Fingerprint spectrum of pills for strengthening body resistance and treating stranguria and construction method and application thereof

Abstract

The invention discloses a fingerprint spectrum of pills for strengthening body resistance and treating stranguria as well as a construction method and application thereof, and belongs to the technical field of quality detection of traditional Chinese medicine preparations. The method comprises the following steps: respectively injecting a test solution, a reference solution and a negative test solution into an ultra-high performance liquid chromatograph for determination by adopting ultra-high performance liquid chromatography, recording chromatograms, establishing a fingerprint spectrum of the pills for strengthening the body resistance and treating stranguria under the same chromatographic condition, calibrating 17 characteristic peaks, and determining the quality of the pills for strengthening the body resistance and treating stranguria. The five components of calycosin-7-glucoside, liquiritin, bisdemethoxycurcumin, demethoxycurcumin and curcumin in the pills for strengthening the body resistance and treating stranguria are quantitatively analyzed, so that content determination and quality detection are realized. The fingerprint spectrum of the pills for strengthening the body resistance and treating stranguria established on the basis of the ultra-high performance liquid chromatography and the multi-index component content determination method are simple, convenient and efficient, and the quality of the pills for strengthening the body resistance and treating stranguria can be comprehensively, systematically and objectively evaluated.

Description

Technical Field

[0001] This invention relates to a fingerprint spectrum of Fuzheng Tonglin Pill, its construction method and application, belonging to the field of quality testing technology of traditional Chinese medicine preparations. Background Technology

[0002] Fuzheng Tonglin Pills are a clinically experienced formula, formulated based on traditional Chinese medicine theory and clinical experience. The formula consists of six herbs: American cockroach, astragalus, ground beetle, aconite, turmeric, and licorice. It has the functions of tonifying qi and warming yang, dispersing nodules and promoting urination. It can be used for symptoms such as frequent urination, nocturia, urgency, difficulty urinating, and dribbling caused by kidney qi deficiency and blood stasis.

[0003] Ultra-high performance liquid chromatography (UHPLC) has advantages such as high resolution, high speed, and high sensitivity. Fingerprint spectroscopy can reflect the overall compositional characteristics of a sample and is a commonly used quality control technology for traditional Chinese medicine. Content determination ensures the quantitative accuracy of key components. The combination of the two can achieve a comprehensive quality evaluation of Fuzheng Tonglin Pill.

[0004] Currently, no research papers or patents have been found related to the establishment method and content determination of the fingerprint spectrum of Fuzheng Tonglin Pills among existing patent technologies. Summary of the Invention

[0005] The purpose of this invention is to provide a fingerprint spectrum of Fuzheng Tonglin Pill and its construction method, which not only constructs the fingerprint spectrum of Fuzheng Tonglin Pill, but also effectively detects the overall quality of Fuzheng Tonglin Pill, and has good repeatability.

[0006] The technical solution adopted in this invention is: The method for constructing the fingerprint spectrum of Fuzheng Tonglin Pill includes the following steps: S1. Prepare samples, test solutions, control solutions and negative test solutions of Fuzheng Tonglin Pills; (1) Preparation of Fuzheng Tonglin Pills: Take 3.0-3.8 parts of American cockroach, 4.0-4.7 parts of Astragalus membranaceus, 2-3 parts of Eupolyphaga sinensis, 2-3 parts of Aconitum carmichaelii, 1.4-1.9 parts of Curcuma longa, and 0.37-0.95 parts of Glycyrrhiza uralensis by weight, pulverize and make into water pills; (2) Preparation of test solution: Take the Fuzheng Tonglin Pill sample, add solvent to extract, filter, and take the filtrate to obtain the test solution; (3) Preparation of reference solution: Weigh accurately the reference standards of verbascoside, glycyrrhizin, bis(demethoxy)curcumin, demethoxy)curcumin and curcumin, dissolve them in solvent and dilute to volume to obtain the solution. (4) Preparation of negative test solution: Take a powder sample lacking any one component of Fuzheng Tonglin Pill, extract it with solvent, filter it, and take the filtrate to prepare negative solutions for those lacking American cockroach, astragalus, ground beetle, aconite, turmeric, or licorice. S2. Using ultra-high performance liquid chromatography (UHPLC), the test solution from step S1 (2), the reference solution from step (3), and the negative test solution from step (4) were injected into the UHPLC instrument for determination, and the chromatograms were recorded: The ultra-high performance liquid chromatography (UHPLC) conditions described herein involve mobile phase A being acetonitrile and mobile phase B being a 0.1% aqueous solution of glacial acetic acid, with gradient elution. The elution gradient is as follows: 0–0.3 min: 5% A; 0.3–19 min: 5% A → 60% A; 19–35 min: 60% A → 85% A; and the flow rate is 0.25–0.35 mL / min. -1 The column temperature is 30~40℃, the detection wavelength is 230~400 nm, and the injection volume is 2~5 µL. S3. Identification of common peaks: Multiple batches of test sample solutions were injected sequentially to obtain multiple batches of test sample chromatograms. These chromatograms were then imported into the Chinese medicine chromatographic fingerprint similarity evaluation system to identify 17 common peaks and generate the fingerprint chromatogram of Fuzheng Tonglin Pill. The common peaks were compared with the chromatograms of the negative test sample solution and the reference solution to assign each common peak to its corresponding chromatogram.

[0007] In the above method, the solvent used in steps (2) to (4) of S1 is 70% to 90% methanol solution or methanol, preferably 80% methanol solution; the extraction method in (2) is heating reflux method or ultrasonic extraction method, preferably ultrasonic extraction method.

