Method for constructing characteristic spectrum of Lygodium japonicum medicinal material

Abstract

The present invention discloses a method for constructing a characteristic spectrum of a Lygodium japonicum medicinal material, comprising: preparing a reference solution, the reference solution comprising a control medicinal material solution and a reference substance solution, the reference substance solution comprising protocatechuic acid, protocatechuic aldehyde, caffeic acid, p-coumaric acid, and vanillic acid; preparing a test solution from the Lygodium japonicum medicinal material; and performing high-performance liquid chromatography analysis on the reference solution and the test solution to establish a characteristic spectrum; wherein the conditions for the high-performance liquid chromatography analysis include: using an octadecylsilane bonded silica gel chromatography column, acetonitrile as mobile phase A, and phosphoric acid solution as mobile phase B. The present invention establishes characteristic spectrums of Lygodium japonicum medicinal materials from different origins, achieves simultaneous characterization of multiple components in the medicinal materials, and identifies multiple characteristic components, providing a reference basis for the formulation of quality control methods for Lygodium japonicum medicinal materials.

Description

Technical Field

[0001] The present invention relates to the technical field of quality analysis and detection of traditional Chinese medicines, and in particular to a method for constructing a characteristic spectrum of a Lygodium japonicum medicinal material. Background Art

[0002] The 2020 edition of the Chinese Pharmacopoeia states that this product is the dried mature spores of Lygodium japonicum (Thunb.) Sw., a plant of the Lygodium family. Lygodium japonicum is primarily found in Sichuan, Jiangxi, Hubei, and Anhui provinces in my country. According to the pharmacopoeia, Lygodium japonicum has a sweet, salty, and cold flavor, and enters the bladder and small intestine meridians. It has the effects of clearing heat and detoxifying, promoting diuresis, and relieving stranguria.

[0003] Lygodium japonicum is rich in phenolic acids, including caffeic acid and vanillic acid. It is primarily used to treat respiratory infections, mumps, and urinary tract infections. Furthermore, relevant research suggests that Lygodium japonicum can also be used as a choleretic, antioxidant, and to treat skin eczema. Overall, these studies indicate that Lygodium japonicum has considerable medicinal value. However, Lygodium japonicum is widely distributed and its quality varies, hindering its clinical application. Therefore, quality control of Lygodium japonicum is necessary, but current research in this area is limited. Ensuring the safety of Traditional Chinese Medicine (TCM) is a key step in its international expansion and a fundamental element in maintaining its core competitiveness. Quality control is a key component of TCM safety, and effective quality control methods are its fundamental guarantee.

[0004] Yuan Xiaolin et al. used a microscope to distinguish Lygodium japonicum from its adulterant, stir-fried pollen; Zhao Penghui et al. determined whether Lygodium japonicum was contaminated with soil by analyzing its ash content; Zhang Daoying et al. used FT-IR technology to distinguish genuine Lygodium japonicum from counterfeit Lygodium japonicum by comparing infrared absorption spectra; Zhang Daoying et al. also studied trace elements in Lygodium japonicum by combining microwave digestion with flame atomic absorption spectrometry; and Ma Wenna et al. conducted quality control studies on Fufang Shilintong Capsules using thin-layer chromatography, content determination, and fingerprint analysis. In summary, current quality control research on Lygodium japonicum primarily focuses on authenticity identification and quality control of traditional Chinese medicines containing Lygodium japonicum. Authenticity identification plays a role in the quality control of traditional Chinese medicines, but the quality of traditional Chinese medicines is primarily related to the chemical composition of the medicinal material. Chemical composition research is an important reference for quality control, and currently, little research has been conducted on the quality of Lygodium japonicum medicinal materials. The quality control research of compound prescriptions can only study the mixed components of decoctions of multiple medicinal materials, and cannot reflect the quality problems of single medicinal materials. The quality assurance of single medicinal materials can ensure the safety and effectiveness of compound prescriptions. Therefore, the quality control research of single medicinal materials is very necessary.

[0005] Traditional quality control methods struggle to comprehensively evaluate traditional Chinese medicines. However, characteristic profile methods can simultaneously reflect multiple chemical components within a traditional Chinese medicine, more comprehensively addressing the complex multi-component system of traditional Chinese medicine. Consequently, they have been widely used and are currently applied to the quality control of various traditional Chinese medicines. Lycopodiella chinensis, a spore-derived medicinal material, has chemical components that are difficult to dissolve, making its characteristic profile study challenging. This study developed a new method that effectively addresses this difficult-to-dissolve component problem in Lycopodiella chinensis. The method also allows for the study of multiple components, providing a better reference for quality control of Lycopodiella chinensis. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for constructing a characteristic spectrum of the medicinal material Lygodium japonicum. The characteristic spectrum of the medicinal material constructed by the method fully demonstrates the chemical composition characteristics of the medicinal material Lygodium japonicum. The method is stable, highly precise and has good reproducibility.

