Method for determining content of chemical components in Sui pill and establishing fingerprint spectrum

Abstract

According to the method for determining the content of the chemical components of the semen perillae and the semen lepidii in the semen perillae pill, high performance liquid chromatography is adopted, chromatographic conditions are reasonably screened, the chemical components of the fructus perillae and the semen lepidii in the semen perillae pill are detected at the same time, and the method has the advantages of being easy and convenient to operate, high in analysis speed, high in specificity and the like. The basis is provided for the quality standard of the classic famous prescription Suqi pill. The invention also provides a method for establishing the fingerprint spectrum of the Sui pill, the qualitative detection of seven characteristic components in the Sui pill is realized by adopting chromatographic conditions which are the same as those in a method for determining the content of the Sui pill, and the obtained fingerprint spectrum is clean in background, good in separation effect of each peak, stable in base line and good in accuracy.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly to a method for determining the content of chemical components in Suding Pills and a method for establishing a fingerprint. Background Art

[0002] Suding Pills is a classic famous prescription, first recorded in "Golden Mirror of Medicine" (by Wu Qian in the Qing Dynasty), with the full name of Suding Dingchuan Pills, composed of semen lepidii and semen perillae, and has the efficacy of purging the lung and relieving asthma. This prescription is included in the "Catalogue of Ancient Classic Famous Prescriptions (Second Batch, Pediatric Part)" announced in 2022, and it can be known from the records of medical books in past dynasties that its clinical effect is good. At present, the research on Suding Pills mostly focuses on clinical application and observation, and there is very little research on its quality control. Although there are not many reports on the research of Suding Pills, as a widely spread classic famous prescription for pediatrics with definite curative effects, it is of great value to improve its quality control method.

[0003] Suding Pills is composed of the compatibility of semen lepidii and semen perillae, with complex components. The content detection of a single active ingredient is difficult to reflect its overall quality characteristics, so the quality cannot be comprehensively controlled by using conventional content determination methods. The fingerprint of traditional Chinese medicine is one of the main means to control the quality of traditional Chinese medicine products at present, and can more comprehensively reflect the overall characteristics and product quality of traditional Chinese medicine products. At present, high performance liquid chromatography, gas chromatography, thin layer chromatography, etc. are commonly used in fingerprint research. Among them, high performance liquid chromatography has good repeatability, high accuracy, and is convenient for popularization and implementation. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for determining the content of chemical components in Suding Pills and a method for establishing a fingerprint and the corresponding fingerprint, and to control the quality of Suding Pills by combining the content of chemical components and the fingerprint of the whole prescription.

[0005] The preparation method of Suding Pills in the present invention includes:

[0006] Take an appropriate amount of stir-fried semen perillae and stir-fried semen lepidii, crush them, and pass through the pharmacopoeia sieve No. 2-4. Mix the medicinal powder with an appropriate amount of jujube paste in a ratio of 1:1 to make pills with a diameter of 3-4 mm; the mass ratio of the medicinal powder to the jujube paste is 1:(0.3-0.5); the Suding Pills prepared from Chinese herbal medicines, calculated by weight, include: equal parts (10-12 parts) of stir-fried semen perillae and stir-fried semen lepidii, and an appropriate amount of Chinese dates (6-12 parts).

[0007] In the first aspect of the present invention, a method for determining the content of chemical components in Suding Pills is provided, including the following steps:

[0008] Z1. Take the powder of Suding Pills and mix it with an alcohol solvent, and obtain a test solution after reflux extraction;

[0009] Z2. Prepare a reference substance solution:

[0010] Using methanol with a volume fraction of 50%-100% as the solvent, prepare a quercetin-3-O-β-D-glucopyranoside-7-O-β-D-gentiobioside reference solution with a concentration of 1-150 μg / mL and a rosmarinic acid reference solution with a concentration of 20-500 μg / mL respectively;

[0011] Z3. Use high performance liquid chromatography to detect the components of the test solution described in step Z1. The specific detection conditions are as follows:

[0012] The chromatographic column is packed with octadecylsilane-bonded silica gel; column temperature: 25-35 °C; detection wavelength: the wavelength for the determination of the content of quercetin-3-O-β-D-glucopyranoside-7-O-β-D-gentiobioside is 250-260 nm, and the wavelength for the determination of the content of rosmarinic acid is 320-330 nm; the flow rate of the mobile phase is 0.8-1.0 mL / min; the injection volume is 1-10 μL; mobile phase A: 0.05-0.2% phosphoric acid aqueous solution, mobile phase B: acetonitrile, gradient elution;

[0013] Z4. According to the injection concentration of the reference substance, the peak area of the reference substance in the chromatogram, and the peak area of the component corresponding to the reference substance in Suting Pills in the chromatogram, and based on the detection conditions in step Z3, calculate the contents of the two active components quercetin-3-O-β-D-glucopyranoside-7-O-β-D-gentiobioside and rosmarinic acid in Suting Pills by the external standard two-point method.

