A method for constructing a characteristic spectrum of pepper and a thin-layer identification method

Abstract

This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for constructing a characteristic spectrum of pepper and a thin-layer chromatography identification method. The method includes the preparation of a test solution, employing a specific gradient elution procedure, and determination. This method lays a solid foundation for the quality stability of pepper products, enables the establishment of feasible quality standards, and achieves effective quality control of pepper. The characteristic spectrum obtained by this method shows good separation between characteristic peaks, good response values ​​of the characteristic peaks, and can effectively identify the active ingredients in pepper, making the method more accurate and reliable. This invention is applicable to both white and black pepper.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for constructing a characteristic spectrum of pepper and a thin-layer chromatography identification method. Background Technology

[0002] Pepper, a perennial woody vine belonging to the genus Piper in the family Piperaceae, is defined in the 2020 edition of the Chinese Pharmacopoeia as the dried, nearly mature or mature fruit of Piper nigrum L. Harvested in late autumn to the following spring when the fruit is dark green, it is sun-dried to produce black pepper; harvested when the fruit turns red, it is soaked in water for several days, the pulp is rubbed off, and then sun-dried to produce white pepper. Current research on pepper is limited, resulting in extremely poor separation of characteristic peaks in the obtained spectra, making peak identification difficult and thus unable to achieve the purpose of assessing the quality of the medicinal material. Summary of the Invention

[0003] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the prior art, such as the difficulty in judging the quality of pepper medicinal materials and the poor separation of characteristic peaks in the obtained pepper characteristic spectrum, so as to provide a method for constructing pepper characteristic spectrum and thin-layer identification method.

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

[0005] The first aspect of this invention provides a method for constructing a pepper feature map, comprising the following steps:

[0006] Preparation of test solution: Take the test sample and prepare a test solution;

[0007] Determination: Measured by high performance liquid chromatography;

[0008] Chromatographic conditions included: acetonitrile as mobile phase A, water as mobile phase B, gradient elution, with the following program: 0-5 min, 20% → 30% mobile phase A, 80% → 70% mobile phase B; 5-6 min, 30% → 40% mobile phase A, 70% → 60% mobile phase B; 6-20 min, 40% mobile phase A, 60% mobile phase B; 20-30 min, 40% → 45% mobile phase A, 60% → 55% mobile phase B.

[0009] The chromatographic conditions also include: a column temperature of 33-37℃; and / or a flow rate of 0.28-0.32 ml / min; and / or a detection wavelength of 285 nm for 0-7 min and 380 nm for 7-30 min; and / or the use of octadecylsilane-bonded silica gel as the packing material; and / or an injection volume of 2-5 μL.

[0010] The chromatographic conditions are as follows: column temperature 35℃; and / or flow rate 0.3 ml / min; and / or theoretical plate number calculated based on piperine peak should be no less than 5000; and / or injection volume 2 μL.

[0011] The pepper is black pepper and / or white pepper;

[0012] Preferably, the test sample is at least one of pepper medicinal material, pepper slices, pepper decoction, pepper formula granules, and pepper standard decoction freeze-dried powder.

[0013] The preparation method of the test sample solution includes: taking the test sample, adding solvent, extracting, and filtering;

[0014] Preferably, the solvent is an organic solvent;

[0015] Preferably, the extraction solvent is a 30-70% aqueous methanol solution or an aqueous ethanol solution;

[0016] Preferably, the ratio of the mass (g) of the test sample to the volume (ml) of the solvent is (0.1-0.5):(10-50).

[0017] The construction method also includes the preparation of a reference solution;

[0018] Preferably, the method for preparing the reference solution includes: taking the reference medicinal material, extracting it with water to obtain an aqueous extract; adding a solvent to the aqueous extract, and then performing a second extraction and filtration;

[0019] Preferably, when preparing the reference solution, the ratio of the mass (g) of the water extract to the volume (ml) of the extraction solvent is (0.4-1.0):(20-100).

[0020] The feature map obtained by the construction method includes 6 feature peaks;

[0021] With peak 3 as the reference peak, the specified values ​​for the relative retention times of peaks 1, 2, 4, 5, and 6 are 0.40, 0.78, 1.50, 1.60, and 1.68, respectively.

[0022] The relative retention time of the characteristic peak is within ±10% of the specified value.

[0023] A second aspect of the present invention provides a thin-layer chromatography method for identifying pepper, comprising the following steps:

[0024] Preparation of the test solution;

[0025] Preparation of reference solution;

[0026] Take the test solution, spot it, develop it using a mixture of chloroform, ethyl acetate and acetone as the developing solvent, remove it and examine it.

[0027] Furthermore, the volume ratio of the trichloromethane, ethyl acetate and acetone is (5-10):(1-5):(1-3).

