Construction method and application of fingerprint spectrum of Xinbao pill

Abstract

The invention provides a construction method of a fingerprint spectrum of Xinbao pills. The construction method comprises the following steps: 1) preparing a test solution and a mixed reference solution; and 2) constructing the fingerprint spectrum of the Xinbao pill: respectively taking the test solution and the mixed reference substance solution, and performing high performance liquid chromatography detection to obtain the fingerprint spectrum of the Xinbao pill. The invention belongs to the technical field of traditional Chinese medicine preparation detection, and provides a construction method of a fingerprint spectrum of a Xinbao pill, the method is simple and easy to implement, has the advantages of good precision, stability and repeatability and the like, can realize detection of multiple components of the Xinbao pill, and well reflects the overall quality condition of the Xinbao pill; meanwhile, the established HPLC fingerprint spectrum of the Xinbao pill is rich in detection components, totally 21 common characteristic peaks are determined and cover anti-heart-failure active components in ginseng, pseudo-ginseng, venenum bufonis and flos daturae medicinal materials in the Xinbao pill, reference can be provided for comprehensively controlling the internal quality of the Xinbao pill, and the clinical curative effect of the product is further ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine preparation detection, and in particular relates to a method for constructing a fingerprint spectrum of Xinbao Pills and its application. Background Art

[0002] The comprehensive effects of various chemical components in traditional Chinese medicine compound are the basis of the efficacy of traditional Chinese medicine compound. Traditional Chinese medicine has the characteristics of multi-component, multi-target and multi-mechanism regulation. Therefore, the evaluation of its quality should adopt the detection method that is suitable for it and can provide rich identification information. Controlling only a single indicator component cannot fully reflect the overall quality of traditional Chinese medicine. Xinbao Pills is a traditional Chinese medicine compound preparation made of nine medicinal materials such as Datura stramonium, ginseng, Panax notoginseng, and Toad venom. It has the effects of warming the heart and kidney, invigorating qi and yang, and promoting blood circulation and unblocking meridians. After years of clinical observation, Xinbao Pills have a definite effect in the treatment of chronic heart failure (CHF), and can be combined with other drugs to enhance the efficacy. It is widely used. Studies have shown that the anti-CHF mechanism of Xinbao Pills is related to the inhibition of β-adrenergic receptor ubiquitination and the improvement of myocardial energy metabolism. Modern pharmacological studies have shown that saponins in ginseng and Panax notoginseng, toad venom in Toad venom, and alkaloids in Datura stramonium have the effect of treating cardiovascular diseases and are the main active ingredients of Xinbao Pills.

[0003] The current quality standard of Xinbao Pills only has identification items for some medicinal materials and index components, and the quality control research methods are relatively single, which is not conducive to the overall quality control of Xinbao Pills. In recent years, the construction and application of fingerprint methods have provided an effective way to evaluate the overall quality of Chinese medicine compound preparations. However, the current public research on the improvement of Xinbao Pills standards is mainly based on the establishment of content determination methods for index components and limit inspection methods for toxic components. There is no report on fingerprint research, and there is a lack of detection methods to comprehensively evaluate its intrinsic quality. In addition, since Xinbao Pills is a Chinese medicine compound preparation containing nine medicinal materials with complex ingredients, how to eliminate the interference of impurity components in the construction of the fingerprint method, and combine multiple pharmacologically active ingredients to make the established fingerprint have a good spectrum-effect relationship, which is of great significance to the intrinsic quality control of the product and to ensure clinical efficacy. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a method for constructing and applying the fingerprint of Xinbao Pills. The method is simple and easy to use, and has the advantages of good precision, stability and repeatability. It can detect multiple components of Xinbao Pills and better reflect the overall quality of Xinbao Pills. At the same time, the detection components of the constructed HPLC fingerprint of Xinbao Pills are rich, and a total of 21 common characteristic peaks are determined, covering the anti-heart failure active ingredients in ginseng, Panax notoginseng, toad venom and datura flower medicinal materials in Xinbao Pills, which can provide a reference for comprehensive control of the intrinsic quality of Xinbao Pills and further ensure the clinical efficacy of the product.

