A method for screening quality markers of red peony root based on anticoagulant spectrum-effect relationship

Abstract

The application discloses a method for screening a quality marker of Radix Paeoniae Rubra based on an anticoagulation spectrum-effect relationship, mainly comprising the following steps: preparing control samples and Radix Paeoniae Rubra sample materials, determining a preparation method of the Radix Paeoniae Rubra sample and optimal chromatographic conditions for separating the Radix Paeoniae Rubra sample, and constructing a fingerprint of the Radix Paeoniae Rubra sample; using a blood coagulation instrument to determine an in-vitro anticoagulation activity of Radix Paeoniae Rubra extracts; analyzing experimental data to determine the quality marker of Radix Paeoniae Rubra based on the anticoagulation spectrum-effect relationship. The efficacy and common peaks of the fingerprint are analyzed by a grey correlation degree analysis method, and the quality marker of Radix Paeoniae Rubra for anticoagulation is screened. The method overcomes the fact that the prior art does not have a related technology for determining the quality marker of Radix Paeoniae Rubra for anticoagulation by the grey correlation degree analysis method.

Description

TECHNICAL FIELD

[0001] The present application relates to a Chinese medicine quality marker screening method, in particular to a Radix Paeoniae Rubra quality marker screening method based on the anti-coagulation spectrum-effect relationship. BACKGROUND

[0002] Radix Paeoniae Rubra is the dried root of Paeonia lactiflora Pall or Paeonia veitchii Lynch, which is distributed in Inner Mongolia, Heilongjiang, Jilin, Liaoning and other places in China, and has the effects of dispersing blood stasis, relieving pain, clearing heat and cooling blood. Radix Paeoniae Rubra contains monoterpenoid glycosides, tannins, triterpenes, volatile oils and other components. The glycoside components are collectively referred to as total paeoniae rubra glycosides (TPG), which are the main effective components. According to literature reports, Radix Paeoniae Rubra has good anti-coagulation efficacy. TPG, paeoniflorin, pentagalloyl glucose, paeonolactone glycoside, protocatechuic acid and paeonol in Radix Paeoniae Rubra are the material basis for the anti-thrombosis effect of Radix Paeoniae Rubra.

[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 index components as the quality standard of medicinal materials, which cannot accurately and comprehensively reflect the quality of medicinal materials. At the same time, the drug-effect correlation is not strong, and the basis for limit setting is insufficient, which are the bottlenecks of traditional Chinese medicine quality control research.

[0004] Currently, some scholars have also studied the drug-effect correlation of traditional Chinese medicine, but there is almost no public literature record on the correlation of Radix Paeoniae Rubra, especially the anti-coagulation efficacy, which to some extent limits the promotion and application of Radix Paeoniae Rubra in the field of anti-coagulation. SUMMARY

[0005] The purpose of the present application is to provide a Radix Paeoniae Rubra quality marker screening method based on the anti-coagulation spectrum-effect relationship, in order to solve the technical problem that the quality marker of Radix Paeoniae Rubra in the field of anti-coagulation cannot be determined based on the drug-effect correlation in the prior art.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0007] In one embodiment, a Radix Paeoniae Rubra quality marker screening method based on the anti-coagulation spectrum-effect relationship is provided, which comprises the following steps:

[0008] Prepare control samples and Radix Paeoniae Rubra sample materials, determine the equipment and instruments including a liquid chromatograph and a centrifuge; construct a fingerprint of the Radix Paeoniae Rubra sample, use experimental animal blood to carry out anti-coagulation activity determination; analyze the experimental data to determine the Radix Paeoniae Rubra quality marker of the anti-coagulation spectrum-effect relationship.

[0009] Further, the Radix Paeoniae Rubra quality marker of the anti-coagulation spectrum-effect relationship comprises the following steps:

[0010] The peak area of the common peaks of the multiple batches of Radix Paeoniae Rubra is obtained, the characteristic value and contribution rate of principal component analysis are determined, and thus the principal component of Radix Paeoniae Rubra is determined; the peak area data of the common peaks are obtained, the factor loading matrix of the principal component is calculated and determined, and finally the contribution rate of the principal component is determined; the anticoagulant efficacy index of Radix Paeoniae Rubra is the associated object, the peak area data of the common peaks in the fingerprint of Radix Paeoniae Rubra are taken as the characteristic sequence variables, the grey correlation analysis is performed on the two, and the quality marker of Radix Paeoniae Rubra is determined.

