A method for constructing a characteristic chromatogram of leaves of Mahonia bealei

Abstract

This invention belongs to the field of medicinal material analysis technology, specifically relating to a method for constructing a characteristic spectrum of Mahonia japonica leaves. The method includes: preparation of a test solution, preparation of a reference solution, determination using ultra-high performance liquid chromatography (UHPLC), and a defined gradient elution procedure. The characteristic peaks obtained by this method exhibit high resolution, good peak shape, and no interference; the retention time distribution of the characteristic peaks is uniform, allowing for good differentiation from impurity peaks. The method provided by this invention discovers three novel indicator substances with high content and strong specificity: methyl 4-O-caffeoylquinic acid, methyl chlorogenic acid, and methyl caffeate. These novel and highly specific indicator substances can identify the authenticity of Mahonia japonica leaves and detect their quality, providing a more comprehensive identification method for Mahonia japonica leaves.

Description

Technical Field

[0001] This invention belongs to the field of medicinal material analysis technology, specifically relating to a method for constructing a characteristic spectrum of Mahonia japonica leaves. Background Technology

[0002] The 2020 edition of the Chinese Pharmacopoeia defines Mahonia as the dried stem of Mahonia bealei (Fort.) Carr. or Mahonia fortunei (Lindl.) Fedde, both belonging to the Berberidaceae family. Mahonia leaves are included in local standards. The "Guizhou Province Standard for the Quality of Traditional Chinese and Ethnic Medicines" specifies that this product is the dried leaf of Mahonia duclouxiana Gagnep., Mahonia bodinieri Gagnep., Mahonia eurybracteata Fedde, Mahonia fortunei (Lindl.) Fedde, and Mahonia bealei (Fort.) Carr., all belonging to the Berberidaceae family. Current techniques for obtaining characteristic chromatograms of Mahonia leaves suffer from several drawbacks, including the proximity of characteristic peaks to impurity peaks, high sensitivity to mobile phase pH, and dispersed peak elution times. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to overcome the defects in the existing technology of poor separation of characteristic peaks in the characteristic spectrum of Mahonia japonica leaves, which are relatively close to impurity peaks and are easily confused with characteristic peaks and impurity peaks, so as to provide a method for constructing characteristic spectrum of Mahonia japonica leaves.

[0004] To this end, the present invention provides the following technical solution.

[0005] This invention provides a method for constructing a feature map of the leaves of the Mahonia fortunei plant, comprising:

[0006] Preparation of test solution: Prepare a test solution from the test sample;

[0007] Preparation of reference solution: Prepare a reference solution from the control medicinal material;

[0008] Determination: The determination was performed using ultra-high performance liquid chromatography.

[0009] The chromatographic conditions for ultra-high performance liquid chromatography include: using acetonitrile as mobile phase A and potassium dihydrogen phosphate aqueous solution as mobile phase B, with gradient elution; the gradient elution conditions are as follows: 0-15 min, 9% mobile phase A, 91% mobile phase B; 15-30 min, 9→17% mobile phase A, 91→83% mobile phase B.

[0010] The ultra-high performance liquid chromatography (UHPLC) conditions also include: using a Waters CORTECS shield RP18 column with the following specifications: inner diameter 2.1 mm, column length 100 mm, and particle size 1.6 μm; and / or,

[0011] Column temperature is 25-35℃; and / or,

[0012] Wavelength of 280-350nm; and / or,

[0013] The injection volume is 1-5 μL; and / or,

[0014] The mobile phase B was a 0.01-0.1 mol / L potassium dihydrogen phosphate aqueous solution.

[0015] The test sample is at least one of the following: Mahonia japonica leaf medicinal material, Mahonia japonica leaf slices, Mahonia japonica leaf standard decoction freeze-dried powder, and Mahonia japonica leaf formula granules.

[0016] The leaves mentioned are those of the small-fruited Mahonia.

[0017] When the test sample is a medicinal material or decoction piece, the preparation method of the test sample solution includes: taking the test sample, adding water, heating under reflux, filtering, evaporating to dryness to obtain an aqueous extract, adding an extraction solvent to the aqueous extract, extracting, and filtering;

[0018] When the test sample is a lyophilized powder or formulation granules, the preparation method of the test sample solution includes: taking the test sample, adding an extraction solvent, extracting, and filtering.

[0019] The extraction process includes organic solvents and inorganic solvents;

[0020] Preferably, the volume ratio of the organic solvent to the inorganic solvent is 100:(1-2.5);

[0021] Preferably, the organic solvent is methanol;

[0022] Preferably, the inorganic solvent is hydrochloric acid.

[0023] When the test sample is a medicinal material or decoction piece, the ratio of the mass (g) of the water extract to the volume (ml) of the extraction solvent in the preparation of the test sample solution is (0.05-0.5):(10-50);

[0024] When the test sample is a lyophilized powder or formulation granules, the ratio of the mass of the test sample (g) to the volume of the extraction solvent (ml) is (0.05-0.5):(10-50) when preparing the test sample solution.