[0005] The purpose of the present invention will be further illustrated by the following detailed description.

[0006] The present invention provides a method for constructing a fingerprint spectrum of Xinbao Pills, comprising the following steps:

[0007] 1) Preparation of test solution and mixed reference solution: Take Xinbao Pill powder in a container, add methanol, extract by ultrasonication, filter, and take the filtrate to obtain the test solution; accurately weigh notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rd, bufotoxin, cinobufogenin, and resifobafugin, respectively, and dilute with methanol to prepare a mixed reference solution;

[0008] 2) Construction of the fingerprint of Xinbao Pills: Take the test solution and the mixed reference solution separately, and perform high performance liquid chromatography detection to obtain the fingerprint of Xinbao Pills; the high performance liquid chromatography detection conditions include:

[0009] Using C 18 The chromatographic column uses acetonitrile as mobile phase A and water as mobile phase B for gradient elution, with a detection wavelength of 201-205 nm and a column temperature of 23-27° C. The gradient elution procedure includes: 0-15 min, mobile phase A 8%→13%, mobile phase B 92%→87%; 15-65 min, mobile phase A 13%→25%, mobile phase B 87%→75%; 65-90 min, mobile phase A 25%→34%, mobile phase B 75%→66%; 90-110 min, mobile phase A 34%→45%, mobile phase B 66%→55%; 110-114 min, mobile phase A 45%, mobile phase B 55%; 114-115 min, mobile phase A 45%→48%, mobile phase B 55%→52%; 115-116 min, mobile phase A 48%→95%, mobile phase B 52%→5%; 116-120min, mobile phase A 95%, mobile phase B 5%; 120-121min, mobile phase A 95%→8%, mobile phase B 5%→92%. The percentage of mobile phase in gradient elution refers to the volume percentage.

[0010] By adopting the above technical scheme, the present invention screens and optimizes to obtain suitable high performance liquid chromatography detection conditions, eliminates the interference of multiple impurity components, the overall peak time of the target component is fast, and the peak shape, separation degree and tailing factor are good, thereby providing a method for constructing the fingerprint of Xinbao Pills, which is simple and easy, and has the advantages of good precision, stability and repeatability. It can carry out multi-component fingerprint research on Xinbao Pills, covering the chemical components in four medicinal materials with anti-heart failure activity, namely ginseng, Panax notoginseng, toad venom and datura flower, and determines 21 common characteristic peaks.

[0011] Preferably, the fingerprint of the Xinbao Pills has 21 common characteristic peaks; among them, peaks 3, 4, 7, 10, 11, 13, 14, and 16 belong to ginseng, peaks 2, 3, 4, 8, 9, 10, 11, 12, 15, 16, 17, and 19 belong to Panax notoginseng, peaks 1 and 5 belong to Datura stramonium, peaks 6, 18, 20, and 21 belong to toad venom, peak 2 is notoginseng saponin R1, peak 3 is ginsenoside Rg1, peak 4 is ginsenoside Re, peak 7 is ginsenoside Rf, peak 10 is ginsenoside Rb1, peak 16 is ginsenoside Rd, peak 18 is bufotoxin, peak 20 is cinobuccal venom toxin base, and peak 21 is resinobufogenin.

[0012] Preferably, taking peak 3 (ginsenoside Rg1) as the reference peak, the relative retention times of the chromatographic peaks are: the relative retention time of peak 1 is 0.598; the relative retention time of peak 2 is 0.933; the relative retention time of peak 3 is 1.000; the relative retention time of peak 4 is 1.013; the relative retention time of peak 5 is 1.034; the relative retention time of peak 6 is 1.050; the relative retention time of peak 7 is 1.341; the relative retention time of peak 8 is 1.355; the relative retention time of peak 9 is 1.393; the relative retention time of peak 10 is 1 .431; the relative retention time of peak 11 is 1.441; the relative retention time of peak 12 is 1.450; the relative retention time of peak 13 is 1.463; the relative retention time of peak 14 is 1.495; the relative retention time of peak 15 is 1.528; the relative retention time of peak 16 is 1.559; the relative retention time of peak 17 is 1.598; the relative retention time of peak 18 is 1.665; the relative retention time of peak 19 is 1.758; the relative retention time of peak 20 is 1.820; the relative retention time of peak 21 is 1.841.

