A characteristic spectrum construction method and quality detection method for milk thistle and its preparation

Abstract

The present invention belongs to the technical field of quality inspection of traditional Chinese medicine preparations, and specifically relates to a characteristic fingerprint spectrum of milk thistle and its aqueous extract pharmaceutical preparation. Further disclosed are methods for constructing the characteristic spectrum and quality inspection methods for milk thistle and its pharmaceutical preparation. The method for constructing the characteristic spectrum of milk thistle and its preparation described in the present invention establishes a characteristic spectrum method for milk thistle raw medicinal material and milk thistle aqueous extract pharmaceutical preparation based on the UPLC method. Furthermore, by optimizing the mobile phase composition, mobile phase gradient, and test sample and reference material processing methods, the method comprehensively displays the chemical components of milk thistle, thereby broadening the application of milk thistle.

Description

Technical Field

[0001] The present invention belongs to the technical field of quality detection of traditional Chinese medicine preparations, and specifically relates to a characteristic fingerprint spectrum of milk thistle and a water extract pharmaceutical preparation thereof. The present invention further discloses a method for constructing the characteristic spectrum, and a quality detection method for milk thistle and a pharmaceutical preparation thereof. Background Art

[0002] Milk thistle (Silybum marianum (L.) Gaertn.) is an annual or biennial herb of the genus Silybum in the Asteraceae family. Its achenes are used as medicine, and are bitter in taste and cool in nature. It has the effects of clearing away heat and detoxifying, promoting lactation, protecting the liver, promoting bile secretion, and resisting X-rays. It can be used to treat acute and chronic hepatitis, cirrhosis, metabolic toxic liver damage, and sand stranguria.

[0003] In the prior art, milk thistle is primarily used as a raw material for extracting the silymarin component. Silymarin is a flavonoid mixture extracted and purified from milk thistle seeds and includes silybin, isosilybin, silybintin, and silymarinine. Modern pharmacological research has shown that this class of compounds exhibits excellent therapeutic effects in scavenging free radicals, combating lipid peroxidation, protecting liver cell membranes, promoting liver cell repair and regeneration, combating liver fibrosis, combating tumors, and lowering blood lipids. It has been widely used in medicine, health products, and cosmetics. Silybin is the most abundant, most active, and possesses the strongest pharmacological effects in the silymarin mixture. Traditional research has used silybin as a quality control indicator for milk thistle medicinal materials. However, using only a single indicator cannot comprehensively evaluate and measure the quality and intrinsic properties of milk thistle medicinal materials. Furthermore, prior research on milk thistle has primarily focused on the efficacy and applications of silymarin-like components, but lacks comprehensive research and application of the pharmacological components of milk thistle. In particular, research on the characteristic spectrum or fingerprint of milk thistle is rarely reported. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a method for constructing a characteristic spectrum that can comprehensively reflect the intrinsic quality and medication safety of milk thistle and its preparations;

[0005] The second technical problem to be solved by the present invention is to provide a quality detection method for milk thistle and its preparation.

[0006] In order to solve the above technical problems, the method for constructing a characteristic spectrum of milk thistle and its preparations described in the present invention includes the steps of performing high performance liquid chromatography on a test solution of milk thistle and its preparations;

[0007] The chromatographic conditions included: octadecylsilane bonded silica gel as the filler, methanol as the mobile phase A, 1% acetic acid solution as the mobile phase B, and gradient elution according to the following procedure:

[0008] 0-15 min, A:B is 15%:85% → 25%:75%;

[0009] 15-18 min, A:B 25%:75% → 28%:72%;

[0010] 18-21 min, A:B is 28%:72%;

[0011] 21-24 min, A:B 28%:72% → 33%:67%;

[0012] 24-29 min, A:B 33%:67% → 36%:64%;

[0013] 29-32 min, A:B 36%:64% → 39%:61%;

[0014] 32-35 min, A:B 39%:61% → 45%:55%;

[0015] 35-54min, A:B is 45%:55%;

[0016] 54-60 min, A:B 45%:55% → 47%:53%;

[0017] At 60-61 min, A:B was 47%:53% → 15%:85%;

[0018] 61-65min, A:B is 15%:85%.

[0019] Specifically, in the method for constructing the characteristic spectrum of milk thistle and its preparation, in the high performance liquid chromatography detection step, the chromatographic conditions also include: column temperature of 25-35°C, flow rate of 0.1-0.3 ml / min, and detection wavelength of 287 nm.

[0020] Specifically, the method for constructing the characteristic spectrum of the milk thistle and its preparation, and the method for preparing the test solution include: taking the test sample and accurately adding an organic solvent, ultrasonically treating it, filtering it and collecting the filtrate to obtain it.

