Method for identifying all components and blood components in Huifeng Qubi Decoction

Abstract

The present application relates to the technical field of drug detection, and in particular to a method for identifying all components and blood-entering components in Huftengqubi Decoction. The method is as follows: preparing XTQBD extract, identifying all components in Huftengqubi Decoction through UPLC-MS / MS detection; giving XTQBD extract to rats by gavage, collecting serum samples of the rats after administration as an experimental group, using serum of rats given ultrapure water as a control group, identifying blood-entering components in Huftengqubi Decoction through UPLC-MS / MS detection. The present application uses LC-MS / MS analysis technology of ultra-high performance liquid chromatography-high resolution mass spectrometry to systematically analyze the chemical composition of Huftengqubi Decoction, and further reveals the blood-entering components in the animal body, which provides a scientific basis for in-depth study of the mechanism of drug efficacy, optimization of prescription compatibility, and establishment of a targeted quality evaluation system.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and particularly relates to a method for identifying all components and blood components in Xueteng Qubi Decoction. Background Art

[0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease mainly characterized by chronic, progressive, and erosive arthritis. The clinical manifestations include symmetrical joint swelling and pain, morning stiffness, and functional impairment. The pathology focuses on synovial inflammation, pannus formation, and destruction of articular cartilage and bone tissue, with characteristics such as high incidence, high disability rate, protracted course, and poor prognosis.

[0003] Traditional Chinese medicine and ethnic medicine have a long history and unique advantages in the treatment of arthralgia. As an important part of traditional Chinese medicine in China, Dong medicine has accumulated rich clinical experience in the treatment of diseases such as rheumatoid arthritis. The prescription of Xueteng Qubi Decoction consists of Sargentodoxa cuneata, Acanthopanax gracilistylus, Diphylleia sinensis, Drynaria fortunei, and Speranskia tuberculata, and it is an empirical prescription for treating rheumatism and arthralgia formed according to the theory of "wind disease" in Dong medicine. It is clinically used for the treatment of diseases such as rheumatoid arthritis and osteoarthritis, with definite curative effect and high safety, and has important clinical application value.

[0004] With the deepening of the research on the modernization of traditional Chinese medicine and ethnic medicine, the systematic characterization of all components of compound prescriptions and the identification of blood pharmacodynamic substances have become key scientific issues for explaining the action mechanism of traditional Chinese medicine compound prescriptions, establishing quality control standards, and promoting new drug research and development. However, the current research on Xueteng Qubi Decoction still mainly focuses on clinical experience application and efficacy observation, and there are obvious deficiencies as follows: (1) The compound prescription of Xueteng Qubi Decoction consists of multiple traditional Chinese medicines, with complex chemical components, and the whole component system has not been systematically elucidated; (2) The research on blood components and in vivo metabolism characteristics has not been carried out, and it is impossible to determine the active substance group that truly enters the body and plays a role; (3) There is still a lack of efficient, accurate, and systematic component identification technical methods.

[0005] Based on this, the present invention provides a method for identifying all components and blood components in Xueteng Qubi Decoction, aiming to systematically analyze the effective components of Xueteng Qubi Decoction through LC-MS / MS analysis technology of ultra-high performance liquid chromatography-high resolution mass spectrometry, and further reveal its blood components in animals, so as to provide a scientific basis for in-depth research on the pharmacodynamic mechanism, optimizing the compatibility of prescriptions, and establishing a targeted quality evaluation system. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for identifying all components and blood components in Xueteng Qubi Decoction.

[0007] To achieve the above purpose, the technical scheme adopted by the present invention is as follows: The prescription of the Xuefeng Qubi Decoction described in the present invention consists of the following components in parts by mass: Sargentodoxa cuneata 30 parts, Acanthopanax gracilistylus 15 parts, Aristolochia mollissima 30 parts, Drynaria fortunei 30 parts, and Speranskia tuberculata 20 parts; the identification method for all components and blood components in the Xuefeng Qubi Decoction is as follows: Prepare the XTQBD extract, detect it by UPLC-MS / MS, and identify all components in the Xuefeng Qubi Decoction; intragastrically administer the XTQBD extract to rats, collect the serum samples after administration as the experimental group, and use the rat serum administered with ultrapure water as the control group, and detect by UPLC-MS / MS to identify the blood components in the Xuefeng Qubi Decoction; the specific steps are as follows: S1. Preparation of the XTQBD extract: Weigh Sargentodoxa cuneata, Acanthopanax gracilistylus, Aristolochia mollissima, Drynaria fortunei, and Speranskia tuberculata according to the prescription composition, decoct twice with water, combine the decoctions and concentrate to obtain it. S2. Preparation of serum samples: Intragastrically administer the XTQBD extract to rats, collect the serum at a predetermined time as the experimental group samples, and use the rat serum administered with ultrapure water as the control group samples. S3. Preparation of test solutions: Preparation of the test solution for identifying all components: Take the XTQBD extract prepared in step S1, add methanol, and vortex to mix evenly; take the mixed solution, add 40% methanol aqueous solution, vortex to mix evenly, centrifuge, and take the supernatant to obtain it. Preparation of the test solution for identifying blood components: Take the serum samples prepared in step S2, add methanol, vortex to mix, let stand under low-temperature conditions, and then centrifuge; take the supernatant and dry it under vacuum, add 40% methanol aqueous solution to the residue, vortex, centrifuge, and take the supernatant to obtain it. S4. LC-MS / MS analysis: Respectively suck the above test solutions and inject them into the liquid chromatography-mass spectrometry system for LC-MS / MS analysis.

[0008] Preferably, in the identification method for all components and blood components in the Xuefeng Qubi Decoction described in the present invention, step S1 is specifically as follows: Weigh Sargentodoxa cuneata, Acanthopanax gracilistylus, Aristolochia mollissima, Drynaria fortunei, and Speranskia tuberculata according to the prescription composition, add water 6-8 times the total mass of the medicinal materials, soak for 20-40 min and then decoct for 0.5-2 h, filter, then add water 6-8 times the total mass of the medicinal materials, decoct for 0.5-1 h, filter; combine the two decoctions and concentrate to a concentration of 4-6 g / mL to obtain it.

[0009] In a further preferred embodiment, the method for identifying all components and blood-entering components in the Xue Teng Qu Bi Tang of the present invention, step S1 specifically comprises: weighing out Da Xue Teng, Wu Jia Pi, Jian Xue Fei, Gu Sui Bu, and Tou Gu Cao according to the prescription composition, adding 7 times the total mass of the medicinal materials in water, soaking for 30 minutes, decocting for 1 hour, filtering, adding another 7 times the total mass of the medicinal materials in water, decocting for 0.5 hours, filtering; combining the two decoctions, concentrating to a concentration of 5 g / mL, thus obtaining the final product.