[0013] Preferably, the C 18 The chromatographic column is AvantorAlltima C 18 The chromatographic column has a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

[0014] Preferably, the detection wavelength is 200-205 nm, the column temperature is 22-28 ° C, and the flow rate is 0.8-1.2 mL·min –1 , the injection volume is 5-15μL.

[0015] Preferably, the detection wavelength is 203 nm, the column temperature is 25 °C, and the flow rate is 1.0 mL min –1 , the injection volume was 10 μL.

[0016] Preferably, the Xinbao Pills are made from the following raw materials: datura flower, ginseng, cinnamon bark, aconite root, pilose antler, borneol, artificial musk, Panax notoginseng, and toad venom.

[0017] Preferably, the ultrasonic extraction time is 20-40 min; and the container is a stoppered conical flask.

[0018] Correspondingly, the present invention also provides the application of the method for constructing the fingerprint of Xinbao Pills in the quality inspection of Xinbao Pills.

[0019] In addition, the present invention also provides a quality evaluation method for Xinbao Pills, comprising the following steps: taking the Xinbao Pills to be tested, constructing a fingerprint spectrum of the Xinbao Pills to be tested according to the construction method, and using a Chinese medicine chromatographic fingerprint spectrum similarity evaluation system to perform similarity analysis; the evaluation criterion for the similarity analysis is: if the similarity between the fingerprint spectrum of the Xinbao Pills to be tested and the control fingerprint spectrum is not less than 0.9, the quality of the Xinbao Pills to be tested meets the requirements.

[0020] Preferably, the quality evaluation method of the Xinbao Pills further comprises the following steps: analyzing and evaluating the Xinbao Pills to be tested in combination with chemometrics; the chemometrics comprises at least one of system cluster analysis, principal component analysis and orthogonal partial least squares discriminant analysis.

[0021] Compared with the prior art, the beneficial effects of the present invention include:

[0022] (1) The technical solution of the present invention screens and optimizes suitable HPLC detection conditions, eliminates interference from multiple impurity components, and the overall peak elution time of the target component is fast, with good peak shape, separation and tailing factor, thereby providing a method for constructing the fingerprint of Xinbao Pills. The constructed fingerprint method of Xinbao Pills has identified a total of 21 common characteristic peaks, with rich detection components, which can realize the detection of multiple components of Xinbao Pills and comprehensively reflect the overall quality of Xinbao Pills; at the same time, the constructed fingerprint of Xinbao Pills covers the anti-heart failure active ingredients in ginseng, Panax notoginseng, toad venom and datura flower in Xinbao Pills, which can provide a reference for comprehensive control of the intrinsic quality of Xinbao Pills and further ensure the clinical efficacy of the product.

[0023] (2) The fingerprint spectrum of the present invention can quickly and accurately identify the main ingredients of Xinbao Pills, especially the anti-heart failure active ingredients, to ensure the stability and uniformity of the product. At the same time, it is simple and easy to operate, and has the advantages of good precision, stability and repeatability, and can be used for quality control of the entire production process of Xinbao Pills. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 HPLC fingerprints of 11 batches of Xinbao Pills.

[0025] Figure 2Identification results of the main chromatographic peaks of Xinbao Pills; among them, 2 notoginsenoside R1; 3 ginsenoside Rg1; 4 ginsenoside Re; 7 ginsenoside Rf; 10 ginsenoside Rb1; 16 ginsenoside Rd; 18 bufotoxin; 20 cintobufacine toxin base; 21 sirotoxin base.

[0026] Figure 3 Comparison results of Xinbao Pills sample and negative sample.

[0027] Figure 4 Comparison results of Xinbao Pills samples and single-herb samples.

[0028] Figure 5 Dendrogram of cluster analysis of 11 batches of Xinbao Pills.

[0029] Figure 6 Principal component analysis score chart for 11 batches of Xinbao Pills.

[0030] Figure 7 OPLS-DA score plot of 11 batches of Xinbao Pills. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below with reference to the accompanying drawings and embodiments.

