Construction method and application of pepper medicinal material UPLC specific chromatogram

Abstract

The invention provides a construction method and application of a pepper medicinal material UPLC characteristic spectrum. The construction method of the pepper medicinal material UPLC characteristic spectrum comprises the following steps: taking hyperoside, hydroxyl-alpha-sanshool and hydroxyl-beta-sanshool to prepare a mixed reference substance solution; preparing a test solution from a pepper medicinal material; and respectively carrying out ultra-high performance liquid chromatography analysis on the mixed reference substance solution and the test solution, and constructing the pepper medicinal material UPLC characteristic spectrum. The construction method of the pepper medicinal material UPLC characteristic spectrum has the advantages of being rapid, stable and high in specificity, the obtained pepper medicinal material characteristic spectrum shows 11 characteristic peaks, spectrum information is rich, the chemical component characteristics of the pepper medicinal material can be fully displayed, a comprehensive and effective rapid evaluation method is provided for quality control of the pepper medicinal material, and the method is suitable for popularization and application. The method can realize identification of different primitives and counterfeit products of Chinese prickly ash, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine identification and pharmaceutical analysis, and particularly relates to a method for constructing a UPLC characteristic fingerprint of Zanthoxylum bungeanum Maxim. and its application in identifying different origins and counterfeits. Background Art

[0002] There are about 250 species of plants in the genus Zanthoxylum L. of the family Rutaceae in the world, which are widely cultivated in the tropical and subtropical regions of Asia, the Americas, Africa and Oceania. The main countries cultivating Zanthoxylum plants in the world are China, Japan and South Korea, among which China has the largest cultivation area, the highest pericarp yield and the richest germplasm resources. Zanthoxylum bungeanum Maxim. is an important aromatic plant. Its pericarp is often used as a condiment and spice for its unique flavor. At the same time, the pericarp of Zanthoxylum bungeanum Maxim. has a variety of active ingredients and has the effects of "warming the middle-jiao to relieve pain, killing insects and relieving itching", so it is recognized as a medicine and food dual-purpose plant.

[0003] The 2020 edition of the Chinese Pharmacopoeia stipulates that the origin of Zanthoxylum bungeanum Maxim. is the dried ripe pericarp of Zanthoxylum schinifolium Sieb. et Zucc. or Zanthoxylum bungeanum Maxim. Although the origin can be identified and distinguished according to the medicinal properties of the herbs, there is still a lack of a comprehensive identification method. In the market, there are often other adulterants circulating as Zanthoxylum bungeanum Maxim.. For example, the common green Zanthoxylum bungeanum Maxim. in the market is mainly Zanthoxylum armatum DC., also known as rattan pepper, which is widely cultivated in southern China and is also often used as a condiment and is deeply loved by consumers. In addition, Zanthoxylum avicennae (Lam.) DC. is also known as wild pepper and is also circulated in the market. Moreover, the extensive planting areas, diverse varieties of Zanthoxylum bungeanum Maxim., and different management, cultivation methods, picking, drying and storage processes in different regions will result in great differences in the quality of Zanthoxylum bungeanum Maxim. in the market.

[0004] Literature research shows that the color, smell and numbness of Zanthoxylum bungeanum Maxim. are the key indicators for evaluating the quality of Zanthoxylum bungeanum Maxim., among which the volatile oil and amide substances of Zanthoxylum bungeanum Maxim. are the main indicators reflecting the internal quality of Zanthoxylum bungeanum Maxim.. The former is the main volatile component of Zanthoxylum bungeanum Maxim., and the latter is the main numbness-causing substance. A large number of previous studies have explored the active ingredients and functions of Zanthoxylum bungeanum Maxim., but usually only from a certain aspect of the quality indicators of Zanthoxylum bungeanum Maxim.. For example, in the Chinese Pharmacopoeia, only the volatile oil is used as the index component for Zanthoxylum bungeanum Maxim., lacking systematicness and comprehensiveness. In the standards related to Zanthoxylum bungeanum Maxim., the methods for judging the appearance color and taste are mainly based on visual observation and smelling, lacking accuracy and objectivity. The only index reflecting the internal quality of Zanthoxylum bungeanum Maxim. is the volatile oil content and the limit requirements are inconsistent. However, the quality characteristics of Zanthoxylum bungeanum Maxim. should also involve more component indicators. The volatile oil can only represent the odor characteristics of Zanthoxylum bungeanum Maxim. and is difficult to comprehensively and truly reflect its internal quality. Therefore, it is very necessary and urgent to establish an accurate method for identifying the types of Zanthoxylum bungeanum Maxim. germplasm resources. Summary of the Invention

[0005] In order to overcome the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide a method for establishing a UPLC characteristic spectrum of Zanthoxylum bungeanum, which is fast, stable and highly specific. The established UPLC characteristic spectrum can be used to identify different origins and counterfeits of Zanthoxylum bungeanum, providing a new scientific method for quality control of Zanthoxylum bungeanum medicinal materials.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for constructing a UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material comprises the following steps:

[0008] Take hyperoside, hydroxy-α-sanshool and hydroxy-β-sanshool to prepare a mixed reference solution;

[0009] Take Zanthoxylum bungeanum medicinal material to prepare the test solution;

[0010] The mixed reference solution and the test solution are subjected to ultra-high performance liquid chromatography analysis respectively to construct the UPLC characteristic spectrum of the Zanthoxylum bungeanum medicinal material;

[0011] The conditions for the ultra-high performance liquid chromatography analysis include: mobile phase A is a mixed solution of acetonitrile and methanol, mobile phase B is a phosphoric acid aqueous solution, and gradient elution is used.

[0012] In one embodiment, the procedure of the gradient elution is:

[0013] From 0 min to 3 min, the volume percentage of mobile phase A changed from 17% to 23%;

[0014] From 3 to 13 minutes, the volume percentage of mobile phase A changed from 23% to 30%;

[0015] 13min~18min, the volume percentage of mobile phase A is 30%;

[0016] From 18 to 23 minutes, the volume percentage of mobile phase A changed from 30% to 40%;

[0017] From 23 to 38 minutes, the volume percentage of mobile phase A changed from 40% to 75%;

[0018] 38min~40min, the volume percentage of mobile phase A is 75%.

