Radix paeoniae rubra quality marker screening method based on anticoagulant spectrum-effect relationship

Abstract

The invention discloses a radix paeoniae rubra quality marker screening method based on an anticoagulant spectrum-effect relationship. The method mainly comprises the following steps: preparing a reference substance and a radix paeoniae rubra sample material, determining a preparation method of a radix paeoniae rubra sample and optimal chromatographic conditions for separation of the radix paeoniae rubra sample, and constructing a fingerprint spectrum of the radix paeoniae rubra sample; carrying out in-vitro anticoagulant activity determination on the red paeony root extract by adopting a coagulometer; and analyzing experimental data, and determining the red paeony root quality marker of the anticoagulation spectrum-effect relationship. And carrying out correlation analysis on the drug effect and the common peak of the fingerprint spectrum through a grey correlation analysis method, and screening out the anticoagulant quality marker of the red paeony root medicinal material. The method overcomes the defect that in the prior art, no related technology is used for specifically researching the determination of the red paeony root anticoagulant quality marker through a grey relational analysis method.

Description

Technical Field

[0001] The invention relates to a method for screening quality markers of traditional Chinese medicine, in particular to a method for screening quality markers of red peony root based on anticoagulant spectrum-effect relationship. Background Art

[0002] Red peony root is the dried root of the Ranunculaceae plant Paeonia lactiflora or Sichuan red peony root, which is distributed in Inner Mongolia, Heilongjiang, Jilin, Liaoning and other places in my country. It has the effects of dispersing blood stasis, relieving pain, clearing heat and cooling blood. Red peony root contains monoterpene glycosides, tannins, triterpenes, and volatile oils. Its glycoside components are collectively referred to as total red peony glucosides (TPG), which are its main active ingredients. According to literature reports, red peony root has good anticoagulant effect. TPG, paeoniflorin, pentagalloyl glucose, paeoniflorin, protocatechuic acid, and paeonol in red peony root are the material basis for red peony root to exert its anti-thrombotic effect.

[0003] The efficacy of traditional Chinese medicine is closely related to its material basis. The current quality control method of traditional Chinese medicine mainly uses the content determination of one or several indicator components as the quality standard of medicinal materials, which cannot accurately and comprehensively reflect the quality of medicinal materials. At the same time, the shortcomings such as weak drug-effect correlation and insufficient basis for setting limits are bottlenecks in the research of quality control of traditional Chinese medicine.

[0004] At present, some scholars have conducted research on the drug-effect correlation of traditional Chinese medicine, but there are almost no public literature records on the research on red peony root, especially the correlation between red peony root and anticoagulant efficacy. This has limited the promotion and application of red peony root in the field of anticoagulation to a certain extent. Summary of the invention

[0005] The purpose of the present invention is to provide a method for screening quality markers of red peony root based on anticoagulant spectrum-effect relationship, so as to solve the technical problem that the existing technology proposed in the above background technology cannot determine the quality markers of red peony root in the anticoagulant field based on drug-effect correlation.

[0006] To achieve the above object, the present invention provides the following technical solutions: In one embodiment, a method for screening red peony root quality markers based on anticoagulant spectrum-effect relationship is provided, comprising the following steps: Prepare materials including reference substances and red peony root samples, and determine equipment including liquid chromatographs and centrifuges; construct fingerprints of red peony root samples, and use blood from experimental animals to carry out anticoagulant activity assays; analyze experimental data and determine quality markers of red peony root for anticoagulant spectrum-activity relationship.

[0007] Further, determining the quality marker of red peony root for the anticoagulant spectrum-effect relationship includes the following steps: The peak areas of common peaks of multiple batches of red peony root were obtained to determine the eigenvalues ​​and contribution rates of principal component analysis, thereby determining the principal components of red peony root; the peak area data of common peaks were obtained, the principal component factor loading matrix was calculated and determined, and finally the contribution rate of the principal component was determined; the anticoagulant efficacy index of red peony root was taken as the associated object, and the peak area data of common peaks in the red peony root fingerprint was taken as the characteristic sequence variable, and the two were subjected to grey correlation analysis to determine the quality marker of red peony root.