[0025] The preparation method of the reference solution includes: taking the reference medicinal material, extracting it with water, filtering it, evaporating it to dryness to obtain an aqueous extract; adding an extraction solvent to the aqueous extract, extracting it, and filtering it.

[0026] Preferably, the extraction includes organic solvents and inorganic solvents;

[0027] Preferably, the volume ratio of the organic solvent to the inorganic solvent is 100:(1-2.5);

[0028] Preferably, the organic solvent is methanol;

[0029] Preferably, the inorganic solvent is hydrochloric acid;

[0030] Preferably, the ratio of the mass of the water extract (g) to the volume of the extraction solvent (ml) is (0.05-0.5):(10-50).

[0031] The feature map obtained by the construction method includes 10 feature peaks;

[0032] With peak 6 as the reference peak, the specified relative retention times for peaks 1, 2, 3, 4, 5, 7, 8, 9, and 10 are 0.32, 0.41, 0.47, 0.61, 0.79, 1.08, 1.20, 1.41, and 1.60, respectively.

[0033] The relative retention time of the characteristic peak is within ±10% of the specified value.

[0034] The construction method also includes the preparation of a reference solution;

[0035] Preferably, the reference standard includes at least one selected from methyl 4-O-caffeoylquinic acid, methyl chlorogenic acid, and methyl caffeate;

[0036] Preferably, each 1 ml of the reference solution contains 10-100 μg of reference standard.

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

[0038] 1. The present invention provides a method for constructing characteristic chromatograms of Mahonia japonica leaves. This method includes: preparing a test solution, preparing a reference solution, and determining the chromatograms using ultra-high performance liquid chromatography (UHPLC), with a defined gradient elution procedure. The characteristic peaks obtained by this method exhibit high resolution, good peak shape, and no interference. The retention time distribution of the characteristic peaks is uniform, allowing for good differentiation from impurity peaks. The method provided by this invention discovers three novel indicator substances with high content and strong specificity: methyl 4-O-caffeoylquinic acid, methyl chlorogenic acid, and methyl caffeate. These novel and highly specific indicator substances can identify the authenticity of Mahonia japonica leaves and detect their quality, providing a more comprehensive identification method for Mahonia japonica leaves.

[0039] Furthermore, the construction method provided by this invention has high accuracy, short time, high precision, good repeatability and stability, and can more comprehensively monitor the quality of Mahonia leaves. Attached Figure Description

[0040] 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.

[0041] Figure 1 This is a characteristic chromatogram of 15 batches of freeze-dried powder of the standard decoction of Mahonia japonica leaves in Example 1 of the present invention;

[0042] Figure 2 This is a comparison chart of the leaves of the small-fruited Mahonia japonica in Example 1 of the present invention;

[0043] Figure 3 These are the characteristic chromatograms of 15 batches of small-fruited Mahonia japonica leaf medicinal materials in Example 2 of this invention;

[0044] Figure 4 These are characteristic chromatograms of 15 batches of small-fruited Mahonia japonica leaf slices from Embodiment 1 of the present invention;

[0045] Figure 5 These are the characteristic spectra obtained from different elution procedures in Experimental Example 1 of this invention;

[0046] Figure 6 These are characteristic spectra obtained for different mobile phase pH values ​​in Experiment Example 2 of this invention;

[0047] Figure 7 These are characteristic chromatograms obtained from different chromatographic columns in Experimental Example 3 of this invention. Detailed Implementation

[0048] 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.

[0049] 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.

[0050] The reagents and instruments used in the following examples

[0051] instrument:

[0052] Chromatograph 1: Ultra-high performance liquid chromatograph: Thermo Vanquish Flex UPLC system, including a quaternary solvent manager (Vanquish Quaternary Pump F VF-P20-A), an autosampler (Vanquish SplitSampler FT VF-A10-A-02), an original imported column oven (Vanquish Column Compartment H VH-C10-A-02), a DAD detector (Vanquish VF-D40-A), and a chromatography workstation;

[0053] Chromatograph 2: Ultra-high performance liquid chromatograph: Agilent 1290 Infinit II chromatography system, including a quaternary solvent manager (G7104A 1290 Flexible pump), an autosampler (G7129B 1290 Vialsampler), an original imported column oven (G7116B 1290MCT), and a DAD detector (G7117A 1290DAD FS);

[0054] Electronic analytical balances: METTLER TOLEDO MS204S, MS36S; Ohaus CP523C;

[0055] Constant Temperature Electric Heating Blower Drying Oven, Shanghai Yiheng Scientific Instruments Co., Ltd. DHG-9240A;

[0056] Ultrasonic Cleaner: KQ-500DB model, Kunshan Ultrasonic Instrument Co., Ltd.

[0057] Column 1: Waters CSH C18 2.1×100mm, 1.7μm;

[0058] Column 2: Waters shield RP18 2.1×100mm, 1.6μm.

