Construction method and detection method of drug effect substance basic database of abnormal sweating stopping traditional Chinese medicine preparation

Abstract

The invention relates to the technical field of drug analysis, in particular to a construction method and a detection method of a drug effect substance basic database of a general debility sweating stopping traditional Chinese medicine preparation, and the construction method comprises the following steps: extracting the general debility sweating stopping traditional Chinese medicine preparation to obtain an extracting solution of the general debility sweating stopping traditional Chinese medicine preparation; constructing a general debility perspiration stopping traditional Chinese medicine preparation administration animal model, and collecting serum of an experimental animal according to set time; purifying the serum to obtain test serum; performing ultra-high performance liquid chromatography-linear ion trap-orbitrap mass spectrum tandem detection on the extracting solution of the abnormal sweating stopping traditional Chinese medicine preparation and the test serum to obtain chemical compositions and mass spectrum information of the abnormal sweating stopping traditional Chinese medicine preparation and the test serum; and comparing the chemical compositions and mass spectrum information of the abnormal sweating stopping traditional Chinese medicine preparation and the test serum, and identifying prototype components and metabolites of the abnormal sweating stopping traditional Chinese medicine preparation. The detection method provided by the invention can be used for more comprehensively and scientifically evaluating the pharmacodynamic material basis of the abnormal sweating stopping traditional Chinese medicine preparation in the to-be-detected sample.

Description

Technical Field

[0001] The present application relates to the technical field of drug analysis, and in particular to a method for constructing and detecting a basic database of pharmacodynamic substances of a Xuhanting Chinese medicine preparation. Background Art

[0002] The material basis of the efficacy of Chinese medicine preparations includes the original ingredients and metabolites of Chinese medicine. The original ingredients of Chinese medicine refer to the ingredients that can be absorbed into the body and may directly participate in the efficacy of the medicine. The metabolites are metabolites formed by the original ingredients that enter the body through various metabolic pathways, such as hydrolysis, desugaring, glucuronidation and sulfation products. By detecting the original ingredients and their metabolites, a scientific basis can be provided for the quality control of Chinese medicine, clarifying which ingredients can enter the body and exert their effects, which helps to optimize the production process of Chinese medicine preparations and improve the effectiveness and safety of drugs.

[0003] Xuhanting Chinese medicine preparation is composed of astragalus, floating wheat, jujube, glutinous rice root, and oyster (calcined). It nourishes qi and yin, consolidates the exterior and restrains sweat. It is used for spontaneous sweating, night sweating and night sweating in children due to qi and yin deficiency. There have been certain research reports on the quality control, pharmacodynamics and clinical application of Xuhanting Chinese medicine preparation, but the pharmacodynamic material basis of Xuhanting Chinese medicine preparation has not been elucidated. At present, the detection method for Xuhanting Chinese medicine preparation basically detects astragaloside IV (the main active ingredient of astragalus), fails to fully consider the dynamic changes of the active ingredients of Chinese medicine in the body, and does not conduct targeted detection of the real pharmacodynamic material basis of the therapeutic effect in the body. Summary of the invention

[0004] Based on this, one or more embodiments of the present application provide a method for constructing and detecting a database of the pharmacological substance basis of the Xuhanting Chinese medicine preparation, which can obtain more prototype components and metabolites and conduct a more comprehensive detection of the pharmacological substance basis of the sample to be tested.

[0005] Specifically, the technical solution of this application is as follows:

[0006] A method for constructing a database of the pharmacological substance basis of a Xuhanting Chinese medicine preparation, wherein the pharmacological substance basis of the Xuhanting Chinese medicine preparation includes its prototype components and metabolites;

[0007] The construction method comprises the following steps:

[0008] Extracting the Xuhanting Chinese medicine preparation to obtain an extract of the Xuhanting Chinese medicine preparation;

[0009] Establish an animal model of Xuhanting Chinese medicine preparation administration, and collect serum from experimental animals at the planned time;

[0010] Purifying the serum to obtain test serum;

[0011] The extract of the Xuhan Ting traditional Chinese medicine preparation and the test serum are subjected to ultra-high performance liquid chromatography-linear ion trap-orbital trap mass spectrometry tandem detection to obtain the chemical compositions and their mass spectrometry information of the Xuhan Ting traditional Chinese medicine preparation and the test serum;

[0012] The chemical compositions and their mass spectrometry information of the Xuhan Ting traditional Chinese medicine preparation and the test serum are compared to identify the prototype components and metabolites of the Xuhan Ting traditional Chinese medicine preparation, and a database of the pharmacodynamic material basis of the Xuhan Ting traditional Chinese medicine preparation is constructed;

[0013] Among them, the conditions for the ultra-high performance liquid chromatography detection include: mobile phase A is an aqueous solution of formic acid, and mobile phase B is acetonitrile; a gradient elution program is adopted; the gradient elution program includes: from 0 min to 10 min, the volume percentage of mobile phase A changes from 97% to 87%; from 10 min to 80 min, the volume percentage of mobile phase A changes from 87% to 55%; from 80 min to 110 min, the volume percentage of mobile phase A changes from 55% to 15%; from 110 min to 120 min, the volume percentage of mobile phase A changes from 15% to 5%.

[0014] A method for detecting the pharmacodynamic material basis of a Xuhan Ting traditional Chinese medicine preparation includes the following steps:

[0015] The database of the pharmacodynamic material basis of the Xuhan Ting traditional Chinese medicine preparation is obtained by using the construction method described above;

[0016] Take a test sample for ultra-high performance liquid chromatography-linear ion trap-orbital trap mass spectrometry tandem detection to obtain the chemical composition and its mass spectrometry information of the test sample;

[0017] The chemical composition and its mass spectrometry information of the test sample are compared with the database of the pharmacodynamic material basis of the Xuhan Ting traditional Chinese medicine preparation to identify the pharmacodynamic material basis of the Xuhan Ting traditional Chinese medicine preparation in the test sample;

[0018] Among them, the conditions for the ultra-high performance liquid chromatography detection include: mobile phase A is an aqueous solution of formic acid, and mobile phase B is acetonitrile; a gradient elution program is adopted; the gradient elution program includes: from 0 min to 10 min, the volume percentage of mobile phase A changes from 97% to 87%; from 10 min to 80 min, the volume percentage of mobile phase A changes from 87% to 55%; from 80 min to 110 min, the volume percentage of mobile phase A changes from 55% to 15%; from 110 min to 120 min, the volume percentage of mobile phase A changes from 15% to 5%.

[0019] The method for constructing the pharmacodynamic substance basis database of the traditional Chinese medicine preparation for stopping sweating of the present application constructs an animal model administered with the traditional Chinese medicine preparation for stopping sweating. Taking the purified serum of experimental animals and the extract of the traditional Chinese medicine preparation for stopping sweating as the detection objects, ultra-high performance liquid chromatography-linear ion trap-orbital trap mass spectrometry tandem detection is adopted to obtain the chemical compositions and their mass spectrometry information of the traditional Chinese medicine preparation for stopping sweating and the test serum. By comparing the chemical compositions and their mass spectrometry information of the traditional Chinese medicine preparation for stopping sweating and the test serum, the prototype components and metabolites of the traditional Chinese medicine preparation for stopping sweating are identified. This detection method breaks through the limitation of the traditional detection method which is limited to a single active ingredient, astragaloside IV, helps to establish a perfect quality control system for the product, and further guarantees the drug quality and clinical pharmacodynamic effect.