[0011] Further, the four anticoagulant indexes of APTT, PT, TT and FIB are taken as the associated objects, the total area data of the 22 common peaks in the fingerprint of Radix Paeoniae Rubra are taken as the input of the computer program, the grey correlation degrees of the two are determined through the set program, and gallic acid, catechin, ethyl gallate, paeoniflorin, benzoic acid, pentagalloyl glucose and benzoylpaeoniflorin are determined as the quality markers of Radix Paeoniae Rubra for anticoagulation.

[0012] Further, through the grey correlation degree analysis of paeoniflorin in the four efficacy indexes, it is determined that paeoniflorin is the most important quality marker of Radix Paeoniae Rubra.

[0013] Further, the peak area of the 22 common peaks in the 73 batches of Radix Paeoniae Rubra is introduced into the computer program as the variable, and the characteristic value, contribution rate and principal component factor loading matrix of the principal component analysis are determined.

[0014] Further, the preparation of the control and Radix Paeoniae Rubra sample materials is prepared, and the determination of the equipment and instruments including the liquid chromatograph and the centrifuge includes the following steps: determining the model of the liquid chromatograph and the centrifuge used for the experiment; determining the control, the multiple reagent kits and reagents; determining the Radix Paeoniae Rubra sample; and determining the experimental animals.

[0015] Further, the fingerprint of the Radix Paeoniae Rubra sample is constructed, and the anticoagulant activity determination of the blood of the experimental animals includes:

[0016] The control is prepared into a control solution, and the Radix Paeoniae Rubra sample is prepared into a sample solution; the fingerprint of the Radix Paeoniae Rubra sample is constructed by using the chromatographic analysis technology, and the precision, repeatability and stability tests are performed; the coagulation four-item kit is used to determine the plasma clotting time.

[0017] Further, in the determination process, the plasma and the sample solution of Radix Paeoniae Rubra are mixed with APTT reagent, PT reagent, TT reagent and FIB reagent respectively, and the clotting time of the plasma under different reagents is determined; at the same time, four control examples are set, the sample solution in which is replaced with a blank solvent, and the clotting time thereof is also determined.

[0018] Further, the precision, repeatability and stability test includes: taking the first sample solution, determining 6 times continuously under the same chromatographic conditions, calculating the relative retention time and relative peak area RSD value of the common peaks, thereby performing the precision experiment; taking the same batch of medicinal materials, preparing 6 sample solutions in parallel, respectively determining under the same chromatographic conditions, calculating the relative retention time and relative peak area RSD value of the common peaks, thereby performing the repeatability experiment; taking the first sample solution, respectively determining at 2, 4, 6, 8, 10 and 12 hours after preparation, calculating the relative retention time and relative peak area RSD value of the common peaks, thereby performing the stability experiment.

[0019] Further, the similarity between samples is evaluated, including: using the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, calculating the similarity of the fingerprint spectrum of different batches of Radix Paeoniae Rubra samples and the control spectrum.

[0020] Compared with the prior art, the beneficial effects of the technical solution are as follows:

[0021] In view of the traditional function of Radix Paeoniae Rubra in dispersing blood stasis and relieving pain, the spectrum-effect relationship between the HPLC fingerprint spectrum of Radix Paeoniae Rubra and the in-vitro anticoagulant activity is established, the multivariate correlation between the efficacy and the fingerprint spectrum peaks is analyzed by the Grey Relation Analysis (GRA), and the anticoagulant quality marker of Radix Paeoniae Rubra is screened out. The present technical solution overcomes the problem in the prior art that there is no related technical solution to determine the anticoagulant quality marker of Radix Paeoniae Rubra by the Grey Relation Analysis. BRIEF DESCRIPTION OF DRAWINGS

[0022] The accompanying drawings are included to provide a further understanding of the present application, and constitute a part of the specification, which together with the embodiments of the present application, serve to explain the present application, and do not constitute a limitation of the present application. In the drawings:

[0023] Figure 1 The flow chart of the Radix Paeoniae Rubra quality marker screening method based on the anticoagulant spectrum-effect relationship of the present application;

[0024] Figure 2 The preparation step chart before the screening of the present application;

[0025] Figure 3 The specific experimental step chart based on the prepared materials of the present application;

[0026] Figure 4 The flow chart of determining the Radix Paeoniae Rubra quality marker based on the experimental data of the present application;

[0027] Figure 5 The fingerprint superimposed spectrum of 73 batches of Radix Paeoniae Rubra samples;

[0028] Figure 6A longitudinal comparison chart of the sample chromatogram (A) of the present application and the chromatogram (B) of the mixed control solution. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work are within the protection scope of the present application.

