46(06):932-937.), three components, namely phloroglucinol AP (peak 1), norphloroglucinol PB (peak 2), and phloroglucinol AB (peak 8), were identified in the characteristic spectrum of Dryopteris crassirhizoma. The compound information is shown in Table 4. The specific analysis process is described in […]. Figures 14-34 .

[0259] Table 4. Mass spectrometry identification results of compounds in Dryopteris crassirhizoma.

[0260]

[0261] ① Peak No. 1

[0262] The first-order mass spectrum of peak 1 was extracted, covering the time interval from 26.48 min to 27.11 min. The results are shown below. Figure 14 , Figure 15 .

[0263] In the first-order mass spectrometry extraction chromatogram of peak 1, the peak at m / z 403.1393 in negative ion mode has the highest response value, which is [MH]. - The peak at m / z 405.1538 in positive ion mode should be [M+H]. + Peak; its molecular formula is inferred to be C based on its precise molecular weight. 21 H 24 O8; The second-order chromatogram of the peak at m / z 403.1393 with a collision energy of 40 was extracted. The results show that the main ion fragments are 195.0653, 151.0752, and 207.0654, etc. For detailed results, see [link to results]. Figure 16 The second-order ion plot of the peak at m / z 405.1538 with a collision energy of 40 was extracted. The results show that the main ion fragments are 197.0809, 179.0702, and 209.0809, etc. For detailed results, see [link to results]. Figure 17 .

[0264] The precise molecular weight and secondary ion fragmentation information of this signal peak were matched with compounds in the literature database. The results showed that the compound with the highest matching degree for the m / z 403.1393 peak was oxaliplatin (AP), the structural formula of which is shown below. Figure 18 Possible pyrolysis modes can be found in [link to relevant documentation]. Figure 19 Based on the comparison of fragment ions and the analysis of the structural fragmentation, it is suggested that the compound may be oxaliplatin (AP).

[0265] ② Peak No. 2

[0266] The first-order mass spectrum of peak 2 was extracted, covering the time interval from 36.63 min to 38.10 min. The results are shown below. Figure 20 , Figure 21 .

[0267] In the primary mass spectrometry extraction chromatogram of peak 2, the peak at m / z 417.1550 in negative ion mode has the highest response value, which is [MH]. - The peak at m / z 419.1693 in positive ion mode should be [M+H]. + Peak; its molecular formula is inferred to be C based on its precise molecular weight. 22 H 26 O8; The second-order chromatogram of the peak at m / z 417.1550 with a collision energy of 40 was extracted. The results show that the main ion fragments are 209.0813, 195.0655, and 165.0910, etc. For detailed results, see [link to results]. Figure 22 The second-order ion plot of the peak at m / z 419.1693 with a collision energy of 40 was extracted. The results show that the main ion fragments are 211.0963, 193.0860, and 137.0596, etc. For detailed results, see [link to relevant documentation]. Figure 23 .

[0268] The precise molecular weight and secondary ion fragmentation information of this signal peak were matched with compounds in the literature database. The results showed that the compound with the highest matching degree at m / z 417.1550 was norfloxacin PB, the structural formula of which is shown below. Figure 24 Possible pyrolysis modes can be found in [link to relevant documentation]. Figure 25 Based on the comparison of fragment ions and the analysis of the structural formula, it is suggested that the compound may be norflavomaric acid (PB).

[0269] ③ Peak No. 8

[0270] The first-order mass spectrum of peak 8 was extracted, covering the time interval from 56.78 min to 58.19 min. The results are shown below. Figure 26 , Figure 27 .

[0271] In the primary mass spectrometry extraction chromatogram of peak 8, the peak at m / z 417.1555 in negative ion mode showed the highest response value, which was [MH]. - The peak at m / z 419.1693 in positive ion mode should be [M+H]. + Peak; its molecular formula is inferred to be C based on its precise molecular weight. 22 H 26 O8; The second-order chromatogram of the peak at m / z 417.1550 with a collision energy of 40 was extracted. The results show that the main ion fragments are 209.0813, 195.0655, and 165.0910, etc. For detailed results, see [link to results]. Figure 28 The second-order ion plot of the peak at m / z 419.1693 with a collision energy of 40 was extracted. The results show that the main ion fragments are 211.0962, 193.0859, and 137.0596, etc. For detailed results, see [link to relevant documentation]. Figure 29 .

[0272] The precise molecular weight and secondary ion fragmentation information of this signal peak were matched with compounds in the literature database. The results showed that the compound with the highest matching degree for the m / z 417.1550 peak was oxaliplatin AB, the structural formula of which is shown below. Figure 30 Possible pyrolysis modes can be found in [link to relevant documentation]. Figure 31 Based on the comparison of fragment ions and the analysis of the structural formula, it is suggested that the compound may be oxaliplatin AB.

[0273] 2.1.5 Confirmation of characteristic peaks

[0274] Given that standards for phloroglucinol compounds from Dryopteris crassirhizoma are difficult to purchase commercially, this application also provides a method for preparing a reference standard of phloroglucinic acid AB, which can obtain high-purity phloroglucinic acid AB with high efficiency and yield. The specific steps are as follows:

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

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

[0277] C) The petroleum ether fraction was purified by chromatographic separation to obtain the compound.

