A method for constructing characteristic maps of safflower medicinal materials and safflower pharmaceutical preparations and their application

Abstract

The present invention belongs to the technical field of quality detection of Chinese medicine preparations, and specifically relates to a characteristic spectrum of safflower medicinal materials and safflower medicinal preparations, and further discloses a method for constructing the characteristic spectrum, and a quality detection method for safflower medicinal materials and safflower medicinal preparations. The method for constructing the characteristic spectrum of safflower medicinal materials and safflower medicinal preparations described in the present invention takes safflower medicinal materials and safflower medicinal preparations as detection objects, establishes a characteristic spectrum method for the pharmaceutical preparation based on high performance liquid chromatography, and uses Chinese medicine chromatographic fingerprints to calibrate 12 common characteristic peaks to determine the characteristics of safflower medicinal materials and safflower medicinal preparations. The method for constructing the characteristic spectrum of safflower medicinal materials and safflower medicinal preparations described in the present invention can fully reflect the intrinsic quality and medication safety of safflower medicinal materials and safflower medicinal preparations, is conducive to its comprehensive quality detection and overall quality control, and has the advantages of high stability, high precision and good repeatability.

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 spectrum of safflower medicinal materials and safflower medicinal preparations, and further discloses a method for constructing the characteristic spectrum, and a quality detection method for safflower medicinal materials and safflower medicinal preparations. Background Art

[0002] Traditional Chinese medicine has complex ingredients, and the effective part is often not a single ingredient. Using a single ingredient as a quality control indicator is increasingly incompatible with the requirements of quality control of traditional Chinese medicine. Therefore, traditional Chinese medicine fingerprint technology came into being. Traditional Chinese medicine fingerprint technology originated from fingerprint identification. It uses modern information technology and quality analysis methods to more comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine. The characteristic spectrum of traditional Chinese medicine is obtained by identifying the common peaks of the characteristics of various component groups. For traditional Chinese medicines, characteristic spectra can be used to identify authenticity and judge the quality; for Chinese patent medicines, characteristic spectra can identify product authenticity, judge the rationality of the preparation process, and effectively control product quality. At present, most of the effective ingredients of traditional Chinese medicines are still unclear. The integrity and ambiguity of the characteristic spectrum of traditional Chinese medicines just adapt to this feature, and are more scientific and comprehensive than the quality control method of a single ingredient. At present, the characteristic spectrum of traditional Chinese medicine has been recognized internationally as a control mode for the quality of traditional Chinese medicine. High performance liquid chromatography has the advantages of high separation efficiency and fast analysis speed, and has become the main analytical method for characteristic spectra today.

[0003] Safflower is the dried flower of Carthamus tinctorius L., a plant of the genus Carthamus in the Asteraceae family. It is also known as red and blue flower, and its aliases include grass safflower, thorny safflower, safflower dish, etc. Safflower is a traditional and commonly used Chinese medicinal material in my country with great medicinal value. Safflower is pungent in taste and warm in nature. It enters the heart and liver meridians. It has the effects of promoting blood circulation and relieving addiction, dispersing addiction and relieving pain, lowering blood pressure and lowering blood lipids. It has certain therapeutic effects in the treatment of coronary heart disease, hypertension, and cerebral hemorrhage. Safflower is included in the 2020 edition of the "Chinese Pharmacopoeia". There are many types of safflower on the market, and the ingredients are complex. The thin layer chromatography identification method is the most common, simple and fast identification method in the country's traditional Chinese medicine preparations. However, there are problems with separation effect, clarity and reproducibility.

[0004] Therefore, establishing a method that can comprehensively and systematically detect safflower medicinal materials and safflower pharmaceutical preparations, especially a method for establishing characteristic maps of safflower medicinal materials and safflower pharmaceutical preparations, is of great significance for their comprehensive quality inspection and overall quality control. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a method for constructing a characteristic spectrum of safflower medicinal materials and safflower medicinal preparations, so as to comprehensively reflect the intrinsic quality and medication safety of safflower medicinal materials and safflower medicinal preparations;

[0006] The second technical problem to be solved by the present invention is to provide a quality detection method for safflower medicinal materials and safflower pharmaceutical preparations.

[0007] In order to solve the above technical problems, the method for constructing a characteristic spectrum of safflower medicinal materials and safflower pharmaceutical preparations described in the present invention comprises the steps of performing high performance liquid chromatography detection on a test solution of safflower medicinal materials and / or safflower pharmaceutical preparations;

[0008] The chromatographic conditions include: using octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and 0.05% trifluoroacetic acid solution as a mobile phase B, and performing gradient elution according to the following procedure:

[0009] 0-8min, A:B is 10%:90%;

[0010] 8-15min, A:B is 10%:90%→15%:85%;

[0011] 15-25min, A:B is 15%:85%→17%:83%;

[0012] 25-35min, A:B is 17%:83%→28%:72%;

[0013] 35-40min, A:B is 28%:72%→42%:58%;

[0014] 40-45min, A:B is 42%:58%→50%:50%.

[0015] Preferably, in the HPLC detection step, the chromatographic conditions further include: column temperature 20-30°C, flow rate 0.5-1.0 ml / min, and detection wavelength 266 nm. Preferably, the column temperature is 25°C, and the flow rate is 0.7 ml / min.

[0016] Specifically, the method for constructing the characteristic spectrum of the safflower medicinal material and the safflower pharmaceutical preparation, the method for preparing the test solution includes: taking the test sample and accurately adding an organic solvent, sealing it with ultrasound, filtering and collecting the filtrate, and obtaining.

[0017] Specifically, the method for constructing the characteristic spectrum of safflower medicinal materials and safflower pharmaceutical preparations also includes the step of preparing a control medicinal material reference solution, which specifically includes: taking safflower control medicinal materials, adding water for heating and reflux extraction, collecting the extract, filtering and adding water-saturated n-butanol solvent for extraction, collecting the n-butanol phase and evaporating it, adding an organic solvent to the residue, plugging it with ultrasound, filtering and collecting the filtrate, and obtaining the desired control medicinal material reference solution.

[0018] Specifically, the method for constructing the characteristic spectrum of safflower medicinal materials and safflower pharmaceutical preparations also includes the steps of preparing a reference substance solution and constructing the characteristic spectrum of the reference substance based on the high performance liquid chromatography method;

[0019] The reference substance includes hydroxysafflor yellow A and / or kaempferol-3-O-rutinoside;

[0020] Preferably, the step of preparing the reference substance solution comprises the step of taking the selected reference substance and accurately adding an organic solvent to prepare the reference substance solution of the selected concentration;

[0021] Preferably, the concentration of each of the reference substances in the reference substance solution includes: 20-30 μg of hydroxysafflor yellow A and 20-30 μg of kaempferol-3-O-rutinoside per 1 ml of solution. Preferably, 25 μg of hydroxysafflor yellow A and 25 μg of kaempferol-3-O-rutinoside per 1 ml of solution.

[0022] Preferably, the organic solvent comprises methanol at a volume concentration of 5-15 v / v%;

[0023] The power of the ultrasonic step is 200-300W and the frequency is 30-50kHz.

[0024] Specifically, the method for constructing the characteristic spectrum of safflower medicinal materials and safflower pharmaceutical preparations, the safflower pharmaceutical preparations include safflower formula granules, safflower pieces or safflower standard decoction.

[0025] The present invention also discloses a characteristic spectrum and / or a reference characteristic spectrum of safflower medicinal materials and safflower pharmaceutical preparations. The characteristic spectrum or the reference characteristic spectrum of safflower medicinal materials and safflower pharmaceutical preparations are constructed by the method.

[0026] The present invention also discloses a method for constructing the characteristic spectrum of the safflower medicinal material and safflower pharmaceutical preparation and / or the application of the characteristic spectrum of the safflower medicinal material and safflower pharmaceutical preparation and / or a reference characteristic spectrum in the field of quality inspection of safflower medicinal material and safflower pharmaceutical preparation.

[0027] The present invention also discloses a quality detection method for safflower medicinal materials and safflower pharmaceutical preparations, comprising the steps of constructing the characteristic spectrum and a reference characteristic spectrum according to the method, and comparing the characteristic spectrum with the reference characteristic spectrum.

[0028] The method for constructing the characteristic spectrum of safflower medicinal materials and safflower pharmaceutical preparations of the present invention takes safflower medicinal materials and safflower pharmaceutical preparations as detection objects, establishes a characteristic spectrum method for the pharmaceutical preparation based on high performance liquid chromatography, calibrates 12 common characteristic peaks by using the Chinese medicine chromatographic fingerprint, confirms that peak 1 is syringin, peak 3 (S) is hydroxysafflor yellow A, peak 4 is tryptophan, peak 7 is kaempferol-3-O-sophoroside, peak 8 is rutin, peak 9 is kaempferol-3-O-rutinoside, peak 12 is safflor glycoside, selects peak 3 hydroxysafflor yellow A as a reference peak, specifies the relative retention time of each characteristic peak and the S peak, and the relative retention time should be within the range of ±10% of the specified value. The specified values ​​are: 0.70 (peak 1), 0.94 (peak 2), 1.20 (peak 4), 1.91 (peak 5), 1.95 (peak 6), 2.43 (peak 7), 2.65 (peak 8), 3.32 (peak 10), 3.39 (peak 11), 4.17 (peak 12), which determine the characteristics of safflower medicinal materials and safflower medicinal preparations. The method for constructing the characteristic spectrum of safflower medicinal materials and safflower medicinal preparations of the present invention can comprehensively reflect the intrinsic quality and drug safety of safflower medicinal materials and safflower medicinal preparations, is beneficial to their comprehensive quality detection and overall quality control, and has the advantages of high stability, high precision and good repeatability.

