Construction method and application of fingerprint spectrum of Xinbao pill

Abstract

The present application provides a method for constructing the fingerprint of Xinbao pills, comprising the following steps: 1) preparation of test sample solution and mixed control solution; 2) construction of the fingerprint of Xinbao pills: taking the test sample solution and the mixed control solution respectively, and performing high performance liquid chromatography detection to obtain the fingerprint of Xinbao pills. The present application belongs to the technical field of Chinese medicine preparation detection, and provides a method for constructing the fingerprint of Xinbao pills, which is simple and easy to operate, has good precision, stability and repeatability, can realize the detection of multiple components of Xinbao pills, and better reflects the overall quality of Xinbao pills; meanwhile, the constructed HPLC fingerprint of Xinbao pills has rich detection components, 21 common characteristic peaks are determined, and the anti-heart failure active components in ginseng, panax notoginseng, toad venom and datura stramonium in Xinbao pills are covered, which can provide a reference for comprehensively controlling the internal quality of Xinbao pills and further ensure the clinical efficacy of the product.

Description

Technical Field

[0001] The present 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 combined effects of multiple chemical components in traditional Chinese medicine (TCM) compounds form the basis of their efficacy. TCMs are characterized by multiple components, multiple targets, and multiple regulatory mechanisms. Therefore, their quality should be evaluated using appropriately tailored testing methods that provide rich identification information. Controlling a single component alone cannot fully reflect the overall quality of a TCM. Xinbao Pills, a TCM compound preparation made from nine herbs—Flos Datura, Panax Notoginseng, Panax Notoginseng, and Toad Venenum—are known to warm and nourish the heart and kidneys, invigorate qi and yang, and activate blood circulation and unblock meridians. Years of clinical observation have demonstrated that Xinbao Pills are effective in treating chronic heart failure (CHF) and can be combined with other medications to enhance their efficacy, resulting in widespread use. Studies have shown that the anti-CHF mechanism of Xinbao Pills is related to pathways involved in inhibiting β-adrenergic receptor ubiquitination and improving myocardial energy metabolism. Modern pharmacological studies have demonstrated that saponins from Panax Notoginseng and Panax Notoginseng, toad venom from Toad Venenum, and alkaloids from Flos Datura have therapeutic effects on cardiovascular disease and are the primary active ingredients in Xinbao Pills.

[0003] The current quality standards for Xinbao Pills only include identification items for some medicinal materials and index ingredients, and the quality control research methods are relatively simple, 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 traditional Chinese medicine compound preparations. However, the current public research on improving the standards of Xinbao Pills focuses on establishing content determination methods for index ingredients and limit inspection methods for toxic ingredients. There are no reports on fingerprint research, and there is a lack of detection methods to comprehensively evaluate its intrinsic quality. In addition, because Xinbao Pills is a traditional Chinese medicine compound preparation containing nine medicinal materials and has a complex composition, how to eliminate the interference of impurities and combine multiple pharmacologically active ingredients in the process of constructing the fingerprint method to ensure that the established fingerprint has a good spectrum-activity relationship is of great significance to the intrinsic quality control of the product and ensure clinical efficacy. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides a method for constructing and applying a fingerprint of Xinbao Pills. The method is simple and easy to use, and has advantages such as high precision, stability, and repeatability. It can detect multiple components of Xinbao Pills and better reflect the overall quality of Xinbao Pills. The constructed HPLC fingerprint of Xinbao Pills is rich in detection components, with a total of 21 common characteristic peaks identified, covering the anti-heart failure active ingredients in the medicinal materials ginseng, Panax notoginseng, toad venom, and datura root in Xinbao Pills. This method can provide a reference for comprehensively controlling the intrinsic quality of Xinbao Pills and further ensure the clinical efficacy of the product.

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

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

[0007] 1) Preparation of test solution and mixed reference solution: Pour Xinbao pill powder into a container, add methanol, perform ultrasonic extraction, filter, and collect the filtrate to obtain the test solution; accurately weigh notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rd, bufalin, cinobufogenin, and resifugin, and dilute with methanol to prepare the mixed reference solution;

[0008] 2) Construction of the fingerprint of Xinbao Pills: The test solution and the mixed reference solution were respectively taken and subjected to high performance liquid chromatography 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 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%; 116-120 min, mobile phase A 95%, mobile phase B 5%; 120-121 min, 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, and the overall peak time of the target component is fast, with good peak shape, separation and tailing factor. Furthermore, a method for constructing the fingerprint of Xinbao Pills is provided, which is simple and easy, and has the advantages of good precision, stability and repeatability. It can perform 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 identifying 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 Venenum, 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 bufalotoxin, peak 20 is cinobufagin, and peak 21 is resifobafugin.

