Construction Method and Application of UPLC Feature Map of Sichuan Pepper Medicinal Material
By constructing a UPLC characteristic map of Sichuan pepper medicinal materials, the problem of identifying the original source and counterfeit products of Sichuan pepper medicinal materials was solved, achieving rapid, stable and highly specific quality control, and providing a scientific quality evaluation method.
Patent Information
- Application Number
- CN202310971422.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-02
- Publication Date
- 2025-10-31
- Estimated Expiration
- 2043-08-02
AI Technical Summary
Existing technologies lack comprehensive methods to identify the original and counterfeit Sichuan pepper medicinal materials, resulting in large differences in the quality of Sichuan pepper on the market, making it difficult to achieve accurate quality control and in-depth processing and utilization.
Ultra-high performance liquid chromatography (UPLC) was used to construct a characteristic chromatogram of Sichuan pepper. By analyzing a mixed reference solution of hyperoside, hydroxy-α-salicornin, and hydroxy-β-salicornin with the test solution, a gradient elution program was established. A mixed solution of acetonitrile and methanol was used as mobile phase A, and an aqueous solution of phosphoric acid was used as mobile phase B. The detection wavelength was 240 nm, the flow rate was 0.28-0.32 mL/min, and the column temperature was 28-32 ℃. UPLC characteristic chromatograms of 11 characteristic peaks were constructed.
It enables rapid, stable, and specific quality control of Sichuan pepper medicinal materials, effectively identifies different origins and counterfeit products, and provides a scientific quality evaluation method.
Smart Images
Figure CN119438408B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of traditional Chinese medicine identification and drug analysis technology, specifically involving a method for constructing UPLC characteristic spectra of Sichuan pepper and its application in the identification of different originals and counterfeits. Background Technology
[0002] There are approximately 250 species of plants in the genus *Zanthoxylum* of the Rutaceae family worldwide, widely cultivated in tropical and subtropical regions of Asia, the Americas, Africa, and Oceania. The main countries cultivating *Zanthoxylum* are China, Japan, and South Korea, with China having the largest cultivation area, highest pericarp yield, and richest germplasm resources. *Zanthoxylum* is an important aromatic plant; its pericarp, with its unique flavor, is often used as a seasoning and spice. Furthermore, the pericarp contains various active ingredients and possesses properties that "warm the stomach and relieve pain, kill parasites and relieve itching," thus it is recognized as a plant with both medicinal and edible uses.
[0003] The 2020 edition of the Chinese Pharmacopoeia specifies that the original source of Sichuan pepper is the dried, mature pericarp of *Zanthoxylum schinifolium* Sieb. et Zucc. or *Zanthoxylum bungeanum* Maxim. While the original source can be identified based on its medicinal properties, a comprehensive identification method is lacking. Furthermore, other adulterants are frequently sold as Sichuan pepper in the market. For example, the common green Sichuan pepper is mainly *Zanthoxylum bungeanum*, also known as rattan pepper, widely cultivated in southern China and commonly used as a condiment, making it popular with consumers. Additionally, *Zanthoxylum bungeanum*, also known as wild Sichuan pepper, is also available in the market. Moreover, the wide range of planting areas, diverse varieties of Sichuan pepper, and varying management, cultivation, harvesting, drying, and storage methods across different regions all contribute to significant differences in the quality of Sichuan pepper sold in the market.
[0004] Literature review indicates that the color, aroma, and numbing sensation of Sichuan pepper are key indicators for evaluating its quality. Among these, volatile oil and amides are the main indicators reflecting the intrinsic quality of Sichuan pepper; the former is the main volatile component, and the latter is the main numbing substance. Previous studies have extensively explored the active ingredients and functions of Sichuan pepper, but these typically focus on only one aspect of its quality indicators. For example, the Chinese Pharmacopoeia only uses volatile oil as an indicator component, lacking systematicity and comprehensiveness. Standards concerning Sichuan pepper primarily rely on visual and olfactory assessment for appearance and taste, lacking accuracy and objectivity. The intrinsic quality of Sichuan pepper is only indicated by volatile oil content, with inconsistent limits. However, the quality characteristics of Sichuan pepper should involve more component indicators; volatile oil only represents the aroma characteristics and cannot comprehensively and accurately reflect its intrinsic quality. Therefore, establishing accurate methods for identifying Sichuan pepper germplasm resource types is both necessary and urgent. Summary of the Invention
[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a method for establishing a UPLC feature map of Sichuan pepper. This method is fast, stable and highly specific. The established UPLC feature map can be used to identify different origins and counterfeits of Sichuan pepper, providing a new scientific method for the quality control of Sichuan pepper medicinal materials.
