A peach branch extract, a preparation method and a detection method thereof

Abstract

This invention belongs to the field of modernization of traditional Chinese medicine, specifically relating to a peach twig extract and its preparation and detection methods. The preparation method of the peach twig extract includes the following steps: (1) taking peach twig medicinal material, adding water and decocting, and separating the solid and liquid to obtain a filtrate; (2) concentrating the filtrate in step (1), and then freeze-drying it, wherein the freeze-drying is divided into three stages: a. pre-freezing: the pre-freezing temperature is -50℃ to -45℃; b. primary drying: the drying temperature is -35℃ to 0℃; c. secondary drying: the drying temperature is 5 to 25℃, to obtain the peach twig extract. The detection methods for the content and / or transfer rate of naringenin in the peach twig extract and the detection methods for the characteristic chromatograms of the peach twig extract provided by this invention are simple, stable, highly precise and reproducible, and the obtained fingerprint chromatograms have many peaks, good peak shapes, are easy to identify, and are accurate and reliable, providing an effective means of controlling the quality of peach twig extract.

Description

Technical Field

[0001] This invention belongs to the field of modern technology of traditional Chinese medicine, specifically relating to a peach twig extract and its preparation and detection methods. Background Technology

[0002] Peach branches are the dried branches of the peach tree (Prunus persica) (L.) Batsch, a plant in the Rosaceae family. They are harvested in summer, cut into sections, and sun-dried. The main medicinal properties of peach branches are to promote blood circulation, detoxify, and kill parasites. They are used to treat abdominal pain, rheumatic pain, injuries from falls, and sores.

[0003] Peach twig extract, also known as standard decoction, is a traditional and widely used form of medicine in clinical practice. The extract is prepared by following the theory of traditional Chinese medicine, decocting according to the standardized method of clinical decoction, separating solids and liquids, and then concentrating or drying it by appropriate methods. It serves as a standard reference for measuring whether the Chinese medicine formula granules are basically consistent with the clinical decoction.

[0004] With the development of new dosage forms of traditional Chinese medicine, the quality control of extracts of Chinese medicinal materials and their preparations has become an urgent problem to be solved. Existing technologies do not have effective indicators and methods for evaluating the quality of peach twig extract, and cannot provide a reference for controlling the quality of end products of Chinese medicinal extracts. The quality control of peach twig extract is not yet perfect, thus limiting its application and development. Summary of the Invention

[0005] To address the aforementioned technical problems, the present invention aims to provide a peach branch extract, its preparation method, and its detection method.

[0006] Specifically, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a peach branch extract, wherein the content of naringenin in the peach branch extract is 7-32 mg / g.

[0008] Preferably, the content of naringenin in the peach branch extract is 7.62–31.35 mg / g.

[0009] Preferably, the content of naringenin in the peach branch extract is 12-24 mg / g.

[0010] Preferably, the content of naringenin in the peach branch extract is 12.87–23.91 mg / g.

[0011] Preferably, the transfer rate of naringenin is 12% to 42%.

[0012] Preferably, the transfer rate of naringenin is 14% to 42%.

[0013] Preferably, the transfer rate of naringenin is 14.21% to 41.98%.

[0014] Secondly, the present invention provides a method for preparing peach branch extract, comprising the following steps:

[0015] (1) Take peach branches as medicinal material, add water and decoct, then separate the solid and liquid to obtain the filtrate;

[0016] (2) The filtrate from step (1) is concentrated and dried, and then freeze-dried. The freeze-drying is divided into three stages: a. Pre-freezing: the pre-freezing temperature is -50℃ to -45℃; b. First drying: the drying temperature is -35℃ to 0℃; c. Second drying: the drying temperature is 5℃ to 25℃, to obtain peach branch extract.

[0017] Preferably, in step (1), the number of times water is added and boiled is 1 to 3.

[0018] More preferably, in step (1), the boiling process is carried out twice.

[0019] Preferably, in step (1), the mass ratio of peach branch medicinal material to water added for each decoction is 1:6 to 8.

[0020] Preferably, in step (1), the boiling time is 40 to 60 minutes each time water is added.

[0021] Preferably, in step (2), the concentration temperature is 50-65°C.

[0022] Preferably, in step (2), the pre-freezing time is 220 to 260 minutes.

[0023] Preferably, in step (2), the drying time is 800 to 1500 min.

[0024] Preferably, in step (2), the secondary drying time is 400-500 min.

[0025] Preferably, in step (2), the vacuum degree of the first drying is -0.2 to 0 mbar.

[0026] Preferably, in step (2), the vacuum degree of the secondary drying is -0.2 to 0 mbar.

[0027] Preferably, the yield of the peach twig extract is 3.0% to 6.3%.

[0028] Thirdly, the present invention provides a method for detecting the content and / or transfer rate of naringenin in peach branch extract, comprising the following steps:

[0029] (1) Preparation of reference solution:

[0030] Naringin was used as a reference standard and a solvent was added to prepare a solution.

[0031] (2) Preparation of the test solution:

[0032] Take the peach branch extract and add a solvent for extraction;

[0033] (3) Liquid chromatography analysis:

[0034] Inject the reference solution and the test solution into the liquid chromatograph, use octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as the mobile phase A and phosphoric acid solution as the mobile phase B, and perform gradient elution to obtain the content and / or transfer rate of naringenin.

[0035] Preferably, in step (1), the solvent is selected from methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol, or water. The percentage of the solvent is a volume percentage.

[0036] Preferably, in step (2), the solvent is selected from methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol, or water. The percentage of the solvent is a volume percentage.

[0037] More preferably, in step (2), the solvent is 75% ethanol.

[0038] Preferably, in step (2), the extraction is performed by ultrasonic extraction, shaking extraction or reflux extraction.

[0039] More preferably, in step (2), the extraction is performed using ultrasonic extraction.

[0040] More preferably, in step (2), the ultrasonic power is 450-550W and / or the frequency is 35-45kHz.

[0041] Preferably, in step (2), the extraction time is 15 to 60 minutes.

[0042] More preferably, in step (2), the extraction time is 30 min.

[0043] Preferably, in step (3), the flow rates of mobile phase A and mobile phase B are 0.28 to 0.32 mL / min.

[0044] More preferably, in step (3), the flow rates of mobile phase A and mobile phase B are 0.30 mL / min.

[0045] Preferably, in step (3), the mobile phase B is a 0.02% to 0.08% phosphoric acid solution.

[0046] More preferably, in step (3), the mobile phase B is a 0.05% phosphoric acid solution.

[0047] Preferably, in step (3), the detection wavelength is 285–295 nm.

[0048] More preferably, in step (3), the detection wavelength is 290 nm.

[0049] Preferably, in step (3), the column temperature is 25-35°C.

[0050] More preferably, in step (3), the column temperature is 30°C.

[0051] Preferably, in step (3), the liquid chromatograph is a high-performance liquid chromatograph.

[0052] Preferably, in step (3), the liquid chromatograph is an ultra-high performance liquid chromatograph.

[0053] Preferably, in step (3), the gradient elution procedure is as follows:

[0054] From 0 to 9 min, the volume percentage of mobile phase A was 8% to 19%, and the volume percentage of mobile phase B was 92% to 81%.

[0055] From 9 to 25 minutes, the volume percentage of mobile phase A changed from 19% to 38%, and the volume percentage of mobile phase B changed from 81% to 62%.

[0056] Over 25–30 minutes, the volume percentage of mobile phase A changed from 38% to 40%, and the volume percentage of mobile phase B changed from 62% to 60%.

[0057] Fourthly, the present invention provides a method for detecting the characteristic spectrum of peach branch extract, comprising the following steps:

[0058] (1) Preparation of reference solution:

[0059] Take peach twig reference material and / or naringin reference standard, and prepare a solution by adding solvent;

[0060] (2) Preparation of the test solution:

[0061] Take the peach branch extract and add a solvent for extraction;

[0062] (3) Liquid chromatography analysis:

[0063] The reference solution and the test solution were injected into the liquid chromatograph, and gradient elution was performed with octadecylsilane-bonded silica gel as the stationary phase to obtain the characteristic chromatogram of the peach twig extract.

[0064] Preferably, in step (1), the solvent is selected from methanol, 75% methanol, 50% methanol, 25% methanol, ethanol (preferably anhydrous ethanol), 75% ethanol, 50% ethanol, 25% ethanol, or water. The percentage of the solvent is a volume percentage.

[0065] Preferably, in step (2), the solvent is selected from methanol, 75% methanol, 50% methanol, 25% methanol, ethanol (preferably anhydrous ethanol), 75% ethanol, 50% ethanol, 25% ethanol, or water. The percentage of the solvent is a volume percentage.

