A method for separating chemical components in corn silk and its preparation or a method for constructing a characteristic map

Abstract

The application belongs to the field of traditional Chinese medicine quality detection, and specifically discloses a method for separating chemical components in corn silk and its preparation or a method for constructing a characteristic chromatogram, which comprises the following steps: (1) preparing a test sample solution; and (2) detecting the test sample solution by high performance liquid chromatography, using octadecylsilane bonded silica gel as a filler, using acetonitrile as mobile phase A, using a water solution containing 1-heptanesulfonic acid sodium and formic acid as mobile phase B, and using a gradient elution procedure as defined in the description. The obtained characteristic chromatogram has a smooth baseline, good peak shape, high resolution, and can accurately locate the peak positions of guanosine, guanine, adenine, adenosine, vanillin, 4-coumaric acid and ferulic acid, fully reflects the integrity and characteristics of corn silk and its preparation (such as formula granules and other preparations), and provides a basis for quality detection and control of corn silk formula granules.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection technology, specifically relating to a method for separating chemical components or constructing characteristic spectra in corn silk and its preparations. Background Technology

[0002] Corn silk is the dried style and stigma of *Zea mays* L., a plant belonging to the Poaceae family. It mainly contains various components such as organic acids, nucleosides, and amino acids, and is primarily used for renal edema, dysuria, damp-heat jaundice, hypertension, and diabetes. The 1977 edition of the *Chinese Pharmacopoeia* includes quality control requirements for corn silk, including the original plant variety, processing of the processed slices, characteristics of the slices, and physicochemical identification. Literature also describes the chemical components of corn silk. The chemical composition of corn silk is complex and diverse. Besides organic acids, it also contains a large amount of purine, pyrimidine, and urea derivative alkaloids with diuretic effects. These components are highly polar and difficult to separate.

[0003] Common formulations of corn silk include powders and granules. For example, granules are obtained from corn silk slices through extraction, concentration, drying, and formulation processes, and their material basis differs significantly from that of the raw medicinal material. Currently, no research has been reported on corn silk granules. Existing detection methods for corn silk medicinal materials suffer from limited shared information and insufficient characteristic features, making them unsuitable for corn silk preparations.

[0004] Furthermore, since traditional Chinese medicine formula granules no longer possess the characteristics for identifying the properties of medicinal materials, the above methods are unsuitable for the quality testing of preparations made from corn silk water extracts, such as corn silk formula granules. They suffer from few characteristic peaks, poor separation effects, and the reference standard, apigenin, is a fat-soluble component, making it unsuitable for the study of formula granules and other preparations. How to improve the testing standards for corn silk and its preparations, and comprehensively control the quality of the finished product, is the technical problem that this invention aims to solve. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a method for separating chemical components or constructing characteristic spectra in corn silk and its preparations. This method establishes characteristic spectra of the variety based on the characteristics of corn silk and its preparations, achieves effective separation of multiple characteristic peaks, improves the characteristicity, and provides a scientific basis for comprehensively establishing quality control standards for corn silk and its preparations.

[0006] Specifically, this invention discloses a method for separating chemical components or constructing characteristic spectra from corn silk and its preparations, comprising the following steps:

[0007] (1) Preparation of the test solution; the extraction solvent used in the preparation of the test solution is selected from water or methanol aqueous solution;

[0008] (2) The test solution was analyzed by high performance liquid chromatography. Octadecylsilane bonded silica gel was used as the stationary phase, acetonitrile was used as the mobile phase A, and an aqueous solution containing sodium 1-heptanesulfonate and formic acid was used as the mobile phase B. Gradient elution was performed according to the following program: 0→42 min, mobile phase A: mobile phase B was 1%:99%→18%:82%.

[0009] In some preferred embodiments, step (2) further satisfies at least one of the following 1)-5):

[0010] 1) The gradient elution also includes the following program: 42→45 min, mobile phase A: mobile phase B is 18%:82%→19%:81%; 45→55 min, mobile phase A: mobile phase B is 19%:81%→60%:40%;

[0011] 2) The flow rate is 0.8-1.2 mL / min, preferably 1.0 mL / min;

[0012] 3) The column temperature is 25-45℃, preferably 40℃;

[0013] 4) The injection volume is 5-15 μL;

[0014] 5) The detection wavelength is 258nm to 322nm. Preferably, the detection wavelength for 0 to 25 minutes is 258-262nm, and the detection wavelength for 25 to 55 minutes is 318-322nm.

[0015] Furthermore, the volume percentage of sodium 1-heptanesulfonate in mobile phase B is 0.03-0.05%, and the volume percentage of formic acid is 0.05-0.15%.

[0016] In this invention, corn silk and its preparations can refer to corn silk medicinal materials, decoction pieces, or preparations. The preparations can be made from water extracts of corn silk, such as powders, granules, tablets, etc.

[0017] In some preferred embodiments, step (1) includes weighing corn silk sample, adding extraction solvent to extract, obtaining extract, solid-liquid separation, and taking the liquid as the sample solution;

[0018] In some preferred embodiments, step (1) further satisfies any one or more of the following AEs:

[0019] A. The ratio of the mass of the corn silk sample to the volume of the extraction solvent is 0.1-0.5:10-50; preferably 0.5:25, and the mass-to-volume relationship is g / mL;

[0020] B. The extraction method is either reflux extraction or ultrasonic extraction;

[0021] C. The extraction time is ≥10 min, preferably 15-45 min, and more preferably 15 min;

[0022] D. The solid-liquid separation is selected from centrifugation or membrane filtration;

[0023] E. The extraction solvent is water or a methanol aqueous solution with a volume percentage ≤15%, preferably a methanol aqueous solution with a volume percentage of 5-15%.

[0024] In some preferred embodiments, the method further includes the step of preparing a reference solution by adding a solvent to at least one of guanosine reference standard, guanine reference standard, adenine reference standard, adenosine reference standard, vanillin reference standard, 4-coumaric acid reference standard and ferulic acid reference standard, and the step of obtaining a reference chromatogram by detecting the reference solution using high performance liquid chromatography in the separation method or characteristic chromatogram construction method of chemical components in corn silk and its preparations according to any of the present invention.

[0025] Preferably, each 1 mL of the reference solution contains 1-100 μg of each reference standard; and / or, the solvent used in the preparation of the reference solution is selected from water, methanol, or an aqueous methanol solution.