[0006] This invention is achieved through the following technical solution:
[0007] A method for constructing a UPLC feature map of Sichuan pepper medicinal material includes the following steps:
[0008] Take hyperoside, hydroxy-α-sanshool, and hydroxy-β-sanshool to prepare a mixed reference solution;
[0009] Prepare a test solution from Sichuan pepper medicinal materials;
[0010] The mixed reference solution and the test solution were analyzed by ultra-high performance liquid chromatography (UPLC) to construct the characteristic UPLC chromatogram of the Sichuan pepper 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 an aqueous solution of phosphoric acid, and gradient elution is used.
[0012] In one embodiment, the gradient elution procedure is as follows:
[0013] From 0 min to 3 min, the volume percentage of mobile phase A changed from 17% to 23%;
[0014] Between 3 and 13 minutes, the volume percentage of mobile phase A changed from 23% to 30%.
[0015] From 13 to 18 minutes, the volume percentage of mobile phase A was 30%.
[0016] Between 18 and 23 minutes, the volume percentage of mobile phase A changed from 30% to 40%.
[0017] Between 23 and 38 minutes, the volume percentage of mobile phase A changed from 40% to 75%.
[0018] From 38 to 40 minutes, the volume percentage of mobile phase A is 75%.
[0019] In one embodiment, the volume ratio of acetonitrile to methanol in the mixed solution of acetonitrile and methanol is 4:3.
[0020] In one embodiment, the volume fraction of phosphoric acid in the aqueous phosphoric acid solution is 0.05% to 0.15%.
[0021] In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography further include: using a YMC Triart C18 column.
[0022] In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography further include a detection wavelength of 240 nm.
[0023] In one embodiment, the chromatographic conditions of the ultra-high performance liquid chromatography further 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 further include a column temperature of 28°C to 32°C.
[0025] In one embodiment, the preparation of the mixed reference solution includes the following steps:
[0026] Hypericin, hydroxy-α-sanshool, and hydroxy-β-sanshool are mixed with 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, the concentration of hyperoside in the mixed reference solution 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 includes the following steps:
[0030] The pepper sample was crushed, sieved, mixed with a second alcohol-containing aqueous solution of 70%–100% by volume, and subjected to ultrasonic extraction. The mixture was then filtered and the filtrate was collected.
[0031] The alcohol in the second alcohol-containing aqueous solution is selected from methanol or ethanol.
[0032] In one embodiment, the conditions for ultrasonic extraction include: power of 250W to 350W; frequency of 35kHz to 45kHz; and time of 25min to 35min.
[0033] In one embodiment, the UPLC characteristic spectrum of the Sichuan pepper medicinal material includes 11 characteristic peaks, among which the peak corresponding to the hyperoside reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is within ±10% of a specified value, which 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] This invention also provides a detection method for identifying different origins of Sichuan pepper and its adulterants, comprising the following steps:
[0035] Take samples of different original peppercorns or counterfeit samples and prepare the sample solution to be tested.
[0036] The sample solution to be tested was analyzed by ultra-high performance liquid chromatography, and the obtained spectrum of the sample solution to be tested was compared with the characteristic spectrum obtained according to the method of constructing the characteristic spectrum of Sichuan pepper medicinal material by UPLC.
[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 an aqueous solution of phosphoric acid, and gradient elution is used.
[0038] In one embodiment of the detection method, the gradient elution procedure is as follows:
[0039] From 0 min to 3 min, the volume percentage of mobile phase A changed from 17% to 23%;
[0040] Between 3 and 13 minutes, the volume percentage of mobile phase A changed from 23% to 30%.
[0041] From 13 to 18 minutes, the volume percentage of mobile phase A was 30%.
[0042] Between 18 and 23 minutes, the volume percentage of mobile phase A changed from 30% to 40%.
[0043] Between 23 and 38 minutes, the volume percentage of mobile phase A changed from 40% to 75%.
[0044] From 38 to 40 minutes, the volume percentage of mobile phase A is 75%.
[0045] In one embodiment of the detection method, the volume ratio of acetonitrile to methanol in the mixed solution of acetonitrile and 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 further include: using a YMC Triart C18 column.
[0048] In one embodiment of the detection method, the chromatographic conditions of the ultra-high performance liquid chromatography further 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 further 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 further 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 includes the following steps:
[0052] Crushed samples of different original peppercorns or counterfeit samples were sieved, mixed with a third alcohol-containing aqueous solution of 70%–100% by volume, and subjected to ultrasonic extraction. The mixture was then filtered and the filtrate was collected.
[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 for ultrasonic extraction include: power of 250W to 350W; frequency of 35kHz to 45kHz; and time of 25min to 35min.
[0055] In one embodiment of the detection method, the different original peppercorns include one or both of Sichuan peppercorns and green peppercorns.
[0056] In one embodiment of the detection method, the counterfeit product includes one or both of Sichuan pepper and Sichuan pepper.