[0008] The ultra-high performance liquid chromatography (UHPLC) column in step S2 is a C18 column. Preferably, the column is a Thermo Acclaim. TM RSLC 120 C 18 (2.1 × 100 mm, 2.2 µm). Preferably, the flow rate of the ultra-high performance liquid chromatography is 0.3 mL·min. -1 The column temperature was 40℃, the detection wavelength was 260 nm, and the injection volume was 3 µL.

[0009] The fingerprint spectrum of Fuzheng Tonglin Pill constructed by the above method includes 17 common peaks, as detailed below; The relative retention time of the characteristic peak No. 1 is 0.439. The relative retention time of the characteristic peak No. 2 is 0.448. The relative retention time of the characteristic peak No. 3 is 0.456. The relative retention time of the characteristic peak No. 4 is 0.654. The relative retention time of the characteristic peak No. 5 is 0.781. The relative retention time of the characteristic peak at peak 6 is 0.805. The relative retention time of the characteristic peak No. 7 is 0.851. The relative retention time of the characteristic peak at peak 8 is 1.000. The relative retention time of the characteristic peak at peak 9 is 1.021. The relative retention time of the characteristic peak at peak number 10 is 1.041. The relative retention time of the characteristic peak at peak 11 is 1.156. The relative retention time of the characteristic peak at peak 12 is 1.184. The relative retention time of the characteristic peak at peak 13 is 1.285. The relative retention time of the characteristic peak at peak 14 is 1.361. The relative retention time of the characteristic peak at peak number 15 is 1.511. The relative retention time of the characteristic peak at peak 16 is 1.523. The relative retention time of the characteristic peak on the 17th is 1.619.

[0010] Each common peak was assigned a specific composition, and the chemical components of some of the common peaks were identified, using the bis(demethoxy)curcumin chromatographic peak as a reference peak. The first characteristic peak in the fingerprint spectrum is verrucoside isoflavone glucoside; The characteristic peak of peak 3 is glycyrrhizin; The characteristic peak of peak 8 is didemethoxycurcumin; The characteristic peak of peak 9 is demethoxycurcumin; The characteristic peak of sample number 10 is curcumin.

[0011] This invention also provides the application of the fingerprint spectrum of Fuzheng Tonglin Pill in content determination. The method is as follows: prepare the test solution, reference solution and negative test solution according to step S1 in the fingerprint spectrum construction method. Under the same chromatographic conditions as step S2 in the fingerprint spectrum construction method, inject the test solution, reference solution and negative test solution into an ultra-high performance liquid chromatograph, record the chromatogram, and calculate the content of five components in the test solution, namely, verrucoside, glycyrrhizin, bisdemethoxycurcumin, methoxycurcumin and curcumin, using the external standard method.

[0012] The application of the fingerprint spectrum of Fuzheng Tonglin Pill in quality testing is as follows: The quality of Fuzheng Tonglin Pill is evaluated using the reference fingerprint spectrum. The similarity between the fingerprint spectrum of each batch of test sample and the reference fingerprint spectrum is calculated using the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system. The similarity is not less than 0.9.

[0013] The beneficial effects of this invention are as follows: (1) The content detection method provided by the present invention has passed the verification of precision, repeatability, stability, linearity and recovery rate. The detection results are accurate and reliable. It can simultaneously and accurately quantify the content of five chemical components in Fuzheng Tonglin Pill: verrucoside glucoside, glycyrrhizin, bis(demethoxy)curcumin, demethoxy)curcumin and curcumin. This is of great significance for the quality control of Fuzheng Tonglin Pill.

[0014] (2) The fingerprint spectrum obtained by applying the mobile phase, elution conditions and detection wavelength provided by the present invention has a clean background, good peak separation effect, stable baseline, is simple and fast, easy to master, and is suitable for establishing the fingerprint spectrum of Fuzheng Tonglin Pill.

[0015] (3) The present invention uses a combination of ultra-high performance liquid chromatography fingerprinting and multi-index component content determination to control the quality of Fuzheng Tonglin Pills, which can provide a basis for formulating quality control standards for Fuzheng Tonglin Pills. Attached Figure Description

[0016] Figure 1 UPLC plots for the wavelength range of 230–400 nm; Figure 2 UPLC plots for different flow rates: where: S1: 0.25 mL·min -1 S2: 0.3 mL·min -1 S3: 0.35 mL·min -1 ; Figure 3 UPLC spectra at different column temperatures; Figure 4 UPLC spectra of different mobile phase systems: where: S1: Acetonitrile-0.1% glacial acetic acid solution, S2: Acetonitrile-0.1% formic acid solution, S3: Acetonitrile-0.1% phosphoric acid solution, S4: Acetonitrile-water solution; Figure 5 UPLC chromatograms for different columns: Where: Column 1: Thermo Acclaim TM RSLC 120 C 18 (2.1×100 mm, 2.2 µm); Column 2: Thermo Scientific Syncronis C 18 (2.1×100 mm, 1.7 µm); Column 3: Waters ACQUITY UPLC® BEH C 18 (2.1×150 mm, 1.7 µm); Figure 6 UPLC spectra for different injection volumes; Figure 7 UPLC spectra for different extraction solvents: S1: 70% methanol; S2: 80% methanol; S3: 90% methanol; S4: methanol; Figure 8 UPLC spectra obtained using different extraction methods; Figure 9 For different ultrasound durations, the atlas was examined; Figure 10 UPLC spectra for different sample sizes; Figure 11 The results are from a precision testing study. Figure 12 The results are from the stability test. Figure 13 This is a result of a repeatability study; Figure 14 Results of 60 min delay for the test sample and blank solvent of Fuzheng Tonglin Pill; Figure 15 The results are from the blank test of the UPLC fingerprint spectrum peaks; Figure 16 Fingerprint spectra of 12 batches of test samples; Figure 17 The fingerprint profiles are for reference of 12 batches of test samples; Figure 18 This is a mixed reference solution; Figure 19 The results show the similarity of UPLC fingerprint spectra of 12 batches of Fuzheng Tonglin Pills. Detailed Implementation