[0021] Specifically, the method for constructing a characteristic spectrum of milk thistle and its preparation also includes the step of preparing a control medicinal material reference solution, which specifically includes: taking milk thistle control medicinal material, adding water for heating and refluxing extraction, collecting the extract, filtering and evaporating to dryness, adding an organic solvent to the residue, ultrasonically treating it, filtering and collecting the filtrate to obtain the required control medicinal material reference solution.

[0022] Specifically, the method for constructing a characteristic spectrum of milk thistle and its preparation further includes the steps of preparing a reference substance solution and constructing a fingerprint spectrum of the reference substance based on the high performance liquid chromatography method;

[0023] The reference substance includes at least one of a silibinin reference substance, a silibinin A reference substance, and a taxifolin reference substance.

[0024] Specifically, in the method for constructing a characteristic spectrum of milk thistle and its preparation, the organic solvent includes methanol at a volume concentration of 40-60 v / v%;

[0025] The power of the ultrasonic treatment step is 200-300W and the frequency is 30-50kHz.

[0026] Specifically, the characteristic spectrum construction method of the milk thistle and its preparations, the milk thistle preparations include milk thistle formula granules, milk thistle slices or milk thistle slice decoctions.

[0027] The present invention also discloses a characteristic spectrum and / or a control characteristic spectrum of milk thistle and its preparation, and the characteristic spectrum and / or the control characteristic spectrum of milk thistle and its preparation are constructed by the method.

[0028] The present invention also discloses a method for constructing a characteristic spectrum of the milk thistle and its preparation and / or application of the characteristic spectrum of the milk thistle and its preparation and / or a control characteristic spectrum in the field of quality detection of the milk thistle and its pharmaceutical preparation.

[0029] The present invention also discloses a quality detection method for milk thistle and its pharmaceutical preparation, comprising the steps of constructing the characteristic spectrum and a control characteristic spectrum according to the method, and comparing the characteristic spectrum with the control characteristic spectrum.

[0030] The method for constructing a characteristic spectrum of milk thistle and its preparations described in the present invention establishes a characteristic spectrum method for milk thistle raw medicinal material and milk thistle water-extracted medicinal preparations based on the UPLC method, and comprehensively displays the chemical components of milk thistle by optimizing the mobile phase composition, mobile phase gradient, and test and reference material processing methods, thereby broadening the use of milk thistle.

[0031] The method for constructing a characteristic spectrum of milk thistle and its preparation described in the present invention is based on the "Technical Requirements for Quality Control and Standardization of Traditional Chinese Medicine Formula Granules" and the technical requirements for research and formulation of traditional Chinese medicine quality standards in the Chinese Pharmacopoeia, establishes chromatographic conditions for determining the characteristic spectrum of a standard decoction of milk thistle slices, studies the determination content of the characteristic spectrum of the milk thistle formula granules, examines the methodology (system adaptability and specificity, precision and repeatability, durability, and solution stability), and establishes a method for determining the characteristic spectrum of the milk thistle formula granules.

[0032] The quality detection method of milk thistle and its pharmaceutical preparation of the present invention obtains a corresponding fingerprint based on the UPLC method, which can not only identify milk thistle and its counterfeits, but also ensure the accuracy of the drug source. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0034] Figure 1 The characteristic spectrum of the reference substance described in Example 1;

[0035] Figure 2 This is the characteristic spectrum of the control medicinal material described in Example 1;

[0036] Figure 3 Characteristic spectrum of the test solution constructed for the milk thistle formula granules described in Example 1;

[0037] Figure 4 is a precision chromatogram of the characteristic spectrum;

[0038] Figure 5 is a repetitive chromatogram of the characteristic spectrum;

[0039] Figure 6 is the intermediate precision (personnel) chromatogram of the characteristic spectrum;

[0040] Figure 7 This is the chromatogram of negative formula particles in the exclusive experiment;

[0041] Figure 8 A stability test chromatogram of the characteristic spectrum;

[0042] Figure 9 The characteristic spectra of the milk thistle formula granules at different flow rates;

[0043] Figure 10 The characteristic spectra of the milk thistle formula granules at different column temperatures are shown below;

[0044] Figure 11 Characteristic spectra of the milk thistle formula granules under different instruments;

[0045] Figure 12 Characteristic spectra of the milk thistle formula granules under different chromatographic columns;

[0046] Figure 13 This is the spectrum of three batches of milk thistle formula granules;

[0047] Figure 14 Characteristic spectra of 18 batches of standard decoctions of milk thistle slices;

[0048] Figure 15 It is the reference medicinal material characteristic spectrum of milk thistle and the reference characteristic spectrum of the standard decoction of milk thistle slices;

