A method for constructing a characteristic spectrum of Lygodium japonicum and its preparations and a method for quality detection

Abstract

The present invention relates to the field of quality detection of traditional Chinese medicine preparations, and specifically provides a method for constructing a characteristic spectrum of Lygodium japonicum and a preparation thereof, and a quality detection method. In the characteristic spectrum construction method, chromatographic conditions include: using octadecylsilane bonded silica gel as a filler, an elution time of less than 13-15 minutes, a detection wavelength of 250-260 nm, an elution time of ≥13-15 minutes, a detection wavelength of 310-330 nm, acetonitrile as a mobile phase A, and a 0.2% formic acid solution as a mobile phase B. Under a specific elution procedure, 11 common characteristic peaks are obtained, and good separation of the common characteristic peaks is achieved. The obtained characteristic spectrum has a stable baseline, good characteristic peak shape, good separation effect, significant characteristic components, and is simple. The method provides a basis for quality detection and control of Lygodium japonicum and a preparation thereof, achieves characterization of the overall components of Lygodium japonicum and a preparation thereof, and has stable reproducibility.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for constructing a characteristic spectrum of Lygodium japonicum and its preparation and a quality detection method. Background Art

[0002] Lygodium japonicum is the dried aerial part of Lygodium japonicum (Thunb.) Sw., a plant of the Lygodium family. Lygodium japonicum has the effects of clearing away heat and toxic substances, promoting diuresis and relieving stranguria, and is used for stranguria caused by damp heat, stranguria with stones, edema, dysuria, mumps, carbuncle and furuncle. Modern research shows that Lygodium japonicum is rich in various compounds such as organic acids, phenolic acids and flavonoids. Among them, the content of phenolic acid components is the highest (caffeic acid). This type of compound is a water-soluble substance with strong pharmacological activity. Lygodium japonicum is the aerial part of a plant of the Lygodium family, and only Lygodium japonicum is included in the "Chinese Pharmacopoeia", while Lygodium japonicum is not included. Some provincial and municipal standards for traditional Chinese medicine and the processing specifications for Chinese herbal medicine slices include Lygodium japonicum, and the research reference is relatively limited.

[0003] Common types of Lygodium chinense preparations include powders and granules. For example, granules are obtained by extracting Lygodium chinense slices through extraction, concentration, drying, and preparation. However, because traditional Chinese medicine granules no longer have the characteristics for identifying medicinal properties, existing methods for identifying Lygodium chinense are not suitable for quality testing of Lygodium chinense granules and other preparations made from Lygodium chinense water extracts, as they have few characteristic peaks and poor separation effects. Qualitative detection of characteristic patterns of Lygodium chinense medicinal materials and their preparations using high-performance liquid chromatography is currently a gap. Improving the testing standards for Lygodium chinense water extracts and their preparations, and comprehensively controlling the quality of the finished products, is a technical problem to be solved by the present invention. Summary of the Invention

[0004] Therefore, the purpose of the present invention is to provide a method for constructing a characteristic spectrum of Lygodium japonicum and its preparations and a quality detection method. This method establishes a characteristic spectrum of this variety based on the characteristics of Lygodium japonicum and its preparations, realizes the effective separation of each characteristic peak, and provides a scientific basis for comprehensively establishing the quality control standards of Lygodium japonicum and its preparations.

[0005] To this end, the present invention provides a method for constructing a characteristic spectrum of Lygodium japonicum and its preparation, comprising the following steps:

[0006] (1) Preparation of the test solution, including the following steps:

[0007] 1) dissolving the test sample in water to obtain a solution, and extracting with water-saturated n-butanol; 2) drying the n-butanol solution, adding an extraction solvent, and extracting to obtain an extract; 3) separating the extract into a solid-liquid state, and taking the liquid to obtain the test sample solution;

[0008] (2) The sample solution was tested by high performance liquid chromatography to obtain a characteristic spectrum of the sample. The chromatographic conditions included: octadecylsilane bonded silica gel as the filler, acetonitrile as the mobile phase A, and formic acid solution as the mobile phase B, and gradient elution was performed according to the following table:

[0009]

[0010] Furthermore, the detection wavelength is 250-330 nm. Preferably, when the elution time is <13-15 min, the detection wavelength is 250-260 nm; when the elution time is ≥13-15 min, the detection wavelength is 310-330 nm.

[0011] Furthermore, step (1) satisfies any one or more of the following AEs:

[0012] A. In step 1), the number of water-saturated n-butanol extractions is 1 to 3 times, and the ratio of the mass of the test sample to the volume of water and the volume of water-saturated n-butanol used each time is 0.1-0.6:10-20:20; the mass-to-volume ratio is g / mL;

[0013] B. In step 2), the mass-to-volume ratio of the test sample to the extraction solvent is 0.1-0.6:20; the mass-to-volume ratio is g / mL;

[0014] C. The extraction solvent is selected from a methanol aqueous solution or an ethanol aqueous solution with a volume percentage of 30-90%, preferably a methanol aqueous solution with a volume percentage of 70%;

[0015] D. In step 2), the extraction method is ultrasonic extraction or reflux extraction, and the extraction time is 30min-60min;

[0016] E. In step 3), the solid-liquid separation is independently selected from centrifugation or filtration.

[0017] Furthermore, a CAPCELL CORE C18 column is used as a chromatographic column; and / or a chromatographic column with a column length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 2.7 μm is used; and / or the Lygodium japonicum and its preparation are selected from Lygodium japonicum herbal materials, Lygodium japonicum decoction pieces, Lygodium japonicum water extracts, or Lygodium japonicum pharmaceutical preparations; and / or the volume percentage of the formic acid solution is 0.05-0.2%; and / or the flow rate is 0.5-0.9 ml / min; and / or the column temperature is 19-30°C.

[0018] Furthermore, the construction method further includes the step of using one or more of protocatechuic acid, caffeic acid, 4-coumarin, isoquercetin, kaempferol-3-O-rutinoside, and linaloside as reference substances and adding solvents to prepare a reference substance solution, and the step of detecting the reference substance solution by high performance liquid chromatography according to the construction method according to any one of claims 1 to 4 to obtain a reference spectrum of the reference substance; preferably, the solvent is selected from methanol or a methanol aqueous solution with a volume percentage of more than 70%; preferably, each 1 mL of the reference substance solution contains 15-100 μg of each reference substance.

