Jianyang capsule fingerprint spectrum and construction method and application thereof

Abstract

The application discloses Jianyang capsule fingerprint spectrum and a construction method and application thereof, and relates to the technical field of quality detection. The construction method of the Jianyang capsule fingerprint spectrum comprises the following steps: preparing a control solution by using ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1, ganoderic acid A, primulaverin and 2,3,5,4'-tetrahydroxystilbene-2-O-beta-D-glucoside; preparing a test solution by using powders of Jianyang capsules of different batches; and performing high performance liquid chromatography analysis on the control solution and the test solution respectively to generate the Jianyang capsule fingerprint spectrum. The main peak and each chromatographic peak of the Jianyang capsule fingerprint spectrum obtained by the application are well separated, the Jianyang capsule fingerprint spectrum is applied to quality control detection of Jianyang capsules, the quality of the products can be comprehensively monitored, the consistency, controllability and stability of the product quality are ensured, and therefore, the quality of the Jianyang capsule products can be more comprehensively monitored.

Description

Technical Field

[0001] This invention relates to the field of quality testing technology, and more specifically, to a fingerprint spectrum of Jianyang capsules, its construction method, and its application. Background Technology

[0002] Perfect Brand Jianyang Capsules are a health food product approved in 2009 by Perfect (Guangdong) Daily Necessities Co., Ltd. The product is made primarily from extracts of traditional Chinese medicines such as Ganoderma lucidum, processed Polygonum multiflorum, and Polygonatum sibiricum. Functional testing has proven that it is a health food product capable of relieving physical fatigue. Currently, the main quality control practice for this product is to conduct thin-layer chromatography identification and marker component testing on each raw material according to the 2020 edition of the Chinese Pharmacopoeia.

[0003] However, the product relies on its multiple chemical components to function, and any single active ingredient or indicator component is insufficient to effectively evaluate the authenticity and quality of traditional Chinese medicine (TCM). The quantitative characteristics of the substances within the product formulation cannot be reflected, nor can the overall medicinal features be revealed. Furthermore, using thin-layer chromatography (TLC) for raw material identification is time-consuming, labor-intensive, and requires a wide variety of organic reagents, failing to achieve both health and environmental friendliness. Currently, there is no comprehensive quality control method that can reflect the overall quality of the product. TCM fingerprinting, on the other hand, refers to a spectrum obtained by using specific analytical methods after appropriate processing of TCM, identifying common peaks representing the characteristics of various component groups. This aims to characterize the overall changes in the intrinsic quality of TCM and represents a new quality control model for TCM.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a fingerprint spectrum of Jianyang capsules and a fingerprint spectrum of Jianyang capsules.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides a method for constructing a fingerprint spectrum of Jianyang capsules, comprising:

[0008] (1) Using ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1, ganoderic acid A, crystal blue glycoside, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside as reference standards, a reference solution was prepared.

[0009] (2) Prepare multiple batches of test solution using powder from multiple different batches of Jianyang capsules as test samples;

[0010] (3) The reference solution and the test solution were analyzed by high performance liquid chromatography, respectively.

[0011] The chromatographic conditions included: using acetonitrile and formic acid aqueous solution as mobile phases, passing the reference solution and the test solution through a Hungpu T3 chromatographic column and performing gradient elution to obtain chromatograms of each batch of Jianyang capsules;

[0012] The elution procedure for gradient elution is as follows:

[0013] When t = 0 min to 5 min, the percentage of acetonitrile in the total amount of the mobile phase is 0%-10%, and the percentage of formic acid aqueous solution in the total amount of the mobile phase is 90%-100%.

[0014] When t = 5 min to 33 min, the percentage of acetonitrile in the total amount of the mobile phase changes from 0%-10% to 30%-40%, and the percentage of formic acid aqueous solution in the total amount of the mobile phase changes from 90%-100% to 60%-70%.

[0015] When t = 33 min to 85 min, the percentage of acetonitrile in the total amount of the mobile phase changes from 30%-40% to 90%-100%, and the percentage of formic acid aqueous solution in the total amount of the mobile phase changes from 60%-70% to 0%-10%.

[0016] When t = 85 min to 90 min, the percentage of acetonitrile in the total amount of the mobile phase is maintained at 90%-100%, and the percentage of formic acid aqueous solution in the total amount of the mobile phase is maintained at 0%-10%.

[0017] When t = 90 min to 95 min, the percentage of acetonitrile in the total amount of the mobile phase changes from 90%-100% to 0%-10%, and the percentage of formic acid aqueous solution in the total amount of the mobile phase changes from 0%-10% to 90%-100%.

[0018] When t = 95 min to 105 min, the percentage of acetonitrile in the total amount of the mobile phase is maintained at 0%-10%, and the percentage of formic acid aqueous solution in the total amount of the mobile phase is maintained at 90%-100%.

[0019] (4) Perform similarity analysis on the chromatograms of each batch of Jianyang capsules, and use the median calculation method to generate a total of 23 common peaks with the relative retention time of the reference peak as 1; and calculate the relative retention time of each common peak with the reference peak to generate the fingerprint spectrum of Jianyang capsules.

[0020] Secondly, the present invention provides a fingerprint spectrum of Jianyang Capsules, which is constructed using the fingerprint spectrum construction method of Jianyang Capsules as described in any of the foregoing embodiments.

[0021] Thirdly, the present invention provides the application of the fingerprint spectrum of Jianyang capsules as described in the foregoing embodiments in the quality control and testing of Jianyang capsules.

