Method for constructing characteristic spectrum of adiantum capillus-veneris and method for determining content of characteristic components of adiantum capillus-veneris

Abstract

The application provides a construction method of a characteristic map of Adiantum capillus-veneris and a content determination method of characteristic components of the Adiantum capillus-veneris. The construction method of the characteristic map comprises the following steps: taking chlorogenic acid, 3-O-p-coumaroyl quinic acid and kaempferol-3-O-beta-D-glucuronide as reference substances to obtain reference substance solutions; mixing the Adiantum capillus-veneris with an extraction solvent and performing heating reflux treatment or ultrasonic treatment, and then filtering the Adiantum capillus-veneris after cooling to obtain a test sample solution; placing the test sample solution and the plurality of reference substance solutions in a high performance liquid chromatograph, determining high performance liquid chromatograms of the test sample solution and the reference substance solutions, taking the high performance liquid chromatograms of the reference substance solutions as a control, selecting common peaks from the high performance liquid chromatogram of the test sample solution, and obtaining a characteristic map of the Adiantum capillus-veneris. The above method has excellent precision, repeatability, stability and durability, and is significantly superior to traditional single component determination or non-standardized methods.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and more specifically, to a method for constructing a characteristic spectrum of Adiantum capillus-veneris and a method for determining the content of characteristic components of Adiantum capillus-veneris. Background Technology

[0002] Maidenhair fern is a plant belonging to the family Adiantaceae, specifically the fine-leaved maidenhair fern. Adiantum venustum The dried whole herb of *Don. var. venustum* is a commonly used medicinal herb in Xinjiang, possessing properties such as clearing heat and detoxifying, promoting urination, relieving cough and resolving phlegm. It is often used to treat colds, coughs, asthma, urinary retention, amenorrhea, and hair loss. Modern pharmacological studies have shown that *Adiantum capillus-venustum* contains various phenolic acids and flavonoid glycosides, such as chlorogenic acid, 3-O-p-coumarylquinic acid, and kaempferol-3-O-β-D-glucuronide. These components are considered important material bases for its medicinal effects, exhibiting antioxidant, anti-inflammatory, hepatoprotective, and antibacterial pharmacological activities.

[0003] Currently, there is a lack of unified quality control standards for the clinical use and pharmaceutical production of maidenhair fern, a traditional Chinese medicine. The current quality standard is the 2010 edition (Volume 1) of the "Xinjiang Uygur Autonomous Region Uygur Medicinal Materials Standard," whose quality evaluation still mainly relies on traditional morphological identification and impurity testing, lacking scientific, objective, and quantifiable detection methods. Although some studies have attempted to use high-performance liquid chromatography (HPLC) to determine the content of individual components (such as chlorogenic acid) in maidenhair fern, these methods generally suffer from the following technical deficiencies:

[0004] 1) Single component detection: Existing methods mostly only quantify chlorogenic acid, which fails to fully reflect the compositional characteristics of the main active components in maidenhair fern and makes it difficult to objectively evaluate the overall quality consistency of the medicinal material.

[0005] 2) Lack of a characteristic chromatographic system: The characteristic chromatographic method based on multi-component synergistic evaluation has not been established, making it impossible to effectively distinguish Adiantum capillus-veneris medicinal materials from different origins, harvesting periods, or processing methods, resulting in difficulty in controlling quality fluctuations;

[0006] 3) Inadequate methodology: Existing detection methods lack systematic optimization and validation in terms of extraction methods, chromatographic conditions, and system applicability. For example, arbitrary solvent selection, unreasonable gradient elution procedures, and lack of robustness data result in poor reproducibility of the methods, making it difficult to promote and apply them in different laboratories.

[0007] 4) Unclear standard references: Some studies failed to identify reference standards for key characteristic peaks or used non-standards for comparison, resulting in insufficient basis for spectral identification and low reliability of results;

[0008] 5) Insufficient stability and uniformity data across multiple batches: There is a lack of systematic investigation into the content of characteristic components in different batches of maidenhair fern, making it impossible to establish reasonable quality control limits.

[0009] Therefore, there is an urgent need to establish an efficient, stable, and repeatable detection method that can simultaneously construct multi-component characteristic maps and determine the content of main active ingredients, so as to comprehensively and accurately evaluate the quality of Adiantum capillus-veneris and provide a scientific basis for the standardized cultivation, processing, preparation, and formulation development of this medicinal material. Summary of the Invention

[0010] The main objective of this invention is to provide a method for constructing a characteristic map of maidenhair fern and a method for determining the content of characteristic components of maidenhair fern, so as to solve the problem of insufficient quality control methods for maidenhair fern in the prior art.

[0011] To achieve the above objectives, according to a first aspect of the present invention, a method for constructing a feature map of *Adiantum capillus-veneris* is provided, the method comprising:

[0012] a) Preparation of reference solutions: Chlorogenic acid, 3-O-p-coumarylquinic acid and kaempferol-3-O-β-D-glucuronide were used as reference standards and dissolved in 80% methanol to obtain the above reference solutions, which included chlorogenic acid solution, 3-O-p-coumarylquinic acid solution and kaempferol-3-O-β-D-glucuronide solution;

[0013] b) Preparation of the test solution: The above-mentioned maidenhair fern was mixed with the extraction solvent and subjected to reflux heating or ultrasonic treatment. After cooling, the mixture was filtered to obtain the above-mentioned test solution.

[0014] c) The above-mentioned test solution and various above-mentioned reference solutions were placed in a high-performance liquid chromatograph, and the high-performance liquid chromatograms of the above-mentioned test solution and the above-mentioned reference solutions were obtained. Using the high-performance liquid chromatograms of the above-mentioned reference solutions as a control, common peaks were selected from the high-performance liquid chromatograms of the above-mentioned test solution to obtain the characteristic chromatograms of the above-mentioned maidenhair fern.

[0015] Furthermore, the concentration of the above chlorogenic acid solution is 16.447~48.03µg / mL;

[0016] Preferably, the concentration of the above-mentioned 3-O-p-coumarylquinic acid solution is 15.868~25.68 μg / mL;

[0017] Preferably, the concentration of the above-mentioned kaempferol-3-O-β-D glucuronide solution is 47.28~163.229 μg / mL.

[0018] Furthermore, the high performance liquid chromatograph mentioned above is selected from any one of the following: Shimadzu LC-20AT, Agilent 1260, or Waters e2695;

[0019] Preferably, the chromatographic column of the above-mentioned high-performance liquid chromatograph is selected from any one of the following: Kromasil C 18 The dimensions are 4.6μm × 250mm; ZORBAX Eclipse XDB-C 18 Specifications are 4.6μm × 250mm; or Symmetry® C 18 The dimensions are 4.6μm × 250mm;

[0020] Preferably, the particle size of the chromatographic column is 5 μm;

[0021] Preferably, the packing material for the above-mentioned chromatographic column is octadecylsilane-bonded silica gel;

[0022] Preferably, the chromatographic conditions of the above-mentioned high performance liquid chromatograph include: methanol as mobile phase A, 0.1% formic acid as mobile phase B, flow rate of 0.8~1.2 mL / min, column temperature of 25~35℃, detection wavelength of 300~330 nm, injection volume of 5~15 μL, and gradient elution;

[0023] Preferably, the elution conditions for the gradient elution described above are as follows:

[0024] From 0 to 40 min, the volume fraction of mobile phase A increased from 10% to 30%, while the volume fraction of mobile phase B decreased from 90% to 70%.