[0278] Step C) involves the chromatographic separation and purification of the petroleum ether fraction.

[0279] The petroleum ether fraction was subjected to column chromatography on silica gel, eluted with a petroleum ether-ethyl acetate-formic acid gradient at a volume ratio of (20:1:0.2) → (10:1:0.2). The fractions were combined to obtain 15 fractions. Fraction 10, after solvent removal, yielded an extract, which was washed with petroleum ether (60℃~90℃) until the washings were clear. Ethyl acetate was then added to dissolve the extract, and crystals were allowed to precipitate. The bottom layer solid was removed, and the extract was washed again with petroleum ether (60℃~90℃) until the washings were clear. The solvent was removed to obtain 415 mg of a bright yellow powder. Fraction 11, after solvent removal, yielded an extract, which was washed with petroleum ether (60℃~90℃) until the washings were clear. Ethyl acetate was then added to wash again until the washings were clear, and the solvent was removed to obtain 1646 mg of a bright yellow powder. High-performance liquid chromatography (HPLC) analysis confirmed that the obtained solids were the same substance, and the fractions were combined. Nuclear magnetic resonance (NMR) analysis identified the structure as oxalic acid AB. Figure 32 , Figure 33 Furthermore, quantitative analysis using nuclear magnetic resonance (NMR) revealed a purity of 94.66%.

[0280] Accurately weigh approximately 1.0 g of the herb *Dryopteris crassirhizoma*. Prepare the test solution according to the method specified in section "2.1.2". Inject 5 μL each of the *Dryopteris crassirhizoma* AB reference solution and the test solution into the liquid chromatograph. Determine the chromatographic results according to the chromatographic conditions specified in section "2.1.1". The results are shown in the figure below. Figure 34 .

[0281] The results showed that the monomeric component of oxaliplatin AB had the same retention time as chromatographic peak 8, and the fragment ions from mass spectrometry fragmentation were the same, confirming that chromatographic peak 8 was oxaliplatin AB.

[0282] 2.1.6 Formulation of Feature Map

[0283] The HPLC characteristic chromatograms of 16 batches of Dryopteris crassirhizoma were matched using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine," and a reference chromatogram was generated using the mean method to establish a reference characteristic chromatogram for Dryopteris crassirhizoma. Figure 35The characteristic spectrum has 8 characteristic peaks. Using the peak corresponding to the reference peak of oxaliplatin AB as the S peak, the relative retention times of peaks 1, 2, 3, 4, 5, 6, and 7 with peak S are calculated. These relative retention times should be within ±10% of the specified values, which 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).

[0284] 2.1.7 Methodological Validation

[0285] (1) Specificity examination

[0286] Accurately weigh approximately 1.0 g of the herb *Dryopteris crassirhizoma* and prepare the test solution according to the method specified in section "2.1.2". Separately, inject 5 μL each of the *Dryopteris crassirhizoma* AB reference solution and blank solvent into the liquid chromatograph and determine the chromatographic conditions according to section "2.1.1". The results are shown below. Figure 36 .

[0287] The results showed that the chromatogram of the test sample had the same chromatographic peak at the corresponding retention time as that of the reference sample, and there was no interference from the blank solvent, indicating that the method has good specificity.

[0288] (2) Precision test

[0289] Approximately 1.0 g of the herb *Dryopteris crassirhizoma* was accurately weighed and prepared into a test solution according to the method specified in section "2.1.2". The solution was injected six times repeatedly under the chromatographic conditions described in section "2.1.1". Using the AB peak of *Dryopteris crassirhizoma* as the reference peak S, the relative retention time and relative peak area of ​​each characteristic peak relative to peak S were calculated, and the RSD values ​​were also calculated. The results showed that for the same test solution, after six consecutive injections, with the AB peak of *Dryopteris crassirhizoma* as the reference peak S, the RSD values ​​of the relative retention time of each characteristic peak relative to peak S were within the range of 0.05%–0.08%, and the RSD values ​​of the relative peak area were within the range of 1.79%–3.39%, all less than 5.0%, indicating good instrument precision.

[0290] (3) Repeated examination

[0291] Approximately 1.0 g of the herb *Dryopteris crassirhizoma* was accurately weighed and prepared into six test solutions according to the preparation method specified in section "2.1.2". These solutions were then injected and analyzed under the chromatographic conditions described in section "2.1.1". Using the AB peak of *Dryopteris crassirhizoma* as the reference peak S, the relative retention time and relative peak area of ​​each characteristic peak relative to peak S were calculated, and the RSD values ​​were also calculated. The results showed that when the same batch of samples was repeatedly measured six times, with the AB peak of *Dryopteris crassirhizoma* as the reference peak S, the RSD values ​​of the relative retention time of each characteristic peak relative to peak S were in the range of 0.07%–0.11%, and the RSD values ​​of the relative peak area were in the range of 1.41%–3.62%, all less than 5.0%, indicating good repeatability of the method.