[0030] Therefore, the present application provides a method for screening a quality marker of Radix Paeoniae Rubra based on an anti-coagulation spectrum-effect relationship. The experimenters can prepare a control solution and a Radix Paeoniae Rubra sample solution in a laboratory, and determine the solution by experiment, specifically, by a chromatographic analysis method. The chromatograms of the sample and the control solution are determined respectively. The peak area data of common peaks in Radix Paeoniae Rubra are used for principal component analysis. The first five principal components are found out, and the cumulative variance contribution rate of the principal components to the sample information is calculated, so as to identify the key components affecting the quality of Radix Paeoniae Rubra. The anti-coagulation efficacy of the Radix Paeoniae Rubra sample is evaluated by a coagulation four-item detection. The anti-coagulation efficacy index of Radix Paeoniae Rubra is subjected to a grey correlation analysis with the peak area data of the common peaks in the fingerprint spectrum, so as to determine the contribution degree of each peak to the efficacy, and identify the peaks strongly related to the efficacy, i.e., the quality markers of Radix Paeoniae Rubra for anti-coagulation. This method can fully consider the correlation influence of different principal components on the anti-coagulation efficacy, and objectively and accurately determine the quality markers of Radix Paeoniae Rubra.

[0031] Please refer to Figure 1 , Figure 1 A method for screening a quality marker of Radix Paeoniae Rubra based on an anti-coagulation spectrum-effect relationship is provided in the embodiments of the present application. The screening of the quality marker is realized by laboratory data and a computer program, as shown in Figure 1 The method for screening a quality marker of Radix Paeoniae Rubra provided in the embodiments of the present application includes the following steps.

[0032] S101, preparing a control solution and a Radix Paeoniae Rubra sample material, and determining a preparation method of the Radix Paeoniae Rubra sample and optimal chromatographic conditions for separation of the Radix Paeoniae Rubra sample.

[0033] The determined optimal chromatographic conditions are as follows: the chromatographic column is Agilent ZORBAX Eclipse XDB-C18 (250 mm x 4.6 mm, 5 µm column), the mobile phase is acetonitrile (A)-0.2% phosphoric acid aqueous solution (B), 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; 50 min-56 min, 17% A-18% A; 56-60 min, 18% A-20% A; 60-70 min, 20% A-25% A; 70 min-80 min, 25% A-34% A; 80 min-90 min, 34% A-36% A); the flow rate is 1.0 mL / min; the column temperature is 30°C; the detection wavelength is 230 nm; and the injection volume is 10 µL.

[0034] S102 Constructing the fingerprint of Radix Paeoniae Rubra sample, and using a blood coagulation instrument to carry out in vitro anti-coagulation activity determination of Radix Paeoniae Rubra sample.

[0035] S103 Analyzing experimental data to determine the quality marker of Radix Paeoniae Rubra with anti-coagulation spectrum-effect relationship.

[0036] The embodiment provides a quality marker screening method of Radix Paeoniae Rubra with anti-coagulation spectrum-effect relationship. Before starting the experiment, experimental materials and instruments are prepared, including selecting appropriate instruments such as a high-performance liquid chromatograph and purchasing high-purity reference substances and reagents; secondly, fingerprint establishment and anti-coagulation activity determination are performed; finally, spectrum-effect relationship is analyzed and a quality marker is screened. The correlation between the peak area of the Radix Paeoniae Rubra fingerprint and the pharmacodynamic index is evaluated by the grey correlation degree analysis method, so as to identify the key chemical components that have a significant contribution to the pharmacodynamic effect. This process not only systematically evaluates the quality of Radix Paeoniae Rubra, but also provides a scientific basis for the quality control of traditional Chinese medicines.

[0037] In step S101, the experimental personnel can select the high-performance liquid chromatograph, centrifuge, reference substances, Radix Paeoniae Rubra samples and experimental animals based on experimental experience. The preparation of these pre-experimental items is an important basis for the later experiment, and the preparation of each item can be carried out in parallel or sequentially.

[0038] In one embodiment, referring to Figure 2 , step S101 preparation includes reference substances and Radix Paeoniae Rubra sample materials, and the determination includes equipment and instruments including a liquid chromatograph and a centrifuge.

[0039] S1011 Determine the model of the liquid chromatograph and the centrifuge used in the experiment.

