A method for constructing a fingerprint of dog spine and a method for determining the content of effective components

Abstract

This invention belongs to the field of drug detection, specifically relating to a method for constructing a fingerprint spectrum of Cibotium barometz and a method for determining the content of its active ingredients. The construction method includes the preparation of a test solution and detection using high-performance liquid chromatography (HPLC) to obtain the fingerprint spectrum of Cibotium barometz. This invention establishes a comprehensive and rapid method for detecting Cibotium barometz drug preparations, which is of great significance for its comprehensive quality detection and overall quality control. The characteristic spectrum obtained by this construction method has at least 12 common characteristic peaks, a stable baseline, and good peak resolution. Furthermore, the relative retention times of each characteristic peak are calculated based on protocatechuic acid, which is beneficial for the comprehensive quality detection and overall quality control of Cibotium barometz preparations, and helps improve the safety and stability of drug use.

Description

Technical Field

[0001] This invention belongs to the field of drug detection, specifically relating to a method for constructing a fingerprint spectrum of Cibotium barometz and a method for determining the content of its active ingredients. Background Technology

[0002] Cibotium barometz (L.) J.Sm., a plant in the family Cibotiumceae, is a dried rhizome that has the functions of dispelling wind and dampness, tonifying the liver and kidneys, and strengthening the waist and knees. It is used for rheumatic pain, soreness and weakness of the waist and knees, and weakness of the lower limbs.

[0003] The 2020 edition of the Chinese Pharmacopoeia includes quality control measures for Cibotium barometz, covering aspects such as the original plant variety, characteristics, physicochemical identification, and content determination. Existing literature also describes the chemical components of Cibotium barometz, including polysaccharides and organic acids. However, on the one hand, determining or identifying the content of a single component in Cibotium barometz granules cannot comprehensively detect and control their quality; on the other hand, combining the determination of a single component with the identification of other components in Cibotium barometz granules is time-consuming and labor-intensive, making it difficult to widely apply in production practice.

[0004] Chinese patent document CN113791164A discloses a method for quality testing of a standard decoction of Cibotium barometz. The method uses methanol as mobile phase A and 0.1% phosphoric acid solution as mobile phase B. The method has a long detection time, the baseline of the obtained characteristic spectrum is not smooth, the number of characteristic peaks is small, the peaks of No. 3, No. 4 and No. 6 have poor peak shapes, and there is obvious peak engulfment phenomenon. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is that the existing technology for quality control of Cibotium barometz is for Cibotium barometz slices, not for Cibotium barometz pharmaceutical preparations. Furthermore, the existing detection methods for Cibotium barometz slices are not applicable to Cibotium barometz pharmaceutical preparations. In addition, the existing detection methods for quality control of Cibotium barometz still have some defects, such as uneven baseline and few characteristic peaks. Therefore, the present invention provides a method for constructing a fingerprint spectrum of Cibotium barometz and a method for determining the content of effective components.

[0006] To this end, the present invention provides the following technical solution.

[0007] The first aspect of this invention provides a method for constructing a fingerprint spectrum of Cibotium barometz, comprising the following steps:

[0008] Preparation of test solution: Cibotium barometz preparation is used as the test sample and is prepared into a test solution;

[0009] The test solution was analyzed by high performance liquid chromatography (HPLC). Chromatographic conditions: methanol as mobile phase A, phosphoric acid solution as mobile phase B, gradient elution. The gradient elution program included: 0-50 min, mobile phase A volume percentage 5% → 10%, mobile phase B volume percentage 95% → 90%; 50-58 min, mobile phase A volume percentage 10%, mobile phase B volume percentage 90%.

[0010] The construction method further includes the following chromatographic conditions: using 0.05–0.2% phosphoric acid aqueous solution as mobile phase B; and / or, a flow rate of 0.9–1.1 ml / min; and / or, a column temperature of 23–27 °C; and / or, a detection wavelength of 258–295 nm; and / or, an injection volume of 5–15 μL; and / or, an Inertil ODS-3 column with dimensions of 250 mm × 4.6 mm and 5 μm.

[0011] The chromatographic conditions for the construction method are as follows: mobile phase B is 0.1% phosphoric acid aqueous solution; flow rate is 1.0 ml / min; column temperature is 25℃; detection wavelength is 295 nm; and injection volume is 10 μL.

[0012] The Cibotium barometz preparation is an aqueous extract.

[0013] Preferably, the dosage form of the Cibotium barometz preparation is tablets, capsules, pills, granules, honey-processed pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, or injectable preparations.