[0007] In order to solve the above technical problems, the present invention provides a method for constructing a characteristic spectrum of the medicinal material Lygodium japonicum, comprising the following steps:

[0008] preparing a reference solution, the reference solution comprising a control medicinal material solution and a reference substance solution, the reference substance solution comprising protocatechuic acid, protocatechuic aldehyde, caffeic acid, p-coumaric acid, and vanillic acid;

[0009] The test solution was prepared from the medicinal material of Lycopodiella ciliata;

[0010] Take the reference solution and the test solution separately for HPLC analysis to establish characteristic spectra;

[0011] The conditions for the high performance liquid chromatography analysis include: using an octadecylsilane bonded silica gel chromatography column, acetonitrile as mobile phase A, and phosphoric acid solution as mobile phase B.

[0012] In one embodiment, the HPLC analysis is performed using the following gradient elution procedure:

[0013] 0min-5min, mobile phase A is 2%-3%, mobile phase B is 98%-97%;

[0014] 5min-15min, mobile phase A is 3%-5%, mobile phase B is 97%-95%;

[0015] 15-20 min, mobile phase A is 5%-12%, mobile phase B is 95%-88%;

[0016] 20-30 min, mobile phase A is 12%, mobile phase B is 88%;

[0017] 30-35 min, mobile phase A is 12%-20%, mobile phase B is 88%-80%;

[0018] 35-45 min, mobile phase A is 20%-25%, mobile phase B is 80%-75%;

[0019] 45min-50min, mobile phase A is 25%, mobile phase B is 75%.

[0020] In one embodiment, the volume concentration of the phosphoric acid solution is 1%-3%.

[0021] In one embodiment, the conditions for the HPLC analysis further include: an injection volume of 0.5 μL-1.5 μL;

[0022] A 100 mm × 2.1 mm, 1.7 μm Waters BEH-C18 column was used;

[0023] Column temperature is 24°C-26°C;

[0024] The flow rate of the mobile phase is 0.2ml / min-0.4ml / min;

[0025] The UV detection wavelength is 250nm-270nm.

[0026] In one embodiment, the test solution is prepared by the following method:

[0027] Take 4.5g-5.5g of the medicinal material of Lycopodiella, add 45ml-55ml of water, boil for 25min-35min, cool, centrifuge, take the supernatant and evaporate to dryness, add 20ml-30ml of methanol to dissolve the residue, ultrasonicate for 25min-35min, cool, centrifuge, take the supernatant and evaporate to dryness, add 20ml-30ml of water, extract with ethyl acetate 2-4 times, each time using 20ml-30ml of ethyl acetate, combine the ethyl acetate solutions, evaporate to dryness, add methanol to dissolve the residue, adjust the volume, filter, and take the filtrate to obtain the product.

[0028] In one embodiment, the control medicinal material solution is prepared by the following method:

[0029] Take 4.5g-5.5g of the control medicinal material of Lycopodiella, add 45ml-55ml of water, boil for 25min-35min, cool, centrifuge, take the supernatant and evaporate to dryness, add 20ml-30ml of methanol to dissolve the residue, ultrasonicate for 25min-35min, cool, centrifuge, take the supernatant and evaporate to dryness, add 20ml-30ml of water, extract with ethyl acetate 2-4 times, each time using 20ml-30ml of ethyl acetate, combine the ethyl acetate solutions, evaporate to dryness, add methanol to dissolve the residue, adjust the volume, filter, and take the filtrate to obtain the product.

[0030] In one embodiment, the reference solution is prepared by the following method:

[0031] Take appropriate amounts of protocatechuic acid, protocatechuic aldehyde, caffeic acid, p-coumaric acid, and vanillic acid reference substances, respectively, and add methanol to prepare a mixed solution containing 80 μg of each substance per 1 ml to obtain the reference substance solution.

[0032] In one embodiment, the characteristic spectrum includes 8 characteristic peaks, wherein peak 1 corresponds to protocatechuic acid, peak 2 corresponds to protocatechuic aldehyde, peak 4 corresponds to vanillic acid, peak 5 corresponds to caffeic acid, and peak 6 corresponds to p-coumaric acid.