[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; and the relative retention time of peak 21 is 1.841.

[0013] Preferably, the C 18 The chromatographic column is Avantor Alltima 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 prepared from the following raw materials: datura flower, ginseng, cinnamon bark, aconite root, deer 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 detection 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 performing similarity analysis using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system; 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 includes at least one of system cluster analysis, principal component analysis, and orthogonal partial least squares discriminant analysis.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The technical solution of the present invention screened and optimized suitable high-performance liquid chromatography detection conditions, eliminated the interference of multiple impurity components, and the overall peak elution time of the target component was 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 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 medicinal materials in Xinbao Pills, which can provide a reference for comprehensively controlling the intrinsic quality of Xinbao Pills and further ensure the clinical efficacy of the product.

[0023] (2) The fingerprint of the present invention can quickly and accurately identify the main components of Xinbao Pills, especially the active ingredients for treating heart failure, and ensure the stability and uniformity of the product. At the same time, it is simple and easy to use, 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 111 HPLC fingerprint of batch 1 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 bufalotoxin; 20 cinobufoxin; 21 bufatoxin genin.

[0026] Figure 3 Comparison results of Xinbao Wan samples and negative samples.

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

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

[0029] Figure 6. Principal component analysis scores for batch 11 of Xinbao Pills.

[0030] Figure 7. OPLS-DA score plot for batch 11 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 this field. Unless otherwise specified, the percentage content in the present invention refers to the percentage content by volume.

[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%), bufalin (batch number: 111981-201501, purity: 99.2%), cinnamon venom (batch number: 110803-20180 7, purity: 99.6%), and lipobuffin (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 them, and dilute them with methanol to make a mixed reference solution containing ginsenoside R120 μg, ginsenoside Rg120 μg, ginsenoside Re 20 μg, ginsenoside Rf 20 μg, ginsenoside Rb120 μg, ginsenoside Rd 20 μg, bufalin 20 μg, cinobufoxin 20 μg, and resifobafignin 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 the chromatographic peaks in the wavelength range of 190 nm to 400 nm, and the detection wavelength was selected at 203 nm. Three mobile phase systems, acetonitrile-water, acetonitrile-0.1% formic acid in water, and acetonitrile-0.1% acetic acid in water, were examined. It was found that acetonitrile-water eluted the peaks faster overall, with better resolution and tailing factor. In addition, by optimizing and adjusting the gradient elution program, eliminating interference from other components and improving the separation 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%; 116-120 min, mobile phase A 95%, mobile phase B 5%; 120-121 min, mobile phase A 95% → 8%, mobile phase B 5% → 92%. Further, the column temperature (25°C, 30°C, 35°C) and column type (Aglient Zorbax, Avantor Alltima, Phenomenex Gemini) were investigated. It was found that when the column type was Avantor Alltima, the column temperature was 25°C, and the flow rate was 1.0 mL·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 the richness of the peaks, 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 had the best effect.

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

[0040] Preparation of negative sample solution: According to the production process and prescription ratio of Xinbao Pills, prepare negative samples lacking ginseng, Panax 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%. The chromatogram is analyzed and processed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" software, and it is found 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] Six test solutions were prepared in parallel from the same batch of Xinbao Pills samples according to the method described in Example 2. Each solution was injected and analyzed under the chromatographic conditions described in Example 1. The peak areas of the 21 common characteristic peaks were recorded, and the RSDs of the relative peak areas were calculated to be less than 5%, indicating good reproducibility of the method.

[0048] 4 Establishment of fingerprint

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

[0050] 1) Preparation of test solution and mixed reference solution: 2.0 g of Xinbao Wan powder was placed in a stoppered conical flask, methanol was added, and ultrasonic extraction was performed for 30 min. The solution was filtered and the filtrate was collected to obtain the test solution. Notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rd, bufalin, cinobufogenin, and resifugine were accurately weighed separately and diluted with methanol to prepare a mixed reference solution containing 20 μg of ginsenoside R1, 120 μg of ginsenoside Rg, 20 μg of ginsenoside Re, 20 μg of ginsenoside Rf, 20 μg of ginsenoside Rb1, 20 μg of ginsenoside Rd, 20 μg of bufalin, 20 μg of cinobufogenin, and 20 μg of resifugine per mL.

