Total component and blood-entering component of cough-relieving powder and detection method of total component and blood-entering component

Abstract

The invention provides a component analysis method of cough relieving powder and a detection method of components entering blood of the cough relieving powder. In component analysis of the cough-relieving powder, an ultra-high performance liquid chromatography-high resolution mass spectrometry technology is adopted to measure a cough-relieving powder liquid medicine, so that 162 chemical components in a cough-relieving powder solution are identified, and a scientific basis is provided for quality control and clinical application of the preparation. In the analysis of the components of the cough relieving powder entering blood, blank serum and administration serum are respectively prepared as test solutions, and are analyzed by using an ultra-high performance liquid chromatography-high resolution mass spectrometry method, and 64 components including 14 prototype components and 50 metabolites are identified in total. The method can comprehensively reflect the composition of the drug-derived component of the drug in vivo, fills up the technical blank of qualitative detection of the blood-entering component of the cough-relieving powder, and lays a foundation for researching the pharmacokinetic characteristics and metabolic pathways of the cough-relieving powder.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to all components and blood components of Zhisousan and a detection method therefor. Background Art

[0002] Zhisousan is a classic prescription in traditional Chinese medicine, which comes from "Medical Insights" written by Cheng Zhongling, a medical expert in the Qing Dynasty. Its core efficacy is to disperse the lung qi, dispel wind, relieve cough and resolve phlegm. It is commonly used to treat exogenous cough, prolonged and unresolved cough, relatively long course of disease, incomplete dissipation of exogenous pathogenic factors, and syndrome of failure of lung qi to disperse.

[0003] The classic compatibility composition of this prescription consists of 7 herbs in total, with clear division of labor among the monarch, minister, assistant, and envoy herbs:

[0004] 1. Monarch herbs: Aster tataricus L. f. and Stemona japonica (Blume) Miq. Aster tataricus L. f.: Bitter, warm, moistening and descending, resolving phlegm and relieving cough, and good at regulating cough caused by stagnation of lung qi; Stemona japonica (Blume) Miq.: Sweet, bitter and slightly warm, moistening the lung and relieving cough, especially good at treating new and old coughs, deficiency and excess coughs, and cough due to pulmonary tuberculosis.

[0005] Both are warm in nature but not dry, with good effect of moistening the lung and relieving cough. For the core pathogenesis of cough, they can be used for both new cough and long-term cough.

[0006] 2. Minister herbs: Platycodon grandiflorus (Jacq.) A. DC. and Cynanchum glaucescens (Decne.) Hand.-Mazz. Platycodon grandiflorus (Jacq.) A. DC.: Pungent, dispersing, bitter and purging, dispersing lung qi and promoting the movement of qi in the throat, and can carry the herbs upward to the lung meridian; Cynanchum glaucescens (Decne.) Hand.-Mazz.: Pungent, sweet and slightly warm, descending qi and resolving phlegm, and can relieve cough and excessive phlegm caused by stagnation of lung qi.

[0007] One promotes and the other descends, regulating the functions of dispersing and descending of lung qi.

[0008] 3. Assistant herbs: Schizonepeta tenuifolia Briq. and Citrus reticulata Blanco Schizonepeta tenuifolia Briq.: Pungent and warm, relieving exterior syndrome and expelling pathogenic wind, aiming at the situation where exogenous wind pathogen has not been completely dissipated; Citrus reticulata Blanco: Regulating qi and strengthening the spleen, drying dampness and resolving phlegm, resolving phlegm-dampness, and at the same time helping the lung qi to flow smoothly.

[0009] 4. Envoy herb: Glycyrrhiza uralensis Fisch. Benefiting qi and tonifying the middle, moistening the lung and relieving cough, harmonizing all herbs in the prescription, and also being able to relieve sore throat and pain.

[0010] At present, there is no relevant report on the analysis of all components and blood components of Zhisousan and its detection method. Summary of the Invention

[0011] The purpose of the present invention is to provide all components and blood components of Zhisousan and a detection method therefor.