[0292] (4) Stability test

[0293] Approximately 1.0 g of the herb *Dryopteris crassirhizoma* was accurately weighed and prepared according to the method specified in section "2.1.2". Chromatographic analysis was performed under the conditions specified in section "2.1.1" at 0, 4, 8, 12, 18, and 24 hours. Using the AB peak of *Dryopteris crassirhizoma* as the reference peak S, the relative retention time and relative peak area of ​​each characteristic peak relative to peak S were calculated, and the RSD values ​​were also calculated. The results showed that for the same sample solution analyzed at 0, 4, 8, 12, 18, and 24 hours, with the AB peak of *Dryopteris crassirhizoma* as the reference peak S, the RSD values ​​of the relative retention time of each characteristic peak relative to peak S were in the range of 0.05%–0.08%, and the RSD values ​​of the relative peak area were in the range of 1.15%–4.45%, all less than 5.0%, indicating that the sample solution had good relative stability within 24 hours.

[0294] (5) Intermediate precision test

[0295] Other analysts in this project team performed the analysis on different dates and using different chromatographs. Approximately 1.0 g of the same batch of *Dryopteris crassirhizoma* was accurately weighed and prepared according to the test sample preparation method determined in section "2.1.2". The analysis was performed under the chromatographic conditions in section "2.1.1", using the AB peak of *Dryopteris crassirhizoma* acid as the reference peak S. The relative retention time and relative peak area of ​​each characteristic peak relative to the S peak were calculated, and the RSD values ​​were also calculated. The results showed that, with different analysts performing the analysis at different times and on different instruments, and with the same batch of samples being measured repeatedly six times, the RSD values ​​of the relative retention time of each characteristic peak relative to the S peak were in the range of 0.12%–0.23%, and the RSD values ​​of the relative peak area were in the range of 1.32%–3.11%, all less than 5.0%. The RSD values ​​of the relative retention time and the six data points of the repeatability test were in the range of 0.10% to 0.75%, and the RSD values ​​of the relative peak area and the six data points of the repeatability test were in the range of 2.06% to 4.34%, both less than 5.0%, indicating that the relative retention time and relative peak area of ​​each characteristic peak have good intermediate precision on different instruments.

[0296] (6) Durability test

[0297] ① Investigation at different column temperatures

[0298] Approximately 1.0 g of the same batch of Dryopteris crassirhizoma was accurately weighed and prepared into a test solution according to the method specified in section "2.1.2". Except for column temperatures of 23℃, 25℃, and 27℃, all other chromatographic conditions were the same as specified in section "2.1.1". The sample was injected and analyzed, using bleaching acid AB as the reference peak S. The relative retention times and relative peak areas of each characteristic peak and peak S were calculated, and the RSD values ​​were calculated. The results are shown below. Figure 37 .

[0299] The results showed that, at different column temperatures, the relative retention time RSD values ​​of each characteristic peak and the S peak were in the range of 0.19% to 1.61%, and the relative peak area RSD values ​​were in the range of 1.38% to 4.60%, both less than 5.0%. This indicates that the column temperature ±2℃ has little effect on the relative retention time and relative peak area of ​​each characteristic peak, and shows good durability at different column temperatures.

[0300] ② Investigation of different flow velocities

[0301] Take approximately 1.0 g of the same batch of Dryopteris crassirhizoma, accurately weigh it, and prepare the test solution according to the test sample preparation method specified in section "2.1.2", except that the flow rate is 0.9 mL·min. -1 1.0 mL·min -1 and 1.1 mL·min -1 All other chromatographic conditions were the same as specified in section "2.1.1". Injection and determination were performed, using oxaliplatin AB as the reference peak S. The relative retention times and relative peak areas of each characteristic peak and peak S were calculated, and the RSD values ​​were calculated. Results are shown below. Figure 38 .

[0302] The results showed that, at different flow rates, the relative retention time RSD values ​​of each characteristic peak and the S peak were in the range of 0.19%–1.15%, and the relative peak area RSD values ​​were in the range of 0.38%–1.86%, all less than 3.0%, indicating that when the flow rate is ±0.1 mL·min -1 At that time, changes in flow rate had little effect on relative retention time and relative peak area.

[0303] ③ Investigation of different chromatographic columns

[0304] Take approximately 1.0 g of the same batch of Dryopteris crassirhizoma herb, accurately weigh it, and prepare the test solution according to the test solution preparation method specified in section "2.1.2". Except for the chromatographic columns (Kromasil 100-5-C18 (4.6 mm × 250 mm, 5 μm), Agilent ZORBAX Eclipse XDB-C18 (4.6 mm × 250 mm, 5 μm), Thermo Scientific™ Acclaim™ 120C18 (4.6 mm × 250 mm, 5 μm), Agilent ZORBAX Extend-C18 (4.6 mm × 250 mm, 5 μm), and Waters XSelect HSS C18 (4.6 mm × 250 mm, 5 μm), all other chromatographic conditions are the same as specified in section "2.1.1". Inject and determine the results. See below. Figure 39 .

[0305] The results showed that, compared with other different packed columns, the Waters XSelect HSS C18 (4.6 mm × 250 mm, 5 μm) column produced better peak shapes and higher resolution in the chromatogram. Therefore, it is recommended to use the Waters XSelect HSS C18 (4.6 mm × 250 mm, 5 μm) column for further research.