[0026] In some preferred embodiments, the characteristic spectra of the corn silk and its preparations are selected from any one of the following (1)-(2):

[0027] (1) It has 9 common characteristic peaks. Peaks 1 and 8 correspond to the retention times of the guanosine reference standard peak and the ferulic acid reference standard peak, respectively. The peak corresponding to the guanosine reference standard peak is designated as peak S1. The relative retention times of peaks 2 to 5 with peak S1 are within ±10% of the specified value. The specified value is:

[0028] Peak 2: 1.14, Peak 3: 1.33, Peak 4: 1.43, Peak 5: 1.61;

[0029] The peak corresponding to the ferulic acid reference standard peak is designated as peak S2. The relative retention times of peaks 6, 7, and 9 with peak S2 are within ±10% of the specified value. The specified value is:

[0030] Peak 6: 0.79, Peak 7: 0.87, Peak 9: 1.13;

[0031] (2) It has 9 common characteristic peaks. Peaks 1 to 4 and peaks 6 to 8 correspond to the retention times of the reference peaks of guanosine, guanine, adenine, adenosine, vanillin, 4-coumaric acid and ferulic acid, respectively.

[0032] In some preferred embodiments, the method further includes constructing a reference characteristic chromatogram of corn silk formula granules. A reference characteristic chromatogram of corn silk formula granules is generated by using a similarity evaluation system for chromatograms of traditional Chinese medicine obtained from the detection of characteristic chromatograms of multiple batches of corn silk formula granules. At least two batches of corn silk formula granules are used, for example, four, seven, eight, or fifteen batches of corn silk formula granules.

[0033] In some preferred embodiments, after generating the control feature spectrum of corn silk formula granules using the software for evaluating the similarity of chromatographic feature spectra of traditional Chinese medicine, the method further includes the step of marking common feature peaks.

[0034] In this invention, the reference characteristic spectrum of corn silk and its preparations can also be obtained by using the characteristic spectrum of corn silk water extract and / or its preparations obtained by a single batch or multiple batches of corn silk and / or its preparations according to any of the separation methods or characteristic spectrum construction methods of corn silk and its preparations described in this invention; optionally, the reference characteristic spectrum of corn silk and its preparations can also be prepared by using the characteristic spectrum of multiple batches of corn silk water extract and / or its preparation test samples obtained according to any of the construction methods described in this invention and then using the average value or median method.

[0035] The present invention also provides the application of the separation method or the characteristic spectrum construction method of the chemical components in corn silk and its preparations as described in any of the above claims in the quality detection of corn silk and its preparations.

[0036] The present invention also provides a method for quality testing of corn silk formulation granules, comprising constructing a characteristic spectrum of the product to be tested according to any of the above-described methods for separating chemical components in corn silk and its preparations or for constructing characteristic spectra.

[0037] Furthermore, it also includes the step of comparing the characteristic spectrum of the corn silk to be tested and / or its preparations with the control characteristic spectrum of corn silk and its preparations.

[0038] In this invention, the product to be tested can be corn silk medicinal material, decoction pieces, or preparations made from corn silk water extract, such as powder, granules, tablets, etc.

[0039] The quality of the corn silk formula granule product under test is evaluated using similarity. If the similarity between the characteristic chromatogram of the corn silk formula granule product under test and the control characteristic chromatogram of corn silk formula granules is not lower than 0.90-1.00 (e.g., 0.90), the quality is qualified; if it is lower than 0.90-1.00 (e.g., 0.90), it is unqualified. Specifically, the similarity is obtained by using software for evaluating the similarity of chromatographic characteristic chromatograms of traditional Chinese medicine.

[0040] The technical solution of this invention has the following advantages:

[0041] 1. The method for separating chemical components or constructing characteristic chromatograms of corn silk and its preparations according to the present invention includes the following steps: (1) preparation of test solution; the extraction solvent used in the preparation of the test solution is selected from water or methanol aqueous solution; (2) the test solution is detected by high performance liquid chromatography, with octadecylsilane bonded silica gel as the packing material, acetonitrile as mobile phase A, and an aqueous solution containing sodium 1-heptanesulfonate and formic acid as mobile phase B, and gradient elution is performed according to the following procedure: 0→42min, mobile phase A: mobile phase B is 1%:99%→18%:82%; the effective components of multiple chemical components are realized, especially the detection wavelength of 0~25min is controlled at 258-262nm, and the detection wavelength of 25~55min is controlled at 318-322nm, finally obtaining 9 common characteristic peaks, and the effective separation of 9 common characteristic peaks is achieved. Moreover, the baseline of the obtained characteristic chromatogram is stable, the characteristic peak shape is good, and the peak height or peak area is uniform, providing a scientific basis for the comprehensive establishment of quality control standards for corn silk formulation granules. Furthermore, it can accurately locate the peak positions of guanosine, guanine, adenine, adenosine, vanillin, 4-coumaric acid, and ferulic acid, fully reflecting the integrity and characteristics of corn silk water extract and water-extracted preparations (such as formulation granules).

[0042] The method for separating chemical components or constructing characteristic spectra of corn silk and its preparations described in this invention can select guanosine (S1 peak) and ferulic acid (S2 peak) as internal reference peaks in the fingerprint spectrum, and can determine 9 common characteristic peaks of corn silk formulation particles. The relative retention times of each common characteristic peak can be calculated based on guanosine (S1 peak) and ferulic acid (S2 peak), thereby helping to improve the safety and stability of the drug.

[0043] The method for separating chemical components or constructing characteristic chromatograms in corn silk and its preparations described in this invention has good separation efficiency and can simultaneously and effectively separate guanosine, guanine, adenine, adenosine, vanillin, 4-coumaric acid, and ferulic acid, thereby determining the content of the above substances. Moreover, the detection method has high precision, good stability, and good repeatability. Therefore, it can comprehensively and rapidly detect the effective components and their content in corn silk formulation granules.

[0044] 2. The method for separating chemical components or constructing characteristic chromatograms in corn silk and its preparations described in this invention, through optimization of chromatographic conditions, extraction solvents, extraction time, and other extraction conditions, has determined the optimal extraction process and chromatographic conditions, resulting in higher peak areas, better separation effects, and more comprehensive quality monitoring of corn silk formulation particles.