[0049] Figure 16This is a comparison of the characteristic spectra of milk thistle and arctium lappa seeds and a comparison of the characteristic spectra of milk thistle and amurense seeds. DETAILED DESCRIPTION

[0050] In the following embodiments of the present invention, the instruments and reagents involved include:

[0051] instrument

[0052] Chromatograph 1: The chromatograph is a Waters H-CLASS UPLC chromatography system, including a quaternary solvent manager (ACQ-QSM), an automatic sample injector (ACQ-FTN), an imported column oven (ACQ-CM), a diode array UV detector (ACQ-PDA), and an Empower chromatography management system;

[0053] Chromatograph 2: Thermo Vanquish Flex UHPLC chromatography system, including a quaternary solvent manager (Vanquish Quaternary Pump F VF-P20-A), an autosampler (Vanquish Split Sampler FTVF-A10-A-02), an imported column oven (Vanquish Column Compartment H VH-C10-A-02), and a DAD detector (Vanquish VF-D40-A).

[0054] Electronic balance: METTLER TOLEDO (Swiss Mettler) ME36S, XS204, XS205, XSE205 (one hundred thousandth);

[0055] Ultrasound instrument: SK5200H Shanghai Kedao Ultrasonic Instrument Co., Ltd.

[0056] Chromatographic column:

[0057] (1)YMC-Triart C18 2.1×100mm, 1.9μm;

[0058] (2)SHIMADZU Shim-pack GIST C18-AQ 2.1x100mm 1.9μm;

[0059] (3)CAPCELL PAK ADME-HR 2.1x100mm 2μm;

[0060] Reagents and test drugs

[0061] Methanol (Thermo Fisher Scientific, chromatographic grade), water was ultrapurified water, and other reagents were of analytical grade;

[0062] Milk thistle control medicinal material (batch number: DSTYS012701, purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.);

[0063] Silybin reference substance (batch number: 110856-201506, for content determination, calculated at 96.3%, purchased from the China Food and Drug Inspection Institutes);

[0064] Taxifolin reference substance (batch number: 111816-201102, for content determination, calculated at 98.9%, purchased from the China Food and Drug Inspection Institutes);

[0065] Silybin A reference substance (batch number: DSTDS031901, purchased from Chengdu Lemeitian Pharmaceutical Technology Co., Ltd.);

[0066] Isosilybin A reference substance (batch number: DST201105-292, purchased from Chengdu Desite Biotechnology Co., Ltd.);

[0067] Milk thistle formula granules (Batch numbers: 211101Y, 211102Y, 211103Y);

[0068] Standard decoction of milk thistle slices (Batch numbers: 1909001Y, 1909002Y, 1909005Y, 1909006Y, 1909008Y, 1909014Y, 1909015Y, 1909016Y, 1909017Y, 1909018Y, 1909019Y, 1909020Y, 1909022Y, 1909024Y, 1909025Y, 211001Y, 211002Y, 211003Y).

[0069] Example 1 Characteristic Spectrum Method Establishment

[0070] Preparation of reference solution

[0071] Take 1g of Silybum marianum reference medicinal material and place it in a stoppered conical flask. Add 100ml of water and heat under reflux for 30 minutes. Filter and evaporate the filtrate to dryness. Add 25ml of 50% methanol to the residue, stopper tightly, and sonicate (power 250W, frequency 40kHz) for 30 minutes. Remove, cool, shake well, filter, and take the filtrate as the reference medicinal material solution. Take appropriate amounts of Silybin A reference substance and Taxifolin reference substance, accurately weigh them, and add methanol to make a mixed solution containing 0.12mg of each per 1ml. This is the reference substance solution. Take an appropriate amount of Silybin reference substance, accurately weigh it, and add methanol to make a solution containing 0.12mg per 1ml. This is the reference substance solution.

[0072] Preparation of test solution

[0073] Take an appropriate amount of the milk thistle formula granules to be tested, grind them into powder, take 0.1g, place it in a stoppered conical flask, add 25ml of 50% methanol, stopper it tightly, and ultrasonically treat it (power 250W, frequency 40KHz) for 30 minutes. Take it out, let it cool, shake it well, filter it, and take the filtrate to obtain the product.

[0074] Chromatographic conditions and system suitability test

[0075] Octadecylsilane bonded silica gel (YMC-Triart C18 2.1×100 mm, 1.9 μm) was used as the packing material. Gradient elution was performed using methanol as the mobile phase A and 1% acetic acid solution as the mobile phase B, as specified in Table 1. The column temperature was 30°C, the flow rate was 0.2 ml / min, and the detection wavelength was 287 nm. The number of theoretical plates, calculated based on the Taxifolin peak, must be no less than 5000.