[0059] Reagents:

[0060] Methanol (chromatographic grade, batch number: 22065203, manufacturer: Honeywell), acetonitrile (chromatographic grade, batch number: W9GA1H, manufacturer: Honeywell), potassium dihydrogen phosphate (analytical grade, batch number: manufacturer:)

[0061] The batch numbers for the freeze-dried powder of *Mahonia japonica* leaf decoction (batch numbers 2103001Y, 2103002Y, 2103003Y, 2103004Y, 2103005Y, 2103006Y, 2103007Y, 2103008Y, 2103009Y, 2103010Y, 2103011Y, 2103012Y, 2103013Y, 2103014Y, and 2103015Y are as follows: Batch numbers for the medicinal material of *Mahonia japonica* leaf (batch numbers 2103001YC-2103015YC); and batch numbers for the processed slices of *Mahonia japonica* leaf (batch numbers 2103001YP-2103015YP).

[0062] Example 1

[0063] This embodiment provides a method for constructing a characteristic spectrum of freeze-dried powder of standard decoction of Mahonia japonica leaves, including the following steps:

[0064] Preparation of the test solution: Take about 0.1g of freeze-dried powder of Mahonia japonica leaf standard decoction, accurately weigh it, place it in a 25mL stoppered conical flask, accurately add 10mL of methanol-hydrochloric acid (volume ratio 100:2.5), weigh it, sonicate it at 200W power and 40KHz frequency for 30min, take it out, cool it, weigh it, add methanol-hydrochloric acid (volume ratio 100:2.5) to make up the lost weight, shake it well, filter it, and take the filtrate to obtain the test solution.

[0065] Preparation of reference solution: Take about 1.0g of Mahonia japonica leaf reference material, place it in a stoppered conical flask, add 50mL of water, heat under reflux for 60min, filter, evaporate the filtrate to dryness, add 10mL of methanol-hydrochloric acid (100:2.5) mixture to the residue, sonicate at 200W power and 40KHz frequency for 30min, remove, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0066] Preparation of reference solutions: Take appropriate amounts of methyl 4-O-caffeoylquinic acid reference standard, methyl chlorogenic acid reference standard, and methyl caffeate reference standard, respectively, accurately weigh them, add methanol to them, and obtain methyl 4-O-caffeoylquinic acid reference solution containing 80 μg of methyl 4-O-caffeoylquinic acid reference standard per 1 mL, methyl chlorogenic acid reference standard solution containing 150 μg of methyl chlorogenic acid reference standard solution, and methyl caffeate reference standard solution containing 100 μg of caffeate reference standard solution per 1 mL of reference solution.

[0067] Determination: Accurately pipette 1 μL each of the test solution, reference solution, and standard solution into the liquid chromatograph and determine. The chromatographic conditions for ultra-high performance liquid chromatography (UHPLC) are as follows: using a Waters CORTECS shield RP18 column (100 mm length, 2.1 mm inner diameter, 1.6 μm particle size), acetonitrile as mobile phase A, and 0.05 mol / L potassium dihydrogen phosphate aqueous solution as mobile phase B, with gradient elution. The gradient elution conditions are as follows: 0-15 min, 9% mobile phase A, 91% mobile phase B; 15-30 min, 9→17% mobile phase A, 91→83% mobile phase B; column temperature 30℃, flow rate 0.3 mL / min; detection wavelength 326 nm. The theoretical plate number, calculated based on the methyl chlorogenic acid peak, should not be less than 5000.

[0068] Confirmation of characteristic peaks

[0069] Fifteen batches of freeze-dried powder of Mahonia japonica leaf standard decoction were taken, and test solutions were prepared according to the above steps. The characteristic spectra of each test solution were obtained under the above conditions. The results are shown in Tables 1-2 and 1-3. Figure 1-2 . Figure 1 S1 in the figure is the control spectrum, and S2-S16 correspond to 15 batches of freeze-dried powder of standard decoction of small-fruited Mahonia japonica leaves. Figure 2 For comparison with the spectrum.

[0070] Ten peaks with good repeatability were selected as common characteristic peaks, namely peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, and peak 10. These ten common characteristic peaks showed relatively small differences in relative retention times, and the average relative retention time was selected as the measured value. Using peak 6 as the reference peak, the specified values ​​for the relative retention times of peaks 1, 2, 3, 4, 5, 7, 8, 9, and 10 were 0.32, 0.41, 0.47, 0.61, 0.79, 1.08, 1.20, 1.41, and 1.60, respectively. The relative retention times of the characteristic peaks were within ±10% of the specified values.

[0071] Table 1. Relative retention time of characteristic spectra of freeze-dried powder of *Mahonia japonica* leaf standard decoction from various batches.

[0072]

[0073]

[0074] Note: t / ts is the relative retention time.

[0075] Table 2. Relative peak areas of characteristic spectra of freeze-dried powder of *Mahonia japonica* leaf standard decoction for various batches.

[0076]

[0077]

[0078] The similarity scores of the characteristic chromatograms of 15 batches of freeze-dried powder of Mahonia japonica leaf standard decoction with the characteristic chromatograms of the common pattern control were 0.992, 0.992, 0.901, 0.996, 0.977, 0.991, 0.995, 0.996, 0.996, 0.996, 0.996, 0.996, 0.999, 0.995, and 0.989, respectively. All similarity scores were above 0.90, indicating a high degree of agreement between the 15 batches of freeze-dried powder of Mahonia japonica leaf standard decoction and the common pattern control chromatograms. The 10 characteristic peaks were relatively stable, demonstrating that this detection method effectively achieved quality control.