[0010] Preferably, in the method for identifying all components and blood-entering components of the Xue Teng Qu Bi Tang of the present invention, the preparation of the test solution for identifying all components in step S3 is as follows: take 600 μL of the XTQBD extract obtained in step S1 into a 1.5 mL EP tube, add 400 μL of methanol, and vortex mix for 10 s; take 200 μL of the mixed solution, add 200 μL of 40% methanol aqueous solution, vortex mix for 10 s, centrifuge at 4 °C for 15 min, and take the supernatant to obtain the product.

[0011] Preferably, in the method for identifying all components and blood-entering components of the Xue Teng Qu Bi Tang of the present invention, the preparation of the test solution for identifying the blood-entering components in step S3 is as follows: Take 600 μL of the serum sample obtained in step S2, add methanol, vortex mix for 60 s, let stand at -20℃ for 30 min, and then centrifuge at 4℃ for 20 min; take the supernatant and vacuum dry it, add 100 μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 4℃ for 20 min, and take the supernatant to obtain the product.

[0012] In a further preferred embodiment, in the method for identifying all components and blood-entering components of the Xue Teng Qu Bi Tang of the present invention, the relative centrifugal force of centrifugation in step S3 is 16000g.

[0013] Preferably, in the method for identifying all components and blood-entering components of the Xue Teng Qu Bi Decoction of the present invention, the chromatographic conditions in step S4 are as follows: The chromatographic column was an ACQUITY UPLC HST3 column with dimensions of 2.1 mm × 100 mm and a diameter of 1.8 μm; the column temperature was 25℃–40℃; the flow rate was 0.2–0.4 mL / min; the injection volume was 6 μL; gradient elution was performed using 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B. The gradient elution program was as follows: .

[0014] In a further preferred embodiment, in the method for identifying all components and blood-entering components of the Xue Teng Qu Bi Tang of the present invention, the column temperature in step S4 is 35°C.

[0015] Further preferably, in the method for identifying all components and blood-incorporated components in the Caulis Spatholobi and Duhuo Decoction of the present invention, in the chromatographic conditions of step S4, the flow rate is 0.3 mL / min.

[0016] Preferably, in the method for identifying all components and blood-incorporated components in the Caulis Spatholobi and Duhuo Decoction of the present invention, the mass spectrometry conditions in step S4 are specifically as follows: ESI ion source, mass spectrometry acquisition is performed in both positive and negative ion modes; spray voltage is 3800 V (ESI+) / 3500 V (ESI-); sheath gas pressure is 45 arb; auxiliary gas pressure is 20 arb; ion transfer tube temperature is 320 °C; atomization temperature is 350 °C; the detection mode is full scan / data-dependent secondary scan mode, and the resolutions of the first and second levels are 60000 FWHM and 15000 FWHM respectively; the collision energy adopts stepped normalized energy levels of 20, 40, and 60; the first-order mass-to-charge ratio scanning range is 90-1300 m / z.

[0017] Advantages of the present invention: 1. The present invention firstly establishes a method for identifying all components and blood-incorporated components in the Caulis Spatholobi and Duhuo Decoction. By using the UPLC-HRMS coupling technology, all components of the Caulis Spatholobi and Duhuo Decoction are characterized, and its blood-incorporated components are analyzed by LC-MS / MS, clarifying the material basis for the Caulis Spatholobi and Duhuo Decoction to treat rheumatoid arthritis, and providing a scientific basis for further carrying out research on the pharmacodynamic mechanism, optimizing the formula compatibility, and establishing a targeted quality evaluation system.

[0018] 2. Through the UPLC-HRMS technology combined with the NPClassifier classification system, a total of 3798 chemical components are identified in the extract of the Caulis Spatholobi and Duhuo Decoction, and their biosynthetic pathways, superclasses, and specific categories are systematically summarized, clearly presenting the distribution characteristics of various components, providing a clear direction for screening active components, and laying a chemical foundation for further understanding the compatibility connotation of the Caulis Spatholobi and Duhuo Decoction.

[0019] 3. By comparing the component differences between the extract of the Caulis Spatholobi and Duhuo Decoction and the serum sample, a total of 626 blood-incorporated components are identified. Combining the identification results of in vitro chemical components and blood-incorporated components, a pharmacodynamic substance network for the Caulis Spatholobi and Duhuo Decoction to treat RA is preliminarily constructed. At the same time, by comparing the component content differences between the serum group and the XTQBD serum group, 20 main blood-incorporated components of the Caulis Spatholobi and Duhuo Decoction are further clarified. The present invention provides a new idea for optimizing the formula compatibility and improving the clinical efficacy, and also provides a chemical basis for the modern interpretation of ethnic medicine. Description of the drawings

[0020] Figure 1 It is a correlation diagram of the sample under positive and negative ion modes (in the figure: A is the positive ion sample correlation diagram of the XTQBD extract group; B is the negative ion sample correlation diagram of the XTQBD extract group); Figure 2 The figures show the total ion chromatograms of each group of samples under positive and negative ion modes (in the figures: A and B are the positive and negative ion chromatograms of the XTQBD extract group; C and D are the positive and negative ion chromatograms of the serum control group; E and F are the positive and negative ion chromatograms of the XTQBD serum sample group). Figures 3-4 Secondary mass spectra of 20 major blood-entering components; Figure 5 The total ion chromatograms of the XTQBD extract sample under positive and negative ion modes are shown in the figure (A is the positive ion chromatogram peak of the XTQBD extract sample; B is the negative ion chromatogram peak of the XTQBD extract sample). Detailed Implementation

[0021] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are for explanation and illustration only, and do not constitute a limitation on the technical solution of the present invention.

[0022] Example 1

[0023] The identification method for all components and blood-entering components in Xue Teng Qu Bi Tang (Blood Vine Nourishing Rheumatism Decoction) is as follows: (1) Preparation of XTQBD extract: Weigh out 30 g of *Sargentodoxa cuneata*, 15 g of *Acanthopanax senticosus*, 30 g of *Sargentodoxa cuneata*, 30 g of *Drynaria fortunei*, and 20 g of *Clematis chinensis* according to the prescription. Add 7 times the total weight of the herbs to water, soak for 30 minutes, decoct for 1 hour, filter, add another 7 times the total weight of the herbs to water, decoct for 0.5 hours, filter again; combine the two decoctions, concentrate to a concentration of 5 g / mL, and the product is obtained.