[0045] 3. The quality testing method for corn silk and its preparations described in this invention, by constructing a characteristic spectrum of the product to be tested using the method of this invention, can comprehensively, clearly, and effectively test the quality of products such as corn silk formulation granules. Attached Figure Description

[0046] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0047] Figure 1 The diagram shows the location of the corn silk granule test sample and different reference standards, from bottom to top: S1: protocatechuic acid; S2: guanosine; S3: guanine; S4: adenine; S5: adenosine; S6: vanillin; S7: 4-coumaric acid; S8: ferulic acid; S9: test sample.

[0048] Figure 2 Characteristic chromatograms of 17 batches of corn silk formula granules and comparative characteristic chromatograms of corn silk formula granules;

[0049] Figure 3 The characteristic spectrum of corn silk as a reference medicinal material; among which, peak 1 (S1): guanosine; peak 2: guanine; peak 3: adenine; peak 4: adenosine; peak 6: vanillin; peak 7: 4-coumaric acid; peak 8 (S2): ferulic acid;

[0050] Figure 4 Characteristic spectrum of corn silk formulation granules;

[0051] Figure 5 The characteristic spectrum of the detection wavelength 220nm in Experiment Example 1;

[0052] Figure 6 The characteristic spectrum of the detection wavelength 240nm in Experiment Example 1;

[0053] Figure 7 The characteristic spectrum of the detection wavelength 260nm in Experiment Example 1;

[0054] Figure 8 The characteristic spectrum of the detection wavelength 280nm in Experiment Example 1;

[0055] Figure 9 The characteristic spectrum of the detection wavelength of 300 nm in Experiment Example 1;

[0056] Figure 10 The characteristic spectrum of the detection wavelength of 320 nm in Experiment Example 1;

[0057] Figure 11 The characteristic spectrum of the detection wavelength of 340 nm in Experiment Example 1;

[0058] Figure 12 The characteristic spectrum of the column temperature at 25℃ in Experiment Example 1;

[0059] Figure 13 The characteristic spectrum of the column temperature of 30℃ in Experiment Example 1;

[0060] Figure 14 The characteristic spectrum of the column temperature of 35℃ in Experiment Example 1;

[0061] Figure 15 The characteristic spectrum of the column temperature of 40℃ in Experiment Example 1;

[0062] Figure 16 The characteristic spectrum of the column temperature of 45℃ in Experiment Example 1;

[0063] Figure 17 The characteristic spectrum is for a flow rate of 0.8 ml / min in Experiment Example 1;

[0064] Figure 18 The characteristic chromatogram for the flow rate of 1.0 ml per minute in Experiment Example 1;

[0065] Figure 19 The characteristic spectrum is for a flow rate of 1.2 ml / min in Experiment Example 1;

[0066] Figure 20 For example, the Penomenex Kinetex XB-C in Experiment 1 18 Characteristic chromatograms of chromatographic column construction;

[0067] Figure 21 For example 1, Cosmosil AR-C 18 Characteristic chromatograms of chromatographic column construction;

[0068] Figure 22 For example, Agela Promosil C in Experiment 1 18 Characteristic chromatograms of chromatographic column construction;

[0069] Figure 23 The characteristic spectrum constructed using ethanol as the extraction solvent in Experiment Example 1;

[0070] Figure 24 The characteristic spectrum of corn silk formulation particles in the specificity experiment of Experiment Example 2;

[0071] Figure 25 This is the chromatogram of the negative blank control in the specificity experiment of Experiment Example 2. Detailed Implementation

[0072] The following embodiments are provided to better understand the present invention and are not intended to limit the preferred embodiments. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention. Where specific experimental steps or conditions are not specified in the embodiments, they can be performed according to the conventional experimental steps or conditions described in the literature in the art. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products. The ratio of mobile phase A to mobile phase B is a volume percentage.

[0073] Example 1

[0074] This embodiment provides a method for separating chemical components or constructing characteristic spectra from corn silk and its preparations, including the following steps:

[0075] (1) Preparation of test solution: Take about 0.5g of corn silk drug preparation powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of 10% methanol, stopper tightly, weigh it, sonicate (power 250W, frequency 35kHz) for 15 minutes, take it out, let it cool, weigh it again, make up the lost weight with 10% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0076] The corn silk pharmaceutical preparation described in this invention is prepared by the following method:

[0077] Corn silk is extracted at least once by heating and reflux, with 6-12 times its weight of water added each time for at least 0.5 hours. The extract is then filtered, and the filtrates are combined. The filtrate is concentrated to a relative density of 1.05-1.10 g / mL at 60°C. Conventional excipients are added, and the product is manufactured according to standard processes to produce clinically acceptable tablets, capsules, pills, granules, honey-processed pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, or injectable preparations. The pharmaceutically acceptable excipients include: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrix, etc. Fillers include: starch, pregelatinized starch, lactose, mannitol, chitosan, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, croscarmellose, low-substituted hydroxypropyl cellulose, croscarmellose 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, cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various flavorings; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chlorethidium acetate, eucalyptus oil, etc.; matrix includes: PEG6000, PEG4000, insect wax, etc.

[0078] In this embodiment, the test sample used is corn silk granules. The specific preparation method of the corn silk granules is as follows: corn silk is taken and extracted twice by heating and reflux. For the first extraction, 12 times the weight of water is added and soaked for 30 minutes, then heated and refluxed for 0.5 hours and filtered. For the second extraction, 10 times the weight of water is added and extracted for 0.5 hours and filtered. The filtrates are combined and concentrated to a relative density of 1.05 g / mL at 60°C. The filtrates are then spray-dried. The dry powder is mixed with the excipient maltodextrin (the mass of maltodextrin accounts for 0.05% of the mass of corn silk), mixed evenly, and then dry-granulated to produce granules.

[0079] Reference solution: Take appropriate amounts of guanosine, guanine, 4-coumaric acid and ferulic acid reference standards, add 10% methanol to prepare a mixed solution containing 10 μg of each reference standard per 1 mL.

[0080] (2) The test solution and the reference solution were analyzed by high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm; brand: Phenomenex Kinetex XB-C). 18 , The mobile phase consisted of acetonitrile (A) and an aqueous solution containing 0.038% (w / v) sodium 1-heptanesulfonate and 0.1% formic acid (B). Gradient elution was performed according to the specifications in the table below. The detection wavelength was 260 nm for 0–25 minutes, and then switched to 320 nm after 25 minutes. The flow rate was 1.0 mL / min, the column temperature was 40 °C, and the injection volume was 10 μL. The theoretical plate number, calculated based on the guanosine peak, should be no less than 5000. The results are shown in Table 1.

