Fingerprint detection method of Mahonia bealei and its identification and extraction method

Abstract

The present application belongs to the technical field of traditional Chinese medicine modernization, and particularly relates to a fingerprint spectrum detection method of Mahonia leaf, and an identification and extraction method thereof. The fingerprint spectrum detection method of Mahonia leaf comprises the following steps: (1) preparation of a reference solution; (2) preparation of a sample solution: taking a Mahonia leaf sample and adding an extraction solvent B for extraction; (3) performing ultra-high performance liquid chromatography analysis on the reference solution and the sample solution; and (4) establishing a fingerprint spectrum of the Mahonia leaf medicinal material. The fingerprint spectrum detection method of Mahonia leaf can identify Mahonia leaf of different origins, and screen common characteristic peaks and / or potential differential components of Mahonia leaf of different origins. In addition, the extraction method can extract index components of Mahonia leaf through column layer analysis. The chemical fingerprint spectrum method of the present application is stable and reliable, can significantly distinguish Mahonia leaf of different origins, and can provide a reference for quality control, evaluation and origin identification of Mahonia leaf, and has important significance for ensuring clinical efficacy.

Description

Technical Field

[0001] This invention belongs to the field of modern technology of traditional Chinese medicine, specifically involving the fingerprint spectrum detection method of Mahonia japonica leaves and its identification and extraction methods. Background Technology

[0002] Folium Mahoniae leaves are primarily used in traditional medicine in Guizhou Province. They are bitter and cold in nature, and enter the lung, liver, and kidney meridians. They possess the effects of nourishing yin, clearing heat, relieving cough and resolving phlegm, and can treat pulmonary tuberculosis cough and night sweats. Modern research shows that plants in the Folium Mahoniae genus mainly contain alkaloids, flavonoids, and phenolic acids, which exert antibacterial, anti-inflammatory, antioxidant, antitumor, and hepatoprotective biological activities. There are over 250 species of Mahonia plants in my country, mainly distributed in Sichuan, Yunnan, Guizhou, and Guangzhou. Due to their numerous origins and the fact that most are wild, adulteration and confusion exist in the current medicinal materials market. The 2003 edition of the "Quality Standards for Traditional Chinese and Ethnic Medicinal Materials of Guizhou Province" includes five species of Mahonia fortunei (Lindl.) Fedde, Mahonia duclouxiana Gagnep., Mahonia bealei (Fort.) Carr., Mahonia bodinieri Gagnep., and Mahonia eurybracteata Fedde as medicinal sources. However, this standard only provides morphological descriptions and microscopic identification of cross-sections of Mahonia eurybracteata leaves. Therefore, establishing reasonable and feasible analytical methods to differentiate Mahonia leaf medicinal materials from multiple origins and to study the differences in chemical composition among different sources are of great significance for interspecific quality evaluation and screening of high-quality resources. Summary of the Invention

[0003] The technical problem solved by this invention is that the existing technology lacks reports on fingerprint spectrum of Mahonia leaves and analytical methods for distinguishing and identifying Mahonia leaves from different origins. This is not conducive to the quality control of Mahonia leaves. There is an urgent need to establish a fingerprint spectrum detection method for Mahonia leaves in order to control the quality of Mahonia leaf-related products.

[0004] To address the aforementioned technical problems, this invention provides a fingerprint spectrum detection method for Mahonia japonica leaves, as well as methods for identification and extraction, providing a reference for the quality control, evaluation, and original identification of Mahonia japonica leaves.

[0005] Specifically, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a method for detecting the fingerprint spectrum of Mahonia leaves, which includes the following steps:

[0007] (1) Preparation of reference solution:

[0008] Weigh appropriate amounts of reference standards for neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, cypermethrin hydrochloride, palmatine hydrochloride, berberine hydrochloride, isochlorogenic acid A, and isochlorogenic acid C, and add solvent A to prepare reference standard solutions.

[0009] (2) Preparation of the test solution:

[0010] Take a sample of Mahonia leaves and add extraction solvent B for extraction to obtain the test solution;

[0011] (3) Ultra-high performance liquid chromatography analysis

[0012] Using octadecylsilane-bonded silica gel as the packing material, mobile phase A was aqueous, and mobile phase B was organic. The reference solution and the test solution were injected into the ultra-high performance liquid chromatograph, and the analysis was performed using a gradient elution program.

[0013] (4) Establish fingerprint maps of the leaves of Mahonia japonica with different origins.

[0014] Preferably, in step (1), solvent A is a mixture of hydrochloric acid and methanol.

[0015] Preferably, in step (1), the volume ratio of hydrochloric acid to methanol is 1:90 to 110.

[0016] Preferably, in step (1), the volume ratio of hydrochloric acid to methanol is 1:100.

[0017] Preferably, in step (1), the concentration of the reference solution is as follows: the mass concentrations of neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, cypermethrin hydrochloride, palmatine hydrochloride, berberine hydrochloride, isochlorogenic acid A and isochlorogenic acid C are 14-15 μg / mL, 8-9 μg / mL, 320-321 μg / mL, 19-20 μg / mL, 35-36 μg / mL, 71-72 μg / mL, 18-19 μg / mL and 5-6 μg / mL, respectively.

[0018] Preferably, in step (1), the concentration of the reference solution is as follows: the mass concentrations of neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, cypermethrin hydrochloride, palmatine hydrochloride, berberine hydrochloride, isochlorogenic acid A and isochlorogenic acid C are 14 μg / mL, 8 μg / mL, 320 μg / mL, 19 μg / mL, 35 μg / mL, 71 μg / mL, 18 μg / mL and 5 μg / mL, respectively.

[0019] Preferably, in step (1), the concentration of the reference solution is as follows: the mass concentrations of neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, cypermethrin hydrochloride, palmatine hydrochloride, berberine hydrochloride, isochlorogenic acid A and isochlorogenic acid C are 14.896 μg / mL, 8.722 μg / mL, 320.977 μg / mL, 19.010 μg / mL, 35.737 μg / mL, 71.520 μg / mL, 18.931 μg / mL and 5.076 μg / mL, respectively.

[0020] Preferably, in step (2), the preparation step of the test solution includes: taking a sample of Mahonia japonica leaves, adding extraction solvent B for extraction, weighing the sample, replenishing the lost weight with extraction solvent B, filtering, and taking the filtrate to obtain the test solution. The Mahonia japonica leaf sample is one or more 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.

[0021] Preferably, in step (2), solvent B is a mixed solvent of hydrochloric acid and lower alcohol.

[0022] Preferably, in step (2), the lower alcohol includes methanol and / or ethanol.

[0023] Preferably, in step (2), the volume ratio of hydrochloric acid to lower alcohol is 1:90-110.

[0024] Preferably, in step (2), the volume ratio of hydrochloric acid to lower alcohol is 1:100.

[0025] Preferably, in step (2), the mass-to-volume ratio of the Mahonia japonica leaf sample to solvent B is 1:30-50, where mass is measured in g and volume in mL.

[0026] Preferably, in step (2), the mass-to-volume ratio of the Mahonia japonica leaf sample to solvent B is 1:40, where the mass unit is g and the volume unit is mL.

[0027] Preferably, in step (2), the extraction is performed using shaking, reflux extraction, or ultrasonic extraction.

[0028] Preferably, in step (2), the extract is obtained by ultrasonic extraction.

[0029] Preferably, in step (2), the extraction time is 15 to 60 minutes.

[0030] Preferably, in step (2), the extraction time is 45 minutes.

[0031] Preferably, in step (3), the gradient elution procedure is as follows:

[0032] From 0 to 21 min, the volume percentage of mobile phase A decreased from 85% to 82%, while the volume percentage of mobile phase B increased from 15% to 18%.

[0033] Between 21 and 21.5 min, the volume percentage of mobile phase A decreased from 82% to 78%, while the volume percentage of mobile phase B increased from 18% to 22%.

[0034] Between 21.5 and 32 minutes, the volume percentage of mobile phase A decreased from 78% to 77%, while the volume percentage of mobile phase B increased from 22% to 23%.

[0035] Over 32–38 minutes, the volume percentage of mobile phase A decreased from 77% to 72%, while the volume percentage of mobile phase B increased from 23% to 28%.

[0036] Over 38–50 minutes, the volume percentage of mobile phase A decreased from 72% to 63%, while the volume percentage of mobile phase B increased from 28% to 37%.

[0037] Between 50 and 50.1 min, the volume percentage of mobile phase A increased from 63% to 85%, while the volume percentage of mobile phase B decreased from 37% to 15%.

[0038] From 50.1 to 55 min, the volume percentage of mobile phase A was 85%, and the volume percentage of mobile phase B was 15%.

[0039] Preferably, in step (3), the detection wavelength of the chromatogram is 330-340 nm.

[0040] Preferably, in step (3), the detection wavelength of the chromatogram is 336 nm;

[0041] Preferably, in step (3), the flow rate is 0.8 to 1.2 mL / min.

[0042] Preferably, in step (3), the flow rate is 1 mL / min;

[0043] Preferably, in step (3), the column temperature is 20-30°C.

[0044] Preferably, in step (3), the column temperature is 25°C;

[0045] Preferably, in step (3), the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0046] Preferably, in step (3), the chromatographic column is 2-JADE-PAKKP-C18-AQ.

[0047] Preferably, in step (3), the organic phase is acetonitrile or methanol.

[0048] Preferably, in step (3), the aqueous phase is water, a solution containing potassium dihydrogen phosphate, a solution containing ammonium dihydrogen phosphate, or a solution containing potassium dihydrogen phosphate and sodium dodecyl sulfate.

[0049] Preferably, in step (3), the pH of the aqueous phase is 2.0 to 4.0.

[0050] Preferably, in step (3), the aqueous phase is a 0.05 mol / L potassium dihydrogen phosphate solution; or the aqueous phase is a 0.05 mol / L ammonium dihydrogen phosphate solution; or the aqueous phase is a 0.05 mol / L potassium dihydrogen phosphate solution and sodium dodecyl sulfate.

[0051] Preferably, in step (3), the aqueous phase is a 0.05 mol / L potassium dihydrogen phosphate solution and sodium dodecyl sulfate, wherein each 100 mL of the aqueous phase contains 0.2 to 0.4 g of sodium dodecyl sulfate.

[0052] Preferably, in step (4), the different original Mahonia leaves are Mahonia long-columnar, Mahonia narrow-leaved, Mahonia broad-leaved, Mahonia small-fruited, and Mahonia broad-bracted, as well as other Mahonia species.