[0020] The detection method for the active ingredients of the traditional Chinese medicine preparation for stopping sweating of the present application can be used to simultaneously detect multiple prototype components and metabolite components, with good separation of each characteristic peak and accurate detection results. Brief Description of the Drawings

[0021] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 It is a comparison chart of the total ion chromatograms in the negative ion mode of the sweating-stopping granules extracted with different extraction solvents (pure water, 100% methanol, 100% ethanol, 50% methanol, and 50% ethanol) in Example 1 of the present application;

[0023] Figure 2 It is a comparison chart of the total ion chromatograms in the negative ion mode of the sweating-stopping granules extracted with different extraction times (30 minutes, 60 minutes, and 120 minutes) in Example 1 of the present application;

[0024] Figure 3 It is a comparison chart of the extracted ion chromatograms of astragaloside IV in serum samples optimized with three different organic solvents (methanol, acetonitrile, and acetone) in Example 1 of the present application;

[0025] Figure 4 It is a comparison chart of the extracted ion chromatograms of calycosin-7-O-β-D-glucoside in serum samples optimized with three different organic solvents (methanol, acetonitrile, and acetone) in Example 1 of the present application;

[0026] Figure 5This is a comparison chart of the total ion current chromatograms of the mixed reference substance (A) and Xuhan Ting granules (B) detected by UPLC-ESI-LTQ-Orbitrap MS / MS in Example 1 of this application under the negative ESI mode;

[0027] Figure 6 This is a comparison chart of the total ion current chromatograms of the mixed reference substance (A) and in vivo serum samples of Xuhan Ting granules (B) detected by UPLC-ESI-LTQ-Orbitrap MS / MS in Example 1 of this application under the negative ESI mode;

[0028] Figure 7 This is a schematic diagram of the metabolic process of Astragaloside IV in serum in Example 1 of this application;

[0029] Figure 8 This is a schematic diagram of the metabolic process of Calycosin in serum in Example 1 of this application;

[0030] Figure 9 This is a schematic diagram of the metabolic process of Zizybeoside I in serum in Example 1 of this application;

[0031] Figure 10 This is a schematic diagram of the metabolic process of Apigenin-7-apioglucoside in serum in Example 1 of this application. Detailed implementation manners

[0032] The present application will be further elaborated below in combination with the implementation manners and examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. In addition, it should be understood that after reading the content taught in the present application, those skilled in the art can make various changes or modifications to the present application, and these equivalent forms also fall within the protection scope of the appended claims of the present application.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0034] Terms

[0035] Unless otherwise stated or there are contradictions, the terms or phrases used herein have the following meanings:

[0036] As used herein, the term "and / or" encompasses any one of two or more of the related listed items, as well as any and all combinations of the related listed items. The said any and all combinations include combinations of any two of the related listed items, any more of the related listed items, or all of the related listed items. It should be noted that when at least three items are connected by at least two conjunctive combinations selected from "and / or", "or / and", "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B.

[0037] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, as well as an open technical solution containing the listed features.

[0038] In this application, regarding the numerical interval (i.e., numerical range), unless otherwise specified, the optional numerical values are considered continuous within the above numerical interval, and include the two numerical endpoints (i.e., the minimum value and the maximum value) of this numerical range, as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within this numerical interval, it includes the two endpoint integers of this numerical range, as well as each integer between the two endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0039] In this application, the weight can be mass units well-known in the pharmaceutical field such as μg, mg, g, kg, etc.

[0040] A method for constructing a database of the effective substance basis of a traditional Chinese medicine preparation for stopping sweating. The effective substance basis of the traditional Chinese medicine preparation for stopping sweating includes its prototype components and metabolites;

[0041] The construction method includes the following steps:

[0042] Subject the traditional Chinese medicine preparation for stopping sweating to extraction treatment to obtain an extract of the traditional Chinese medicine preparation for stopping sweating;

[0043] Construct an animal model administered with the traditional Chinese medicine preparation for stopping sweating, and collect the serum of the experimental animals at the scheduled time;

[0044] Purify the serum to obtain the test serum;

[0045] Perform ultra-high performance liquid chromatography-linear ion trap-orbitrap mass spectrometry tandem detection on the extract of the traditional Chinese medicine preparation for stopping sweating and the test serum to obtain the chemical composition and its mass spectrometry information of the traditional Chinese medicine preparation for stopping sweating and the test serum;

[0046] Compare the chemical compositions and mass spectrometry information of the Xuhan Ting traditional Chinese medicine preparation and the test serum to identify the prototype components and metabolites of the Xuhan Ting traditional Chinese medicine preparation, and construct a database of the effective substance basis of the Xuhan Ting traditional Chinese medicine preparation;

[0047] Among them, the conditions for ultra-high performance liquid chromatography detection include: mobile phase A is an aqueous solution of formic acid, and mobile phase B is acetonitrile; a gradient elution program is adopted; the gradient elution program includes: from 0 min to 10 min, the volume percentage of mobile phase A changes from 97% to 87%; from 10 min to 80 min, the volume percentage of mobile phase A changes from 87% to 55%; from 80 min to 110 min, the volume percentage of mobile phase A changes from 55% to 15%; from 110 min to 120 min, the volume percentage of mobile phase A changes from 15% to 5%.

[0048] In some embodiments, the composition of the Xuhan Ting traditional Chinese medicine preparation includes Astragalus membranaceus, Fructus Tritici Levis, Fructus Jujubae, Rhizoma Oryzae Glutinosae and Concha Ostreae Usta.

[0049] In some embodiments, the effective substance basis of the Xuhan Ting traditional Chinese medicine preparation includes prototype components and metabolites. The prototype components include Calycosin-7-glucoside, Ononin, Astraisoflavan-7-O-β-D-glucoside, Calycosin, Astragaloside IV, Isoastragaloside II, Astragaloside II, Zizybeoside I and Apigenin-7-apioglucoside;

[0050] The parent compounds of the metabolites sequentially include: AstragalosideIV, AstragalosideII, Apiin, Astraisoflavan-7-O-β-D-glucoside, Ononin, ZizybeosideI, ZizybeosideI, AstragalosideIV, AstragalosideIV, AstragalosideII, Astraisoflavan-7-O-β-D-glucoside, Ononin, ZizybeosideI, AstragalosideIV, AstragalosideII, Astraisoflavan-7-O-β-D-glucoside, Ononin, ZizybeosideI, AstragalosideIV, ZizybeosideI, ZizybeosideI, AstragalosideIV, AstragalosideII, AstragalosideIV, AstragalosideIV, AstragalosideII, AstragalosideIV, Apiin, Apiin, Apiin, AstragalosideIV, ZizybeosideI, AstragalosideIV, AstragalosideII, AstragalosideIV, AstragalosideII, ZizybeosideI, AstragalosideII, calycosin, Formononetin, Ononin, Ononin, Ononin, Ononin, Ononin, calycosin, calycosin, calycosin, Apiin, Ononin, Ononin, AstragalosideIV, AstragalosideII, AstragalosideII, AstragalosideIV, AstragalosideII, AstragalosideIV, AstragalosideII, AstragalosideII, ZizybeosideI, AstragalosideII, Ononin, ZizybeosideI, AstragalosideII, AstragalosideIV, Astraisoflavan-7-O-β-D-glucoside, AstragalosideIV, AstragalosideII, Astraisoflavan-7-O-β-D-glucoside,Ononin, Zizybeoside I, Astragaloside II, Apiin, Astragaloside II, Zizybeoside I, Astragaloside II, Astraisoflavan-7-O-β-D-glucoside, Zizybeoside I, Zizybeoside I, Astragaloside II, Zizybeoside I, Astraisoflavan-7-O-β-D-glucoside, Astragaloside II, Astragaloside IV, Astragaloside II, Astragaloside IV, Astragaloside II, Apiin, Astraisoflavan-7-O-β-D-glucoside, Zizybeoside I, Astraisoflavan-7-O-β-D-glucoside, Zizybeoside I, Astragaloside II, Zizybeoside I, Astragaloside IV, Astragaloside II, Zizybeoside I, Astragaloside IV, Zizybeoside I, Astragaloside IV, Astragaloside II, Zizybeoside I, Astragaloside II, Zizybeoside I, Zizybeoside I, Zizybeoside I and Zizybeoside I.,

[0051] In some embodiments, the step of extracting the Xuhan Ting traditional Chinese medicine preparation to obtain the extract of the Xuhan Ting traditional Chinese medicine preparation includes:

[0052] After mixing the Xuhan Ting traditional Chinese medicine preparation and the extraction solvent, perform ultrasonic treatment to obtain a mixed solution;

[0053] Perform solid-liquid separation on the mixed solution, collect the liquid, and obtain the extract of the Xuhan Ting traditional Chinese medicine preparation.