[0306] 2.1.8 Comparison of characteristic chromatograms of different adulterants

[0307] Approximately 1.0 g of each of the following herbs were accurately weighed: *Cypripedium simulans*, *Dryopteris japonicus*, *Dryopteris serratus*, *Cycas revoluta*, *Dryopteris brevicornu*, *Dryopteris crassirhizoma*, *Dryopteris ossificans*, *Dryopteris apicalis*, *Dryopteris bulbifera*, and *Dryopteris buergerianum*. The test solution was prepared according to the method specified in section "2.1.2" and analyzed under the chromatographic conditions specified in section "2.1.1". The results are as follows: Figure 40 As shown.

[0308] The results showed that there were differences in the characteristic chromatograms of *Dryopteris indicus*, *Dryopteris bladderii*, *Dryopteris bulbifera*, *Cibotium barometz*, *Cibotium japonicum*, *Cibotium barometz*, *Cibotium barometz*, *Cibotium barometz*, *Dryopteris septum*, *Dryopteris indicus*, and *Dryopteris pubescens*. *Dryopteris indicus* contained only characteristic peak 3, *Dryopteris bladderii* contained characteristic peaks 3, 4, 5, 7, and 8, *Dryopteris bulbifera* contained peaks 4 and 6, *Cibotium barometz* contained only characteristic peak 4, *Cibotium barometz* contained characteristic peaks 4 and 6, *Cibotium barometz* contained only characteristic peak 6, *Cibotium barometz* contained characteristic peaks 4, 5, 6, and 7, *Dryopteris septum* contained only characteristic peak 4, and *Dryopteris indicus* contained characteristic peaks 4 and 5. The peak area ratios of each chromatographic peak in the adulterant and the raw *Dryopteris crassirhizoma* were calculated. Only *Dryopteris crassirhizoma* contained characteristic peaks 1 and 2. The characteristic peak 3 in *Dryopteris sibiricum* and *Dryopteris brevicornu* had an area close to that of *Dryopteris crassirhizoma*. Except for the characteristic peak 4 in *Cibotium barometz*, which had an area close to that of *Dryopteris crassirhizoma*, the characteristic peak 4 in *Dryopteris brevicornu*, *Cibotium barometz*, *Cibotium japonicum*, *Dryopteris nigra*, *Dryopteris septemlobus*, and *Dryopteris crassirhizoma* was significantly smaller than that of *Dryopteris crassirhizoma*. The characteristic peak 5 in *Cibotium barometz* had an area close to that of *Dryopteris crassirhizoma*, but the characteristic peak 5 in *Dryopteris brevicornu* and *Dryopteris crassirhizoma* was significantly smaller than that of *Dryopteris crassirhizoma*. The characteristic peak 6 in *Cibotium barometz* had an area close to that of *Dryopteris crassirhizoma*, but the characteristic peak 6 in *Dryopteris brevicornu*, *Cibotium japonicum*, and *Dryopteris cycad* was significantly smaller than that of *Dryopteris crassirhizoma*. The characteristic peak 7 in *Cibotium barometz* had an area close to that of *Dryopteris crassirhizoma*, but the characteristic peak 7 in *Dryopteris brevicornu* was significantly smaller than that of *Dryopteris crassirhizoma In summary, the established feature map method can effectively distinguish *Dryopteris crassirhizoma* from various adulterants.

[0309] 2.2 Establishment of a method for determining the content of alpha-acid AB

[0310] 2.2.1 Preparation of reference solution

[0311] Accurately weigh 2.304 mg of oxalic acid AB reference standard, place it in a 10 mL volumetric flask, and add methanol to prepare a reference standard solution containing 218.097 μg per mL.

[0312] 2.2.2 Optimization of chromatographic conditions

[0313] Same as the chromatographic conditions for the characteristic chromatograms under section “2.1.1”.

[0314] 2.2.3 Investigation of sample treatment conditions

[0315] (1) Investigation of extraction solvent types

[0316] The sample size of the medicinal material was 1.0 g. 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 to investigate the different types of extraction solvents for the same batch of test samples. The results showed that methanol was used as the extraction solvent, and therefore methanol was ultimately selected as the extraction solvent for determining the content of AB in Dryopteris crassirhizoma.

[0317] (2) Examination of extraction methods

[0318] The sample size of the medicinal material was 1.0 g. Methanol solution was used as the extraction solvent. Extraction methods were investigated using both ultrasonic treatment and reflux extraction on the same batch of samples. The results showed that there was no significant difference between the content of AB obtained from reflux extraction and ultrasonic extraction. Based on the pretreatment conditions of the characteristic chromatogram, ultrasonic treatment was ultimately selected as the extraction method for determining the AB content of *Dryopteris crassirhizoma*.

[0319] (3) Examination of extraction time

[0320] The sample size of the medicinal material was 1.0 g. Methanol solution was used as the extraction solvent, and ultrasonic treatment was used as the extraction method. The extraction time was investigated at 15 min, 30 min, and 45 min for the same batch of test samples. The results showed that AB of ramie acid could be completely extracted at an extraction time of 30 min. Therefore, 30 min was finally selected as the extraction time for determining the AB content of ramie acid in Dryopteris crassirhizoma.