[0012] The detection method for all components of Zhisousan provided by the present invention includes the following steps: 1) Mix the various medicinal materials used to make the cough-relieving powder, decoct and extract, collect the extract and concentrate it, freeze-dry the concentrated extract to obtain freeze-dried powder. 2) Add the lyophilized powder to the extraction solution for extraction. After extraction, take the supernatant and filter it through a microporous membrane (such as a 0.22μm microporous membrane). Detect the filtrate using ultra-high performance liquid chromatography-high resolution mass spectrometry. The cough-relieving powder contains 162 ingredients, including 91 shikimic acid and phenylpropionic acid derivatives, 27 terpenes, 14 fatty acids, 12 alkaloids, 6 amino acids and short peptides, 5 carbohydrates, 5 polyketides, and 2 acetophenones. Among them, 9 are derived from Aster tataricus, 12 from Stemona japonica, 14 from Platycodon grandiflorus, 5 from Cynanchum paniculatum, 26 from Schizonepeta tenuifolia, 19 from Citrus reticulata peel, 52 from Glycyrrhiza uralensis, and 25 from various medicinal herbs.

[0013] Specifically, the complete component analysis of the cough-relieving powder is shown in Table 1.

[0014] In step 1) of the above method, the cough-relieving powder is made from the following medicinal materials in parts by weight: 10 parts Platycodon grandiflorus, 10 parts Schizonepeta tenuifolia, 10 parts Aster tataricus, 10 parts Stemona japonica, 10 parts Cynanchum paniculatum, 5 parts Glycyrrhiza uralensis, and 5 parts Citrus reticulata.

[0015] In step 1) of the above method, the decoction extraction is performed at least twice. In each extraction, the mass ratio of water to mixed medicinal materials in the first decoction extraction is (8-12):1, and the extraction time can be 1-2 hours. In the second and subsequent decoction extractions, the mass ratio of water to mixed medicinal materials is (6-8):1, and the extraction time can be 0.67-1 hour.

[0016] According to a specific embodiment of the present invention, the specific method of decoction extraction is as follows: the mixed medicinal materials are soaked in 10 times the amount of distilled water for 30 min, decocted for 1 h, filtered, the residue is added to 8 times the amount of water, decocted for 1 h, filtered, and the two filtrates are combined.

[0017] In step 1) of the above method, the specific method for freeze-drying the concentrated extract is as follows: Place the concentrated extract at room temperature, pour it evenly into a freeze-drying dish, cover the top with plastic film and poke a few small holes, then freeze at -80℃. Place the frozen extract into a freeze dryer for freeze-drying. The specific operation of the freeze dryer is as follows: tighten the vent valve and pre-cool to -40℃. After 20 minutes, put the extract into the freeze dryer, cover it with an acrylic glass lid, select the vacuum mode, and start freeze-drying. After 3 days, take out the sample to obtain the product.

[0018] In step 2) of the above method, the extract is a mixed solvent composed of methanol, acetonitrile and water, with a volume ratio of (1.8-2.2): (1.8-2.2): (0.8-1.2) as follows, specifically 2:2:1.

[0019] In step 2) of the above method, the extract contains isotopically labeled internal standards, and the internal standards and their corresponding concentrations are as follows: L-leucine-5,5,5-d3 (concentration: 5.20 μg / mL), L-2-chlorophenylalanine (concentration: 2.01 μg / mL), glycocholic acid-D4 (concentration: 0.09 μg / mL), diisobutyl phthalate-D4 (concentration: 3.54 μg / mL), decanoic acid-D19 (concentration: 0.16 μg / mL), [2H5]-trans-zeatin (concentration: 0.51 μg / mL), and indole-3-acet-2,2-d2 acid (concentration: 2.38 μg / mL).

[0020] In step 2) of the above method, the ratio of the lyophilized powder to the extract is (10-100) mg : 500 μL.

[0021] In step 2) of the above method, the specific extraction method is as follows: Take 50 mg of sample powder into a 2 mL EP tube, add homogenizing beads and 500 μL of extraction solution, vortex and mix for 30 s; place in a homogenizer for homogenization (35 Hz, 240 s), then transfer to an ice-water bath for sonication for 5 min, repeat the treatment 3 times; let stand for 30 min, centrifuge at 4℃ and 12000 rpm for 15 min, take the supernatant, let stand again at -40℃ for 10 min, centrifuge for 15 min, and take the supernatant.

[0022] In step 2) of the above method, the ultra-high performance liquid chromatography uses a Vanquish ultra-high performance liquid chromatograph.