[0057] The present invention has at least the following beneficial effects:
[0058] This invention provides a method for constructing a UPLC characteristic spectrum of Sichuan pepper medicinal material. This method is rapid, stable, and highly specific. The obtained characteristic spectrum of Sichuan pepper medicinal material exhibits 11 characteristic peaks, with rich spectral information, which can fully demonstrate the chemical composition characteristics of Sichuan pepper medicinal material. Furthermore, the constructed UPLC characteristic spectrum of Sichuan pepper medicinal material can be used to identify different original materials and their counterfeits, thus providing a more comprehensive, effective, and rapid evaluation method for the quality control of Sichuan pepper medicinal material. Attached Figure Description
[0059] Figure 1 Chromatograms were investigated for different extraction solvents to determine the characteristic chromatograms of Sichuan pepper (Zanthoxylum bungeanum) medicinal material;
[0060] Figure 2 Chromatograms were examined for different extraction methods to determine the characteristic chromatograms of Sichuan pepper (Zanthoxylum bungeanum) medicinal material;
[0061] Figure 3 Chromatograms were examined at different extraction times to determine the characteristic chromatograms of Sichuan pepper (Zanthoxylum bungeanum) medicinal material.
[0062] Figure 4 To investigate the chromatograms of different detection wavelengths for the characteristic chromatograms of Sichuan pepper (Zanthoxylum bungeanum) medicinal material;
[0063] Figure 5 3D spectral scan of Sichuan pepper (Zanthoxylum bungeanum) medicinal material;
[0064] Figure 6 Optimize the chromatogram of the mobile phase for Sichuan pepper (Zanthoxylum bungeanum) medicinal material;
[0065] Figure 7 To investigate the specificity of the characteristic spectral data of Sichuan pepper (Zanthoxylum bungeanum) as a medicinal material;
[0066] Figure 8 This is an overlay of characteristic maps of 21 batches of Sichuan pepper (Zanthoxylum bungeanum) medicinal materials;
[0067] Figure 9 The characteristic spectrum of Sichuan pepper reference medicinal material is shown; among them, peak 5 (S): hyperoside; peak 6: isoquercitrin; peak 8: narcissin; peak 9: hydroxy-ε-salicylic acid; peak 10: hydroxy-α-salicylic acid; peak 11: hydroxy-β-salicylic acid;
[0068] Figure 10 The reference characteristic spectrum of Sichuan pepper (Zanthoxylum bungeanum) medicinal material; among them, peak 5 (S): hyperoside; peak 6: isoquercitrin; peak 8: narcissin; peak 9: hydroxy-ε-salicylic acid; peak 10: hydroxy-α-salicylic acid; peak 11: hydroxy-β-salicylic acid;
[0069] Figure 11 Comparative chromatograms of the characteristics of two batches of green pepper and Sichuan pepper medicinal materials;
[0070] Figure 12 Comparative chromatograms of the characteristics of four batches of Chinese prickly ash, two batches of Chinese prickly ash, and Chinese prickly ash medicinal materials. Detailed Implementation
[0071] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.
[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0073] The 2020 edition of the Chinese Pharmacopoeia specifies that the basic source of Sichuan pepper includes both green pepper and Sichuan pepper. However, due to differences in planting management practices in different producing areas and variations in Sichuan pepper germplasm resources, the quality of Sichuan pepper varies greatly among different germplasm resources. Furthermore, other adulterants are often sold as Sichuan pepper in the market. Therefore, it is difficult to accurately identify them based solely on appearance and odor. The lack of effective methods for quality control and intensive processing of Sichuan pepper seriously affects the quality of Sichuan pepper and its processed products, hindering their application in various fields and 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 feature map of Sichuan pepper medicinal material, comprising the following steps:
[0075] Take hyperoside, hydroxy-α-sanshool, and hydroxy-β-sanshool to prepare a mixed reference solution;
[0076] Prepare a test solution from Sichuan pepper medicinal materials;
[0077] The mixed reference solution and the test solution were analyzed by ultra-high performance liquid chromatography (UPLC) to construct the characteristic UPLC chromatogram of the Sichuan pepper 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 an aqueous solution of phosphoric acid, and gradient elution is used.
[0079] In a specific example of the feature map construction method, the gradient elution procedure is as follows:
[0080] From 0 min to 3 min, the volume percentage of mobile phase A changed from 17% to 23%;
[0081] Between 3 and 13 minutes, the volume percentage of mobile phase A changed from 23% to 30%.
[0082] From 13 to 18 minutes, the volume percentage of mobile phase A was 30%.
[0083] Between 18 and 23 minutes, the volume percentage of mobile phase A changed from 30% to 40%.
[0084] Between 23 and 38 minutes, the volume percentage of mobile phase A changed from 40% to 75%.
[0085] From 38 to 40 minutes, the volume percentage of mobile phase A is 75%.
[0086] In a specific example of the method for constructing the feature map, the volume ratio of acetonitrile to methanol in the mixed solution of acetonitrile and methanol is 4:3.