[0054] In some embodiments, the extraction solvent is an aqueous solution of alcohol; optionally, the volume ratio of alcohol in the aqueous solution of alcohol is 40% - 60%; examples of the volume ratio of alcohol in the aqueous solution of alcohol are 40%, 50%, 60%, etc.

[0055] In some embodiments, the mass-volume ratio of the Xuhan Ting granules to the extraction solvent can be selected from 500 mg: 4 - 6 mL; examples of the mass-volume ratio of the Xuhan Ting granules to the extraction solvent are 125 mg: 1 mL, 100 mg: 1 mL, 250 mg: 3 mL, etc.

[0056] In some embodiments, the ultrasonic power is 130W to 140W, the frequency is 40kHz to 45kHz, and the time is 20min to 40min. Alternatively, the ultrasonic power can be selected from 130W to 140W, such as 130W, 135W, 140W, etc., the ultrasonic frequency can be 40kHz to 45kHz, such as 40kHz, 41kHz, 42kHz, 43kHz, 44kHz, 45kHz, etc., and the ultrasonic time can be 20min to 40min, such as 20min, 25min, 30min, 35min, 40min, etc.

[0057] In some embodiments, the chromatographic column is an octadecylsilane bonded silica gel column.

[0058] In some embodiments, the flow rate is 0.2 mL / min to 0.4 mL / min. Alternatively, the flow rate may be 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, etc.

[0059] In some embodiments, the injection volume is 1 μL to 3 μL. Alternatively, the injection volume can be 1 μL, 2 μL, 3 μL, etc.

[0060] In some embodiments, the column temperature of the chromatographic column is 30° C. to 40° C. Alternatively, the column temperature may be 30° C., 35° C., 40° C., etc.

[0061] In some embodiments, the length of the chromatographic column is 140 mm to 160 mm, the diameter is 2 mm to 2.2 mm, and the particle size of the filler is 2 μm to 4 μm.

[0062] In some embodiments, the conditions for linear ion trap-orbital trap mass spectrometry detection include: using negative ion mode, the ion spray voltage is -4kV to -6kV; the capillary temperature is 300°C to 400°C; the capillary voltage is 30V to 40V; the tube lens voltage is 90V to 110V; the sheath gas and auxiliary gas are nitrogen; and the collision gas is helium.

[0063] In some embodiments, the conditions for linear ion trap-orbitrap mass spectrometry detection include: using negative ion mode, ion spray voltage is -5kV; capillary temperature is 350°C; capillary voltage is 35V; tube lens voltage is 100V; sheath gas and auxiliary gas are nitrogen; collision gas is helium.

[0064] In some embodiments, the step of comparing the chemical composition and mass spectrometry information of the Xuhanting Chinese medicine preparation and the test serum to identify the prototype components and metabolites of the Xuhanting Chinese medicine preparation comprises:

[0065] Compare the chemical compositions of the Xuhan Ting traditional Chinese medicine preparation and the test serum to obtain the prototype components of the Xuhan Ting traditional Chinese medicine preparation;

[0066] Use computer software to statistically analyze the fragmentation of the chemical composition of the Xuhan Ting traditional Chinese medicine preparation, obtain the mass spectrometry information of the fragmentation products, and match it with the mass spectrometry information of the chemical composition of the test serum to screen and obtain the mass spectrometry information of the metabolites of the Xuhan Ting traditional Chinese medicine preparation;

[0067] Deduce the structure of the metabolite by combining the chromatographic information and mass spectrometry information of the metabolite of the Xuhan Ting traditional Chinese medicine preparation.

[0068] In some embodiments, the step of purifying the serum to obtain the test serum includes:

[0069] Vortex-mix the serum and acetonitrile for 4 min to 6 min, then centrifuge at 10000 rpm to 15000 rpm for 4 min to 6 min, and collect the liquid;

[0070] Vortex-mix the liquid and methanol for 50 s to 70 s, then sonicate for 2 min to 4 min, and then centrifuge at 10000 rpm to 15000 rpm for 4 min to 6 min to obtain the test serum.

[0071] In the above steps, the centrifugation speed can be selected from 10000 rpm to 15000 rpm, for example, 10000 rpm, 12000 rpm, 15000 rpm, etc.

[0072] In the above steps, the time for vortex-mixing the serum and acetonitrile can be selected from 4 min to 6 min, for example, 4 min, 5 min, 6 min, etc.

[0073] This application also provides a method for detecting the pharmacodynamic material basis of the Xuhan Ting traditional Chinese medicine preparation. This detection method can at least achieve the following uses (1) to (3):

[0074] (1) For detecting various active ingredients in the Xuhan Ting traditional Chinese medicine preparation;

[0075] (2) For detecting various metabolites and prototype components in the serum components after administration. Here, the metabolites and prototype components are independently selected from the metabolites and prototype components of the Xuhan Ting traditional Chinese medicine preparation described above;

[0076] (3) For identifying the clinical effectiveness of the formula with added or subtracted ingredients and the formula with similar pharmacodynamic effects. Here, the formula with added or subtracted ingredients and the formula with similar pharmacodynamic effects are the formula with added or subtracted ingredients of the Xuhan Ting traditional Chinese medicine preparation and the formula with similar pharmacodynamic effects to it.

[0077] In some embodiments, the active ingredients include the prototype components described above.

[0078] In some embodiments, the method for detecting the pharmacodynamic substance basis of the Xuhanting Chinese medicine preparation comprises the following steps:

[0079] The above construction method is used to obtain the pharmacodynamic substance basic database of Xuhanting Chinese medicine preparation;

[0080] The sample to be tested is taken for ultra-high performance liquid chromatography-linear ion trap-orbital trap mass spectrometry tandem detection to obtain the chemical composition and mass spectrum information of the sample to be tested;

[0081] Comparing the chemical composition and mass spectrum information of the sample to be tested with the database of the pharmacological substance basis of the Xuhanting Chinese medicine preparation to identify the pharmacological substance basis of the Xuhanting Chinese medicine preparation in the sample to be tested;

[0082] Among them, the conditions for ultra-high performance liquid chromatography detection include: mobile phase A is an aqueous solution of formic acid, and mobile phase B is acetonitrile; a gradient elution program is adopted; the gradient elution program includes: from 0min to 10min, the volume percentage of mobile phase A changes from 97% to 87%; from 10min to 80min, the volume percentage of mobile phase A changes from 87% to 55%; from 80min to 110min, the volume percentage of mobile phase A changes from 55% to 15%; from 110min to 120min, the volume percentage of mobile phase A changes from 15% to 5%.

[0083] Preferably, the conditions for ultra-high performance liquid chromatography-linear ion trap-orbitrap mass spectrometry tandem detection are the same as the relevant conditions in the construction method of the basic database of medicinal substances of the Xuhanting traditional Chinese medicine preparation described above.

