Method for constructing specific chromatogram of caulis clematidis armandii or derivative products thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method of a specific chromatogram of caulis clematidis armandii or derivative products thereof. The invention provides a construction method of a characteristic chromatogram of caulis clematidis armandii or a derivative product thereof, which comprises the following steps: by taking octadecylsilane chemically bonded silica as a filler and a mobile phase comprising a formic acid-containing aqueous solution and acetonitrile, carrying out a specific gradient elution procedure to obtain 22 common characteristic peaks, and realizing effective separation of the common characteristic peaks, so that the separation degree is good, and the accuracy is high. The obtained characteristic spectrum has more characteristic peaks and better baseline separation, and is easier to position and qualitatively analyze; the precision, the stability and the repeatability are relatively good; compared with the prior art, the method has the advantages that the characteristic peaks are more, six characteristic components including the straight clematis seed B, the lariciresinol, the secoisolariciresinol, the lariciresinol-4-O-beta-D-glucoside, the caffeic acid and the ferulic acid are identified, and a basis is provided for quality detection and content determination of the caulis clematidis armandii or derivative products of the caulis clematidis armandii.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method of a characteristic spectrum of clematis armandi or a derivative product thereof. BACKGROUND

[0002] Clematis armandi is the dried stem of Clematis armandii Franch. or Clematis montana Buch.-Ham. of Ranunculaceae. Its clinical efficacy is diuresis, clearing heart and relieving restlessness, and promoting menstruation and lactation. It is used for treating stranguria, edema, restlessness and red urine, sore mouth and tongue, and dysmenorrhea and less lactation.

[0003] Through the study of the fingerprint spectrum of clematis armandi formula granules, the efficacy material basis and biological basis of the patient taking the decoction medicine can be more clearly known. On the one hand, through the quality control of the components of clematis armandi formula granules, the accuracy and consistency of the dose of the medicine can be ensured. On the other hand, the establishment of the clematis armandi formula granule fingerprint spectrum determination can be widely applied to production practice.

[0004] The prior art has few characteristic peaks for identifying clematis armandi, and the qualitative and quantitative analysis accuracy is poor. SUMMARY

[0005] Therefore, the first object of the present application is to provide a construction method of a characteristic spectrum of clematis armandi or a derivative product thereof. The method establishes the characteristic spectrum of the variety according to the characteristics of clematis armandi or its derivative product, has more characteristic peaks and high separation degree, obvious characteristics, better precision, stability and repeatability, and can comprehensively control the quality of clematis armandi or its derivative product.

[0006] To this end, the present application provides the following technical solutions.

[0007] The present application provides a construction method of a characteristic spectrum of clematis armandi or a derivative product thereof, which comprises detecting by using ultra-high performance liquid chromatography, and the chromatographic conditions comprise:

[0008] octadecylsilane-bonded silica gel as the filler, acetonitrile as the mobile phase A, and a water solution containing formic acid as the mobile phase B, and the gradient elution program comprises:

[0009] 0→10min, the volume percentage of the mobile phase A is 4%→9%, and the volume percentage of the mobile phase B is 96%→91%;

[0010] 10min→20min, the volume percentage of the mobile phase A is 9%, and the volume percentage of the mobile phase B is 91%;

[0011] 20 min→ 45 min, volume percentage of mobile phase A: 9%→ 26%, volume percentage of mobile phase B: 91%→ 74%.

[0012] In an alternative embodiment, the chromatographic conditions comprise at least one of the following:

[0013] (1) detection wavelength is 240-280 nm; alternatively, detection wavelength is 280 nm;

[0014] (2) flow rate is 0.29-0.31 ml / min; alternatively, flow rate is 0.3 ml / min;

[0015] (3) column temperature is 28-32℃; alternatively, column temperature is 30℃;

[0016] (4) injection volume is 1-5 μL; alternatively, injection volume is 2 μL;

[0017] (5) concentration of formic acid in mobile phase B is 0.18-0.22% v / v; alternatively, concentration of formic acid is 0.2% v / v.

[0018] In an alternative embodiment, the method further comprises a step of preparing the test sample solution, comprising: weighing the test sample, extracting with solvent, separating solid and liquid, and taking the liquid, which is the test sample solution.

[0019] In an alternative embodiment, the preparation of the test sample solution satisfies at least one of the following:

[0020] A. the ratio of the mass of the test sample of Caulis Lonicerae Sichuanensis to the volume of the solvent is (0.2-1.0):(10-50); the unit of the mass of the test sample is g, and the unit of the volume of the solvent is mL;

[0021] B. the extraction method is ultrasonic extraction;

[0022] C. the extraction time is ≥ 30 min, preferably 30 min;

[0023] D. the solid-liquid separation is selected from centrifugation or filtration;

[0024] E. the solvent is selected from one or more of methanol, ethanol, and water; preferably, the solvent is 50-70% methanol aqueous solution.

[0025] In an alternative embodiment, the method further comprises a step of preparing a reference solution of at least one of straight Tsukushin B, Larici Resinol, Open-ring Isolarici Resinol, Larici Resinol-4-O-β-D-glucoside, Caffeic Acid, and Ferulic Acid with solvent, and a step of detecting the reference solution according to the method to obtain a reference chromatogram of the reference solution.

[0026] Preferably, the concentration of the direct iron lily ning B reference substance in the direct iron lily ning B reference substance solution is 5-50 μg / ml; preferably 50 μg;

[0027] Preferably, the concentration of the larch resin alcohol reference substance in the larch resin alcohol reference substance solution is 5-50 μg / ml; preferably 50 μg;

[0028] Preferably, the concentration of the open ring isomer of larch resin alcohol reference substance in the open ring isomer of larch resin alcohol reference substance solution is 5-50 μg / ml; preferably 50 μg;

[0029] Preferably, the concentration of the larch resin alcohol-4-O-β-D-glucoside reference substance in the larch resin alcohol-4-O-β-D-glucoside reference substance solution is 5-50 μg / ml; preferably 50 μg;

[0030] Preferably, the concentration of the caffeic acid reference substance in the caffeic acid reference substance solution is 5-50 μg / ml; preferably 50 μg;

[0031] Preferably, the concentration of the ferulic acid reference substance in the ferulic acid reference substance solution is 5-50 μg / ml; preferably 50 μg;

[0032] Preferably, the solvent used in the preparation of the reference substance solution is selected from methanol or a methanol aqueous solution with a volume fraction of not less than 50-70%; more preferably, the solvent used in the preparation of the reference substance solution is selected from a 50% methanol aqueous solution;

[0033] Preferably, the construction method further comprises the step of preparing a reference medicinal material solution using the Cynanchum thesioides reference medicinal material, and the step of obtaining a reference medicinal material reference map by detecting the reference medicinal material solution according to the ultra-high performance liquid chromatography in the construction method.

