Method for constructing characteristic spectrum and method for quality inspection of trichosanthis pericarpium pharmaceutical preparation

Abstract

The present invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a method for constructing a characteristic chromatogram and a quality detection method for a trichosanthes peel pharmaceutical preparation. The construction method comprises the following steps: (1) preparation of a test solution; (2) taking the test solution and detecting it by ultra-high performance liquid chromatography using an Agilent InfinityLab Poroshell 120SB-C18 chromatographic column, wherein the mobile phase comprises an aqueous solution containing acetic acid and acetonitrile, and the detection wavelength and gradient elution program are shown in the specification. Through the above method, 12 common characteristic peaks are obtained, and the peak positions of rutin, luteoloside, chrysoeriol-7-O-glucoside, luteolin and Arvenin III can also be accurately located, fully reflecting the integrity and characteristics of the trichosanthes peel pharmaceutical preparation (such as formula granules and other preparations).

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for constructing a characteristic chromatogram and a quality detection method for a trichosanthis pericarpium pharmaceutical preparation. Background Art

[0002] Trichosanthis pericarpium is a traditional Chinese medicine name. It is the dried ripe pericarp of the cucurbitaceous plant Trichosanthes kirilowii Maxim. or Trichosanthes rosthornii Harms. In autumn, the ripe fruits are picked, cut open, the pulp and seeds are removed, and then dried in the shade. Its functions and indications are: clearing heat and resolving phlegm, promoting qi circulation and relieving chest oppression. It is used for phlegm-heat cough, chest distress and hypochondriac pain. Common preparation types of trichosanthis pericarpium preparations include powder, formula granules, etc. Among them, the formula granules are obtained by steps such as extraction, concentration, drying, and granulation of trichosanthis pericarpium, and there are significant differences in the material basis between them and the material basis of the medicinal materials. At present, there is no report on the research of the characteristic chromatogram of trichosanthis pericarpium formula granules. The literature research report "Study on the HPLC Fingerprint of Trichosanthis Pericarpium Medicinal Materials", by Shi Guoyu et al. This literature mainly focuses on the research of trichosanthis pericarpium medicinal materials. Since traditional Chinese medicine formula granules no longer have the characteristics of medicinal material character identification, the above method is also not suitable for the quality detection of preparations made from trichosanthis pericarpium drugs such as trichosanthis pericarpium formula granules, and has defects such as few characteristic peaks, poor separation effect, and long detection and analysis time. Summary of the Invention

[0003] Therefore, the purpose of the present invention is to provide a method for constructing a characteristic chromatogram and a quality detection method for a trichosanthis pericarpium pharmaceutical preparation. According to the characteristics of the trichosanthis pericarpium pharmaceutical preparation, this method establishes the characteristic chromatogram of this variety, realizes the effective separation of each characteristic peak, increases the number of characteristic peaks, shortens the detection and analysis time at the same time, improves the separation effect, and provides a scientific basis for comprehensively establishing the quality control standard of trichosanthis pericarpium formula granules.

[0004] To this end, the present invention provides a method for constructing a characteristic chromatogram of a trichosanthis pericarpium pharmaceutical preparation, including the following steps,

[0005] (1) Preparation of the test solution;

[0006] (2) The test sample solution is detected by ultra-high performance liquid chromatography. An Agilent InfinityLab Poroshell 120SB-C18 chromatographic column is used. The mobile phase includes acetonitrile and also includes an aqueous solution of acetic acid at 0.05 - 0.2 vt% or an aqueous solution of formic acid at 0.2 - 0.3 vt%. The gradient elution program includes: 0 → 15 min → 25 min → 50 min → 55 min. The volume percentage of acetonitrile in the mobile phase is: 13% → 15% → 20% → 30% → 30%. The elution time < 30 - 34 min, the detection wavelength is 348 - 352 nm. When the elution time ≥ 30 - 34 min, the detection wavelength is 248 - 252 nm.

[0007] Further, step (2) also satisfies at least one of the following 1) - 3):

[0008] 1) The flow rate is 0.29 - 0.31 mL / min, the column temperature is 23 - 27 °C, and the injection volume is 1 - 4 μL;

[0009] 2) The volume percentage of acetic acid in the aqueous solution containing acetic acid is 0.05 - 0.2%;

[0010] 3) The elution time < 32 min, the detection wavelength is 350 nm. When the elution time ≥ 32 min, the detection wavelength is 250 nm.

[0011] Further, the gradient elution program also includes: 55 - 60 min, and the volume percentage of acetonitrile in the mobile phase is: 30% → 13%.

[0012] Further, the gradient elution program also includes: 60 - 65 min, and the volume percentage of acetonitrile in the mobile phase is: 13% → 13%.

[0013] Further, step (1) includes weighing the test sample, extracting with a solvent to obtain an extract, performing solid-liquid separation, and taking the liquid, which is the test sample solution.

[0014] Further, step (1) also satisfies any one or more of the following A - E:

[0015] A. The ratio of the mass of the test sample to the volume of the solvent is 1 - 5:20; the relationship between mass and volume is g / mL;

[0016] B. The extraction method is reflux extraction or ultrasonic extraction;

[0017] C. The extraction time is ≥ 30 min, preferably 30 - 120 min;

[0018] D. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0019] E. The solvent is selected from water or an aqueous methanol solution with a volume percentage not higher than 50%.

[0020] Furthermore, step (1) includes weighing 1 - 5 g of Trichosanthis Pericarpium formula granules, placing them in a stoppered conical flask, precisely adding 20 ml of water or an aqueous methanol solution with a volume percentage not higher than 50%, tightly stopper, weigh, ultrasonically treat for 30 - 120 min, weigh again, make up the lost weight with water or an aqueous methanol solution with a volume percentage not higher than 50%, shake well, filter, and thus obtain the test solution.

[0021] Furthermore, the described construction method also includes the steps of preparing a reference solution by using at least one of rutin, luteoloside, chrysoeriol - 7 - O - glucoside, luteolin, and Arvenin III plus a solvent, and detecting the reference solution by the ultra - performance liquid chromatography method in any of the above - described construction methods to obtain a reference spectrum.

[0022] Furthermore, each 1 mL of the reference solution contains at least one of 1 - 100 μg of rutin, 1 - 100 μg of luteoloside, 1 - 100 μg of chrysoeriol - 7 - O - glucoside, 1 - 100 μg of luteolin, and 1 - 100 μg of Arvenin III; and / or the solvent used in the preparation of the reference solution is selected from methanol or an aqueous methanol solution.

[0023] Furthermore, the described construction method also includes the steps of preparing a reference solution of the control crude drug by using the extract obtained by water extraction of the Trichosanthis Pericarpium control crude drug according to the preparation method of the test solution in any of the above - described construction methods, and detecting the reference solution of the control crude drug by the ultra - performance liquid chromatography method in any of the above - described construction methods to obtain a reference spectrum of the control crude drug.