[0032] In the present invention, the medicinal materials, reagents and instruments involved are all conventional commercial products, or can be obtained by conventional technical means in the art. Unless otherwise specified, the percentage content in the present invention refers to the volume percentage content.

[0033] Specifically, Agilent 1260 high performance liquid chromatograph, purchased from Agilent Technologies China Co., Ltd., Infinity I model. Reference substances: notoginsenoside R1 (batch number: D-2307001, purity: 97.8%), ginsenoside Rg1 (batch number: 110703-202236, purity: 97.0%), ginsenoside Re (batch number: D-2404001, purity: 96.9%), ginsenoside Rf (batch number: D-2307001, purity: 99.6%), ginsenoside Rb1 (batch number: 110704-202231, purity: 93.8%), ginsenoside Rd (batch number: 110718-201809, purity: 98.0%), bufotoxin (batch number: 111981-201501, purity: 99.2%), and cinnamon venom (batch number: 110803-20180 7, purity: 99.6%), resifolinone (batch number: 110718-201809, purity: 98.0%) were purchased from China Food and Drug Inspection Institute; chromatographic grade acetonitrile was (purchased from Shanghai Xingke High Purity Solvent Co., Ltd.); purified water; analytical grade methanol, formic acid, and glacial acetic acid (all purchased from Guangdong Guanghua Technology Co., Ltd.); Xinbao Pills (No. S1-S11, batch numbers are 20190701, 20200506, 20200601, 20200602, 20200605, 20200905, 20200906, 20211204, 20230302, 20230402, and 20230428, respectively) were produced by Guangdong Xinbao Pharmaceutical Technology Co., Ltd.

[0034] Preparation of mixed reference solution: accurately weigh appropriate amounts of notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1 and ginsenoside Rd reference substances, accurately weigh, and dilute with methanol to make a mixed reference solution containing ginsenoside R1 20 μg, ginsenoside Rg 120 μg, ginsenoside Re 20 μg, ginsenoside Rf 20 μg, ginsenoside Rb1 20 μg, ginsenoside Rd 20 μg, bufalotoxin 20 μg, cinobucini 20 μg, and resifobacterin 20 μg per 1 mL.

[0035] Example 1 Screening and optimization of high performance liquid chromatography detection conditions

[0036] The DAD detector was used to scan in the wavelength range of 190nm to 400nm to examine the number and shape of chromatographic peaks, and 203nm was selected as the detection wavelength. In the investigation of three mobile phase systems, acetonitrile-water, acetonitrile-0.1% formic acid water, and acetonitrile-0.1% acetic acid water, it was found that when acetonitrile-water was used for elution, the overall peak elution time was fast, and the separation and tailing factor were good. In addition, by optimizing and adjusting the gradient elution program, eliminating interference from other components and improving the separation degree of components, the following optimal mobile phase gradient elution program was obtained: with acetonitrile as mobile phase A and water as mobile phase B, the gradient elution program included: 0-15 min, mobile phase A 8%→13%, mobile phase B 92%→87%; 15-65 min, mobile phase A 13%→25%, mobile phase B 87%→75%; 65-90 min, mobile phase A 25%→34%, mobile phase B 75%→66%; 90-110 min, mobile phase A 34%→45%, mobile phase B 66%→55%; 110-114 min, mobile phase A 45%, mobile phase B 55%; 114-115 min, mobile phase A 45%→48%, mobile phase B 55%→52%; 115-116 min, mobile phase A 48%→95%, mobile phase B 52%→5%; 116~120min, mobile phase A 95%, mobile phase B 5%; 120~121min, mobile phase A95%→8%, mobile phase B5%→92%. Further, the column temperature (25℃, 30℃, 35℃), column type (Aglient Zorbax, AvantorAlltima, Phenomenex Gemini) and other parameters were investigated. It was found that when the column type was Avantor Alltima, the column temperature was 25℃, and the flow rate was 1.0mL·min -1 The method has good durability.