[0028] The volume ratio of chloroform, ethyl acetate and acetone is 7:2:1.

[0029] The technical solution of this invention has the following advantages:

[0030] 1. The present invention provides a method for constructing a characteristic spectrum of pepper, comprising the preparation of a test solution, employing a specific gradient elution procedure, and determination. This method lays a solid foundation for the quality stability of pepper products, enables the establishment of feasible quality standards, and achieves effective control of pepper quality. The characteristic spectrum obtained by this method exhibits good separation between characteristic peaks, good response values ​​of characteristic peaks, and can effectively identify the active ingredients in pepper, making the method more accurate and reliable. This invention is applicable to white pepper and black pepper.

[0031] 2. The pepper thin-layer identification method provided by the present invention uses piperine as a positive control for identification. The Rf value of the spots is moderate, the spots are clear, and the separation is good. Attached Figure Description

[0032] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 This is the chromatogram of the reference medicinal material; Figure 2 It is a diagram of freeze-dried powder of standard black pepper broth; Figure 3 This is the chromatogram obtained from comparative experiments 1-7 in Experimental Example 1 of this invention; Figure 4 These are chromatograms obtained at different wavelengths in Experiment Example 1 of this invention; Figure 5 These are the spectra of each characteristic peak in Experiment Example 1 of this invention under different ultraviolet absorption light; Figure 6 These are the spectra obtained at different column temperatures during the durability test of Experiment Example 2 of this invention; Figure 7 These are the spectra obtained at different flow rates during the durability test of Experiment Example 2 of this invention; Figure 8 These are the spectra obtained from different chromatographic columns during the robustness test of Experimental Example 2 of this invention; Figure 9These are the spectra obtained by different chromatographs during the durability test of Experiment Example 2 of this invention; Figure 10 This is a control spectrum obtained from black pepper medicinal materials, black pepper slices, and freeze-dried powder of black pepper standard decoction in Experiment 3 of this invention; Figure 11 This is a chromatogram of different batches of freeze-dried white pepper standard decoction powder from Example 3 of the present invention; Figure 12 This is a thin-layer chromatography identification spectrum of the freeze-dried powder of black pepper standard decoction in Example 4 of the present invention. Detailed Implementation

[0034] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0035] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0036] instrument:

[0037] Chromatograph 1: Waters UHPLC; Chromatograph 2: Agilent UHPLC; Chromatograph 3: Thermo Fisher UHPLC. Column 1: Agilent Eclipse Plus C18 (2.1×100mm, 1.8μm); Column 2: Agilent Extend C18 (2.1×100mm, 1.8μm); Column 3: Waters ACQUITY CSH C18 (2.1×100mm, 1.7μm); Column 4: Agilent SB C18 (2.1×100mm, 1.8μm). Little Bear Decoction Pot (Mechanical Type 500W) - Clay Pot Decoction Pot JYH-C30A1, Little Bear Electric Appliances Co., Ltd. Silicone G Prefabricated Plate: Qingdao Ocean Chemical Plant Branch; Silicone G Prefabricated Plate: Yantai Dexin Biotechnology Co., Ltd. ZF-20D Dark Box Ultraviolet Analyzer: Shanghai Baoshan Gucun Electro-Optical Instrument Factory. TH-II type thin-layer heater: Shanghai Kezhe Biochemical Technology Co., Ltd. Quantitative capillary tubes: Hirschmann; 1μL, 3μL, 5μL.

[0038] Medicine:

[0039] Piperine reference standard, purity ≥98.9%, batch number: 110775-201706, China National Institutes for Food and Drug Control; Pepper reference material (black pepper), batch number: 121440-200401, China National Institutes for Food and Drug Control; Methanol (chromatographic grade, 34860-4L-R, Sigma-Aldrich), Methanol (analytical grade, 20211008, Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.), Anhydrous ethanol (AR, 20211001420, Tianjin Zhiyuan Chemical Reagent Co., Ltd.); Acetonitrile (chromatographic grade, 34851-4, Sigma-Aldrich).

[0040] The batch numbers of the 18 batches of black pepper slices are: 2103001Y, 2103002Y, 2103003Y, 2103004Y, 2103005Y, 2103006Y, 2103007Y, 2103008Y, 2103009Y, 2103010Y, 2103011Y, 2103012Y, 2103013Y, 2103014Y, 2103015Y, 220701Y, 220702Y, and 220703Y; respectively designated as Y1-Y18; where Y1-Y The origin of the processed black pepper slices is Wenchang City, Hainan Province; Y4-Y6 are from Qionghai City, Hainan Province; Y7-Y9 are from Gaozhou City, Guangdong Province; Y10-Y12 are from Tengchong County, Yunnan Province; Y13-Y15 are from Lüchun County, Yunnan Province; and Y16-Y18 are from Qionghai City, Hainan Province. S1-S18 are the batch numbers of the freeze-dried black pepper standard decoction powder made from batches Y1-Y18 of black pepper processed black pepper slices.