[0024] Preferably, after water extraction of the Trichosanthis Pericarpium control crude drug, it is filtered, the liquid is taken and dried, and then the reference solution of the control crude drug is prepared according to the preparation method of the test solution in any of the above - described construction methods.

[0025] Furthermore, take 2 - 5 g of the Trichosanthis Pericarpium control crude drug, place it in a stoppered conical flask, add 20 - 100 ml of water, heat under reflux for 10 - 60 minutes, filter, evaporate the filtrate to dryness, add 20 ml of water or an aqueous methanol solution with a volume percentage not higher than 50% to the residue, ultrasonically treat for 30 - 120 min, cool, shake well, filter, and take the subsequent filtrate as the reference solution of the control crude drug.

[0026] Furthermore, the characteristic fingerprint of the Trichosanthes peel pharmaceutical preparation has 12 common characteristic peaks. The peak corresponding to the orientin-7-O-glucoside reference substance peak is the S peak, and the relative retention times of each characteristic peak and the S peak are within the range of ±10% of the specified values. The specified values are respectively: 0.48 (peak 1), 0.55 (peak 2), 1.10 (peak 4), 1.16 (peak 5), 1.44 (peak 6), 1.67 (peak 7), 1.80 (peak 8), 1.90 (peak 9), 2.05 (peak 10), 2.19 (peak 11), 2.27 (peak 12).

[0027] In the present invention, the Trichosanthes peel pharmaceutical preparation can be Trichosanthes peel formula granules.

[0028] In certain embodiments, it further includes the construction of the control characteristic fingerprint of the Trichosanthes peel pharmaceutical preparation. For the characteristic fingerprints obtained by detecting the test samples of multiple batches of Trichosanthes peel pharmaceutical preparations, the control characteristic fingerprint of the Trichosanthes peel pharmaceutical preparation is generated by using the similarity evaluation system for traditional Chinese medicine chromatographic characteristic fingerprints. At least 3 batches of Trichosanthes peel formula granules are used, such as 3 batches, 8 batches, 15 batches, 18 batches of Trichosanthes peel formula granules.

[0029] In certain embodiments, after generating the control characteristic fingerprint of the Trichosanthes peel pharmaceutical preparation by using the similarity evaluation software for traditional Chinese medicine chromatographic characteristic fingerprints, it further includes the step of marking the common characteristic peaks.

[0030] The present invention also provides an application of the method for constructing the characteristic fingerprint of the Trichosanthes peel pharmaceutical preparation as described in any one of the above in the quality inspection of the Trichosanthes peel pharmaceutical preparation.

[0031] The present invention also provides a quality inspection method for the Trichosanthes peel pharmaceutical preparation, including the step of comparing the characteristic fingerprint of the Trichosanthes peel product to be tested with the control characteristic fingerprint of the Trichosanthes peel pharmaceutical preparation; the characteristic fingerprint of the Trichosanthes peel product to be tested is constructed by using the Trichosanthes peel product to be tested according to the construction method described in any one of the above, and the control characteristic fingerprint of the Trichosanthes peel pharmaceutical preparation is selected from any one of the following (1)-(3):

[0032] (1) It has 12 common characteristic peaks. The peak corresponding to the orientin-7-O-glucoside reference substance peak is the S peak, and the relative retention times of each characteristic peak and the S peak are within the range of ±10% of the specified values. The specified values are respectively: 0.48 (peak 1), 0.55 (peak 2), 1.10 (peak 4), 1.16 (peak 5), 1.44 (peak 6), 1.67 (peak 7), 1.80 (peak 8), 1.90 (peak 9), 2.05 (peak 10), 2.19 (peak 11), 2.27 (peak 12);

[0033] (2) The characteristic chromatogram of the Trichosanthis Pericarpium pharmaceutical preparation obtained by using a single batch or multiple batches of Trichosanthis Pericarpium pharmaceutical preparation according to the construction method described in any one of the above.

[0034] (3) The characteristic chromatogram obtained by using multiple batches of Trichosanthis Pericarpium pharmaceutical preparation according to the construction method described in any one of the above is made into a reference characteristic chromatogram by the average value or median method.

[0035] In the present invention, the "peak corresponding to the scopoletin-7-O-glucoside reference peak" is a characteristic peak with a coincidence degree of not less than 50% with the scopoletin-7-O-glucoside reference peak.

[0036] % methanol represents the volume percentage of methanol in the methanol aqueous solution. % acetic acid represents the volume percentage of acetic acid in the acetic acid aqueous solution, and % phosphoric acid represents the volume percentage of phosphoric acid in the phosphoric acid aqueous solution.

[0037] The technical solution of the present invention has the following advantages:

[0038] 1. For the construction method of the characteristic chromatogram of the Trichosanthis Pericarpium pharmaceutical preparation described in the present invention, the ultra-high performance liquid chromatography method is adopted. Using an Agilent InfinityLab Poroshell 120SB-C18 column as the chromatographic column, the mobile phase includes acetonitrile, and also includes an aqueous solution of 0.05 - 0.2 vt% acetic acid or an aqueous solution of 0.2 - 0.3 vt% formic acid. The gradient elution program includes: 0 → 15 min → 25 min → 50 min → 55 min. The volume percentage of acetonitrile in the mobile phase is: 13% → 15% → 20% → 30% → 30%. The elution time is < 30 - 34 min, and the detection wavelength is 348 - 352 nm. When the elution time ≥ 30 - 34 min, the detection wavelength is 248 - 252 nm; finally, 12 common characteristic peaks are obtained, and the effective separation of these common characteristic peaks is achieved. Moreover, the obtained characteristic chromatogram has a stable baseline, good peak shapes of the characteristic peaks, and a short detection time, providing a scientific basis for comprehensively establishing the quality control standard of Trichosanthis Pericarpium formula granules. Moreover, the peak positions of rutin, luteoloside, scopoletin-7-O-glucoside, luteolin, and Arvenin III can be accurately located, fully reflecting the integrity and characteristics of the Trichosanthis Pericarpium pharmaceutical preparation (such as formula granules and other preparations).

[0039] 2. The method for constructing the characteristic spectrum of the Gualou peel pharmaceutical preparation of the present invention is to investigate the extraction conditions such as the optimization of the chromatographic conditions, the extraction solvent, and the amount of the extraction solvent, and determine the optimal extraction process and chromatographic conditions, so that the peak area is higher and the separation effect is better, and the quality of the Gualou peel formula granules can be more comprehensively monitored. The effective separation of rutin, luteolin, kuilisu-7-O-glucoside, luteolin and Arvenin III is achieved, and the contents of rutin, luteolin, kuilisu-7-O-glucoside, luteolin and Arvenin III can be determined simultaneously, and the effective ingredients and their contents in the Gualou peel (Trichosanthes) formula granules can be comprehensively and quickly detected.