[0019] Furthermore, the construction method also includes extracting the control medicinal material of Lygodium japonicum with water, filtering the obtained filtrate, and extracting it with water-saturated n-butanol; drying the n-butanol solution, adding an extraction solvent, and extracting to obtain an extract; separating the extract into a solid and liquid, and taking the liquid to obtain a control medicinal material reference solution, and the step of detecting the control medicinal material reference solution by high performance liquid chromatography according to the construction method described in any one of claims 1 to 4 to obtain a control medicinal material reference atlas.

[0020] Furthermore, the characteristic spectrum of the seaweed and its preparation has 11 characteristic peaks, the peak corresponding to the caffeic acid reference peak is the S peak, peak 4 is the S peak, and the relative retention times of peaks 1 to 3 and peaks 5 to 11 and the S peak are within ±10% of the specified values, and the specified values ​​of peaks 1 to 3 and peaks 5 to 11 are: 0.48, 0.77, 0.94, 1.06, 1.45, 1.93, 2.17, 2.32, 2.52, and 3.16, respectively.

[0021] Furthermore, the characteristic spectrum of the Lygodium japonicum and its preparation has 11 characteristic peaks, peak 1 is protocatechuic acid, peak 4 is caffeic acid, peak 6 is 4-coumaric acid, peak 7 is isoquercetin, peak 8 is kaempferol-3-O-rutinoside, and peak 11 is linaloside.

[0022] The present invention also provides the application of any of the methods for constructing a characteristic spectrum of Lygodium japonicum and its preparations in the quality detection of Lygodium japonicum and its preparations.

[0023] The present invention also provides a quality detection method for Lygodium japonicum and its preparation products, characterized in that it includes the step of obtaining a characteristic spectrum of the product to be tested according to any of the construction methods described above.

[0024] The technical solution of the present invention has the following advantages:

[0025] 1. The method for constructing a characteristic spectrum of Lygodium japonicum and its preparation provided by the present invention is that the test solution is prepared by the following method: 1) taking a test sample, adding water to dissolve it to obtain a solution, and extracting it with water-saturated n-butanol; 2) taking the n-butanol solution, drying it, and adding an extraction solvent to extract it to obtain an extract; 3) separating the extract into solid and liquid, and taking the liquid to obtain the test solution; the above-mentioned test solution is detected by high performance liquid chromatography under the following chromatographic conditions, using octadecylsilane bonded silica gel as a filler, acetonitrile as mobile phase A, and formic acid solution as mobile phase B, and 11 common characteristic peaks are obtained under a specific elution program, and these common characteristic peaks are well separated. The obtained characteristic spectrum has a stable baseline, good characteristic peak shape, good separation effect, significant characteristic components, and is simple, providing a basis for quality detection and control of Lygodium japonicum and its preparation, realizing the characterization of the overall components of Lygodium japonicum and its preparation, and having stable reproducibility.

[0026] 2. The method for constructing a characteristic spectrum of Lygodium japonicum and its preparations provided by the present invention, using medicinal materials, decoction pieces, and related preparations as research objects, confirmed 11 common characteristic peaks and identified six characteristic components: protocatechuic acid, caffeic acid, 4-coumarin, isoquercetin, kaempferol-3-O-rutinoside, and montanside. The results displayed by the spectrum are extremely easy to determine, providing a rapid and reliable detection method for the characteristic spectrum identification of Lygodium japonicum medicinal materials. This method overcomes the misjudgment that may occur when evaluating only a single indicator content, and enables more comprehensive quality monitoring of Lygodium japonicum formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 This is the characteristic spectrum of the freeze-dried powder of the standard decoction of Lygodium japonicum slices obtained in Example 1;

[0029] Figure 2 This is a chromatogram of the COSMOSIL-5C18-PAQ chromatographic column in the investigation experiment of the chromatographic column in Example 2;

[0030] Figure 3 This is the chromatogram of the CAPCELL CORE C18 column in the investigation experiment of the chromatographic column in Example 2;

[0031] Figure 4 This is the chromatogram of the COSMOSIL-5C18-AR-Ⅱ column in the investigation experiment of the chromatographic column in Example 2;

[0032] Figure 5 This is the chromatogram obtained by using ethyl acetate as the extraction solvent in the investigation experiment of the extraction solvent in Example 2;

[0033] Figure 6 This is the investigation chromatogram obtained by using n-butanol as the extraction solvent in the investigation experiment of the extraction solvent in Example 2;

[0034] Figure 7 This is the chromatogram at a wavelength of 254 nm in the investigation experiment of the detection wavelength in Example 2;

[0035] Figure 8 This is the chromatogram at a wavelength of 320 nm in the investigation experiment of the detection wavelength in Example 2;

[0036] Figure 9 This is the chromatogram of the Agilent 1260 instrument used in the chromatograph investigation experiment of Example 2;

[0037] Figure 10 This is the chromatogram of the waters e2695 instrument in the chromatograph investigation experiment of Example 2;

[0038] Figure 11 This is the chromatogram of the Thermo Fisher U3000 instrument used in the chromatograph investigation experiment of Example 2;

[0039] Figure 12 This is the characteristic spectrum of 15 batches of Lycopodiella herb in Example 2;

[0040] Figure 13 This is the reference characteristic spectrum of the Herba Lycopodii in Example 2;

[0041] Figure 14 The characteristic spectra of 15 batches of Lygodium japonicum decoction pieces in Example 2;

[0042] Figure 15 This is the reference characteristic spectrum of the Herba Lygodii in Example 2;

[0043] Figure 16 The characteristic spectra of 15 batches of freeze-dried powder of standard decoction of Lygodium japonicum slices in Example 2;

[0044] Figure 17 This is the reference characteristic spectrum of the freeze-dried powder of the standard decoction of the Herba Lycopodii in Example 2;

[0045] Figure 18 The reference characteristic maps of the Herba Lygodii, decoction pieces, and decoction piece standard decoctions in Example 2 are shown below: S1: a reference map of the Herba Lygodii; S2: a reference map of the Herba Lygodii decoction pieces; S3: a reference map of the Herba Lygodii decoction pieces standard decoctions;

[0046] Figure 19The results of the comparative positioning of the characteristic map of Lygodium japonicum in Example 2 are shown in the figure; from bottom to top, they are the test sample, protocatechuic acid, caffeic acid, 4-coumarin, kaempferol-3-O-rutinoside, isoquercitrin, and montanin;