[0023] In step 2) of the above method, the conditions for ultra-high performance liquid chromatography-high resolution mass spectrometry detection are as follows: Chromatographic separation was performed using a Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 μm) liquid chromatography column. The mobile phase A was aqueous containing 0.01% acetic acid, and the mobile phase B was a 1:1 mixture of isopropanol and acetonitrile. Gradient elution was used. The flow rate was 0.300 mL / min. - ¹; Sample plate temperature 4 ℃, injection volume 2 μL; column oven temperature set to 25°C; total analysis time 12 min.

[0024] Mass spectrometry was performed using an Orbitrap Exploris 120 mass spectrometer, with data acquired in both positive and negative ion modes. The parameters were set as follows: sheath gas flow rate 50 Arb, auxiliary gas flow rate 15 Arb, capillary temperature 320 °C, first-order resolution 60,000, second-order resolution 15,000, collision energy SNCE 20 / 30 / 40, and spray voltages of 3.8 kV (positive ion) and -3.4 kV (negative ion).

[0025] Furthermore, the gradient elution procedure is as follows: 0 ~ 1.0 min, the volume fraction of mobile phase B is 1%; 1.0 ~ 8.0 min, the volume fraction of mobile phase B changes from 1% to 99%; 8.0 ~ 9.0 min, the volume fraction of mobile phase B is 99%; 9.1 ~ 12.0 min, the volume fraction of mobile phase B is 1%.

[0026] The method for detecting the blood-entering components of the cough-relieving powder provided by the present invention includes the following steps: A. Rats were given Zhike San (a traditional Chinese medicine for relieving cough), and serum was collected after a predetermined time as the experimental group. The serum of rats that were not given Zhike San was used as the control group. B. Add the serum to the extraction solution for extraction. After extraction, take the supernatant and filter it through a microporous membrane (such as a 0.22 μm microporous membrane). Detect the filtrate using ultra-high performance liquid chromatography-high resolution mass spectrometry. There are 64 components that enter the bloodstream, including 14 in their original form and 50 in their metabolite form. Among them, 2 come from Aster tataricus, 9 from Stemona japonica, 3 from Platycodon grandiflorus, 1 from Cynanchum paniculatum, 7 from Citrus reticulata peel, 8 from Schizonepeta tenuifolia, 27 from Glycyrrhiza uralensis, and 7 from various other medicinal herbs.

[0027] Specifically, the blood component analysis of the cough-relieving powder is shown in Table 2.

[0028] In step B of the above method, the extraction solution is a mixed solvent composed of methanol, acetonitrile and water, with the volume ratio of the three being (1.8-2.2): (1.8-2.2): (0.8-1.2), specifically 2:2:1.

[0029] In step B of the above method, the extract contains isotopically labeled internal standards, and the internal standards and their corresponding concentrations are as follows: L-leucine-5,5,5-d3 (concentration: 5.20 μg / mL), L-2-chlorophenylalanine (concentration: 2.01 μg / mL), glycocholic acid-D4 (concentration: 0.09 μg / mL), diisobutyl phthalate-D4 (concentration: 3.54 μg / mL), decanoic acid-D19 (concentration: 0.16 μg / mL), [2H5]-trans-zeatin (concentration: 0.51 μg / mL), and indole-3-acet-2,2-d2 acid (concentration: 2.38 μg / mL).

[0030] In step B of the above method, the specific extraction method is as follows: 200 μL of serum sample is taken into a 2 mL EP tube, 20 μL of hydrochloric acid solution is added, vortexed for 30 s, sonicated in an ice-water bath for 5 min, 780 μL of acetonitrile is added, vortexed for 30 s, sonicated again for 5 min, and allowed to stand at -40℃ for 30 min, then centrifuged for 15 min; 800 μL of supernatant is taken into a 2 mL EP tube, evaporated at low temperature, 80 μL of extraction solution is added to reconstitute, vortexed for 30 s, sonicated in an ice-water bath for 1 min, centrifuged for 15 min, and the supernatant is collected.

[0031] In step B) of the above method, the ultra-high performance liquid chromatography is performed using a Vanquish ultra-high performance liquid chromatograph.