[0022] The present invention has the following beneficial effects:

[0023] The fingerprint spectrum of this invention can comprehensively describe and reflect the product quality of Jianyang Capsules, effectively controlling the overall product quality. Using peak 9 (2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside) as a control peak, the fingerprint spectrum of Jianyang Capsules exhibits a total of 23 common peaks, and the relative retention time of each peak is provided. By applying the established fingerprint spectrum of Jianyang Capsules to quality control testing, this invention facilitates comprehensive monitoring of product quality, ensuring consistency, controllability, and stability, thereby enabling more comprehensive quality control of Jianyang Capsules.

[0024] The detection conditions selected in this invention have been repeatedly compared and verified experimentally, enabling semi-quantitative determination. This invention is the first to employ a traditional Chinese medicine fingerprinting method to detect this product. Through optimization of the mobile phase and gradient elution conditions, the main peak and other chromatographic peaks in the chromatogram are well separated, allowing for comprehensive control of the product's overall quality while meeting the requirements of large-scale industrial production. By establishing a reference fingerprint chromatogram for this product using the method provided by this invention, the quality control of the product is improved, further promoting its international development. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The chromatograms of 10 batches of Jianyang Capsule test solutions provided in Example 1 of this application;

[0027] Figure 2 The chromatograms of the reference standard and the Jianyang Capsule test solution provided in Example 1 of this application are shown below. From bottom to top, they are S1-ganoderic acid A, S2-2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, S3-ginsenoside Rb1, S4-ginsenoside Rg1, S5-panax notoginsenoside R1, S6-monochlorophyll, and S7-Jianyang Capsule test solution.

[0028] Figure 3 This is a reference chromatogram of Jianyang Capsules provided in Example 1 of this application;

[0029] Figure 4 The chromatograms of the extraction method provided in Example 2 of this application are examined, wherein, from bottom to top, S1 is ultrasonic extraction; S2 is reflux extraction;

[0030] Figure 5 The chromatogram for the extraction solvent analysis provided in Example 2 of this application shows that, from bottom to top, S1 - 30% methanol extraction; S2 - 50% methanol extraction; S3 - 80% methanol extraction; S4 - 100% methanol extraction; and S4 - anhydrous ethanol extraction.

[0031] Figure 6 The chromatogram for ultrasonic extraction time provided in Example 2 of this application shows that, from bottom to top, S1 - ultrasonic 30 min; S2 - ultrasonic 45 min; S3 - ultrasonic 60 min;

[0032] Figure 7 The chromatogram for the extraction material-liquid ratio analysis provided in Example 2 of this application shows, from bottom to top, S1-0.5g:15mL; S2-0.5g:30mL; S3-1g:15mL; S4-1g:30mL;

[0033] Figure 8 This is a 3D image of the fingerprint spectrum detection wavelength of Jianyang Capsules provided in Embodiment 3 of this application;

[0034] Figure 9 The fingerprint spectrum detection wavelength selection chromatogram of Jianyang capsules provided in Example 3 of this application is shown, wherein, from bottom to top, S1-205nm; S2-255nm; S3-280nm; S4-290nm;

[0035] Figure 10 The chromatograms for different mobile phases of the fingerprint chromatogram of Jianyang Capsules provided in Example 3 of this application are as follows: from bottom to top, S1-methanol-water; S2-acetonitrile-water; S3-acetonitrile-0.1% formic acid aqueous solution; S4-acetonitrile-0.01% formic acid aqueous solution; S5-acetonitrile-0.001% formic acid aqueous solution.

[0036] Figure 11 The fingerprint chromatograms of Jianyang Capsules provided in Example 3 of this application are chromatograms of different gradients, wherein, from bottom to top, they are S1-gradient 1, S2-gradient 2, S3-gradient 3, S4-gradient 4, S5-gradient 5, S6-gradient 6, S7-gradient 7, S8-gradient 8, S9-gradient 9, and S10-gradient 10.

[0037] Figure 12The chromatograms for different flow rates of the fingerprint chromatogram of Jianyang capsules provided in Example 3 of this application are shown, where from bottom to top, S1-0.6mL / min, S2-0.8mL / min, and S3-1.0mL / min.

[0038] Figure 13 The chromatograms of the fingerprint spectrum of Jianyang capsules provided in Example 3 of this application are investigated at different column temperatures, where, from bottom to top, they are S1-25℃, S2-30℃, S3-35℃, and S4-40℃;

[0039] Figure 14 The fingerprint chromatograms of Jianyang capsules provided in Example 3 of this application are chromatograms for different injection volumes, wherein, from bottom to top, they are S1-2μL, S2-5μL, S3-7μL, and S4-10μL;

[0040] Figure 15 The chromatograms of the fingerprint chromatogram of Jianyang capsules provided in Example 3 of this application were examined using different chromatographic columns. From bottom to top, they are S1-Hungpu T3; S2-Hungpu C18-AQ(2); S3-Hungpu XBT C18;

[0041] Figure 16 This is a chromatogram for the suitability study of the fingerprinting system for Jianyang Capsules provided in Example 4 of this application;

[0042] Figure 17 The specificity investigation chromatogram provided in Example 4 of this application is shown, wherein, from bottom to top, S1 is the sample solution and S2 is 80% methanol solvent;

[0043] Figure 18 This is the precision evaluation chromatogram provided in Example 4 of this application;

[0044] Figure 19 This is the repeatability test chromatogram provided in Example 4 of this application;

[0045] Figure 20 The chromatograms for stability testing provided in Example 4 of this application are shown from bottom to top, representing measurements taken at 0, 2, 4, 8, 12, and 24 hours. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0047] This invention provides a method for constructing the fingerprint spectrum of Jianyang capsules, which includes the following steps:

[0048] (1) Preparation of reference solution

[0049] A reference solution was prepared using ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1, ganoderic acid A, crystal blue glycoside, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside as reference standards.