[0014] Preferably, the alcohol solvent described in step Z1 is a methanol solvent with a volume fraction of 30%-100%, the mass-volume ratio of the Suting Pills powder to the alcohol solvent is (0.4-0.7) g:(20-50) mL, and the reflux extraction time is 30-120 min;

[0015] Preferably, the gradient elution conditions in step Z3 are as follows:

[0016] At 0-6 min, the volume content of mobile phase A in the mobile phase is 93%-91%, and the volume content of mobile phase B in the mobile phase is 7%-9%;

[0017] At 6-30 min, the volume content of mobile phase A in the mobile phase is 91%-57%, and the volume content of mobile phase B in the mobile phase is 9%-43%;

[0018] At 30-32 min, the volume content of mobile phase A in the mobile phase is 57%-10%, and the volume content of mobile phase B in the mobile phase is 43%-90%;

[0019] From 32 to 40 minutes, the mobile phase A and the mobile phase B are isocratic eluted. The volume content of the mobile phase A in the mobile phase is 10%, and the volume content of the mobile phase B in the mobile phase is 90%.

[0020] Preferably, in step Z4, the injection concentration of the quercetin-3-O-β-D-glucopyranoside-7-O-β-D-gentiobioside reference substance solution is 41.108 μg / mL; the injection concentration of the rosmarinic acid reference substance solution is 162.619 μg / mL. The present application does not particularly limit the preparation method and concentration of the reference substance solution, as long as the purpose of the present application can be achieved.

[0021] In the second aspect of the present invention, a method for establishing a fingerprint of Suting Pills is provided, including the following steps:

[0022] (1) Mix the Suting Pills powder with an alcohol solvent, and obtain a test solution after reflux extraction;

[0023] (2) Detect the test solution in step (1) by high performance liquid chromatography. The specific detection conditions are as follows:

[0024] The chromatographic column is packed with octadecylsilane-bonded silica gel; column temperature: 25 - 35 °C; detection wavelength: 250 - 260 nm; mobile phase flow rate is 0.8 - 1.0 mL / min; injection volume is 1 - 10 μL; mobile phase A: 0.05 - 0.2% phosphoric acid aqueous solution, mobile phase B: acetonitrile, gradient elution;

[0025] (3) Analyze the chromatogram of the Suting Pills by using the similarity evaluation software for traditional Chinese medicine chromatographic fingerprints to obtain the fingerprint of the Suting Pills.

[0026] Preferably, the alcohol solvent in step (1) is a methanol solvent with a volume fraction of 30% - 100%. The mass-to-volume ratio of the Suting Pills powder to the alcohol solvent is (0.4 - 0.7) g∶(20 - 50) mL, and the reflux extraction time is 30 - 120 minutes.

[0027] Preferably, the gradient elution conditions in step (2) are specifically as follows:

[0028] From 0 to 6 minutes, the volume content of the mobile phase A in the mobile phase is 93% - 91%, and the volume content of the mobile phase B in the mobile phase is 7% - 9%;

[0029] From 6 to 30 minutes, the volume content of the mobile phase A in the mobile phase is 91% - 57%, and the volume content of the mobile phase B in the mobile phase is 9% - 43%;

[0030] At 30 to 32 minutes, the volume content of the mobile phase A in the mobile phase is 57% to 10%, and the volume content of the mobile phase B in the mobile phase is 43% to 90%;

[0031] At 32 to 40 minutes, the mobile phase A and the mobile phase B are subjected to isocratic elution, the volume content of the mobile phase A in the mobile phase is 10%, and the volume content of the mobile phase B in the mobile phase is 90%.

[0032] The method for detecting chemical components in Su Ting Pills and the method for establishing fingerprints provided by the present invention have the following specific technical effects:

[0033] 1. The precision, repeatability, stability and sample recovery rate of the content detection method provided by the present invention all meet the requirements of the current edition of the Pharmacopoeia. The detection results are accurate and reliable, and the contents of two chemical components in Su Ting Pills can be accurately quantified at the same time. This is of great significance for promoting the inspection, control and evaluation of the quality of Su Ting Pills and the modernization of the prescription, and lays a foundation for the subsequent research and new applications of this classic prescription.