[0040] In this step, the experimenter prepared an LC-20AD high performance liquid chromatograph (Shimadzu Corporation, Japan); a Heraeus Megafuge 8R centrifuge (Thermo Scientific, USA); an MS205DU electronic balance [Mettler Toledo (China) Co., Ltd.]; a KQ-500DB digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); a four-channel blood coagulation instrument (MC-4000Plus, Germany Meier) and the like. The model numbers and manufacturers of each device are identified, which can help subsequent experimenters consistently complete the related experiments in this example.

[0041] S1012 Determine the control, multiple kit reagents and reagents.

[0042] In this step, the experimenters determined the control, kit specifications as follows: gallic acid (batch number: 20230227), catechin (batch number: 20230320), ethyl gallate (batch number: 230510), paeonol (batch number: 20230509), benzoic acid (batch number: 20220904), 1,2,3,4,6-O-penta-galloyl glucose (batch number: 230214), p-hydroxy benzoic acid (batch number: AP200314-10), paeonol (batch number: 20230224), benzoyl paeonol (batch number: 230412) control purchased from Beijing Beifang Weiyeh Measurement Technology Research Institute, the mass fraction is all > 98%; methanol, acetonitrile (chromatographic pure, Fisher Scientific, USA); purified water purchased from Hangzhou Wahaha Group Co., Ltd.; the rest of the reagents are all analytical pure; APTT kit (batch number: 112270), PT kit (batch number: 105520), TT kit (batch number: 121338) and FIB kit (batch number: 1322193) are all from Shanghai Sunshine Biotechnology Co., Ltd., Kangweishi disposable vacuum blood collection tube (sodium citrate 1:9, 5 mL; batch number: 230212), ethyl carbamate (batch number: 20220810, Shanghai Zhan Yun Chemical Co., Ltd.).

[0043] S1013 Determine the sample of red peony root.

[0044] In this step, the experimenters prepared 73 batches of red peony root samples (S1-S73) collected from different producing areas in Inner Mongolia, Heilongjiang, Shandong, Hebei and the like (see Table 1), all the samples were crushed and passed through a No. 2 sieve for standby.

[0045] Table 1 Source information of 73 batches of red peony root samples

[0046]

[0047]

[0048]

[0049]

[0050] S1014 determining the experimental animals.

[0051] In this step, the experimenter selects SPF level Japanese white rabbits, male, body weight > 1 kg, purchased from Inner Mongolia Medical University Animal Experiment Center, license number: SCXK (Meng) 2020-0003. White rabbits are raised in the SPF level animal room of Inner Mongolia Medical University Experimental Animal Center. One week before the experiment, the rabbits were adaptively fed with standard SPF feed and free access to pure water.

[0052] In the above step, the experimenter determines the samples, controls and materials used in the experimental steps. The determination of the fingerprint and the anticoagulant activity is carried out in S102. The materials used in the determination process are determined in S101.

[0053] In step S102, the experimenter uses centrifuge and other equipment to physically process the red peony root samples and controls to prepare control solution and sample solution. Chromatography is used to determine the control solution and sample solution, and the corresponding chromatogram is formed. The fingerprint of the sample is determined by analyzing the chromatogram. At the same time, the sample solution is subjected to precision, repeatability and stability tests. The experimental results show that the instrument precision is good, the method repeatability is good, and the sample solution has good stability within 12h. The method uses a coagulation four-item kit to measure plasma clotting time. Each sample solution is measured in triplicate during the determination process and compared with the blank sample to improve the reliability of the test

[0054] In one embodiment, referring to Figure 3 , step S102 constructs the fingerprint of the red peony root sample, and the anticoagulant activity determination is carried out using the blood of experimental animals, including:

[0055] S1021 preparing the control into a control solution and the red peony root sample into a sample solution.

[0056] In this step, the experimenter prepares the control solution and the sample solution. The preparation method of the control solution is as follows: take gallic acid, gallic acid ethyl ester, benzoic acid, paeoniflorin, 1,2,3,4,6-O-penta-galloyl glucose, catechin, benzoyl paeoniflorin, paeonol, and p-hydroxy benzoic acid control, respectively, and dissolve them in methanol to prepare a mixed control solution with a concentration of 20.077, 19.000, 38.508, 852.308, 38.846, 38.308, 38.769, 19.731, and 34.938 µg / mL, respectively.