[0050] Specifically, ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1, ganoderic acid A, cynomolgus glycoside, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside were respectively mixed with 70%-100% methanol solution to prepare single standard solutions with concentrations of 18-27 μg / mL for ginsenoside Rg1, 18-27 μg / mL for ginsenoside Rb1, 18-27 μg / mL for notoginsenoside R1, 6-10 μg / mL for ganoderic acid A, 18-27 μg / mL for cynomolgus glycoside, and 18-27 μg / mL for 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

[0051] (2) Preparation of the test solution.

[0052] Multiple batches of Jianyang capsule powder were used as test samples to prepare multiple batches of test sample solutions.

[0053] Specifically, the powder of Jianyang capsules is mixed with methanol with a volume concentration of 70%-80% at a mass-volume ratio of 1g:10-20mL, and extracted by ultrasonication or reflux for 20-40min. After cooling to room temperature, the lost weight is replenished with methanol with a volume concentration of 70-80%, shaken well, and the supernatant is filtered through a 0.22μm microporous membrane to obtain the final product.

[0054] The study conducted in this application found that extraction with methanol of the above concentration at a specific volume ratio resulted in high response values ​​of the main component peaks in the obtained chromatograms.

[0055] (3) High performance liquid chromatography analysis.

[0056] The reference solution and the test solution were analyzed by high performance liquid chromatography to obtain the chromatograms of each batch of Jianyang capsules.

[0057] The chromatographic conditions included: column: Hungpu T3 (250mm×4.6mm, 5μm), flow rate: 0.8~1.2mL / min, detection wavelength: 200-220nm, column temperature: 25℃-40℃.

[0058] Using acetonitrile (A) and 0.001% formic acid aqueous solution (B) as the mobile phase, gradient elution was performed (see Table 1 below). The theoretical plate number, calculated based on 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, should not be less than 30,000.

[0059] The elution procedure for gradient elution can be found in Table 1:

[0060] Table 1. Mobile Phase Gradient Elution Program

[0061] Time (min) Acetonitrile (%) 0.001% formic acid aqueous solution (%) 0 0-10 90-100 5 0-10 90-100 33 30-40 60-70 85 90-100 0-10 90 90-100 0-10 95 0-10 90-100 105 0-10 90-100

[0062] Preferably, the elution procedure can be found in Table 2:

[0063] Table 2. Optimal mobile phase gradient elution program

[0064] Time (min) Acetonitrile (%) 0.001% formic acid aqueous solution (%) 0 0 100 5 0 100 33 35 65 85 100 0 90 100 0 95 0 100 105 0 100

[0065] Elution was performed according to the chromatographic conditions and elution procedures provided in this application. The resulting chromatograms contained a large amount of analytical signal peak information, had a stable baseline, large peak response values, good peak shapes, and good separation between peaks.

[0066] (4) Construction of fingerprint spectrum of Jianyang capsule.

[0067] Similarity analysis was performed on the chromatograms of each batch of Jianyang Capsules. With the relative retention time of the reference peak as 1, a total of 23 common peaks were generated using the median calculation method. The relative retention time of each common peak was then calculated using the reference peak to generate the fingerprint chromatogram of Jianyang Capsules.

[0068] Among them, peak 2 is crystallizing glycoside, peak 9 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, peak 11 is notoginsenoside R1, peak 12 is ginsenoside Rg1, peak 14 is ginsenoside Rb1, and peak 18 is ganoderic acid A.

[0069] The retention times of the common peaks are as follows: Peak 1: 0.439±0.5%; Peak 2: 0.452±0.5%; Peak 3: 0.476±0.5%; Peak 4: 0.496±0.5%; Peak 5: 0.505±0.5%; Peak 6: 0.585±0.5%; Peak 7: 0.749±0.5%; Peak 8: 0.755±0.5%; Peak 9: 1.000; Peak 10: 1.066±0.5%; Peak 11: 1.086±0.5%; Peak 12: 1.000. Peak 121±0.5%; Peak 13: relative retention time 1.280±0.5%; Peak 14: relative retention time 1.355±0.5%; Peak 15: relative retention time 1.422±0.5%; Peak 16: relative retention time 1.444±0.5%; Peak 17: relative retention time 1.472±0.5%; Peak 18: relative retention time 1.549±0.5%; Peak 19: relative retention time 1.742±0.5%; Peak 20: relative retention time 1.958±0.5%; Peak 21: relative retention time 2.016±0.5%; Peak 22: relative retention time 2.044±0.5%; Peak 23: relative retention time 2.755±0.5%.

[0070] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0071] The instruments and equipment used in the embodiments of this application are shown in Table 3 below.

[0072] Table 3. Statistics of Instruments Used

[0073] Instrument Name model Manufacturer Electronic balance ML 204 METTLER TOLEOD Electronic balance MS205 METTLER TOLEOD High Performance Liquid Chromatography Waters e2695+2998 Waters Technology (Shanghai) Co., Ltd. ultrasonic cleaner JM-16D-28 / 45 Shenzhen Jiemeng Technology Co., Ltd. Ultrapure water system Genie Purist Shanghai Lefeng Biotechnology Co., Ltd. chromatographic column Hungpu T3(250mm×4.6mm, 5μm) Guangzhou Hengpu Technology Co., Ltd.

[0074] The reagents used in the embodiments of this invention are: acetonitrile and methanol (chromatographic grade, CNW GmbH, Germany); methanol and formic acid (analytical grade, Sinopharm Chemical Reagent Co., Ltd.); formic acid (mass spectrometry grade, CNW GmbH, Germany); and ultrapure water (self-made).