[0008] Furthermore, four anticoagulant indicators, APTT, PT, TT, and FIB, were used as correlation objects, and the total area data of 22 common peaks in the red peony root fingerprint were used as the input of the computer program. The grey correlation between the two was determined through the set program, and gallic acid, catechins, ethyl gallate, paeoniflorin, benzoic acid, pentagalloylglucose, and benzoylpeoniflorin were determined as anticoagulant quality markers of red peony root.

[0009] Furthermore, through the grey correlation analysis of paeoniflorin in four efficacy indicators, paeoniflorin was determined to be the most important quality marker of red peony root.

[0010] Furthermore, the peak areas of 22 common peaks in 73 batches of red peony root were imported into a computer program as variables to determine the principal component analysis eigenvalues, contribution rates and principal component factor loading matrix.

[0011] Furthermore, preparing materials including reference substances and red peony root sample materials, and determining equipment and instruments including a liquid chromatograph and a centrifuge includes the following steps: determining the machine models of the liquid chromatograph and centrifuge used in the experiment; determining reference substances, multiple test kit drugs and reagents; determining the red peony root samples; and determining the experimental animals.

[0012] Furthermore, the fingerprint of red peony root samples was constructed, and the anticoagulant activity was determined using the blood of experimental animals, including: The reference substance is prepared into a reference substance solution, and the red peony root sample is prepared into a sample solution; a chromatographic analysis technique is used to construct a fingerprint of the red peony root sample, and precision, repeatability and stability tests are performed; and four coagulation test kits are used to measure plasma coagulation time respectively.

[0013] Furthermore, during the measurement process, the sample solutions of plasma and red peony root were mixed with APTT reagent, PT reagent, TT reagent, and FIB reagent, respectively, and the coagulation time of plasma under different reagents was measured; at the same time, 4 control examples were set up, and the sample solutions in them were replaced with blank solvents, and their coagulation times were also measured.

[0014] Furthermore, the precision, repeatability and stability tests include: taking the first sample solution, continuously sampling and measuring it 6 times under the chromatographic conditions, calculating the relative retention time and relative peak area RSD value of the common peak, and conducting a precision experiment; taking the same batch of medicinal materials, preparing 6 sample solutions in parallel, sampling and measuring them separately under the same chromatographic conditions, calculating the relative retention time and relative peak area RSD value of the common peak, and conducting a repeatability experiment; taking the first sample solution, sampling and measuring it 2, 4, 6, 8, 10, and 12 hours after preparation, respectively, calculating the relative retention time and relative peak area RSD value of the common peak, and conducting a stability experiment.

[0015] Furthermore, the method includes evaluating the similarity between samples, including: using a Chinese medicine chromatographic fingerprint similarity evaluation system to calculate the similarity between fingerprints of different batches of red peony root samples and control fingerprints.

[0016] Compared with the prior art, the beneficial effects of this technical solution are as follows: In view of the traditional efficacy of red peony root in "dispersing blood stasis and relieving pain", with the anticoagulant effect as the guide, the spectrum-effect relationship between the HPLC fingerprint of red peony root and the in vitro anticoagulant activity was established, and the multivariate correlation analysis of the efficacy and fingerprint peaks was performed by grey correlation analysis (GRA) to screen out the anticoagulant quality markers of red peony root. This overcomes the problem that there is no relevant technology in the prior art to conduct targeted research on the determination of anticoagulant quality markers of red peony root by grey correlation analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 The present invention is a flow chart of the method for screening quality markers of red peony root based on the anticoagulant spectrum-effect relationship; Figure 2 A diagram of the steps for preparing the pre-screening experiments of the present invention; Figure 3 A diagram of specific experimental steps for preparing materials according to the present invention; Figure 4 A flow chart for determining quality markers of red peony root based on experimental data of the present invention; Figure 5 Fingerprint superposition of 73 batches of red peony root samples; Figure 6 It is a longitudinal comparison diagram of the sample chromatogram (A) of the present invention and the chromatogram (B) of the mixed reference solution. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Based on this, the embodiment of the present application provides a method for screening red peony root quality markers based on the anticoagulant spectrum-effect relationship. The experimenter can prepare reference substances and red peony root sample experimental solutions in the laboratory, and perform experimental determination on the experimental solutions, specifically the method of chromatographic analysis, respectively determine the chromatograms of the samples and the reference substances, and use the peak area data of the common peaks in red peony root to perform principal component analysis, find out the first 5 principal components and calculate their cumulative variance contribution rate to the sample information, so as to identify the key components affecting the quality of red peony root. The anticoagulant efficacy of red peony root samples is evaluated by four coagulation tests, and the anticoagulant efficacy index of red peony root is subjected to gray correlation analysis with the common peak peak area data in the fingerprint spectrum to determine the contribution of each peak to the efficacy, and identify the peaks strongly correlated with the efficacy, i.e., red peony root anticoagulant quality markers. This method can fully consider the correlation between different principal components on the anticoagulant efficacy, and objectively and accurately determine the red peony root quality markers.