[0025] For 40-55 minutes, the volume fraction of mobile phase A is 30%, and the volume fraction of mobile phase B is 70%.

[0026] Over 55–80 min, the volume fraction of mobile phase A increased from 30% to 45%, while the volume fraction of mobile phase B decreased from 70% to 55%.

[0027] For 80-90 minutes, the volume fraction of mobile phase A is 45%, and the volume fraction of mobile phase B is 55%.

[0028] From 90 to 90.01 min, the volume fraction of mobile phase A decreased from 45% to 10%, while the volume fraction of mobile phase B increased from 55% to 90%.

[0029] 90.01~100 min, the volume fraction of mobile phase A is 10%, and the volume fraction of mobile phase B is 90%.

[0030] Furthermore, the extraction solvent is selected from any one of the following: methanol solution, ethanol solution, or water;

[0031] Preferably, the volume fraction of methanol in the above methanol solution is 50% to 100%;

[0032] More preferably, the volume fraction of methanol in the above methanol solution is 50%, 80%, or 100%;

[0033] Preferably, the volume fraction of ethanol in the above ethanol solution is 50% to 100%;

[0034] More preferably, the volume fraction of ethanol in the above-mentioned ethanol solution is 50%, 80%, or 100%;

[0035] Preferably, the mass-to-volume ratio of the above-mentioned maidenhair fern to the above-mentioned extraction solvent is 1 g: (10~50) mL;

[0036] Preferably, the heating and reflux time is 30 min to 90 min;

[0037] More preferably, the heating reflux time is selected from 30 min, 60 min, or 90 min;

[0038] Preferably, the ultrasound duration is 30 min to 90 min;

[0039] More preferably, the duration of the ultrasound is selected from 30 min, 60 min, or 90 min.

[0040] To achieve the above objectives, according to a second aspect of the present invention, a method for quality detection of maidenhair fern is provided. The method includes: constructing a feature spectrum of a maidenhair fern sample to be tested using the aforementioned method for constructing a feature spectrum of maidenhair fern, thereby obtaining a feature spectrum of the maidenhair fern sample to be tested; and comparing the feature spectrum of the maidenhair fern sample to be tested with a control feature spectrum obtained under the same feature spectrum detection conditions to achieve quality detection of the maidenhair fern sample to be tested.

[0041] Furthermore, the characteristic peaks in the above-mentioned comparative characteristic spectrum are three; among them, peak 1 is the peak of the above-mentioned chlorogenic acid, peak 2 is the peak of the above-mentioned 3-O-p-coumarylquinic acid, and peak 3 is the peak of the above-mentioned kaempferol-3-O-β-D glucuronide.

[0042] Preferably, peak No. 2 is used as the positioning peak, with a relative retention time of 1.00; the average relative retention times of the other two characteristic peaks are: peak No. 1 is 0.66~0.8; peak No. 3 is 2.03~2.49.

[0043] To achieve the above objectives, according to a third aspect of the present invention, a feature map constructed using the above-described method for constructing a feature map of Adiantum capillus-veneris is provided;

[0044] Preferably, the above characteristic spectrum has three characteristic peaks; wherein, peak 1 is the peak of the above chlorogenic acid, peak 2 is the peak of the above 3-O-p-coumarylquinic acid, and peak 3 is the peak of the above kaempferol-3-O-β-D glucuronide.

[0045] More preferably, peak No. 2 is used as the positioning peak, with a relative retention time of 1.00; the average relative retention times of the other two characteristic peaks are: peak No. 1 is 0.66~0.8; peak No. 3 is 2.03~2.49.

[0046] To achieve the above objectives, according to a fourth aspect of the present invention, a method for determining the content of characteristic components of maidenhair fern is provided, wherein the characteristic components are selected from any one of the following: chlorogenic acid, 3-O-p-coumarylquinic acid or kaempferol-3-O-β-D-glucuronide.

[0047] The above determination method includes: calculating the peak area of ​​the characteristic peak in the characteristic spectrum; calculating the content of the characteristic component corresponding to the characteristic peak in the above test solution using the external standard method; wherein the above characteristic spectrum is a characteristic spectrum constructed using the above-mentioned method for constructing the characteristic spectrum of Adiantum capillus-veneris.

[0048] To achieve the above objectives, according to a fifth aspect of the present invention, the application of the above-described method for constructing a characteristic spectrum of maidenhair fern, the above-described quality detection method, the above-described characteristic spectrum, or the above-described method for determining the content of characteristic components of maidenhair fern in the quality control of maidenhair fern is provided.

[0049] The technical solution of this invention uses 3-O-p-coumarylquinic acid as a reference peak, which has a moderate retention time, symmetrical chromatographic peak shape, and good separation. Under gradient elution conditions, it can effectively distinguish the main active components such as chlorogenic acid, 3-O-p-coumarylquinic acid, and kaempferol-3-O-β-D-glucuronide in Adiantum capillus-veneris. For the first time, it achieves the simultaneous presentation of three characteristic peaks in a single high-performance liquid chromatogram at a single detection wavelength (e.g., 300 nm), comprehensively and objectively reflecting the chemical characteristics of Adiantum capillus-veneris.

[0050] Methodological validation has demonstrated that this application has excellent precision (RSD < 0.2%), repeatability (RSD < 0.2%), stability (RSD < 0.3% within 24 h), and robustness (relative retention time is stable under different instruments, columns, flow rates, and column temperatures), which is significantly better than traditional single-component determination or non-standardized methods.

[0051] Therefore, the characteristic spectrum and content determination method established in this invention not only solves the industry problem of the lack of unified quality standards for maidenhair fern, but also realizes the integrated control of the quality of the medicinal material through "fingerprint identification + quantitative evaluation". It is simple to operate and the results are reliable. It is expected to be widely used in the quality evaluation and control of the entire process of planting, harvesting, processing, circulation quality inspection, formulation research and development and clinical use of maidenhair fern, and provide core technical support for the standardization, modernization and industrialization of this medicinal material. Attached Figure Description

[0052] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0053] Figure 1 The following is a characteristic spectrum of the Adiantum capillus-veneris medicinal material according to an embodiment of the present invention; wherein, peak 1 is chlorogenic acid; peak 2 (S) is 3-O-p-coumarylquinic acid; and peak 3 is kaempferol-3-O-β-D-glucuronide.

[0054] Figure 2 A characteristic atlas of maidenhair fern according to an embodiment of the present invention is shown, wherein S15-S1 sequentially represent maidenhair fern medicinal material samples with batch numbers 20250320, 20250413, 20250825, 20251202-1, 20251202-2, 20251202-3, 20251202-4, YL-545-2408001, YL-545-2408002, YL-545-2408003, YL-545-2408004, YL-202509-020, YL-202510-020, YL-202511-020 and YL-202512-020, respectively.