[0041] Example 1

[0042] This embodiment provides a method for constructing a black pepper feature map, including the following steps:

[0043] Preparation of the test solution: Weigh approximately 0.1 g of lyophilized black pepper standard decoction powder accurately, place it in a stoppered conical flask, add 20 ml of 50% methanol accurately, sonicate (power 250 W, frequency 40 kHz) for 30 min, remove, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0044] Preparation of reference solution: Take 0.6g of pepper (black pepper) reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 30 minutes, remove, cool, filter, evaporate the filtrate to dryness, add 20ml of 50% methanol to the residue, sonicate (power 250W, frequency 40kHz) for 30 minutes, remove, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0045] Preparation of reference solution: Weigh an appropriate amount of piperine reference standard accurately, place it in a brown volumetric flask, and add 50% methanol to prepare a solution containing 30 μg of reference standard per 1 ml.

[0046] Determination: The determination was performed using high-performance liquid chromatography (HPLC), with octadecylsilane-bonded silica gel as the packing material (specifications: column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A and water as mobile phase B; gradient elution; column temperature 35℃; flow rate 0.3 ml / min; detection wavelength 285 nm from 0 to 7 min, 380 nm from 7 min to the end; the theoretical plate number, calculated based on the piperine peak, should not be less than 5000; gradient elution program: 0-5 min, 20% → 30% mobile phase A, 80% → 70% mobile phase B; 5-6 min, 30% → 40% mobile phase A, 70% → 60% mobile phase B; 6-20 min, 40% mobile phase A, 60% mobile phase B; 20-30 min, 40% → 45% mobile phase A, 60% → 55% mobile phase B.

[0047] Accurately pipette 2 μL each of the test solution, reference solution, and standard solution, inject them into the liquid chromatograph, and determine the result.

[0048] Identification and designation of characteristic peaks

[0049] Eighteen batches of lyophilized black pepper standard decoction powder were tested using the method described above. The retention time and peak area of ​​the 18 batches of lyophilized black pepper standard decoction powder are shown in the table below. The fingerprint chromatogram similarity evaluation software "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" compiled by the Pharmacopoeia Commission was used to generate a reference characteristic chromatogram.

[0050] Peak matching was performed on 18 batches of lyophilized black pepper standard decoction powder. Common peaks were found in the characteristic chromatograms of the test solutions from these 18 batches. Based on the characteristic peaks of the reference herb, the separation of each peak, and the response values, six characteristic peaks were selected. These were then compared with the reference standard. Peak identification and assignment were performed using UPLC and LC / MS / MS. Peak number 3 was identified as piperine. The chromatograms of the 18 batches of lyophilized black pepper standard decoction powder were compared with those of the reference herb (chromatograms are shown in...). Figure 1 Corresponding to the solution, using peak 3 as the reference peak, the relative retention times and relative peak areas of the six characteristic peaks in the characteristic spectrum relative to peak S were statistically analyzed. The relative retention times of each characteristic peak with peak S were calculated. The specified values ​​for the relative retention times of peaks 1, 2, 4, 5, and 6 were 0.40, 0.78, 1.50, 1.60, and 1.68, respectively, all within the range of ±10% of the mean. The spectrum of the freeze-dried powder of the black pepper standard decoction is shown below. Figure 2 .

[0051] Table 1. Relative retention times of characteristic chromatograms of 18 batches of black pepper slices in standard decoction (freeze-dried powder).

[0052]

[0053]

[0054] Table 2. Relative peak areas of characteristic spectra of 18 batches of black pepper slices in standard decoction (freeze-dried powder)

[0055]

[0056]

[0057] Black pepper samples were analyzed using ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-TOF / MS). Based on the multi-stage mass spectrometry information of the samples, combined with the natural product high-resolution mass spectrometry database and relevant literature, the target peaks were identified. Using reference standards for localization and spectral studies, peak 1 was confirmed to be N-trans-feruloyltyramine, peak 2 to piperine, peak 3 to piperine, and peak 4 to piperine. The specific localization chromatograms of the LC / MS / MS analyses are shown in the table below.

[0058] Table 3 Mass Spectrometry Analysis of Black Pepper

[0059]

[0060] Investigation of chromatographic conditions in Experiment Example 1

[0061] (1) This experimental example investigated chromatographic conditions, providing 7 sets of comparative experiments with chromatographic conditions as the variable. The conditions for each comparative experiment are as follows, and the results are shown in [the table below]. Figure 3 The rest is the same as in Example 1.