[0047] Figure 20 This is the characteristic spectrum of the standard decoction of the Herba Lycopodii in Example 3;

[0048] Figure 21 This is the negative control chromatogram in Example 3 (70% methanol). DETAILED DESCRIPTION

[0049] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0050] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0051] In the present invention, the freeze-dried powder of the standard decoction of Lygodium japonicum is prepared from Lygodium japonicum slices as raw materials according to conventional methods in the art. For example, in the present invention, the sample is prepared according to the following decoction method: take Lygodium japonicum slices, soak for 30 minutes, decoct twice, add 12 times the weight of water for the first decoction, boil and extract for 30 minutes, add 10 times the weight of water for the second decoction, boil and extract for 20 minutes, filter, and concentrate the filtrate under reduced pressure to a pure extract with a relative density of 1.00 to 1.04 (60°C), place it in a freeze dryer for freezing to dryness, and grind it into powder to obtain. 70% methanol refers to a methanol aqueous solution with a volume percentage of 70%.

[0052] Example 1

[0053] This embodiment provides a method for constructing a characteristic spectrum of a lyophilized powder of a standard decoction of Lygodium japonicum, comprising the following steps:

[0054] Preparation of test solution: Use freeze-dried powder of standard decoction of Lygodium japonicum as the test sample, take about 0.1 g of the test sample, add 10 ml of water to dissolve it, extract twice with water-saturated n-butanol, 20 ml each time, combine the n-butanol solutions, evaporate to dryness, cool, add 20 ml of 70% methanol to the residue, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate to obtain.

[0055] Preparation of reference solution: Take an appropriate amount of caffeic acid reference, accurately weigh it, and add methanol to make a solution containing 40 μg of caffeic acid per 1 ml, which is used as the reference solution;

[0056] HPLC: Inject the reference sample solution and the test sample solution into an HPLC instrument using the following chromatographic conditions: octadecylsilane bonded silica gel as the packing (column length, 150 mm, inner diameter, 4.6 mm, particle size, 2.7 μm); acetonitrile as mobile phase A, 0.2% formic acid solution as mobile phase B, gradient elution as specified in the table below; elution at 254 nm (elution time <14 minutes), 320 nm (elution time ≥14 minutes), column temperature, 20°C, flow rate, 0.7 ml / min. The number of theoretical plates, calculated based on the caffeic acid peak, should be no less than 3000.

[0057]

[0058] The results are shown in the table below and Figure 1 As shown, the characteristic spectrum of the freeze-dried powder of the standard decoction of Lygodium japonicum has a stable baseline, good characteristic peak shape, good separation effect, and significant characteristic components. It has 11 characteristic peaks, peak 4 corresponds to the caffeic acid reference substance, and the peak corresponding to the reference peak of the caffeic acid reference substance is the S peak. The relative retention times of peaks 1 to 3 and peaks 5 to 11 to the S peak are 0.48, 0.77, 0.94, 1.06, 1.45, 1.93, 2.17, 2.31, 2.52, and 3.15, respectively.

[0059] Table 1 Information of each peak of Lygodium japonicum standard decoction freeze-dried powder

[0060]

[0061] Example 2

[0062] 1. Instruments and test drugs

[0063] 1.1 Instrument

[0064] High-performance liquid chromatograph 1: Thermo U3000 chromatography system, including a quaternary solvent manager (Pump), an autosampler (Autosampler), an imported column compartment (Column Compartment), a diode array UV detector (Detector), and a Chromeleon chromatography management system;

[0065] High performance liquid chromatograph 2: Waters E2695, including G7104A quaternary pump, 132-position autosampler, G7116B column oven, and diode array detector;

[0066] High performance liquid chromatograph 3: Agilent 1260 chromatography system, including G1311B quaternary pump, G1367E autosampler, G1316A PDA diode array detector, and G1330B column oven;

[0067] METTLER TOLEDO (Swiss Mettler) ME36S, XS204, XSE205 (1 / 100,000); SK5200H Shanghai Kedao Ultrasonic Instrument Co., Ltd.;

[0068] Chromatographic column: (1) COSMOSIL-5C18-AR-Ⅱ 4.6×250mm 5μm

[0069] (2)CAPCELL CORE C18 4.6×150mm 2.7μm

[0070] (3)COSMOSIL-5TC18-PAQ 4.6×250mm 5μm.

[0071] 1.2 Reagents

[0072] Acetonitrile was of chromatographic grade, water was ultrapure water; other reagents such as glacial acetic acid, formic acid, trifluoroacetic acid, n-butanol, ethyl acetate, and methanol were of analytical grade.

[0073] 1.3 Drug testing

[0074] Caffeic acid reference substance: purchased from China Food and Drug Inspection Institute, batch number 110885-201703, purity: 99.7%.

[0075] Lygodium japonicum control medicinal material: purchased from Shanghai Hongyong Biotechnology Co., Ltd., batch number 280067-202103.

[0076] The batch information of Lygodium japonicum is as follows:

[0077]

[0078]

[0079] 2. Investigation of chromatographic conditions and extraction solvents

[0080] (1) Investigation of mobile phase system

[0081] The effects of methanol-water and acetonitrile-water systems on chromatographic peak separation and peak shape were investigated. The test solution was prepared as follows: 0.1 g of lyophilized powder of Lygodium japonicum decoction was placed in a 50 mL stoppered conical flask. 20 mL of 70% methanol was added and ultrasonicated (power 250 W, frequency 40 kHz) for 30 minutes. The solution was cooled, weighed, and the weight loss was made up with 70% methanol. The solution was shaken and filtered to obtain the test solution. Gradient elution was performed on the same aliquot of the test solution as specified in the table below. The chromatographic column was COSMOSIL-5C18-AR-Ⅱ, 4.6 × 250 mm, 5 μm; the detection wavelength was 320 nm; the column temperature was 30°C; and the injection volume was 10 μL.

[0082]

[0083] Table 2 System suitability parameters for mobile phase methanol-acetonitrile

[0084]

[0085] Note: "*" indicates that the peak separation is extremely poor or the peaks are crossed. The instrument does not calculate the parameters such as resolution, symmetry factor or theoretical plate number by default.

