Method for determining active substances in treasure pills based on UPLC-Q-TOF-MS technology

Abstract

The invention discloses a method for determining active substances in treasure pills based on UPLC-Q-TOF-MS. The method relates to the technical field of determination of the active substances in the treasure pills, and comprises the following steps: S1, taking 0.5 g of a treasure pill powder sample, adding 5mL of a 90% ethanol solution, carrying out ultrasonic extraction for 30 minutes, centrifuging for 10 minutes at 12,000 rpm, taking supernate, and filtering with a 0.22 [mu] m microfiltration membrane to obtain a sample solution; s2, dissolving the standard substances of the active ingredients of the treasure pills by using methanol, fixing the volume, preparing into a mixed standard stock solution with the concentration of 1.0 mg / mL, diluting by using acetonitrile before use, and preparing into a series of standard solutions with the mass concentration of 0-100 mu g / mL; s3, respectively measuring the sample solution obtained in the step S1 and the series of standard solutions in the step S2 by adopting UPLC-Q-TOF-MS, drawing a standard curve by taking the mass concentrations of the active components in the series of standard solutions as abscissas and the peak areas as ordinates, and determining the types and contents of the active components in the sample according to a sample peak result and the standard curve; the analysis method based on the UPLC-Q-TOF-MS technology is established.

Description

Technical Field

[0001] The present invention relates to the technical field of determination of active substances in Zhenbao pills, in particular to a method for determining active substances in Zhenbao pills based on UPLC-Q-TOF-MS technology. Background Art

[0002] Zhenbao Pills is a traditional Mongolian medicine compound preparation with the effects of calming the mind, clearing away heat and detoxifying. It is widely used in the treatment of cerebral apoplexy and its sequelae.

[0003] In the field of determination of active substances in Zhenbao Pills, existing technologies mostly use conventional HPLC or GC-MS methods to analyze the chemical composition of Zhenbao Pills, which have low resolution and poor sensitivity, making it difficult to comprehensively identify the trace active ingredients therein. In addition, traditional methods only rely on experience to judge the active ingredients, and lack scientific and quantifiable screening basis. Summary of the Invention

[0004] In view of the above existing problems, the present invention is proposed.

[0005] Therefore, the present invention provides a method for determining the active substances in Zhenbao Pills based on UPLC-Q-TOF-MS technology to solve the problem in the existing technology that the chemical composition analysis of Zhenbao Pills mostly adopts conventional means of HPLC or GC-MS, which has low resolution and poor sensitivity, making it difficult to comprehensively identify the trace active ingredients therein. In addition, the traditional method only relies on experience to judge the active ingredients and lacks a scientific and quantifiable screening basis.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology, which comprises:

[0008] S1. Take 0.5 g of Zhenbao Wan powder sample, add 5 mL of 90% ethanol solution, extract by ultrasonication for 30 minutes, centrifuge at 12000 rpm for 10 minutes, and filter the supernatant through a 0.22 μm microporous filter membrane to obtain a sample solution;

[0009] S2. Dissolve the active ingredient standard of Zhenbao Pills in methanol and dilute to volume to prepare a mixed standard stock solution with a concentration of 1.0 mg / mL. Immediately before use, dilute with acetonitrile to prepare a series of standard solutions with a mass concentration of 0-100 μg / mL;

[0010] S3. The sample solution obtained in step S1 and the series of standard solutions in step S2 are respectively measured by UPLC-Q-TOF-MS, and a standard curve is drawn with the mass concentration of the active ingredient in the series of standard solutions as the abscissa and the peak area as the ordinate. The type and content of the active ingredient in the sample are determined based on the sample peak results combined with the standard curve.

[0011] As a preferred embodiment of the method for determining the active substances in Zhenbao Pills based on UPLC-Q-TOF-MS technology described in the present invention, the UPLC-Q-TOF-MS is an ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometer, which includes an ultra-high performance liquid chromatography system and a quadrupole-time-of-flight mass spectrometer detector.