[0053] Preferably, the fingerprint spectrum of the Mahonia leaves contains 7 common fingerprint characteristic peaks; the relative retention time A of each common peak is calculated with reference to the retention time of the second common fingerprint characteristic peak (peak 9), wherein the second fingerprint characteristic peak is the fingerprint characteristic peak of chlorogenic acid;

[0054] The relative retention time of the first shared fingerprint feature peak (peak 5) is 0.61.

[0055] The relative retention time of the second shared fingerprint feature peak (peak 9) is 1.0.

[0056] The relative retention time of the third shared fingerprint feature peak (peak 11) is 1.41.

[0057] The relative retention time of the fourth shared fingerprint feature peak (peak 14) is 1.79.

[0058] The relative retention time of the fifth shared fingerprint feature peak (peak 15) is 1.9.

[0059] The relative retention time of the sixth shared fingerprint feature peak (peak 16) is 2.16, and

[0060] The relative retention time of the shared fingerprint feature peak (peak 17) is 2.23.

[0061] The error in the relative retention time of the fingerprint feature peaks is within ±10%.

[0062] Preferably, the fingerprint characteristic peaks are assigned as follows: peak 4 is neochlorogenic acid, peak 7 is cryptochlorogenic acid, peak 9 is chlorogenic acid, peak 15 is cypermethrin hydrochloride, peak 16 is palmatine hydrochloride, peak 17 is berberine hydrochloride, peak 18 is isochlorogenic acid A, and peak 19 is isochlorogenic acid C.

[0063] Preferably, the similarity between the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the control sample shall not be less than 0.90, calculated according to the similarity evaluation system of chromatographic fingerprint chromatogram of traditional Chinese medicine.

[0064] Secondly, the present invention provides an application of the fingerprint spectrum detection method of Mahonia japonica leaves in the quality control of Mahonia japonica leaves, in the identification of Mahonia japonica leaves of different origins, or in the detection of the content of indicative components in Mahonia japonica leaves.

[0065] Preferably, the present invention provides an application of the fingerprint spectrum detection method of Mahonia japonica leaves in the quality control of Mahonia japonica leaf medicinal materials, Mahonia japonica leaf slices, Mahonia japonica leaf standard decoction freeze-dried powder, or Mahonia japonica leaf formula granules; in the identification of Mahonia japonica leaf medicinal materials, Mahonia japonica leaf slices, Mahonia japonica leaf standard decoction freeze-dried powder, or Mahonia japonica leaf formula granules from different origins; or in the detection of the content of indicative components in Mahonia japonica leaf medicinal materials, Mahonia japonica leaf slices, Mahonia japonica leaf standard decoction freeze-dried powder, or Mahonia japonica leaf formula granules.

[0066] Thirdly, the present invention provides a method for identifying characteristic markers of Mahonia leaves from different basal origins, wherein the method for identifying characteristic markers of Mahonia leaves from different basal origins is performed by a fingerprint spectrum detection method for Mahonia leaves.

[0067] Preferably, peaks 3, 12, 13, 15, 16, 7, and 14 are potential differential components; the relative retention time of each common peak is calculated with reference to the retention time of peak 9, which is a fingerprint characteristic peak of chlorogenic acid; the relative retention time of peak 3 is 0.41, the relative retention time of peak 12 is 1.52, the relative retention time of peak 13 is 1.64, the relative retention time of peak 15 is 1.90, the relative retention time of peak 16 is 2.16, the relative retention time of peak 7 is 0.78, and the relative retention time of peak 14 is 1.79; the error of the relative retention time of the fingerprint characteristic peaks is within ±10%.

[0068] Preferably, peak 3 is exclusive to broadleaf Mahonia leaves and can distinguish broadleaf Mahonia leaves from other original Mahonia leaves.

[0069] Preferably, peak 12 is missing only the broad-bracted Mahonia leaves, which can distinguish the broad-bracted Mahonia leaves from other original Mahonia leaves.

[0070] Preferably, the leaves containing peak 13 but not peak 3 are small-fruited Mahonia leaves, which can distinguish small-fruited Mahonia leaves from other original Mahonia leaves.

[0071] Preferably, the leaves containing peak 12 but not peak 7 are fine-leaved Mahonia leaves, which can distinguish fine-leaved Mahonia leaves from other original Mahonia leaves.

[0072] Preferably, the leaves containing peak 7 but not peak 13 are Mahonia longifolia leaves, which can distinguish Mahonia longifolia leaves from other Mahonia primordia leaves.

[0073] Fourthly, the present invention provides a chromatographic column extraction method for the indicative components of Mahonia japonica leaves, characterized in that the chromatographic column extraction method includes the following steps:

[0074] (2) Preparation of the test solution

[0075] Take a sample of Mahonia leaves and add extraction solvent B for extraction to obtain the test solution;

[0076] (5) Extraction by chromatographic column analysis

[0077] Using octadecylsilane-bonded silica gel as the packing material, mobile phase A as the aqueous phase and mobile phase B as the organic phase, extraction was performed using a gradient elution process to obtain one or more components containing neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, purslane hydrochloride, palmatine hydrochloride, small wall wall alkaloid, isochlorogenic acid A, and isochlorogenic acid C.

[0078] Preferably, in step (2), solvent B is a mixed solvent of hydrochloric acid and lower alcohol.

[0079] Preferably, in step (2), the lower alcohol includes methanol and / or ethanol.

[0080] Preferably, in step (2), the volume ratio of hydrochloric acid to lower alcohol is 1:90-110.

[0081] Preferably, in step (2), the volume ratio of hydrochloric acid to lower alcohol is 1:100.

[0082] Preferably, in step (2), the mass-to-volume ratio of the Mahonia japonica leaf sample to solvent B is 1:30-50, where mass is measured in g and volume in mL.

[0083] Preferably, in step (2), the mass-to-volume ratio of the Mahonia japonica leaf sample to solvent B is 1:40, where the mass unit is g and the volume unit is mL.

[0084] Preferably, in step (2), the extraction is performed using shaking, reflux extraction, or ultrasonic extraction.

[0085] Preferably, in step (2), the extract is obtained by ultrasonic extraction.

[0086] Preferably, in step (2), the extraction time is 15 to 60 minutes.

[0087] Preferably, in step (2), the extraction time is 45 minutes.

[0088] Preferably, in step (5), the gradient elution procedure is as follows:

[0089] From 0 to 21 min, the volume percentage of mobile phase A decreased from 85% to 82%, while the volume percentage of mobile phase B increased from 15% to 18%.

[0090] Between 21 and 21.5 min, the volume percentage of mobile phase A decreased from 82% to 78%, while the volume percentage of mobile phase B increased from 18% to 22%.

[0091] Between 21.5 and 32 minutes, the volume percentage of mobile phase A decreased from 78% to 77%, while the volume percentage of mobile phase B increased from 22% to 23%.

[0092] Over 32–38 minutes, the volume percentage of mobile phase A decreased from 77% to 72%, while the volume percentage of mobile phase B increased from 23% to 28%.

[0093] Over 38–50 minutes, the volume percentage of mobile phase A decreased from 72% to 63%, while the volume percentage of mobile phase B increased from 28% to 37%.

[0094] Between 50 and 50.1 min, the volume percentage of mobile phase A increased from 63% to 85%, while the volume percentage of mobile phase B decreased from 37% to 15%.

[0095] From 50.1 to 55 min, the volume percentage of mobile phase A was 85%, and the volume percentage of mobile phase B was 15%.

[0096] Preferably, in step (5), the detection wavelength of the chromatogram is 330-340 nm.

[0097] Preferably, in step (5), the detection wavelength of the chromatogram is 336 nm;

[0098] Preferably, in step (5), the flow rate is 0.8 to 1.2 mL / min.

[0099] Preferably, in step (5), the flow rate is 1 mL / min;

[0100] Preferably, in step (5), the column temperature is 20-30°C.

[0101] Preferably, in step (5), the column temperature is 25°C;

[0102] Preferably, in step (5), the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0103] Preferably, in step (5), the chromatographic column is 2-JADE-PAKKP-C18-AQ.

[0104] Preferably, in step (5), the organic phase is acetonitrile or methanol.

[0105] Preferably, in step (5), the aqueous phase is water, a solution containing potassium dihydrogen phosphate, a solution containing ammonium dihydrogen phosphate, or a solution containing potassium dihydrogen phosphate and sodium dodecyl sulfate.

[0106] Preferably, in step (5), the pH of the aqueous phase is 2.0 to 4.0.

[0107] Preferably, in step (5), the aqueous phase is a 0.05 mol / L potassium dihydrogen phosphate solution; or the aqueous phase is a 0.05 mol / L ammonium dihydrogen phosphate solution; or the aqueous phase is a 0.05 mol / L potassium dihydrogen phosphate solution and sodium dodecyl sulfate.

[0108] Preferably, in step (5), the aqueous phase is a 0.05 mol / L potassium dihydrogen phosphate solution and sodium dodecyl sulfate, wherein each 100 mL of the aqueous phase contains 0.2 to 0.4 g of sodium dodecyl sulfate.

[0109] Beneficial effects of the present invention

[0110] (1) Based on the structural properties of the active ingredients contained in Mahonia japonica leaves, the present invention includes neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, cypermethrin hydrochloride, palmatine hydrochloride, berberine hydrochloride, isochlorogenic acid A and isochlorogenic acid C, etc. Through a large number of experiments, the optimal mobile phase composition, gradient elution program, flow rate, detection wavelength, chromatographic column and column temperature and other analytical conditions were screened. After multiple experimental verifications, it is shown that the fingerprint detection method of Mahonia japonica leaves provided by the present invention can comprehensively, objectively and accurately detect and evaluate the quality of Mahonia japonica leaves, which is of great significance for ensuring clinical efficacy.

[0111] (2) The HPLC fingerprint spectrum of Mahonia leaves provided by the present invention showed that: the leaves of Mahonia fasciata with narrow leaves contained 13 fingerprint characteristic peaks, the leaves of Mahonia fasciata with long columns contained 17 fingerprint characteristic peaks, the leaves of Mahonia fasciata with broad leaves contained 11 fingerprint characteristic peaks, the leaves of Mahonia fasciata with small fruits contained 14 fingerprint characteristic peaks, and the leaves of Mahonia fasciata with broad bracts contained 10 fingerprint characteristic peaks; and the chemical composition of 8 of the fingerprint characteristic peaks was identified.

