Method for detecting blood entering components of Mongolian medicine Tiangui 3 for treating asthma

Abstract

The invention discloses a detection method of a blood entering component of a Mongolian medicine Tiangu-3 for treating asthma, and relates to the technical field of medicine in-vivo metabolism, and the detection method specifically comprises the following steps: S1, collection of a biological sample: respectively collecting initial blank serum and drug-containing serum for later use; s2, preparation of a biological sample: treating the initial blank serum and the drug-containing serum to obtain blank serum and drug administration serum samples for later use; s3, preparing an in-vitro test sample, namely treating daily check-3 to obtain a traditional Chinese medicine sample for later use; and S4, respectively taking the blank serum, the administration serum and 10 mu L of the traditional Chinese medicine sample, carrying out LC-MS / MS analysis, and determining the in-blood components based on the detection result. The method disclosed by the invention can more comprehensively reveal the characteristics of the drug-derived components after the drug enters the blood, and fills the blank in the fields of qualitative analysis of the components entering the blood and quantitative detection of all the effective components in the Jiaogue-3 (Jiaogue-3) (Jiaogue-3) (Jiaogue-3).

Description

Technical Field

[0001] The present invention relates to the technical field of drug metabolism in vivo, and in particular to a method for detecting blood-entering components of the Mongolian medicine Cha Ri Cha-3 used for treating asthma. Background Art

[0002] Cha Ri Cha-3 powder is a traditional Mongolian medicine made from ephedra, licorice, and grasshoppers. It has been used in Mongolian medicine for thousands of years to treat colds, influenza, upper respiratory tract infections, and other ailments. Its proven efficacy, safety, natural properties, lack of toxic side effects, and affordability make it a popular choice among patients. Its primary efficacy is clearing away heat and relieving symptoms. Modern pharmacology demonstrates this benefit through anti-inflammatory, antibacterial, and antiviral effects. In Mongolian medicine, asthma, known as "husile," is a common chronic airway inflammatory disease. Asthma is primarily associated with an imbalance in the three roots of "hei," "xila," and "badagan." Excessive "hei" can cause airway spasm, resulting in symptoms such as wheezing and shortness of breath; excessive "xila" can cause congestion and increased secretions in the airway mucosa; and excessive "badagan" can lead to mucus stagnation and airway obstruction.

[0003] Current research focuses on analyzing the chemical composition of Charicha-3, but the underlying substance and mechanism of action of its active ingredients remain largely unexplained. Currently, methods for measuring the concentration and distribution of these components in blood are inadequate. Future research should focus on further elucidating the basis for its efficacy and developing blood testing technologies.

[0004] Liquid chromatography-mass spectrometry (LC-MS) is a very important means and method in metabolomics research. It uses high-performance liquid chromatography to separate metabolites and then uses high-resolution mass spectrometry to detect metabolites. Compared with other detection technologies, LC-MS is more suitable for detecting and analyzing metabolites that are difficult to volatilize or have poor thermal stability in samples. Summary of the Invention

[0005] In view of the above problems, the present invention provides a method for detecting blood-entering components of the Mongolian medicine Cha Ri Cha-3 for treating asthma.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a method for detecting blood components of the Mongolian medicine Cha Ri Cha-3 for treating asthma, such as Figure 26 、 Figure 27 , specifically including the following steps: S1: Biological sample collection: Collect initial blank serum and drug-containing serum separately for future use; S2: Biological sample preparation: The initial blank serum and drug-containing serum are processed to obtain blank serum and drug-containing serum samples for later use; S3: Preparation of in vitro test samples: Take Cha Ri Cha-3 and process it to obtain Chinese medicine samples for later use; S4: Take 10 μL of blank serum, drug-treated serum, and Chinese medicine sample for LC-MS / MS analysis, and determine the blood components based on the test results.