[0084] In some embodiments, the sample to be tested is an extract of the Chinese medicine preparation to be tested.

[0085] Furthermore, the extract of the Chinese medicine preparation to be tested is prepared by subjecting the Chinese medicine preparation to be tested to an extraction process. Preferably, the conditions of the extraction process are the same as the conditions in the construction method of the pharmacodynamic substance basic database of the Xuhanting Chinese medicine preparation described above.

[0086] In some embodiments, the sample to be tested is a serum sample.

[0087] Furthermore, the preparation method of the serum sample is to purify the serum. Preferably, the purification conditions are the same as the relevant conditions in the construction method of the pharmaceutical substance basis database of the Xuhanting Chinese medicine preparation described above.

[0088] In some embodiments, the step of identifying the clinical effectiveness of the increased and decreased prescriptions and prescriptions with similar efficacy comprises:

[0089] Performing extraction treatment on the target prescription (selected from the increased and decreased prescription and prescriptions with similar medicinal effects) to obtain an extract of the target prescription;

[0090] Ultra-high performance liquid chromatography-linear ion trap-orbitrap mass spectrometry tandem detection was used to obtain the chemical composition of the target prescription and its mass spectrometry information;

[0091] The chemical composition of the target prescription and its mass spectrometry information were compared with the pharmacodynamic substance basis database of the above-mentioned Xuhan Ting traditional Chinese medicine preparation to identify whether the active ingredients were the same; and / or,

[0092] An animal model administered with the target prescription was constructed, and the serum of the experimental animals was collected at the prescribed time;

[0093] The serum was purified to obtain the target serum;

[0094] The target serum was subjected to ultra-high performance liquid chromatography-linear ion trap-orbitrap mass spectrometry tandem detection to obtain the chemical composition of the target and its mass spectrometry information;

[0095] The chemical composition of the target and its mass spectrometry information were compared with the pharmacodynamic substance basis database of the above-mentioned Xuhan Ting traditional Chinese medicine preparation to identify whether the prototype components and metabolites were the same.

[0096] If the target prescription has basically the same active ingredients, prototype components and metabolites as the Xuhan Ting traditional Chinese medicine preparation, it can be considered for clinical application as a substitute for the Xuhan Ting traditional Chinese medicine preparation.

[0097] The following are some specific examples.

[0098] For the experimental parameters not specified in the following specific examples, the guidance given in this application document should be preferentially referred to, and the experimental manuals in this field or other experimental methods known in this field, or the experimental conditions recommended by the manufacturer can also be referred to.

[0099] The raw materials and reagents involved in the following specific examples can be obtained commercially, or can be prepared by those skilled in the art according to known means.

[0100] I. Detection of the components in vivo of the Xuhan Ting traditional Chinese medicine preparation

[0101] 1. Instruments, reagents and drugs

[0102] Instruments: Dionex Ultimate 3000RSLC ultra-high performance liquid chromatograph (Dionex, Thermo Fisher Scientific, USA), including: SRD-3600 degasser, DGP-3600RS dual ternary pump (left and right pumps), WPS-3000TRS autosampler, TCC-3000RS chromatographic column thermostat and DAD-3000RS detector; LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany); high-speed low-temperature centrifuge (Thermo Fisher Scientific, USA); Branson 8510 ultrasonic cleaner (Emerson, USA); Milli-Q ultrapure water treatment system (Millipore, USA).

[0103] Reagents: Methanol and acetonitrile used in liquid chromatography were of chromatography grade (Merck, Darmstadt, Germany); formic acid (Aladdin, China) used as a chromatography additive was of chromatography grade. Ultrapure water (18.2 Mohm.cm) was prepared by a Milli-Q ultrapure water system.

[0104] Drugs: Xuhanting granules were provided by Guangzhou Baiyunshan Qixing Pharmaceutical Co., Ltd. Reference substances such as kaempferol, quercetin, isorhamnetin, isoliquiritigenin, formononetin, formononetin, isoflavones, isoflavone glycosides, meditartin, astragaloside, astragaloside I, isoastragaloside I, isoastragaloside II, astragaloside II, astragaloside III, astragaloside IV, cycloastragenol, ferulic acid, chlorogenic acid, caffeic acid, palmitic acid, betaine, and nicotinamide were purchased from Chengdu Munster Biotechnology Co., Ltd. with a purity of ≥98%. All drugs were confirmed by ultraviolet absorption spectroscopy, high-resolution mass spectrometry, and multi-stage fragmentation data before use.

[0105] 2. Experimental Animals

[0106] Male SD rats, weighing 250–270 g, were provided by the Faculty of Health Sciences, University of Macau. The animal experiments in this study have been approved by the Animal Ethics Committee of the University of Macau (application number: UMARE-007-2023). The animals were housed under standard conditions of 25 °C, 50%–60% humidity, and a 12-h light–dark cycle. Food and water were freely available.

[0107] 3. Experimental Methods

[0108] 3.1 Identification of chemical components of Xuhanting granules in vitro

[0109] 3.1.1 Preparation of test solution

[0110] In order to obtain the best extraction efficiency, different extraction solvents (water, 50% ethanol, 100% ethanol, 50% methanol and 100% methanol) and different extraction times (30 min, 60 min and 120 min) were tested, and the results are compared in Figure 1 and Figure 2 According to the comparison results, the optimized sample extraction method is to accurately weigh 500 mg of Xuhanting granules, put it in a stoppered conical bottle, accurately add 5 mL of 50% ethanol, seal it, weigh the mass, ultrasonically treat (power / frequency, 135W / 42kHz) for 30 minutes, cool it, weigh it, make up the weight loss with 50% ethanol, and shake it well. Take the extract and centrifuge it at 12000r / min for 10 minutes, take the supernatant, and filter it through a 0.22μm micropore filter to obtain it.

[0111] 3.1.2 Preparation of reference solution

[0112] Accurately weigh an appropriate amount of each standard substance, dissolve it in chromatographic methanol and prepare a 0.1 mg / mL stock solution as the standard stock solution. Take an appropriate amount of each standard stock solution, mix and dilute it to a 1 μg / mL mixed standard solution, and store it in a refrigerator at 4°C for later use.

[0113] 3.1.3 Chromatographic and mass spectrometry conditions

[0114] Chromatographic column: ACE Excel 2C18-AMIDE column (2.1 mm × 150 mm, 3 μm, Avantor, USA)

[0115] Mobile phase: A: 0.1% formic acid aqueous solution, B: acetonitrile;

[0116] Flow rate: 0.3 mL / min;

[0117] Injection volume: 2 μL;

[0118] Column temperature: 35°C;

[0119] The elution program is shown in Table 1.

[0120] Table 1 Gradient elution program

[0121]

[0122] Mass spectrometry conditions:

[0123] Data-dependent scanning was performed in the ESI negative ion mode. The parameter settings were as follows: ion spray voltage, -5 kV; capillary temperature, 350 °C; capillary voltage, 35 V; tube lens voltage, 100 V. Nitrogen (N2) was used as the sheath gas and auxiliary gas, and high-purity helium (He) was used as the collision gas in the linear ion trap. Mass spectrometry data were recorded in six ranges of mass-to-charge ratio (m / z) of 100 - 1500, 100 - 350, 300 - 550, 500 - 750, 700 - 950, and 900 - 1500. One cycle of scanning consisted of six events, including one full-scan event and five data-dependent acquisition (DDA) events. In the full-scan event, the resolution of the Orbitrap mass analyzer was set to 30,000 (FWHM defined at m / z 400), and in the DDA events, the two strong ions detected in the full-scan event were selected to acquire MS 2 data. The activation of collision-induced dissociation (CID) was adjusted to a normalized collision energy of 35%, and the isolation width for all DDA events was m / z 2.0. A series of strong background interference ions were added to the rejection mass list, and after enabling the dynamic exclusion function, more relatively low-intensity ions could become the targets for MS 2 detection. External mass calibration of the Orbitrap mass analyzer was performed every three days according to the manufacturer's guidelines.

