A method for establishing HPLC fingerprint and determining content of Zhida Hu

Abstract

The application discloses a HPLC fingerprint spectrum establishing and content determination method of Zha Dihu, and belongs to the technical field of drug chemical component analysis. The application establishes a HPLC fingerprint spectrum, confirms that six common peaks in test sample solutions of 14 batches are p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shatavari glycoside, paeoniflorin and p-hydroxycinnamic acid, adopts SPSS 27 and SIMCA14.1 analysis software, carries out cluster analysis, principal component analysis and orthogonal partial least square discriminant analysis on peak areas of the six identified common peaks in the fingerprint spectrum of the Zha Dihu medicinal materials of the 14 batches, and carries out main component content determination. The chemical component composition of the Zha Dihu is determined, the characteristic components are revealed, and the main components are quantitatively analyzed, so as to provide a scientific basis for quality evaluation, efficacy research and subsequent development and utilization of the Zha Dihu.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemical component analysis technology, specifically, it relates to a method for establishing an HPLC fingerprint spectrum and determining the content of *Gnaphalium affine*. Background Technology

[0002] Sorghum bicolor (L.) Moench., a plant belonging to the genus Sorghum of the Poaceae family, is used in traditional Chinese medicine by ethnic minorities in Guizhou Province. It is listed in the 2019 edition of the "Standards for Traditional Chinese and Ethnic Medicines of Guizhou Province" and is also recorded in classic Chinese medicine texts such as the "Compendium of Materia Medica," "Dictionary of Traditional Chinese Medicine," and "Chinese Materia Medica." It is sweet, astringent, and cold in nature, and possesses the effects of clearing heat and detoxifying, cooling the blood and stopping bleeding, and promoting diuresis and relieving cough.

[0003] As a traditional medicinal resource, *Gnaphalium affine* is currently subject to limited quality standards that only cover routine inspections and extractive analysis. It lacks specific qualitative and quantitative control indicators for its chemical components, and systematic research on its chemical composition is scarce, severely hindering its in-depth development and quality evaluation. Given the synergistic effects of multiple components and targets in traditional Chinese medicine, establishing a systematic and comprehensive chemical composition analysis method is crucial for the quality control of *Gnaphalium affine*.

[0004] This invention established an HPLC fingerprint chromatogram, combined with chemometric analysis of 14 batches of samples from different origins, screened key quality markers, and determined the content of major components. Through the above research, the aim is to clarify the chemical composition of *Gnaphalium affine*, reveal its characteristic components, and quantitatively analyze its major components, in order to provide a scientific basis for the quality evaluation, efficacy research, and subsequent development and utilization of *Gnaphalium affine*. Summary of the Invention

[0005] This invention aims to provide a method for establishing an HPLC fingerprint and determining the content of *Gnaphalium affine*. By identifying and analyzing the components in the medicinal material, the chemical composition of *Gnaphalium affine* can be clarified, providing support for further elucidating the pharmacodynamic material basis of *Gnaphalium affine*.

[0006] This invention is achieved using the following technical solution: a method for establishing an HPLC fingerprint of *Gnaphalium affine* and determining its content, comprising the following steps: (1) Preparation of test solution: Pulverize the dried tiger herb, weigh 0.5-1.5g of tiger powder into a round bottom flask, add 10-20mL of 50% methanol, weigh, reflux extract in a water bath at 70-90°C for 1-2h, cool to room temperature, add weight, mix well, centrifuge at 12000-16000r / min at 3-5°C for 5-15min, take the supernatant, and the test solution is obtained. (2) Preparation of mixed reference solution: Accurately weigh hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, schafotaside, paeoniflorin and p-hydroxycinnamic acid reference standards, add 50% methanol to prepare a mixed reference solution containing 38.000-42.000µg / mL p-hydroxybenzoic acid, 38.000-42.000µg / mL vanillic acid, 38.000-42.000µg / mL p-hydroxybenzaldehyde, 38.000-42.000µg / mL schafotaside, 38.000-42.000µg / mL paeoniflorin and 38.000-42.000µg / mL p-hydroxycinnamic acid; (3) Preparation of reference solutions: Accurately weigh p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, schafodine, paeoniflorin and p-hydroxycinnamic acid respectively, dissolve them in methanol to prepare 0.900-1.100 mg / mL p-hydroxybenzoic acid reference stock solution, 0.900-1.100 mg / mL vanillic acid reference stock solution, 0.900-1.100 mg / mL p-hydroxybenzaldehyde reference stock solution, 0.900-1.100 mg / mL schafodine reference stock solution, 0.900-1.100 mg / mL paeoniflorin reference stock solution and 0.900-1.100 mg / mL p-hydroxycinnamic acid reference stock solution; accurately pipette the above 6 reference stock solutions, dilute them to prepare 5 series of standard concentration reference solutions, refrigerate and use them for later use; (4) Chromatographic conditions: Hypersil GOLD™ column, 4.1×250mm, 5μm; column temperature 30℃; injection volume 10µL; detection wavelength 270nm; mobile phase A was 0.2% formic acid in water, mobile phase B was 0.2% formic acid in acetonitrile, and the elution gradient program was as follows: ; (5) Establishment of fingerprint spectrum: Take the medicinal material of *Gnaphalium affine* and prepare the test solution according to the method of step (1). Inject the test solution and the mixed reference solution prepared in step (2) into the chromatographic conditions in step (4) for detection. Based on the chromatographic peaks of the mixed reference solution, identify 6 common peaks in the test solution and construct the fingerprint spectrum of *Gnaphalium affine*. The 6 common peaks are p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin and p-hydroxycinnamic acid. (6) Fingerprint cluster analysis: SPSS 27 and SIMCA14.1 analysis software were used to perform cluster analysis, principal component analysis and orthogonal partial least squares discriminant analysis on the peak area of ​​the six identified common peaks in the fingerprint spectrum of the medicinal material *Gnaphalium affine*. (7) Preparation of standard curve: Take the standard solutions of the 6 reference solutions in step (3) with 5 series of standard concentrations respectively, and inject them for detection according to the chromatographic conditions in step (4). Plot the standard curve with concentration x as the abscissa and peak area y as the ordinate. (8) Content determination: Take the test sample solution and inject it according to the chromatographic conditions in step (4). Calculate the content of the 6 components contained in the sample based on the standard curve.