[0029] The method for constructing characteristic spectra of safflower medicinal materials and safflower pharmaceutical preparations described in the present invention can be applied to the construction of characteristic spectra of safflower pieces, safflower standard decoction freeze-dried powder, and safflower formula granules, and can realize quality detection and quality control of the above preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0031] Figure 1 The detection spectra under different chromatographic columns in Example 1;

[0032] Figure 2 Detection spectra at different detection wavelengths in Example 1;

[0033] Figure 3 The detection spectra under different mobile phase gradient control programs in Example 1;

[0034] Figure 4 Detection spectra under different mobile phase systems in Example 1;

[0035] Figure 5 The detection spectra at different mobile phase flow rates in Example 1;

[0036] Figure 6 The detection spectra at different column temperatures in Example 1;

[0037] Figure 7 Detection spectra under different instruments in Example 1;

[0038] Figure 8 It is the characteristic spectrum of the test sample in Example 2;

[0039] Fig. 9 This is the chromatogram of the test sample in Example 2 under ion mode;

[0040] Fig.10 is the specific localization map of the compound in Example 2;

[0041] Fig.11 The chromatograms of safflower and various reference substances in Example 2;

[0042] Fig.12 is the ultraviolet absorption spectrum of each compound in Example 2;

[0043] Fig.13 The chromatogram of the safflower medicinal material reference characteristic spectrum, the chromatogram of the safflower medicinal material test sample and the chromatogram of the hydroxysafflor yellow A reference substance in Example 3;

[0044] Fig.14 This is a common pattern diagram of instrument precision in Example 3;

[0045] Fig.15 This is a repetitive common pattern diagram in Example 3;

[0046] Fig.16 This is the common mode chromatogram of the intermediate precision of different personnel in Example 3;

[0047] Fig.17 The common mode chromatograms under different batches of chromatographic columns in Example 3;

[0048] Fig.18 It is the characteristic spectrum of the negative blank test product in Example 3;

[0049] Fig.19 The chromatogram is a common stability pattern diagram in Example 3;

[0050] Fig. 20 It is the chromatogram at different flow rates in Example 3;

[0051] Fig.21 The chromatograms at different column temperatures in Example 3 are shown below:

[0052] Fig. 22 The chromatograms of different instruments in Example 3 are shown below;

[0053] Fig.23 The characteristic spectra of multiple batches of safflower medicinal materials in Example 3;

[0054] Fig.24 The chromatogram of the safflower pieces for reference characteristic and the safflower pieces for test sample in Example 4;

[0055] Fig.25 The characteristic spectra of multiple batches of safflower pieces in Example 4;

[0056] Fig.26 The chromatogram of the safflower standard decoction and the safflower freeze-dried powder test sample in Example 5;

[0057] Fig. 27 This is a common pattern diagram of instrument precision in Example 5;

[0058] Fig.28 This is a repetitive common pattern diagram in Example 5;

[0059] Fig.29 This is the common mode chromatogram of the intermediate precision of different personnel in Example 5;

[0060] Fig.30 The common mode chromatograms under different batches of chromatographic columns in Example 5;

[0061] Fig.31 It is the characteristic spectrum of the negative blank test product in Example 5;

[0062] Fig.32 The chromatogram of the stability common pattern diagram in Example 5;

[0063] Fig.33 It is the chromatogram at different flow rates in Example 5;

[0064] Fig.34 The chromatograms at different column temperatures in Example 5 are shown below:

[0065] Fig.35 Characteristic spectra of multiple batches of safflower standard decoction freeze-dried powder in Example 5;

[0066] Fig.36 The chromatogram of the control characteristic spectrum of the safflower formula granules and the chromatogram of the test sample of the safflower formula granules in Example 6;

[0067] Fig.37 This is a common pattern diagram of instrument precision in Example 6;

[0068] Fig.38This is a repetitive common pattern diagram in Example 6;

[0069] Fig.39 This is the common mode chromatogram of the intermediate precision of different personnel in Example 6;

[0070] Fig.40 The common mode chromatograms under different batches of chromatographic columns in Example 6;

[0071] Fig.41 It is the characteristic spectrum of the negative blank test product in Example 6;

[0072] Fig.42 The chromatogram of the stability common pattern diagram in Example 6;

[0073] Fig.43 is the chromatogram at different flow rates in Example 6;

[0074] Fig.44 It is the chromatogram under different column temperatures in Example 6. DETAILED DESCRIPTION

[0075] In the following embodiments of the present invention, the instruments and reagents involved include:

[0076] High performance liquid chromatograph 1: Thermo U3000 chromatography system, including quaternary solvent manager (Pμmp), autosampler (Autosampler), original imported chromatography column oven (Colμmn Compartment), diode array ultraviolet detector (Detector), Chromeleon chromatography management system;

[0077] High performance liquid chromatograph 2: Waters e2695 chromatography system, including quaternary gradient infusion pump (Alliance 2695 model), 120-position high performance autosampler, imported column oven, Waters 2998 diode array UV detector, Empower chromatography management system;

[0078] High performance liquid chromatograph 3: Agilent 1260 chromatography system, including G1311B quaternary pump, G1367E autosampler, G1316A PDA diode array detector, G1330B column oven;

[0079] METTLER TOLEDO (Swiss Mettler) ME36S, XS204, XS205, XSE205 (one hundred thousandth); SK5200H Shanghai Kedao Ultrasonic Instrument Co., Ltd.;

[0080] Chromatographic column: Waters CORTECS HPLC T3 4.6×150nm 2.7μm; CAPCELL CORE C18 4.6×150nm 2.7μm; Waters CORTECS HPLC T3 2.1×150nm 1.6μm;

[0081] Acetonitrile was of chromatographic grade, water was ultrapure water; other reagents such as phosphoric acid, formic acid, trifluoroacetic acid, n-butanol, and methanol were of analytical grade;

[0082] Hydroxysafflor yellow A; reference substance (batch number: 111637-202111, purchased from China Food and Drug Inspection Institutes)

[0083] Kaempferol-3-O-rutinoside reference substance (batch number: 112007-202103, purchased from China Food and Drug Inspection Institute);

[0084] Carthamus tinctorius control medicinal material (batch number: 120907-201713, purchased from China Food and Drug Inspection Institute);

[0085] Safflower formula granules (batch numbers: 1903001Y, 1903002Y, 1903003Y).

[0086] Example 1 Characteristic Spectrum Construction Method Development

[0087] 1. Chromatographic column screening

[0088] Preliminary studies have confirmed that the main components of safflower slices after being decocted in water to prepare a standard decoction are flavonoids and flavonoid glycosides. These components in safflower freeze-dried powder are numerous and complex, with good water solubility and strong polarity. Therefore, the T3 chromatographic column with strong retention capacity was first considered, and comparisons were attempted in HPLC and UPLC at the same time. The specific chromatographic columns selected are as follows:

[0089] Waters CORTECS T3 4.6×150nm 2.7μm;

[0090] CAPCELL CORE C18 4.6×150nm 2.7μm;

[0091] Waters CORTECS C18 4.6×150nm 2.7μm;

[0092] Waters CORTECS T3 2.1x150mm 1.6um;

[0093] Waters HSS T3 2.1x150mm 1.8um.

[0094] The spectra formed by each chromatographic column are shown in the attached Figure 1 As shown, (a)-(e) represent the spectra of Waters CORTECS T3, CAPCELL CORE C18, Waters CORTECS C18, Waters CORTECS T3, and Waters HSS T3, respectively.

[0095] The results showed that when UPLC was used, the overall peak elution time was also longer, and the chromatographic peak shape and separation degree were not much better than those of HPLC. Therefore, HPLC was tentatively selected for method development. After comparison of chromatographic columns, the chromatographic peak distribution was more uniform under the CAPCELL CORE C18 chromatographic column. Therefore, the method of the present invention selected the CAPCELL CORE C18 (4.6×150nm 2.7μm) chromatographic column to continue the next step of optimization.

[0096] 2. Detection wavelength selection

[0097] First, the absorption spectrum of the main chromatographic peaks in the lyophilized powder of safflower standard decoction at 190-400nm was extracted. The results are shown in the attached figure. Figure 2 As shown, (a)-(d) represent the detection spectra at wavelengths of 220nm, 266nm, 280nm, and 403nm, respectively. It is found that the maximum absorption of the chromatographic peak is mainly concentrated near four bands (220nm, 266nm, 280nm, and 403nm), as shown below. At 266nm, the response of each characteristic peak is relatively average, and the baseline is relatively stable, so the detection wavelength is selected as 266nm.

[0098] 3. Gradient program optimization

[0099] Under the above detection conditions, the mobile phase gradient program was optimized with the information content of the chromatographic peaks, the separation degree of the chromatographic peaks, the analysis time, etc. as indicators. The specific optimized gradient is shown in Table 1.

[0100] Table 1 Gradient program control

[0101]

[0102] The detection spectrum under the above gradient program is attached. Figure 3 As shown, (a)-(c) respectively represent the detection spectra of gradient programs 1- 3. After the above investigation, gradient 2 has the best separation effect, so this method selects gradient 2 as the characteristic spectrum method.

[0103] 4. Mobile phase system selection

[0104] This example compares the separation effect of different mobile phase components on chromatographic peaks, and compares the chromatograms of different mobile phase systems such as acetonitrile-0.05% phosphoric acid, acetonitrile-0.05% formic acid, and acetonitrile-0.05% trifluoroacetic acid, using the information content of the chromatographic peaks and the system adaptability parameters as the selection indicators. The detection results under different mobile phases are shown in Tables 2-4, and the detection spectra are shown in Attachment Figure 4 As shown, (a)-(c) respectively represent the detection spectra under the mobile phase systems of acetonitrile-0.05% phosphoric acid, acetonitrile-0.05% formic acid, and acetonitrile-0.05% trifluoroacetic acid.

[0105] Table 2 Detection results of acetonitrile-0.05% phosphoric acid mobile phase system

[0106] name Retention time area high width Separation Symmetry Factor Theoretical plates 1 5.908 27481 4070 31.198 - 1.75 19947 2 6.553 70063 10356 48.496 3.98 1.56 25061 3 8.414 34867 3345 55.209 9.57 0.98 22638 4 11.226 54166 4107 48.51 9.9 1.1 16311 5 12.985 42913 3986 38.208 5.58 1.06 31932 6 17.824 22646 4284 14.903 22.61 1.2 254064 7 18.116 61734 9757 28.806 2.03 1.65 229080 8 22.81 23639 2473 30.406 23.8 1.8 137836 9 25.168 41753 2577 72.515 8.35 1.08 91633 10 31.668 140219 17917 32.707 25.04 1.22 385169 11 32.281 41560 6244 24.805 3.28 1.13 527585 12 33.357 38092 4610 25.005 5.52 0.97 392579 13 40.38 63958 13275 24.005 42.77 1.55 1992191 14 41.316 49873 9423 18.904 7.62 1.23 1556843 15 42.294 53824 11572 24.005 8.32 0.89 2795914

[0107] Table 3 Detection results of acetonitrile-0.05% formic acid mobile phase system

[0108]

[0109]

[0110] Table 4 Detection results of acetonitrile-0.05% trifluoroacetic acid mobile phase system

[0111] name Retention time area high width Separation Symmetry Factor Theoretical plates 1 6.654 66068 10262 20.898 - 1.25 26032.11 2 8.79 28874 3385 29.206 11.04 1.19 24245.01 3 9.543 52225 4021 37.508 2.7 1.24 12811.8 4 11.314 25290 2218 35.007 5.63 1.37 21431.67 5 13.655 48265 4411 33.407 8.07 1.09 36634.94 6 17.889 30695 5576 15.603 19.89 1.29 254643.8 7 18.196 59546 10623 22.205 2.23 1.83 282782.8 8 22.991 26231 2705 23.905 24.9 1.31 129331.5 9 25.379 33063 2717 42.309 8.94 1.05 121619.1 10 31.736 143690 19797 27.806 27.09 1.22 460676.3 11 32.322 43247 6832 25.305 3.36 1.15 614102.7 12 33.101 60505 7138 26.606 4.06 0.93 350433.8 13 40.384 56315 13237 14.503 44 1.12 2070108 14 41.325 62637 10974 22.805 7.5 1.44 1386263 15 42.303 53272 11624 24.105 8.08 0.81 2665514

[0112] Based on the above results, this method determines acetonitrile-0.05% trifluoroacetic acid as the mobile phase system for the characteristic spectrum detection method.