[0087] In a specific example of the method for constructing the feature map, the volume fraction of phosphoric acid in the phosphoric acid aqueous solution is 0.05% to 0.15%.
[0088] Understandably, in this 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%.
[0089] In a specific example of the method for constructing the characteristic spectrum, the chromatographic conditions of the ultra-high performance liquid chromatography also include: using a YMC Triart C18 column.
[0090] In a specific example of the method for constructing the feature map, 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 the 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 the feature map, the chromatographic conditions of the ultra-high performance liquid chromatography also include: a column temperature of 28℃~32℃.
[0093] Understandably, in the method for constructing the feature map 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] Understandably, in the method for constructing the feature map 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 feature map of the present invention, the injection volume is 1 μL to 2 μL.
[0096] More specifically, in the method for constructing the characteristic chromatogram of the present invention, the dimensions of the chromatographic column include the following conditions: column length of 150 mm, inner diameter of 2.1 mm, and particle size of the packing material of 1.9 μm.
[0097] In a specific example of the method for constructing the characteristic spectrum, the preparation of the mixed reference solution includes the following steps:
[0098] Hypericin, hydroxy-α-sanshool, and hydroxy-β-sanshool are mixed with 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] Understandably, 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 the 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] Understandably, the concentration of 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 hydroxy-α-sanshool includes, but is not limited to, 245 μg / mL, 250 μg / mL, and 255 μg / mL; and the concentration of 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 the feature map, the preparation of the test solution includes the following steps:
[0104] The pepper sample was crushed, sieved, mixed with a second alcohol-containing aqueous solution of 70%–100% by volume, and subjected to ultrasonic extraction. The mixture was then filtered and the filtrate was collected.
[0105] Understandably, the volume percentage of the second alcohol-containing aqueous solution includes, but is not limited to, 70%, 80%, and 100%.
[0106] Preferably, the pulverized pepper sample, sieving, refers to making the pepper sample into powder and passing it through a No. 3 sieve to collect a powder sample with a particle size of about 50 mesh.
[0107] In a specific example of the feature map construction method, the conditions for ultrasonic extraction include: power of 250W to 350W; frequency of 35kHz to 45kHz; and time of 25min to 35min.
[0108] Understandably, the power includes, but is not limited to, 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.
[0109] Understandably, the frequency includes, but is not limited to, 35 kHz, 40 kHz, and 45 kHz; preferably, the frequency is 40 kHz.
[0110] Understandably, the time includes, but is not limited to, 25 min, 30 min, and 35 min; preferably, the time is 30 min.
[0111] Understandably, the filtration includes, but is not limited to, filtration using filter paper; the filtrate is preferably the filtrate collected after the initial filtrate has been removed in a single filtration process.
[0112] In a specific example of the method for constructing the feature spectrum, the UPLC feature spectrum of the Sichuan pepper medicinal material includes 11 feature peaks, among which the peak corresponding to the hyperoside reference peak is the S peak. The relative retention time of each feature peak and the S peak is within ±10% of a specified value, which 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] This invention provides a method for constructing UPLC feature maps of Sichuan pepper medicinal materials. This method is fast, stable and highly specific. The constructed feature maps of Sichuan pepper medicinal materials exhibit 11 characteristic peaks and are rich in information, which can fully demonstrate the chemical composition characteristics of Sichuan pepper medicinal materials.
[0114] This invention also provides a detection method for identifying different origins of Sichuan pepper and its adulterants, comprising the following steps:
[0115] Take samples of different original peppercorns or counterfeit samples and prepare the sample solution to be tested.
[0116] The sample solution to be tested was analyzed by ultra-high performance liquid chromatography, and the obtained spectrum of the sample solution to be tested was compared with the characteristic spectrum obtained according to the method of constructing the characteristic spectrum of Sichuan pepper medicinal material by UPLC.
[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 an aqueous solution of phosphoric acid, and gradient elution is used.
[0118] In a specific example of the detection method, the gradient elution procedure is as follows:
[0119] From 0 min to 3 min, the volume percentage of mobile phase A changed from 17% to 23%;
[0120] Between 3 and 13 minutes, the volume percentage of mobile phase A changed from 23% to 30%.
[0121] From 13 to 18 minutes, the volume percentage of mobile phase A was 30%.
[0122] Between 18 and 23 minutes, the volume percentage of mobile phase A changed from 30% to 40%.
[0123] Between 23 and 38 minutes, the volume percentage of mobile phase A changed from 40% to 75%.
[0124] From 38 to 40 minutes, the volume percentage of mobile phase A is 75%.
[0125] In one specific example of the detection method, the volume ratio of acetonitrile to methanol in the mixed solution of acetonitrile and methanol is 4:3.
[0126] In one 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] Understandably, 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 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℃~32℃.
[0132] Understandably, 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, and 0.32 mL / min; preferably, the flow rate is 0.30 mL / min.