[0079] Example 2

[0080] This embodiment provides a method for constructing a characteristic map of the leaves of Mahonia japonica, including the following steps:

[0081] Preparation of the test solution: Take approximately 1.0 g of Mahonia japonica leaf material from 15 batches, place it in a stoppered conical flask, add 50 mL of water, heat under reflux for 60 min, filter, and evaporate the filtrate to dryness to obtain the water extract. Add 10 mL of a methanol-hydrochloric acid (volume ratio 100:2.5) mixture to the water extract, and sonicate at 250 W and 40 kHz for 30 min. Remove, cool, shake well, filter, and collect the filtrate to obtain the test solution. When preparing the test solution, the mass of the water extract obtained by heating and refluxing different batches of Mahonia japonica leaf material is generally 6.5%-12.5% ​​of the material weight.

[0082] Determination: Accurately pipette 1 μL of each test solution and inject it into the liquid chromatograph for determination. The chromatographic conditions for ultra-high performance liquid chromatography (UHPLC) are as follows: using a Waters CORTECS shield RP18 column (100 mm length, 2.1 mm inner diameter, 1.6 μm particle size), acetonitrile as mobile phase A, and 0.05 mol / L potassium dihydrogen phosphate aqueous solution as mobile phase B, with gradient elution. The gradient elution conditions are as follows: 0-15 min, 9% mobile phase A, 91% mobile phase B; 15-30 min, 9→17% mobile phase A, 91→83% mobile phase B; column temperature 30℃, flow rate 0.3 mL / min; detection wavelength 326 nm. The theoretical plate number, calculated based on the methyl chlorogenic acid peak, should not be less than 5000.

[0083] Table 3. Relative retention times of characteristic chromatograms of 15 batches of small-fruited Mahonia japonica leaf medicinal materials

[0084]

[0085]

[0086] Table 4. Peak areas of characteristic spectra of 15 batches of small-fruited Mahonia japonica leaf medicinal materials

[0087]

[0088]

[0089] Figure 3 S1 in the diagram represents the control chromatogram, while S2-S16 correspond to 15 batches of *Mahonia divaricata* leaf medicinal materials. The similarity scores between the characteristic chromatograms of these 15 batches of *Mahonia divaricata* leaf medicinal materials and the characteristic chromatograms of the common pattern control are 0.913, 0.980, 0.973, 0.971, 0.978, 0.983, 0.984, 0.983, 0.987, 0.987, 0.984, 0.979, 0.985, 0.979, and 0.981, respectively. All similarity scores are above 0.90, indicating a high degree of fit between the 15 batches of *Mahonia divaricata* leaf medicinal materials and the common pattern control chromatograms. The 10 characteristic peaks are relatively stable, demonstrating that this detection method effectively achieves quality control.

[0090] Example 3

[0091] This embodiment provides a method for constructing a characteristic map of Mahonia japonica leaf slices, including the following steps:

[0092] Preparation of the test solution: Take approximately 1.0 g of Mahonia japonica leaf slices from 15 batches, place them in a stoppered conical flask, add 50 mL of water, heat under reflux for 60 min, filter, and evaporate the filtrate to dryness to obtain the water extract. Add 10 mL of a methanol-hydrochloric acid (volume ratio 100:2.5) mixture to the water extract, and sonicate at 250 W and 40 kHz for 30 min. Remove, cool, shake well, filter, and collect the filtrate to obtain the test solution. When preparing the test solution, the mass of the water extract obtained by heating and refluxing different batches of Mahonia japonica leaf slices is generally 6.5%-12.5% ​​of the weight of the slices.

[0093] Determination: Accurately pipette 1 μL of each test solution and inject it into the liquid chromatograph for determination. The chromatographic conditions for ultra-high performance liquid chromatography (UHPLC) are as follows: using a Waters CORTECS shield RP18 column (100 mm length, 2.1 mm inner diameter, 1.6 μm particle size), acetonitrile as mobile phase A, and 0.05 mol / L potassium dihydrogen phosphate aqueous solution as mobile phase B, with gradient elution. The gradient elution conditions are as follows: 0-15 min, 9% mobile phase A, 91% mobile phase B; 15-30 min, 9→17% mobile phase A, 91→83% mobile phase B; column temperature 30℃, flow rate 0.3 mL / min; detection wavelength 326 nm. The theoretical plate number, calculated based on the methyl chlorogenic acid peak, should not be less than 5000.

[0094] Table 5. Relative retention times of characteristic chromatograms of 15 batches of small-fruited Mahonia japonica leaf slices.