[0017] To make the technical means, creative features, objectives, and effects of this invention easier to understand, the invention is further illustrated below with reference to specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention.

[0018] The instruments and reagents used in this invention are as follows: Table 1. Instruments, reagents and their sources

[0019] Table 2 Sample Information

[0020] Table 3. Information on processed medicinal slices

[0021] Table 4 Reference Standard Information

[0022] Example 1: Fingerprint spectroscopy detection of Fuzheng Tonglin Pills: (1) Preparation of Fuzheng Tonglin Pill Sample Take 4.0-4.7 g of Astragalus membranaceus, 3.0-3.8 g of American cockroach, 2-3 g of Aconitum carmichaelii, 2-3 g of Eupolyphaga sinensis, 1.4-1.9 g of Curcuma longa, and 0.37-0.95 g of Glycyrrhiza uralensis, grind them into powder and make them into water pills.

[0023] (2) Preparation of the test solution Take 0.4 g of Fuzheng Tonglin Pill powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 80% methanol, seal tightly, weigh, and extract ultrasonically for 30 min (power 250 W, frequency 40 kHz). Cool, weigh, replenish the lost weight with 80% methanol, shake well, filter, and take the filtrate. Filter through a 0.22 μm microporous membrane to obtain the final product.

[0024] (3) Sample injection detection The test solution was scanned across the entire wavelength range. Based on the results, the wavelength range of 230–400 nm was determined as the screening condition. Tests were conducted at wavelengths of 230 nm, 245 nm, 260 nm, 270 nm, 285 nm, 290 nm, 310 nm, and 400 nm, respectively. (See attached image.) Figure 1-2 The results showed that the baseline was uneven at a wavelength of 230 nm, the peak separation was poor at a wavelength of 245 nm, and the number of peaks was low at wavelengths of 270 nm, 285 nm, 290 nm, 310 nm and 400 nm.

[0025] Column: Thermo Acclaim TM RSLC 120 C 18 (2.1 × 100 mm, 2.2 µm); Mobile phase: acetonitrile (A) - 0.1% glacial acetic acid aqueous solution (B), elution gradient: 0–0.3 min: 5% A; 0.3–19 min: 5% A → 60% A; 19–35 min: 60% A → 85% A; Flow rate: 0.3 mL·min -1 The column temperature was 30 °C and the injection volume was 3 µL.

[0026] Taking into account the overall spectral quality, and considering factors such as a stable baseline, a large number of peaks, good peak separation, and relatively balanced peak areas, 260 nm was ultimately selected as the detection wavelength. (See below) Figure 1 .

[0027] Example 2: Fingerprint analysis of Fuzheng Tonglin Pills: (1) Preparation of the test solution: Same as step (2) in Example 1.

[0028] (2) Sample injection and detection: Column: Thermo Acclaim TM RSLC 120 C 18 (2.1×100 mm, 2.2 µm); Mobile phase: acetonitrile (A) - 0.1% glacial acetic acid aqueous solution (B), elution gradient: 0~0.3 min: 5% A; 0.3~19 min: 5% A → 60% A; 19~35 min: 60% A → 85% A; Column temperature: 30 ℃; Injection volume: 3 µL; Detection wavelength: 260 nm.

[0029] Take the same test solution and administer it at 0.25 mL / min. -1 0.3 mL·min -1 and 0.35 mL·min -1 Three different flow rates were used for injection analysis to investigate the effect of different flow rates on the chromatogram. (See...) Figure 2 .

[0030] The results showed that the slower the flow rate, the longer the retention time of each chromatographic peak. Considering both peak shape and separation effect, the optimal flow rate was determined to be 0.3 mL / min. -1 .

[0031] Example 3: Fingerprint spectroscopy detection of Fuzheng Tonglin Pills: (1) Preparation of the test solution: Same as step (2) in Example 1.

[0032] (2) Sample injection and detection: Column: Thermo Acclaim TM RSLC 120 C 18 (2.1 × 100 mm, 2.2 µm); Mobile phase: acetonitrile (A) - 0.1% glacial acetic acid aqueous solution (B), elution gradient: 0–0.3 min: 5% A; 0.3–19 min: 5% A → 60% A; 19–35 min: 60% A → 85% A; Flow rate: 0.3 mL·min -1 The injection volume was 3 µL; the detection wavelength was 260 nm.

[0033] Using the same test solution and keeping other chromatographic conditions constant, the sample was injected and analyzed at three column temperatures: 30 ℃, 35 ℃, and 40 ℃. The effect of different column temperatures on the chromatogram was investigated. (See attached table.) Figure 3 .

[0034] The results showed that the column temperature mainly affected the number of peaks, retention time, and peak shape. Based on the number of peaks and the separation effect, the optimal column temperature was selected as 30 ℃.