[0055] Figure 3 The results of the linear relationship study of chlorogenic acid according to an embodiment of the present invention are shown.

[0056] Figure 4 The results of the linearity study of 3-O-p-coumarylquinic acid according to an embodiment of the present invention are shown.

[0057] Figure 5 The results of the linearity study of kaempferol-3-O-β-D glucuronide according to an embodiment of the present invention are shown. Detailed Implementation

[0058] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0059] As mentioned in the background section, while Adiantum capillus-veneris possesses medicinal value such as clearing heat and detoxifying, and promoting diuresis, current quality control methods are lacking. Existing technologies mostly rely on single-component determination or unverified chromatographic conditions, making it impossible to achieve systematic identification and characteristic characterization of its multiple active components, and difficult to objectively evaluate the consistency and authenticity of the medicinal material's quality. In this invention, the inventors attempted to establish a method using chlorogenic acid, 3-O-p-coumarylquinic acid, and kaempferol-3-O-β-D-glucuronide as reference standards. A reference solution was prepared using 80% methanol, and the Adiantum capillus-veneris test sample solution was extracted by ultrasonication or heating and reflux. The test sample and reference solution were then measured in parallel under the same chromatographic conditions using high-performance liquid chromatography. Based on the chromatographic peaks of the reference standards, common peaks were identified and locked from the chromatogram of the test sample, thereby constructing a characteristic chromatogram of Adiantum capillus-veneris. Therefore, the protection scheme of this invention is proposed.

[0060] In a first typical embodiment of the present invention, a method for constructing a feature map of maidenhair fern is provided, the method comprising:

[0061] a) Preparation of reference solutions: Chlorogenic acid, 3-O-p-coumarylquinic acid and kaempferol-3-O-β-D-glucuronide were used as reference standards and dissolved in 80% methanol to obtain the above reference solutions, which included chlorogenic acid solution, 3-O-p-coumarylquinic acid solution and kaempferol-3-O-β-D-glucuronide solution;

[0062] b) Preparation of the test solution: The above-mentioned maidenhair fern was mixed with the extraction solvent and subjected to reflux heating or ultrasonic treatment. After cooling, the mixture was filtered to obtain the above-mentioned test solution.

[0063] c) The above-mentioned test solution and various above-mentioned reference solutions were placed in a high-performance liquid chromatograph, and the high-performance liquid chromatograms of the above-mentioned test solution and the above-mentioned reference solutions were obtained. Using the high-performance liquid chromatograms of the above-mentioned reference solutions as a control, common peaks were selected from the high-performance liquid chromatograms of the above-mentioned test solution to obtain the characteristic chromatograms of the above-mentioned maidenhair fern.

[0064] The above-mentioned construction method is based on high-performance liquid chromatography (HPLC). By identifying and comparing characteristic peaks of multiple components, a chemical fingerprint chromatogram of *Adiantum capillus-veneris* (a type of fern) is established. Chlorogenic acid, 3-O-p-coumarylquinic acid, and kaempferol-3-O-β-D-glucuronide are representative phenolic acids and flavonoid glycosides in *Adiantum capillus-veneris*, exhibiting relatively stable distribution and good resolution and response characteristics under selected chromatographic conditions. Using the reference standard chromatogram as a reference, common peaks with consistent retention times in the test sample are identified, enabling the construction of a multi-peak chromatogram reflecting the overall chemical composition of *Adiantum capillus-veneris*. Compared to single-component detection, this method is more effective in capturing subtle compositional differences caused by the origin, harvesting period, or processing method of the medicinal material, providing a more comprehensive basis for quality evaluation.

[0065] Furthermore, using 80% methanol (by volume) as the reference solvent helps improve the solubility and stability of these moderately polar compounds, avoiding peak broadening or retention time drift caused by solvent polarity mismatch. After ultrasonic or reflux extraction of the test sample, a clear solution is obtained through a reasonable filtration procedure, which can effectively reduce matrix interference and improve the clarity and reproducibility of the chromatogram.

[0066] In a preferred embodiment of the present invention, the concentration of the chlorogenic acid solution is 16.447~48.03µg / mL; in a preferred embodiment of the present invention, the concentration of the 3-O-p-coumarylquinic acid solution is 15.868~25.68µg / mL; in a preferred embodiment of the present invention, the concentration of the kaempferol-3-O-β-D-glucuronide solution is 47.28~163.229µg / mL.

[0067] The concentration ranges of chlorogenic acid, 3-O-p-coumarylquinic acid, and kaempferol-3-O-β-D-glucuronide in the above reference solution cover the typical content ranges of each component in the tested samples, ensuring that the peak areas of the reference and test samples are comparable, which is beneficial for establishing a stable and linear quantitative correlation. Too low a concentration may lead to a decrease in the signal-to-noise ratio, affecting the identification of low-content components; too high a concentration may cause quantitative errors due to column overload or peak overlap.

[0068] In a preferred embodiment of the present invention, the high-performance liquid chromatograph (HPLC) is selected from any one of the following: Shimadzu LC-20AT, Agilent 1260, or Waters e2695. These HPLC instruments, such as the Shimadzu LC-20AT, Agilent 1260, or Waters e2695, are all high-stability devices widely used in laboratories, possessing precise gradient control capabilities and good system pressure resistance, and can support the execution requirements of this method for complex elution procedures. Although there are hardware differences between instruments of different brands, by using relative retention times rather than absolute retention times for chromatographic comparison, the systematic errors caused by instrument differences can be effectively reduced. This method has good transferability and cross-platform applicability.

[0069] In a preferred embodiment of the present invention, the chromatographic column of the above-mentioned high-performance liquid chromatograph is selected from any of the following: Kromasil C 18 The dimensions are 4.6μm × 250mm; ZORBAX Eclipse XDB-C 18 Specifications are 4.6μm × 250mm; or Symmetry® C 18 The specifications are 4.6μm×250mm; in a preferred embodiment of the present invention, the particle size of the above-mentioned chromatographic column is 5μm; in a preferred embodiment of the present invention, the packing material of the above-mentioned chromatographic column is octadecylsilane-bonded silica gel;

[0070] The above chromatographic column used is Kromasil C10. 18 ZORBAX Eclipse XDB-C 18 Or Symmetry®C 18 The packing material is octadecylsilane-bonded silica gel, with a particle size of 4.6 μm × 250 mm. This type of packing material has a uniform particle size distribution, a stable silica matrix, and good batch reproducibility, making it suitable for separating phenolic acids and flavonoid glycosides with similar structures. This packing material has a moderate retention capacity for the target components, enabling effective separation of chlorogenic acids and kaempferol glycosides within a reasonable time, and exhibits good peak symmetry, which helps improve the accuracy of characteristic peak identification.

