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CN-117110464-B - Separation method, characteristic spectrum and construction method of 5 substances in fritillaria cirrhosa

CN117110464BCN 117110464 BCN117110464 BCN 117110464BCN-117110464-B

Abstract

The invention discloses a separation method, a characteristic spectrum and a construction method of 5 substances in fritillaria cirrhosa. The separation method comprises detecting the sample solution with high performance liquid chromatography-evaporative light scattering detector to separate the components to be detected, wherein the components to be detected comprise fritillary bulb octyl, tendril-base glycoside, ibuprside A, fritillary bulb alkali and fusiform fritillary bulb alkali, the preparation method of the sample solution comprises extracting alkaloid from standard decoction or decoction pieces of the fritillary bulb with ammonia water solution to obtain mixed solution, and heating and refluxing the mixed solution and the mixed solvent. The separation method can effectively separate all substances at the same time, and the separation time is short. The invention establishes the quality evaluation method for qualitatively evaluating the fritillary bulb standard decoction, which is relatively systematic, simple and easy to implement, and has good specificity and repeatability. The decoction pieces have high similarity with the characteristic patterns of the standard decoction. The method can be used for quality control of fritillary bulb standard decoction, and provides basic data support for subsequent formula granules and other researches.

Inventors

  • YIN QIANG
  • SONG FEI
  • YIN HAILONG
  • MU DANDAN
  • Kang Menxuan
  • DONG YUWEI
  • TIAN FANG
  • ZHOU QIN
  • Du Mengge
  • LI CHAOJIE

Assignees

  • 新疆维吾尔药业有限责任公司

Dates

Publication Date
20260512
Application Date
20230815

Claims (13)