[0040] 3. The quality detection method of the Trichosanthes peel pharmaceutical preparation described in the present invention can comprehensively, clearly and effectively detect the quality of the Trichosanthes peel formula granules by comparing the characteristic spectrum of the Trichosanthes peel formula granules to be tested with the control characteristic spectrum of the Trichosanthes peel formula granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0042] Figure 1 This is the chromatogram of method 1 in the mobile phase gradient optimization experiment of Experimental Example 1;

[0043] Figure 2 This is the chromatogram of method 2 in the mobile phase gradient optimization experiment of Experimental Example 1;

[0044] Figure 3 This is the chromatogram of method 3 in the mobile phase gradient optimization experiment of Experimental Example 1;

[0045] Figure 4 This is the chromatogram of method 4 in the mobile phase gradient optimization experiment of Experimental Example 1;

[0046] Figure 5 This is the chromatogram of method 5 in the mobile phase gradient optimization experiment of Experimental Example 1;

[0047] Figure 6 This is the chromatogram at 250nm wavelength in the investigation of detection wavelength in Experimental Example 1;

[0048] Figure 7 This is the chromatogram at 280nm wavelength in the investigation of detection wavelength in Experimental Example 1;

[0049] Figure 8Chromatogram at a wavelength of 300 nm in the investigation of the detection wavelength for Experimental Example 1;

[0050] Figure 9 Chromatogram at a wavelength of 350 nm in the investigation of the detection wavelength for Experimental Example 1;

[0051] Figure 10 Chromatogram at wavelengths of 0 - 32 min (350 nm) and 32 - 55 min (250 nm) in the investigation of the detection wavelength for Experimental Example 1;

[0052] Figure 11 Chromatogram of the 0.1% acetic acid - methanol mobile phase system in the investigation of the mobile phase for Experimental Example 1;

[0053] Figure 12 Chromatogram of the 0.1% acetic acid - acetonitrile mobile phase system in the investigation of the mobile phase for Experimental Example 1;

[0054] Figure 13 Chromatogram of the 0.1% phosphoric acid - acetonitrile mobile phase system in the investigation of the mobile phase for Experimental Example 1;

[0055] Figure 14 Chromatogram of the 0.1% formic acid - acetonitrile mobile phase system in the investigation of the mobile phase for Experimental Example 1;

[0056] Figure 15 Chromatogram of the Agilent Poroshell C18 - SB column in the investigation of the column for Experimental Example 1;

[0057] Figure 16 Chromatogram of the Waters HSS T3 column in the investigation of the column for Experimental Example 1;

[0058] Figure 17 Chromatogram of the Shim - pack GIST - HP C18 - AQ column in the investigation of the column for Experimental Example 1;

[0059] Figure 18 Chromatogram of the waters ultra - high performance liquid chromatography in the investigation of the chromatograph for Experimental Example 1;

[0060] Figure 19 Chromatogram of the Agilent ultra - high performance liquid chromatography in the investigation of the chromatograph for Experimental Example 1;

[0061] Figure 20 Chromatogram of the Thermo Fisher ultra - high performance liquid chromatography in the investigation of the chromatograph for Experimental Example 1;

[0062] Figure 21 Chromatogram extracted with water as the extraction solvent for Experimental Example 1;

[0063] Figure 22Chromatogram extracted with ethanol as the extraction solvent in Experimental Example 1;

[0064] Figure 23 Chromatogram extracted with methanol as the extraction solvent in Experimental Example 1;

[0065] Figure 24 Chromatogram extracted with 50% methanol as the extraction solvent in Experimental Example 1;

[0066] Figure 25 Chromatogram extracted with 30% methanol as the extraction solvent in Experimental Example 1;

[0067] Figure 26 Chromatogram extracted with 50% ethanol as the extraction solvent in Experimental Example 1;

[0068] Figure 27 Chromatogram for positioning reference substances in Experimental Example 1. S2 is the rutin reference substance solution; S3 is the luteoloside reference substance solution; S4 is the chrysoeriol-7-O-glucoside reference substance solution; S5 is the luteolin reference substance solution; S6 is the Arvenin III reference substance solution;

[0069] Figure 28 Chromatogram of the reference medicinal material of Trichosanthis Fructus (Gualoupi) in Experimental Example 1;

[0070] Figure 29 Characteristic chromatogram of 18 batches of Trichosanthis Fructus (Gualoupi) formula granules in Experimental Example 1;

[0071] Figure 29 Reference characteristic chromatogram of Trichosanthis Fructus formula granules in Experimental Example 1;

[0072] Among them, peak 1: rutin; peak 2: luteoloside; peak 3(S): chrysoeriol-7-O-glucoside; peak 6: luteolin; peak 7: Arvenin III;

[0073] Figure 30 Chromatogram of the negative blank solution for specificity investigation in Experimental Example 2;

[0074] Figure 31 Characteristic chromatogram of Trichosanthis Fructus formula granules in Example 1. Detailed implementation method

[0075] The following embodiments are provided to better understand the present invention further. It is not limited to the best mode, and does not limit the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration 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. For those not specifying specific experimental steps or conditions in the embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments not indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0076] Experimental Example 1 Investigation of the Construction Method

[0077] 1. Instruments, Reagents and Medicinal Agents

[0078] Ultra-high performance liquid chromatograph: Waters Hchass, including a quaternary gradient infusion pump (Alliance 2695 type), a 120-position high-performance automatic sampler, an originally imported chromatographic column thermostat, a Waters 2998 diode array ultraviolet detector, and an Empower chromatographic management system.

[0079] Other instruments: ten-thousandth electronic balance (Shanghai Shunyu Hengping Scientific Instrument Co., Ltd., FA1004); one-hundred-thousandth electronic balance (Sartorius Scientific Instrument Co., Ltd., SQP); thermostatic magnetic stirrer with heating mantle (Gongyi Yuhua Instrument Co., Ltd., DF-101D); medical centrifuge (Changzhou Jintan Kexing Instrument, TG16G); temperature-controlled electric heater (Nantong Tongzhou Shengtong Electric Heater Factory); freeze dryer (Beijing Boyikang Experimental Instrument Co., Ltd., FD-1-50); SHZ-DⅢ circulating water vacuum pump (Gongyi Yuhua Instrument Co., Ltd.); rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, RE-52 series); ultrasonic cleaner: WB400US Shanghai Wangbiao Instrument Co., Ltd.