[0086] The results showed that the number of chromatographic peaks of the freeze-dried powder of the standard decoction of Herba Lygodii was relatively small, the baseline of the characteristic spectrum was unstable, the separation was poor, the peak shape was poor, and the difference in the peak height of the chromatographic peaks was also relatively large; overall, the acetonitrile-water system was better than the methanol-water system, and the acetonitrile-water system was used in subsequent exploration.

[0087] (2) Investigation of acid types

[0088] The elution gradient was adjusted to investigate the effect of the acid type in the mobile phase on chromatographic peak separation and peak shape. The test solution was prepared as follows: 0.1 g of lyophilized powder of Lygodium japonicum decoction was placed in a 50 mL stoppered conical flask. 20 mL of 70% methanol was added and ultrasonicated (power 250 W, frequency 40 kHz) for 30 minutes. The solution was cooled, weighed, and the weight loss was made up with 70% methanol. The mixture was shaken and filtered to prepare the test solution. The same sample solution was tested using acetonitrile as mobile phase A and 0.1% formic acid aqueous solution, 0.1% glacial acetic acid aqueous solution, or 0.1% trifluoroacetic acid aqueous solution as mobile phase B, using gradient elution as specified in the table below. The chromatographic column was COSMOSIL-5C18-AR-Ⅱ, 4.6 × 250 mm, 5 μm; the detection wavelength was 320 nm; the column temperature was 30°C; and the injection volume was 10 μL.

[0089]

[0090] Table 3 System suitability parameters for different mobile phases

[0091]

[0092]

[0093] Note: "*" indicates that the peak separation is extremely poor or there are cross peaks, so the instrument cannot calculate the parameters such as resolution, symmetry factor or theoretical plate number by default;

[0094] The results show that when using formic acid, the separation degree and response value of the corresponding peaks are better than those of trifluoroacetic acid and glacial acetic acid, and the separation effect is significantly improved. However, from the perspective of the retention time of the characteristic peaks, there are too many characteristic peaks (some baseline peaks are still not integrated), the baseline is not stable, and the peak time is too dense, which is not conducive to the overall positioning of the peaks and the judgment of the results.

[0095] (3) Investigation of acid concentration

[0096] The effect of acid concentration on chromatographic peak separation and peak shape was investigated. The test solution was prepared as follows: 0.1 g of lyophilized powder of Lygodium japonicum decoction was placed in a 50 mL stoppered conical flask. 20 mL of 70% methanol was added and ultrasonicated (power 250 W, frequency 40 kHz) for 30 minutes. The solution was cooled, weighed, and the weight loss was made up with 70% methanol. The mixture was shaken and filtered to prepare the test solution. The same sample solution was tested using acetonitrile as mobile phase A and 0.1% formic acid aqueous solution, 0.05% formic acid aqueous solution, or 0.2% formic acid aqueous solution as mobile phase B, using gradient elution as specified in the table below. The chromatographic column was COSMOSIL-5C18-AR-Ⅱ, 4.6 × 250 mm, 5 μm, with a detection wavelength of 320 nm. The column temperature was 30°C, and the injection volume was 10 μL.

[0097]

[0098] Table 4 System suitability parameters for different mobile phase acid concentrations

[0099]

[0100]

[0101] The results showed that the use of 0.05-0.2% formic acid can achieve the separation of multiple characteristic peaks, especially 0.2% formic acid has the best separation effect.

[0102] (4) Inspection of chromatographic column

[0103] The effect of the chromatographic column on the separation and peak shape of the chromatographic peaks was investigated. The test solution was prepared as follows: 0.1 g of the freeze-dried powder of the standard decoction of Lycopodiella cava was placed in a 50 mL stoppered conical flask, 20 mL of 70% methanol was added, and ultrasonic treatment (power 250 W, frequency 40 kHz) was performed for 30 minutes. The mixture was cooled, weighed, and the weight loss was supplemented with 70% methanol. The mixture was shaken and filtered to obtain the test solution. The same test solution was tested at a detection wavelength of 320 nm, a column temperature of 30 ° C, an injection volume of 10 μL, acetonitrile as mobile phase A, and 0.2% formic acid aqueous solution as mobile phase B. The following three chromatographic columns were used for detection:

[0104] 1. COSMOSIL-5C18-PAQ 4.6x250mm, 5μm

[0105] 2. CAPCELL CORE C18 4.6x150mm, 2.7μm;

[0106] 3. COSMOSIL-5C18-AR-Ⅱ 4.6×250mm 5μm; perform gradient elution according to the regulations in the following table.

[0107]

[0108] See the results Figure 2-4 The chromatographic peak distribution is more uniform and the baseline is more uniform when using the CAPCELL CORE C18 chromatographic column, so the CAPCELL CORE C18 4.6×150mm 2.7μm chromatographic column is temporarily selected.

[0109] (5) Investigation of extraction agents

[0110] After the above optimization investigation, the separation was improved, but it was found that there were too many characteristic peaks (some baseline impurities were still not integrated) and the peak elution time was too close, which was not conducive to the overall positioning of the peaks and the judgment of the results. Therefore, it was considered to use different organic solvents (n-butanol, ethyl acetate) to extract the samples in order to remove the water-soluble impurities in the samples and make the baseline more stable.

[0111] The specific method is as follows: take about 0.1 g of the freeze-dried powder of the standard decoction of Lycopodiella cava, add 10 mL of water to dissolve it, extract it twice with water-saturated n-butanol or ethyl acetate, 20 mL each time, combine the n-butanol or ethyl acetate liquids, evaporate to dryness, cool, add 20 mL of 70% methanol to the residue, ultrasonically treat it (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0112] See the results Figure 5-6, indicating that the overall distribution of the characteristic chromatographic peaks obtained by extraction with different organic reagents is more uniform than that before extraction, but there are certain differences in the total peak area. The response value of the chromatographic peak detected after n-butanol extraction is significantly larger than the chromatographic peak detected by ethyl acetate extraction. Taking comprehensive consideration, it is determined that the pretreatment method of the test sample uses n-butanol as the extraction solvent to continue to optimize the gradient.