[0020] See also Figure 1 , Figure 1 The flowchart of the method for screening quality markers of red peony root based on the anticoagulant spectrum-effect relationship provided in the embodiment of the present application is as follows. The quality marker screening is realized by laboratory data and computer programs, such as Figure 1 The method for screening red peony root quality markers provided in the embodiments of the present application comprises:

[0021] S101 Prepare reference materials and red peony root sample materials, determine the preparation method of red peony root samples and the optimal chromatographic conditions for separation of red peony root samples.

[0022] The optimal chromatographic conditions were as follows: the chromatographic column was Agilent ZORBAX Eclipse XDB-C18 (250 mm × 4.6 mm, 5 µm column, the mobile phase was acetonitrile (A)-0.2% phosphoric acid aqueous solution (B), and the gradient elution (0-10 min, 7% A-10% A; 10-25 min, 10% A-11% A; 25 min-33 min, 11% A-11% A; 33 min-43 min, 11% A-15% A; 43 min-50 min, 15% A- 17%A; 50min~56min, 17%A~18%A; 56~60min, 18%A~20%A; 60~70min, 20A%~25A%; 70min~80min, 25%A~34A%; 80min~90min, 34A%~36A%)); the flow rate was 1.0mL / min; the column temperature was 30℃; the detection wavelength was 230nm; the injection volume was 10µL.

[0023] S102 Construct the fingerprint of red peony root samples and use a coagulometer to determine the in vitro anticoagulant activity of red peony root samples.

[0024] S103 Analyze the experimental data and determine the quality markers of red peony root for the anticoagulant spectrum-effect relationship.

[0025] This embodiment provides a method for screening quality markers of red peony root for anticoagulant spectrum-effect relationship. Before starting the experiment, prepare experimental materials and set up instruments, including selecting suitable instruments such as high-performance liquid chromatography and purchasing high-purity reference substances and reagents; secondly, perform fingerprint establishment and anticoagulant activity determination; finally, analyze the spectrum-effect relationship and screen quality markers, and evaluate the correlation between the peak area of ​​the red peony root fingerprint spectrum and the efficacy index through the gray correlation analysis method, so as to identify the key chemical components that contribute significantly to the efficacy. This process not only systematically evaluates the quality of red peony root medicinal materials, but also provides a scientific basis for the quality control of traditional Chinese medicine.

[0026] In step S101, the experimenter can select the HPLC, centrifuge, reference materials, red peony root samples and experimental animals based on experimental experience. The preparation of these early experimental items is an important basis for the later experiments. The preparation of each item has no particular order and can be carried out in parallel or one by one.

[0027] In one embodiment, see Figure 2 Step S101 prepares reference materials and red peony root sample materials, and determines that the equipment and instruments including a liquid chromatograph and a centrifuge include: S1011 Determine the machine models of liquid chromatograph and centrifuge used in the experiment.

[0028] In this step, the experimenters prepared LC-20AD high performance liquid chromatograph (Shimadzu Corporation, Japan); Heraeus Megafuge 8R centrifuge (Thermo Scientific, USA); MS205DU electronic balance (Mettler Toledo Technology (China) Co., Ltd.); KQ-500DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); four-channel coagulometer (MC-4000Plus, Metron, Germany), etc. The models and manufacturers of each device are clarified, which can help subsequent experimenters to consistently complete the relevant experiments in this embodiment.

[0029] S1012 Identify reference materials, multiple test kit reagents and reagents.