[0124] 3.1.4 Data processing

[0125] A local database was established. Through literature research and database queries, the structural information of the compounds of the five herbs (Astragalus membranaceus, Concha Ostreae Usta, Ziziphus jujuba, Fructus Tritici Levis, and Rhizoma Oryzae Glutinosae) in Xuhan Ting granules was collected and organized to establish a local database. Then, Compound Discoverer 3.0 software (Thermo Fisher Scientific, San Jose, CA, USA) was used to compare the self-built database with the ion information of the acquired high-resolution mass spectrometry data, and screening parameters (mass error < 5 ppm, isotope abundance ratio < 5) were set to match known compounds. Then, by analyzing the fragmentation behavior of the reference substances and the fragments matched from the local database, the characteristic fragments and fragmentation rules of each class of compounds were summarized. Finally, through searching for characteristic fragments, further annotation and analysis were carried out. However, some unknown compounds that did not satisfy the fragmentation rules were further annotated through the online secondary database mzCloud.

[0126] 3.1.5 Identification results

[0127] Under the above chromatographic and mass spectrometric conditions, sample analysis was carried out to obtain the total ion current chromatograms of the mass spectrometry scans of the mixed reference substances and the Xuhan Ting granule samples (as Figure 5). By comparing with reference substances, local databases, online databases and literature, 296 chemical components were preliminarily identified, including 75 flavonoids, 57 terpenoids, 53 organic acids, 25 sugars and glycosides, 26 phenols, 9 amino acids, 5 nucleotides, 3 polypeptides, 2 lignins and other substances.

[0128] 3.1.6 Analysis of mass spectrometry fragmentation patterns of main components

[0129] Flavonoids are a class of natural polyphenolic compounds widely found in plants. Abundant flavonoids were detected in Xuhanting Granules, which are mainly divided into isoflavones, flavonoids, pterodactyls and isoflavans according to the structural type of the parent nucleus. They are often connected with functional groups such as phenolic hydroxyl, methoxy, methyl, and isopentenyl, and the possible substitution positions are C-3, C-5, C-6, C-7, C-8 and C-2', C-3', C-4', C-5', and C-6'. In addition, it is often combined with sugars to form glycosides, and the possible substitution sugars are rhamnose, glucose, etc., and there may also be derived acetate and malonate groups on the sugar chain. By summarizing the fragment ion information, it was found that the main mass spectrometry fragmentation patterns of flavonoids are glycosidic bond cleavage, reverse Diels-Alder reaction (RDA) cleavage, and neutral loss (such as CO, H2O, CH2O, and C2H2O).

[0130] The main flavonoids in Astragalus are isoflavone compounds, and their main characteristic fragments are m / z 283[C 16 H 11 O5] - and m / z 268[C 16 H 11 O4] - For example, the molecular formula of peak 87 is C 22 H 22 O 10 In negative ion mode, the main secondary ion fragments are m / z 223.08, 227.08, 251.01, 255.20, 268.03, 283.06. Parent ion m / z 445.1134 [MH] - After the glycosidic bond of the parent ion is broken, m / z 283.06 [MH-Glc] is generated - , and then lose two molecules of CO to obtain m / z 227.08 [MH-Glc-2CO] - . At m / z 283.06 [MH-Glc] - On the basis of the above, a CH3 molecule was broken off on the B ring to obtain the ion fragment 268.03 [MH-Glc-CH3] -After that, the fragment continues to remove the OH on ring B, generating a fragment with m / z 251.01 [M-H-Glc-CH3-OH] - Then, it removes CO, generating a fragment with m / z 223.08 [M-H-Glc-CH3-OH-CO] - Based on the above mass spectrometry data and literature reports, this compound was identified as Calycosin-7-glucoside. The glycosylation patterns of flavonoids in floating wheat are mainly C-glycosides and O-glycosides, and the main aglycones are apigenin, luteolin, and tricin. For example, the molecular formula of peak 80 is C 26 H 28 O 14 , and the main secondary ion fragments are 443.20, 473.07, 503.31, 383.05, 353.14, 545.27. The parent ion is m / z563.1404 [M-H] - , and after removing one molecule of H2O, the ion fragment m / z 545.27 [M-H-H2O] is obtained - After that, the fragment continues to remove GlcA, generating a fragment with m / z 503.31 [M-H-H2O-GlcA] - Based on m / z 503.31 [M-H-H2O-GlcA] - , by continuing to remove CH2O, the ion fragments m / z 473.07 [M-H-H2O-CH2O] - and m / z 443.20 [M-H-H2O-2CH2O] - are obtained. Based on the above mass spectrometry data and literature reports, this compound was identified as Apigenin-7-O-apioglucoside. Similarly, the cleavage behavior can be extended to other flavonoids.

[0131] Saponins are a class of glycosides with triterpenoids or spirostanoids as aglycones. There is a rich variety of saponin compounds in Astragalus membranaceus and Ziziphus jujuba. The saponin compounds in Astragalus membranaceus belong to triterpenoid saponins and their derivatives, and their structures are mainly composed of the parent aglycone and different numbers and types of sugar groups. The saponin compounds in Ziziphus jujuba are mainly dammarane-type triterpenoid saponins. Sugars mostly substitute other groups at the C-3, C-20, and C-23 positions of saponins, and the sugar groups are mainly D-glucose, D-galactose, D-xylose, L-rhamnose, etc. By analyzing saponin standards, we found that Astragaloside I, Isoastragaloside I, Astragaloside II, Isoastragaloside II, Astragaloside IV, etc. all have the same main chain structure, and their differences are only limited to the number and position of the acetyl groups linked to xylose. Further comparing the MS of their ionic products 2Data showed that the fragmentation patterns were mostly glycosidic bond cleavages, and during the cleavage process, one to multiple H2O (18), Glc (162), Xyl (132), GlcA (42), and Rha (146) might be neutrally lost. Due to the special structure of the acetyl group, fragment ions such as Ac (42), Ac + H2O (60), and Ac + Ac (84) would also be generated. These fragmentation rules and characteristic fragments can be used to determine the structural framework of saponins and simplify the structure analysis.

[0132] In the negative ion mode, for peak 200, the parent ion was m / z 829.4599 [M + COOH]-. The parent ion fragmented to generate m / z 783.45 [M - H] - , followed by glycosyl cleavage, losing one molecule of Xyl and one molecule of Glu to generate m / z 651.40 [M - H - Xyl] - , 621.39 [M - H - Glu] - , 489.37 [M - H - Glc - Xyl] - . Based on the above mass spectrometry data, this compound was identified as Astragaloside IV. Astragaloside I and Astragaloside IV have the same parent nucleus, with two molecules of acetyl substitution on the xylose connected to the 3 - hydroxyl group, m / z 867.4797 [M - H - Ac - H2O] - , and its structural formula can be determined as C 45 H 72 O 16 . Therefore, in the secondary mass spectrometry, fragments generated by consecutive loss of acetyl groups, such as 825.06 [M - H - Ac - H2O] - , 783.56 [M - H - 2(Ac - H2O)] - , 765.21 [M - H - Ac - (Ac - H2O)], 693.80 [M - H - Ac - H2O - Xyl] - , 645.28 [M - H - Ac - H2O - 180] - .