[0066] Preferably, in step (2), the solvent is 75% ethanol.

[0067] Preferably, in step (2), the extraction is performed using one of ultrasonic extraction, shaking extraction, or reflux extraction.

[0068] Preferably, in step (2), the extraction is performed using ultrasonic extraction.

[0069] Preferably, in step (2), the ultrasonic power is 450-550W and the frequency is 35-45kHz.

[0070] Preferably, in step (2), the extraction time is 30 to 60 minutes.

[0071] Preferably, in step (3), the detection wavelength is 190–400 nm.

[0072] Preferably, in step (3), the detection wavelength is 290 nm.

[0073] Preferably, in step (3), the liquid chromatograph is a high-performance liquid chromatograph.

[0074] Preferably, in step (3), the liquid chromatograph is an ultra-high performance liquid chromatograph.

[0075] Preferably, in step (3), the gradient elution procedure is as follows:

[0076] From 0 to 9 min, the volume percentage of mobile phase A was 8% to 19%, and the volume percentage of mobile phase B was 92% to 81%.

[0077] From 9 to 25 minutes, the volume percentage of mobile phase A changed from 19% to 38%, and the volume percentage of mobile phase B changed from 81% to 62%.

[0078] Over 25–30 minutes, the volume percentage of mobile phase A changed from 38% to 40%, and the volume percentage of mobile phase B changed from 62% to 60%.

[0079] Preferably, in step (3), the mobile phase comprises mobile phase A and mobile phase B.

[0080] Preferably, in step (3), mobile phase A is an organic phase and mobile phase B is an aqueous phase.

[0081] Preferably, in step (3), the organic phase is selected from acetonitrile and / or methanol.

[0082] More preferably, in step (3), the organic phase is acetonitrile.

[0083] Preferably, in step (3), the aqueous phase is selected from one or more of the following: 0.05% formic acid solution, 0.05% phosphoric acid solution, 0.05% acetic acid solution, or water. The percentage of the aqueous phase is a volume percentage.

[0084] More preferably, in step (3), the aqueous phase is a 0.05% phosphoric acid solution.

[0085] More preferably, in step (3), the mobile phase is acetonitrile-0.05% phosphoric acid solution.

[0086] Preferably, in step (3), the flow rate of the mobile phase is 0.25 to 0.35 mL / min.

[0087] More preferably, in step (3), the flow rate of the mobile phase is 0.30 mL / min.

[0088] Preferably, in step (3), the column temperature is 25-35°C.

[0089] More preferably, in step (3), the column temperature is 30°C.

[0090] Preferably, the characteristic spectrum has no less than 5 characteristic peaks, and the peak corresponding to the naringenin reference standard is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time is within ±10% of a specified value. The specified value includes 0.60 (peak 1), 0.64 (peak 2), 0.69 (peak 3), and 0.75 (peak 4).

[0091] Fifthly, the present invention provides a standard decoction of peach twigs, wherein the standard decoction of peach twigs contains the aforementioned peach twig extract.

[0092] Sixthly, the present invention provides a peach branch formula granule, wherein the peach branch formula granule contains the aforementioned peach branch extract.

[0093] Preferably, the peach branch formula granules also contain excipients.

[0094] More preferably, the excipient is selected from one or more of maltodextrin, silicon dioxide, or magnesium stearate.

[0095] In a seventh aspect, the present invention provides a method for detecting the content and / or transfer rate of naringenin in the peach twig extract, and its application in controlling the quality of the peach twig extract and its preparations.

[0096] Eighthly, the present invention provides an application of the detection method of the characteristic spectrum of the peach twig extract in controlling the quality of the peach twig extract and its preparations.

[0097] The beneficial effects of this invention are:

[0098] (1) This invention provides a peach branch extract and its preparation and detection methods to standardize the quality control and standard research of traditional Chinese medicine formula granules, realize the overall quality control and effective supervision of traditional Chinese medicine formula granules, and provide a reference for the quality control of peach branch formula granules.

[0099] (2) The present invention uses peach branch medicinal material to prepare peach branch extract, and the yield of the standard peach branch decoction is relatively high.

[0100] (3) The characteristic chromatographic detection method for peach branch extract provided by this invention uses ultra-high performance liquid chromatography (UHPLC), which is simple, stable, highly precise, and reproducible. Furthermore, the obtained fingerprint chromatogram has numerous peaks with good peak shapes, making it easy to identify and accurate. It can provide a quality control method for the quality evaluation of peach branch extract.

[0101] (4) The present invention provides a method for quality control of peach branches by using ultra-high performance liquid chromatography to determine the content of naringenin in peach branch extract as an indicator and formulating an internal control standard for the content of extract. Attached Figure Description

[0102] Figure 1 The figures shown are the specificity test chromatograms for the content determination method validation. Among them, (a) shows the specificity test chromatogram with 75% ethanol, (b) shows the specificity test chromatogram with naringin reference solution, and (c) shows the specificity test chromatogram with peach twig standard decoction solution.

[0103] Figure 2 The figures shown are chromatograms of peak purity, where (a) is the chromatogram of peak purity of naringenin reference solution and (b) is the chromatogram of peak purity of peach twig standard decoction solution.

[0104] Figure 3 The figure shows the linear regression equation for naringin.

[0105] Figure 4 The figures shown are chromatograms of peach twig standard decoction on different chromatographic columns. Among them, (a) shows the Waters ACQUITY column. (a) shows the UPLC chromatogram of HSS T3 column 1, (b) shows the UPLC chromatogram of Agilent ZORBAX SB-Aq column 2, and (c) shows the UPLC chromatogram of Endeavorsil column 3.

[0106] Figure 5 The figures show chromatograms of the standard peach twig decoction under different chromatographic instruments. (a) shows the effect of the Agilent 1290 Infinity II chromatographic instrument on the determination of the target component content in the peach twig extract, and (b) shows the effect of the Waters Acquity H class chromatographic instrument on the determination of the target component content in the peach twig extract.

[0107] Figure 6 The figures show chromatograms of peach twig standard decoction at different column temperatures. (a) shows the effect of column temperature at 25℃ on the determination of the target component content in peach twig extract, (b) shows the effect of column temperature at 30℃ on the determination of the target component content in peach twig extract, and (c) shows the effect of column temperature at 35℃ on the determination of the target component content in peach twig extract.

[0108] Figure 7 The figures show the chromatograms of the standard peach twig decoction at different flow rates. (a) shows the effect of a flow rate of 0.28 mL / min on the determination of the target component in the peach twig extract, (b) shows the effect of a flow rate of 0.30 mL / min on the determination of the target component in the peach twig extract, and (c) shows the effect of a flow rate of 0.32 mL / min on the determination of the target component in the peach twig extract.

[0109] Figure 8 The figures shown are the chromatograms and DAD diagrams of the peach twig standard decoction and the naringenin reference standard. (a) shows the chromatogram of the peach twig standard decoction, (b) shows the chromatogram of the naringenin reference standard, (c) shows the chromatogram of the maximum absorption wavelength of the naringenin peak in the peach twig standard decoction, and (d) shows the chromatogram of the maximum absorption wavelength of the naringenin reference standard peak.

[0110] Figure 9 The image shows UPLC spectra of different mobile phase systems.

[0111] Figure 10 The figures shown are chromatograms for different mobile phase gradients. (a) shows the chromatogram for chromatographic condition 1, (b) shows the chromatogram for chromatographic condition 2, and (c) shows the chromatogram for chromatographic condition 3.

[0112] Figure 11The figures shown are characteristic spectra of peach twig standard decoction with different mobile phase compositions. Among them, (a) shows the characteristic spectra of peach twig with acetonitrile-0.05% formic acid solution as the mobile phase, (b) shows the characteristic spectra of peach twig with acetonitrile-0.05% phosphoric acid solution as the mobile phase, (c) shows the characteristic spectra of peach twig with acetonitrile-0.05% acetic acid solution as the mobile phase, (d) shows the characteristic spectra of peach twig with acetonitrile-water as the mobile phase, and (e) shows the characteristic spectra of peach twig with methanol-water as the mobile phase.

[0113] Figure 12 The figures shown are characteristic spectra of peach twig standard decoction at different flow rates. Among them, (a) shows the characteristic spectra of peach twig standard decoction at a flow rate of 0.30 ml / min, (b) shows the characteristic spectra of peach twig standard decoction at a flow rate of 0.25 ml / min, and (c) shows the characteristic spectra of peach twig standard decoction at a flow rate of 0.35 ml / min.