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

[0322] 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, and the extraction time was 30 min. The extraction solvent dosage was investigated using 15 mL, 25 mL, and 50 mL of the same batch of test sample. The results showed that 25 mL of extraction solvent could completely extract AB from the sample. Therefore, 25 mL was selected as the extraction solvent dosage for the determination of AB content in Dryopteris crassirhizoma.

[0323] (5) Examination of the number of extractions

[0324] The sample size of the medicinal material was 1.0 g. Methanol solution was used as the extraction solvent, ultrasonic treatment was used for extraction, the extraction time was 30 min, and the extraction solvent volume was 25 mL. The number of extractions was investigated for the same batch of test samples at 2, 3, 4, and 5 times. The results showed that AB of the herbaceous oxalis acid could be completely extracted at 3 extractions. Therefore, 3 extractions were ultimately selected as the extraction number for determining the AB content of the herbaceous oxalis acid in *Dryopteris crassirhizoma*.

[0325] (6) Investigation on 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 for extraction, the extraction time was 30 min, the extraction solvent volume was 25 mL, and the extraction was performed three times. The extraction solvent volume was investigated using 15 mL, 25 mL, and 40 mL of the same batch of sample. The results showed that 25 mL of extraction solvent was sufficient for complete extraction of oxalis AB. Therefore, 25 mL was ultimately selected as the extraction solvent volume for determining the oxalis AB content in *Dryopteris crassirhizoma*.

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

[0328] Based on the above experimental results, the sample pretreatment method for determining the content of AB-yellow cottony herb in Dryopteris crassirhizoma is determined as follows:

[0329] Accurately weigh approximately 1.0g of powdered Dryopteris crassirhizoma (passed through a No. 3 sieve), place it in a stoppered conical flask, accurately add 25mL of methanol solution, weigh, sonicate for 30min, cool, weigh again, replenish the lost weight with methanol, shake well, filter, evaporate to dryness under reduced pressure using a rotary evaporator, dissolve the residue in 25mL of water, add 25mL of petroleum ether (60℃~90℃) and extract 3 times, combine the solvents, evaporate to dryness under reduced pressure using a rotary evaporator, dilute the residue to 10mL with methanol, shake well, filter, and collect the filtrate to obtain the final product.

[0330] 2.2.4 Methodological Validation

[0331] (1) Specificity examination

[0332] Accurately weigh approximately 1.0 g of the herb *Dryopteris crassirhizoma* and prepare the test solution according to the method specified in section "2.2.3". Separately, inject 5 μL each of the *Dryopteris crassirhizoma* AB reference solution and blank solvent into the liquid chromatograph and determine the chromatographic conditions according to section "2.2.2". The results are shown below. Figure 41 .

[0333] The results showed that the chromatogram of the test sample had the same chromatographic peak at the corresponding retention time as that of the reference sample, and there was no interference from the blank solvent, indicating that the method has good specificity.

[0334] (2) Peak purity investigation

[0335] Accurately weigh approximately 1.0 g of the herb *Dryopteris crassirhizoma*. Prepare the test solution according to the method specified in section "2.2.3". Accurately pipette the test solution and the reference solution of oxaliplatin A and B, inject them into the liquid chromatograph, and perform a DAD detector scan from 210 nm to 400 nm under the chromatographic conditions specified in section "2.2.2". Calculate the peak purity. The results are shown in [Figure 1]. Figure 42 .

[0336] The results showed that no impurity peaks were detected in the AB peak of ramie acid in the sample, and the peak purity met the requirements, indicating that the purity of the AB peak of ramie acid met the requirements under the chromatographic conditions.

[0337] (3) Examination of linear relationships

[0338] Accurately weigh 25.392 mg of oxaliplatin AB reference standard and place it in a 10 mL volumetric flask. Add methanol to prepare a reference standard stock solution containing 2403.630 μg per 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 the mark to prepare reference standard 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 mL, respectively. Accurately pipette the linear solution of the above reference standard solution and inject it according to the chromatographic conditions under section “4..1”. Record the chromatographic peak area. A standard curve was plotted with peak area as the ordinate (y) and reference concentration as the abscissa (x), as shown in Table 5. Figure 43 .

[0339] Table 5 Results of the linearity study of alpha-1-hydroxyl acid (Alanine A)

[0340]

[0341] The results showed that the regression equation for oxalis AB was y = 14,991.1720x - 166,093.8476, with a correlation coefficient r = 1.0000, indicating that oxalis AB at a concentration of 19.229 μg·mL⁻¹ was effective. -1 ~2403.630 μg·mL -1 Within the specified range, the injection concentration and peak area showed a good linear relationship.

[0342] (4) Precision test

[0343] Accurately pipette the reference solution from section "2.2.1" (C = 218.097 μg·mL). -1 The sample was injected six times under the chromatographic conditions described in section "2.2.2", and the RSD value was calculated based on the peak area of ​​AB oxaliplatin. The results showed that the RSD value of the peak area of ​​AB oxaliplatin was 0.77% after six consecutive injections of the same reference solution, which is less than 3.0%, indicating that the instrument precision was good.