[0034] In an alternative embodiment, the Cynanchum thesioides or its derivative products include one or more of Cynanchum thesioides medicinal materials, Cynanchum thesioides decoction pieces, or Cynanchum thesioides preparations.

[0035] In an alternative embodiment, the Cynanchum thesioides preparation includes at least one of Cynanchum thesioides standard decoction, Cynanchum thesioides dispensing granules; preferably, the Cynanchum thesioides standard decoction includes Cynanchum thesioides standard decoction concentrated extract, Cynanchum thesioides standard decoction freeze-dried powder, Cynanchum thesioides standard decoction water decoction.

[0036] The preparation method of the Aristolochia debilis granules of the present invention includes: taking Aristolochia debilis, heating and refluxing for extraction at least once, adding 6 to 12 times the weight of water for extraction for at least 0.5 hours each time, filtering, combining the filtrates, concentrating the filtrate to a relative density of 1.05-1.10 g / mL at 60°C, and preparing a clinically acceptable formulation according to conventional processes in the art;

[0037] The types of the formulations include tablets, capsules, pills, granules, controlled-release formulations, oral liquid formulations, or injectable formulations; the pills include honey-processed pills;

[0038] The excipients of a pharmaceutical preparation include fillers, disintegrants, lubricants, suspending agents, binders, flavoring agents, preservatives, and matrix.

[0039] The filler includes at least one of starch, pregelatinized starch, lactose, mannitol, microcrystalline cellulose, and sucrose; the disintegrant includes at least one of starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, and croscarmellose sodium; the lubricant includes at least one of magnesium stearate, talc, and silica; the suspending agent includes at least one of polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, and hydroxypropyl methylcellulose; the binder includes... The flavoring agent comprises at least one of starch paste, polyvinylpyrrolidone, and hydroxypropyl methylcellulose; the flavoring agent comprises at least one of sweetener and flavoring; the flavoring can be selected conventionally in the art; the sweetener comprises at least one of sodium saccharin, aspartame, sucrose, cyclamate, and glycyrrhetinic acid; the preservative comprises at least one of parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorhexidine acetate, and eucalyptus oil; the matrix comprises at least one of PEG6000, PEG4000, and insect wax.

[0040] This invention also provides a method for determining the content of active ingredients in Aristolochia debilis or its derivatives, comprising:

[0041] Take the test solution and the reference solution, and detect them by ultra-high performance liquid chromatography in the above-mentioned method for constructing characteristic chromatograms of Aristolochia debilis or its derivatives;

[0042] The reference standards include at least one of the following: thiamethoxam B, larch resin alcohol, open-ring isolararch resin alcohol, larch resin alcohol-4-O-β-D-glucoside, caffeic acid, and ferulic acid.

[0043] The present invention also provides a quality testing method for Aristolochia debilis or its derivative products, including the step of comparing the feature spectrum of the product to be tested with the feature spectrum of Aristolochia debilis or its derivative products;

[0044] The feature map of the product under test is obtained according to the above construction method;

[0045] The characteristic spectrum of Aristolochia debilis or its derivative products is selected from any one of the following (1)-(4):

[0046] (1) It has 22 common characteristic peaks. The peak corresponding to the reference peak of the thiamethoxam B standard is the S peak. The relative retention times of peaks 1-11 and 13-22 with the S peak are within ±10% of the specified values. The specified values ​​of peak 1-11 are: 0.29, 0.33, 0.37, 0.50, 0.53, 0.65, 0.67, 0.70, 0.80, 0.92, 0.96. The specified values ​​of peak 13-22 are: 1.08, 1.16, 1.38, 1.59, 1.72, 1.82, 1.89, 2.09, 2.14, 2.25.

[0047] (2) It has 22 common characteristic peaks, of which 6 peaks correspond to the retention times of reference peaks of caffeic acid, clematisin B, ferulic acid, larch resin alcohol-4-O-β-D-glucoside, open-ring isolararch resin alcohol, and larch resin alcohol reference standards, respectively. The peak corresponding to the reference peak of clematisin B reference standard is the S peak. The relative retention times of peaks 1-11 and 13-22 with the S peak are within ±10% of the specified values. The specified values ​​of peaks 1-11 are: 0.29, 0.33, 0.37, 0.50, 0.53, 0.65, 0.67, 0.70, 0.80, 0.92, and 0.96, respectively. The specified values ​​of peaks 13-22 are: 1.08, 1.16, 1.38, 1.59, 1.72, 1.82, 1.89, 2.09, 2.14, and 2.25, respectively.

[0048] (3) Characteristic chromatograms of Aristolochia chuanxiong and / or its derivatives obtained by using single or multiple batches of Aristolochia chuanxiong and / or its derivatives as test samples according to the above construction method;

[0049] (4) Using multiple batches of Aristolochia debilis and / or its derivatives as test samples, the characteristic spectra obtained according to the above construction method are used to construct control characteristic spectra by means of average or median.

[0050] In one alternative embodiment, peak 5 corresponds to caffeic acid; peak 12(S) corresponds to clematin B; peak 13 corresponds to ferulic acid; peak 16 corresponds to larch resin alcohol-4-O-β-D-glucoside; peak 20 corresponds to open-ring isolararch resin alcohol; and peak 21 corresponds to larch resin alcohol.

[0051] The technical solution of this invention has the following advantages:

[0052] 1. The method for constructing characteristic spectra of Aristolochia debilis or its derivatives provided by the present invention uses octadecylsilane-bonded silica gel as a packing material, and the mobile phase includes an aqueous solution containing formic acid and acetonitrile. Through a specific gradient elution program, 22 common characteristic peaks are obtained, and the common characteristic peaks are effectively separated with good separation. Moreover, the obtained characteristic spectra have more characteristic peaks and better baseline separation, making them easier to locate and qualitatively analyze. Furthermore, the precision, stability, and repeatability are good.

[0053] Compared with existing technologies, it has more characteristic peaks and identifies six characteristic components: straight iron clematisin B, larch resin alcohol, open-ring isolararch resin alcohol, larch resin alcohol-4-O-β-D-glucoside, caffeic acid, and ferulic acid, providing a basis for the quality detection and content determination of Aristolochia debilis or its derivatives.