[0037] Example 2 Screening and Optimization of Test Solution Preparation Methods

[0038] In the study of the test sample solution preparation method, the sample extraction method (ultrasonic extraction, hot reflux extraction), extraction solvent (75% ethanol aqueous solution, ethanol, 75% methanol aqueous solution, methanol), extraction time (30, 60 and 90 min), and sampling amount (0.5, 1.0 and 2.0 g) were investigated. Taking into account factors such as peak richness, extraction rate, solution stability, preparation time, and method convenience, it was finally determined that ultrasonic extraction of 2.0 g of Xinbao Pills powder with methanol for 30 min was the best.

[0039] Preparation of test solution: Take an appropriate amount of Xinbao Pills, crush and grind, take about 2.0g of Xinbao Pills powder, accurately weigh, put it in a stoppered conical flask, accurately add 25mL of methanol, weigh the weight, extract by ultrasonic for 30min, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain the solution.

[0040] Preparation of negative sample solution: According to the production process and prescription ratio of Xinbao Pills, prepare negative samples lacking ginseng, notoginseng, toad venom and datura flower respectively. Take appropriate amount and operate according to the preparation method of the test solution.

[0041] Example 3 Construction and methodological investigation of the fingerprint of Xinbao Pills

[0042] 1. Instrument precision test

[0043] Take the same batch of Xinbao Pills samples, prepare the test solution according to the method in Example 2, and inject and measure 6 times under the chromatographic conditions in Example 1. Take ginsenoside Rg1 as the reference peak, record the peak area and retention time of each common characteristic peak, and measure the RSD of the relative retention time of each common characteristic peak to be less than 1%, and the RSD of the relative peak area to be less than 5%. Use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software to analyze and process the chromatogram, and find that the similarity between the spectrum obtained by 6 consecutive injections and the control spectrum is ≥0.99, indicating that the instrument has good precision and meets the requirements of fingerprint determination.

[0044] 2. Stability test

[0045] The same batch of Xinbao Pills samples were taken, and the test solution was prepared according to the method in Example 2. The samples were injected and measured under the chromatographic conditions in Example 1 at 0, 18, 36, and 54 hours after preparation, and the peak areas of 21 common characteristic peaks were recorded. The RSDs of the relative peak areas were calculated to be <5%, indicating that the test solution had good stability within 54 hours.

[0046] 3 Repeatability test

[0047] Take the same batch of Xinbao Pills samples, prepare 6 test solutions in parallel according to the method in Example 2, and inject and measure them respectively under the chromatographic conditions in Example 1. Record the peak areas of 21 common characteristic peaks, and calculate that the RSD of each relative peak area is <5%, indicating that the method has good repeatability.

[0048] 4 Establishment of fingerprint

[0049] The method for constructing the fingerprint spectrum of Xinbao Pills comprises the following steps:

[0050] 1) Preparation of test solution and mixed reference solution: 2.0 g Xinbao Pill powder was placed in a stoppered conical flask, methanol was added, ultrasonic extraction was performed for 30 min, and then the solution was filtered and the filtrate was taken to obtain the test solution; notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rd, bufotoxin, cinobucoid venom toxin base, and resinobufogenin were accurately weighed respectively, and diluted with methanol to prepare a mixed reference solution containing 20 μg ginsenoside R1, 120 μg ginsenoside Rg, 20 μg ginsenoside Re, 20 μg ginsenoside Rf, 20 μg ginsenoside Rb1, 20 μg ginsenoside Rd, 20 μg bufotoxin, 20 μg cinobucoid venom toxin base, and 20 μg resinobufogenin per 1 mL;

[0051] 2) Construction of the fingerprint of Xinbao Pills: Take the test solution and the mixed reference solution separately, and perform high performance liquid chromatography detection to obtain the fingerprint of Xinbao Pills; the high performance liquid chromatography detection conditions include:

[0052] Using AvantorAlltima C 18 The chromatographic column uses acetonitrile as mobile phase A and water as mobile phase B for gradient elution, the detection wavelength is 203 nm, and the column temperature is 25°C; the gradient elution program includes: 0-15 min, mobile phase A 8%→13%, mobile phase B 92%→87%; 15-65 min, mobile phase A 13%→25%, mobile phase B 87%→75%; 65-90 min, mobile phase A 25%→34%, mobile phase B 75%→66%; 90-110 min, mobile phase A 34%→45%, mobile phase B 66%→55%; 110-114 min, mobile phase A 45%, mobile phase B 55%; 114-115 min, mobile phase A 45%→48%, mobile phase B 55%→52%; 115-116 min, mobile phase A 48%→95%, mobile phase B 52%→5%; 116-120min, mobile phase A 95%, mobile phase B 5%; 120-121min, mobile phase A 95%→8%, mobile phase B 5%→92%. The percentage of mobile phase in gradient elution refers to the volume percentage.