[0057] The preparation method of the sample solution of Radix Paeoniae Rubra is as follows: about 1.0 g of Radix Paeoniae Rubra powder is accurately weighed, placed in a stoppered triangular flask, 10 mL of 40% methanol is accurately added, tightly stoppered, the weight is determined, ultrasonic extraction (350 W, 40 KHz) is performed for 45 min, allowed to cool, the weight is determined again, the lost weight is made up with 40% methanol, shaken well, and placed to take an appropriate amount of solution in a covered centrifuge tube, centrifuged at 12,000 r / min for 3 min, the supernatant is sucked, filtered through a microporous filter membrane (0.45 μm), and the sample solution is obtained.

[0058] S1022 The chromatographic analysis technology is used to construct the fingerprint of the sample of Radix Paeoniae Rubra, and the precision, repeatability and stability tests are performed.

[0059] The experimenters respectively determine the chromatograms of the sample solutions of 73 batches of Radix Paeoniae Rubra by the scheme recorded in S1021, and form the component fingerprint of the 73 batches of Radix Paeoniae Rubra samples. The chromatographic data is introduced into “Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.1307023 version)”, the chromatogram of sample S1 is set as the reference chromatogram, the median method is used, the time window width is 0.6, and the multi-point correction method is used to generate the control chromatogram and the HPLC fingerprint of the 73 batches of Radix Paeoniae Rubra samples, which is shown in Figure 5 By comparison with the mixed control (see Figure 6 ), 9 components are identified, the peak No. 5 is gallic acid, the peak No. 7 is p-hydroxybenzoic acid, the peak No. 9 is catechin, the peak No. 14 is ethyl gallate, the peak No. 15 is paeoniflorin, the peak No. 17 is benzoic acid, the peak No. 18 is 1,2,3,4,6-O-penta-galloyl glucose, the peak No. 19 is benzoyl paeoniflorin, and the peak No. 21 is paeonol.

[0060] Before or after the formation of the component fingerprint of the 73 batches of Radix Paeoniae Rubra samples, the precision, repeatability and stability tests can also be simultaneously performed. The experimenters take the first sample solution (i.e. the sample solution numbered S1), continuously sample and determine 6 times under the same chromatographic conditions, calculate the RSD values of the relative retention time and the relative peak area of the common peaks, and thus perform the precision experiment. According to the experimental data analysis, the RSD values of the relative retention time and the relative peak area of the common peaks are less than 0.26% and 2.85% respectively, indicating that the precision of the instrument is good.

[0061] The experimenters take the first sample solution (i.e. the sample solution numbered S1), prepare 6 sample solutions in parallel, sample and determine respectively under the same chromatographic conditions, calculate the RSD values of the relative retention time and the relative peak area of the common peaks, and thus perform the repeatability experiment. According to the experimental data analysis, the RSD values of the relative retention time and the relative peak area of the common peaks are less than 0.21% and 2.88% respectively, indicating that the repeatability of the method is good.

[0062] The experimenters took the first sample solution (i.e., sample solution numbered S1), respectively, at 2, 4, 6, 8, 10, and 12 h after preparation, to determine the relative retention time and relative peak area RSD value of the common peaks, to perform the stability experiment. According to the experimental data analysis, the relative retention time and relative peak area RSD value of each common peak were calculated to be less than 0.26% and 2.85%, respectively, indicating that the stability of the test sample solution within 12 h was good.

[0063] The above is a single sample solution from the perspective of demonstrating the accuracy, reliability, and timeliness of experimental data in the case of a single sample. At the same time, in order to ensure that there is no obvious difference between the information of the samples, the similarity between the samples can be selectively evaluated.

[0064] In an optional embodiment, the experimenters perform comparative analysis of the chromatographic data of different sample solutions. The method used is the similarity comparison of the chromatograms of different red peony root sample solutions and the control solution (i.e., S1 sample), forming the data in Table 2.

[0065] Table 2 Similarity calculation results of 73 batches of red peony root fingerprint chromatograms

[0066]

[0067] From the above table, it can be clearly seen that there are 66 batches of samples with a similarity greater than 0.9 between the 73 batches of red peony root samples and the control chromatogram, indicating that the chemical components of red peony root samples from different origins are similar, and the overall quality of the medicinal materials is relatively stable. Except for the similarity of S31 sample 0.355 and the similarity of S43 sample 0.726, the similarity of the remaining samples is above 0.8, and the highest similarity is 0.997. Through traceability investigation and analysis, S31 red peony root medicinal material is artificially planted on black land, and S43 is a transplanted variety, which may be related to factors such as the growth environment, growth method, and origin, etc. The similarity result proves that the quality of each batch of red peony root medicinal material is relatively stable.

[0068] The above preliminary analysis comparison further demonstrates that the basic data can stably and reproducibly reflect the medicinal properties of red peony root, thereby further stabilizing and determining the quality markers of red peony root.