[0080] Chromatographic column: Agilent InfinityLab Poroshell 120SB-C18, 2.1×100 mm, 1.9 μm; methanol and acetonitrile were of chromatographic grade (Merck); phosphoric acid was of analytical grade; water (Milli-Q). Chrysoeriol-7-O-glucoside reference substance (batch number: 19993-32-9, purity 98.98%, Jiangxi Baicaoyuan Biotechnology Co., Ltd.); rutin reference substance (batch number: 100080-202012, National Institutes for Food and Drug Control); luteoloside reference substance (batch number: 111720-202111, National Institutes for Food and Drug Control); Arvenin III reference substance (batch number: 001728-202108, Jiangxi Baicaoyuan Biotechnology Co., Ltd.); Fructus Trichosanthis peel reference crude drug (batch number: 121184-202106, National Institutes for Food and Drug Control);

[0081] The pharmaceutical preparation of Fructus Trichosanthis peel (Trichosanthes kirilowii Maxim.) in the present invention is prepared by the following method: Take Fructus Trichosanthis peel (Trichosanthes kirilowii Maxim.), heat under reflux for extraction for at least 1 time, each time adding 6-12 times the weight of water for extraction for at least 0.5 h, filter, combine the filtrates, concentrate the filtrates to a relative density of 1.05-1.10 g / mL at 60 °C, add conventional excipients, and prepare into clinically acceptable tablets, capsules, pills, granules, honeyed pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations or injection preparations according to conventional processes. The pharmaceutically acceptable excipients are: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, matrices, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked carboxymethyl cellulose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silica, etc.; suspending agents include: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include, starch paste, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: sodium saccharin, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various flavors; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorhexidine acetate, eucalyptus oil, etc.; matrices include: PEG6000, PEG4000, beeswax, etc.

[0082] In this example, the test sample selected was Trichosanthis Fructus formula granules. The specific preparation method of the Trichosanthis Fructus formula granules was as follows: Take Trichosanthis Fructus, extract it by heating under reflux 2 times. For the first time, add 10 times the weight of water and soak for 30 min, then extract by heating under reflux for 0.5 h, filter. For the second time, add 8 times the weight of water and extract for 0.5 h, filter. Combine the filtrates, concentrate the filtrates to a relative density of 1.05 g / mL at 60 °C, perform spray drying, add the auxiliary material maltodextrin to the dry powder, mix evenly and then perform dry granulation to make granule agents. The batches were respectively: 1903001W, 1906001S, 1909001W, 1912001W, 2001001S, 2002001W, 2005001S, 2008001W, 2011001S, 2102001S, 2105001S, 2108001W, 2110001S, 2112001S, 2202001W, 2206001W, 2210001S, 2212001S.

[0083] 2. Preparation of the test sample solution

[0084] Precisely weigh about 3 g of Trichosanthis Fructus formula granules, accurately weigh, place in a stoppered conical flask, precisely add 20 mL of 30% methanol, tightly stopper, weigh, ultrasonically treat (power 250 W, frequency 35 kHz) for 1 hour, take out, let cool, weigh again, make up the lost weight with 30% methanol, shake well, filter, and take the subsequent filtrate to obtain the test sample solution.

[0085] 3. Optimization of the chromatographic conditions

[0086] (1) Mobile phase gradient optimization experiment

[0087] Use ultra-high performance liquid chromatography to detect the test sample solution prepared according to item 2 of this experimental example. Use an Agilent InfinityLab Poroshell 120SB-C18 chromatographic column, and elute according to the following gradient conditions respectively. Mobile phase A is acetonitrile; mobile phase B is 0.2% formic acid solution; the flow rate is 0.3 ml per minute; the column temperature is 25 °C. When the elution time < 32 min, the detection wavelength is 350 nm. When the elution time ≥ 32 min, the detection wavelength is 250 nm. The injection volume is 2 μl.

[0088] Table 1 Gradient conditions for methods one to three

[0089]

[0090]

[0091] Table 2 Gradient conditions for methods four to five

[0092]

[0093] Table 3 System suitability parameters of chromatographic peaks at each gradient

[0094]

[0095]

[0096] The results are as Figure 1 - 5 shown in Table 3. Compared with other methods, the chromatogram presented under Method 4 has richer chromatographic information, and the number of characteristic peaks with good separation effect increases significantly, the peak shape is better, and the baseline is relatively stable.

[0097] (2) Investigation of detection wavelength

[0098] Take the Fructus Trichosanthis formula granules to prepare the test solution of Fructus Trichosanthis formula granules according to the method under Item 2 of this experimental example. Inject it into the liquid chromatograph, use the Agilent InfinityLab Poroshell 120SB-C18 chromatographic column, and perform elution using the gradient elution program of Method 4 under Item 3(1) of this experimental example. Mobile phase A is acetonitrile; mobile phase B is 0.2% formic acid; the flow rate is 0.3 ml per minute; the column temperature is 25°C, and the injection volume is 2 μl. Select and compare the chromatograms of Fructus Trichosanthis (Trichosanthes kirilowii Maxim.) under 5 different absorption wavelengths (230 nm; 250 nm; 300 nm; 350 nm; the detection wavelength is 350 nm when the elution time < 32 min, and the detection wavelength is 250 nm when the elution time ≥ 32 minutes), and use the detected chromatographic peak information amount and system suitability parameters as the selection indicators to optimize the best absorption wavelength. Determine that the detection wavelength is 350 nm when the elution time < 32 min and 250 nm when the elution time ≥ 32 minutes as the best detection wavelength of the characteristic chromatogram of Fructus Trichosanthis (Trichosanthes kirilowii Maxim.) formula granules.

[0099] Table 4 Investigation results of different detection wavelengths

[0100]

[0101]

[0102] As Figure 6 - 10The results in the above table show that, compared with other wavelengths, switching the wavelength from 350 nm to 250 nm at 32 minutes, i.e., the elution time < 32 min, the detection wavelength is 350 nm, the elution time ≥ 32 minutes, the detection wavelength is 250 nm, results in the largest amount of chromatographic peak information. At a wavelength of 250 nm, there is more peak information in the second half of the method and less in the first half; under the condition of 350 nm, there is more peak information in the first half of the method and less in the second half. Therefore, it is finally preferred that the detection wavelength from 0 to 32 minutes is 350 nm, and the detection wavelength from 32 to 55 minutes is 250 nm (i.e., elution time < 32 min, detection wavelength is 350 nm, elution time ≥ 32 min, detection wavelength is 250 nm) as the detection wavelength for the characteristic chromatogram of Trichosanthis Pericarpium (Trichosanthes kirilowii Maxim.) formula granules.

[0103] (3) Investigation experiment of mobile phase

[0104] Take the Trichosanthis Pericarpium formula granules to prepare the test solution of Trichosanthis Pericarpium formula granules according to the method under item 2 of this experimental example. Inject it into the liquid chromatograph, use an Agilent InfinityLab Poroshell 120SB-C18 chromatographic column, and perform gradient elution according to the gradient elution program of method four under item 3(1) of this experimental example, with a flow rate of 0.3 ml per minute; the column temperature is 25 °C, the elution time < 32 min, the detection wavelength is 350 nm, the elution time ≥ 32 minutes, the detection wavelength is 250 nm, and the injection volume is 2 μl. Compare the effects of different mobile phase systems on the separation effect of the characteristic chromatogram of Trichosanthis Pericarpium formula granules. The mobile phase systems are set as follows: ① Mobile phase A: methanol, mobile phase B: 0.1% acetic acid; ② Mobile phase A: acetonitrile, mobile phase B: 0.1% acetic acid; ③ Mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid; ④ Mobile phase A: acetonitrile, mobile phase B: 0.1% formic acid.