[0053] Take 11 batches of Xinbao Pills (numbered S1-S11, batch numbers are 20190701, 20200506, 20200601, 20200602, 20200605, 20200905, 20200906, 20211204, 20230302, 20230402, 20230428 respectively), crush and grind, take about 2.0 g respectively, accurately weigh, prepare test solutions according to the method in Example 2, sample injection and measurement under the chromatographic conditions in Example 1, record the chromatogram of each batch of samples, and import them into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software in CDF format for processing. Taking S1 (batch number: 20190701) as the reference spectrum, the time window width was 0.1 min, the multi-point calibration method was used to match the chromatogram, and the reference fingerprint (R) was generated by the median method to obtain the HPLC fingerprints of 11 batches of Xinbao Pills, as shown in Figure 2. Figure 1 As shown, a total of 21 common characteristic peaks were identified.

[0054] 5. Identification and attribution of common characteristic peaks

[0055] Take Xinbao Pills sample, single herb, negative sample, prepare test solution according to the method in Example 2; take Xinbao Pills sample solution, reference solution, single herb solution, negative sample solution, respectively, inject and measure under the chromatographic conditions in Example 1, record chromatograms, and the identification results of the main chromatographic peaks of Xinbao Pills, the comparison results of Xinbao Pills sample and negative sample, and the comparison results of Xinbao Pills sample and single herb sample are as follows: Figure 2 , Figure 3 , Figure 4 As shown. According to the results of the stability test, the 21 common characteristic peaks were numbered. By comparing the medicinal material spectrum and its negative spectrum and combining the chromatographic peak retention time, peak 2 was notoginseng saponin R1, peak 3 was ginsenoside Rg1, peak 4 was ginsenoside Re, peak 7 was ginsenoside Rf, peak 10 was ginsenoside Rb1, peak 16 was ginsenoside Rd, peak 18 was bufotoxin, peak 20 was cinobufogenin, and peak 21 was sirobufogenin. The characteristic peaks were attributed and analyzed using positive medicinal materials and control solutions. The results showed that the 21 common characteristic peaks of the established Xinbao Pills for the treatment of heart failure could be attributed to the medicinal materials, and there was a good correlation between the preparation and the medicinal materials. Among them, peaks 3, 4, 7, 10, 11, 13, 14, and 16 belong to ginseng, peaks 2, 3, 4, 8, 9, 10, 11, 12, 15, 16, 17, and 19 belong to Panax notoginseng, peaks 1 and 5 belong to Datura stramonium, and peaks 6, 18, 20, and 21 belong to Toad venom.

[0056] 6 Fingerprint similarity evaluation

[0057] The fingerprint data of the above 11 batches of Xinbao Pills samples were analyzed, and the similarity between the samples was calculated. The results are shown in Table 1. The similarity between the 11 batches of Xinbao Pills was 0.992-1.000, indicating that the quality similarity of different batches of Xinbao Pills was high and the chemical components contained were basically the same, indicating that the established HPLC fingerprint method is suitable for the qualitative analysis of Xinbao Pills.