[0030] In this step, the reference substances and kit specifications determined by the experimenters are as follows: gallic acid (batch number: 20230227), catechin (batch number: 20230320), ethyl gallate (batch number: 230510), paeoniflorin (batch number: 20230509), benzoic acid (batch number: 20220904), 1,2,3,4,6-O-pentagalloylglucose (batch number: 230214), p-hydroxybenzoic acid (batch number: AP200314-10), paeonol (batch number: 20230224), benzoylpeoniflorin (batch number: 230412) were purchased from Beijing North Weiye Institute of Metrology and Technology, with a mass fraction of >98%; methanol and acetonitrile (chromatographically pure, Fisher, USA) were used as the reference substances. Scientific Company); purified water was purchased from Hangzhou Wahaha Group Co., Ltd.; the other reagents were of analytical grade; APTT kit (batch number: 112270), PT kit (batch number: 105520), TT kit (batch number: 121338) and FIB kit (batch number: 1322193) were all from Shanghai Sun Biotechnology Co., Ltd., Kangweishi disposable vacuum blood collection tube (sodium citrate 1:9, 5 mL; batch number: 230212), ethyl formate (batch number: 20220810, Shanghai Zhanyun Chemical Co., Ltd.).

[0031] S1013 Identify red peony root samples.

[0032] In this step, the experimenters prepared 73 batches of red peony root samples (S1~S73) collected from different production areas such as Inner Mongolia, Heilongjiang, Shandong, and Hebei (see Table 1). All samples were crushed and passed through a No. 2 sieve for later use.

[0033] Table 1 Source information of 73 batches of red peony root samples

[0034]

[0035]

[0036]

[0037]

[0038] S1014 Identify experimental animals.

[0039] In this step, the experimenters selected SPF Japanese large-eared white rabbits, male, weighing >1 kg, purchased from the Animal Experiment Center of Inner Mongolia Medical University, license number: SCXK (Mongolia) 2020-0003. The rabbits were raised in the SPF animal room of the Experimental Animal Center of Inner Mongolia Medical University, and were adaptively fed for one week before the experiment, fed with standard SPF feed and free access to purified water.

[0040] In the above steps, the experimenter determined the samples, reference materials and materials needed in the relevant steps of the experiment. Subsequently, the fingerprint and anticoagulant activity were measured in S102. The materials used in the measurement process were all the materials prepared and determined in step S101.

[0041] In step S102, the experimenter uses a centrifuge and other equipment to physically process the red peony root sample and the reference substance to prepare a reference substance solution and a sample solution, and uses a chromatograph to perform chromatographic determination on the reference substance solution and the sample solution to form a corresponding chromatogram, and determines the fingerprint of the sample by analyzing the chromatogram. At the same time, the sample solution is tested for precision, repeatability and stability. The experimental results show that the instrument has good precision, the method has good repeatability, and the sample solution has good stability within 12 hours. The method uses a four-item coagulation kit to measure the plasma clotting time separately. During the determination process, each sample solution is measured in parallel three times and compared with a blank sample, which improves the reliability of the test. In one embodiment, see Figure 3 Step S102 constructs the fingerprint of the red peony root sample, and uses the blood of experimental animals to carry out anticoagulant activity determination, including: S1021 Prepare the reference substance into a reference substance solution, and prepare the red peony root sample into a sample solution.

[0042] In this step, the experimenters prepared reference solution and sample solution respectively. The reference solution was prepared by taking appropriate amount of gallic acid, ethyl gallate, benzoic acid, paeoniflorin, 1,2,3,4,6-O-pentagalloylglucose, catechin, benzoylpeoniflorin, paeonol, and p-hydroxybenzoic acid reference substances, dissolving them in methanol, and preparing mixed reference solutions with concentrations of 20.077, 19.000, 38.508, 852.308, 38.846, 38.308, 38.769, 19.731, and 34.938 µg / mL, respectively.

[0043] The preparation method of the red peony root sample solution is as follows: take about 1.0 g of red peony root powder, accurately weigh it, put it in a stoppered conical flask, accurately add 10 mL of 40% methanol, stopper it, weigh it, extract it by ultrasound (350W, 40KHz) for 45 minutes, let it cool, weigh it again, make up the lost weight with 40% methanol, shake it well and let it stand, take an appropriate amount of solution into a centrifuge tube with a lid, centrifuge it at 12000r / min for 3 minutes, aspirate the supernatant, and filter it through a microporous filter membrane (0.45μm) to obtain the sample solution.

[0044] S1022 Chromatographic analysis technology was used to construct the fingerprint of red peony root samples, and precision, repeatability and stability tests were carried out.