[0007] Furthermore, in step S1, the biological sample collection is specifically as follows: after one week of adaptive feeding of SD rats, 1 mL of blood is collected from the orbits of each rat before administration, and the blood is centrifuged at 3500 r / min for 10 minutes to separate the plasma to obtain the initial blank serum, which is frozen at -80°C for later use; after continuous administration for 37 days, 1 mL of blood is collected from the orbits of each rat 1 hour after administration on the last day, and the blood is centrifuged at 3500 r / min for 10 minutes to separate the plasma to obtain the initial drug-containing serum, which is frozen at -80°C for later use.

[0008] Furthermore, the dosage of Cha Ri Cha-3 is 1.08 g / (kg·d).

[0009] Furthermore, in step S2, the biological sample preparation is specifically as follows: 100 μL of initial blank serum and drug-containing serum are taken respectively, 400 μL of pre-cooled pure methanol is added, the mixture is shaken for 1 min, ultrasonicated in an ice bath for 20 min, allowed to stand at -20°C for 1 h, centrifuged at 16000g at 4°C for 20 min, the supernatant is taken, lyophilized, 100 μL of pre-cooled 50% methanol is added for reconstitution, and the mixture is centrifuged at 20000g at 4°C for 15 min. The supernatant is taken to obtain blank serum and drug-containing serum samples for later use.

[0010] Furthermore, in step S3, the in vitro test sample preparation is specifically as follows: weigh 100 mg of Cha Ri Cha-3 powder, add 1 mL of pure water to dissolve, add 3 mL of ethanol, ultrasonicate at room temperature for 10 minutes, let stand at 4°C for 12 hours, centrifuge at 4000g for 10 minutes at 4°C, take the supernatant, blow dry the ethanol with nitrogen to obtain a refined solution, add ultrapure water to make up the volume to 1 mL, filter through a 0.22 μm filter membrane to obtain a traditional Chinese medicine sample for later use.

[0011] Furthermore, in step S4, the chromatographic conditions are as follows: the chromatographic column is an ACQUITY UPLC® HSS T3 chromatographic column, 2.1×100 mm, 1.8 µm; the column temperature is 40°C, the flow rate is 0.3 mL / min; the chromatographic mobile phase A is 0.1% formic acid in water, and the mobile phase B is 0.1% formic acid in acetonitrile; the chromatographic gradient elution program is as follows: 0---1 min, 0% B; 1---2 min, B changes linearly from 0% to 30%; 2---12 min, B changes linearly from 30% to 50%; 12---21 min, B changes linearly from 50% to 100%; 21---26 min, B is maintained at 100%; 26---26.1 min, B changes linearly from 100% to 0%; 26.1---30 min, B is maintained at 0%.

[0012] Furthermore, in step S4, the mass spectrometry conditions are as follows: electrospray ionization is used for positive ion (+) and negative ion (-) mode detection, and the sample is separated by UPLC and then subjected to mass spectrometry analysis using TripleTOF6600 (ABSciex). The source gas parameters are as follows: ion spray voltage: +5500 / -4500 V; temperature: 500°C; ion source gas 1: 60 psi; ion source gas 2: 60 psi; curtain gas: 45 psi; declustering voltage: 60 V; TOF MS scan collision: 10 V; product ion scan collision energy: 40 V; collision energy expansion: 20 V.