[0024] (2) Preparation of serum samples: Rats were administered XTQBD extract (1.29 g / mL, 4 mL / day, for 7 consecutive days) by gavage. One hour after the last administration, blood was collected aseptically via the abdominal aorta and centrifuged at 4°C and 3500 r / min for 10 min to obtain the experimental group sample. Rat serum administered with ultrapure water was used as the control group sample. The supernatants from both groups were mixed and stored at -80°C for later use.

[0025] (3) Preparation of the test solution: Preparation of the test solution for full component analysis: Take 600 μL of the XTQBD extract obtained in step S1 into a 1.5 mL EP tube, add 400 μL of methanol, and vortex for 10 s; take 200 μL of the mixed solution, add 200 μL of 40% methanol aqueous solution, vortex for 10 s, centrifuge at 4℃ and 16000 g for 15 min, and take the supernatant.

[0026] Preparation of test solutions for blood component analysis: Take 600 μL of each of the two serum samples obtained in step S2, add methanol, vortex mix for 60 s, let stand at -20℃ for 30 min, and then centrifuge at 4℃ and 16000 g for 20 min; take the supernatant and vacuum dry it, add 100 μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 4℃ and 16000 g for 20 min, and take the supernatant to obtain the solution.

[0027] (4) LC-MS / MS analysis: 6 μL of each of the above test solutions was injected into a liquid chromatography-mass spectrometry (LC-MS / MS) system for analysis. The specific chromatographic and mass spectrometric conditions are as follows: 1) Chromatographic conditions: The chromatographic column was an ACQUITY UPLC HSS T3 column with dimensions of 2.1 mm × 100 mm and a diameter of 1.8 μm; the column temperature was 35℃; the flow rate was 0.3 mL / min; gradient elution was performed using 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B. The gradient elution program is as follows: .

[0028] 2) Mass spectrometry conditions: ESI ion source, mass spectrometry acquisition in both positive and negative ion modes; spray voltage 3800 V (ESI+) / 3500 V (ESI-); sheath gas pressure 45 arb; auxiliary gas pressure 20 arb; ion transmission tube temperature 320℃; nebulization temperature 350℃; detection mode is full scan / data-dependent two-stage scan mode, with first-stage and second-stage resolutions of 60000 FWHM and 15000 FWHM, respectively; collision energies are normalized to step levels 20, 40, and 60; first-stage mass-to-charge ratio scan range is 90-1300 m / z.

[0029] Example 2

[0030] The identification method for all components and blood-entering components in Xue Teng Qu Bi Tang (Blood Vine Nourishing Rheumatism Decoction) is as follows: (1) Preparation of XTQBD extract: Weigh out 30 g of *Sargentodoxa cuneata*, 15 g of *Acanthopanax senticosus*, 30 g of *Sargentodoxa cuneata*, 30 g of *Drynaria fortunei*, and 20 g of *Clematis chinensis* according to the prescription. Add 6 times the total weight of the herbs to water, soak for 20 minutes, decoct for 2 hours, filter, add another 6 times the total weight of the herbs to water, decoct for 1 hour, filter again, combine the two decoctions, and concentrate to a concentration of 6 g / mL to obtain the final product.

[0031] (2) Preparation of serum samples: Rats were administered XTQBD extract (1.29 g / mL, 4 mL / day, for 7 consecutive days) by gavage. One hour after the last administration, blood was collected aseptically via the abdominal aorta and centrifuged at 4°C and 3500 r / min for 10 min to obtain the experimental group sample. Rat serum administered with ultrapure water was used as the control group sample. The supernatants from both groups were mixed and stored at -80°C for later use.

[0032] (3) Preparation of the test solution: Preparation of the test solution for full component analysis: Take 600 μL of the XTQBD extract obtained in step S1 into a 1.5 mL EP tube, add 400 μL of methanol, and vortex for 10 s; take 200 μL of the mixed solution, add 200 μL of 40% methanol aqueous solution, vortex for 10 s, centrifuge at 4℃ and 16000 g for 15 min, and take the supernatant.

[0033] Preparation of test solutions for blood component analysis: Take 600 μL of each of the two serum samples obtained in step S2, add methanol, vortex mix for 60 s, let stand at -20℃ for 30 min, and then centrifuge at 4℃ and 16000 g for 20 min; take the supernatant and vacuum dry it, add 100 μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 4℃ and 16000 g for 20 min, and take the supernatant to obtain the solution.

[0034] (4) LC-MS / MS analysis: 6 μL of each of the above test solutions was injected into a liquid chromatography-mass spectrometry (LC-MS / MS) system for analysis. The specific chromatographic and mass spectrometric conditions are as follows: 1) Chromatographic conditions: The chromatographic column was an ACQUITY UPLC HSS T3 column with dimensions of 2.1 mm × 100 mm and a diameter of 1.8 μm; the column temperature was 35℃; the flow rate was 0.3 mL / min; gradient elution was performed using 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B. The gradient elution program is as follows: .

[0035] 2) Mass spectrometry conditions: ESI ion source, mass spectrometry acquisition in both positive and negative ion modes; spray voltage 3800 V (ESI+) / 3500 V (ESI-); sheath gas pressure 45 arb; auxiliary gas pressure 20 arb; ion transmission tube temperature 320℃; nebulization temperature 350℃; detection mode is full scan / data-dependent two-stage scan mode, with first-stage and second-stage resolutions of 60000 FWHM and 15000 FWHM, respectively; collision energies are normalized to step levels 20, 40, and 60; first-stage mass-to-charge ratio scan range is 90-1300 m / z.

[0036] Example 3

[0037] The identification method for all components and blood-entering components in Xue Teng Qu Bi Tang (Blood Vine Nourishing Rheumatism Decoction) is as follows: (1) Preparation of XTQBD extract: Weigh out 30 g of *Sargentodoxa cuneata*, 15 g of *Acanthopanax senticosus*, 30 g of *Sargentodoxa cuneata*, 30 g of *Drynaria fortunei*, and 20 g of *Clematis chinensis* according to the prescription. Add 8 times the total weight of the herbs to water, soak for 40 minutes, then decoct for 0.5 hours. Filter, add another 8 times the total weight of the herbs to water, decoct for 0.5 hours, and filter again. Combine the two decoctions and concentrate to a concentration of 4 g / mL to obtain the final product.