[0045] The experimenters will use the scheme recorded in the steps of S1021 to perform chromatographic determination on 73 batches of red peony sample solutions, record the corresponding chromatograms, and form the component fingerprints of 73 batches of red peony samples. The chromatographic data will be imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.1307023 Edition)", and the chromatogram of sample S1 will be set as the reference spectrum. The median method, the time window width is 0.6, and the multi-point correction method will be used to generate the reference spectrum and the HPLC fingerprints of 73 batches of red peony medicinal materials. Figure 5 By mixing with reference substances (see Figure 6 ) were compared and 9 components were identified, peak 5 was gallic acid, peak 7 was p-hydroxybenzoic acid, peak 9 was catechin, peak 14 was ethyl gallate, peak 15 was paeoniflorin, peak 17 was benzoic acid, peak 18 was 1,2,3,4,6-O-pentagalloylglucose, peak 19 was benzoylpeoniflorin and peak 21 was paeonol.

[0046] Before or after forming the component fingerprint of 73 batches of red peony root samples, precision, repeatability and stability tests can also be carried out simultaneously. The experimenter took the first sample solution (i.e., the sample solution numbered S1), and continuously injected and measured 6 times under chromatographic conditions, and calculated the relative retention time and relative peak area RSD value of the common peak to conduct precision experiments. According to the analysis of experimental data, the relative retention time and relative peak area RSD values ​​of each common peak were calculated to be less than 0.26% and 2.85%, respectively, indicating that the instrument has good precision.

[0047] The experimenter took the first sample solution (i.e., the sample solution numbered S1), prepared 6 sample solutions in parallel, injected and measured them separately under the same chromatographic conditions, and calculated the relative retention time and relative peak area RSD values ​​of the common peaks to conduct a repeatable experiment. According to the analysis of the experimental data, the relative retention time and relative peak area RSD values ​​of each common peak were calculated to be less than 0.21% and 2.88%, respectively, indicating that the method has good repeatability.

[0048] The experimenter took the first sample solution (i.e., the sample solution numbered S1) and injected it for measurement at 2, 4, 6, 8, 10, and 12 hours after preparation, and calculated the relative retention time and relative peak area RSD values ​​of the common peaks to conduct a stability experiment. According to the analysis of the experimental data, the relative retention time and relative peak area RSD values ​​of each common peak were calculated to be less than 0.26% and 2.85%, respectively, indicating that the test solution had good stability within 12 hours.

[0049] The above is a demonstration of the accuracy, reliability and timeliness of experimental data from a single sample solution. At the same time, in order to ensure that there is no obvious difference in information between samples, the similarity evaluation between samples can be selectively carried out.

[0050] In an optional embodiment, the experimenter compares and analyzes the chromatographic data of different sample solutions by comparing the chromatographic similarities of different red peony root sample solutions with the control solution (ie, S1 sample), thereby forming the data in Table 2.

[0051] Table 2 Similarity calculation results of 73 batches of red peony root fingerprints

[0052] From the table above, we can clearly see that among the 73 batches of red peony samples, 66 batches have a similarity greater than 0.9 with the control spectrum, indicating that the chemical composition of red peony samples from different origins is similar, and the overall quality of the medicinal materials is relatively stable. Except for the similarity of 0.355 for the S31 sample and 0.726 for the S43 sample, the similarities of the remaining samples are all above 0.8, with the highest similarity being 0.997. According to the traceability investigation and analysis, the S31 red peony medicinal material was artificially planted in black soil, and S43 was a transplanted variety, which may be related to factors such as the growth environment, growth method and origin of the collection. The similarity results prove that the quality of each batch of red peony medicinal materials is relatively stable.

[0053] The above preliminary analysis and comparison further demonstrated that the basic data can stably and reproducibly reflect the medicinal properties of red peony root, thereby further stably determining the quality markers of red peony root.

[0054] S1023 Use four coagulation test kits to measure plasma coagulation time.

[0055] In this step, the experimenters collected plasma from big-eared white rabbits and sample solutions of red peony root, then took a certain amount of plasma and sample solutions of red peony root and mixed them with APTT reagent, PT reagent, TT reagent, and FIB reagent, respectively, to measure the coagulation time of plasma under different reagents. During the determination process, 4 control examples were set up, and the sample solutions were replaced with blank solvents, and their coagulation time was also measured. By using control examples and different reagents to measure the coagulation time of red peony root samples, the final verification data was provided for the subsequent determination of the red peony root quality marker for the anticoagulant spectrum-effect relationship.