[0034] 2. The fingerprint spectrum obtained by using the mobile phase, elution conditions and detection wavelength provided by the present invention has a clean background, good peak separation effect, stable baseline, good precision, strong repeatability and high stability, which is suitable for the establishment of the fingerprint spectrum of Su Ting Wan.

[0035] 3. The present invention adopts a method combining chemical component content determination with fingerprint spectrum to control the quality of the classic prescription Su Ting Wan, providing a basis for the quality control standard of the classic prescription Su Ting Wan.

[0036] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0038] Figure 1 This is a specificity investigation spectrum in Example 1 of the present invention.

[0039] Figure 2 This is a linear result graph of quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside in Example 1 of the present invention.

[0040] Figure 3 This is the linear result diagram of rosmarinic acid in Example 1 of the present invention.

[0041] Figure 4 Control fingerprint pattern R of Suding Pills in Example 21 of the present invention: Among them, the 2nd peak is quercetin-3-O-β-D-glucoside-7-O-β-D-gentiobioside, and the 6th peak is rosmarinic acid.

[0042] Figure 5 Fingerprint patterns of Suding Pills numbered S1-S10 in Examples 11-20 of the present invention: Among them, the 2nd peak is quercetin-3-O-β-D-glucoside-7-O-β-D-gentiobioside, and the 6th peak is rosmarinic acid.

[0043] Figure 6 Mixed reference substance chromatogram of Suding Pills in Example 21 of the present invention: Among them, the 2nd peak is quercetin-3-O-β-D-glucoside-7-O-β-D-gentiobioside, and the 6th peak is rosmarinic acid. Detailed implementation manners

[0044] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention.

[0045] All other implementation manners obtained by those of ordinary skill in the art based on the implementation manners in the present invention without creative efforts fall within the scope of protection of the present invention.

[0046] The instruments, reagents and materials used in the present invention are as follows:

[0047] Instruments: Agilent 1260 high performance liquid chromatograph, Agilent Company, USA; XP205 type one hundred thousandth electronic analytical balance, METTLER TOLEDO Company, Switzerland; AX224ZH type ten thousandth balance, Ohaus Instruments (Changzhou) Co., Ltd.; YP5001N type electronic balance, Shanghai Qinghai Instruments Co., Ltd.; ZDHW type electric heating mantle, Beijing Zhongxing Weiye Instruments Co., Ltd.; DLSB-5 / 25 type low temperature cooling circulating pump, Tianjin Kepu Instrument Equipment Co., Ltd.; Milli-Q academic ultrapure water system, Miliipore Company, USA; 1000C type multi-functional pulverizer, Yongkang Hongtaiyang Electromechanical Co., Ltd.; Similarity evaluation system for traditional Chinese medicine chromatographic fingerprint (2012 version), Pharmacopoeia Commission of the People's Republic of China;

[0048] Reagents: Methanol and acetonitrile are both chromatographically pure, Fisher Company, USA; water is ultrapure water; phosphoric acid, chromatographically pure, Acid85%, Thermo Fisher Scientific Co., Ltd.;

[0049] Materials: Quercetin-3-O-β-D-glucopyranoside-7-O-β-D-gentiobioside (hereinafter referred to as QGG) (batch number: 111854-201905, purity 94.5%), purchased from the National Institutes for Food and Drug Control; Rosmarinic acid (batch number: A20IB223325, purity 98.2%), purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; Stir-fried Lepidii Semen, Stir-fried Perillae Fructus, and Chinese dates were all purchased from their authentic producing areas or main production areas. After identifying the medicinal materials, they all complied with the provisions of the Chinese Pharmacopoeia 2020 Edition: Stir-fried Lepidii Semen is the dried ripe seeds of Descurainia sophia (L.) Webb. ex Prantl. of the Brassicaceae family, prepared by the method of stir-frying (General Rule 0213); Stir-fried Perillae Fructus is the dried ripe fruits of Perilla frutescens (L.) Britt. of the Lamiaceae family, prepared by the method of stir-frying (General Rule 0213); Chinese dates are the dried ripe fruits of Ziziphus jujuba Mill. of the Rhamnaceae family; The origin and batch number information of each batch of medicinal materials are shown in Table 1.

