A whole ingredient and blood ingredient of yinling qushi granules and a detection method thereof

Abstract

The application discloses a kind of yinling quzhi granules whole component and blood component and its detection method.A, yinling quzhi granules is decocted, and the decocting liquid is filtered to obtain filtrate, and the filtrate is concentrated to obtain concentrated solution;B, concentrated solution is detected using liquid chromatography-mass spectrometry;The liquid chromatography condition is: using reversed-phase C18 chromatographic column, column temperature 40 DEG C, mobile phase: A is 0.1% formic acid water by volume fraction, B is acetonitrile, flow rate 0.3ml / min.This study investigates the whole component of dampness-removing prescription yinling quzhi granules, and investigates the blood component of dampness-removing prescription yinling quzhi granules after long-term intervention of dampness syndrome patients in clinical practice.To inject new present theoretical system for the research of TCM dampness syndrome and dampness-removing prescription clinical intervention, provide reference for the research of other TCM syndromes.

Description

Technical fields:

[0001] This invention belongs to the field of traditional Chinese medicine, specifically relating to the complete composition of Yinling Qushi Granules, its blood-entering components, and their detection methods. Background technology:

[0002] In recent years, scholars have summarized the medication experience of renowned traditional Chinese medicine practitioners in treating dampness syndrome through data mining. Meng Peipei et al., using association analysis and other methods, discovered that Mr. Pu Fuzhou frequently used single herbs in treating dampness syndrome, including Poria cocos, licorice root, Tetrapanax papyriferus, Artemisia capillaris, and Coix lacryma-jobi, emphasizing the importance of dispelling dampness. Commonly used paired herbs included Tetrapanax papyriferus-Artemisia capillaris, Tetrapanax papyriferus-Coix lacryma-jobi, and Pinellia ternata-Poria cocos, while complementary herbs included talc-Tetrapanax papyriferus-Prunus armeniaca, and Tetrapanax papyriferus-Poria cocos-Coix lacryma-jobi. Mr. Pu added ingredients to classic formulas, such as Prunus armeniaca and Talc Decoction with Artemisia capillaris and Alisma plantago-aquatica, to clear damp-heat and harmonize the spleen and stomach. Zhang Xinwen et al. summarized Li Shimou's experience in treating dampness. Mr. Li advocated "nourishing yin and dispelling dampness," frequently using Ophiopogon japonicus, Rehmannia glutinosa, and Scrophularia ningpoensis to nourish yin fluids, and Atractylodes macrocephala and Poria cocos to dry and promote urination. Zeng Chuiyi summarized Mao Dexi's experience, noting that Mao believed dampness should be treated by differentiating between the three jiaos: clearing heat and drying dampness in the upper jiao, aromatically dispelling dampness in the middle jiao, and promoting diuresis and eliminating dampness in the lower jiao. Commonly used herbs included Coptis chinensis, Scutellaria baicalensis, Pogostemon cablin, Atractylodes lancea, Poria cocos, Polyporus umbellatus, and Alisma plantago-aquatica. Zhang Junhua et al., using complex network analysis, discovered that Poria cocos, Atractylodes macrocephala, Alisma plantago-aquatica, and Citrus reticulata peel are core herbs for dispelling dampness, and summarized classic formulas and commonly used treatments for dampness. These studies are of great significance for inheriting the dampness-treating thoughts, medication rules, and clinical guidance of renowned TCM practitioners.

[0003] Yinling Dampness-Clearing Granules are based on the formula of Fuling Zexie Decoction (from *Synopsis of Prescriptions of the Golden Chamber*), with added or subtracted ingredients. Yinling Dampness-Clearing Granules consist of eight medicinal herbs: Poria cocos, Atractylodes macrocephala, Polyporus umbellatus, Cinnamomum cassia, Zingiber officinale (dried), Glycyrrhiza uralensis, Artemisia capillaris, and Adenophora stricta. Their [source], [efficacy], [indications], and [formula explanation] are as follows:

[0004] [Source] Guangdong Provincial Hospital of Traditional Chinese Medicine.