[0035] Example 4: Fingerprint spectroscopy detection of Fuzheng Tonglin Pills: (1) Preparation of the test solution: Same as step (2) in Example 1.

[0036] (2) Sample injection and detection: Column: Thermo Acclaim TM RSLC 120 C 18 (2.1 × 100 mm, 2.2 µm); elution gradient: 0–0.3 min: 5% A; 0.3–19 min: 5% A → 60% A; 19–35 min: 60% A → 85% A; flow rate: 0.3 mL / min -1 The injection volume was 3 µL; the column temperature was 30 ℃; and the detection wavelength was 260 nm.

[0037] The effects of different mobile phase systems on the chromatograms were investigated by using mobile phases 1 (acetonitrile-0.1% glacial acetic acid aqueous solution), 2 (acetonitrile-0.1% formic acid aqueous solution), 3 (acetonitrile-0.1% phosphoric acid aqueous solution), and 4 (acetonitrile-water), respectively. (See figure). Figure 4 .

[0038] The results showed that the mobile phase system had a certain impact on the number of peaks, peak shape, retention time and resolution. Among them, the mobile phase 1 (acetonitrile-0.1% glacial acetic acid aqueous solution) had a stable baseline and ideal peak number, peak shape and separation effect. Finally, the mobile phase was determined to be acetonitrile-0.1% glacial acetic acid aqueous solution.

[0039] Example 5: Fingerprint analysis of Fuzheng Tonglin Pills: (1) Preparation of the test solution: Same as step (2) in Example 1.

[0040] (2) Sample injection and detection: Mobile phase: acetonitrile (A) - 0.1% glacial acetic acid aqueous solution (B); elution gradient: 0–0.3 min: 5% A; 0.3–19 min: 5% A → 60% A; 19–35 min: 60% A → 85% A; flow rate: 0.3 mL / min. -1 The injection volume was 3 µL; the column temperature was 30℃; and the detection wavelength was 260 nm.

[0041] The effects of different chromatographic columns on the chromatograms were investigated by analyzing samples injected using the following three different chromatographic columns. Figure 5 .

[0042] Column 1: Thermo Acclaim TM RSLC 120 C 18 (2.1×100 mm, 2.2 µm), Column 2: Thermo Scientific Syncronis C 18 (2.1×100 mm, 1.7 µm), Column 3: Waters ACQUITY UPLC® BEH C 18 (2.1×150 mm, 1.7 µm), The results showed that the brand and model of the chromatographic column had a certain impact on retention time, number of peaks, peak area, and peak shape. The Thermo Acclaim column was selected for its best separation performance. TM RSLC 120 C 18 Column (2.1×100 mm, 2.2 µm).

[0043] Example 6: Fingerprint spectroscopy detection of Fuzheng Tonglin Pills: (1) Preparation of the test solution: Same as step (2) in Example 1.

[0044] (2) Sample injection and detection: Column: Thermo Acclaim TM RSLC 120 C 18 (2.1 × 100 mm, 2.2 µm); Mobile phase: acetonitrile (A) - 0.1% glacial acetic acid aqueous solution (B), elution gradient: 0–0.3 min: 5% A; 0.3–19 min: 5% A → 60% A; 19–35 min: 60% A → 85% A; Flow rate: 0.3 mL·min -1 The column temperature was 30 ℃; the detection wavelength was 260 nm.

[0045] Injecting 2 µL, 3 µL, and 5 µL respectively to investigate the effect of different injection volumes on the chromatogram, see [reference needed]. Figure 6 .

[0046] The results showed that the injection volume only affected the peak area of ​​the chromatographic peak. Taking all factors into consideration, 3 µL, with a moderate peak area, was selected as the injection volume.

[0047] In summary, the chromatographic conditions were determined as follows: the chromatographic column was a Thermo Acclaim. TM RSLC 120 C 18(2.1 × 100 mm, 2.2 µm, Thermo Fisher Scientific); mobile phase: acetonitrile (A) - 0.1% glacial acetic acid aqueous solution (B) gradient elution; detection wavelength: 260 nm; flow rate: 0.3 mL·min -1 The column temperature was 30 °C and the injection volume was 3 µL.

[0048] Example 7: Fingerprint spectroscopy detection of Fuzheng Tonglin Pills: (1) Preparation of the test solution Weigh four portions of Fuzheng Tonglin Pill powder, each approximately 0.4 g, accurately, and place them in stoppered conical flasks. Accurately add 25 mL each of 70% methanol, 80% methanol, 90% methanol, and methanol, respectively. Seal tightly, weigh, and extract ultrasonically for 30 min (power 250 W, frequency 40 kHz). Cool, weigh, and replenish the lost weight with the corresponding extraction solvent. Shake well, filter, and take the filtrate. Filter through a 0.22 μm microporous membrane to obtain the final product.

[0049] (2) Sample injection and detection: The chromatographic column was Thermo Acclaim. TM RSLC 120 C 18 (2.1 × 100 mm, 2.2 µm, Thermo Fisher Scientific); mobile phase: acetonitrile (A) - 0.1% glacial acetic acid aqueous solution (B) gradient elution; detection wavelength: 260 nm; flow rate: 0.3 mL·min -1 The column temperature was 30℃; the injection volume was 3 µL.

[0050] The effects of different extraction solvents on the chromatograms were investigated by analyzing samples under the same chromatographic conditions. (See attached image.) Figure 7 .

[0051] The results showed that using 80% methanol as the extraction solvent was optimal.