[0007] In step (1) above, the preparation of the test solution is as follows: the dried *Gnaphalium affine* herb is pulverized, passed through a 60-80 mesh sieve, 0.8-1.1 g of *Gnaphalium affine* powder is weighed into a round-bottom flask, 12-18 mL of 50% methanol is added, the mixture is weighed, and the mixture is refluxed in a water bath at 75-85°C for 1.2-1.8 h. After cooling to room temperature, the weight is added, the mixture is mixed, and the mixture is centrifuged at 12000-16000 r / min at 3-5°C for 8-12 min. The supernatant is then collected to obtain the test solution.

[0008] Specifically, in the aforementioned step (1), the preparation of the test solution is as follows: the dried *Gnaphalium affine* medicinal material is pulverized, passed through a 60-mesh sieve, 1g of *Gnaphalium affine* powder is weighed into a 50mL round-bottom flask, 15mL of 50% methanol is added, the weight is weighed, and the mixture is refluxed in an 80°C water bath for 1.5h. After cooling to room temperature, the weight is added, the mixture is mixed, and the mixture is centrifuged at 14000r / min for 10min at 4°C. The supernatant is then collected to obtain the test solution.

[0009] In step (2) above, the preparation of the mixed reference solution is as follows: accurately weigh hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin and p-hydroxycinnamic acid reference standards, add 50% methanol to prepare a mixed reference solution containing 40.367µg / mL p-hydroxybenzoic acid, 40.320µg / mL vanillic acid, 40.052µg / mL p-hydroxybenzaldehyde, 40.200µg / mL shampooside, 40.480µg / mL paeoniflorin and 40.518µg / mL p-hydroxycinnamic acid.

[0010] In step (3) above, the preparation of the reference solutions is as follows: p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, schafodine, paeoniflorin and p-hydroxycinnamic acid are accurately weighed and dissolved in methanol to prepare 1.091 mg / mL p-hydroxybenzoic acid reference stock solution, 0.960 mg / mL vanillic acid reference stock solution, 1.054 mg / mL p-hydroxybenzaldehyde reference stock solution, 1.005 mg / mL schafodine reference stock solution, 1.012 mg / mL paeoniflorin reference stock solution and 1.121 mg / mL p-hydroxycinnamic acid reference stock solution; the above 6 reference stock solutions are accurately pipetted and diluted to prepare 5 series of standard concentration reference solutions, which are then refrigerated for later use.

[0011] Specifically, in step (3) above, the five series of standard concentrations of the six reference solutions are as follows: .

[0012] In step (5) above, the fingerprint spectrum is established as follows: 14 batches of *Gnaphalium affine* medicinal materials are taken and 14 batches of test solutions are prepared according to the method in step (1). The 14 batches of test solutions and the mixed reference solution prepared in step (2) are injected and detected according to the chromatographic conditions in step (4). Based on the chromatographic peaks of the mixed reference solution, 6 common peaks in the 14 batches of test solutions are identified, and a fingerprint spectrum of *Gnaphalium affine* is constructed. The 6 common peaks are p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin and p-hydroxycinnamic acid.

[0013] In step (6) above, fingerprint cluster analysis: SPSS 27 and SIMCA14.1 analysis software were used to perform cluster analysis, principal component analysis and orthogonal partial least squares discriminant analysis on the peak areas of the six identified common peaks in the fingerprint spectra of 14 batches of Tiger Herb.

[0014] In step (8) above, the six components are p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin and p-hydroxycinnamic acid.

[0015] Beneficial effects of this invention: 1. This invention established HPLC fingerprint chromatograms of 14 batches of *Gnaphalium affine*. The fingerprint chromatograms identified 6 common peaks, namely p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin, and p-hydroxycinnamic acid, with similarity ranging from 0.850 to 0.983. The results indicate that the reproducibility of different batches of samples is relatively good.

[0016] 2. This invention, by determining the content of six identified common peaks in various batches of *Gnaphalium affine*, found that the content of these six components varied among different origins and batches of *Gnaphalium affine* medicinal materials. The content of paeoniflorin showed the greatest variation, followed by p-hydroxycinnamic acid. Significant differences in content were observed between medicinal materials from different origins, and the data distribution was relatively dispersed, which may be related to factors such as growth environment and processing technology. This suggests that in the quality control of *Gnaphalium affine* medicinal materials, the impact of origin and processing technology on the content of the main active ingredients should be comprehensively considered.

[0017] 3. This invention establishes an HPLC fingerprint of *Gnaphalium affine*, which, combined with chemical pattern recognition and multi-component content determination methods, helps to improve the quality evaluation and control level of *Gnaphalium affine*, and provides a scientific basis for its pharmacodynamic material basis research and subsequent development and utilization. Attached Figure Description

[0018] Figure 1 : Overlay of 14 batches of characteristic images and comparison images of groundhogs; Figure 2 HPLC chromatograms of the test solution (A) and the mixed reference solution (B) (6 is p-hydroxybenzoic acid; 7 is vanillic acid; 8 is p-hydroxybenzaldehyde; 9 is shampooside; 10 is paeoniflorin; 12 is p-hydroxycinnamic acid). Figure 3 Dendrogram of cluster analysis of 14 batches of *Gnaphalium affine*; Figure 4 Principal component score chart of 14 batches of ground tigers; Figure 5 : 14 batches of Grappling Tiger OPLS-DA score chart; Figure 6 Permutation test chart of OPLS-DA model of 14 batches of ground tiger; Figure 7 : 14 batches of ground tigers have a total of peak VIPs; Figure 8 Graph showing the changes in the content of 6 components in 14 batches of *Gnaphalium affine*; Figure 9 Box plot of component content. Detailed Implementation

[0019] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention.