[0112] (3) This invention establishes an HPLC fingerprint of Mahonia japonica leaves and applies cluster analysis and pattern recognition techniques to identify the differences between medicinal materials from different origins. This effectively distinguishes Mahonia japonica leaves from different origins and identifies seven significantly different components among the species. This chemical fingerprinting method is stable and reliable, and can significantly distinguish five different Mahonia japonica leaves from different origins, providing a reference for their quality control, evaluation, and origin identification. Attached Figure Description

[0113] Figure 1 HPLC chromatogram of organic phase for fingerprint analysis of Mahonia japonica leaf (long column) medicinal material.

[0114] Figure 2 HPLC chromatograms of different types of buffer salts were used to investigate the fingerprint chromatograms of Mahonia japonica leaves (long column).

[0115] Figure 3 HPLC chromatograms of the aqueous phase at different pH values ​​were used to investigate the fingerprinting of Mahonia japonica leaf (long column) medicinal material.

[0116] Figure 4 HPLC chromatogram for fingerprint gradient analysis of Mahonia japonica leaf (long column) medicinal material.

[0117] Figure 5 HPLC chromatograms of different brands of chromatographic columns were investigated to obtain fingerprint chromatograms of Mahonia japonica leaves (long column).

[0118] Figure 6 HPLC chromatograms of the fingerprint spectrum of Mahonia japonica leaf (long column) at different column temperatures were investigated.

[0119] Figure 7 HPLC chromatograms were used to investigate the fingerprinting of Mahonia japonica leaves (long column) at different flow rates.

[0120] Figure 8 HPLC chromatograms of different solvents were used to investigate the fingerprint chromatograms of Mahonia japonica leaves (long column) medicinal material.

[0121] Figure 9 HPLC chromatograms were used to investigate the fingerprinting of Mahonia japonica leaves (long column) under different solvent concentrations.

[0122] Figure 10 HPLC fingerprints of 18 batches of Mahonia japonica leaf medicinal materials.

[0123] Figure 11 HPLC fingerprints of 25 batches of Mahonia japonica leaf medicinal materials.

[0124] Figure 12 HPLC fingerprints of 15 batches of broadleaf Mahonia leaf medicinal materials.

[0125] Figure 13 HPLC fingerprints of 16 batches of small-fruited Mahonia japonica leaf medicinal materials.

[0126] Figure 14 HPLC fingerprints of 13 batches of Mahonia japonica leaf medicinal materials.

[0127] Figure 15 This is the HPLC chromatogram showing the common peak assignments.

[0128] Figure 16 Cluster analysis diagram of 87 batches of Mahonia japonica leaf samples.

[0129] Figure 17 PCA(A) and OPLS-DA(B) score plots for Mahonia japonica leaf samples.

[0130] Figure 18 VIP value plot for potential interspecific differences.

[0131] Figure 19 HPLC chromatogram for fingerprinting specificity of Mahonia japonica leaf (long column) medicinal material.

[0132] Figure 20 HPLC chromatogram for the overall fingerprint analysis of Mahonia japonica leaf (long column) medicinal material. Detailed Implementation

[0133] As described above, the purpose of this invention is to provide a fingerprint spectrum detection method for Mahonia japonica leaves, as well as a method for identification and extraction.

[0134] The potential differential components, also known as characteristic markers, are compounds that can be used to distinguish the differences between different original Mahonia leaves.

[0135] In one specific embodiment, the present invention provides a fingerprint spectrum detection method for the leaves of Mahonia japonica, comprising the following steps:

[0136] (1) Chromatographic conditions

[0137] A KP-C18-AQ column (250 mm × 4.6 mm, 5 μm) was used with a flow rate of 1.0 mL / min, a column temperature of 25 °C, and a detection wavelength of 336 nm. Gradient elution was performed using acetonitrile (B) - 0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid) (A).

[0138] The gradient elution procedure is as follows:

[0139] From 0 to 21 min, the volume percentage of mobile phase A decreased from 85% to 82%, while the volume percentage of mobile phase B increased from 15% to 18%.

[0140] Between 21 and 21.5 min, the volume percentage of mobile phase A decreased from 82% to 78%, while the volume percentage of mobile phase B increased from 18% to 22%.

[0141] Between 21.5 and 32 minutes, the volume percentage of mobile phase A decreased from 78% to 77%, while the volume percentage of mobile phase B increased from 22% to 23%.

[0142] Over 32–38 minutes, the volume percentage of mobile phase A decreased from 77% to 72%, while the volume percentage of mobile phase B increased from 23% to 28%.

[0143] Over 38–50 minutes, the volume percentage of mobile phase A decreased from 72% to 63%, while the volume percentage of mobile phase B increased from 28% to 37%.

[0144] Between 50 and 50.1 min, the volume percentage of mobile phase A increased from 63% to 85%, while the volume percentage of mobile phase B decreased from 37% to 15%.

[0145] From 50.1 to 55 min, the volume percentage of mobile phase A was 85%, and the volume percentage of mobile phase B was 15%.

[0146] (2) Preparation of reference solution

[0147] Accurately weigh appropriate amounts of neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, purslane hydrochloride, palmatine hydrochloride, berberine hydrochloride, isochlorogenic acid A, and isochlorogenic acid C reference standards, and add hydrochloric acid-methanol (hydrochloric acid and methanol volume ratio of 1:100) to prepare mixed reference standard solutions with mass concentrations of 14.896 μg / mL, 8.722 μg / mL, 320.977 μg / mL, 19.010 μg / mL, 35.737 μg / mL, 71.520 μg / mL, 18.931 μg / mL, and 5.076 μg / mL, respectively.

[0148] (3) Preparation of test solution

[0149] Accurately weigh approximately 0.5 g of sample powder (passed through a No. 3 sieve, pore size 355±13 μm) and place it in a stoppered conical flask. Accurately add 20 mL of hydrochloric acid-methanol mixture (hydrochloric acid to methanol volume ratio 1:100), and sonicate (power 500W, frequency 40kHz) for 45 min. After cooling, replenish the lost mass with hydrochloric acid-methanol mixture (hydrochloric acid to methanol volume ratio 1:100), shake well, filter, and filter the filtrate through a 0.45 μm microporous membrane. The hydrochloric acid used in this invention is commercially available hydrochloric acid with a concentration of 36%–38%.

[0150] In a second specific embodiment, the present invention provides a method for identifying characteristic markers of leaves from different basal origins of Mahonia japonica, comprising the following steps:

[0151] (1) Preparation of reference solution

[0152] Weigh appropriate amounts of reference standards for neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, cypermethrin hydrochloride, palmatine hydrochloride, berberine hydrochloride, isochlorogenic acid A, and isochlorogenic acid C, and add solvent A to prepare reference standard solutions.

[0153] (2) Preparation of the test solution

[0154] Take a sample of Mahonia leaves and add extraction solvent B for extraction to obtain the test solution;

[0155] (3) Ultra-high performance liquid chromatography analysis

[0156] Using octadecylsilane-bonded silica gel as the packing material, mobile phase A was aqueous, and mobile phase B was organic. The reference solution and the test solution were injected into the ultra-high performance liquid chromatograph, and the analysis was performed using a gradient elution program.

[0157] (4) Establish fingerprint maps of the leaves of Mahonia japonica with different origins.

[0158] Preferably, in step (1), solvent A is a mixed solvent of hydrochloric acid and methanol.

[0159] Preferably, in step (1), the volume ratio of hydrochloric acid to methanol is 1:90 to 110.

[0160] Preferably, the test solution preparation step in step (2) includes: taking a sample of Mahonia leaves, adding extraction solvent B for extraction, weighing the sample, replenishing the lost weight with extraction solvent B, filtering, and taking the filtrate to obtain the test solution.

[0161] Preferably, in step (2), solvent B is a mixed solvent of hydrochloric acid and lower alcohol.

[0162] Preferably, in step (2), the lower alcohol includes methanol and / or ethanol.

[0163] Preferably, in step (2), the volume ratio of hydrochloric acid to lower alcohol is 1:90-110.

[0164] Preferably, in step (2), the mass-to-volume ratio of the Mahonia japonica leaf sample to solvent B is 1:30-50, where mass is measured in g and volume in mL.

[0165] Preferably, in step (2), the extraction is performed using shaking, reflux extraction, or ultrasonic extraction.

[0166] Preferably, in step (2), the extraction time is 15 to 60 minutes.

[0167] Preferably, in step (3), the gradient elution procedure is as follows:

[0168] From 0 to 21 min, the volume percentage of mobile phase A decreased from 85% to 82%, while the volume percentage of mobile phase B increased from 15% to 18%.

[0169] Between 21 and 21.5 min, the volume percentage of mobile phase A decreased from 82% to 78%, while the volume percentage of mobile phase B increased from 18% to 22%.

[0170] Between 21.5 and 32 minutes, the volume percentage of mobile phase A decreased from 78% to 77%, while the volume percentage of mobile phase B increased from 22% to 23%.

[0171] Over 32–38 minutes, the volume percentage of mobile phase A decreased from 77% to 72%, while the volume percentage of mobile phase B increased from 23% to 28%.

[0172] Over 38–50 minutes, the volume percentage of mobile phase A decreased from 72% to 63%, while the volume percentage of mobile phase B increased from 28% to 37%.

[0173] Between 50 and 50.1 min, the volume percentage of mobile phase A increased from 63% to 85%, while the volume percentage of mobile phase B decreased from 37% to 15%.

[0174] From 50.1 to 55 min, the volume percentage of mobile phase A was 85%, and the volume percentage of mobile phase B was 15%.

[0175] Preferably, in step (3), the detection wavelength of the ultra-high performance liquid chromatography is 330-340 nm.

[0176] Preferably, in step (3), the flow rate is 0.8 to 1.2 mL / min.

[0177] Preferably, in step (3), the column temperature is 20-30°C.

[0178] Preferably, in step (3), the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0179] Preferably, in step (3), the chromatographic column is 2-JADE-PAK KP-C18-AQ.

[0180] Preferably, in step (3), the organic phase is acetonitrile or methanol.

[0181] Preferably, in step (3), the aqueous phase is water, a solution containing potassium dihydrogen phosphate, a solution containing ammonium dihydrogen phosphate, or a solution containing potassium dihydrogen phosphate and sodium dodecyl sulfate.

[0182] Preferably, in step (3), the pH of the aqueous phase is 2.0 to 4.0.

[0183] Preferably, in step (3), the aqueous phase is a 0.05 mol / L potassium dihydrogen phosphate solution; or the aqueous phase is a 0.05 mol / L ammonium dihydrogen phosphate solution; or the aqueous phase is a 0.05 mol / L potassium dihydrogen phosphate solution and sodium dodecyl sulfate.