[0038] (2) Preparation of serum samples: Rats were administered XTQBD extract (1.29 g / mL, 4 mL / day, for 7 consecutive days) by gavage. One hour after the last administration, blood was collected aseptically via the abdominal aorta and centrifuged at 4°C and 3500 r / min for 10 min to obtain the experimental group sample. Rat serum administered with ultrapure water was used as the control group sample. The supernatants from both groups were mixed and stored at -80°C for later use.

[0039] (3) Preparation of the test solution: Preparation of the test solution for full component analysis: Take 600 μL of the XTQBD extract obtained in step S1 into a 1.5 mL EP tube, add 400 μL of methanol, and vortex for 10 s; take 200 μL of the mixed solution, add 200 μL of 40% methanol aqueous solution, vortex for 10 s, centrifuge at 4℃ and 16000 g for 15 min, and take the supernatant.

[0040] Preparation of test solutions for blood component analysis: Take 600 μL of each of the two serum samples obtained in step S2, add methanol, vortex mix for 60 s, let stand at -20℃ for 30 min, and then centrifuge at 4℃ and 16000 g for 20 min; take the supernatant and vacuum dry it, add 100 μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 4℃ and 16000 g for 20 min, and take the supernatant to obtain the solution.

[0041] (4) LC-MS / MS analysis: 6 μL of each of the above test solutions was injected into a liquid chromatography-mass spectrometry (LC-MS / MS) system for analysis. The specific chromatographic and mass spectrometric conditions are as follows: 1) Chromatographic conditions: The chromatographic column was an ACQUITY UPLC HSS T3 column with dimensions of 2.1 mm × 100 mm and a diameter of 1.8 μm; the column temperature was 35℃; the flow rate was 0.3 mL / min; gradient elution was performed using 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B. The gradient elution program is as follows: .

[0042] 2) Mass spectrometry conditions: ESI ion source, mass spectrometry acquisition in both positive and negative ion modes; spray voltage 3800 V (ESI+) / 3500 V (ESI-); sheath gas pressure 45 arb; auxiliary gas pressure 20 arb; ion transmission tube temperature 320℃; nebulization temperature 350℃; detection mode is full scan / data-dependent two-stage scan mode, with first-stage and second-stage resolutions of 60000 FWHM and 15000 FWHM, respectively; collision energies are normalized to step levels 20, 40, and 60; first-stage mass-to-charge ratio scan range is 90-1300 m / z.

[0043] Example 4

[0044] The identification method for all components and blood-entering components in Xue Teng Qu Bi Tang (Blood Vine Nourishing Rheumatism Decoction) is as follows: (1) Preparation of XTQBD extract: Weigh out 30 g of *Sargentodoxa cuneata*, 15 g of *Acanthopanax senticosus*, 30 g of *Sargentodoxa cuneata*, 30 g of *Drynaria fortunei*, and 20 g of *Clematis chinensis* according to the prescription. Add 7 times the total weight of the herbs to water, soak for 30 minutes, decoct for 1 hour, filter, add another 7 times the total weight of the herbs to water, decoct for 0.5 hours, filter again; combine the two decoctions, concentrate to a concentration of 5 g / mL, and the product is obtained.

[0045] (2) Preparation of serum samples: Rats were administered XTQBD extract (1.29 g / mL, 4 mL / day, for 7 consecutive days) by gavage. One hour after the last administration, blood was collected aseptically via the abdominal aorta and centrifuged at 4°C and 3500 r / min for 10 min to obtain the experimental group sample. Rat serum administered with ultrapure water was used as the control group sample. The supernatants from both groups were mixed and stored at -80°C for later use.

[0046] (3) Preparation of the test solution: Preparation of the test solution for full component analysis: Take 600 μL of the XTQBD extract obtained in step S1 into a 1.5 mL EP tube, add 400 μL of methanol, and vortex for 10 s; take 200 μL of the mixed solution, add 200 μL of 40% methanol aqueous solution, vortex for 10 s, centrifuge at 4℃ and 16000 g for 15 min, and take the supernatant.

[0047] Preparation of test solutions for blood component analysis: Take 600 μL of each of the two serum samples obtained in step S2, add methanol, vortex mix for 60 s, let stand at -20℃ for 30 min, and then centrifuge at 4℃ and 16000 g for 20 min; take the supernatant and vacuum dry it, add 100 μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 4℃ and 16000 g for 20 min, and take the supernatant to obtain the solution.

[0048] (4) LC-MS / MS analysis: 6 μL of each of the above test solutions was injected into a liquid chromatography-mass spectrometry (LC-MS / MS) system for analysis. The specific chromatographic and mass spectrometric conditions are as follows: 1) Chromatographic conditions: The chromatographic column was an ACQUITY UPLC HSS T3 column with dimensions of 2.1 mm × 100 mm and a diameter of 1.8 μm; the column temperature was 25℃; the flow rate was 0.4 mL / min; gradient elution was performed using 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B. The gradient elution program is as follows: .

[0049] 2) Mass spectrometry conditions: ESI ion source, mass spectrometry acquisition in both positive and negative ion modes; spray voltage 3800 V (ESI+) / 3500 V (ESI-); sheath gas pressure 45 arb; auxiliary gas pressure 20 arb; ion transmission tube temperature 320℃; nebulization temperature 350℃; detection mode is full scan / data-dependent two-stage scan mode, with first-stage and second-stage resolutions of 60000 FWHM and 15000 FWHM, respectively; collision energies are normalized to step levels 20, 40, and 60; first-stage mass-to-charge ratio scan range is 90-1300 m / z.

[0050] Example 5

[0051] The identification method for all components and blood-entering components in Xue Teng Qu Bi Tang (Blood Vine Nourishing Rheumatism Decoction) is as follows: (1) Preparation of XTQBD extract: Weigh out 30 g of *Sargentodoxa cuneata*, 15 g of *Acanthopanax senticosus*, 30 g of *Sargentodoxa cuneata*, 30 g of *Drynaria fortunei*, and 20 g of *Clematis chinensis* according to the prescription. Add 7 times the total weight of the herbs to water, soak for 30 minutes, decoct for 1 hour, filter, add another 7 times the total weight of the herbs to water, decoct for 0.5 hours, filter again; combine the two decoctions, concentrate to a concentration of 5 g / mL, and the product is ready.

[0052] (2) Preparation of serum samples: Rats were administered XTQBD extract (1.29 g / mL, 4 mL / day, for 7 consecutive days) by gavage. One hour after the last administration, blood was collected aseptically via the abdominal aorta and centrifuged at 4°C and 3500 r / min for 10 min to obtain the experimental group sample. Rat serum administered with ultrapure water was used as the control group sample. The supernatants from both groups were mixed and stored at -80°C for later use.