[0005] [Efficacy] It strengthens the spleen, promotes diuresis and eliminates dampness, warms the middle and benefits the stomach, and also eliminates damp heat.

[0006]

Indications

[0007] [Explanation of the formula] The chief herb in this formula is Poria cocos, which is sweet and bland in nature and has a neutral nature. It is used to strengthen the spleen, promote diuresis and eliminate dampness. It is combined with the adjuvant herb Cinnamomum cassia, which is pungent and warm, to open the bladder and promote the transformation of qi and diuresis, so that the dampness can be eliminated.

[0008] The assistant herb, Atractylodes macrocephala, is sweet and bitter in taste and warm in nature. It can strengthen the middle jiao (middle burner) and assist the principal herb in enhancing its dampness-removing power, thus eliminating the source of dampness. Polyporus umbellatus is sweet and bland in nature and neutral in nature. It drains dampness and promotes urination, assisting the principal herb in removing dampness without harming the true yin. Dried ginger is pungent and hot, and can strengthen the spleen, warm the middle jiao, and resolve dampness.

[0009] Artemisia capillaris, an adjuvant herb, is bitter, pungent, and slightly cold. It can clear heat and promote diuresis, while also counteracting the warming and drying properties of the other herbs. Glehnia littoralis, sweet, bitter, and slightly cold, is sweet and can nourish yin, preventing excessive diuresis from damaging yin fluids.

[0010] The herb licorice root, sweet in taste and neutral in nature, can strengthen the spleen, clear heat, promote diuresis, and harmonize the effects of other herbs. The entire formula works synergistically to strengthen the spleen, promote diuresis, eliminate dampness, warm the middle jiao and benefit the stomach, and also eliminate damp-heat. Summary of the Invention:

[0011] The purpose of this invention is to provide a complete list of the components of Yinling Qushi Granules, the components that enter the bloodstream, and a method for their detection.

[0012] The method for detecting all components of Yinling Dehumidifying Granules of the present invention includes the following steps:

[0013] A. Decoction of Yinling dampness-removing granules, filtration of the decoction to obtain filtrate, and concentration of the filtrate to obtain concentrated solution.

[0014] B. Analyze the concentrated solution using liquid chromatography-mass spectrometry;

[0015] The liquid chromatography conditions are as follows: reversed-phase C18 column, column temperature 40℃, mobile phase: A is 0.1% formic acid water, B is acetonitrile, flow rate 0.3 ml / min;

[0016] The mobile phase A decreased from 95% to 50% within 0-20 minutes;

[0017] The mobile phase A decreased from 50% to 5% over 20-27 minutes.

[0018] Mobile phase A was maintained at 5% for 27-32 minutes;

[0019] The mobile phase A increased from 5% to 95% over 32-33 minutes.

[0020] Mobile phase A was maintained at 95% for 33-38 minutes;

[0021] Mass spectrometry conditions were as follows: ESI ion source, carrier gas 1 / 2: 50psi, CUR: 35psi, temperature: 500℃, ISVF: 5500V(+) and -4500V(-), CE and CES: 35±15eV, m / z: 50~1500Da, DP: 80eV.

[0022] The complete composition of Yinling Dehumidifying Granules includes triterpenoids, flavonoids, saponins, amino acids, and phenolic acids.

[0023] Preferably, the particle analysis of all components is shown in Table 2.

[0024] Preferably, the concentrate obtained by filtrate concentration is a concentrate obtained by vacuum concentration to a specific gravity of 1.08 to 1.10.

[0025] The second objective of this invention is to provide a method for detecting the blood-entering components of Yinling Qushi Granules, comprising the following steps:

[0026] A. Patients with dampness syndrome were given Yinling Qushi Granules, and their serum was collected after a predetermined time as the experimental group. The serum of patients with dampness syndrome who were not given Yinling Qushi Granules was used as the control group.