[0076] Table 1 Gradient elution program

[0077] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-15 15→25 85→75 15-18 25→28 75→72 18-21 28 72 21-24 28→33 72→67 24-29 33→36 67→64 29-32 36→39 64→61 32-35 39→45 61→55 35-54 45 55 54-60 45→47 55→53

[0078] Assay

[0079] Accurately pipette 2 μl each of the reference solution, test solution and reference medicinal material solution, inject them into the liquid chromatograph, measure, and record the chromatogram.

[0080] In this example, the test solution and the reference solution were prepared according to the solution method according to the test solution preparation method, and UHPLC analysis was performed under the described chromatographic conditions to evaluate the system applicability of the characteristic spectrum method of the milk thistle formula granules.

[0081] The chromatogram of the reference substance solution obtained in this example is shown in Figure 1 As shown, the chromatograms of silicilin, silybin, silybin A, silybin B, isosilybin A, and silybin are shown in the attached Figure 1 As shown in (a)-(f), the corresponding spectrum data are shown in Table 2 below.

[0082] Table 2 Chromatogram data of reference substance

[0083]

[0084] The chromatogram of the test solution obtained in this embodiment is shown in Figure 2 The corresponding spectrum data are shown in Table 3 below.

[0085] Table 3 Chromatogram data of milk thistle control medicinal material solution

[0086]

[0087] The chromatogram of the test solution of the milk thistle formula granules constructed in this example is shown in Figure 3 The corresponding spectrum data are shown in Table 4 below.

[0088] Table 4 Spectral data of the test solution of milk thistle formula granules

[0089]

[0090]

[0091] It can be seen from the chart that the chromatographic method has good system adaptability and specificity, and can be used as a detection method for the characteristic spectrum of milk thistle formula granules.

[0092] Example 2 Characteristic Spectrum Characteristic Peak S Peak Selection Basis

[0093] According to the above Example 1, taxifolin in the standard decoction and granules of silymarin slices has a higher response in the characteristic spectrum of the standard decoction and granules of silymarin slices, so taxifolin is selected as the S1 peak, and the relative retention times of peaks 1-6 are calculated. At the same time, since the analysis method is relatively time-consuming, silybin A is added as the S2 peak to calculate the relative retention times of peaks 7-13.

[0094] Example 3 Precision Test

[0095] Take the same test solution and repeat the injection 6 times according to the chromatographic conditions of the milk thistle formula granules in Example 1. The relative retention time and relative peak area of ​​13 common peaks and reference peaks are measured respectively. The results are shown in Tables 5-6 below. The precision chromatogram is shown in Table 5-6 below. Figure 4 As shown, from top to bottom it represents the precision of 1-6 needles.

[0096] Table 5 Instrument precision relative retention time test results

[0097]

[0098]

[0099] Table 6 Instrument precision relative peak area test results

[0100]

[0101]

[0102] The test results showed that the RSD of the relative retention time of each characteristic peak was less than 2%, indicating that the instrument had good precision.

[0103] Example 4 Method Repeatability Test

[0104] Take 6 samples of the same batch number for testing, and measure the relative retention time and relative peak area of ​​13 common peaks and reference peaks according to the characteristic spectrum method in the milk thistle formula granules in Example 1. The results are shown in Tables 7-8 below. The repeatability chromatograms are shown in Tables 7-8 below. Figure 5 As shown, from top to bottom, renaturation samples 1-6 are shown.

[0105] Table 7 Method repeatability relative retention time test results

[0106]

[0107]

[0108] Table 8 Method repeatability relative peak area test results

[0109]

[0110]

[0111] The test results showed that the RSD of the relative retention time of each characteristic peak was less than 2%, indicating that the method has good reproducibility.

[0112] Example 5 Intermediate Precision (Different Operators)

[0113] Three inspectors, at different times, used the same equipment to measure the relative retention times and relative peak areas of the 13 common peaks and the reference peaks according to the characteristic spectrum method of the milk thistle formula granules in Example 1. The results are shown in Tables 9-10 below. The intermediate precision (personnel) chromatogram is shown in the attached Figure 6 As shown, samples 1-6 of different persons are represented from top to bottom.

[0114] Table 9 Intermediate precision relative retention time test results (different operators)

[0115]

[0116] Table 10 Intermediate precision relative peak area test results (different operators)

[0117]

[0118]

[0119] The test results showed that the RSDs of the relative retention times of the characteristic peaks were all less than 2%, indicating that the intermediate precision of this method was good.