[0033] In one embodiment, taking peak 5 corresponding to caffeic acid as reference peak S, the specified values ​​of the relative retention times of peaks 1, 2, 3, 4, 6, 7, and 8 relative to peak S are as follows:

[0034] Peak 1 was 0.326, peak 2 was 0.533, peak 3 was 0.578, peak 4 was 0.891, peak 6 was 1.271, peak 7 was 1.544, and peak 8 was 2.210, with RSD% <2.0%.

[0035] In one embodiment, taking peak 5 corresponding to caffeic acid as reference peak S, the specified values ​​of the relative peak areas of peaks 1, 2, 3, 4, 6, 7, 8 and S are as follows:

[0036] Peak 1 was 0.077, peak 2 was 0.075, peak 3 was 0.099, peak 4 was 0.080, peak 6 was 0.160, peak 7 was 0.140, and peak 8 was 0.052, with RSD% <5.0%.

[0037] The implementation of the present invention has the following beneficial effects:

[0038] The present invention addresses the technical issues of quality control for Lygodium japonicum medicinal materials and provides a method for establishing a characteristic spectrum for Lygodium japonicum medicinal materials. This method, based on the properties of Lygodium japonicum medicinal materials, rationally controls the mobile phase conditions of high-performance liquid chromatography to construct a characteristic spectrum method for Lygodium japonicum medicinal materials. This method provides a scientific experimental basis for the research of a quality evaluation system for Lygodium japonicum medicinal materials, can compensate for the defects of inaccurate identification caused by the lack of appearance and microscopic characteristics of raw materials, reduce the adverse effects of counterfeit and adulterated products on the traditional Chinese medicine market, and ensure that the public has access to high-quality Lygodium japonicum medicinal materials. The present invention makes the method simple and efficient, and the results are objective, precise, and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the reference characteristic spectrum of Lygodium japonicum medicinal materials;

[0040] Figure 2Characteristic maps of 18 batches of Lygodium japonicum medicinal materials;

[0041] Figure 3 This is the total ion current and ultraviolet absorption chromatogram of the test solution of Lycopodiella chinensis medicinal material;

[0042] Figure 4 is the primary mass spectrometry scan of peak 1 (negative ion mode);

[0043] Figure 5 The secondary mass spectrometry scan of the peak m / z = 153.0182 (negative ion mode);

[0044] Figure 6 is the structural formula of protocatechuic acid;

[0045] Figure 7 is the primary mass spectrometry scan of peak 2 (negative ion mode);

[0046] Figure 8 The secondary mass spectrometry scan pattern of the peak m / z = 137.0232 (negative ion mode);

[0047] Figure 9 is the structural formula of protocatechuic aldehyde;

[0048] Figure 10 This is the primary mass spectrometry scan of peak 5 (negative ion mode);

[0049] Figure 11 The secondary mass spectrometry scan pattern of the peak m / z = 179.0340 (negative ion mode);

[0050] Figure 12 is the structural formula of caffeic acid;

[0051] Figure 13 This is the primary mass spectrometry scan of peak 6 (negative ion mode);

[0052] Figure 14 The secondary mass spectrometry scan of the peak m / z = 163.0390 (negative ion mode);

[0053] Figure 15 is the structural formula of p-coumaric acid;

[0054] Figure 16 Characteristic spectra for the control and test products;

[0055] Figure 17 3D comparison diagram of common peaks and reference substances;

[0056] Figure 18 Characteristic spectra of different wavelengths;

[0057] Figure 19 Characteristic spectra at different column temperatures;

[0058] Figure 20 Characteristic spectra of different chromatographic columns. DETAILED DESCRIPTION

[0059] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0060] Example 1

[0061] A method for establishing a characteristic spectrum of Lygodium japonicum medicinal material and its application

[0062] 1. Instruments and test drugs

[0063] 1.1 Instrument

[0064] Instruments: Thermo high performance liquid chromatograph (Thermo Vanquish), Waters high performance liquid chromatograph (e2695, Waters), Agilent high performance liquid chromatograph (1290, Agilent), Waters BEH C18 column (2.1 mm × 100 mm, 1.7 μm, BH-374), Waters Cortecs T3 column (2.1 mm × 100 mm, 1.6 μm, BH-185), Agilent SB A C18 chromatographic column (2.1 mm × 100 mm, 1.7 μm, BH-342) was used; a ten-thousandth analytical electronic balance (ME204E, Mettler-Toledo), a millionth analytical electronic balance (XP26, Mettler-Toledo), an electric constant-temperature water bath (HWS-28, Shanghai Yiheng Technology Co., Ltd.), a CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), and an ultrapure water system (Milli-Q Direct, Merck KGaA).