[0069] S1023 Using a blood coagulation four-item kit, respectively, to determine the plasma clotting time.

[0070] In this step, the experimenters collected the plasma of the big-eared white rabbits, the sample solution of the red peony root, and then took a certain amount of plasma and red peony root sample solution and mixed them with APTT reagent, PT reagent, TT reagent, and FIB reagent, respectively, to measure the coagulation time of the plasma under different reagents. During the measurement process, four control examples were set, in which the sample solution was replaced with a blank solvent, and the coagulation time was also measured. By using the control examples and different reagents to measure the coagulation time of the red peony root sample, the final verification data for determining the quality markers of the red peony root with anticoagulant spectrum effect relationship were provided.

[0071] In step S1023, the experimenters used three large experimental steps to measure the final coagulation time, including blood collection, sample preparation, and coagulation four reagent kit determination.

[0072] In an embodiment, the blood collection adopts the following scheme: the injection amount of the anesthetic solution of ethyl carbamate for the big-eared white rabbits should be 5 mL·kg-1 body weight, and the blood is taken from the common carotid artery after the ear marginal vein of the rabbit is anesthetized. The collected blood is placed in a 5 mL blood collection tube with sodium citrate 1:9, each blood collection tube is collected to the scale line, and after mixing, it is centrifuged at 3000 rpm for 15 min, and the upper liquid (plasma) is collected and stored at 4 ℃ for standby use. Do not freeze-thaw repeatedly when using.

[0073] In an embodiment, the sample preparation adopts the following scheme: filter the sample solution with a microporous filter membrane, precisely take 5 mL of the filtered solution, dry it in a water bath, add physiological saline solution for redissolution, place it in a 5 mL volumetric flask for constant volume to obtain the red peony root extract stock solution, and reserve it for standby use.

[0074] In an embodiment, the coagulation four reagent kit determination adopts the following scheme:

[0075] APTT test method: take 50 μL of the measured plasma and place it in a blood coagulation cup, add 50 μL of the sample solution, add 50 μL of the APTT reagent, incubate at 37 ℃ for 5 minutes, and finally add 50 μL of the pre-warmed calcium chloride solution (0.025 mol·L-1) at 37 ℃, and measure the coagulation time. Replace the sample with a blank solvent, and measure the coagulation time according to the above method, which is the control.

[0076] PT test method: take 50 μL of the measured plasma and place it in a blood coagulation cup, add 50 μL of the sample solution, add 100 μL of the PT reagent, incubate at 37 ℃ for 3 minutes, and measure the coagulation time. Replace the sample with a blank solvent, and measure the coagulation time according to the above method, which is the control.

[0077] TT test method: 100 μL of the test plasma was taken into a blood clotting cup, 100 μL of the sample solution was added, 100 μL of the TT reagent was added, and the mixture was incubated at 37 ℃ for 5 minutes, and the clotting time was measured. The sample was replaced with the blank solvent, and the method was the same as above, and the clotting time was measured, which was the control.

[0078] FIB test method: 100 μL of the test plasma was taken into a blood clotting cup, 50 μL of the sample solution was added, 50 μL of the thrombin solution was added, and the mixture was incubated at 37 ℃ for 5 minutes, and the clotting time was measured. The sample was replaced with the blank solvent, and the method was the same as above, and the clotting time was measured, which was the control.

[0079] All the above coagulation test steps were determined in triplicate for each sample solution.

[0080] S103 Analyzing the experimental data to determine the quality marker of Chishaba for anticoagulant spectrum.

[0081] The foregoing steps determine reliable chromatographic data based on experiments, and on this basis, the principal components of Chishaba are determined using principal component eigenvalues and contribution rates, and the principal component loading matrix and its comprehensive score are used to determine that multiple components jointly determine the quality of Chishaba. On this basis, the peak area data of the common peaks in the fingerprint chromatogram are used as characteristic sequence variables, and the principal component is finally determined to be the quality marker of Chishaba for anticoagulant.

[0082] Specifically, it includes two parts of experimental data principal component analysis and quality marker, and in the principal component analysis part, in addition to determining what the principal component is, the interaction between the principal components also needs to be determined, therefore, the following steps are included:

[0083] S1031 Obtaining the peak area of the common peaks of multiple batches of Chishaba to determine the eigenvalues and contribution rates of principal component analysis, thereby determining the principal components of Chishaba.