[0133] Understandably, 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: column length is 150 mm, inner diameter is 2.1 mm, and particle size of the packing material is 1.9 μm.
[0136] In a specific example of the detection method, the preparation of the sample solution to be tested includes the following steps:
[0137] Crush different original pepper samples or counterfeit samples, sieve them, mix them with a third alcohol-containing aqueous solution of 70% to 100% by volume, perform ultrasonic extraction, filter, and collect the filtrate.
[0138] Understandably, the volume percentage of the third alcohol-containing aqueous solution includes, but is not limited to, 70%, 80%, and 100%. Preferably, the pulverization of different original pepper samples or counterfeit samples, and sieving, refers to preparing the pepper samples or counterfeit samples into powder and passing them through a No. 3 sieve to collect powder samples with a particle size of approximately 50 mesh.
[0139] In a specific example of the detection method, the conditions for ultrasonic extraction include: power of 250W to 350W; frequency of 35kHz to 45kHz; and time of 25min to 35min.
[0140] Understandably, the power includes, but is not limited to, 250W, 280W, 300W, 320W, and 350W; preferably, the power is 300W.
[0141] Understandably, the frequency includes, but is not limited to, 35 kHz, 40 kHz, and 45 kHz; preferably, the frequency is 40 kHz.
[0142] Understandably, the time includes, but is not limited to, 25 min, 30 min, and 35 min; preferably, the time is 30 min.
[0143] Understandably, the filtration includes, but is not limited to, filtration using filter paper; the filtrate is preferably the filtrate collected after the initial filtrate has been removed in a single filtration process.
[0144] In a specific example of the detection method, the different original peppercorns include one or both of Sichuan peppercorns and green peppercorns; and / or,
[0145] The counterfeit products include one or both of the following: Sichuan peppercorns (specifically, bamboo leaf peppercorns) and Sichuan peppercorns (specifically, prickly ash peppercorns).
[0146] This invention compares the established UPLC characteristic spectrum of Sichuan pepper with the spectrum of the sample to be tested, enabling the identification of different origins and counterfeits of Sichuan pepper, and providing a new scientific method for the quality control of Sichuan pepper.
[0147] The following detailed description, in conjunction with specific embodiments, illustrates the method for constructing UPLC feature maps of Sichuan pepper medicinal materials according to the present invention. Unless otherwise specified, all raw materials used in the following embodiments are commercially available products.
[0148] Example 1
[0149] This embodiment provides a method for constructing UPLC feature maps of Sichuan pepper medicinal materials, as detailed below:
[0150] 1. Instruments and reagents
[0151] Instruments: Thermo UHPLC (Vanquish, Thermo Fisher Scientific), Agilent UHPLC (1290, Agilent Technologies), Waters UHPLC (H-class, Waters Corporation), 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 column (2.1 mm × 150 mm, 1.9 μm), 0.0001 g balance (Mettler-Toledo, ME204E), 0.1 million g balance (Mettler-Toledo, XP26), electric thermostatic water bath (Shanghai Yiheng Technology Co., Ltd., HWS-28), CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water system (Merck, Milli-Q Direct).
[0152] Reagents: Ethanol (analytical grade, Guangdong Guanghua Science & Technology Co., Ltd.), methanol (analytical grade, Guangdong Guanghua Science & Technology Co., Ltd.); acetonitrile (chromatographic grade, Merck Inc.), methanol (chromatographic grade, Merck Inc.), phosphoric acid (Tianjin Kemeio Chemical Reagent Co., Ltd., chromatographic grade), and ultrapure water (laboratory-made).
[0153] Test reagents: hyperoside (China National Institutes for Food and Drug Control, batch number: 111521-201809, content: 94.9%); hydroxy-α-sanshool (Shanghai Shidander Standard Technology Co., Ltd., batch number: 13212, content: 92.41%); hydroxy-β-sanshool (Shanghai Shidander Standard Technology Co., Ltd., batch number: 14149, content: 96.40%); Sichuan pepper reference material (China National Institutes for Food and Drug Control, batch number: 121106-201906); 21 batches of Sichuan pepper medicinal materials were identified by Professor Huang Haibo of Guangzhou University of Traditional Chinese Medicine as the dried mature pericarp of Zanthoxylum bungeanum Maxim. (Rutaceae family), and 2 batches of green pepper medicinal materials were identified as the dried mature pericarp of Zanthoxylum schinifolium Sieb. et Zucc. (Rutaceae family). Origin and batch number are shown in Table 1.
[0154] Table 1 Sample Information Table
[0155]
[0156]
[0157] 2. Chromatographic conditions and preparation of test solution
[0158] 2.1 Chromatographic conditions
[0159] A YMC Triart C18 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 specifications in Table 2. 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 amounts of hyperoside, hydroxy-α-salicornin reference standard and hydroxy-β-salicornin reference standard, accurately weigh them, and add methanol to prepare a mixed solution containing 35 μg of hyperoside, 250 μg of hydroxy-α-salicornin and 25 μg of hydroxy-β-salicornin per 1 ml.