[0054] 2. The quality testing method for Akebia trifoliata or its derivative products provided by this invention selects the S-peak straight ferroline B as the internal reference peak in the fingerprint spectrum, and identifies 22 common characteristic peaks in the fingerprint spectrum of Akebia trifoliata or its derivative products. The relative retention time of each common characteristic peak is calculated based on the S-peak straight ferroline B, which is beneficial for comprehensive quality testing and overall quality control of Akebia trifoliata or its derivative products, thereby helping to improve the safety and stability of Akebia trifoliata or its derivative products in use. Attached Figure Description

[0055] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0056] Figure 1 The characteristic spectrum of Aristolochia debilis formula granules; specifically, Figure 1 -(1) is the test sample; Figure 1 -(2) is a mixed reference standard; Figure 1 -(3) is the reference herb for Aristolochia debilis;

[0057] Figure 2 The UPLC chromatogram for gradient condition 1 in Experimental Example 1;

[0058] Figure 3 The UPLC chromatogram for gradient condition 2 in Experimental Example 1;

[0059] Figure 4 The UPLC chromatogram for gradient condition 3 in Experimental Example 1;

[0060] Figure 5The UPLC chromatogram for the flow rate of 0.29 ml / min in Experiment Example 1 is shown.

[0061] Figure 6 The UPLC chromatogram for the flow rate investigation in Experiment Example 1 is shown at a flow rate of 0.30 ml / min.

[0062] Figure 7 The UPLC chromatogram for the flow rate of 0.31 ml / min in Experiment Example 1 is shown.

[0063] Figure 8 The UPLC chromatogram for the column temperature investigation in Experiment Example 1 is shown at 28℃.

[0064] Figure 9 The UPLC chromatogram for the column temperature investigation in Experiment Example 1 is the column temperature at 30℃.

[0065] Figure 10 The UPLC chromatogram for the column temperature investigation in Experiment Example 1 is the column temperature at 32℃.

[0066] Figure 11 The UPLC chromatogram for the mobile phase in Experimental Example 1, where the mobile phase was acetonitrile-0.2% v / v phosphoric acid aqueous solution;

[0067] Figure 12 The UPLC chromatogram for the mobile phase in Experimental Example 1, which was acetonitrile-0.2% v / v formic acid aqueous solution, is shown.

[0068] Figure 13 The UPLC chromatogram for the mobile phase in Experimental Example 1, which was acetonitrile-0.2% v / v acetic acid aqueous solution, is shown.

[0069] Figure 14 The UPLC chromatogram for the detection wavelength of 230 nm is shown in Experiment Example 1.

[0070] Figure 15 The UPLC chromatogram for the detection wavelength of 240 nm is shown in Experiment Example 1.

[0071] Figure 16 The UPLC chromatogram for the detection wavelength of 250 nm is shown in Experiment Example 1.

[0072] Figure 17 The UPLC chromatogram for the detection wavelength of 260 nm in Experiment Example 1 is shown.

[0073] Figure 18 The UPLC chromatogram for the detection wavelength of 270 nm is shown in Experiment Example 1.

[0074] Figure 19 The UPLC chromatogram for the detection wavelength of 280 nm is shown in Experiment Example 1.

[0075] Figure 20 The UPLC chromatogram for the detection wavelength of 300 nm is shown in Experiment Example 1.

[0076] Figure 21 The UPLC chromatogram for the detection wavelength of 320 nm is shown in Experiment Example 1.

[0077] Figure 22 The UPLC chromatogram of column 1 in Experimental Example 1;

[0078] Figure 23 The UPLC chromatogram of column 2 in Experimental Example 1;

[0079] Figure 24 The UPLC chromatogram of column 3 in Experimental Example 1;

[0080] Figure 25 The characteristic spectrum of the Akebia trifoliata control material in Experiment Example 3;

[0081] Figure 26 The characteristic spectrum is the control spectrum in Experiment Example 3; among which, peak 5: caffeic acid; peak 12: straight iron-clematin B; peak 13: ferulic acid; peak 16: larch resin alcohol-4-O-β-D-glucoside; peak 20: open-ring isolararch resin alcohol; peak 21: larch resin alcohol;

[0082] Figure 27 This is a superimposed comparison of the characteristic chromatograms of 18 batches of Akebia trifoliata granules and the control chromatogram in Experimental Example 3; where S1: 1803001C; S2: 1803003C; S3: 1905002C; S4: 1907001C; S5: 1910001C; S6: 2006002W; S7: 2006003W; S8: 2011001C; S9 S10: 2108001W; S11: 2203001C; S12: 2203002C; S13: 2203003C; S14: 2310004W; S15: 2310005W; S16: 2404001C; S17: 2404002C; S18: 2404003C; R: Reference characteristic spectrum;

[0083] Figure 28The localization chromatogram of the reference standard in Experimental Example 3 is shown below; where S1: 2404001C; S2: caffeic acid; S3: straight iron-clematin B; S4: ferulic acid; S5: larch resin alcohol-4-O-β-D-glucoside; S6: open-ring isolararch resin alcohol; S7: larch resin alcohol;

[0084] Figure 29 This is the chromatogram of the negative blank solution in Experiment Example 4;

[0085] Figure 30 The UPLC chromatogram of chromatograph 1 in Experiment Example 4;

[0086] Figure 31 The image shows the UPLC chromatogram of chromatograph 2 in Experiment Example 4. Detailed Implementation

[0087] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0088] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0089] The test sample used in the following examples and experimental cases was Aristolochia debilis granules. The specific preparation method of Aristolochia debilis granules is as follows: Aristolochia debilis was taken and extracted twice by heating and reflux. For the first extraction, 10 times the weight of water was added and soaked for 30 minutes, and then heated and refluxed for 0.5 hours. After filtration, 8 times the weight of water was added and extracted for 0.5 hours. After filtration, the filtrates were combined and concentrated at 60°C to a relative density of 1.05-1.10 g / mL. Aristolochia debilis granules were then prepared by the formulation process.

[0090] The main instruments and reagents involved in this invention are as follows:

[0091] 1. Instruments:

[0092] Chromatograph 1: Waters Hclass chromatography system, including Quaternary Solvent Manager quaternary pump, FTN-H autosampler, PDA diode array detector, and Empower chromatography management system;

[0093] Chromatograph 2: Thermo Fisher Vanquish chromatography system, including VF-P20-A quaternary pump, VF-A10-A autosampler, VF-D11-A DAD detector, and Chromeleon chromatography workstation;

[0094] Chromatographic columns: Thermo Fisher Hypersil GOLD 120C18 (column length 100 mm, inner diameter 2.1 mm, particle size 1.9 μm); Waters ACQUITY UPLC HSS T3 (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); Shimadzu Shim-pack GIST_HP C18-Aq (column length 100 mm, inner diameter 2.1 mm, particle size 1.9 μm).