[0113] (6) Further optimization of gradient elution program

[0114] Gradient Optimization: The chromatogram shows that some characteristic peaks are poorly separated and require further separation optimization. Based on the above, the gradient was optimized using the information content of the chromatographic peaks, the resolution of the chromatographic peaks, and the analysis time as indicators. The test solution was prepared as follows: approximately 0.1 g of lyophilized powder of the standard decoction of Lygodium japonicum was dissolved in 10 mL of water. Extraction was performed twice with 20 mL of water-saturated n-butanol. The n-butanol solutions were combined, evaporated to dryness, and cooled. The residue was added with 20 mL of 70% methanol and sonicated (power 250 W, frequency 40 kHz) for 30 minutes. The residue was cooled, shaken, filtered, and the filtrate was obtained. The same test solution was tested using a CAPCELL CORE column with a detection wavelength of 320 nm and a column temperature of 25°C. The injection volume was 10 μL. Acetonitrile was used as mobile phase A, and 0.2% formic acid aqueous solution was used as mobile phase B. Gradient elution was performed as specified in the table below.

[0115] Table 5 Gradient elution program

[0116]

[0117] Table 6 Gradient elution program

[0118]

[0119] Table 7 System suitability parameters for different gradient inspections

[0120]

[0121]

[0122] After the above investigation, gradient 6 has the best separation effect, with relatively uniform distribution of peaks, a large number of peaks, and a stable baseline. Therefore, gradient 6 is tentatively selected as the characteristic spectrum method.

[0123] (7) Investigation of detection wavelength

[0124] The absorption spectrum of the main chromatographic peaks in the chromatogram of the freeze-dried powder of the standard decoction of Herba Cercidiphyllae under the gradient 6 at 190-400 nm was extracted. Figure 7-8The peak absorption peak for the first 14 minutes is primarily concentrated at 254nm, with caffeic acid and 4-coumaric acid maximally absorbing at 320nm. After 14 minutes, the peak absorption is more evenly distributed at 320nm. At 254nm, protocatechuic acid exhibits a strong absorption wavelength before 14 minutes, while caffeic acid exhibits a lower response. At 320nm, the wavelength change after 14 minutes is primarily due to the selection of components with strong Lygodium japonicum specificity.

[0125] (8) Inspection of chromatograph

[0126] The test solution prepared according to item (6) of this embodiment was injected into chromatographs of different brands (Agilent 1260, Waters E2695, and Thermo Fisher U3000) to investigate the durability of different instruments. Other chromatographic conditions were as follows:

[0127] The chromatographic column was CAPCELL CORE, with an elution time of <14 min and a detection wavelength of 254 nm; the elution time was ≥14 min and a detection wavelength of 320 nm; the column temperature was 25°C; the injection volume was 10 μL, acetonitrile was used as mobile phase A, and 0.2% formic acid aqueous solution was used as mobile phase B. Gradient elution was performed according to the above gradient 6, respectively. The results are shown in FIG. Figure 9-11 , indicating that different instruments have good durability.

[0128] (9) Investigation of flow rate

[0129] The test solution prepared according to item (6) of this embodiment was taken and examined at flow rates of 0.5 ml / min, 0.7 ml / min, and 0.9 ml / min, respectively. The other chromatographic conditions were the same as item (8) of this embodiment. The results showed that the characteristic peaks could be well separated at a flow rate of 0.5 ml / min-0.9 ml / min, among which the separation effect was the best at 0.7 ml / min.

[0130] Table 8 Peak results at different flow rates

[0131]

[0132]

[0133]

[0134]

[0135] (10) Investigation of column temperature

[0136] The test solution prepared according to item (6) of this embodiment was taken and examined at column temperatures of 20°C, 25°C, and 30°C, respectively. The other chromatographic conditions were the same as item (8) of this embodiment. The results showed that the characteristic peaks could be well separated at a column temperature of 20-30°C, among which the separation effect at 20°C was the best.

[0137] Table 9 Peak results at different column temperatures

[0138]

[0139]

[0140]

[0141] 3. Establishment of feature maps and parameters

[0142] The characteristic spectrum similarity evaluation software "Chinese Herbal Chromatographic Characteristic Spectrum Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate control characteristic spectra using multiple batches of representative freeze-dried powder samples of standard decoction of Lygodium japonicum. The chemical properties of each characteristic peak of the freeze-dried powder of standard decoction of Lygodium japonicum were analyzed by consulting literature, and caffeic acid was used to locate and identify them.

[0143] (1) Establishment of characteristic profiles for 15 batches of Lycopodiella herb, herbal slices, and freeze-dried powder of standard decoctions

[0144] Preparation of reference solution: Take caffeic acid, accurately weigh it, and add 70% methanol to make a solution of 20μg caffeic acid per 1ml, which is used as the reference solution. Take 0.6g of Lygodium japonicum reference medicinal material, place it in a stoppered conical flask, add 25ml of water, heat and reflux for 30 minutes, filter, and extract the filtrate by shaking twice with water-saturated n-butanol, 25ml each time, combine the n-butanol solutions, evaporate to dryness, cool, add 20ml of 70% methanol to the residue, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, filter, and take the filtrate as the reference solution of the reference medicinal material. Separately, take the reference solution under [Assay] as the reference solution;

[0145] Preparation of test solution: 15 batches of Lygodium japonicum herbs, decoction pieces and freeze-dried powder of decoction piece standard decoction were used as test samples to prepare test solution according to the method of Example 1.

[0146] High performance liquid chromatography: The test solution and the reference solution were subjected to high performance liquid chromatography (HPLC) under the same chromatographic conditions as in Example 1.

[0147] like Figure 12As shown in Figure 2, the characteristic spectrum test results of 15 batches of Lygodium japonicum medicinal materials were analyzed, and the characteristic spectrum similarity evaluation software "Chinese Herbal Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate a control characteristic spectrum, as shown in Figure 2. Figure 13 Eleven chromatographic peaks with good resolution were identified in the HPLC characteristic spectrum of Lygodium japonicum, and the peaks were rearranged from 1 to 11 according to their order. The results showed that all 15 batches of Lygodium japonicum exhibited 11 characteristic peaks in their characteristic spectrum, corresponding to the 11 characteristic peaks in the chromatogram of the control herbal material. The peak corresponding to the caffeic acid reference peak was designated the S peak. The relative retention times of Peaks 1, 2, 3, 5, 6, 7, 8, 9, 10, and 11 relative to the S peak were within ±10% of the specified values. The specified values ​​for Peaks 1 to 3 and Peaks 5 to 11 were, respectively, 0.48, 0.77, 0.94, 1.06, 1.45, 1.93, 2.17, 2.32, 2.52, and 3.16.