[0014] In this invention, the preparation method of Cibotium barometz preparation includes: taking Cibotium barometz slices, heating and refluxing for extraction at least once, adding 6 to 12 times the weight of water each time, extracting for at least 0.5 hours, filtering, combining the filtrates, concentrating the filtrate to a relative density of 1.05-1.10 g / mL at 60°C, adding conventional excipients, and preparing clinically acceptable tablets, capsules, pills, granules, honey-refined pills, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, or injectable preparations according to conventional processes.

[0015] Pharmaceutically acceptable excipients include: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, and matrices. 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.

[0016] The preparation process of Cibotium barometz formula granules and Cibotium barometz ointment is listed here, but it is not limited to this method. The preparation method of Cibotium barometz formula granules includes: taking Cibotium barometz slices, heating and refluxing to extract twice. For the first extraction, add 9 times the weight of water and soak for 30 minutes, then heat and reflux to extract for 0.5 hours, filter, and for the second extraction, add 7 times the weight of water and extract for 0.5 hours, filter, combine the filtrates, concentrate the filtrate to a relative density of 1.05 g / mL at 60℃, spray dry, add maltodextrin as an excipient to the dry powder, mix evenly, and then dry granulate to make granules.

[0017] Preparation method of Cibotium barometz ointment: Take Cibotium barometz slices, add 10 times the amount of water, decoct twice, 1 hour each time, filter, combine the filtrates, and concentrate the filtrate under reduced pressure at 60℃ to form a thick ointment with a relative density of 1.30~1.40 (60℃), which is the final product.

[0018] The preparation of the test solution includes:

[0019] Take the test sample, add solvent, extract, filter, and collect the filtrate;

[0020] Preferably, the solvent is water;

[0021] Preferably, the ratio of the mass (in g) of the test sample to the volume (in ml) of the solvent is 0.5-1.5:10-25.

[0022] The feature map obtained by the construction method includes at least 12 feature peaks;

[0023] Using peak 10 as the reference peak, the specified relative retention times of each characteristic peak are as follows: peak 1: 0.18, peak 2: 0.37, peak 3: 0.53, peak 4: 0.57, peak 5: 0.63, peak 6: 0.77, peak 7: 0.82, peak 8: 0.88, peak 9: 0.94, peak 11: 1.13, and peak 12: 1.38.

[0024] The relative retention time of each characteristic peak is within ±10% of the specified value.

[0025] Furthermore, peak 4 is 5-hydroxymethylfurfural; peak 5 is 1-O-caffeoyl-β-D-glucose; peak 6 is protocatechuic acid-4-O-glucosinolate; peak 10 is protocatechuic acid; and peak 12 is protocatechuic aldehyde.

[0026] The construction method also includes the preparation of a reference solution;

[0027] Preferably, the preparation of the reference solution specifically includes: taking the reference substance, adding a solvent, extracting, filtering, evaporating the filtrate to dryness to obtain a residue; adding water to the residue, extracting, filtering, and collecting the filtrate. For example, the solvent can be water.

[0028] The construction method also includes the preparation of a reference solution;

[0029] Preferably, each 1 ml of the reference solution contains 5–20 μg of reference standard;

[0030] Preferably, the reference standard is at least one selected from 5-hydroxymethylfurfural, protocatechuic acid, protocatechuic aldehyde, 1-O-caffeoyl-β-D-glucose, and protocatechuic acid-4-O-glucosinolate.

[0031] A second aspect of the present invention provides a method for determining the content of active ingredients in Cibotium barometz, using the above-described construction method;

[0032] Preferably, the active ingredient includes at least one of 5-hydroxymethylfurfural, protocatechuic acid, protocatechuic aldehyde, 1-O-caffeoyl-β-D-glucose, and protocatechuic acid-4-O-glucosinolate.

[0033] The technical solution of this invention has the following advantages:

[0034] 1. This invention provides a method for constructing a fingerprint spectrum of Cibotium barometz, comprising the preparation of a test solution and detection using high-performance liquid chromatography (HPLC) to obtain the fingerprint spectrum of Cibotium barometz. This invention establishes a comprehensive and rapid method for detecting Cibotium barometz drug preparations, which is of great significance for comprehensive quality testing and overall quality control. The characteristic spectrum obtained by this method has at least 12 common characteristic peaks, a stable baseline, and good peak resolution. Furthermore, the relative retention times of each characteristic peak are calculated based on protocatechuic acid, which is beneficial for comprehensive quality testing and overall quality control of Cibotium barometz preparations, and helps improve the safety and stability of drug use.