[0095] Ultrasonic instrument: KQ-500DE (Kunshan Ultrasonic Instrument Co., Ltd.)

[0096] High-power numerically controlled ultrasonic instrument (KQ-400KDB, Kunshan Ultrasonic Instrument Co., Ltd.);

[0097] One ten-thousandth electronic balance (Shimadz μAY120, Shimadzu Corporation, Japan);

[0098] One ten-thousandth electronic balance (Sartoriμs SQP SECMRA225D-1CN, Sartorius Scientific Instruments (Beijing) Co., Ltd.);

[0099] Digital display constant temperature water bath (HH-S6, Jiangsu Jinyi Instrument Technology Co., Ltd.)

[0100] 2. Reagents and reagents:

[0101] Methanol and acetonitrile were chromatographic grade (Merck); phosphoric acid (chromatographic grade, 85-90%, Aladdin); glacial acetic acid (HPLC, >99.9%, Aladdin); formic acid (chromatographic grade, ≥98%, Aladdin); water (Milli-Q); and other reagents were analytical grade.

[0102] Straight iron-ladenine B reference standard (Jiangxi Baicaoyuan Biotechnology Co., Ltd., batch number: 003767-202405); Larch resin alcohol reference standard (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 120319-202401); Secocyclic isolararch resin alcohol reference standard (Shanghai Hongyong Biotechnology Co., Ltd., batch number: 11084-202406); Larch resin alcohol-4-O-β-D-glucoside reference standard (Shanghai Stande Technology Co., Ltd., batch number: 143663-00-7); Caffeic acid reference standard (China National Institutes for Food and Drug Control, batch number: 110885-201703); Ferulic acid reference standard (China National Institutes for Food and Drug Control, batch number: 110773-202316);

[0103] Reference material for Akebia trifoliata (small Akebia trifoliata) (China National Institutes for Food and Drug Control, batch number: 121409-201402);

[0104] Aristolochia debilis (small Aristolochia) formula granules (batch numbers: 1803001C, 1803003C, 1905002C, 1907001C, 1910001C, 2006002W, 2006003W, 2011001C, 2108001W, 2108002W, 2203001C, 2203002C, 2203003C, 2310004W, 2310005W, 2404001C, 2404002C, 2404003C).

[0105] Example 1

[0106] This embodiment provides a method for constructing a feature map of Aristolochia debilis or its derivatives, including:

[0107] (1) Preparation of test solution: Using the Akebia quinata formula granules as the test sample, take about 0.2g of the test sample powder, accurately weigh it, place it in a stoppered conical flask, add 10ml of 50% v / v methanol aqueous solution, weigh it, sonicate (power 250W, frequency 40kHz) for 30 minutes, take it out, cool it, shake it well, filter it, and take the filtrate to obtain the solution.

[0108] Preparation of reference solution for reference medicinal material: Take 2g of Akebia trifoliata (small Akebia trifoliata) reference medicinal material, place it in a stoppered conical flask, add 25ml of water, heat under reflux for 1h, filter, evaporate the filtrate to dryness, add 5ml of 50% v / v methanol aqueous solution to the residue, sonicate (power 250W, frequency 40kHz) for 30 minutes, filter, and take the filtrate to obtain the reference solution;

[0109] Preparation of reference solutions: Accurately weigh appropriate amounts of clematisin B, larch resin alcohol, ferulic acid, larch resin alcohol-4-O-β-D-glucoside, caffeic acid, and open-ring isolararch resin alcohol reference standards, and add 50% v / v methanol aqueous solution to prepare solutions containing 50 μg per ml, thus obtaining the following reference solutions: clematisin B reference solution, larch resin alcohol reference solution, ferulic acid reference solution, larch resin alcohol-4-O-β-D-glucoside reference solution, caffeic acid reference solution, and open-ring isolararch resin alcohol reference solution.

[0110] (2) Take 2 μL each of the test solution, reference material solution, and reference standard solution and determine them by ultra-high performance liquid chromatography (UHPLC). The chromatographic conditions are as follows: use octadecylsilane-bonded silica gel as the packing material (Thermo Fisher Hypersil GOLD 120C18 column with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.9 μm); use acetonitrile as mobile phase A and 0.2% (v / v) formic acid solution as mobile phase B, and perform gradient elution according to the specifications in the table below; the flow rate is 0.3 ml per minute; the column temperature is 30℃; and the detection wavelength is 280 nm. The theoretical plate number, calculated based on the B peak of cinnamyl ether, should not be less than 5000.

[0111] Table 1 Gradient Elution Table

[0112]

[0113]

[0114] The results are shown in Table 2 and Figure 1 As shown, by Figure 1 -(1) to Figure 1-(3) It can be seen that the characteristic chromatogram of the *Aristolochia debilis* formula granules corresponds to the characteristic chromatogram of the reference medicinal material. The characteristic chromatogram of the *Aristolochia debilis* formula granules has 22 characteristic peaks, all of which are effectively separated. Each characteristic peak has a good peak shape, stable baseline, short detection time, and uniform peak height or area. Peaks 5, 12, 13, 16, 20, and 21 correspond to the retention times of the caffeic acid reference peak, the *Aristolochia debilis* B reference peak, the ferulic acid reference peak, the larch resin alcohol-4-O-β-D-glucoside reference peak, the open-ring iso-larch resin alcohol reference peak, and the larch resin alcohol reference peak, respectively. The peak corresponding to the retention time of the *Aristolochia debilis* B reference peak is S. Calculate the relative retention times of peaks 1-11 and 13-22 with peak S. The relative retention times should be within ±10% of the specified values, which are: 0.29 (peak 1), 0.33 (peak 2), 0.37 (peak 3), 0.50 (peak 4), 0.53 (peak 5), 0.65 (peak 6), 0.67 (peak 7), 0.70 (peak 8), 0.80 (peak 9), 0.92 (peak 10), 0.96 (peak 11), 1.08 (peak 13), 1.16 (peak 14), 1.38 (peak 15), 1.59 (peak 16), 1.72 (peak 17), 1.82 (peak 18), 1.89 (peak 19), 2.09 (peak 20), 2.14 (peak 21), and 2.25 (peak 22). Among them, peak 5 corresponds to caffeic acid; peak 12(S) corresponds to clematin B; peak 13 corresponds to ferulic acid; peak 16 corresponds to larch resin alcohol-4-O-β-D-glucoside; peak 20 corresponds to open-ring isolararch resin alcohol; and peak 21 corresponds to larch resin alcohol.