[0114] Figure 13 Characteristic spectra of peach twig standard decoction at different column temperatures, wherein (a) shows the characteristic spectra of peach twig standard decoction at a column temperature of 25℃, (b) shows the characteristic spectra of peach twig standard decoction at a column temperature of 35℃, and (c) shows the characteristic spectra of peach twig standard decoction at a column temperature of 30℃.

[0115] Figure 14 The figures shown are characteristic chromatograms of different extraction solvents for the standard peach twig decoction. Among them, (a) shows the characteristic chromatogram of methanol as the extraction solvent, (b) shows the characteristic chromatogram of 75% methanol as the extraction solvent, (c) shows the characteristic chromatogram of 50% methanol as the extraction solvent, (d) shows the characteristic chromatogram of 25% methanol as the extraction solvent, (e) shows the characteristic chromatogram of ethanol as the extraction solvent, (f) shows the characteristic chromatogram of 75% ethanol as the extraction solvent, (g) shows the characteristic chromatogram of 50% ethanol as the extraction solvent, (h) shows the characteristic chromatogram of 25% ethanol as the extraction solvent, and (i) shows the characteristic chromatogram of water as the extraction solvent.

[0116] Figure 15 The figure shown is a comparison of the extraction efficiency of peach twig standard decoction using different extraction solvents (the vertical axis represents peak area / sample weight).

[0117] Figure 16 The figures show the specificity of the characteristic chromatograms of the peach twig standard decoction. (a) shows the specificity of the characteristic chromatograms of the peach twig standard decoction using 75% ethanol blank solvent, and (b) shows the specificity of the peach twig standard decoction solution.

[0118] Figure 17 The image shown is a comprehensive analysis of the characteristic spectrum of the standard peach twig decoction.

[0119] Figure 18The figures shown are comparisons of UPLC results from different chromatographic columns. (a) shows the UPLC chromatogram from column 1, (b) shows the UPLC chromatogram from column 2, and (c) shows the UPLC chromatogram from column 3.

[0120] Figure 19 The figures shown are comparisons of UPLC at different column temperatures. (a) shows the UPLC spectrum at a column temperature of 25℃, (b) shows the UPLC spectrum at a column temperature of 30℃, and (c) shows the UPLC spectrum at a column temperature of 35℃.

[0121] Figure 20 The following are characteristic chromatograms of standard decoctions at different flow rates: (a) shows the characteristic chromatogram of the standard decoction at a flow rate of 0.28 mL / min, (b) shows the characteristic chromatogram of the standard decoction at a flow rate of 0.30 mL / min, and (c) shows the characteristic chromatogram of the standard decoction at a flow rate of 0.32 mL / min.

[0122] Figure 21 The image shows the characteristic peak pattern of the standard peach twig decoction (R is the control spectrum).

[0123] Figure 22 The figure shows the common peak pattern of 15 batches of peach twig standard decoction (R is the control spectrum).

[0124] Figure 23 The images show the characteristic spectra of peach twig reference material and naringin reference standard. Among them, (a) shows the characteristic spectra of peach twig reference material and (b) shows the characteristic spectra of naringin reference standard. Detailed Implementation

[0125] As described above, the purpose of this invention is to provide a peach branch extract and its preparation and detection methods.

[0126] The first objective of this invention is to provide a method for preparing a standard peach twig decoction, comprising the following steps:

[0127] (1) Take peach branches and decoct them with water 1 to 3 times. The mass ratio of peach branches to water is 1:6 to 8 each time. The decoction time is 40 to 60 minutes each time. The solid and liquid are separated to obtain the filtrate.

[0128] (2) The filtrate from step (1) is concentrated and dried, and then freeze-dried. The freeze-drying is divided into three stages: a. Pre-freezing: the pre-freezing temperature is -50℃ to -45℃, and the time is 220 to 260 min; b. First drying: the drying temperature is -35℃ to 0℃, and the time is 800 to 1500 min; c. Second drying: the drying temperature is 5 to 25℃, and the time is 400 to 500 min, to obtain the standard peach twig decoction.

[0129] The second objective of this invention is to provide a method for detecting the content and / or transfer rate of naringenin in a standard peach twig decoction, comprising the following steps:

[0130] (1) Preparation of reference solution:

[0131] Naringin was used as a reference standard and a solvent was added to prepare a solution.

[0132] (2) Preparation of the test solution:

[0133] Take the peach branch extract and add a solvent for extraction;

[0134] (3) Liquid chromatography analysis:

[0135] Inject the reference solution and the test solution into the liquid chromatograph, use octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as the mobile phase A and phosphoric acid solution as the mobile phase B, and perform gradient elution to obtain the content and / or transfer rate of naringenin.

[0136] The third objective of this invention is to provide a method for detecting the characteristic chromatogram of a standard peach twig decoction, comprising the following steps:

[0137] (1) Preparation of reference solution:

[0138] Take peach twig reference material and / or naringin reference standard, and prepare a solution by adding solvent;

[0139] (2) Preparation of the test solution:

[0140] Take the peach branch extract and add a solvent for extraction;

[0141] (3) Liquid chromatography analysis:

[0142] The reference solution and the test solution were injected into the liquid chromatograph, and gradient elution was performed with octadecylsilane-bonded silica gel as the stationary phase to obtain the characteristic chromatogram of the peach twig extract.

[0143] Unless otherwise specified, all reagents used in the embodiments of this invention are conventional reagents.

[0144] To better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0145] Example 1: Preparation of Standard Peach Twig Decoction

[0146] 1 Instrument

[0147] RE-5205A rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), ceramic health pot (Huzhou Gangdian Craft Manufacturing Factory), HLD-30002 electronic balance (Hangzhou Youheng Weighing Equipment Company), AL104 0.01% electronic analytical balance [Mettler-Toledo Instruments (Shanghai) Co., Ltd.], SHZ-D III circulating water vacuum pump (Gongyi Yuhua Instrument Co., Ltd.), DLSB-5 / 20B low-temperature coolant circulating water pump (Zhengzhou Changcheng Science & Industry Trade Co., Ltd.), LYO-0.5 vacuum freeze dryer (Shanghai Dongfulong Technology Co., Ltd.), 202-2AB electric thermostatic drying oven (Tianjin Taist Instrument Co., Ltd.), 101-4A electric blast thermostatic drying oven (Hunan Electric Furnace Oven Factory), HH-S6A electric thermostatic water bath (Beijing Kewei Yongxing Instrument Co., Ltd.), etc.

[0148] 2 materials

[0149] Information on the medicinal materials used is shown in Table 1-1:

[0150] Table 1-1 Information on Peach Twig Medicinal Materials

[0151]

[0152]

[0153] 3. Standard Preparation Method of Peach Twig Decoction

[0154] Take 100g of peach twig slices, place them in an electric ceramic kettle, add water, and decoct twice. For the first decoction, add 8 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 60 minutes. Filter the decoction while hot through a 300-mesh sieve, and record the mass of the decoction after it cools to room temperature. For the second decoction, add 6 times the amount of water, heat to a boil over high heat, then simmer over low heat for 40 minutes. Filter the decoction while hot through a 300-mesh sieve, and record the mass of the decoction after it cools to room temperature. Combine the two decoctions. The decoction was transferred to a 2000ml round-bottom flask and concentrated under reduced pressure at low temperature using a rotary evaporator (temperature: 65℃; vacuum: -0.080~-0.090MPa) to 100ml of extract. Its density and yield were measured. Under magnetic stirring, the extract was dispensed into 10ml amber vials, 2ml per vial, half-stopped, and then transferred to a vacuum freeze dryer for lyophilization. The lyophilization parameters were: pre-freezing temperature -45℃, pre-freezing time 240 minutes; primary drying temperature -30℃, 420 minutes; -20℃, 120 minutes; -10℃, 120 minutes; 0℃, 180 minutes; vacuum -0.2mbar; secondary drying temperature 5℃, 120 minutes; 15℃, 120 minutes; 25℃, 180 minutes; vacuum -0.2mbar. The extract was then removed, capped with an aluminum cap, and the final product was obtained. Fifteen batches of standard peach twig decoction were prepared using the above method. Raw material information and detailed preparation process parameters are shown in Tables 1-2 below.

[0155] 4. Method for determining the yield of ointment

[0156] The yield of the extract was calculated based on the standard decoction dosage. 10g of the concentrated solution was placed in a pre-weighed evaporating dish, dried in a water bath, and then dried in a 105℃ drying oven for 3 hours. After cooling in a desiccator for half an hour, the solution was quickly weighed. The yield of the extract was then calculated.