[0062] Comparative Experiment 1: An Agilent SB C18 column (2.1 mm × 100 mm, 1.8 μm) was used; the column temperature was 30℃, the flow rate was 0.2 ml / min, the detection wavelength was 333 nm, and gradient elution was performed using the following program:

[0063] Time (min) Mobile phase A: Acetonitrile (%) Mobile phase B (water) (%) 0～5 23→30 77→70 5～7 30→39 70→61 7～10 39→40 61→60 10～20 40→42 60→58 20～35 42→43 58→57

[0064] Comparative Experiment 2: An Agilent Extend C18 column (2.1 mm × 100 mm, 1.8 μm) was used; the column temperature was 30℃, the flow rate was 0.2 ml / min, the detection wavelength was 333 nm, and gradient elution was performed using the following program:

[0065] Time (min) Mobile phase A: Acetonitrile (%) Mobile phase B (water) (%) 0～5 20→30 80→70 5～7 30→35 70→65 7～10 35→38 65→62 10～20 38→41 62→59 20～35 41→42 59→58

[0066] Comparative Experiment 3: An Agilent Extend C18 column (2.1 mm × 100 mm, 1.8 μm) was used; the column temperature was 35℃, the flow rate was 0.2 ml / min, the detection wavelength was 285 nm before 7 min and 380 nm after 7 min, with gradient elution and the following program:

[0067] Time (min) Mobile phase A: Acetonitrile (%) Mobile phase B (water) (%) 0～5 22→30 78→70 5～6 30→36 70→64 6～20 36→37 64→63 20～25 37→41 63→59 25～40 41→42 59→58

[0068] Comparative Experiment 4: An Agilent Extend C18 column (2.1 mm × 100 mm, 1.8 μm) was used; the column temperature was 35℃, the flow rate was 0.3 ml / min, and the detection wavelength was 285 nm before 7 min and 380 nm after 7 min, with gradient elution and the following program:

[0069] Time (min) Mobile phase A: Acetonitrile (%) Mobile phase B (water) (%) 0～5 20→30 80→70 5～6 30→40 70→60 6～15 40 60 15～20 40→45 60→55

[0070] Comparative Experiment 5: An Agilent Extend C18 column (2.1 mm × 100 mm, 1.8 μm) was used; the column temperature was 35℃, the flow rate was 0.3 ml / min, and the detection wavelength was 285 nm before 7 min and 380 nm after 7 min, with gradient elution. The program was as follows:

[0071] Time (min) Mobile phase A: Acetonitrile (%) Mobile phase B (water) (%) 0～5 20→30 80→70 5～6 30→40 70→60 6～20 40 60 20～30 40→45 60→55

[0072] Comparative Experiment 6: A Waters CSH C18 column with dimensions of 2.1 mm × 100 mm and a diameter of 1.7 μm was used. The column temperature was 35 ℃, the flow rate was 0.3 ml / min, the detection wavelength was 285 nm before 7 min and 380 nm after 7 min, and gradient elution was performed. The gradient elution program was the same as in Comparative Experiment 5.

[0073] Comparative Experiment 7: An Eclipse plus C18 column with dimensions of 2.1 mm × 100 mm and 1.8 μm was used. The column temperature was 35 ℃, the flow rate was 0.3 ml / min, the detection wavelength was 285 nm before 7 min and 380 nm after 7 min, and gradient elution was performed. The gradient elution program was the same as in Comparative Experiment 5.

[0074] The results above show that the separation of chromatographic peaks in the chromatogram obtained in Comparative Experiment 1 was poor after 30 min. The separation between chromatographic peaks obtained in Comparative Experiment 2 needs further optimization. The results of Comparative Experiment 3 show that each peak has significant absorption at 285 nm and 380 nm. Based on this, further optimization was carried out in Comparative Experiments 4 and 5. Comparing these two sets of experiments, it can be seen that the characteristic chromatogram obtained in Comparative Experiment 5 is better. Based on Comparative Experiment 5, the chromatographic column was adjusted, and Comparative Experiments 6 and 7 were carried out. The results show that the separation of each chromatographic peak in the chromatogram obtained in Comparative Experiment 7 is optimal. Therefore, the final chromatographic conditions are determined as follows: the elution program in Comparative Experiment 5 is optimized, the absorption wavelength is 285 nm before 7 min and 380 nm after 7 min, the flow rate is 0.3 ml / min, and the column temperature is 35 ℃.