[0105] The results are shown in the following table and Figure 11 - 14 As shown, the results indicate that different mobile phase components have a great influence on the separation effect of chromatographic peaks. When the mobile phase components are 0.1% acetic acid - acetonitrile, the separation effect of chromatographic peaks is the best. Therefore, it is tentatively determined to use 0.1% acetic acid - acetonitrile mobile phase for subsequent screening.

[0106] Table 5 Investigation results of different mobile phases

[0107]

[0108]

[0109] (4) Investigation experiment of mobile phase system with different acetic acid concentrations

[0110] Take the Trichosanthis Pericarpium formula granule and prepare the test solution of Trichosanthis Pericarpium formula granule according to the method under item 2 of this experimental example. Inject it into the liquid chromatograph. Use an Agilent InfinityLab Poroshell 120SB-C18 chromatographic column and carry out elution using the gradient elution program of method four under item 3(1) of this experimental example. Mobile phase A is acetonitrile; mobile phase B is acetic acid aqueous solution with different concentrations; the flow rate is 0.3 ml per minute; the column temperature is 25 °C, the elution time < 32 min, the detection wavelength is 350 nm, when the elution time ≥ 32 minutes, the detection wavelength is 250 nm, and the injection volume is 2 μl. Compare the influence of different concentrations of mobile phase B on the separation effect of the characteristic chromatogram of Trichosanthis Pericarpium formula granule. The mobile phase system settings are as follows: ① Mobile phase A: acetonitrile, mobile phase B: 0.1% acetic acid; ② Mobile phase A: acetonitrile, mobile phase B: 0.05% acetic acid; ③ Mobile phase A: acetonitrile, mobile phase B: 0.2% acetic acid.

[0111] The results are shown in the following table. The results show that different concentrations of acetic acid (volume percentage 0.05 - 0.2%) have little influence on the information content of chromatographic peaks and the system suitability parameters.

[0112] Table 6 Investigation results of mobile phase systems with different acetic acid concentrations

[0113]

[0114]

[0115] (5) Investigation experiment on flow rate

[0116] Take the Trichosanthis Pericarpium formula granule (1903001W) and prepare the test solution of Trichosanthis Pericarpium formula granule according to the method under item 2 of this experimental example. Inject it into the liquid chromatograph. Use an Agilent InfinityLab Poroshell 120SB-C18 chromatographic column and carry out elution using the gradient elution program of method four under item 3(1) of this experimental example. Mobile phase A is acetonitrile; mobile phase B is 0.1% acetic acid solution; the column temperature is 25 °C, the elution time < 32 min, the detection wavelength is 350 nm, when the elution time ≥ 32 minutes, the detection wavelength is 250 nm, and the injection volume is 2 μl. Compare the influence of different flow rates (0.29 mL / min, 0.3 mL / min, 0.31 mL / min) on the durability of the characteristic chromatogram of the freeze-dried powder of Trichosanthis Pericarpium formula granule. The results show that the separation effects of each characteristic peak are good at different flow rates (0.29 mL / min, 0.3 mL / min, 0.31 mL / min). When the flow rate is 0.3 mL / min, the separation effect of each chromatographic peak is relatively better. Therefore, the tentative flow rate is 0.3 mL / min.

[0117] Table 7 Investigation results of different flow rates

[0118]

[0119]

[0120] (6) Investigation experiment on column temperature

[0121] Take Trichosanthis Pericarpium formula granules (1903001W) to prepare the test solution of Trichosanthis Pericarpium formula granules according to the method under item 2 of this experimental example. Inject it into the liquid chromatograph, use an Agilent InfinityLab Poroshell 120SB-C18 chromatographic column, and perform elution using the gradient elution program of method four under item 3(1) of this experimental example. Mobile phase A is acetonitrile; mobile phase B is 0.1% acetic acid solution; the flow rate is 0.3 mL / min, the elution time < 32 min, the detection wavelength is 350 nm, the elution time ≥ 32 minutes, the detection wavelength is 250 nm, and the injection volume is 1 μl. Compare the influence of different column temperatures (25 °C, 23 °C, 27 °C) on the durability of the characteristic chromatogram of Trichosanthis Pericarpium formula granules. The results show that the separation effects of each characteristic peak are good at different column temperatures (25 °C, 23 °C, 27 °C). When the column temperature is 25 °C, the separation effect of each chromatographic peak is relatively better. Therefore, the column temperature is tentatively set at 25 °C.

[0122] Table 8 Results of investigation on different column temperatures

[0123]

[0124] (7) Investigation experiment on chromatographic column

[0125] Take Trichosanthis Pericarpium formula granules to prepare the test solution of Trichosanthis Pericarpium formula granules according to the method under item 2 of this experimental example. Inject it into the liquid chromatograph, and perform elution using the gradient elution program of method four under item 3(1) of this experimental example. Mobile phase A is acetonitrile; mobile phase B is 0.1% acetic acid solution; the flow rate is 0.3 mL / min, the column temperature is 25 °C, the elution time < 32 min, the detection wavelength is 350 nm, the elution time ≥ 32 minutes, the detection wavelength is 250 nm, and the injection volume is 2 μl. Compare the influence of different chromatographic columns on the durability of the characteristic chromatogram of Trichosanthis Pericarpium formula granules, using octadecylsilane-bonded silica chromatographic columns of different brands respectively (Chromatographic column 1: Agilent InfinityLab Poroshell 120SB-C18, 2.1×100 mm, 1.9 μm; Chromatographic column 2: Waters HSST3 (1.8 μm, 2.1×100 mm); Chromatographic column 3: Shim-pack GIST-HP C18-AQ (1.9 μm, 2.1×100 mm). The results are shown in the following table and Figure 15 - 17Shown as follows: Different chromatographic columns have a certain influence on the separation effect of each chromatographic peak. The Agilent InfinityLab Poroshell 120SB-C18, 2.1×100mm, 1.9μm chromatographic column has the best separation effect on each chromatographic peak. Therefore, the Agilent InfinityLab Poroshell 120SB-C18, 2.1×100mm, 1.9μm chromatographic column was selected as the chromatographic column for the characteristic chromatogram method of Trichosanthis Pericarpium (Trichosanthes kirilowii Maxim.) formula granules.

[0126] Table 9 Investigation results of different chromatographic columns

[0127]

[0128] (8) Investigation experiment of chromatograph

[0129] Take the Trichosanthis Pericarpium formula granules and prepare the test solution of Trichosanthis Pericarpium formula granules according to the method under item 2 of this experimental example. Inject them into three different models of ultra-high performance liquid chromatographs, namely Waters, Agilent, and Thermo, respectively. The chromatographic conditions are the same. Specifically: Use the gradient elution program of method four under item 3(1) of this experimental example for elution. Mobile phase A is acetonitrile; mobile phase B is 0.1% acetic acid; the flow rate is 0.3 mL / min, the column temperature is 25°C, the elution time is <32 min, the detection wavelength is 350 nm, the elution time is ≥32 minutes, the detection wavelength is 250 nm, and the injection volume is 1 μL. Compare the influence of different chromatographs on the durability of the characteristic chromatogram of Trichosanthis Pericarpium formula granules. The results are shown in Figure 18 - 20 Shown as follows: The chromatographic information presented by the ultra-high performance liquid chromatography methods of Waters, Agilent, and Thermo models is relatively complete, and each characteristic peak is not missing. Therefore, it is not necessary to fix the model of the liquid chromatograph to determine the characteristic chromatogram of Trichosanthis Pericarpium (Trichosanthes kirilowii Maxim.) formula granules.

