Detection method and application of concha haliotidis medicinal material or decoction pieces, standard decoction and formula granules thereof

Abstract

The invention discloses a detection method and application of concha haliotidis medicinal materials or decoction pieces, standard decoction and formula granules thereof, and relates to the technical field of analysis and detection. The detection method comprises the following steps: performing enzymolysis extraction on concha haliotidis medicinal materials or decoction pieces thereof, standard decoction and formula granules to obtain a test solution; the basis of the concha haliotidis medicinal material or the decoction pieces, the standard decoction and the formula granules thereof is haliotis discus hannai; extracting or dissolving the concha haliotidis polypeptide reference substance by adopting an extraction solvent to obtain a reference substance solution; and / or performing enzymolysis extraction on a concha haliotidis reference medicinal material to obtain a reference medicinal material solution; the amino acid sequence of the concha haliotidis polypeptide reference substance is as shown in SEQ ID NO: 1; and detecting the test solution, the reference substance solution and / or the reference medicinal material solution by adopting an ultra-high performance liquid chromatograph-mass spectrometer. The detection method disclosed by the invention is high in specificity, can realize effective identification of different original concha haliotidis and counterfeit products through the organic matter part of the concha haliotidis, and provides a reliable basis for quality control and evaluation of the concha haliotidis and products thereof.

Description

Technical Field

[0001] The present invention relates to the field of analysis and detection technology, and in particular to a detection method and application of Semen Cassiae Herba or its decoction pieces, standard decoction, and formula granules. Background Art

[0002] The 2020 edition of the Chinese Pharmacopoeia stipulates that Shijueming is the shell of the abalone Haliotis diversicolor Reeve, Haliotis discus hannai Ino, Haliotis ovina Gmelin, Haliotis ruber (Leach), Haliotis asinina Linnaeus or Haliotis laevigata (Donovan). It has the effects of calming the liver and suppressing yang, clearing the liver and improving eyesight. It is used to treat headaches and dizziness, red eyes, cataracts, blurred vision, and amblyopia.

[0003] Under the Pharmacopoeia section on Lithospermum officinale, quality control is performed through properties, microscopic identification, and determination of calcium carbonate content. Current research in the literature and patents primarily focuses on infrared spectroscopy, Raman spectroscopy, X-ray diffraction, inductively coupled plasma-mass spectrometry, and titration, primarily analyzing its crystal form, trace elements, and calcium carbonate content. A few studies have also focused on its protein composition. Property identification requires experienced professionals, while other studies such as microscopic identification and determination of calcium carbonate content have not been able to distinguish between Lithospermum officinale of different origins, counterfeits, and other shell-based Chinese medicines. When Lithospermum officinale is decocted into standard Chinese medicine decoctions or formulated into granules, it loses its appearance, making identification even more difficult.

[0004] On the other hand, the main component of Shijuemin is calcium carbonate, with very little organic matter. Furthermore, during the decoction process, only water-soluble substances are retained, and heating destroys the structures of proteins and peptides. Therefore, the extraction and identification of proteins and peptides in Shijuemin is difficult. Therefore, how to identify Shijuemin with high specificity to meet quality control requirements is an urgent technical challenge. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a detection method for Semen Cassiae medicinal materials or their decoction pieces, standard decoctions, and formula granules, which has strong specificity and can effectively distinguish different Semen Cassiae origins and counterfeit Semen Cassiae products.

[0006] The technical problem that the present invention also aims to solve is to provide an application of the detection method of the above-mentioned Semen Cassiae medicinal material or its decoction pieces, standard decoction, and formula granules.

[0007] The technical problem that the present invention needs to solve is to provide a method for identifying Semen Cassiae and its counterfeits.

[0008] The technical problem that the present invention also aims to solve is to provide a method for identifying the origin of Semen Cassiae medicinal materials or their decoction pieces, standard decoctions, and formula granules.

[0009] The technical problem that the present invention also aims to solve is to provide a method for identifying Semen Cassiae and its counterfeits, which can identify common counterfeits of Semen Cassiae.

[0010] The technical problem that the present invention also aims to solve is to provide a Semen Cassiae polypeptide that can identify Semen Cassiae from different origins and common counterfeits.

[0011] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned Semen Cassiae polypeptide.

[0012] Another technical problem to be solved by the present invention is to provide an ion pair that can realize the detection of the above-mentioned Semen Cassiae polypeptide.

[0013] Another technical problem to be solved by the present invention is to provide an application of the above-mentioned ion pair.