[0184] Preferably, in step (3), the aqueous phase is a 0.05 mol / L potassium dihydrogen phosphate solution and sodium dodecyl sulfate, wherein each 100 mL of the aqueous phase contains 0.2 to 0.4 g of sodium dodecyl sulfate.

[0185] In a third specific embodiment, the present invention provides a chromatographic column extraction method for the indicative components of Mahonia japonica leaves, wherein the indicative components include one or more of neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, isochlorogenic acid A, and isochlorogenic acid C, comprising the following steps:

[0186] (2) Preparation of the test solution

[0187] Take a sample of Mahonia leaves and add extraction solvent B for extraction to obtain the test solution;

[0188] (5) Ultra-high performance liquid chromatography analysis

[0189] Using octadecylsilane-bonded silica gel as the packing material, mobile phase A was aqueous, and mobile phase B was organic. The reference solution and the test solution were injected into the ultra-high performance liquid chromatograph, and the analysis and extraction were performed using a gradient elution program.

[0190] Preferably, the test solution preparation step in step (2) includes: taking a sample of Mahonia leaves, adding extraction solvent B for extraction, weighing the sample, replenishing the lost weight with extraction solvent B, filtering, and taking the filtrate to obtain the test solution.

[0191] Preferably, in step (2), solvent B is a mixed solvent of hydrochloric acid and lower alcohol.

[0192] Preferably, in step (2), the lower alcohol includes methanol and / or ethanol.

[0193] Preferably, in step (2), the volume ratio of hydrochloric acid to lower alcohol is 1:90-110.

[0194] Preferably, in step (2), the mass-to-volume ratio of the Mahonia japonica leaf sample to solvent B is 1:30-50, where mass is measured in g and volume in mL.

[0195] Preferably, in step (2), the extraction is performed using shaking, reflux extraction, or ultrasonic extraction.

[0196] Preferably, in step (2), the extraction time is 15 to 60 minutes.

[0197] Preferably, in step (5), the gradient elution procedure is as follows:

[0198] From 0 to 21 min, the volume percentage of mobile phase A decreased from 85% to 82%, while the volume percentage of mobile phase B increased from 15% to 18%.

[0199] Between 21 and 21.5 min, the volume percentage of mobile phase A decreased from 82% to 78%, while the volume percentage of mobile phase B increased from 18% to 22%.

[0200] Between 21.5 and 32 minutes, the volume percentage of mobile phase A decreased from 78% to 77%, while the volume percentage of mobile phase B increased from 22% to 23%.

[0201] Over 32–38 minutes, the volume percentage of mobile phase A decreased from 77% to 72%, while the volume percentage of mobile phase B increased from 23% to 28%.

[0202] Over 38–50 minutes, the volume percentage of mobile phase A decreased from 72% to 63%, while the volume percentage of mobile phase B increased from 28% to 37%.

[0203] Between 50 and 50.1 min, the volume percentage of mobile phase A increased from 63% to 85%, while the volume percentage of mobile phase B decreased from 37% to 15%.

[0204] From 50.1 to 55 min, the volume percentage of mobile phase A was 85%, and the volume percentage of mobile phase B was 15%.

[0205] Preferably, in step (5), the detection wavelength of the ultra-high performance liquid chromatography is 330-340 nm.

[0206] Preferably, in step (5), the flow rate is 0.8 to 1.2 mL / min.

[0207] Preferably, in step (5), the column temperature is 20-30°C.

[0208] Preferably, in step (5), the specifications of the chromatographic column are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0209] Preferably, in step (5), the chromatographic column is 2-JADE-PAK KP-C18-AQ.

[0210] Preferably, in step (5), the organic phase is acetonitrile or methanol.

[0211] Preferably, in step (5), the aqueous phase is water, a solution containing potassium dihydrogen phosphate, a solution containing ammonium dihydrogen phosphate, or a solution containing potassium dihydrogen phosphate and sodium dodecyl sulfate.

[0212] Preferably, in step (5), the pH of the aqueous phase is 2.0 to 4.0.

[0213] Preferably, in step (5), the aqueous phase is a 0.05 mol / L potassium dihydrogen phosphate solution; or the aqueous phase is a 0.05 mol / L ammonium dihydrogen phosphate solution; or the aqueous phase is a 0.05 mol / L potassium dihydrogen phosphate solution and sodium dodecyl sulfate.

[0214] Preferably, in step (5), the aqueous phase is a 0.05 mol / L potassium dihydrogen phosphate solution and sodium dodecyl sulfate, wherein each 100 mL of the aqueous phase contains 0.2 to 0.4 g of sodium dodecyl sulfate.

[0215] Unless otherwise stated, all reagents / instruments used in the embodiments and comparative examples of this invention are conventional commercially available products. Information on the experimental materials and instruments used in this invention is shown in the table below:

[0216] Table 1 Experimental Materials / Instruments and Manufacturers

[0217]

[0218]

[0219] To better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0220] Example 1: Screening of chromatographic conditions

[0221] 1. Optimization of the mobile phase

[0222] (1) Investigation of the organic phase

[0223] Mobile phase A was 0.05 mol / L potassium dihydrogen phosphate (adjusted to pH 3.0 with phosphoric acid), and mobile phase B was an organic phase. The gradient elution program is shown in Table 2 below.

[0224] Table 2 Gradient elution program

[0225] Time (min) Mobile phase A (volume %) Mobile phase B (volume %) 0～60 95→50 5→50 60～60.1 50→95 50→5 60.1～65 95 5

[0226] The organic phases are methanol and acetonitrile, and the chromatogram obtained by the gradient elution program described above is as follows. Figure 1 As shown. From Figure 1 It can be seen that the elution ability of acetonitrile-0.05 mol / L potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid) solution is stronger than that of methanol-0.05 mol / L potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid) solution, with earlier peak elution, better overall peak shape and better separation. Therefore, acetonitrile-0.05 mol / L potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid) solution was selected for condition exploration. In the later stage, various buffer salts used in the mobile phase and different pH values ​​will be investigated.

[0227] (2) Investigation of the aqueous phase

[0228] The organic phase was acetonitrile, and the aqueous phase was investigated with different buffer salts. Mobile phase A consisted of different types of buffer salts, and mobile phase B consisted of acetonitrile. The chromatograms obtained according to the gradient elution program shown in Table 2 are as follows. Figure 2 As shown. From Figure 2 It can be seen that adding a buffer salt is more effective than not adding a buffer salt. The mobile phases with the best peak shapes are acetonitrile-0.05 mol / L potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid) and acetonitrile-0.05 mol / L potassium dihydrogen phosphate (0.4 g sodium dodecyl sulfate per 100 ml) (pH adjusted to 3.0 with phosphoric acid). However, the latter has a later peak time. Therefore, the acetonitrile-0.05 mol / L potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid) solution is selected for elution.

[0229] (3) The pH value of the aqueous phase was investigated.

[0230] The organic phase was acetonitrile, and the aqueous phase was a 0.05 mol / L potassium dihydrogen phosphate solution. Chromatograms were investigated with and without pH adjustment, and with pH adjusted to 2.0, 3.0, and 4.0 using phosphoric acid. Chromatograms obtained according to the gradient elution program shown in Table 2 are as follows: Figure 3 As shown. From Figure 3 It can be seen that adjusting the pH with phosphoric acid is more effective than not adjusting it, and there is obvious tailing at pH 4.0. When the pH is adjusted to 3.0 and 2.0 with phosphoric acid, the overall peak shape is the best and the resolution is the best. However, considering that pH 2.0 will cause some wear on the chromatographic column, acetonitrile-0.05mol / L potassium dihydrogen phosphate (adjusted to pH 3.0 with phosphoric acid) was chosen as the final elution solution.

[0231] 2. Selection of gradient elution program

[0232] Different elution gradients were investigated. Mobile phase A was 0.05 mol / L potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid), and mobile phase B was acetonitrile. The following four gradient elution programs were investigated.

[0233] Table 3 First gradient elution procedure

[0234] Time (min) Mobile phase A (volume %) Mobile phase B (volume %) 0～19 85→81.5 15→18.5 19～19.5 81.5→78 18.5→22 19.5～26 78→75 22→25 26～30 75→72 25→28 30～42 72→63 28→37 42～42.1 63→85 37→15 42.1～48 85 15

[0235] Table 4. Second gradient elution procedure

[0236] Time (min) Mobile phase A (volume %) Mobile phase B (volume %) 0～8 85→78 15→22 8～18 78→76 22→24 18～28 76→68 24→32 28～28.1 68→85 32→15 28.1～32 85 15

[0237] Table 5. Third Gradient Elution Procedure

[0238] Time (min) Mobile phase A (%) Mobile phase B (%) 0～21 85→82 15→18 21～21.5 82→78 18→22 21.5～34 78→76 22→24 34～38 76→72 24→28 38～50 72→63 28→37 50～50.1 63→85 37→15 50.1～55 85 15

[0239] Table 6. Fourth Gradient Elution Procedure

[0240]

[0241]

[0242] The chromatograms obtained using the four gradient elution procedures described above are as follows: Figure 4 As shown. From Figure 4 It can be seen that the fourth gradient elution program has the best peak separation, appropriate overall peak elution time and stable baseline. Therefore, the fourth gradient elution program is selected as the elution gradient for the fingerprint spectrum of Mahonia japonica leaf (long column).

[0243] 3. Investigation of different brands of chromatographic columns

[0244] Three chromatographic columns were investigated: column 1 - Diamonsil C18 (250 mm length, 4.6 mm inner diameter, 5 μm particle size); column 2 - JADE-PAKKP-C18-AQ (250 mm length, 4.6 mm inner diameter, 5 μm particle size); and column 3 - Waters Atlantis T3 (250 mm length, 4.6 mm inner diameter, 5 μm particle size). The effects of these three columns on the peak elution of the fingerprint chromatogram of Mahonia japonica leaf (long column) were examined. Figure 5 As shown. From Figure 5 It can be seen that the chromatographic column has a significant impact on the fingerprint chromatogram. Elution using a 2-JADE-PAKKP-C18-AQ ultra-high performance liquid chromatography (HPLC) column (250 mm length, 4.6 mm inner diameter, 5 μm particle size) yielded the best peak shape and separation effect. Therefore, this method uses a 2-JADE-PAKKP-C18-AQ HPLC column (250 mm length, 4.6 mm inner diameter, 5 μm particle size).