[0035] Furthermore, this construction method also has the advantages of good separation, high precision, good stability and repeatability, and can comprehensively and rapidly detect the effective components and contents in Cibotium barometz drugs, such as 5-hydroxymethylfurfural, protocatechuic acid, protocatechuic aldehyde, 1-O-caffeoyl-β-D-glucose and protocatechuic acid-4-O-glucosinolate.

[0036] 2. The method for determining the content of effective components in Cibotium barometz provided by the present invention can simultaneously determine the content of 5-hydroxymethylfurfural, protocatechuic acid, protocatechuic aldehyde, 1-O-caffeoyl-β-D-glucose and protocatechuic acid-4-O-glucosinolate, and the test results have high accuracy. Attached Figure Description

[0037] 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.

[0038] Figure 1 This is the chromatogram of the negative control sample in Experiment Example 1 of this invention; Figure 2 These are the spectra obtained from different gradient elution procedures in Section 2.1 of Experimental Example 2 of this invention; where a is the spectra obtained from gradient elution procedure 1, b is the spectra obtained from gradient elution procedure 2, and c is the spectra obtained from gradient elution procedure 3. Figure 3 This is the full-wavelength scan spectrum in section 2.2 of Experimental Example 2 of this invention; Figure 4 These are the spectra obtained from different mobile phases B in Section 2.3 of Experimental Example 2 of this invention; a is the spectra obtained with 0.05% phosphoric acid solution as mobile phase B, b is the spectra obtained with 0.1% phosphoric acid solution as mobile phase B, and c is the spectra obtained with 0.2% phosphoric acid solution as mobile phase B. Figure 5 These are the spectra obtained at different column temperatures in Section 2.4 of Experimental Example 2 of this invention; a is the spectra obtained at a column temperature of 25℃, b is the spectra obtained at a column temperature of 23℃, and c is the spectra obtained at a column temperature of 27℃. Figure 6 These are the spectra obtained at different flow rates in Section 2.5 of Experimental Example 2 of this invention; a is the spectra obtained at a flow rate of 0.95 ml / min, b is the spectra obtained at a flow rate of 1.0 ml / min, and c is the spectra obtained at a flow rate of 1.05 ml / min. Figure 7 These are the chromatograms obtained from different chromatographic columns in Section 2.6 of Experimental Example 2 of this invention; a is the chromatogram obtained with a Waters XSelect HSS T3 column, b is the chromatogram obtained with an Agilent ZORBAX SB-C18 column, and c is the chromatogram obtained with an Inertsil ODS-3 column. Figure 8 These are the spectra obtained by different instruments in Section 2.7 of Experimental Example 2 of this invention; a is the spectra obtained by the Waters chromatograph, and b is the spectra obtained by the Thermo Fisher chromatograph. Figure 9 This is a chromatogram of different batches of Cibotium barometz formula granules from Example 3 of the present invention; Figure 10 This is a comparative characteristic spectrum of the Cibotium barometz formula granules from Example 3 of the present invention; Figure 11 These are the chromatograms of different reference standards and test samples from Example 3 of the present invention; Figure 12 This is a chromatogram of the control medicinal material in Example 3 of the present invention; Figure 13 This is the chromatogram of the negative control sample in section 4.4 of Example 4 of this invention. Detailed Implementation

[0039] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0040] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0041] Instruments and reagents

[0042] Instruments and equipment:

[0043] Chromatograph 1: Thermo Fisher U3000 chromatography system, including LPG-3400A quaternary pump, WPS-3000TSL autosampler, PDA diode array detector, and Chromeleon chromatography workstation;

[0044] Chromatograph 2: Waters Arc chromatography system, including a quaternary gradient pump, a 120-position high-performance autosampler, an original imported column oven, a Waters 2998 diode array UV detector, and an Empower chromatography management system;

[0045] High-power numerically controlled ultrasonic instrument (KQ-400KDB, Kunshan Ultrasonic Instrument Co., Ltd.), 0.001g electronic balance (Shimadzu AY120, Shimadzu Corporation, Japan), 0.001g electronic balance (Sartorius SQP SECURA225D-1CN, Sartorius Scientific Instruments (Beijing) Co., Ltd.);

[0046] Chromatographic column:

[0047] Waters XSelect HSS T3 (4.6mm×250mm, 5μm)

[0048] Agilent ZORBAX SB-C18 (4.6mm×250mm, 5μm)

[0049] GL Sciences Inertsil ODS-3 (4.6mm×250mm, 5μm)

[0050] reagents

[0051] Methanol and acetonitrile were chromatographic grade (Merck); phosphoric acid (chromatographic grade, 85-90%, Aladdin); glacial acetic acid (HPLC, >99.9%, Aladdin); formic acid (chromatographic grade, ≥98%, Aladdin); water (Milli-Q).