[0120] Example 6 Specificity

[0121] Preparation of negative blank samples

[0122] Take the negative particles, grind them into powder, take about 0.1g, weigh accurately, put it into a stoppered conical flask, add 25ml of 50% methanol, stopper it tightly, and ultrasonically treat it (power 250W, frequency 40KHz) for 30 minutes. Take it out, let it cool, shake it well, filter it, and take the filtrate to obtain it.

[0123] Accurately draw 1 μl of the test solution and the negative blank sample solution, respectively, and inject them into the high performance liquid chromatograph. Test according to the method of [characteristic spectrum] of the milk thistle formula granules in Example 1. The chromatogram of the negative granule solution is as follows: Figure 7 As shown. Figure 3 Compared with the spectrum of the test solution of the milk thistle formula granules shown, the results showed that it was negative and had no interference.

[0124] Example 7 Stability Study

[0125] Take the same batch of test samples, after preparation, according to the method of [characteristic spectrum] of milk thistle formula granules in Example 1, and inject samples at 0, 4, 8, 12, 18, and 24 hours respectively, and measure the relative retention time and relative peak area of ​​13 common peaks and reference peaks respectively. The results are shown in Tables 11-12, and the stability test chromatogram is shown in Figure 8 As shown, from top to bottom are samples at each time point in stability 1-6.

[0126] Table 11 Stability relative retention time test results

[0127]

[0128] Table 12 Stability relative peak area test results

[0129]

[0130]

[0131] The test results showed that the RSD of the relative retention time of each characteristic peak was less than 2%. The results showed that the test solution was stable within 24 hours and met the test requirements.

[0132] Example 8 Investigation of different flow rates

[0133] Take the same batch of test solution (2111001Y) and measure the characteristic spectrum of the milk thistle formula granules in Example 1 at flow rates of 0.18 ml / min, 0.2 ml / min, and 0.22 ml / min, respectively. The relative retention time and relative peak area of ​​each characteristic peak and the reference S peak when the flow rate changes slightly are investigated. The results are shown in Tables 13-14, respectively. The spectra under different flow rate conditions are shown in Tables 13-14. Figure 9As shown, (a), (b), and (c) represent the spectra at flow rates of 0.18 ml / min, 0.2 ml / min, and 0.22 ml / min, respectively.

[0134] Table 13 Results of relative retention time tests at different flow rates

[0135]

[0136]

[0137] Table 14 Experimental results of relative peak area at different flow rates

[0138]

[0139] The results show that the separation of peak 11 is affected at different flow rates. Therefore, this method recommends a fixed flow rate (0.2 ml / min) for measurement.

[0140] Example 9 Investigation of different column temperatures

[0141] Take the same batch of test solution (2111001Y), according to the determination method of milk thistle formula granules [characteristic spectrum] in Example 1, set the column temperature to 23 ° C, 25 ° C, and 27 ° C, and investigate the relative retention time and relative peak area of ​​each characteristic peak and the reference S peak when the column temperature changes. The results are shown in Tables 15-16, and the chromatograms at different column temperatures are shown in Tables 15-16. Figure 10 As shown, (a), (b), and (c) represent the spectra at column temperatures of 23°C, 25°C, and 27°C, respectively.

[0142] Table 15 Comparison of relative retention time determination results at different column temperatures

[0143]

[0144] Table 16 Results of relative peak area tests at different column temperatures

[0145]

[0146]

[0147] The results show that the separation of peak 12 is affected at different column temperatures. Therefore, this method recommends a fixed column temperature (25°C) for measurement.

[0148] Example 10 Investigation of different instruments

[0149] The same batch of test solution was taken, and the characteristic spectrum of the milk thistle formula granules was determined according to the method of Example 1. Different models of high performance liquid chromatography instruments were set, namely Waters H-CLASS UPLC and Thermo UPLC chromatographs. The relative retention time and relative peak area of ​​each characteristic peak and the reference S peak were investigated when the high performance liquid chromatography instrument was changed. The results are shown in Tables 17-18. The spectra under different instruments are shown in Tables 17-18. Figure 11 As shown, (a) and (b) represent the chromatograms under liquid phase waters and liquid phase Thermo UPLC conditions, respectively.

[0150] Table 17 Comparison of relative retention time determination results of characteristic spectra of different HPLC instruments

[0151]

[0152] Table 18 Test results of relative peak areas of characteristic spectra of different HPLC instruments

[0153]

[0154]

[0155] The results show that the characteristic peaks of the milk thistle formula granules were well resolved between different instruments, and differences between different instruments should be considered when specifying relative retention times. The average deviation of the relative retention times of the characteristic peaks in the characteristic spectra detected using the Thermo Vanquish, Waters ACQUITY UPLC H-Class, and Ailgent 1290infinity II chromatographs was less than 5%. The relative retention times of each characteristic peak were within ±10% of the specified values ​​for the relative retention times of the formula granules control. It is recommended that these values ​​be considered within the specified relative retention time range.