[0245] 4. Investigation at different column temperatures

[0246] The elution performance at different column temperatures (20℃, 25℃, 30℃) was investigated. Figure 6 As shown. From Figure 6It can be seen that column temperature has a certain impact on peak elution. The separation effect is best and the peak shape is better when the column temperature is 25℃. Therefore, it is recommended to use a column temperature of 25℃ for the determination.

[0247] 5. Investigation of different flow velocities

[0248] The effects of different flow rates (0.80 ml / min, 1.00 ml / min, and 1.20 ml / min) on the fingerprint chromatogram of Mahonia japonica leaf (long column) were compared. Figure 7 As shown. From Figure 7 It can be seen that the flow rate has a relatively small impact on the peak shape, but mainly affects the peak elution time. A flow rate of 1.00 ml / min produces the best peak shape and a suitable peak elution time, resulting in the most stable column efficiency. Therefore, it is recommended to use 1.00 ml / min as the measurement flow rate.

[0249] 6. Determination of final chromatographic conditions

[0250] After a series of condition explorations, octadecylsilane-bonded silica gel was finally selected as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); 0.05 mol / L potassium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid) was used as mobile phase A, acetonitrile was used as mobile phase B, and the gradient elution program is shown in Table 6; the flow rate was 1.0 ml per minute; the column temperature was 25 ℃; and the detection wavelength was 336 nm.

[0251] Example 2: Investigation of the extraction process

[0252] 1. Investigation of the types of extraction solvents

[0253] Accurately weigh 0.5g of Mahonia japonica leaves (long column) and place them in a 50ml stoppered Erlenmeyer flask. Accurately transfer 20ml of hydrochloric acid-methanol solution (1:100, v / v) and hydrochloric acid-ethanol solution (1:100, v / v) to each flask, weigh them, and extract using ultrasound for 45min. Weigh again, and replenish the lost weight with hydrochloric acid-methanol solution (1:100, v / v) and hydrochloric acid-ethanol solution (1:100, v / v). Shake thoroughly, filter, and collect the filtrate for later use. Detect using the chromatographic conditions described in section 6 of Example 1. Figure 8 As shown. From Figure 8 As can be seen from the data, when the extraction solvent is hydrochloric acid-ethanol, the peak shape is asymmetrical and the number of peaks is significantly less than that extracted with hydrochloric acid-methanol. Therefore, hydrochloric acid-methanol solution was chosen as the extraction solvent for the fingerprint chromatogram of Mahonia japonica leaf (long column).

[0254] 2. Investigation of solvent concentration during extraction

[0255] Accurately weigh 0.5g of Mahonia japonica leaf (long column) and place it in a 50ml stoppered Erlenmeyer flask. Accurately transfer 20ml of each of the following solutions: hydrochloric acid-30% methanol solution (1:100, v / v), hydrochloric acid-50% methanol solution (1:100, v / v), hydrochloric acid-75% methanol solution (1:100, v / v), hydrochloric acid-methanol solution (1:100, v / v), and hydrochloric acid-aqueous solution (1:100, v / v). Weigh each solution, extract ultrasonically for 45 minutes, weigh again, replenish the lost weight with the corresponding solution, shake thoroughly, filter, and collect the filtrate for later use. Detect the chromatographic conditions described in section 6 of Example 1. Figure 9 As shown. From Figure 9 As can be seen from the data, the chromatographic separation and response value are best when using hydrochloric acid-methanol solution (1:100, v / v) as the solvent for extraction, and more compounds are extracted than with other concentrations of solvent. Therefore, hydrochloric acid-methanol solution (1:100, v / v) was selected as the extraction solvent for Mahonia japonica leaf (long column) medicinal material.

[0256] 3. Optimal Extraction Method

[0257] (1) Shaking method

[0258] Take 0.5g of Mahonia japonica leaf (long column) medicinal material, accurately weigh it, and place it in a 50ml Erlenmeyer flask with a stopper. Accurately transfer 20ml of hydrochloric acid-methanol solution (1:100, v / v) solution, weigh it, shake it at room temperature for 45min, weigh it again, make up the lost weight with hydrochloric acid-methanol solution (1:100, v / v) solution, shake it thoroughly, filter it, and collect the filtrate for later use.

[0259] (2) Ultrasonic extraction method

[0260] Take 0.5g of Mahonia japonica leaf (long column) medicinal material, accurately weigh it, place it in a 50ml Erlenmeyer flask with a stopper, accurately transfer 20ml of hydrochloric acid-methanol solution (1:100, v / v) solution, weigh it, sonicate at room temperature for 45min, weigh it again, make up the lost weight with hydrochloric acid-methanol solution (1:100, v / v) solution, shake well, filter, and collect the filtrate for later use.

[0261] (3) Reflux extraction method

[0262] Take 0.5g of Mahonia japonica leaf (long column) medicinal material, accurately weigh it, place it in a 50ml Erlenmeyer flask with a stopper, accurately transfer 20ml of hydrochloric acid-methanol solution (1:100, v / v) solution, weigh it, reflux for 45min, weigh it again, make up the lost weight with hydrochloric acid-methanol solution (1:100, v / v) solution, shake well, filter, and collect the filtrate for later use.

[0263] The chromatographic conditions described in subsection 6 of Example 1 were used for detection, as shown in Table 7.

[0264] Table 7 Peak areas of different extraction methods

[0265] Peak number Shaking ultrasound reflux 1 1269.324 57.638 35.682 2 44.982 88.832 102.197 3(S) 2073.172 3537.704 3421.717 4 93.274 156.591 109.023 5 637.118 665.240 699.565 6 5203.429 5068.422 4990.781 7 70.492 175.913 150.476 8 25.554 45.975 74.543 Total peak area 9417.345 9796.315 9583.984

[0266] As can be seen from Table 7, by comparing the area of ​​each peak and the total peak area, the total peak area of ​​ultrasound is the highest. Therefore, the fingerprint extraction method for Mahonia japonica leaf (long column) medicinal material is determined to be ultrasonic extraction.

[0267] (4) Extraction time

[0268] Accurately weigh 0.5g of Mahonia japonica leaf (long column) and place it in a 50ml stoppered Erlenmeyer flask. Accurately transfer 20ml of hydrochloric acid-methanol solution (1:100, v / v), weigh, and sonicate for 15min, 30min, 45min, and 60min respectively, weighing each time. Recover the lost weight with hydrochloric acid-methanol solution (1:100, v / v), shake thoroughly, filter, and collect the filtrate for later use. Detect the chromatographic conditions described in section 6 of Example 1, as shown in Table 8.

[0269] Table 8 Peak areas at different extraction times

[0270] Peak number 15min 30min 45min 60min 1 59.491 59.773 57.638 64.177 2 84.639 85.331 88.832 90.405 3 3478.265 3569.774 3637.704 3678.368 4 138.463 128.306 156.591 83.910 5 680.371 679.072 685.240 663.192 6 5245.638 5235.906 5498.422 5456.816 7 165.478 160.415 175.913 172.195 8 41.649 38.139 45.975 45.969 Total peak area 9893.994 9956.716 10346.315 10255.032

[0271] As can be seen from Table 8, the target peak and total peak area are the largest when the ultrasonic time is 45 min. Therefore, 45 min of ultrasonic treatment was selected as the extraction time for the fingerprint spectrum of Mahonia japonica leaf (long column).

[0272] 4. Determination of the final extraction method

[0273] Take 0.5g of Mahonia japonica leaf (long column) medicinal material, accurately weigh it, place it in a 50ml Erlenmeyer flask with a stopper, accurately transfer 20ml of hydrochloric acid-methanol solution (1:100, v / v) solution, weigh it, sonicate for 45min, weigh it again, make up the lost weight with hydrochloric acid-methanol solution (1:100, v / v) solution, shake well, filter, and collect the filtrate for later use.

[0274] Example 3

[0275] This embodiment collected 87 batches of Mahonia fortunei leaves, which were identified by Professor Wei Shenghua of Guizhou University of Traditional Chinese Medicine as follows: *Mahonia fortunei* (Lindl.) Fedde, *M. duclouxiana* Gagnep., *M. bealei* (Fort.) Carr., *M. bodinieri* Gagnep., and *M. eurybracteata* Fedde, all belonging to the genus *Mahonia* of the family Berberidaceae. Detailed sample information is shown in Table 9.

[0276] Table 9 Information on samples of Mahonia japonica leaves

[0277]

[0278]

[0279]

[0280] The fingerprint analysis method for the leaves of the Mahonia japonica includes the following steps:

[0281] 1. Preparation of reference solution

[0282] Accurately weigh appropriate amounts of neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, cypermethrin hydrochloride, palmatine hydrochloride, berberine hydrochloride, isochlorogenic acid A, and isochlorogenic acid C reference standards, and prepare mixed reference solutions with mass concentrations of 14.896, 8.722, 320.977, 19.010, 35.737, 71.520, 18.931, and 5.076 μg / mL by adding hydrochloric acid-methanol (1:100, v / v).

[0283] 2. Preparation of the test solution

[0284] Accurately weigh approximately 0.5 g of sample powder and place it in a stoppered conical flask. Accurately add 20 mL of hydrochloric acid-methanol (1:100, v / v), sonicate (500 W power, 40 kHz frequency) for 45 min, cool, replenish the lost mass with hydrochloric acid-methanol (1:100, v / v), shake well, filter, and filter the filtrate through a 0.45 μm microporous membrane to obtain the final product.

[0285] The preparation of the sample powder includes the following steps: drying the fresh Mahonia japonica leaves, grinding them into powder, and then passing them through a No. 3 sieve (50 mesh) to pulverize them into powder.

[0286] 3. Ultra-high performance liquid chromatography analysis

[0287] KP-C18-AQ column (250mm×4.6mm, 5μm), flow rate 1.0mL / min, column temperature 25℃, detection wavelength 336nm, mobile phase acetonitrile (B)-0.05mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid) (A);

[0288] Gradient elution program: 0–21 min, the volume percentage of mobile phase A is reduced from 85% to 82%, and the volume percentage of mobile phase B is increased from 15% to 18%.

[0289] Between 21 and 21.5 min, the volume percentage of mobile phase A decreased from 82% to 78%, while the volume percentage of mobile phase B increased from 18% to 22%.

[0290] Between 21.5 and 32 minutes, the volume percentage of mobile phase A decreased from 78% to 77%, while the volume percentage of mobile phase B increased from 22% to 23%.