[0344] (5) Repeated examination

[0345] Approximately 1.0 g of the herb *Dryopteris crassirhizoma* was accurately weighed and prepared in six parallel portions. Six test solutions were prepared according to the method specified in section "2.2.3". The solutions were then analyzed under the chromatographic conditions specified in section "2.2.2". The content of anaerobic acid AB and the RSD value were calculated. The results showed that, in six repeated determinations of the same batch of samples, the RSD value of anaerobic acid AB was 0.81%. According to the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is between 0.1% and 1%, the repeatability RSD limit is <2%, indicating that the analytical method has good repeatability.

[0346] (6) Stability test

[0347] Approximately 1.0 g of *Dryopteris crassirhizoma* was accurately weighed and prepared in six parallel portions. The test solution was prepared according to the method specified in section "2.2.3". Chromatographic analysis was performed under the conditions specified in section "2.2.2" at 0, 4, 8, 12, 16, 20, and 24 hours. The peak area of ​​*Dryopteris crassirhizoma* AB in the test solution was determined, and the peak area RSD was calculated. The results showed that for the same test solution, the RSD of the peak area of ​​*Dryopteris crassirhizoma* AB was 2.75% after injection at 0, 4, 8, 12, 16, 20, and 24 hours, indicating that the test solution under this method had good stability within 24 hours.

[0348] (7) Intermediate precision test

[0349] Different personnel were selected to perform the assay at different times and using different instruments. Approximately 1.0 g of *Dryopteris crassirhizoma* (a type of medicinal herb) was accurately weighed and prepared in six parallel portions. The test solution was prepared according to the method specified in section "2.2.3," and the chromatographic conditions in section "2.2.2" were followed. The samples were injected separately to determine the content of *Dryopteris crassirhizoma* AB in the test solution. The content of *Dryopteris crassirhizoma* AB and the RSD value were calculated and compared with the results of the repeatability test. The results showed that when the same batch of samples was measured six times by different personnel at different times using different instruments, the RSD value of *Dryopteris crassirhizoma* AB was 1.42%, compared to an RSD value of 2.51% for the six data points in the repeatability test. According to the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in the 2020 edition of the Chinese Pharmacopoeia, when the content of the analyte in the sample is between 0.1% and 1%, the intermediate precision RSD limit is <4%. Therefore, this method demonstrates good intermediate precision even when different analysts operate on different dates and using different chromatograms.

[0350] (8) Accuracy assessment

[0351] Three experimental groups were designed using the spiking recovery method, with the ratio of the amount of reference standard added to the content of the analyte in the test sample controlled at approximately 0.5:1, 1:1, and 1.5:1, with each group having three replicates. An appropriate amount of azoxystrobin AB reference standard was accurately weighed into a stoppered conical flask, and approximately 0.5 g of the powdered *Dryopteris crassirhizoma* (with its content already determined) was also accurately weighed into the conical flask. The test solution was prepared according to the method described in section "2.2.3". The solution was injected and determined under the chromatographic conditions described in section "2.2.2". The results are shown in Table 6.

[0352] Table 6. Results of Recovery Rate of Acid AB in Dryopteris crassirhizoma (a medicinal herb)

[0353]

[0354]

[0355] The results showed that the recovery rate of oxaliplatin AB was 96.15%. According to the "Guiding Principles for Validation of Analytical Methods for Drug Quality Standards" in the 2020 edition of the Chinese Pharmacopoeia, the recovery limit is 92% to 105% when the content of the analyte in the sample is between 0.1% and 1%. The recovery rate of oxaliplatin AB ranged from 92.79% to 100.85%, with an average recovery rate of 96.15% and an RSD of 2.91%, indicating that the method has good accuracy.

[0356] (9) Durability test

[0357] ① Investigation at different column temperatures

[0358] Approximately 1.0 g of *Dryopteris crassirhizoma* was accurately weighed and prepared according to the method specified in section "2.2.3". Except for column temperatures of 23℃, 25℃, and 27℃, all other chromatographic conditions were as specified in section "2.2.2". The sample was injected for analysis to determine the content of oxaliplatin AB and the RSD value in the test solution. The results showed that the RSD value of oxaliplatin AB in *Dryopteris crassirhizoma* was 1.48% at different column temperatures (±2℃), indicating that the analytical method has good robustness within the column temperature range of ±2℃.

[0359] ② Investigation of different flow velocities

[0360] Take approximately 1.0 g of the herb *Dryopteris crassirhizoma*, accurately weigh it, and prepare the test solution according to the method specified in section "2.2.3", except that the flow rate is 0.9 mL·min. -1 1.0 mL·min -1 and 1.1 mL·min -1 Except for the chromatographic conditions described in section "2.2.2", all other conditions were performed according to the chromatographic conditions, and the sample was injected and analyzed to determine the content of alpha-linolenic acid AB and the RSD value in the test solution. The results showed that at different flow rates (±0.1 mL·min...-1 The RSD value of AB content of ramie acid in the herb *Dryopteris crassirhizoma* was 2.07% as measured by the method, indicating that the analytical method is effective at flow rates ±0.1 mL / min. -1 It exhibits good durability within its range.