[0053] (3) Preparation of the test solution: Preparation of the test solution for full component analysis: Take 600 μL of the XTQBD extract obtained in step S1 into a 1.5 mL EP tube, add 400 μL of methanol, and vortex for 10 s; take 200 μL of the mixed solution, add 200 μL of 40% methanol aqueous solution, vortex for 10 s, centrifuge at 4℃ and 16000 g for 15 min, and take the supernatant.

[0054] Preparation of test solutions for blood component analysis: Take 600 μL of each of the two serum samples obtained in step S2, add methanol, vortex mix for 60 s, let stand at -20℃ for 30 min, and then centrifuge at 4℃ and 16000 g for 20 min; take the supernatant and vacuum dry it, add 100 μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 4℃ and 16000 g for 20 min, and take the supernatant to obtain the solution.

[0055] (4) LC-MS / MS analysis: 6 μL of each of the above test solutions was injected into a liquid chromatography-mass spectrometry (LC-MS / MS) system for analysis. The specific chromatographic and mass spectrometric conditions are as follows: 1) Chromatographic conditions: The chromatographic column was an ACQUITY UPLC HSS T3 column, with dimensions of 2.1 mm × 100 mm and a diameter of 1.8 μm; the column temperature was 40℃; the flow rate was 0.2 mL / min; gradient elution was performed using 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B. The gradient elution program is as follows: .

[0056] 2) Mass spectrometry conditions: The ESI ion source was used to collect mass spectra in both positive and negative ion modes; the spray voltage was 3800 V (ESI+) / 3500 V (ESI-); the sheath gas pressure was 45 arb; the auxiliary gas pressure was 20 arb; the temperature of the ion transfer tube was 320 °C; the nebulization temperature was 350 °C; the detection method was full scan / data-dependent MS / MS scan mode, and the resolutions of the first and second stages were 60000 FWHM and 15000 FWHM respectively; the collision energy adopted stepped normalized energy levels of 20, 40, and 60; the scan range of the first-stage mass-to-charge ratio was 90 - 1300 m / z.

[0057] To further verify the reliability of the present invention and screen out the optimal solution, the inventors conducted a series of experiments as follows: 1. Experimental materials 1.1 Experimental subjects Twelve 6-week-old female Sprague-Dawley (SD) rats, of SPF grade, with a body weight of 200 ± 20 g, were provided by the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, certificate number: SYXK (Beijing) 2024-0003.

[0058] 1.2 Reagents and instruments The main reagents are shown in Table 1, and the main instruments are shown in Table 2.

[0059] ; .

[0060] 2. Experimental methods 2.1 Preparation of XTQBD extract Weigh 30 g of Sargentodoxa cuneata, 15 g of Acanthopanax gracilistylus, 30 g of Aristolochia mollissima, 30 g of Drynaria fortunei, and 20 g of Speranskia tuberculata in proportion, add 7 times the amount of water of the medicinal materials, soak for 30 min, then decoct for 1 h, filter out the medicinal liquid with gauze, add 7 times the amount of water again and decoct for 0.5 h, filter the medicinal liquid with gauze, combine the two medicinal liquids and concentrate to a mass concentration of 5 g / mL, and store at 4 °C for later use.

[0061] 2.2 Feeding of experimental animals Rat feeding: temperature 23 ± 2 °C, humidity 35% - 60%, light / dark (12 h / 12 h) day-night alternation, adaptively fed for one week, and keep the environment dry. The rats were fed in separate cages and groups, supplied with animal drinking water and feed, and the activities, drinking water, and foraging behaviors of the rats were not restricted.

[0062] 2.3 Grouping The twelve SD rats were divided into a serum control group and an XTQBD serum group by the random number table method, and together with the XTQBD extract group, this experiment was divided into 3 groups in total.

[0063] 2.4 Administration method and dosage SD rats were administered the drug via gavage, with the rat dose being approximately 6.17 times the clinical dose for a 60 kg adult. The XTQBD serum group was administered the drug at a dose of 1.29 g / mL via gavage, 4 mL / day for 7 consecutive days. The serum control group was given an equal volume of ultrapure water.

[0064] 2.5 Preparation of serum samples One hour after the last administration, anesthesia was administered via intraperitoneal injection of 30 mg / kg pentobarbital. Blood was collected via the abdominal aorta under aseptic conditions, centrifuged at 4°C for 10 min at 3500 r / min, and the supernatants from the same group were mixed and stored at -80°C for later use.

[0065] 2.6 LC-MS / MS Analysis 2.6.1 Sample processing for use XTQBD aqueous extract: Take 600 μL of XTQBD aqueous extract into a 1.5 mL EP tube, add 400 μL of pure methanol, and vortex to mix for 10 s; take 200 μL of the above solution, add 200 μL of 40% methanol solution, vortex to mix for 10 s, centrifuge at 16000 g for 15 min at 4℃, and take the supernatant.

[0066] Serum control group and XTQBD serum group: Take an appropriate amount of serum sample, add methanol, vortex mix for 60 s, let stand at -20℃ for 30 min, and centrifuge at 16000 g for 20 min at 4℃; take the supernatant and vacuum dry it, add 100 μL of 40% methanol solution to the residue, vortex, centrifuge at 16000 g for 20 min at 4℃, and take the supernatant.

[0067] 2.6.2 On-machine testing (1) Chromatographic conditions Samples were separated using a Vanquish UHPLC ultra-high performance liquid chromatography system combined with an ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 µm) column. The injection volume was 6 μL, the column temperature was 35 °C, and the flow rate was 0.3 mL / min. Gradient elution was performed using 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid acetonitrile solution as mobile phase B, according to Table 3.

[0068] .

[0069] (2) Mass spectrometry conditions The Q-Exactive HFX mass spectrometer was used to acquire primary and secondary spectra of the samples. The Q-Exactive HFX mass spectrometer was coupled with a UHPLC system, and mass spectrometry was performed using both positive and negative electrospray ionization (ESI) modes. The spray voltage was 3800 V (ESI+) / 3500 V (ESI-), the sheath gas pressure was 45 arb, the auxiliary gas pressure was 20 arb, the ion transfer tube temperature was 320℃, and the nebulization temperature was 350℃. The detection mode was full-scan / data-dependent secondary scan (Full-MS / dd-MS2) mode, with primary and secondary resolutions of 60000 FWHM and 15000 FWHM, respectively. The top 10 MS1 ions were used to obtain MS / MS spectra, and the collision energies (CEs) were normalized to levels 20, 40, and 60. The primary mass-to-charge ratio scan range was 90–1300 m / z.