[0013] Compared with the prior art, the present invention has the following beneficial effects: This study developed a method for detecting the blood-entering components of Cha Ri Cha-3 based on liquid chromatography-mass spectrometry (LC-MS). Using this method, 780 substances were detected in both positive and negative ion modes, including 614 traditional Chinese medicine components, 38 of which entered the bloodstream. This method can more comprehensively reveal the characteristics of the drug's medicinal components after entering the bloodstream, filling a gap in the field of qualitative analysis of Cha Ri Cha-3's blood-entering components and quantitative detection of all active ingredients in Cha Ri Cha-3. It lays a solid foundation for in-depth research into the drug's metabolic cycle and mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The positive (upper) and negative (lower) ion mode Base Peak spectra of the blank serum group KB-1 of the present invention are shown; Figure 2 The positive (upper) and negative (lower) ion mode Base Peak spectra of the blank serum group KB-2 of the present invention are shown; Figure 3 The positive (upper) and negative (lower) ion mode Base Peak spectra of the blank serum group KB-3 of the present invention are shown; Figure 4 The positive (upper) and negative (lower) ion mode Base Peak spectra of the blank serum group KB-4 of the present invention are shown; Figure 5 The positive (upper) and negative (lower) ion mode Base Peak spectra of the blank serum group KB-5 of the present invention are shown; Figure 6 The positive (upper) and negative (lower) ion mode Base Peak spectra of the blank serum group KB-6 of the present invention are shown; Figure 7 The positive (upper) and negative (lower) ion mode Base Peak spectra of the GY-1 serum group administered according to the present invention are shown; Figure 8The positive (upper) and negative (lower) ion mode Base Peak spectra of the serum group GY-2 administered in the present invention; Figure 9 The positive (upper) and negative (lower) ion mode Base Peak spectra of the serum group GY-3 administered in the present invention are shown; Figure 10 The positive (upper) and negative (lower) ion mode Base Peak spectra of the serum group GY-4 administered in the present invention are shown; Figure 11 The Base Peak spectra of the positive (upper) and negative (lower) ion modes of the serum group GY-5 administered according to the present invention are shown; Figure 12 The Base Peak spectra of the positive (upper) and negative (lower) ion modes of the serum group GY-6 administered according to the present invention are shown; Figure 13 The base peak spectra of the Chinese medicine group CRC-1 in the positive (upper) and negative (lower) ion modes of the present invention are shown; Figure 14 The base peak spectra of the Chinese medicine group CRC-2 in the positive (upper) and negative (lower) ion modes of the present invention are shown; Figure 15 The base peak spectra of the Chinese medicine group CRC-3 in positive (upper) and negative (lower) ion modes of the present invention are shown; Figure 16 The positive (upper) and negative (lower) ion mode Base Peak spectra of Ephedra MH of the present invention are shown; Figure 17 The positive (upper) and negative (lower) ion mode Base Peak spectra of the GC of Glycyrrhizae Radix of the present invention are shown; Figure 18 The base peak spectra of the grasshopper MZ in the positive (upper) and negative (lower) ion modes of the present invention are shown; Figure 19 It is a Venn diagram of blood components of the Chinese medicine of the present invention (CRC), blank serum (KB), and drug-administered serum (GY); Figure 20 It is the Upset diagram of the CRC of the Chinese medicine of the present invention and related medicinal materials; Figure 21 It is a fan diagram of compound classification of the Chinese medicine CRC of the present invention; Figure 22 This is a fan-shaped diagram of the classification of blood components of the Chinese medicine CRC of the present invention; Figure 23 The KEGG enrichment of the blood-entering components of the Chinese medicine CRC of the present invention in the model plant Arabidopsis thaliana; Figure 24 The KEGG enrichment of the blood components of the Chinese medicine CRC in rats; Figure 25 This is the KEGG pathway diagram of the CRC blood components of the traditional Chinese medicine of the present invention in rats; Figure 26 It is a technical roadmap for identifying the components of Chinese medicines of the present invention; Figure 27 It is a flow chart for analyzing the components of Chinese medicine entering blood. DETAILED DESCRIPTION

[0015] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0016] Unless otherwise specified, the instruments, reagents, and materials used in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0017] Example 1 1. Experimental instruments and reagents Table 1 Experimental instruments and reagents

[0018] 2. Extraction of metabolites (1) Experimental animal grouping and drug administration Six female Sprague-Dawley (SD) rats, weighing 200-230 g, were SPF-qualified. They were housed in a clean animal room at the GLP Center of the School of Pharmacy, Inner Mongolia Medical University. The room temperature was 22 ± 2°C, the humidity was 40% ± 5%, and the light and dark cycles were 12 h each. The rats had free access to food and water. After one week of acclimatization, orbital blood was drawn before dosing to obtain blank plasma. On the day of dosing, 1.08 g / kg / day of Chacha-3 was administered for 7 consecutive days, and orbital blood was drawn to obtain drug-containing plasma.