[0291] Over 32–38 minutes, the volume percentage of mobile phase A decreased from 77% to 72%, while the volume percentage of mobile phase B increased from 23% to 28%.

[0292] Over 38–50 minutes, the volume percentage of mobile phase A decreased from 72% to 63%, while the volume percentage of mobile phase B increased from 28% to 37%.

[0293] Between 50 and 50.1 min, the volume percentage of mobile phase A increased from 63% to 85%, while the volume percentage of mobile phase B decreased from 37% to 15%.

[0294] From 50.1 to 55 min, the volume percentage of mobile phase A was 85%, and the volume percentage of mobile phase B was 15%.

[0295] 4. Establishment of fingerprint patterns

[0296] (1) Establishment of fingerprint spectrum and assignment of common peaks

[0297] The HPLC chromatograms of 87 batches of medicinal material samples were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software (version 130723, 2012) for analysis in AIA format.

[0298] like Figure 10-14As shown, the chromatographic peak information of the leaves of *Mahonia fortunei* (a type of medicinal herb) is the richest, and 17 common peaks were identified. The relative retention times of each common peak were calculated using the retention time of the 8th fingerprint characteristic peak as a reference. The 8th fingerprint characteristic peak is the fingerprint characteristic peak of chlorogenic acid. The relative retention times of the 1st common fingerprint characteristic peak (peak 1) are 0.31, the 2nd common fingerprint characteristic peak (peak 2) are 0.35, the 3rd common fingerprint characteristic peak (peak 4) are 0.52, the 4th common fingerprint characteristic peak (peak 5) are 0.61, the 5th common fingerprint characteristic peak (peak 6) are 0.68, the 6th common fingerprint characteristic peak (peak 7) are 0.78, the 7th common fingerprint characteristic peak (peak 8) are 0.91, and the 8th common fingerprint characteristic peak (peak 9) has a relative retention time of... The relative retention times of the fingerprint feature peaks are as follows: peak 10 (9th shared fingerprint peak) is 1.08, peak 11 (10th shared fingerprint peak) is 1.41, peak 12 (11th shared fingerprint peak) is 1.52, peak 14 (12th shared fingerprint peak) is 1.77, peak 15 (13th shared fingerprint peak) is 1.90, peak 16 (14th shared fingerprint peak) is 2.16, peak 17 (15th shared fingerprint peak) is 2.23, peak 18 (16th shared fingerprint peak) is 2.57, and peak 19 (17th shared fingerprint peak) is 2.64. The error of the relative retention times of the fingerprint feature peaks is within ±10%.

[0299] Thirteen common peaks were identified in the leaves of *Mahonia japonica*. The relative retention times of each common peak were calculated with reference to the retention time of the sixth fingerprint characteristic peak, which is the fingerprint characteristic peak of chlorogenic acid. The relative retention times of the first common fingerprint characteristic peak (peak 1) were 0.32, the second common fingerprint characteristic peak (peak 4) were 0.52, the third common fingerprint characteristic peak (peak 5) were 0.61, the fourth common fingerprint characteristic peak (peak 6) were 0.68, the fifth common fingerprint characteristic peak (peak 8) were 0.91, and the sixth common fingerprint characteristic peak (peak 9) was... The retention times of the shared fingerprint feature peaks are as follows: 1.00 for peak 7 (peak 10), 1.08 for peak 8 (peak 11), 1.41 for peak 9 (peak 12), 1.52 for peak 10 (peak 14), 1.78 for peak 11 (peak 15), 1.87 for peak 12 (peak 16), 2.16 for peak 13 (peak 17), and 2.22 for peak 13 (peak 17). The relative retention times of the fingerprint feature peaks are within ±10%.

[0300] Eleven common peaks were identified in the leaves of *Mahonia alatafolia*. The relative retention times of each common peak were calculated with reference to the retention time of the fourth fingerprint characteristic peak, which is the fingerprint characteristic peak of chlorogenic acid. The relative retention times of the first common fingerprint characteristic peak (peak 3) were 0.41, the second common fingerprint characteristic peak (peak 5) were 0.60, the third common fingerprint characteristic peak (peak 7) were 0.78, the fourth common fingerprint characteristic peak (peak 9) were 1.00, and the fifth common fingerprint characteristic peak (peak 11) was... 1.41, the relative retention time of the sixth shared fingerprint feature peak (peak 12) is 1.52, the relative retention time of the seventh shared fingerprint feature peak (peak 13) is 1.64, the relative retention time of the eighth shared fingerprint feature peak (peak 14) is 1.77, the relative retention time of the ninth shared fingerprint feature peak (peak 15) is 1.90, the relative retention time of the tenth shared fingerprint feature peak (peak 16) is 2.23, and the relative retention time of the eleventh shared fingerprint feature peak (peak 17) is 2.28; the error of the relative retention time of the fingerprint feature peaks is within ±10%.

[0301] Fourteen common peaks were identified in the leaves of *Mahonia fortunei*. The relative retention times of each common peak were calculated with reference to the retention time of the sixth fingerprint characteristic peak, which is the fingerprint characteristic peak of chlorogenic acid. The relative retention times of the first common fingerprint characteristic peak (peak 4) were 0.52, the second common fingerprint characteristic peak (peak 5) were 0.61, the third common fingerprint characteristic peak (peak 6) were 0.68, the fourth common fingerprint characteristic peak (peak 7) were 0.78, the fifth common fingerprint characteristic peak (peak 8) were 0.91, the sixth common fingerprint characteristic peak (peak 9) were 1.00, and the seventh common fingerprint characteristic peak (peak 1) was 0.52. The relative retention time of the fingerprint feature peak (peak 10) is 1.08, the relative retention time of the shared fingerprint feature peak (peak 11) is 1.41, the relative retention time of the shared fingerprint feature peak (peak 12) is 1.52, the relative retention time of the shared fingerprint feature peak (peak 13) is 1.64, the relative retention time of the shared fingerprint feature peak (peak 14) is 1.80, the relative retention time of the shared fingerprint feature peak (peak 15) is 1.89, the relative retention time of the shared fingerprint feature peak (peak 16) is 2.17, and the relative retention time of the shared fingerprint feature peak (peak 17) is 2.23; the error of the relative retention time of the fingerprint feature peaks is within ±10%.

[0302] Ten common peaks were identified in the leaves of *Mahonia scabra*. The relative retention times of each common peak were calculated with reference to the retention time of the fourth fingerprint characteristic peak, which is the fingerprint characteristic peak of chlorogenic acid. The relative retention times of the first common fingerprint characteristic peak (peak 5) were 0.61, the second common fingerprint characteristic peak (peak 6) were 0.68, the third common fingerprint characteristic peak (peak 8) were 0.91, the fourth common fingerprint characteristic peak (peak 9) were 1.00, and the fifth common fingerprint characteristic peak (peak 10)... The relative retention time of the fingerprint feature peak is 1.08, the relative retention time of the sixth common fingerprint feature peak (peak 11) is 1.41, the relative retention time of the seventh common fingerprint feature peak (peak 14) is 1.79, the relative retention time of the eighth common fingerprint feature peak (peak 15) is 1.88, the relative retention time of the ninth common fingerprint feature peak (peak 16) is 2.16, and the retention time of the tenth common fingerprint feature peak (peak 17) is 2.23; the error of the relative retention time of the fingerprint feature peaks is within ±10%.

[0303] There are 7 common peaks among the 5 common Mahonia japonica leaves. The relative retention time A of each common peak is calculated with reference to the retention time of the second common fingerprint characteristic peak, where the second common fingerprint characteristic peak is the fingerprint characteristic peak of chlorogenic acid. The relative retention time of the first common fingerprint characteristic peak (peak 5) is 0.61, the relative retention time of the second common fingerprint characteristic peak (peak 9) is 1.0, the relative retention time of the third common fingerprint characteristic peak (peak 11) is 1.41, the relative retention time of the fourth common fingerprint characteristic peak (peak 14) is 1.79, the relative retention time of the fifth common fingerprint characteristic peak (peak 15) is 1.9, the relative retention time of the sixth common fingerprint characteristic peak (peak 16) is 2.16, and the relative retention time of the seventh common fingerprint characteristic peak (peak 17) is 2.23. The error of the relative retention time of the fingerprint characteristic peaks is within ±10%.

[0304] Five types of medicinal materials (Mahonia japonica leaves, namely, Mahonia japonica long-column leaf, Mahonia japonica narrow-leaf leaf, Mahonia japonica broad-leaf leaf, Mahonia japonica small-fruited leaf, and Mahonia japonica broad-bracted leaf) were tested, and a total of 19 fingerprint characteristic peaks were obtained. The relative retention time of each common peak was calculated with the retention time of peak 9 as a reference. Peak 9 is the fingerprint characteristic peak of chlorogenic acid. The relative retention time of peak 1 is 0.31; the relative retention time of peak 2 is 0.35, peak 3 is 0.41, peak 4 is 0.52, peak 5 is 0.61, peak 6 is 0.68, peak 7 is 0.78, peak 8 is 0.91, peak 9 is 1.00, peak 10 is 1.08, peak 11 is 1.41, peak 12 is 1.52, peak 13 is 1.64, peak 14 is 1.79, peak 15 is 1.90, peak 16 is 2.16, peak 17 is 2.23, peak 18 is 2.57, and peak 19 is 2.64. Eight characteristic peaks from five raw medicinal materials were assigned using retention times and isoabsorption spectra of various reference standards. Peak 4 was identified as neochlorogenic acid, peak 7 as cryptochlorogenic acid, peak 9 as chlorogenic acid, peak 15 as berberine hydrochloride, peak 16 as palmatine hydrochloride, peak 17 as berberine hydrochloride, peak 18 as isochlorogenic acid A, and peak 19 as isochlorogenic acid C. Figure 15A). Peak 4 is neochlorogenic acid with a relative retention time of 0.52; peak 7 is cryptochlorogenic acid with a relative retention time of 0.78; peak 9 is chlorogenic acid with a relative retention time of 1.00; peak 15 is berberine hydrochloride with a relative retention time of 1.90; peak 16 is palmatine hydrochloride with a relative retention time of 2.16; peak 17 is berberine hydrochloride with a relative retention time of 2.23; peak 18 is isochlorogenic acid A with a relative retention time of 2.57; peak 19 is isochlorogenic acid C with a relative retention time of 2.64. The error of the relative retention time of the fingerprint characteristic peaks is within ±10%.