[0032] In step B) of the above method, the conditions for ultra-high performance liquid chromatography-high resolution mass spectrometry detection are as follows: Chromatographic separation was performed using a Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 μm) liquid chromatography column. The mobile phase A was aqueous containing 0.01% acetic acid, and the mobile phase B was a 1:1 mixture of isopropanol and acetonitrile. Gradient elution was used. The flow rate was 0.300 mL / min. - ¹; Sample tray temperature 4 ℃, injection volume 2 μL; column oven temperature set to 25°C; total analysis time 12 min.

[0033] Mass spectrometry was performed using an Orbitrap Exploris 120 mass spectrometer, with data acquired in both positive and negative ion modes. The parameters were set as follows: sheath gas flow rate 50 Arb, auxiliary gas flow rate 15 Arb, capillary temperature 320 °C, first-order resolution 60,000, second-order resolution 15,000, collision energy SNCE 20 / 30 / 40, and spray voltages of 3.8 kV (positive ion) and -3.4 kV (negative ion).

[0034] Furthermore, the elution program for gradient elution is as follows: from 0 to 1.0 min, the volume fraction of mobile phase B is 1%; from 1.0 to 8.0 min, the volume fraction of mobile phase B changes from 1% to 99%; from 8.0 to 9.0 min, the volume fraction of mobile phase B is 99%; from 9.1 to 12.0 min, the volume fraction of mobile phase B is 1%.

[0035] In the component analysis of Zhisousan, ultra-high performance liquid chromatography-high resolution mass spectrometry was used to detect the Zhisousan medicinal liquid, thereby identifying 162 chemical components in the Zhisousan solution, providing a scientific basis for the quality control and clinical application of this preparation. In the analysis of the components of Zhisousan entering the blood, blank serum and administered serum were respectively prepared as test sample solutions, and analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry. A total of 64 compounds were identified, including 1 four kinds of prototype components and 50 metabolites. This method can comprehensively reflect the composition of drug-induced components in the body, filling the technical gap in the qualitative detection of the components of Zhisousan entering the blood, and laying a foundation for studying its pharmacokinetic characteristics and metabolic pathways. Brief Description of the Drawings

[0036] Figure 1 Total ion chromatograms of UPLC-Q-TOF-MS of Zhisousan extract in positive and negative ion modes (A. Positive ion mode; B. Negative ion mode).

[0037] Figure 2 Characterization of chemical components in Zhisousan and drug-containing serum. A-B. Positive / negative ion modes of blank serum; C-D. Positive / negative ion modes of drug-containing serum; E. Venn diagram analysis of prototype and metabolite components of Zhisousan; F. Analysis of the proportion of prototype and metabolite components in the components entering the blood; G. Analysis of the proportion of prototype and metabolite compound types. Detailed Embodiments

[0038] The present invention will be further described in detail below in combination with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0039] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0040] Example 1. Component Analysis of Zhisousan and Detection of Its Components Entering the Blood I. Experimental Method 1. Medicinal materials Platycodon grandiflorum (batch number: 241201, Bozhou Shenghai Traditional Chinese Medicine Decoction Pieces Co., Ltd.), Schizonepeta tenuifolia (batch number: 310240902, Anguo Yuanguang Pharmaceutical Co., Ltd.), Aster tataricus (serial number: 20210901, Hebei Anguo Traditional Chinese Medicine Market), Stemona sessilifolia (batch number: 24030601, Hebei Mingzhu Pharmaceutical Co., Ltd.), Cynanchum glaucescens (batch number: 8230820301, Hebei Lily Health Pharmaceutical Co., Ltd.), Glycyrrhiza glabra (batch number: 24112501, Hebei Kangyiqiang Pharmaceutical Co., Ltd.), Citrus reticulata Blanco (batch number: 250201, Bozhou Shenghai Traditional Chinese Medicine Decoction Pieces Co., Ltd.).

[0041] 2. Reagents

[0042] 3. Instruments

[0043] 4. Preparation of medicinal liquid Weigh the medicinal materials for Zhisousan according to the proportion, that is, 10 g of Platycodon grandiflorum, 10 g of Schizonepeta tenuifolia, 10 g of Aster tataricus, 10 g of Stemona sessilifolia, 10 g of Cynanchum glaucescens, 5 g of Glycyrrhiza glabra, and 5 g of Citrus reticulata Blanco. After soaking in 10 times the amount (i.e., 6 L) of distilled water for 30 min, boil for 1 h, filter. Add 8 times the amount of distilled water (4.8 L) to the residue, boil and extract for 1 h, filter. Combine the two filtrates, concentrate. Place the concentrated medicinal liquid at room temperature, pour it evenly into a freeze-drying dish, cover the top with a plastic sealing film and make several small holes, then place it in a -80°C refrigerator for freezing. Place the frozen medicinal liquid in a freeze dryer for freeze-drying (specific operation of the freeze dryer: tighten the air release valve and pre-cool to -40°C, after 20 min, put the medicinal liquid in and cover it with an organic glass cover, select the vacuum pumping mode, and start freeze-drying). Take out the sample after 3 d to obtain the product.