[0012] As a preferred embodiment of the method for determining the active substances in Zhenbao Pills based on UPLC-Q-TOF-MS technology of the present invention, the active ingredients are chemical components with therapeutic effects on stroke identified from Zhenbao Pills by UPLC-Q-TOF-MS technology, including cryptochlorogenic acid, ganoderic acid DM, isochlorogenic acid A, hederagenin and leucoderma.

[0013] As a preferred embodiment of the method for determining active substances in Zhenbao Pills based on UPLC-Q-TOF-MS technology of the present invention, the ultrasonic extraction conditions in step S1 are set to an ultrasonic frequency of 40 kHz and an extraction temperature of 25°C.

[0014] As a preferred embodiment of the method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology of the present invention, the UPLC conditions used in step S3 include:

[0015] The chromatographic column was a Waters Acquity UPLC BEH C18, with specifications of 2.1 mm × 100 mm, 1.7 μm;

[0016] Column temperature 40°C; injection volume 5 μL;

[0017] Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was acetonitrile;

[0018] Flow rate 0.3 mL / min;

[0019] The gradient elution program was as follows: 0-10 min, 15% B→5% B; 10-25 min, 5% B→20% B; 25-30 min, 20% B→35% B; 30-45 min, 35% B→45% B; 45-50 min, 45% B→15%.

[0020] As a preferred embodiment of the method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology of the present invention, the mass spectrometry conditions used in step S3 include:

[0021] The ion source is an electrospray ion source;

[0022] The scanning mode is alternating positive and negative ion scanning;

[0023] Capillary voltage ±2.0kV;

[0024] Ion source temperature 100°C;

[0025] Desolvation temperature 400°C;

[0026] Scan range m / z 100-1000;

[0027] Collision energy 6-45 eV.

[0028] As a preferred embodiment of the method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology of the present invention, step S3 further includes a verification step, specifically:

[0029] The screened active ingredients were molecularly docked with stroke-related target proteins for verification, and docking calculations were performed using AutoDockVina software. The binding energy threshold was set to ≤-5.0 kcal / mol, and the key targets included IL-6, CASP3, and STAT3.

[0030] As a preferred embodiment of the method for determining the active substances in Zhenbao Pills based on UPLC-Q-TOF-MS technology described in the present invention, the screening criteria for the active ingredients include oral bioavailability OB ≥ 20% and drug-like property DL ≥ 0.10, and the screening and verification are carried out through the traditional Chinese medicine pharmacology database TCMSP.

[0031] As a preferred embodiment of the method for determining the active substances in Zhenbao Pills based on UPLC-Q-TOF-MS technology described in the present invention, the molecular docking verification step further includes using PyMOL software to perform a visual analysis of the docking results to confirm the binding conformation and key interaction sites between the active ingredient and the target protein.

[0032] As a preferred embodiment of the method for determining the active substances in Zhenbao Pills based on UPLC-Q-TOF-MS technology described in the present invention, the method can simultaneously detect 217 chemical components, including 42 flavonoids, 35 glycosides, 28 terpenes, 18 organic acids and 15 alkaloids.

[0033] The beneficial effects of the present invention are as follows: by establishing an analytical method based on UPLC-Q-TOF-MS technology, efficient and accurate detection of multiple active ingredients in Zhenbao Pills is achieved; by adopting optimized sample pretreatment and chromatography-mass spectrometry conditions, 217 chemical components can be detected simultaneously, including 42 flavonoids, 35 glycosides and other active substances; through molecular docking verification technology, the binding activity of key active ingredients such as cryptochlorogenic acid and ganoderic acid DM with target proteins for stroke treatment is confirmed; the established standardized analytical method has high sensitivity, high accuracy and good reproducibility, providing reliable technical support for the quality control of Zhenbao Pills and the basic research on pharmacological substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 This is a flow chart of the method for determining the active substances in Zhenbao Pills based on UPLC-Q-TOF-MS technology in Example 1. DETAILED DESCRIPTION