[0058] Table 1 Comparison of similarity of 11 batches of Xinbao Pills samples

[0059]

[0060] Example 4 Quality Evaluation Method of Xinbao Pills

[0061] On the basis of the analysis of the fingerprint data of the above 11 batches of Xinbao Pills samples in Example 3, the Xinbao Pills were analyzed and evaluated in combination with the chemometric method, so as to further analyze the differential components between different batches and ensure the quality of the product in a more instructive manner. After processing the peak area values ​​of the 21 common characteristic peaks of the 11 batches of Xinbao Pills samples, the samples were analyzed using the chemometric method to evaluate the similarities and differences between different samples and to examine the main differential components. First, hierarchical clustering analysis (HCA) was used to establish the original data matrix with the peak areas of the 21 common characteristic peaks of the 11 batches of Xinbao Pills samples as variables, which was then imported into the SPSS26.0 software. The squared Euclidean distance was used as the measure to evaluate the quality differences and similarities of different batches of Xinbao Pills. The results are as follows: Figure 5 As shown. The results show that when the classification distance is 5, 11 batches of Xinbao Pills samples can be divided into 4 categories, batches 1, 4, 5, 6, and 8 are classified into one category, batches 9 and 10 are classified into one category, batches 2, 3, and 11 are classified into one category, and batch 7 is classified into one category; when the classification distance is greater than 5, batch 7 is in one category and the remaining samples are in one category, indicating that the peak area difference of the common characteristic peaks between batch 7 and other samples is relatively large, while the quality differences between the remaining batches of samples are relatively small. The clustering results have a certain correlation with the production date. Preliminary analysis shows that some quality differences caused by different production batches may be related to factors such as medicinal material batches and storage. SIMCA-P14.1 software was used for principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (PLS-DA), and the results are shown as follows. Figure 6 , Figure 7 PCA analysis can intuitively show the degree of natural specificity between different samples and evaluate their similarity. Figure 6It can be seen that the samples of batches 1, 4, 5, 6, and 8 are clustered into one category, the samples of batches 2, 3, and 11 are clustered into one category, the samples of batches 9 and 10 are clustered into one category, and the samples of batch 7 are clustered into one category, which is consistent with the results of HCA. According to the results of HCA and PCA, the 11 batches of Xinbao pill samples were divided into 4 groups, and OPLS-DA was used to maximize the difference between the 4 groups. The results are shown in the figure. Figure 7 As shown; combined with the variable importance in the projection (VIP) marker difference analysis, the fitting parameter R of the established OPLS-DA model 2 X = 0.813, R 2 Y=0.987, prediction parameter Q 2 =0.5, indicating that the established model is stable and has a good prediction accuracy. Taking VIP>1 as the evaluation standard, the potential chemical components that cause the quality differences of different batches of Xinbao Pills were screened, and the results are shown in Table 2. As shown in Table 2, there are 13 statistically significant difference components, namely peak 2 (notoginseng saponin R1), 3 (ginsenoside Rg1), 4 (ginsenoside Re), 6, 10 (ginsenoside Rb1), 12, 13, 14, 16 (ginsenoside Rd), 18, 19, 20 (cinonabufagin), and 21 (resinogenin).

[0062] Table 2 VIP values ​​of 21 common characteristic peaks

[0063]

[0064]

[0065] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A method for constructing the fingerprint of Xinbao Pills, characterized in that: The steps include: 1) Preparation of test solution and mixed reference solution: Take Xinbao Pill powder in a container, add methanol, extract by ultrasonication, filter, and take the filtrate to obtain the test solution; accurately weigh notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rd, bufotoxin, cinobufogenin, and resifobafugin, respectively, and dilute with methanol to prepare a mixed reference solution; 2) Construction of the fingerprint of Xinbao Pills: The test solution and the mixed reference solution were taken separately and tested by high performance liquid chromatography to obtain the fingerprint of Xinbao Pills; The HPLC detection conditions include: Using C 18 The chromatographic column uses acetonitrile as mobile phase A and water as mobile phase B for gradient elution, with a detection wavelength of 201-205 nm and a column temperature of 23-27° C. The gradient elution procedure includes: 0-15 min, mobile phase A 8%→13%, mobile phase B 92%→87%; 15-65 min, mobile phase A 13%→25%, mobile phase B 87%→75%; 65-90 min, mobile phase A 25%→34%, mobile phase B 75%→66%; 90-110 min, mobile phase A 34%→45%, mobile phase B 66%→55%; 110-114 min, mobile phase A 45%, mobile phase B 55%; 114-115 min, mobile phase A 45%→48%, mobile phase B 55%→52%; 115-116 min, mobile phase A 48%→95%, mobile phase B 52%→5%; 116-120min, mobile phase A 95%, mobile phase B 5%; 120-121min, mobile phase A 95%→8%, mobile phase B 5%→92%.