[0044] 5. Experimental animals 12 SPF-grade male SD rats, with a body weight of 180 ± 20 g, purchased from Beijing Spearf Bio-Technology Co., Ltd., certificate number 110324251104294728, license number SCXK (Beijing) 2024-0001. This experiment was approved by the Medical Ethics Committee of Shanxi University of Traditional Chinese Medicine (approval number: AWE202507342), and all experimental operations were carried out strictly in accordance with the guiding principles for animal research.

[0045] After 7 days of acclimatization, 12 rats were randomly divided into two groups of 6 rats each. The rats were administered the drug via gavage at a dose of 1 mL / 100 g body weight. 6.47 g of lyophilized powder (prepared in step 4 above) was weighed and diluted to 50 mL with physiological saline to prepare a homogeneous suspension with a concentration of 0.1294 g / mL. The gavage volume was calculated based on the rats' actual body weight, and the corresponding volume of drug solution was aspirated, thoroughly resuspended, and administered via gavage. The control group and the cough-relieving powder group (ZSS, 2.7 g / kg) received the same volume of physiological saline via gavage for 7 consecutive days. One hour after the last administration, 0.1 mL of 10% chloral hydrate was injected intraperitoneally into each 100 g rat. Once the anesthesia took effect, the rats were placed in a supine position and secured on the operating table. Subsequently, the abdomen was cut open, the abdominal aorta was located, blood was drawn, and after standing for 30 minutes, it was centrifuged at 3500 r / min for 10 minutes. The supernatant was collected and used for later use.

[0046] 6 Sample Preprocessing 6.1 Dried samples of medicinal materials Accurately weigh 50 mg of the sample powder (prepared in step 4 above) into a 2 mL EP tube using an analytical balance. Add a homogenizing bead and 500 μL of extraction buffer (methanol:acetonitrile:water = 2:2:1, v / v / v). The extraction buffer contains isotopically labeled internal standards (L-leucine-5,5,5-d3, concentration: 5.20 μg / mL; L-2-chlorophenylalanine, concentration: 2.01 μg / mL; glycocholic acid-D4, concentration: 0.09 μg / mL; diisobutyl phthalate-D4, concentration: 3.54 μg / mL; decanoic acid-D19, concentration: 0.16 μg / mL; [2H5]-trans-zeatin, concentration: 0.51 μg / mL; indole-3-acet-2,2-d2 acid, concentration: 2.38 μg / mL). Vortex to mix for 30 s. Homogenize in a homogenizer (35 Hz, 240 s), then transfer to an ice-water bath and sonicate for 5 min, repeating this process 3 times. Let stand for 30 min, then centrifuge at 12000 rpm for 15 min at 4℃, collect the supernatant, let stand again at -40℃ for 10 min, centrifuge for 15 min, collect the supernatant, filter through a 0.22 μm microporous membrane into a vial, and analyze using the instrument.

[0047] 6.2 Animal serum Thaw the serum sample, aspirate 200 μL of the sample into a 2 mL EP tube, add 20 μL of hydrochloric acid solution, vortex for 30 s, sonicate in an ice-water bath for 5 min, add 780 μL of acetonitrile, vortex for 30 s, sonicate again for 5 min, incubate at -40℃ for 30 min, and centrifuge for 15 min. Aspirate 800 μL of the supernatant into a 2 mL EP tube, evaporate to dryness at low temperature, add 80 μL of extraction buffer (methanol:acetonitrile:water = 2:2:1, v / v / v) to reconstitute, vortex for 30 s, sonicate in an ice-water bath for 1 min, centrifuge for 15 min, transfer the supernatant to a vial, and perform analysis.

