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CN-122017084-A - Full component and blood-entering component of cough relieving powder and detection method thereof

CN122017084ACN 122017084 ACN122017084 ACN 122017084ACN-122017084-A

Abstract

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

Inventors

  • CHENG YANGANG
  • TAN JINYAN
  • WANG YINGLI
  • ZHANG YI
  • Zang yun

Assignees

  • 山西中医药大学

Dates

Publication Date
20260512
Application Date
20260326

Claims (10)

  1. 1. The method for detecting the total ingredients of the cough relieving powder comprises the following steps: 1) Mixing the above materials, decocting, collecting extractive solution, concentrating, and lyophilizing to obtain lyophilized powder; 2) Extracting the freeze-dried powder with an extracting solution, taking a supernatant after the extraction is finished, filtering the supernatant with a microporous filter membrane, and detecting the filtrate by adopting ultra-high performance liquid chromatography-high resolution mass spectrometry; The cough relieving powder comprises 162 components including 91 shikimic acid and phenylpropionic acid, 27 terpenes, 14 fatty acids, 12 alkaloids, 6 amino acids and short peptides, 5 carbohydrates, 5 polyketones and 2 acetophenones, wherein 9 components from aster, 12 components from stemona root, 14 components from platycodon root, 5 components from white front, 26 components from schizonepeta, 19 components from dried orange peel and 52 components from liquorice, and 25 components from multiple medicinal materials.
  2. 2. The method according to claim 1, wherein the analysis of the total ingredients of the cough relieving powder is shown in Table 1; TABLE 1 Wherein ZW represents radix Asteris, BB represents radix Stemonae, BQ represents rhizoma Cynanchi Stauntonii, JG represents radix Platycodi, JJ represents herba Schizonepetae, CP represents pericarpium Citri Tangerinae, and GC represents Glycyrrhrizae radix.
  3. 3. The method according to claim 1 or 2, wherein in the step 1), the cough relieving powder is prepared from the following medicinal materials, by mass, 10 parts of platycodon grandiflorum, 10 parts of schizonepeta, 10 parts of aster, 10 parts of stemona root, 10 parts of cynanchum atratum, 5 parts of liquorice and 5 parts of dried orange peel; and/or, in the step 1), the decoction and extraction are carried out at least 2 times, and each time of the extraction, the mass ratio of the water of the first decoction and the mixed medicinal material is (8-12) 1, the extraction time is 1-2 hours, the mass ratio of the water of the second decoction and the above decoction and the mixed medicinal material is (6-8) 1, and the extraction time is 0.67-1 hour.
  4. 4. The detection method according to any one of claims 1 to 3, wherein in the step 2), the extracting solution is a mixed solvent of methanol, acetonitrile and water, and the volume ratio of the extracting solution to the mixed solvent is (1.8-2.2): 0.8-1.2; And/or the extracting solution in the step 2) contains an isotope-labeled internal standard, wherein the internal standard and the corresponding concentration are shown as L-leucine-5, 5-D3.20 mug/mL, L-2-chlorophenylalanine 2.01 mug/mL, glycocholic acid-D4.09 mug/mL, diisobutyl phthalate-D4.54 mug/mL, capric acid-D19.16 mug/mL, [2H5] -trans-zeatin 0.51 mug/mL and indole-3-ethyl-2, 2-D2 acid 2.38 mug/mL; And/or, in the step 2), the dosage ratio of the freeze-dried powder to the extracting solution is (10-100) mg/500 mu L.
  5. 5. The detection method according to claim 4, wherein the specific extraction method comprises the steps of sampling 50 mg parts of the powder in a 2mL EP tube, adding homogenized grinding beads and 500 μl of the extract, mixing by vortex for 30 s parts, homogenizing (35 Hz, 240 s) in a homogenizer, transferring to ice water bath ultrasonic 5min parts, repeatedly treating for 3 times, standing for 30min parts, centrifuging for 15min parts at 4 ℃ and 12000 rpm parts, collecting supernatant, standing for 10min parts at-40 ℃ again, centrifuging for 15min parts, and collecting supernatant.
  6. 6. A method for detecting blood-entering components of cough relieving powder comprises the following steps: A. giving the anti-cough powder to rats, collecting serum after a predetermined time as an experimental group, and taking the serum of the rats without the anti-cough powder as a control group; B. adding the serum into an extracting solution for extraction, taking supernatant after the extraction is finished, filtering the supernatant by a microporous filter membrane, and detecting the filtrate by adopting ultra-high performance liquid chromatography-high resolution mass spectrometry; the blood is filled with 64 components, wherein 14 components are filled into the blood in the form of prototype components, 50 components are filled into the blood in the form of metabolites, wherein 2 components are filled from aster, 9 components are filled from radix stemonae, 3 components are filled from platycodon grandiflorum, 1 component is filled from cynanchum glaucescenum, 7 components are filled from pericarpium citri reticulatae, 8 components are filled from schizonepeta, 27 components are filled from liquorice, and 7 components are filled from multiple medicinal materials.