[0071] In a preferred embodiment of the present invention, the chromatographic conditions of the above-mentioned high-performance liquid chromatograph include: using methanol as mobile phase A, 0.1% formic acid as mobile phase B, a flow rate of 0.8~1.2 mL / min (e.g., 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min or 1.2 mL / min), a column temperature of 25~35℃ (e.g., 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃ or 35℃), a detection wavelength of 300~330 nm (e.g., 300 nm, 310 nm, 320 nm or 330 nm), an injection volume of 5~15 μL (e.g., 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL or 15 μL), and gradient elution;

[0072] In the above chromatographic conditions, the mobile phase consisted of a gradient system of methanol and 0.1% formic acid aqueous solution. The introduction of formic acid effectively suppressed the ionization of acidic components, improved peak tailing, and enhanced separation efficiency. The detection wavelength of 300-330 nm covered the typical UV absorption peaks of chlorogenic acid and its derivatives, while also taking into account the absorption characteristics of kaempferol glycosides, ensuring stable responses for all three types of components. The flow rate, column temperature, and injection volume were optimized by balancing separation efficiency, analysis time, and system pressure to ensure stable operation under standard laboratory conditions.

[0073] In a preferred embodiment of the present invention, the elution conditions for the gradient elution are as follows: 0-40 min, the volume fraction of mobile phase A increases from 10% to 30%, and the volume fraction of mobile phase B decreases from 90% to 70%; 40-55 min, the volume fraction of mobile phase A is 30%, and the volume fraction of mobile phase B is 70%; 55-80 min, the volume fraction of mobile phase A increases from 30% to 45%, and the volume fraction of mobile phase B decreases from 70% to 55%; 80-90 min, the volume fraction of mobile phase A is 45%, and the volume fraction of mobile phase B is 55%; 90-90.01 min, the volume fraction of mobile phase A decreases from 45% to 10%, and the volume fraction of mobile phase B increases from 55% to 90%; 90.01-100 min, the volume fraction of mobile phase A is 10%, and the volume fraction of mobile phase B is 90%.

[0074] The above elution conditions, achieved through segmented control of the organic phase ratio, enable the orderly elution of components with significant polar differences in Adiantum capillus-veneris: an initial low proportion of methanol helps retain highly polar water-soluble components; a slow increase to 30%–45% methanol in the middle stage gradually elutes moderately polar phenolic acids and flavonoid glycosides; and a rapid recovery system at the end ensures column cleaning and stability for subsequent injections. This procedure has been validated with multiple batches of samples and can complete the separation of all target components within 100 minutes, with a stable baseline and no significant tailing or extraneous peak interference.

[0075] In a preferred embodiment of the present invention, the extraction solvent is selected from any one of the following: methanol solution, ethanol solution, or water; the extraction solvent is selected from methanol or ethanol solution, both of which are commonly used solvents for extracting plant active ingredients and have good solubility for phenolic acids and flavonoid glycosides. Among them, methanol has slightly stronger permeability, while ethanol is more in line with the trend of environmental protection and safety, and both can be flexibly selected according to actual conditions.

[0076] In a preferred embodiment of the present invention, the volume fraction of methanol in the methanol solution is 50% to 100%; in a more preferred embodiment of the present invention, the volume fraction of methanol in the methanol solution is 50%, 80%, or 100%; in a preferred embodiment of the present invention, the volume fraction of ethanol in the ethanol solution is 50% to 100%; in a more preferred embodiment of the present invention, the volume fraction of ethanol in the ethanol solution is 50%, 80%, or 100%; the volume fraction of methanol or ethanol in the range of 50% to 100% can adjust the polarity of the extraction system, thereby affecting the dissolution efficiency of the target component and the degree of co-extraction of impurities, and achieving optimization of extraction selectivity.

[0077] In a preferred embodiment of the present invention, the mass-to-volume ratio of the above-mentioned maidenhair fern to the above-mentioned extraction solvent is 1 g : (10~50) mL (e.g., 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50). This ratio range ensures that the solvent is sufficient to fully wet the herbal powder without significantly diluting the sample, allowing the target components to dissolve fully. A ratio that is too low may result in incomplete extraction, while a ratio that is too high increases the burden on subsequent concentration and sample injection. This range conforms to standard laboratory operating practices.

[0078] In a preferred embodiment of the present invention, the heating reflux time is 30 min to 90 min (e.g., 30 min, 40 min, 50 min, 60 min, 70 min, 80 min or 90 min); in a more preferred embodiment of the present invention, the heating reflux time is selected from 30 min, 60 min or 90 min; the setting of the heating reflux time can achieve effective extraction of characteristic components and avoid degradation of heat-sensitive components that may be caused by prolonged heating.

[0079] In a preferred embodiment of the present invention, the ultrasound duration is 30 min to 90 min (e.g., 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, or 90 min); in a more preferred embodiment of the present invention, the ultrasound duration is selected from 30 min, 60 min, or 90 min. The mechanism of action of the ultrasound treatment is that the ultrasonic cavitation effect accelerates solvent penetration and cell rupture, thereby promoting the release of components. This time range can serve as an operational flexibility range, balancing efficiency and resource consumption.

[0080] In a second typical embodiment of the present invention, a quality detection method for maidenhair fern is provided. The quality detection method includes: constructing a feature spectrum of a maidenhair fern sample to be tested using the above-mentioned method for constructing a feature spectrum of maidenhair fern, thereby obtaining a feature spectrum of the maidenhair fern sample to be tested; and comparing the feature spectrum of the maidenhair fern sample to be tested with a control feature spectrum obtained under the same feature spectrum detection conditions to achieve quality detection of the maidenhair fern sample to be tested.

[0081] The aforementioned quality testing method is based on the similarity comparison of characteristic spectra. It assesses the consistency of retention times and relative peak area ratios of common peaks by overlaying and analyzing the chromatogram of the sample under the same conditions with a reference chromatogram. This method does not rely on absolute content but focuses on the degree of matching of the overall chemical profile. It is suitable for quality monitoring of medicinal materials where single standards are lacking or where component content fluctuates significantly, and it demonstrates good practicality, especially in tracing origin, identifying authenticity, and evaluating process consistency.

[0082] In a preferred embodiment of the present invention, the above-mentioned comparative characteristic spectrum has three characteristic peaks; wherein, peak 1 is the peak of the above-mentioned chlorogenic acid, peak 2 is the peak of the above-mentioned 3-O-p-coumarylquinic acid, and peak 3 is the peak of the above-mentioned kaempferol-3-O-β-D-glucuronide.

[0083] In a more preferred embodiment of the present invention, peak No. 2 is used as the positioning peak, and its relative retention time is 1.00. The average relative retention times of the other two characteristic peaks are: peak No. 1 is 0.66~0.8 (e.g., 0.729, 0.730, 0.731 or 0.732); peak No. 3 is 2.03~2.49 (e.g., 2.252, 2.253, 2.254, 2.255, 2.256, 2.257, 2.258, 2.259, 2.260, 2.261, 2.262). The three characteristic peaks in the above-mentioned comparative characteristic chromatograms correspond to chlorogenic acid, 3-O-p-coumarylquinic acid and kaempferol-3-O-β-D-glucuronide, respectively. They appeared stably in 15 batches of maidenhair fern samples, and were well separated under the selected chromatographic conditions with sharp peaks. They have high reproducibility and identifiability and can be used as a characteristic chemical marker for maidenhair fern.