[0019] In one of the embodiments, in the mixed solution of acetonitrile and methanol, the volume ratio of acetonitrile to methanol is 4:3.

[0020] In one of the embodiments, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.15%.

[0021] In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography also include: using a YMC TriartC18 chromatographic column.

[0022] In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a detection wavelength of 240 nm.

[0023] In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a flow rate of 0.28 mL / min to 0.32 mL / min.

[0024] In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a column temperature of 28°C to 32°C.

[0025] In one embodiment, the preparation of the mixed reference solution comprises the following steps:

[0026] Mixing hyperoside, hydroxy-α-sanshool, hydroxy-β-sanshool and a first alcohol-containing aqueous solution with a volume percentage of 70% to 100%;

[0027] The alcohol in the first alcohol-containing aqueous solution is selected from methanol or ethanol.

[0028] In one embodiment, in the mixed reference solution, the concentration of hyperoside is 30 μg / mL to 40 μg / mL, the concentration of hydroxy-α-sanshool is 245 μg / mL to 255 μg / mL, and the concentration of hydroxy-β-sanshool is 20 μg / mL to 30 μg / mL.

[0029] In one embodiment, the preparation of the test solution comprises the following steps:

[0030] The pepper sample is crushed, sieved, mixed with a second alcohol-containing aqueous solution with a volume percentage of 70% to 100%, subjected to ultrasonic extraction, filtered, and a filtrate is collected;

[0031] The alcohol in the second alcohol-containing aqueous solution is selected from methanol or ethanol.

[0032] In one embodiment, the conditions of ultrasonic extraction include: power of 250W to 350W; frequency of 35kHz to 45kHZ; time of 25min to 35min.

[0033] In one embodiment, the UPLC characteristic spectrum of the Zanthoxylum bungeanum medicinal material includes 11 characteristic peaks, among which the peak corresponding to the hyperoside reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is within ±10% of the specified value, and the specified value is: peak 1 is 0.55, peak 2 is 0.60, peak 3 is 0.75, peak 4 is 0.85, peak 6 is 1.04, peak 7 is 1.30, peak 8 is 1.33, and peak 9 is 2.47.

[0034] The present invention also provides a detection method for identifying different origins of Zanthoxylum bungeanum medicinal materials and their counterfeits, comprising the following steps:

[0035] Take different original Zanthoxylum bungeanum samples or counterfeit samples to prepare sample solutions to be tested;

[0036] The sample solution to be tested is subjected to ultra-high performance liquid chromatography analysis, and the obtained spectrum of the sample solution to be tested is compared with the characteristic spectrum obtained according to the method for constructing the UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material;

[0037] The conditions for the ultra-high performance liquid chromatography analysis include: mobile phase A is a mixed solution of acetonitrile and methanol, mobile phase B is a phosphoric acid aqueous solution, and gradient elution is used.

[0038] In one embodiment of the detection method, the procedure of the gradient elution is:

[0039] From 0 min to 3 min, the volume percentage of mobile phase A changed from 17% to 23%;

[0040] From 3 to 13 minutes, the volume percentage of mobile phase A changed from 23% to 30%;

[0041] 13min~18min, the volume percentage of mobile phase A is 30%;

[0042] From 18 to 23 minutes, the volume percentage of mobile phase A changed from 30% to 40%;

[0043] From 23 to 38 minutes, the volume percentage of mobile phase A changed from 40% to 75%;

[0044] 38min~40min, the volume percentage of mobile phase A is 75%.

[0045] In one embodiment of the detection method, in the mixed solution of acetonitrile and methanol, the volume ratio of acetonitrile to methanol is 4:3.

[0046] In one embodiment of the detection method, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.15%.

[0047] In one embodiment of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: using a YMC Triart C18 chromatographic column.

[0048] In one embodiment of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a detection wavelength of 240 nm.

[0049] In one embodiment of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a flow rate of 0.28 mL / min to 0.32 mL / min.

[0050] In one embodiment of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a column temperature of 28°C to 32°C.

[0051] In one embodiment of the detection method, the preparation of the sample solution to be tested comprises the following steps:

[0052] Crush the original pepper samples of different origins or counterfeit samples, sieve, mix with 70% to 100% by volume of the third alcohol-containing aqueous solution, perform ultrasonic extraction, filter, and collect the filtrate;

[0053] The alcohol in the third alcohol-containing aqueous solution is selected from methanol or ethanol.

[0054] In one embodiment of the detection method, the conditions of the ultrasonic extraction include: power of 250W to 350W; frequency of 35kHz to 45kHZ; time of 25min to 35min.

[0055] In one embodiment of the detection method, the different origin Zanthoxylum bungeanum includes one or both of Zanthoxylum bungeanum and green pepper.

[0056] In one embodiment of the detection method, the counterfeit product includes one or two of Zanthoxylum bungeanum and Zanthoxylum bungeanum.

[0057] The present invention has at least the following beneficial effects:

[0058] The present invention provides a method for constructing a UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material. The method is rapid, stable and highly specific. The obtained characteristic spectrum of Zanthoxylum bungeanum medicinal material presents 11 characteristic peaks, and the spectrum information is rich, which can fully display the chemical composition characteristics of Zanthoxylum bungeanum medicinal material. Furthermore, the constructed UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material can realize the identification of different origins and their counterfeits, thereby providing a relatively comprehensive and effective rapid evaluation method for the quality control of Zanthoxylum bungeanum medicinal material. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 To investigate the chromatograms of different extraction solvents for the characteristic spectrum of Zanthoxylum bungeanum (Zanthoxylum bungeanum);

[0060] Figure 2 To investigate the chromatograms of different extraction methods for the characteristic spectrum of Zanthoxylum bungeanum (Zanthoxylum bungeanum);

[0061] Figure 3 Investigate chromatograms at different extraction times for Zanthoxylum bungeanum (Zanthoxylum bungeanum) medicinal material characteristic profile;