[0027] B. The experimental and control groups were detected by liquid chromatography-mass spectrometry.

[0028] The liquid chromatography conditions are as follows: reversed-phase C18 column, column temperature 40℃, mobile phase: A is 0.1% formic acid water, B is acetonitrile, flow rate 0.3 ml / min;

[0029] The mobile phase A decreased from 95% to 50% within 0-20 minutes;

[0030] The mobile phase A decreased from 50% to 5% over 20-27 minutes.

[0031] Mobile phase A was maintained at 5% for 27-32 minutes;

[0032] The mobile phase A increased from 5% to 95% over 32-33 minutes.

[0033] Mobile phase A was maintained at 95% for 33-38 minutes;

[0034] Mass spectrometry conditions were as follows: ESI ion source, carrier gas 1 / 2: 50psi, CUR: 35psi, temperature: 550℃, ISVF: 5500V(+) and -4500V(-), CE and CES: 35±15eV, m / z: 20~1500Da, DP: 80eV.

[0035] The blood-entering components of Yinling Qushi Granules are: L-Valin, Liquiditigenin, Ononin, Formononetin, Sulfation of Formononetin, Daidzein, calycosin, Sulfation of calycosin, poricoicacid G, Glycyrrhizic acid, Daedaleanic acid B, Poricoic acid B, Poricoic Acid DM, Dehydroeburic acid, α-linolenic acid, linoleic acid, and oleic acid.

[0036] Preferably, the serum undergoes pretreatment, and the pretreatment steps are as follows:

[0037] (1) Take 90 μL of human serum from patients who have been treated for 4 weeks and put it into a centrifuge tube. Add 10 μL of IS solution containing 10 μg / mL of deuterated naringenin.

[0038] (2) Add 1 mL of ethyl acetate solution, vortex for 5 min, and centrifuge at 10,000 rpm for 15 min at 4℃;

[0039] (3) Take the supernatant, blow it dry with nitrogen, and add 100 μL of 80% methanol-water mixture;

[0040] (4) Ultrasound for 5 minutes, vortex for 5 minutes, centrifuge at 13000 rpm for 15 minutes at 4℃, and then test on the machine.

[0041] This study investigated the complete composition of Yinling Qushi Granules, a traditional Chinese medicine formula for dispelling dampness, and examined its blood components after long-term intervention in patients with dampness-related metabolic syndrome. The aim is to inject a new modern theoretical framework into the research of TCM dampness syndrome and the clinical intervention of dampness-dispelling formulas, and to provide a reference for the research of other TCM syndromes. Attached image description:

[0042] Figure 1 This describes the metabolic transformation pathway of the components of Yinling Qushi Granules entering the bloodstream;

[0043] Figure 2 These are secondary fragmentation diagrams of gentianin and its sulfated products: (A) gentianin; (B) gentianin sulfated esterified products. Detailed implementation method:

[0044] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0045] Example 1:

[0046] I. Methods

[0047] 1. Complete component analysis of Yinling Dehumidifying Granules

[0048] 1.1 Preparation of Yinling Dehumidifying Granules and Single Herbal Ingredients

[0049] The preparation method of Yinling Dehumidifying Granules is as follows:

[0050] (1) Poria cocos 77.76g, Atractylodes macrocephala 29.16g, ginger 12.96g, Polyporus umbellatus 19.44g, Cinnamomum cassia 19.44g, Artemisia capillaris 24.3g, Adenophora stricta 19.44g, Glycyrrhiza uralensis 19.44g.

[0051] (2) Mix all the Chinese medicinal materials and decoct them. For the first decoction, add 10 times the volume of water, heat to boiling, keep simmering for 1.5 hours, and filter (using a 200-mesh sieve).