[0020] Example 1: Establishment of HPLC fingerprint of *Gnaphalium affine* (1) Preparation of test solution: The dried tiger thorn medicinal material was pulverized and passed through a 60-mesh sieve. 1g of tiger thorn powder was weighed into a 50mL round bottom flask, 15mL of 50% methanol was added, and after weighing, it was extracted by reflux in an 80°C water bath for 1.5h. After cooling to room temperature, the weight was added, the mixture was mixed, and centrifuged at 14000r / min at 4°C for 10min. The supernatant was taken to obtain the test solution. (2) Preparation of mixed reference solution: Accurately weigh hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampodoside, paeoniflorin and p-hydroxycinnamic acid reference standards, add 50% methanol to prepare a mixed reference solution containing 40.000µg / mL p-hydroxybenzoic acid, 40.000µg / mL vanillic acid, 40.000µg / mL p-hydroxybenzaldehyde, 40.000µg / mL shampodoside, 40.000µg / mL paeoniflorin and 40.000µg / mL p-hydroxycinnamic acid; (3) Chromatographic conditions: Hypersil GOLD™ column, 4.1×250mm, 5μm; column temperature 30℃; injection volume 10µL; detection wavelength 270nm; mobile phase A was 0.2% formic acid in water, mobile phase B was 0.2% formic acid in acetonitrile, and the elution gradient program was as follows: ; (4) Establishment of fingerprint spectrum: The test solution and the mixed reference solution were injected and detected according to the chromatographic conditions. Based on the chromatographic peaks of the mixed reference solution, the 6 common peaks in the test solution were identified and the fingerprint spectrum of the tiger was constructed. The 6 common peaks were p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin and p-hydroxycinnamic acid. (5) Fingerprint cluster analysis: SPSS 27 and SIMCA14.1 analysis software were used to perform cluster analysis, principal component analysis and orthogonal partial least squares discriminant analysis on the peak area of ​​the six identified common peaks in the fingerprint spectrum of the medicinal material *Gnaphalium affine*.

[0021] Example 2: Method for determining the content of *Gnaphalium affine* (1) Preparation of test solution: Pulverize the dried tiger herb, weigh 0.5-1.5g of tiger powder into a round bottom flask, add 10-20mL of 50% methanol, weigh, reflux extract in a water bath at 70-90°C for 1-2h, cool to room temperature, add weight, mix well, centrifuge at 12000-16000r / min at 3-5°C for 5-15min, take the supernatant, and the test solution is obtained. (2) Preparation of reference solutions: Accurately weigh p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin and p-hydroxycinnamic acid respectively, dissolve in methanol to prepare 1.000 mg / mL p-hydroxybenzoic acid reference stock solution, 1.000 mg / mL vanillic acid reference stock solution, 1.000 mg / mL p-hydroxybenzaldehyde reference stock solution, 1.000 mg / mL shampooside reference stock solution, 1.000 mg / mL paeoniflorin reference stock solution and 1.000 mg / mL p-hydroxycinnamic acid reference stock solution respectively; accurately pipette the above 6 reference stock solutions, dilute them to prepare 5 series of standard concentration reference solutions respectively, refrigerate and use for later use; The five series of standard concentrations for the six reference solutions are as follows: ; (3) Chromatographic conditions: Hypersil GOLD™ column, 4.1×250mm, 5μm; column temperature 30℃; injection volume 10µL; detection wavelength 270nm; mobile phase A was 0.2% formic acid in water, mobile phase B was 0.2% formic acid in acetonitrile, and the elution gradient program was as follows: ; (4) Preparation of standard curve: Take five series of standard concentration reference solutions, inject and detect them according to chromatographic conditions, and plot the standard curve with concentration x as the abscissa and peak area y as the ordinate. (5) Content determination: Take the test sample solution and inject it according to the chromatographic conditions. Calculate the content of the 6 components contained in the sample based on the standard curve.

[0022] Example 3: Establishment of HPLC fingerprint of *Gnaphalium affine* (1) Preparation of test solution: The dried tiger cactus medicinal material was pulverized, and 0.5 g of tiger cactus powder was weighed into a round bottom flask. 20 mL of 50% methanol was added, and after weighing, the mixture was extracted by reflux in a 90°C water bath for 2 h. After cooling to room temperature, the weight was added, the mixture was mixed, and centrifuged at 16000 r / min at 5°C for 15 min. The supernatant was taken to obtain the test solution. (2) Accurately weigh hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampodophyllin, paeoniflorin and p-hydroxycinnamic acid reference standards, add 50% methanol to prepare a mixed reference solution containing 38.000µg / mL p-hydroxybenzoic acid, 42.000µg / mL vanillic acid, 42.000µg / mL p-hydroxybenzaldehyde, 38.000µg / mL shampodophyllin, 42.000µg / mL paeoniflorin and 38.000µg / mL p-hydroxycinnamic acid; (3) Chromatographic conditions: Hypersil GOLD™ column, 4.1×250mm, 5μm; column temperature 30℃; injection volume 10µL; detection wavelength 270nm; mobile phase A was 0.2% formic acid in water, mobile phase B was 0.2% formic acid in acetonitrile, and the elution gradient program was as follows: ; (4) Establishment of fingerprint spectrum: The test solution and the mixed reference solution were injected and detected according to the chromatographic conditions. Based on the chromatographic peaks of the mixed reference solution, the 6 common peaks in the test solution were identified and the fingerprint spectrum of the tiger was constructed. The 6 common peaks were p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin and p-hydroxycinnamic acid. (5) Fingerprint cluster analysis: SPSS 27 and SIMCA14.1 analysis software were used to perform cluster analysis, principal component analysis and orthogonal partial least squares discriminant analysis on the peak area of ​​the six identified common peaks in the fingerprint spectrum of the medicinal material *Gnaphalium affine*.