[0052] Drug testing

[0053] Protocatechuic acid reference standard (batch number: 110809-202207, purity: 97.5%, purchased from China National Institutes for Food and Drug Control);

[0054] Cibotium barometz reference material (batch number: 121071-201706, purchased from the National Institutes for Food and Drug Control);

[0055] Cibotium barometz formula granules (batch numbers: 1903001W, 1905002S, 1909001S, 2003001S, 2005001W, 2011001W, 2102001S, 2106002W, 2108001W, 2112002S, 2201002S, 2203001W, 2207002W, 2209003S, 2211001W, 2302001W, 2305002S, 2308003W, designated as S1~S18).

[0056] Example 1

[0057] This embodiment provides a method for constructing a fingerprint map of canine spine, including the following steps:

[0058] Preparation of the test solution: Weigh approximately 1g of Cibotium barometz formula granules accurately, place them in a stoppered conical flask, add 20mL of water accurately, seal tightly, sonicate (power 250W, frequency 40kHz) for 30min, remove, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0059] Preparation of reference solution: Accurately weigh 2g of Cibotium barometz reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 45min, filter, evaporate the filtrate to dryness, add 10ml of water to the residue, sonicate (power 250W, frequency 40kHz) for 30min, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0060] Preparation of reference solution: Take an appropriate amount of protocatechuic acid reference standard, add water to prepare a solution containing 10 μg protocatechuic acid per 1 ml, and use it as the reference solution.

[0061] The test solution, reference solution, and standard solution were taken separately and analyzed by high performance liquid chromatography (HPLC) to obtain characteristic chromatograms. The chromatographic conditions were as follows: an Inertil ODS-3 column was used, packed with octadecylsilane-bonded silica gel (250 mm length, 4.6 mm inner diameter, 5 μm particle size); methanol was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution. The gradient elution program included: 0-50 min, mobile phase A volume percentage 5% → 10%, mobile phase B volume percentage 95% → 90%; 50-58 min, mobile phase A volume percentage 10%, mobile phase B volume percentage 90%; flow rate 1.0 mL / min; column temperature 25 °C; detection wavelength 295 nm; theoretical plate number calculated based on the protocatechuic acid peak should be no less than 3000; injection volume 10 μL.

[0062] Example 2

[0063] This embodiment provides a method for constructing a fingerprint map of canine spine, including the following steps:

[0064] Preparation of the test solution: Weigh about 1g of Cibotium barometz extract, place it in a 20mL volumetric flask, add 15mL of water, seal tightly, sonicate (power 250W, frequency 40kHz) for 30min, remove, cool, add water to volume, shake well, filter, and collect the filtrate to obtain the test solution.

[0065] Preparation of reference solution: Accurately weigh 2g of Cibotium barometz reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 45min, filter, evaporate the filtrate to dryness, add 10ml of water to the residue, sonicate (power 250W, frequency 40kHz) for 30min, cool, shake well, filter, and take the filtrate as the reference solution for the reference material.

[0066] Preparation of reference solution: Take an appropriate amount of protocatechuic acid reference standard, add water to prepare a solution containing 10 μg protocatechuic acid per 1 ml, and use it as the reference solution.

[0067] The test solution, reference solution, and standard solution were taken separately and analyzed by high performance liquid chromatography (HPLC) to obtain characteristic chromatograms. The chromatographic conditions were as follows: an Inertil ODS-3 column was used, packed with octadecylsilane-bonded silica gel (250 mm length, 4.6 mm inner diameter, 5 μm particle size); methanol was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution. The gradient elution program included: 0-50 min, mobile phase A volume percentage 5% → 10%, mobile phase B volume percentage 95% → 90%; 50-58 min, mobile phase A volume percentage 10%, mobile phase B volume percentage 90%; flow rate 1.0 mL / min; column temperature 25 °C; detection wavelength 295 nm; theoretical plate number calculated based on the protocatechuic acid peak should be no less than 3000; injection volume 10 μL.

[0068] Experimental Example 1: Validation of Chromatographic Conditions and System Suitability

[0069] The test solution of Cibotium barometz formula granules was prepared according to Example 1, and the chromatographic conditions and system suitability of Cibotium barometz formula granules were determined to verify whether they were suitable for Cibotium barometz formula granules, and to investigate whether negative samples of Cibotium barometz formula granules would cause interference.

[0070] Figure 1 The results show that the negative control sample (the negative control sample is an excipient used in the production process of the formulation granules, namely maltodextrin) has no interference with the characteristic chromatogram. The chromatographic method has good system adaptability and specificity and can be used as a method for detecting the characteristic chromatogram of Cibotium barometz formulation granules.