[0084] Peak 2 (3-O-p-coumarylquinic acid) was used as the reference peak, and its relative retention time was set to 1.00. The average relative retention time ranges of the other two peaks were 0.66~0.8 and 2.03~2.49, respectively. These ranges were derived from statistical analysis of multiple batches of measured data and took into account factors such as instrument fluctuations and column aging. They can be used as reasonable judgment intervals for chromatographic matching.

[0085] In a third typical embodiment of the present invention, a characteristic spectrum constructed using the above-described method for constructing characteristic spectra of *Adiantum capillus-veneris* is provided. In a preferred embodiment of the present invention, the characteristic spectrum contains three characteristic peaks; wherein peak 1 is the peak of chlorogenic acid, peak 2 is the peak of 3-O-p-coumarylquinic acid, and peak 3 is the peak of kaempferol-3-O-β-D-glucuronide. In a more preferred embodiment of the present invention, peak 2 is used as... The location peak has a relative retention time of 1.00; the average relative retention times of the other two characteristic peaks are: peak 1 is 0.66~0.8 (e.g., 0.729, 0.730, 0.731 or 0.732); peak 3 is 2.03~2.49 (e.g., 2.252, 2.253, 2.254, 2.255, 2.256, 2.257, 2.258, 2.259, 2.260, 2.261, 2.262).

[0086] The aforementioned characteristic chromatogram serves as the chemical fingerprint of Adiantum capillus-veneris, integrating the relative retention relationships of multiple components. Compared to single-component content determination, this chromatogram can more sensitively reflect the overall chemical composition fluctuations of the medicinal material under changes in growth environment, harvesting season, drying method, or storage and transportation conditions, providing a more systematic and robust evaluation method for quality control.

[0087] In a fourth typical embodiment of the present invention, a method for determining the content of characteristic components of maidenhair fern is provided, wherein the characteristic components are selected from any one of the following: chlorogenic acid, 3-O-p-coumarylquinic acid, or kaempferol-3-O-β-D-glucuronide; the determination method includes: calculating the peak area of ​​the characteristic peak in the above characteristic spectrum; and calculating the content of the characteristic component corresponding to the above characteristic peak in the above test solution using the external standard method; wherein the above characteristic spectrum is the aforementioned characteristic spectrum.

[0088] The above-mentioned method for determining the content of characteristic components is based on the external standard method. It utilizes three identified characteristic peaks in the characteristic chromatogram and calculates the content of chlorogenic acid, 3-O-p-coumarylquinic acid, or kaempferol-3-O-β-D-glucuronide in the sample by analyzing the linear relationship between peak area and standard concentration. This method eliminates the need for a separate quantitative method, achieving a "dual-method" approach for both characteristic chromatogram analysis and content determination. While ensuring methodological consistency, it improves detection efficiency, reduces analytical costs, and is suitable for the comprehensive evaluation of pharmaceutical material quality.

[0089] In a fifth typical embodiment of the present invention, the application of the above-mentioned method for constructing the characteristic spectrum of maidenhair fern, the above-mentioned quality detection method, the above-mentioned characteristic spectrum, or the above-mentioned method for determining the content of characteristic components of maidenhair fern in the quality control of maidenhair fern is provided.

[0090] The aforementioned construction methods, quality testing methods, characteristic chromatograms, and content determination methods can be applied to the raw material acceptance, processing technology monitoring, finished product batch release, and market sampling of Adiantum capillus-veneris. Through the synergistic analysis of chromatogram similarity and component content, these methods provide technical support for the stability, consistency, and safety of the medicinal material. This series of methods does not rely on a single indicator but constructs a multi-dimensional quality evaluation system, which helps to promote the transformation of traditional medicinal material quality control from "experience-based judgment" to "data-driven" approaches, enhancing its applicability and scientific rigor within the modern standardization system of traditional Chinese medicine.

[0091] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0092] Example 1

[0093] I. Experimental Materials and Instruments

[0094] (1) Source of reference standard

[0095] Chlorogenic acid (110753-202520) was purchased from the National Institutes for Food and Drug Control; 3-O-p-coumarylquinic acid (DSTDD037901) and kaempferol-3-O-β-D-glucuronide (DSTDS017501) were purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.

[0096] (2) Reagents

[0097] Methanol was of chromatographic grade, methanol, formic acid, ethanol, etc. were of analytical grade, and water was purified water. All reagents used were those specified in the pharmacopoeia, and the preparation methods were all those specified in Part IV of the 2025 edition of the Chinese Pharmacopoeia.

[0098] (3) Main instruments

[0099] Shimadzu LC-20AT HPLC system (Shimadzu, Japan); Agilent 1260 HPLC system (Agilent Technologies, USA); Waters e2695 HPLC system (Waters); ME204T electronic balance (Mettler-Toledo); ME55 electronic balance (Mettler-Toledo); Kromasil C10 column 18 (4.6μm×250mm), ZORBAX Eclipse XDB-C 18 (4.6μm×250mm), Symmetry®C 18 (4.6μm×250mm); KH5200B ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.); XMTD-7000 electric thermostatic water bath (Beijing Yongguangming Medical Instrument Co., Ltd.).

[0100] II. Feature Map

[0101] (1) Chromatographic conditions

[0102] Octadecylsilane-bonded silica gel was used as the packing material; methanol was used as mobile phase A and 0.1% formic acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the specifications in Table 1; the column temperature was 30℃; the detection wavelength was 300nm; the flow rate was 1.0mL per minute and the injection volume was 10μl.

[0103] Table 1 Gradient elution program

[0104]

[0105] (2) Confirmation of characteristic peaks in characteristic spectrum

[0106] Chromatography using reference standards identified three characteristic peaks: chlorogenic acid, 3-O-p-coumarylquinic acid, and kaempferol-3-O-β-D-glucuronide. Chromatograms of the Adiantum capillus-veneris medicinal material and each reference solution are shown below. Figure 1 .

[0107] (3) Investigation of the preparation method of the test sample

[0108] Extraction method investigation: Approximately 0.5g of *Adiantum capillus-veneris* (batch number: 20251202-1, purchased from Yunnan Traditional Chinese Medicine Market, origin: Lijiang, Yunnan) was accurately weighed and placed in a stoppered conical flask. 10mL of 80% methanol was added, and the weight was recorded. The flask was then subjected to ultrasonic and reflux treatment for 30 minutes each, cooled, and weighed again. The weight loss was replenished with 80% methanol, the flask was shaken well, filtered, and the filtrate was injected into a liquid chromatograph for analysis. The results are shown in Table 2. It should be noted that the contents in Tables 2-5, 11, and 20 were calculated using the external standard method, with the formula: peak area of ​​the analyte / peak area of ​​the reference standard. Reference concentration / sample weight / 1,000,000 100.

[0109] Table 2 Results of the Extraction Method Examination

[0110]

[0111] The experimental results showed that the content of each characteristic peak in the samples treated with ultrasound and heating reflux did not change significantly. Ultrasound is more convenient, so ultrasound was selected as the extraction method.