[0052] Example 8: Fingerprint spectroscopy detection of Fuzheng Tonglin Pills: (1) Preparation of the test solution: Weigh two portions of Fuzheng Tonglin Pill powder, each approximately 0.4 g, accurately, and place them in a stoppered conical flask. Accurately add 25 mL of 80% methanol, seal tightly, and weigh. Extract one portion by sonication for 30 min (power 250 W, frequency 40 kHz), and extract the other portion by reflux for 30 min. Cool, weigh, and replenish the lost weight with 80% methanol. Shake well, filter, and filter the filtrate through a 0.22 μm microporous membrane to obtain the final product.

[0053] (2) Sample injection and detection: Chromatographic conditions were the same as in Example 7. Injection and analysis were performed under the same chromatographic conditions to investigate the effect of different extraction methods on the chromatogram. (See attached image) Figure 8 .

[0054] The results showed no significant difference between ultrasonic extraction and reflux extraction. For ease of operation, ultrasonic extraction was chosen.

[0055] Example 9: Fingerprint spectroscopy detection of Fuzheng Tonglin Pills: (1) Preparation of the test solution: Weigh three portions of Fuzheng Tonglin Pill powder, each approximately 0.4 g, accurately, and place them in a stoppered conical flask. Accurately add 25 mL of 80% methanol, seal tightly, and weigh. Extract by ultrasonication for 20 min, 30 min, and 40 min respectively (power 250 W, frequency 40 kHz). Cool, weigh, and replenish the lost weight with 80% methanol. Shake well, filter, and take the filtrate. Filter through a 0.22 μm microporous membrane to obtain the final product.

[0056] (2) Sample injection and detection: Chromatographic conditions were the same as in Example 7. Injection and analysis were performed under the same chromatographic conditions to investigate the effect of different extraction times on the chromatogram. (See attached image.) Figure 9 .

[0057] The results showed that the extraction time had no significant impact on the chromatogram information. To ensure sufficient extraction, the final extraction time was selected as 30 min.

[0058] Example 10: Fingerprint spectroscopy detection of Fuzheng Tonglin Pills: (1) Preparation of the test solution: Take 0.3 g, 0.4 g, and 0.5 g of Fuzheng Tonglin Pill powder respectively, weigh accurately, place them in a stoppered conical flask, add 25 mL of 80% methanol accurately, stopper tightly, weigh, and extract ultrasonically for 30 min (power 250 W, frequency 40 kHz). Cool, weigh, replenish the lost weight with 80% methanol, shake well, filter, and take the filtrate. Filter through a 0.22 μm microporous membrane to obtain the final product.

[0059] (2) Sample injection and detection: The chromatographic conditions were the same as in Example 7. Sample injection and analysis were performed under the same chromatographic conditions to investigate the effect of different sample volumes on the chromatogram. (See attached image.) Figure 10 .

[0060] The results showed that the separation effect of each chromatographic peak was the best when the sample amount was 0.4 g, and the peak area was moderate and relatively balanced. Therefore, the sample amount was determined to be 0.4 g.

[0061] In summary, the preparation method of the test solution is determined as follows: Take 0.4 g of Fuzheng Tonglin Pill powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 80% methanol, seal tightly, weigh, and ultrasonically extract for 30 min (power 250 W, frequency 40 kHz). Cool, weigh, replenish the lost weight with 80% methanol, shake well, filter, and take the filtrate. Filter through a 0.22 μm microporous membrane to obtain the test solution.

[0062] Example 11: Method for constructing the fingerprint spectrum of Fuzheng Tonglin Pill: S1. Solution preparation: (1) Preparation of the reference solution, Take appropriate amounts of verbascoside, glycyrrhizin, bis(demethoxy)curcumin, demethoxy)curcumin, and curcumin reference standards, accurately weigh them, dissolve them in 80% methanol, and prepare a single reference standard solution.

[0063] Each of the above single reference solutions was precisely transferred to a volumetric flask and diluted to the mark with 80% methanol to obtain a mixed reference solution containing 4.74 μg of verbascoside, 12.75 μg of glycyrrhizin, 21.3 μg of bis(demethoxy)curcumin, 25.26 μg of demethoxy)curcumin, and 31.85 μg of curcumin per 1 mL.

[0064] (2) Preparation of the test solution, Take 0.4 g of Fuzheng Tonglin Pill powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 80% methanol, seal tightly, weigh, and extract ultrasonically for 30 min (power 250 W, frequency 40 kHz). Cool, weigh, replenish the lost weight with 80% methanol, shake well, filter, and take the filtrate. Filter through a 0.22 μm microporous membrane to obtain the final product.

[0065] (3) Preparation of negative test solution, Negative solutions were prepared for each of the following deficiencies: American cockroach, Astragalus membranaceus, Eupolyphaga sinensis, Aconitum carmichaelii, Curcuma longa, and Glycyrrhiza uralensis. The powdered medicinal slices lacking any one of the components in the prescription were accurately weighed, added to 80% methanol, sonicated, cooled, shaken, filtered, and the filtrate was filtered through a membrane to obtain the final product.

[0066] S2. Using ultra-high performance liquid chromatography (UHPLC), the test solution from step S1 (1), the reference solution from step (2), and the negative test solution from step (3) were injected into the UHPLC instrument for determination, and the chromatograms were recorded: The chromatographic column was Thermo Acclaim. TM RSLC 120 C 18(2.1 × 100 mm, 2.2 µm); mobile phase: acetonitrile (A) - 0.1% glacial acetic acid aqueous solution (B); elution gradient: 0–0.3 min: 5% A; 0.3–19 min: 5% A → 60% A; 19–35 min: 60% A → 85% A; detection wavelength: 260 nm; flow rate: 0.3 mL·min -1 The column temperature was 30 °C and the injection volume was 3 µL.

