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Determination method for absorption and transportation amount of six components in rhizoma bletillae in Caco-2 cell model

A cell model and measurement method technology, which is applied in the field of determination of the absorption and transport of six components in the Caco-2 cell model, can solve the problem that the absorption characteristics of the extract of P. chinensis have not been reported.

Active Publication Date: 2016-09-21
GUIZHOU MEDICAL UNIV
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Problems solved by technology

[0004] At present, there is no report on the absorption characteristics of white and its extracts

Method used

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  • Determination method for absorption and transportation amount of six components in rhizoma bletillae in Caco-2 cell model
  • Determination method for absorption and transportation amount of six components in rhizoma bletillae in Caco-2 cell model
  • Determination method for absorption and transportation amount of six components in rhizoma bletillae in Caco-2 cell model

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Experimental program
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Embodiment 1

[0025] 1 Experimental equipment

[0026] 1.1 Instrument Ultra-high liquid chromatography-triple quadrupole mass spectrometry (Waters, USA); CO 2 Incubator (Thermo sci-entific); ultra-clean bench (Suzhou Purification Equipment General Factory); functional microplate reader (Thermo3001VARIOSKAN FLASH); inverted phase contrast microscope (Olympus, Japan).

[0027] 1.2 Reagents and reagents α-Isobutylmalic acid (B1), Blestroside (B2), Militarine (B3), Gymnoside (B4), Dihydrophenanthrene 1 (B5) and Gymnoside IX (B6) were all isolated in the laboratory (batch number 20150305); PSS buffer solution (Solarbio); fetal bovine serum (Gibco); DMEM medium (high glucose, Gibco); trypsin (Gibco); double antibody (Hyclone); Coomassie brilliant blue stain (Nanjing Jiancheng Bioengineering Institute); DMSO, methanol, and acetonitrile are all chromatographically pure; experimental water is ultrapure water; other reagents are analytically pure.

[0028] 1.3 Cell line Caco-2 cell line (Shanghai A...

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Abstract

The invention discloses a determination method for the absorption and transportation amount of six components in rhizoma bletillae in a Caco-2 cell model. The method comprises the steps of: preparing a rhizoma bletillae extract solution, a standard solution serving as a reference substance and an internal standard solution; establishing a human-derived colon adenocarcinoma cell line Caco-2 cell model; preparing a cell suspension through a Caco-2 cell model; determining the content of the six components by UPLC-MS / MS; determining the total protein content according to a Coomassie brilliant blue dye liquor protein determination kit method, and calculating the cell uptake X=the total protein of a to-be-determined substance. The invention adopts UPLC-MS / MS to establish the analysis method for the 6 components in a rhizoma bletillae extract, determines the influence of the rhizoma bletillae extract to absorption and uptake of Caco-2 cells under the conditions of time, concentration, temperature, pH and P-gh inhibitors, preliminarily evaluates the in vivo absorption characteristics of the rhizoma bletillae extract, and provides scientific basis for the research and development of the oral preparation of the rhizoma bletillae extract.

Description

technical field [0001] The invention belongs to the technical field of detection of traditional Chinese medicines, and in particular relates to a method for measuring the absorption and transport amount of six components of bletilla chinensis in a Caco-2 cell model. Background technique [0002] The human colon adenocarcinoma cell line (Caco-2 cell line) has become a classic cell model in the in vitro study of the absorption properties of orally administered drugs. After being cultured for a period of time in vitro, the Caco-2 monolayer cell model can differentiate into a microvilli structure with similar functions to intestinal epithelial cells, and contains small intestinal epithelial cell brush border-related enzymes. It can spontaneously form differentiated epithelial and tight junction tissues. Its cell morphology, specific marker enzymes and permeability characteristics are similar to those of small intestinal epithelial cells, and the reproducibility is good. It has a...

Claims

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Application Information

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IPC IPC(8): G01N30/02
CPCG01N30/02G01N2030/027
Inventor 黄勇李勇军郑林陆苑兰燕宇王爱民孙佳巩仔鹏李月婷陈思颖杨畅
Owner GUIZHOU MEDICAL UNIV
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