[0048] 7. On-machine testing The cough-relieving powder was separated by chromatography using a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph with a Phenomenex Kinetex C18 column (2.1 mm × 100 mm, 2.6 μm). Phase A of the liquid chromatography was aqueous containing 0.01% acetic acid, and phase B was isopropanol:acetonitrile (1:1, v / v). The sample pan temperature was 4 ℃, and the injection volume was 2 μL. Gradient elution was used with the following program: 0 ~ 1.0 min, 1% B; 1.0 ~ 8.0 min, 1% B → 99% B; 8.0 ~ 9.0 min, 99% B; 9.1 ~ 12.0 min, 1% B. The flow rate was 0.300 mL / min. - ¹, The column oven temperature was set to 25°C. The total analysis time was 12 min.

[0049] Mass spectrometry was performed using an Orbitrap Exploris 120 mass spectrometer, with data acquired in both positive and negative ion modes. The parameters were set as follows: sheath gas flow rate 50 Arb, auxiliary gas flow rate 15 Arb, capillary temperature 320 °C, first-order resolution 60,000, second-order resolution 15,000, collision energy SNCE 20 / 30 / 40, and spray voltages of 3.8 kV (positive ion) and -3.4 kV (negative ion).

[0050] II. Experimental Results 1. Identification results of the chemical components of the cough-relieving powder UHPLC-Orbitrap-MS was used to characterize the whole components of the Zhisousan extract, and the accurate molecular information and secondary fragment information of the Zhisousan extract were obtained. By comparing with the BiotreeDB (V3.0) database, a total of 162 components were identified, including 91 shikimic acid and phenylpropionic acid derivatives, 27 terpenoids, 14 fatty acids, 12 alkaloids, 6 amino acids and short peptides, 5 carbohydrates, 5 polyketides, and 2 acetophenones. Among them, 9 components were from Aster tataricus L.f., 12 from Stemona japonica (Blume) Miq., 14 from Platycodon grandiflorus (Jacq.) A. DC., 5 from Cynanchum glaucescens (Decne.) Hand.-Mazz., 26 from Schizonepeta tenuifolia Briq., 19 from Citrus reticulata Blanco cv. Tankan, 52 from Glycyrrhiza uralensis Fisch., and 25 from multiple herbs, see Figure 1 and Table 1.

[0051] Table 1 Chemical Composition Table of Zhisousan Based on UHPLC-Orbitrap-MS

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] Note: ZW represents Aster tataricus; BB represents Stemona japonica; BQ represents Cynanchum paniculatum; JG represents Platycodon grandiflorus; JJ represents Schizonepeta tenuifolia; CP represents Citrus reticulata peel; GC represents Glycyrrhiza uralensis. 2. Screening results of blood components of cough-relieving powder Comparing the results of component identification of medicinal materials, blank serum, and drug-containing serum, and using a response intensity in drug-containing serum that is more than 1.2 times that of blank serum as the standard, further research was conducted on the blood-entering components based on the complete component analysis of Zhike San. A total of 64 components were found to have entered the bloodstream, including 14 in their original form and 50 in their metabolite form. Among these, 2 were from Aster tataricus, 9 from Stemona japonica, 3 from Platycodon grandiflorus, 1 from Cynanchum paniculatum, 7 from Citrus reticulata peel, 8 from Schizonepeta tenuifolia, 27 from Glycyrrhiza uralensis, and 7 from multiple medicinal materials. (See below) Figure 2 And Table 2.

[0073] Table 2. Identification of Cough-Relieving Powder Components and Metabolites Entering the Bloodstream Based on UHPLC-Orbitrap-MS

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] Note: ZW represents Aster tataricus; BB represents Stemona japonica; BQ represents Cynanchum paniculatum; JG represents Platycodon grandiflorus; JJ represents Schizonepeta tenuifolia; CP represents Citrus reticulata peel; GC represents Glycyrrhiza uralensis; I-metabolized as the original component; II-metabolized as metabolites.