[0023] Example 4: Method for determining the content of *Gnaphalium affine* (1) Preparation of test solution: The dried tiger cactus medicinal material was pulverized, and 0.5 g of tiger cactus powder was weighed into a round bottom flask. 20 mL of 50% methanol was added, and after weighing, the mixture was extracted by reflux in a 90°C water bath for 2 h. After cooling to room temperature, the weight was added, the mixture was mixed, and centrifuged at 16000 r / min at 5°C for 15 min. The supernatant was taken to obtain the test solution. (2) Preparation of reference solutions: Accurately weigh p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, schafodine, paeoniflorin and p-hydroxycinnamic acid respectively, dissolve them in methanol to prepare 0.900 mg / mL p-hydroxybenzoic acid reference stock solution, 1.100 mg / mL vanillic acid reference stock solution, 0.900 mg / mL p-hydroxybenzaldehyde reference stock solution, 0.900 mg / mL schafodine reference stock solution, 1.100 mg / mL paeoniflorin reference stock solution and 1.100 mg / mL p-hydroxycinnamic acid reference stock solution respectively; accurately pipette the above 6 reference stock solutions, dilute them to prepare 5 series of standard concentration reference solutions respectively, refrigerate and use them for later use; The five series of standard concentrations for the six reference solutions are as follows: ; (3) Chromatographic conditions: Hypersil GOLD™ column, 4.1×250mm, 5μm; column temperature 30℃; injection volume 10µL; detection wavelength 270nm; mobile phase A was 0.2% formic acid in water, mobile phase B was 0.2% formic acid in acetonitrile, and the elution gradient program was as follows: ; (4) Preparation of standard curve: Take five series of standard concentration reference solutions, inject and detect them according to chromatographic conditions, and plot the standard curve with concentration x as the abscissa and peak area y as the ordinate. (5) Content determination: Take the test sample solution and inject it according to the chromatographic conditions. Calculate the content of the 6 components contained in the sample based on the standard curve.

[0024] Example 5: Establishment of HPLC fingerprint of *Gnaphalium affine* (1) Pulverize the dried tiger herb, weigh 1.5g of tiger powder into a round bottom flask, add 10mL of 50% methanol, weigh, reflux extract for 1h in a 70°C water bath, cool to room temperature, add weight, mix well, centrifuge at 12000r / min for 5min at 3°C, take the supernatant, and obtain the test solution. (2) Accurately weigh hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampodoside, paeoniflorin and p-hydroxycinnamic acid reference standards, add 50% methanol to prepare a mixed reference solution containing 42.000µg / mL p-hydroxybenzoic acid, 38.000µg / mL vanillic acid, 38.000µg / mL p-hydroxybenzaldehyde, 42.000µg / mL shampodoside, 38.000µg / mL paeoniflorin and 42.000µg / mL p-hydroxycinnamic acid; (3) Chromatographic conditions: Hypersil GOLD™ column, 4.1×250mm, 5μm; column temperature 30℃; injection volume 10µL; detection wavelength 270nm; mobile phase A was 0.2% formic acid in water, mobile phase B was 0.2% formic acid in acetonitrile, and the elution gradient program was as follows: ; (4) Establishment of fingerprint spectrum: The test solution and the mixed reference solution were injected and detected according to the chromatographic conditions. Based on the chromatographic peaks of the mixed reference solution, the 6 common peaks in the test solution were identified and the fingerprint spectrum of the tiger was constructed. The 6 common peaks were p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin and p-hydroxycinnamic acid. (5) Fingerprint cluster analysis: SPSS 27 and SIMCA14.1 analysis software were used to perform cluster analysis, principal component analysis and orthogonal partial least squares discriminant analysis on the peak area of ​​the six identified common peaks in the fingerprint spectrum of the medicinal material *Gnaphalium affine*.

[0025] Example 6: Method for determining the content of *Gnaphalium affine* (1) Pulverize the dried tiger herb, weigh 1.5g of tiger powder into a round bottom flask, add 10mL of 50% methanol, weigh, reflux extract for 1h in a 70°C water bath, cool to room temperature, add weight, mix well, centrifuge at 12000r / min for 5min at 3°C, take the supernatant, and obtain the test solution. (2) Preparation of reference solutions: Accurately weigh p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin and p-hydroxycinnamic acid respectively, dissolve in methanol to prepare 1.100 mg / mL p-hydroxybenzoic acid reference stock solution, 0.900 mg / mL vanillic acid reference stock solution, 1.100 mg / mL p-hydroxybenzaldehyde reference stock solution, 1.100 mg / mL shampooside reference stock solution, 0.900 mg / mL paeoniflorin reference stock solution and 0.900 mg / mL p-hydroxycinnamic acid reference stock solution respectively; accurately pipette the above 6 reference stock solutions, dilute them to prepare 5 series of standard concentration reference solutions respectively, refrigerate and use for later use; The five series of standard concentrations for the six reference solutions are as follows: ; (3) Chromatographic conditions: Hypersil GOLD™ column, 4.1×250mm, 5μm; column temperature 30℃; injection volume 10µL; detection wavelength 270nm; mobile phase A 0.2% formic acid in water, mobile phase B 0.2% formic acid in acetonitrile, elution gradient program as follows: ; (4) Preparation of standard curve: Take five series of standard concentration reference solutions, inject and detect them according to chromatographic conditions, and plot the standard curve with concentration x as the abscissa and peak area y as the ordinate. (5) Content determination: Take the test sample solution and inject it according to the chromatographic conditions. Calculate the content of the 6 components contained in the sample based on the standard curve.

[0026] To obtain the solution and verify the technical effects of this invention, the inventors conducted extensive experimental research, some of which are recorded below: 1. Materials 1.1 Sample Collection The sources and information of the ground tiger are shown in Table 1.