[0112] Extraction time investigation: Approximately 0.5 g of maidenhair fern (batch number: 20251202-1) was accurately weighed and placed in a stoppered conical flask. 10 mL of 80% methanol was added, and the weight was measured. The mixture was ultrasonically treated for 30, 60, and 90 minutes respectively, cooled, and weighed again. The weight loss was replenished with 80% methanol, shaken well, filtered, and the filtrate was injected into a liquid chromatograph for analysis. The experimental results are shown in Table 3.

[0113] Table 3 Results of the investigation on extraction time

[0114]

[0115] The experimental results showed that the extraction time had no significant effect on the content of each characteristic peak in the sample, so the extraction time was determined to be 30 minutes.

[0116] Extraction solvent investigation: Approximately 0.5 g of maidenhair fern (batch number: 20251202-1) was accurately weighed and placed in a stoppered conical flask. 10 mL each of water, 50% methanol, 80% methanol, methanol, 50% ethanol, 80% ethanol, and anhydrous ethanol were added, and the mixture was weighed. The mixture was sonicated for 30 minutes, cooled, and weighed again. The lost weight was replenished with the appropriate reagents, the mixture was shaken well, filtered, and the filtrate was injected into a liquid chromatograph for analysis. The results are shown in Table 4.

[0117] Table 4 Results of the extraction solvent investigation

[0118]

[0119] The experimental results show that when the extraction solvent is 80% ethanol, the content of each characteristic peak in the sample is the highest, but the peak shape of each chromatographic peak is poor. When the extraction solvent is 80% methanol, the content of each characteristic peak in the sample is higher, and the peak shape of each chromatographic peak is better. Therefore, the extraction solvent is determined to be 80% methanol.

[0120] Material-liquid ratio determination: Approximately 0.5 g of maidenhair fern (batch number: 20251202-1) was accurately weighed and placed in a stoppered conical flask. 5 mL, 10 mL, 15 mL, 20 mL, and 25 mL of 80% methanol were added respectively, and the weights were measured. The mixture was sonicated for 30 minutes, cooled, and weighed again. The weight loss was replenished with 80% methanol, the mixture was shaken well, filtered, and the filtrate was injected into a liquid chromatograph for analysis. The experimental results are shown in Table 5.

[0121] Table 5 Results of the study on the feed-to-liquid ratio

[0122]

[0123] The experimental results show that when the material-to-liquid ratio is 1:50, the content of each characteristic peak in the sample is the highest, but the peak area of ​​the sample is relatively small in the chromatogram. Considering that the response of each chromatographic peak in the chromatogram is moderate, the material-to-liquid ratio is determined to be 1:20.

[0124] The final determined method for processing the test sample is as follows: Take about 0.5g of maidenhair fern (batch number: 20251202-1), accurately weigh it, place it in a stoppered conical flask, add 10mL of 80% methanol, weigh it, sonicate it for 30 minutes, cool it, weigh it again, replenish the lost weight with 80% methanol, shake it well, filter it, and take the filtrate.

[0125] (4) Methodological investigation

[0126] Preparation of reference solution: Accurately weigh appropriate amounts of chlorogenic acid reference standard, 3-O-p-coumarylquinic acid reference standard, and kaempferol-3-O-β-D-glucuronide reference standard, and add 80% methanol to prepare a solution containing 48.03 µg of chlorogenic acid, 25.68 µg of 3-O-p-coumarylquinic acid, and 47.28 µg of kaempferol-3-O-β-D-glucuronide per mL.

[0127] Preparation of the test solution: Take about 0.5g of maidenhair fern (batch number: 20251202-1), accurately weigh it, place it in a stoppered conical flask, add 10mL of 80% methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, replenish the lost weight with 80% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0128] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0129] Durability test

[0130] Take approximately 0.5g of maidenhair fern (batch number: 20251202-1), accurately weigh it, place it in a stoppered conical flask, add 10mL of 80% methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, replenish the lost weight with 80% methanol, shake well, filter it, and take the filtrate. Inject it under different column temperatures, different flow rates, different chromatographic columns, and different high-performance liquid chromatographs, determine and record the retention times of each characteristic peak. Using the chromatographic peak of 3-O-p-coumarylquinic acid (No. 2) as the reference peak, calculate the relative retention times of the characteristic peaks. The results are shown in Table 6.

[0131] Table 6. Durability Relative Retention Time Results

[0132]

[0133] The experimental results show that the flow rate has a significant impact on the method, so the flow rate was determined to be 1.0 mL per minute. Different column temperatures, different chromatographic columns, and different high-performance liquid chromatographs have no significant impact on the method, indicating that the method has good robustness.

[0134] Precision test

[0135] Take about 0.5g of maidenhair fern (batch number: 20251202-1), prepare a test solution according to the test sample processing method, and inject 10μl of the solution 6 times consecutively under the final determined chromatographic conditions. Measure and record the retention time of each chromatographic peak. Using the chromatographic peak of 3-O-p-coumarylquinic acid No. 2 as the reference peak, calculate the relative retention time of the characteristic peaks. The results are shown in Table 7.

[0136] Table 7. Precision Test Chromatographic Peak Relative Retention Time Table

[0137]

[0138] The experimental results show that the RSD of the relative retention time of each characteristic peak is less than 2%, indicating that the instrument has good precision.

[0139] Repeatability test

[0140] Take about 0.5g of maidenhair fern (batch number: 20251202-1) in 6 portions, prepare 6 test solutions according to the test sample processing method, and inject 10μl of each solution 6 times consecutively under the final determined chromatographic conditions. Record the retention time of each chromatographic peak. Using the chromatographic peak of 3-O-p-coumarylquinic acid No. 2 as the reference peak, calculate the relative retention time of the characteristic peak. The results are shown in Table 8.

[0141] Table 8. Relative Retention Table of Chromatographic Peaks in Repeatability Tests

[0142]

[0143] The experimental results show that the RSD of the relative retention time of each characteristic peak is less than 2%, indicating that the method has good repeatability.

[0144] Stability test

[0145] Take about 0.5g of maidenhair fern (batch number: 20251202-1), prepare a test solution according to the test sample processing method, and inject 10μl at 0, 2, 4, 8, 12 and 24h according to the final determined chromatographic conditions. Record the retention time of each chromatographic peak. Using the chromatographic peak of 3-O-p-coumarylquinic acid No. 2 as the reference peak, calculate the relative retention time of the characteristic peak. The results are shown in Table 9.

[0146] Table 9. Stability Test Chromatographic Peak Relative Retention Time Table

[0147]

[0148] The experimental results show that the RSD of the relative retention time of each characteristic peak is less than 2%, indicating that the test solution is stable within 24 hours.

[0149] Characteristic maps of multiple batches of maidenhair fern

[0150] Take approximately 0.5g of maidenhair fern medicinal material from 15 batches, prepare a test solution according to the sample processing method, inject the sample under the final determined chromatographic conditions, record the retention time of the chromatographic peak, and calculate the RSD value. The results are shown in Table 10. Figure 2 .

[0151] Table 10 Results of relative retention time determination for 15 batches of Adiantum capillus-veneris medicinal samples

[0152]

[0153] The chromatogram of the test sample should show three characteristic peaks, each with a retention time consistent with the corresponding reference peak. The peak corresponding to the 3-O-p-coumarylquinic acid reference peak is taken as the S peak. The relative retention times of the three characteristic peaks and the S peak should be calculated. The relative retention times should be within ±10% of the specified values, which are: 0.73 (peak 1), 1.00 (peak 2, S), and 2.26 (peak 3).