[0067] Take the reference solution and each batch of test solution separately, inject them into the chromatographic sample under the above-described chromatographic conditions, and record the chromatograms.

[0068] (I) Methodological Examination: 1.1 Precision Examination Six consecutive injections were performed according to the determined extraction method and chromatographic conditions, and the chromatograms were recorded. Peak 8 (bisdemethoxycurcumin), with a suitable response value and retention time, was used as the reference peak S. The relative retention times and relative peak areas of each common peak were calculated. The calculated RSD values ​​of the relative retention times of the 17 common peaks all did not exceed 0.048%, and the RSD values ​​of the relative peak areas of each common peak all did not exceed 2.988%. The results are shown in [Figure number missing]. Figure 11 Tables 5 and 6.

[0069] Table 5. Precision test results (relative retention time) .

[0070] Table 6. Precision test results (relative peak area) .

[0071] 1.2 Stability Test The samples were injected and analyzed at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after preparation, and the chromatograms were recorded. Using peak 8 (bisdemethoxycurcumin) as the reference peak S, the relative retention time and relative peak area of ​​each common peak were calculated. The calculated RSD values ​​of the relative retention times of the 17 common peaks all did not exceed 0.120%, and the RSD values ​​of the relative peak areas of each common peak all did not exceed 3.919%. The results are shown in [Figure number missing]. Figure 12 Tables 7 and 8.

[0072] Table 7. Stability Study Results (Relative Retention Time) .

[0073] Table 8. Stability test results (relative peak area) .

[0074] 1.3 Repeatability Test Six parallel aliquots of the test solution were prepared from the same batch, injected and analyzed separately, and the chromatograms were recorded. Using peak 8 (bisdemethoxycurcumin) as the reference peak S, the relative retention time and relative peak area of ​​each common peak were calculated. The calculated RSD values ​​of the relative retention times of the 17 common peaks all did not exceed 0.240%, and the RSD values ​​of the relative peak areas of each common peak all did not exceed 4.789%. The results are shown in [Figure number missing]. Figure 13 Tables 9 and 10.

[0075] Table 9. Results of repeatability studies (relative retention time) .

[0076] Table 10. Results of repeatability studies (relative peak area) .

[0077] 1.4 Delay Test With the endpoint gradient of the chromatographic conditions remaining constant, the acquisition time was extended to 60 min, and chromatographic information was analyzed. Comparison with the chromatogram of the 80% methanol blank solvent showed that the original chromatographic conditions could cover a relatively complete range of chromatographic information. Results are as follows: Figure 14 .

[0078] 1.5 Blank Test Take 3 μL each of the test solution, negative control solution of each medicinal slice, and 80% methanol, inject them into the sample, and record the chromatogram information. The results are shown in [the table below]. Figure 15 .

[0079] S3. Standardization of chromatographic peaks and establishment of fingerprint chromatograms: Twelve batches of Fuzheng Tonglin Pills were used to prepare test solutions, which were then injected and analyzed. The obtained chromatograms were imported into the "Traditional Chinese Medicine Fingerprint Similarity Evaluation System (2012 Edition)" software in CDF format. Using S1 (FZTLW-01) as the reference chromatogram, the mean method was used, with a time window width of 0.1 min, for multi-point correction. After Mark peak matching, fingerprint chromatograms and reference chromatograms R for each batch of samples were generated. See [link to relevant documentation]. Figure 16 and Figure 17 Using the generated control chromatogram R as a reference, the similarity between the fingerprint chromatogram of each sample and R was calculated, see [link to relevant documentation]. Figure 19 The similarity of all 12 batches of Fuzheng Tonglin Pills samples was above 0.900, indicating good consistency among the batches. The chromatogram of the mixed reference standard is shown below. Figure 18 .

[0080] After comparison with the reference standard, five chromatographic peaks were identified: peak 1 (verrucin glucoside), peak 3 (glycyrrhizin), peak 8 (bisdemethoxycurcumin), peak 9 (demethoxycurcumin), and peak 10 (curcumin). Based on the comparison of the chromatograms of the test solution and the negative control solution, the peaks were assigned as follows: peaks 1 and 4 were assigned to Astragalus membranaceus; peaks 2-3, 6-7, and 12 were assigned to Glycyrrhiza uralensis; and peaks 5, 8-11, and 13-17 were assigned to Curcuma longa.

[0081] Example 12: Determination of the content of multiple components in Fuzheng Tonglin Pills: S1. Solution preparation: (1) Preparation of reference solution Take appropriate amounts of verbascoside, glycyrrhizin, bis(demethoxy)curcumin, demethoxy)curcumin, and curcumin reference standards, accurately weigh them, dissolve them in 80% methanol, and prepare a single reference standard solution.

[0082] Each of the above single reference solutions was precisely transferred to a volumetric flask and diluted to the mark with 80% methanol to obtain a mixed reference solution containing 4.74 μg of verbascoside, 12.75 μg of glycyrrhizin, 21.3 μg of bis(demethoxy)curcumin, 25.26 μg of demethoxy)curcumin, and 31.85 μg of curcumin per 1 mL.