[0157]

[0158] Table 1-2 Summary of Peach Twig Standard Decoction Preparation

[0159]

[0160]

[0161]

[0162] Based on the test results of 15 batches of peach twig standard decoction, the extract yield was calculated, and the results are shown in Table 1-3 below:

[0163] Table 1-3 Determination of the yield of 15 batches of peach twig standard decoction

[0164]

[0165] As shown in Table 1-3 above, the yield of the standard peach twig decoction, based on 15 batches, fluctuated between 4.08% and 5.87%, with a mean of 4.69% and a SD of 0.55. The mean yield ranged from 70% to 130% from 3.29% to 6.10%, the ±2 SD ranged from 3.60% to 5.79%, and the ±3 SD ranged from 3.05% to 6.33%. The yield range of the standard peach twig decoction is tentatively set at 3.05% to 6.33%, approximately rounded to 3.0% to 6.3%.

[0166] Example 2: Determination of the content of peach twig in standard decoction

[0167] Using naringin content as an indicator, a quantitative standard for this traditional Chinese medicine was established, providing a scientific basis for the establishment of quality standards for peach twig medicinal materials.

[0168] 1. Instruments, reagents and reagents

[0169] Instruments: Agilent 1290Infinity II UHPLC [Agilent Technologies (China) Co., Ltd.], Waters Acquity H class UHPLC [Waters Technologies (China) Co., Ltd.], KQ-500DA / DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), ML204, XRPARXD / R electronic analytical balance [Mettler-Toledo Technologies (China) Co., Ltd.]; Column: ACQUITY HSS T3C18 (2.1×150mm, 1.8μm) [Waters Technology (China) Co., Ltd.], ZORBAX SB-Aq (2.1×150mm, 1.8μm) [Agilent Technologies (China) Co., Ltd.], Endeavorsil (2.1×150mm, 1.8μm) (Beijing Dicoma Technology Co., Ltd.).

[0170] Reagents: Methanol and acetonitrile were of chromatographic grade, with batch numbers 216571 and F22M3S203, respectively, purchased from Fisher Scientific. Water was ultrapure water, and all other reagents were of analytical grade.

[0171] Test reagent: Naringenin (batch number: DST23515-100, purity ≥98%), purchased from Lemeitian Pharmaceutical Desite Biotechnology Co., Ltd. For content determination, no pretreatment is required.

[0172] The standard decoction of peach twigs prepared in Example 1 was used. The batch numbers of the 15 batches of the standard decoction of peach twigs were as follows: BT220316-1, BT220316-2, BT220316-3, BT220316-4, BT220316-5, BT220316-6, BT220316-7, BT220316-8, BT220316-9, BT220316-10, BT220316-11, BT220316-12, BT220316-13, BT220316-14, and BT220316-15.

[0173] 2. Inspection of the source and purity of the reference standard

[0174] Naringenin (batch number: DST23515-100, purity ≥98%) was purchased from Lemeitian Pharmaceutical Desite Biotechnology Co., Ltd. It is for content determination purposes and requires no pretreatment.

[0175] 3. Determination of chromatographic conditions

[0176] The column was packed with octadecylsilane-bonded silica gel (150 mm column length, 2.1 mm inner diameter, 1.8 μm particle size); acetonitrile was used as mobile phase A, and 0.05% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in the table below; the flow rate was 0.30 mL / min; the column temperature was 30 °C; and the detection wavelength was 290 nm. The theoretical plate number, calculated based on the naringin peak, should not be less than 10,000. The elution conditions are shown in Table 2-1 below.

[0177] Table 2-1 Elution conditions

[0178]

[0179] 4. Preparation of reference solution

[0180] Take an appropriate amount of naringenin reference standard, accurately weigh it, and add 75% ethanol to prepare a solution containing 0.16 mg of naringenin per 1 ml, which is used as the reference solution.

[0181] 5. Preparation of the test solution

[0182] (1) Investigation of different extraction solvents

[0183] Take an appropriate amount of peach twig standard decoction (BT220316-8), grind it into a fine powder, and accurately weigh approximately 0.2g. Divide the powder into nine portions, with two parallel samples per portion. Place each portion in a stoppered conical flask and accurately add methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol, and 25ml of water sequentially. Seal the flasks tightly, weigh them, and sonicate (500W, 40kHz) for 30 minutes. Remove the flasks, cool them, and weigh them again. Make up the weight loss with the appropriate solvent, shake well, and filter to obtain the test solutions in different solvents. Accurately pipette 1μl of each of the above test solutions and inject them under the chromatographic conditions described above. Record the peak area of ​​naringenin and calculate the naringenin content (mg / g). Determine the optimal extraction solvent based on the naringenin content analysis. The results are shown in Table 2-2 below.

[0184] Table 2-2 Results of Naringin Content Determination with Different Extraction Solvents (n=2)

[0185]

[0186]

[0187] As shown in Table 2-2, the extraction rates of naringenin in the standard peach twig decoction varied significantly among different solvents. Methanol, 75% methanol, and 75% ethanol had comparable extraction rates of naringenin. Considering the economic, environmental, and safety factors, as well as the results of the investigation on extraction solvents for peach twig medicinal materials, 75% ethanol was selected as the extraction solvent for preparing the test solution of the standard peach twig decoction.

[0188] (2) Examination of extraction methods

[0189] Take an appropriate amount of peach twig standard decoction (BT220316-8), grind it into a fine powder, and accurately weigh about 0.2g. Divide the powder into three portions, two in parallel for each portion. Place each portion in a stoppered conical flask, accurately add 25ml of 75% ethanol to each, seal tightly, and weigh. Extract one portion by reflux in a water bath for 30 minutes, another by sonication (500W, 40kHz) for 30 minutes, and the third by shaking for 30 minutes. Remove, cool, and weigh again. Make up the weight loss with 75% ethanol, shake well, and filter to obtain the test solutions for different extraction methods. Accurately pipette 1μl of each of the above test solutions and inject according to the above chromatographic conditions. Record the peak area of ​​naringenin and calculate the naringenin content (mg / g). Determine the optimal extraction method based on the naringenin content analysis. The results are shown in Table 2-3 below.

[0190] Table 2-3 Results of Naringenin Content Determination by Different Extraction Methods (n=2)

[0191]

[0192]

[0193] As shown in Table 2-3, different extraction methods result in different extraction rates of naringenin in the peach twig standard decoction. Considering the advantages of ultrasonic extraction, such as lower temperature, simpler operation, and faster speed, and taking into account the overall peaks of the characteristic chromatograms, ultrasonic extraction was finally selected as the extraction method for preparing the test solution of the peach twig standard decoction.

[0194] (3) Examination of different extraction times

[0195] Take an appropriate amount of peach twig standard decoction (BT220316-8), grind it into a fine powder, and accurately weigh approximately 0.2g. Divide the powder into four portions, with two samples per portion. Place each portion in a stoppered conical flask, accurately add 25ml of 75% ethanol, seal tightly, and weigh. Sonicate the flasks (500W power, 40kHz frequency) for 15, 30, 45, and 60 minutes respectively. After cooling, weigh again, replenish the lost weight with 75% ethanol, shake well, and filter to obtain the test solutions for different extraction times. Accurately pipette 1μl of each of the above test solutions and inject them under the chromatographic conditions described above. Record the peak area of ​​naringenin and calculate the naringenin content (mg / g). Determine the optimal extraction time based on the naringenin content analysis. The results are shown in Table 2-4 below.

[0196] Table 2-4 Results of naringin content determination at different extraction times (n=2)

[0197]

[0198] As shown in Table 2-4, the results indicate that different extraction times do not significantly affect the naringin content in the peach twig standard decoction. Considering the time cost, ultrasonic extraction for 30 minutes was ultimately chosen as the extraction time for preparing the peach twig standard decoction test solution.

[0199] (4) Determination of the preparation method of the test solution

[0200] Based on the results of the sample pretreatment experiment, the preparation method of the test sample can be determined as follows: Take an appropriate amount of peach twig standard decoction, about 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 75% ethanol, seal tightly, weigh it, sonicate (power 500W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with 75% ethanol, shake well, filter it, and the test sample is obtained.

[0201] 6. Validation of content determination method

[0202] (1) Exclusivity

[0203] Accurately pipette 1 μl each of the prepared peach twig standard decoction (BT220316-8) test solution, naringin reference solution, and 75% ethanol blank solvent, and inject them under the chromatographic conditions described above. Record the chromatograms. Results are shown below. Figure 1 .

[0204] Depend on Figure 1 The chromatogram results show that this analytical method has good specificity for the determination of naringenin content in peach twig standard decoction.