[0115] Table 2 System Adaptability Results

[0116]

[0117] Experimental Example 1

[0118] I. Investigation of chromatographic conditions

[0119] 1. Selection of mobile phase gradient

[0120] Take the same sample solution prepared according to Example 1, and inject 2 μL; use octadecylsilane-bonded silica gel as the packing material (Thermo Fisher Hypersil column, length 100 mm, inner diameter 2.1 mm, particle size 1.9 μm); use acetonitrile as mobile phase A and 0.2% (v / v) formic acid solution as mobile phase B, and perform gradient elution according to the specifications in Table 3; the flow rate is 0.3 mL / min; the column temperature is 30 °C; the detection wavelength is 240 nm. The results are shown in Table 3. Figure 2 .

[0121] Table 3 Gradient Condition 1

[0122] Time (min) Mobile phase A (% v / v) Mobile phase B (% v / v) 0～60 2→35 98→65

[0123] Depend on Figure 2 It can be seen that gradient elution under gradient condition 1 resulted in poor peak resolution and limited chromatographic information. Therefore, the mobile phase ratio was further optimized, and gradient elution was performed according to the specifications in Table 4. The results are shown in [Table 4]. Figure 3 ;

[0124] Table 4 Gradient Condition 2

[0125] Time (min) Mobile phase A (% v / v) Mobile phase B (% v / v) 0～10 12 88 10～40 12→15 88→85 40～60 15→40 85→60

[0126] Depend on Figure 3 It can be seen that elution using gradient condition 2 resulted in poor peak separation and limited chromatographic information. Further optimization was performed using gradient elution as specified in Table 5, and the results are shown below. Figure 4 ;

[0127] Table 5 Gradient Condition 3

[0128] Time (min) Mobile phase A (% v / v) Mobile phase B (% v / v) 0～10 96→91 0～10 10～20 91 10～20 20～45 91→74 20～45

[0129] Depend on Figure 4 It can be seen that using gradient condition 3 for elution improves the resolution of each chromatographic peak, resulting in better separation and richer chromatographic information. Therefore, gradient condition 3 is tentatively set as the elution gradient, and the flow rate, column temperature, and detection wavelength will be further investigated.

[0130] 2. Examination of flow velocity

[0131] Except for the flow rate, under the chromatographic conditions determined according to the selection of the mobile phase gradient in section 1, with the flow rate as the variable, the same sample solution prepared according to Example 1 was measured at flow rates of 0.29 ml / min, 0.30 ml / min, and 0.31 ml / min, respectively. The resulting chromatograms are shown below. Figures 5-7 As shown. By Figures 5-7 It is known that changes in elution flow rate have a certain impact on the information content of chromatographic peaks and system adaptability parameters. Therefore, the flow rate is tentatively set at 0.30 ml / min for subsequent condition screening.

[0132] 3. Examination of column temperature

[0133] Except for column temperature, the chromatographic conditions determined according to section 2 (flow rate investigation) were used, with column temperature as the variable. The same sample solution prepared according to Example 1 was measured at column temperatures of 28°C, 30°C, and 32°C, respectively. The resulting chromatograms are shown below. Figures 8-10 As shown. By Figures 8-10It is known that changes in column temperature have a certain impact on the information content of chromatographic peaks and system adaptability parameters. Therefore, the column temperature is tentatively set at 30℃ for subsequent condition screening and investigation.

[0134] 4. Investigation of the mobile phase

[0135] Except for the mobile phase, under the chromatographic conditions determined according to section 3 (column temperature), with the mobile phase as the variable, the same sample solution prepared according to Example 1 was tested under different mobile phases (acetonitrile-0.2% v / v phosphoric acid aqueous solution, acetonitrile-0.2% v / v formic acid aqueous solution, and acetonitrile-0.2% v / v acetic acid aqueous solution). The resulting chromatograms are shown below. Figures 11-13 As shown. By Figures 11-13 It can be seen that different mobile phases have a significant impact on the characteristic chromatograms. When phosphoric acid or acetic acid is used as the mobile phase, it has a certain impact on the information content of the chromatographic peaks and the system adaptability parameters. When 0.2% formic acid aqueous solution is used as the mobile phase, the chromatographic peak resolution and peak shape are better. Therefore, the mobile phase is tentatively set as acetonitrile-0.2% formic acid aqueous solution for subsequent condition screening and investigation.

[0136] 5. Examination of the detection wavelength

[0137] In addition to the detection wavelength, under the chromatographic conditions determined according to section 4 (mobile phase investigation), and with the detection wavelength as a variable, the same sample solution prepared according to Example 1 was measured at different detection wavelengths of 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 300 nm, and 320 nm. The resulting chromatograms are shown below. Figures 14-21 As shown. By Figures 14-21 It can be seen that different detection wavelengths have a significant impact on the chromatographic peak response. At wavelengths of 240nm-280nm, the characteristic peaks of the characteristic spectrum respond well, and there are differences in the peak response size, but no peaks are missing. Therefore, the detection wavelength is tentatively set at 280nm for subsequent condition screening and investigation.

[0138] 6. Investigation of different brands of chromatographic columns

[0139] Based on the chromatographic conditions determined in section 5 regarding the detection wavelength, and using different brands of chromatographic columns as variables, the same sample solution prepared according to Example 1 was analyzed using different chromatographic columns (Column 1: Thermo Fisher Hypersil GOLD120C18; Column 2: Waters ACQUITY UPLC HSS T3; Column 3: Shimadzu Shim-pack GIST_HP C18-Aq). The resulting chromatograms are shown below. Figures 22-24 As shown. By Figures 22-24It is known that different chromatographic columns have a certain impact on the information content of chromatographic peaks and system adaptability parameters; therefore, Thermo Fisher Hypersil GOLD 120C18 column is tentatively selected for subsequent condition screening and investigation.

[0140] 7. Determination of optimal chromatographic conditions

[0141] An octadecylsilane-bonded silica gel column (Thermo Fisher Hypersil GOLD 120C18, 100 mm long, 2.1 mm inner diameter, 1.9 μm particle size) was used as the packing material. Acetonitrile was used as mobile phase A, and 0.2% (v / v) formic acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in Table 6. The flow rate was 0.3 mL / min; the column temperature was 30 °C; and the detection wavelength was 280 nm. The injection volume was 2 μL.