[0071] Experimental Example 2: Investigation of Chromatographic Conditions

[0072] 2.1 Examination of gradient elution procedures

[0073] Three test solutions were prepared according to Example 1. Using a gradient elution program as the variable, the results were determined according to Example 1. The gradient elution program is shown in the table below, and the results are shown in the table below. Figure 2 and the table below.

[0074] The optimal gradient elution program was determined based on the richness of chromatographic information, the separation of major chromatographic peaks, baseline, and other parameters. Results showed that gradient elution programs 1 and 2 yielded poorer peak information and resolution, while gradient elution program 3 provided better peak separation and richer chromatographic information. Therefore, gradient elution program 3 was the preferred option.

[0075] A. Gradient elution procedure 1

[0076] Table 1 Gradient elution program 1

[0077] Time (minutes) Methanol (%) 0.1% Phosphoric Acid (%) 0～50 0～5 100～95 50～60 5～20 95～80

[0078] B. Gradient elution program 2

[0079] Table 2 Gradient elution program 2

[0080] Time (minutes) Methanol (%) 0.1% Phosphoric Acid (%) 0～20 8～18 92～82 20～55 18～15 82～85 55～60 15～25 85～75

[0081] C. Gradient elution procedure 3

[0082] Table 3 Gradient elution program 3

[0083] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～50 5～10 95～90 50～58 10 90

[0084] Table 4 Results of the investigation of different gradient elution programs

[0085]

[0086]

[0087] 2.2 Wavelength Selection

[0088] A full-wavelength scan of protocatechuic acid reference standard was performed, and the results are shown below. Figure 3 The protocatechuic acid reference standard was found to have maximum absorption at wavelengths of 260.07 nm and 294.05 nm. However, the resolution and peak response of the characteristic peaks in the chromatogram of the Cibotium barometz formula granules were worse at 260.07 nm than at 294.05 nm. Therefore, 294.05 nm was selected and rounded down to 295 nm as the detection wavelength for the characteristic chromatogram method of Cibotium barometz formula granules.

[0089] 2.3 Selection of mobile phase B

[0090] Three test solutions were prepared according to Example 1. Using mobile phase B as the variable, the tests were performed according to Example 1. The results are shown below. Figure 4 The results are shown in the table below. Mobile phase B was 0.05% phosphoric acid, 0.1% phosphoric acid, and 0.2% phosphoric acid, respectively. The results indicate that different concentrations of phosphoric acid have a certain impact on the information content of chromatographic peaks and system adaptability parameters; 0.1% phosphoric acid is preferred as mobile phase B.

[0091] Table 5. Results of using phosphoric acid solutions of different concentrations as mobile phase B

[0092]

[0093]

[0094] 2.4 Column Temperature Selection

[0095] Three test solutions were prepared according to Example 1, and the results were determined according to Example 1, with column temperature as the variable. The results are shown in [Figure 1]. Figure 5 The results are shown in the table below. The column temperatures were 25℃, 23℃, and 27℃. The results indicate that different column temperatures have a certain impact on the information content of chromatographic peaks and system adaptability parameters, with 25℃ being the optimal column temperature.

[0096] Table 6 Results of the investigation at different column temperatures

[0097]

[0098] 2.5 Selection of Flow Rate

[0099] Three test solutions were prepared according to Example 1, and the flow rate was measured according to Example 1. The results are shown in [Figure 1]. Figure 6 The results are shown in the table below. The flow rates were 0.95 ml / min, 1.0 ml / min, and 1.05 ml / min. The results indicate that different flow rates have a certain impact on the information content of the chromatographic peaks and the system adaptability parameters; the optimal flow rate is 1.0 ml / min.

[0100] Table 7 Results of investigation at different flow velocities

[0101]

[0102]

[0103] 2.6 Selection of Chromatographic Column

[0104] Three test solutions were prepared according to Example 1, and the chromatographic column was used as a variable for determination according to Example 1. The results are shown in [Figure 1]. Figure 7The results are shown in the table below. Columns 1-3 were: Waters XSelect HSS T3 (4.6mm × 250mm, 5μm), Agilent ZORBAX SB-C18 (4.6mm × 250mm, 5μm), and GL Sciences Inertsil ODS-3 (4.6mm × 250mm, 5μm), respectively. The results indicate that different columns have a certain impact on the information content of chromatographic peaks and system suitability parameters, with the GL Sciences Inertsil ODS-3 (4.6mm × 250mm, 5μm) column being the preferred choice.