[0050] Table 1 Origin and batch number information of each batch of medicinal materials

[0051]

[0052] Preparation of Suting Pills:

[0053] Randomly combine the different batches of decoction pieces of each medicinal material to obtain 10 batches of Suting Pills samples. Respectively, take the stir-fried Perillae Fructus and stir-fried Lepidii Semen of each batch and crush them. Among them, the stir-fried Perilla powder passes through the No. 2 sieve of the pharmacopoeia, and the stir-fried Lepidii Semen powder passes through the No. 4 sieve of the pharmacopoeia. Remove the cores of the Chinese dates and steam them into date paste; Then, take 20 g of stir-fried Perilla powder and 20 g of stir-fried Lepidii Semen powder respectively, mix them evenly with 18 g of date paste, and make pills with a diameter of 3 mm using a pill-making board to obtain 10 batches of Suting Pills, numbered S1-S10 in sequence, and store them in a desiccator.

[0054] Example 1

[0055] A method for determining the content of chemical components in Suting Pills, comprising the following steps:

[0056] Z1. Preparation of test solution: Take the Suting Pills numbered S1, grind them into powder, accurately weigh 0.6 g and place it in a 100 mL round-bottom flask, accurately add 25 mL of methanol aqueous solution with a volume fraction of 50%, reflux and extract for 60 min, cool it, make up the weight loss, shake well, take the supernatant and filter it through a 0.22 μm filter membrane to obtain the test solution;

[0057] Z2. Preparation of reference solution:

[0058] Accurately weigh an appropriate amount of QGG reference substance, dissolve it by ultrasonic wave with 50% methanol and make up the volume to obtain a reference solution containing 41.108 μg of QGG per 1 mL;

[0059] Accurately weigh an appropriate amount of rosmarinic acid reference substance, add 50% methanol, ultrasonically dissolve and make up to volume to prepare a reference substance solution containing 162.619 μg of rosmarinic acid per 1 mL;

[0060] Accurately weigh appropriate amounts of QGG and rosmarinic acid reference substances, add 50% methanol to dissolve by ultrasonication and make up to volume to prepare a mixed reference substance solution containing 32.886 μg of QGG per 1 mL and 162.619 μg of rosmarinic acid per 1 mL;

[0061] Z3. Use high performance liquid chromatography to detect the test solution described in step Z1. The specific detection conditions are as follows:

[0062] Column temperature: 30°C; injection volume: 8 μL; detection wavelength: 254 nm for QGG content and 320 nm for rosmarinic acid content;

[0063] The chromatographic column was InfinttyLab Poroshell 120EC-C18, 4.6 × 150 mm, 4 μm;

[0064] The mobile phase flow rate was 0.8 mL / min;

[0065] Mobile phase A: 0.1% phosphoric acid aqueous solution; Mobile phase B: acetonitrile

[0066] The procedure of the gradient elution is:

[0067] At 0 to 6 minutes, the volume content of the mobile phase A in the mobile phase is 93% to 91%, and the volume content of the mobile phase B in the mobile phase is 7% to 9%;

[0068] In 6 to 30 minutes, the volume content of the mobile phase A in the mobile phase is 91% to 57%, and the volume content of the mobile phase B in the mobile phase is 9% to 43%;

[0069] At 30 to 32 minutes, the volume content of the mobile phase A in the mobile phase is 57% to 10%, and the volume content of the mobile phase B in the mobile phase is 43% to 90%;

[0070] At 32 to 40 minutes, the mobile phase A and the mobile phase B are subjected to isocratic elution, the volume content of the mobile phase A in the mobile phase is 10%, and the volume content of the mobile phase B in the mobile phase is 90%;

[0071] Z4. Determine the content of the index component in the Suting Pills to be tested:

[0072] According to the injection concentration of the reference substance, the peak area of the reference substance in the chromatogram, and the peak area of the component corresponding to the reference substance in the Su-ting Pills in the chromatogram, and based on the detection conditions in step Z3, the contents of QGG and rosmarinic acid in the Su-ting Pills numbered S1 are calculated by the external standard two-point method.

[0073] Preparation of negative test sample solution: According to the proportion of Chinese medicinal materials in the Su-ting Pills, negative samples lacking stir-fried Lepidii Semen, stir-fried Perillae Fructus, and jujube were taken respectively, and prepared according to the method in step Z1, and then obtained; the operation methods of steps Z2-Z4 are the same as above.

[0074] Example 2-10

[0075] Except for taking the Su-ting Pills numbered S2-S10 to replace the Su-ting Pills numbered 1 in Example 1 respectively, the rest are the same as Example 1, and the contents of QGG and rosmarinic acid in the Su-ting Pills numbered S2-S10 are obtained respectively. The results are shown in Table 2.

