Specific chromatogram construction method and application of corn stigma and medicinal preparation thereof

Abstract

The invention belongs to the technical field of quality detection of traditional Chinese medicine preparations, particularly relates to a specific chromatogram of a corn stigma medicinal material and a corn stigma medicinal preparation, and further discloses a construction method of the specific chromatogram and a quality detection method of the corn stigma medicinal material and the corn stigma medicinal preparation. According to the construction method of the characteristic chromatogram of the corn stigma medicinal material and the medicinal preparation of the corn stigma medicinal material, the corn stigma medicinal material and the medicinal preparation of the corn stigma medicinal material are taken as detection objects, the characteristic chromatogram method aiming at the medicinal preparation is established on the basis of high performance liquid chromatography, and the method is relatively high in precision and relatively good in stability and repeatability; the method can be used for comprehensively and quickly detecting the active ingredients and the contents thereof in the medicinal preparations such as the corn stigma formula granules.

Description

Technical Field

[0001] The present invention belongs to the technical field of quality inspection of traditional Chinese medicine preparations, and particularly relates to a characteristic fingerprint of corn stigma and corn stigma pharmaceutical preparations, and further discloses a method for constructing the characteristic fingerprint, and a method for quality inspection of corn stigma and corn stigma pharmaceutical preparations. Background Art

[0002] Traditional Chinese medicine has complex components, and the effective parts are often not single components. Using a single component as a quality control index is increasingly unable to meet the requirements of quality control of traditional Chinese medicine. Therefore, the technology of traditional Chinese medicine fingerprint has emerged as the times require. The technology of traditional Chinese medicine fingerprint originated from fingerprint identification science, and comprehensively reflects the types and quantities of chemical components contained in traditional Chinese medicine by using modern information technology and quality analysis means. The characteristic fingerprint of traditional Chinese medicine is obtained by identifying the common peaks that mark the group characteristics of various components. For Chinese medicinal materials, the characteristic fingerprint can be used to identify the authenticity and evaluate the quality; for Chinese patent medicines, the characteristic fingerprint can identify the authenticity of products, evaluate the rationality of the preparation process, and effectively control the product quality. At present, most of the effective components of traditional Chinese medicine are not clear, and the integrity and ambiguity of the characteristic fingerprint of traditional Chinese medicine just meet this characteristic, which is more scientific and comprehensive than the quality control method of single component. At present, the international community has recognized the use of the characteristic fingerprint of traditional Chinese medicine as a quality control mode for traditional Chinese medicine. High performance liquid chromatography has the advantages of high separation efficiency and fast analysis speed, and has become the main analysis means for characteristic fingerprints today.

[0003] Corn stigma is the dried style and stigma of Zea mays L. of the Gramineae family, mainly containing various components such as flavonoids, organic acids, nucleosides, amino acids, etc., and is mainly used for renal edema, dysuria, damp-heat jaundice, hypertension, diabetes. Corn stigma formula granules are obtained by extracting, concentrating and granulating traditional Chinese medicine corn stigma.

[0004] The quality control of corn stigma in the 1977 edition of the Chinese Pharmacopoeia includes items such as original plant variety, processing of decoction pieces, traits of decoction pieces, physical and chemical identification, etc. The literature also elaborates on the chemical components in corn stigma, including components such as organic acids, nucleosides, amino acids, etc. However, on the one hand, by measuring the content of the above single component or identifying corn stigma formula granules, the quality cannot be detected and controlled as a whole; on the other hand, by measuring the content of a single component in combination with the identification of other components of corn stigma formula granules, it is also time-consuming and laborious, and it is difficult to be widely applied in production practice. In addition, most of the existing technologies for quality control of corn stigma are aimed at corn stigma decoction pieces, rather than corn stigma pharmaceutical preparations, and the detection methods for corn stigma decoction pieces in the existing technologies are not applicable to corn stigma pharmaceutical preparations.

[0005] Therefore, there is an expectation in the art to develop a method that is comprehensively applicable to corn silk and its pharmaceutical preparations and can comprehensively and rapidly detect the flavonoid components of corn silk pharmaceutical preparations, which is of great significance for its comprehensive quality detection and overall quality control. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for constructing a characteristic chromatogram of corn silk medicinal materials and their pharmaceutical preparations to comprehensively reflect the internal quality and medication safety of corn silk medicinal materials and their pharmaceutical preparations;

[0007] The second technical problem to be solved by the present invention is to provide a method for quality detection of corn silk medicinal materials and their pharmaceutical preparations.

[0008] To solve the above technical problems, a method for constructing a characteristic chromatogram of corn silk and its pharmaceutical preparations according to the present invention includes the step of performing high-performance liquid chromatography detection on the test solution of corn silk medicinal materials and / or corn silk pharmaceutical preparations;

[0009] The chromatographic conditions include: using octadecylsilane-bonded silica gel as the filler, using acetonitrile as mobile phase A, and using 0.1% formic acid solution as mobile phase B, and performing gradient elution according to the following program:

[0010] 0 - 10 min, A:B is 7%:93% → 10%:90%;

[0011] 10 - 22 min, A:B is 10%:90% → 12%:88%;

[0012] 22 - 40 min, A:B is 12%:88% → 14%:86%;

[0013] 40 - 45 min, A:B is 14%:86%;

[0014] Preferably, the gradient elution further includes the following program:

[0015] 45 - 60 min, A:B is 14%:86% → 16%:84%;

[0016] 60 - 65 min, A:B is 16%:84%.

[0017] Specifically, for the method for constructing a characteristic chromatogram of corn silk and its pharmaceutical preparations, in the high-performance liquid chromatography detection step, the chromatographic conditions further include:

[0018] The column temperature is 20 - 30 °C, preferably 25 °C; and / or,

[0019] The flow rate is 0.1 - 0.5 mL / min, preferably 0.29 - 0.31 mL / min, more preferably 0.3 mL / min; and / or,

[0020] The detection wavelength is 330 nm; and / or,

[0021] The injection volume is 1-5 μL, preferably 3 μL.

[0022] Specifically, the characteristic spectrum construction method of corn silk and its pharmaceutical preparation, the preparation method of the test solution comprises: taking the test sample and adding an organic solvent for extraction, collecting the extract for solid-liquid separation, and collecting the filtrate to obtain.

[0023] Specifically, the method for constructing a characteristic spectrum of corn silk and its pharmaceutical preparation also includes the step of preparing a reference medicinal material solution, which specifically includes: taking corn silk reference medicinal material, adding water to perform heating reflux extraction, collecting the extract for solid-liquid separation, collecting the residue, adding an organic solvent for extraction, collecting the extract for solid-liquid separation, collecting the filtrate, and obtaining a reference medicinal material solution.