[0148] Table 10 Relative retention time results of characteristic spectra of 15 batches of Lycopodiella herb

[0149]

[0150]

[0151] Table 11 Relative peak area results of characteristic spectra of 15 batches of Lycopodiella herb

[0152]

[0153] like Figure 14 As shown in Figure 2, by analyzing the detection results of the characteristic spectra of 15 batches of Lycopodiella chinensis slices, the characteristic spectra similarity evaluation software "Chinese Herbal Chromatographic Characteristic Spectrum Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate a control characteristic spectrum, as shown in Figure 2. Figure 15 The determination results showed that the characteristic spectra of 15 batches of Herba Lygodii presented 11 characteristic peaks, and the chromatographic peak numbers 1 to 11 were rearranged according to the order of the chromatographic peaks. Corresponding to the 11 characteristic peaks in the chromatogram of the reference medicinal material, the peak corresponding to the caffeic acid reference peak was designated as the S peak, and the relative retention times of peaks 1, 2, 3, 5, 6, 7, 8, 9, 10, and 11 with the S peak were within ±10% of the specified values. The specified values ​​of peaks 1 to 3 and peaks 5 to 11 were, respectively, 0.48, 0.77, 0.94, 1.06, 1.45, 1.93, 2.17, 2.32, 2.52, and 3.16.

[0154] Table 12 Relative retention time results of characteristic spectra of 15 batches of Lygodium japonicum slices

[0155]

[0156]

[0157] Table 13 Relative peak area results of characteristic spectrum determination of 15 batches of Lygodium japonicum slices

[0158]

[0159]

[0160] like Figure 16 As shown in the figure, by analyzing the detection results of the characteristic spectra of 15 batches of freeze-dried powder of standard decoction of Lycopodiella quinata slices, the characteristic spectra similarity evaluation software "Chinese Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Committee was used to generate a reference characteristic spectrum, as shown in the figure. Figure 17 The results showed that the characteristic chromatograms of 15 batches of freeze-dried powder of standard decoction of Herba Lygodii presented 11 characteristic peaks, and the chromatographic peak numbers 1 to 11 were rearranged according to the order of the chromatographic peaks. Corresponding to the 11 characteristic peaks in the chromatogram of the reference medicinal material, the peak corresponding to the caffeic acid reference peak was designated as the S peak. The relative retention times of peaks 1, 2, 3, 5, 6, 7, 8, 9, 10, and 11 with respect to the S peak were within the range of ±10% of the specified values. The specified values ​​of peaks 1 to 3 and peaks 5 to 11 were, respectively, 0.48, 0.77, 0.94, 1.06, 1.45, 1.93, 2.17, 2.32, 2.52, and 3.16.

[0161] Table 14 Relative retention time results of 15 batches of standard decoctions of Lygodium japonicum slices

[0162]

[0163] Table 15 Relative peak area results of 15 batches of standard decoctions of Lygodium japonicum slices

[0164]

[0165]

[0166] (2) Transfer relationship between characteristic spectra of medicinal materials, decoction pieces and freeze-dried powder of standard decoction

[0167] like Figure 18As shown, according to the 15 batches of Herba Lygodii selected for the study and the corresponding 15 batches of Herba Lygodii slices and 15 batches of standard decoctions of decoction slices, the number of characteristic peaks obtained was consistent, with 11 characteristic peaks appearing. This indicates that the characteristic spectrum method constructed by the present invention is used. During the processing of Herba Lygodii from the medicinal material to Herba Lygodii slices, and in the freeze-dried powder of the standard decoction prepared from the Herba Lygodii slices according to the traditional preparation method, the detected substance components have not changed, and the quantitative transfer relationship is good. The relative retention time specified for each characteristic peak is within the range of ±10% of the specified value of the Herba Lygodii slices standard decoction reference spectrum, corresponding to the retention time of the 11 characteristic peaks in the chromatogram of the reference medicinal material reference.

[0168] Table 16 Comparison of relative retention times of medicinal materials, decoction pieces and standard decoctions

[0169]

[0170] (3) Peak identification

[0171] Preparation of reference solution: Take appropriate amounts of protocatechuic acid, caffeic acid, 4-coumarin, isoquercetin, kaempferol-3-O-rutinoside, and montanaside, accurately weigh them, and add 70% methanol to prepare a solution containing 40 μg protocatechuic acid, 40 μg caffeic acid, 15 μg 4-coumarin, 40 μg isoquercetin, 40 μg kaempferol-3-O-rutinoside, or 20 μg montanaside per 1 mL of reference solution.

[0172] Take the test solution of the lyophilized powder of the standard decoction of Lygodium japonicum prepared in Example 1 above, and compare the reference solution and the test solution by high performance liquid chromatography according to the method of Example 1. The results are as follows: Figure 19 .

[0173] Summary: In the chromatogram of the freeze-dried powder of the standard decoction of Lycopodiella cava, peak 1 is protocatechuic acid, peak 4 is caffeic acid, peak 6 is 4-coumaric acid, peak 7 is isoquercetin, peak 8 is kaempferol-3-O-rutinoside, and peak 11 is montmorillonate.

[0174] Example 3 Methodology Verification

[0175] 1. Instrument precision test

[0176] A test solution was prepared using the herb Lygodium japonicum as a test sample according to the method of Example 1. The same sample solution of the herb Lygodium japonicum was taken and injected 6 times according to the chromatographic conditions of Example 1. The relative retention times and relative peak areas of the 11 characteristic peaks were measured and analyzed. The results are shown in the table below.

[0177] Table 17 Instrument precision relative retention time test results

[0178]

[0179] Table 18 Instrument precision relative peak area test results

[0180]

[0181] The results showed that the RSDs of the relative retention times of the characteristic peaks and the reference S peak were less than 2%, and the RSDs of the relative peak areas of the characteristic peaks and the reference S peak were less than 4.5%, indicating that the instrument had good precision.

[0182] The instrument precision test was conducted using the same method with freeze-dried powder of standard decoction of Lygodium japonicum as the test sample. The results showed that the RSDs of the relative retention times of each characteristic peak and the reference S peak were all less than 2.0%, and the RSDs of the relative peak areas were all less than 4.0%, indicating good instrument precision.