[0062] Figure 4 Investigate chromatograms at different detection wavelengths for the characteristic spectrum of Zanthoxylum bungeanum (Zanthoxylum bungeanum) medicinal material;

[0063] Figure 5 This is a 3D spectrum scan of Zanthoxylum bungeanum (Zanthoxylum bungeanum) medicinal material;

[0064] Figure 6 Optimize the chromatogram for the mobile phase of Zanthoxylum bungeanum (Zanthoxylum bungeanum);

[0065] Figure 7 To investigate the specificity of the characteristic spectrum of Zanthoxylum bungeanum (Zanthoxylum bungeanum);

[0066] Figure 8 This is an overlay of characteristic spectra of 21 batches of Zanthoxylum bungeanum (Zanthoxylum bungeanum) medicinal materials;

[0067] Fig. 9 This is the characteristic spectrum of Zanthoxylum bungeanum reference medicinal material; among them, Peak 5 (S): hyperoside; Peak 6: isoquercetin; Peak 8: narcissusin; Peak 9: hydroxy-ε-sanshool; Peak 10: hydroxy-α-sanshool; Peak 11: hydroxy-β-sanshool;

[0068] Fig.10 This is a reference characteristic spectrum of Zanthoxylum bungeanum (Zanthoxylum bungeanum); wherein, peak 5 (S): hyperoside; peak 6: isoquercetin; peak 8: narcissusin; peak 9: hydroxy-ε-sanshool; peak 10: hydroxy-α-sanshool; peak 11: hydroxy-β-sanshool;

[0069] Fig.11 This is a comparison chart of the characteristic spectra of two batches of green pepper and Sichuan pepper;

[0070] Fig.12 This is a comparison chart of the characteristic spectra of 4 batches of Zanthoxylum bungeanum, 2 batches of Zanthoxylum bungeanum and Zanthoxylum bungeanum medicinal materials. DETAILED DESCRIPTION

[0071] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0073] The 2020 edition of the Chinese Pharmacopoeia stipulates that the origin of Sichuan pepper includes green pepper and Sichuan pepper. Due to the influence of planting management measures in different origins and differences in Sichuan pepper germplasm resources, the quality of Sichuan pepper from different germplasm resources varies greatly. In addition, other confusing products are often circulated on the market as Sichuan pepper. Therefore, it is difficult to accurately identify it based on appearance and smell alone. The lack of effective methods for quality control and intensive processing, development and utilization of Sichuan pepper has seriously affected the quality of Sichuan pepper and its processed products and their application in more fields, restricting the sustainable, high-quality and healthy development of the Sichuan pepper industry.

[0074] Based on this, the present invention provides a method for constructing a UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material, comprising the following steps:

[0075] Take hyperoside, hydroxy-α-sanshool and hydroxy-β-sanshool to prepare a mixed reference solution;

[0076] Take Zanthoxylum bungeanum medicinal material to prepare the test solution;

[0077] The mixed reference solution and the test solution are subjected to ultra-high performance liquid chromatography analysis respectively to construct the UPLC characteristic spectrum of the Zanthoxylum bungeanum medicinal material;

[0078] The conditions for the ultra-high performance liquid chromatography analysis include: mobile phase A is a mixed solution of acetonitrile and methanol, mobile phase B is a phosphoric acid aqueous solution, and gradient elution is used.

[0079] In a specific example of the method for constructing a characteristic spectrum, the procedure of the gradient elution is:

[0080] From 0 min to 3 min, the volume percentage of mobile phase A changed from 17% to 23%;

[0081] From 3 to 13 minutes, the volume percentage of mobile phase A changed from 23% to 30%;

[0082] 13min~18min, the volume percentage of mobile phase A is 30%;

[0083] From 18 to 23 minutes, the volume percentage of mobile phase A changed from 30% to 40%;

[0084] From 23 to 38 minutes, the volume percentage of mobile phase A changed from 40% to 75%;

[0085] 38min~40min, the volume percentage of mobile phase A is 75%.

[0086] In a specific example of the method for constructing a characteristic spectrum, in the mixed solution of acetonitrile and methanol, the volume ratio of acetonitrile to methanol is 4:3.

[0087] In a specific example of the method for constructing a characteristic spectrum, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.15%.

[0088] It can be understood that in the present invention, in the phosphoric acid aqueous solution, the volume fraction of the phosphoric acid includes but is not limited to 0.05%, 0.08%, 0.1%, 0.12%, 0.15%; preferably, the volume fraction of the phosphoric acid is 0.1%.

[0089] In a specific example of the method for constructing a characteristic spectrum, the chromatographic conditions of the ultra-high performance liquid chromatography also include: using a YMC Triart C18 chromatographic column.

[0090] In a specific example of the method for constructing a characteristic spectrum, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a detection wavelength of 240 nm.

[0091] In a specific example of the method for constructing a characteristic spectrum, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a flow rate of 0.28 mL / min to 0.32 mL / min.

[0092] In a specific example of the method for constructing a characteristic spectrum, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a column temperature of 28°C to 32°C.

[0093] It can be understood that in the method for constructing the characteristic spectrum of the present invention, the flow rate includes but is not limited to 0.28 mL / min, 0.29 mL / min, 0.30 mL / min, 0.31 mL / min, and 0.32 mL / min; preferably, the flow rate is 0.30 mL / min.

[0094] It can be understood that in the method for constructing the characteristic spectrum of the present invention, the column temperature includes but is not limited to 28°C, 29°C, 30°C, 31°C, and 32°C; preferably, the column temperature is 30°C.

[0095] More specifically, in the method for constructing the characteristic spectrum of the present invention, the injection volume is 1 μL to 2 μL.

[0096] More specifically, in the method for constructing the characteristic spectrum of the present invention, the size of the chromatographic column includes the following conditions: the column length is 150 mm, the inner diameter is 2.1 mm, and the particle size of the filler is 1.9 μm.