[0024] Specifically, the method for constructing the characteristic spectrum of corn silk and its pharmaceutical preparation further comprises the steps of preparing a reference substance solution and constructing the characteristic spectrum of the reference substance based on the high performance liquid chromatography method;

[0025] The reference substance includes ferulic acid;

[0026] The method for preparing the reference substance solution comprises the steps of adding the reference substance into an organic solvent and mixing;

[0027] The mass concentration of the reference substance solution is 1-20 μg of the reference substance per 1 ml.

[0028] Specifically, in the method for constructing the characteristic spectrum of corn silk and its pharmaceutical preparation, the organic solvent includes a methanol aqueous solution, preferably at a concentration of 30-80 v / v%, more preferably 50 v / v%;

[0029] Preferably, the extraction step comprises reflux extraction or ultrasonic extraction;

[0030] Preferably, the solid-liquid separation comprises filtration or centrifugation.

[0031] Specifically, the characteristic spectrum construction method of corn silk and its pharmaceutical preparation, the corn silk pharmaceutical preparation includes corn silk formula granules, corn silk decoction pieces, corn silk standard decoction or corn silk standard decoction freeze-dried powder.

[0032] The present invention also discloses a characteristic spectrum and / or a reference characteristic spectrum of a corn silk medicinal material and a pharmaceutical preparation thereof, wherein the characteristic spectrum or the reference characteristic spectrum of the corn silk medicinal material and a pharmaceutical preparation thereof is constructed by the method;

[0033] Preferably, there are 13 characteristic peaks presented in the characteristic chromatogram, which correspond to the retention times of 13 characteristic peaks in the reference chromatogram of the control medicinal material. Among them, peak 3 corresponds to the retention time of the corresponding reference substance peak of the reference substance; the peak corresponding to the retention time of the ferulic acid reference substance peak is peak S, and the relative retention times of each characteristic peak and peak S are calculated, and the relative retention times should be within the range of ±10% of the specified value; the specified values are:

[0034] Peak 1: 0.76, Peak 2: 0.90, Peak 4: 1.37, Peak 5: 1.75, Peak 6: 1.92, Peak 7: 2.08, Peak 8: 2.13, Peak 9: 2.19, Peak 10: 2.66, Peak 11: 3.07, Peak 12: 3.15, Peak 13: 3.51.

[0035] The present invention also discloses a method for constructing a characteristic chromatogram of the corn stigma officinalis medicinal material and its pharmaceutical preparations and / or the application of the characteristic chromatogram and / or the control characteristic chromatogram of the corn stigma officinalis medicinal material and its pharmaceutical preparations in the field of quality detection of the corn stigma officinalis medicinal material and its pharmaceutical preparations.

[0036] The present invention also discloses a method for quality detection of a corn stigma officinalis medicinal material and its pharmaceutical preparations, including the steps of constructing the characteristic chromatogram and the control characteristic chromatogram according to the method, and the step of comparing the characteristic chromatogram with the control characteristic chromatogram.

[0037] The method for constructing the characteristic chromatogram of the corn stigma officinalis medicinal material and its pharmaceutical preparations according to the present invention takes the corn stigma officinalis medicinal material and its pharmaceutical preparations as the detection object, and establishes a characteristic chromatogram method for the pharmaceutical preparation based on high performance liquid chromatography. The method for constructing the characteristic chromatogram according to the present invention has good resolution, can simultaneously determine the contents of 4-coumaric acid, wicenin-2, ferulic acid, isoorientin 2''-O-rhamnoside, chrysoeriol 7-O-glucoside-2-O-apioside, N-trans-feruloyltyramine, and the method has high precision, good stability and repeatability, and can comprehensively and rapidly detect the active ingredients and their contents in pharmaceutical preparations such as corn stigma officinalis formula granules.

[0038] The method for constructing the characteristic chromatogram of the corn stigma officinalis medicinal material and its pharmaceutical preparations according to the present invention selects ferulic acid as the internal reference peak in the fingerprint chromatogram, calibrates 13 common characteristic peaks by using the traditional Chinese medicine chromatographic fingerprint, and calculates the relative retention times of each common characteristic peak according to peak S ferulic acid, which is beneficial to the comprehensive quality detection and overall quality control of the corn stigma officinalis formula granules, thereby contributing to improving the safety and stability of the use of the drug.

[0039] The method for constructing the characteristic chromatogram of the corn stigma officinalis medicinal material and its pharmaceutical preparations according to the present invention is applicable to the construction of characteristic chromatograms of pharmaceutical preparations such as corn stigma officinalis decoction pieces, freeze-dried powder of standard corn stigma officinalis decoction, and corn stigma officinalis formula granules, and can realize the quality detection and quality control of the above preparations.

[0040] The present invention has established a method for determining the characteristic chromatogram (high performance liquid chromatography) of corn stigma formula granules. The sample preparation method is simple to make, the chromatographic conditions are easy to achieve, and the results of the analysis method verification show that the method is simple to operate, the results are accurate, and the method has good reproducibility. Description of the Drawings

[0041] In order to make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to the specific embodiments of the present invention and in conjunction with the drawings, wherein,

[0042] Figure 1 It is the chromatogram under different elution procedures in Example 2;

[0043] Figure 2 It is the chromatogram under different detection wavelengths in Example 2;

[0044] Figure 3 It is the chromatogram under different mobile phase systems in Example 2;

[0045] Figure 4 It is the chromatogram under different column temperatures in Example 2;

[0046] Figure 5 It is the chromatogram under different flow rates in Example 2;

[0047] Figure 6 It is the chromatogram under different chromatographic columns in Example 2;

[0048] Figure 7 It is the chromatogram under different extraction solvents in Example 3;

[0049] Figure 8 It is the chromatogram under different extraction methods in Example 3;

[0050] Figure 9 It is the chromatogram under different extraction times in Example 3;

[0051] Figure 10 It is the chromatogram under different extraction powers in Example 3;

[0052] Figure 11 It is the chromatogram under different extraction solvent volumes in Example 3;

[0053] Figure 12 It is the chromatogram of ferulic acid reference substance in Example 4;

[0054] Figure 13 It is the chromatogram of the test sample of corn stigma formula granules in Example 4;

[0055] Figure 14 It is the characteristic chromatogram of the reference medicinal material of corn stigma in Example 5;

[0056] Figure 15 Chromatograms of different reference substances in Example 5;

[0057] Figure 16 Characteristic reference chromatogram of the corn stigma formula granules in Example 5;

[0058] Figure 17 Negative blank chromatogram in Example 6;

[0059] Figure 18 Chromatograms under different chromatographic columns in Example 6;

[0060] Figure 19 Chromatograms under different liquid phase apparatuses in Example 6;

[0061] Figure 20 Characteristic chromatogram of 20 batches of corn stigma formula granules in Example 7. Detailed implementation manners

[0062] In the following examples of the present invention, the instruments and reagents involved include:

[0063] High performance liquid chromatograph 1: Waters ACQUITY UPLC I-Class chromatographic system, including a quaternary solvent manager (ACQ-QSM), an autosampler (ACQ-FTN), an imported chromatographic column oven (ACQ-CM), a diode array ultraviolet detector (ACQ-TUV), and an Empower chromatographic management system;

[0064] High performance liquid chromatograph 2: Thermo Fisher Vanquish chromatographic system, including an LPG-3400A quaternary pump, a WPS-3000TSL autosampler, a PDA diode array detector, and a chromatographic workstation;

[0065] One ten-thousandth electronic balance (Shanghai Shunyu Hengping Scientific Instrument Co., Ltd., FA1004);

[0066] One hundred-thousandth electronic balance (Sartorius Scientific Instrument Co., Ltd., SQP);

[0067] Ultrasonic cleaner: WB400US, Shanghai Wangbiao Instrument Co., Ltd.;

[0068] Chromatographic column 1: Shim-pack GIST-HP C18-AQ (2.1×100 mm, 1.9 μm);

[0069] Chromatographic column 2: ACQUITY UPLC HSS T3 (2.1×100 mm, 1.8 μm);

[0070] Chromatographic column 3: Poroshell 120SB-C18 (2.1×100 mm, 1.9 μm);

[0071] 4-Coumaric acid reference substance (Batch number: 112037-202102, provided by the National Institutes for Food and Drug Control);

[0072] Vicenin-2 reference substance (Batch number: 112096-202201, provided by the National Institutes for Food and Drug Control);

[0073] Ferulic acid reference substance (Batch number: 110773-202316, provided by the National Institutes for Food and Drug Control);

[0074] Isorientin 2''-O-rhamnoside reference substance (Batch number: 005375-202402, provided by Jiangxi Baicaoyuan Biotechnology Co., Ltd.);

[0075] Apigenin-7-O-glucoside-2-O-apioside: (Batch number: 110085-202407, provided by Shanghai Hongyong Biotechnology Co., Ltd.);

[0076] N-trans-Feruloyltyramine (Batch number: 001151-202201, provided by Jiangxi Baicaoyuan Biotechnology Co., Ltd.);

[0077] Corn silk reference medicinal material (Batch number: 121659-202103, provided by the National Institutes for Food and Drug Control);

[0078] Acetonitrile is of chromatographic grade, and water is ultrapure water; other reagents are of analytical grade.

[0079] In the following examples of the present invention, the corn silk pharmaceutical preparation can be prepared according to the processes known in the art or can be detected based on commercially available products.

[0080] As an exemplary preparation protocol, the corn stigma pharmaceutical preparation can be prepared by the following method: Take corn stigma, heat under reflux for extraction at least once, each time adding 10 - 15 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 according to conventional processes, prepare clinically acceptable tablets, capsules, pills, granules, honeyed pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations or injection preparations. For example, 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, crosslinked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, crosslinked carboxymethyl cellulose sodium, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc powder, silicon dioxide, 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.

[0081] As an exemplary embodiment, the test samples selected in the following examples of the present invention include corn stigma formula granules, and the specific preparation method of the corn stigma formula granules can be: Take corn stigma, heat under reflux for extraction 2 times, the first time adding 15 times the weight of water to soak for 30 min, heat under reflux for extraction for 0.5 h, filter, the second time adding 12 times the weight of water for extraction for 0.5 h, filter, combine the filtrates, concentrate the filtrates to a relative density of 1.05 g / mL at 60 °C, carry out spray drying, add the excipient maltodextrin to the dry powder, mix evenly and then carry out dry granulation to prepare granules.

[0082] In the following examples of the present invention, the information on corn stigma formula granules is shown in Table 1 below.

[0083] Table 1 Information on corn stigma formula granules

[0084] Batch number Place of origin 1903001W Guangyuan City, Sichuan Province 1906001S Guangyuan City, Sichuan Province 1909001W Guangyuan City, Sichuan Province 1912001W Shangluo City, Shaanxi Province 2001001S Shangluo City, Shaanxi Province 2002001W Shangluo City, Shaanxi Province 2005001S Lower Village, Jiefang Village, Qiubei County, Yunnan Province 2008001W Pojiao Town, Maguan County, Wenshan City, Yunnan Province 2011001S Xiangyang District, Xiangyang City, Hubei Province 2102001S Xiangyang District, Xiangyang City, Hubei Province 2105001S Xiangyang District, Xiangyang City, Hubei Province 2108001W Zhecheng County, Shangqiu City, Henan Province 2110001S Zhecheng County, Shangqiu City, Henan Province 2112001S Zhecheng County, Shangqiu City, Henan Province 2202001W Anqiu City, Weifang City, Shandong Province 2206001W Anqiu City, Weifang City, Shandong Province 2212001S Anqiu City, Weifang City, Shandong Province 2309001W Anqiu City, Weifang City, Shandong Province 2311001S Anqiu City, Weifang City, Shandong Province 2312001W Anqiu City, Weifang City, Shandong Province

[0085] Example 1

[0086] In this example, a fingerprint of flavonoids in corn stigma and its pharmaceutical preparation was constructed.

[0087] Preparation of the test solution: Take about 0.5 g of the corn stigma formula granule powder, place it in a stoppered conical flask, accurately add 10 ml of 50% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, take it out, let it cool, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0088] Preparation of the reference solution: Take 4 g of the reference crude drug of corn stigma, place it in a stoppered conical flask, add 100 ml of water, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, dissolve the residue in 10 ml of 50% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, take it out, let it cool, shake well, filter, and take the subsequent filtrate as the reference solution of the reference crude drug.

[0089] Take an appropriate amount of ferulic acid reference substance, dissolve it in methanol to prepare a solution containing 10 μg per 1 ml as the reference solution of the reference substance.

[0090] Example 2

[0091] This example is based on the establishment of chromatographic conditions under different conditions.

[0092] (1) Selection of gradient

[0093] In this example, referring to different chromatographic conditions, octadecylsilane chemically bonded silica gel is used as the filler; acetonitrile is used as mobile phase A, and 0.1% formic acid solution is used as mobile phase B, and gradient elution is carried out according to the regulations in Table 2 below; the flow rate is 0.3 ml per minute; the column temperature is 25 °C; the detection wavelength is 330 nm.

[0094] Table 2 Gradient program

[0095]

[0096] In this example, the chromatograms formed under different gradient elution programs (gradient 1 - gradient 3) are respectively shown as (a) - (c) in the appendix, and the results of the system suitability parameters under each condition are shown in Table 3 below. Figure 1 as shown in (a) - (c) below, and the results of the system suitability parameters under each condition are shown in Table 3 below.

[0097] Table 3 System suitability parameters

[0098]

[0099]

[0100] It can be seen that the characteristic peak information amount is relatively large and the resolution is good under the condition of gradient 3. Therefore, the mobile phase gradient elution program is tentatively selected as condition 3.