[0065] 1.2 Reagents

[0066] Reagents: Ethanol (Xilong Scientific Co., Ltd.) and methanol (Xilong Scientific Co., Ltd.) were of analytical grade; phosphoric acid was of chromatographic grade (Tianjin Komiou Chemical Reagent Co., Ltd.); acetonitrile (Merck Co., Ltd.) was used for liquid phase analysis; and ultrapure water (homemade in the laboratory) was used.

[0067] 1.3 Drug testing

[0068] The 18 batches of Lygodium japonicum medicinal materials were mainly sourced from Jiangxi, Hubei, Sichuan, and Anhui provinces, see Table 1, Lygodium japonicum reference medicinal materials (121607-201202).

[0069] Table 1 Origin information of Lycopodiella ciliata medicinal materials

[0070]

[0071]

[0072] Reference substances: protocatechuic aldehyde (batch number: 110810-201909, content: 99.6%, China Food and Drug Inspection Institute); protocatechuic acid (batch number: 110809-201906, content: 97.7%, China Food and Drug Inspection Institute); vanillic acid (batch number: 110776-201503, content: 99.8%, China Food and Drug Inspection Institute); caffeic acid (batch number: 110885-201703, content: 99.7%, China Food and Drug Inspection Institute); p-coumaric acid (batch number: 112037-201801, content: 99.3%, China Food and Drug Inspection Institute).

[0073] 2. Methods and Results

[0074] 2.1 Chromatographic conditions

[0075] Chromatographic column: Waters BEH-C18 (2.1×100 mm, 1.7 μm); mobile phase: acetonitrile as mobile phase A, 0.2% by volume phosphoric acid aqueous solution as mobile phase B, gradient elution according to the requirements in Table 2; column temperature: 25°C; flow rate: 0.3 ml / min; detection wavelength: 260 nm; injection volume: 1 μL.

[0076] Table 2 Gradient elution table

[0077]

[0078] 2.2 Preparation of test solution

[0079] Take an appropriate amount of this product, about 5.0g, place it in a stoppered conical flask, add 50ml of water, boil for 30 minutes, cool, centrifuge, take the supernatant and evaporate to dryness, add 25ml of methanol to dissolve the residue, treat with ultrasound (power 250W, frequency 45kHz) for 30 minutes, let cool, centrifuge, take the supernatant and evaporate to dryness, add 25ml of water, extract with ethyl acetate 3 times, 25ml each time, combine the ethyl acetate, evaporate to dryness, add methanol to dissolve the residue, make up to the volume in a 5ml volumetric flask, filter, and take the filtrate to obtain.

[0080] 2.3 Preparation of reference solution

[0081] Take an appropriate amount of reference medicinal material, about 5.0 g, place it in a stoppered conical flask, add 50 ml of water, boil for 30 minutes, cool, centrifuge, take the supernatant and evaporate to dryness, add 25 ml of methanol to dissolve the residue, treat with ultrasound (power 250 W, frequency 45 kHz) for 30 minutes, cool, centrifuge, take the supernatant and evaporate to dryness, add 25 ml of water, extract with ethyl acetate 3 times, 25 ml each time, combine the ethyl acetate liquid, evaporate to dryness, add methanol to dissolve the residue, make up to the volume in a 5 ml volumetric flask, filter, and take the filtrate to obtain the reference medicinal material solution.

[0082] Take appropriate amounts of protocatechuic acid, protocatechuic aldehyde, caffeic acid, p-coumaric acid, and vanillic acid reference substances, respectively, and add methanol to make a mixed solution containing 80 μg of each substance per 1 ml to obtain the reference substance solution.

[0083] 2.4 Assay

[0084] Accurately pipette 1 μL of the reference solution and the test solution respectively and inject them into the high performance liquid chromatograph.

[0085] 2.5 Common peak confirmation and similarity evaluation

[0086] The chromatograms of 18 batches of Lygodium japonicum were imported into the “Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition” and 8 common peaks were identified. Figure 1 ; 18 batches of medicinal materials have common peak identifications in their characteristic spectra. For specific results, see Figure 2 The specific results of the similarity of 18 batches of Lycopodiella medicinal materials are shown in Table 3.

[0087] Table 3 Similarity calculation results of 18 batches of Lygodium japonicum samples

[0088]

[0089] 3. Common peak identification

[0090] 3.1 High-resolution mass spectrometry identification

[0091] (1) Ultra-high performance liquid chromatography-mass spectrometry conditions

[0092] Liquid chromatography conditions: A Waters BEH C18 column (150 mm × 2.1 mm, 1.7 μm) was used; acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B, with gradient elution as specified in Table 2; the flow rate was 0.3 ml per minute; the column temperature was 25°C; the injection volume was 1 μl; and the detection wavelength was 260 nm.