[0305] Depend on Figure 15 From B, we know that peaks 2, 18 (isochlorogenic acid A), and 19 (isochlorogenic acid C) are specific to long-column plants, while peak 3 is specific to broad-leaved plants; peaks 6, 8, and 10 are only absent in broad-leaved plants, and peak 12 is only absent in broad-bracted plants. Peaks 5, 14, 15 (berberine hydrochloride) and 16 (palmatine hydrochloride) have higher peak areas than other basal sites for small-leaved plants; peak 17 (berberine hydrochloride) has a higher peak area than other basal sites for small-fruited plants.

[0306] (2) Similarity evaluation results and analysis

[0307] The HPLC fingerprint data of 87 batches of medicinal materials from 5 different sources were processed using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software (version 130723, 2012). The median, time window of 0.2, multi-point correction, and Mark peak matching were used to calculate the similarity coefficient. The similarity of the leaves of 18 batches of *Mahonia stenoptera* ranged from 0.889 to 0.993, the similarity of the leaves of 25 batches of *Mahonia longifolia* ranged from 0.809 to 1.000, the similarity of the leaves of 15 batches of *Mahonia broadifolia* ranged from 0.953 to 1.000, the similarity of the leaves of 16 batches of *Mahonia septemlobata* ranged from 0.864 to 0.991, and the similarity of the leaves of *Mahonia scabra* ranged from 0.954 to 0.997. Only 7 out of 87 batches had a similarity below 0.9, indicating that *Mahonia* medicinal materials from different sources but with the same origin exhibit good consistency.

[0308] The reference chromatograms of five original samples were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software (version 130723, 2012). Pairwise comparisons were performed. The similarities between the fine-leaved variety and the broad-leaved variety, small fruit, and broad bract were 0.754, 0.708, 0.664, and 0.658, respectively; the similarities between the long column and the broad-leaved variety, small fruit, and broad bract were 0.953, 0.269, and 0.120, respectively; the similarities between the broad-leaved variety and the small fruit and broad bract were 0.320 and 0.155, respectively; and the similarity between the small fruit and broad bract was 0.938. These results indicate that the fingerprint chromatograms of different original samples within the same genus of medicinal materials show differences in both common peak information and similarity. The fine-leaved variety of Mahonia japonica leaves and the other four original samples all had similarities below 0.76 and can be completely isolated. Figure 15 It can be seen that the alkaloid content (peaks 15 and 16) in the fine leaves is also higher than that of other genes.

[0309] 5. Cluster analysis

[0310] The peak area data of 19 common peaks (with the area of ​​differential peaks counted as 0) selected from the common patterns of the fingerprint spectrum of Mahonia japonica leaves were standardized. SPSS software was used to perform systematic clustering on 87 batches of medicinal material samples, employing the between-group mean linkage method with cosine distance as the metric. Figure 16 As shown, the samples clustered into three groups: XY1–XY18 clustered into group I, CZ1–CZ25 clustered into group II, and all KB, XG, and KY samples clustered into group III. The results indicate that the narrow-leaved and long-columned samples are significantly different from other samples, while the broad-leaved, small-fruited, and broad-bracted samples are quite similar. All samples clustered into group III can be further clustered into three groups: KB1–KB13 clustered into group IIIa, XG1–XG16 clustered into group IIIb, and KY1–KY15 clustered into group IIIc. These results show that the broad-leaved, small-fruited, and broad-bracted samples have clear cluster boundaries, exhibit good consistency, and can be distinguished from each other.

[0311] 6. Pattern Recognition Analysis

[0312] (1) PCA (Principal Component Analysis) analysis

[0313] The standardized common peak area data were imported into SIMCA14.1 software for pattern recognition analysis. The independent variable in the analysis was the fit index (R²). 2 x) is 1.000, and the model prediction index (Q) is 1.000. 2 The value is 0.993, R 2 x and Q 2 All values ​​are greater than 0.5, indicating that the model has good predictive reliability. See the PCA score chart below. Figure 17 Consistent with the cluster analysis results, narrow-leaved and long-columned varieties each clustered into one group, showing good separation from other primordia. Broad-leaved, small-fruited, and broad-bracted varieties showed high similarity, but primordia of the same species clustered into one group, exhibiting good consistency. The standardization process referred to was performed using SPSS software, employing z-score standardization to achieve a normal distribution with a mean of 0 and a standard deviation of 1.

[0314] (2) OPLS-DA (Orthogonal Partial Least Squares Discriminant Analysis) analysis

[0315] To further analyze the differences among different basal sources of Mahonia japonica leaves, a supervised pattern recognition method, OPLS-DA, was used to analyze all samples based on PCA. The independent variable in the analysis was the fit index (R²). 2 x) is 0.969, and the dependent variable fit index (R) is...2 The model prediction index (Q) is 0.831. 2 The scores were 0.721, all greater than 0.5, indicating that the model is stable, reliable, and makes good predictions. See the OPLS-DA score chart. Figure 17 The results of this method are more clustered among the samples of each primordium than those of PCA, and the out-of-domain samples are closer to the confidence boundary, further indicating that the chemical composition of the narrow-leaved and long-columned primordiums differs from that of the other three primordia (broad-leaved, small-fruited, and broad-bracted).

[0316] Variable importance inprojection (VIP) was used to screen for potential differential components distinguishing the five basal components of Mahonia japonica leaves, with a VIP > 1.0 as the screening criterion. Figure 18 It was found that seven potential differentially expressed components were identified, arranged from highest to lowest VIP value as follows: Peak 3 > Peak 12 > Peak 13 > Peak 15 (Phytoretin hydrochloride) > Peak 16 (Pamatine hydrochloride) > Peak 7 (Cryptochlorogenic acid) > Peak 14. Among the seven identified components, Peak 3 is specific to broadleaf and can distinguish it from the other four types of basalts; Peak 12 is distinguished from the other four types of basalts only by the absence of broad bracts; those containing Peak 13 but not Peak 3 are small fruits; those containing Peak 12 but not Peak 7 are narrow leaves; and those containing Peak 7 but not Peak 13 are long columns, which can be distinguished from the other four types of basalts, thus meeting the criteria... Figure 15 A direct analysis is needed. Therefore, peaks 3, 12, 13, and 7 (cryptochlorogenic acid) can be used as components to distinguish the interspecific differences among the five native species of Mahonia japonica leaves in Guizhou Province. In addition, peaks 15 (paracetamol hydrochloride), 16 (palmatine hydrochloride), and 14 are potential components of difference shared by the five species, but it is not possible to directly determine whether the differences are statistically significant. Therefore, it is necessary to further confirm the significance of their differences.

[0317] This invention employs a combination of cluster analysis and chemical pattern recognition to distinguish and identify five different primordia of Mahonia leaves. The results of the two chemical identification methods are basically consistent. The narrow leaves and long columns can each be clustered into one category, showing significant differences from the other three primordia. The broad leaves, broad bracts, and small fruits show high similarity among the three, but the clustering results of the three also have clear boundaries, allowing them to be distinguished from each other.

[0318] Seven differentially expressed components were identified through variable importance projection and analysis of variance: peaks 3, 12, 13, and 7 (cryptochlorogenic acid) were the individual differential peaks of the five medicinal materials, and their presence or absence could be used to distinguish the five materials. Peaks 15 (paracetamol hydrochloride), 16 (palmatine hydrochloride), and 14 were common peaks of the five medicinal materials, with significant differences in peak area among the five species (P < 0.5). Peaks 3, 13, and 7, being small peaks, are often allowed to have large variations or can be ignored in fingerprint analysis. However, some studies have indicated that subtle differences in fingerprints may be key to quality evaluation. Therefore, these seven components can be used as markers of interspecific differences in Mahonia japonica leaves. For the currently unidentified common peaks, future analyses will employ phytochemical separation and HPLC-MS techniques for further identification.

[0319] Example 4: Investigation of Fingerprint Mapping Methodology

[0320] (1) Precision

[0321] Take one sample solution of Mahonia japonica leaf (long column) medicinal material, inject it repeatedly 6 times, record the chromatogram, and evaluate it using the 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software. Tentatively identify 8 common peaks, using chlorogenic acid (peak 3) as the reference peak, calculate the relative retention time and relative peak area, and the experimental results are shown in the table below.

[0322] Table 9. Precision results of fingerprint spectroscopy of Mahonia japonica leaves (long column) (relative peak area)

[0323]

[0324] Table 10. Fingerprint precision results (relative retention time) of Mahonia japonica leaf (long column) medicinal material.

[0325]

[0326]

[0327] Table 11. Fingerprint Precision Results (Similarity) of Mahonia japonica Leaf (Long Column) Medicinal Material

[0328]

[0329] Experimental results: The RSD of the relative retention time and relative peak area of ​​each peak is less than 3.0%, indicating that the instrument has good precision and meets the requirements of fingerprint spectrum, with similarity between 0.990 and 1.00.

[0330] (2) Repeatability

[0331] Accurately weigh 0.5g of Mahonia japonica leaves (long column) from the same batch, prepare six parallel samples, and inject them into the test solution according to the test solution preparation method. Record the chromatograms and evaluate them using the 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software. Tentatively identify eight common peaks, using chlorogenic acid (peak 3) as the reference peak, calculate the relative retention time and relative peak area. The experimental results are shown in the table below.

[0332] Table 12 Repeatability results of fingerprint chromatograms of Mahonia japonica leaves (long column) (relative peak area)

[0333]

[0334]

[0335] Table 13. Repeatability results of fingerprint chromatograms of Mahonia japonica leaves (long column) (relative retention time)

[0336]

[0337] Table 14. Repeatability results (similarity) of fingerprint chromatograms of Mahonia japonica leaves (long column).

[0338]

[0339] Experimental results: The RSD of the relative retention time and relative peak area of ​​each peak is less than 3.0%. The instrument has good repeatability and meets the requirements of fingerprint spectrum. The similarity is between 0.990 and 1.00.

[0340] (3) Stability

[0341] The leaves (long column) of Mahonia japonica were injected into the test solution at 0, 2, 4, 8, 12, and 24 hours, and the chromatograms were recorded. The results were evaluated using the 2012 version of the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software. Eight common peaks were tentatively identified, with chlorogenic acid (peak 3) as the reference peak. The relative retention time and relative peak area were calculated. The experimental results are shown in the table below.

[0342] Table 15. Stability results of fingerprint spectral analysis of Mahonia japonica leaves (long columnar) (relative peak area)

[0343]

[0344] Table 16. Stability results of fingerprint chromatograms of Mahonia japonica leaves (long column) (relative retention time)

[0345]

[0346] Table 17. Stability results (similarity) of fingerprint chromatograms of Mahonia japonica leaves (long column).