[0101] (2) Selection of detection wavelength

[0102] The test solution of the corn stigma formula granules (batch number: 2202001W) was scanned at full wavelength. The results showed that there was more characteristic peak information at 4 different absorption wavelengths (270nm, 290nm, 310nm, 330nm). Therefore, the chromatograms and system suitability parameters at these 4 wavelengths were further evaluated.

[0103] In this example, the chromatograms at different detection wavelengths (270nm, 290nm, 310nm, 330nm) are shown in Appendix Figure 2 as shown in (a)-(d) below, and the results of the system suitability parameters are shown in Table 4 below.

[0104] Table 4 System suitability parameters

[0105]

[0106]

[0107]

[0108] The results showed that when the wavelength was 330nm, the peak responses of each characteristic peak were larger and the system suitability parameters were relatively better. Therefore, 330nm was determined as the detection wavelength for the characteristic chromatogram of the corn stigma formula granules.

[0109] (3) Selection of mobile phase

[0110] In this example, the separation effects of the characteristic chromatograms of the test solution of the corn stigma formula granules were compared among three mobile phase systems: acetonitrile - 0.1% formic acid, acetonitrile - 0.1% acetic acid, and acetonitrile - 0.1% phosphoric acid solution.

[0111] In this example, the chromatograms under different mobile phases (acetonitrile - 0.1% formic acid, acetonitrile - 0.1% acetic acid, acetonitrile - 0.1% phosphoric acid solution) are shown in Appendix Figure 3 as shown in (a)-(c) below.

[0112] The results showed that the separation effect of the mobile phase system of acetonitrile - 0.1% formic acid solution was better and the peak information was more abundant. Therefore, acetonitrile - 0.1% formic acid solution was selected as the system mobile phase.

[0113] (4) Selection of different column temperatures

[0114] In this example, the effects of different column temperatures on the durability of the characteristic chromatogram of the corn stigma formula granules were compared. The column temperatures were set at 20°C, 25°C, and 30°C. An appropriate amount of the corn stigma formula granules (batch number: 2202001W) was taken to observe the effects of different column temperatures on the separation effect of the test solution of the corn stigma formula granules.

[0115] In this example, the chromatograms at different column temperatures (20°C, 25°C, 30°C) are shown in AppendixFigure 4 As shown in (a)-(c) below, the investigation results at different column temperatures are shown in Table 5 below.

[0116] Table 5 Investigation Results at Different Column Temperatures

[0117]

[0118]

[0119]

[0120] The results show that the separation effect of each chromatographic peak at a column temperature of 25°C is relatively good. Therefore, the column temperature is tentatively set at 25°C.

[0121] (5) Selection of Different Flow Rates

[0122] In this example, the effects of different flow rates on the durability of the characteristic chromatogram of the corn silk formula granules were compared. The flow rates were set as follows: 0.2 mL / min, 0.25 mL / min, and 0.3 mL / min. An appropriate amount of the corn silk formula granules (batch number: 2202001W) was taken and determined by the method determined above to observe the effects of different flow rates on the separation effect of the test samples of the corn silk formula granules.

[0123] In this example, the chromatograms at different flow rates (0.2 mL / min, 0.25 mL / min, 0.3 mL / min) are shown in Appendix Figure 5 As shown in (a)-(c) below, the investigation results at different flow rates are shown in Table 6 below.

[0124] Table 6 Investigation Results at Different Flow Rates

[0125]

[0126]

[0127] The results show that the separation effect of each chromatographic peak is relatively good when the flow rate is 0.3 mL / min. Therefore, the flow rate is tentatively set at 0.3 mL / min.

[0128] (6) Selection of Different Chromatographic Columns

[0129] Take the same test solution of corn stigma formula granules (2202001W), and determine the characteristic chromatogram on different types of chromatographic columns (Chromatographic column 1: Shim-pack GIST-HP C18-AQ, 2.1×100 mm, 1.9 μm; Chromatographic column 2: ACQUITY UPLC HSS T3, 2.1×100 mm, 1.8 μm; Chromatographic column 3: Poroshell 120SB-C18, 2.1×100 mm, 1.9 μm) respectively, and compare the chromatograms and relative retention time results measured on different types of chromatographic columns.

[0130] In this example, the chromatograms under different chromatographic columns (Chromatographic column 1, Chromatographic column 2, Chromatographic column 3) are shown in the appendix Figure 6 , and the investigation results of chromatographic columns of different brands are shown in Table 7 below.

[0131] Table 7 Investigation Results of Chromatographic Columns of Different Brands

[0132]

[0133]

[0134]

[0135] It was found that the Poroshell 120SB-C18 chromatographic column had better separation effect, and there were significant differences in relative retention time among chromatographic columns of different brands. Therefore, the Poroshell 120SB-C18, 2.1 * 100 mm, 1.9 μm chromatographic column was selected.

[0136] Determination of chromatographic conditions

[0137] To sum up, the chromatographic conditions and system suitability test confirmed in this example are as follows: using octadecylsilane chemically bonded silica gel as the filler (column length is 100 mm, column inner diameter is 2.1 mm, particle size is 1.9 μm), using acetonitrile as mobile phase A, using 0.1% formic acid solution as mobile phase B, and performing gradient elution according to the procedure specified in Table 8 below; the column temperature is 25 °C, the flow rate is 0.3 ml per minute, and the detection wavelength is 330 nm. The number of theoretical plates calculated based on the ferulic acid peak should not be less than 5000.

[0138] Table 8 Elution Procedure

[0139] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～10 7→10 93→90 10～22 10→12 90→88 22～40 12→14 88→86 40～45 14 86 45～60 14→16 86→84 60～65 16 84

[0140] Precisely pipette 3 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and measure to obtain.

[0141] Example 3

[0142] Based on the chromatographic conditions determined in the foregoing Example 2, this example further examines the preparation method of the test solution.

[0143] (1) Selection of extraction solvent

[0144] Referring to the preparation method of the test solution in the foregoing Example 1, this example examines the extraction effects of different extraction solvents, namely methanol, 50% methanol, and water, on the corn silk formula granules. By comparing the test solution chromatograms and system suitability parameters obtained with different solvents, a suitable extraction solvent is selected.

[0145] In this example, the chromatograms under different extraction solvents (water, 50% methanol, methanol) are shown in the appendix Figure 7 , and the examination results under different solvents are shown in Table 9 below.

[0146] Table 9 Examination results of different extraction methods

[0147]

[0148]

[0149]

[0150] The results show that the system suitability parameters of the test solution extracted with 50% methanol are relatively better, so 50% methanol is selected as the extraction solvent.

[0151] (2) Selection of extraction method

[0152] Referring to the preparation method of the test solution in Example 1, this example examines the extraction effects of ultrasonic extraction and reflux on the corn silk formula granules. By comparing the test solution chromatograms and system suitability parameters obtained with different extraction methods, a suitable extraction method is selected.