[0027] Table 1 Sample Information Table

[0028] 1.2 Reagents p-Hydroxybenzaldehyde, p-hydroxybenzoic acid, vanillic acid, p-hydroxycinnamic acid, and paeoniflorin were purchased from Chengdu Efa Biotechnology Co., Ltd. (batch numbers: AFCC0754, AFCH2253, AFCA0952, AFCA3052, AFCE0452, all with a purity ≥98%); shampodoside was purchased from Sichuan Weikeqi Biotechnology Co., Ltd. (batch number: WP2111107, all with a purity ≥98%); mass spectrometry grade methanol and acetonitrile (Merck, Germany, batch numbers: I1353307 428, JB155230); Wahaha drinking water (Hangzhou Wahaha Group Co., Ltd.); chromatographic grade methanol and acetonitrile (Texas, USA, batch numbers: 25106275, 25086082).

[0029] 1.3 Instruments and Equipment High-performance liquid chromatograph Ultimate 3000 (Thermo Fisher Scientific, USA); EL204 0.0001 g electronic balance (Mettler Toledo, Shanghai); AE-240 0.0001 g balance (Mettler-Toledo Instruments Co., Ltd.); CQ250A-TS ultrasonic cleaner (Shanghai Yuejin Medical Optical Instrument Factory); Water purifier laboratory ultrapure water system (Wotel Water Treatment Equipment Co., Ltd.); Allegra 64R low-temperature high-speed centrifuge (Beckman-Coulter, USA); micropipette (Eppendorf AG, Germany).

[0030] 2 Methods 2.1 Establishment of HPLC fingerprint chromatogram 2.1.1 Preparation of the test solution Weigh 1g of *Gnaphalium affine* powder (No.: S4) into a 50 mL round-bottom flask, add 15 mL of 50% methanol (v / v), reflux extract at 80°C for 1.5 h, cool to room temperature, add weight, mix well, centrifuge at 14000 r / min at 4°C for 10 min, and take the supernatant for analysis.

[0031] 2.1.2 Preparation of mixed reference solution Accurately weigh appropriate amounts of each reference standard to prepare a mixed reference solution containing 40.367 µg / mL of p-hydroxybenzoic acid, 40.320 µg / mL of vanillic acid, 40.052 µg / mL of p-hydroxybenzaldehyde, 40.200 µg / mL of shampooside, 40.480 µg / mL of paeoniflorin, and 40.518 µg / mL of p-hydroxycinnamic acid.

[0032] 2.1.3 Chromatographic conditions Hypersil GOLD™ column (4.1×250mm, 5μm); column temperature: 30℃; injection volume: 10 µL; detection wavelength: 270nm; mobile phase: 0.2% formic acid water (A) - 0.2% formic acid acetonitrile (B), elution gradient program as follows.

[0033] Table 2 Elution gradient program

[0034] 2.2 Chemometric Analysis SPSS 27 and SIMCA 14.1 analysis software were used to perform cluster analysis, principal component analysis and orthogonal partial least squares discriminant analysis using the peak areas of the six identified common peaks in the fingerprint spectra of 14 batches of *Gnaphalium affine* medicinal materials as variables.

[0035] 2.3 Determination of the content of multiple components 2.3.1 Preparation of the test solution Prepare the test solution according to the same method as in section "2.1.1".

[0036] 2.3.2 Preparation of reference solution Accurately weigh appropriate amounts of p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin, and p-hydroxycinnamic acid reference standards, dissolve them in methanol to prepare a single reference stock solution containing 1.091 mg / mL p-hydroxybenzoic acid, 0.960 mg / mL vanillic acid, 1.054 mg / mL p-hydroxybenzaldehyde, 1.005 mg / mL shampooside, 1.012 mg / mL paeoniflorin, and 1.121 mg / mL p-hydroxycinnamic acid.

[0037] Accurately take appropriate amounts of the stock solutions of the above 7 reference standards and gradually dilute them into 5 series of standard concentrations (see Table 3 for series of standard concentrations), refrigerate, and keep for later use.

[0038] Table 3 Standard Concentrations of a Series of Reference Solutions

[0039] 2.3.3 Chromatographic conditions Same as the chromatographic conditions in item “2.1.3”.

[0040] 3 Results 3.1 HPLC fingerprint results 3.1.1 Precision Test Take the same sample solution (number: S4) and inject it 6 times under the chromatographic conditions in section "2.1.3". Using the common peak No. 8 as the reference peak, the RSD of the relative retention time and relative peak area of ​​each common peak is less than 3%, indicating that the instrument has good precision.

[0041] 3.1.2 Repeatability Test Take the S4 sample of *Gnaphalium affine* and prepare 6 parallel samples according to the method in section "2.1.1". Analyze the samples according to the chromatographic conditions in section "2.1.3". Using the common peak No. 8 as the reference peak, the RSD of the relative retention time and relative peak area of ​​each common peak is less than 3%, indicating that the analytical method has good repeatability.

[0042] 3.1.3 Stability Test Take the same sample (number: S4) and analyze it at 0, 2, 4, 8, 12 and 24 h according to the chromatographic conditions under section “2.1.3”. Using the common peak No. 8 as the reference peak, the RSD of the relative retention time and relative peak area of ​​each common peak is less than 3%, indicating that the sample has good stability within 24 h.

[0043] 3.1.4 Fingerprint pattern establishment and similarity evaluation Fourteen batches of *Gnaphalium affine* were prepared according to method “2.1.1” and analyzed using method “2.1.3”. The resulting chromatograms were recorded and imported into the *Traditional Chinese Medicine Fingerprint Similarity Evaluation System* (2012 version). Using the chromatogram of sample S1 as the reference chromatogram, a time window width of 0.1 min was set. A control chromatogram R was generated using the mean method. After multi-point correction and Mark peak matching, similarity evaluation was performed. The results showed that the similarity of the 14 batches of samples (numbered S1~S14) ranged from 0.850 to 0.983, indicating relatively stable overall quality, although some differences existed in individual batches. Figure 1 ).