[0070] 2.6.3 Chromatographic Condition Investigation Experiment (1) Selection of mobile phase The chromatographic separation effect under different aqueous and organic phase compositions was investigated using pure water, 0.1% formic acid, 0.2% formic acid, and 0.3% formic acid as the aqueous phase, and methanol and acetonitrile as the organic phase, respectively. Finally, 0.1% formic acid aqueous solution was determined as mobile phase A and 0.1% formic acid acetonitrile solution as mobile phase B, and chromatographic separation was performed according to the chromatographic conditions and gradient elution program in section (1) of "2.6.1 Instrumental Detection". Under these conditions, the chromatographic peaks of each component were symmetrical and well-separated, meeting the analytical requirements.

[0071] (2) Selection of column temperature and flow rate This study investigated the effects of different column temperatures (25℃, 30℃, 35℃, 40℃) and flow rates (0.2 mL / min, 0.3 mL / min, 0.4 mL / min) on chromatographic separation. The results showed that at a column temperature of 35℃ and a flow rate of 0.3 mL / min, the obtained chromatographic peaks exhibited good shape, number, and resolution.

[0072] 2.7 Data Processing After converting the data format, software was used for peak alignment, retention time correction, and peak extraction. Compound identification was performed by searching the CAS New Life Local Traditional Chinese Medicine High-Resolution Mass Spectrometry Database. The first-order mass error was less than 25 ppm, and the second-order fragmentation spectrum matching score was greater than 0.7. A higher matching score indicates higher spectral similarity; generally, a score above 0.7 is considered reliable.

[0073] 3. Experimental Results 3.1 Evaluation of Experimental Data This experiment evaluated the stability and data reliability of the instrument, and performed Pearson correlation analysis on the detection results of traditional Chinese medicine samples. The correlation of samples under positive and negative ion modes is shown in [the table below]. Figure 1 Generally, a correlation coefficient greater than 0.9 indicates a good correlation. The results of this experiment show that the correlation coefficients between samples are all above 0.9, indicating good repeatability and stable, reliable data.

[0074] 3.2 Chromatograms of positive and negative total ion peaks for each group of samples This experiment used UHPLC-HRMS to collect data from the XTQBD extract group, serum control group, and XTQBD serum group, and compared their base peak chromatograms (BPC). Figure 2 As shown in the spectrum, it is clear from the spectrum that there are certain differences between the XTQBD extract group, the serum control group, and the XTQBD serum group.

[0075] 3.3 Identification of Chemical Composition of XTQBD The chemical components in the XTQBD extract were analyzed and identified. Compounds identified in this experiment (including those identified by both positive and negative ions) were annotated according to the NPClassifier classification method, with the number of compounds identified in each pathway and major superclass. The NPClassifier classification system divides the structure of natural products into three levels: 7 pathways, 70 superclasses, and 672 classes. The pathways include fatty acids, polyketides, shikimates and phenylpropanoids, terpenoids, alkaloids, amino acids and peptides, and carbohydrates, used to represent different biosynthetic pathways. A natural product can belong to multiple pathways. Superclasses represent subcategories of pathways, representing general categories of metabolites (e.g., flavonoids, terpenoids, or steroids), general chemical / molecular shapes (e.g., tryptophan, macrolides), or biosynthetic information (e.g., tryptophan alkaloids, aromatic polyketides, or pseudoalkaloids). Classes are further subdivided from superclasses, representing specific compound families (e.g., erythromycin, penicillin, or cannabinoids), characteristic functional groups (e.g., chromones, isoflavones, or indole alkaloids), or superclass skeletal diversity (e.g., flavanones, flavonoids, and chalcones among flavonoids). Statistical results showed that 2444 were detected and identified in the XTQBD extract under positive ion mode and 1477 under negative ion mode, resulting in a total of 3798 chemical components after removing duplicates. According to Pathway statistics, Alkaloids comprise 793 (23%), Terpenoids comprise 658 (19%), Shikimates and Phenylpropanoids comprise 894 (26%), Fatty acids comprise 411 (12%), Aminoacids and Peptides comprise 280 (8%), Polyketides comprise 250 (7%), Carbohydrates comprise 94 (3%), and there are 111 other substances (3%). Table 4 further shows the top 5 substances with the highest count values ​​in each category's superclass within the Pathway.

[0076] .

[0077] 3.4 Identification of XTQBD components entering the bloodstream Comparing the XTQBD decoction extract group with the XTQBD serum group, 352 compounds were identified in positive ion mode and 280 compounds were identified in negative ion mode. After removing duplicates, a total of 626 chemical components were identified that entered the bloodstream. According to Pathway statistics, Alkaloids accounted for 147 (24%), Terpenoids for 134 (22%), Shikimates and Phenylpropanoids for 147 (24%), Fatty acids for 28 (5%), Aminoacids and Peptides for 49 (8%), Polyketides for 54 (9%), and Carbohydrates for 18 (3%). Table 5 further shows the top 5 most frequently counted substances in the superclass of each category in Pathway and the top 3 substances with the highest count in the class.

[0078] ; ; ; In the BPC chromatogram of the XTQBD decoction extract, peaks eluted within 2-18 min, with a median peak area of ​​1×10⁻⁶. 9 The above substances. Further comparison with the serum group showed that substances with a median peak area three times or more than that in the XTQBD serum group were identified as the main components of XTQBD entering the bloodstream. A total of 20 main components were found, and detailed information on these substances is shown in Table 6. The 20 chromatographic peaks were marked on the BPC chromatogram of the XTQBD extract, and their secondary mass spectra are shown below. Figures 3-4 As shown, the overall positive and negative ion diagram is as follows: Figure 5 As shown.

[0079] .

[0080] 4. Discussion The pharmacodynamic material basis of traditional Chinese medicine (TCM) compound formulas is the core of their clinical efficacy. Clarifying the in vitro and in vivo chemical composition of these formulas, as well as their active components entering the bloodstream, is crucial for revealing their pharmacodynamic mechanisms, optimizing compatibility, and establishing a scientific quality control system. XTQBD is an effective TCM formula for treating rheumatoid arthritis (RA). Its chemical composition is complex and diverse, with numerous isomers and a wide range of component content and polarity, posing many challenges to the study of its pharmacodynamic material basis. This study used LC-MS / MS technology to systematically analyze the in vitro extract of XTQBD and its components entering the bloodstream of animals, elucidating the material basis of XTQBD's therapeutic effect on RA.