[0133] Another type of characteristic component in Chinese jujube is benzyl glycosides, represented by Zizybeoside II (peak 51), whose [M - H] - peak was m / z 639.2130 [C 25 H 37 O 16 - , and the main secondary ion fragments were m / z 431.09, 584.01, 269.16, 297.05, 535.18. In the negative ion mode, after the glycosidic bond of the parent ion was cleaved, m / z 431.09 [M - H - Glc] was generated​- , and 269.16[MH-Glc-Glc] - At the same time, the parent ion loses C4H7, giving m / z 584.01[MH-C4H7] - .

[0134] 3.2 Identification of the components of Xuhanting Granules that enter the blood

[0135] 3.2.1 Dosage regimen and sample collection

[0136] (1) Dosage regimen

[0137] After one week of adaptive feeding, the rats were randomly divided into a blank control group and a Xuhanting granule group, with 6 rats in each group. The blank group was gavaged with drinking water, and the drug group was gavaged with 5.4 g / kg Xuhanting granules, once a day, for one week.

[0138] (2) Collection of serum samples

[0139] The rats were fasted for 12 hours before the last administration and were allowed to drink water freely. Then, about 1.0 mL of blood was collected from the orbital vein of the rats at 0, 0.5, 1, and 2 hours after the corresponding drug solution was administered by gavage to each group, and immediately transferred into EP tubes, left to stand for 1 hour, centrifuged at 3000 r / min for 10 minutes, and serum was collected and stored in a -80°C refrigerator for testing.

[0140] 3.2.2 Serum sample pretreatment

[0141] In order to obtain a more comprehensive metabolite component spectrum, the sample pretreatment method was optimized. Three different organic solvents (methanol, acetonitrile, and acetone) were used to optimize the protein precipitation of serum samples. According to the comparison results, the serum sample precipitated with acetonitrile had the highest response value when extracting the chromatographic peaks of the representative components astragaloside IV and verbascoside-7-O-glucoside ( Figure 3 and Figure 4 ). Therefore, the optimized pretreatment method is to mix the serum of each group of rats at each time point, take 1.0 mL of the mixed serum, add 3.0 mL of acetonitrile, vortex mix for 5 minutes, centrifuge at 12000 rpm for 5 minutes, take the supernatant and put it in another EP tube, blow dry with nitrogen, add 100 μL of methanol to the residue, vortex for 1 minute, ultrasonicate for 3 minutes, centrifuge at 12000 rpm for 5 minutes, and take the supernatant for analysis according to "3.1.3".

[0142] 3.2.3 Data Analysis

[0143] In this experiment, Compound Discoverer 3.0 software was used to process the raw mass spectrometry data of serum obtained after oral administration of Xuhanting granules. The "Metabolism w Stats Expected w FISh Scoring and Background Removal" module was selected to quickly find and identify expected metabolites, automatically annotate fragment ion structures and background removal blanks (using blank files) using FISh scoring; generate group ratios and trend line graphs at time points; perform retention time calibration, detect expected metabolites, dealkylation and dealkylation products, and biotransformation products with resolution-aware isotope pattern matching, and group expected compounds in all samples. The molecular formula and structure of the metabolites were deduced based on the chromatographic retention time, calculation formula, and information of molecular ions and fragment ions of the obtained metabolites. Xcalibur 2.3 software was used to extract secondary fragments in the raw mass spectrometry data, and the metabolites calculated by Compound Discoverer 3.0 software were verified one by one according to the fragmentation rules.

[0144] 3.2.4 Identification results

[0145] Based on the mass spectrometry data of blank and dosed rat serum ( Figure 5 and Figure 6), 116 components were discovered and identified from the serum, including 9 prototypes and 107 metabolites. Among them, the prototype components were mainly flavonoids and related glycosides, pentacyclic triterpenoid saponins, and monoterpenoid components. Their biotransformation reaction methods mainly included oxidation, hydration, dehydration, reduction, stearoylation, aminoacylation, palmitoylation, methylation, acetylation, and glucuronidation conjugation. The parent compounds of the metabolites were successively: AstragalosideIV, AstragalosideII, Apiin, Astraisoflavan-7-O-β-D-glucoside, Ononin, ZizybeosideI, ZizybeosideI, AstragalosideIV, AstragalosideIV, AstragalosideII, Astraisoflavan-7-O-β-D-glucoside, Ononin, ZizybeosideI, AstragalosideIV, AstragalosideII, Astraisoflavan-7-O-β-D-glucoside, Ononin, ZizybeosideI, AstragalosideIV, ZizybeosideI, ZizybeosideI, AstragalosideIV, AstragalosideII, AstragalosideIV, AstragalosideIV, AstragalosideII, AstragalosideIV, Apiin, Apiin, Apiin, AstragalosideIV, ZizybeosideI, AstragalosideIV, AstragalosideII, AstragalosideIV, AstragalosideII, ZizybeosideI, AstragalosideII, calycosin, Formononetin, Ononin, Ononin, Ononin, Ononin, Ononin, calycosin, calycosin, calycosin, Apiin, Ononin, Ononin, AstragalosideIV, AstragalosideII, AstragalosideII, AstragalosideIV, AstragalosideII, AstragalosideIV, AstragalosideII, AstragalosideII, ZizybeosideI, AstragalosideII, Ononin, ZizybeosideI,AstragalosideII, AstragalosideIV, Astraisoflavan-7-O-β-D-glucoside, AstragalosideIV, AstragalosideII, Astraisoflavan-7-O-β-D-glucoside, Ononin, ZizybeosideI, AstragalosideII, Apiin, AstragalosideII, ZizybeosideI, AstragalosideII, Astraisoflavan-7-O-β-D-glucoside, ZizybeosideI, ZizybeosideI, AstragalosideII, ZizybeosideI, Astraisoflavan-7-O-β-D-glucoside, AstragalosideII, AstragalosideIV, AstragalosideII, AstragalosideIV, AstragalosideII, Apiin, Astraisoflavan-7-O-β-D-glucoside, ZizybeosideI, Astraisoflavan-7-O-β-D-glucoside, ZizybeosideI, AstragalosideII, ZizybeosideI, AstragalosideIV, AstragalosideII, ZizybeosideI, AstragalosideIV, ZizybeosideI, AstragalosideIV, AstragalosideII, ZizybeosideI, AstragalosideII, ZizybeosideI, ZizybeosideI, ZizybeosideI and ZizybeosideI.,

[0146] 3.2.5 Analysis of the Mass Spectrometric Fragmentation Rules of the Main Metabolites

[0147] Based on the accurate molecular weight and MS 2 fragment information, and comparison with reference substances, literature, databases such as PubChem and HMDB, a total of 9 prototype components were identified in the serum samples (the specific structures are shown in Table 2).

[0148] Table 2 Nine Prototype Components

[0149]

[0150] These ingredients in Table 2 mainly come from the astragalus, jujube and floating wheat in the formula.

[0151] The following is an example of the process of identifying the prototype components using P1. In the negative ion mode, the molecular ion peak m / z 445.1139 was detected. In addition, the fragmentation ions of m / z 224.10, 227.08, 251.01, 255.20, 268.15, 283.06, and 305.18 behaved the same as the standard mass spectrum. Other components were identified based on fragmentation rules and literature reports. The fragmentation information of all other prototype components is shown in Table 3.

[0152] Table 3

[0153]

[0154]

[0155] The original mass spectrometry data of serum samples and the chemical structure of the prototype components of Xuhanting Granules were imported into CompoundDiscoverer 3.0 software for calculation. The retention time, molecular mass, chemical formula, mass error and inferred reaction were obtained by analysis and collation. At the same time, secondary fragments were extracted from the original mass spectrometry data and verified one by one according to the fragmentation rules. A total of 107 metabolites were identified. These metabolites can be divided into three categories according to their sources: (1) Astragalus metabolites; (2) Jujube metabolites; (3) Floating wheat metabolites. The following are examples of the structural description process of some representative metabolites.