[0014] In order to solve the above technical problems, the present invention provides a method for detecting Semen Cassiae Herba or its decoction pieces, standard decoctions, and formula granules, which is characterized by comprising:

[0015] The medicinal material of Semen Cassiae or its decoction pieces, standard decoction, or formula granules is enzymatically extracted to obtain a test solution; the medicinal material of Semen Cassiae or its decoction pieces, standard decoction, or formula granules is derived from Haliotis discus henna;

[0016] The reference substance of Shijueming polypeptide is extracted or dissolved with an extraction solvent to obtain a reference substance solution; and / or the reference medicinal material of Shijueming is subjected to enzymatic extraction to obtain a reference medicinal material solution; the amino acid sequence of the reference substance of Shijueming polypeptide is shown in SEQ ID NO: 1; wherein the enzyme used for enzymatic extraction can be a hydrolase commonly used in the art, such as nuclease, trypsin, pepsin, etc., but is not limited thereto. The extraction solvent can be a bicarbonate solution, such as sodium bicarbonate solution, ammonium bicarbonate solution, etc., but is not limited thereto. The concentration of bicarbonate in the extraction solvent is 0.5wt% to 10wt%, exemplified by 1wt%, 1.5wt%, 3wt%, 5wt%, 6.5wt%, 8wt% or 9.5wt%, but is not limited thereto.

[0017] The test solution, reference solution and / or reference medicinal material solution are detected by ultra-high performance liquid spectrometry to obtain;

[0018] The detection ion pairs include m / z 517.27→432.26 and m / z 517.27→618.36. The amino acid sequence of the polypeptide corresponding to the detection ion pairs is shown in SEQ ID NO: 1.

[0019] It should be noted that, in the present invention, only one of the control medicinal material solution or the reference substance solution can be used together with the test solution for determination, or the control medicinal material solution, the reference substance solution and the test solution can be used together for determination.

[0020] As an improvement of the above technical solution, the detection ion pairs also include m / z 540.30→333.19 and m / z 540.30→819.44.

[0021] Based on this technical solution, a control solution and a test sample solution need to be used for determination, or a control medicinal material solution, a reference sample solution and a test sample solution can be used for determination.

[0022] As an improvement to the above technical solution, the chromatographic conditions of the ultra-high performance liquid spectrometer include:

[0023] The chromatographic column is filled with octadecylsilane bonded silica gel, and the column length is 50 mm to 150 mm, exemplified by 50 mm, 75 mm, 100 mm or 150 mm, but not limited thereto. The column diameter is 2 mm to 3 mm, exemplified by 2.1 mm or 3 mm, but not limited thereto. The particle size of the filler is 1.6 μm to 1.9 μm, exemplified by 1.7 μm or 1.8 μm, but not limited thereto. The column temperature is 20°C to 40°C, exemplified by 22°C, 25°C, 28°C, 32°C, 35°C or 38°C, but not limited thereto. The flow rate is 0.1 mL / min to 0.4 mL / min, exemplified by 0.14 mL / min, 0.18 mL / min, 0.22 mL / min, 0.26 mL / min, 0.3 mL / min, 0.34 mL / min or 0.38 mL / min, but not limited thereto. The injection volume is 1 μL to 3 μL, exemplified by 1.2 μL, 1.6 μL, 2.0 μL, 2.5 μL or 2.8 μL, but not limited thereto.

[0024] Mobile phase A is acetonitrile, and mobile phase B is a 0.05 vol% to 0.2 vol% formic acid aqueous solution; illustratively, the concentration of mobile phase B is 0.08 vol%, 0.11 vol%, 0.13 vol%, 0.15 vol% or 0.18 vol%, but is not limited thereto. The elution curve is:

[0025] 0 min to 7 min, mobile phase A from 6% to 40%, mobile phase B from 94% to 60%;

[0026] 7min to 10min, mobile phase A changes from 40% to 80%, and mobile phase B changes from 60% to 20%.

[0027] As an improvement of the above technical solution, the chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry instrument are:

[0028] The chromatographic column has a length of 100 mm, a column diameter of 2.1 mm, a filler particle size of 1.8 μm, a column temperature of 30° C., a flow rate of 0.3 mL / min, and an injection volume of 2 μL; and / or

[0029] Mobile phase A is acetonitrile, mobile phase B is 0.1 vol% formic acid aqueous solution; the elution curve is:

[0030] 0 min to 7 min, mobile phase A from 6% to 40%, mobile phase B from 94% to 60%;

[0031] 7min to 10min, mobile phase A changes from 40% to 80%, and mobile phase B changes from 60% to 20%.

[0032] As an improvement to the above technical solution, the mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry instrument are as follows: electrospray power supply, positive ion mode, multiple reaction monitoring, capillary voltage of 1 kV to 2 kV, exemplarily 1.2 kV, 1.4 kV, 1.6 kV, or 1.8 kV; ion source temperature of 300° C. to 400° C., exemplarily 310° C., 340° C., 370° C., or 390° C.; desolvation gas flow rate of 600 L / hr to 700 L / hr, exemplarily 615 L / hr, 630 L / hr, 650 L / hr, 670 L / hr, or 690 L / hr, but not limited thereto; ion source cone voltage of 15 V to 25 V, exemplarily 16 V, 18 V, 20 V, 22 V, or 24 V, but not limited thereto. The collision energy is 15V to 25V, exemplified by 16V, 18V, 20V, 22V or 24V, but not limited thereto.