[0083] (2) Preparation of the test solution Take 0.4 g of Fuzheng Tonglin Pill powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of 80% methanol, seal tightly, weigh, and extract ultrasonically for 30 min (power 250 W, frequency 40 kHz). Cool, weigh, replenish the lost weight with 80% methanol, shake well, filter, and take the filtrate. Filter through a 0.22 μm microporous membrane to obtain the final product.

[0084] S2. Sample injection and determination: The chromatographic column was Thermo Acclaim. TM RSLC 120 C 18 (2.1 × 100 mm, 2.2 µm); mobile phase: acetonitrile (A) - 0.1% glacial acetic acid aqueous solution (B); elution gradient: 0–0.3 min: 5% A; 0.3–19 min: 5% A → 60% A; 19–35 min: 60% A → 85% A; detection wavelength: 260 nm; flow rate: 0.3 mL·min -1 The column temperature was 30℃; the injection volume was 3 µL.

[0085] S3. Take the reference solution and each batch of test solution separately, inject them into the chromatographic sample under the above conditions, record the chromatogram, and calculate the contents of verbascoside, glycyrrhizin, bis(demethoxy)curcumin, demethoxy)curcumin and curcumin in the sample.

[0086] (I) Methodological Examination 1.1 Examination of Linear Relationships Take appropriate amounts of verbascoside, glycyrrhizin, bis(demethoxy)curcumin, demethoxy)curcumin, and curcumin reference standards, and dissolve them in 80% methanol to prepare solutions with concentrations of 5.688 μg·mL, respectively. -1 8.5 μg·mL -1 51.12 μg·mL -1 67.36 μg·mL -1 and 76.44 μg·mL -1 The mixed reference solution was serially diluted and then injected for analysis. Standard curves for each component were plotted with the reference concentration as the x-axis (X) and the target peak area as the y-axis (Y). Linear regression was performed, and the results are shown in Table 11. The five components showed good linearity within their respective concentration ranges.

[0087] Table 11 Results of Linearity Examination .

[0088] 1.2 Precision Test The same mixed reference solution was injected six times consecutively, and the RSD values ​​of the peak areas of each chromatographic peak were calculated. The results are shown in Table 12. The RSD values ​​of the peak areas of verrucoside, glycyrrhizin, bis(demethoxy)curcumin, demethoxy)curcumin, and curcumin were 0.489%, 0.465%, 0.243%, 1.100%, and 1.770%, respectively, indicating that the instrument precision was good.

[0089] Table 12 Precision Test Results .

[0090] 1.3 Repeatability Test Six parallel test solutions were prepared from the same batch of samples, and each solution was injected and analyzed. The RSD values ​​of the peak areas of each chromatographic peak were calculated. The results are shown in Table 13. The RSD values ​​of the peak areas of verrucoside glucoside, glycyrrhizin, bis(demethoxy)curcumin, demethoxy)curcumin, and curcumin were 2.770%, 1.415%, 1.691%, 1.971%, and 2.069%, respectively, indicating that the method has good repeatability.

[0091] Table 13 Repeatability Test Results .

[0092] 1.4 Stability Test A test solution was prepared using the same sample, and the sample was injected and analyzed at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after preparation. The RSD values ​​of the peak areas of each chromatographic peak were calculated. The results are shown in Table 14. The RSD values ​​of the peak areas of verrucoside glucoside, glycyrrhizin, bis(demethoxy)curcumin, demethoxy)curcumin, and curcumin were 2.513%, 0.548%, 0.750%, 1.846%, and 2.645%, respectively, indicating that the test solution had good stability within 12 h.

[0093] Table 14 Stability Test Results .

[0094] 1.5 Recovery Test Six test samples with known index component contents were weighed, each approximately 0.2 g, and accurately weighed. Reference solutions of vernix isoflavone glucoside, glycyrrhizin, bis(demethoxy)curcumin, demethoxy)curcumin, and curcumin were added to each sample in a ratio of approximately 1:1 according to the corresponding component contents in the sample. After preparing the solutions, the samples were injected for analysis. The peak areas of each component were recorded and the recovery rates were calculated. The results are shown in Table 15.

[0095] Table 15 Recovery Test .

[0096] S. Content determination of 12 batches of samples Take 0.4 g of powder from 12 batches of Fuzheng Tonglin Pills, accurately weigh it, prepare the test solution, inject it for determination, and calculate the contents of isoflavone glucoside, glycyrrhizin, bisdemethoxycurcumin, demethoxycurcumin and curcumin in the 12 batches of samples. The results are shown in Table 16.

[0097] Table 16 Results of content determination in 12 batches of Fuzheng Tonglin Pills .