[0095]

[0096] Table 6. Peak area of ​​characteristic chromatograms of 15 batches of small-fruited Mahonia japonica leaf slices

[0097]

[0098]

[0099] Figure 4 In the diagram, R represents the control chromatogram, and S1-S15 correspond to 15 batches of small-fruited Mahonia japonica leaf decoction pieces. The similarity between the characteristic chromatograms of the 15 batches of small-fruited Mahonia japonica leaf decoction pieces and the characteristic chromatograms of the common pattern control is 0.918, 0.981, 0.974, 0.972, 0.981, 0.984, 0.984, 0.984, 0.988, 0.988, 0.984, 0.982, 0.986, 0.981, and 0.980, respectively. All similarities are above 0.90, indicating a high degree of fit between the 15 batches of small-fruited Mahonia japonica leaf decoction pieces and the common pattern control chromatograms. The 10 characteristic peaks are relatively stable, and this detection method effectively achieves quality control.

[0100] Experiment Example 1: Examination of Gradient Procedure

[0101] This experimental example investigated the gradient elution procedure, specifically including: taking five portions of the same batch of lyophilized powder of Mahonia japonica leaf standard decoction, preparing the test solution according to the "Test Solution" section of Example 1, using the gradient elution procedure as a variable, and obtaining the characteristic chromatogram according to the "Determination Method" section of Example 1. The results of elution procedures 1-5 are shown in [the table / example]. Figure 5 The gradient elution procedure and results are as follows:

[0102] (1) Elution program 1: 0-10 min, 25→28% mobile phase A, 75→72% mobile phase B; 10-18 min, 28→50% mobile phase A, 72→50% mobile phase B; 18-22 min, 50% mobile phase A, 50% mobile phase B. The chromatograms obtained by this gradient program indicate that most components in the sample were eluted within the first 4 min, and good separation was not achieved.

[0103] (2) Elution program 2: 0-60 min, 5 → 95% mobile phase A, 95 → 5% mobile phase B. The chromatograms obtained by this gradient program show that most of the components in the sample are eluted within the first 25 min, and the separation effect between characteristic peaks is poor.

[0104] (3) Elution program 3: 0-60 min, 10→40% mobile phase A, 90→60% mobile phase B. The spectrum obtained by this gradient program shows that the retention time of the characteristic peaks is relatively concentrated and the peak separation is poor.

[0105] (4) Elution program 4: isocratic elution, acetonitrile-0.05mol / L potassium dihydrogen phosphate solution volume ratio 10:90, mobile phase pH 3. As can be seen from the spectrum, this spectrum mainly presents 7 characteristic peaks. Compared with elution programs 1-3, the peak presentation information is better, but there are still problems with the number of peaks and poor peak symmetry, indicating that the elution separation ability at this ratio still needs to be improved.

[0106] (5) Elution program 5: 0-15 min, 9% mobile phase A, 91% mobile phase B; 15-30 min, 9→17% mobile phase A, 91→83% mobile phase B. As can be seen from the spectrum, the separation of each characteristic peak is good.

[0107] In summary, the gradient elution procedure provided by this invention produces a feature spectrum with 10 characteristic peaks, exhibiting good separation and symmetry. The retention time of the characteristic peaks is evenly distributed, providing rich information and allowing for good differentiation from impurity peaks.

[0108] Experiment Example 2: Investigation of the pH value of the mobile phase

[0109] This experimental example investigated the mobile phase pH value. Specifically, the following steps were taken: The same batch of freeze-dried powder of Mahonia japonica leaf standard decoction was used to prepare a test solution according to the "Test Solution" section of Example 1. Using the mobile phase pH value as a variable, the characteristic chromatogram was obtained according to the "Determination Method" section of Example 1. The mobile phase pH values ​​were 2.8, 2.9, 3.0, 3.1, 3.2, 3.5, and 4.5. The results are shown in [Figure Number]. Figure 6 The pH of the mobile phase was adjusted using phosphoric acid.

[0110] Figure 6 It can be seen that a mobile phase pH of 4.5 corresponds to a technical solution where the acetonitrile-0.05mol / L potassium dihydrogen phosphate solution does not require pH adjustment. From the above results, it can be seen that the peak information content is similar when the mobile phase pH is between 2.8 and 4.5. When the mobile phase pH is 4.5, that is, the pH adjustment with phosphoric acid is not required, and no additional process is needed. Therefore, this is the optimal solution under this condition.

[0111] Experimental Example 3: Investigation of the Chromatographic Column

[0112] This experimental example investigated chromatographic columns, specifically including: taking the same batch of freeze-dried powder of Mahonia japonica leaf standard decoction, preparing the test solution according to Example 1, using different chromatographic columns as variables, and determining the characteristic chromatograms according to Example 1. See [example details]. Figure 7 The chromatographic columns were Waters shield RP18 1.6μm 2.1mm×100mm and Waters CSH C18 1.7μm 2.1mm×100mm, respectively.

[0113] The results showed that the Waters shield RP18 column produced more chromatographic peaks, a more uniform time distribution, and better peak symmetry in the characteristic chromatogram. Therefore, this column is preferred as the column for the construction method of this invention.

[0114] Experiment Example 4: Investigation of Wavelength

[0115] This experimental example investigated the detection wavelength. The results showed that the maximum absorption wavelength of most characteristic peaks was around 326 nm. Therefore, 326 nm was selected as the preferred detection wavelength.