[0205] (2) Peak purity

[0206] Accurately pipette the prepared peach twig standard decoction (BT220316-8) test solution and naringenin reference solution, and inject them according to the above chromatographic conditions. Detect the naringenin peak purity using the peak purity detection method built into the Agilent 1290 Infinity II ultra-high performance liquid chromatograph. The results are shown in [Figure number missing]. Figure 2 See Table 2-5.

[0207] Table 2-5 Matching values ​​for target peaks and peak purity

[0208]

[0209] Depend on Figure 2 As shown in Table 2-5, the peak purity matching value of the index component naringenin is 999≥960, which indicates that its peak purity meets the analytical requirements.

[0210] (3) Linear

[0211] Accurately weigh an appropriate amount of naringenin reference standard, place it in a 50ml volumetric flask, add 75% ethanol to prepare a solution containing 0.3mg of naringenin per 1ml, shake well, and the naringenin reference standard stock solution is obtained.

[0212] Accurately pipette 2 ml, 4 ml, 6 ml, 8 ml, and 10 ml of naringenin reference standard stock solution into 10 ml volumetric flasks, and dilute to the mark with 75% ethanol to obtain naringenin reference standard solutions of different concentrations. Inject the solutions under the chromatographic conditions described above. Plot the concentration on the x-axis and the peak area on the y-axis to investigate the linear range of naringenin. The linearity test results are shown in Tables 2-6 and 2-6 below. Figure 3 .

[0213] Table 2-6 Linearity Study of Naringenin

[0214]

[0215] From Table 2-6 and Figure 3 Experimental results showed that the concentration of naringenin and the peak area exhibited a good linear relationship within the concentration range of 60.352 μg / ml to 301.760 μg / ml, with a correlation coefficient R0. 2 =0.9998, and the linear regression equation is y = 11.393x + 28.141.

[0216] (4) Precision test

[0217] ① Instrument precision test

[0218] Accurately pipette the test solution prepared from the standard peach twig decoction (BT220316-8), inject it 6 times under the above chromatographic conditions, record the peak area of ​​naringenin, and calculate the naringenin content (mg / g) and RSD (%). The results are shown in Table 2-7 below.

[0219] Table 2-7 Results of Instrument Precision Experiment for Peach Twig Standard Decoction

[0220]

[0221] As shown in Table 2-7, the RSD (%) of naringenin was 1.09%, which is less than 2%, indicating that the instrument has good precision.

[0222] ② Repeatability test

[0223] Take approximately 0.2 g of the same batch of peach twig standard decoction (BT220316-8), accurately weigh it, and prepare 6 parallel solutions according to the established test solution preparation method. Inject the samples under the above chromatographic conditions, record the peak area of ​​naringenin, and calculate the naringenin content (mg / g) and RSD (%). The results are shown in Table 2-8 below.

[0224] Table 2-8 Results of Repeatability Experiments on Peach Twig Standard Decoction

[0225]

[0226] As shown in Table 2-8, the RSD (%) of naringenin content was 1.58%. According to the "Guiding Principles for Analytical Method Validation" in General Chapter 9101 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, when the content of the test sample is between 10 mg / g and 100 mg / g, the RSD value of repeatability should be less than 2%. Therefore, the results show that the repeatability of this method is good.

[0227] ③ Intermediate precision

[0228] Other analysts in this project team operated on different dates and under different chromatograms, taking approximately 0.2 g of the same batch of peach twig standard decoction (BT220316-8), accurately weighing it, and preparing 6 parallel aliquots according to the determined test solution preparation method. The samples were injected under the above chromatographic conditions, the peak area of ​​naringenin was recorded, and the naringenin content (mg / g) and RSD (%) were calculated. The results are shown in Table 2-9 below.

[0229] Table 2-9 Results of Intermediate Precision Experiment for Peach Twig Standard Decoction

[0230]

[0231]

[0232] As shown in Table 2-9, the RSD (%) of naringenin content was 1.53%. According to the "Guiding Principles for Analytical Method Validation" in General Chapter 9101 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, when the content of the test sample is between 10 mg / g and 100 mg / g, the RSD of reproducibility should be less than 3%. The results show that the intermediate precision of this method is good.

[0233] (5) Accuracy test

[0234] Take 0.1g of peach twig standard decoction (BT220316-8, naringenin content 21.00%) with known content, accurately weigh 6 portions, and accurately add 5ml of naringenin reference solution prepared with 75% ethanol (concentration 0.42748mg / ml) to each portion. Then accurately add 20ml of 75% ethanol, seal tightly, weigh, and prepare the test solution according to the test solution preparation method. Inject the sample under the above chromatographic conditions, record the peak area of ​​naringenin, calculate the naringenin content (mg), and calculate the recovery rate (%) and RSD (%) according to the following formulas. The results are shown in Table 2-10 below.

[0235]

[0236] Table 2-10 Results of the naringenin recovery experiment in peach twig medicinal material

[0237]

[0238]

[0239] As shown in Table 2-10, the recovery rate of naringenin in peach twig medicinal material was 96.78%, with an RSD of 0.89%. According to the "Guiding Principles for Validation of Analytical Methods" in General Chapter 9101 of Part IV of the 2020 edition of the Chinese Pharmacopoeia, when the content of the test sample is between 10 mg / g and 100 mg / g, the recovery rate should be between 95% and 102%, and the RSD% should be less than 2%. The results show that the recovery rate of naringenin is within the range, and the RSD% is less than 2%, indicating that the accuracy of the detection method is good.

[0240] (6) Stability test

[0241] Accurately pipette the prepared test solution and inject it at 0h, 2h, 4h, 6h, 8h, 12h, 24h and 36h according to the above chromatographic conditions. Record the peak area of ​​naringenin and calculate the naringenin content (mg / g) and RSD (%). The results are shown in Table 2-11 below.

[0242] Table 2-11 Results of the stability test of peach twig standard decoction

[0243]

[0244] Table 2-11 shows that the RSD (%) of naringin content within 36 hours was 1.70%, which is less than 3%, indicating that the test solution of peach twig standard decoction had relatively good stability within 36 hours.

[0245] (7) Durability test

[0246] ① Investigation of different chromatographic columns

[0247] Three brands of chromatographic columns were examined: 1-Waters ACQUITY column. The effects of three chromatographic columns—HSS T3 (2.1×150mm, 1.8μm), column 2—Agilent ZORBAX SB-Aq (2.1×100mm, 1.8μm), and column 3—Endeavorsil (2.1×150mm, 1.8μm)—on the peak shape and resolution of naringenin in peach twig standard decoction were investigated.

[0248] Take the test solution prepared under the [Content Determination] section of the peach twig standard decoction (BT220316-8), and determine it according to the above chromatographic conditions. Record the resolution value, theoretical plate number, peak area, and chromatogram of naringenin, and calculate the naringenin content (mg / g) based on the peak area. The experimental results are shown below. Figure 4 and Table 2-12 below.

[0249] Table 2-12 Results of the study on the effects of different chromatographic columns on the standard decoction of peach twig.

[0250]

[0251] Depend on Figure 4 The experimental results in Table 2-12 show that the separation effect of the three chromatographic columns is good, which can meet the requirements for the determination of naringin content in peach twig standard decoction. However, ACQUITY The HSS T3 (2.1×150mm, 1.8μm) column has superior resolution and theoretical plate number, therefore ACQUITY was chosen. HSS T3 (2.1×150mm, 1.8μm) was used as the chromatographic column for the determination of the content of peach twig standard decoction.

[0252] ② Exploration using different chromatographs

[0253] Based on the existing equipment in the laboratory, the Waters Ultra High Performance Liquid Chromatography (WatersAcquity Hclass) and the Agilent Ultra High Performance Liquid Chromatography (Agilent 1290Infinity II) were selected to compare the effects of the two chromatographs on the content, resolution and peak shape of naringenin in the peach twig standard decoction.

[0254] Take the test solution prepared under the [Content Determination] section of the peach twig standard decoction (BT220316-8), and determine it according to the above chromatographic conditions. Record the resolution value, theoretical plate number, peak area, and chromatogram of naringenin, and calculate the naringenin content (mg / g) based on the peak area. The experimental results are shown below. Figure 5 and Table 2-13 below.

[0255] Table 2-13 Results of Chromatographic Examination of Peach Twig Standard Decoction Using Different Instruments

[0256]

[0257] Depend on Figure 5 The experimental results in Table 2-13 show that this analytical method exhibits good durability with different chromatographs. Variations in the chromatograph can meet the system adaptability requirements.

[0258] ③ Investigation at different column temperatures

[0259] The effects of different column temperatures (25℃, 30℃, and 35℃) on the peak shape and resolution of naringenin in peach twig medicinal materials were compared.