[0105] Table 8 Results of investigation using different chromatographic columns

[0106]

[0107]

[0108] 2.7 Instrument Selection

[0109] Two test solutions were prepared according to Example 1, and the chromatograms were used as the variable for determination according to Example 1. The results are shown in [Figure 1]. Figure 8 The results are shown in the table below. Chromatographs 1 and 2 are Waters and Thermo Fisher, respectively. The results indicate that different chromatographs have little impact on the information content of chromatographic peaks and system adaptability parameters; therefore, a stationary liquid chromatograph is not required at this time, and all chromatographs are applicable.

[0110] Table 9. Results of the investigation using different instruments

[0111]

[0112] Based on the above, the optimal chromatographic parameters can be determined as follows: Octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, with gradient elution. The gradient elution program includes: 0-50 min, mobile phase A volume percentage 5% → 10%, mobile phase B volume percentage 95% → 90%; 50-58 min, mobile phase A volume percentage 10%, mobile phase B volume percentage 90%; flow rate 1.0 mL / min, column temperature 25℃; detection wavelength 295 nm. The theoretical plate number, calculated based on the protocatechuic acid peak, should be no less than 3000.

[0113] Example 3: Establishment of Feature Map

[0114] Preparation of the test solution: Weigh approximately 1g of Cibotium barometz formula granules accurately, place them in a stoppered conical flask, add 20ml of water, seal tightly, sonicate (power 250W, frequency 40kHz) for 30 minutes, remove, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0115] Preparation of reference solution: Accurately weigh 2g of Cibotium barometz reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 45min, filter, evaporate the filtrate to dryness, add 10ml of water to the residue, sonicate (power 250W, frequency 40kHz) for 30min, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0116] Take an appropriate amount of protocatechuic acid reference standard, add water to prepare a solution containing 10 μg per 1 ml, and use it as the reference solution.

[0117] Take appropriate amounts of 5-hydroxymethylfurfural, protocatechuic acid-4-O-glucoside, and protocatechuic aldehyde reference standards, add 50% methanol to prepare a mixed solution containing 10 μg of each per ml, and use it as a mixed reference solution.

[0118] Accurately pipette 10 μL of each sample and inject it into the liquid chromatograph for determination. 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); methanol was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution. The gradient elution program included: 0-50 min, mobile phase A volume percentage 5% → 10%, mobile phase B volume percentage 95% → 90%; 50-58 min, mobile phase A volume percentage 10%, mobile phase B volume percentage 90%; flow rate 1.0 mL / min, column temperature 25 °C; detection wavelength 295 nm; and the theoretical plate number, calculated based on the protocatechuic acid peak, should be no less than 3000.

[0119] The fingerprint chromatograms of 18 representative batches of Cibotium barometz formula granules were analyzed. The fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission was used to generate reference characteristic chromatograms. Protocatechuic acid, 5-hydroxymethylfurfural, protocatechuic acid-4-O-glucoside, and protocatechuic aldehyde reference standards were used for localization and identification. Through the identification and assignment of characteristic peaks, a total of 12 characteristic peaks were obtained from the HPLC characteristic chromatograms of Cibotium barometz formula granules. The fingerprint chromatograms of the 18 representative batches of Cibotium barometz formula granules are shown below. Figure 9 See the table below for comparison with the characteristic maps. Figure 10 The chromatograms of the reference standard and the test sample are shown in [reference]. Figure 11 The reference chromatogram of the medicinal material can be found in [the image]. Figure 12 .

[0120] Using peak 10 as a reference peak, the relative retention times of each characteristic peak were calculated. The relative retention times were within ±10% of the specified values: peak 1 was 0.18, peak 2 was 0.37, peak 3 was 0.53, peak 4 was 0.57, peak 5 was 0.63, peak 6 was 0.77, peak 7 was 0.82, peak 8 was 0.88, peak 9 was 0.94, peak 11 was 1.13, and peak 12 was 1.38. Based on peak identification and reference standard localization, peak 4 was confirmed as 5-hydroxymethylfurfural; peak 5 as 1-O-caffeoyl-β-D-glucose; peak 6 as protocatechuic acid-4-O-glucoside; peak 10 as protocatechuic acid; and peak 12 as protocatechuic aldehyde.

[0121] Table 10. Characteristic chromatograms of different batches of Cibotium barometz formulation granules, with relative retention times of each chromatographic peak.

[0122]

[0123]

[0124]

[0125] The fingerprint similarity results of 18 batches of Cibotium barometz formula granules were 0.995, 0.991, 0.989, 0.992, 0.993, 0.989, 0.977, 0.987, 1.000, 0.974, 0.993, 0.999, 0.975, 0.981, 0.965, 0.974, 0.966, and 0.981, respectively.