[0116] In summary, the optimal method for constructing the characteristic chromatogram of Mahonia japonica leaves is as follows: using a Waters CORTECS Shield RP18 column and octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.6 μm); using acetonitrile as mobile phase A and 0.05 mol / L potassium dihydrogen phosphate solution as mobile phase B, with gradient elution under the following conditions: 0-15 min, 9% mobile phase A, 91% mobile phase B; 15-30 min, 9→17% mobile phase A, 91→83% mobile phase B; column temperature 30℃, flow rate 0.3 ml / min; detection wavelength 326 nm. The theoretical plate number, calculated based on the methyl chlorogenic acid peak, should be no less than 5000.

[0117] Experimental Example 5: Methodological Investigation of Freeze-dried Powder of Standard Decoction of Mahonia japonica Leaves

[0118] (1) Precision test

[0119] The same sample solution (2103001Y) of the lyophilized powder of Mahonia japonica leaf standard decoction was injected six times according to the method in Example 1, and the characteristic chromatograms were obtained, as shown in the table below. The RSD of the relative retention time of each characteristic peak and the reference S peak (peak 6) in the chromatogram is less than 2%, and the RSD of the relative peak area of ​​each characteristic peak and the reference S peak (peak 6) is less than 2%, indicating that the instrument has good precision.

[0120] Table 7 Relative Retention Time

[0121]

[0122]

[0123] Table 8 Relative Peak Area

[0124]

[0125] (2) Repeated examination

[0126] Six samples (2103001Y) from the same batch were tested and their spectra were determined according to the method in Example 1. The results are shown in the table below. The RSD values ​​of the relative retention times of each characteristic peak were all less than 2.0%, and the RSD values ​​of the relative peak areas of each characteristic peak were all less than 2.0%, indicating that the method has good repeatability.

[0127] Table 9 Results of the method repeatability relative retention time test

[0128]

[0129]

[0130] Table 10 Results of the method repeatability relative peak area test

[0131]

[0132] (3) Different operators have different intermediate precision requirements

[0133] Three inspectors, at different times, took the same sample of freeze-dried powder of Mahonia japonica leaf standard decoction (batch number: 2103001Y), prepared the test solution according to Example 1, and measured the chromatograms using the same equipment. The results are shown in the table below. The results show that the RSD values ​​of the relative retention times of each characteristic peak are all less than 2.0%, and the RSD values ​​of the relative retention times of each characteristic peak are all less than 2%, indicating that the intermediate precision of this method is good.

[0134] Table 11. Results of intermediate precision relative retention time tests (by different operators)

[0135]

[0136]

[0137] Table 12 Results of Intermediate Precision Relative Peak Area Test

[0138]

[0139] (4) Stability

[0140] The same sample solution was injected at 0, 2, 4, 6, 12, and 24 hours according to the method in Example 1, and the resulting spectra are shown in the table below. The results show that the relative retention time of each characteristic peak and the reference S peak is less than 2.0%, and the relative peak area of ​​each characteristic peak and the reference S peak is less than 2%, indicating that the sample solution is stable within 24 hours and meets the determination requirements.

[0141] Table 13 Results of the relative retention time test for stability

[0142]

[0143]

[0144] Table 14 Results of Stability Relative Peak Area Test

[0145]

[0146] (5) Examination of flow velocity

[0147] The same sample of lyophilized powder of Mahonia japonica leaf standard decoction was used to prepare a test solution according to Example 1. Using flow rate as a variable, the characteristic chromatograms were obtained according to Example 1. The flow rates were 0.27 ml / min, 0.30 ml / min, and 0.33 ml / min, and the results are shown in the table below. The results show that the relative retention time RSD values ​​of each characteristic peak and the reference S peak are all less than 2.0%, and the relative peak area RSD values ​​of each characteristic peak and the reference S peak are all less than 2.0%. This indicates that small variations in flow rate have little impact on the retention time of each characteristic peak and do not affect the determination of the characteristic peak results. A flow rate of 0.3 ml / min is the optimal solution.

[0148] Table 15 Retention time of spectra obtained at different flow rates

[0149]

[0150] Table 16 Peak areas of spectra obtained at different flow rates

[0151]

[0152]

[0153] (6) Investigation of column temperature

[0154] The same sample of lyophilized powder of Mahonia japonica leaf standard decoction was used to prepare a test solution according to the "Test Solution" section of Example 1. Using column temperature as a variable, the characteristic chromatograms were obtained according to the "Determination Method" section of Example 1. The flow rates were 27℃, 30℃, and 33℃. The results showed that the relative retention time RSD values ​​of each characteristic peak and the reference S peak were all less than 3.0%, and the relative peak area RSD values ​​of each characteristic peak and the reference S peak were all less than 2.0%. This indicates that small changes in column temperature have little impact on the relative peak area of ​​each characteristic peak and do not affect the determination of the characteristic peak results. A column temperature of 30℃ is the optimal column temperature for this invention.