[0153] In this example, the chromatograms under different extraction methods (reflux, ultrasonic) are shown in the appendix Figure 8 , and the examination results under different extraction methods are shown in Table 10 below.

[0154] Table 10 Examination results of different extraction methods

[0155]

[0156]

[0157] The results show that the system suitability parameters of the test solution extracted by ultrasonic extraction and reflux are both good. Considering that ultrasonic extraction is easy to operate, ultrasonic extraction is selected as the extraction method.

[0158] (3) Selection of extraction time

[0159] In this example, referring to the preparation method of the test solution in Example 1, the extraction effects of different extraction times (15 minutes, 30 minutes, 45 minutes) on the corn stigma formula granules were investigated respectively. By comparing the chromatograms of the test solutions obtained at different extraction times and the system suitability parameters, the appropriate extraction time was selected.

[0160] In this example, the chromatograms at different extraction times (15 minutes, 30 minutes, 45 minutes) are shown in the appendix Figure 9 , and the investigation results at different extraction times are shown in Table 11 below.

[0161] Table 11 Investigation Results at Different Extraction Times

[0162]

[0163]

[0164] The results show that the system suitability parameters of the test solution are relatively better when the extraction time is 30 minutes. Therefore, 30 minutes was selected as the extraction time.

[0165] (4) Selection of Extraction Power

[0166] In this example, referring to the preparation method of the test solution in Example 1, the extraction effects of different ultrasonic extraction powers (100W, 250W, 500W) on the corn stigma formula granules were investigated respectively. By comparing the chromatograms of the test solutions obtained by ultrasonic extraction at different powers and the system suitability parameters, the appropriate ultrasonic extraction power was selected.

[0167] In this example, the chromatograms at different extraction powers (100W, 250W, 500W) are shown in the appendix Figure 10 , and the investigation results at different extraction powers are shown in Table 12 below.

[0168] Table 12 Investigation Results at Different Extraction Powers

[0169]

[0170]

[0171] The results show that the system suitability parameters of the test solution are relatively better when the ultrasonic extraction power is 250W. Therefore, 250W was selected as the ultrasonic extraction power.

[0172] (5) Selection of Extraction Solvent Volume

[0173] In this example, referring to the preparation method of the test solution in Example 1, the extraction effects of different extraction solvent volumes (10ml, 25ml, 50ml) on the freeze-dried powder were investigated respectively. By comparing the chromatograms of the test solutions obtained with different extraction solvent volumes and the system suitability parameters, the appropriate sampling volume was selected.

[0174] In this example, the chromatograms under different extraction solvent volumes (10 ml, 25 ml, 50 ml) are shown in the appendix Figure 11 , and the investigation results under different extraction solvent volumes are shown in Table 13 below

[0175] Table 13 Investigation Results of Different Extraction Solvent Volumes

[0176]

[0177]

[0178]

[0179] The results show that the system suitability parameters of the test sample are relatively better when the different extraction solvent volume is 10 ml. Therefore, 10 ml is selected as the different extraction solvent volume

[0180] Confirmation of preparation method of test sample

[0181] In summary, the preparation method of the test solution for the corn stigma formula granules optimized by the present invention is as follows: Take about 0.5 g of the powder of this product, place it in a stoppered conical flask, accurately add 10 ml of 50% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, take it out, let it cool, shake well, filter, and take the continuous filtrate to obtain the solution

[0182] Example 4

[0183] In this example, the fingerprint of flavonoids in corn stigma and its pharmaceutical preparations is constructed based on the previously determined conditions

[0184] Preparation of the test solution: Take about 0.5 g of the powder of the corn stigma formula granules, place it in a stoppered conical flask, accurately add 10 ml of 50% methanol, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, take it out, let it cool, shake well, filter, and take the continuous filtrate to obtain the solution

[0185] Preparation of the reference solution: Take 4 g of the reference crude drug of corn stigma, place it in a stoppered conical flask, add 100 ml of water, heat under reflux for 1 hour, filter, evaporate the filtrate to dryness, add 10 ml of 50% methanol to the residue, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, take it out, let it cool, shake well, filter, and take the continuous filtrate as the reference solution of the reference crude drug

[0186] Take an appropriate amount of ferulic acid reference substance, add methanol to make a solution containing 10 μg per 1 ml as the reference solution of the reference substance

[0187] High performance liquid UPLC chromatographic analysis conditions

[0188] Chromatographic column: Packed with octadecylsilyl silica gel (column length 100 mm, column inner diameter 2.1 mm, particle size 1.9 μm), using acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B, gradient elution was carried out according to the regulations in Table 8 above; the column temperature was 25 °C, the flow rate was 0.3 ml per minute, and the detection wavelength was 330 nm. The number of theoretical plates calculated by the ferulic acid peak should be not less than 5000.

[0189] Precisely pipette 3 μl each of the reference substance solution and the test solution, inject into the liquid chromatograph, and determine to obtain the results.

[0190] This example is based on the method established above, using the fingerprint similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission, to generate the chromatogram of ferulic acid reference substance, that is, the reference characteristic fingerprint, as Figure 12 shown, and the chromatogram of the test solution of the corn silk formula granules is as attached Figure 13 shown. According to the reference characteristic fingerprint, the detection results of the characteristic fingerprint of the formula granules can be analyzed and compared for the quality control of the formula granules.

[0191] In this example, the results of the system adaptability parameters are shown in Table 14 below.

[0192] Table 14 System adaptability parameters

[0193]

[0194] Selection of main chemical components in characteristic chromatogram

[0195] According to the existing literature reports, the main active ingredients of corn silk are flavonoid components, and also contain polysaccharides, organic acids, sterols, amino acids and other compounds. The efficacy of corn silk is diuretic and detumescence, and lowering blood pressure. It is used for nephritis edema, dysuria, damp-heat jaundice, and hypertension. Existing research mainly focuses on the total flavonoids, polysaccharides and organic acids of corn silk. Through the analysis of the flavonoid components of the corn silk formula granules, it is found that 4-coumaric acid, ferulic acid, vicenin-2, isoorientin 2”-O-rhamnoside, chrysoeriol-7-O-glucoside-2-O-apioside, N-trans-feruloyltyramine, etc. are all detected, and the peaks of 4-coumaric acid and ferulic acid have higher responses and better resolution. Therefore, the main active ingredient flavonoid component is selected as the main developed component of the characteristic fingerprint of the corn silk formula granules.

[0196] As can be seen from the above characteristic chromatogram, there are 13 chromatographic peaks in the UPLC characteristic fingerprint chromatogram of the corn stigma formula granule, among which 6 peaks (peak 1, 2, 3, 4, 11, 13) are peaks of known components; the peak corresponding to the ferulic acid reference substance peak is selected as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and its relative retention time should be within the range of ±10% of the specified value. The specified values are: 0.76 (peak 1), 0.90 (peak 2), 1.37 (peak 4), 1.75 (peak 5), 1.92 (peak 6), 2.08 (peak 7), 2.13 (peak 8), 2.19 (peak 9), 2.66 (peak 10), 3.07 (peak 11), 3.15 (peak 12), 3.51 (peak 13).