[0126] Experiment Example 4: Methodological Validation

[0127] 4.1 Instrument Precision

[0128] The same Cibotium barometz formula granules (1903001W) were used to prepare a test solution according to Example 1. The sample was injected 6 times in accordance with Example 1. The RSD of the relative retention time of the characteristic peak was less than 2.0%, which indicates that the instrument has good precision.

[0129] Table 11 Results of instrument precision relative retention time

[0130]

[0131] Table 12 Results of Instrument Precision Relative Peak Area

[0132]

[0133]

[0134] 4.2 Repeatability Experiment

[0135] Six test solutions were prepared from the same batch of Cibotium barometz formula granules (1903001W) according to Example 1, and the RSD of the relative retention time of the characteristic peak was less than 2.0%, indicating that the method has good repeatability.

[0136] Table 13 Results of Relative Retention Times in Repeatability Experiments

[0137]

[0138] Table 14 Results of Relative Peak Area in Repeatability Experiments

[0139]

[0140] 4.3 Stability Test

[0141] The same Cibotium barometz formula granules (1903001W) were used to prepare a test solution according to Example 1. The solution was measured at 0h, 2h, 4h, 8h, 12h and 24h according to the method of Example 1. The RSD of the relative retention time of the characteristic peak was calculated to be less than 2.0%. The results showed that the test solution was stable within 24h and met the measurement requirements.

[0142] Table 15 Results of relative retention times in stability experiments

[0143]

[0144]

[0145] Table 16 Results of relative peak area in stability test

[0146]

[0147] 4.4 Specificity

[0148] Accurately pipette 10 μL each of the test solution and the negative control sample solution (maltodextrin, an excipient used in the production of Cibotium barometz formula granules) and inject them separately into the high-performance liquid chromatograph. Analyze the results according to Example 1. Figure 13 The spectrum of the negative control sample shows that there is no interference from the negative control.

[0149] 4.5 Intermediate Precision

[0150] Three different researchers prepared the Cibotium barometz formula granule test solution (Cibotium barometz formula granule batch number: 1903001W) according to Example 1, and measured and calculated the RSD of the relative retention time of the characteristic peak. The results showed that the intermediate precision was good.

[0151] Table 17 Intermediate Precision (Personnel) Relative Retention Time

[0152]

[0153]

[0154] Table 18 Intermediate Precision (Personnel) Relative Peak Area

[0155]

[0156] 4.6 Chromatographic columns of different brands

[0157] The same Cibotium barometz granules (1903001W) were used to prepare a test solution according to Example 1. The chromatographic column was used as a variable, and the determination was performed according to Example 1. Columns 1-3 were Waters XSelect HSS T3, Agilent ZORBAX SB-C18, and Inertil ODS-3, respectively. The results showed that different columns had a certain impact on the information content of the chromatographic peaks and the system suitability parameters. After comparative analysis of the above results, the Inertil ODS-3 column was preferred. Where t is the retention time, and t / ts is the relative retention time.

[0158] Table 19 Comparison of relative retention times for different brands of chromatographic columns

[0159]

[0160] 4.7 Different models of high performance liquid chromatographs

[0161] Take the same Cibotium barometz formula granules (1903001W), prepare a test solution according to Example 1, and determine the characteristic chromatograms on Thermo Fisher Scientific and Waters high-performance liquid chromatography (HPLC). Compare the chromatograms and relative retention times obtained by different instruments. Among them, chromatographs 1-2 are Waters and Thermo Fisher Scientific, respectively.

[0162] The results showed that the chromatographic information presented by different brands of high performance liquid chromatography was relatively complete, and none of the characteristic peaks were missing. Different brands of high performance liquid chromatographs showed good instrument durability for this method.

[0163] Table 20 Relative Retention Time for Instrument Durability Assessment

[0164]

[0165] Example 3

[0166] This embodiment provides a method for determining the content of active ingredients in Cibotium barometz, including the following steps:

[0167] Preparation of the test solution: Weigh approximately 1g of Cibotium barometz formula granules accurately, place them in a stoppered conical flask, add 20mL of water accurately, seal tightly, sonicate (power 250W, frequency 40kHz) for 30min, remove, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0168] Preparation of reference solution: Take an appropriate amount of protocatechuic acid reference standard, add water to prepare a solution containing 10 μg protocatechuic acid per 1 ml, and use it as the reference solution.