[0093] Mass spectrometry conditions:

[0094] Table 4 Mass spectrometry parameters

[0095]

[0096] (2) Preparation of test solution

[0097] Take an appropriate amount of Lycopodiella splendens medicinal material, about 5.0 g, put it in a stoppered conical flask, add 50 ml of water, boil for 30 minutes, cool, centrifuge, take the supernatant and evaporate to dryness, add 25 ml of methanol to dissolve the residue, treat with ultrasound (power 250 W, frequency 45 kHz) for 30 minutes, let cool, centrifuge, take the supernatant and evaporate to dryness, add 25 ml of water, extract with ethyl acetate 3 times, 25 ml each time, combine the ethyl acetate, evaporate to dryness, add methanol to dissolve the residue, make up to the volume in a 5 ml volumetric flask, filter, and take the filtrate to obtain the test solution.

[0098] (3) Sample measurement

[0099] Accurately draw 1 μl of the test solution and inject it into the liquid chromatography-mass spectrometer. Use the above liquid chromatography conditions and mass spectrometry conditions to detect the test solution. The total ion current and ultraviolet absorption chromatogram of the test solution are shown in Figure 3 .

[0100] (4) Result analysis

[0101] Through mass spectrometry accurate molecular weight, fragment ion comparison analysis, and matching with the Thermo Fisher Scientific mzVault standard database, four components were confirmed in the characteristic spectrum of Lycopodiella chinensis: protocatechuic acid (peak 1), protocatechuic aldehyde (peak 2), caffeic acid (peak 5), and p-coumaric acid (peak 6). The compound information is shown in Table 5, and the specific analysis process is shown in Figures 4 to 15 .

[0102] Table 5 Mass spectrometry identification results of compounds in Lycopodiella

[0103]

[0104] ① Peak 1

[0105] Extract the primary chromatogram of peak 1, the time period is 5.93min-5.98min. Figure 4 .

[0106] In the primary chromatographic extraction diagram of peak 1, the response value of the peak m / z = 153.024 in negative ion mode is [MH] - Peak; from its precise molecular weight, it is speculated that its molecular formula may be C7H6O4; extract the secondary map of the peak m / z = 153.024 with a collision energy of 60, and the results show that the main ion fragments are 153.02, 109.03, etc. Detailed results are shown in Figure 5 .

[0107] The precise molecular weight and secondary ion fragment information of the signal peak were matched with the compound spectra in the local mass spectrometry database. The results showed that the compound with the highest peak matching degree of m / z = 153.02 was protocatechuic acid. Based on the fragment ion comparison results and the fragmentation inference analysis of the structural formula, it was speculated that the compound was protocatechuic acid. The structural formula of protocatechuic acid is shown in Figure 6 .

[0108] ② Peak 2

[0109] Extract the primary chromatogram of peak 2, the time period is 9.18min-9.23min. Figure 7 .

[0110] In the primary chromatographic extraction diagram of peak 2, the response value of the peak m / z = 137.02 in negative ion mode is [MH] - Peak; from its precise molecular weight, it is speculated that its molecular formula may be C7H6O3; extract the secondary graph of the peak m / z = 137.024 with a collision energy of 60, and the results show that the main ion fragments are 137.02, 109.03, etc. Detailed results are shown in Figure 8 .

[0111] The precise molecular weight and secondary ion fragment information of the signal peak were matched with the compound spectra in the local mass spectrometry database. The results showed that the compound with the highest peak match of m / z = 137.02 was protocatechuic aldehyde. Based on the fragment ion comparison results and the fragmentation inference analysis of the structural formula, it was speculated that the compound was protocatechuic aldehyde. The structural formula of protocatechuic aldehyde is shown in Figure 9 .

[0112] ③ Peak 5

[0113] Extract the primary chromatogram of peak 5, the time period is 17.15min-17.23min. Figure 10 .

[0114] In the primary chromatographic extraction diagram of peak 5, the response value of the peak m / z = 179.03 in negative ion mode is [MH] - Peak; from its precise molecular weight, it is speculated that its molecular formula may be C7H6O3; extract the secondary graph of the peak m / z = 179.03 with a collision energy of 60, and the results show that the main ion fragments are 179.03, 135.04, etc. Detailed results are shown in Figure 11 .