[0052] (3) Add 10 times the volume of water to the second decoction, heat to boiling, maintain a gentle boil for 1.0 h, and filter (using a 200-mesh sieve).

[0053] (4) Combine the two filtrates and concentrate them under reduced pressure to a specific gravity of 1.08 to 1.10.

[0054] (5) After spray drying, the mixture is sieved and mixed to obtain the final product.

[0055] (6) The extracts of each single herb were prepared in the same manner as described above.

[0056] (7) Accurately measure 1 mL of Yinling Qushi Granules Concentrate and 1 mL of each single herb concentrate into a 5 mL volumetric flask, add water to make up to the mark, mix well and then test on the instrument.

[0057] 1.2 Liquid Chromatography-Mass Spectrometry Detection Conditions

[0058] Liquid chromatography conditions: A reversed-phase C18 column (3×150mm, 2.6μm) of pheromone (Phenonmenex, USA) was used at a column temperature of 40℃. The mobile phases were: A was 0.1% formic acid in water and B was acetonitrile. The elution parameters are shown in Table 1.

[0059] Table 1 Gradient elution program

[0060]

[0061] Detection was performed using an AB Sciex 5600plus mass spectrometer system. Optimized parameters were as follows: ESI ion source, carrier gas 1 / 2: 50 psi, CUR: 35 psi, temperature: 500 °C, ISVF: 5500 V (+), -4500 V (-), CE and CES: 35 ± 15 eV, m / z: 50–1500 Da, DP: 80 eV. The detection was performed using an AB Sciex-developed mass spectrometer in IDA mode. 1.2 The software (AB Sciex, USA) was used for data collection and detection.

[0062] 1.3 Data Analysis

[0063] The steps for analyzing the complete composition of Yinling Dehumidifying Granules are as follows:

[0064] (1) Self-built database construction: Chemical composition information of Yinling Qushi Granules was collected using PubMed and CNKI, and integrated into a Peakview (version 1.2, AB Sciex) compatible data format. The specific steps are as follows: Using the built-in XIC Manager module in Peakview, after clicking "New session", select the Wiff download file, select "Asastandard TIC", import the previously collected single herb information into the XIC manager, and save this information as a file with the XIClist extension, thus obtaining the self-built database. Then, in subsequent analysis, after importing the data using Peakview, simply import this XIClist file, click "show XIC" to obtain the comparison results. Finally, secondary fragment information comparison was performed, and components where both Mass and Isotope were red were deleted to obtain the final comparison results.

[0065] (2) High-resolution natural product database comparison: Natural Products HR MS was used. 2 Further alignment was performed using the SpectralLibrary (Version 1.0.3, AB Sciex) database. The specific steps are as follows: Using Peakview (version 2.2, AB Sciex), which is compatible with the natural product database developed by AB Sciex, click "File" to import the downloaded .wiff file. Use the "Masterview" module with the following alignment parameters: Precursor Mass: 0.4 Da, Mass Tolerence: 0.4 Da, Maximum Number of Hits: 5, Intensity Threshold: 0.05, Minimal Purity: 10%. All parameters are set to default. Click "Import Compounds from LibraryViewDatabase." After loading, select the "TCM MS / MS Library" database and check "Import FragmentMass (Da) for compounds." Confirm to start the search. Finally, the data was cleaned up, deleting components where both Mass and Isotope were red, and retaining compounds with an alignment score above 0.8. This yields the final alignment results.

[0066] 2. Blood component analysis of Yinling Dampness-Clearing Granules

[0067] 2.1 Preparation of serum samples after long-term intervention with Yinling Qushi Granules

[0068] Thirty patients with dampness-induced metabolic syndrome were selected for clinical trials. They were given one dose of Yinling Qushi Granules (137g of total raw medicinal material) orally daily for 4 weeks, and serum samples were collected.