[0075] The reference standards used in the embodiments of this invention are: ginsenoside Rg1 (batch number: 110703-201128, China National Institute for Food and Drug Control, 20 mg / bottle, purity 93.4%); ginsenoside Rb1 (batch number: GD1O11Y126429, Shanghai Yuanye Biotechnology Co., Ltd., 20 mg / bottle, purity 98%); and notoginsenoside R1 (batch number: 110745-201318, China National Institute for Food and Drug Control, 20 mg / bottle, purity 98%). 20mg / bottle, purity 94.0%; Crystallized glycoside (batch number: 111870-201302, China Food and Drug Inspection Institute, 20mg / bottle, purity 94.2%); Ganoderic acid A (batch number: 21080931, TAUTO, 10mg / bottle, purity 98.0%); 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucoside (batch number: I2107570, Aladdin, 20mg / bottle, purity 98%).

[0076] The Jianyang Capsules with batch numbers 104013(JYJN1), 124013(JYJN2), 144013(JYJN3), 164013(JYJN4), 184013(JYJN5), 184803(JYJN6), 123403(JYJN7), 123013(JYJN8), 123803(JYJN9), and 104803(JYJN10) were supplied by Perfect (Guangdong) Daily Necessities Co., Ltd.

[0077] Example 1

[0078] This embodiment provides a method for constructing the fingerprint spectrum of Jianyang capsules, which includes:

[0079] S1. Preparation of the reference solution:

[0080] Accurately weigh 26.86 mg of ginsenoside Rg1 reference standard, 18.36 mg of ginsenoside Rb1 reference standard, 6.44 mg of ganoderic acid A reference standard, 18.41 mg of notoginsenoside R1 reference standard, 23.30 mg of crystallizing glycoside reference standard, and 21.96 mg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference standard. Dissolve these in methanol to prepare single-standard solutions with the following concentrations: ginsenoside Rg1: 0.2686 mg / mL, ginsenoside Rb1: 0.1836 mg / mL, ganoderic acid A: 0.0644 mg / mL, notoginsenoside R1: 0.1814 mg / mL, crystallizing glycoside: 0.233 mg / mL, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside: 0.2196 mg / mL.

[0081] S2. Preparation of the test solution of Jianyang Capsules:

[0082] Take an appropriate amount of Jianyang capsules, remove the capsule shells, accurately weigh 1g, place it in a stoppered conical flask, add 15mL of 80% methanol, weigh it, extract by sonication for 30min, remove it, cool it to room temperature, replenish the lost weight with 80% methanol, shake well, take 2mL of the supernatant, filter it through a 0.22μm microporous membrane, and you will get the product.

[0083] S3. Fingerprint analysis of Jianyang Capsules:

[0084] High-performance liquid chromatography (HPLC) was used to obtain chromatograms of the test solution and the reference solution under the same detection conditions.

[0085] The detection conditions included: a Hungpu T3 column (250 mm × 4.6 mm, 5 μm), gradient elution with acetonitrile as mobile phase A and 0.001% formic acid aqueous solution as mobile phase B, a flow rate of 1.0 mL / min, a detection wavelength of 205 nm, a column temperature of 35 ℃, and an injection volume of 5 μL.

[0086] During gradient elution, the changes in mobile phase A and mobile phase B are as follows:

[0087] 0 min, mobile phase A 0%, mobile phase B 100%;

[0088] 5 min, mobile phase A 0%, mobile phase B 100%;

[0089] 33 min, mobile phase A 35%, mobile phase B 65%;

[0090] 85 min, mobile phase A 100%, mobile phase B 0%;

[0091] 90 min, mobile phase A 100%, mobile phase B 0%;

[0092] 95 min, mobile phase A 0%, mobile phase B 100%;

[0093] 105 min, mobile phase A 0%, mobile phase B 100%;

[0094] Take the test solution and reference solution of each batch of Jianyang capsules separately, and determine them according to the above liquid chromatography conditions. The chromatograms are shown below. Figure 1 and Figure 2 As shown.

[0095] S4. Generate the fingerprint spectrum of Jianyang Capsules:

[0096] The chromatograms of each batch of Jianyang capsules were analyzed using the National Pharmacopoeia Commission's "Similarity Evaluation System for Chromatographic Characteristic Chromatography of Traditional Chinese Medicine" (2012 version). A time window width of 0.1 was set, and peak 9 (2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside) was used as the reference peak. A total of 23 common peaks were used to generate a reference fingerprint chromatogram (see [link to relevant documentation]). Figure 3 The similarity calculation results are shown in Table 4.

[0097] The relative retention times of each characteristic peak in the JYJN1-JYJN10 Jianyang Capsule test solution were calculated using a reference peak. The average relative retention times of the 23 characteristic peaks and the reference peak were as follows: 0.439 (peak 1), 0.452 (peak 2), 0.476 (peak 3), 0.496 (peak 4), 0.505 (peak 5), 0.585 (peak 6), 0.749 (peak 7), 0.755 (peak 8), 1.000 (peak 9), 1.066 (peak 10), 1.086 (peak 11), 1.121 (peak 12), 1.280 (peak 13), 1.355 (peak 14). The peaks were 1.422 (peak 15), 1.444 (peak 16), 1.472 (peak 17), 1.549 (peak 18), 1.742 (peak 19), 1.958 (peak 20), 2.016 (peak 21), 2.044 (peak 22), and 2.755 (peak 23). The results are detailed in Table 5. In this chromatogram, peak 2 is crystallizing glycoside, peak 9 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, peak 11 is notoginsenoside R1, peak 12 is ginsenoside Rg1, peak 14 is ginsenoside Rb1, and peak 18 is ganoderic acid A.