[0033] As an improvement of the above technical solution, the mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry instrument are as follows: electrospray power supply, positive ion mode, multiple reaction monitoring, capillary voltage of 1.5 kV, ion source temperature of 350°C, desolvation gas flow rate of 650 L / hr, ion source cone voltage of 20 V, and collision energy of 20 V.

[0034] As an improvement to the above technical solution, the step of enzymatically extracting the Semen Cassiae Herba or its decoction pieces, standard decoction, or formula granules to obtain a test solution comprises:

[0035] Semen Cassiae Herba or its decoction pieces, standard decoction, and formula granules are sequentially subjected to ammonium bicarbonate solution extraction, dithiothreitol solution extraction, iodoacetamide solution treatment, trypsin solution enzymatic hydrolysis, and solid phase extraction column desalting treatment to obtain a test solution;

[0036] The dosage ratio of the ammonium bicarbonate solution to the herbal medicine Cassiae glomerata or its decoction pieces, standard decoction, or formula granules is 40 mL to 80 mL: 1 g to 2 g; exemplary ratios are 43 mL: 1.1 g, 53 mL: 1.3 g, 57 mL: 1.5 g, 68 mL: 1.6 g; 74 mL: 1.7 g, or 79 mL: 1.9 g, but are not limited thereto. The concentration of the ammonium bicarbonate solution is 1 wt% to 5 wt%; exemplary ratios are 1.4 wt%, 2 wt%, 2.6 wt%, 3.2 wt%, 3.8 wt%, or 4.4 wt%, but are not limited thereto.

[0037] The dosage ratio of the dithiothreitol solution to the herbal medicine Cassiae Cassiae or its decoction pieces, standard decoction, or formula granules is 100 μL to 200 μL: 1 g to 2 g; exemplary ratios are 113 μL: 1.1 g, 127 μL: 1.3 g, 133 μL: 1.5 g, 149 μL: 1.6 g, 167 μL: 1.6 g, or 185 μL: 1.8 g, but are not limited thereto. The concentration of the dithiothreitol solution is 0.5 mol / L to 3 mol / L, exemplary ratios are 1.2 mol / L, 1.9 mol / L, 2.3 mol / L, or 2.8 mol / L, but are not limited thereto.

[0038] The dosage ratio of the iodoacetamide solution to the herbal medicine Cassiae Herba or its decoction pieces, standard decoction, or formula granules is 300 μL to 800 μL: 1 g to 2 g; exemplary ratios are 355 μL: 1.1 g, 447 μL: 1.3 g, 503 μL: 1.4 g, 667 μL: 1.7 g, or 786 μL: 1.8 g, but are not limited thereto. The concentration of the iodoacetamide solution is 0.2 mol / L to 2 mol / L; exemplary ratios are 0.6 mol / L, 1 mol / L, 1.3 mol / L, or 1.9 mol / L, but are not limited thereto.

[0039] The dosage ratio of the trypsin solution to the Cassiae Herba medicinal materials or their decoction pieces, standard decoctions, and formula granules is 200μL~1000μL:1g~2g; exemplary ratios are 239μL:1.1g, 456μL:1.3g, 569μL:1.5g, 773μL:1.6g, 799μL:1.7g, or 889μL:1.8g, but are not limited thereto.

[0040] The trypsin solution is prepared by dissolving trypsin in ammonium bicarbonate solution, and the dosage ratio of trypsin to ammonium bicarbonate solution is 3mg-10mg:0.5mL-2mL, exemplified by 3.4mg:0.7mL, 4.6mg:0.9mL, 5.7mg:1.2mL, 6.9mg:1.4mL, 8.7mg:1.8mL or 9.1mg:0.9mL, but not limited thereto. The concentration of the ammonium bicarbonate solution is 0.5wt%-2wt%; exemplified by 0.7wt%, 1.3wt%, 1.5wt% or 1.8wt%, but not limited thereto.

[0041] The solid phase extraction column desalination process comprises:

[0042] The enzymatic hydrolyzate obtained by enzymatic hydrolysis is centrifuged, and the resulting supernatant is added to an HLB column and eluted with a 50 vol% to 90 vol% methanol solution. The eluate is collected and filtered to obtain the product. Exemplarily, the concentration of the methanol solution used for elution is 55 vol%, 60 vol%, 70 vol%, 80 vol%, or 87 vol%, but is not limited thereto. The methanol solution is a methanol-water solution, but is not limited thereto. Preferably, in one embodiment, before eluting with a 50 vol% to 90 vol% methanol solution, purified water is used for elution, and the eluate is discarded.

[0043] As an improvement of the above technical solution, the preparation method of the control medicinal material solution in the present invention is the same as the preparation method of the test sample solution.