[0069] (1) Take 90 μL of clinical human serum after 4 weeks of administration into a centrifuge tube and add 10 μL of IS solution (containing 10 μg / mL of deuterated naringenin);

[0070] (2) Add 1 mL of ethyl acetate solution, vortex for 5 min, and centrifuge at 4℃ for 15 min (10000 rpm);

[0071] (3) Take the supernatant, blow it dry with nitrogen, and add 100 μL of 80% methanol-water mixture;

[0072] (4) After sonication for 5 minutes, vortexing for 5 minutes, centrifugation at 4℃ for 15 minutes (13000 rpm), and then testing, the sample was tested.

[0073] 2.2 Liquid Chromatography-Mass Spectrometry Detection Conditions

[0074] A reversed-phase C18 column (2.1×100mm, 2.6μm) with pheromone (Phenonmenex, USA) was used, and the elution parameters were the same as those in "1. Analysis of all components of Yinling Qushi Granules, 1.2 Liquid Chromatography-Mass Spectrometry".

[0075] Detection was performed using an AB Sciex 7600 Zeno-TOF mass spectrometer system. Optimized parameters were as follows: ESI as the ion source, carrier gas 1 / 2: 50 psi, CUR: 35 psi, temperature: 550 °C, ISVF: 5500 V (+), -4500 V (-), CE and CES: 35 ± 15 eV, m / z: 20–1500 Da, DP: 80 eV. Data acquisition was performed in IDA mode, and data analysis was conducted using SCIEXOS 2.0 software (AB Sciex, USA).

[0076] 2.3 Data Analysis

[0077] The steps for analyzing the blood components of Yinling Qushi Granules are the same as those in "1.3 Data Analysis under the Item 1. Analysis of All Components of Yinling Qushi Granules". For the identification of metabolites of blood-entering components, under mass spectrometry detection, secondary fragments and fragments of the parent nucleus exhibit similar fragmentation patterns. Furthermore, if a phase I metabolic reaction occurs in vivo, such as methylation (CH2), the mass of the metabolite is 14 Da greater than that of the parent nucleus; if a phase II metabolic reaction occurs, such as sulfation (SO3), the mass of the metabolite is 80 Da greater than that of the parent nucleus. Based on these patterns, a self-built library of the in vivo metabolic pathways of each chemical component can be constructed using the self-built library steps, and the in vivo metabolite results can be obtained through retrieval and comparison.

[0078] II. Experimental Results

[0079] In order to fully analyze the chemical composition of Yinling Qushi Granules, the chemical composition of individual medicinal materials was collected by consulting literature, including Poria cocos, Polyporus umbellatus, dried ginger and other medicinal materials.

[0080] The chemical components of Yinling Qushi Granules and its eight constituent medicinal herbs were analyzed using UFLC-Q-TOF-MS / MS technology. Based on information from a self-built database and secondary fragment information from a natural product database, a total of 106 chemical components of Yinling Qushi Granules were identified under positive and negative detection modes. After comparison and component attribution with individual medicinal herbs, 43 components were found to be from Poria cocos, 6 from dried ginger, and 8 from Polyporus umbellatus. The number of components detected in the remaining medicinal herbs is detailed in Table 2. The chemical components were then structurally classified. The results showed that the components of Yinling Qushi Granules include triterpenoids, flavonoids, saponins, amino acids, phenolic acids, and others. These results indicate that Yinling Qushi Granules have a complex and diverse composition, suggesting the multi-component characteristic of traditional Chinese medicine.

[0081] Table 2. Component Analysis of Yinling Dehumidifying Granules

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] Note: FL: Poria cocos, BZ: Atractylodes macrocephala, ZL: Polyporus umbellatus, GJ: Dried ginger, YC: Artemisia capillaris, GZ: Cinnamomum cassia, BSS: Adenophora stricta, GC: Glycyrrhiza uralensis

[0099] In our previous study, we conducted a comprehensive analysis of all components of Yinling Qushi Granules and constructed a chemical composition database for the granules. Based on this database, we identified the in vivo blood-entering components of Yinling Qushi Granules. The results are shown in Table 3. Through comparison with secondary fragments, a total of 17 blood-entering components were detected in the patient's serum, of which 14 were the original components, classified into four categories according to their parent nucleus structure: terpenes, flavonoids, saponins, and amino acids; and 3 metabolites (M4-1, M4-2, and M5-1).