[0142] Table 6 Gradient Elution Table

[0143] Time (min) Mobile phase A (% v / v) Mobile phase B (% v / v) 0～10 4→9 96→91 10～20 9 91 20～45 9→26 91→74

[0144] Example 2: Construction of characteristic chromatograms of 18 batches of Akebia trifoliata granules

[0145] Eighteen batches of Akebia trifoliata granule formulation samples were taken as test samples (1803001C, 1803003C, 1905002C, 1907001C, 1910001C, 2006002W, 2006003W, 2011001C, 2108001W, 2108002W, 2203001C, 2203002C, 2203003C, 2310004W, 2310005W, 2404001C, 2404002C, 2404003C), and test sample solutions were prepared according to the method in Example 1.

[0146] The above solution was analyzed using the ultra-high performance liquid chromatography method described in Example 1, and characteristic chromatograms of 18 batches of Aristolochia debilis formulation granules were obtained. The results are shown in Table 7. The relative retention times of all 18 batches of Aristolochia debilis formulation granules were within ±10% of the specified values, which meets the requirements.

[0147] Table 7. Relative retention time results of 18 batches of Akebia trifoliata (small Akebia trifoliata) formulation granules

[0148]

[0149]

[0150]

[0151] Example 3: Establishment of control feature maps

[0152] (1) Generation of comparative feature maps

[0153] Eighteen batches of *Akebia trifoliata* (Chuanmutong) formula granules were taken as test samples (1803001C, 1803003C, 1905002C, 1907001C, 1910001C, 2006002W, 2006003W, 2011001C, 2108001W, 2108002W, 2203001C, 2203002C, 2203003C, 2310004W, 2310005W, 2404001C, 2404002C, 2404003C). Test sample solutions were prepared according to the method in Example 1, and the results are shown in Example 2. *Akebia trifoliata* reference material was taken and a reference solution of *Akebia trifoliata* reference material was prepared according to the method in Example 1.

[0154] The above solution was analyzed using the ultra-high performance liquid chromatography method described in Example 1. The results are shown in Table 8. Figure 25 The results showed that the characteristic chromatograms of 18 batches of *Aristolochia debilis* formula granules contained 22 characteristic peaks, corresponding to the retention times of the 22 characteristic peaks in the chromatograms of the reference medicinal material. The characteristic chromatograms of the 18 batches of *Aristolochia debilis* formula granules were fitted using the fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission to generate reference characteristic chromatograms. Through UPLC identification of the characteristic peaks, 22 common characteristic peaks with suitable response values, good separation, and high purity were obtained. The chromatographic peak numbers were rearranged according to the order of the chromatographic peaks as peak 1-peak 22. Figure 26 As shown.

[0155] The characteristic chromatograms of 18 batches of Aristolochia debilis formula granules were superimposed and compared with the control characteristic chromatograms, such as... Figure 27 As shown; the fingerprint spectrum similarity results are shown in Table 9; as shown in Table 9, the similarity is >0.9; among them, S1: 1803001C; S2: 1803003C; S3: 1905002C; S4: 1907001C; S5: 1910001C; S6: 2006002W; S7: 2006003W; S8: 2011001C; S9: 2 108001W; S10: 2108002W; S11: 2203001C; S12: 2203002C; S13: 2203003C; S14: 2310004W; S15: 2310005W; S16: 2404001C; S17: 2404002C; S18: 2404003C; R: Reference characteristic spectrum.

[0156] Table 8 Results of relative retention time determination of Aristolochia debilis (a reference herb).

[0157]

[0158] Table 9. Similarity results of fingerprint spectra of granular characteristics of Aristolochia debilis from 18 batches.

[0159] Batch number Similarity Batch number Similarity R 1 R 1 S1 0.99 S10 0.90 S2 0.99 S11 0.92 S3 0.99 S12 0.95 S4 0.99 S13 0.97 S5 0.99 S14 0.90 S6 0.99 S15 0.96 S7 0.99 S16 0.96 S8 0.99 S17 0.96 S9 0.99 S18 0.95

[0160] (3) Identify characteristic peaks in the characteristic spectrum

[0161] Reference solutions of clematisin B, larch resin alcohol, seco-isolararch resin alcohol, larch resin alcohol-4-O-β-D-glucoside, caffeic acid, and ferulic acid were prepared according to the method in Example 1. The above solutions were analyzed using the ultra-high performance liquid chromatography method of Example 1 to obtain the reference chromatograms of the reference standards. The chromatograms were then compared with the characteristic chromatograms of the reference standards, and the results are shown in [Figure 1]. Figure 28 Ten characteristic peaks were located and identified using reference standards. The retention times of peaks 5, 12, 13, 16, 20, and 21 corresponded to the retention times of reference peaks of caffeic acid, clematin B, ferulic acid, larch resin alcohol-4-O-β-D-glucoside, open-ring isolararch resin alcohol, and larch resin alcohol, respectively. Peak 5 was identified as caffeic acid, peak 12 as clematin B, peak 13 as ferulic acid, peak 16 as larch resin alcohol-4-O-β-D-glucoside, peak 20 as open-ring isolararch resin alcohol, and peak 21 as larch resin alcohol.

[0162] In summary, the chromatogram of the test sample should show 22 characteristic peaks, and the retention times should correspond to the 22 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 5, 12, 13, 16, 20, and 21 should correspond to the retention times of the corresponding reference peaks. Using the peak corresponding to the retention time of the reference standard thiazoline B as peak S, the relative retention times of peaks 1-11 and 13-22 with peak S were calculated. The relative retention times were within ±10% of the specified values, which were: 0.29 (peak 1), 0.33 (peak 2), 0.37 (peak 3), 0.50 (peak 4), 0.53 (peak 5), 0.65 (peak 6), 0.67 (peak 7), 0.70 (peak 8), 0.80 (peak 9), 0.92 (peak 10), 0.96 (peak 11), 1.08 (peak 13), 1.16 (peak 14), 1.38 (peak 15), 1.59 (peak 16), 1.72 (peak 17), 1.82 (peak 18), 1.89 (peak 19), 2.09 (peak 20), 2.14 (peak 21), and 2.25 (peak 22); similarity > 0.9. Among them, peak 5 corresponds to caffeic acid; peak 12(S) corresponds to clematin B; peak 13 corresponds to ferulic acid; peak 16 corresponds to larch resin alcohol-4-O-β-D-glucoside; peak 20 corresponds to open-ring isolararch resin alcohol; and peak 21 corresponds to larch resin alcohol.