[0044] As an improvement to the above technical solution, the steps of extracting or dissolving the Semen Cassiae polypeptide reference substance with an extraction solvent to obtain a reference solution; and / or extracting the Semen Cassiae reference medicinal material by enzymatic hydrolysis to obtain a reference medicinal material solution include:

[0045] Dissolve 10 μL to 30 μL of the Cassiae Oleracea polypeptide reference substance in a sodium bicarbonate solution with a concentration of 1 wt% to 5 wt% to obtain a reference substance solution.

[0046] Specifically, the Shijueming slices can be purified Shijueming, calcined Shijueming (see page 91 of the first part of the 2020 edition of the Chinese Pharmacopoeia) or salt-processed Shijueming, but are not limited to this. Shijueming standard decoction refers to the preparation made by decocting, concentrating and freeze-drying the above-mentioned slices with reference to the provisions of the "Technical Requirements for Quality Control and Standardization of Chinese Herbal Formula Granules". Shijueming formula granules refer to the preparation made by extracting, separating, concentrating, drying and granulating the above-mentioned slices with reference to the provisions of the "Technical Requirements for Quality Control and Standardization of Chinese Herbal Formula Granules", but are not limited to this.

[0047] Correspondingly, the present invention also discloses the application of the above-mentioned method for detecting the medicinal material Semen Cassiae or its prepared slices, standard decoction, or formula granules in the following (1) or (2):

[0048] (1) Identify the origin of Semen Cassiae Herba or its decoction pieces, standard decoctions, and formula granules;

[0049] (2) Identify Semen Cassiae Herba or its decoction pieces, standard decoctions, formula granules and their counterfeits.

[0050] As an improvement of the above technical solution, the origin of the Shijueming medicinal material or its decoction pieces, standard decoction, and formula granules includes variegated abalone, sheep abalone, Australian abalone, ear abalone, and white abalone;

[0051] The counterfeit products include black gold abalone, half-marked abalone, and oysters, but are not limited to these.

[0052] Correspondingly, the present invention also discloses a method for identifying the origin of a Shijueming medicinal material or its decoction pieces, standard decoction, or formula granules, which comprises: providing a sample to be tested, and detecting the sample to be tested using the above-mentioned detection method for the Shijueming medicinal material or its decoction pieces, standard decoction, or formula granules; if, in a mass spectrum obtained by detecting a test solution prepared from the sample to be tested under a preset detection ion pair, no ion is detected at the ion peak retention time of a control medicinal material solution and / or a control substance solution, then the origin of the sample to be tested is abalone, sheep abalone, Australian abalone, ear abalone, and / or white abalone.

[0053] Correspondingly, the present invention also discloses a method for identifying Semen Cassiae and its counterfeits, which comprises: providing a sample to be tested, and detecting the sample to be tested using the above-mentioned detection method for Semen Cassiae medicinal materials or its decoction pieces, standard decoctions, and formula granules; if, in the mass spectrum of the test solution prepared from the sample to be tested, no ion peak is detected at the retention time of the ion peak of the control medicinal material solution and / or the control substance solution, then the sample to be tested is a counterfeit.

[0054] Correspondingly, the present invention also discloses a Semen Cassiae polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 1. Based on the Semen Cassiae polypeptide, different Semen Cassiae origins and counterfeit products can be identified.

[0055] Correspondingly, the present invention also discloses the use of the above-mentioned Semen Cassiae polypeptide in the following (1) or (2):

[0056] (1) Identify the origin of Semen Cassiae Herba or its decoction pieces, standard decoctions, and formula granules;

[0057] (2) Identify Semen Cassiae Herba or its decoction pieces, standard decoctions, formula granules and their counterfeits.

[0058] Correspondingly, the present invention also discloses an ion pair for detecting the above-mentioned Semen Cassiae polypeptide, wherein the mass-to-charge ratio of the parent ion is 517.27, and the mass-to-charge ratios of the daughter ions are 432.26 and 618.36.

[0059] Correspondingly, the present invention also discloses the application of the above ion pair in the following (1) or (2):

[0060] (1) Identify the origin of Semen Cassiae Herba or its decoction pieces, standard decoctions, and formula granules;

[0061] (2) Identify Semen Cassiae Herba or its decoction pieces, standard decoctions, formula granules and their counterfeits.

[0062] It should be noted that, unless otherwise specified, the solutions referred to in the present invention are all aqueous solutions. For example, the ammonium bicarbonate solution refers to a solution obtained by dissolving ammonium bicarbonate in water.