[0097] In a specific example of the method for constructing a characteristic spectrum, the preparation of a mixed reference solution comprises the following steps:

[0098] Mixing hyperoside, hydroxy-α-sanshool, hydroxy-β-sanshool and a first alcohol-containing aqueous solution with a volume percentage of 70% to 100%;

[0099] The alcohol in the first alcohol-containing aqueous solution is selected from methanol or ethanol.

[0100] It can be understood that the volume percentage of the first alcohol-containing aqueous solution includes but is not limited to 70%, 80%, and 100%.

[0101] In a specific example of the method for constructing a characteristic spectrum, in the mixed reference solution, the concentration of hyperoside is 30 μg / mL to 40 μg / mL, the concentration of hydroxy-α-sanshool is 245 μg / mL to 255 μg / mL, and the concentration of hydroxy-β-sanshool is 20 μg / mL to 30 μg / mL.

[0102] It can be understood that the concentration of the hyperoside in the mixed reference solution includes but is not limited to 30 μg / mL, 35 μg / mL, and 40 μg / mL; the concentration of the hydroxy-α-sanshool includes but is not limited to 245 μg / mL, 250 μg / mL, and 255 μg / mL; the concentration of the hydroxy-β-sanshool includes but is not limited to 20 μg / mL, 25 μg / mL, and 30 μg / mL.

[0103] In a specific example of the method for constructing a characteristic spectrum, the preparation of the test solution comprises the following steps:

[0104] The pepper sample is crushed, sieved, mixed with a second alcohol-containing aqueous solution with a volume percentage of 70% to 100%, subjected to ultrasonic extraction, filtered, and a filtrate is collected.

[0105] It can be understood that the volume percentage of the second alcohol-containing aqueous solution includes but is not limited to 70%, 80%, and 100%.

[0106] Preferably, the crushing of the pepper sample and sieving means making the pepper sample into powder, passing it through a No. 3 sieve, and collecting a powder sample with a particle size of about 50 mesh.

[0107] In a specific example of the method for constructing a characteristic spectrum, the conditions for ultrasonic extraction include: power of 250W to 350W; frequency of 35kHz to 45kHZ; time of 25min to 35min.

[0108] It can be understood that the power includes but is not limited to 250W, 280W, 300W, 320W, 350W; preferably, the power is 300W;

[0109] It can be understood that the frequency includes but is not limited to 35kHZ, 40kHZ, 45kHZ; preferably, the frequency is 40kHZ;

[0110] It can be understood that the time includes but is not limited to 25 minutes, 30 minutes, and 35 minutes; preferably, the time is 30 minutes.

[0111] It can be understood that the filtration includes but is not limited to filtration using filter paper; the filtrate is preferably the filtrate collected after removing the initial filtrate in a single filtration.

[0112] In a specific example of the method for constructing a characteristic spectrum, the UPLC characteristic spectrum of the Zanthoxylum bungeanum medicinal material includes 11 characteristic peaks, among which the peak corresponding to the hyperoside reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is within ±10% of the specified value, and the specified value is: peak 1 is 0.55, peak 2 is 0.60, peak 3 is 0.75, peak 4 is 0.85, peak 6 is 1.04, peak 7 is 1.30, peak 8 is 1.33, and peak 9 is 2.47.

[0113] The present invention provides a method for constructing a UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material. The method is rapid, stable and highly specific. The constructed characteristic spectrum of Zanthoxylum bungeanum medicinal material presents 11 characteristic peaks, has rich spectrum information, and can fully display the chemical composition characteristics of Zanthoxylum bungeanum medicinal material.

[0114] The present invention also provides a detection method for identifying different origins of Zanthoxylum bungeanum medicinal materials and their counterfeits, comprising the following steps:

[0115] Take different original Zanthoxylum bungeanum samples or counterfeit samples to prepare sample solutions to be tested;

[0116] The sample solution to be tested is subjected to ultra-high performance liquid chromatography analysis, and the obtained spectrum of the sample solution to be tested is compared with the characteristic spectrum obtained according to the method for constructing the UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material;

[0117] The conditions for the ultra-high performance liquid chromatography analysis include: mobile phase A is a mixed solution of acetonitrile and methanol, mobile phase B is a phosphoric acid aqueous solution, and gradient elution is used.

[0118] In a specific example of the detection method, the gradient elution procedure is:

[0119] From 0 min to 3 min, the volume percentage of mobile phase A changed from 17% to 23%;

[0120] From 3 to 13 minutes, the volume percentage of mobile phase A changed from 23% to 30%;

[0121] 13min~18min, the volume percentage of mobile phase A is 30%;

[0122] From 18 to 23 minutes, the volume percentage of mobile phase A changed from 30% to 40%;

[0123] From 23 to 38 minutes, the volume percentage of mobile phase A changed from 40% to 75%;

[0124] 38min~40min, the volume percentage of mobile phase A is 75%.

[0125] In a specific example of the detection method, in the mixed solution of acetonitrile and methanol, the volume ratio of acetonitrile to methanol is 4:3.

[0126] In a specific example of the detection method, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.15%.

[0127] It can be understood that in the detection method of the present invention, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution includes but is not limited to 0.05%, 0.08%, 0.1%, 0.12%, and 0.15%; preferably, the volume fraction of phosphoric acid is 0.1%.

[0128] In a specific example of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: using a YMCTriart C18 chromatographic column.

[0129] In a specific example of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a detection wavelength of 240 nm.

[0130] In a specific example of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a flow rate of 0.28 mL / min to 0.32 mL / min.

[0131] In a specific example of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a column temperature of 28°C to 32°C.

[0132] It can be understood that in the detection method of the present invention, the flow rate includes but is not limited to 0.28 mL / min, 0.29 mL / min, 0.30 mL / min, 0.31 mL / min, 0.32 mL / min; preferably, the flow rate is 0.30 mL / min.

[0133] It can be understood that in the detection method of the present invention, the column temperature includes but is not limited to 28°C, 29°C, 30°C, 31°C, and 32°C; preferably, the column temperature is 30°C.

[0134] More specifically, in the detection method of the present invention, the injection volume is 1 μL to 2 μL.