[0197] It can be seen that through the detection method in this example, a fingerprint chromatogram with good resolution of each characteristic peak can be effectively obtained, and the contents of 4-coumaric acid, vicenin-2, ferulic acid, isoorientin 2''-O-rhamnoside, chrysoeriol 7-O-glucoside-2-O-apioside, and N-trans-feruloyltyramine can be simultaneously determined. Moreover, by selecting ferulic acid as the internal reference peak in the fingerprint chromatogram, the relative retention times of the common characteristic peaks 1, peak 2, and peaks 4-13 of the corn stigma formula granule can be determined. Therefore, the corn stigma formula granule can be comprehensively and rapidly detected, which is beneficial to the comprehensive quality detection and overall quality control of the corn stigma formula granule, thereby contributing to improving the safety and stability of the drug use.

[0198] Example 5

[0199] Confirmation of characteristic peaks and identification of characteristic peaks in characteristic chromatogram

[0200] Based on the method established above, this example analyzes the detection results of the fingerprint chromatograms of multiple batches of test samples, and uses the fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Chromatogram Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission to generate a reference characteristic chromatogram.

[0201] In this example, through the identification and assignment of characteristic peaks, there are 13 chromatographic peaks in the UPLC characteristic chromatogram of the corn stigma formula granule. The remaining chromatographic peaks are all chromatographic peaks with relatively small responses or poor resolution. Therefore, only 13 chromatographic peaks with relatively large responses are selected as characteristic peaks, and the chromatographic peaks are rearranged according to the order of the chromatographic peaks. The characteristic chromatogram of the corn stigma reference medicinal material is as attached Figure 14 .

[0202] Based on the results of peak identification and reference substance localization, six known peaks, namely 4-coumaric acid (peak 1), vicenin-2 (peak 2), ferulic acid (peak 3), isoorientin 2''-O-rhamnoside (peak 4), chrysoeriol 7-O-glucoside-2-O-apioside (peak 11), and N-trans-feruloyltyramine (peak 13), were confirmed to be flavonoid and organic acid components. This characteristic chromatogram contains the main chemical components of corn silk.

[0203] Basis for selection of characteristic peak S in characteristic chromatogram

[0204] According to the results of peak identification and peak selection of characteristic peaks in the corn silk formula granules, in the characteristic chromatogram of the corn silk formula granules, the response of flavonoid components is relatively low, while the response of ferulic acid is relatively high and the peak emergence time is moderate. It belongs to organic acid components and is one of the main active components of corn silk. It is temporarily specified that the peak corresponding to the ferulic acid reference substance is the S peak, and the relative retention times of peaks 1-2 and peaks 4-13 are calculated.

[0205] Confirmation of characteristic peaks in characteristic chromatogram

[0206] Through the study of reference substance localization by UPLC, in the UPLC chromatogram of the corn silk formula granules (as shown in the appendix Figure 13 ), the molecular weights and molecular structures of peaks 1-14 were analyzed by LC / MS / MS to provide a reference basis for the subsequent qualitative analysis of characteristic component peaks. Four of the peaks were identified as known chromatographic peaks by reference substances: peak 1 (4-coumaric acid), peak 2 (vicenin-2), peak 3 (ferulic acid), peak 4 (isoorientin 2''-O-rhamnoside), peak 11 (chrysoeriol 7-O-glucoside-2-O-apioside), and peak 13 (N-trans-feruloyltyramine). The LC / MS / MS analysis results are shown in Table 15 below.

[0207] Among them, the chromatograms of 4-coumaric acid, vicenin-2, ferulic acid, isoorientin 2''-O-rhamnoside, chrysoeriol 7-O-glucoside-2-O-apioside, and N-trans-feruloyltyramine are shown in (a)-(f) of the appendix Figure 15 respectively.

[0208] Table 15 LC / MS / MS Analysis Results of Corn Silk Formula Granules

[0209]

[0210]

[0211] Specified value of characteristic peaks in characteristic chromatogram

[0212] The relative retention time of the above characteristic chromatogram is determined according to the research results: there should be 13 characteristic peaks in the characteristic chromatogram of the corn stigma formula granules, and they should correspond to the retention times of the corresponding characteristic peaks of the control medicinal material. The results are shown in Tables 16 - 17 below.

[0213] In this example, the control chromatogram of the corn stigma formula granules is as attached Figure 16 as shown. Among them, Peak 1: 4 - Coumaric acid; Peak 2: Vicenin - 2; Peak 3(S): Ferulic acid; Peak 4: Isorientin 2'' - O - rhamnoside; Peak 11: Chrysoeriol - 7 - O - glucoside - 2 - O - apioside; Peak 13: N - trans - Feruloyltyramine. Chromatographic column: Agilent Poroshell 120SB - C18, 2.1 * 100 mm, 1.9 μm.

[0214] Table 16 Relative retention time of the control chromatogram of the corn stigma formula granules

[0215] Peak 1 2 3(S) 4 5 6 7 Retention time 12.752 15.203 16.874 23.081 29.548 32.436 35.107 Relative retention time 0.76 0.90 1.00 1.37 1.75 1.92 2.08 Peak 8 9 10 11 12 13 Retention time 35.951 36.932 44.817 51.751 53.149 59.245 Relative retention time 2.13 2.19 2.66 3.07 3.15 3.51

[0216] Table 17 Relative peak area of the control chromatogram of the corn stigma formula granules

[0217] Peak 1 2 3(S) 4 5 6 7 Peak area 494.286 80.432 899.421 43.711 602.122 244.615 146.996 Relative peak area 0.55 0.09 1.00 0.05 0.67 0.27 0.16 Peak 8 9 10 11 12 13 Peak area 69.774 94.547 94.808 75.97 253.849 33.219 Relative peak area 0.08 0.11 0.11 0.08 0.28 0.04

[0218] Example 6

[0219] In this example, the methodological investigation of the characteristic chromatogram is carried out based on the previously determined construction method.

[0220] (1) Verification of specificity, integrity and system suitability

[0221] Prepare the test solution and the negative blank solution according to the previous method, and perform UPLC analysis according to the [Characteristic Chromatogram] method of the corn stigma formula granules to verify whether the chromatographic conditions and system suitability of the corn stigma formula granules are applicable to the corn stigma formula granules, and investigate whether the negative samples of the corn stigma formula granules will cause interference. The results are shown in the attachment Figure 17 .

[0222] It can be seen from the results that the negative blank control has no interference on the characteristic chromatogram, and the system suitability and specificity of the chromatographic method are good, which can be used for the detection of the characteristic chromatogram of the corn stigma formula granules.