[0075] (2) Examination of wavelength

[0076] Using the same batch of lyophilized black pepper standard decoction powder, and with the detection wavelength as a variable, test solutions were prepared and measured according to Example 1 to obtain characteristic spectra at different wavelengths, see [reference needed]. Figure 4 The wavelengths are 220 nm, 285 nm, 320 nm, and 380 nm, respectively. The characteristic peaks are then compared with the spectra of different ultraviolet absorption wavelengths (see...). Figure 5 The results showed that the maximum absorption was 285 nm in the first 7 minutes and 380 nm after 7 minutes. Therefore, the preferred detection wavelength is 285 nm before 7 minutes and 380 nm after 7 minutes.

[0077] Methodological Investigation of Experiment Example 2

[0078] 2.1 Precision Experiment

[0079] The same batch of lyophilized black pepper standard decoction powder was used to prepare the test sample according to Example 1. The sample was injected six times consecutively, and the chromatograms were recorded. Using piperine (peak 3) as the reference peak, the relative retention times and peak areas of the other peaks were calculated. The results are shown in the table below. The results indicate that the RSD of the relative retention time and relative peak area of ​​each chromatographic peak is less than 1.0%, indicating good instrument precision.

[0080] Table 4. Results of relative retention times of chromatographic peaks in the instrument precision experiment.

[0081]

[0082] Table 5. Results of relative peak areas in the instrument precision experiment.

[0083]

[0084]

[0085] 2.2 Intermediate Precision Experiments by Different Operators

[0086] The same batch of black pepper standard decoction freeze-dried powder was used, and three different inspectors prepared test solutions at different times according to Example 1. The relative retention time and relative peak area of ​​each common peak were measured using the same equipment. The results showed that the RSD of the relative retention time of each characteristic peak was small, and the relative peak areas of some characteristic peaks were close. Overall, the method has good intermediate precision.

[0087] Table 6. Experimental results of intermediate precision relative retention time

[0088]

[0089] Table 7. Experimental results of intermediate precision relative peak area

[0090]

[0091] 2.3 Repeatability Experiment

[0092] Six test solutions were prepared using the same batch of black pepper standard decoction freeze-dried powder, following Example 1. The results are shown in the table below. The results indicate that the method of the present invention has good repeatability.

[0093] Table 8 Repeatability Experiments - Relative Retention Time

[0094]

[0095]

[0096] Table 9 Repeatability Experiments - Relative Peak Area

[0097]

[0098] 2.4 Stability Test

[0099] The same batch of lyophilized black pepper standard decoction powder was used to prepare test solutions according to Example 1. Samples were injected at 0, 4, 8, 12, 16, and 24 hours for analysis. The results showed that the relative retention time RSD of each common peak was less than 1.0%, and the average relative peak area of ​​each characteristic peak was less than 6.2%, indicating that the test solution was stable within 24 hours.

[0100] Table 10 Stability Experiment - Relative Retention Time

[0101]

[0102]

[0103] Table 11 Stability Experiment - Relative Peak Area

[0104]

[0105] 2.5 Durability Test

[0106] (1) Different column temperatures

[0107] Approximately 0.1 g of the same batch of black pepper standard decoction freeze-dried powder was accurately weighed. Using column temperature as a variable, the column temperatures were 33℃, 35℃, and 37℃. The test solution was prepared and measured according to Example 1. The results are shown in the table below. Figure 6 The results showed that different column temperatures had little effect on the relative retention time of chromatographic peaks, indicating that the method has good robustness.

[0108] Table 12 Relative Retention Times at Different Column Temperatures

[0109]

[0110] Table 13 Relative Peak Areas at Different Column Temperatures

[0111]

[0112]

[0113] (2) Different flow velocities

[0114] Approximately 0.1 g of the same batch of lyophilized black pepper standard decoction powder was accurately weighed. The flow rate was varied to 0.28 mL / min, 0.30 mL / min, and 0.32 mL / min. The test solution was prepared according to Example 1 and the results are shown in the table below. Figure 7 The results showed that different flow rates had little effect on the relative retention time and relative peak area of ​​the chromatographic peaks, indicating that the method of the present invention has good robustness.

[0115] Table 14 Relative Retention Time in Experiments at Different Flow Rates

[0116]

[0117] Table 15 Relative Peak Area in Experiments at Different Flow Rates

[0118]

[0119] (3) Different chromatographic columns

[0120] Approximately 0.1 g of the same batch of lyophilized black pepper standard decoction powder was accurately weighed. Using the chromatographic column as the variable (columns 1-3 being Agilent Eclipse Plus C18, Agilent Extend C18, and Waters CSH-C18 respectively), the test solution was prepared and measured according to Example 1. The results are shown in the table below. Figure 8Considering the influence of different chromatographic columns on the relative peak area of ​​peak 1, column 1 is preferred: Agilent Eclipse Plus.