[0044] 3.1.5 Identification of Common Peaks The characteristic chromatogram identified 13 common peaks, of which 6 were identified using reference standards: peak 6 is p-hydroxybenzoic acid, peak 7 is vanillic acid, peak 8 is p-hydroxybenzaldehyde, peak 9 is shampoostoside, peak 10 is paeoniflorin, and peak 12 is p-hydroxycinnamic acid. Results are shown below. Figure 2 .

[0045] 3.2 Results of Chemical Pattern Recognition Analysis 3.2.1 Cluster Analysis Using the peak areas of six identified common peaks in the fingerprint chromatograms of 14 batches of *Gnaphalium affine* medicinal materials as variables, cluster analysis was performed using SPSS 27 software. The results are shown below. Figure 3 Fourteen batches of *Gnaphalium affine* medicinal materials were divided into two categories: Category A included S1, S3, S5, S8, S9, S10, and S12, while Category B included S2, S4, S6, S7, S11, S13, and S14. In terms of geographical distribution, both Category A and Category B covered samples from Shandong, Jiangsu, and Guizhou provinces. Clustering results showed some differences even within the same production area, indicating that subtle differences between batches of medicinal materials are not entirely determined by geographical origin. However, samples from Shandong were relatively concentrated in Category B, suggesting good overall stability in their chemical quality.

[0046] 3.2.2 Principal Component Analysis The peak areas of six identified common peaks in the fingerprint spectra of 14 batches of *Gnaphalium affine* medicinal materials were imported into SIMCA14.1 software for principal component analysis. The results showed that ( Figure 4 All data points are within the 95% confidence interval, and the cumulative explained rate R of the established model is [value missing]. 2 X cum The cumulative model predictive power Q is 0.852. 2 X cum It is 0.504, and R 2 X cum and Q 2 X cumAll values ​​are greater than 0.5, indicating a high model fit, no outliers, and reliable data quality. The scatter plot shows that Class A samples are mainly distributed to the left of the first principal component, while Class B samples are mainly distributed to the right, suggesting a difference between the two groups, consistent with the clustering results.

[0047] 3.2.3 Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) The peak areas of six identified common peaks in the fingerprint spectra of 14 batches of *Cephalotaxus fortunei* medicinal materials were imported into SIMCA14.1 software for orthogonal partial least squares discriminant analysis. An orthogonal partial least squares discriminant model was established to further analyze the differences among the 14 batches of *Cephalotaxus fortunei* medicinal materials. The results are as follows: Figure 5 The solidity parameter R of the data matrix 2 X is 0.832, and the model's discriminant parameter R0 2 Y is 0.881, and the model prediction parameter Q is... 2 The values ​​were 0.804, all greater than 0.5 and close to 1, indicating a high good fit and good predictive ability of the model. The model data were randomly permuted 200 times, and a permutation test was performed on it. Figure 6 The results show that R 2 and Q 2 The intercept values ​​are 0.231 and -0.637, respectively. (Figure R) 2 and Q 2 All values ​​are greater than the permutation value, indicating that the model has not overfitted. Based on the VIP value, the components influencing the differences were selected. A VIP > 1 indicates that the component has a large contribution rate and high correlation. A total of three quality difference components were selected. Figure 7 The components, arranged in order of VIP value, are p-hydroxybenzoic acid, p-hydroxybenzaldehyde, and paeoniflorin. These three components are the main factors causing the differences in the composition of *Gnaphalium affine* medicinal materials from different producing areas.

[0048] 3.3 Content determination results 3.3.1 Examination of Linear Relationships Take the series of reference solutions from section "2.3.2" and analyze them according to the chromatographic conditions from section "2.1.3". Plot a standard curve with concentration x (μg / mL) as the abscissa and peak area y as the ordinate. The six components, including p-hydroxybenzoic acid, showed good linearity within their respective concentration ranges, with correlation coefficients R0. 2 ≥0.9996, the results are shown in Table 4.

[0049] Table 4. Examination of linear relationships among the six components

[0050] 3.3.2 Precision Test Take the same sample solution (No.: S4), and inject it six times consecutively under the chromatographic conditions described in section "2.1.3". Record the peak areas of the six components, including p-hydroxybenzoic acid, and calculate the RSD values. The results show that the RSDs of the peak areas of the six components, including p-hydroxybenzoic acid, are between 1.35% and 2.86%, all less than 3%, indicating good instrument precision.

[0051] 3.3.3 Repeatability Test Take sample S4 (a type of wild worm), prepare the test solution according to the above method, and prepare 6 parallel samples. Inject and analyze the samples according to the chromatographic conditions in section "2.1.3", and record the peak area of ​​each chromatographic peak. Calculate the content and RSD value of each component. The results show that the RSD of the contents of the 6 components, including p-hydroxybenzoic acid, are between 1.34% and 1.93%, all less than 3%, indicating that the method has good repeatability.

[0052] 3.5.4 Stability Test The sample solution of *Gnaphalium affine* (S4) was taken and analyzed at room temperature at 0, 2, 4, 8, 12, and 24 hours. The peak areas of each component were recorded, and the RSD was calculated. The results showed that the RSD of the peak areas of the six components, including p-hydroxybenzoic acid, were between 0.53% and 2.23%, all less than 3%, indicating that the sample solution had good stability within 24 hours.

[0053] 3.5.5 Recovery Experiment Accurately weigh the determined amount of *Gnaphalium affine* (sample number: S4), and accurately add the reference standard at amounts of 80%, 100%, and 120% of the sample mass, respectively. Prepare the test solution according to the test solution preparation method. The results show that the average recovery rate of p-hydroxybenzoic acid (80%) ranges from 101.79% to 103.23%. The RSD ranged from 1.37 to 2.45%; the average recovery rate of vanillic acid ranged from 98.24 to 101.88%, with an RSD range of 0.61 to 1.69%; the average recovery rate of p-hydroxybenzaldehyde ranged from 103.27 to 104.38%, with an RSD of 1.30 to 2.58%; the average recovery rate of shampoosin was 103.86 to 104.54%, with an RSD of 0.56 to 2.29%; the average recovery rate of paeoniflorin was 102.03 to 103.55%, with an RSD of 0.65 to 2.41%; and the average recovery rate of p-hydroxycinnamic acid was 100.45 to 103.55%, with an RSD of 1.02 to 2.51%, indicating that the recovery rate of this experiment was good and the method accuracy was high.