[0156] (1) Astragalus metabolites

[0157] PM86 is one of the metabolites obtained by importing the structural formula of Astragaloside IV into Compound Discoverer 3.0 software. [MH] can be extracted at 89.84min. - The parent ion is m / z 975.20679, and m / z 972.78076 is [MH] - The decrease is 3Da (-H3), m / z 371.31445 is [MH]-the decrease is 604Da (-H 3- C 34 H 49 O9). Other characteristic fragments are m / z 480.3472 and m / z 540.2010, which are similar to the fragments of Astragaloside IV. Therefore, it is speculated that PM86 is the result of phase I metabolism (desaturation and oxidation reaction) and phase II metabolism (palmitoyl conjugation) of Astragaloside IV in the body. Figure 7 .

[0158] PM39 is one of the metabolites obtained by importing the structural formula of Calycosin into Compound Discoverer 3.0 software, and can be extracted at 30.67 min with [M-H] - as the parent ion with m / z 441.2765, and m / z 284.1713 is [M-H] - decreased by 258 Da (-C4H4O3), and m / z 254.6426 is [M-H]- decreased by 287 Da (-C4H4O 3- C1H1O1). Other characteristic fragments are m / z 267.1857 and m / z 283.1155, which are similar to the fragments of Calycosin. Therefore, it is speculated that PM39 is the result of the phase I metabolism (hydration and reduction reactions) and phase II metabolism (acetylation conjugation) of Calycosin in vivo. The metabolic process of Calycosin in serum is as Figure 8 .

[0159] (2) Jujube metabolites

[0160] PM78 is one of the metabolites obtained by importing the structural formula of Zizybeoside I into Compound Discoverer 3.0 software, and can be extracted at 86.01 min with [M-H] - as the parent ion with m / z 528.3723, and m / z 457.0967 is [M-H] - decreased by 71 Da (-C3H3O2), and m / z 356.1273 is [M-H]- decreased by 172 Da (-C3H3O 2- C4H5O3). Other characteristic fragments are m / z246.0115 and m / z 328.6751, which are similar to the fragments of Zizybeoside I. Therefore, it is speculated that PM78 is the result of the phase I metabolism (dealkylation and dehydration reactions) and phase II metabolism (palmitoylation conjugation) of Zizybeoside I in vivo. The metabolic process of Zizybeoside I in serum is as Figure 9 .

[0161] (3) Fructus Tritici Levis metabolites

[0162] PM28 is one of the metabolites obtained by importing the structural formula of Apigenin-7-apioglucoside into Compound Discoverer 3.0 software, and can be extracted at 12.98 min with [M-H+H2O] - as the parent ion with m / z 811.40271, and m / z767.25122 is [M-H+H2O] -44Da (-C2H4O) is reduced, m / z 749.52716 is [M-H+H2O] - The other characteristic fragments are m / z 548.83105, m / z 711.55505 and m / z 789.93951, which are similar to the characteristic fragments of Apigenin-7-apioglucoside. Therefore, it is speculated that PM28 is the result of Apigenin-7-apioglucoside undergoing phase I metabolism (reduction reaction) and phase II metabolism (stearoyl conjugation) in vivo. The metabolic process of Apigenin-7-apioglucoside in serum is as follows Figure 10 .

[0163] This experiment studied the migratory components of Xuhanting granules in serum. After analysis, it was found that after oral administration of Xuhanting granules, a few chemical components were absorbed by serum and remained in the prototype without bile metabolism. After oral administration, most Chinese medicine ingredients will be excreted as hydrophilic prototypes, or metabolized into more polar molecules by the liver and then excreted in urine. When passing through the digestive tract, due to the effects of gastric acid, various digestive enzymes and intestinal flora, a variety of metabolic reactions may occur, which will inactivate some drugs in intestinal metabolism and reduce the amount of prototype drugs absorbed into the body. Most drugs are mainly absorbed in the small intestine. Due to the presence of a large number of enzymes and intestinal flora in the small intestine, they will metabolize the glycoside components to a certain extent and convert the glycoside components into aglycones.

[0164] In this study, 107 metabolites were identified in the serum after administration, of which 9.3% (10) were due to phase I reactions, mainly oxidation, dehydration and desaturation reactions, while 90.7% (97) were due to phase I and phase II reactions, mainly stearoylation, amino acidification, palmitoylation, methylation, acetylation and glucuronidation reactions. These reactions relatively reduced the polarity of the metabolites, making them easier to pass through biological membranes and be absorbed by tissues.

[0165] In summary, Example 1 of the present application characterized the chemical components in the water extract of Xuhanting Granules and the serum of rats after oral administration of Xuhanting Granules by UPLC-MS / MS method based on linear ion trap-orbital trap (LTQ-Orbitrap XL) technology, and finally preliminarily identified 296 chemical components of Xuhanting Granules, mainly flavonoids, terpenes, organic acids, sugars and glycosides. A total of 9 prototype components and 107 metabolic components were identified in the serum samples after administration, of which phase I reactions accounted for 9.3% (10), mainly oxidation, dehydration and desaturation reactions, while phase I and II reactions accounted for 90.7% (97), mainly stearoylation, amino acidization, palmitoylation, methylation, acetylation and glucuronidation reactions.

[0166] The present application constructs a basic library of pharmacological substances of Xuhanting Granules. By testing the extract of the Chinese medicine preparation to be tested and the serum of the modeled mice, the corresponding mass spectrometry information is obtained and compared with the basic database of pharmacological substances, which can be used for the effectiveness identification of the increase or decrease prescription, the efficacy investigation, the active ingredient investigation and other aspects.

[0167] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0168] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0169] The above-described embodiments only express several implementation methods of the present application, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can also be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solutions provided in the present application through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for constructing a database of the effective substance basis of a traditional Chinese medicine preparation for stopping sweating due to debility, characterized in that, The pharmacodynamic material basis of the Xuhan Ting traditional Chinese medicine preparation includes its prototype components and metabolites; The construction method includes the following steps: Extract the Xuhan Ting traditional Chinese medicine preparation to obtain an extract of the Xuhan Ting traditional Chinese medicine preparation; Construct an animal model administered with the Xuhan Ting traditional Chinese medicine preparation, and collect the serum of the experimental animals at the designated time; Purify the serum to obtain the test serum; Perform ultra-high performance liquid chromatography-linear ion trap-orbitrap mass spectrometry tandem detection on the extract of the Xuhan Ting traditional Chinese medicine preparation and the test serum to obtain the chemical composition and mass spectrometry information of the Xuhan Ting traditional Chinese medicine preparation and the test serum; Compare the chemical composition and mass spectrometry information of the Xuhan Ting traditional Chinese medicine preparation and the test serum, identify the prototype components and metabolites of the Xuhan Ting traditional Chinese medicine preparation, and construct a pharmacodynamic material basis database for the Xuhan Ting traditional Chinese medicine preparation; Among them, the conditions for the ultra-high performance liquid chromatography detection include: mobile phase A is an aqueous solution of formic acid, and mobile phase B is acetonitrile; a gradient elution program is adopted; the gradient elution program includes: from 0 min to 10 min, the volume percentage of mobile phase A changes from 97% to 87%; from 10 min to 80 min, the volume percentage of mobile phase A changes from 87% to 55%; from 80 min to 110 min, the volume percentage of mobile phase A changes from 55% to 15%; from 110 min to 120 min, the volume percentage of mobile phase A changes from 15% to 5%.