[0163] 2.3 Preparation of the test solution: Weigh approximately 0.5 g of the powder (passed through a No. 3 sieve) accurately, place it in a stoppered conical flask, add 25 ml of methanol accurately, weigh the solution, sonicate (300 W power, 40 kHz frequency) for 30 minutes, cool, replenish the lost weight with methanol, shake well, filter, and collect the filtrate to obtain the test solution.
[0164] 2.4 Determination Method: Accurately pipette 2 μl of the test solution and the reference solution into the liquid chromatograph for determination.
[0165] 3. Investigation of the preparation method of the test solution
[0166] 3.1 Investigation of extraction solvent
[0167] Accurately weigh approximately 0.5g of the same batch of Sichuan pepper powder (batch number: S21, passed through a No. 3 sieve), place it in a stoppered conical flask, and accurately add 25ml each of methanol, 70% methanol, 50% methanol, ethanol, 70% ethanol, and 50% ethanol. Sonicate the mixture (300W power, 40kHz frequency) for 30 minutes, cool, replenish the lost weight with the appropriate solvent, shake well, filter, and collect the filtrate. Determine the chromatographic results according to the conditions specified in section "2.1". Figure 1 As shown in the figure. The results indicate 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 impact, and the extraction efficiency is relatively consistent. Therefore, methanol was selected as the extraction solvent.
[0168] 3.2 Examination of Extraction Methods
[0169] Take approximately 0.5g of the same batch of Sichuan pepper powder (batch number: S21, passed through a No. 3 sieve), accurately weigh it, and place it in a stoppered conical flask. Accurately add 25ml of methanol, and sonicate (300W, 40kHz) for 30 minutes, then reflux for 30 minutes. Cool, replenish the lost weight with methanol, shake well, filter, and collect the filtrate. Determine the chromatographic results according to the conditions specified in section "2.1". Figure 2 As shown in the figure. The results indicate that different extraction methods have little effect on the characteristic spectrum of Sichuan pepper. Considering ease of operation, ultrasonic treatment was chosen.
[0170] 3.3 Examination of extraction time
[0171] Accurately weigh approximately 0.5g of the same batch of Sichuan pepper powder (batch number: S21, passed through a No. 3 sieve), place it in a stoppered conical flask, and accurately add 25ml of methanol. Sonicate the flasks (300W power, 40kHz frequency) for 15 minutes, 30 minutes, and 60 minutes respectively. After cooling, replenish the lost weight with methanol, shake well, filter, and collect the filtrate. Determine the chromatographic results according to the conditions specified in section "2.1". Figure 3 As shown in the figure. The results indicate that different extraction times have little effect on the characteristic spectrum of Sichuan pepper (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 approximately 0.5 g of Sichuan pepper powder (batch number: S21, passed through a No. 3 sieve), prepare a test solution according to the method in section "2.3", and analyze it under the chromatographic conditions in section "2.1". Record the absorption spectrum in the range of 190–400 nm. Figure 4 As shown in the figure. The results show that by comparing the chromatograms of different detection wavelengths, it was found that the peak response and information content were richer and the interference was smaller at a wavelength of 240 nm, with better separation. Therefore, considering all factors, 240 nm was selected as the detection wavelength.
[0175] 4.2 Mobile phase optimization
[0176] Take approximately 0.5 g of Sichuan pepper powder (batch number: S21, passed through a No. 3 sieve) and prepare a test solution according to the method in section "2.3". Investigate the effect of different organic phase ratios, such as... Figure 6 As shown in the figure. The results indicate that acetonitrile-methanol (4:3) is better in terms of characteristic peak shapes and separation performance when used as the mobile phase. Considering all factors, acetonitrile-methanol (4:3) is selected as the organic phase.
[0177] 5. Methodological Examination
[0178] 5.1 Specificity Examination
[0179] Take approximately 0.5 g of Sichuan pepper (prickly ash) powder (batch number: S21, passed through a No. 3 sieve), prepare the test solution according to the method in section "2.3", accurately pipette 2 μl each of the test solution, the reference solution in section "2.2", and the blank solvent, and inject them for analysis according to the chromatographic conditions in section "2.1". Figure 7 As shown in the figure. The results indicate that the chromatogram of the test sample shows the same chromatographic peak at the corresponding retention time as that of the reference sample, and there is no interference from the blank solvent, indicating that the method has good specificity.