[0056] In step S1023, the experimenter used three major experimental steps to determine the final coagulation time, including blood collection, sample preparation, and coagulation four-item kit measurement.

[0057] In one embodiment, the blood collection adopts the following scheme: the injection volume of the urethane solution of the big-eared white rabbit anesthetic should be 5 mL kg-1 body weight, and the rabbit's ear vein is anesthetized and blood is collected from the common carotid artery. The collected blood is placed in a 5 mL blood collection tube with 1:9 sodium citrate, and each blood collection tube is collected to the scale line. After mixing, centrifuge at 3000 rpm for 15 minutes, and the upper layer (plasma) is collected and stored at 4 ° C for use. It cannot be repeatedly frozen and thawed during use.

[0058] In one embodiment, the sample is prepared by the following scheme: filtering with a microporous membrane, accurately aspirating 5 mL of the filtrate, evaporating it in a water bath, adding physiological saline solution to dissolve it, placing it in a 5 mL volumetric flask to make a fixed volume to obtain a red peony root extract stock solution for use.

[0059] In one embodiment, the coagulation four-item kit is measured using the following scheme: APTT test method: Pipette 50 μL of the plasma to be tested into a blood coagulation cup, add 50 μL of sample solution, add 50 μL of APTT reagent, incubate at 37°C for 5 minutes, and finally add 50 μL of 37°C pre-warmed calcium chloride solution (0.025 mol·L-1) to measure the coagulation time. Replace the sample with a blank solvent and measure the coagulation time in the same way as above, which is the control.

[0060] PT test method: Pipette 50 μL of plasma to be tested into a blood coagulation cup, add 50 μL of sample solution, add 100 μL of PT reagent, incubate at 37°C for 3 minutes, and measure the coagulation time. Replace the sample with blank solvent, and measure the coagulation time in the same way as above, which is the control.

[0061] TT test method: Pipette 100 μL of the plasma to be tested into a blood coagulation cup, add 100 μL of sample solution, add 100 μL of TT reagent, incubate at 37°C for 5 minutes, and measure the coagulation time. Replace the sample with a blank solvent and measure the coagulation time in the same manner as above, which is the control.

[0062] FIB test method: Pipette 100 μL of plasma to be tested into a blood coagulation cup, add 50 μL of sample solution, add 50 μL of thrombin solution, incubate at 37°C for 5 minutes, and measure the coagulation time. Replace the sample with a blank solvent, and measure the coagulation time in the same way as above, which is the control.

[0063] Each sample solution in all the above coagulation test steps was measured in parallel three times.

[0064] S103 Analyze the experimental data and determine the quality markers of red peony root for the anticoagulant spectrum-effect relationship.

[0065] The above steps determined reliable chromatographic data based on experiments. On this basis, the principal components of red peony root were determined by using the principal component eigenvalue and contribution rate. The principal component loading matrix and its comprehensive score were used to determine that the quality of red peony root was jointly determined by multiple components. On this basis, the peak area data of the common peaks in the fingerprint spectrum were used as characteristic sequence variables, and the anticoagulant quality marker of red peony root was finally determined in combination with the principal components.

[0066] Specifically, it includes two parts: principal component analysis of experimental data and quality markers. In the principal component analysis part, in addition to determining the specific substance of the principal component, it is also necessary to determine the interaction between the principal components, so it includes the following steps: S1031 Obtain the peak areas of common peaks of multiple batches of red peony root, determine the principal component analysis eigenvalues ​​and contribution rates, and thus determine the principal components of red peony root.

[0067] In this step, the experimenters used the peak areas of 22 common peaks in 73 batches of red peony root as variables and imported them into SPSS26.0 software for principal component analysis. The results are shown in Table 3. The eigenvalues ​​of the first five principal components were greater than 1, and the cumulative variance contribution rate was 79.152%, indicating that the first five principal components could represent 79.152% of the information in the 22 common peaks of 73 batches of red peony root samples.

[0068] Table 3 Principal component analysis eigenvalues ​​and contribution rates

[0069] S1032 obtains the peak area data of the common peak, calculates and determines the principal component factor loading matrix, and finally determines the contribution rate of the principal component.