2. The construction method according to claim 1, characterized in that The composition of the Xuhan Ting traditional Chinese medicine preparation includes Astragalus membranaceus, Fructus Tritici Levis, Fructus Jujubae, Rhizoma Oryzae Glutinossae and Concha Ostreae Usta.

3. The construction method according to claim 1, characterized in that, The prototype components include Calycosin-7-glucoside, Ononin, Astraisoflavan-7-O-β-D-glucoside, Calycosin, Astragaloside IV, Isoastragaloside II, Astragaloside II, Zizybeoside I and Apigenin-7-apioglucoside; and / or The parent compounds of the metabolites sequentially include: AstragalosideIV, AstragalosideII, Apiin, Astraisoflavan-7-O-β-D-glucoside, Ononin, ZizybeosideI, ZizybeosideI, AstragalosideIV, AstragalosideIV, AstragalosideII, Astraisoflavan-7-O-β-D-glucoside, Ononin, ZizybeosideI, AstragalosideIV, AstragalosideII, Astraisoflavan-7-O-β-D-glucoside, Ononin, ZizybeosideI, AstragalosideIV, ZizybeosideI, ZizybeosideI, AstragalosideIV, AstragalosideII, AstragalosideIV, AstragalosideIV, AstragalosideII, AstragalosideIV, Apiin, Apiin, Apiin, AstragalosideIV, ZizybeosideI, AstragalosideIV, AstragalosideII, AstragalosideIV, AstragalosideII, ZizybeosideI, AstragalosideII, calycosin, Formononetin, Ononin, Ononin, Ononin, Ononin, Ononin, calycosin, calycosin, calycosin, Apiin, Ononin, Ononin, AstragalosideIV, AstragalosideII, AstragalosideII, AstragalosideIV, AstragalosideII, AstragalosideIV, AstragalosideII, AstragalosideII, ZizybeosideI, AstragalosideII, Ononin, ZizybeosideI, AstragalosideII, AstragalosideIV, Astraisoflavan-7-O-β-D-glucoside, AstragalosideIV, AstragalosideII, Astraisoflavan-7-O-β-D-glucoside,Ononin, Zizybeoside I, Astragaloside II, Apiin, Astragaloside II, Zizybeoside I, Astragaloside II, Astraisoflavan-7-O-β-D-glucoside, Zizybeoside I, Zizybeoside I, Astragaloside II, Zizybeoside I, Astraisoflavan-7-O-β-D-glucoside, Astragaloside II, Astragaloside IV, Astragaloside II, Astragaloside IV, Astragaloside II, Apiin, Astraisoflavan-7-O-β-D-glucoside, Zizybeoside I, Astraisoflavan-7-O-β-D-glucoside, Zizybeoside I, Astragaloside II, Zizybeoside I, Astragaloside IV, Astragaloside II, Zizybeoside I, Astragaloside IV, Zizybeoside I, Astragaloside IV, Astragaloside II, Zizybeoside I, Astragaloside II, Zizybeoside I, Zizybeoside I, Zizybeoside I and Zizybeoside I., 4. The construction method according to any one of claims 1 to 3, characterized in that, The step of extracting the Xuhan Ting traditional Chinese medicine preparation to obtain an extract of the Xuhan Ting traditional Chinese medicine preparation includes: Mix the Xuhan Ting traditional Chinese medicine preparation with an extraction solvent and perform ultrasonic treatment to obtain a mixed solution; Perform solid-liquid separation on the mixed solution, collect the liquid, and obtain the extract of the Xuhan Ting traditional Chinese medicine preparation.

5. The construction method according to claim 4, wherein Meet at least one of the following conditions: (1) The extraction solvent is an aqueous solution of alcohol; optionally, the volume ratio of alcohol in the aqueous solution of alcohol is 40% to 60%; (2) The mass-volume ratio of the Xuhan Ting granules to the extraction solvent is 500 mg: 4 to 6 mL; (3) The power of ultrasonic treatment is 130 W to 140 W, the frequency is 40 kHz to 45 kHz, and the time is 20 min to 40 min.

6. The construction method according to any one of claims 1 to 3, characterized in that The conditions for the ultra-high performance liquid chromatography detection also include at least one of the following conditions: (1) The chromatographic column is an octadecylsilyl silica gel column; (2) The flow rate is 0.2 mL / min to 0.4 mL / min; (3) The injection volume is 1 μL to 3 μL; and (4) The column temperature of the chromatographic column is 30 °C to 40 °C.

7. The construction method according to claim 6, characterized in that The length of the chromatographic column is 140 mm to 160 mm, the diameter is 2 mm to 2.2 mm, and the particle size of the packing is 2 μm to 4 μm.

8. The construction method according to any one of claims 1 to 3, characterized in that The conditions for the linear ion trap-orbitrap mass spectrometry detection include: using the negative ion mode, the ion spray voltage is -4 kV to -6 kV; the capillary temperature is 300 °C to 400 °C; the capillary voltage is 30 V to 40 V; the tube lens voltage is 90 V to 110 V; the sheath gas and auxiliary gas are nitrogen; the collision gas is helium.

9. The construction method according to claim 8, characterized in that, The steps of comparing the chemical compositions and their mass spectrometry information of the Xuhanling traditional Chinese medicine preparation and the test serum to identify the effective substance basis of the Xuhanling traditional Chinese medicine preparation include: Comparing the chemical compositions of the Xuhanling traditional Chinese medicine preparation and the test serum to obtain the prototype components of the Xuhanling traditional Chinese medicine preparation; Using computer software to statistically analyze the fragmentation of the chemical composition of the Xuhanling traditional Chinese medicine preparation, obtaining the mass spectrometry information of the fragmentation fragments and matching it with the mass spectrometry information of the chemical composition of the test serum, and screening to obtain the mass spectrometry information of the metabolites of the Xuhanling traditional Chinese medicine preparation; Deriving the structure of the metabolite based on the chromatographic information and mass spectrometry information of the metabolite of the Xuhanling traditional Chinese medicine preparation.

10. The construction method according to any one of claims 1 to 3, characterized in that The steps of purifying the serum to obtain the test serum include: Vortex-mixing the serum and acetonitrile for 4 min to 6 min, then centrifuging at 10000 rpm to 15000 rpm for 4 min to 6 min, and collecting the liquid; Vortex-mixing the liquid and methanol for 50 s to 70 s, then ultrasonically treating for 2 min to 4 min, and then centrifuging at 10000 rpm to 15000 rpm for 4 min to 6 min to obtain the test serum.

11. A method for detecting the pharmacodynamic material basis of a traditional Chinese medicine preparation for stopping night sweats, characterized in that, Include the following steps: Obtaining the effective substance basis database of the Xuhanling traditional Chinese medicine preparation by using the construction method according to any one of claims 1 to 10; Taking a test sample for ultra-high performance liquid chromatography-linear ion trap-orbitrap mass spectrometry tandem detection to obtain the chemical composition and its mass spectrometry information of the test sample; Comparing the chemical composition and its mass spectrometry information of the test sample with the effective substance basis database of the Xuhanling traditional Chinese medicine preparation to identify the effective substance basis of the Xuhanling traditional Chinese medicine preparation in the test sample; Among them, the conditions for the ultra-high performance liquid chromatography detection include: mobile phase A is an aqueous solution of formic acid, mobile phase B is acetonitrile; using a gradient elution program; the gradient elution program includes: 0 min to 10 min, the volume percentage of mobile phase A changes from 97% to 87%; 10 min to 80 min, the volume percentage of mobile phase A changes from 87% to 55%; 80 min to 110 min, the volume percentage of mobile phase A changes from 55% to 15%; 110 min to 120 min, the volume percentage of mobile phase A changes from 15% to 5%.

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