[0163] Experiment Example 4: Methodological Validation

[0164] 1. Precision

[0165] The same sample solution of Aristolochia debilis granules (2404001C) prepared according to the method of Example 1 was injected 6 times repeatedly, the chromatograms were recorded, and the relative retention times of 22 characteristic peaks were determined. The results are shown in Table 10. The RSD of the relative retention times of the 22 characteristic peaks were all less than 2%, indicating good precision.

[0166] Table 10 Relative retention time results from precision tests

[0167]

[0168]

[0169] 2. Repeatability test

[0170] The same sample of Akebia quinata granules was taken and repeated 6 times according to the method in Example 1. The chromatographic analysis was performed under the chromatographic conditions described in Example 1, and the chromatograms were recorded. The results are shown in Table 11. The RSD of the relative retention times of the 10 characteristic peaks was less than 2%, indicating good repeatability.

[0171] Table 11 Results of Relative Retention Time in Repeatability Tests

[0172]

[0173]

[0174] 3. Intermediate precision (for different operators)

[0175] Three inspectors, at different times, used the same equipment to determine the relative retention times of the same batch of Akebia trifoliata granules according to the test solution preparation method and chromatographic conditions described in Example 1. The results are shown in Table 12. The RSD of the relative retention times of the 22 characteristic peaks were all less than 2%, indicating that the method has good intermediate precision.

[0176] Table 12 Intermediate Precision (Personnel) Relative Retention Time

[0177]

[0178]

[0179] 4. Stability test

[0180] Take the same sample solution prepared according to Example 1, and analyze it at 0, 2, 4, 8, 12 and 24 h according to the chromatographic conditions described in Example 1. Record the chromatogram, determine the relative retention time of 22 characteristic peaks and analyze them. The results are shown in Table 13. The RSD of the relative retention time of the 22 characteristic peaks is less than 2%, which indicates that the method has good stability and can meet the needs of the determination.

[0181] Table 13 Results of relative retention times in stability tests

[0182]

[0183]

[0184] 5. Exclusivity

[0185] Preparation of negative blank solution: Take the negative granules prepared from the excipients in the preparation of Aristolochia debilis granules, grind them finely, take about 0.2g, weigh accurately, place in a stoppered conical flask, add 10ml of 50% v / v methanol aqueous solution, weigh, sonicate (power 250W, frequency 40kHz) for 30 minutes, take out, cool, shake well, filter, and take the filtrate to obtain the negative blank solution.

[0186] Accurately pipette 2 μL of the test solution and 2 μL of the negative blank solution obtained in Example 1, and inject them separately into the ultra-high performance liquid chromatograph. Perform the test according to the chromatographic conditions of Example 1. Figure 29 and Figure 1 As shown, the results indicate that negative results do not cause interference.

[0187] 6. Durability test

[0188] 6.1 Investigation of different flow velocities

[0189] Take the same sample solution prepared according to Example 1, and measure it at flow rates of 0.29 ml / min, 0.30 ml / min and 0.31 ml / min respectively under the chromatographic conditions described in Example 1. Record the chromatograms, measure the relative retention times at different flow rates and analyze them. The results are shown in Table 14.

[0190] Table 14 Results of relative retention time for different flow velocities

[0191]

[0192]

[0193] Different flow rates have a certain impact on the information content of chromatographic peaks and system adaptability parameters. After comparing and analyzing the above results, the separation effect of each chromatographic peak is good and the system adaptability parameters are relatively similar under different flow rates. However, the relative retention time of each characteristic peak shows significant fluctuations. Considering that the characteristic spectrum specifies the relative retention time of each characteristic peak, the present invention selects a flow rate of 0.30 ml / min as the optimal value.

[0194] 6.2 Investigation at different column temperatures

[0195] Take the same sample solution prepared according to Example 1, and measure it at column temperatures of 28℃, 30℃ and 32℃ according to the chromatographic conditions described in Example 1. Record the chromatograms, measure the relative retention times at different column temperatures and analyze them. The results are shown in Table 15.

[0196] Table 15 Results of relative retention times at different column temperatures

[0197]

[0198] Different column temperatures have a certain impact on the information content of chromatographic peaks and system adaptability parameters. After comparing and analyzing the above results, the separation effect of each chromatographic peak is good and the system adaptability parameters are relatively similar under different column temperatures. However, the relative retention time of each characteristic peak shows significant fluctuations. Considering that the characteristic chromatogram specifies the relative retention time of each characteristic peak, this invention selects a column temperature of 30℃ as the optimal value.

[0199] 6.3 Investigation of different chromatographs

[0200] Take the same sample solution prepared according to Example 1, and perform chromatographic analysis under the chromatographic conditions described in Example 1 using different chromatograms (chromatogram 1: Waters Hclass chromatographic system; chromatogram 2: Thermo Dionex chromatographic system). Record the chromatograms, determine the relative retention times of different instruments, and analyze the results. The results are shown in Table 16 and... Figures 30-31 ;

[0201] Table 16 Relative Retention Time for Instrument Durability Assessment

[0202]

[0203]

[0204] After comparing and analyzing the above results, it was found that the reproducibility of each chromatographic peak was good for different chromatograms, the chromatographic information was relatively complete, and none of the characteristic peaks were missing, indicating that the method provided by the present invention has good robustness.

[0205] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a feature map of Aristolochia debilis or its derivatives, characterized in that, This includes detection using ultra-high performance liquid chromatography (UHPLC), with chromatographic conditions including: Using octadecylsilane-bonded silica gel as the packing material, acetonitrile as mobile phase A, and an aqueous solution containing formic acid as mobile phase B, the gradient elution program includes: From 0 to 10 min, the volume percentage of mobile phase A changed from 4% to 9%, and the volume percentage of mobile phase B changed from 96% to 91%. From 10 min to 20 min, the volume percentage of mobile phase A was 9%, and the volume percentage of mobile phase B was 91%. From 20 min to 45 min, the volume percentage of mobile phase A changed from 9% to 26%, and the volume percentage of mobile phase B changed from 91% to 74%.

2. The construction method according to claim 1, characterized in that, The chromatographic conditions include at least one of the following: (1) The detection wavelength is 240-280nm; optionally, the detection wavelength is 280nm. (2) The flow rate is 0.29-0.31 ml / min; optionally, the flow rate is 0.3 ml / min; (3) Column temperature 28-32℃; optional, column temperature is 30℃; (4) The injection volume is 1-5 μL; optionally, the injection volume is 2 μL. (5) The concentration of formic acid in the aqueous solution containing formic acid in mobile phase B is 0.18-0.22% v / v; optionally, the concentration of formic acid is 0.2% v / v.