[0098] Table 4. Similarity results of fingerprint spectra of 10 batches of Jianyang capsules

[0099]

[0100] Table 5. Relative retention times of characteristic peaks in 10 batches of Jianyang Capsules

[0101]

[0102]

[0103] Example 2 - Investigation of Extraction Conditions

[0104] Following the chromatographic detection conditions described in Example 1 above, the factors influencing the extraction efficiency of chemical components from Jianyang capsules were investigated from four aspects: extraction method (ultrasound, reflux), extraction solvent (anhydrous ethanol, 30%, 50%, 80%, and 100% methanol), extraction time (30 min, 45 min, and 60 min), and extraction solid-liquid ratio (0.5 g: 15 mL, 0.5 g: 30 mL, 1 g: 15 mL, and 1 g: 30 mL). The results are as follows: Figure 4 , Figure 5 , Figure 6 and Figure 7 .

[0105] The results showed that there was no significant difference in the number of peaks obtained from ultrasonic and reflux extraction methods, and the main peak types were consistent. The only difference was in the response values ​​of the main component peaks. Ultrasonic extraction was chosen as the preferred method due to its speed and ease of operation. Comparison of extraction with ethanol and different concentrations of methanol solvents revealed that the response values ​​of the main component peaks extracted with 80% methanol were higher than those extracted with other solvents, while the number of peaks was the same. Therefore, 80% methanol was selected as the extraction solvent for Jianyang capsules. Regarding extraction time, ultrasonic extraction time had no significant impact on the extraction effect. Considering time and cost savings, 30 min was ultimately chosen as the extraction time for preparing the sample solution. Comparison of extraction with different solid-liquid ratios revealed that the main peak types were consistent across all ratios, but the response values ​​of each main peak showed significant differences. The sample obtained with a solid-liquid ratio of 1 g:15 mL had higher response values ​​for the main component peaks. Furthermore, considering solvent conservation and environmental protection, a solid-liquid ratio of 1 g:15 mL was ultimately selected. Furthermore, this application can achieve similar results with slight fluctuations within the above range. Therefore, the final extraction conditions are as follows: accurately weigh 1g of Jianyang capsule powder, place it in a stoppered conical flask, add 10-20mL of 70%-80% methanol, weigh it, extract ultrasonically for 20-40min, remove it, cool it to room temperature, replenish the lost weight with 70%-80% methanol, shake well, take 2mL of the supernatant, filter it through a 0.22μm microporous membrane, and the product is obtained.

[0106] Example 3 - Optimization of Chromatographic Conditions

[0107] 3.1 Selection of detection wavelength

[0108] According to literature reports, the commonly used detection wavelengths for the analysis of various traditional Chinese medicines in Jianyang Capsules are 205nm, 255nm, 280nm, and 290nm. The Jianyang Capsule test solution was prepared according to the method described in Example 1. The chromatographic conditions were as follows: a Waterse2695+2998 high-performance liquid chromatograph was used; the column was a Hungpu T3 (250mm × 4.6mm, 5μm); the mobile phase was acetonitrile (A) - 0.1% formic acid aqueous solution (B); gradient elution was used (0–60 min, 0%–100% A; 60–70 min, 100%–100% A; 70–75 min, 100%–0% A; 75–85 min, 0%–0% A); the flow rate was 1.0 mL / min; and the column temperature was 35℃. The injection volume of the test sample was 5 μL. The test solution was scanned across all wavelengths, with the selection criteria being that the wavelength contained abundant analytical signal peak information, had a stable baseline, and exhibited large peak response values. This was achieved by accessing the 3D spectrum (see...). Figure 8 Analysis revealed that the chromatographic peak information content of Jianyang capsules was high at 205 nm, with each peak showing a relatively high response value. Therefore, 205 nm was ultimately selected as the analytical wavelength for the fingerprint spectrum of Jianyang capsules (see...). Figure 9 ).

[0109] 3.2 Selection of Mobile Phase

[0110] The selection criteria for mobile phase systems were based on the number of peaks, high resolution between peaks, stable baseline, and short total elution time. The effects of different mobile phase systems on the separation of chemical components in the test solution were compared. The mobile phase systems are as follows:

[0111] (1) Methanol (A)-Water (B); (2) Acetonitrile (A)-Water (B); (3) Acetonitrile (A)-0.1% formic acid aqueous solution (B); (4) Acetonitrile (A)-0.01% formic acid aqueous solution (B); (5) Acetonitrile (A)-0.001% formic acid aqueous solution (B).

[0112] The test solution was prepared according to the preparation method in Example 1. The chromatographic conditions were as follows: Hungpu T3 (250 mm × 4.6 mm, 5 μm) column; mobile phases were the above 5 types; gradient elution (0–60 min, 0%–100% A; 60–70 min, 100%–100% A; 70–75 min, 100%–0% A; 75–85 min, 0%–0% A); flow rate was 1.0 mL / min; column temperature was 35 °C; detection wavelength was 205 nm; and sample injection volume was 5 μL.

[0113] The results are as follows Figure 10As shown, the component separation effects of the five mobile phases differed significantly. The samples obtained from methanol-water and acetonitrile-0.1% formic acid aqueous solution exhibited uneven baselines, the sample obtained from acetonitrile-water showed poor peak shapes, and the sample obtained from acetonitrile-0.01% formic acid aqueous solution showed a more stable baseline. The sample obtained from acetonitrile-0.001% formic acid aqueous solution showed a stable baseline, good peak shape, moderate response values ​​of the main component peaks, and good separation between peaks. Therefore, acetonitrile-0.001% formic acid aqueous solution was ultimately selected as the mobile phase. Even when the concentration of the formic acid aqueous solution was adjusted within the range of 0.001%-0.005%, good peak shapes, response values ​​of the main component peaks, and separation were still obtained.