[0260] Take the test solution prepared under the [Content Determination] section of the peach twig standard decoction (BT220316-8), and determine it according to the above chromatographic conditions. Record the resolution value, theoretical plate number, peak area, and chromatogram of naringenin, and calculate the naringenin content (mg / g) based on the peak area. The experimental results are shown below. Figure 6 and Table 2-14 below.

[0261] Table 2-14 Results of naringenin in peach twig decoction at different column temperatures

[0262]

[0263] Figure 6 The results in Table 2-14 show that under chromatographic conditions with a column temperature variation of ±5℃, the peak shape and separation were good, and the naringenin content remained stable. Considering the column's tolerance and the analysis time required, a column temperature of 30℃ was selected for this study.

[0264] ④ Investigation of different flow velocities

[0265] The effects of different flow rates of 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min on the peak shape and resolution of naringenin in peach twig medicinal material were compared.

[0266] Take the test solution prepared under the [Content Determination] section of the peach twig standard decoction (BT220316-8), and determine it according to the above chromatographic conditions. Record the resolution value, theoretical plate number, peak area, and chromatogram of naringenin, and calculate the naringenin content (mg / g) based on the peak area. The experimental results are shown below. Figure 7 and Table 2-15 below.

[0267] Table 2-15 Results of the determination of peach twig content in standard decoction by different flow rates

[0268]

[0269] Figure 7 The results in Table 2-15 show that the chromatographic peak shape and separation effect are good under the chromatographic conditions of a flow rate with a small variation of ±0.02 ml / min. However, considering the overall characteristic chromatogram, the flow rate of 0.30 ml / min was selected for this experiment.

[0270] Results of content determination in 7 different batches of peach twig standard decoction

[0271] Based on the results of naringin content in 15 batches of peach twig decoction pieces, the transfer rate (%) of 15 batches of standard decoctions was calculated.

[0272] The results are shown in Table 2-16 below.

[0273]

[0274] Table 2-16 Results of content and transfer rate of standard decoction of peach twigs in different batches

[0275]

[0276]

[0277] Based on the results of naringenin content determination in 15 batches of peach twig decoction pieces and 15 batches of standard decoctions (Table 2-16), the mean and range of naringenin content transfer rate in peach twig standard decoction were calculated. The mean naringenin content in peach twig standard decoction was 18.39 mg / g. Calculated as 70%–130% of the mean naringenin content, its fluctuation range is 12.87 mg / g–23.91 mg / g. Calculated using ±2 times SD of the mean naringenin content, its fluctuation range is 14.85 mg / g–21.94 mg / g. Calculated using ±3 times SD of the mean naringenin content, its fluctuation range is 13.08 mg / g–23.71 mg / g. Therefore, 12.87 mg / g–23.91 mg / g is considered as the limit range for peach twig standard decoction.

[0278] The content of naringenin per 1g of this product is tentatively set at 12.87mg to 23.91mg.

[0279] Based on the results of naringenin transfer rate in the standard peach twig decoction, the mean transfer rate was 28.09%, with a SD of 4.63. The fluctuation range was calculated as 19.66%–36.5% based on the mean transfer rate of 70%–130%, 18.84%–37.35% based on ±2 times the SD, and 14.21%–41.98% based on ±3 times the SD. Therefore, the wider range of 14.21%–41.98% is considered the acceptable limit for the transfer rate of the standard peach twig decoction.

[0280] The transfer rate of naringenin in the provisional standard decoction of peach twigs is between 14.21% and 41.98%.

[0281] Example 3: Characteristic chromatographic analysis of peach twig standard decoction

[0282] 1. Instruments, reagents and reagents

[0283] Instruments: Agilent 1290 Infinity II UHPLC system [Agilent Technologies (China) Co., Ltd.], KQ-500DA / DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), ML204, XRPARXD / R electronic analytical balance [Mettler-Toledo Technologies (China) Co., Ltd.]; Column: ACQUITY HSS T3C18 (2.1×150mm, 1.8μm) [Waters Technology (China) Co., Ltd.], ZORBAX SB-Aq (2.1×150mm, 1.8μm) [Agilent Technologies (China) Co., Ltd.], Endeavorsil (2.1×150mm, 1.8μm) (Beijing Dicoma Technology Co., Ltd.).

[0284] Reagents: Acetonitrile was of chromatographic grade; water was ultrapure water; all other reagents were of analytical grade.

[0285] Test reagent: Naringenin (batch number: DST230515-100, purity 98%), purchased from Lemeitian Pharmaceutical Desite Biotechnology. For content determination, no pretreatment is required. Peach twig reference material (batch number: 121601-202002, purchased from China National Institutes for Food and Drug Control). The batch numbers of the 15 batches of Taozhi Standard Soup are as follows: BT220321-1, BT220321-2, BT220321-3, BT220321-4, BT220321-5, BT220321-6, BT220321-7, BT220321-8, BT220321-9, BT220321-10, BT220321-11, BT220321-12, BT220321-13, BT220321-14, and BT220321-15 (provided by the laboratory process group).

[0286] 2. Preparation of reference solution

[0287] Accurately weigh approximately 0.5g of peach twig reference material and place it in a stoppered conical flask. Accurately add 25ml of 75% ethanol, seal tightly, and weigh. Sonicate (500W, 40kHz) for 60 minutes, cool, and weigh again. Make up the lost weight with 75% ethanol, shake well, filter, and use the filtrate as the reference solution. Accurately weigh an appropriate amount of naringenin reference standard and add 75% ethanol to prepare a solution containing 0.16mg of naringenin per ml, which will be used as the reference solution.

[0288] 3. Preparation of the test solution

[0289] Take an appropriate amount of peach twig standard decoction (BT220316-8), grind it into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 75% ethanol, seal tightly, weigh it, sonicate (power 500W, frequency 40kHz) for 60 minutes, cool it, weigh it again, make up the lost weight with 75% ethanol, shake well, filter it, and take the filtrate to obtain the decoction.

[0290] 4. Determination of chromatographic conditions

[0291] 4.1 Determination of detection wavelength

[0292] Accurately pipette 1 μl of the test solution prepared from the peach twig standard decoction into the high-performance liquid chromatograph and record the absorption spectrum in the range of 190–400 nm, as shown below. Figure 8 .

[0293] Figure 8Experimental results show that naringenin has the maximum absorption at a wavelength of 290 nm. The standard peach twig decoction sample solution has more detectable chromatographic peak information and higher peak response value at a wavelength of 290 nm, and the baseline noise interference is smaller. Therefore, 290 nm was selected as the detection wavelength.

[0294] 4.2 Optimization of the mobile phase

[0295] (1) Investigation of the full gradient of the mobile phase

[0296] Chromatographic conditions: The chromatographic column was ACQUITY. HSS T3C18 (2.1×150mm, 1.8μm); acetonitrile as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, gradient elution was performed according to the specifications in Table 3-1; the flow rate was 0.30 mL / min; the column temperature was 30℃; and the detection wavelength was 290 nm. Results are as follows... Figure 9 As shown.

[0297] Table 3-1 Gradient Elution

[0298]

[0299] Figure 9 Experimental results showed that the chromatographic peaks in the peach twig standard decoction were mostly of medium polarity, and the mobile phase consisted of about 50% aqueous phase. Therefore, subsequent studies adjusted the gradient conditions.

[0300] (2) Investigation of adjusting the mobile phase gradient

[0301] Chromatographic conditions 1: The chromatographic column is ACQUITY. HSS T3C18 (2.1×150mm, 1.8μm); acetonitrile was used as mobile phase A, and 0.05% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 3-2; the flow rate was 0.30 mL / min; the column temperature was 30℃; and the detection wavelength was 290 nm. Results are as follows: Figure 10 As shown in (a).

[0302] Table 3-2 Gradient Elution

[0303]

[0304] Chromatographic condition 2: The chromatographic column is ACQUITY. HSS T3C18 (2.1×150mm, 1.8μm); acetonitrile was used as mobile phase A, and 0.05% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 3-3; the flow rate was 0.30 mL / min; the column temperature was 30℃; and the detection wavelength was 290 nm. Results are as follows... Figure 10 As shown in (b).

[0305] Table 3-3 Gradient Elution

[0306]

[0307] Chromatographic condition 3: The chromatographic column is ACQUITY. HSS T3C18 (2.1×150mm, 1.8μm); acetonitrile as mobile phase A and 0.05% phosphoric acid solution as mobile phase B, gradient elution was performed according to the specifications in Table 3-4; the flow rate was 0.30 mL / min; the column temperature was 30℃; and the detection wavelength was 290 nm. Results are as follows... Figure 10 As shown in (c).