[0156] Example 11 Investigation of different chromatographic columns

[0157] The same batch of test solution was taken and the method for determining the characteristic spectrum of the milk thistle formula granules in Example 1 was used to set different types of chromatographic columns, namely YMC-Triart C18 (chromatographic column 1), SHIMADZU Shim-pack GIST C18-AQ (chromatographic column 2), and CAPCELL PAK ADME-HR (chromatographic column 3). The separation effect of each characteristic peak and the reference S peak was investigated when the chromatographic column was changed. The results are shown in Tables 19-20, and the separation results under different chromatographic columns are shown in Tables 19-20, respectively. Figure 12 As shown, (a), (b), and (c) represent the chromatographic columns YMC-Triart C 18、SHIMADZU Shim-pack GIST C 18 -AQ and CAPCELLPAK ADME-HR columns.

[0158] Table 19 Comparison of relative retention time determination results of different chromatographic columns

[0159]

[0160]

[0161] Table 20 Results of relative peak area tests on different chromatographic columns

[0162]

[0163] The results showed that when the chromatographic column was changed, the separation effect of each characteristic peak was affected, and the retention time was affected by the change of the chromatographic column model. In particular, the peak shape of the chromatographic peak on chromatographic column 3 (CAPCELL PAK ADME-HR) was poor. Therefore, it is recommended that the fixed chromatographic column YMC-Triart C18 of this method be used as the chromatographic column for subsequent research and investigation.

[0164] In summary, based on the above methodological findings, 13 common peaks were established for the characteristic spectrum of milk thistle granules. Each peak is affected to some extent by changes in chromatographic conditions, including the chromatographic column, HPLC instrument, column temperature, and flow rate. The relative retention times of each characteristic peak are within a ±10% range. This method uses Peak 4 (dithyrolactone) and Peak 11 (silybin A) as double S peaks and calculates the relative retention times of the two characteristic peaks before and after them. The relative retention times of each characteristic peak are significantly affected by instrument status and experimental environment. Based on comprehensive analysis, it is recommended that the relative retention time of each characteristic peak be controlled within a ±10% range.

[0165] Based on the above research results, we recommend using a YMC-Triart C18 2.1×100mm, 1.9μm column; using methanol as mobile phase A and 1% acetic acid solution as mobile phase B, with gradient elution according to the procedure specified in Table 1. The detection wavelength should be 287nm; the column temperature should be 30°C; and the flow rate should be 0.2ml / min. The theoretical plate number, calculated based on the Taxifolin peak, should be no less than 5000.

[0166] The chromatogram of the silymarin sample should show 13 characteristic peaks, which should correspond to the 13 characteristic peaks in the chromatogram of the control medicinal material. Among them, Peak 4, Peak 11, and Peak 12 should correspond to the retention time of the corresponding reference peaks. The peak corresponding to the taxifolin reference peak is the S1 peak, and the relative retention times of Peaks 1 to 6 and the S1 peak are calculated. The peak corresponding to the silymarin A reference peak is the S2 peak, and the relative retention times of Peaks 7 to 13 and the S2 peak are calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are 0.28 (Peak 1), 0.48 (Peak 2), 0.88 (Peak 3), 1.14 (Peak 5), 1.17 (Peak 6), 0.66 (Peak 7), 0.72 (Peak 8), 0.76 (Peak 9), 0.79 (Peak 10), and 1.18 (Peak 13).

[0167] Example 12 Characteristic Spectrum Determination of Multiple Batches of Milk Thistle Formula Granules

[0168] According to the characteristic spectrum method determined above, three batches of milk thistle formula granules (batch numbers: 211101Y, 211102Y, and 211103Y) were measured according to this method. The results are shown in Tables 21-22 below, and the spectra of each batch of milk thistle formula granules are shown in the attached Figure 13 As shown, (a), (b), and (c) represent the spectra of samples with batch numbers 211101Y, 211102Y, and 211103Y, respectively.

[0169] Table 21 Relative retention time test results of three batches of milk thistle formula granules

[0170]

[0171] Table 22 Relative peak areas of characteristic spectra of three batches of milk thistle formula granules

[0172]

[0173]

[0174] The above results show that the relative retention times of the three batches of milk thistle formula granules are all within the required range.

[0175] Example 13 Characteristic Spectrum Data and Fitting of Standard Decoction of Milk Thistle Pieces

[0176] 18 batches of standard decoction of milk thistle slices (lyophilized powder) (batch numbers: 1909001Y, 1909002Y, 1909005Y, 1909006Y, 1909008Y, 1909014Y, 1909015Y, 1909016Y, 1909017Y, 1909018Y, 1909019Y, 1909020Y, 1909022Y, 1909024Y, 1909025Y, 211001Y, 211002Y, 211003Y) were taken and operated according to the aforementioned [characteristic spectrum] determination method. The determination results are shown in Tables 23-24 below.