[0081] 4.1 Diversity and characteristics of in vitro chemical components of XTQBD The clinical efficacy of traditional Chinese medicine (TCM) compound formulas relies on the synergistic effects of multiple chemical components, and comprehensive characterization of their in vitro chemical components is a prerequisite for research on the pharmacodynamic material basis. This study, using UPLC-HRMS technology combined with the NPClassifier classification system, identified a total of 3798 chemical components in the XTQBD extract, covering seven major biosynthetic pathways: fatty acids, polyketides, shikimate-phenylpropanoids, terpenes, alkaloids, amino acids / peptides, and carbohydrates. This fully demonstrates the "multi-component, multi-target" characteristics of this compound formula.

[0082] In terms of compound category distribution, shikimate-phenylpropanoids accounted for the highest proportion (26%), with 239 flavonoids among them. These components are widely present in traditional Chinese medicine and have been proven to have significant anti-inflammatory, antioxidant, and immunomodulatory effects. The pathological process of RA is closely related to excessive activation of the inflammatory response and imbalance of oxidative stress. The abundant flavonoids in XTQBD may exert their anti-inflammatory effects by inhibiting the release of inflammatory mediators such as prostaglandin E2 (PGE2) and scavenging oxidative free radicals. Alkaloids (23%) and terpenes (19%) were the second and third dominant components, respectively. Among the alkaloids, tryptophan alkaloids (105) and tyrosine alkaloids (93) and among the terpenes, steroids (154) and sesquiterpenes (124) have been reported in previous studies to exert anti-inflammatory, analgesic, and immune cell-regulating pharmacological activities by regulating the balance of Janus kinase 2 (JAK2), osteoprotegerin (OPG), and T helper 17 / regulatory T cell (Th17 / Treg). It is speculated that they may be an important material basis for XTQBD in the treatment of RA. Furthermore, the presence of fatty acids (12%), amino acids / peptides (8%), polyketides (7%), and carbohydrates (3%) further enriches the chemical composition of this compound. These components may exert their therapeutic effects indirectly through pathways such as participating in energy metabolism, regulating intestinal flora balance, and enhancing the body's immunity, demonstrating the overall advantage of the synergistic effect of multiple components in traditional Chinese medicine compound formulas. This experiment systematically categorized the biosynthetic pathways, superclasses, and specific categories of XTQBD compounds, clearly presenting the distribution characteristics of various components. This provides a clear direction for subsequent targeted screening of active ingredients and lays a chemical foundation for a deeper understanding of the compatibility of XTQBD.

[0083] 4.2 Screening of XTQBD Components Entering the Bloodstream and Correlation Analysis with Efficacy After oral administration of traditional Chinese medicine, only the original components or their metabolites absorbed into the bloodstream can exert direct pharmacological effects in the body. Therefore, the identification of blood-injected components is a core step in revealing the material basis of pharmacodynamic efficacy. This study identified 626 blood-injected components by comparing the compositional differences between XTQBD extract and serum samples. Among them, alkaloids, shikimate-phenylpropanoids, and terpenoids accounted for 24%, 24%, and 22% respectively, becoming the three dominant categories of blood-injected components. This is largely consistent with the dominant categories of in vitro chemical components, suggesting that these three categories of components have good oral bioavailability and may be the core substance group for XTQBD to exert its pharmacological effects in vivo.

[0084] Further screening identified 20 key blood-entry components, whose peak areas were significantly higher than those of serum background components, indicating stronger in vivo exposure characteristics, suggesting they are the key pharmacodynamic components of this formula. Among them, the coumarin compounds 7-Hydroxy-4-(methoxymethyl)coumarin and 7,8-Dimethoxycoumarin have been shown to exert anti-inflammatory effects by inhibiting the activation of the nuclear factor kappa-B (NF-κB) signaling pathway, reducing the release of pro-inflammatory cytokines such as TNF-α and IL-6. The terpene component Toddalolactone has clear anti-inflammatory and analgesic activity, and can alleviate inflammatory responses by regulating the mitogen-activated protein kinase (MAPK) signaling pathway. The alkaloid component Tyramine can participate in regulating the neuro-immune-endocrine network, indirectly improving the immune dysregulation state in RA patients. The pharmacological activities of these components are highly consistent with the pathological mechanisms of RA, further confirming the close correlation between the blood-entry components screened in this study and the anti-RA efficacy of XTQBD.

[0085] 4.3 Comprehensive Analysis of the Pharmacodynamic Material Basis of XTQBD Combined with the identification results of in vitro chemical components and blood components, a pharmacodynamic substance network for XTQBD in the treatment of RA can be preliminarily constructed. Among the 3,798 chemical components identified in vitro, some components were not absorbed into the blood due to reasons such as excessive molecular weight, unsuitable polarity, or the existence of first-pass effect. However, they may play a local role in the intestine, such as regulating the intestinal flora structure and protecting the intestinal mucosal barrier, and indirectly participate in immune regulation. Among the 626 components absorbed into the blood, especially the 20 main blood components, they reach the lesion site through blood circulation and directly play a pharmacological role. Core components such as flavonoids, alkaloids, and terpenoids may achieve the anti-RA effect through the synergistic effects of multiple targets, such as inhibiting inflammatory signaling pathways, regulating the functions of immune cells, antioxidative stress, and inhibiting osteoclast activity. In addition, the sources and compatibility logics of some blood components in XTQBD deserve attention. As an ethnic medicine compound, the synergistic effect of its formula herbs may be achieved through the complementarity of chemical components. For example, flavonoid and alkaloid components from different herb sources cooperate with each other to enhance the anti-inflammatory effect; amino acid / peptide components provide nutritional support for the body and assist in the repair of immune function. This discovery provides a new idea for optimizing the formula compatibility and improving the clinical efficacy, and also provides a chemical basis for the modern interpretation of ethnic medicine.

[0086] 5. Summary In this study, UPLC-HRMS coupling technology was used to perform LC-MS / MS analysis on the active components and blood components of XTQBD. The experimental results showed that the chemical composition of XTQBD covered various compounds such as fatty acids, polyketides, shikimate ester-phenylpropanoids, terpenoids, alkaloids, amino acids / peptides, and carbohydrates, suggesting that its therapeutic effect on RA stems from the synergistic effects of multiple components. Further screening and identification of 20 main blood components, which are very likely to be the key substances for XTQBD to exert anti-RA pharmacodynamic effects, thus revealing the pharmacodynamic substance basis of this formula in the treatment of RA. This experiment preliminarily clarified the substance basis of XTQBD in the treatment of RA, provided a theoretical basis for its pharmacological mechanism research, and laid a foundation for further exploring its mechanism of action.