2. The method for constructing the fingerprint of Xinbao Pills according to claim 1, characterized in that: The fingerprint of the Xinbao Pills has 21 common characteristic peaks; among them, peaks 3, 4, 7, 10, 11, 13, 14, and 16 belong to ginseng, peaks 2, 3, 4, 8, 9, 10, 11, 12, 15, 16, 17, and 19 belong to Panax notoginseng, peaks 1 and 5 belong to Datura stramonium, peaks 6, 18, 20, and 21 belong to toad venom, peak 2 is notoginseng saponin R1, peak 3 is ginsenoside Rg1, peak 4 is ginsenoside Re, peak 7 is ginsenoside Rf, peak 10 is ginsenoside Rb1, peak 16 is ginsenoside Rd, peak 18 is bufotoxin, peak 20 is cinobuccal venom toxin base, and peak 21 is resinobufogenin.

3. The method for constructing the fingerprint of Xinbao Pills according to claim 2, characterized in that: Taking peak 3 as the reference peak, the relative retention times of the chromatographic peaks are as follows: the relative retention time of peak 1 is 0.598; the relative retention time of peak 2 is 0.933; the relative retention time of peak 3 is 1.000; the relative retention time of peak 4 is 1.013; the relative retention time of peak 5 is 1.034; the relative retention time of peak 6 is 1.050; the relative retention time of peak 7 is 1.341; the relative retention time of peak 8 is 1.355; the relative retention time of peak 9 is 1.393; the relative retention time of peak 10 is 1.431; the relative retention time of peak 11 is 1.6 The relative retention time of peak 11 is 1.441; the relative retention time of peak 12 is 1.450; the relative retention time of peak 13 is 1.463; the relative retention time of peak 14 is 1.495; the relative retention time of peak 15 is 1.528; the relative retention time of peak 16 is 1.559; the relative retention time of peak 17 is 1.598; the relative retention time of peak 18 is 1.665; the relative retention time of peak 19 is 1.758; the relative retention time of peak 20 is 1.820; and the relative retention time of peak 21 is 1.

841.

4. The method for constructing the fingerprint of Xinbao Pills according to any one of claims 1 to 3, characterized in that: The C 18 The chromatographic column is AvantorAlltima C 18 The chromatographic column has a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

5. The method for constructing the fingerprint of Xinbao Pills according to any one of claims 1 to 3, characterized in that: The detection wavelength is 200-205 nm, the column temperature is 22-28° C., and the flow rate is 0.8-1.2 mL·min –1 , the injection volume is 5-15μL.

6. The method for constructing the fingerprint of Xinbao Pills according to claim 5, characterized in that: The detection wavelength was 203 nm, the column temperature was 25° C., and the flow rate was 1.0 mL·min –1 , the injection volume was 10 μL.

7. The method for constructing the fingerprint of Xinbao Pills according to any one of claims 1 to 3, characterized in that: The Xinbao pills are prepared from the following raw materials: datura flower, ginseng, cinnamon bark, aconite root, pilose antler, borneol, artificial musk, Panax notoginseng and toad venom.

8. Application of the method for constructing the fingerprint of Xinbao Pills according to any one of claims 1 to 7 in the quality inspection of Xinbao Pills.

9. A quality evaluation method for Xinbao Pills, characterized in that: The method comprises the following steps: taking a Xinbao pill to be tested, constructing a fingerprint of the Xinbao pill to be tested according to the construction method described in any one of claims 1 to 7, and performing similarity analysis using a Chinese medicine chromatographic fingerprint similarity evaluation system; the evaluation criterion for the similarity analysis is: if the similarity between the fingerprint of the Xinbao pill to be tested and the control fingerprint is not less than 0.9, the quality of the Xinbao pill to be tested meets the requirements.

10. The quality evaluation method of Xinbao Pills according to claim 9, characterized in that: The method also includes the following steps: analyzing and evaluating the Xinbao pills to be tested by combining chemical metrology; the chemical metrology method includes at least one of system cluster analysis, principal component analysis and orthogonal partial least squares discriminant analysis.

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