[0135] More specifically, in the detection method of the present invention, the dimensions of the chromatographic column include the following conditions: the column length is 150 mm, the inner diameter is 2.1 mm, and the particle size of the filler is 1.9 μm.

[0136] In a specific example of the detection method, the preparation of the sample solution to be tested comprises the following steps:

[0137] The original Zanthoxylum bungeanum samples or counterfeit samples are crushed, sieved, mixed with a third alcohol-containing aqueous solution with a volume percentage of 70% to 100%, subjected to ultrasonic extraction, filtered, and the filtrate is collected.

[0138] It is understandable that the volume percentage of the third alcohol-containing aqueous solution includes but is not limited to 70%, 80%, and 100%. Preferably, the crushing of different origin pepper samples or counterfeit samples and sieving refers to making the pepper samples or counterfeit samples into powder, passing them through a No. 3 sieve, and collecting powder samples with a particle size of about 50 mesh.

[0139] In a specific example of the detection method, the conditions for the ultrasonic extraction include: power of 250W to 350W; frequency of 35kHz to 45kHZ; and time of 25min to 35min.

[0140] It can be understood that the power includes but is not limited to 250W, 280W, 300W, 320W, 350W; preferably, the power is 300W;

[0141] It can be understood that the frequency includes but is not limited to 35kHZ, 40kHZ, 45kHZ; preferably, the frequency is 40kHZ;

[0142] It can be understood that the time includes but is not limited to 25 minutes, 30 minutes, and 35 minutes; preferably, the time is 30 minutes.

[0143] It can be understood that the filtration includes but is not limited to filtration using filter paper; the filtrate is preferably the filtrate collected after removing the initial filtrate in a single filtration.

[0144] In a specific example of the detection method, the different origin Zanthoxylum bungeanum includes one or both of Zanthoxylum bungeanum and green pepper; and / or,

[0145] The counterfeit products include one or two of Zanthoxylum bungeanum and Zanthoxylum bungeanum.

[0146] The present invention can identify different origins and counterfeits of Zanthoxylum bungeanum by comparing the established UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material with the spectrum of the sample to be tested, thereby providing a new scientific method for quality control of Zanthoxylum bungeanum medicinal material.

[0147] The construction method of the Zanthoxylum bungeanum UPLC characteristic spectrum of the present invention is further described in detail below in conjunction with specific examples. The raw materials used in the following examples, unless otherwise specified, are all commercially available products.

[0148] Example 1

[0149] This embodiment provides a method for constructing a UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material, which is as follows:

[0150] 1. Instruments and reagents

[0151] Instruments: Thermo ultra-high performance liquid chromatograph (Vanquish, Thermo Fisher Scientific), Agilent ultra-high performance liquid chromatograph (1290, Agilent), Waters ultra-high performance liquid chromatograph (H-class, Waters), Waters HSST3 C18 column (2.1 mm × 150 mm, 1.8 μm), Waters BEH C18 column (2.1 mm × 150 mm, 1.7 μm), YMCTriart C18 column (2.1 mm × 150 mm, 1.9 μm), SHIMADZU Shim-pack Scepter C18 chromatographic column (2.1 mm × 150 mm, 1.9 μm), one-tenth balance (Mettler-Toledo, ME204E), one-millionth balance (Mettler-Toledo, XP26), electric constant temperature water bath (Shanghai Yiheng Technology Co., Ltd., HWS-28), CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water system (Merck Co., Ltd., Milli-Q Direct).

[0152] Reagents: ethanol (analytical grade, Guangdong Guanghua Science and Technology Co., Ltd.), methanol (analytical grade, Guangdong Guanghua Science and Technology Co., Ltd.); acetonitrile (chromatographic grade, Merck & Co., Ltd.), methanol (chromatographic grade, Merck & Co., Ltd.), phosphoric acid (Tianjin Komiou Chemical Reagent Co., Ltd., chromatographic grade), and ultrapure water (self-made in the laboratory).

[0153] Test drugs: hyperoside (China Food and Drug Inspection Institute, batch number: 111521-201809, content: 94.9%); hydroxy-α-sanshool (Shanghai Shidande Standard Technology Co., Ltd., batch number: 13212, content: 92.41%); hydroxy-β-sanshool (Shanghai Shidande Standard Technology Co., Ltd., batch number: 14149, content: 96.40%); Zanthoxylum bungeanum Maxim., a Rutaceae plant, was identified as dried mature peel by Professor Huang Haibo of Guangzhou University of Chinese Medicine, and 2 batches of green pepper were identified as dried mature peel of Zanthoxylum schinifolium Sieb.et Zucc., a Rutaceae plant. See Table 1 for the origin and number.

[0154] Table 1 Sample information table

[0155]

[0156]

[0157] 2 Chromatographic conditions and test solution preparation

[0158] 2.1 Chromatographic conditions

[0159] A YMC Triart C18 chromatographic column (2.1 mm × 150 mm, 1.9 μm) was used, with acetonitrile:methanol (4:3) as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, and gradient elution was performed according to the provisions in Table 2 below; the flow rate was 0.30 ml per minute; the column temperature was 30°C; and the detection wavelength was 240 nm.

[0160] Table 2 Gradient elution table

[0161]

[0162] 2.2 Preparation of reference solution Take appropriate amount of hyperoside, hydroxy-α-sanshool reference substance and hydroxy-β-sanshool reference substance, weigh accurately, and add methanol to prepare a mixed solution containing 35 μg of hyperoside, 250 μg of hydroxy-α-sanshool and 25 μg of hydroxy-β-sanshool per 1 ml.

[0163] 2.3 Preparation of test solution Take about 0.5 g of the powder of the product (passed through No. 3 sieve), weigh it accurately, put it in a stoppered conical flask, add 25 ml of methanol accurately, weigh it, treat it with ultrasound (power 300 W, frequency 40 kHz) for 30 minutes, cool it, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain the product.

[0164] 2.4 Determination method: Accurately pipette 2 μl of the test solution and the reference solution respectively, inject into the liquid chromatograph for determination.