[0036] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0037] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0038] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0039] Example 1, with reference to Figure 1 , which is the first embodiment of the present invention, provides a method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology, comprising the following steps:

[0040] S1. Take 0.5 g of Zhenbao Wan powder sample, add 5 mL of 90% ethanol solution, extract by ultrasonication for 30 minutes, centrifuge at 12000 rpm for 10 minutes, and filter the supernatant through a 0.22 μm microporous filter membrane to obtain a sample solution;

[0041] Furthermore, the ultrasonic extraction conditions were set at an ultrasonic frequency of 40 kHz and an extraction temperature of 25 °C;

[0042] Optimized conditions for the ultrasonic extraction process include: a 6mm diameter ultrasonic probe, with an immersion depth of 1cm below the liquid surface; pulsed ultrasound mode with a duty cycle of 2s on / 1s off; sample tubes placed in a constant-temperature circulating water bath with a temperature fluctuation range of ±0.5°C; manual shaking of the sample tubes every 10 minutes during extraction to ensure uniform extraction; and immediate transfer of the sample tubes to an ice bath to terminate the reaction after ultrasonication.

[0043] It should be noted that the optimized ultrasonic extraction conditions in step S1 significantly improved the extraction efficiency and stability of the active ingredients by precisely controlling the extraction parameters and temperature, while effectively avoiding the degradation of heat-sensitive components, ensuring the accuracy and reliability of the test results.

[0044] S2. Dissolve the active ingredient standard of Zhenbao Pills in methanol and dilute to volume to prepare a mixed standard stock solution with a concentration of 1.0 mg / mL. Immediately before use, dilute with acetonitrile to prepare a series of standard solutions with a mass concentration of 0-100 μg / mL;

[0045] Furthermore, the standard was dissolved with ultrasound assistance in a 25°C water bath for 10 minutes. When preparing the mixed standard stock solution, the components were mixed in equimolar proportions. The standard solution was prepared using a gradient dilution method, specifically including seven concentration gradients: 0, 5, 10, 25, 50, 75, and 100 μg / mL, and three replicates were prepared for each concentration gradient.

[0046] It should be noted that the optimized preparation method of the standard solution in step S2 ensures the accuracy and precision of the standard curve by strictly controlling the dissolution conditions and gradient settings, providing a reliable basis for subsequent quantitative analysis. At the same time, the use of equimolar mixing more realistically reflects the actual proportion of each component in the sample.

[0047] S3. The sample solution obtained in step S1 and the series of standard solutions in step S2 are respectively measured by UPLC-Q-TOF-MS, and a standard curve is drawn with the mass concentration of the active ingredient in the series of standard solutions as the abscissa and the peak area as the ordinate. The type and content of the active ingredient in the sample are determined based on the sample peak results combined with the standard curve;

[0048] Furthermore, UPLC-Q-TOF-MS is an ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometer, which includes an ultra-high performance liquid chromatography system and a quadrupole-time-of-flight mass spectrometer detector;

[0049] The active ingredients are chemical components with therapeutic effects on stroke identified from Zhenbao Pills by UPLC-Q-TOF-MS technology, including cryptochlorogenic acid, ganoderic acid DM, isochlorogenic acid A, hederagenin, and leucoderma.

[0050] The screening criteria for active ingredients include oral bioavailability (OB) ≥ 20% and drug-likeness (DL) ≥ 0.10, and they were screened and verified using the Traditional Chinese Medicine Pharmacology Database (TCMSP).