[0081]

[0082] Table 1 System Adaptability Parameters

[0083]

[0084] The results showed that the characteristic chromatogram of the test sample exhibited nine common characteristic peaks, and these nine common characteristic peaks were effectively separated. Furthermore, the baseline of the obtained characteristic chromatogram was stable, and the peak shapes, heights, or areas were uniform. Peaks 1 and 8 corresponded to the retention times of the guanosine and ferulic acid reference standards, respectively. The peak corresponding to the guanosine reference standard peak was designated as peak S1. The relative retention times of peaks 2–5 with peak S1 were within ±10% of the specified values; the specified values ​​were: peak 2: 1.14, peak 3: 1.33, peak 4: 1.43, peak 5: 1.61. The peak corresponding to the ferulic acid reference standard peak was designated as peak S2. The relative retention times of peaks 6, 7, and 9 with peak S2 were within ±10% of the specified values; the specified values ​​were: peak 6: 0.79, peak 7: 0.87, peak 9: 1.13.

[0085] Example 2: Establishment of Feature Maps and Their Parameters

[0086] (1) Establishment of feature maps and their parameters

[0087] Preparation of reference solutions: Take appropriate amounts of guanosine, adenine, and adenosine reference standards, and add 10% methanol to prepare mixed solutions containing 40 μg per 1 mL, which is mixed reference solution 1; Take an appropriate amount of guanine reference standard, add 10 mL of 0.1 mol / L NaOH to dissolve it, and then dilute with water to 50 mL to obtain guanine reference solution; Take appropriate amounts of vanillin, protocatechuic acid, 4-coumaric acid, and ferulic acid reference standards, and add methanol to prepare mixed solutions containing 10 μg per 1 mL, which is mixed reference solution.

[0088] Take 2g of corn silk reference material, place it in a stoppered conical flask, add 100ml of water, heat under reflux for 2 hours, filter, evaporate the filtrate to dryness, cool, add 25ml of 10% methanol to the residue, seal tightly, sonicate (power 250W, frequency 35kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution for the reference material.

[0089] Seventeen batches of corn silk formula granule samples (the preparation method of corn silk formula granules is the same as in Example 1) were taken. Test solutions and reference solutions were prepared according to the method in Example 1, and determined by high-performance liquid chromatography (HPLC) according to Example 1. The fingerprint similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission was used to generate control characteristic fingerprint spectra. The results are as follows: Figure 2-4 As shown. Figure 2 In the diagram, S1 to S17 are batch numbers, respectively: 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: 2212001S; R: reference characteristic chromatogram.

[0090] (2) Selection criteria and specified values ​​of characteristic peak S-peak in characteristic spectrum

[0091] pass Figure 1 It is known that guanine and ferulic acid have high responses in the characteristic spectrum of corn silk medicinal materials, and they belong to purine alkaloids and phenolic acids, respectively. Purine alkaloids and phenolic acids are the main chemical components of corn silk medicinal materials. Considering that purine alkaloids are effective diuretic components, and that guanosine, 4-coumaric acid, and ferulic acid are index peaks in the content determination, with ferulic acid having a higher peak than 4-coumaric acid, and that the peak times of guanosine and ferulic acid are relatively moderate, guanosine and ferulic acid are used as the S1 and S2 peaks of this characteristic spectrum, respectively, and the relative retention times of characteristic peaks 1 to 9 are calculated.

[0092] pass Figure 2-4 It was found that the HPLC characteristic chromatograms of all 17 batches of corn silk formulation granules had 9 common chromatographic peaks. The peaks corresponding to the reference peaks of guanosine and ferulic acid were selected as S1 and S2, respectively. The relative retention times of each characteristic peak and peaks S1 and S2 were calculated, and all relative retention times were within ±10% of the specified values. The specified values ​​were: 1.14 (peak 2), 1.33 (peak 3), 1.43 (peak 4), 1.61 (peak 5), 0.79 (peak 6), 0.87 (peak 7), and 1.13 (peak 9). The results are shown in Tables 2-4.

[0093] Table 2. Relative retention time of common patterns in corn silk formulation granules.

[0094]

[0095] Table 3 Common Pattern Matching Data for Corn Silk Granule Formulation

[0096]

[0097] Table 4. Results of fingerprint analysis of 18 batches of corn silk formulation granules

[0098]

[0099]

[0100] (3) Results of peak identification and reference standard positioning

[0101] Positioning of reference standards with different characteristic peaks, such as Figure 1 As shown, seven known peaks were identified through comparison: guanosine (peak 1), guanine (peak 2), adenine (peak 3), adenosine (peak 4), vanillin (peak 6), 4-coumaric acid (peak 7), and ferulic acid (peak 8). Among them, guanosine, guanine, adenine, and adenosine are purine alkaloids and are the main diuretic active components of corn silk. Ferulic acid, 4-coumaric acid, and vanillin are phenolic acids and have anti-inflammatory and antioxidant effects. This characteristic spectrum contains the main active chemical components of corn silk.

[0102] Nine characteristic peaks were identified and located using LC / MS / MS. The chromatographic column was a Phenomenex Kinetex C18, 250 mm × 20 mm, 5 μm, with a flow rate of 10 mL / min. The gradient was amplified according to the analytical conditions, and the detection wavelengths were 260 nm and 320 nm. The corresponding chromatographic peaks were collected. After solid-phase extraction for desalting, sample concentration and drying, molecular structure analysis was performed using NMR and ESI-MS to confirm that the molecular structure was consistent with that of the reference standard. The results are shown in the table below.

[0103] Table 5. Analysis results of characteristic peaks in corn silk.

[0104]

[0105] (4) Similarity

[0106] According to the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the characteristic chromatogram of the test sample and the characteristic chromatogram of the reference sample, calculated by Mark peak similarity, should not be less than 0.90. The similarity of the HPLC characteristic chromatograms of 17 batches of corn silk formula granules was all greater than 0.90.