[0169] The test solution and reference solution were taken separately and determined by high performance liquid chromatography (HPLC) to obtain characteristic chromatograms. The chromatographic conditions were as follows: an Inertil ODS-3 column was used, packed with octadecylsilane-bonded silica gel (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); methanol was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution. The gradient elution program included: 0-50 min, mobile phase A volume percentage 5% → 10%, mobile phase B volume percentage 95% → 90%; 50-58 min, mobile phase A volume percentage 10%, mobile phase B volume percentage 90%; flow rate 1.0 mL / min; column temperature 25 °C; detection wavelength 295 nm; theoretical plate number calculated based on the protocatechuic acid peak should be no less than 3000; injection volume 10 μL.

[0170] Table 21 Results of determination of protocatechuic acid and protocatechuic aldehyde content in Cibotium barometz formula granules

[0171]

[0172]

[0173] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a fingerprint spectrum of Cibotium barometz, characterized in that, Includes the following steps: Preparation of the test solution: The Cibotium barometz preparation is used as the test sample and is prepared into a test solution; the Cibotium barometz preparation is an aqueous extract; water is used as the solvent in the preparation of the test solution. The reference standards are 5-hydroxymethylfurfural, protocatechuic acid, protocatechuic aldehyde, 1-O-caffeoyl-β-D-glucose and protocatechuic acid-4-O-glucosidase; The test solution was analyzed by high performance liquid chromatography (HPLC). Chromatographic conditions: Inertil ODS-3 column, detection wavelength 295 nm, methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution, gradient elution program included: 0-50 min, mobile phase A volume percentage 5%→10%, mobile phase B volume percentage 95%→90%. 50-58 min, the volume percentage of mobile phase A is 10%, and the volume percentage of mobile phase B is 90%.

2. The construction method according to claim 1, characterized in that, Chromatographic conditions also include a flow rate of 0.9–1.1 ml / min; and / or a column temperature of 23–27 °C; and / or an injection volume of 5–15 μL; and / or a column size of 250 mm × 4.6 mm and 5 µm.

3. The construction method according to claim 1, characterized in that, The chromatographic conditions were as follows: flow rate 1.0 ml / min; column temperature 25 ℃; injection volume 10 μL.

4. The construction method according to any one of claims 1 to 3, characterized in that, The dosage forms of the Cibotium barometz preparations are tablets, capsules, pills, granules, sustained-release preparations, immediate-release preparations, controlled-release preparations, oral liquid preparations, or injectable preparations.

5. The construction method according to any one of claims 1 to 3, characterized in that, The preparation of the test solution includes: Take the sample, add solvent, extract, filter, and collect the filtrate.

6. The construction method according to claim 5, characterized in that, The ratio of the mass of the test sample to the volume of the solvent is 0.5~1.5:10~25, where the mass of the test sample is in g and the volume of the solvent is in ml.

7. The construction method according to any one of claims 1 to 3, characterized in that, The feature map obtained by the construction method includes at least 12 feature peaks; Using peak 10 as the reference peak, the specified values ​​for the relative retention times of each characteristic peak are as follows: peak 1: 0.18, peak 2: 0.37, peak 3: 0.53, peak 4: 0.57, peak 5: 0.63, peak 6: 0.77, peak 7: 0.82, peak 8: 0.88, peak 9: 0.94, peak 11: 1.13, and peak 12: 1.

38. The relative retention time of each characteristic peak is within ±10% of the specified value.

8. The construction method according to claim 7, characterized in that, Peak 4 is 5-hydroxymethylfurfural; peak 5 is 1-O-caffeoyl-β-D-glucose; peak 6 is protocatechuic acid-4-O-glucoside; peak 10 is protocatechuic acid; and peak 12 is protocatechuic aldehyde.

9. The construction method according to any one of claims 1 to 3, characterized in that, The construction method also includes the preparation of a reference solution.

10. The construction method according to claim 9, characterized in that, The preparation of the reference solution specifically includes: taking the reference substance, adding solvent, extracting, filtering, evaporating the filtrate to dryness to obtain the residue; adding water to the residue, extracting, filtering, and taking the filtrate.

11. The construction method according to any one of claims 1 to 3, characterized in that, The reference standard was prepared into a reference standard solution.

12. The construction method according to claim 11, characterized in that, Each 1 ml of the reference solution contains 5-20 μg of reference standard.

13. A method for determining the content of active ingredients in Cibotium barometz, characterized in that, The construction method described in any one of claims 1 to 12 is adopted; The active ingredients include 5-hydroxymethylfurfural, protocatechuic acid, protocatechuic aldehyde, 1-O-caffeoyl-β-D-glucose and protocatechuic acid-4-O-glucosinolate.

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