[0165] 3 Investigation of the preparation method of test solution

[0166] 3.1 Investigation of extraction solvent

[0167] Take about 0.5g of the same batch of Zanthoxylum bungeanum powder (batch number: S21, passed through No. 3 sieve), weigh accurately, put into a stoppered conical flask, accurately add 25ml of methanol, 70% methanol, 50% methanol, ethanol, 70% ethanol, 50% ethanol, respectively, and treat with ultrasound (power 300W, frequency 40kHz) for 30 minutes, let cool, make up the weight loss with the corresponding solvent, shake well, filter, and take the filtrate. Determine according to the chromatographic conditions specified in "2.1". Figure 1 The results show that by comparing the characteristic spectra of the six extraction solvents, it can be found that the characteristic peak shapes of the six extraction solvents have little effect, and the extraction efficiency is relatively consistent. Therefore, methanol is selected as the extraction solvent.

[0168] 3.2 Investigation of extraction methods

[0169] Take about 0.5g of the same batch of Zanthoxylum bungeanum powder (batch number: S21, passed through No. 3 sieve), weigh accurately, put into a stoppered conical flask, add 25ml of methanol accurately, treat with ultrasound (power 300W, frequency 40kHz) for 30 minutes, heat under reflux for 30 minutes, cool, make up the weight loss with methanol, shake well, filter, and take the filtrate. Determine according to the chromatographic conditions specified in "2.1". Figure 2 The results show that different extraction methods have little effect on the characteristic spectrum of Zanthoxylum bungeanum. Considering the convenience of operation, ultrasonic treatment is selected.

[0170] 3.3 Extraction time investigation

[0171] Take about 0.5g of the same batch of Zanthoxylum bungeanum powder (batch number: S21, passed through No. 3 sieve), weigh accurately, put into a stoppered conical bottle, add 25ml of methanol accurately, and treat with ultrasound (power 300W, frequency 40kHz) for 15 minutes, 30 minutes, and 60 minutes respectively, cool, make up the weight loss with methanol, shake well, filter, and take the filtrate. Determine according to the chromatographic conditions specified in "2.1". Figure 3 The results show that different extraction times have little effect on the characteristic spectrum of Zanthoxylum bungeanum (Zanthoxylum bungeanum). To ensure complete extraction, ultrasonic treatment for 30 minutes was selected.

[0172] 4 Optimization of chromatographic conditions

[0173] 4.1 Determination of detection wavelength

[0174] Take about 0.5g of Zanthoxylum bungeanum powder (batch number: S21, passed through No. 3 sieve), prepare the test solution according to the method in "2.3", inject and analyze according to the chromatographic conditions in "2.1", and record the absorption spectrum in the range of 190-400nm. Figure 4 The results show that by comparing the chromatograms of different detection wavelengths, it is found that at a wavelength of 240nm, the peak response and information are richer, the interference is small, and the separation is better. Therefore, after comprehensive consideration, 240nm is selected as the detection wavelength.

[0175] 4.2 Mobile phase optimization

[0176] Take about 0.5g of Zanthoxylum bungeanum powder (batch number: S21, passed through No. 3 sieve) and prepare the test solution according to the method under "2.3" to investigate the effects of different organic phase ratios. Figure 6 The results show that acetonitrile-methanol (4:3) as the mobile phase has better characteristic peak shapes and separation effects. After comprehensive consideration, acetonitrile-methanol (4:3) was selected as the organic phase.

[0177] 5 Methodological considerations

[0178] 5.1 Specificity Investigation

[0179] Take about 0.5g of Zanthoxylum bungeanum (Zanthoxylum bungeanum) powder (batch number: S21, passed through No. 3 sieve), prepare the test solution according to the method under "2.3", accurately pipette 2μl of the test solution, the reference solution under "2.2" and the blank solvent, and inject and analyze according to the chromatographic conditions under "2.1". Figure 7 The results show that the test sample chromatogram has the same chromatographic peak at the corresponding retention time as the reference sample chromatogram, and the blank solvent has no interference, indicating that the method has good specificity.

[0180] 5.2 Precision investigation

[0181] Take about 0.5g of Zanthoxylum bungeanum (Zanthoxylum bungeanum) powder (batch number: S21, passed through No. 3 sieve), prepare the test solution according to the method under "2.3", and repeat the injection 6 times according to the chromatographic conditions under "2.1". Take hyperoside as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak and S peak, and calculate the RSD value. The relative retention time RSD value of 11 common peaks is in the range of 0.00% to 0.20%, and the relative peak area RSD value is in the range of 0.22% to 2.60%, indicating that the instrument has good precision.

[0182] 5.3 Reproducibility study

[0183] Take about 0.5g of Zanthoxylum bungeanum (Zanthoxylum bungeanum) powder (batch number: S21, passed through No. 3 sieve), prepare 6 test solutions according to the method under "2.3", and inject and measure according to the chromatographic conditions under "2.1". Taking hyperoside as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak and S peak, and calculate the RSD value. The relative retention time RSD value of 11 common peaks is in the range of 0.00% to 0.15%, and the relative peak area RSD value is in the range of 0.40% to 2.66%, indicating that the method has good repeatability.

[0184] 5.4 Stability investigation

[0185] Take about 0.5g of Zanthoxylum bungeanum (Zanthoxylum bungeanum) powder (batch number: S21, passed through No. 3 sieve), prepare the test solution according to the method under "2.3", and inject and measure at 0, 2, 4, 6, 12, 20, and 24 hours according to the chromatographic conditions under "2.1". Take hyperoside as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak and S peak, and calculate the RSD value. The relative retention time RSD value of 11 common peaks is in the range of 0.00% to 0.27%, and the relative peak area RSD value is in the range of 0.48% to 2.94%, indicating that the test solution is relatively stable within 24 hours.