[0100] Table 3. Blood-entry components and metabolites of Yinling Qushi Granules

[0101]

[0102]

[0103]

[0104] Note: Glc = glucosinolate; P: original compound, M: metabolite; BZ, Atractylodes macrocephala; GC, Glycyrrhiza uralensis; FL, Poria cocos.

[0105] Therefore, we constructed the metabolic pathways of the components entering the bloodstream and their metabolites in the body. For example... Figure 1As shown, the results indicate that terpenoids and saponins in Yinling Qushi Granules mainly exist in their original form in vivo, but at low abundance. After absorption and transformation in vivo, some flavonoids remain in their original form, while others undergo metabolic transformation, primarily sulfation in phase II metabolism. Specifically, P3, P4, and P5 were confirmed, after comparison with secondary fragments, to be ononin, formononetin, and calycosin, respectively. Unlike terpenoids, these flavonoids underwent extensive phase I and phase II metabolic reactions in vivo. Among them, gentianin (P3) undergoes hydrolysis in vivo to remove its glycoside portion, forming its aglycone, gentianin (P4). Gentianin can be demethylated in vivo to generate daidzein (M4-2), and conversely, daidzein can be methylated to generate gentianin (P4). In addition, gentianin can be dehydroxylated to generate verbascoflavonoids (P5), and similarly, verbascoflavonoids can be hydroxylated to generate gentianin (P5). For the phase II reaction, the sulfation reaction resulted in a loss of 80 Da (SO3) in the characteristic fragments of mass spectrometry. Based on this fragmentation pattern, M4-1 and M5-1 were preliminarily identified as the sulfation products of gentianin (P4) and verbascoflavonoids (P5) in vivo, respectively. For example, under negative mode detection, mongospermin (molecular weight: 268.26) generates a precursor ion 267.0665[MH]- under ESI source, then loses CH3 (15 Da) to generate 252.0433[MH-CH3]-, and then further cleaves to generate 223.0404[MHO-CO]-, 208.0526[MHO-CO-CH3]- and 195.0455 characteristic fragments. As a flavonoid compound, mongospermin ( Figure 2 In the case of A), a ring-opening reaction (retro Diels-Alder, RDA) occurs under the action of an ESI source, generating the characteristic ionic fragment 132.0218[1,3B]-. Given this fragmentation mode, M4-1 was identified as a metabolite of sulfation of gentianin. Figure 2As shown in Figure B), under the action of an ESI source, it generates a precursor ion 347.0227 [MH]-, which then loses 80 Da (SO3) to give a fragment ion 267.0666 [MH-SO3]-. This fragment ion is also a precursor ion of the original monganin compound in negative mode, and then generates the same characteristic fragments, such as 252.0429 [MH-SO3-CH3]-, 223.0397 [MH-SO3-O-CO]-, 208.0526 [MH-SO3-O-CO-CH3]-, and 195.0457. Finally, the sulfation product of monganin (M4-1) also undergoes an RDA ring-opening reaction and generates the characteristic fragment 132.0233 [1,3B]-. In summary, after administration of Yinling Qushi Granules, the components entering the bloodstream are absorbed into the body and undergo phase I or phase II metabolic reactions. Furthermore, high-resolution Zeno-TOF-MS / MS analysis revealed that the metabolites exhibited highly similar secondary fragmentation and lysis patterns to the corresponding blood-entered components. This suggests that the aforementioned blood-entered components and metabolites may be the active compounds of Yinling Qushi Granules.