[0063] The implementation of the present invention has the following beneficial effects:

[0064] The present invention is based on an in-depth analysis of the medicinal material Semen Cassiae and other raw medicinal materials of Semen Cassiae, and establishes a detection method for the medicinal material Semen Cassiae or its decoction pieces, standard decoctions, and formula granules. The detection method has strong specificity and can effectively identify Semen Cassiae of different bases and counterfeits through the organic matter part of Semen Cassiae, thereby solving the problem that products such as standard decoctions and formula granules of Semen Cassiae are difficult to identify due to the lack of appearance characteristics, and provides a reliable basis for the quality control and evaluation of Semen Cassiae and its products. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is the mass spectrum of batch 17 of Semen Cassiae medicinal materials in Example 1 (m / z 517.27), with the left side being m / z 517.27→432.26 and the right side being m / z 517.27→618.36;

[0066] Figure 2 17 batches of Cassiae Herba Decoction pieces in Example 1 (m / z 517.27), with m / z 517.27→432.26 on the left and m / z 517.27→618.36 on the right;

[0067] Figure 3 This is the mass spectrum of batch 17 of the standard decoction of Semen Cassiae in Example 1 (m / z 517.27), with the left side being m / z 517.27→432.26 and the right side being m / z 517.27→618.36;

[0068] Figure 4 The mass spectra of three batches of Shijueming granules in Example 1 (m / z 517.27) are as follows: m / z 517.27→432.26 on the left and m / z 517.27→618.36 on the right;

[0069] Figure 5This is the mass spectrum of batch 17 of Semen Cassiae medicinal materials in Example 1 (m / z 540.30), with the left side being m / z 540.30→333.19 and the right side being m / z 540.30→819.44;

[0070] Figure 6 17 batches of Cassiae Herba Decoction pieces in Example 1 are mass spectra (m / z 540.30), with m / z 540.30→333.19 on the left and m / z 540.30→819.44 on the right;

[0071] Figure 7 The mass spectrum of batch 17 of the standard decoction of Semen Cassiae in Example 1 (m / z 540.30) is shown on the left side as m / z 540.30→333.19, and on the right side as m / z 540.30→819.44.

[0072] Figure 8 The mass spectra of three batches of Shijueming granules in Example 1 (m / z 540.30) are as follows: m / z 540.30→333.19 on the left and m / z 540.30→819.44 on the right;

[0073] Figure 9 The mass spectra of 11 batches of other raw Cassiae and 1 batch of counterfeit products in Example 2 (m / z 517.27) are as follows: m / z 517.27→432.26 on the left and m / z 517.27→618.36 on the right;

[0074] Figure 10 This is the mass spectrum (m / z 540.30) of 11 batches of other raw Cassiae and 1 batch of counterfeit products in Example 2, with the left side being m / z 540.30→333.19 and the right side being m / z 540.30→819.44. DETAILED DESCRIPTION

[0075] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0076] Example 1 Detection method of Semen Cassiae

[0077] 1. Preparation of test solution

[0078] Take 1-2g of the powder of this product, place it in a stoppered conical flask, add 50mL of 1% ammonium bicarbonate solution, heat under reflux or ultrasonically treat for 30 minutes, cool to room temperature, add 150μL of dithiothreitol (DTT) solution (1mol / L), mix well, and place in a 56℃ water bath for 1 hour. Take it out, let it cool, add 500μL of iodoacetamide (IAA) solution (0.6mol / L), mix well, protect from light for 30 minutes, and add 500μL of trypsin solution (take trypsin for sequence analysis and add 1% ammonium bicarbonate solution to make a 500μL iodoacetamide (IAA) solution). Each 1 ml contains 5 mg of the solution, prepared just before use), shake well, enzymatically hydrolyze at 37 ° C for 12 hours, centrifuge (speed is 4000 rpm) for 5 minutes, take the supernatant, slowly pass it through an activated and balanced HLB column [specification: 6 mL (200 mg), eluted with 6 mL of methanol and 1% ammonium bicarbonate solution in sequence], add 2 mL of purified water for elution, discard, and then elute with 4 mL of 70% methanol solution. Collect the eluate, shake well, and filter with a microporous filter membrane (0.22 μm) as the test solution.

[0079] 2. Preparation of control medicinal material solution

[0080] Take 1-2 g of Cassiae cassiae control medicinal material powder, place it in a stoppered conical flask, add 50 mL of 1% ammonium bicarbonate solution, heat reflux or ultrasonicate for 30 minutes, cool to room temperature, add 150 μL of dithiothreitol (DTT) solution (1 mol / L), mix well, and place in a 56°C water bath for 1 hour. Take it out, let it cool, add 500 μL of iodoacetamide (IAA) solution (0.6 mol / L), mix well, protect from light for 30 minutes, add 500 μL of trypsin solution (take trypsin for sequence analysis, add 1% ammonium bicarbonate solution Prepare a solution containing 5 mg per 1 ml, prepare it when used), shake it, keep it enzymatically hydrolyzed at 37°C for 12 hours, centrifuge it (at 4000 revolutions per minute) for 5 minutes, take the supernatant, slowly pass it through an activated and balanced HLB column [specification: 6 mL (200 mg), eluted with 6 mL each of methanol and 1% ammonium bicarbonate solution], add 2 mL of purified water for elution, discard it, and then elute it with 4 mL of 70% methanol solution, collect the eluate, shake it, and filter it with a microporous filter membrane (0.22 μm) as the test solution.