[0019] (2) Collection of biological samples: After one week of adaptive feeding, 1 mL of blood was collected from the orbits of rats before administration, and the blood was centrifuged at 3500 r / min for 10 min to separate the plasma. The initial blank serum was obtained and stored at -80°C for future use. After continuous administration of Chadai Cha-3 1.08 g / (kg·d) for 7 days, 1 mL of blood was collected from the orbits of rats 1 hour after administration on the last day. The blood was centrifuged at 3500 r / min for 10 min to separate the plasma. The initial drug serum was obtained and stored at -80°C for future use. (3) Biological sample preparation: 100 μL of initial blank serum and drug-containing serum were taken respectively, and 400 μL of pre-cooled pure methanol was added. The mixture was shaken for 1 min, ultrasonicated in an ice bath for 20 min, allowed to stand at -20°C for 1 h, centrifuged at 16,000 g and 4°C for 20 min, the supernatant was taken, freeze-dried, and re-dissolved in 100 μL of pre-cooled 50% methanol. The mixture was centrifuged at 20,000 g and 4°C for 15 min, and 50 μL of supernatant was taken for later use. Blank serum and drug-containing serum samples were obtained. 10 μL of each sample was injected into the mass spectrometer, and each was injected once. The blank serum group was recorded as the KB group, including KB-1, KB-2, KB-3, KB-4, KB-5, and KB-6, and the drug-containing serum group was recorded as the GY group, including GY-1, GY-2, GY-3, GY-4, GY-5, and GY-6. (4) Preparation of in vitro test samples: Weigh 100 mg of Cha Ri Cha-3 powder, add 1 mL of pure water to dissolve, add 3 mL of ethanol, ultrasonicate at room temperature for 10 min, let stand at 4°C for 12 h, centrifuge at 4000 g for 10 min at 4°C, take the supernatant, blow dry the ethanol with nitrogen to obtain the refined solution, add ultrapure water to make up the volume to 1 mL, filter through a 0.22 μm filter membrane, and obtain the traditional Chinese medicine sample for use. Inject 10 μL of the sample into the mass spectrometer, repeat the technique for 3 injections, and the traditional Chinese medicine group is recorded as the CRC group, including CRC-1, CRC-2, and CRC-3. Weigh ephedra, licorice and grasshopper separately and process them according to the above method to obtain ephedra, licorice and grasshopper samples, which are recorded as MH, GC and MZ.

[0020] 3. LC-MS / MS analysis (1) Chromatographic separation Throughout the analysis, samples were placed in an autosampler at 4°C and analyzed using a SHIMADZU-LC30 ultra-high-performance liquid chromatography (UHPLC) system with an ACQUITY UPLC® HSS T3 (2.1 × 100 mm, 1.8 µm) column (Waters, Milford, MA, USA). The column temperature was 40°C, and the flow rate was 0.3 mL / min. The mobile phases A and B consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The chromatographic gradient elution program was as follows: 0---1 min, 0% B; 1---2 min, B changed linearly from 0% to 30%; 2---12 min, B changed linearly from 30% to 50%; 12---21 min, B changed linearly from 50% to 100%; 21---26 min, B was maintained at 100%; 26---26.1 min, B changed linearly from 100% to 0%; 26.1---30 min, B was maintained at 0%.

[0021] (2) Mass spectrometry acquisition Each sample was detected using electrospray ionization (ESI) in both positive (+) and negative (-) modes. Following UPLC separation, the samples were analyzed by mass spectrometry using a TripleTOF 6600 (ABSciex). The source gas parameters were as follows: Table 2

[0022] 4. Data Preprocessing A library of ephedra, licorice, and grasshopper was established, and related medicinal materials with special marks (origin, subspecies) were uniformly classified into a certain medicinal material category in the results.