[0087] Although the present invention has been described in detail above with general descriptions, specific embodiments and experiments, some modifications or improvements can be made based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A method for identifying all components and blood-entering components of a Xue Teng Qu Bi Tang (Blood Vine Nourishing Pain Relief Decoction), wherein the prescription of Xue Teng Qu Bi Tang consists of the following components in parts by weight: 30 parts of large-stemmed vine, 15 parts of Acanthopanax bark, 30 parts of *Ligusticum striatum*, 30 parts of Drynaria fortunei, and 20 parts of Clematis armandii; The method is characterized by the following steps: preparing XTQBD extract, identifying all components of the Xue Teng Qu Bi Tang (a traditional Chinese medicine formula) using UPLC-MS / MS; administering XTQBD extract to rats by gavage, collecting serum samples after administration as the experimental group, and using rat serum administered with ultrapure water as the control group; identifying the blood-entering components of the Xue Teng Qu Bi Tang using UPLC-MS / MS; the specific steps are as follows: Preparation of S1 and XTQBD extracts: Weigh out the following ingredients according to the prescription: large-stemmed vine, five-adzu root bark, blood-sucking herb, bone-drying herb, and clematis root. Add water and decoct twice. Combine the decoctions and concentrate them to obtain the final product. Preparation of S2 serum samples: Rats were administered XTQBD extract by gavage, and serum was collected at predetermined times as the experimental group sample, while the serum of rats given ultrapure water was used as the control group sample. S3. Preparation of the test solution: Preparation of test solution for full component identification: Take the XTQBD extract obtained in step S1, add methanol, and vortex to mix; take the mixed solution, add 40% methanol aqueous solution, vortex to mix, centrifuge, and take the supernatant to obtain the solution; Preparation of test solution for blood component identification: Take the serum sample obtained in step S2, add methanol, vortex mix, let stand at low temperature, and then centrifuge; take the supernatant and vacuum dry, add the residue to 40% methanol aqueous solution, vortex, centrifuge, and take the supernatant to obtain the solution; S4, LC-MS / MS analysis: Each of the above test solutions was separately injected into a liquid chromatography-mass spectrometry (LC-MS / MS) system for analysis.

2. The method for identifying all components and blood-entering components of the Xue Teng Qu Bi Decoction according to claim 1, characterized in that, Step S1 is as follows: Weigh out the following herbs according to the prescription: large-leaved vine, five-leaved bark, blood-sucking herb, bone-drying herb, and clematis root. Add 6-8 times the total weight of the herbs in water, soak for 20-40 minutes, then decoct for 0.5-2 hours. Filter, then add another 6-8 times the total weight of the herbs in water, decoct for 0.5-1 hours, and filter. Combine the two decoctions and concentrate to a concentration of 4-6 g / mL to obtain the final product.

3. The method for identifying all components and blood-entering components of the Xue Teng Qu Bi Decoction according to claim 2, characterized in that, Step S1 is as follows: Weigh out the following herbs according to the prescription: large blood vine, five-adzu root bark, blood-sucking herb, bone-drying herb, and clematis root. Add water equal to 7 times the total weight of the herbs, soak for 30 minutes, decoct for 1 hour, filter, add water equal to 7 times the total weight of the herbs again, decoct for 0.5 hours, filter, combine the two decoctions, and concentrate to a concentration of 5 g / mL to obtain the final product.

4. The method for identifying all components and blood-entering components of the Xue Teng Qu Bi Decoction according to claim 1, characterized in that, The preparation of the test solution for full component identification in step S3 is as follows: take 600 μL of the XTQBD extract obtained in step S1 into a 1.5 mL EP tube, add 400 μL of methanol, and vortex mix for 10 s. Take 200 μL of the mixed solution, add 200 μL of 40% methanol aqueous solution, vortex to mix for 10 s, centrifuge at 4℃ for 15 min, and take the supernatant to obtain the solution.

5. The method for identifying all components and blood-entering components of the Xue Teng Qu Bi Decoction according to claim 1, characterized in that, The preparation of the test solution for identifying blood components in step S3 is as follows: Take 600 μL of the serum sample obtained in step S2, add methanol, vortex mix for 60 s, let stand at -20℃ for 30 min, and then centrifuge at 4℃ for 20 min; take the supernatant and vacuum dry it, add 100 μL of 40% methanol aqueous solution to the residue, vortex, centrifuge at 4℃ for 20 min, and take the supernatant to obtain the solution.

6. The method for identifying all components and blood-entering components of the Xue Teng Qu Bi Decoction according to any one of claims 4 or 5, characterized in that, The relative centrifugal force in step S3 is 16000 g.

7. The method for identifying all components and blood-entering components of the Xue Teng Qu Bi Decoction according to claim 1, characterized in that, The specific chromatographic conditions for step S4 are as follows: The chromatographic column was an ACQUITY UPLC HSS T3 column, with dimensions of 2.1 mm × 100 mm and a diameter of 1.8 μm; the column temperature was 25℃–40℃; the flow rate was 0.2–0.4 mL / min; the injection volume was 6 μL; gradient elution was performed using 0.1% formic acid aqueous solution as mobile phase A and 0.1% formic acid acetonitrile as mobile phase B. The gradient elution program was as follows: 。 8. The method for identifying all components and blood-entering components of the Xue Teng Qu Bi Decoction according to claim 7, characterized in that, In the chromatographic conditions of step S4, the column temperature is 35°C.

9. The method for identifying all components and blood-entering components of the Xue Teng Qu Bi Decoction according to claim 7, characterized in that, In the chromatographic conditions of step S4, the flow rate is 0.3 mL / min.

10. The method for identifying all components and blood-entering components of the Xue Teng Qu Bi Decoction according to claim 1, characterized in that, The specific mass spectrometry conditions for step S4 are as follows: ESI ion source, mass spectrometry acquisition in both positive and negative ion modes; spray voltage 3800 V (ESI+) / 3500 V (ESI-); sheath gas pressure 45 arb; auxiliary gas pressure 20 arb; ion transmission tube temperature 320℃; nebulization temperature 350℃; detection mode is full scan / data-dependent secondary scan mode, with primary and secondary resolutions of 60000 FWHM and 15000 FWHM, respectively. The collision energies were normalized to step levels 20, 40, and 60; the first-order mass-to-charge ratio scan range was 90-1300 m / z.

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