[0084] In this step, the experimental personnel imported the peak area of 22 common peaks in 73 batches of Chishaba into SPSS 26.0 software as variables for principal component analysis, and the results are shown in Table 3. The characteristic values of the first five principal components are greater than 1, and the cumulative variance contribution rate is 79.152%, indicating that the first five principal components can represent 79.152% of the information of 22 common peaks in 73 batches of Chishaba samples.

[0085] Table 3 Eigenvalues and contribution rates of principal component analysis

[0086]

[0087] S1032 Obtaining the peak area data of the common peaks, calculating and determining the principal component factor loading matrix, and finally determining the contribution rate of the principal component.

[0088] The peak area of 22 common peaks in 73 batches of red peony root was introduced into SPSS 26.0 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 is shown that the quality of red peony root is not affected by a single component, but by 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 by taking the contribution rate of 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, S70 with positive comprehensive score are classified into one class, and those with negative comprehensive score are classified into another class. Among them, S34-S40 red peony root from Chifeng Balin Right Banner of Inner Mongolia have higher comprehensive score, indicating that the quality of red peony root from Chifeng Balin Right Banner is excellent and stable.

[0089] Table 4 Correlation between common peaks and efficacy

[0090]

[0091] Table 5 Principal component score and comprehensive score

[0092]

[0093] The anti-coagulation efficacy index of S1033 red peony root was taken as the correlation object, and the peak area data of common peaks in the fingerprint of red peony root was taken as the characteristic sequence variable for gray correlation analysis to determine the quality marker of red peony root.

[0094] The anti-coagulation index (APTT, PT, TT, FIB) of Radix Paeoniae Rubra was taken as the correlation object, i.e. quantitative variable, and the peak area data of common peaks in HPLC fingerprint of Radix Paeoniae Rubra were taken as characteristic sequence variable, which were input into SPSS PRO (1.1.21 version) software for grey correlation analysis. Since the determination units of mother sequence and characteristic sequence were different, non-dimensionalization treatment (mean value) was needed before analysis, and p was set as 0.5. The data of four coagulation indexes were analyzed respectively. The grey correlation degree and ranking were obtained under the algorithm configuration, as shown in Table 6. The calculation results of GRA showed that the greater the correlation degree, the greater the contribution to the efficacy. According to the results, the correlation degrees of 22 common peaks in the fingerprint of Radix Paeoniae Rubra and four efficacy indexes were all greater than 0.800, indicating that the 22 common peaks had different degrees of influence on the four efficacy indexes. The correlation degrees of peaks No. 1, 2, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19 and 20 with efficacy were all above 0.900, indicating that the 16 common peaks were strongly correlated with the changes of the four efficacy indexes. It was confirmed by standard identification that peaks No. 5 (gallic acid), No. 9 (catechin), No. 14 (ethyl gallate), No. 15 (paeoniflorin), No. 17 (benzoic acid), No. 18 (pentagalloyl glucose) and No. 19 (benzoylpaeoniflorin) were the quality markers for anti-coagulation of Radix Paeoniae Rubra, among which peak No. 15 (paeoniflorin) ranked first in the grey correlation degree ranking of the four efficacy indexes, which also verified the scientificity of its serving as the index component of Radix Paeoniae Rubra.

[0095] Table 6 Correlation degree between common peaks and efficacy

[0096]

[0097] Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope of the application being indicated by the claims.

Claims

1. A method for screening quality markers of Radix Paeoniae Rubra based on anticoagulant profile-effect relationship, characterized in that, It comprises the following steps: Preparation includes reference substance and Radix Paeoniae Rubra sample material, determine the preparation method of Radix Paeoniae Rubra sample and the chromatographic conditions of Radix Paeoniae Rubra sample separation; Constructing the fingerprint of Radix Paeoniae Rubra, using blood coagulation instrument to carry out in vitro anti-coagulation activity determination of Radix Paeoniae Rubra sample; Analyzing the experimental data to determine the anti-coagulation spectrum efficacy relationship of Radix Paeoniae Rubra quality marker; Among them, the anti-coagulation spectrum efficacy relationship of Radix Paeoniae Rubra quality marker includes the following steps: Constructing the fingerprint of Radix Paeoniae Rubra, according to the chromatographic conditions of Radix Paeoniae Rubra sample, carrying out methodological investigation, determining 22 common peaks, obtaining the peak area data of common peaks of multiple batches of Radix Paeoniae Rubra samples, determining the principal component analysis characteristic value and contribution rate, so as to determine the principal component of Radix Paeoniae Rubra; Obtaining the peak area data of common peaks of multiple batches of Radix Paeoniae Rubra samples, calculating and determining the principal component factor loading matrix, and finally determining the contribution rate of the principal component; The anti-coagulation efficacy index of Radix Paeoniae Rubra is the associated object, the common peak area data in the fingerprint of Radix Paeoniae Rubra is the characteristic sequence variable, the gray correlation analysis is carried out on the two, and the quality marker of Radix Paeoniae Rubra is determined; The chromatographic conditions are as follows: The chromatographic column is Agilent ZORBAX Eclipse XDB-C18 column, The mobile phase is acetonitrile (A)-0.2% phosphoric acid aqueous solution (B), 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; 50 min-56 min, 17% A-18% A; 56-60 min, 18% A-20% A; 60-70 min, 20% A-25% A; 70 min-80 min, 25% A-34% A; 80 min-90 min, 34% A-36% A; The flow rate is 1.0 mL / min; The detection wavelength is 230 nm.