[0070] The peak areas of 22 common peaks in 73 batches of red peony root were used as variables to import into SPSS26.0 software, and the principal component factor loading matrix data was calculated and determined. The data export table is shown in Table 4. Based on the absolute value, the first principal component mainly reflects the information of chromatographic peaks 1, 6, 9-16, the second principal component mainly reflects the information of chromatographic peaks 1, 2, 4, 5, 18-20, the third principal component mainly reflects the information of chromatographic peak 22, the fourth principal component mainly reflects the information of chromatographic peak 21, and the fifth principal component mainly reflects the information of chromatographic peak 7. It shows that it is not a single component that affects the quality of red peony root, but the result of the interaction of multiple components. According to the principal component matrix data, the weight coefficient of the corresponding factor is obtained by dividing the peak coefficient in the loading matrix by the square root of the eigenvalue of the principal component. Then, the standardized original data is substituted into the principal component expression to calculate the principal component score of each sample, and the comprehensive score is calculated with the contribution rate corresponding to each principal component as the weight coefficient. The results are shown in Table 5. According to the results, S4, S5, S811, S13, S17~19, S24, S28, S32, S34~45, S49, S53~58, S61, S65, and S70 with positive comprehensive scores are clustered into one category, and those with negative comprehensive scores are clustered into one category. Among them, S34~S40 red peony roots from Balin Youqi, Chifeng, Inner Mongolia all ranked relatively high in comprehensive scores, indicating that the quality of red peony roots from Balin Youqi, Chifeng is excellent and stable.

[0071] Table 4 Correlation between common peaks and drug efficacy

[0072] Table 5 Principal component scores and comprehensive scores

[0073] S1033 The anticoagulant efficacy index of red peony root was taken as the correlation object, and the common peak area data in the red peony root fingerprint was taken as the characteristic sequence variable. Grey correlation analysis was performed on the two to determine the quality marker of red peony root.

[0074] The anticoagulant efficacy indexes (APTT, PT, TT, and FIB) of red peony root were taken as the associated objects, i.e., quantitative variables, and the peak area data of the common peaks in the HPLC fingerprint of red peony root were taken as the characteristic sequence variables. The data were input into SPSSPRO (version 1.1.21) software to perform grey correlation analysis on the two. Since the measurement units of the parent sequence and the characteristic sequence are different, dimensionless processing (average) is required before analysis. ρ is set to 0.5, and data analysis is performed on the four coagulation indicators respectively. The grey correlation degree and ranking are obtained under the algorithm configuration, as shown in Table 6. The GRA calculation results show that the greater the correlation degree, the greater its contribution to the efficacy. It can be seen from the results that the correlation degree between the 22 common peaks in the fingerprint of red peony root and the four efficacy indicators is greater than 0.800, indicating that the 22 common peaks have different degrees of influence on the four efficacy indicators. The correlation between peaks 1, 2, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, and 20 and the efficacy was above 0.900, indicating that these 16 common peaks were strongly correlated with the changes in the four efficacy indicators. Peak 5 (gallic acid), peak 9 (catechins), peak 14 (ethyl gallate), peak 15 (peonidin), peak 17 (benzoic acid), peak 18 (pentagalloyl glucose), and peak 19 (benzoylpeonidin) were identified as anticoagulant quality markers of red peony through standard identification. Among them, peak 15 (peonidin) ranked first in the grey correlation ranking of the four efficacy indicators, which also verified its scientificity as an indicator component of red peony.

[0075] Table 6 Correlation between common peaks and drug efficacy

[0076] Those skilled in the art will readily appreciate other embodiments of the present application after considering the specification and practicing the application disclosed herein. The present application is intended to cover any variations, uses or adaptations of the present application, which follow the general principles of the present application and include common knowledge or customary technical means in the art disclosed in the present application. The specification and examples are to be regarded as exemplary only, and the true scope of the present application is indicated by the claims.