[0308] Table 3-4 Gradient Elution

[0309]

[0310] Based on the above investigation of chromatographic conditions, such as Figure 10 The results show that the peak separation in the chromatogram under chromatographic condition 3 is relatively better, therefore this chromatographic condition was chosen for the experiment in this study.

[0311] (3) Investigation of the composition of the mobile phase system

[0312] Chromatographic conditions: The chromatographic column was ACQUITY. HSS T3C18 (2.1×150mm, 1.8μm); using acetonitrile (methanol) as mobile phase A and water (acidic water) as mobile phase B, gradient elution was performed according to the specifications in the table below; the flow rate was 0.30 ml per minute; the column temperature was 30℃; and the detection wavelength was 290 nm.

[0313] Based on chromatographic conditions 3, the effects of mobile phases acetonitrile-0.05% phosphoric acid, acetonitrile-0.05% formic acid, acetonitrile-0.05% acetic acid, acetonitrile-water, and methanol-water systems on the chromatogram of peach twig standard decoction were investigated. Gradient elution was performed according to the specifications in Table 3-4. The results are as follows: Figure 11 As shown.

[0314] Figure 11 Experimental results showed that the acetonitrile-0.05% phosphoric acid system had the best peak shape, separation effect and overall number of peaks, so the acetonitrile-0.05% phosphoric acid system was selected for elution.

[0315] (4) Investigation of different flow velocities

[0316] The effects of different flow rates on the characteristic peaks of the standard peach twig decoction were compared.

[0317] Chromatographic conditions: The chromatographic column was ACQUITY. HSS T3C18 (2.1×150mm, 1.8μm); acetonitrile was used as mobile phase A, and 0.05% acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 3-4; flow rates were 0.25 ml, 0.30 ml, and 0.35 ml per minute; column temperature was 30℃; detection wavelength was 290 nm. Results are as follows: Figure 12 As shown.

[0318] Figure 12 Experimental results show that flow rate has a certain influence on the overall characteristic peaks of peach twig standard decoction. When the flow rate is 0.30 ml / min, the peak shape and separation effect of each characteristic peak are the best. Therefore, the flow rate of 0.30 ml / min was selected.

[0319] (5) Investigation at different column temperatures

[0320] The effects of different column temperatures on the characteristic peaks of the standard peach twig decoction were compared.

[0321] Chromatographic conditions: The chromatographic column was ACQUITY. HSS T3C18 (2.1×150mm, 1.8μm); acetonitrile was used as mobile phase A, and 0.05% acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 3-4; the flow rate was 0.30 mL / min; the column temperature was 25℃, 30℃, and 35℃; the detection wavelength was 290 nm. Results are as follows... Figure 13 As shown.

[0322] Figure 13 Experimental results show that the flow rate has a certain influence on the overall characteristic peaks of the peach twig standard decoction. The peak shape and separation effect of each characteristic peak are best at a temperature of 30℃. Therefore, the column temperature is selected as 30℃.

[0323] (6) Determination of chromatographic conditions

[0324] The column was packed with octadecylsilane-bonded silica gel (150 mm column length, 2.1 mm inner diameter, 1.8 μm particle size); acetonitrile was used as mobile phase A, and 0.05% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 3-5; the flow rate was 0.30 mL / min; the column temperature was 30 °C; and the detection wavelength was 290 nm. The theoretical plate number, calculated based on the naringin peak, should be no less than 10,000.

[0325] Table 3-5

[0326]

[0327] 5. Preparation of the test solution

[0328] This experiment investigated the effects of different extraction solvents on the characteristic chromatograms of peach twig standard decoction. Methanol, 75% methanol, 50% methanol, 25% methanol, anhydrous ethanol, 75% ethanol, 50% ethanol, and 25% ethanol-water were selected as extraction solvents. The characteristic chromatograms of different extraction solvents were compared by using the total peak area / sample weight of five tentatively determined chromatographic peaks and the chromatograms.

[0329] Take an appropriate amount of peach twig standard decoction (BT220316-8), approximately 0.2g, accurately weigh it, and place it in a stoppered conical flask. Accurately add methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol, and 25ml of water, respectively. Weigh the flask, sonicate (500W power, 40kHz frequency) for 30 minutes, cool, and weigh again. Make up the lost weight with the appropriate solvent, shake well, filter, and collect the filtrate. Under the chromatographic conditions determined above, record the peak areas of the five characteristic peaks. Calculate the total amount by dividing the peak area by the sample weight and plot a column chart. See the chromatograms and column charts for different extraction solvents of the peach twig standard decoction characteristic chromatograms. Figure 14 and Figure 15 .

[0330] like Figure 14 and Figure 15 The experimental results show that when the extraction solvent is 75% ethanol, the peak area / sample weight of the five characteristic peaks is relatively high. Therefore, 75% ethanol was chosen as the extraction solvent.

[0331] Based on the above experimental results and the results of the investigation on the preparation of the test solution for content determination, the preparation method of the test solution for the characteristic spectrum of peach twig standard decoction was determined as follows: Take an appropriate amount of peach twig standard decoction, about 0.2g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 75% ethanol, weigh it, sonicate it (power 500W, frequency 40kHz) for 30 minutes, cool it, weigh it again, replenish the lost weight with 75% ethanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0332] 6. Methodological Validation of Feature Map Analysis Method

[0333] (1) Specificity examination

[0334] Accurately pipette 1 μl each of the test solution and blank solvent from the [Characteristic Chromatography] section of the peach twig standard decoction (BT220316-8) and inject them into the liquid chromatograph. Determine the chromatographic results under the conditions described above. See results below. Figure 16 .

[0335] Depend on Figure 16 The experimental results show that the solvent does not interfere with the characteristic peaks in the chromatogram of the peach twig standard decoction.

[0336] (2) Holistic Examination

[0337] The test solution prepared under the [Characteristic Chromatography] section of the peach twig standard decoction (BT220316-8) was injected into the liquid chromatograph. The elution time was doubled under the above chromatographic conditions at the gradient endpoint mobile phase ratio, and the characteristic chromatogram was analyzed. The results are as follows: Figure 17 As shown.

[0338] Figure 17 The results showed that no obvious chromatographic peaks were observed after doubling the elution time under these chromatographic conditions, indicating that the chromatographic conditions basically met the principle of maximizing information content.

[0339] (3) Precision test

[0340] Take the test solution prepared under the [Characteristic Chromatography] section of the standard decoction of peach twigs (BT220613-8), inject it into the liquid chromatograph, and determine it under the chromatographic conditions described above. Repeat the injection 6 times. Record the retention times of the 5 calibrated characteristic peaks. Using peak 5 as the reference peak, calculate the relative retention times of the other 4 peaks. The experimental results are shown in Table 3-6 below.

[0341] Table 3-6 Precision results of characteristic chromatograms of peach twig standard decoction (relative retention time)

[0342]

[0343] The experimental results in Table 3-6 show that the RSD of the relative retention time of each chromatographic peak is less than 3.0%, indicating that the instrument has good precision.

[0344] (4) Stability test

[0345] Take the test solution prepared under the [Characteristic Chromatography] section of the peach twig standard decoction (BT220613-8) and inject it into the liquid chromatograph. Under the chromatographic conditions described above, inject the sample at 0, 2, 4, 6, 8, 12, 24, and 36 hours. Record the retention times of the five calibrated characteristic peaks. Using peak 5 as the reference peak, calculate the relative retention times of the remaining four peaks. The experimental results are shown in Table 3-7 below.

[0346] Table 3-7 Stability results of the characteristic spectra of peach twig standard decoction (relative retention time)

[0347]

[0348]

[0349] The experimental results in Table 3-7 show that the RSD of the relative retention time of the chromatographic peak is less than 3.0%, indicating that the sample solution is relatively stable.

[0350] (5) Repeated examination

[0351] Accurately weigh approximately 2.0 g of the same batch of peach twig standard decoction (BT220316-8), prepare six parallel portions, and prepare test solutions according to the method for preparing test solutions under the [Characteristic Chromatography] section. Inject 1 μl of each solution under the chromatographic conditions described above. Record the retention times of the five calibrated characteristic peaks. Using peak 5 as the reference peak, calculate the relative retention times of the remaining four peaks. The experimental results are shown in Table 3-8 below.

[0352] Table 3-8 Repeatability results of characteristic spectra of peach twig standard decoction (relative retention time)

[0353]

[0354] The experimental results in Table 3-8 show that the relative retention time (RSD) of each chromatographic peak is less than 3.0%, indicating that the method has good repeatability.