[0107] Table 6. Similarity results of characteristic fingerprint spectra of 18 batches of corn silk formulation granules

[0108]

[0109]

[0110] The detection method described in this embodiment can effectively obtain fingerprint spectra with good separation of various characteristic peaks. It can also simultaneously determine the contents of guanosine, guanine, adenine, adenosine, vanillin, 4-coumaric acid, and ferulic acid. Furthermore, by selecting guanosine peak S1 and ferulic acid peak S2 as internal reference peaks in the fingerprint spectrum, the relative retention times of the common characteristic peaks 1-9 of the corn silk formulation granules can be determined. Therefore, it enables comprehensive and rapid detection of corn silk formulation granules, which is beneficial for comprehensive quality testing and overall quality control of corn silk formulation granules, thereby helping to improve the safety and stability of the drug.

[0111] Examination of the construction method in Experiment Example 1

[0112] 1. Instruments, reagents and reagents

[0113] High Performance Liquid Chromatography System 1: Agilent 1200 system, including: degasser G1379B, quaternary pump G1311A; autosampler G1329B; DAD detector G1315D.

[0114] High Performance Liquid Chromatograph 2: UltiMate 3000 Chromatography System, including LPG-3400SDN quaternary pump, WPS-3000SL autosampler, DAD-3000 diode array detector, and chromatography workstation.

[0115] High Performance Liquid Chromatograph 3: Shimadzu LC20-A, including: CBM-20A system controller, DGU-20A degasser, LC20-AT quaternary pump, SIL-20A autosampler, CTO-20A column oven, and SPD-M20A PDA detector.

[0116] KQ-5200V CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 0.001% balance (CP114, Ohaus Instrument Co., Ltd.); 0.001% balance (AB265-S, METTLER TOLEDO); rotary evaporator (Shanghai Ailang Instrument Co., Ltd., OSB-2200); decoction equipment: AUX fully automatic decoction pot (500W); freeze dryer: Christ Alpha 2-4.

[0117] Column 1: Phenomenex Kinetex XB-C18 (4.6×250mm, 5μm);

[0118] Column 2: Cosmosil AR C18 (4.6 × 250 mm, 5 μm);

[0119] Column 3: Agela Promosil C18 (4.6×250mm, 5μm, ).

[0120] Guanosine reference standard (China National Institutes for Food and Drug Control, batch number: 111977-201501, purity: 93.6%);

[0121] Adenosine reference standard (China National Institutes for Food and Drug Control, batch number: 110879-201703, purity: 99.7%);

[0122] Adenine reference standard (Shanghai Titan Technology Co., Ltd., batch number: P1660265, purity: 99%);

[0123] Guanine reference standard (Shanghai Titan Technology Co., Ltd., batch number: P1834009, purity: 99%);

[0124] Vanillin reference standard (Afaeza (Tianjin) Chemical Co., Ltd., batch number: 10165532, purity: 99%);

[0125] Protocatechuic acid (Yuanye Biotechnology batch number: W09A11B111046, purity 97%);

[0126] 4-Coumaric acid reference standard (Shanghai Aladdin Biochemical Technology Co., Ltd., batch number: F2122054, purity: 98%)

[0127] Ferulic acid reference standard (China National Institutes for Food and Drug Control, batch number: 110773-201915, purity: 99.4%).

[0128] Corn silk reference material (China National Institutes for Food and Drug Control, batch number: 121659-202103).

[0129] Methanol and acetonitrile were of chromatographic grade (TEDIA), and water was ultrapure water; all other reagents were of analytical grade.

[0130] Sodium 1-heptanylsulfonate (Bide Pharmaceuticals batch number: BD235726, purity: 98%).

[0131] Test drug: Corn silk granules were formulated in the same way as in Example 1.

[0132] 2. Optimization of chromatographic conditions

[0133] (1) Optimization of mobile phase gradient

[0134] The test solution prepared according to the method in Example 1 using corn silk granules was used for detection. A Phenomenex Kinetex XB-C18 column was used. (Column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), acetonitrile was used as mobile phase A and 0.1% formic acid water was used as mobile phase B. Elution was performed using the following gradients, with a flow rate of 1.0 ml per minute; column temperature was 40 ℃, detection wavelength was 260 nm, and 10 μl of sample was injected.

[0135] Table 7 Gradient conditions 1-3

[0136]

[0137] Table 8 System Adaptability Parameters

[0138]

[0139] The results are shown in Table 8. Under gradient condition 3, the characteristic peak information is more abundant and the separation is more ideal. Therefore, the gradient is tentatively set as condition 3 for further investigation.

[0140] (2) Investigation of the mobile phase

[0141] The effects of different mobile phase systems on the separation effect of characteristic chromatograms of corn silk formulation granules were compared. High-performance liquid chromatography (HPLC) was performed using corn silk formulation granules as the test sample, prepared according to the method in Example 1. The mobile phase systems were set as follows: Mobile phase ①: acetonitrile as mobile phase A, water as mobile phase B; Mobile phase ②: acetonitrile as mobile phase A, 0.1% (v / v) formic acid aqueous solution as mobile phase B; Mobile phase ③: acetonitrile as mobile phase A, 0.038% (v / v) 1-heptanesulfonate sodium and 0.1% (v / v) formic acid aqueous solution as mobile phase B. Gradient elution was performed using the above gradient condition 3. A Phenomenex Kinetex XB-C18 column was used. (Column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), flow rate 1.0 ml / min; column temperature 40 ℃, detection wavelength 260 nm, injection 10 μl.

[0142] Table 9 System Adaptability Parameters

[0143]

[0144] The results are shown in Table 9. The chromatogram obtained under mobile phase system ③ has the most chromatographic peak information and the overall chromatogram is relatively stable. Therefore, it is tentatively decided to use an aqueous solution of 0.038% sodium 1-heptanesulfonate and 0.1% formic acid as mobile phase B for subsequent investigation.

[0145] (3) Investigation of detection wavelength

[0146] The test solution prepared according to Example 1 was detected by high performance liquid chromatography (HPLC) at wavelengths of 220 nm, 240 nm, 260 nm, 280 nm, 300 nm, 320 nm, and 340 nm. Acetonitrile was used as mobile phase A, and an aqueous solution of 0.038% (v / v) sodium 1-heptanesulfonate and 0.1% (v / v) formic acid was used as mobile phase B. Gradient elution was performed under the aforementioned gradient conditions (3). A Phenomenex Kinetex XB-C18 column was used. (Column length 250 mm, inner diameter 4.6 mm, particle size 5 μm), flow rate 1.0 ml / min; column temperature 40 ℃, injection 10 μl.