[0361] (10) Sample determination

[0362] Sixteen batches of Dryopteris crassirhizoma were collected, and sixteen test solutions were prepared according to the test solution preparation method under section "2.2.3". The solutions were injected and determined according to the chromatographic conditions under section "2.2.2". The peak areas were recorded, and the content of AB yellow cottony herb acid was calculated using the external standard method. The results are shown in Table 7.

[0363] Table 7. Results of determination of Acid AB in 16 batches of Dryopteris crassirhizoma.

[0364]

[0365] The results showed that the content of oxaliplatin AB in 16 batches of *Dryopteris crassirhizoma* ranged from 1.1846 mg / g to 2.7330 mg / g, with a mean of 1.6884 mg / g and a standard deviation (SD) of 0.3874 mg / g. The mean ± 30% ranged from 1.1819 mg / g to 2.1949 mg / g, and the mean ± 3 times SD ranged from 0.5261 mg / g to 2.8507 mg / g. Considering the representativeness of the 16 batches, the limit was set at -3 times SD of the mean, and the content of oxaliplatin AB (C) in *Dryopteris crassirhizoma* was determined to be within the limit range. 22 H 26 O8) shall not be less than 0.050%.

[0366] 2.2.5 Determination of counterfeit medicinal material samples

[0367] Approximately 1.0 g of each of the following herbs were accurately weighed: single-budd Cynodon dactylon, Japanese Cynodon dactylon, small Cynodon dactylon, Cycas revoluta, Blanchus brevis, Cynodon dactylon, terminal-budd Cynodon dactylon, bulbous-budd Cynodon dactylon, and Dryopteris crassirhizoma. Test solutions were prepared according to the method described in section "2.2.3". The solutions were injected and analyzed under the chromatographic conditions described in section "2.2.2". The peak areas were recorded, and the content of oxalis acid (AB) was calculated using the external standard method. The results are shown in Table 8.

[0368] Table 8. Results of determination of Acid AB in Dryopteris crassirhizoma and its adulterants.

[0369]

[0370] The results showed that the content of oxalis AB in *Brownia argentea* was significantly lower than that in *Dryopteris crassirhizoma*, and the other adulterants did not contain oxalis AB, indicating that using oxalis AB as an indicator for the content determination of *Dryopteris crassirhizoma* has good specificity.

[0371] Example 2

[0372] This embodiment provides a method for constructing a characteristic spectrum of resorcinols in Dryopteris crassirhizoma and determining the content of active ingredients, as well as its application in identifying adulterants, as detailed below:

[0373] Among them, the batch number of the medicinal material *Dryopteris crassirhizoma* is S1;

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

[0375] Preparation of reference solution: Accurately weigh 2.304 mg of oxalic acid AB reference standard, place it in a 10 mL volumetric flask, and add methanol to prepare a reference solution containing 218.097 μg per mL.

[0376] Preparation of the test solution: Weigh approximately 1.0 g of Dryopteris crassirhizoma powder (passed through a No. 3 sieve), place it in a stoppered conical flask, accurately add 25 mL of methanol solution, weigh, sonicate for 30 min, cool, weigh again, replenish the lost weight with methanol, shake well, filter, evaporate to dryness under reduced pressure using a rotary evaporator, dissolve the residue in 25 mL of water, add 25 mL of petroleum ether (60℃~90℃) and extract 3 times, combine the solvents, evaporate to dryness under reduced pressure using a rotary evaporator, dilute the residue to 10 mL with methanol, shake well, filter, and collect the filtrate to obtain the test solution.

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

[0378] Results: The *Dryopteris crassirhizoma* herb in batch S1 had eight characteristic peaks. Peak 8, corresponding to the reference peak of oxalis AB, was designated as peak S. The relative retention times of peaks 1, 2, 3, 4, 5, 6, and 7 with peak S were calculated as follows: 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), respectively. The oxalis AB content in batch S1 of *Dryopteris crassirhizoma* was 0.1637%, which is within the specified range for herb content. Results are shown in Tables 9 and 10. Figure 44 .

[0379] Table 9. Calculation results of the relative retention times of each characteristic peak in the characteristic spectrum of Dryopteris crassirhizoma (batch number: S1).

[0380]

[0381]

[0382] Table 10. Results of determination of AB content of *Dryopteris crassirhizoma* (batch number: S1)

[0383]

[0384] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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 embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic spectrum of resorcinol components in the medicinal herb Dryopteris crassirhizoma, characterized in that, Includes the following steps: The medicinal material Dryopteris crassirhizoma was extracted using an extraction solvent to prepare a test solution; the extraction solvent was methanol or an aqueous methanol solution, and the extraction method was ultrasonic extraction or reflux extraction. A reference solution was prepared by dissolving the phloroglucinol components of Dryopteris crassirhizoma using a dissolving agent. The test solution and the reference solution were analyzed by high performance liquid chromatography to construct a characteristic spectrum of phloroglucinol components in Dryopteris crassirhizoma. The conditions for high-performance liquid chromatography (HPLC) detection include: mobile phase A is a mixture of acetonitrile, methanol, and an aqueous solution of phosphoric acid; mobile phase B is an aqueous solution of phosphoric acid; gradient elution is used; and the chromatographic column is a C18 column. The gradient elution procedure is as follows: From 0 to 60 minutes, the volume percentage of mobile phase A increased from 60% to 64%. The volume percentage of mobile phase A was maintained at 64% for 60-65 minutes. The phloroglucinol compounds include phloroglucinol AP, norphloroglucinol PB, and phloroglucinol AB.