  7. 7. The method according to claim 6, wherein the analysis of the antitussive blood component is as shown in Table 2; TABLE 2 Wherein ZW represents radix Asteris, BB represents radix Stemonae, BQ represents radix Cynanchi Stauntonii, JG represents radix Platycodi, JJ represents herba Schizonepetae, CP represents pericarpium Citri Tangerinae, GC represents Glycyrrhrizae radix, I-is metabolized as prototype component, and II-is metabolized as metabolite.
  8. 8. The method according to claim 6 or 7, wherein in the step A, the preparation method of the cough relieving powder comprises the steps of mixing the raw materials, decocting, extracting, collecting the extracting solution, concentrating, and freeze-drying the concentrated extracting solution to obtain freeze-dried powder; Further, the cough relieving powder is prepared from the following medicinal materials, by mass, 10 parts of platycodon grandiflorum, 10 parts of schizonepeta, 10 parts of aster, 10 parts of radix stemonae, 10 parts of cynanchum glaucescens, 5 parts of liquorice and 5 parts of dried orange peel; Further, the decoction and extraction are carried out at least 2 times, and each time of the extraction, the mass ratio of the water of the first decoction and the mixed medicinal material is (8-12) 1, the extraction time is 1-2 hours, the mass ratio of the water of the second decoction and the mixed medicinal material is (6-8) 1, and the extraction time is 0.67-1 hour.
  9. 9. The detection method according to any one of claims 6 to 8, wherein in the step B, the extracting solution is a mixed solvent of methanol, acetonitrile and water, and the volume ratio of the extracting solution to the mixed solvent is (1.8-2.2): 0.8-1.2; And/or in the step B, the extracting solution contains an isotope labeled internal standard, wherein the internal standard and the corresponding concentration are shown as L-leucine-5, 5-D3.20 mug/mL, L-2-phenylalanine 2.01 mug/mL, glycocholic acid-D4.09 mug/mL, diisobutyl phthalate-D4.54 mug/mL, capric acid-D19.16 mug/mL, [2H5] -trans-zeatin 0.51 mug/mL and indole-3-ethyl-2, 2-D2 acid 2.38 mug/mL; And/or in the step B, the specific method for extraction comprises the steps of absorbing 200 mu L of serum sample in a2 mL EP pipe, adding 20 mu L of hydrochloric acid solution, swirling 30 s, adding 780 mu L of acetonitrile into ice water bath ultrasonic 5min, swirling 30 s, again ultrasonic 5min, standing 30 min at-40 ℃ for 15min, absorbing 800 mu L of supernatant in a2 mL EP pipe, spin-drying at low temperature, adding 80 mu L of extract for re-dissolution, swirling 30 s, ice water bath ultrasonic 1min, centrifuging 15min, and taking supernatant.
  10. 10. The method according to any one of claims 1 to 9, wherein in the step 2) or the step B), the ultra performance liquid chromatography is performed by a Vanquish ultra performance liquid chromatograph; In the step 2) or in the step B), the conditions for the ultra-high performance liquid chromatography-high resolution mass spectrometry detection are as follows: Carrying out chromatographic separation by adopting Phenomenex Kinetex C18 (2.1 mm multiplied by 100 mm, 2.6 mu m) liquid chromatographic column, wherein the mobile phase A of the liquid chromatographic column is water phase, contains 0.01% acetic acid, the mobile phase B is mixed solvent of isopropanol and acetonitrile in a volume ratio of 1:1, the elution mode is gradient elution, the flow rate is 0.300 mL.min - 1, the temperature of a sample tray is 4 ℃, the sample injection volume is 2 mu L, the temperature of a column temperature box is set to 25 ℃, and the total analysis time is 12 min; The mass spectrum detection uses Orbitrap Exploris mass spectrometer, data are collected under positive and negative ion modes, parameters are set, namely, the sheath air flow rate is 50 Arb, the auxiliary air flow rate is 15 Arb, the capillary temperature is 320 ℃, the primary resolution is 60000, the secondary resolution is 15000, the collision energy is SNCE/30/40, and the spray voltages are respectively 3.8 kV (positive ions) and-3.4 kV (negative ions); Further, the gradient elution comprises the following elution procedures of 0-1.0 min, 1% of mobile phase B, 1.0-8.0 min, 1% -99% of mobile phase B, 8.0-9.0 min, 99% of mobile phase B, 9.1-12.0 min and 1% of mobile phase B.