[0147] See results Figure 5-11 As shown, the information content and signal are relatively strong at a wavelength of 260nm, but the signal becomes weaker after 25 minutes. In this range, the signal is stronger at a wavelength of 320nm. Therefore, the wavelength switching procedure of 0-25 minutes, 260nm, and 320nm after 25 minutes is determined as the detection wavelength for the characteristic spectrum of corn silk formula particles. Under this condition, the system adaptability parameters are relatively superior.

[0148] (4) Investigation of column temperature

[0149] The effect of different column temperatures on the robustness of the characteristic chromatograms of corn silk granule formulations was compared. Column temperatures were set at 25℃, 30℃, 35℃, 40℃, and 45℃. The same corn silk granule formulation sample solution (prepared according to the method in Example 1) was used as the research object for detection. The only difference was the column temperature; all other chromatographic conditions were the same as in Example 1. The effect of different column temperatures on the separation effect of the corn silk granule formulation sample was observed.

[0150] The results are as follows Figure 12-16 As shown, at 40℃, the position of the impurity peak is far away from the main peak, and the separation degree of the nine main peaks is greater than 1.5. Therefore, it is recommended to select a column temperature of 40℃.

[0151] (5) Examination of flow velocity

[0152] The effect of different flow rates on the robustness of the characteristic chromatograms of corn silk granules was compared. Flow rates were set at 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min. The same corn silk granule test solution (prepared according to the method in Example 1) was used as the research object for detection, with the only difference being the flow rate; all other chromatographic conditions were the same as in Example 1. The effect of different flow rates on the separation effect of the corn silk granule test sample was observed.

[0153] The results are as follows Figure 17-19 As shown, the separation effect of each chromatographic peak is better when the flow rate is 1.0 mL / min, so the preferred flow rate is 1.0 mL / min.

[0154] (6) Examination of the chromatographic column

[0155] To compare the effects of different brands of chromatographic columns on the robustness of the characteristic chromatograms of corn silk formulation granules, different brands of octadecylsilane-bonded silica columns were used (Column 1: Penomenex Kinetex XB-C). 18 Column 2: Cosmosil AR-C 18 Column 3: Agela Promosil C 18 The same corn silk granule test solution (prepared according to the method in Example 1) was tested, with all other chromatographic conditions consistent with those in Example 1. The effect of different chromatographic columns on the separation effect of the corn silk granule test solution was observed.

[0156] The results are as follows Figure 20-22 As shown, column 1 has the highest peak information content and the best separation effect for each peak, therefore, Penomenex Kinetex XB-C is preferred. 18 A chromatographic column for the characteristic chromatographic method of corn silk formulation particles.

[0157] (7) Examination of the chromatograph

[0158] The same corn silk granule sample solution (prepared according to the method in Example 1) was used to determine characteristic chromatograms on different instruments under the same chromatographic conditions as in Example 1. The system suitability parameters of the chromatograms obtained by different instruments were compared. Comparison of characteristic chromatograms obtained from different brands of high-performance liquid chromatography (HPLC) revealed that the chromatographic information presented by the Shimadzu LC-20A, Agilent 1200, and Thermo Ultimate U3000 HPLC models was relatively complete, and none of the characteristic peaks were missing. Therefore, it is not necessary to fix the HPLC model to determine the characteristic chromatograms of corn silk granules.

[0159] 3. Preparation of the test solution

[0160] (1) Selection of extraction method

[0161] Take approximately 0.5g of corn silk granules, accurately weigh them, place them in a stoppered conical flask, accurately add 25mL of 10% methanol, seal tightly, weigh, sonicate (power 250W, frequency 35kHz) or reflux extract for 15 minutes, remove, cool, weigh again, replenish the lost weight with 10% methanol, shake well, filter, accurately pipette 10μl of the filtrate, and inject into a high-performance liquid chromatograph; determine according to the chromatographic conditions of Example 1, and the results are shown in the table below.

[0162] Table 10 Reagents for Extraction of Lyophilized Corn Silk Powder

[0163]

[0164] The results showed that reflux extraction had a lower extraction rate than ultrasonic extraction. Considering the ease of operation and the recovery rate, and since corn silk granules are traditional Chinese medicine granules prepared by decocting, concentrating, drying, and granulating corn silk slices, which have already undergone a water heating extraction process, ultrasonic extraction is the preferred extraction method for the corn silk granules.

[0165] (2) Examination of ultrasonic power

[0166] The extraction effect of different ultrasonic powers (150W, 35kHz, 200W, 35kHz, and 250W, 35kHz) on corn silk granules (1903001W) was investigated. Approximately 0.5g of corn silk granules was accurately weighed and placed in a stoppered conical flask. 25mL of 10% methanol was accurately added, the flask was sealed, and the weight was measured. The flask was then ultrasonically treated at different powers (150W, 35kHz, 200W, 35kHz, and 250W, 35kHz) for 15 minutes each. After removal and cooling, the flask was weighed again, and the weight loss was replenished with 10% methanol. The flask was shaken well, filtered, and 10μl of the filtrate was accurately injected into the high-performance liquid chromatograph (HPLC). Chromatographic analysis was performed according to the conditions in Example 1. The results are shown in the table below. The results indicate that ultrasonic power has a relatively small effect on the extraction of the sample. When the power is above 200W, the system adaptability parameters are relatively better. Therefore, an ultrasonic power of 250W and a frequency of 35kHz are preferred.

[0167] Table 11 Results of different ultrasonic powers

[0168]

[0169]

[0170] (3) Selection of extraction solvent

[0171] The extraction effects of eight extraction solvents (ethanol, water, methanol, 10% methanol, 30% methanol, 50% methanol, 70% methanol, and 90% methanol) on corn silk granules (1903001W) were investigated. Approximately 0.5g of corn silk granules was accurately weighed and placed in a stoppered conical flask. 25mL of each extraction solvent was accurately added, the flask was sealed, and the flask was weighed. The flask was then ultrasonically treated at different powers (250W, 35kHz) for 15 minutes each. After removal and cooling, the flask was weighed again, and the lost weight was replenished with the corresponding solvent. The flask was shaken well, filtered, and 10μl of the filtrate was accurately injected into a high-performance liquid chromatograph (HPLC). Chromatographic analysis was performed according to the conditions in Example 1. The results showed that ethanol had extremely poor extraction ability and was not used (see Example 1). Figure 23As shown in the table below, the results, excluding ethanol, indicate that methanol, when used as the extraction solvent, resulted in a very low extraction rate for the main components of the formulation particles and was therefore not adopted. In the water-methanol mixed solvent system, low-concentration methanol showed high extraction rates for alkaloids and organic acids, and the system adaptability parameters were relatively superior. Among these, water and 10% methanol yielded the highest extraction rates. However, since the water extract is prone to deterioration, 10% methanol was chosen as the extraction solvent.