[0186] 5.5 Intermediate precision study

[0187] The operation was performed by different analysts in different laboratories and on instruments of different brands. About 0.5 g of Zanthoxylum bungeanum (Zanthoxylum bungeanum) powder (batch number: S21, passed through No. 3 sieve) was taken, and 6 test solutions were prepared according to the method under "2.3", and the samples were injected and measured according to the chromatographic conditions under "2.1". Taking hyperoside as the reference peak, the relative retention time and relative peak area of ​​each characteristic peak and the S peak were calculated, and the RSD value was calculated. The relative retention time RSD values ​​of the 11 common peaks were in the range of 0.14% to 2.91%, and the relative peak area RSD values ​​were in the range of 0.71% to 3.58%, indicating that the established method can be reproduced under different conditions such as different equipment and laboratories, and the intermediate precision of this method is good.

[0188] 6 Establishment of characteristic spectrum of Zanthoxylum bungeanum (Zanthoxylum bungeanum)

[0189] 6.1 Results of Zanthoxylum bungeanum (Zanthoxylum bungeanum) characteristic spectrum determination

[0190] Take 21 batches of Zanthoxylum bungeanum (Zanthoxylum bungeanum) medicinal materials, according to the chromatographic conditions under "2.1" and the test solution preparation method determined under "2.3", take hyperoside as the reference peak, calculate the relative retention time of each characteristic peak and the S peak, and calculate the RSD value. The experimental results are shown in Table 3. Combined with the established characteristic spectrum analysis, the types of components of Zanthoxylum bungeanum (Zanthoxylum bungeanum) from different origins are basically the same. The characteristic spectrum of 21 batches of Zanthoxylum bungeanum (Zanthoxylum bungeanum) medicinal materials is shown in Figure 8 .

[0191] Table 3 Characteristic spectra of 21 batches of Zanthoxylum bungeanum (Zanthoxylum bungeanum) medicinal materials (relative retention time)

[0192]

[0193] 6.2 Establishment of the common pattern of Zanthoxylum bungeanum (Zanthoxylum bungeanum) characteristic profile

[0194] Take Zanthoxylum bungeanum reference medicinal material (batch number: 121106-201906, China Food and Drug Inspection Institute) and 21 batches of Zanthoxylum bungeanum (Zanthoxylum bungeanum) medicinal materials, and inject and measure according to the chromatographic conditions under "2.1" and the preparation method of the test solution under "2.3" above, and obtain the characteristic spectrum of Zanthoxylum bungeanum reference medicinal material ( Fig. 9 ); the Chinese medicine chromatographic fingerprint similarity evaluation software was used to generate a reference map by the average method, and a reference characteristic map of Zanthoxylum bungeanum (Zanthoxylum bungeanum) was established ( Fig.10 ).

[0195] It is determined that the characteristic spectrum of Zanthoxylum bungeanum (Zanthoxylum bungeanum) should present 11 characteristic peaks, and the retention times of the 11 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other. Peak 5, peak 10, and peak 11 should correspond to the retention times of the corresponding reference substance peaks, respectively. The peak corresponding to the peak of the hyperoside reference substance is the S peak. The relative retention times of peaks 1 to 4, peaks 6 to 9 and the S peak are calculated. The relative retention times should be within the range of ±10% of the specified values. The specified values ​​are: 0.55 (peak 1), 0.60 (peak 2), 0.75 (peak 3), 0.85 (peak 4), 1.04 (peak 6), 1.30 (peak 7), 1.33 (peak 8), and 2.47 (peak 9).

[0196] 6.3 Identification of different origins and their counterfeits

[0197] 6.3.1 Comparison of characteristic spectra of different origin peppers (Zanthoxylum bungeanum and green pepper)

[0198] The characteristic spectra of the two original peppers and green peppers were compared and studied. Fig.11 The results showed that the characteristic spectra of the two bases were quite different, and green pepper did not have 11 characteristic peaks of the characteristic spectrum of Sichuan pepper, indicating that the chemical components contained in the two were quite different.

[0199] 6.3.2 Comparison of counterfeit feature maps

[0200] The preparation method of Zanthoxylum bungeanum (Zanthoxylum bungeanum) medicinal material and the characteristic spectrum detection method were used to measure 4 batches of Zanthoxylum bungeanum and 2 batches of Zanthoxylum bungeanum, and compared with the characteristic spectrum of Zanthoxylum bungeanum (Zanthoxylum bungeanum). The results are as follows Fig.12The results show that the characteristic spectra of Zanthoxylum bungeanum and Zanthoxylum bungeanum samples are quite different, and they do not have 11 characteristic peaks of Zanthoxylum bungeanum samples, and the characteristic spectrum of Zanthoxylum bungeanum has a chromatographic peak with a higher response at 25 minutes; the characteristic spectra of Zanthoxylum bungeanum and Zanthoxylum bungeanum are relatively consistent, but Zanthoxylum bungeanum lacks characteristic peak 7. Therefore, the characteristic spectrum method established by the present invention can well distinguish fake Zanthoxylum bungeanum products.

[0201] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0202] The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in detail, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to interpret the contents of the claims.

Claims

1. A method for constructing a UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material, characterized in that: The steps include: Take hyperoside, hydroxy-α-sanshool and hydroxy-β-sanshool to prepare a mixed reference solution; Take Zanthoxylum bungeanum medicinal material to prepare the test solution; The mixed reference solution and the test solution are subjected to ultra-high performance liquid chromatography analysis respectively to construct the UPLC characteristic spectrum of the Zanthoxylum bungeanum medicinal material; The conditions for the ultra-high performance liquid chromatography analysis include: mobile phase A is a mixed solution of acetonitrile and methanol, mobile phase B is a phosphoric acid aqueous solution, and gradient elution is used.

2. The method for constructing the UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material according to claim 1, characterized in that: The procedure of the gradient elution is: From 0 min to 3 min, the volume percentage of mobile phase A changed from 17% to 23%; From 3 to 13 minutes, the volume percentage of mobile phase A changed from 23% to 30%; 13min~18min, the volume percentage of mobile phase A is 30%; From 18 to 23 minutes, the volume percentage of mobile phase A changed from 30% to 40%; From 23 to 38 minutes, the volume percentage of mobile phase A changed from 40% to 75%; 38min~40min, the volume percentage of mobile phase A is 75%.