[0130] (9) Finally determined chromatographic conditions

[0131] Using octadecylsilane chemically bonded silica gel as the filler (column length is 100 mm, inner diameter is 2.1 mm, particle size is 1.9 μm); using acetonitrile as mobile phase A and 0.1% acetic acid solution as mobile phase B, perform gradient elution according to the regulations in the following table; the flow rate is 0.3 ml per minute; the column temperature is 25°C; the detection wavelength is 350 nm for 0 - 32 minutes and 250 nm for 32 - 55 minutes (that is, the elution time is <32 min, the detection wavelength is 350 nm, and the elution time is ≥32 min, the detection wavelength is 250 nm). The theoretical plate number calculated by coumestrol-7-O-glucoside should not be less than 10000.

[0132]

[0133]

[0134] 4. Preparation of Test Solution

[0135] (1) Investigation of Extraction Solvent

[0136] Take about 3 g of Trichosanthis Pericarpium formula granules, accurately weigh, place in a stoppered conical flask, accurately add 20 ml of extraction solvents (water, methanol, ethanol, 50% methanol, 30% methanol, 50% ethanol) respectively, stopper tightly, weigh, ultrasonically treat (power 250 W, frequency 35 kHz) for 1 hour, take out, cool, weigh again, make up the lost weight with 30% methanol, shake well, filter, take the subsequent filtrate to obtain the solution, and determine according to the chromatographic conditions under item 3(9) of this experimental example. The results are shown in the following table and Figure 21 - 26 As shown, 30% methanol, 50% methanol and water have better separation effects on each chromatographic peak. Especially for the test solution extracted with 30% methanol, the system suitability parameters are relatively better. Therefore, 30% methanol is preferably selected as the extraction solvent.

[0137] Table 10 Results of Investigation of Different Extraction Solvents

[0138]

[0139]

[0140] (2) Investigation of Extraction Time

[0141] Take about 3 g of Trichosanthis Pericarpium formula granules, accurately weigh, place in a stoppered conical flask, accurately add 20 ml of 30% methanol respectively, stopper tightly, weigh, ultrasonically treat (power 250 W, frequency 35 kHz) for different extraction times (30 minutes, 60 minutes, 90 minutes) respectively, take out, cool, weigh again, make up the lost weight with 30% methanol, shake well, filter, take the subsequent filtrate to obtain the solution, and determine according to the chromatographic conditions under item 3(9) of this experimental example. The results show that the separation effects on each chromatographic peak are better, the peak shapes are better, and the baseline is stable within 30 - 90 minutes, all of which can meet the requirements of chromatographic separation. Among them, the system suitability parameters are the best at 60 minutes (the total peak area is the largest and the total peak height is the largest).

[0142] (3) Investigation of Sampling Quantity

[0143] To investigate the effects of different sampling amounts (1 g, 3 g, 5 g) on the extraction efficiency of Trichosanthis Fructus formula granules, take Trichosanthis Fructus formula granules (1 g, 3 g, 5 g), accurately weigh them, place them in a stoppered conical flask, accurately add 20 ml of 30% methanol respectively, stopper tightly, weigh, ultrasonically treat them respectively (power 250 W, frequency 35 kHz) for 60 minutes, take them out, let them cool, weigh again, make up the lost weight with 30% methanol, shake well, filter, take the continuous filtrate, and you will get it. Determine according to the chromatographic conditions under item 3(9) of this experimental example. The results show that 1 g, 3 g, and 5 g can all meet the requirements of chromatographic separation. The peak response of the 1 g sampling amount is relatively small, and it is difficult to dissolve with a large sample amount of 5 g. When the sampling amount is 3 g, the system suitability parameters of the test sample are relatively better. Therefore, the sampling amount is preferably determined to be 3 g.

[0144] (4) Investigation of injection volume

[0145] Take Trichosanthis Fructus formula granules, about 3 g, accurately weigh them, place them in a stoppered conical flask, accurately add 20 ml of 30% methanol respectively, stopper tightly, weigh, ultrasonically treat them respectively (power 250 W, frequency 35 kHz) for different extraction times (30 minutes, 60 minutes, 90 minutes), take them out, let them cool, weigh again, make up the lost weight with 30% methanol, shake well, filter, take the continuous filtrate, accurately absorb 1 μl, 2 μl, 4 μl of the continuous filtrate respectively, and determine according to the chromatographic conditions under item 3(9) of this experimental example. It can be seen from the following table that injection volumes of 1 μl, 2 μl, and 4 μl can all meet the requirements of chromatographic separation. After comparative analysis, it is found that when the injection volume is 2 μl, the system suitability parameters of the test sample are relatively better. Therefore, the preferred injection volume is 2 μl.

[0146] (5) Determination of the preparation method of the test solution

[0147] According to the above research results, the preparation method of the test solution for Trichosanthis Fructus formula granules is determined as follows: Take 3 g of the powder of this product, accurately weigh it, place it in a stoppered conical flask, stopper tightly, accurately add 20 ml of 30% methanol, ultrasonically treat it (power 250 W, frequency 40 kHz) for 1 hour, let it cool, shake well, filter, and use it as the test solution.

[0148] 5. Determination of characteristic peaks and establishment of reference chromatogram

[0149] (1) Construction method

[0150] Take 3 g of the reference medicinal material of Trichosanthis Fructus, place it in a stoppered conical flask, add 50 ml of water, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 20 ml of 30% methanol to the residue, ultrasonically treat it (power 250 W, frequency 40 kHz) for 1 hour, let it cool, shake well, filter, and use it as the reference medicinal material reference solution.

[0151] Take 18 batches of Trichosanthis Pericarpium formula granules to prepare test solutions according to the method of Example 1 respectively, and detect the test solutions of 18 batches of Trichosanthis Pericarpium formula granules and the reference medicinal material reference solution according to the method of Example 1. The results are as Figure 28 - 29 shown, among which Figure 29 in, S1-S18 are the characteristic fingerprints of 18 batches of Trichosanthis Pericarpium formula granules, and the batch numbers are S1: 1903001W, S2: 1906001S, S3: 1909001W, S4: 1912001W, S5: 2001001S, S6: 2002001W, S7: 2005001S, S8: 2008001W, S9: 2011001S, S10: 2102001S, S11: 2105001S, S12: 2108001W, S13: 2110001S, S14: 2112001S, S15: 2202001W, S16: 2206001W, S17: 2210001S, S18: 2212001S, and R(11) is the reference characteristic fingerprint.