[0154] III. Content Determination

[0155] (1) Chromatographic conditions

[0156] Chromatographic conditions for the same characteristic chromatogram.

[0157] (2) Method for preparing the test sample

[0158] Methods for preparing test samples with the same characteristic chromatograms.

[0159] (3) Methodological investigation

[0160] Preparation of reference solution: Accurately weigh appropriate amounts of chlorogenic acid reference standard, 3-O-p-coumarylquinic acid reference standard, and kaempferol-3-O-β-D-glucuronide reference standard, and add 80% methanol to prepare a solution containing 48.03 µg of chlorogenic acid, 25.68 µg of 3-O-p-coumarylquinic acid, and 47.28 µg of kaempferol-3-O-β-D-glucuronide per mL.

[0161] Preparation of the test solution: Take about 0.5g of maidenhair fern (batch number: 20251202-1), accurately weigh it, place it in a stoppered conical flask, add 10mL of 80% methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, replenish the lost weight with 80% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0162] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0163] Durability test

[0164] Take approximately 0.5g of maidenhair fern (batch number: 20251202-1), accurately weigh it, place it in a stoppered conical flask, add 10mL of 80% methanol, weigh it, sonicate for 30 minutes, cool it, weigh it again, replenish the lost weight with 80% methanol, shake well, filter it, take the filtrate, and inject it under different column temperatures, different flow rates, different chromatographic columns, and different high performance liquid chromatographs, determine the peak area, and calculate the content. The results are shown in Table 11.

[0165] Table 11 Results of Durability Relative Retention Time

[0166]

[0167] The experimental results show that column temperature has a significant impact on the method, so the column temperature was determined to be 30℃. Different flow rates, different chromatographic columns, and different high-performance liquid chromatographs have no significant impact on the method, indicating that the method has good robustness.

[0168] Precision test

[0169] Take about 0.5g of maidenhair fern (batch number: 20251202-1), prepare a test solution according to the test sample processing method, and inject 10μl of the sample 6 times consecutively under the final determined chromatographic conditions. Measure and record the peak area. The results are shown in Table 12.

[0170] Table 12 Precision Test Results

[0171]

[0172] The experimental results show that the RSD of the peak areas of chlorogenic acid, 3-O-p-coumarylquinic acid, and kaempferol-3-O-β-D-glucuronide is less than 2%, indicating that the instrument has good precision.

[0173] Repeatability test

[0174] Take about 0.5g of maidenhair fern (batch number: 20251202-1) in 6 portions, prepare 6 test solutions according to the test sample processing method, and inject 10μl of each solution 6 times consecutively under the final determined chromatographic conditions. Measure and record the peak area. The results are shown in Table 13.

[0175] Table 13 Repeatability Test Results

[0176]

[0177] The experimental results showed that the RSD of the peak areas of chlorogenic acid, 3-O-p-coumarylquinic acid, and kaempferol-3-O-β-D-glucuronide was <2%, indicating that the method had good reproducibility.

[0178] Stability test

[0179] Take about 0.5g of maidenhair fern (batch number: 20251202-1), prepare a test solution according to the test sample processing method, and inject 10μl of sample at 0, 2, 4, 8, 12 and 24h according to the final determined chromatographic conditions. Measure and record the peak area. The results are shown in Table 14.

[0180] Table 14 Stability Test Results

[0181]

[0182] The experimental results showed that the RSD of the peak areas of chlorogenic acid, 3-O-p-coumarylquinic acid, and kaempferol-3-O-β-D-glucuronide was <2%, indicating that the test solution was stable within 24 hours.

[0183] Examining the linear range

[0184] Take appropriate amounts of chlorogenic acid, 3-O-p-coumarylquinic acid, and kaempferol-3-O-β-D-glucuronide reference standards, and dilute them serially with 80% methanol to prepare a series of reference solutions (see Table 15). Accurately pipette 10 μl of each series of reference solutions into the liquid chromatograph, record the peak area, and plot a standard curve with reference standard concentration as the abscissa and peak area as the ordinate. The results are shown in Table 16. Figures 3 to 5 .

[0185] Table 15 Concentrations of Linear Series Solutions

[0186]

[0187] Table 16 Results of Linear Relationship Examination

[0188]

[0189] Accuracy test

[0190] Take approximately 0.25 g of maidenhair fern (batch number: 20251202-1) with known content, accurately weigh it, and place it in a stoppered conical flask, making a total of 9 portions. For 3 portions, accurately add 4 mL of reference solution (each 1 mL contains 16.447 μg of chlorogenic acid, 15.868 μg of 3-O-p-coumarylquinic acid, and 163.229 μg of kaempferol-3-O-β-D-glucuronide). For 3 portions, accurately add 5 mL of reference solution. For 3 portions, accurately add 6 mL of reference solution. Proceed in the same manner as the test solution preparation method. Inject 10 μl of the sample, record the peak area, and calculate the content. The results are shown in Tables 17-19.

[0191] Table 17 Results of Chlorogenic Acid Accuracy Test

[0192]

[0193] Table 18 Accuracy Test Results of 3-O-p-Coumaroylquinic Acid

[0194]

[0195] Table 19. Accuracy Test Results of Kaempferol-3-O-β-D-glucuronide

[0196]

[0197] The experimental results showed that the average recoveries of chlorogenic acid, 3-O-p-coumarylquinic acid, and kaempferol-3-O-β-D-glucuronide were 100.67% and RSD < 3%, respectively, indicating that the method had good accuracy.

[0198] Multiple batches of maidenhair fern content determination

[0199] Take about 0.5g of maidenhair fern powder from 15 batches, prepare a test solution according to the test sample processing method, inject 10μl of the solution under the above chromatographic conditions, record the peak area, calculate the content, and the results are shown in Table 20.

[0200] Table 20 Content determination results

[0201]

[0202] The results of content determination of 15 batches of maidenhair fern medicinal materials showed that the content of chlorogenic acid ranged from 0.012% to 0.044%, with an average of 0.033%; the content of 3-O-p-coumarylquinic acid ranged from 0.015% to 0.050%, with an average of 0.038%; and the content of kaempferol-3-O-β-D-glucuronide ranged from 0.192% to 0.285%, with an average of 0.279%.

[0203] As can be seen from the above description, the method for constructing the characteristic spectrum of Adiantum capillus-veneris in this application has for the first time achieved multi-peak synchronous identification and stable characterization of three representative components as core indicators: chlorogenic acid, 3-O-p-coumarylquinic acid and kaempferol-3-O-β-D-glucuronide. This method breaks through the limitations of traditional single-component content determination in evaluating the quality of medicinal materials, which is one-sided and lagging.

[0204] This method, through high-performance liquid chromatography combined with gradient elution and relative retention time comparison, can not only fully reflect the characteristic components of maidenhair fern in a single analysis, but also establish a spectral judgment standard with high reproducibility and cross-instrument compatibility through systematic validation of 15 batches of samples, enabling quality control to shift from "passive sampling" to "active fingerprint identification".