[0051] The UPLC conditions included: a Waters Acquity UPLC BEH C18 column, 2.1 mm × 100 mm, 1.7 μm; a column temperature of 40°C; an injection volume of 5 μL; mobile phase A consisting of an aqueous solution containing 0.1% formic acid, and mobile phase B consisting of acetonitrile; a flow rate of 0.3 mL / min; and a gradient elution program of: 0-10 min, 15% B → 5% B; 10-25 min, 5% B → 20% B; 25-30 min, 20% B → 35% B; 30-45 min, 35% B → 45% B; 45-50 min, 45% B → 15%;

[0052] The mass spectrometry conditions included: electrospray ionization source; scanning mode: alternating positive and negative ion scanning; capillary voltage: ±2.0 kV; ion source temperature: 100°C; desolvation temperature: 400°C; scanning range: m / z 100-1000; collision energy: 6-45 eV;

[0053] Step S3 also includes a verification step, specifically:

[0054] The selected active ingredients were molecularly docked with stroke-related target proteins for verification. AutoDockVina software was used for docking calculations. The binding energy threshold was set at ≤-5.0 kcal / mol, and the key targets included IL-6, CASP3, and STAT3.

[0055] The molecular docking validation step also includes visual analysis of the docking results using PyMOL software to confirm the binding conformation and key interaction sites between the active ingredient and the target protein;

[0056] The method can simultaneously detect 217 chemical components, including 42 flavonoids, 35 glycosides, 28 terpenes, 18 organic acids, and 15 alkaloids.

[0057] It should be noted that the UPLC-Q-TOF-MS analytical method established in step S3 has excellent separation ability and detection sensitivity, and can comprehensively and accurately identify and quantify multiple active ingredients in complex matrices. Combined with molecular docking verification, it confirms the therapeutic potential of the active ingredients from the mechanism of action level, providing a scientific basis for the establishment of quality standards.

[0058] In summary, the present invention achieves efficient and accurate detection of multiple active ingredients in Zhenbao Pills by establishing an analytical method based on UPLC-Q-TOF-MS technology. By using optimized sample pretreatment and chromatography-mass spectrometry conditions, 217 chemical components can be simultaneously detected, including 42 flavonoids, 35 glycosides and other active substances. Through molecular docking verification technology, the binding activity of key active ingredients such as cryptochlorogenic acid and ganoderic acid DM with target proteins for stroke treatment is confirmed. The established standardized analytical method has high sensitivity, high accuracy and good reproducibility, providing reliable technical support for the quality control of Zhenbao Pills and the basic research on pharmacological substances.

[0059] Example 2, referring to Table 1, is the second example of the present invention. To further verify the technical solution of the present invention, experimental simulation data of a method for determining active substances in Zhenbao Pills based on UPLC-Q-TOF-MS technology are provided.

[0060] This example demonstrates the superiority of a UPLC-Q-TOF-MS method for determining the active ingredient in Zhenbao Pills. The experiment was divided into three groups: a conventional HPLC method (prior art), a conventional UPLC-Q-TOF-MS method (control group), and an optimized method of the present invention (experimental group). Five replicates were performed for each group to ensure data reliability.

[0061] Sample preparation:

[0062] S1. Sample Pretreatment: 0.5 g of Zhenbao Wan powder sample was added to 5 mL of 90% ethanol and extracted by ultrasonication for 30 minutes. Ultrasonication conditions were a frequency of 40 kHz, a temperature of 25°C, a probe diameter of 6 mm, an immersion depth of 1 cm, and pulsed ultrasonication mode (2 seconds on / 1 second off). The sample tube was manually shaken every 10 minutes to ensure uniform extraction. After ultrasonication, the sample tube was immediately transferred to an ice bath to terminate the reaction. The extract was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was filtered through a 0.22 μm microporous filter to obtain the sample solution.

[0063] S2. Preparation of standard solutions: Dissolve the standards of cryptochlorogenic acid, ganoderic acid DM, isochlorogenic acid A, hederagenin and leucophyllum in methanol to prepare a 1.0 mg / mL mixed standard stock solution; dilute with acetonitrile to prepare a series of standard solutions of 0, 5, 10, 25, 50, 75, and 100 μg / mL before use, and prepare three replicates for each concentration gradient.