2. The method for constructing the characteristic spectrum of phloroglucinol components in the medicinal herb Dryopteris crassirhizoma 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) The column temperature is 20℃~40℃; (3) The flow rate is 0.5 mL / min. -1 ~1.5 mL•min -1 ; (4) The injection volume is 1 μL to 10 μL.

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

4. The method for constructing the characteristic spectrum of resorcinol components in Dryopteris crassirhizoma according to claim 1, characterized in that, The characteristic peaks of the characteristic spectrum of the medicinal material Dryopteris crassirhizoma were identified by high performance liquid chromatography-mass spectrometry. The conditions for the high performance liquid chromatography-mass spectrometry (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 gradient elution procedure is as follows: From 0 to 60 minutes, the volume percentage of mobile phase A increased from 60% to 64%. The volume percentage of mobile phase A was maintained at 64% for 60-65 minutes. The characteristic peaks of the characteristic spectrum of the herbal medicine Dryopteris crassirhizoma include: peak 1 is oxalic acid AP, peak 2 is noroxalic acid PB, and peak 8 is oxalic acid AB.

5. The method for constructing the characteristic spectrum of phloroglucinol components in the medicinal herb Dryopteris crassirhizoma according to claim 4, characterized in that, The conditions for the high-performance liquid chromatography-mass spectrometry (HPLC-MS) method further include: in the mobile phase A, the volume percentage of acetonitrile and methanol is (1~8):(1~2); and / or The total volume ratio of the acetonitrile and methanol to the formic acid aqueous solution is 0.1%~0.3%; and / or The formic acid aqueous solution contains formic acid at a volume percentage of 0.05% to 0.3%.

6. The method for constructing the characteristic spectrum of phloroglucinol components in the medicinal herb Dryopteris crassirhizoma according to claim 1, characterized in that, The step of extracting Dryopteris crassirhizoma using an extraction solvent satisfies at least one of the following conditions: (1) In the methanol aqueous solution, the volume percentage of methanol is 30%~70%; (2) The extraction time is 15 min to 60 min; (3) The mass-volume ratio of the medicinal material *Dryopteris crassirhizoma* and the extraction solvent is 1 g: (15~50) mL.

7. A method for determining the content of phloroglucinol components in the medicinal herb Dryopteris crassirhizoma, characterized in that, Includes the following steps: The medicinal material Dryopteris crassirhizoma was extracted using an extraction solvent to prepare a test solution; The reference standard was dissolved using a dissolving reagent to prepare reference standard solutions of different concentrations; The reference standard solutions of different concentrations were injected separately and detected by high performance liquid chromatography. 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 the linear regression equation. Obtain the peak area corresponding to the active ingredient in the test sample solution, and substitute it into the linear regression equation to calculate the content of the active ingredient in the test sample solution; Wherein, the phloroglucinol component includes oxaliplatin AB; the reference standard includes oxaliplatin AB; the preparation method of the test solution and the conditions for high-performance liquid chromatography detection are the same as those in any one of claims 1 to 4.

8. A method for identifying the medicinal material Dryopteris crassirhizoma and its adulterants, characterized in that, Includes the following steps: Extraction solvents were used to extract the medicinal material Dryopteris crassirhizoma and its adulterants to prepare test solutions; The test solution was subjected to high performance liquid chromatography to construct characteristic chromatograms of Dryopteris crassirhizoma and its adulterants, and to observe whether the characteristic chromatograms of each adulterant showed characteristic peaks of phloroglucinol components corresponding to the characteristic chromatogram of Dryopteris crassirhizoma. in ； The preparation method of the test solution and the conditions for high-performance liquid chromatography detection are the same as those in any one of claims 1 to 4.

9. A method for preparing oxaliplatin AB, characterized in that, Includes the following steps: The powder of Dryopteris crassirhizoma was extracted using an extraction solvent. The resulting extract was then concentrated and dried to prepare an extract of Dryopteris crassirhizoma. The extraction solvent was an aqueous ethanol solution. The extract of Dryopteris crassirhizoma was mixed with water, and the resulting mixture was extracted using an extraction solvent, namely petroleum ether, ethyl acetate, and n-butanol. The obtained extract was separated by column chromatography, and the chromatographic solution containing the phloroglucinol component of Dryopteris crassirhizoma was collected; the elution solution used in the column chromatography included a mixed solution of petroleum ether, ethyl acetate and formic acid; The chromatography solution was purified to prepare oxaliplatin AB.

10. The method for preparing oxalisic acid AB according to claim 9, characterized in that, The volume percentage of ethanol in the ethanol-water solution is 80% to 99%.

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