[0177] Table 23 Relative retention time determination results of characteristic spectra of 18 batches of standard decoction of milk thistle slices (lyophilized powder)

[0178]

[0179]

[0180]

[0181] Table 24 Relative peak area results of characteristic spectra of 18 batches of standard decoction of milk thistle slices (lyophilized powder)

[0182]

[0183]

[0184]

[0185]

[0186] The results showed that the characteristic spectra of 18 batches of standard decoctions (lyophilized powder) of milk thistle slices had a total of 13 characteristic peaks. Among them, peaks 4, 11, and 12 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the taxifolin reference peak is designated as peak S1, and the relative retention times of peaks 1-6 to S1 are calculated. The peak corresponding to the silybin A reference peak is designated as peak S2, and the relative retention times of peaks 7-13 to S2 are calculated. The relative retention times should be within ±10% of the specified values. According to the chromatographic peak identification results, peak 2 is chlorogenic acid, peak 4 is taxifolin, peak 9 is silybin, peak 11 is silybin A, peak 12 is silybin B, and peak 13 is isosilybin A. The remaining characteristic peaks have not yet been confirmed. Among them, peak 9 has a higher response and is the component with a higher concentration in silymarin, the main active ingredient in milk thistle that exerts a hepatoprotective effect. Therefore, by specifying the relative peak area of ​​Peak 9 and S2 (silybin A, a content determination indicator), semi-quantitative control of silybin is added to further strengthen the quality control of milk thistle. The relative peak area range of Peak 9 and S2 is: 2.289-3.152, with a minimum value of 80% and no less than 1.8.

[0187] Specified values ​​of characteristic maps

[0188] Based on the research results, the chromatogram of the silymarin sample should show 13 characteristic peaks, which should correspond to the chromatogram of the reference medicinal material. Peaks 4, 11, and 12 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the taxifolin reference peak is designated as S1, and the relative retention times of Peaks 1-6 to S1 are calculated. The peak corresponding to the silybin A reference peak is designated as S2, and the relative retention times of Peaks 7-13 to S2 are calculated. These relative retention times should be within ±10% of the specified values. The specified values ​​are 0.28 (Peak 1), 0.48 (Peak 2), 0.88 (Peak 3), 1.14 (Peak 5), 1.17 (Peak 6), 0.66 (Peak 7), 0.72 (Peak 8), 0.76 (Peak 9), 0.79 (Peak 10), and 1.18 (Peak 13).

[0189] In this embodiment, the relative retention time and relative peak area of ​​the obtained control characteristic spectrum and the control medicinal material characteristic spectrum results are shown in Tables 25-28 below. The characteristic spectra of 18 batches of standard decoction of milk thistle slices (lyophilized powder) (in the order of batch numbers 1909001Y, 1909002Y, 1909005Y, 1909006Y, 1909008Y, 1909014Y, 1909015Y, 1909016Y, 1909017Y, 1909018Y, 1909019Y, 1909020Y, 1909022Y, 1909024Y, 1909025Y, 211001Y, 211002Y, and 211003Y) are shown in the attached Figure 14 As shown in (a)-(r), the characteristic spectrum of the reference medicinal material of milk thistle and the characteristic spectrum of the reference decoction of milk thistle slices are shown in the attached Figure 15 As shown in (a)-(b), the reference spectrum was fitted using Mark peak fitting.

[0190] Table 25 Relative retention time of the comparison chart of milk thistle formula granules

[0191]

[0192] Table 26 Relative peak areas of the comparison spectrum of milk thistle formula granules

[0193]

[0194] Table 27 Relative retention time of characteristic spectrum of milk thistle control medicinal materials

[0195]

[0196] Table 28 Relative peak areas of characteristic spectrum of milk thistle control medicinal materials

[0197]

[0198] Example 14 is distinguished from burdock seeds and cloud wood fragrance seeds

[0199] The present invention consulted the literature and found that the common counterfeit products of milk thistle in the market are burdock seeds and cloud costus seeds. The appearance of these two traditional Chinese medicines is similar to milk thistle. Therefore, three batches of burdock seeds and three batches of cloud costus seeds were collected and prepared into standard decoctions of medicinal slices. The decoctions were then tested according to the aforementioned milk thistle characteristic spectrum method. The comparison of milk thistle-burdock seed characteristic spectrum and milk thistle-cloud costus seed characteristic spectrum are shown in the attached figure. Figure 16 As shown in (a)-(b), S1 is the characteristic spectrum of milk thistle.