[0121] Table 16 Relative Retention Times in Experiments with Different Chromatographic Columns

[0122]

[0123]

[0124] Table 17 Relative Peak Areas in Experiments with Different Chromatographic Columns

[0125]

[0126] (4) Different chromatographs

[0127] Approximately 0.1 g of the same batch of freeze-dried black pepper standard decoction powder was accurately weighed. Using the chromatograph as the variable (chromatographs 1-3 were Waters, Agilent, and Thermo Fisher, respectively), the test solution was prepared and measured according to Example 1. The results are shown in the table below. Figure 9 The results showed that different chromatographs had little effect on the relative retention time of the chromatographic peaks, and the method was generally reliable across different instruments.

[0128] Table 18 Relative Retention Times in Experiments Using Different Chromatographs

[0129]

[0130] Table 19 Relative Peak Areas in Experiments Using Different Chromatographs

[0131]

[0132] Experimental Example 3 examines the chromatograms obtained from different formulations.

[0133] Eighteen batches of black pepper medicinal materials, eighteen batches of black pepper slices, and eighteen batches of black pepper standard decoction freeze-dried powder were taken. Test solutions were prepared and measured according to Example 1. The fitted characteristic spectra were obtained, as shown in the table below. Figure 10 , Figure 10 S1 in the diagram represents the reference chromatogram obtained from raw black pepper, S2 represents the reference chromatogram obtained from processed black pepper slices, and S3 represents the reference chromatogram obtained from freeze-dried standard black pepper decoction powder. Comparison of the characteristic chromatograms obtained from different formulations shows that the number of characteristic peaks varies little among the different formulations. The process of processing raw black pepper into slices and then decocting it in water to produce freeze-dried powder does not significantly alter the main chemical components of black pepper in different dosage forms, resulting in good transmissibility of values. The method of this invention is applicable to different dosage forms of black pepper, such as freeze-dried standard black pepper decoction powder, raw black pepper, processed black pepper slices, and black pepper granules.

[0134] Table 20: Comparison chromatograms of relative retention times for black pepper medicinal materials, black pepper slices, and freeze-dried powder of standard black pepper decoction.

[0135]

[0136] Table 21. Relative peak areas of comparative chromatograms of black pepper medicinal materials, black pepper slices, and freeze-dried powder of standard black pepper decoction.

[0137]

[0138] Example 2

[0139] This embodiment provides a method for preparing freeze-dried powder of standard black pepper soup stock, including the following steps:

[0140] Take black pepper medicinal materials and process them to remove impurities to make black pepper slices. Take about 200g of black pepper slices, crush them to a crushing degree of not less than 90%, put them in a clay pot, add 8 times the amount of water and soak for 30 minutes. First, bring to a boil over high heat (500W), then simmer over low heat (300W) for 30 minutes. Filter while hot through a 200-mesh filter cloth. Add 6 times the amount of water to the dregs, bring to a boil over high heat (500W), then simmer over low heat (300W) for 25 minutes. Filter while hot through a 200-mesh filter cloth. Combine the filtrates and concentrate them under reduced pressure (65℃) to a concentrated extract with a material-to-liquid ratio of about 1:4. Place the extract in a freeze dryer and freeze dry (-40℃, 110kPa) until dry. Grind it into powder to obtain the freeze-dried powder of standard black pepper decoction.

[0141] Eighteen batches of black pepper slices (Y1-Y18) were processed into 18 batches of standard black pepper decoction freeze-dried powder (batch numbers S1-S18) using the method described above. Two parallel samples of standard decoction freeze-dried powder were prepared from each batch of slices. The average yield was calculated, and the yield range of black pepper was specified according to the requirements of the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules". The yield of each batch of slices and the specified yield range are shown in the table below:

[0142] Table 22. Extract yield from different batches of medicinal slices.

[0143]

[0144] The above results indicate that the measured yield of extract was between 10.4% and 16.8%, with an average yield of 14.0%, a SD of 1.4%, and a mean fluctuation range of 9.8% to 18.2% within ±30%. The specified yield range for pepper slices in the preparation of standard decoctions is 10% to 17%, which meets the requirements of the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules".

[0145] The characteristics of the freeze-dried powder of black pepper standard decoction prepared by the above method are as follows: This product is a yellow-brown to brown powder; it has a slightly fragrant odor and a spicy taste; the yield is 10%-17% for the standard decoction of black pepper slices (freeze-dried powder); the solubility is tested according to the solubility test method (General Chapter 0104 of Chinese Pharmacopoeia 2020 edition), add 200ml of hot water, stir for 5 minutes (heat to boiling if necessary), and observe immediately. It should be completely dissolved or slightly turbid, and there should be no charred residue; the extract is taken as follows: take an appropriate amount of this product, grind it into a fine powder, take about 2g, accurately weigh it, place it in a stoppered conical flask, accurately add 100ml of ethanol, and determine it according to the hot extraction method under the alcohol-soluble extract determination method (General Chapter 2201 of Chinese Pharmacopoeia 2020 edition). It should not be less than 6.0%; each 1g of this product should contain 12.0mg-30.0mg of piperine (C17H19NO3).