[0054] 3.5.6 Results of Sample Content Determination The content results of p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampodophyllin, paeoniflorin, and p-hydroxycinnamic acid are shown in Table 5. The determination results of 14 batches of samples showed significant batch- and origin-specific differences in the content of the six components, with the greatest difference in paeoniflorin content among batches (e.g., ...). Figure 8 As shown in the figure, the content ranges from 1.545 to 6.800 mg / g, followed by p-hydroxycinnamic acid at 0.113 to 0.822 mg / g, p-hydroxybenzaldehyde at 0.025 to 0.402 mg / g, p-hydroxybenzoic acid at 0.031 to 0.398 mg / g, shampoostoside at 0.059 to 0.379 mg / g, and vanillic acid at the lowest content at 0.003 to 0.082 mg / g.

[0055] Table 5. Results of content determination of six components (mg / g)

[0056] Import the content determination results into Origin 2024 software and draw a box plot. Figure 9 The results showed that the five components, p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, paeoniflorin, and p-hydroxycinnamic acid, were found to be most abundant in Jiangsu, while shampootinamide was found to be most abundant in Guizhou.

[0057] 4. Discussion Since the components of traditional Chinese medicine are relatively complex, it is crucial to select a detection wavelength that can comprehensively reflect the characteristics of the sample. In this study, the sample was scanned across the entire wavelength range using a UV detector. It was found that the detection wavelength in the range of 260-280 nm contained the most information. Further comparison of the HPLC chromatograms at 265 nm, 270 nm, and 275 nm revealed that the chromatographic peak separation was the best at 270 nm, with a stable baseline and a high peak signal response value. Therefore, 270 nm was selected as the detection wavelength.

[0058] The fingerprint spectrum of this invention identified six common peaks: p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin, and p-hydroxycinnamic acid. Literature studies have shown that p-hydroxybenzoic acid possesses anti-inflammatory, antibacterial, and antiviral pharmacological effects; vanillic acid has heat-clearing and detoxifying, anti-inflammatory, antibacterial, hepatoprotective, and choleretic effects; p-hydroxybenzaldehyde has antitussive, antiasthmatic, sedative, and analgesic effects; shampooside has anti-inflammatory, heat-clearing, and diuretic effects; paeoniflorin has anti-inflammatory, analgesic, immunomodulatory, hepatoprotective, nephroprotective, and musculoskeletal pain-relieving effects; and p-hydroxycinnamic acid has anti-inflammatory, antiviral, and analgesic effects. These pharmacological effects are consistent with the heat-clearing, detoxifying, blood-cooling, hemostatic, meridian-clearing, diuretic, and antitussive effects of *Gnaphalium affine*, corroborating the scientific validity of this study and providing literature support for p-hydroxybenzoic acid and vanillic acid as potential active ingredients in *Gnaphalium affine*. The content of six identified common peaks in various batches of *Gnaphalium affine* was determined. It was found that the content of these six components varied among different origins and batches of *Gnaphalium affine* medicinal materials. Paeoniflorin showed the greatest variation, followed by p-hydroxycinnamic acid. Significant differences in content were observed between different origins, and the data were dispersed, which may be related to factors such as growth environment and processing technology. This suggests that the impact of origin and processing technology on the content of major active ingredients should be comprehensively considered in the quality control of *Gnaphalium affine* medicinal materials.

[0059] In summary, this invention establishes an HPLC fingerprint spectrum, which, combined with chemical pattern recognition and multi-component content determination methods, helps to improve the quality evaluation and control level of *Gnaphalium affine*, and provides a scientific basis for its pharmacodynamic material basis research and subsequent development and utilization.

Claims

1. A method for establishing an HPLC fingerprint and determining the content of *Gnaphalium affine*, characterized in that: Follow these steps: (1) Preparation of test solution: Pulverize the dried tiger herb, weigh 0.5-1.5g of tiger powder into a round bottom flask, add 10-20mL of 50% methanol, weigh, reflux extract in a water bath at 70-90°C for 1-2h, cool to room temperature, add weight, mix well, centrifuge at 12000-16000r / min at 3-5°C for 5-15min, take the supernatant, and the test solution is obtained. (2) Preparation of mixed reference solution: Accurately weigh hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, schafotaside, paeoniflorin and p-hydroxycinnamic acid reference standards, add 50% methanol to prepare a mixed reference solution containing 38.000-42.000µg / mL p-hydroxybenzoic acid, 38.000-42.000µg / mL vanillic acid, 38.000-42.000µg / mL p-hydroxybenzaldehyde, 38.000-42.000µg / mL schafotaside, 38.000-42.000µg / mL paeoniflorin and 38.000-42.000µg / mL p-hydroxycinnamic acid; (3) Preparation of reference solutions: Accurately weigh p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, schafodine, paeoniflorin and p-hydroxycinnamic acid respectively, dissolve them in methanol to prepare 0.900-1.100 mg / mL p-hydroxybenzoic acid reference stock solution, 0.900-1.100 mg / mL vanillic acid reference stock solution, 0.900-1.100 mg / mL p-hydroxybenzaldehyde reference stock solution, 0.900-1.100 mg / mL schafodine reference stock solution, 0.900-1.100 mg / mL paeoniflorin reference stock solution and 0.900-1.100 mg / mL p-hydroxycinnamic acid reference stock solution; accurately pipette the above 6 reference stock solutions, dilute them to prepare 5 series of standard concentration reference solutions, refrigerate and use them for later use; (4) Chromatographic conditions: Hypersil GOLD™ column, 4.1×250mm, 5μm; column temperature 30℃; injection volume 10µL; detection wavelength 270nm; mobile phase A was 0.2% formic acid in water, mobile phase B was 0.2% formic acid in acetonitrile, and the elution gradient program was as follows: ； (5) Establishment of fingerprint spectrum: Take the medicinal material of *Gnaphalium affine* and prepare the test solution according to the method of step (1). Inject the test solution and the mixed reference solution prepared in step (2) into the chromatographic conditions in step (4) for detection. Based on the chromatographic peaks of the mixed reference solution, identify 6 common peaks in the test solution and construct the fingerprint spectrum of *Gnaphalium affine*. The 6 common peaks are p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin and p-hydroxycinnamic acid. (6) Fingerprint cluster analysis: SPSS 27 and SIMCA14.1 analysis software were used to perform cluster analysis, principal component analysis and orthogonal partial least squares discriminant analysis on the peak area of ​​the six identified common peaks in the fingerprint spectrum of the medicinal material *Gnaphalium affine*. (7) Preparation of standard curve: Take the standard solutions of the 6 reference solutions in step (3) with 5 series of standard concentrations respectively, and inject them for detection according to the chromatographic conditions in step (4). Plot the standard curve with concentration x as the abscissa and peak area y as the ordinate. (8) Content determination: Take the test sample solution and inject it according to the chromatographic conditions in step (4). Calculate the content of the 6 components contained in the sample based on the standard curve.