[0152] Use the fingerprint similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission to generate the reference characteristic fingerprint, as Figure 30 shown. According to Figure 23 the shown reference characteristic fingerprint, the detection results of the formula granule characteristic fingerprints can be analyzed and compared for the quality control of formula granules. The specific method for quality control using the reference characteristic fingerprint is as follows:

[0153] The HPLC characteristic fingerprint of Trichosanthis Pericarpium (Trichosanthes kirilowii Maxim.) formula granules has 12 common chromatographic peaks, among which 5 peaks (peak 1, 2, 3, 6, 7) are peaks of known components; select the peak corresponding to the reference peak of chrysoeriol-7-O-glucoside as the S peak, and calculate the relative retention time of each characteristic peak to the S peak, and its relative retention time should be within the range of ±10% of the specified value. The specified values are: the specified values are: 0.48 (peak 1), 0.55 (peak 2), 1.10 (peak 4), 1.16 (peak 5), 1.44 (peak 6), 1.67 (peak 7), 1.80 (peak 8), 1.90 (peak 9), 2.05 (peak 10), 2.19 (peak 11), 2.27 (peak 12). The obtained results are shown in Tables 11-14.

[0154] Table 11 Relative retention time of the common pattern of Trichosanthis Pericarpium (Trichosanthes kirilowii Maxim.) formula granules

[0155]

[0156] Table 12 Matching data of the common pattern of Trichosanthis Pericarpium (Trichosanthes kirilowii Maxim.) formula granules

[0157]

[0158] Table 13 Relative retention times of characteristic chromatograms of 18 batches of Trichosanthis Pericarpium (Trichosanthes kirilowii Maxim.) formula granules

[0159]

[0160]

[0161]

[0162] Table 14 Relative peak areas of characteristic chromatograms of 18 batches of Trichosanthis Pericarpium (Trichosanthes kirilowii Maxim.) formula granules

[0163]

[0164]

[0165] As can be seen from the above table, the similarity of the characteristic fingerprint chromatograms of 18 batches of Trichosanthis Pericarpium (Trichosanthes kirilowii Maxim.) formula granules is between 0.915 and 0.87, all greater than 0.90.

[0166] 6. Identification of characteristic peaks

[0167] Take appropriate amounts of chrysoeriol-7-O-glucoside and rutin reference substances, and prepare solutions containing 5 μg of each reference substance per 1 ml with methanol; take appropriate amounts of luteoloside, luteolin, and Arvenin III reference substances, accurately weigh them, and prepare solutions containing 2 μg of each reference substance per 1 mL with 50% methanol, respectively.

[0168] Prepare the test solution of Trichosanthis Pericarpium formula granules according to the preparation method of Example 1. After detecting and comparing the rutin reference substance solution, luteoloside reference substance solution, chrysoeriol-7-O-glucoside reference substance solution, Arvenin III reference substance solution, and the test solution of Trichosanthis Pericarpium formula granules by ultra-high performance liquid chromatography according to the method of Example 1, the results are as Figure 27 , where S1 is Trichosanthis Pericarpium formula granules, S2 is rutin, S3 is luteoloside, S4 is chrysoeriol-7-O-glucoside, S5 is luteolin, and S6 is Arvenin III.

[0169] Summary: The retention times of the 1st, 2nd, 3rd, 6th, and 7th peaks in the chromatogram of the test solution of Trichosanthis Pericarpium formula granules are consistent with those of the rutin, luteoloside, chrysoeriol-7-O-glucoside, luteolin, and Arvenin III reference substance chromatograms, respectively. It can be confirmed that the 1st peak is rutin, the 2nd peak is luteoloside, the 3rd peak is chrysoeriol-7-O-glucoside, the 6th peak is luteolin, and the 7th peak is Arvenin III.

[0170] Through the study of UPLC reference substance positioning, in the UPLC chromatogram of Trichosanthis Fructus formula granules, 5 of the peaks are known chromatographic peaks, namely peak 1 (rutin), peak 2 (luteoloside), peak 3 (chrysoeriol-7-O-glucoside), peak 6 (luteolin), and peak 7 (Arvenin III). Molecular weight and molecular structure analysis were carried out on peaks 4-5 and peaks 7-11 by LC / MS / MS to provide a reference basis for the subsequent qualitative analysis of characteristic component peaks. The analysis results of LC / MS / MS are shown in the following table.

[0171] Table 15 Analysis Results of LC / MS / MS of Trichosanthis Fructus Formula Granules

[0172]

[0173]

[0174] Experimental Example 2 Methodology Verification

[0175] 1. Specificity

[0176] Precisely pipette 2 μL of the test solution (prepared according to the method of Example 1) and 2 μL of the negative blank solution (prepared by extracting negative granules with 30% methanol), and inject them into the ultra-high performance liquid chromatograph respectively. Test according to the method of Example 1, and the results are shown in Figure 31 , and there is no interference from the negative.

[0177] 2. Instrument Precision

[0178] Take Trichosanthis Fructus formula granules to prepare the test solution according to the method of Example 1. Take the same test solution and test it by the ultra-high performance liquid chromatography method of Example 1. Inject the sample repeatedly 6 times, and calculate that the RSD of the relative retention time of 12 characteristic peaks is less than 2.0%. The results show that the instrument precision is good.

[0179] 3. Repeatability

[0180] Take Trichosanthis Fructus formula granules and prepare 6 test solutions in parallel according to the method of Example 1 and determine them. Calculate that the RSD of the relative retention time of 12 characteristic peaks is less than 2.0%. The results show that the method repeatability is good.

[0181] 4. Intermediate Precision (Personnel)

[0182] Three experimental personnel respectively prepared the test solution of Trichosanthis Fructus formula granules according to the method of Example 1 and determined it. Calculate that the RSD of the relative retention time of 12 characteristic peaks is less than 2.0%, and the intermediate precision is good.

[0183] 5. Robustness

[0184] Take the Trichosanthis Pericarpium formula granule to prepare the test solution according to the method of Example 1, and test it by the ultra-high performance liquid chromatography method of Example 1 at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h respectively. Calculate that the RSD of the relative retention time of 12 characteristic peaks is less than 2.0%. The results show that the test solution is stable within 24 h and meets the determination requirements.

[0185] Example 1

[0186] This example provides a method for constructing a characteristic fingerprint of Trichosanthis Pericarpium formula granule, which includes the following steps:

[0187] Preparation of test solution: Weigh accurately 3 g of Trichosanthis Pericarpium formula granule powder, weigh accurately, place it in a stoppered conical flask, stopper tightly, accurately add 20 ml of 30% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 1 hour, let it cool, shake well, filter, and use it as the test solution.

[0188] Preparation of reference solution: Take appropriate amounts of Chrysoeriol-7-O-glucoside and Rutin reference substances, and prepare solutions containing 5 μg of each reference substance per 1 ml with methanol; take appropriate amounts of Luteoloside, Luteolin, and Arvenin III reference substances, weigh accurately, and prepare solutions containing 2 μg of each reference substance per 1 mL with 50% methanol respectively to obtain.