Claims

1. A method for screening quality markers of red peony root based on anticoagulant spectrum-effect relationship, characterized in that: The following steps are involved: Prepare reference materials and red peony root sample materials, determine the preparation method of red peony root samples and the best chromatographic conditions for separation of red peony root samples; Fingerprints of red peony root samples were constructed, and the anticoagulant activity of red peony root samples in vitro was determined using a coagulometer. Analyze the experimental data and determine the quality markers of red peony root for the anticoagulant spectrum-effect relationship; Among them, determining the quality markers of red peony root for the anticoagulant spectrum-effect relationship includes the following steps: The fingerprint of red peony root was constructed, and the methodological investigation was carried out according to the optimal chromatographic conditions of red peony root samples to determine 22 common peaks, obtain the peak area data of the common peaks of multiple batches of red peony root samples, determine the characteristic value and contribution rate of principal component analysis, and thus determine the main components of red peony root; Obtain the peak area data of the common peaks of multiple batches of red peony root samples, calculate and determine the principal component factor loading matrix, and finally determine the contribution rate of the principal component; The anticoagulant efficacy index of red peony root was taken as the correlation object, and the common peak area data in the red peony root fingerprint was taken as the characteristic sequence variable. Grey correlation analysis was performed on the two to determine the quality marker of red peony root.

2. The method for screening red peony root quality markers based on anticoagulant spectrum-effect relationship according to claim 1, characterized in that: Four anticoagulant indicators, APTT, PT, TT and FIB, were used as correlation objects, and the peak area data of 22 common peaks in the red peony root fingerprint were used as the input layer of the computer program. The grey correlation degree between the two was determined by the set program, and gallic acid, catechin, ethyl gallate, paeoniflorin, benzoic acid, pentagalloylglucose and benzoylpeoniflorin were determined as the anticoagulant quality markers of red peony root.

3. The method for screening red peony root quality markers based on anticoagulant spectrum-effect relationship according to claim 2, characterized in that: Through the grey correlation analysis of four efficacy indicators of paeoniflorin, paeoniflorin was determined to be the most important quality marker of red peony root.

4. The method for screening red peony root quality markers based on anticoagulant spectrum-effect relationship according to claim 1, characterized in that: The peak areas of 22 common peaks in 73 batches of red peony root were imported into a computer program as variables to determine the principal component analysis eigenvalues, contribution rates and principal component factor loading matrix.

5. The method for screening red peony root quality markers based on anticoagulant spectrum-effect relationship according to claim 1, characterized in that: Preparation of reference materials and red peony root sample materials, and determination of equipment including liquid chromatograph and centrifuge include the following steps: Determine the models of liquid chromatograph and centrifuge used in the experiment; Identify reference materials, multiple test kits and reagents; Determine the optimal extraction conditions for red peony root samples; Identify experimental animals.

6. The method for screening red peony root quality markers based on anticoagulant spectrum-effect relationship according to claim 1, characterized in that: The fingerprint of red peony root samples was constructed and the anticoagulant activity was determined using the blood of experimental animals, including: The reference substance is prepared into a reference substance solution, and the red peony root sample is prepared into a sample solution; The fingerprint of red peony root samples was constructed by chromatographic analysis, and the precision, repeatability and stability of the method were tested. Four coagulation test kits were used to measure the plasma coagulation time.

7. The method for screening red peony root quality markers based on anticoagulant spectrum-effect relationship according to claim 6, characterized in that: During the determination process, plasma and red peony root sample solutions were mixed with APTT reagent, PT reagent, TT reagent, and FIB reagent, respectively, and the coagulation time of plasma under different reagents was measured; at the same time, 4 control examples were set up, and the sample solutions in them were replaced with blank solvents, and their coagulation times were also measured.

8. The method for screening red peony root quality markers based on anticoagulant spectrum-effect relationship according to claim 6, characterized in that: The precision, repeatability and stability tests include: Take the first sample solution, inject it continuously for 6 times under the chromatographic conditions, calculate the relative retention time and relative peak area RSD value of the common peak, and conduct precision experiments; Take the same batch of medicinal materials, prepare 6 sample solutions in parallel, and inject them separately under the same chromatographic conditions to calculate the relative retention time and relative peak area RSD value of the common peak, so as to conduct a repeatable experiment; Take the first sample solution, and inject it for measurement at 2, 4, 6, 8, 10, and 12 hours after preparation, and calculate the relative retention time and relative peak area RSD value of the common peak to conduct a stability experiment.

9. The method for screening red peony root quality markers based on anticoagulant spectrum-effect relationship according to claim 8, characterized in that: Further included is the evaluation of similarity between samples, including: The similarity between fingerprints of different batches of red peony root samples and the control was calculated using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system.

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