[0180] 5.2 Precision test
[0181] Take approximately 0.5 g of Sichuan pepper (batch number: S21, passed through a No. 3 sieve) powder and prepare the test solution according to the method in section "2.3". Inject the sample 6 times repeatedly under the chromatographic conditions in section "2.1". Using hyperoside as the reference peak, calculate the relative retention time and relative peak area of each characteristic peak and the S peak, and calculate the RSD value. The RSD values of the relative retention time of the 11 common peaks are in the range of 0.00% to 0.20%, and the RSD values of the relative peak area are in the range of 0.22% to 2.60%, indicating that the instrument precision is good.
[0182] 5.3 Reproducibility Test
[0183] Take approximately 0.5 g of Sichuan pepper (prickly ash) powder (batch number: S21, passed through a No. 3 sieve) and prepare 6 test solutions according to the method in section "2.3". Inject the solutions under the chromatographic conditions in section "2.1". Using hyperoside as the reference peak, calculate the relative retention time and relative peak area of each characteristic peak and the S peak, and calculate the RSD value. The RSD values of the relative retention time of the 11 common peaks are in the range of 0.00% to 0.15%, and the RSD values of the relative peak area are in the range of 0.40% to 2.66%, indicating that the method has good repeatability.
[0184] 5.4 Stability Test
[0185] Take approximately 0.5 g of Sichuan pepper (batch number: S21, passed through a No. 3 sieve) powder and prepare the test solution according to the method in section "2.3". Inject the sample at 0, 2, 4, 6, 12, 20, and 24 hours according to the chromatographic conditions in section "2.1". Using hyperoside as the reference peak, calculate the relative retention time and relative peak area of each characteristic peak and the S peak, and calculate the RSD value. The RSD values of the relative retention time of the 11 common peaks are in the range of 0.00% to 0.27%, and the RSD values of the relative peak area are 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 Examination
[0187] The method was performed by different analysts in different laboratories and on instruments of different brands. Approximately 0.5 g of Sichuan pepper (batch number: S21, passed through a No. 3 sieve) powder was used to prepare six test solutions according to the method described in section "2.3". These solutions were then injected and analyzed under the chromatographic conditions described in section "2.1". Using hyperoside as a reference peak, the relative retention times and relative peak areas of each characteristic peak and the S peak were calculated, and the RSD values were calculated. The RSD values of the relative retention times of the 11 common peaks ranged from 0.14% to 2.91%, and the RSD values of the relative peak areas ranged from 0.71% to 3.58%, indicating that the established method can be reproduced under different conditions and with different equipment and laboratories, demonstrating good intermediate precision.
[0188] 6. Establishment of the characteristic map of Sichuan pepper (Zanthoxylum bungeanum)
[0189] 6.1 Results of Characteristic Spectrum Determination of Sichuan Pepper
[0190] Twenty-one batches of Sichuan pepper (Zanthoxylum bungeanum) medicinal materials were collected and prepared according to the chromatographic conditions in section "2.1" and the test solution preparation method determined in section "2.3". Using hyperoside as a reference peak, the relative retention times of each characteristic peak and the S peak were calculated, and the RSD values were also calculated. The experimental results are shown in Table 3. Based on the established characteristic chromatogram analysis, the component types of Sichuan pepper (Zanthoxylum bungeanum) from different producing areas are basically consistent. The characteristic chromatograms of the 21 batches of Sichuan pepper (Zanthoxylum bungeanum) medicinal materials are shown in Table 3. Figure 8 .
[0191] Table 3. Characteristic chromatograms of 21 batches of Sichuan pepper (Zanthoxylum bungeanum) medicinal materials (relative retention time)
[0192]
[0193] 6.2 Establishment of shared patterns in the characteristic map of Sichuan pepper (Zanthoxylum bungeanum)
[0194] The reference material of Sichuan pepper (batch number: 121106-201906, China National Institutes for Food and Drug Control) and 21 batches of Sichuan pepper (Sichuan pepper) medicinal materials were sampled and analyzed according to the chromatographic conditions in section "2.1" and the test solution preparation method in section "2.3" above. The characteristic chromatogram of the reference material of Sichuan pepper (Sichuan pepper) was obtained. Figure 9 The software "Similarity Evaluation Software for Chromatographic Fingerprints of Traditional Chinese Medicine" was used to generate a reference chromatogram using the mean method, and a reference characteristic chromatogram of Sichuan pepper (Zanthoxylum bungeanum) was established. Figure 10 ).
[0195] The characteristic chromatogram of Sichuan pepper (Zanthoxylum bungeanum) should show 11 characteristic peaks, which should correspond to the retention times of the 11 characteristic peaks in the chromatogram of the reference medicinal material. Peaks 5, 10, and 11 should correspond to the retention times of the corresponding reference standard peaks, respectively. The peak corresponding to the hyperoside reference peak is the S peak. The relative retention times of peaks 1-4, 6-9 and S peak should be calculated. The relative retention times should be within ±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 origins of Sichuan pepper (Sichuan pepper, green pepper)
[0198] A comparative study was conducted using the medicinal characteristic spectra of two original sources: Sichuan pepper and green pepper. Figure 11 As shown in the figure. The results show that there are significant differences in the characteristic spectra between the two sources. Green pepper does not have the 11 characteristic peaks of Sichuan pepper, indicating that there are significant differences in the chemical composition between the two.