[0355] (6) Durability test

[0356] ① Column analysis

[0357] Three brands of chromatographic columns were examined: Column 1 - Waters ACQUITY The effects of three chromatographic columns on the peak characteristics of the peach twig standard decoction were investigated: HSS T3 (2.1×150mm, 1.8μm); column 2 - Agilent ZORBAX SB-Aq (2.1×100mm, 1.8μm); column 3 - Endeavorsil (2.1×150mm, 1.8μm).

[0358] Take the test solution prepared under the [Content Determination] section of the peach twig standard decoction (BT220316-8) and determine its content according to the above chromatographic conditions. Record the retention times and chromatograms of the five characteristic peaks. Based on the calibrated retention times of the five characteristic peaks, and using peak 5 as the reference peak, calculate the relative retention times of the remaining four peaks. The experimental results are shown in [Figure 1]. Figure 18 and Table 3-9 below.

[0359] Table 3-9 Chromatographic column analysis results of the characteristic chromatogram of peach twig decoction (relative retention time)

[0360]

[0361] Figure 18 The experimental results in Table 3-9 show that different chromatographic columns have a certain impact on the peak elution. Using the ACQUITY column... Elution with an HSS T3 (2.1×150mm, 1.8μm) ultra-high performance liquid chromatography column yielded good peak shapes and optimal separation. Therefore, it is recommended to use an ACQUITY column for this method. The HSS T3 (2.1×150mm, 1.8μm) ultra-high performance liquid chromatography column was used to determine the characteristic chromatogram of peach twig standard decoction.

[0362] ② Column temperature investigation

[0363] The effects of different column temperatures (25℃, 30℃, and 35℃) on the peak shape and separation of five characteristic peaks in the characteristic spectrum of peach twig standard decoction were compared.

[0364] Take the test solution prepared under the [Content Determination] section of the peach twig standard decoction (BT220316-8) and determine its content according to the above chromatographic conditions. Record the retention times and chromatograms of the five characteristic peaks. Based on the calibrated retention times of the five characteristic peaks, and using peak 5 as the reference peak, calculate the relative retention times of the remaining four peaks. The experimental results are shown in [Figure 1]. Figure 19 and Table 3-10 below.

[0365] Table 3-10 Results of column temperature analysis of characteristic spectra of peach twig decoction (relative retention time)

[0366]

[0367] Figure 19 The experimental results in Table 3-10 show that column temperature has a certain impact on peak elution, but the separation effect is best and the peak shape is better when the column temperature is 30℃. Therefore, it is recommended to use a column temperature of 30℃ for the determination.

[0368] ③ Flow velocity investigation

[0369] The effects of different flow rates (0.28 ml / min, 0.30 ml / min, and 0.32 ml / min) on the peak shape and resolution of five characteristic peaks in the characteristic spectrum of peach twig standard decoction were compared.

[0370] Take the test solution prepared under the [Content Determination] section of peach twig medicinal material (BT220316-8) and determine its content according to the above chromatographic conditions. Record the retention times and chromatograms of the five characteristic peaks. Based on the calibrated retention times of the five characteristic peaks, and using peak 5 as the reference peak, calculate the relative retention times of the remaining four peaks. The experimental results are shown in […]. Figure 20 and Table 3-11 below.

[0371] Table 3-11 Results of flow velocity study (relative retention time) of Taozhi Standard Decoction Characteristic Spectrum

[0372]

[0373] Figure 20 The experimental results in Table 3-11 show that flow rate has a certain impact on peak formation, but the peak shape is optimal when the flow rate is 0.30 ml / min. Therefore, it is recommended to use 0.30 ml / min as the measurement flow rate.

[0374] 7. Identification and designation of common peaks

[0375] UPLC chromatograms of different batches of peach twig standard decoction samples were determined, and the results were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission, and common peaks were selected.

[0376] The results were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission, and five common peaks were identified. The results are shown in Table 3-12.

[0377] Table 3-12 Test results (relative retention time) of 15 batches of standard decoctions

[0378]

[0379]

[0380] The results in Table 3-12 show that, taking peak 5 as the reference peak, the relative retention time (RSD) values ​​of the five characteristic peaks in the characteristic chromatograms of the 15 batches of peach twig standard decoction ranged from 0.00% to 0.30%, all less than 3.0%, which meets the standard requirements for the characteristic chromatograms of peach twig standard decoction.

[0381] UPLC chromatograms of different batches of peach twig standard decoction samples were determined, and the results were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission. Peak 5 was used as the reference peak to establish a characteristic chromatogram for the peach twig standard decoction. Figure 21 As shown.

[0382] Figure 21 The results showed that the characteristic spectrum of the peach twig standard decoction contained five relatively obvious common peaks. Specifically, as follows... Figure 22 As shown. Figure 23 The image shows the characteristic chromatograms of peach twig reference material and naringin reference standard (chromatographic column: ACQUITY). HSST3C18, 2.1×150mm, 1.8μm).

[0383] After establishing and validating a method for determining characteristic spectra using UPLC, characteristic spectra of 15 batches of peach twig standard decoction and peach twig reference material were determined. Based on the analysis of common peaks in the characteristic spectra of the 15 batches of standard decoction, peaks in peach twig reference material, and peaks in naringenin reference standard, the standard for the characteristic spectra of peach twig standard decoction was finally determined: the characteristic spectra of the test sample should show 5 characteristic peaks, corresponding to the retention times of the 5 characteristic peaks in the chromatogram of the reference material. Among them, peak 5 corresponds to the retention time of the naringenin reference peak, and the peak corresponding to the naringenin reference peak is the S peak. The relative retention times of peaks 1, 2, 3, and 4 with peak S were calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.60 (peak 1), 0.64 (peak 2), 0.69 (peak 3), and 0.75 (peak 4).

[0384] While the specific embodiments of the present invention have been described above, they are not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. A method for detecting the characteristic chromatogram of a standard peach twig decoction, characterized in that, Includes the following steps: (1) Preparation of reference solution: Take peach twig reference material and naringin reference standard, add solvent to prepare solution; (2) Preparation of the test solution: Take the standard decoction of peach twigs, add 75% ethanol, and sonicate to obtain the product. (3) Liquid chromatography analysis: The reference solution and the test solution were injected into the high-performance liquid chromatograph (HPLC), which was a T3 C18 column (2.1 × 150 mm, 1.8 μm) with a detection wavelength of 290 nm. Acetonitrile was used as mobile phase A, and 0.02-0.08% phosphoric acid solution was used as mobile phase B. The flow rates of mobile phases A and B were 0.25-0.35 mL / min. Gradient elution was performed to obtain the characteristic chromatogram of the peach branch extract. The gradient elution program was as follows: From 0 to 9 min, the volume percentage of mobile phase A changed from 8% to 19%, and the volume percentage of mobile phase B changed from 92% to 81%. From 9 to 25 minutes, the volume percentage of mobile phase A changed from 19% to 38%, and the volume percentage of mobile phase B changed from 81% to 62%. After 25-30 minutes, the volume percentage of mobile phase A changed from 38% to 40%, and the volume percentage of mobile phase B changed from 62% to 60%.

2. The method for detecting the characteristic chromatogram of the peach twig standard decoction according to claim 1, The ultrasonic power is 450-550W and the frequency is 35-45kHz.

3. The method for detecting the characteristic spectrum of the peach twig standard decoction according to claim 2, wherein the extraction time is 30-60 min.

4. The method for detecting the characteristic chromatogram of the peach twig standard decoction according to claim 1, characterized in that, In step (3), the column temperature is 25-35℃.

5. The method for detecting the characteristic chromatogram of the peach twig standard decoction according to claim 4, characterized in that, In step (3), the column temperature is 30℃.

6. The method for detecting the characteristic chromatogram of the peach twig standard decoction according to claim 1, characterized in that, In step (3), the flow rates of mobile phase A and mobile phase B are 0.30 mL / min.

7. The method for detecting the characteristic chromatogram of the peach twig standard decoction according to any one of claims 1-6, characterized in that, The characteristic spectrum shall have no fewer than 5 characteristic peaks, with the peak corresponding to the naringenin reference standard being designated as the S peak. The relative retention time of each characteristic peak and the S peak shall be calculated, and the relative retention time shall be within ±10% of a specified value. The specified value includes peak 1: 0.60, peak 2: 0.64, peak 3: 0.69, peak 4: 0.

75.

8. The application of the method for detecting the characteristic chromatogram of the peach twig standard decoction according to any one of claims 1-7 in controlling the quality of the peach twig standard decoction.

Images