[0023] The collected mass spectrometry raw data were peak aligned, retention time corrected, and peak area extracted using MS DIAL software. Metabolite structure identification parameters included a mass tolerance of <0.01 Da for primary spectrum matching, a mass tolerance of <0.02 Da for secondary spectrum matching, and a secondary mass spectrum matching score greater than 70%.

[0024] 5. Data visualization Identified metabolites were mapped into superclass and class fan and ring diagrams using R. Pathway enrichment was performed using the KEGG IDs of the identified target components. Fisher's exact test with a p < 0.05 was used to screen for significantly enriched functions and pathways, and plotted using the ggplot2 package.

[0025] 6. Analysis of experimental results (1) Base Peak graph of the sample Two mass spectrometry raw files (positive ion mode and negative ion mode) were obtained for each sample after UPLC-Q-TOF (LC-MS / MS) detection and analysis. Figure 1-18 The mass spectrometry BasePeak graphs of blank serum KB group, drug-treated serum GY group, traditional Chinese medicine CRC group, ephedra MH, licorice GC, and grasshopper MZ in positive and negative ion detection modes are shown respectively.

[0026] (2) Identification results of TCM components entering blood The results of TCM blood components were obtained by comparing the TCM CRC group, blank serum KB group, and drug serum GY group. The analysis principle is as follows: Table 3

[0027] After comparative analysis, a total of 780 substances were detected in the positive and negative modes, including 614 traditional Chinese medicine components, of which 38 entered the blood, as shown in Tables 4 and 5 .

[0028] Table 4 List of the top 30 substances with the highest relative response in CRC component identification of traditional Chinese medicine ,

[0029] Table 5 Chinese herbal medicine components entering the blood ,

[0030] (3) Venn diagram analysis of blood components Figure 19 The Venn plot of traditional Chinese medicine (CRC), blank serum (KB), and drug-administered serum (GY) was displayed, and a preliminary statistical analysis of the entry of medicinal ingredients into the blood was conducted.

[0031] (4) Ownership of Chinese medicine ingredients and medicinal materials Figure 20 The Upset diagram of the traditional Chinese medicine CRC and the medicinal materials ephedra, licorice, and grasshopper is displayed.

[0032] The bar graph on the left of the figure represents the number of Chinese herbal ingredients detected in each sample, the bar graph above represents the number of common ingredients in each sample, and the dotted line below represents the samples where the common ingredients are located. CRC stands for compound medicine daily check, MH stands for ephedra, GC stands for licorice, and MZ stands for grasshopper.

[0033] (5) Classification of Chinese medicine substances Figure 21 The compound classification ring diagram of the Chinese medicine CRC is shown. Figure 22 A classification ring diagram of the components of traditional Chinese medicine that enter the blood for CRC is shown.

[0034] (6) KEGG analysis of substances entering the blood The 38 blood-entering components of the compound drug CRC were subjected to KEGG analysis using Arabidopsis thaliana and rats as references to analyze the synthesis of the compounds in plants and the metabolic pathways in animals. Figure 23 、 Figure 24 The KEGG enrichment results of blood-entering components in the model plant Arabidopsis and rats are shown. The pathway with the most significant P value is selected and a pathway diagram is drawn to show the following: Figure 25 .