[0081] 3. Preparation of reference solution

[0082] Take an appropriate amount of Semen Cassiae polypeptide reference substance (sequence: GTADASVSAVR), add 1% sodium bicarbonate to prepare a solution containing 20 μL per 1 mL as the reference substance solution.

[0083] 4. Chromatographic and mass spectrometry conditions

[0084] 4.1 Chromatographic conditions

[0085] Chromatographic column: C18 column (100 mm × 2.1 mm, 1 μm); mobile phase: acetonitrile (A)-0.1 vol% formic acid solution (B); elution mode: gradient elution; flow rate: 0.3 mL / min, column temperature: 30°C. The mobile phase gradient elution program is shown in Table 1.

[0086] Table 1 Elution program

[0087]

[0088] 4.2 Mass spectrometry conditions

[0089] An electrospray ionization source (ESI+) was used with a capillary voltage of 1.5 kV, a source temperature of 350°C, and a desolvation gas flow rate of 650 L / hr. Multiple reaction monitoring (MRM) was performed using the mass-to-charge ratios of m / z 517.27 (doubly charged) → 432.26, m / z 517.27 (doubly charged) → 618.36, m / z 540.30 (doubly charged) → 333.19, and m / z 540.30 (doubly charged) → 819.44 as detection ion pairs. A 2 μL injection of the control herbal solution was performed. The signal-to-noise ratio of the MRM peaks measured using the above detection ion pairs should be greater than 3:1.

[0090] Table 2 Detection ion pair information

[0091]

[0092] 5. Determination

[0093] Accurately pipette 2 μL of control medicinal material solution, 2 μL of reference substance solution, and 2 μL of test solution respectively, inject them into ultra-high performance liquid spectrometry and measure.

[0094] 6. Testing of Semen Cassiae medicinal materials, decoction pieces, standard decoctions, and formula granules

[0095] This embodiment studies a total of 55 batches of samples, including 1 batch of control medicinal materials of Shijueming (Handanoids discus). 17 batches of Shijueming (Handanoids discus) medicinal materials (batch numbers: G1-G17), 17 batches of Shijueming (Handanoids discus) decoction pieces (batch numbers: GP1-GP17), 17 batches of Shijueming (Handanoids discus) standard decoctions (batch numbers: GT1-GT17), and 3 batches of Shijueming (Handanoids discus) formula granules (batch numbers: CG1-CG3). It should be noted that the Shijueming decoction pieces here refer to the decoction pieces obtained by washing, drying, and crushing the Shijueming medicinal materials. The Shijueming standard decoction refers to the product obtained by water decoction, solid-liquid separation, concentration, and drying of the above-mentioned Shijueming decoction pieces; the Shijueming formula granules refer to the granules obtained by water heating extraction, separation, concentration, drying, and granulation of the above-mentioned Shijueming decoction pieces.

[0096] Prepare the test solution of each sample according to the method under "1" and test it according to the conditions under "4". The specific test results are shown in Table 3 and Figures 1 to 8 As shown in the figure, in the ion current chromatogram of the test sample extracted with the mass-to-charge ratio m / z 517.27 (doubly charged) → 432.26 and m / z 517.27 (doubly charged) → 618.36 ion pairs, Shijueming (Handabane) medicinal materials, decoction pieces, standard decoctions, and formula granules all showed chromatographic peaks with the same retention time as the Shijueming (Handabane) control medicinal materials and peptide reference substances; in the ion current chromatogram of the test sample extracted with the ion pairs m / z 540.30 (doubly charged) → 333.19 and m / z 540.30 (doubly charged) → 819.44, Shijueming (Handabane) medicinal materials, decoction pieces, standard decoctions, and formula granules all showed chromatographic peaks with the same retention time as the Shijueming (Handabane) control medicinal materials.

[0097] Table 3 Mass spectrometry results of Semen Cassiae Herba, decoction pieces, standard decoctions, and formula particles

[0098]

[0099]

[0100] Example 2 Identification of Different Origins and Counterfeit Products of Semen Cassiae

[0101] In this embodiment, a total of 11 batches of other raw materials of Cassia tora were studied, among which the other raw materials included variegated abalone (batch number: ZSB1~ZSB2), sheep abalone (batch number: YB), Australian abalone (batch number: AZB1~AZB4), ear abalone (batch number: EB1~EB2), and white abalone (batch number: BB), and the counterfeit products included black gold abalone (batch number: HJB1~HJB2).