[0199] 6.3.2 Comparison of Counterfeit Product Feature Maps
[0200] Using the preparation method and characteristic chromatographic detection method for Sichuan pepper (Zanthoxylum bungeanum) medicinal materials, four batches of Zanthoxylum bungeanum and two batches of Zanthoxylum bungeanum were tested, and the results were compared with the characteristic chromatograms of Zanthoxylum bungeanum. The results are as follows: Figure 12As shown in the figure. The results show that the characteristic chromatograms of *Zanthoxylum bungeanum* and *Zanthoxylum bungeanum* samples are significantly different. *Zanthoxylum bungeanum* does not have the 11 characteristic peaks of *Zanthoxylum bungeanum* samples, and the characteristic chromatogram of *Zanthoxylum bungeanum* has a chromatographic peak with a high response at 25 minutes. The characteristic chromatograms of *Zanthoxylum bungeanum* are more consistent with those of *Zanthoxylum bungeanum*, but *Zanthoxylum bungeanum* lacks characteristic peak 7. Therefore, the characteristic chromatogram method established in this invention can effectively distinguish *Zanthoxylum bungeanum* adulterants.
[0201] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above 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 embodiments described above are merely illustrative of several implementations of the present invention, designed to facilitate a detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this invention patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.
Claims
1. A method for constructing a UPLC feature map of Sichuan pepper medicinal material, characterized in that, Includes the following steps: Take hyperoside, hydroxy-α-sanshool, and hydroxy-β-sanshool to prepare a mixed reference solution; Preparation of the test solution: Crush the pepper sample, sieve it, mix it with a second alcohol-containing aqueous solution of 70% to 100% by volume, perform ultrasonic extraction, filter it, and take the filtrate; the alcohol in the second alcohol-containing aqueous solution is selected from methanol or ethanol. The mixed reference solution and the test solution were analyzed by ultra-high performance liquid chromatography (UPLC) to construct the characteristic UPLC chromatogram of the Sichuan pepper medicinal material. The conditions for ultra-high performance liquid chromatography analysis include: using a YMC Triart C18 column with the following specifications: column length of 150 mm, inner diameter of 2.1 mm, particle size of packing material of 1.9 μm, mobile phase A being a mixed solution of acetonitrile and methanol, mobile phase B being an aqueous solution of phosphoric acid, gradient elution, and detection wavelength of 240 nm. The gradient elution procedure is as follows: From 0 min to 3 min, the volume percentage of mobile phase A changed from 17% to 23%; Between 3 and 13 minutes, the volume percentage of mobile phase A changed from 23% to 30%. From 13 to 18 minutes, the volume percentage of mobile phase A was 30%. Between 18 and 23 minutes, the volume percentage of mobile phase A changed from 30% to 40%. Between 23 and 38 minutes, the volume percentage of mobile phase A changed from 40% to 75%. From 38 to 40 minutes, the volume percentage of mobile phase A is 75%. In the mixed solution of acetonitrile and methanol, the volume ratio of acetonitrile to methanol is 4:3; in the aqueous solution of phosphoric acid, the volume fraction of phosphoric acid is 0.05% to 0.15%. The UPLC characteristic spectrum of the Sichuan pepper medicinal material includes 11 characteristic peaks, of which peak 5 is hyperoside, peak 6 is isoquercitrin, peak 8 is narcissin, peak 9 is hydroxy-ε-salicornin, peak 10 is hydroxy-α-salicornin, and peak 11 is hydroxy-β-salicornin. The peak corresponding to the hyperoside reference peak is the S peak. The relative retention time of each characteristic peak and the S peak is within ±10% of the specified value, which 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.
2. The method for constructing the UPLC feature map of Sichuan pepper medicinal material according to claim 1, characterized in that, The chromatographic conditions for the ultra-high performance liquid chromatography include a flow rate of 0.28 mL / min to 0.32 mL / min.
3. The method for constructing the UPLC feature map of Sichuan pepper medicinal material according to claim 1, characterized in that, The chromatographic conditions for the ultra-high performance liquid chromatography include a column temperature of 28℃ to 32℃.
4. The method for constructing the UPLC feature map of Sichuan pepper medicinal material according to any one of claims 1 to 3, characterized in that, The preparation of the mixed reference solution includes the following steps: Hypericin, hydroxy-α-sanshool, and hydroxy-β-sanshool are mixed with 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.
5. The method for constructing the UPLC feature map of Sichuan pepper medicinal material according to claim 4, 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.
6. The method for constructing the UPLC feature map of Sichuan pepper medicinal material according to any one of claims 1 to 3 and 5, 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.