[0347]

[0348]

[0349] Experimental results: The RSD of the relative retention time and relative peak area of ​​each peak is less than 3.0%, indicating that the instrument has good stability and meets the requirements of fingerprint spectrum, with similarity between 0.990 and 1.00.

[0350] (4) Exclusivity

[0351] Accurately pipette 10 μL each of the test solution and blank solvent from the leaves (long column) of Mahonia japonica, inject them into the liquid chromatograph, and determine the chromatographic conditions as described above. The results are as follows: Figure 19 As shown in the figure. The experimental results show that the solvent did not interfere with the chromatographic peaks in the fingerprint spectrum of Mahonia japonica leaf (long column) medicinal material.

[0352] (5) Holistic

[0353] A solution of Mahonia japonica leaf (long column) medicinal material was injected into a liquid chromatograph. The elution time was doubled under a gradient endpoint mobile phase ratio. The fingerprint chromatogram was analyzed, and the results are as follows: Figure 20 As shown in the figure. The results indicate that no obvious chromatographic peaks were observed after doubling the elution time under these chromatographic conditions, suggesting that these conditions basically meet the principle of maximizing information content.

[0354] While the specific embodiments of the present invention have been described above, they are not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. A method for detecting the fingerprint spectrum of Mahonia japonica leaves, characterized in that, The detection method includes the following steps: (1) Preparation of reference solution Weigh appropriate amounts of reference standards for neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, cypermethrin hydrochloride, palmatine hydrochloride, berberine hydrochloride, isochlorogenic acid A, and isochlorogenic acid C, and add solvent A to prepare reference standard solutions. (2) Preparation of the test solution Extraction was performed by adding extraction solvent B to a sample of Mahonia japonica leaves to obtain a test solution; extraction solvent B was a solution of 1% hydrochloric acid and methanol; the extraction was performed by ultrasonic extraction. (3) Ultra-high performance liquid chromatography analysis Using octadecylsilane-bonded silica gel as the packing material, mobile phase A was aqueous, and mobile phase B was organic. Reference solution and test solution were injected into the ultra-high performance liquid chromatograph (UHPLC) and analyzed using a gradient elution program; the flow rate was 0.8–1.2 mL / min; the organic phase was acetonitrile; the aqueous phase was a 0.05 mol / L potassium dihydrogen phosphate solution with a pH of 3.0; the column specifications were: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm; column temperature 20–30 °C; and the detection wavelength was 336 nm. (4) Establish fingerprint patterns of Mahonia leaves from different origins; The gradient elution procedure is as follows: From 0 to 21 min, the volume percentage of mobile phase A decreased from 85% to 82%, while the volume percentage of mobile phase B increased from 15% to 18%. Between 21 and 21.5 minutes, the volume percentage of mobile phase A decreased from 82% to 78%, while the volume percentage of mobile phase B increased from 18% to 22%. Between 21.5 and 32 minutes, the volume percentage of mobile phase A decreased from 78% to 77%, while the volume percentage of mobile phase B increased from 22% to 23%. Over 32-38 minutes, the volume percentage of mobile phase A decreased from 77% to 72%, while the volume percentage of mobile phase B increased from 23% to 28%. Over 38–50 minutes, the volume percentage of mobile phase A decreased from 72% to 63%, while the volume percentage of mobile phase B increased from 28% to 37%. Over a period of 50–50.1 min, the volume percentage of mobile phase A increased from 63% to 85%, while the volume percentage of mobile phase B decreased from 37% to 15%. From 50.1 to 55 min, the volume percentage of mobile phase A was 85%, and the volume percentage of mobile phase B was 15%.

2. The fingerprint spectrum detection method for Mahonia japonica leaves according to claim 1, wherein, In step (1), the concentrations of the reference solution are as follows: the mass concentrations of neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, cypermethrin hydrochloride, palmatine hydrochloride, berberine hydrochloride, isochlorogenic acid A and isochlorogenic acid C are 14~15 μg / mL, 8~9 μg / mL, 320~321 μg / mL, 19~20 μg / mL, 35~36 μg / mL, 71~72 μg / mL, 18~19 μg / mL and 5~6 μg / mL, respectively.

3. The fingerprint spectrum detection method for Mahonia japonica leaves according to claim 1, wherein, In step (1), the concentrations of the reference solution are as follows: the mass concentrations of neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, cypermethrin hydrochloride, palmatine hydrochloride, berberine hydrochloride, isochlorogenic acid A and isochlorogenic acid C are 14 μg / mL, 8 μg / mL, 320 μg / mL, 19 μg / mL, 35 μg / mL, 71 μg / mL, 18 μg / mL and 5 μg / mL, respectively.

4. The fingerprint spectrum detection method for Mahonia japonica leaves according to claim 1, wherein, In step (2), the test solution preparation steps include: taking a sample of Mahonia japonica leaves, adding extraction solvent B for extraction, weighing the sample, replenishing the lost weight with extraction solvent B, filtering, and taking the filtrate to obtain the test solution; wherein, the Mahonia japonica leaf sample is one or more of Mahonia japonica leaf medicinal material, Mahonia japonica leaf slices, Mahonia japonica leaf standard decoction freeze-dried powder, and Mahonia japonica leaf formula granules.

5. The fingerprint spectrum detection method for Mahonia japonica leaves according to claim 1, wherein, In step (2), the mass-to-volume ratio of the Mahonia japonica leaf sample to the extraction solvent B is 1:30~50, where the mass unit is g and the volume unit is mL.

6. The fingerprint spectrum detection method for Mahonia japonica leaves according to claim 4, wherein, In step (2), the mass-to-volume ratio of the Mahonia japonica leaf sample to the extraction solvent B is 1:30~50, where the mass unit is g and the volume unit is mL.

7. The fingerprint spectrum detection method for Mahonia japonica leaves according to claim 5, wherein, In step (2), the mass-to-volume ratio of the Mahonia japonica leaf sample to the extraction solvent B is 1:40, where mass is in g and volume is in mL.

8. The fingerprint spectrum detection method for Mahonia japonica leaves according to claim 1, wherein, In step (2), the extraction time is 15~60 min.

9. The fingerprint spectrum detection method for Mahonia japonica leaves according to claim 1, wherein, In step (2), the extraction time is 45 minutes.

10. The fingerprint spectrum detection method for Mahonia japonica leaves according to claim 1, wherein, In step (3), the chromatographic column is JADE-PAK KP-C18-AQ.

11. The fingerprint spectrum detection method for Mahonia japonica leaves according to any one of claims 1-10, wherein, The Mahonia leaves mentioned refer to Mahonia leaves from different phyla, including Mahonia long-columnar, Mahonia narrow-leaved, Mahonia broad-leaved, Mahonia small-fruited, and Mahonia broad-bracted, as well as other Mahonia species.

12. The fingerprint spectrum detection method for Mahonia japonica leaves according to claim 11, wherein, The fingerprint patterns of the ten Mahonia leaves of different origins contain seven common fingerprint feature peaks. The relative retention time A of each common peak is calculated with reference to the retention time of the second common fingerprint feature peak 9, wherein the second common fingerprint feature peak 9 is the fingerprint feature peak of chlorogenic acid. The relative retention time of fingerprint feature peak 5, which is shared by fingerprint #1, is 0.

61. The relative retention time of fingerprint feature peak 9, which is shared by fingerprint #2, is 1.

0. The relative retention time of fingerprint feature peak 11, which is shared by fingerprint #3, is 1.

41. The relative retention time of fingerprint feature peak 14, which is shared by fingerprint #4, is 1.

79. The relative retention time of fingerprint feature peak 15, which is common to fingerprint number 5, is 1.

9. The relative retention time of fingerprint feature peak 16, which is common to fingerprint number 6, is 2.16, and... The relative retention time of fingerprint feature peak 17 shared by fingerprint number 7 is 2.23; The error in the relative retention time of the fingerprint feature peaks is within ±10%.

13. The fingerprint spectrum detection method for Mahonia japonica leaves according to any one of claims 1-10, wherein, The fingerprint characteristic peaks were assigned as follows: peak 4 is neochlorogenic acid, peak 7 is cryptochlorogenic acid, peak 9 is chlorogenic acid, peak 15 is cypermethrin hydrochloride, peak 16 is palmatine hydrochloride, peak 17 is berberine hydrochloride, peak 18 is isochlorogenic acid A, and peak 19 isochlorogenic acid C.

14. The fingerprint spectrum detection method for Mahonia japonica leaves according to claim 11, wherein, The fingerprint characteristic peaks were assigned as follows: peak 4 is neochlorogenic acid, peak 7 is cryptochlorogenic acid, peak 9 is chlorogenic acid, peak 15 is cypermethrin hydrochloride, peak 16 is palmatine hydrochloride, peak 17 is berberine hydrochloride, peak 18 is isochlorogenic acid A, and peak 19 isochlorogenic acid C.

15. The fingerprint spectrum detection method for Mahonia japonica leaves according to claim 12, wherein, The fingerprint characteristic peaks were assigned as follows: peak 4 is neochlorogenic acid, peak 7 is cryptochlorogenic acid, peak 9 is chlorogenic acid, peak 15 is cypermethrin hydrochloride, peak 16 is palmatine hydrochloride, peak 17 is berberine hydrochloride, peak 18 is isochlorogenic acid A, and peak 19 isochlorogenic acid C.

16. The fingerprint spectrum detection method for Mahonia japonica leaves according to any one of claims 1-10, wherein, According to the similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine, the similarity between the fingerprint chromatogram of the test sample and the fingerprint chromatogram of the reference sample shall not be less than 0.

90.

17. The application of the fingerprint spectrum detection method for Mahonia japonica leaves according to any one of claims 1-16 in the quality control of Mahonia japonica leaves from different origins, wherein, The different types of Mahonia leaves are: long-columnar Mahonia leaves, narrow-leaved Mahonia leaves, broad-leaved Mahonia leaves, small-fruited Mahonia leaves, and broad-bracted Mahonia leaves, totaling five types.

18. The application of the fingerprint spectrum detection method for Mahonia japonica leaves according to any one of claims 1-16 in the detection of the content of indicative components in Mahonia japonica leaves from different origins, wherein, The different types of Mahonia leaves are five types: long-columnar Mahonia leaves, narrow-leaved Mahonia leaves, broad-leaved Mahonia leaves, small-fruited Mahonia leaves, and broad-bracted Mahonia leaves; the indicative components include neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, isochlorogenic acid A, and isochlorogenic acid C.

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