[0113] 5. Investigation of different flow rates

[0114] Since safflower drug preparations have many ingredients and complex chromatographic peaks, considering that the flow rate may have a certain impact on its separation effect, the test solution of the same standard decoction freeze-dried powder sample was taken and tested at flow rates of 0.6ml / min, 0.7ml / min, and 0.8ml / min according to the above selected mobile phase and gradient program. The results are shown in Tables 5-7 below and the detection spectrum results are shown in the attached Figure 5 , where (a)-(c) represent the detection spectra at flow rates of 0.6 ml / min, 0.7 ml / min, and 0.8 ml / min, respectively.

[0115] Table 5 Test results at flow rate 0.6 ml / min

[0116]

[0117]

[0118] Table 6 Test results at flow rate 0.7 ml / min

[0119] Peak Retention time Peak area Peak height Peak width (50%) Asymmetry Separation Number of plates 1 5.88 0.9721 11.16 0.078 1.26 8.22 31257 2 7.38 0.4596 2.72 0.137 2.25 2.88 16085 3 8.117 2.024 11.03 0.165 1.23 6.64 13395 4 10.173 0.3938 1.86 0.2 1.75 26.29 14275 5 16.603 0.4684 4.12 0.088 na 2.22 196549 6 16.92 1.1184 11.16 0.08 1.52 22.53 245941 7 21.037 0.5576 3.91 0.135 1.25 8.19 133953 8 22.857 0.4722 3.13 0.127 1.56 24.44 179197 9 28.473 1.6007 10 0.144 1.21 3.48 216348 10 29.26 1.0199 7.44 0.123 1.19 4.16 315615 11 30.237 1.7916 10.22 0.155 1.18 36.87 211556 12 37.97 0.9381 8.98 0.093 1.4 17.27 927837

[0120] Table 7 Test results at flow rate 0.8 ml / min

[0121]

[0122] The above results show that when the flow rate is 0.7 ml / min, the separation effect of each proposed characteristic peak is the best. This method selects a flow rate of 0.7 ml / min for subsequent research.

[0123] 6. Investigation of different column temperatures

[0124] Take the standard decoction freeze-dried powder test solution and inject it into the liquid chromatograph to investigate the separation effect of the standard decoction freeze-dried powder at column temperatures of 20°C, 25°C, and 30°C. The results are shown in Tables 8-10 below, and the detection spectrum results are shown in the attached Figure 6 , where (a)-(c) represent the detection spectra at column temperatures of 20°C, 25°C, and 30°C, respectively.

[0125] Table 8 Test results at column temperature 20°C

[0126] Peak Retention time Peak area Peak height Peak width (50%) Asymmetry Separation Number of plates 1 6.083 0.989 10.62 0.083 1.22 11.9 29706 2 8.103 0.4245 3.22 0.117 1.3 3.31 26466 3 8.89 1.9243 10.72 0.163 1.31 7.69 16477 4 11.283 0.5592 2.21 0.204 1.32 24.71 16945 5 17.227 0.3585 4.01 0.08 1.39 2.61 258213 6 17.577 0.9697 10.97 0.079 1.22 27.82 277104 7 22.18 0.3784 3 0.117 1.11 9.09 200281 8 24.073 0.3539 2.54 0.129 1.19 27.26 192173 9 29.7 1.6514 12.7 0.114 1.35 1.99 373802 10 30.063 0.9881 8.59 0.101 1.13 3.93 488953 11 30.933 1.5227 8.61 0.16 0.84 34.52 206832 12 38.13 0.9583 9.28 0.086 1.58 na 1091746

[0127] Table 9 Test results at column temperature 25°C

[0128] Peak Retention time Peak area Peak height Peak width (50%) Asymmetry Separation Number of plates 1 5.88 0.9721 11.16 0.078 1.26 8.22 31257 2 7.38 0.4596 2.72 0.137 2.25 2.88 16085 3 8.117 2.024 11.03 0.165 1.23 6.64 13395 4 10.173 0.3938 1.86 0.2 1.75 26.29 14275 5 16.603 0.4684 4.12 0.088 na 2.22 196549 6 16.92 1.1184 11.16 0.08 1.52 22.53 245941 7 21.037 0.5576 3.91 0.135 1.25 8.19 133953 8 22.857 0.4722 3.13 0.127 1.56 24.44 179197 9 28.473 1.6007 10 0.144 1.21 3.48 216348 10 29.26 1.0199 7.44 0.123 1.19 4.16 315615 11 30.237 1.7916 10.22 0.155 1.18 36.87 211556 12 37.97 0.9381 8.98 0.093 1.4 17.27 927837

[0129] Table 10 Detection results at column temperature 30°C

[0130]

[0131]

[0132] The above test results show that when the column temperature is 25°C, the separation effect of each characteristic peak is better. Therefore, this method selects a column temperature of 25°C for detection.

[0133] 7. Durability test of different instruments

[0134] According to the above determined method and parameter conditions, the samples were injected on chromatographs of different brands (Agilent 1260, Waters e2695 and Thermo Fisher U3000) to investigate the durability of different instruments. The detection spectrum results are shown in the attached Figure 7 , where (a)-(c) represent the detection spectra of Agilent 1260, waters e2695 and Thermo U3000 instruments respectively. The results show that the durability of different instruments is good.

[0135] In summary, based on the above investigation, the chromatographic conditions selected in this embodiment are: octadecylsilane bonded silica gel as filler; acetonitrile as mobile phase A, 0.05% trifluoroacetic acid solution as mobile phase B, gradient elution according to the provisions in Table 11 below; detection wavelength of 266nm; flow rate of 0.7ml / min, and the theoretical plate number calculated based on the hydroxysafflor yellow A peak should be not less than 5000.

[0136] Table 11 Mobile phase gradient program

[0137] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0-8 10 90 8-15 10→15 90→85 15-25 15→17 85→83 25-35 17→28 83→72 35-40 28→42 72→58 40-45 42→50 58→50

[0138] Example 2 Identification of characteristic peaks in [Characteristic spectrum]

[0139] According to the characteristic spectrum method determined in the above embodiment 1, a sample of safflower formula granules to be tested was taken for testing. The test spectrum is shown in the attached Figure 8 As shown, the chromatogram under ion mode is attached Fig. 9 shown.

[0140] Combined with the results of multiple batches of measurements, the characteristic peaks were reselected, and the identification of the characteristic peaks was studied in depth. The safflower samples were analyzed by ultra performance liquid chromatography-high resolution mass spectrometry (UPLC-Q-TOF / MS). According to the multi-level mass spectrometry information of the samples, combined with the natural product high-resolution mass spectrometry database and related literature, the target peaks were identified. The reference substances were used for positioning and spectral research. The identification results of the target components are shown in Table 12 below, confirming that Peak 1 is syringin, Peak 3 is hydroxysafflor yellow A, Peak 4 is tryptophan, Peak 7 is kaempferol-3-O-sophoroside, Peak 8 is rutin, Peak 9 is kaempferol-3-O-rutinoside, and Peak 12 is safflower acetyl glycoside.

[0141] The structure was analyzed by LC / MS / MS, and the specific localization map is attached. Fig.10 As shown, (a)-(h) represent the MS corresponding to compound 1 (RT = 6.70min), compound 5 (RT = 17.00min), compound 6 (RT = 17.28min), compound 7 (RT = 21.42min), compound 8 (RT = 23.37min), compound 9 (RT = 29.06min), compound 10 (RT = 29.76min), and compound 12 (RT = 37.79min), respectively. 1 and MS 2 .

[0142] Table 12 Identification results of safflower target components

[0143]

[0144] Based on the characteristic peaks determined above, the chromatograms of safflower and various reference substances are shown in the attached Fig.11 As shown, in which, from top to bottom in Figure (b) are tryptophan reference, safflower formula granule sample, hydroxysafflor yellow A reference, respectively, in Figure (c) from top to bottom are safflower acetyl glucoside reference and safflower formula granule sample, respectively, Figure (d) is the chromatogram of hydroxysafflor yellow A reference, and Figure (e) is the chromatogram of tryptophan reference.

[0145] The UV absorption spectra of hydroxysafflor yellow A, tryptophan, syringin, kaempferol-3-O-sophoroside, rutin, kaempferol 3-O-rutinoside, and saffloridine are shown in the attached figure. Fig.12 As shown in (a)-(g), 1 represents the spectrum of the reference substance and 2 represents the spectrum of the compound contained in the sample.

[0146] In the following examples of the present invention, in order to clarify the transfer of quantity values ​​in the entire research process, it is necessary to verify the analytical method and conduct multiple batch tests on safflower medicinal materials and safflower pharmaceutical preparations in different dosage forms with reference to the optimized HPLC characteristic spectrum determination method. The verification results show that the method is simple to operate, the results are accurate, and the method has good reproducibility.

[0147] Example 3 Characteristic spectrum of safflower medicinal material

[0148] 1. Feature map creation

[0149] [Characteristic spectrum] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0150] Chromatographic conditions and system suitability test

[0151] Octadecylsilane bonded silica gel is used as filler, chromatographic column CAPCELL CORE C18 (4.6×150nm, 2.7μm); acetonitrile is used as mobile phase A, 0.05% trifluoroacetic acid solution is used as mobile phase B, gradient elution is performed according to the provisions in the above Table 11; detection wavelength is 266nm; flow rate is 0.7ml / min. The theoretical plate number calculated based on the hydroxysafflor yellow A peak should not be less than 5000.

[0152] Preparation of reference solution

[0153] Take 0.5 g of safflower reference medicinal material, place it in a stoppered conical flask, add 25 ml of water, heat and reflux for 30 minutes, take it out, filter, and extract the filtrate twice with water-saturated n-butanol, 25 ml each time, combine the n-butanol liquids, evaporate to dryness, cool, add 20 ml of 10% methanol to the residue, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate to obtain the reference medicinal material reference solution.

[0154] Take an appropriate amount of hydroxysafflor yellow A and kaempferol-3-O-rutinoside reference substances, weigh them accurately, add 10% methanol to make solutions containing 25 μg of hydroxysafflor yellow A and 25 μg of kaempferol-3-O-rutinoside per ml, respectively, as reference substance solutions.