[0183] 2. Method repeatability test

[0184] Six samples of the same batch of Lycopodiella herb were taken for testing. The relative retention times and relative peak areas of the 11 common peaks were measured according to the following method of Example 1, and the results were analyzed. The results are shown in the table below.

[0185] Table 19 Method repeatability relative retention time test results (n=6)

[0186]

[0187]

[0188] Table 20 Method repeatability relative peak area test results (n=6)

[0189]

[0190] The results showed that the RSD of the relative retention time between each characteristic peak and the reference S peak was less than 2.0%, and the RSD of the relative peak area was less than 5.09%, indicating that the method had good repeatability.

[0191] The repeatability test was carried out using the freeze-dried powder of the standard decoction of Lygodium japonicum as the test sample. The results showed that the RSD values ​​of the relative retention times of the 11 common peaks and the reference peak (peak S) were less than 2.0%, indicating that the method had good repeatability.

[0192] 3. Intermediate precision (different operators)

[0193] Three inspectors, at different times, used the same equipment to measure the same batch of Lycopodiella herb according to the chromatographic conditions of Example 1. The relative retention times and relative peak areas of the 11 common peaks were measured and analyzed. The results are shown in the table below.

[0194] Table 21 Intermediate precision relative retention time test results (different operators)

[0195]

[0196] Table 22 Intermediate precision relative peak area test results (different operators)

[0197]

[0198]

[0199] The results showed that the RSDs of the relative retention times of the characteristic peaks and the reference S peak were all less than 2.0%, and the RSDs of the relative peak areas were all less than 2.0%, indicating that the intermediate precision (different personnel) of the method was good.

[0200] The intermediate precision test was carried out using the freeze-dried powder of the standard decoction of Lycopodiella cava as the test sample. The results showed that the RSD values ​​of the relative retention times of the 11 common peaks and the reference peak (peak S) were less than 2.0%, indicating that the intermediate precision of this method was good.

[0201] 4. Durability inspection

[0202] (1) Stability investigation

[0203] The same Herba Lygodii was used as the test sample, and the test solution was prepared and tested by high performance liquid chromatography according to Example 1. The samples were injected 0, 4, 8, 12, 16, and 24 hours after preparation, and the relative retention times and relative peak areas of the 11 common peaks were measured. The stability of the test solution was determined by analysis. The results are shown in the table below.

[0204] Table 23 Stability relative retention time test results

[0205]

[0206] Table 24 Stability relative peak area test results

[0207]

[0208] The results showed that the RSDs of the relative retention times of the characteristic peaks and the reference S peak were less than 2%, and the RSDs of the relative peak areas were less than 6%, indicating that the test solution was stable within 24 hours and met the determination requirements.

[0209] (2) Investigation of different flow rates

[0210] The same Herba Lygodii was used as the test sample and the test sample solution was prepared according to Example 1. The same batch of test sample solution was taken and measured according to the determination method of Example 1 at flow rates of 0.6 ml / min, 0.7 ml / min, and 0.8 ml / min, respectively. The relative retention time and relative peak area of ​​each characteristic peak of this product and the reference S peak when the flow rate changed slightly were examined, and the results were analyzed. The table below shows the results.

[0211] Table 25 Relative retention time results at different flow rates

[0212]

[0213] Table 26 Relative peak area results at different flow rates

[0214]

[0215] The relative retention time and relative peak area of ​​each characteristic peak and the reference S peak were investigated when the flow rate changed slightly. The results showed that the RSD% of the relative retention time was less than 8%, and the RSD% of the relative peak area of ​​each peak was large, indicating that the flow rate had a slight effect on the relative retention time of each peak and a greater effect on the peak area. Therefore, the flow rate was preferably 0.7 ml / min.

[0216] The flow rate was tested using the same method using freeze-dried powder of the standard decoction of Lygodium japonicum as the test sample. The results showed that the RSD value of the relative retention time of the 11 common peaks and the reference peak (peak S) was less than or equal to 8.0%. The results showed that different flow rates had a certain effect on the relative retention time of the characteristic peaks of peaks 2, 10, and 11. When the flow rate changed slightly, the retention time of each characteristic chromatographic peak changed slightly. Therefore, the flow rate is preferably 0.7 ml / min.

[0217] (3) Investigation of different column temperatures

[0218] The herb of Lycopodiella cava was used as the test sample, and the test solution was prepared and detected by high performance liquid chromatography according to Example 1. The column temperature was set to 19°C, 20°C, and 22°C, respectively, and the sample was injected and measured. The chromatogram was recorded, and the relative retention time and relative peak area of ​​the characteristic peaks were analyzed. The results are shown in the table below.

[0219] Table 27 Relative retention time results at different column temperatures

[0220]

[0221] Table 28 Relative peak area results at different column temperatures

[0222]

[0223] Result analysis: The relative retention time and relative peak area of ​​each characteristic peak and the reference S peak were investigated when the column temperature changed. The results showed that the RSD% of the relative retention time was less than 4%, indicating good durability at different column temperatures.

[0224] Taking all factors into consideration, the column temperature of this method is preferably 20°C.

[0225] The column temperature was tested using the same method using the freeze-dried powder of the standard decoction of Lycopodiella ciliata as the test sample. The results showed that the relative retention time of 11 common peaks was less than 2%, indicating that slight changes in column temperature had little effect on the relative retention time of characteristic peaks, and the durability under different column temperatures was good.

[0226] (4) Investigation of different chromatographic columns

[0227] Following the chromatographic conditions determined in the method study, the effects of different batches of chromatographic columns on the characteristic spectrum of Lygodium japonicum were investigated. Samples were prepared and tested by HPLC as in Example 1. Chromatographic analysis was performed, and the relative retention times and peak areas of the characteristic peaks were analyzed. The results are shown in the table below.

[0228] Table 29 Relative retention time results of different batches of chromatographic columns

[0229]

[0230] Table 30 Relative peak area results of different batches of chromatographic columns

[0231]

[0232] The results of the investigation on different chromatographic column batches showed that the relative retention time RSD% was less than 2%, and the relative peak area RSD% was less than 2%, indicating that different chromatographic column batches had little effect on the relative retention time of the characteristic spectrum.

[0233] (5) Investigation of different liquid phases

[0234] The same sample solution of Lycopodiella herb prepared by the method of Example 1 was taken and measured on Agilent, Thermo Fisher, and Waters chromatographs according to the determination method of Example 1. The chromatograms were recorded, and the relative retention times and relative peak areas of the characteristic peaks were analyzed. The results are shown in the table below.