[0200] As can be seen, the chemical composition of the seeds of Arctium lappa and Aucklandia lappa differs significantly from that of Milk Thistle. Specifically, the chromatographic peaks at 20-27 minutes and 49-60 minutes are unique to Milk Thistle. Peak 4 is taxifolin, and peaks 11, 12, and 13 are silybin A, silybin B, and isosilybin A, respectively. Arctium lappa and Aucklandia lappa exhibit a maximum peak around 36 minutes, representing arctiin, a unique component of these two varieties, with no corresponding characteristic peak in Milk Thistle. Chemical component analysis and reference standard localization revealed that the chromatographic peak following arctiin is 4,5-O-dicaffeoylquinic acid. A chromatographic peak in Milk Thistle with a similar elution time to 4,5-O-dicaffeoylquinic acid was confirmed as silybintin by HPLC-MS and reference standard localization. Therefore, despite similar retention times, these components are actually different chemical components. Arctium lappa and Aucklandia lappa exhibit a unique peak at 46 minutes (identified by LC / MS analysis as arctigenin). The presence or absence of arctiin and arctigenin can be used to distinguish milk thistle from common counterfeits. However, the presence of numerous chromatographic peaks near arctiin can easily lead to misidentification, so the absence of arctigenin is used as a distinguishing factor between milk thistle and common counterfeits.

[0201] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for constructing a characteristic spectrum of milk thistle and its preparation, characterized in that: The method comprises the steps of conducting ultra-high performance liquid chromatography on a test solution of milk thistle and its preparation; The chromatographic conditions included: methanol as mobile phase A, 1% acetic acid solution as mobile phase B, and gradient elution according to the following procedure: 0-15min, A:B is 15%:85%→25%:75%; 15-18 min, A:B 25%:75% → 28%:72%; 18-21 min, A:B is 28%:72%; 21-24 min, A:B 28%:72% → 33%:67%; 24-29 min, A:B 33%:67% → 36%:64%; 29-32 min, A:B 36%:64% → 39%:61%; 32-35 min, A:B 39%:61% → 45%:55%; 35-54min, A:B is 45%:55%; 54-60 min, A:B 45%:55% → 47%:53%; 60-61 min, A:B 47%:53% → 15%:85%; 61-65min, A:B is 15%:85%; The chromatographic column was YMC-Triart C18 2.1 × 100 mm, 1.9 μm; The detection wavelength was 287 nm, the flow rate was 0.2 ml / min, and the column temperature was 25°C; In the characteristic spectrum, peak 2 is chlorogenic acid, peak 4 is taxifolin, peak 9 is silybin, peak 11 is silybin A, peak 12 is silybin B, and peak 13 is isosilybin A; The preparation method of the test solution comprises: taking the test sample and precisely adding an organic solvent, ultrasonically treating it, filtering it and collecting the filtrate to obtain the test solution; the organic solvent comprises methanol with a volume concentration of 40-60 v / v%.

2. The method for constructing a characteristic spectrum of milk thistle and its preparation according to claim 1, characterized in that: The method further includes the step of preparing a control medicinal material reference solution, specifically comprising: taking a milk thistle control medicinal material, adding water to perform heating and reflux extraction, collecting the extract, filtering and evaporating to dryness, adding an organic solvent to the residue, ultrasonically treating it, filtering and collecting the filtrate to obtain the desired control medicinal material reference solution.

3. The method for constructing a characteristic spectrum of milk thistle and its preparation according to claim 1, characterized in that: The method further comprises the steps of preparing a reference substance solution and constructing a reference substance fingerprint based on the ultra-high performance liquid chromatography detection; The reference substance includes at least one of a silybin reference substance, a silybin A reference substance, and a taxifolin reference substance; The silybin reference substance consists of silybin A and silybin B.

4. The method for constructing a characteristic spectrum of milk thistle and its preparation according to claim 1, characterized in that: The power of the ultrasonic treatment step is 200-300W and the frequency is 30-50kHz.

5. The method for constructing a characteristic spectrum of milk thistle and its preparation according to any one of claims 1 to 4, characterized in that: The milk thistle preparation includes milk thistle formula granules, milk thistle slices or milk thistle slice decoction.

6. Application of the characteristic spectrum construction method of milk thistle and its preparation according to any one of claims 1 to 5 in the field of quality inspection of milk thistle and its pharmaceutical preparations.

7. A quality detection method for milk thistle and its pharmaceutical preparation, characterized in that: The method comprises the steps of constructing the characteristic map and the control characteristic map according to the method according to any one of claims 1 to 5, and comparing the characteristic map with the control characteristic map.

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