[0102] Prepare the test solution of each sample according to the method under item "1" in Example 1, and inject the sample and test it according to the conditions under item "4" in Example 1. The specific test results are as follows: Figures 9 and 10 As shown in the figure, in the ion current chromatogram of the test sample extracted with the mass-to-charge ratios of m / z 517.27 (doubly charged)→432.26, m / z 517.27 (doubly charged)→618.36 and m / z 540.30 (doubly charged)→333.19, m / z 540.30 (doubly charged)→819.44, other raw materials of Shijueming (abalone, abalone, abalone, abalone, white abalone) and the counterfeit (black gold abalone) do not show chromatographic peaks with the same retention time as the Shijueming (haliotis discus henna) control medicinal material and peptide reference substance.

[0103] The above is a preferred embodiment of the invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for detecting Semen Cassiae or its prepared slices, standard decoctions, or formula granules, characterized in that: include: The medicinal material of Semen Cassiae or its decoction pieces, standard decoction, or formula granules is enzymatically extracted to obtain a test solution; the medicinal material of Semen Cassiae or its decoction pieces, standard decoction, or formula granules is derived from Haliotis discus henna; Extracting or dissolving the Shijueming polypeptide reference substance with an extraction solvent to obtain a reference substance solution; and / or extracting the Shijueming reference medicinal material by enzymatic hydrolysis to obtain a reference medicinal material solution; the amino acid sequence of the Shijueming polypeptide reference substance is shown in SEQ ID NO: 1; The test solution, reference solution and / or reference medicinal material solution are detected by ultra-high performance liquid spectrometry to obtain; The detection ion pairs include m / z 517.27→432.26 and m / z 517.27→618.

36.

2. The method for detecting the herbal medicine of Semen Cassiae or its prepared slices, standard decoction, or formula granules according to claim 1, wherein: The detection ion pairs also include m / z 540.30→333.19 and m / z 540.30→819.

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3. The method for detecting the herbal medicine of Semen Cassiae or its prepared slices, standard decoction, or formula granules according to claim 1, wherein: The chromatographic conditions of the ultra-high performance liquid spectrometer include: The chromatographic column is filled with octadecylsilane bonded silica gel, with a column length of 50 mm to 150 mm, a column diameter of 2 mm to 3 mm, a filler particle size of 1.6 μm to 1.9 μm, a column temperature of 20°C to 40°C, a flow rate of 0.1 mL / min to 0.4 mL / min, and an injection volume of 1 μL to 3 μL; and / or Mobile phase A is acetonitrile, mobile phase B is 0.05 vol% to 0.2 vol% formic acid aqueous solution; the elution curve is: 0 min to 7 min, mobile phase A from 6% to 40%, mobile phase B from 94% to 60%; 7min to 10min, mobile phase A changes from 40% to 80%, and mobile phase B changes from 60% to 20%.

4. The method for detecting the herbal medicine of Semen Cassiae or its prepared slices, standard decoction, or formula granules according to claim 1 or 3, wherein: The chromatographic conditions of the ultra-high performance liquid chromatography-mass spectrometry instrument are: The chromatographic column has a length of 100 mm, a column diameter of 2.1 mm, a filler particle size of 1.8 μm, a column temperature of 30° C., a flow rate of 0.3 mL / min, and an injection volume of 2 μL; and / or Mobile phase A is acetonitrile, mobile phase B is 0.1 vol% formic acid aqueous solution; the elution curve is: 0 min to 7 min, mobile phase A from 6% to 40%, mobile phase B from 94% to 60%; 7min to 10min, mobile phase A changes from 40% to 80%, and mobile phase B changes from 60% to 20%.

5. The method for detecting Semen Cassiae Herba or its prepared slices, standard decoction, or formula granules according to claim 1, wherein: The mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry instrument are as follows: electrospray power supply, positive ion mode, multiple reaction monitoring, capillary voltage of 1 kV to 2 kV, ion source temperature of 300°C to 400°C, desolvation gas flow rate of 600 L / hr to 700 L / hr, ion source cone voltage of 15 V to 25 V, and collision energy of 15 V to 25 V.

6. The method for detecting the Semen Cassiae Herba or its prepared slices, standard decoction, or formula granules according to claim 1 or 5, wherein: The mass spectrometry conditions of the ultra-high performance liquid chromatography-mass spectrometry instrument are as follows: electrospray power supply, positive ion mode, multiple reaction monitoring, capillary voltage of 1.5 kV, ion source temperature of 350° C., desolvation gas flow rate of 650 L / hr, ion source cone voltage of 20 V, and collision energy of 20 V.