[0155] Preparation of test solution

[0156] Take 0.5g of safflower, put it in a stoppered conical flask, add 25ml of water, heat and reflux for 30 minutes, take it out, filter, extract the filtrate twice with water-saturated n-butanol, 25ml each time, combine the n-butanol liquid, evaporate to dryness, cool, add 20ml of 10% methanol to the residue, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, filter, and take the filtrate to obtain.

[0157] Determination method

[0158] Accurately pipette 5 μl each of the reference solution, control medicinal solution, and test solution, inject into the liquid chromatograph for determination.

[0159] The obtained safflower medicinal material reference characteristic spectrum, safflower medicinal material test sample chromatogram and hydroxysafflower yellow A reference sample chromatogram are shown in the attached Fig.13 As shown in (a)-(c), peak 1: syringin; peak 3 (S): hydroxysafflor yellow A; peak 4: tryptophan; peak 7: kaempferol-3-O-sophoroside; peak 8: rutin; peak 9: kaempferol-3-O-rutinoside; peak 12: saffloridine.

[0160] As can be seen from the results in the attached figure, there should be 12 characteristic peaks in the chromatogram of the test sample, and they should correspond to the 12 characteristic peaks in the chromatogram of the reference medicinal material. The retention time of peak 3 and peak 9 should correspond to the retention time of the corresponding reference peak of the reference substance. The peak of the hydroxysafflor yellow A reference substance is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, which should be within the range of ±10% of the specified value. The specified values ​​are: 0.70 (peak 1), 0.94 (peak 2), 1.20 (peak 4), 1.91 (peak 5), 1.95 (peak 6), 2.43 (peak 7), 2.65 (peak 8), 3.32 (peak 10), 3.39 (peak 11), 4.17 (peak 12). The relative peak area of ​​peak 9, peak 12 and S peak should not be less than 1.0 (peak 9) and 0.40 (peak 12).

[0161] 2. System adaptability

[0162] Based on the above detection method, the system adaptability parameter results of the method described in this embodiment are shown in Table 13 below.

[0163] Table 13 System adaptability parameters

[0164]

[0165]

[0166] 3. Precision verification

[0167] Take 0.5g of safflower, weigh accurately, prepare the test sample according to the method of "Preparation of sample solution" above, inject 6 times continuously, record the chromatogram, calculate the relative retention time and relative peak area RSD% of each characteristic peak, and the results are shown in Tables 14-15 below. The common pattern diagram of instrument precision is shown in the attached figure. Fig.14 As shown, from bottom to top, they correspond to the spectra of precision 1, 2, 3, 4, 5, and 6 respectively.

[0168] Table 14 Instrument precision relative retention time test results

[0169]

[0170] Table 15 Instrument precision relative peak area test results

[0171]

[0172]

[0173] The above results show that the relative retention time RSD% of the 12 characteristic peaks is less than 2%, and the relative peak area RSD% is less than 5%. Comprehensively judging, the precision of the method is good and meets the requirements of the characteristic spectrum.

[0174] 4. Method repeatability test

[0175] Take 0.5g of the same batch of safflower medicinal materials, weigh 6 portions in parallel, accurately weigh, prepare the test sample according to the method of "Preparation of Test Sample Solution" mentioned above, inject and analyze, record the chromatogram, calculate the relative retention time and relative peak area RSD% of each characteristic peak, the results are shown in Tables 16-17 below, and the reproducibility common pattern diagram is shown in the attached Fig.15 As shown, from bottom to top, they correspond to the spectra of repeatability 1, 2, 3, 4, 5, and 6 respectively.

[0176] Table 16 Method repeatability relative retention time test results (n = 6)

[0177]

[0178] Table 17 Method repeatability relative peak area test results (n = 6)

[0179]

[0180]

[0181] The above results show that the relative retention time RSD% of the 12 characteristic peaks is less than 2%, and the relative peak area RSD% is less than 3%. Comprehensively judging, the repeatability of this method is good and meets the requirements.

[0182] 5. Intermediate precision (different operators)

[0183] The same batch of safflower medicinal materials was taken and independently operated by experimenters A, B and C. The test samples were prepared according to the test sample preparation method, sampled and analyzed, chromatograms were recorded, and the relative retention time and relative peak area RSD% of each characteristic peak were calculated. The results are shown in Tables 18-19 below. The chromatograms of the common modes of intermediate precision among different personnel are shown in the attached table. Fig.16 As shown, from bottom to top they correspond to personnel A, B, and C respectively.

[0184] Table 18 Intermediate precision relative retention time test results (different operators)

[0185]

[0186] Table 19 Intermediate precision relative peak area test results (different operators)

[0187]

[0188] The above results show that the relative retention time RSD% of the 12 characteristic peaks is less than 2%, and the relative peak area RSD% is less than 5%. Comprehensively judging, the intermediate precision (different personnel) of the method is good and meets the requirements of the characteristic spectrum.

[0189] 6. Intermediate precision (different column batches)

[0190] Through the above-mentioned chromatographic condition optimization and chromatographic column selection results, it can be known that the chromatographic condition reproduction requires a fixed CAPCELLCORE C18; 4.6mm×150mm, 2.7um chromatographic column. Therefore, the same test solution was taken and tested with chromatographic columns of different production batches (batch number A34AB10165; batch number A34AB10192), and the chromatograms were recorded. The relative retention time and relative peak area RSD% of each characteristic peak were calculated. The results are shown in Tables 20-21 below. The chromatograms under different chromatographic column conditions are shown in Tables 20-21 below. Fig.17 As shown, (a) and (b) represent the test results of batch number A34AB10165 and batch number A34AB10192, respectively.

[0191] Table 20 Relative retention time results of different chromatographic column batches

[0192]

[0193] Table 21 Relative peak area results of different chromatographic column batches

[0194]

[0195] The results show that the relative retention time RSD% of the 12 characteristic peaks is less than 2%, and the relative peak area RSD% of the 12 characteristic peaks is less than 4%. It can be seen from the figure that different chromatographic column batches have little effect on peak separation.

[0196] 7. Exclusive

[0197] Prepare the test solution according to the test solution preparation method, and examine whether the negative sample of safflower medicinal material will cause interference. Perform HPLC analysis according to the chromatographic conditions described under [Characteristic Spectrum] and record the chromatogram as shown in the attached Fig.18 As shown, the results showed that the negative had no interference and the method had good specificity.

[0198] 8. Stability test

[0199] Take the same sample solution and inject it at 0, 4, 8, 12, 16, and 24 hours after preparation. Record the chromatogram, calculate the relative retention time and RSD% of the relative peak area of ​​each characteristic peak, and examine the stability of the sample solution. The results are shown in Tables 22-23 below. The stability common pattern chromatogram is attached. Fig.19 As shown, from bottom to top they correspond to stability 0h, 4h, 8h, 12h, 16h, and 24h respectively.

[0200] Table 22 Stability relative retention time test results

[0201]

[0202]

[0203] Table 23 Stability relative peak area test results

[0204]

[0205] The results showed that the relative retention time of the 12 characteristic peaks was less than 2%, and the relative peak area RSD% was less than 6%, indicating that the test solution was stable within 24 hours.

[0206] 9. Investigation of different flow rates

[0207] Take the same test solution and test it at different flow rates of 0.68ml / min, 0.70ml / min and 0.72ml / min. Calculate the RSD% of the relative retention time and relative peak area of ​​each characteristic peak. The results are shown in Tables 24-25 below. Record the chromatogram as shown in the attached figure. Fig. 20 As shown, (a)-(c) represent the spectra at flow rates of 0.68 ml / min, 0.70 ml / min, and 0.72 ml / min, respectively.

[0208] Table 24 Relative retention time results at different flow rates

[0209]

[0210] Table 25 Relative peak area results at different flow rates

[0211]

[0212] The results show that the relative retention time RSD% of the 12 characteristic peaks is less than 2%, and the relative peak area RSD% of the 12 characteristic peaks is between 0% and 5%. It can be seen from the figure that a slight change in flow rate has little effect on the separation of the characteristic peaks. In order to ensure the separation and reproducibility of the characteristic spectrum, it is shown that the present invention selects 0.70 ml / min as the flow rate effect of the method for determining the characteristic spectrum of safflower medicinal materials for the best effect.

[0213] 10. Investigation of different column temperatures

[0214] Take the same test solution and test it at 23℃, 25℃ and 27℃ respectively, record the chromatogram, calculate the RSD% of the relative retention time and relative peak area of ​​each characteristic peak, and the results are shown in Table 26-27 below. The chromatograms at different column temperatures are shown in Table 26-27 below. Fig.21 As shown, (a)-(c) represent the chromatograms at column temperatures of 23°C, 25°C and 27°C, respectively.

[0215] Table 26 Relative retention time results of different column temperatures

[0216]

[0217]

[0218] Table 27 Relative peak area results at different column temperatures

[0219]

[0220] The results show that the relative retention time of the 12 characteristic peaks is affected by the flow rate to a certain extent, and the relative peak area RSD% of the 12 characteristic peaks is between 0% and 5%. It can be seen from the figure that different column temperatures have a slight effect on the separation of the characteristic peaks. In order to ensure the reproducibility of the separation of the characteristic spectrum, it is proved that the present invention selects a column temperature of 25°C for the best effect.

[0221] 11. Investigation of different instruments

[0222] The same test solution was tested under Thermo U3000, waters e2695 and Agilent 1260 respectively. The chromatograms were recorded and the relative retention time and relative peak area RSD% of each characteristic peak were calculated. The results are shown in Tables 28-29 below. The detection spectra under different instruments are shown in the attached table. Fig. 22 As shown, (a)-(c) represent the chromatograms under the conditions of Waters e2695, Thermo U3000 or Agilent 1260 instruments, respectively.

[0223] Table 28 Relative retention time results of different instruments

[0224]

[0225] Table 29 Relative peak area results measured by different instruments

[0226]

[0227] The above results show that the relative retention times of the 12 characteristic peaks are all within the range of ±10% of the specified values, and the relative peak area RSD% of the 12 characteristic peaks is between 0% and 8.39%. This may be because the response values ​​of the characteristic peaks on different instruments are slightly different. As can be seen from the figure, different instrument conditions have little effect on the separation of the characteristic peaks.