[0223] (2) Instrument precision

[0224] Take the test solution of the corn stigma formula granules (2202001W), inject samples repeatedly 6 times according to the method under the characteristic chromatogram, and calculate that the RSD of the relative retention time of the characteristic peaks should be less than 2.0%. The results are shown in Tables 18 - 19 below.

[0225] Table 18 Results of relative retention time in the instrument precision test

[0226]

[0227] Table 19 Results of Relative Peak Areas in Instrument Precision Test

[0228]

[0229]

[0230] The results showed that the instrument precision was good.

[0231] (3) Repeatability

[0232] Take the sample of corn stigma formula granules (batch number: 2202001W), prepare 6 portions of test solutions in parallel according to the method under the characteristic chromatogram and determine. The RSD of the relative retention time of each characteristic peak should be less than 2.0%. The results are shown in the following Tables 20 - 21.

[0233] Table 20 Results of Relative Retention Times in Repeatability Test

[0234]

[0235] Table 21 Results of Relative Peak Areas in Repeatability Test

[0236]

[0237]

[0238] The results showed that the method repeatability was good.

[0239] (4) Intermediate Precision (Personnel)

[0240] Three experimenters respectively prepared the test solutions of corn stigma formula granules (2202001W) according to the characteristic chromatogram method and determined. The RSD of the relative retention time of each characteristic peak should be less than 2.0%. The results are shown in the following Tables 22 - 23.

[0241] Table 22 Relative Retention Times in Intermediate Precision (Personnel)

[0242]

[0243] Table 23 Relative Peak Areas in Intermediate Precision (Personnel)

[0244]

[0245] The results showed that the intermediate precision was good.

[0246] (5) Robustness - Stability Investigation

[0247] Take the test solution of corn stigma formula granules (2202001W), and determine it according to the method under the characteristic chromatogram at 0h, 4h, 8h, 12h, 18h, and 24h respectively. The RSD of the relative retention time of each characteristic peak should be less than 2.0%. The results are shown in Tables 24 - 25 below.

[0248] Table 24 Results of relative retention time in the stability test

[0249]

[0250] Table 25 Results of relative peak area in the stability test

[0251]

[0252] The results show that the test solution is stable within 24 hours and meets the determination requirements.

[0253] (6) Robustness - Investigation at different column temperatures

[0254] Compare the effects of different column temperatures on the characteristic chromatogram of corn stigma formula granules (2202001W). The column temperatures are set at 24°C, 25°C, and 26°C. Take an appropriate amount of corn stigma formula granules and determine them according to the method determined above. Observe the effects of different column temperatures on the separation effect of the test solution of corn stigma formula granules. The results are shown in Tables 26 - 27 below.

[0255] Table 26 Results of relative retention time at different column temperatures

[0256]

[0257] Table 27 Results of relative peak area at different column temperatures

[0258]

[0259] The results show that the column temperature with small fluctuations has little effect on the specified values of the relative retention time of each characteristic peak, indicating that this method has good robustness to small fluctuations in temperature.

[0260] (7) Robustness - Investigation at different flow rates

[0261] Compare the effects of different flow rates on the robustness of the characteristic chromatogram of corn stigma formula granules (2202001W). The flow rates are set at 0.29 mL / min, 0.30 mL / min, and 0.31 mL / min respectively. Take an appropriate amount of corn stigma formula granules and determine them according to the method determined above. Observe the effects of different flow rates on the separation effect of the test solution of corn stigma formula granules. The results are shown in Tables 28 - 29 below.

[0262] Table 28 Results of relative retention time at different flow rates

[0263]

[0264] Table 29 Results of relative peak areas at different flow rates

[0265]

[0266] The results show that small fluctuations in the flow rate have little impact on the specified values of the relative retention times of each characteristic peak, indicating that this method has good durability against small fluctuations in the flow rate.

[0267] (8) Durability - Investigation of different batches of chromatographic columns

[0268] Take the test solution of the same corn silk formula granules (2202001W), and use the same model chromatographic columns produced by the same manufacturer in different batches (brand model: Agilent Poroshell 120SB-C18 (100mm×2.1mm, 1.9μm); chromatographic column 1: S / N: USKEC01239; chromatographic column 2: S / N: USKEC01312; chromatographic column 3: S / N: USKEC01527) for determination. The results are shown in Tables 30 - 31 below. The chromatograms under different chromatographic columns (chromatographic columns 1, 2, and 3) are respectively shown in Appendix Figure 18 as shown in (a)-(c).

[0269] Table 30 Results of relative retention times for the investigation of different brand chromatographic columns

[0270]

[0271] Table 31 Results of specified values of relative peak areas for the investigation of different brand chromatographic columns

[0272]

[0273] The results show that the specified values of the relative retention times of each characteristic peak fluctuate little for the chromatographic columns of this manufacturer in different batches, indicating that this method has good durability for different batches of this model of chromatographic columns.

[0274] (9) Durability - Investigation of different liquid phase equipment

[0275] Compare the effects of different brand instruments on the durability of the characteristic chromatograms of corn silk formula granules (2202001W). The instrument brands include: Waters Arc of Waters and Vanquish of Thermo Fisher. Take an appropriate amount of corn silk formula granules and perform the determination according to the method determined above. Observe the effects of different instruments on the separation effect of the test solution of corn silk formula granules. The results are shown in Tables 32 - 33 below. The chromatograms under different liquid phase equipment (Waters Arc, Vanquish of Thermo Fisher) are respectively shown in Appendix Figure 19 as shown in (a)-(b).

[0276] Table 32 Results of relative retention times of instruments of different brands

[0277]

[0278] Table 33 Results of relative peak areas of instruments of different brands

[0279]

[0280] The results show that the specified values of the relative retention times of the characteristic peaks by liquid chromatographs of different brands have little influence.

[0281] To sum up, from the results of the above-mentioned methodology investigation in this example, among the 13 common peaks of the characteristic chromatogram of the corn stigma formula granules established in the present invention, factors such as different high-performance liquid chromatographs, column temperatures, flow rates, and chromatographic columns all have a certain degree of influence on each characteristic chromatogram. The relative retention time values are within the range of ±10%. To adapt to its durability, it is recommended to control the specified value range within ±10%. The influence of the remaining chromatographic conditions changes little.

[0282] Example 7

[0283] Based on the method established above, this example measures the characteristic chromatograms of 20 batches of corn stigma formula granules.

[0284] Take 20 batches of corn stigma formula granules and operate according to the established method for measuring the characteristic chromatogram of corn stigma formula granules. The results are shown in Tables 34 - 37 below. The characteristic chromatograms of 20 batches of corn stigma formula granules are as attached Figure 20 , where S1 - 20: 1903001W, 1906001S, 1909001W, 1912001W, 2001001S, 2002001W, 2005001S, 2008001W, 2011001S, 2102001S, 2105001S, 2108001W, 2110001S, 2112001S, 2202001W, 2206001W, 2212001S, 2309001W, 2311001S, 2312001W; R: reference chromatogram.