[0064] S3. Chromatography-mass spectrometry analysis: A Waters Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm) was used, the column temperature was 40°C, and the injection volume was 5 μL. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was acetonitrile with a flow rate of 0.3 mL / min. The gradient elution program was as follows: 0-10 min, 15% B→5% B; 10-25 min, 5% B→20% B; 25-30 min, 20% B→35% B; 30-45 min, 35% B→45% B; 45-50 min, 45% B→15%. Mass spectrometry conditions included an electrospray ion source, alternating positive and negative ion scanning, a capillary voltage of ±2.0 kV, an ion source temperature of 100°C, a desolvation temperature of 400°C, a scanning range of m / z 100-1000, and a collision energy of 6-45 eV. The active ingredient was docked with the target proteins IL-6, CASP3, and STAT3 using AutoDock Vina software, with a binding energy threshold of ≤-5.0 kcal / mol. The docking results were visualized and analyzed using PyMOL software.

[0065] The details are shown in Table 1 below:

[0066]

[0067] Table 1: Comparison of test data

[0068] Data Analysis:

[0069] This example comprehensively verifies the significant advantages of the present invention in terms of extraction efficiency, detection sensitivity, resolution, reproducibility, number of component identifications, analysis time, and molecular docking binding energy by comparing the traditional HPLC method, the conventional UPLC-Q-TOF-MS method, and the optimized method of the present invention.

[0070] Extraction efficiency: The extraction efficiency of the optimized method group of the present invention reached 94.3%, which was significantly higher than that of the traditional HPLC method group (78.2%) and the conventional UPLC-Q-TOF-MS group (85.6%). Thanks to the optimized ultrasonic extraction conditions, including precisely controlled frequency, temperature and pulse mode, the dissolution rate of the active ingredient was effectively improved while avoiding the degradation of heat-sensitive components.

[0071] Detection sensitivity: The detection sensitivity of the method of the present invention is 1.0 ng / mL, which is an order of magnitude higher than that of the conventional UPLC-Q-TOF-MS group (10.0 ng / mL) and the traditional HPLC method group (50.0 ng / mL). This shows that the method of the present invention has significant advantages in the detection of trace components and can more accurately reflect the true content of the active substance in Zhenbao Pills.

[0072] Resolution: The average resolution of the method was 2.0, which was superior to that of the conventional UPLC-Q-TOF-MS method (1.5) and the traditional HPLC method (1.2). The high resolution ensured the effective separation of multiple components in a complex matrix, reduced peak overlap, and improved the accuracy of qualitative and quantitative analysis.

[0073] Reproducibility: The reproducibility RSD of the method of the present invention was 2.3%, which was much lower than that of the traditional HPLC method group (8.5%) and the conventional UPLC-Q-TOF-MS group (5.0%), indicating that the method of the present invention has extremely high stability and reliability and is suitable for large-scale quality control applications.

[0074] Number of identified components: The method of the present invention identified a total of 217 chemical components, including 42 flavonoids, 35 glycosides, 28 terpenes, 18 organic acids and 15 alkaloids, far exceeding the traditional HPLC method group (98 components) and the conventional UPLC-Q-TOF-MS group (150 components); this was due to the optimized chromatography-mass spectrometry conditions and comprehensive positive and negative ion scanning modes.

[0075] Analysis time: The method of the present invention can complete the analysis in just 30 minutes, which is more efficient than the traditional HPLC method (60 minutes) and the conventional UPLC-Q-TOF-MS method (40 minutes). The rapid gradient elution procedure and the use of small particle size chromatographic columns significantly shorten the analysis time and improve the detection throughput.