[0115] The precise molecular weight and secondary ion fragment information of the signal peak were matched with the compound spectra in the local mass spectrometry database. The results showed that the compound with the highest peak match of m / z = 179.03 was caffeic acid. Based on the fragment ion comparison results and the fragmentation inference analysis of the structural formula, it was speculated that the compound was caffeic acid. The structural formula of caffeic acid is shown in Figure 12 .

[0116] ④ Peak 6

[0117] Extract the primary chromatogram of peak 6, the time period is 22.17min-17.25min. Figure 13 .

[0118] In the primary chromatographic extraction diagram of peak 6, the response value of the peak m / z = 163.04 in negative ion mode is [MH] - Peak; from its precise molecular weight, it is speculated that its molecular formula may be C7H6O3; extract the secondary graph of the peak m / z = 163.04 with a collision energy of 60, and the results show that the main ion fragments are 163.04, 119.05, etc. Detailed results are shown in Figure 14 .

[0119] The precise molecular weight and secondary ion fragment information of the signal peak were matched with the compound spectra in the local mass spectrometry database. The results showed that the compound with the highest peak matching degree of m / z = 163.039 was p-coumaric acid. Based on the fragment ion comparison results and the fragmentation inference analysis of the structural formula, it was speculated that the compound was p-coumaric acid. The p-coumaric acid structural formula is shown in Figure 15 .

[0120] 3.2 Identification of reference substances

[0121] Protocatechuic acid, protocatechuic aldehyde, caffeic acid, p-coumaric acid, and vanillic acid were used as reference substances for positioning. Figure 16 The results showed that peak 1 was protocatechuic acid, peak 2 was protocatechuic aldehyde, peak 4 was vanillic acid, peak 5 was caffeic acid, and peak 6 was p-coumaric acid. Figure 17 .

[0122] 4. Methodological Investigation

[0123] 4.1 Precision test

[0124] One sample of Lycopodiella quinata (Batch No. YG1902003) was prepared according to the method in "2.2 Test Solution Preparation." Accurately pipette 1 μL of the same test solution and inject it six times. The sample was measured according to the method in "2.1 Chromatographic Conditions." The chromatogram was recorded, and the relative retention time and peak area of ​​each characteristic peak were calculated using the caffeic acid reference peak as the reference peak. The results are shown in Tables 6 and 7. The precision test results showed that the RSD values ​​of the relative retention times and peak areas of each characteristic peak were all less than 2.0%, indicating good instrument precision.

[0125] Table 6 Precision test - relative retention time ratio

[0126] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Precision -1 0.326 0.533 0.578 0.890 1.000 1.271 1.544 2.210 Precision-2 0.326 0.532 0.577 0.890 1.000 1.272 1.544 2.211 Precision-3 0.326 0.533 0.578 0.890 1.000 1.272 1.544 2.211 Precision -4 0.326 0.532 0.577 0.890 1.000 1.271 1.544 2.210 Precision -5 0.326 0.532 0.577 0.891 1.000 1.273 1.545 2.213 Precision -6 0.326 0.532 0.577 0.891 1.000 1.273 1.545 2.214 RSD% 0.000 0.097 0.089 0.058 0.000 0.070 0.033 0.074

[0127] Table 7 Precision test - relative peak area ratio

[0128] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Precision -1 0.076 0.075 0.099 0.080 1.000 0.160 0.140 0.051 Precision-2 0.077 0.074 0.099 0.080 1.000 0.162 0.140 0.052 Precision-3 0.076 0.075 0.099 0.080 1.000 0.160 0.140 0.052 Precision -4 0.076 0.074 0.099 0.080 1.000 0.160 0.139 0.052 Precision -5 0.077 0.075 0.099 0.080 1.000 0.161 0.139 0.052 Precision -6 0.077 0.074 0.099 0.080 1.000 0.160 0.139 0.052 RSD% 0.233 0.611 0.125 0.138 0.000 0.555 0.247 0.224

[0129] 4.2 Repeatability test

[0130] Six samples of Lycopodiella cinerea (Batch No. 20161102) were prepared according to the method described in "2.2 Test Solution Preparation." Accurately pipette 5 μL of the test solution into each sample and measure the sample according to the method described in "2.1 Chromatographic Conditions." Chromatograms were recorded, and the relative retention times and peak areas of each characteristic peak were calculated using the caffeic acid reference peak as the reference peak. The results are shown in Tables 8 and 9. Repeatability test results showed that the RSD values ​​of the relative retention times of the characteristic peaks ranged from 0.000 to 1.448%, and the RSD values ​​of the relative peak areas ranged from 0.000 to 4.992%, both less than 5%, indicating good repeatability of the method.