[0228] In summary, the above methodological investigation results show that among the 12 common peaks of the established safflower medicinal material [characteristic spectrum], different column temperatures and different flow rates have a certain degree of influence on each chromatographic peak, and the influence of other chromatographic conditions is not significant. The relative retention time value is within the range of ±10%. In order to adapt to its durability, the specified value range is controlled within ±10%. 12. Determination of characteristic spectra of multiple batches of safflower medicinal materials

[0229] Take safflower medicinal materials (batch numbers: K17030801, K17030802, K17030803, G1611197, G1611198, G1611199, Q2017-01, Q2017-02, Q2017-03, T2017-01, T2017-02, T2017-03, J2017-01, J2017-02, J2017-03) and operate according to the proposed safflower medicinal material [characteristic spectrum] determination method. The test results are shown in Tables 30-31 below. The characteristic spectra of multiple batches of safflower medicinal materials are shown in the attached Fig.23As shown, the corresponding medicinal material batch numbers from bottom to top are K17030801, K17030802, K17030803, G1611197, G1611198, G1611199, Q2017-01, Q2017-02, Q2017-03, T2017-01, T2017-02, T2017-03, J2017-01, J2017-02, and J2017-03.

[0230] The measurement results show that the relative retention times of the characteristic peaks of the characteristic spectra of multiple batches of safflower medicinal materials are all within the range of ±10% of the values ​​specified in the reference spectra.

[0231] Table 30 Relative retention time of multiple batches of safflower medicinal materials

[0232]

[0233]

[0234] Table 31 Relative peak areas of multiple batches of safflower medicinal materials

[0235]

[0236]

[0237] Relative retention time specified value: Determined based on the results of characteristic spectra of multiple batches of safflower medicinal materials: 12 characteristic peaks should be present in the chromatogram of the test sample, and should correspond to the 12 characteristic peaks in the chromatogram of the reference medicinal material, among which the retention time of peak 3 and peak 9 should correspond to the retention time of the corresponding reference peak of the reference material; the peak of the hydroxysafflor yellow A reference material is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, which should be within the range of ±10% of the specified value. The specified values ​​are: 0.70 (peak 1), 0.94 (peak 2), 1.20 (peak 4), 1.91 (peak 5), 1.95 (peak 6), 2.43 (peak 7), 2.65 (peak 8), 3.32 (peak 10), 3.39 (peak 11), 4.17 (peak 12).

[0238] Relative peak area ratio specified value: The main chemical components of safflower include flavonoids, alkaloids, amino acids, fatty acids, and polysaccharides. The flavonoid components of safflower are currently recognized as the main active ingredients, mainly including safflower quinone glycosides, safflower yellow A, hydroxysafflower yellow A, hydroxysafflower yellow B, neokaempferol, kaempferol, kaempferol-3-O-rutinoside, 6-hydroxykaempferol, kaempferol-3-O-β-D-glucosyl-(1→2)-β-D-glucoside, quercetin-3,7-di-O-β-D-glucoside, etc. Among them, safflower yellow is currently the most important active ingredient of safflower. Through the detection of characteristic spectra of multiple batches of safflower combined with the characteristic spectrum peak identification information, peak 1 is syringin, peak 3 is hydroxysafflor yellow A, peak 7 is kaempferol-3-O-sophoroside, peak 8 is rutin, peak 9 is kaempferol-3-O-rutinoside, peak 12 is safflower amygdalus glycoside, all of which are flavonoid components, peak 4 is tryptophan, which is an amino acid component. Hydroxysafflor yellow A has good stability throughout the research process. At the same time, as the main efficacy indicator, it is more suitable as the S peak to calculate the relative peak areas of the other characteristic peaks for control. As identification ingredients, syringin and tryptophan are not very specific in safflower, and the relative peak areas of kaempferol-3-O-sophoroside and rutin to the S peak are relatively too small. Therefore, stipulating their relative peak area values ​​will make them of little significance in the final product standard; the relative peak area range of peak 9 is 1.32-1.63, and the relative peak area range of peak 12 is 0.54-0.68. From the results, it can be seen that in the process of preparing medicinal materials into decoction pieces and then into safflower standard decoction freeze-dried powder, peaks 9 and 12 have good transferability. Therefore, based on multiple batches of data, the relative peak area of ​​peak 9 is set to 1.049, which is rounded to 1.0, based on 80% of the lower limit of multiple batches, and the relative peak area of ​​peak 12 is set to 0.432, which is rounded to 0.40, based on 80% of the lower limit of multiple batches.

[0239] Embodiment 4 Carthamus tinctorius decoction pieces

[0240] 1. Feature map creation

[0241] [Characteristic spectrum] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0242] Chromatographic conditions and system suitability

[0243] Octadecylsilane bonded silica gel was used as filler, chromatographic column was CAPCELL CORE C18 (4.6×150nm, 2.7μm); acetonitrile was used as mobile phase A, 0.05% trifluoroacetic acid solution was used as mobile phase B, gradient elution was performed according to the provisions in the above Table 11; detection wavelength was 266nm; flow rate was 0.7mL / min. The theoretical plate number calculated based on the hydroxysafflor yellow A peak should not be less than 5000.

[0244] Preparation of reference solution

[0245] Take 0.5 g of safflower reference medicinal material, place it in a stoppered conical flask, add 25 ml of water, heat and reflux for 30 minutes, take it out, filter, and extract the filtrate twice with water-saturated n-butanol, 25 ml each time, combine the n-butanol liquids, evaporate to dryness, cool, add 20 ml of 10% methanol to the residue, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate to obtain the reference medicinal material reference solution.

[0246] Take an appropriate amount of hydroxysafflor yellow A and kaempferol-3-O-rutinoside reference substances, weigh them accurately, add 10% methanol to prepare solutions containing 25 μg of hydroxysafflor yellow A and 25 μg of kaempferol-3-O-rutinoside per 1 mL, respectively, as reference substance solutions.

[0247] Preparation of test solution

[0248] Take 0.5g of safflower slices, put it in a stoppered conical flask, add 25ml of water, heat and reflux for 30 minutes, take it out, filter, extract the filtrate twice with water-saturated n-butanol, 25ml each time, combine the n-butanol liquids, evaporate to dryness, cool, add 20ml of 10% methanol to the residue, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, filter, and take the filtrate to obtain.

[0249] Determination method: Accurately pipette 5 μL of reference solution, control medicinal material solution, and test solution, inject into liquid chromatograph, and determine.

[0250] The obtained safflower slices reference characteristic spectrum and safflower slices test sample chromatogram are shown in the attached Fig.24 As shown in (a)-(b), peak 1: syringin; peak 3 (S): hydroxysafflor yellow A; peak 4: tryptophan; peak 7: kaempferol-3-O-sophoroside; peak 8: rutin; peak 9: kaempferol-3-O-rutinoside; peak 12: saffloridine.

[0251] It can be seen that there should be 12 characteristic peaks in the chromatogram of the test sample, and they should correspond to the 12 characteristic peaks in the chromatogram of the reference medicinal material. The retention time of peak 3 and peak 9 should correspond to the retention time of the corresponding reference peak of the reference substance. The peak of the hydroxysafflor yellow A reference substance is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, which should be within the range of ±10% of the specified value. The specified values ​​are: 0.70 (peak 1), 0.94 (peak 2), 1.20 (peak 4), 1.91 (peak 5), 1.95 (peak 6), 2.43 (peak 7), 2.65 (peak 8), 3.32 (peak 10), 3.39 (peak 11), 4.17 (peak 12). The relative peak area of ​​peak 9, peak 12 and S peak should not be less than 1.0 (peak 9) and 0.40 (peak 12).

[0252] 2. System adaptability

[0253] The chromatographic conditions were verified by referring to the method of Safflower Medicinal Material [Characteristic Spectrum]. The specific results are shown in Table 32 below. The results show that this method is applicable to Safflower Pieces.

[0254] Table 32 System adaptability parameters

[0255]

[0256] 3. Determination of characteristic spectra of multiple batches of safflower slices

[0257] Take multiple batches of safflower slices (batch numbers: K17030801, K17030802, K17030803, G1611197, G1611198, G1611199, Q2017-01, Q2017-02, Q2017-03, T2017-01, T2017-02, T2017-03, J2017-01, J2017-02, J2017-03) and operate according to the proposed safflower slice [characteristic spectrum] determination method. The determination results are shown in Tables 33-34 respectively. The characteristic spectra of multiple batches of safflower slices are shown in the attached Fig.25 As shown, from bottom to top they correspond to samples K17030801, K17030802, K17030803, G1611197, G1611198, G1611199, Q2017-01, Q2017-02, Q2017-03, T2017-01, T2017-02, T2017-03, J2017-01, J2017-02, and J2017-03.

[0258] Table 33 Relative retention time results of characteristic spectra of multiple batches of safflower slices

[0259]

[0260] Table 34 Results of relative peak area determination of characteristic spectra of multiple batches of safflower slices

[0261]

[0262]

[0263] The measurement results show that the relative retention times of the characteristic peaks in the characteristic spectrum of safflower pieces are all within the range of ±10% of the values ​​specified in the reference spectrum of safflower pieces.

[0264] Relative retention time specified value: Determined based on the results of characteristic spectra of multiple batches of safflower slices: 12 characteristic peaks should be present in the chromatogram of the test sample, and should correspond to the 12 characteristic peaks in the chromatogram of the reference medicinal material, among which the retention time of peak 3 and peak 9 should correspond to the retention time of the corresponding reference peak of the reference substance; the peak of the hydroxysafflor yellow A reference substance is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, which should be within the range of ±10% of the specified value. The specified values ​​are: 0.70 (peak 1), 0.94 (peak 2), 1.20 (peak 4), 1.91 (peak 5), 1.95 (peak 6), 2.43 (peak 7), 2.65 (peak 8), 3.32 (peak 10), 3.39 (peak 11), 4.17 (peak 12).

[0265] Relative peak area regulations: Safflower is prepared into decoction pieces after simple purification. The processing process has little effect on the number and peak area of ​​characteristic peaks, so the relative peak area value of safflower decoction pieces is implemented with reference to the medicinal materials. That is, the relative peak area of ​​characteristic peaks 9, 12 and S peak should not be less than 1.0 (peak 9) and 0.40 (peak 12).

[0266] Example 5: Safflower Standard Decoction Freeze-dried Powder

[0267] 1. Feature Spectrum Method

[0268] [Characteristic spectrum] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0269] Chromatographic conditions and system suitability

[0270] Octadecylsilane bonded silica gel is used as filler, chromatographic column: CAPCELL CORE C18; 4.6mm×150mm, 2.7um; acetonitrile is used as mobile phase A, 0.05% trifluoroacetic acid solution is used as mobile phase B, gradient elution is performed according to the provisions in the above Table 11; detection wavelength is 266nm; flow rate is 0.7ml / min. The theoretical plate number calculated based on the hydroxysafflor yellow A peak should not be less than 5000.

[0271] Preparation of reference solution

[0272] Take 0.5 g of safflower reference medicinal material, place it in a stoppered conical flask, add 25 ml of water, heat and reflux for 30 minutes, take it out, filter, and extract the filtrate twice with water-saturated n-butanol, 25 ml each time, combine the n-butanol liquids, evaporate to dryness, cool, add 20 ml of 10% methanol to the residue, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate to obtain the reference medicinal material reference solution.