[0098] Finally, the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for constructing the fingerprint spectrum of Fuzheng Tonglin Pill, characterized by: The steps include the following: S1. Prepare samples, test solutions, control solutions and negative test solutions of Fuzheng Tonglin Pills; (1) Preparation of Fuzheng Tonglin Pills: Take 3.0-3.8 parts of American cockroach, 4.0-4.7 parts of Astragalus membranaceus, 2-3 parts of Eupolyphaga sinensis, 2-3 parts of Aconitum carmichaelii, 1.4-1.9 parts of Curcuma longa, and 0.37-0.95 parts of Glycyrrhiza uralensis by weight, pulverize and make into water pills; (2) Preparation of test solution: Take the Fuzheng Tonglin Pill sample, add solvent to extract, filter, and take the filtrate to obtain the test solution; (3) Preparation of reference solution: Weigh accurately the reference standards of verbascoside, glycyrrhizin, bis(demethoxy)curcumin, demethoxy)curcumin and curcumin, dissolve them in solvent and dilute to volume to obtain the solution. (4) Preparation of negative test solution: Take a powder sample lacking any one component of Fuzheng Tonglin Pill, extract it with solvent, filter it, and take the filtrate to prepare negative solutions for those lacking American cockroach, astragalus, ground beetle, aconite, turmeric, or licorice. S2. Using ultra-high performance liquid chromatography (UHPLC), the test solution from step S1 (2), the reference solution from step (3), and the negative test solution from step (4) were injected into the UHPLC instrument for determination, and the chromatograms were recorded: In the ultra-high performance liquid chromatography (UHPLC) chromatographic conditions, mobile phase A is acetonitrile, mobile phase B is 0.1% glacial acetic acid aqueous solution, and gradient elution is performed. The elution gradient is: 0~0.3 min: 5% A; 0.3–19 min: 5% A → 60% A; 19–35 min: 60% A → 85% A; flow rate: 0.25–0.35 mL / min -1 The column temperature is 30~40℃, the detection wavelength is 230~400 nm, and the injection volume is 2~5 µL. S3. Identification of common peaks: Multiple batches of test sample solutions were injected sequentially to obtain multiple batches of test sample chromatograms. These chromatograms were then imported into the Chinese medicine chromatographic fingerprint similarity evaluation system to identify 17 common peaks and generate the fingerprint chromatogram of Fuzheng Tonglin Pill. The common peaks were compared with the chromatograms of the negative test sample solution and the reference solution to assign each common peak to its corresponding chromatogram.

2. The method for constructing the fingerprint spectrum of Fuzheng Tonglin Pill according to claim 1, characterized in that, The solvent used in steps S1 (2) to (4) is 70% to 90% methanol solution or methanol.

3. The method for constructing the fingerprint spectrum of Fuzheng Tonglin Pill according to claim 1, characterized in that, The extraction method described in step S1 (2) is either heating reflux or ultrasonic extraction.

4. The method for constructing the fingerprint spectrum of Fuzheng Tonglin Pill according to claim 3, characterized in that, The extraction method described in step S1 (2) is ultrasonic extraction.

5. The method for constructing the fingerprint spectrum of Fuzheng Tonglin Pill according to claim 1, characterized in that, The ultra-high performance liquid chromatography column used in step S2 is C16. 18 Chromatographic column.

6. The method for constructing the fingerprint spectrum of Fuzheng Tonglin Pill according to claim 1, characterized in that, The flow rate for the ultra-high performance liquid chromatography method is 0.3 mL·min. -1 The column temperature was 40℃, the detection wavelength was 260 nm, and the injection volume was 3 µL.

7. The fingerprint spectrum of Fuzheng Tonglin Pill constructed by the method described in claims 1-6, characterized in that, It includes 17 common peaks, as follows: The relative retention time of the characteristic peak No. 1 is 0.

439. The relative retention time of the characteristic peak No. 2 is 0.

448. The relative retention time of the characteristic peak No. 3 is 0.

456. The relative retention time of the characteristic peak No. 4 is 0.

654. The relative retention time of the characteristic peak No. 5 is 0.

781. The relative retention time of the characteristic peak at peak 6 is 0.

805. The relative retention time of the characteristic peak No. 7 is 0.

851. The relative retention time of the characteristic peak at peak 8 is 1.

000. The relative retention time of the characteristic peak at peak 9 is 1.

021. The relative retention time of the characteristic peak at peak number 10 is 1.

041. The relative retention time of the characteristic peak at peak 11 is 1.

156. The relative retention time of the characteristic peak at peak 12 is 1.

184. The relative retention time of the characteristic peak at peak 13 is 1.

285. The relative retention time of the characteristic peak at peak 14 is 1.

361. The relative retention time of the characteristic peak at peak number 15 is 1.

511. The relative retention time of the characteristic peak at peak 16 is 1.

523. The relative retention time of the characteristic peak on the 17th is 1.

619.

8. The fingerprint spectrum of Fuzheng Tonglin Pill according to claim 7, characterized in that the fingerprint... The common characteristic peak 1 in the spectrum is verrucoside isoflavone glucoside; The characteristic peak of peak 3 is glycyrrhizin; The characteristic peak of peak 8 is didemethoxycurcumin; The characteristic peak of peak 9 is demethoxycurcumin; The characteristic peak of sample number 10 is curcumin.

9. The application of the fingerprint spectrum of Fuzheng Tonglin Pill according to claim 7 in content determination, characterized in that the method... The procedure is as follows: Prepare the test solution, reference solution, and negative test solution according to step S1 of the fingerprint chromatogram construction method described in claim 1. Using the same chromatographic conditions as step S2 of the fingerprint chromatogram construction method described in claim 1, inject the test solution, reference solution, and negative test solution into an ultra-high performance liquid chromatograph, record the chromatograms, and calculate the content of five components in the test solution—vermiculin, glycyrrhizin, bis(demethoxy)curcumin, methoxycurcumin, and curcumin—using the external standard method.

10. The application of the fingerprint spectrum of Fuzheng Tonglin Pill as described in claim 7 in quality testing, characterized in that the method is as follows: the quality of Fuzheng Tonglin Pill is evaluated using the reference fingerprint spectrum, and the similarity between the fingerprint spectrum of each batch of test sample and the reference fingerprint spectrum is not less than 0.9, calculated using a similarity evaluation system for chromatographic fingerprint spectrum of traditional Chinese medicine.

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