[0131] Table 8 Repeatability test - characteristic peak relative retention time ratio

[0132] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Repeatability 1 0.327 0.537 0.583 0.898 1 1.249 1.541 2.212 Repeatability 2 0.339 0.535 0.581 0.893 1 1.243 1.533 2.2 Repeatability 3 0.328 0.538 0.582 0.896 1 1.248 1.541 2.212 Repeatability 4 0.335 0.536 0.583 0.895 1 1.246 1.536 2.205 Repeatability 5 0.333 0.537 0.58 0.894 1 1.245 1.536 2.206 Repeatability 6 0.328 0.536 0.583 0.897 1 1.25 1.541 2.213 RSD 1.448 0.195 0.217 0.209 0 0.212 0.225 0.234

[0133] Table 9 Repeatability test - relative peak area ratio

[0134] Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Repeatability 1 0.216 0.052 0.219 0.131 1.000 0.263 0.047 0.104 Repeatability 2 0.215 0.052 0.223 0.139 1.000 0.251 0.042 0.094 Repeatability 3 0.202 0.048 0.202 0.128 1.000 0.232 0.043 0.092 Repeatability 4 0.218 0.049 0.212 0.128 1.000 0.261 0.047 0.098 Repeatability 5 0.229 0.052 0.219 0.142 1.000 0.260 0.047 0.097 Repeatability 6 0.201 0.054 0.202 0.128 1.000 0.245 0.042 0.094 RSD 4.948 4.486 4.328 4.614 0.000 4.793 4.992 4.243

[0135] 4.3 Stability test

[0136] One sample of Lycopodiella cava (Batch No. 20161102) was prepared according to the method in "2.2 Test Solution Preparation." The sample was assayed according to the method in "2.1 Chromatographic Conditions" at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 21 h. The chromatograms were recorded, and the relative retention time and peak area of ​​each characteristic peak were calculated using the caffeic acid reference peak as the reference peak. The results are shown in Tables 10 and 11. The stability test results showed that the RSD values ​​of the relative retention times and peak areas of each characteristic peak ranged from 0.00 to 0.082%, indicating that the sample was stable within 21 hours.

[0137] Table 10 Stability test - relative retention time ratio

[0138]

[0139]

[0140] Table 11 Stability test - relative peak area ratio

[0141]

[0142] 4.4 Durability inspection

[0143] Wavelength investigation: The chromatographic peaks at the detection wavelengths of 210nm, 230nm, 260nm, 280nm, 300nm, and 330nm were compared. The chromatographic peaks at 260nm were more numerous, the separation between the peaks was better, the amount of information was large, and the baseline was stable. Therefore, the detection wavelength was determined to be 260nm. Figure 18 .

[0144] Column temperature investigation: 23, 25, and 27 were investigated, showing that the method has good durability under different column temperature conditions. Figure 19 .

[0145] Chromatographic column investigation: Different chromatographic columns were also investigated. The results showed that the method has good durability under different chromatographic column conditions. Figure 20 .

[0146] In summary, the present invention addresses the technical issues of quality control of Lygodium japonicum medicinal materials and provides a method for establishing a characteristic spectrum of Lygodium japonicum medicinal materials. This method, based on the properties of Lygodium japonicum medicinal materials, rationally controls the mobile phase conditions of high-performance liquid chromatography to construct a characteristic spectrum method for Lygodium japonicum medicinal materials, thereby providing a scientific experimental basis for the research of a quality evaluation system for Lygodium japonicum medicinal materials. This method can compensate for the defects of inaccurate identification caused by the lack of appearance and microscopic characteristics of raw materials, reduce the adverse effects of counterfeit and adulterated products on the traditional Chinese medicine market, and ensure that the public uses high-quality Lygodium japonicum medicinal materials. The present invention makes the method simple and efficient, the results objective, and the accuracy and reliability.