[0114] 3.3 Investigation of elution gradient, column temperature and flow rate

[0115] Based on the criteria of moderate elution time, stable baseline, and good resolution of major chromatographic peaks, the effects of elution gradients (Table 6), flow rates of 0.6 mL / min, 0.8 mL / min, and 1.0 mL / min, column temperatures of 25℃, 30℃, 35℃, and 40℃, and injection volumes of 2 μL, 5 μL, 7 μL, and 10 μL on the separation of chemical components in the test solution were compared. The results are as follows: Figure 11 , Figure 12 , Figure 13 and Figure 14 As shown.

[0116] Table 6. Schematic diagram of different elution gradients

[0117]

[0118]

[0119] 3.4 Investigation of different chromatographic columns

[0120] Based on the criteria of numerous peaks, stable baseline, and high resolution of major chromatographic peaks in the sample chromatogram, the effects of chromatographic columns H1: Hungpu T3 (250mm × 4.6mm, 5μm); H2: Hungpu C18-AQ(2) (250mm × 4.6mm, 5μm); and H3: Hungpu XBT C18 (250mm × 4.6mm, 5μm) on the separation of chemical components in the test solution were compared. The results are as follows: Figure 15 As shown.

[0121] The results showed that different chromatographic columns had different separation effects on the chemical components in the test solution. Among them, the Hungpu T3 column separated a large number of chromatographic peaks, and the resolution between the chromatographic peaks was high, with a moderate total elution time. Therefore, the Hungpu T3 column is preferred for the fingerprint chromatographic spectrum of Jianyang capsules.

[0122] Example 4 - Methodological Validation

[0123] 4.1 System adaptability assessment

[0124] Take an appropriate amount of Jianyang capsules as a sample, prepare the test solution and reference solution according to the sample preparation method in Example 1, and perform the detection according to the chromatographic conditions in Example 1. The results are shown in [Figure 1]. Figure 16 The theoretical plate number of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside is greater than 30,000.

[0125] 4.2 Specificity Examination

[0126] The test solution of Jianyang capsules and 80% methanol solution were analyzed under the chromatographic conditions described in Example 1. The results are as follows: Figure 17 As shown, there were no chromatographic peaks interfering with the corresponding retention times of each peak in the 80% methanol solvent chromatogram, which meets the technical requirements of traditional Chinese medicine fingerprinting, indicating that the established method has good specificity.

[0127] 4.3 Precision Test

[0128] Take an appropriate amount of Jianyang capsule sample, prepare the test solution according to the sample preparation method in Example 1, and continuously determine it 6 times under the chromatographic conditions in Example 1. The chromatogram is shown in the figure. Figure 18 The relative retention time (RSD) values ​​of the 23 characteristic peaks were less than 1%, and the RSD values ​​of the peak areas were less than 10% (see Tables 7 and 8 for details), indicating that the instrument's precision meets the requirements.

[0129] Table 7 Precision Study - Relative Retention Time of Characteristic Peaks

[0130]

[0131]

[0132] Table 8 Precision Examination - Peak Area of ​​Characteristic Peaks

[0133] Element 1 time 2 times 3 times 4 times 5 times 6 times RSD value (%) Peak 1 124327 128687 130564 133012 134144 132237 2.74 Peak 2 295758 305398 305128 342643 318500 302064 5.43 Peak 3 155092 176204 158869 187813 184627 161379 8.23 Peak 4 523752 535288 530974 565054 547297 542027 2.68 Peak 5 207806 208559 208934 224055 217799 217375 3.10 Peak 6 94496 82192 89044 93667 93777 87724 5.32 Peak 7 82778 79149 99845 89737 95762 94229 8.83 Peak 8 362355 355700 365850 392934 372671 376039 3.50 Peak 9 2417544 2372344 2437062 2497699 2470988 2455087 1.79 Peak 10 46538 48515 51792 58406 53460 56463 8.66 Peak 11 91279 88535 85518 90563 73044 80216 8.31 Peak 12 339273 338256 340761 355587 347973 348912 1.98 Peak 13 63809 63757 65055 68798 66651 65845 2.91 Peak 14 254280 254736 255876 267018 261087 261749 1.94 Peak 15 127374 126983 128010 133997 131021 131267 2.13 Peak 16 98391 99106 99422 103032 100851 100885 1.67 Peak 17 97833 96344 97072 106891 103570 104723 4.47 Peak 18 49637 51474 50739 52785 53488 51557 2.68 Peak 19 198074 198265 200248 213148 208778 202489 3.02 Peak 20 121043 121725 121103 127049 124980 122743 1.97 Peak 21 46162 46732 47851 48495 47523 47768 1.77 Peak 22 80173 79572 79571 84174 81366 79746 2.23 Peak 23 103868 100803 97258 100463 99822 102351 2.24

[0134] 4.4 Repeatability Test

[0135] Take an appropriate amount of Jianyang capsule sample and prepare 6 test solutions in parallel according to the sample preparation method in Example 1. Perform the determination under the chromatographic conditions in Example 1. The chromatogram is shown in [Figure 1]. Figure 19 The relative retention time RSD of the 23 characteristic peaks was less than 1%, and the peak area RSD was less than 10% (see Table 9-10 for details), indicating that the repeatability of the method meets the requirements.