[0235] Table 31 Relative retention time results of different liquid phases

[0236]

[0237]

[0238] Table 32 Results of relative peak areas of different liquid phases

[0239]

[0240] The relative retention times of the characteristic peaks and the reference S peak were examined during the liquid phase changes. The RSD% of the relative retention times was less than 4%. The results showed that different brands of liquid chromatographs (Agilent, Thermo Fisher, and Waters) had little effect on the relative retention times of the characteristic spectra, indicating that the method is robust across different brands of liquid chromatographs.

[0241] The influence of different instruments was investigated using the same method using freeze-dried powder of standard decoction of Lycopodiella ciliata as the test sample. The results showed that the relative retention time of 11 common peaks was less than 4%, indicating that the instrument had little effect on the relative retention time and relative peak area of ​​the characteristic peaks, and the durability under different instruments was good.

[0242] 5. Exclusivity

[0243] The test sample was extracted with methanol as the extraction solvent. 10 μL of the test solution and negative control solution (70% methanol) prepared in Example 1 were accurately drawn and injected into the liquid chromatograph respectively. The test was carried out according to the method of Example 1. Figure 20 and 21 As shown, the result is negative and there is no interference.

[0244] According to the results of the above methodological investigation, among the 11 characteristic peaks of the characteristic spectrum of the herbal medicine or standard decoction freeze-dried powder of Herba Lycopodii, each chromatographic peak is slightly affected by different liquid phase instruments, chromatographic columns, column temperatures and flow rates, but the relative retention time values ​​are in the range of -10% to 10%. Therefore, it is recommended to control the specified value range within ±10%.

[0245] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic spectrum of Lygodium japonicum and its preparation, characterized in that: The following steps are included: (1) Preparation of test solution, including the following steps: 1) dissolving the test sample in water to obtain a solution, and extracting with water-saturated n-butanol; 2) drying the n-butanol solution, and then extracting with an extraction solvent to obtain an extract; 3) separating the extract into a solid-liquid state, and obtaining the liquid to obtain the test sample solution; the extraction solvent is selected from a 30-90% by volume methanol-water solution or an ethanol-water solution; (2) The test sample solution and the reference sample solution were respectively subjected to high performance liquid chromatography to obtain a characteristic spectrum of the test sample. The chromatographic conditions included: acetonitrile as mobile phase A and formic acid solution as mobile phase B, and the volume percentage of the formic acid solution was 0.05-0.2%; Perform gradient elution as specified in the table below: The detection wavelength was 250-330 nm, and a CAPCELL CORE C18 column with a column length of 150 mm, an inner diameter of 4.6 mm, and a particle size of 2.7 μm was used as the chromatographic column; Protocatechuic acid, caffeic acid, 4-coumarin, isoquercetin, kaempferol-3-O-rutinoside and montanol were used as reference substances and solvents were added to prepare reference substance solutions.

2. The construction method according to claim 1, characterized in that If the elution time is <13-15min, the detection wavelength is 250-260nm; if the elution time is ≥13-15min, the detection wavelength is 310-330nm.

3. The construction method according to claim 2, characterized in that Step (1) satisfies any one or more of the following AEs: A. In step 1), the number of water-saturated n-butanol extractions is 1 to 3 times, and the ratio of the mass of the test sample to the volume of water and the volume of water-saturated n-butanol used each time is 0.1-0.6:10-20:20; the mass-to-volume ratio is g / mL; B. In step 2), the mass-to-volume ratio of the test sample to the extraction solvent is 0.1-0.6:20; the mass-to-volume ratio is g / mL; C. In step 2), the extraction method is ultrasonic extraction or reflux extraction, and the extraction time is 30 min-60 min; D. In step 3), the solid-liquid separation is independently selected from centrifugation or filtration.

4. The construction method according to claim 3, characterized in that The extraction solvent is a methanol aqueous solution with a volume percentage of 70%.

5. The construction method according to claim 1, characterized in that The flow rate is 0.5-0.9 ml / min; and / or the column temperature is 19-30°C.

6. The construction method according to claim 1, characterized in that The solvent used in the preparation of the reference solution is selected from methanol or a methanol aqueous solution with a volume percentage of more than 70%.

7. The construction method according to claim 1, characterized in that Each 1 mL of reference solution contains 15-100 μg of each reference substance.

8. The construction method according to any one of claims 1 to 7, characterized in that: The construction method also includes extracting a control medicinal material of Lygodium japonicum with water, filtering the obtained filtrate, and extracting it with water-saturated n-butanol; drying the n-butanol solution, adding an extraction solvent, and extracting to obtain an extract; separating the extract into a solid and liquid, and taking the liquid to obtain a control medicinal material reference solution; and detecting the control medicinal material reference solution by high performance liquid chromatography according to the construction method described in any one of claims 1 to 7 to obtain a control medicinal material reference atlas.

9. The construction method according to any one of claims 1 to 7, characterized in that: The characteristic spectrum of the seaweed and its preparation has 11 characteristic peaks, the peak corresponding to the caffeic acid reference peak is the S peak, peak 4 is the S peak, the relative retention times of peaks 1 to 3 and peaks 5 to 11 and the S peak are within ±10% of the specified values, and the specified values ​​of peaks 1 to 3 and peaks 5 to 11 are: 0.48, 0.77, 0.94, 1.06, 1.45, 1.93, 2.17, 2.32, 2.52, and 3.16, respectively.

10. The construction method according to any one of claims 1 to 7, characterized in that: The characteristic spectrum of the Lygodium japonicum and its preparation has 11 characteristic peaks, peak 1 is protocatechuic acid, peak 4 is caffeic acid, peak 6 is 4-coumaric acid, peak 7 is isoquercetin, peak 8 is kaempferol-3-O-rutinoside, and peak 11 is montanside.

11. Use of the method for constructing a characteristic spectrum of Lygodium japonicum and its preparations according to any one of claims 1 to 10 in quality inspection of Lygodium japonicum and its preparations.

12. A quality detection method for Lygodium japonicum and its preparation products, characterized in that: The method comprises the steps of obtaining a characteristic spectrum of the product to be tested according to the construction method described in any one of claims 1 to 10.

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