3. The method for constructing the UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material according to claim 1, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography satisfy at least one of the following (1) to (2): (1) In the mixed solution of acetonitrile and methanol, the volume ratio of acetonitrile to methanol is 4:3; (2) In the phosphoric acid aqueous solution, the volume fraction of phosphoric acid is 0.05% to 0.15%.

4. The method for constructing the UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material according to any one of claims 1 to 3, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography satisfy at least one of the following (3) to (6): (3) Using YMC Triart C18 chromatographic column; (4) The detection wavelength is 240nm; (5) Flow rate: 0.28 mL / min to 0.32 mL / min; (6) The column temperature is 28℃~32℃.

5. The method for constructing the UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material according to any one of claims 1 to 3, characterized in that: The preparation of the mixed reference substance solution comprises the following steps: Mixing hyperoside, hydroxy-α-sanshool, hydroxy-β-sanshool and a first alcohol-containing aqueous solution with a volume percentage of 70% to 100%; The alcohol in the first alcohol-containing aqueous solution is selected from methanol or ethanol.

6. The method for constructing the UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material according to claim 5, characterized in that: In the mixed reference solution, the concentration of hyperoside is 30 μg / mL to 40 μg / mL, the concentration of hydroxy-α-sanshool is 245 μg / mL to 255 μg / mL, and the concentration of hydroxy-β-sanshool is 20 μg / mL to 30 μg / mL.

7. The method for constructing the UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material according to any one of claims 1 to 3, characterized in that: The preparation of the test solution comprises the following steps: The pepper sample is crushed, sieved, mixed with a second alcohol-containing aqueous solution with a volume percentage of 70% to 100%, subjected to ultrasonic extraction, filtered, and a filtrate is collected; The alcohol in the second alcohol-containing aqueous solution is selected from methanol or ethanol.

8. The method for constructing the UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material according to claim 7, characterized in that: The conditions for ultrasonic extraction include: power of 250W to 350W; frequency of 35kHz to 45kHZ; and time of 25min to 35min.

9. The method for constructing the UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material according to any one of claims 1 to 3, characterized in that: The UPLC characteristic spectrum of the Zanthoxylum bungeanum medicinal material includes 11 characteristic peaks, among which the peak corresponding to the hyperoside reference peak is the S peak, and the relative retention time of each characteristic peak and the S peak is within ±10% of the specified value, and the specified value is: peak 1 is 0.55, peak 2 is 0.60, peak 3 is 0.75, peak 4 is 0.85, peak 6 is 1.04, peak 7 is 1.30, peak 8 is 1.33, and peak 9 is 2.

47.

10. A detection method for identifying different origins of Zanthoxylum bungeanum medicinal materials and their counterfeits, characterized in that: The steps include: Take different original Zanthoxylum bungeanum samples or counterfeit samples to prepare sample solutions to be tested; The sample solution to be tested is subjected to ultra-high performance liquid chromatography analysis, and the obtained spectrum of the sample solution to be tested is compared with the characteristic spectrum obtained by the method for constructing the UPLC characteristic spectrum of Zanthoxylum bungeanum medicinal material according to any one of claims 1 to 9; The conditions for the ultra-high performance liquid chromatography analysis include: mobile phase A is a mixed solution of acetonitrile and methanol, mobile phase B is a phosphoric acid aqueous solution, and gradient elution is used.

11. The detection method for identifying different origins of Zanthoxylum bungeanum medicinal materials and their counterfeits according to claim 10, characterized in that: The procedure of the gradient elution is: From 0 min to 3 min, the volume percentage of mobile phase A changed from 17% to 23%; From 3 to 13 minutes, the volume percentage of mobile phase A changed from 23% to 30%; 13min~18min, the volume percentage of mobile phase A is 30%; From 18 to 23 minutes, the volume percentage of mobile phase A changed from 30% to 40%; From 23 to 38 minutes, the volume percentage of mobile phase A changed from 40% to 75%; 38min~40min, the volume percentage of mobile phase A is 75%.

12. The detection method for identifying different origins of Zanthoxylum bungeanum medicinal materials and their counterfeits according to claim 10, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography satisfy at least one of the following (1) to (2): (1) In the mixed solution of acetonitrile and methanol, the volume ratio of acetonitrile to methanol is 4:3; (2) In the phosphoric acid aqueous solution, the volume fraction of phosphoric acid is 0.05% to 0.15%.

13. The detection method for identifying different origins of Zanthoxylum bungeanum medicinal materials and their counterfeits according to any one of claims 10 to 12, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatography satisfy at least one of the following (3) to (6): (3) Using YMC Triart C18 chromatographic column; (4) The detection wavelength is 240nm; (5) Flow rate: 0.28 mL / min to 0.32 mL / min; (6) The column temperature is 28℃~32℃.

14. The detection method for identifying different origins of Zanthoxylum bungeanum medicinal materials and their counterfeits according to any one of claims 10 to 12, characterized in that: The preparation of the sample solution to be tested comprises the following steps: Crush the original pepper samples of different origins or counterfeit samples, sieve, mix with 70% to 100% by volume of the third alcohol-containing aqueous solution, perform ultrasonic extraction, filter, and collect the filtrate; The alcohol in the third alcohol-containing aqueous solution is selected from methanol or ethanol.

15. The detection method for identifying different origins of Zanthoxylum bungeanum medicinal materials and their counterfeits according to claim 14, characterized in that: The conditions for ultrasonic extraction include: power of 250W to 350W; frequency of 35kHz to 45kHZ; and time of 25min to 35min.

16. The detection method for identifying different origins of Zanthoxylum bungeanum medicinal materials and their counterfeits according to any one of claims 10 to 12 or claim 15, characterized in that: Satisfy at least one of the following (7) to (8): (7) The different origin Zanthoxylum bungeanum comprises one or both of Zanthoxylum bungeanum and green pepper; (8) The counterfeit products include one or both of Zanthoxylum bungeanum and Zanthoxylum bungeanum.

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