[0136] Table 9 Repeatability Tests - Relative Retention Times of Characteristic Peaks

[0137]

[0138]

[0139] Table 10 Repeatability Tests - Peak Area of ​​Characteristic Peaks

[0140] Element Parallel Sample 1 Parallel Sample 2 Parallel Sample 3 Parallel Sample 4 Parallel Sample 5 Parallel Sample 6 RSD value (%) Peak 1 116055 102931 118925 124427 125390 106103 8.07 Peak 2 310745 285162 324884 314726 345011 286418 7.38 Peak 3 198180 171983 212470 204290 216765 183653 8.71 Peak 4 588419 513184 591774 606690 573128 478867 9.10 Peak 5 224602 195650 222221 224310 219185 183149 8.36 Peak 6 75672 66317 74289 76279 77572 63360 8.18 Peak 7 86385 80696 93070 94430 92323 79140 7.56 Peak 8 371705 336440 385415 394768 372474 319129 8.08 Peak 9 2533303 2273276 2640030 2686728 2490143 2207345 7.84 Peak 10 46684 41667 51015 49349 50901 41853 9.14 Peak 11 72862 92094 91024 93157 93873 91534 9.00 Peak 12 337443 319052 354663 363688 354694 312954 6.12 Peak 13 64395 60099 69770 73686 68141 58053 9.08 Peak 14 254646 237929 266965 286704 281247 231541 8.67 Peak 15 126412 119308 130943 135666 133995 115760 6.35 Peak 16 96663 89999 98279 99482 95962 88594 4.73 Peak 17 88406 86109 94693 74405 75520 86496 9.31 Peak 18 62862 50121 56059 63852 54925 53852 9.41 Peak 19 195671 184292 204372 214625 199978 182436 6.22 Peak 20 126114 118441 133543 135765 127733 109512 7.83 Peak 21 46566 43240 48007 48970 48027 42691 5.76 Peak 22 79844 72846 81939 84879 83186 71591 7.03 Peak 23 97822 92959 95691 97993 95831 81134 6.80

[0141] 4.5 Stability Test

[0142] Take an appropriate amount of Jianyang capsule sample, prepare the test solution according to the sample preparation method in Example 1, and determine the chromatogram at 0, 2, 4, 8, 12, and 24 hours according to the chromatographic conditions in Example 1. See the chromatogram below. Figure 20 The relative retention time RSD of the 23 characteristic peaks was less than 1%, and the peak area RSD was less than 10% (see Tables 11 and 12 for details), indicating that the test solution was stable within 24 hours.

[0143] Table 11 Stability Study - Relative Retention Time of Characteristic Peaks

[0144] Element 0h 2h 4h 8h 12h 24h RSD value (%) Peak 1 0.447 0.447 0.448 0.447 0.447 0.446 0.14 Peak 2 0.458 0.458 0.458 0.459 0.458 0.458 0.07 Peak 3 0.479 0.479 0.480 0.480 0.479 0.479 0.07 Peak 4 0.500 0.500 0.501 0.498 0.497 0.495 0.46 Peak 5 0.510 0.509 0.510 0.510 0.509 0.509 0.08 Peak 6 0.588 0.588 0.589 0.588 0.588 0.587 0.12 Peak 7 0.750 0.750 0.749 0.749 0.749 0.748 0.10 Peak 8 0.756 0.756 0.756 0.756 0.756 0.755 0.09 Peak 9 1.000 1.000 1.000 1.000 1.000 1.000 0.00 Peak 10 1.066 1.066 1.065 1.067 1.067 1.067 0.07 Peak 11 1.084 1.084 1.084 1.090 1.090 1.093 0.37 Peak 12 1.120 1.120 1.119 1.126 1.126 1.129 0.38 Peak 13 1.279 1.279 1.279 1.282 1.283 1.284 0.20 Peak 14 1.352 1.353 1.352 1.361 1.362 1.366 0.44 Peak 15 1.419 1.419 1.419 1.427 1.428 1.433 0.42 Peak 16 1.442 1.442 1.442 1.450 1.451 1.456 0.42 Peak 17 1.468 1.469 1.469 1.478 1.480 1.485 0.48 Peak 18 1.547 1.548 1.547 1.553 1.554 1.557 0.28 Peak 19 1.738 1.740 1.739 1.750 1.751 1.757 0.44 Peak 20 1.956 1.958 1.957 1.959 1.960 1.961 0.10 Peak 21 2.011 2.013 2.012 2.024 2.026 2.032 0.44 Peak 22 2.039 2.041 2.040 2.052 2.054 2.060 0.43 Peak 23 2.752 2.755 2.755 2.757 2.758 2.761 0.11

[0145] Table 12 Stability Study - Peak Area of ​​Characteristic Peaks

[0146]

[0147]

[0148] In summary, the fingerprint spectrum of this invention can comprehensively describe and reflect the product quality of Jianyang Capsules, effectively controlling the overall product quality. Using peak 9 (2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside) as a control peak, the fingerprint spectrum of Jianyang Capsules exhibits a total of 23 common peaks, and the relative retention time of each peak is provided. By applying the established fingerprint spectrum of Jianyang Capsules to quality control testing, this invention facilitates comprehensive monitoring of product quality, ensuring consistency, controllability, and stability, thereby enabling more comprehensive quality control of Jianyang Capsules.

[0149] The detection conditions selected in this invention have been repeatedly compared and verified experimentally, enabling semi-quantitative determination. This invention is the first to employ a traditional Chinese medicine fingerprinting method to detect this product. Through optimization of the mobile phase and gradient elution conditions, the main peak and other chromatographic peaks in the chromatogram are well separated, allowing for comprehensive control of the product's overall quality while meeting the requirements of large-scale industrial production. By establishing a reference fingerprint chromatogram for this product using the method provided by this invention, the quality control of the product is improved, further promoting its international development.