[0146] This embodiment also provides a method for determining the content of active ingredients in black pepper slices, including:

[0147] Pepper contains a variety of components, mainly including amide alkaloids, volatile oils, organic acids, lignans, phenols, and trace elements. Among these, alkaloids and volatile oils are the most important active ingredients, with piperine being the most abundant and widely active among the alkaloids. The Chinese Pharmacopoeia uses piperine as the content indicator for pepper medicinal materials, and it is tentatively set as the content indicator for pepper medicinal materials and standard decoction freeze-dried powder.

[0148] Eighteen batches of lyophilized pepper standard decoction powder samples were collected (two parallel samples for each batch). The piperine content in each batch of lyophilized standard decoction powder was determined and the transfer rate was calculated according to the method and chromatographic conditions provided in Example 1. The transfer rate range of piperine content in the lyophilized standard decoction powder was specified in accordance with the requirements of the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules". The piperine content and transfer rate of each batch of lyophilized standard decoction powder, as well as the specified range, are shown in the table below.

[0149] Table 23. Transfer rate and range of content in freeze-dried powder of 18 batches of standard decoction pieces.

[0150]

[0151]

[0152] Conclusion: As shown in the table above, the measured average transfer rate of piperine content in the freeze-dried powder of standard decoctions in each batch ranged from 3.5% to 7.9%, with a mean of 5.5% and a SD of 1.4%. The fluctuation range of the mean ± 30% was 3.9% to 7.2%. The specified transfer rate range of piperine content in freeze-dried powder of standard pepper decoctions is 4.0% to 8.0%, which meets the requirements of the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules". The mean piperine content of the freeze-dried powder of standard pepper decoctions was 17.8 mg / g, with a minimum of 11 mg / g and a maximum of 30.2 mg / g.

[0153] Example 3

[0154] This embodiment provides a method for constructing a feature map of white pepper, including the following steps:

[0155] Preparation of the test solution: Weigh approximately 0.1 g of lyophilized white pepper standard decoction powder accurately, place it in a stoppered conical flask, add 20 ml of 50% methanol accurately, sonicate (power 250 W, frequency 40 kHz) for 30 min, remove, cool, shake well, filter, and collect the filtrate to obtain the test solution.

[0156] Preparation of reference solution: Take 0.6g of pepper (white pepper) reference material, place it in a stoppered conical flask, add 50ml of water, heat under reflux for 30 minutes, remove, cool, filter, evaporate the filtrate to dryness, add 20ml of 50% methanol to the residue, sonicate (power 250W, frequency 40kHz) for 30 minutes, remove, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0157] Preparation of reference solution: Weigh an appropriate amount of piperine reference standard accurately, place it in a brown volumetric flask, and add 50% methanol to prepare a solution containing 30 μg of reference standard per 1 ml.

[0158] Determination: The determination was performed using high-performance liquid chromatography (HPLC), with octadecylsilane-bonded silica gel as the packing material (specifications: column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A and water as mobile phase B; gradient elution; column temperature 35℃; flow rate 0.3 ml / min; detection wavelength 285 nm from 0 to 7 min, 380 nm from 7 min to the end; the theoretical plate number, calculated based on the piperine peak, should not be less than 5000; gradient elution program: 0-5 min, 20% → 30% mobile phase A, 80% → 70% mobile phase B; 5-6 min, 30% → 40% mobile phase A, 70% → 60% mobile phase B; 6-20 min, 40% mobile phase A, 60% mobile phase B; 20-30 min, 40% → 45% mobile phase A, 60% → 55% mobile phase B.

[0159] Accurately pipette 2 μL each of the test solution, reference solution, and standard solution into the liquid chromatograph and determine the concentration. Fifteen batches of lyophilized white pepper standard decoction powder were analyzed using the above method, and the resulting chromatograms are shown in the table below. Figure 11 The characteristic spectra of 15 batches of standard decoction lyophilized powder test solutions were matched to identify common peaks. The spectra of the 15 batches of standard decoction lyophilized powder were compared with those of the reference medicinal material solution, using peak 3 as the reference peak. The relative retention times and relative peak areas of the six characteristic peaks in the characteristic spectra relative to peak S were statistically analyzed, and the relative retention times of each characteristic peak and peak S were calculated. The experimental results indicate that the method of this invention is applicable to white pepper.