3. The construction method according to claim 1 or 2, characterized in that, It also includes the preparation of the test sample solution, including: weighing the test sample, extracting with solvent, separating solid and liquid, and taking the liquid, which is the test sample solution.

4. The construction method according to claim 3, characterized in that, The preparation of the test solution satisfies at least one of the following: A. The ratio of the mass of the *Aristolochia debilis* sample to the volume of the solvent is (0.2-1.0):(10-50); the mass of the sample is in g, and the volume of the solvent is in mL. B. The extraction method is ultrasonic extraction; C. The extraction time is ≥30 min, preferably 30 min; D. The solid-liquid separation is selected from centrifugation or filtration; E. The solvent is selected from one or more of methanol, ethanol, and water; preferably, it is a methanol aqueous solution with a volume percentage of 50-70%.

5. The construction method according to any one of claims 1-4, characterized in that, The construction method further includes the steps of preparing a reference solution by adding at least one of the following: straight iron-clematisin B, larch resin alcohol, open-ring isolararch resin alcohol, larch resin alcohol-4-O-β-D-glucoside, caffeic acid, and ferulic acid to a solvent, and the step of obtaining a reference standard reference spectrum by detecting the reference solution according to the ultra-high performance liquid chromatography method in the construction method. Preferably, the concentration of clematisin B reference standard in the clematisin B reference solution is 5-50 μg / ml; preferably 50 μg. Preferably, the concentration of larch resin alcohol reference standard in the larch resin alcohol reference solution is 5-50 μg / ml; more preferably 50 μg. Preferably, the concentration of the open-ring isolaricol reference standard in the open-ring isolaricol reference solution is 5-50 μg / ml; preferably 50 μg. Preferably, the concentration of larch resin alcohol-4-O-β-D-glucoside reference standard in the larch resin alcohol-4-O-β-D-glucoside reference standard solution is 5-50 μg / ml; preferably 50 μg. Preferably, the concentration of caffeic acid reference standard in the caffeic acid reference solution is 5-50 μg / ml; more preferably 50 μg. Preferably, the concentration of ferulic acid reference standard in the ferulic acid reference solution is 5-50 μg / ml; more preferably 50 μg. Preferably, the solvent used in the preparation of the reference solution is selected from methanol or an aqueous methanol solution with a volume fraction of not less than 50-70%; more preferably, the solvent used in the preparation of the reference solution is selected from a 50% aqueous methanol solution. Preferably, the construction method further includes the step of preparing a reference herb solution using Aristolochia debilis as a reference herb, and the step of obtaining a reference herb reference spectrum by detecting the reference herb solution using ultra-high performance liquid chromatography in the construction method.

6. The construction method according to any one of claims 1-5, characterized in that, The *Aristolochia debilis* or its derivatives include one or more of the following: *Aristolochia debilis* medicinal material, *Aristolochia debilis* slices, or *Aristolochia debilis* preparations.

7. The construction method according to claim 6, characterized in that, The preparation of Aristolochia decoction includes at least one of Aristolochia decoction slices and Aristolochia granules; preferably, the Aristolochia decoction standard preparation includes concentrated extract of Aristolochia decoction standard preparation, freeze-dried powder of Aristolochia decoction standard preparation, and decoction of Aristolochia decoction standard preparation.

8. A method for determining the content of active ingredients in Aristolochia debilis or its derivatives, characterized in that, include: Take the test solution and the reference solution, and detect them respectively using the ultra-high performance liquid chromatography method in the method for constructing the characteristic spectrum of Aristolochia debilis or its derivatives as described in any one of claims 1-7; The reference standards include at least one of the following: thiamethoxam B, larch resin alcohol, open-ring isolararch resin alcohol, larch resin alcohol-4-O-β-D-glucoside, caffeic acid, and ferulic acid.

9. A method for quality testing of Aristolochia debilis or its derivatives, characterized in that, This includes the step of comparing the feature map of the product to be tested with the feature map of Aristolochia debilis or its derivatives; The feature map of the product under test is obtained by the construction method according to any one of claims 1-7; The characteristic spectrum of Aristolochia debilis or its derivative products is selected from any one of the following (1)-(4): (1) It has 22 common characteristic peaks. The peak corresponding to the reference peak of the thiamethoxam B standard is peak S. The relative retention times of peaks 1-11 and 13-22 with peak S are within ±10% of the specified values. The specified values ​​for peaks 1-11 are: 0.29, 0.33, 0.37, 0.50, 0.53, 0.65, 0.67, 0.70, 0.80, 0.92, and 0.96, respectively. The specified values ​​for peaks 13-22 are: 1.08、1.16、1.38、1.59、1.72、1.82、1.89、2.09、2.14、2.25； (2) It has 22 common characteristic peaks, of which 6 peaks correspond to the retention times of reference peaks of caffeic acid, clematisin B, ferulic acid, larch resin alcohol-4-O-β-D-glucoside, open-ring isolararch resin alcohol, and larch resin alcohol reference standards, respectively. The peak corresponding to the reference peak of clematisin B reference standard is peak S. The relative retention times of peaks 1-11 and 13-22 with peak S are within ±10% of the specified values. The specified values ​​for peaks 1-11 are: 0.29, 0.33, 0.37, 0.50, 0.53, 0.65, 0.67, 0.70, 0.80, 0.92, and 0.96, respectively. The specified values ​​for peaks 13-22 are: 1.08、1.16、1.38、1.59、1.72、1.82、1.89、2.09、2.14、2.25； (3) The characteristic chromatograms of Aristolochia chuanxiong and / or its derivatives obtained by the construction method according to any one of claims 1-7 using a single batch or multiple batches of Aristolochia chuanxiong and / or its derivatives as test samples; (4) Using multiple batches of Aristolochia debilis and / or its derivatives as test samples, the characteristic spectra obtained by the construction method according to any one of claims 1-7 are used to prepare a control characteristic spectra by means of average or median.

10. The quality testing method for Aristolochia debilis or its derivatives according to claim 9, characterized in that, Peak 5 corresponds to caffeic acid; peak 12(S) corresponds to clematin B; peak 13 corresponds to ferulic acid; peak 16 corresponds to larch resin alcohol-4-O-β-D-glucoside; peak 20 corresponds to open-ring isolararch resin alcohol; peak 21 corresponds to larch resin alcohol.

Images