[0273] Take an appropriate amount of hydroxysafflor yellow A and kaempferol-3-O-rutinoside reference substances, weigh them accurately, add 10% methanol to prepare a solution containing 25 ug of hydroxysafflor yellow A and kaempferol-3-O-rutinoside per 1 ml, which serves as the reference substance solution.

[0274] Preparation of test solution

[0275] Take an appropriate amount of safflower standard decoction freeze-dried powder, grind it into powder, take about 0.2g, add 10ml of water to dissolve it, extract it twice with water-saturated n-butanol, 10ml each time, combine the n-butanol liquids, evaporate to dryness, cool, add 20ml of 10% methanol to the residue, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, filter, and take the filtrate to obtain.

[0276] Determination method: Accurately pipette 5μl of reference solution, control medicinal material solution and test solution, inject into liquid chromatograph, and determine.

[0277] The above-mentioned safflower standard decoction freeze-dried powder reference characteristic spectrum and safflower standard decoction freeze-dried powder test sample chromatogram are respectively attached Fig.26 As shown in (a) and (b), peak 1: syringin; peak 3 (S): hydroxysafflor yellow A; peak 4: tryptophan; peak 7: kaempferol-3-O-sophoroside; peak 8: rutin; peak 9: kaempferol-3-O-rutinoside; peak 12: saffloridine.

[0278] It can be seen that there should be 12 characteristic peaks in the chromatogram of the test sample, and they should correspond to the 12 characteristic peaks in the chromatogram of the reference medicinal material. The retention time of peak 3 and peak 9 should correspond to the retention time of the corresponding reference peak of the reference substance. The peak of the hydroxysafflor yellow A reference substance is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, which should be within the range of ±10% of the specified value. The specified values ​​are: 0.70 (peak 1), 0.94 (peak 2), 1.20 (peak 4), 1.91 (peak 5), 1.95 (peak 6), 2.43 (peak 7), 2.65 (peak 8), 3.32 (peak 10), 3.39 (peak 11), 4.17 (peak 12). The relative peak area of ​​peak 9, peak 12 and S peak should not be less than 0.60 (peak 9) and 0.35 (peak 12).

[0279] 2. System adaptability

[0280] Referring to the method of [Characteristic Spectrum] determined above, the chromatographic conditions were verified. The specific results are shown in Table 35 below. The results show that this method is applicable to safflower standard decoction freeze-dried powder.

[0281] Table 35 System adaptability parameters

[0282] name Retention time area high width Separation Symmetry Factor Theoretical plate number 1 6.498 52431 7658 31.3 - 1.25 21505 2 8.711 26823 3198 24.6 11.31 1.22 26194 3 9.313 65375 5105 46.6 2.22 1.29 13178 4 11.177 20547 1906 27.8 6.16 1.15 25351 5 17.813 15807 2733 18.6 32.09 1.69 284224 6 18.121 37631 6165 24.9 2.26 1.95 263338 7 22.617 23226 2713 20.9 24.36 1.09 158101 8 24.636 21703 2003 33.5 8.53 0.84 149611 9 30.27 91472 12797 24 26.29 1.18 427188 10 30.807 21629 3419 19.7 3.09 1.28 556557 11 31.514 79437 7975 28.6 3.37 1.24 226271 12 38.723 59501 10597 26.9 36.01 1.14 1096221

[0283] 3. Precision

[0284] Take 0.2g of safflower freeze-dried powder, weigh accurately, prepare the test sample according to the method of "Test sample solution preparation", inject 6 times continuously, record the chromatogram, calculate the relative retention time and relative peak area RSD% of each characteristic peak, the results are shown in Tables 36-37 below, and the common pattern diagram of instrument precision is shown in the attached Fig. 27 , from bottom to top correspond to precision 1, 2, 3, 4, 5, 6 respectively.

[0285] Table 36 Instrument precision relative retention time test results

[0286]

[0287]

[0288] Table 37 Instrument precision relative peak area test results

[0289]

[0290] The results showed that the relative retention time RSD% of the 12 characteristic peaks was less than 2%, and the relative peak area RSD% was less than 3.5%. Comprehensively judging, the method has good precision and meets the requirements of the characteristic spectrum.

[0291] 4. Method repeatability test

[0292] Take 0.2g of the same batch of safflower freeze-dried powder, weigh 6 portions in parallel, weigh accurately, prepare the test sample according to the method of "Preparation of Test Sample Solution", inject and analyze, record the chromatogram, calculate the relative retention time and relative peak area RSD% of each characteristic peak, the results are shown in Tables 38-39 below, and the reproducibility common pattern diagram is shown in the attached Fig.28 , from bottom to top they correspond to repeatability 1, 2, 3, 4, 5, and 6 respectively.

[0293] Table 38 Method repeatability relative retention time test results (n = 6)

[0294]

[0295] Table 39 Method repeatability relative peak area test results (n = 6)

[0296]

[0297] The results showed that the relative retention time RSD% of the 12 characteristic peaks was less than 2%, and the relative peak area RSD% was less than 4%. Comprehensively judging, the repeatability of this method was good and met the requirements.

[0298] 5. Intermediate precision (different operators)

[0299] The same batch of safflower freeze-dried powder was taken and independently operated by experimenters A, B and C. The test samples were prepared according to the test sample preparation method, sampled and analyzed, chromatograms were recorded, and the relative retention time and relative peak area RSD% of each characteristic peak were calculated. The results are shown in Tables 40-41 below. The chromatograms of the common modes of intermediate precision among different personnel are shown in the attached table. Fig.29 , from bottom to top they correspond to personnel A, B, and C respectively.

[0300] Table 40 Intermediate precision relative retention time test results (different operators)

[0301]

[0302] Table 41 Intermediate precision relative peak area test results (different operators)

[0303]

[0304] The results show that the relative retention time RSD% of the 12 characteristic peaks is less than 2%, and the relative peak area RSD% is less than 4%. Comprehensively judging, the intermediate precision (different personnel) of the method is good and meets the requirements of the characteristic spectrum.

[0305] 6. Intermediate precision (different column batches)

[0306] Through the results of chromatographic condition optimization and chromatographic column selection, it can be seen that the chromatographic condition reproduction requires a fixed CAPCELL COREC18; 4.6mm×150mm, 2.7um chromatographic column. Therefore, the same test solution was taken and tested with chromatographic columns of different production batches (batch number A34AB10165 and batch number A34AB10192), the chromatograms were recorded, and the relative retention time and relative peak area RSD% of each characteristic peak were calculated. The results are shown in Tables 42-43 below. The chromatograms of different chromatographic column batch numbers A34AB10165 and A34AB10192 are shown in the attached Fig.30 As shown in (a) and (b).

[0307] Table 42 Relative retention time results of different chromatographic column batches

[0308]

[0309]

[0310] Table 43 Relative peak area results of different chromatographic column batches

[0311]

[0312] The results show that the relative retention time RSD% of the 12 characteristic peaks is less than 2%, and the relative peak area RSD% of the 12 characteristic peaks is less than 2%. It can be seen from the figure that different chromatographic column batches have little effect on peak separation.

[0313] 7. Exclusive

[0314] Prepare the test solution according to the test solution preparation method, and examine whether the negative sample of safflower freeze-dried powder will cause interference. Perform HPLC analysis according to the chromatographic conditions described under [Characteristic Spectrum] and record the chromatogram. The characteristic spectrum of the negative blank test sample is as attached. Fig.31 The results showed that the negative had no interference and the method had good specificity.

[0315] 8. Stability test

[0316] Take the same sample solution and inject it at 0, 4, 8, 12, 16, and 24 hours after preparation. Record the chromatogram, calculate the relative retention time and RSD% of the relative peak area of ​​each characteristic peak to examine the stability of the sample solution. The results are shown in Tables 44-45 below. The stability common pattern chromatogram is as follows Fig.32 As shown, from bottom to top they correspond to stability 0h, 4h, 8h, 12h, 16h, and 24h respectively.

[0317] Table 44 Stability relative retention time test results

[0318]

[0319]

[0320] Table 45 Stability relative peak area test results

[0321]

[0322] The results showed that the relative retention time of the 12 characteristic peaks was less than 2%, and the relative peak area RSD% was less than 4%, indicating that the test solution was stable within 24 hours.

[0323] 9. Investigation of different flow rates

[0324] Take the same test solution and test it at different flow rates of 0.68ml / min, 0.70ml / min and 0.72ml / min, record the chromatogram, calculate the relative retention time and relative peak area RSD% of each characteristic peak, and the results are shown in Tables 46-47 below. The chromatograms at flow rates of 0.68ml / min, 0.70ml / min and 0.72ml / min are shown in the attached table. Fig.33 As shown in the figure, the flow rates from bottom to top are 0.70 ml / min, 0.68 ml / min, and 0.72 ml / min respectively.

[0325] Table 46 Relative retention time results at different flow rates

[0326]

[0327]

[0328] Table 47 Relative peak area results at different flow rates

[0329]

[0330] The results showed that the relative retention time RSD% of the 12 characteristic peaks was 0% to 2%, and the relative peak area RSD% of the 12 characteristic peaks was between 0% and 3.4%. It can be seen from the figure that different flow rates have little effect on the separation of the characteristic peaks. In order to ensure the separation and reproducibility of the characteristic spectrum, the flow rate of 0.70 ml / min is the best method for determining the characteristic spectrum of safflower freeze-dried powder.

[0331] 10. Investigation of different column temperatures

[0332] Take the same test solution and test it at 20℃, 25℃ and 30℃ respectively, record the chromatogram, calculate the relative retention time and relative peak area RSD% of each characteristic peak. The results are as follows Table 48-49. The chromatograms at 20℃, 25℃ and 30℃ are attached. Fig.34 As shown in (a)-(c).

[0333] Table 48 Relative retention time results of different column temperatures

[0334]

[0335] Table 49 Relative peak area results at different column temperatures

[0336]

[0337]

[0338] The results show that the relative retention time RSD% of the 12 characteristic peaks is less than 8%, and the relative peak area RSD% of the 12 characteristic peaks is between 0% and 4.7%. It can be seen from the figure that different column temperature conditions have a certain influence on the separation degree of the characteristic peaks. In order to ensure the separation and reproducibility of the characteristic spectrum, it is recommended to fix the column temperature at 25℃.

[0339] From the above methodological investigation results, it can be seen that among the 12 common peaks of the established safflower freeze-dried powder [characteristic spectrum], each chromatographic peak is affected to a certain extent by different column temperatures and different flow rates, and the influence of other chromatographic conditions is not significant. In order to adapt to its durability, it is recommended to control the specified value range within ±10%.