  1. 1. A separation method of 5 substances in fritillaria cirrhosa is characterized by comprising the following steps of detecting a sample solution by a high performance liquid chromatography-evaporative light scattering detector and separating a component to be detected; Wherein the components to be tested comprise fritillary, fritillary glycoside, ibandroside A, fritillary base and fusiform fritillary base; The preparation method of the sample solution comprises the following steps of S1 extracting alkaloid from the standard decoction of the fritillary bulb or the decoction pieces of the fritillary bulb by adopting an ammonia water solution to obtain a mixed solution, S2 heating and refluxing the mixed solution and the mixed solvent to obtain the sample solution, wherein in the preparation method of the sample solution, the volume mass ratio of the ammonia water solution to the standard decoction of the fritillary bulb is (6-8) mL (7.5-10.0) g; In S2, the mixed solvent is a mixed solution of haloalkane and an alcohol solvent, wherein the haloalkane is dichloromethane or trichloromethane, the alcohol solvent is methanol, and the volume ratio of the haloalkane to the alcohol solvent is (3.5-4.5): 1; the mobile phase A is a triethylamine water solution with the concentration of 0.1-0.5%, and the mobile phase B is a mixed solution of acetonitrile and triethylamine, wherein the volume ratio of acetonitrile to triethylamine in the mobile phase B is (99.50-99.90) (0.10-0.50); The gradient elution conditions of the mobile phase A and the mobile phase B are 0-20 min,35% -42% of the mobile phase B, 20-40 min,42% -64% of the mobile phase B, 40-50 min,64% -90% of the mobile phase B, 50-55 min,90% -35% of the mobile phase B, and the chromatographic column is Zafex Supfex AQ-C18 chromatographic column.
  2. 2. The method for separating 5 substances from fritillary bulb according to claim 1, wherein the separation method satisfies one or more of the following conditions (1) to (6): (1) The mobile phase A is a triethylamine water solution with the concentration of 0.2-0.3%; (2) In the mobile phase B, the volume ratio of acetonitrile to triethylamine is 99.75 (0.20-0.30); (3) The column temperature is 25-40 ℃; (4) The flow rate of the sample solution is 0.6-1.5 mL/min; (5) The sample injection volume is 15-25 mu L; (6) The parameters of the evaporative light scattering detector are that the temperature of a drift tube is 102 ℃, the gas flow rate is 3.0L/min, and the gain value is 2.
  3. 3. The method for separating 5 substances from fritillary bulb according to claim 1, wherein the separation method satisfies one or more of the following conditions (1) to (5): (1) The mobile phase A is a triethylamine water solution with the concentration of 0.25%; (2) In the mobile phase B, the volume ratio of acetonitrile to triethylamine is 99.75:0.25; (3) The column temperature is 30 ℃ or 35 ℃; (4) The flow rate of the sample solution is 1mL/min; (5) The sample volume was 20. Mu.L.
  4. 4. The method for separating 5 substances from fritillaria cirrhosa according to claim 1, wherein the detection conditions of the high performance liquid chromatography-evaporative light scattering detector are that the chromatographic column is Zafex Supfex AQ-C18 chromatographic column with the specification of 4.6 mm multiplied by 150 mm and 5 μm, the mobile phase A is a mixed solution with the concentration of 0.25% of triethylamine and the volume ratio of acetonitrile to triethylamine is 99.75:0.25, the gradient elution conditions are that the gradient elution is 0-20 min,35% -42% of mobile phase B, 20-40 min,42% -64% of mobile phase B, 40-50 min,64% -90% of mobile phase B, 50-55 min,90% -35% of mobile phase B, the column temperature is 30 ℃, the flow rate is 1 mL/min, the ELSD detector is 102 ℃ in drift tube temperature, the gas flow rate is 3.0L/min, the gain value is 2 and the sample injection amount is 20 mu L.
  5. 5. The method for separating 5 substances from fritillaria cirrhosa according to claim 1, wherein the method for detecting by using a high performance liquid chromatography-evaporative light scattering detector comprises the following steps: s11, respectively preparing a mixed reference substance solution and the test sample solution, and adopting the detection method to analyze and detect the mixed reference substance solution and the test sample solution to respectively obtain high performance liquid chromatograms of the mixed reference substance solution and the test sample solution; s12, determining the compound corresponding to each peak in the high performance liquid chromatogram of the sample solution according to the high performance liquid chromatograms of the mixed reference solution, and obtaining the peak areas of the corresponding compounds in the mixed reference solution and the sample solution.
  6. 6. The method for separating 5 substances from fritillary bulb as set forth in claim 5, wherein the concentration of the mixed reference solution in S11 is 30-500 μg/mL.
  7. 7. The method for separating 5 substances from fritillary bulb as set forth in claim 5, wherein the concentration of said mixed reference solution in S11 is 200 μg/mL.
  8. 8. The method for separating 5 substances from fritillary bulb according to claim 1, wherein one or more of the following conditions (1) to (7) are satisfied in the preparation method of the sample solution: (1) In S1, the volume-mass ratio of the ammonia water solution to the fritillary bulb standard decoction is 6mL:7.5g, 8mL:7.5g or 8mL:10g; (2) In S1, the volume-mass ratio of the ammonia water solution to the fritillary bulb decoction pieces is (2-10) mL, 3.0g; (3) S1, the extraction method comprises the following steps of standing the fritillary bulb standard decoction or the fritillary bulb decoction pieces in the ammonia water solution; (4) S2, the mass-volume ratio of the fritillary bulb standard decoction to the mixed solvent is 7.5g (15-65) mL; (5) In S2, the mass-volume ratio of the fritillary bulb decoction pieces to the mixed solvent is 3.0g (15-65) mL; (6) S2, heating and refluxing at 45-85 ℃; (7) In S2, the heating reflux time is 0.3-3.5h.
  9. 9. The method for separating 5 substances from fritillary bulb according to claim 8, wherein one or more of the following conditions (1) to (3) are satisfied in the preparation method of the sample solution: (1) In S1, the volume-mass ratio of the ammonia water solution to the fritillary bulb decoction pieces is 2mL:3.0 g, 4mL:3.0g, 6mL:3.0g or 8mL:3.0g; (2) Standing for 0.3-2.5h; (3) The volume ratio of the haloalkane to the alcohol solvent is 4:1.
  10. 10. The method for separating 5 substances from fritillary bulb according to claim 8, wherein the standing time is 0.5h, 1 h or 2 h.
  11. 11. The method for separating 5 substances from fritillary bulb according to claim 8, wherein one or more of the following conditions (1) to (4) are satisfied in the preparation method of the sample solution: (1) In S2, the mass-volume ratio of the fritillary bulb standard decoction to the mixed solvent is 7.5g to 20mL, 7.5g to 40 mL or 7.5g to 60 mL; (2) In S2, the mass-volume ratio of the fritillary bulb decoction pieces to the mixed solvent is 3.0g to 20 mL, 3.0g to 40 mL or 3.0g to 60 mL; (3) S2, the temperature of the heating reflux is 50 ℃, 60 ℃ or 80 ℃; (4) In S2, the heating reflux time is 0.5 h, 1 h, 2h or 3 hours.
  12. 12. The method for separating 5 substances from fritillary bulb according to claim 1, wherein the preparation method of the sample solution comprises the following steps: Adding ammonia water 8 mL into 7.5 g g of standard decoction of fritillaria or 3.0g of decoction pieces of fritillaria, standing for 0.5 h, adding mixed solution of dichloromethane and methanol 20 mL in a volume ratio of 4:1, mixing uniformly, heating and refluxing for 2h on a water bath at 60 ℃, cooling, filtering, concentrating the filtrate under reduced pressure until the filtrate is dry, adding methanol into the residue for dissolution, fixing the volume in a 5mL volumetric flask, shaking uniformly, filtering with an organic microporous filter membrane with a volume of 0.45 μm, and taking the subsequent filtrate.
  13. 13. A method for constructing characteristic spectrum of Bulbus Fritillariae Cirrhosae comprises detecting sample solution with high performance liquid chromatography-evaporative light scattering detector, and generating characteristic spectrum; the test solution and the detection method of the high performance liquid chromatography-evaporative light scattering detector are all as defined in any one of claims 1 to 12.