Claims

1. A method for detecting all components of Yinling Dehumidifying Granules, characterized in that, Includes the following steps: A. Decoction of Yinling dampness-removing granules, filtration of the decoction to obtain filtrate, and concentration of the filtrate to obtain concentrated solution. B. Analyze the concentrated solution using liquid chromatography-mass spectrometry; The liquid chromatography conditions are as follows: reversed-phase C18 column, column temperature 40 ℃, mobile phase: A is 0.1% formic acid water (v / v), B is acetonitrile, flow rate 0.3 ml / min; Within 0-20 minutes, mobile phase A decreased from 95% to 50%; The mobile phase A decreased from 50% to 5% over 20-27 minutes. Mobile phase A was maintained at 5% for 27-32 minutes; The mobile phase A increased from 5% to 95% over 32-33 minutes. Mobile phase A was maintained at 95% for 33-38 minutes; Mass spectrometry conditions were as follows: ESI ion source, carrier gas 1 / 2: 50 psi, CUR: 35 psi, temperature: 500 ℃, ISVF: 5500 V (+), -4500 V (-), CE and CES: 35 ± 15 eV. m / z :50~1500 Da, DP: 80 eV; The complete composition of Yinling Dehumidifying Granules includes triterpenoids, flavonoids, saponins, amino acids, and phenolic acids; The analysis of the complete components is shown below.

2. The detection method according to claim 1, characterized in that, The concentrate obtained by filtrate concentration is a concentrate obtained by vacuum concentration to a specific gravity of 1.08 to 1.

10.

3. A method for detecting the blood-entering components of Yinling Qushi Granules, characterized in that, Includes the following steps: A. Patients with dampness syndrome were given Yinling Qushi Granules, and their serum was collected after a predetermined time as the experimental group. The serum of patients with dampness syndrome who were not given Yinling Qushi Granules was used as the control group. B. The experimental and control groups were detected by liquid chromatography-mass spectrometry. The liquid chromatography conditions are as follows: reversed-phase C18 column, column temperature 40 ℃, mobile phase: A is 0.1% formic acid water (v / v), B is acetonitrile, flow rate 0.3 ml / min; Within 0-20 minutes, mobile phase A decreased from 95% to 50%; The mobile phase A decreased from 50% to 5% over 20-27 minutes. Mobile phase A was maintained at 5% for 27-32 minutes; The mobile phase A increased from 5% to 95% over 32-33 minutes. Mobile phase A was maintained at 95% for 33-38 minutes; Mass spectrometry conditions were as follows: ESI ion source, carrier gas 1 / 2: 50 psi, CUR: 35 psi, temperature: 550 ℃, ISVF: 5500 V (+), -4500 V (-), CE and CES: 35 ± 15 eV. m / z :20~1500 Da, DP: 80 eV; The blood-entering components of Yinling Dehumidifying Granules are: L-Valin, Liquiditigenin, Ononin, Formononetin, Sulfation of Formononetin, Daidzein, calycosin, Sulfation of calycosin, poricoicacid G, Glycyrrhizic acid, Daedaleanic acid B, Poricoic acid B, Poricoic Acid DM, Dehydroeburic acid, α-linolenic acid, linoleic acid, and oleic acid. The serum underwent pretreatment, and the pretreatment steps are as follows: (1) Take 90 μL of human serum from patients who have been treated for 4 weeks and put it into a centrifuge tube. Add 10 μL of IS solution containing 10 μg / mL of deuterated naringenin. (2) Add 1 mL of ethyl acetate solution, vortex for 5 min, and centrifuge at 10,000 rpm for 15 min at 4 ℃; (3) Take the supernatant, blow it dry with nitrogen, and add 100 μL of 80% methanol-water mixture; (4) Ultrasound for 5 min, vortex for 5 min, centrifuge at 13000 rpm for 15 min at 4 ℃, and then test on the machine.

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