[0051] 2) Construction of the fingerprint of Xinbao Pills: The test solution and the mixed reference solution were respectively taken and subjected to high performance liquid chromatography 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, with a detection wavelength of 203 nm and a column temperature of 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%; 116-120 min, mobile phase A 95%, mobile phase B 5%; 120-121 min, 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), crush and grind, take about 2.0 g respectively, accurately weigh, prepare the test solution according to the method in Example 2, sample injection and determination under the chromatographic conditions in Example 1, record the chromatogram of each batch of samples, and import them into the "Traditional 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 correction method was used to match the chromatogram, and the control fingerprint (R) was generated by the median method. The HPLC fingerprints of 11 batches of Xinbao Pills were obtained, 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 pill samples, single medicinal materials, and negative samples, and prepare test solutions according to the method in Example 2 respectively; take Xinbao pill sample solutions, reference solution, single medicinal material solution, and negative sample solutions respectively, and inject and measure under the chromatographic conditions in Example 1, record the chromatograms, and the identification results of the main chromatographic peaks of Xinbao pills, the comparison results of Xinbao pill samples and negative samples, and the comparison results of Xinbao pill samples and single medicinal material samples are shown as follows: Figure 2 、 Figure 3 、 Figure 4 As shown in the figure. The 21 shared characteristic peaks were numbered based on the results of the stability test. By comparing the herbal material spectrum with its negative spectrum and combining the chromatographic peak retention times, peak 2 was identified as notoginsenoside R1, peak 3 as ginsenoside Rg1, peak 4 as ginsenoside Re, peak 7 as ginsenoside Rf, peak 10 as ginsenoside Rb1, peak 16 as ginsenoside Rd, peak 18 as bufalin, peak 20 as cinobufagin, and peak 21 as resifobafine. Characteristic peak attribution analysis was performed using positive herbal materials and control solutions. The results showed that all 21 shared characteristic peaks of the established Xinbao Pills component characteristic spectrum for the treatment of heart failure can be attributed to the herbal materials, demonstrating a good correlation between the preparation and the herbal 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 Venenum.

[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 ranged from 0.992 to 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 Similarity comparison of 11 batches of Xinbao pill 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 way. 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, and then imported into SPSS26.0 software. The square Euclidean distance was used as the measure to evaluate the quality differences 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 samples of the remaining batches 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 batch and storage. SIMCA-P14.1 software was used to perform 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 as follows Figure 7 As shown; combined with the variable importance 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 good predictive accuracy. Using VIP>1 as the evaluation criterion, potential chemical components that cause quality differences between batches of Xinbao Pills were screened. The results are shown in Table 2. As shown in Table 2, 13 statistically significant differential components were detected, namely peaks 2 (notoginsenoside R1), 3 (ginsenoside Rg1), 4 (ginsenoside Re), 6, 10 (ginsenoside Rb1), 12, 13, 14, 16 (ginsenoside Rd), 18, 19, 20 (cinobufagin), and 21 (resiobufagin).

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

[0063]

[0064]

[0065] The above is a further detailed description of the present invention in conjunction with specific preferred embodiments, and the specific implementation of the present invention should not be considered to be limited to these descriptions. For those skilled in the art of the present invention, without departing from the concept of the present invention, several simple deductions or substitutions can be made, which should be considered to fall within the scope of protection of the present invention.

Claims

1. A method for constructing the fingerprint of Xinbao Pills, characterized by: The steps include: 1) Preparation of test solution and mixed reference solution: Pour Xinbao pill powder into a container, add methanol, perform ultrasonic extraction, filter, and collect the filtrate to obtain the test solution; accurately weigh notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rf, ginsenoside Rb1, ginsenoside Rd, bufalin, cinobufogenin, and resifugin, and dilute with methanol to prepare the mixed reference solution; 2) Construction of the fingerprint of Xinbao Pills: The test solution and the mixed reference solution were taken separately and subjected to 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 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%; 116-120 min, mobile phase A 95%, mobile phase B 5%; 120-121 min, 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 Venenum, peak 2 is Panax 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 cinobufagin, and peak 21 is resifobafugin.

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 1.606; the relative retention time of peak 12 is 1.660; the relative retention time of peak 13 is 1.641; the relative retention time of peak 14 is 1.697 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 Avantor Alltima 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 pill is prepared from the following raw materials: datura flower, ginseng, cinnamon bark, aconite root, deer antler, borneol, artificial musk, Panax notoginseng and toad venom.

8. Use 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 method for evaluating the quality of Xinbao Pills, characterized by: 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 traditional 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 further includes the following steps: analyzing and evaluating the Xinbao pills to be tested by combining chemometrics; the chemometrics method includes at least one of system cluster analysis, principal component analysis, and orthogonal partial least squares discriminant analysis.

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