[0155] Table 17 Results of relative retention times at different column temperatures

[0156]

[0157] Table 18 Results of relative peak areas at different column temperatures

[0158]

[0159] (7) Different instruments

[0160] Take the same sample solution and determine it according to the method in Example 1. The difference is that the ultra-high performance liquid chromatography instruments are different brands: Agilent 1290 and Thermo Vanquish chromatograph. The results are shown in the table below.

[0161] Table 19 Results of relative retention time determination for characteristic spectra of different ultra-high performance liquid chromatography instruments

[0162]

[0163] Table 20 Relative peak area results of characteristic spectra from different ultra-high performance liquid chromatography instruments

[0164]

[0165] The above results indicate that the method of the present invention is durable for chromatographs, and both types of chromatographs are suitable for this invention.

[0166] Experimental Example 6: Methodological Investigation of the Small-Fruited Mahonia Leaf Medicinal Material

[0167] (1) Precision

[0168] The same sample solution of Mahonia japonica leaf (2103001YC) was used for testing. Following Example 2, the sample was injected six times. The RSD of the relative retention time of each characteristic peak and the reference peak (peak 6) was less than 2.0%, and the RSD of the relative peak area of ​​each characteristic peak and the reference peak (peak 6) was less than 2.0%, indicating good instrument precision. The results are shown in the table below.

[0169] Table 21 Results of Instrument Precision and Relative Retention Time Tests

[0170]

[0171]

[0172] Table 22 Results of Instrument Precision Relative Peak Area Test

[0173]

[0174] (2) Repeatability

[0175] The same sample solution of Mahonia japonica leaf was used for testing, and the results were obtained according to Example 2. The results showed that the RSD values ​​of the relative retention times of each characteristic peak were all less than 2.0%, and the RSD values ​​of the relative peak areas of each characteristic peak were all less than 2.0%, indicating that the method has good repeatability. The results are shown in the table below.

[0176] Table 23 Results of the method repeatability relative retention time test

[0177]

[0178] Table 24 Results of Method Repeatability Relative Peak Area Test

[0179]

[0180] (3) Different operators have different intermediate precision requirements

[0181] Three inspectors collected the same sample of Mahonia japonica leaves at different times, prepared test solutions according to Example 2, and used the same equipment to determine the relative retention time and relative peak area of ​​each common peak according to the method in Example 2. The results showed that the RSD values ​​of the relative retention times and relative peak areas of each characteristic peak were all less than 2.0%, indicating good intermediate precision of the method. The results are shown in the table below.

[0182] Table 25 Intermediate Precision Relative Retention Time Test Results (Different Operators)

[0183]

[0184] Table 26 Intermediate Precision Relative Peak Area Test Results (Different Operators)

[0185]

[0186] (4) Stability test

[0187] The same sample solution of Mahonia japonica leaf was taken and injected at 0, 2, 4, 6, 12, and 24 hours according to the method in Example 2. The relative retention time and relative peak area of ​​10 common peaks were measured. The relative retention time of each characteristic peak and the reference S peak were less than 2.0%, and the relative peak area of ​​each characteristic peak and the reference S peak were less than 2.0%. The results show that the sample solution is stable within 24 hours and meets the determination requirements. The results are shown in the table below.

[0188] Table 27 Results of the relative retention time test for stability

[0189]

[0190] Table 28 Results of Stability Relative Peak Area Test

[0191]

[0192]

[0193] (5) Flow rate

[0194] The same sample of Mahonia japonica leaf material was used to prepare a test solution according to the "Test Solution" section of Example 2. Using flow rate as a variable, the characteristic chromatograms were obtained according to the "Determination Method" section of Example 2. The flow rates were 0.27 ml / min, 0.30 ml / min, and 0.33 ml / min. The results showed that the relative retention time RSD values ​​of each characteristic peak and the reference S peak were all less than 4.0%, and the relative peak area RSD values ​​were all less than 2.0%. This indicates that small variations in flow rate have little impact on the relative retention time and relative peak area of ​​each characteristic peak, and do not affect the determination of the characteristic peak results. A flow rate of 0.3 ml / min was the optimal solution. The results are shown in the table below.

[0195] Table 29 Results of relative retention time for different flow velocities

[0196]

[0197] Table 30 Results of relative peak area at different flow velocities

[0198]

[0199]

[0200] (6) Column temperature

[0201] The same sample of Mahonia japonica leaf material was used to prepare a test solution according to the "Test Solution" section of Example 2. Using column temperature as a variable, the characteristic chromatograms were obtained according to the "Determination Method" section of Example 2. The flow rates were 27℃, 30℃, and 33℃. The results showed that the relative retention time RSD values ​​of each characteristic peak and the reference S peak were all less than 3.0%, and the relative peak area RSD values ​​were less than 2.0%. This indicates that slight variations in column temperature have little effect on the relative retention time and relative peak area of ​​each characteristic peak, and do not affect the chromatogram results. The results are shown in the table below.