[0076] Table 2 Contents of QGG and rosmarinic acid in the Su-ting Pills numbered S1-S10 (mean ± standard deviation, mg / g, n = 2)

[0077]

[0078] Methodology verification of the method for determining the chemical composition content of Su-ting Pills

[0079] Specificity analysis: Take the mixed reference substance solution of QGG and rosmarinic acid, the test sample solution, the blank solvent (methanol with a volume fraction of 50%), and the negative test sample solution respectively, and inject and analyze according to the chromatographic conditions in Example 1, and record the chromatogram (see Figure 1 ). The results show that the blank solvent and the negative test sample solution have no interference at the peak positions of the target components, indicating that the method has good specificity.

[0080] Linearity investigation: Weigh appropriate amounts of QGG and rosmarinic acid reference substances precisely, dissolve them ultrasonically with 50% methanol and make up the volume to obtain a stock solution containing 103.194 μg of QGG and 469.396 μg of rosmarinic acid per 1 mL for standby. Pipette the QGG stock solution and dilute it with 50% methanol to prepare QGG reference substance linear solutions with concentrations of 1.032, 5.160, 20.639, 41.278, and 51.146 μg / ml respectively; Pipette the rosmarinic acid stock solution and dilute it with 50% methanol to prepare rosmarinic acid reference substance linear solutions with concentrations of 23.470, 46.940, 93.879, 117.349, and 187.758 μg / ml respectively. Inject and analyze according to the chromatographic conditions in Example 1, and perform regression analysis with the concentration (μg / mL) as the abscissa (X) and the peak area A (Y) as the ordinate, and draw the standard curve as shown in Figure 2 and Figure 3, the regression equations are shown in Table 3. As can be seen from Table 3, the linear relationships of QGG are good in the range of 1.032 - 51.146 μg / mL and of rosmarinic acid are good in the range of 23.470 - 187.758 μg / mL.

[0081] Table 3 Regression equations of the target components

[0082]

[0083] Precision test: Take the mixed reference substance solution of QGG with a concentration of 32.886 μg / mL and rosmarinic acid with a concentration of 162.619 μg / mL in Example 1, inject samples continuously 6 times according to the chromatographic conditions in Example 1, record the peak areas, and calculate the RSD. The results are shown in Table 4 below. As can be seen from Table 4, the RSDs of the peak areas of QGG and rosmarinic acid are both less than 3%, indicating good precision of the instrument.

[0084] Table 4 Precision results

[0085]

[0086] Stability: Take the Suting Pills numbered S1 (the batch is not limited), prepare the test solution according to the method in Example 1, and inject samples for analysis at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, and 48 h respectively according to the chromatographic conditions in Example 1, record the peak areas of the target components, and calculate the RSD of the content. The results are shown in Table 5 below. As can be seen from Table 5, the RSDs of the contents of QGG and rosmarinic acid are less than 3% within 48 h, indicating good stability of the test solution when stored at room temperature for 48 h.

[0087] Table 5 Stability test

[0088]

[0089] Repeatability: Take the Suting Pills numbered S1 (the batch is not limited), prepare 6 portions of the test solution according to the method in Example 1, inject samples for analysis according to the chromatographic conditions in Example 1 respectively, record the peak areas of the target components, and calculate the RSD of the content. The results are shown in Table 6 below. As can be seen from Table 6, the RSDs of the contents of QGG and rosmarinic acid are both less than 3%, indicating good repeatability of this method.

[0090] Table 6 Repeatability test

[0091]

[0092] Sample addition recovery rate: Based on the repeatability results, the content of the target components in 0.3 g of the powdered Suting Pills with serial number S1 can be calculated. Take 0.3 g of the powdered Suting Pills with serial number S1 of known content, weigh 6 aliquots in parallel, precisely add the QGG reference substance solution and rosmarinic acid reference substance solution respectively, prepare the test solution according to the method of Example 1, inject the sample for analysis under the chromatographic conditions of Example 1, calculate the sample addition recovery rate according to the following formula, and the obtained results are shown in Tables 7 and 8 below.

[0093]

[0094] Table 7 Test data of QGG sample addition recovery rate

[0095]

[0096] Table 8 Test data of rosmarinic acid sample addition recovery rate

[0097]

[0098] As can be seen from Tables 7 and 8, the sample addition recovery rates of QGG are all between 85% and 110%, and the sample addition recovery rates of rosmarinic acid are all between 90% and 108%, indicating that the method has good accuracy.