[0160] Table 24. Relative retention times of characteristic chromatograms of 15 batches of white pepper slices in standard decoction (freeze-dried powder).

[0161]

[0162]

[0163] Table 25. Relative peak areas of characteristic spectra of 15 batches of white pepper slices in standard decoction (freeze-dried powder)

[0164]

[0165] Example 4

[0166] This embodiment provides a thin-layer chromatography method for identifying freeze-dried powder of black pepper standard soup concentrate, including the following steps:

[0167] Preparation of test solution: Take 0.5g of 18 batches of black pepper freeze-dried powder (S1-S18), add 15ml of anhydrous ethanol, sonicate for 30min, filter, and obtain the filtrate, which is the test solution corresponding to the 18 batches of freeze-dried powder.

[0168] Preparation of reference solution: Take piperine reference standard, add anhydrous ethanol, and prepare a solution containing 4 mg of reference standard per 1 ml.

[0169] Take 5 μL of the test solution and 1 μL of the reference solution, and spot them separately on the same silica gel G thin-layer plate. Use a mixture of chloroform, ethyl acetate, and acetone (v / v) as the developing solvent. Develop, remove, and air-dry. Spray with 10% sulfuric acid ethanol solution and heat until the spots are clearly visible. Examine under a UV lamp (365 nm). The results are shown in the figure. Figure 12 The figures from left to right correspond to the chromatograms of D1-D18 and the piperine reference standard, respectively. The results show that the chromatogram of the test sample and the piperine reference standard show the same light yellow fluorescent spots at the same positions.

[0170] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a pepper feature map, characterized in that, Includes the following steps: Preparation of the test solution: Take the test sample and prepare a test solution; when preparing the test solution, the extraction solvent is a 30-70% methanol aqueous solution or an ethanol aqueous solution; Determination: Measured by ultra-high performance liquid chromatography; detection wavelength: 285 nm for 0-7 min, 380 nm for 7-30 min; octadecylsilane-bonded silica gel was used as the packing material; specifications: column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm. Chromatographic conditions included: acetonitrile as mobile phase A, water as mobile phase B, gradient elution, with the following program: 0-5 min, 20%→30% mobile phase A, 80%→70% mobile phase B; 5-6 min, 30%→40% mobile phase A, 70%→60% mobile phase B; 6-20 min, 40% mobile phase A, 60% mobile phase B; 20-30 min, 40%→45% mobile phase A, 60%→55% mobile phase B. The characteristic spectrum obtained by the construction method includes 6 characteristic peaks, of which peak 1 is N-trans-feruloyltyramine, peak 2 is piperine, peak 3 is piperine, and peak 4 is piperine.

2. The construction method according to claim 1, characterized in that, The chromatographic conditions also include: a column temperature of 33-37℃; and / or a flow rate of 0.28-0.32 ml / min; and / or an injection volume of 2-5 μL.

3. The construction method according to claim 1 or 2, characterized in that, The chromatographic conditions are as follows: column temperature 35℃; and / or flow rate 0.3 ml / min; and / or theoretical plate number calculated based on piperine peak should be no less than 5000; and / or injection volume 2 μL.

4. The construction method according to claim 1 or 2, characterized in that, The pepper is black pepper and / or white pepper.

5. The construction method according to claim 4, characterized in that, The test sample is at least one of pepper medicinal material, pepper slices, pepper decoction, pepper formula granules, and pepper standard decoction freeze-dried powder.

6. The construction method according to claim 1 or 2, characterized in that, The preparation method of the test sample solution includes: taking the test sample, adding solvent, extracting, and filtering.

7. The construction method according to claim 6, characterized in that, The ratio of the mass of the test sample to the volume of the solvent is (0.1-0.5) g:(10-50) ml.

8. The construction method according to claim 1 or 2, characterized in that, It also includes the preparation of reference solutions.

9. The construction method according to claim 8, characterized in that, The preparation method of the reference solution includes: taking the reference medicinal material, extracting it with water to obtain an aqueous extract; adding a solvent to the aqueous extract, and then performing a second extraction and filtration.

10. The construction method according to claim 9, characterized in that, When preparing the reference solution, the ratio of the mass of the water extract to the volume of the extraction solvent is (0.4-1.0) g:(20-100) ml.

11. The construction method according to claim 1 or 2, characterized in that, The feature map obtained by the construction method includes 6 feature peaks; With peak 3 as the reference peak, the specified values ​​for the relative retention times of peaks 1, 2, 4, 5, and 6 are 0.40, 0.78, 1.50, 1.60, and 1.68, respectively. The relative retention time of the characteristic peak is within ±10% of the specified value.

Images