Description

Full component and blood-entering component of cough relieving powder and detection method thereof Technical Field The invention belongs to the field of medicines, and particularly relates to a full component of cough relieving powder, a blood entering component and a detection method thereof. Background Cough relieving powder is a traditional Chinese medicine prescription and comes from medical heart understanding of a Qing dynasty doctor Cheng Zhongling, and has the core effects of dispersing lung qi, dispelling wind, relieving cough and reducing phlegm, and is commonly used for treating exogenous cough, lasting and relatively long course of disease, inexhaustible exterior evil and lung qi failure and dispersing symptoms. The prescription has the classical compatibility of 7 medicines, and has definite monarch, minister, assistant and guide division of labor: 1. Monarch drug of aster and stemona root Radix Asteris, bitter with the effects of warming and moistening the lung, resolving phlegm and relieving cough, and is good at regulating cough caused by lung qi stagnation; stemona root: sweet and bitter with slight warmth, lung moistening and cough stopping are good for treating cough due to deficiency and excess, and cough due to tuberculosis. Both are warm and not dry, and have good effects of moistening lung and relieving cough, and can be used for treating cough with core pathogenesis, new cough and chronic cough. 2. Ministerial drugs including radix Platycodi and rhizoma Cynanchi Stauntonii Radix Platycodi, with the effects of dispersing bitter and diarrhea, dispersing lung qi, relieving sore throat, and ascending to lung meridian; before white, pungent, sweet and slightly warm, can reduce qi and phlegm, and can relieve cough and excessive phlegm caused by lung qi stagnation. Both of them can promote the dispersing and descending functions of the lung qi. 3. Adjuvant drug including herba Schizonepetae and pericarpium Citri Tangerinae Herba Schizonepetae, pungent and warm in nature, can relieve exterior syndrome, dispel wind and eliminate pathogenic factors, and aims at the condition that exogenous wind pathogenic factors are not completely dissipated; pericarpium Citri Tangerinae, having effects of regulating qi-flowing, invigorating spleen, eliminating dampness, resolving phlegm dampness, and promoting lung qi circulation. 4. Radix Glycyrrhizae Tonify qi, nourish middle energizer, moisten lung, relieve cough, harmonize the Chinese medicines, relieve sore throat and alleviate pain. At present, no report is made on analysis of the whole ingredients of the cough relieving powder and the blood entering ingredients and detection methods thereof. Disclosure of Invention The invention aims to provide a full component of cough relieving powder, a blood entering component and a detection method thereof. The invention provides a method for detecting the total ingredients of cough relieving powder, which comprises the following steps: 1) Mixing the above materials, decocting, collecting extractive solution, concentrating, and lyophilizing to obtain lyophilized powder; 2) Extracting the lyophilized powder with extractive solution, collecting supernatant, filtering with microporous membrane (such as 0.22 μm microporous membrane), and detecting filtrate with ultra-high performance liquid chromatography-high resolution mass spectrometry; The cough relieving powder comprises 162 components including 91 shikimic acid and phenylpropionic acid, 27 terpenes, 14 fatty acids, 12 alkaloids, 6 amino acids and short peptides, 5 carbohydrates, 5 polyketones and 2 acetophenones, wherein 9 components from aster, 12 components from stemona root, 14 components from platycodon root, 5 components from white front, 26 components from schizonepeta, 19 components from dried orange peel and 52 components from liquorice, and 25 components from multiple medicinal materials. Specifically, the whole ingredient analysis of the cough relieving powder is shown in table 1. In the step 1) of the method, the cough relieving powder is prepared from the following medicinal materials, by mass, 10 parts of platycodon grandiflorum, 10 parts of schizonepeta, 10 parts of aster, 10 parts of radix stemonae, 10 parts of cynanchum glaucescens, 5 parts of liquorice and 5 parts of dried orange peel. In the step 1), the decoction and extraction are carried out at least 2 times, and each time of the extraction, the mass ratio of the water of the first decoction and the mixed medicinal material is (8-12): 1, the extraction time can be 1-2 hours, the mass ratio of the water of the second decoction and the mixed medicinal material is (6-8): 1, and the extraction time can be 0.67-1 hour. According to the specific embodiment of the invention, the specific method for decoction and extraction comprises the steps of adding 10 times of distilled water into the mixed medicinal materials for 30min times of distilled water, decocting for 1: 1h, filtering, adding 8 times o