[0099] Example 11

[0100] A method for constructing a fingerprint of Suting Pills, comprising the following steps:

[0101] a. Preparation of test solution: Take the Suting Pills with serial number S1, grind them into powder, precisely weigh 0.6 g each, place them in 100 mL round-bottom flasks respectively, precisely add 25 mL of methanol aqueous solution with a volume fraction of 50%, reflux and extract for 60 min, let it cool, make up the weight loss, shake well, take the supernatant and filter it through a 0.22 μm filter membrane to obtain the test solution;

[0102] b. Use high performance liquid chromatography to detect the test solution prepared in step a, and the specific detection conditions are as follows:

[0103] Column temperature: 30 °C; Injection volume: 8 μL; Detection wavelength: 254 nm;

[0104] Chromatographic column: InfinttyLab Poroshell 120EC-C18, 4.6×150 mm, 4 μm;

[0105] Mobile phase flow rate: 0.8 mL / min;

[0106] Mobile phase A: 0.1% phosphoric acid aqueous solution; Mobile phase B: acetonitrile

[0107] The gradient elution program is as follows:

[0108] From 0 to 6 minutes, the volume content of mobile phase A in the mobile phase is 93% to 91%, and the volume content of mobile phase B in the mobile phase is 7% to 9%;

[0109] From 6 to 30 minutes, the volume content of mobile phase A in the mobile phase is 91% to 57%, and the volume content of mobile phase B in the mobile phase is 9% to 43%;

[0110] From 30 to 32 minutes, the volume content of mobile phase A in the mobile phase is 57% to 10%, and the volume content of mobile phase B in the mobile phase is 43% to 90%;

[0111] From 32 to 40 minutes, isocratic elution is performed on mobile phase A and mobile phase B. The volume content of mobile phase A in the mobile phase is 10%, and the volume content of mobile phase B in the mobile phase is 90%;

[0112] c. Import the chromatogram of Suding Pills numbered S1 into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (2012 version) to establish the fingerprint of Suding Pills numbered S1, as shown in S1 in Figure 5 .

[0113] Examples 12 - 20

[0114] Except for separately taking Suding Pills numbered S2 - S10 to replace Suding Pills numbered S1 in Example 11, the rest are the same as in Example 11, and the fingerprints of Suding Pills numbered S2 - S10 are obtained respectively. Among them, the fingerprints of Suding Pills numbered S2 - S10 are respectively as shown in Figure 5 S2 - S10 in

[0115] Example 21

[0116] Take the fingerprints of Suding Pills numbered S1 - S10 obtained in Examples 11 - 20, and perform data analysis using the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints" (2012 version). Taking the fingerprint of Suding Pills numbered S1 as the reference fingerprint, using the median method, with a time window width of 0.1, perform multi-point calibration and Mark peak matching to generate the control fingerprint of Suding Pills (see Figure 4 ), the fingerprints of 10 batches of Suding Pills (see Figure 5 ). A total of 7 common peaks are calibrated. Comparing with the reference substance chromatogram, 2 chromatographic peaks are identified, namely peak 2 QGG and peak 6 rosmarinic acid, and the reference substance chromatogram (see Figure 6 ); Based on the control fingerprint, calculate the similarity between the fingerprints of Suding Pills numbered S1 - S10 and the control fingerprint.

[0117] The similarities between the fingerprint spectra of Suding Pills numbered S1 - S10 and the control fingerprint spectrum were 0.973, 0.989, 0.988, 0.983, 0.991, 0.989, 0.986, 0.969, 0.968, and 0.986 respectively. All of them were above 0.96, indicating good similarity, which shows that the established fingerprint spectrum of Suding Pills can reflect its fingerprint characteristics. See Table 9 for details.

[0118] Table 9 Similarity Results

[0119]

[0120]

[0121] The HPLC fingerprint spectrum of Suding Pills was established for the first time in this invention. This method is efficient, simple, and has good repeatability, providing a better reference basis for the effective substances and overall quality control of Suding Pills.

[0122] Methodology investigation of the method for establishing the fingerprint spectrum of Suding Pills:

[0123] Precision test: Take the Suding Pills numbered S1, prepare the test solution according to the method in Example 11, inject samples continuously 6 times under the chromatographic conditions in Example 11, record the HPLC chromatogram, take QGG (peak 2) as the reference peak, calculate the relative retention time, relative peak area of each chromatographic peak and their RSD. The obtained results are shown in Table 9 and Table 10. The results in Table 10 and Table 11 show that the RSD of the relative peak area and relative retention time of each common peak are both less than 3.0%, indicating good instrument precision.