2. The Radix Paeoniae Rubra quality marker screening method based on anti-coagulation spectrum efficacy relationship according to claim 1, characterized in that: Using APTT, PT, TT and FIB four anti-coagulation indexes as the associated object, taking the peak area data of 22 common peaks in the fingerprint of Radix Paeoniae Rubra as the input layer of the computer program, determining the gray correlation degree of the two through the set program, and determining gallic acid, catechin, gallic acid ethyl ester, paeoniflorin, benzoic acid, pentagalloyl glucose and benzoylpaeoniflorin as the anti-coagulation quality markers of Radix Paeoniae Rubra.

3. The method according to claim 2, wherein the method is based on the anti-coagulant spectrum-effect relationship. Through the gray correlation degree analysis of paeoniflorin in the four efficacy indexes, it is determined that paeoniflorin is the most important quality marker of Radix Paeoniae Rubra.

4. The Radix Paeoniae Rubra quality marker screening method based on anti-coagulation spectrum efficacy relationship according to claim 1, characterized in that: The peak area of 22 common peaks in 73 batches of Radix Paeoniae Rubra is introduced into the computer program as a variable to determine the principal component analysis characteristic value, contribution rate and principal component factor loading matrix.

5. The method according to claim 1, wherein the method is based on the anti-coagulant spectrum-effect relationship. Preparation includes reference substance and Radix Paeoniae Rubra sample material, determination of equipment and instruments including liquid chromatograph and centrifuge includes the following steps: Determine the model of the liquid chromatograph and centrifuge used in the experiment; Determination of control, reagent and reagent; Determination of extraction conditions of red peony root sample; Determination of experimental animals.

6. The method according to claim 1, wherein the method is based on the anti-coagulant spectrum-effect relationship. Construction of fingerprint of red peony root sample, blood of experimental animals was used to carry out anticoagulant activity determination, including: The control was prepared into a control solution, and the red peony root sample was prepared into a sample solution; Fingerprint of red peony root sample was constructed by chromatographic analysis technology, and precision, repeatability and stability tests of methodology were carried out; Coagulation four kits were used to determine plasma clotting time.

7. The red peony root quality marker screening method based on anticoagulant spectrum-effect relationship according to claim 6, characterized in that, During the determination process, the plasma and the sample solution of red peony root were mixed with APTT reagent, PT reagent, TT reagent and FIB reagent respectively to determine the clotting time of plasma under different reagents; at the same time, four control examples were set, in which the sample solution was replaced with a blank solvent, and the clotting time was also determined.

8. The method according to claim 6, wherein the anti-coagulant spectrum relationship based quality marker screening method of Radix Paeoniae Rubra is characterized by, The precision, repeatability and stability tests include: Take the first sample solution, determine the relative retention time and relative peak area RSD value of the common peak by chromatographic conditions continuous sampling determination for 6 times, to carry out precision experiment; Take the same batch of medicinal materials, prepare 6 sample solutions in parallel, and determine them by the same chromatographic conditions, calculate the relative retention time and relative peak area RSD value of the common peak, to carry out repeatability experiment; Take the first sample solution, respectively at 2, 4, 6, 8, 10, 12h after preparation, determine the relative retention time and relative peak area RSD value of the common peak, to carry out stability experiment.

9. The method according to claim 8, wherein the anti-coagulant spectrum is selected from the group consisting of: aPTT, PT, TT, INR, APTT-PT, APTT-TT, APTT-INR, PT-TT, PT-INR, and APTT-PT-INR. Further including the evaluation of similarity between samples, including: Using traditional Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity of fingerprint of different batches of red peony root sample and control fingerprint was calculated.

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