[0205] Building upon this foundation, this application further combines characteristic chromatograms with external standard quantification, enabling simultaneous determination of the content of multiple characteristic components under the same chromatographic conditions. This eliminates the need for repeated sample preparation or switching of detection methods, significantly improving detection efficiency and data consistency.

[0206] This application provides a quantifiable, reproducible, and scalable scientific tool for tracing the origin of maidenhair fern, optimizing the harvest period, improving the processing technology, and identifying authenticity. It significantly enhances the objectivity, systematicness, and modern adaptability of the traditional Chinese medicine quality evaluation system and has substantial practical value for promoting the standardization and international application of ethnic medicine resources.

[0207] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic map of maidenhair fern, characterized in that, The construction method includes: a) Preparation of reference solutions: Chlorogenic acid, 3-O-p-coumarylquinic acid and kaempferol-3-O-β-D-glucuronide were used as reference standards and dissolved in 80% methanol to obtain the reference solutions, which included chlorogenic acid solution, 3-O-p-coumarylquinic acid solution and kaempferol-3-O-β-D-glucuronide solution; b) Preparation of the test solution: The maidenhair fern is mixed with the extraction solvent and subjected to reflux heating or ultrasonic treatment. After cooling, the mixture is filtered to obtain the test solution. c) The test solution and various reference solutions are placed in a high-performance liquid chromatograph, and the high-performance liquid chromatograms of the test solution and the reference solutions are obtained. Using the high-performance liquid chromatogram of the reference solutions as a control, common peaks are selected from the high-performance liquid chromatograms of the test solution to obtain the characteristic chromatogram of the maidenhair fern.

2. The construction method according to claim 1, characterized in that, The concentration of the chlorogenic acid solution is 16.447~48.03µg / mL; Preferably, the concentration of the 3-O-p-coumarylquinic acid solution is 15.868~25.68 μg / mL; Preferably, the concentration of the kaempferol-3-O-β-D glucuronide solution is 47.28~163.229 μg / mL.

3. The construction method according to claim 1, characterized in that, The high-performance liquid chromatograph is selected from any of the following: Shimadzu LC-20AT, Agilent 1260 or Waters e2695; Preferably, the chromatographic column of the high-performance liquid chromatograph is selected from any one of the following: Kromasil C 18 The dimensions are 4.6μm × 250mm; ZORBAX Eclipse XDB-C 18 Specifications are 4.6μm × 250mm; or Symmetry® C 18 The dimensions are 4.6μm × 250mm; Preferably, the particle size of the chromatographic column is 5 μm; Preferably, the chromatographic column is packed with octadecylsilane-bonded silica gel; Preferably, the chromatographic conditions of the high performance liquid chromatograph include: methanol as mobile phase A, 0.1% formic acid as mobile phase B, flow rate of 0.8~1.2 mL / min, column temperature of 25~35℃, detection wavelength of 300~330 nm, injection volume of 5~15 μL, and gradient elution. Preferably, the elution conditions for the gradient elution are as follows: From 0 to 40 min, the volume fraction of mobile phase A increased from 10% to 30%, while the volume fraction of mobile phase B decreased from 90% to 70%. For 40-55 minutes, the volume fraction of mobile phase A is 30%, and the volume fraction of mobile phase B is 70%. Over 55–80 min, the volume fraction of mobile phase A increased from 30% to 45%, while the volume fraction of mobile phase B decreased from 70% to 55%. For 80-90 minutes, the volume fraction of mobile phase A is 45%, and the volume fraction of mobile phase B is 55%. From 90 to 90.01 min, the volume fraction of mobile phase A decreased from 45% to 10%, while the volume fraction of mobile phase B increased from 55% to 90%. 90.01~100 min, the volume fraction of mobile phase A is 10%, and the volume fraction of mobile phase B is 90%.

4. The construction method according to claim 3, characterized in that, The extraction solvent is selected from any one of the following: methanol solution, ethanol solution, or water; Preferably, the volume fraction of methanol in the methanol solution is 50% to 100%. More preferably, the methanol solution has a methanol volume fraction of 50%, 80%, or 100%. Preferably, the volume fraction of ethanol in the ethanol solution is 50% to 100%. More preferably, the volume fraction of ethanol in the ethanol solution is 50%, 80%, or 100%. Preferably, the mass-to-volume ratio of the maidenhair fern to the extraction solvent is 1 g : (10~50) mL; Preferably, the heating reflux time is 30 min to 90 min; More preferably, the heating reflux time is selected from 30 min, 60 min, or 90 min; Preferably, the ultrasound duration is 30 min to 90 min; More preferably, the duration of the ultrasound is selected from 30 min, 60 min, or 90 min.

5. A method for quality testing of maidenhair fern, characterized in that, The quality detection method includes: constructing a feature spectrum of the maidenhair fern sample to be tested using the feature spectrum construction method of any one of claims 1 to 4, and obtaining the feature spectrum of the maidenhair fern sample to be tested; comparing the feature spectrum of the maidenhair fern sample to be tested with a control feature spectrum obtained under the same feature spectrum detection conditions, thereby realizing the quality detection of the maidenhair fern sample to be tested.

6. The quality inspection method according to claim 5, characterized in that, The characteristic peaks in the reference feature spectrum are 3; Among them, peak 1 is the peak of chlorogenic acid, peak 2 is the peak of 3-O-p-coumarylquinic acid, and peak 3 is the peak of kaempferol-3-O-β-D-glucuronide. Preferably, peak No. 2 is used as the positioning peak, with a relative retention time of 1.00; the average relative retention times of the other two characteristic peaks are: peak No. 1 is 0.66~0.8; peak No. 3 is 2.03~2.

49.

7. A feature map constructed using the method for constructing the feature map of Adiantum capillus-veneris according to any one of claims 1-4; Preferably, the feature spectrum has three characteristic peaks; wherein, Peak 1 is the peak of chlorogenic acid, peak 2 is the peak of 3-O-p-coumarylquinic acid, and peak 3 is the peak of kaempferol-3-O-β-D-glucuronide. More preferably, peak No. 2 is used as the positioning peak, with a relative retention time of 1.00; the average relative retention times of the other two characteristic peaks are: peak No. 1 is 0.66~0.8; peak No. 3 is 2.03~2.

49.

8. A method for determining the content of characteristic components of maidenhair fern, characterized in that, The characteristic component is selected from any one of the following: chlorogenic acid, 3-O-p-coumarylquinic acid, or kaempferol-3-O-β-D-glucuronide; The determination method includes: calculating the peak area of ​​the characteristic peak in the characteristic spectrum; and calculating the content of the characteristic component corresponding to the characteristic peak in the test sample solution using the external standard method. The feature map is the feature map described in claim 7.

9. The application of the method for constructing the characteristic spectrum of Adiantum capillus-veneris according to any one of claims 1-4, the quality testing method according to claim 5 or 6, the characteristic spectrum according to claim 7, or the method for determining the content of characteristic components of Adiantum capillus-veneris according to claim 8 in the quality control of Adiantum capillus-veneris.

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