[0202] Table 31 Comparison of relative retention times at different column temperatures

[0203]

[0204] Table 32 Results of relative peak area at different column temperatures

[0205]

[0206] (7) Investigation with different instruments

[0207] Take the same sample solution 2 and determine it according to the method of Example 2. The difference is that the ultra-high performance liquid chromatography (UHPLC) instruments are different. The UHPLC instruments are Agilent 1290UPLC and Thermo Vanquish chromatograph. The relative retention time of each characteristic peak and the reference S peak is investigated when the UHPLC instrument changes. In order to ensure applicability on different instruments, the specified range of relative retention time is set as ±10%.

[0208] Table 33 Relative retention times of characteristic spectra from different ultra-high performance liquid chromatography instruments

[0209]

[0210] Table 34 Relative peak area results of characteristic spectra from different ultra-high performance liquid chromatography instruments

[0211]

[0212] Based on the above methodological investigation results, among the 10 common peaks in the characteristic chromatogram of Mahonia japonica leaf, each chromatographic peak is affected to a certain extent by column temperature and mobile phase flow rate. The influence of each chromatographic condition is not significant, and the relative retention time values ​​are within ±10%, which is suitable for its robustness.

[0213] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a feature map of the leaves of the Mahonia japonica, characterized in that, include: Preparation of test solution: Prepare a test solution from the test sample; When the test sample is Mahonia japonica leaf medicinal material or Mahonia japonica leaf slices, the preparation method of the test sample solution includes: taking the test sample, adding water, heating under reflux, filtering, evaporating to dryness to obtain an aqueous extract, adding an extraction solvent to the aqueous extract, extracting, and filtering. When the test sample is a freeze-dried powder of Mahonia japonica leaf standard decoction or Mahonia japonica leaf formula granules, the preparation method of the test sample solution includes: taking the test sample, adding extraction solvent, extracting, and filtering; When preparing the test solution, the extraction solvent used includes an organic solvent and an inorganic solvent, wherein the volume ratio of the organic solvent to the inorganic solvent is 100:(1-2.5), the organic solvent is methanol, and the inorganic solvent is hydrochloric acid; Preparation of reference solution: Prepare a reference solution from the control medicinal material; Preparation of reference solutions: The reference solutions include methyl 4-O-caffeoylquinic acid, methyl chlorogenic acid, and methyl caffeate; Determination: The determination was performed using ultra-high performance liquid chromatography. The chromatographic conditions for ultra-high performance liquid chromatography (UHPLC) include: acetonitrile as mobile phase A, 0.01-0.1 mol / L potassium dihydrogen phosphate aqueous solution as mobile phase B, and gradient elution; the gradient elution conditions are as follows: 0-15 min, 9% mobile phase A, 91% mobile phase B; 15-30 min, 9→17% mobile phase A, 91→83% mobile phase B; a Waters CORTECS shield RP18 column is used, with the following specifications: inner diameter 2.1 mm, column length 100 mm, particle size 1.6 μm, and wavelength 280-350 nm.

2. The construction method according to claim 1, characterized in that, The ultra-high performance liquid chromatography conditions also include: a column temperature of 25-35℃; and / or, The injection volume is 1-5 μL.

3. The construction method according to claim 1 or 2, characterized in that, The leaves mentioned are those of the small-fruited Mahonia.

4. The construction method according to claim 1, characterized in that, When the test sample is a medicinal material or decoction piece, the ratio of the mass of the water extract to the volume of the extraction solvent in the preparation of the test sample solution is (0.05-0.5):(10-50), with the unit being g / ml; When the test sample is a freeze-dried powder of Mahonia japonica leaf standard decoction or Mahonia japonica leaf formula granules, the ratio of the mass of the test sample to the volume of the extraction solvent is (0.05-0.5):(10-50) when preparing the test sample solution, with the unit being g / ml.

5. The construction method according to claim 1, characterized in that, The preparation method of the reference solution includes: taking the reference medicinal material, extracting it with water, filtering it, evaporating it to dryness to obtain an aqueous extract; adding an extraction solvent to the aqueous extract, extracting it, and filtering it.

6. The construction method according to claim 5, characterized in that, The extraction solvents include organic solvents and inorganic solvents.

7. The construction method according to claim 5, characterized in that, The volume ratio of the organic solvent to the inorganic solvent is 100:(1-2.5).

8. The construction method according to claim 5, characterized in that, The organic solvent is methanol.

9. The construction method according to claim 5, characterized in that, The inorganic solvent is hydrochloric acid.

10. The construction method according to claim 5, characterized in that, The ratio of the mass of the water extract to the volume of the extraction solvent is (0.05-0.5):(10-50), with units of g / ml.

11. The construction method according to any one of claims 1, 2, 4-10, characterized in that, The feature map obtained by the construction method includes 10 feature peaks; With peak 6 as the reference peak, the specified relative retention times for peaks 1, 2, 3, 4, 5, 7, 8, 9, and 10 are 0.32, 0.41, 0.47, 0.61, 0.79, 1.08, 1.20, 1.41, and 1.60, respectively. The relative retention time of the characteristic peak is within ±10% of the specified value.

12. The construction method according to any one of claims 1, 2, 4-10, characterized in that, Each 1 ml of the reference solution contains 10-100 μg of reference standard.

Images