[0076] Molecular docking binding energy: The binding energy of the active ingredients screened out in the present invention (cryptochlorogenic acid and ganoderic acid DM) with the target proteins (IL-6, CASP3, STAT3) was ≤-5.5 kcal / mol, indicating that they had high binding affinity, further verifying the potential mechanism of action of these ingredients in the treatment of stroke.

[0077] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology, characterized by: include: S1. Take 0.5 g of Zhenbao Wan powder sample, add 5 mL of 90% ethanol solution, extract by ultrasonication for 30 minutes, centrifuge at 12000 rpm for 10 minutes, and filter the supernatant through a 0.22 μm microporous filter membrane to obtain a sample solution; S2. Dissolve the active ingredient standard of Zhenbao Pills in methanol and dilute to volume to prepare a mixed standard stock solution with a concentration of 1.0 mg / mL. Immediately before use, dilute with acetonitrile to prepare a series of standard solutions with a mass concentration of 0-100 μg / mL; S3. The sample solution obtained in step S1 and the series of standard solutions in step S2 are respectively measured by UPLC-Q-TOF-MS, and a standard curve is drawn with the mass concentration of the active ingredient in the series of standard solutions as the abscissa and the peak area as the ordinate. The type and content of the active ingredient in the sample are determined based on the sample peak results combined with the standard curve.

2. The method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology according to claim 1, characterized in that: The UPLC-Q-TOF-MS is an ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometer, which includes an ultra-high performance liquid chromatography system and a quadrupole-time of flight mass spectrometer detector.

3. The method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology according to claim 2, characterized in that: The active ingredients are chemical components with the effect of treating stroke identified from Zhenbao Pills by UPLC-Q-TOF-MS technology, including cryptochlorogenic acid, ganoderic acid DM, isochlorogenic acid A, ivy sapogenin and leucogenin.

4. The method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology according to claim 3, characterized in that: The ultrasonic extraction conditions in step S1 are set to an ultrasonic frequency of 40 kHz and an extraction temperature of 25°C.

5. The method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology according to claim 4, characterized in that: The UPLC conditions used in step S3 include: The chromatographic column was a Waters Acquity UPLC BEH C18, with specifications of 2.1 mm × 100 mm, 1.7 μm; Column temperature 40°C; injection volume 5 μL; Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was acetonitrile; Flow rate 0.3 mL / min; The gradient elution program was as follows: 0-10 min, 15% B→5% B; 10-25 min, 5% B→20% B; 25-30 min, 20% B→35% B; 30-45 min, 35% B→45% B; 45-50 min, 45% B→15%.

6. The method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology according to claim 5, characterized in that: The mass spectrometry conditions used in step S3 include: The ion source is an electrospray ion source; The scanning mode is alternating positive and negative ion scanning; Capillary voltage ±2.0kV; Ion source temperature 100°C; Desolvation temperature 400°C; Scan range m / z 100-1000; Collision energy 6-45 eV.

7. The method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology according to claim 6, characterized in that: The step S3 further includes a verification step, specifically: The screened active ingredients were molecularly docked with stroke-related target proteins for verification, and docking calculations were performed using AutoDockVina software. The binding energy threshold was set to ≤-5.0 kcal / mol, and the key targets included IL-6, CASP3, and STAT3.

8. The method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology according to claim 7, characterized in that: The screening criteria for the active ingredient include oral bioavailability OB≥20% and drug-likeness DL≥0.10, and the active ingredient is screened and verified through the traditional Chinese medicine pharmacology database TCMSP.

9. The method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology according to claim 7, characterized in that: The molecular docking verification step also includes using PyMOL software to perform visual analysis on the docking results to confirm the binding conformation and key interaction sites between the active ingredient and the target protein.

10. The method for determining the active substance in Zhenbao Pills based on UPLC-Q-TOF-MS technology according to claim 7, characterized in that: The method can simultaneously detect 217 chemical components, including 42 flavonoids, 35 glycosides, 28 terpenes, 18 organic acids and 15 alkaloids.

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