[0150] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a fingerprint spectrum of Jianyang capsules, characterized in that, It includes: (1) Using ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1, ganoderic acid A, crystal blue glycoside and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside as reference standards, prepare reference standard solutions; (2) Prepare multiple batches of test solution using powder from multiple batches of Jianyang capsules as test samples. The preparation method of the test solution includes: mixing the powder from Jianyang capsules with methanol of 70-80% volume concentration at a mass-volume ratio of 1g:10-20mL, extracting by ultrasonication or reflux for 20-40min, cooling to room temperature, replenishing the lost weight with methanol of 70-80% volume concentration, shaking well, taking the supernatant, and filtering it through a 0.22μm microporous membrane to obtain the solution. (3) The reference solution and the test solution were analyzed by high performance liquid chromatography, respectively; The chromatographic conditions included: using acetonitrile and formic acid aqueous solution as mobile phases, passing the reference solution and the test solution through a Hungpu T3 column and performing gradient elution to obtain chromatograms of each batch of Jianyang capsules; the detection wavelength was 200-220 nm; and the volume concentration of the formic acid aqueous solution in the mobile phase was 0.001%-0.005%. The elution procedure for gradient elution is as follows: When t=0min~5min, the percentage of acetonitrile in the total amount of the mobile phase is 0%, and the percentage of formic acid aqueous solution in the total amount of the mobile phase is 100%. When t=5min~33min, the percentage of acetonitrile in the total amount of the mobile phase changes from 0% to 35%, and the percentage of formic acid aqueous solution in the total amount of the mobile phase changes from 100% to 65%. During the period from t=33min to 85min, the percentage of acetonitrile in the total amount of the mobile phase changes from 35% to 100%, and the percentage of formic acid aqueous solution in the total amount of the mobile phase changes from 65% to 0%. When t=85min~90min, the percentage of acetonitrile in the total amount of the mobile phase is maintained at 100%, and the percentage of formic acid aqueous solution in the total amount of the mobile phase is maintained at 0%. When t=90min~95min, the percentage of acetonitrile in the total amount of the mobile phase changes from 100% to 0%, and the percentage of formic acid aqueous solution in the total amount of the mobile phase changes from 0% to 100%. When t=95min~105min, the percentage of acetonitrile in the total amount of the mobile phase is maintained at 0%, and the percentage of formic acid aqueous solution in the total amount of the mobile phase is maintained at 100%. (4) Perform similarity analysis on the chromatograms of each batch of Jianyang capsules, and use the median calculation method to generate a total of 23 common peaks with the relative retention time of the reference peak as 1; and calculate the relative retention time of each common peak with the reference peak to generate the fingerprint spectrum of Jianyang capsules.

2. The method for constructing the fingerprint spectrum of Jianyang capsules according to claim 1, characterized in that, Among the common peaks, peak 2 is crystallizing glycoside, peak 9 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, peak 11 is notoginsenoside R1, peak 12 is ginsenoside Rg1, peak 14 is ginsenoside Rb1, and peak 18 is ganoderic acid A.

3. The method for constructing the fingerprint spectrum of Jianyang capsules according to claim 2, characterized in that, The reference peak is peak 9: 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside. The theoretical plate number, calculated based on 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, is not less than 30,000.

4. The method for constructing the fingerprint spectrum of Jianyang capsules according to claim 3, characterized in that, The retention times of the common peaks are as follows: Peak 1: relative retention time 0.439±0.5%, Peak 2: relative retention time 0.452±0.5%, Peak 3: relative retention time 0.476±0.5%, Peak 4: relative retention time 0.496±0.5%, Peak 5: relative retention time 0.505±0.5%, Peak 6: relative retention time 0.585±0.5%, Peak 7: relative retention time 0.749±0.5%, Peak 8: relative retention time 0.755±0.5%, Peak 9: relative retention time 1.000%, Peak 10: relative retention time 1.066±0.5%, Peak 11: relative retention time 1.086±0.5%, Peak 12: relative retention time Peak 13: Relative retention time 1.280±0.5%; Peak 14: Relative retention time 1.355±0.5%; Peak 15: Relative retention time 1.422±0.5%; Peak 16: Relative retention time 1.444±0.5%; Peak 17: Relative retention time 1.472±0.5%; Peak 18: Relative retention time 1.549±0.5%; Peak 19: Relative retention time 1.742±0.5%; Peak 20: Relative retention time 1.958±0.5%; Peak 21: Relative retention time 2.016±0.5%; Peak 22: Relative retention time 2.044±0.5%; Peak 23: Relative retention time 2.755±0.5%.

5. The method for constructing the fingerprint spectrum of Jianyang capsules according to any one of claims 1-4, characterized in that, The method for preparing the reference solution includes: mixing ginsenoside Rg1, ginsenoside Rb1, notoginsenoside R1, ganoderic acid A, citronellol, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside with methanol solution to prepare single standard solutions with concentrations of ginsenoside Rg1 (18-27 μg / mL), ginsenoside Rb1 (18-27 μg / mL), notoginsenoside R1 (18-27 μg / mL), ganoderic acid A (6-10 μg / mL), citronellol (18-27 μg / mL), and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (18-27 μg / mL).

6. The method for constructing the fingerprint spectrum of Jianyang capsules according to any one of claims 1-4, characterized in that, The chromatographic conditions also include: a flow rate of 0.8~1.0 mL / min and a column temperature of 25℃-40℃.

7. The application of the fingerprint spectrum of Jianyang Capsules constructed by the method of any one of claims 1-6 in the quality control and testing of Jianyang Capsules.

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