[0285] Table 34 Results of relative retention times of 20 batches of corn stigma formula granules

[0286]

[0287]

[0288] Table 35 Results of relative peak areas of 20 batches of corn stigma formula granules

[0289]

[0290] Table 36 Similarity Results of 20 Batches of Corn Silk Formula Granules

[0291] Batch number Similarity Batch number Similarity Batch number Similarity R 1.000 2005001S 0.907 2112001S 0.989 1903001W 0.988 2008001W 0.981 2202001W 0.950 1906001S 0.959 2011001S 0.983 2206001W 0.961 1909001W 0.960 2102001S 0.919 2212001S 0.978 1912001W 0.980 2105001S 0.987 2309001W 0.984 2001001S 0.993 2108001W 0.990 2311001S 0.984 2002001W 0.902 2110001S 0.994 2312001W 0.983

[0292] Table 37 Matching Data of the Common Pattern of Corn Silk Formula Granules

[0293]

[0294] It can be seen that the determination results show that the characteristic chromatograms of 20 batches of corn silk formula granules present 13 characteristic peaks, corresponding to the 13 characteristic peaks in the reference chromatogram of the control crude drug. The relative retention times of each characteristic peak and the S peak are calculated within the range of ±10% of the specified value.

[0295] It can be seen that the construction method of the present invention meets the requirements.

[0296] In summary, through the detection method in this embodiment, a fingerprint chromatogram with good separation of each characteristic peak can be effectively obtained, and the contents of 4-coumaric acid, vicenin-2, ferulic acid, isoorientin 2''-O-rhamnoside, chrysoeriol 7-O-glucoside-2-O-apioside, and N-trans-feruloyltyramine can be determined simultaneously. Moreover, by selecting ferulic acid as the internal reference peak in the fingerprint chromatogram, the relative retention times of the common characteristic peaks 1, 2, 4-13 of the corn silk formula granules can be determined. Therefore, the corn silk formula granules can be comprehensively and rapidly detected, which is beneficial to the comprehensive quality detection and overall quality control of the corn silk formula granules, thus contributing to improving the safety and stability of the drug use.

[0297] Obviously, the above embodiments are only examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of corn silk and its pharmaceutical preparation, characterized in that: The method comprises the steps of conducting high performance liquid chromatography on a test solution of corn silk medicinal material and / or corn silk pharmaceutical preparation; The chromatographic conditions include: using octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and 0.1% formic acid solution as a mobile phase B, and performing gradient elution according to the following procedure: 0-10min, A:B is 7%:93%→10%:90%; 10-22min, A:B is 10%:90%→12%:88%; 22-40min, A:B is 12%:88%→14%:86%; 40-45min, A:B is 14%:86%; Preferably, the gradient elution further comprises the following procedure: 45-60min, A:B 14%:86% → 16%:84%; 60-65min, A:B is 16%:84%.

2. The method for constructing a characteristic spectrum of corn silk and its pharmaceutical preparation according to claim 1, characterized in that: In the high performance liquid chromatography detection step, the chromatographic conditions also include: The column temperature is 20-30°C, preferably 25°C; and / or, The flow rate is 0.1-0.5 mL / min, preferably 0.29-0.31 mL / min, more preferably 0.3 mL / min; and / or, The detection wavelength is 330 nm; and / or, The injection volume is 1-5 μL, preferably 3 μL.

3. The method for constructing characteristic spectrum of corn silk and its pharmaceutical preparation according to claim 1 or 2, characterized in that: The preparation method of the test solution comprises: adding the test sample into an organic solvent for extraction, collecting the extract for solid-liquid separation, and collecting the subsequent filtrate to obtain the test solution.

4. The method for constructing characteristic spectrum of corn silk and its pharmaceutical preparation according to claim 1 or 2, characterized in that: The method also includes the step of preparing a control medicinal material reference solution, specifically including: taking corn silk control medicinal material, adding water to perform heating reflux extraction, collecting the extract for solid-liquid separation, collecting the residue, adding an organic solvent for extraction, collecting the extract for solid-liquid separation, and collecting the filtrate to obtain a control medicinal material reference solution.

5. The method for constructing characteristic spectrum of corn silk and its pharmaceutical preparation according to claim 1 or 2, characterized in that: The method further comprises the steps of preparing a reference substance solution and constructing a reference substance characteristic spectrum based on the high performance liquid chromatography method; The reference substance includes ferulic acid; The method for preparing the reference substance solution comprises the steps of adding the reference substance into an organic solvent and mixing; The mass concentration of the reference substance solution is 1-20 μg of the reference substance per 1 ml.

6. The method for constructing characteristic spectrum of corn silk and its pharmaceutical preparation according to any one of claims 3 to 5, characterized in that: The organic solvent comprises methanol aqueous solution, preferably at a concentration of 30-80 v / v%, more preferably 50 v / v%; Preferably, the extraction step comprises reflux extraction or ultrasonic extraction; Preferably, the solid-liquid separation comprises filtration or centrifugation.

7. The method for constructing characteristic spectrum of corn silk and its pharmaceutical preparation according to any one of claims 1 to 6, characterized in that: The corn silk medicinal preparation includes corn silk formula granules, corn silk decoction pieces, corn silk standard decoction or corn silk standard decoction freeze-dried powder.

8. A characteristic spectrum and / or a comparative characteristic spectrum of a corn silk medicinal material and a pharmaceutical preparation thereof, characterized in that: The characteristic spectrum or reference characteristic spectrum of the corn silk medicinal material and its pharmaceutical preparation is constructed by the method according to any one of claims 1 to 7; Preferably, the characteristic chromatogram presents 13 characteristic peaks, and the retention times of the 13 characteristic peaks in the chromatogram of the reference medicinal material reference substance correspond to each other, wherein peak 3 corresponds to the retention time of the corresponding reference substance peak; the peak corresponding to the retention time of the ferulic acid reference substance peak is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within the range of ±10% of the specified value; the specified value is: Peak 1: 0.76, peak 2: 0.90, peak 4: 1.37, peak 5: 1.75, peak 6: 1.92, peak 7: 2.08, peak 8: 2.13, peak 9: 2.19, peak 10: 2.66, peak 11: 3.07, peak 12: 3.15, peak 13: 3.

51.

9. The method for constructing the characteristic spectrum of the corn silk medicinal material and its pharmaceutical preparation according to any one of claims 1 to 7 and / or the application of the characteristic spectrum and / or the reference characteristic spectrum of the corn silk medicinal material and its pharmaceutical preparation according to claim 8 in the field of quality inspection of corn silk medicinal material and its pharmaceutical preparation.

10. A quality detection method for corn silk medicinal materials and pharmaceutical preparations thereof, characterized in that: The method comprises the steps of constructing the characteristic map and the reference characteristic map according to the method of any one of claims 1 to 7, and comparing the characteristic map with the reference characteristic map.

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