[0081] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for detecting all ingredients of a cough-relieving powder, comprising the following steps: 1) Mix the various medicinal materials used to make the cough-relieving powder, decoct and extract, collect the extract and concentrate it, freeze-dry the concentrated extract to obtain freeze-dried powder. 2) The freeze-dried powder was added to the extraction solution for extraction. After extraction, the supernatant was collected, filtered through a microporous membrane, and the filtrate was detected by ultra-high performance liquid chromatography-high resolution mass spectrometry. The cough-relieving powder contains 162 ingredients, including 91 shikimic acid and phenylpropionic acid derivatives, 27 terpenes, 14 fatty acids, 12 alkaloids, 6 amino acids and short peptides, 5 carbohydrates, 5 polyketides, and 2 acetophenones. Among them, 9 are derived from Aster tataricus, 12 from Stemona japonica, 14 from Platycodon grandiflorus, 5 from Cynanchum paniculatum, 26 from Schizonepeta tenuifolia, 19 from Citrus reticulata peel, 52 from Glycyrrhiza uralensis, and 25 from various medicinal herbs.

2. The detection method according to claim 1, characterized in that: The complete component analysis of the cough-relieving powder is shown in Table 1; Table 1 Among them, ZW represents Aster tataricus; BB represents Stemona japonica; BQ represents Cynanchum paniculatum; JG represents Platycodon grandiflorus; JJ represents Schizonepeta tenuifolia; CP represents Citrus reticulata peel; and GC represents Glycyrrhiza uralensis.

3. The detection method according to claim 1 or 2, characterized in that: In step 1), the cough-relieving powder is made from the following medicinal materials in the indicated weight proportions: 10 parts Platycodon grandiflorus, 10 parts Schizonepeta tenuifolia, 10 parts Aster tataricus, 10 parts Stemona japonica, 10 parts Cynanchum paniculatum, 5 parts Glycyrrhiza uralensis, and 5 parts Citrus reticulata. And / or, in step 1), the decoction extraction is performed at least twice. In each extraction, the mass ratio of water to mixed medicinal materials in the first decoction extraction is (8-12):1, and the extraction time is 1-2 hours. In the second and subsequent decoction extractions, the mass ratio of water to mixed medicinal materials is (6-8):1, and the extraction time is 0.67-1 hour.

4. The detection method according to any one of claims 1-3, characterized in that: In step 2), the extract is a mixed solvent composed of methanol, acetonitrile, and water, with a volume ratio of (1.8-2.2):(1.8-2.2):(0.8-1.2). And / or, in step 2), the extract contains an isotopically labeled internal standard; the internal standard and its corresponding concentration are as follows: L-leucine-5,5,5-d3 5.20 μg / mL, L-2-chlorophenylalanine 2.01 μg / mL, glycocholic acid-D4 0.09 μg / mL, diisobutyl phthalate-D4 3.54 μg / mL, decanoic acid-D19 0.16 μg / mL, [2H5]-trans-zeatin 0.51 μg / mL, indole-3-acet-2,2-d2 acid 2.38 μg / mL; And / or, in step 2), the ratio of the lyophilized powder to the extract is (10-100) mg : 500 μL.

5. The detection method according to claim 4, characterized in that: The specific extraction method is as follows: Take 50 mg of sample powder into a 2 mL EP tube, add homogenizing beads and 500 μL of extraction solution, vortex and mix for 30 s; homogenize in a homogenizer (35 Hz, 240 s), then transfer to an ice-water bath and sonicate for 5 min, repeat the process 3 times; let stand for 30 min, centrifuge at 4℃ and 12000 rpm for 15 min, take the supernatant, let stand again at -40℃ for 10 min, centrifuge for 15 min, and take the supernatant.

6. A method for detecting the blood-entering components of a cough-relieving powder, comprising the following steps: A. Rats were given Zhike San (a traditional Chinese medicine for cough relief), and serum was collected after a predetermined time as the experimental group. The serum of rats that were not given Zhike San was used as the control group. B. The serum was added to the extraction solution for extraction. After extraction, the supernatant was collected, filtered through a microporous membrane, and the filtrate was detected by ultra-high performance liquid chromatography-high resolution mass spectrometry. There are 64 components that enter the blood, including 14 in their original form and 50 in their metabolite form. Among them, 2 come from Aster tataricus, 9 from Stemona japonica, 3 from Platycodon grandiflorus, 1 from Cynanchum paniculatum, 7 from Citrus reticulata peel, 8 from Schizonepeta tenuifolia, 27 from Glycyrrhiza uralensis, and 7 from various medicinal herbs.