[0189] Ultra-high performance liquid chromatography test: Precisely pipette the reference solution and the test solution of Trichosanthis Pericarpium formula granule, inject them into the liquid chromatograph, and determine. The chromatographic conditions are as follows: Use octadecylsilane chemically bonded silica as the filler (column length is 100 mm, inner diameter is 2.1 mm, particle size is 1.9 μm); use an Agilent InfinityLab Poroshell120SB-C18 chromatographic column, use acetonitrile as mobile phase A, and 0.1% acetic acid solution as mobile phase B, and perform gradient elution according to the regulations in the following table; the flow rate is 0.3 ml per minute; the column temperature is 25 °C; the elution time < 32 min, the detection wavelength is 350 nm, and the elution time ≥ 32 minutes, the detection wavelength is 250 nm. The theoretical plate number calculated by Chrysoeriol-7-O-glucoside should be not less than 10000.

[0190] Table 16 Gradient program

[0191]

[0192] Table 17 System suitability parameters

[0193]

[0194] From the above table and Figure 32 it can be seen that Figure 24 the separation effect of the 12 characteristic peaks inFigure 25 Peak 1 is rutin; Peak 2 is luteoloside; Peak 3 is chrysoeriol-7-O-glucoside; Peak 6 is luteolin; Peak 7 is Arvenin III.

[0195] Obviously, the above embodiments are merely examples given for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or alterations derived therefrom still fall within the protection scope of the present invention.

Claims

1. A method for constructing a characteristic fingerprint of a Trichosanthis Pericarpium pharmaceutical preparation, characterized in that, It includes the following steps: (1) Preparation of the test solution: Weigh the test sample, extract it with a solvent to obtain an extract, separate the solid from the liquid, and take the liquid as the test solution. The solvent is selected from water or a methanol aqueous solution with a volume percentage not higher than 50%; Preparation of the reference solution: Use rutin, luteoloside, chrysoeriol-7-O-glucoside, luteolin, and Arvenin III to prepare the reference solution with a solvent; (2) Take the test solution and the reference solution and detect them by ultra-high performance liquid chromatography. Use an Agilent InfinityLab Poroshell 120 SB-C18 chromatographic column. Mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of acetic acid with a volume percentage of 0.05 - 0.2 vt% or an aqueous solution of formic acid with a volume percentage of 0.2 - 0.3 vt%. The gradient elution program includes: 0 → 15 min → 25 min → 50 min → 55 min. The volume percentage of acetonitrile in the mobile phase is: 13% → 15% → 20% → 30% → 30%. The elution time < 30 - 34 min, the detection wavelength is 348 - 352 nm, and when the elution time ≥ 30 - 34 min, the detection wavelength is 248 - 252 nm.

2. The construction method according to claim 1, characterized in that Step (2) also satisfies at least one of the following 1) - 3): 1) The flow rate is 0.29 - 0.31 mL / min, the column temperature is 23 - 27 °C, and the injection volume is 1 - 4 μL; 2) The volume percentage of acetic acid in the aqueous solution containing acetic acid is 0.05 - 0.2%; 3) The elution time < 32 min, the detection wavelength is 350 nm, and when the elution time ≥ 32 min, the detection wavelength is 250 nm.

3. The construction method according to claim 1, wherein The said step (1) also satisfies any one or more of the following A - E: A. The ratio of the mass of the test sample to the volume of the solvent is 1 - 5:20; the relationship between mass and volume is g / mL; B. The extraction method is reflux extraction or ultrasonic extraction; C. The extraction time is ≥ 30 min; D. The solid-liquid separation is selected from centrifugation or membrane filtration.

4. The construction method according to claim 3, characterized in that The extraction time is 30 - 120 min.

5. The construction method according to claim 1, characterized in that, Each 1 mL of the reference solution contains at least one of 1 - 100 μg of rutin, 1 - 100 μg of luteoloside, 1 - 100 μg of chrysoeriol-7-O-glucoside, 1 - 100 μg of luteolin, and 1 - 100 μg of Arvenin III; and / or, the solvent used in the preparation of the reference solution is selected from methanol or a methanol aqueous solution.

6. The construction method according to any one of claims 1-4, characterized in that The said construction method also includes the step of preparing a reference solution of the control medicinal material by using the extract obtained by water extraction of the control medicinal material of Trichosanthes kirilowii Maxim. pericarp according to the preparation method of the test solution in any one of claims 1 - 4, and detecting the reference solution of the control medicinal material by the ultra-high performance liquid chromatography method in any one of claims 1 - 4 to obtain a reference chromatogram of the control medicinal material.

7. The construction method according to claim 6, wherein After water extraction of the control medicinal material of Trichosanthes kirilowii Maxim. pericarp, it is filtered, the liquid is taken and dried, and then a reference solution of the control medicinal material is prepared according to the preparation method of the test solution in any one of claims 1 - 4.

8. The construction method according to any one of claims 1-4, characterized in that, The characteristic fingerprint of the pericarpium trichosanthis drug preparation has 12 common characteristic peaks. The peak corresponding to the orientin-7-O-glucoside reference peak is the S peak. The relative retention times of peaks 1, 2, 4 - 12 with respect to the S peak are within the range of ±10% of the specified values. The specified values are respectively: 0.48, 0.55, 1.10, 1.16, 1.44, 1.67, 1.80, 1.90, 2.05, 2.19, 2.

27.

9. Use of the method for constructing the characteristic fingerprint of the pericarpium trichosanthis drug preparation according to any one of claims 1 - 8 in the quality inspection of the pericarpium trichosanthis drug preparation.

10. A quality detection method for a trichosanthis pericarp drug preparation, characterized in that, It includes the step of comparing the characteristic fingerprint of the pericarpium trichosanthis product to be tested with the control characteristic fingerprint of the pericarpium trichosanthis drug preparation; the characteristic fingerprint of the pericarpium trichosanthis product to be tested is obtained by using the pericarpium trichosanthis product to be tested and constructing it according to the construction method described in any one of claims 1 - 6, and the control characteristic fingerprint of the pericarpium trichosanthis drug preparation is selected from any one of the following (1) - (3): (1) It has 12 common characteristic peaks. The peak corresponding to the orientin-7-O-glucoside reference peak is the S peak. The relative retention times of peaks 1, 2, 4 - 12 with respect to the S peak are within the range of ±10% of the specified values. The specified values are respectively: 0.48, 0.55, 1.10, 1.16, 1.44, 1.67, 1.80, 1.90, 2.05, 2.19, 2.27; (2) The characteristic fingerprint of the pericarpium trichosanthis drug preparation obtained by using a single batch or multiple batches of the pericarpium trichosanthis drug preparation and constructing it according to the construction method described in any one of claims 1 - 8; (3) The characteristic fingerprint obtained by using multiple batches of the pericarpium trichosanthis drug preparation and constructing it according to the construction method described in any one of claims 1 - 8 is made into a control characteristic fingerprint by the average value or median method.

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