2. The method for establishing and determining the content of *Gnaphalium affine* using HPLC fingerprinting according to claim 1, characterized in that: In step (1), the preparation of the test solution is as follows: the dried *Gnaphalium affine* medicinal material is pulverized, passed through a 60-80 mesh sieve, and 0.8-1.1 g of *Gnaphalium affine* powder is weighed into a round-bottom flask. 12-18 mL of 50% methanol is added, and after weighing, the mixture is refluxed in a water bath at 75-85°C for 1.2-1.8 h. After cooling to room temperature, the weight is added, the mixture is mixed, and the mixture is centrifuged at 12000-16000 r / min at 3-5°C for 8-12 min. The supernatant is then collected to obtain the test solution.

3. The method for establishing and determining the content of *Gnaphalium affine* using HPLC fingerprinting according to claim 2, characterized in that: In step (1), the preparation of the test solution is as follows: the dried tiger thorn medicinal material is pulverized, passed through a 60-mesh sieve, 1g of tiger thorn powder is weighed into a 50mL round bottom flask, 15mL of 50% methanol is added, and after weighing, it is refluxed in an 80°C water bath for 1.5h, cooled to room temperature, the weight is added, mixed well, and centrifuged at 14000r / min at 4°C for 10min. The supernatant is then taken to obtain the test solution.

4. The method for establishing and determining the content of *Gnaphalium affine* using HPLC fingerprinting according to claim 1, characterized in that: In step (2), the preparation of the mixed reference solution is as follows: accurately weigh hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, schafod, paeoniflorin and p-hydroxycinnamic acid reference standards, add 50% methanol to prepare a mixed reference solution containing 40.367µg / mL p-hydroxybenzoic acid, 40.320µg / mL vanillic acid, 40.052µg / mL p-hydroxybenzaldehyde, 40.200µg / mL schafod, 40.480µg / mL paeoniflorin and 40.518µg / mL p-hydroxycinnamic acid.

5. The method for establishing and determining the content of *Gnaphalium affine* using HPLC fingerprinting according to claim 1, characterized in that: In step (3), the preparation of the reference solutions is as follows: p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin, and p-hydroxycinnamic acid are accurately weighed and dissolved in methanol to prepare 1.091 mg / mL p-hydroxybenzoic acid reference stock solution, 0.960 mg / mL vanillic acid reference stock solution, 1.054 mg / mL p-hydroxybenzaldehyde reference stock solution, 1.005 mg / mL shampooside reference stock solution, 1.012 mg / mL paeoniflorin reference stock solution, and 1.121 mg / mL p-hydroxycinnamic acid reference stock solution; the above 6 reference stock solutions are accurately pipetted and diluted to prepare 5 series of standard concentration reference solutions, which are then refrigerated for later use.

6. The method for establishing and determining the content of *Gnaphalium affine* using HPLC fingerprinting according to claim 1 or 5, characterized in that: In step (3), the five series of standard concentrations of the six reference solutions are as follows: 。 7. The method for establishing and determining the content of *Gnaphalium affine* using HPLC fingerprinting according to claim 1, characterized in that: In step (5), the fingerprint spectrum is established as follows: 14 batches of *Gnaphalium affine* medicinal materials are taken and 14 batches of test solutions are prepared according to the method in step (1). The 14 batches of test solutions and the mixed reference solution prepared in step (2) are injected and detected according to the chromatographic conditions in step (4). Based on the chromatographic peaks of the mixed reference solution, 6 common peaks in the 14 batches of test solutions are identified, and a fingerprint spectrum of *Gnaphalium affine* is constructed. The 6 common peaks are p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin and p-hydroxycinnamic acid.

8. The method for establishing and determining the content of *Gnaphalium affine* using HPLC fingerprinting according to claim 1, characterized in that: In step (6), fingerprint spectrum cluster analysis: SPSS 27 and SIMCA14.1 analysis software were used to perform cluster analysis, principal component analysis and orthogonal partial least squares discriminant analysis on the peak areas of the six identified common peaks in the fingerprint spectrum of 14 batches of Tiger Herb.

9. The method for establishing and determining the content of *Gnaphalium affine* using HPLC fingerprinting according to claim 1, characterized in that: In step (8), the six components are p-hydroxybenzoic acid, vanillic acid, p-hydroxybenzaldehyde, shampooside, paeoniflorin and p-hydroxycinnamic acid.

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