[0340] 11. Determination of characteristic spectra of multiple batches of safflower freeze-dried powder

[0341] Multiple batches of safflower freeze-dried powder (batch numbers: K17030801, K17030802, K17030803, G1611197, G1611198, G1611199, Q2017-01, Q2017-02, Q2017-03, T2017-01, T2017-02, T2017-03, J2017-01, J2017-02, J2017-03) were taken and operated according to the proposed safflower freeze-dried powder [characteristic spectrum] determination method. The determination results are shown in Tables 50-51, respectively. The characteristic spectra of multiple batches of safflower freeze-dried powder are shown in the attached Fig.35 , from bottom to top, they correspond to samples K17030801, K17030802, K17030803, G1611197, G1611198, G1611199, Q2017- 01, Q2017-02, Q2017-03, T2017-01, T2017-02, T2017-03, J2017-01, J2017-02, and J2017- 03.

[0342] Table 50 Relative retention time of safflower freeze-dried powder

[0343]

[0344]

[0345] Table 51 Relative peak area of ​​safflower standard decoction freeze-dried powder

[0346]

[0347]

[0348] The measurement results show that the relative retention time of each characteristic peak of the characteristic spectrum of safflower freeze-dried powder is within the range of ±10% of the specified value of the reference spectrum of safflower freeze-dried powder.

[0349] Relative retention time specified value: Determined based on the results of characteristic spectra of multiple batches of safflower label medicinal materials: 12 characteristic peaks should be present in the chromatogram of the test sample, and should correspond to the 12 characteristic peaks in the chromatogram of the reference medicinal material, among which the retention time of peak 3 and peak 9 should correspond to the retention time of the corresponding reference peak of the reference material; the peak of the hydroxysafflor yellow A reference material is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, which should be within the range of ±10% of the specified value. The specified values ​​are: 0.70 (peak 1), 0.94 (peak 2), 1.20 (peak 4), 1.91 (peak 5), 1.95 (peak 6), 2.43 (peak 7), 2.65 (peak 8), 3.32 (peak 10), 3.39 (peak 11), 4.17 (peak 12).

[0350] Relative peak area ratio specified value: The main chemical components of safflower include flavonoids, alkaloids, amino acids, fatty acids, and polysaccharides. The flavonoid components of safflower are currently recognized as the main active ingredients. The reported components mainly include safflower quinone glycosides, safflower yellow A, hydroxysafflower yellow A, hydroxysafflower yellow B, neokaempferol, kaempferol, kaempferol-3-O-rutinoside, 6-hydroxykaempferol, kaempferol-3-O-β-D-glucosyl-(1→2)-β-D-glucoside, quercetin-3,7-di-O-β-D-glucoside, etc. Among them, safflower yellow is currently the most important active ingredient of safflower. Through the detection of characteristic spectra of multiple batches of safflower and combined with the peak identification information of the characteristic spectra, Peak 1 is syringin, Peak 3 is hydroxysafflor yellow A, Peak 7 is kaempferol-3-O-sophoroside, Peak 8 is rutin, and Peak 9 is kaempferol-3-O-rutinoside, all of which are flavonoid components, Peak 4 is tryptophan, which is an amino acid component. Hydroxysafflor yellow A has good stability throughout the research process. At the same time, as the main efficacy indicator, it is more suitable as the S peak to calculate the relative peak areas of the other characteristic peaks for control. As identification components, syringin and tryptophan are not specific to safflower, and the relative peak areas of kaempferol-3-O-sophoroside and rutin to S peak are relatively too small. Therefore, combining the three batches of safflower formula granules, the transmission of peaks 9 and 12 from safflower medicinal materials to decoction pieces, intermediates and then to formula granules is good. Therefore, the relative peak areas of peaks 9 and 12 are specified, that is, the relative peak area of ​​peak 9 is not less than 0.60, and the relative peak area value of peak 12 is not less than 0.35.

[0351] Example 6 Safflower Formula Granules

[0352] 1. Feature Spectrum

[0353] [Characteristic spectrum] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0354] Chromatographic conditions and system suitability

[0355] Octadecylsilane bonded silica gel is used as filler, chromatographic column: CAPCELL CORE C18; 4.6mm×150mm, 2.7um; acetonitrile is used as mobile phase A, 0.05% trifluoroacetic acid solution is used as mobile phase B, gradient elution is performed according to the provisions in the above Table 11; detection wavelength is 266nm; flow rate is 0.7ml / min. The theoretical plate number calculated based on the hydroxysafflor yellow A peak should not be less than 5000.

[0356] Preparation of reference solution

[0357] Take 0.5 g of safflower reference medicinal material, place it in a stoppered conical flask, add 25 ml of water, heat and reflux for 30 minutes, take it out, filter, and extract the filtrate twice with water-saturated n-butanol, 25 ml each time, combine the n-butanol liquids, evaporate to dryness, cool, add 20 ml of 10% methanol to the residue, ultrasonically treat (power 250 W, frequency 40 kHz) for 30 minutes, filter, and take the filtrate to obtain the reference medicinal material reference solution.

[0358] Take an appropriate amount of hydroxysafflor yellow A and kaempferol-3-O-rutinoside reference substances, weigh them accurately, add 10% methanol to prepare a solution containing 25 ug of hydroxysafflor yellow A and kaempferol-3-O-rutinoside per 1 ml, which serves as the reference substance solution.

[0359] Preparation of test solution

[0360] Take an appropriate amount of this product, grind it into powder, take about 0.2g, add 10ml of water to dissolve it, extract it twice with water-saturated n-butanol, 10ml each time, combine the n-butanol liquids, evaporate to dryness, cool, add 20ml of 10% methanol to the residue, ultrasonically treat (power 250W, frequency 40kHz) for 30 minutes, filter, and take the filtrate to obtain.

[0361] Determination method

[0362] Accurately pipette 5 μl each of the reference solution, control medicinal solution, and test solution, inject into the liquid chromatograph, and measure to obtain the result.

[0363] The reference characteristic spectrum of the safflower formula granules and the chromatogram of the safflower granules test sample are attached. Fig.36 As shown in (a) and (b), peak 1: syringin; peak 3 (S): hydroxysafflor yellow A; peak 4: tryptophan; peak 7: kaempferol-3-O-sophoroside; peak 8: rutin; peak 9: kaempferol-3-O-rutinoside; peak 12: saffloridine.

[0364] It can be seen that there should be 12 characteristic peaks in the chromatogram of the test sample, and they should correspond to the 12 characteristic peaks in the chromatogram of the reference medicinal material. The retention time of peak 3 and peak 9 should correspond to the retention time of the corresponding reference peak of the reference substance. The peak of the hydroxysafflor yellow A reference substance is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, which should be within the range of ±10% of the specified value. The specified values ​​are: 0.70 (peak 1), 0.94 (peak 2), 1.20 (peak 4), 1.91 (peak 5), 1.95 (peak 6), 2.43 (peak 7), 2.65 (peak 8), 3.32 (peak 10), 3.39 (peak 11), 4.17 (peak 12). The relative peak area of ​​peak 9, peak 12 and S peak should not be less than 0.60 (peak 9) and 0.35 (peak 12).

[0365] 2. System adaptability

[0366] Referring to the method of [Characteristic Spectrum] determined above, the chromatographic conditions were verified. The specific results are shown in Table 52 below. The results show that this method is applicable to safflower formula granules.

[0367] Table 52 System adaptability parameters

[0368]

[0369] 3. Precision

[0370] Take 0.2g of safflower granules, weigh accurately, prepare the test sample according to the method of "Preparation of test sample solution", inject 6 times continuously, record the chromatogram, calculate the relative retention time and relative peak area RSD% of each characteristic peak, and the results are shown in Tables 53-54 below. The common pattern diagram of instrument precision is shown in the attached Fig.37 , from bottom to top correspond to precision 1, 2, 3, 4, 5, 6 respectively.

[0371] Table 53 Instrument precision relative retention time test results

[0372]

[0373]

[0374] Table 54 Instrument precision relative peak area test results

[0375]

[0376] The results showed that the relative retention time RSD% of the 12 characteristic peaks was less than 2%, and the relative peak area RSD% was less than 4%. Comprehensively speaking, the method had good precision and met the requirements of the characteristic spectrum.

[0377] 4. Method repeatability test

[0378] Take 0.2g of the same batch of safflower granules, weigh 6 portions in parallel, accurately weigh, prepare the test sample according to the method of "Preparation of test sample solution", inject and analyze, record the chromatogram, calculate the relative retention time and relative peak area RSD% of each characteristic peak, and the results are shown in Tables 55-56 below. The repeatability common pattern diagram is shown in Fig.38 As shown, from bottom to top they correspond to repeatability 1, 2, 3, 4, 5, and 6 respectively.

[0379] Table 55 Method repeatability relative retention time test results (n = 6)

[0380]

[0381]

[0382] Table 56 Method repeatability relative peak area test results (n = 6)

[0383]

[0384] The results showed that the relative retention time RSD% of the 12 characteristic peaks was less than 2%, and the relative peak area RSD% was less than 4%. Comprehensively judging, the repeatability of this method was good and met the requirements.

[0385] 5. Intermediate precision (different operators)

[0386] The same batch of safflower granules was taken and independently operated by experimenters A, B, and C. The test samples were prepared according to the test sample preparation method, sampled and analyzed, the chromatograms were recorded, and the relative retention time and relative peak area RSD% of each characteristic peak were calculated. The results are shown in Tables 57-58 below. The chromatograms with the common mode of intermediate precision among different personnel are shown in the attached table. Fig.39 As shown, from bottom to top they correspond to personnel A, B, and C respectively.

[0387] Table 57 Intermediate precision relative retention time test results (different operators)

[0388]

[0389] Table 58 Intermediate precision relative peak area test results (different operators)

[0390]

[0391] The results show that the relative retention time RSD% of the 12 characteristic peaks is less than 2%, and the relative peak area RSD% is less than 4%. Comprehensively judging, the intermediate precision (different personnel) of the method is good and meets the requirements of the characteristic spectrum.

[0392] 6. Intermediate precision (different column batches)

[0393] Through the results of chromatographic condition optimization and chromatographic column selection, it can be seen that the chromatographic conditions need to be reproduced with a fixed CAPCELL COREC18; 4.6mm×150mm, 2.7um chromatographic column. Therefore, the same test solution was taken and tested with chromatographic columns of different production batches (batch number A34AB10165; batch number A34AB10192), the chromatograms were recorded, and the relative retention time and relative peak area RSD% of each characteristic peak were calculated. The results are shown in Tables 59-60 below. The chromatograms under the conditions of chromatographic columns A34AB10165 and A34AB10192 are shown in the attached table. Fig.40 As shown in (a)-(b).