Description

Separation method, characteristic spectrum and construction method of 5 substances in fritillaria cirrhosa Technical Field The invention particularly relates to a separation method, a characteristic spectrum and a construction method of 5 substances in fritillaria cirrhosa. Background The Bulbus Fritillariae Cirrhosae is used as one of rare and genuine medicinal materials in Uygur autonomous region of Xinjiang, and is mainly distributed in Yili river basin, and is dry bulb of Sinkiang fritillaria FRITILLARIA WALUJEWII REGEL or Yili fritillaria FRITILLARIA PALLIDIFLORA SCHRENK of Fritillaria genus of Liliaceae. Yi Bei mu is slightly cold in nature, bitter in flavor, enters lung and heart meridians. In clinic, it is often used for treating cough with phlegm, dry cough due to lung heat, etc., and is a common medicinal material in traditional Chinese medicine production and prescription. The Bulbus Fritillariae Cirrhosae contains alkaloid, saponin, polysaccharide, coumarin, starch, flavonoid, etc. The research on pharmaceutically active component of the steroid alkaloids is deeper, and the pharmacological effects of clearing heat and moistening lung, resolving phlegm and relieving cough, relieving asthma, relieving spasm, inhibiting bacteria, resisting oxidation and the like are achieved. The standard decoction of the traditional Chinese medicine decoction pieces is used as an important bridge for improving the traditional Chinese medicine formula granules, and can ensure accurate clinical medication and consistent dosage. The quality standard of the traditional Chinese medicine standard decoction combines quantitative and qualitative aspects, wherein the quantitative aspects mainly comprise parameters such as content and transfer rate of index components, ointment yield, pH, relative density and the like, and the qualitative aspects begin from HPLC-ELSD characteristic spectrum and thin-layer chromatography identification which can reflect the condition of the total components. The research of the prior literature on the fritillary bulb is mainly focused on the aspects of medicinal materials, artificial cultivation and the like, and no related report is found on the research of the standard decoction, so that the formation of a standard decoction quality evaluation system is beneficial to ensuring the medication safety and the curative effect, and is also beneficial to inheritance and research of traditional Chinese medicine decoction pieces, formula particles, classical formulas and the like. The quality control of the characteristic spectrum as a whole is more obvious than that of a single component or even a multi-component qualitative and quantitative method. However, research reports on the characteristic spectrum of the fritillary bulb are not yet seen in the current literature. Therefore, in order to better monitor the content of each component in the fritillary bulb, the art needs to establish a simple and convenient characteristic spectrum of the fritillary bulb. Meanwhile, the method has great significance for more effectively controlling the quality of the fritillary bulb and comprehensively and objectively evaluating the fritillary bulb. Disclosure of Invention The invention aims to overcome the defect that the prior art lacks of research on standard decoction of fritillaria cirrhosa tablets, and provides a separation method, a characteristic spectrum and a construction method of 5 substances in fritillaria cirrhosa. The invention solves the technical problems through the following technical proposal. The invention provides a separation method of 5 substances in fritillaria cirrhosa, which comprises the following steps of detecting a sample solution by a high performance liquid chromatography-evaporative light scattering detector and separating components to be detected; Wherein the components to be tested comprise fritillary, fritillary glycoside, ibandroside A, fritillary base and fusiform fritillary base; The preparation method of the sample solution comprises the following steps of S1, extracting alkaloid from standard decoction of Bulbus Fritillariae Cirrhosae or decoction pieces of Bulbus Fritillariae Cirrhosae with ammonia water solution to obtain mixed solution, S2, heating and refluxing the mixed solution and mixed solvent to obtain the sample solution; the mobile phase A is a triethylamine water solution with the concentration of 0.1-0.5%, the mobile phase B is a mixed solution of acetonitrile and triethylamine, and the volume ratio of acetonitrile to triethylamine in the mobile phase B is (99.50-99.90) (0.10-0.50); the gradient elution conditions of the mobile phase A and the mobile phase B are 0-20min, 35% -42% of the mobile phase B, 20-40 min,42% -64% of the mobile phase B, 40-50 min, 64% -90% of the mobile phase B, and 50-55 min,90% -35% of the mobile phase B. In the present invention, the mobile phase A is preferably a 0.2 to 0.3% aqueous triethylamine solution, for example, a 0.25% a