[0172] Table 12 Results of the investigation of different extraction solvents

[0173]

[0174]

[0175]

[0176] (4) Selection of extraction solvent amount

[0177] The extraction effect of different amounts (10 ml, 25 ml, and 50 ml) of 10% methanol on corn silk granules (batch number: 1903001W) was investigated. Approximately 0.5 g of corn silk granules was accurately weighed and placed in a stoppered conical flask. 10 ml, 25 ml, or 50 ml of 10% methanol was accurately added, the flask was sealed, and the flask was weighed. The flask was then ultrasonically treated at different powers (250 W, 35 kHz) for 15 minutes each. After removal and cooling, the flask was weighed again, and the weight was replenished with 10% methanol. The flask was shaken well, filtered, and 10 μl of the filtrate was accurately injected into the high-performance liquid chromatograph (HPLC). Chromatographic analysis was performed according to the conditions in Example 1. The results are shown in the table below. The results indicate that the system adaptability parameters of the test sample were relatively better when the amount of 10% methanol was 25 ml. Therefore, 25 ml is the preferred amount of 10% methanol as the extraction solvent.

[0178] Table 13 Results of the investigation on the amount of different extraction solvents used

[0179]

[0180]

[0181] (5) Selection of extraction time

[0182] The effects of different extraction times (15 min, 30 min, and 45 min) on the extraction efficiency of corn silk granules (batch number: 1903001W) were investigated. Approximately 0.5 g of corn silk granules was accurately weighed and placed in a stoppered conical flask. 25 ml of 10% methanol was accurately added, the flask was sealed, and the weight was measured. The flask was then ultrasonically treated at different powers (250 W, 35 kHz) for 15 min, 30 min, or 45 min. After removal and cooling, the flask was weighed again, and the weight loss was replenished with 10% methanol. The flask was shaken well, filtered, and 10 μl of the filtrate was accurately injected into the high-performance liquid chromatograph (HPLC). Chromatographic analysis was performed according to the conditions in Example 1. The results are shown in the table below. The results indicate that the differences between different extraction times are small. The system adaptability parameters of the test sample were relatively better when the extraction time was 15 minutes; therefore, 15 minutes is the preferred extraction time.

[0183] Table 14 Results of the investigation at different extraction times

[0184]

[0185]

[0186] (6) Selection of sampling size

[0187] The extraction effect of different sample amounts (0.1g, 0.2g, 0.3g, 0.5g) on ​​corn silk granules (batch number: 1903001W) was investigated. Accurately weigh 0.1g, 0.2g, 0.3g, or 0.5g of corn silk granules, place them in a stoppered conical flask, accurately add 25ml of 10% methanol, seal tightly, and weigh. Sonicate at different powers (250W, 35kHz) for 15 minutes each, remove, cool, and weigh again. Make up the weight loss with 10% methanol, shake well, filter, and accurately inject 10μl of the filtrate into the high-performance liquid chromatograph (HPLC). Chromatographic analysis was performed according to the conditions in Example 1. The results are shown in the table below. The results show that the sample amount has little effect on the chromatographic results. A sample amount of 0.5g results in better system adaptability parameters and a stronger chromatographic signal response; therefore, 0.5g is the preferred sample amount.

[0188] Table 15 Results of the study with different sampling sizes

[0189]

[0190]

[0191] (7) Selection of injection volume

[0192] The effects of different injection volumes (5 μl, 10 μl, and 15 μl) on the liquid chromatography separation of corn silk granules (batch number: 1903001W) were investigated. 0.5 g of corn silk granules was accurately weighed and placed in a stoppered conical flask. 25 ml of 10% methanol was accurately added, the flask was sealed, and the weight was measured. The flask was then ultrasonically treated at different powers (250 W, 35 kHz) for 15 minutes each. After removal and cooling, the weight was measured again, and the lost weight was replenished with 10% methanol. The flask was shaken well, filtered, and 5 μl, 10 μl, and 15 μl of the filtrate were accurately injected into the high-performance liquid chromatograph (HPLC). Chromatographic analysis was performed according to the conditions in Example 1. The results showed that the injection volume had a relatively small impact on the chromatographic results. A sample volume of 10 μl resulted in better system adaptability parameters and a stronger chromatographic signal response; therefore, 10 μl was the preferred injection volume.

[0193] Table 16 Results of different injection volumes

[0194]

[0195]

[0196] Experiment Example 2: Methodological Validation

[0197] 1. Exclusivity

[0198] Take 10 μL each of the test solution and negative control solution (10% methanol) prepared in Example 1, and inject them into the high-performance liquid chromatograph. The chromatographic conditions are the same as in Example 1. The results are as follows: Figure 24 and 25 As shown, the result was negative and there was no interference.

[0199] 2. Completeness

[0200] 10 μL of the test solution prepared in Example 1 was injected into a high-performance liquid chromatograph under the same chromatographic conditions as in Example 1. The chromatographic acquisition time was extended by one time at the highest organic phase time point. The results showed that there were no obvious chromatographic peaks on the chromatogram during the extended time period, indicating that the method has good integrity.

[0201] 3. Repeatability

[0202] Six test solutions were prepared in parallel using the same batch of corn silk granule samples according to the method in Example 1. The RSDs of the relative retention times of the nine characteristic peaks were all less than 2.0%. The corn silk granules were then analyzed using the Mark peak similarity calculation system for chromatographic fingerprints of traditional Chinese medicine. The similarity scores were all higher than 0.90. The results indicate that the method has good repeatability.

[0203] 4. Instrument precision test

[0204] The same corn silk formula granule test solution (1903001W) prepared according to the method in Example 1 was injected six times repeatedly according to the method in Example 1. The RSD of the relative retention times of the nine characteristic peaks was less than 2.0%. Furthermore, the corn silk formula granules were analyzed using the Mark peak similarity evaluation system for traditional Chinese medicine chromatographic fingerprints. The similarity between the test sample characteristic chromatogram and the control characteristic chromatogram of the corn silk formula granules was calculated, and the similarity was higher than 0.90. The results indicate that the instrument precision is good.