[0394] Table 59 Relative retention time results of different chromatographic column batches

[0395]

[0396]

[0397] Table 60 Relative peak area results of different chromatographic column batches

[0398]

[0399] The results show that the relative retention time RSD% of the 12 characteristic peaks is less than 2%, and the relative peak area RSD% of the 12 characteristic peaks is less than 4%. It can be seen from the figure that different chromatographic column batches have little effect on peak separation.

[0400] 7. Exclusive

[0401] Prepare the test solution according to the test solution preparation method, and examine whether the safflower negative granule sample will cause interference. Perform HPLC analysis according to the chromatographic conditions described under [Characteristic spectrum], record the chromatogram, and attach the negative blank test sample characteristic spectrum. Fig.41 As shown, the results showed that the negative had no interference and the method had good specificity.

[0402] 8. Stability test

[0403] Take the same sample solution and inject it at 0, 4, 8, 12, 16, and 24 hours after preparation. Record the chromatogram, calculate the relative retention time of each characteristic peak and the RSD% of the relative peak area to examine the stability of the sample solution. The results are shown in Tables 61-62 below. The stability common pattern chromatogram is shown in the attached Fig.42 As shown, from bottom to top they correspond to stability 0h, 4h, 8h, 12h, 16h, and 24h respectively.

[0404] Table 61 Stability relative retention time test results

[0405]

[0406] Table 62 Stability relative peak area test results

[0407]

[0408] The results showed that the relative retention time of the 12 characteristic peaks was less than 2%, and the relative peak area RSD% was less than 4%, indicating that the test solution was stable within 24 hours.

[0409] 9. Investigation of different flow rates

[0410] Take the same test solution and test it at different flow rates of 0.68ml / min, 0.70ml / min and 0.72ml / min, record the chromatogram, calculate the relative retention time and relative peak area RSD% of each characteristic peak, and the results are shown in Tables 63-64 below. The chromatograms at flow rates of 0.68ml / min, 0.70ml / min and 0.72ml / min are shown in the attached table. Fig.43 As shown in (a)-(c).

[0411] Table 63 Relative retention time results at different flow rates

[0412]

[0413] Table 64 Relative peak area results at different flow rates

[0414]

[0415] The results show that the relative retention time RSD% of the 12 characteristic peaks is 0% to 3%, and the relative peak area RSD% of the 12 characteristic peaks is between 5%. It can be seen from the figure that different flow rates have a certain influence on the separation degree of the characteristic peaks. In order to ensure the separation and reproducibility of the characteristic spectrum, the present invention selects 0.70ml / min as the optimal effect of the safflower granule characteristic spectrum determination method.

[0416] 10. Investigation of different column temperatures

[0417] Take the same test solution and test it at 23℃, 25℃ and 27℃ respectively, record the chromatogram, calculate the relative retention time and relative peak area RSD% of each characteristic peak, and the results are shown in Table 65-66 below. The chromatograms at 23℃, 25℃ and 27℃ column temperatures are shown in Table 65-66 below. Fig.44 As shown in (a)-(c).

[0418] Table 65 Relative retention time results of different column temperatures

[0419]

[0420] Table 66 Relative peak area results at different column temperatures

[0421]

[0422]

[0423] The results show that the relative retention time RSD% of the 12 characteristic peaks is less than 8%, and the relative peak area RSD% of the 12 characteristic peaks is between 0% and 4.5%. It can be seen from the figure that different column temperature conditions have a certain influence on the separation degree of the characteristic peaks. In order to ensure the separation and reproducibility of the characteristic spectrum, this method has the best effect when the column temperature is fixed at 25℃.

[0424] In summary, the above methodological investigation results show that among the 12 common peaks of the established safflower granules [characteristic spectrum], each chromatographic peak is affected to a certain extent by different column temperatures and different flow rates, and the influence of other chromatographic conditions is not significant. The relative retention time value is within the range of ±10%. In order to adapt to its durability, it is recommended to control the specified value range within ±10%.

[0425] 11. Determination of characteristic spectra of 3 batches of safflower granules

[0426] Three batches of safflower freeze-dried powder (batch numbers: 1903001Y, 1903002Y, 1903003Y) were taken and operated according to the proposed safflower granule [characteristic spectrum] determination method. The determination results are shown in Tables 67-68 below.

[0427] The measurement results show that the relative retention times of the characteristic peaks in the characteristic spectra of the three batches of safflower granules are all within ±10% of the specified values, and the relative peak areas meet the requirements.

[0428] Table 67 Relative retention time of three batches of safflower granules

[0429]

[0430] Table 68 Relative peak areas of 3 batches of safflower granules

[0431]

[0432]

[0433] Relative retention time specified value: Determined based on the characteristic spectrum measurement results of 3 batches of safflower formula granules: 12 characteristic peaks should be present in the chromatogram of the test sample, and should correspond to the 12 characteristic peaks in the chromatogram of the reference medicinal material, among which the retention time of peak 3 and peak 9 should correspond to the retention time of the corresponding reference peak of the reference substance; the peak of the hydroxysafflor yellow A reference substance is taken as the S peak, and the relative retention time of each characteristic peak and the S peak is calculated, which should be within the range of ±10% of the specified value. The specified values ​​are: 0.70 (peak 1), 0.94 (peak 2), 1.20 (peak 4), 1.91 (peak 5), 1.95 (peak 6), 2.43 (peak 7), 2.65 (peak 8), 3.32 (peak 10), 3.39 (peak 11), 4.17 (peak 12).

[0434] Relative peak area ratio specified value: The main chemical components of safflower include flavonoids, alkaloids, amino acids, fatty acids, and polysaccharides. The flavonoid components of safflower are currently recognized as the main active ingredients. The reported components mainly include safflower quinone glycosides, safflower yellow A, hydroxysafflower yellow A, hydroxysafflower yellow B, neokaempferol, kaempferol, kaempferol-3-O-rutinoside, 6-hydroxykaempferol, kaempferol-3-O-β-D-glucosyl-(1→2)-β-D-glucoside, quercetin-3,7-di-O-β-D-glucoside, etc. Among them, safflower yellow is currently the most important active ingredient of safflower. Through the detection of characteristic spectra of multiple batches of safflower and combined with the peak identification information of the characteristic spectra, Peak 1 is syringin, Peak 3 is hydroxysafflor yellow A, Peak 7 is kaempferol-3-O-sophoroside, Peak 8 is rutin, and Peak 9 is kaempferol-3-O-rutinoside, all of which are flavonoid components, Peak 4 is tryptophan, which is an amino acid component. Hydroxysafflor yellow A has good stability throughout the research process. At the same time, as the main efficacy indicator, it is more suitable as the S peak to calculate the relative peak areas of the other characteristic peaks for control. As identification components, syringoside and tryptophan are not specific in safflower, and the relative peak areas of kaempferol-3-O-sophoroside and rutin to the S peak are relatively too small. Therefore, combining the three batches of safflower formula granules, the relative peak area range of peak 9 is 0.77-1.95, and the relative peak area range of peak 12 is 0.46-1.57. From the results, it can be seen that in the process of preparing the medicinal materials into decoction pieces and then into the safflower standard decoction freeze-dried powder, peaks 9 and 12 have good transferability. Therefore, the relative peak area of ​​peak 9 is set as 0.616 based on 80% of the lower limit of multiple batches, rounded to 0.60, and the relative peak area of ​​peak 12 is set as 0.368 based on 80% of the lower limit of multiple batches, rounded to 0.35.

[0435] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for constructing a characteristic spectrum of safflower medicinal materials and safflower pharmaceutical preparations, characterized in that: The method comprises the steps of conducting high performance liquid chromatography on a test solution of safflower medicinal material and / or safflower pharmaceutical preparation; The chromatographic conditions include: using octadecylsilane bonded silica gel as a filler, with a specification of 4.6×150 mm 2.7 μm, using acetonitrile as mobile phase A, using 0.05% trifluoroacetic acid solution as mobile phase B, and detecting at a wavelength of 266 nm, and performing gradient elution according to the following procedure: 0-8min, A:B is 10%:90%; 8-15min, A:B is 10%:90%→15%:85%; 15-25min, A:B is 15%:85%→17%:83%; 25-35min, A:B is 17%:83%→28%:72%; 35-40min, A:B is 28%:72%→42%:58%; 40-45min, A:B is 42%:58%→50%:50%; The preparation method of the test solution comprises: adding water to the test sample for heating and reflux, filtering the filtrate, adding water-saturated n-butanol for extraction, combining the n-butanol solution and evaporating to dryness, adding 10% methanol to the residue for ultrasonic treatment, filtering and taking the filtrate to obtain the solution.

2. The method for constructing the characteristic spectrum of safflower medicinal materials and safflower pharmaceutical preparations according to claim 1, characterized in that: In the high performance liquid chromatography detection step, the chromatographic conditions also include: column temperature of 20-30°C and flow rate of 0.5-1.0 ml / min.

3. The method for constructing the characteristic spectrum of safflower medicinal materials and safflower pharmaceutical preparations according to claim 1 or 2, characterized in that: The method also includes the step of preparing a control medicinal material reference solution, which specifically includes: taking safflower control medicinal material, adding water to perform heating reflux extraction, collecting the extract, filtering and adding water-saturated n-butanol solvent to perform extraction, collecting the n-butanol phase and evaporating to dryness, adding an organic solvent to the residue, sealing it with ultrasonic treatment, filtering and collecting the filtrate to obtain the desired control medicinal material reference solution.

4. The method for constructing the characteristic spectrum of safflower medicinal materials and safflower pharmaceutical preparations according to claim 1 or 2, characterized in that: The method further comprises the steps of preparing a reference substance solution and constructing a reference substance characteristic spectrum based on high performance liquid chromatography; The reference substance includes hydroxysafflor yellow A and / or kaempferol-3-O-rutinoside.

5. The method for constructing the characteristic spectrum of safflower medicinal materials and safflower pharmaceutical preparations according to claim 3, characterized in that: The power of the ultrasonic step is 200-300W and the frequency is 30-50kHz.

6. The method for constructing the characteristic spectrum of safflower medicinal materials and safflower pharmaceutical preparations according to claim 1 or 2, characterized in that: The safflower medicinal preparation includes safflower formula granules, safflower pieces or safflower standard decoction.

7. Application of the characteristic spectrum construction method of safflower medicinal materials and safflower pharmaceutical preparations according to any one of claims 1 to 6 in the field of quality inspection of safflower medicinal materials and safflower pharmaceutical preparations.

8. A method for quality inspection of safflower medicinal materials and safflower pharmaceutical preparations, characterized in that: The method comprises the steps of constructing the characteristic map and the reference characteristic map according to the method of any one of claims 1 to 6, and comparing the characteristic map with the reference characteristic map.

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