[0124] Table 10 Precision Test Results (Relative Retention Time)

[0125]

[0126] Table 11 Precision Investigation Results (Relative Peak Area)

[0127]

[0128]

[0129] Stability test: Take the Suding Pills numbered S1, prepare the test solution according to the method in Example 11, under the chromatographic conditions in Example 11, inject samples at 0h, 4h, 8h, 12h, 24h, and 48h respectively, record the HPLC chromatogram, take QGG (peak 2) as the reference peak, calculate the relative retention time, relative peak area of each chromatographic peak and their RSD. The obtained results are shown in Table 12 and Table 13. The results in Table 11 and Table 12 show that the RSD of the relative peak area and relative retention time of each common peak are both less than 3.0%, indicating that the sample is relatively stable within 48h.

[0130] Table 12 Stability test results (relative retention time)

[0131]

[0132] Table 13 Stability test results (relative peak area)

[0133]

[0134] Repeatability test: Take the Suting Pills numbered S1, prepare 6 portions of the test solution according to the method of Example 11, inject samples for detection under the chromatographic conditions of Example 11, record the HPLC chromatogram, take QGG (peak 2) as the reference peak, calculate the relative retention time, relative peak area and their RSDs of each common peak. The results are shown in Table 14 and Table 15. The results in Table 14 and Table 15 show that the RSDs of the relative peak area and relative retention time of each common peak are less than 3.0%, indicating that the repeatability of this method is good.

[0135] Table 14 Repeatability test results (relative retention time)

[0136]

[0137] Table 15 Repeatability test results (relative peak area)

[0138]

[0139] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the given examples, those of ordinary skill in the art can modify or make equivalent substitutions to the technical solutions of the present invention according to needs, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for determining the content of chemical components in Su Ting Wan, comprising the following steps: Z1. Mix the Suting Pill powder with an alcohol solvent, and reflux and extract to obtain a test solution; Z2. Prepare reference solution: using 50%-100% methanol as solvent, prepare 1-150 μg / mL quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside reference solution and 20-500 μg / mL rosmarinic acid reference solution; Z3. Detect the components of the solution to be tested in step Z1 by high performance liquid chromatography. The specific detection conditions are as follows: the chromatographic column is filled with octadecylsilane bonded silica gel; column temperature: 25-35°C; detection wavelength: the content determination wavelength of quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside is 250-260nm, and the content determination wavelength of rosmarinic acid is 320-330nm; the mobile phase flow rate is 0.8-1.0mL / min; the injection volume is 1-10μL; mobile phase A: 0.05-0.2% phosphoric acid aqueous solution, mobile phase B: acetonitrile, gradient elution; Z4. According to the injection concentration of the reference substance, the peak area of ​​the reference substance in the chromatogram and the peak area of ​​the component corresponding to the reference substance in the chromatogram of Su Ting Pills, and based on the detection conditions in step Z3, calculate the contents of two active ingredients in Su Ting Pills, quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside and rosmarinic acid, by the external standard two-point method.

2. The method for determining the chemical composition content of Su Ting Wan according to claim 1, characterized in that: The alcohol solvent in step Z1 is a methanol solvent with a volume fraction of 30%-100%.

3. The method for determining the content of chemical components of Su Ting Wan according to claim 1 or 2, characterized in that: The mass volume ratio of the Su Ting Pill powder to the alcohol solvent in step Z1 is (0.4-0.7) g: (20-50) mL.

4. The method for determining the content of chemical components of Su Ting Wan according to any one of claims 1 to 3, characterized in that: The reflux extraction time in step Z1 is 30-120 min.

5. The method for determining the content of chemical components of Su Ting Wan according to any one of claims 1 to 4, characterized in that: The gradient elution conditions in step Z3 are: At 0 to 6 minutes, the volume content of the mobile phase A in the mobile phase is 93% to 91%, and the volume content of the mobile phase B in the mobile phase is 7% to 9%; In 6 to 30 minutes, the volume content of the mobile phase A in the mobile phase is 91% to 57%, and the volume content of the mobile phase B in the mobile phase is 9% to 43%; At 30 to 32 minutes, the volume content of the mobile phase A in the mobile phase is 57% to 10%, and the volume content of the mobile phase B in the mobile phase is 43% to 90%; At 32 to 40 minutes, the mobile phase A and the mobile phase B are subjected to isocratic elution, the volume content of the mobile phase A in the mobile phase is 10%, and the volume content of the mobile phase B in the mobile phase is 90%.

6. The method for determining the content of chemical components of Su Ting Wan according to any one of claims 1 to 5, characterized in that: In step Z4, the injection concentration of the quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside reference solution is 1-150 μg / mL; the injection concentration of the rosmarinic acid reference solution is 20-500 μg / mL.