[0205] 5. Intermediate precision (personnel)

[0206] Three researchers prepared and measured the corn silk formula granule test solution according to the method in Example 1. The RSD of the relative retention times of the nine characteristic peaks was less than 2.0%. The corn silk formula granules were then compared with the control chromatographic fingerprint of traditional Chinese medicine using the Mark peak similarity evaluation system, and the similarity was calculated to be higher than 0.90. The results indicate good intermediate precision.

[0207] 6. Solution stability study

[0208] A sample solution of the same corn silk granule formulation (1903001W) (prepared according to the method in Example 1) was tested at 0h, 2h, 4h, 8h, 12h, and 24h according to the method in Example 1. The RSD of the relative retention times of the nine characteristic peaks was less than 2.0%. The similarity between the sample solution and the control sample solution, calculated using the Mark peak similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, was also found to be higher than 0.90. The results indicate that the sample solution is stable within 24h and meets the requirements for determination.

[0209] The examples provided are not intended to limit the implementation of the invention. Those skilled in the art will recognize that various variations and modifications can be made based on the foregoing description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations and modifications derived therefrom remain within the scope of this invention.

Claims

1. A method for constructing a characteristic spectrum of corn silk and its formulations, characterized in that, Includes the following steps, (1) Preparation of the test solution; When the test sample is a corn silk preparation, the preparation method of the test sample solution includes: weighing the corn silk test sample, adding an extraction solvent to extract it, obtaining an extract, separating the solid and liquid, and taking the liquid, which is the test sample solution; the extraction solvent used in the preparation of the test sample solution is selected from water or methanol aqueous solution; When the test sample is corn silk reference material, the preparation method of the test sample solution includes taking 2 g of corn silk reference material, placing it in a stoppered conical flask, adding 100 ml of water, heating under reflux for 2 hours, filtering, evaporating the filtrate to dryness, cooling, adding 25 ml of 10% methanol to the residue, sealing tightly, sonicating for 30 minutes with an ultrasonic power of 250 W and a frequency of 35 kHz, cooling, shaking well, filtering, and taking the filtrate as the test sample solution; Preparation of reference solutions: Reference solutions were prepared by adding solvents to guanosine reference, guanine reference, adenine reference, adenosine reference, vanillin reference, 4-coumaric acid reference and ferulic acid reference. (2) The test solution and reference solution were detected by high performance liquid chromatography. Octadecylsilane bonded silica gel was used as the stationary phase, acetonitrile was used as mobile phase A, and an aqueous solution containing sodium 1-heptanesulfonate and formic acid was used as mobile phase B. Gradient elution was performed according to the following program: 0→42 min, mobile phase A: mobile phase B was 1%:99%→18%:82%; 42→45 min, mobile phase A: mobile phase B was 18%:82%→19%:81%; 45→55 min, mobile phase A: mobile phase B was 19%:81%→60%:40%; the mass-volume percentage of sodium 1-heptanesulfonate in mobile phase B was 0.03-0.05%, the volume percentage of formic acid was 0.05-0.15%, the detection wavelength was 258-262 nm from 0 to 25 min, and the detection wavelength was 318-322 nm from 25 to 55 min.

2. The method for constructing the characteristic spectrum of corn silk and its formulations according to claim 1, characterized in that, Step (2) also satisfies at least one of the following 1)-3): 1) The flow rate is 0.8-1.2 mL / min; 2) Column temperature is 25-45℃; 3) The injection volume is 5-15µL.

3. The method for constructing the characteristic spectrum of corn silk and its formulations according to claim 1, characterized in that, Step (1) also satisfies any one or more of the following AEs: A. The ratio of the mass of the corn silk sample to the volume of the extraction solvent is 0.1-0.5:10-50; the mass-volume relationship is g / mL. B. The extraction method is either reflux extraction or ultrasonic extraction; C. Extraction time is ≥10 min; D. The solid-liquid separation is selected from centrifugation or membrane filtration; E. The extraction solvent is water or a methanol aqueous solution with a volume percentage ≤15%.

4. The method for constructing the characteristic spectrum of corn silk and its formulations according to claim 1, characterized in that, In step (1), the extraction time is 15-45 min.

5. The method for constructing the characteristic spectrum of corn silk and its formulations according to claim 1, characterized in that, In step (1), the extraction time is 15 minutes.

6. The method for constructing the characteristic spectrum of corn silk and its formulations according to claim 1, characterized in that, Each 1 mL of the reference solution contains 1-100 µg of reference standard; and / or, the solvent used in the preparation of the reference solution is selected from water, methanol, or an aqueous methanol solution.

7. The method for constructing the characteristic spectrum of corn silk and its formulations according to any one of claims 1-6, characterized in that, The characteristic spectra of the corn silk and its preparations are selected from any one of the following (1)-(2): (1) It has 9 common characteristic peaks. Peaks 1 and 8 correspond to the retention times of the guanosine reference standard peak and the ferulic acid reference standard peak, respectively. The peak corresponding to the guanosine reference standard peak is designated as peak S1. The relative retention times of peaks 2 to 5 with peak S1 are within ±10% of the specified value. The specified value is: Peak 2: 1.14, Peak 3: 1.33, Peak 4: 1.43, Peak 5: 1.61; The peak corresponding to the ferulic acid reference standard peak is designated as peak S2. The relative retention times of peaks 6, 7, and 9 with peak S2 are within ±10% of the specified value. The specified value is: Peak 6: 0.79, Peak 7: 0.87, Peak 9: 1.13; (2) It has 9 common characteristic peaks. Peaks 1 to 4 and peaks 6 to 8 correspond to the retention times of the reference peaks of guanosine, guanine, adenine, adenosine, vanillin, 4-coumaric acid and ferulic acid, respectively.

8. The method for constructing the characteristic spectrum of corn silk and its preparations according to any one of claims 1-7 is used in the quality detection of corn silk and its preparations.

9. A method for quality testing of corn silk and its preparations, characterized in that, This includes constructing a characteristic spectrum of the product to be tested using the method for constructing characteristic spectra of corn silk and its formulations according to any one of claims 1-7.

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