7. The method for detecting Semen Cassiae Herba or its prepared slices, standard decoction, or formula granules according to claim 1, wherein: The step of enzymatically extracting the Semen Cassiae Herba medicinal materials or their decoction pieces, standard decoctions, or formula granules to obtain a test solution comprises: Semen Cassiae Herba or its decoction pieces, standard decoction, and formula granules are sequentially subjected to ammonium bicarbonate solution extraction, dithiothreitol solution extraction, iodoacetamide solution treatment, trypsin solution enzymatic hydrolysis, and solid phase extraction column desalting treatment to obtain a test solution; The ratio of the ammonium bicarbonate solution to the Shijueming medicinal material or its decoction pieces, standard decoction, or formula granules is 40 mL to 80 mL: 1 g to 2 g; the concentration of the ammonium bicarbonate solution is 1 wt% to 5 wt%; The dosage ratio of the dithiothreitol solution to the herbal medicine Cassiae radix or its decoction pieces, standard decoction, or formula granules is 100 μL to 200 μL: 1 g to 2 g; the concentration of the dithiothreitol solution is 0.5 mol / L to 3 mol / L; The dosage ratio of the iodoacetamide solution to the herbal medicine Cassiae glomerata or its prepared slices, standard decoction, or formula granules is 300 μL to 800 μL: 1 g to 2 g; the concentration of the iodoacetamide solution is 0.2 mol / L to 2 mol / L; The dosage ratio of the trypsin solution to the herbal medicine Cassiae radix or its decoction pieces, standard decoction, or formula granules is 200 μL to 1000 μL: 1 g to 2 g; The trypsin solution is prepared by dissolving trypsin in ammonium bicarbonate solution, the dosage ratio of trypsin to ammonium bicarbonate solution is 3mg-10mg:0.5mL-2mL, and the concentration of the ammonium bicarbonate solution is 0.5wt%-2wt%; The solid phase extraction column desalination process comprises: The enzymatic hydrolyzate obtained by enzymatic hydrolysis is centrifuged, and the obtained supernatant is added to an HLB column, eluted with a 50 vol% to 90 vol% methanol solution, and the eluate is collected and filtered to obtain the product.

8. The method for detecting Semen Cassiae Herba or its prepared slices, standard decoction, or formula granules according to claim 1, wherein: The Cassiae Semen polypeptide reference substance is extracted or dissolved with an extraction solvent to obtain a reference substance solution; And / or the step of enzymatically extracting the Semen Cassiae as a control medicinal material to obtain a control medicinal material solution comprises: Dissolve 10 μL to 30 μL of the Cassiae Oleracea polypeptide reference substance in a sodium bicarbonate solution with a concentration of 1 wt% to 5 wt% to obtain a reference substance solution.

9. Use of the method for detecting the herbal material Semen Cassiae or its prepared slices, standard decoction, or formula granules as claimed in any one of claims 1 to 8 in the following (1) or (2): (1) Identify the origin of Semen Cassiae Herba or its decoction pieces, standard decoctions, and formula granules; (2) Identify Semen Cassiae Herba or its decoction pieces, standard decoctions, formula granules and their counterfeits.

10. The use according to claim 9, characterized in that The origins of the Shijueming medicinal material or its decoction pieces, standard decoctions, and formula granules include abalone, abalone, abalone, abalone, and white abalone; The counterfeits include black abalone.

11. A method for identifying the origin of Semen Cassiae or its decoction pieces, standard decoctions, and formula granules, characterized in that: include: A sample to be tested is provided, and the sample to be tested is detected by the detection method of the Semen Cassiae medicinal material or its decoction pieces, standard decoction, and formula granules as described in any one of claims 1 to 8; if, in a mass spectrum obtained by detecting a test solution prepared from the sample to be tested under a preset detection ion pair, no ion is detected at the ion peak retention time of the control medicinal material solution and / or the reference substance solution, then the origin of the sample to be tested is abalone, sheep abalone, Australian abalone, ear abalone and / or white abalone.

12. A method for identifying Semen Cassiae and its counterfeits, characterized in that: include: A sample to be tested is provided, and the sample to be tested is tested using the detection method for the Semen Cassiae medicinal material or its decoction pieces, standard decoction, or formula granules as described in any one of claims 1 to 8; if, in a mass spectrum obtained by detecting a test solution prepared from the sample to be tested under a preset detection ion pair, no ion peak is detected at the retention time of the ion peak of the control medicinal material solution and / or the reference substance solution, the sample to be tested is a counterfeit.

13. A Semen Cassiae polypeptide, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

14. Use of the Semen Cassiae polypeptide according to claim 13 in the following (1) or (2): (1) Identify the origin of Semen Cassiae Herba or its decoction pieces, standard decoctions, and formula granules; (2) Identify Semen Cassiae Herba or its decoction pieces, standard decoctions, formula granules and their counterfeits.

15. An ion pair for detecting the Semen Cassiae polypeptide according to claim 13, characterized in that: The mass-to-charge ratio of the parent ion was 517.27, and the mass-to-charge ratios of the product ions were 432.26 and 618.

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16. Use of the ion pair according to claim 15 in the following (1) or (2): (1) Identify the origin of Semen Cassiae Herba or its decoction pieces, standard decoctions, and formula granules; (2) Identify Semen Cassiae Herba or its decoction pieces, standard decoctions, formula granules and their counterfeits.

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