[0035] Using liquid chromatography-mass spectrometry (LC-MS) and ultra-performance liquid chromatography (UPLC) coupled with quadrupole-orbitrap (Q-Exactive) mass spectrometry, the present invention accurately identified and quantified the main components of Charicha-3 and its blood-entering components. This method enables precise identification and quantitative determination of the various compounds in Charicha-3. These compounds encompass a wide range of bioactive molecules that play a crucial role in Charicha-3's pharmacological mechanisms, laying a solid foundation for subsequent drug metabolism research.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting blood components of the Mongolian medicine Cha Ri Cha-3 for treating asthma, characterized by: The specific steps include: S1: Biological sample collection: Collect initial blank serum and drug-containing serum separately for future use; Specifically, after one week of adaptive feeding, 1 mL of blood was collected from each SD rat's orbit before administration, and the blood was centrifuged at 3500 r / min for 10 minutes to separate the plasma to obtain the initial blank serum, which was then frozen at -80°C for future use. After 7 consecutive days of Cha Ri Cha-3 administration, 1 mL of blood was collected from each rat's orbit 1 hour after administration on the last day, and the blood was centrifuged at 3500 r / min for 10 minutes to separate the plasma to obtain the initial drug-containing serum, which was then frozen at -80°C for future use. The dosage of Cha Ri Cha-3 was 1.08 g / (kg·d). S2: Biological sample preparation: The initial blank serum and drug-containing serum are processed to obtain blank serum and drug-containing serum samples for later use; Specifically, 100 μL of initial blank serum and drug-containing serum were taken, 400 μL of pre-cooled pure methanol was added, shaken for 1 min, sonicated in an ice bath for 20 min, allowed to stand at -20°C for 1 h, centrifuged at 16,000 g at 4°C for 20 min, the supernatant was taken, lyophilized, and reconstituted with 100 μL of pre-cooled 50% methanol. The supernatant was centrifuged at 20,000 g at 4°C for 15 min, and the blank serum and drug-containing serum samples were obtained for later use. S3: Preparation of in vitro test samples: Take Cha Ri Cha-3 and process it to obtain Chinese medicine samples for later use; S4: Take 10 μL of blank serum, drug-treated serum, and Chinese medicine sample for LC-MS / MS analysis, and determine the blood components based on the test results.

2. The method for detecting blood components of the Mongolian medicine Cha Ri Cha-3 for treating asthma according to claim 1, characterized in that: In step S3, the in vitro test sample preparation is specifically as follows: weigh 100 mg of Cha Ri Cha-3 powder, add 1 mL of pure water to dissolve, add 3 mL of ethanol, ultrasonicate at room temperature for 10 minutes, let stand at 4°C for 12 hours, centrifuge at 4000g at 4°C for 10 minutes, take the supernatant, blow dry the ethanol with nitrogen to obtain a refined solution, add ultrapure water to make up the volume to 1 mL, filter through a 0.22 μm filter membrane to obtain a traditional Chinese medicine sample for later use.

3. The method for detecting blood components of the Mongolian medicine Cha Ri Cha-3 for treating asthma according to claim 1, characterized in that: In step S4, the chromatographic conditions are as follows: the chromatographic column is an ACQUITY UPLC® HSS T3 column, 2.1×100 mm, 1.8 µm; the column temperature is 40°C, the flow rate is 0.3 mL / min; the chromatographic mobile phase A is 0.1% formic acid in water, and the mobile phase B is 0.1% formic acid in acetonitrile; the chromatographic gradient elution program is as follows: 0---1 min, 0% B; 1---2 min, B is linearly changed from 0% to 30%; 2---12 min, B is linearly changed from 30% to 50%; 12---21 min, B is linearly changed from 50% to 100%; 21---26 min, B is maintained at 100%; 26---26.1 min, B is linearly changed from 100% to 0%; 26.1---30min, B is maintained at 0%.

4. The method for detecting blood components of the Mongolian medicine Cha Ri Cha-3 for treating asthma according to claim 1, characterized in that: In step S4, the mass spectrometry conditions are as follows: electrospray ionization is used for positive ion (+) and negative ion (-) mode detection, and the sample is separated by UPLC and then analyzed by mass spectrometry using TripleTOF6600 (ABSciex). The source gas parameters are as follows: ion spray voltage: +5500 / -4500 V; temperature: 500°C; ion source gas 1: 60 psi; ion source gas 2: 60 psi; curtain gas: 45 psi; declustering voltage: 60 V; TOF MS scanning collision: 10 V; Product ion scan collision energy: 40 V; collision energy expansion: 20 V.

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