7. The method for determining the content of chemical components of Su Ting Wan according to claim 6, characterized in that: In step Z4, the injection concentration of the quercetin-3-O-β-D-glucose-7-O-β-D-gentiobioside reference solution is 41.108 μg / mL; the injection concentration of the rosmarinic acid reference solution is 162.619 μg / mL.

8. The assay method according to any one of claims 1 to 7, characterized in that The preparation method of the Suting pills comprises: Take appropriate amounts of fried perilla seed and fried scutellaria seed slices, grind them, pass through a pharmacopoeia sieve No. 2-4 to obtain medicinal powder, take fried perilla seed powder and fried scutellaria seed powder, mix them in a ratio of 1:1 to obtain mixed medicinal powder, and mix them with jujube paste to form 3-4 mm pills; the mass ratio of the mixed medicinal powder to the jujube paste is 1:(0.3-0.5); the Su Ting pill medicinal pieces, in parts by weight, include: 10-12 parts of fried perilla seed and fried scutellaria seed, and 6-12 parts of jujube.

9. A method for establishing a fingerprint spectrum of Su Ting Wan, characterized in that: The following steps are involved: (1) Mixing Suting Wan powder with an alcohol solvent, and reflux extraction to obtain a test solution; (2) The test solution described in step (1) is detected by high performance liquid chromatography, and the specific detection conditions are as follows: The chromatographic column is filled with octadecylsilane bonded silica gel; column temperature: 25-35°C; detection wavelength: 250-260nm; mobile phase flow rate: 0.8-1.0mL / min; injection volume: 1-10μL; mobile phase A: 0.05-0.2% phosphoric acid aqueous solution, mobile phase B: acetonitrile, gradient elution; (3) The chromatogram of the Su Ting Pills is analyzed using a traditional Chinese medicine chromatographic fingerprint similarity evaluation software to obtain a fingerprint of the Su Ting Pills.

10. The method for establishing the fingerprint spectrum of Su Ting Wan according to claim 9, characterized in that: The alcohol solvent in step (1) is a methanol solvent with a volume fraction of 30%-100%.

11. The method for establishing the fingerprint spectrum of Su Ting Wan according to claim 9 or 10, characterized in that: The mass volume ratio of the Su Ting Wan powder to the alcohol solvent in step (1) is (0.4-0.7) g: (20-50) mL.

12. The method for establishing the fingerprint spectrum of Su Ting Wan according to any one of claims 9 to 11, characterized in that: The reflux extraction time in step (1) is 30-120 min.

13. The method for establishing the fingerprint spectrum of Su Ting Wan according to any one of claims 9 to 12, characterized in that: The gradient elution conditions in step (2) are specifically: At 0 to 6 minutes, the volume content of the mobile phase A in the mobile phase is 93% to 91%, and the volume content of the mobile phase B in the mobile phase is 7% to 9%; In 6 to 30 minutes, the volume content of the mobile phase A in the mobile phase is 91% to 57%, and the volume content of the mobile phase B in the mobile phase is 9% to 43%; At 30 to 32 minutes, the volume content of the mobile phase A in the mobile phase is 57% to 10%, and the volume content of the mobile phase B in the mobile phase is 43% to 90%; At 32 to 40 minutes, the mobile phase A and the mobile phase B are subjected to isocratic elution, the volume content of the mobile phase A in the mobile phase is 10%, and the volume content of the mobile phase B in the mobile phase is 90%.

14. The method for establishing the fingerprint spectrum of Su Ting Wan according to any one of claims 9 to 12, characterized in that: In step (3), the 2012 version of "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" is used for analysis.

15. The method for establishing the fingerprint spectrum of Su Ting Wan according to any one of claims 9 to 12, characterized in that: The preparation method of the Suting pills comprises: Take appropriate amounts of fried perilla seed and fried scutellaria seed slices, grind them, pass them through a pharmacopoeia sieve No. 2-4, take fried perilla seed and fried scutellaria seed powders, mix them in a ratio of 1:1 to obtain mixed powders, and mix them with jujube paste to form 3-4 mm pills; the mass ratio of the mixed powder to the jujube paste is 1:(0.3-0.5); the Su Ting pill slices include, by weight, 10-12 parts of fried perilla seed and fried scutellaria seed, and 6-12 parts of jujube.

16. The fingerprint of Su Ting Wan established according to the method of any one of claims 9 to 15, as shown in FIG4 or FIG5.

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