[0147] The above is a preferred embodiment of the invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic spectrum of the medicinal material Lygodium japonicum, characterized in that: The following steps are involved: preparing a reference solution, the reference solution comprising a control medicinal material solution and a reference substance solution, the reference substance solution comprising protocatechuic acid, protocatechuic aldehyde, caffeic acid, p-coumaric acid and vanillic acid; The test solution was prepared from the medicinal material of Lycopodiella ciliata; Take the reference solution and the test solution for liquid chromatography analysis respectively to establish the characteristic spectrum; The liquid chromatography analysis conditions include: using a 100 mm × 2.1 mm, 1.7 μm Waters BEH-C18 column, acetonitrile as mobile phase A, phosphoric acid solution as mobile phase B; ultraviolet detection wavelength of 250 nm-270 nm; The liquid chromatography analysis was performed using the following gradient elution procedure: 0min-5min, mobile phase A is 2%~3%, mobile phase B is 98%~97%; 5min-15min, mobile phase A is 3%~5%, mobile phase B is 97%~95%; 15-20 min, mobile phase A is 5%-12%, mobile phase B is 95%-88%; 20-30 min, mobile phase A is 12%, mobile phase B is 88%; 30-35 min, mobile phase A is 12%-20%, mobile phase B is 88%-80%; 35-45 min, mobile phase A is 20%-25%, mobile phase B is 80%-75%; 45-50 min, mobile phase A: 25%, mobile phase B: 75%; The volume concentration of the phosphoric acid solution is 1%-3%; The test solution was prepared by the following method: Take 4.5g-5.5g of the medicinal material of Lycopodiella, add 45ml-55ml of water, boil for 25min-35min, cool, centrifuge, take the supernatant and evaporate to dryness, add 20ml-30ml of methanol to dissolve the residue, ultrasonicate for 25min-35min, cool, centrifuge, take the supernatant and evaporate to dryness, add 20ml-30ml of water, extract with ethyl acetate 2-4 times, each time using 20ml-30ml of ethyl acetate, combine the ethyl acetate solutions, evaporate to dryness, add methanol to dissolve the residue, adjust the volume, filter, and take the filtrate to obtain the product.

2. The method for constructing the characteristic spectrum of the medicinal material Lygodium japonicum according to claim 1, wherein: The conditions for the liquid chromatography analysis also include: an injection volume of 0.5 μL-1.5 μL; Column temperature is 24°C-26°C; The flow rate of the mobile phase is 0.2 ml / min-0.4 ml / min.

3. The method for constructing the characteristic spectrum of the medicinal material Lygodium japonicum according to claim 1, wherein: The control medicinal material solution was prepared by the following method: Take 4.5g-5.5g of the control medicinal material of Lycopodiella, add 45ml-55ml of water, boil for 25min-35min, cool, centrifuge, take the supernatant and evaporate to dryness, add 20ml-30ml of methanol to dissolve the residue, ultrasonicate for 25min-35min, cool, centrifuge, take the supernatant and evaporate to dryness, add 20ml-30ml of water, extract with ethyl acetate 2-4 times, each time using 20ml-30ml of ethyl acetate, combine the ethyl acetate solutions, evaporate to dryness, add methanol to dissolve the residue, adjust the volume, filter, and take the filtrate to obtain the product.

4. The method for constructing the characteristic spectrum of the medicinal material Lygodium japonicum according to claim 1, wherein: The reference solution was prepared by the following method: Take appropriate amounts of protocatechuic acid, protocatechuic aldehyde, caffeic acid, p-coumaric acid, and vanillic acid reference substances, respectively, and add methanol to prepare a mixed solution containing 80 μg of each substance per 1 ml to obtain the reference substance solution.

5. The method for constructing the characteristic spectrum of the medicinal material Lygodium japonicum according to claim 1, wherein: The characteristic spectrum includes 8 characteristic peaks, wherein peak 1 corresponds to protocatechuic acid, peak 2 corresponds to protocatechuic aldehyde, peak 4 corresponds to vanillic acid, peak 5 corresponds to caffeic acid, and peak 6 corresponds to p-coumaric acid.

6. The method for constructing the characteristic spectrum of the medicinal material Lygodium japonicum according to claim 5, characterized in that: Taking peak 5 corresponding to caffeic acid as the reference peak S, the specified values ​​of the relative retention times of peaks 1, 2, 3, 4, 6, 7, 8 and S are as follows: Peak 1 was 0.326, peak 2 was 0.533, peak 3 was 0.578, peak 4 was 0.891, peak 6 was 1.271, peak 7 was 1.544, and peak 8 was 2.210, with RSD% <2.0%.

7. The characteristic spectrum of the Lygodium japonicum medicinal material according to claim 5, characterized in that: Taking peak 5 corresponding to caffeic acid as the reference peak S, the specified values ​​of the relative peak areas of peaks 1, 2, 3, 4, 6, 7, 8 and S peak are as follows: Peak 1 was 0.077, peak 2 was 0.075, peak 3 was 0.099, peak 4 was 0.080, peak 6 was 0.160, peak 7 was 0.140, and peak 8 was 0.052, with RSD% <5.0%.

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