7. The detection method according to claim 6, characterized in that: The blood component analysis of the cough-relieving powder is shown in Table 2; Table 2 Among them, ZW represents Aster tataricus; BB represents Stemona japonica; BQ represents Cynanchum paniculatum; JG represents Platycodon grandiflorus; JJ represents Schizonepeta tenuifolia; CP represents Citrus reticulata peel; GC represents Glycyrrhiza uralensis; I- is metabolized as the original component; II- is metabolized in the form of metabolites.

8. The detection method according to claim 6 or 7, characterized in that: In step A, the preparation method of the cough-relieving powder is as follows: after mixing the various medicinal materials for making the cough-relieving powder, decoct and extract, collect the extract and concentrate it, freeze-dry the concentrated extract to obtain freeze-dried powder. Furthermore, the cough-relieving powder is made from the following medicinal materials in the indicated weight proportions: 10 parts Platycodon grandiflorus, 10 parts Schizonepeta tenuifolia, 10 parts Aster tataricus, 10 parts Stemona japonica, 10 parts Cynanchum paniculatum, 5 parts Glycyrrhiza uralensis, and 5 parts Citrus reticulata. Furthermore, the decoction extraction is performed at least twice. In each extraction, the mass ratio of water to mixed medicinal materials in the first decoction extraction is (8-12):1, and the extraction time is 1-2 hours. In the second and subsequent decoction extractions, the mass ratio of water to mixed medicinal materials in the second decoction extraction is (6-8):1, and the extraction time is 0.67-1 hour.

9. The detection method according to any one of claims 6-8, characterized in that: In step B, the extract is a mixed solvent composed of methanol, acetonitrile, and water, with a volume ratio of (1.8-2.2):(1.8-2.2):(0.8-1.2). And / or, in step B, the extract contains an isotopically labeled internal standard; the internal standard and its corresponding concentration are as follows: L-leucine-5,5,5-d3 5.20 μg / mL, L-2-chlorophenylalanine 2.01 μg / mL, glycocholic acid-D4 0.09 μg / mL, diisobutyl phthalate-D4 3.54 μg / mL, decanoic acid-D19 0.16 μg / mL, [2H5]-trans-zeatin 0.51 μg / mL, indole-3-acet-2,2-d2 acid 2.38 μg / mL; And / or, in step B, the specific extraction method is as follows: 200 μL of serum sample is drawn into a 2 mL EP tube, 20 μL of hydrochloric acid solution is added, vortexed for 30 s, sonicated in an ice-water bath for 5 min, 780 μL of acetonitrile is added, vortexed for 30 s, sonicated again for 5 min, and allowed to stand at -40℃ for 30 min, then centrifuged for 15 min; 800 μL of supernatant is drawn into a 2 mL EP tube, evaporated at low temperature, 80 μL of extraction solution is added to reconstitute, vortexed for 30 s, sonicated in an ice-water bath for 1 min, centrifuged for 15 min, and the supernatant is collected.

10. The detection method according to any one of claims 1-9, characterized in that: In step 2) or step B, the ultra-high performance liquid chromatography uses a Vanquish ultra-high performance liquid chromatograph. In step 2) or step B, the conditions for ultra-high performance liquid chromatography-high resolution mass spectrometry detection are as follows: Chromatographic separation was performed using a Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 μm) liquid chromatography column. The mobile phase A was aqueous containing 0.01% acetic acid, and the mobile phase B was a 1:1 mixture of isopropanol and acetonitrile. Gradient elution was used. The flow rate was 0.300 mL / min. - ¹; Sample tray temperature 4 ℃, injection volume 2 μL; column oven temperature set to 25°C; total analysis time 12 min; Mass spectrometry was performed using an Orbitrap Exploris 120 mass spectrometer, with data acquired in both positive and negative ion modes. The parameters were set as follows: sheath gas flow rate 50 Arb, auxiliary gas flow rate 15 Arb, capillary temperature 320 °C, first-order resolution 60,000, second-order resolution 15,000, collision energy SNCE 20 / 30 / 40, and spray voltages of 3.8 kV (positive ion) and -3.4 kV (negative ion). Furthermore, the gradient elution procedure is as follows: 0 ~ 1.0 min, the volume fraction of mobile phase B is 1%; 1.0 ~ 8.0 min, the volume fraction of mobile phase B changes from 1% to 99%; 8.0 ~ 9.0 min, the volume fraction of mobile phase B is 99%; 9.1 ~ 12.0 min, the volume fraction of mobile phase B is 1%.

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