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Cellular membrane chromatography for sifting analgesic traditional Chinese medicine active ingredient

A technology of active ingredients and cell membranes, which is applied in the field of screening of active ingredients of traditional Chinese medicine and research on the mechanism of action, can solve problems such as no reports on in vitro culture, and achieve the effect of avoiding contamination by hybrid cells and saving time.

Inactive Publication Date: 2008-10-15
JINAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, some people have successfully cultured rat vas deferens smooth muscle cells (Deferential Smooth Muscle Cell, DSMC), but the in vitro culture of mouse DSMC and rabbit DSMC has not been reported.

Method used

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  • Cellular membrane chromatography for sifting analgesic traditional Chinese medicine active ingredient
  • Cellular membrane chromatography for sifting analgesic traditional Chinese medicine active ingredient
  • Cellular membrane chromatography for sifting analgesic traditional Chinese medicine active ingredient

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: the establishment method (rabbit vas deferens smooth muscle cell) of the vas deferens cell membrane chromatography model of screening analgesic Chinese medicine active ingredient

[0040] (1) Establishment of fingerprints of Angelica dahurica

[0041] Accurately weigh 20g of the dry root of Angelica dahurica (Fisch.ex Hoffm.) Benth.etHook.f.var.formosana (Boiss.) Shan et Yuan, add 160ml of 95% ethanol, reflux for 2h, evaporate the ethanol to dryness, and the residue Dissolve it with methanol to 20ml, dilute 250μl with methanol to 7.5ml, pass through a 0.45μm microporous membrane, and the obtained filtrate is the alcohol extract of Angelica dahurica, which is used as the test solution for HPLC analysis. The filtrate was drawn and determined by high-performance liquid chromatography. Chromatographic analysis conditions: Lichrospher-C18 (4.6×250mm), using methanol-water as mobile phase, detection wavelength 254nm, injection volume 20μl, column temperature 30°...

Embodiment 2

[0070] Embodiment 2: the establishment method of the vas deferens cell membrane chromatography model of screening traditional Chinese medicine analgesic active ingredient (mouse vas deferens smooth muscle cell)

[0071] (1) Establishment of fingerprints of Angelica dahurica

[0072] The method is the same as step (1) in Example 1.

[0073] (2) Primary culture of vas deferens smooth muscle cells

[0074] The vas deferens of adult male mice were taken, the inner and outer membrane tissues were stripped, and cells were cultured in culture flasks by enzymatic digestion. Method is with embodiment 1.

[0075] (3) Subculture of vas deferens smooth muscle cells

[0076] When the cells cover about 80% of the bottom area of ​​the bottle, pour off the cell culture medium, wash 2 times with PBS (phosphate buffered saline) solution at 37°C, and add trypsin containing 0.2% (mass percentage) + 0.02% (mass percentage) EDTA cell digestion solution, let it stand for 1-3 minutes, observe und...

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Abstract

The invention provides a cell membrane chromatography capable of synchronously sieving active ingredients of antalgic traditional Chinese medicine and determining an opium receptor subtype combined with the active ingredients. The method comprises the following steps of: the establishment of a fingerprint spectrum of extract liquid of angelica dahurica; the primary cultivation and the serial subcultivation of smooth muscle cells in a vas deferens; the combination and the dissociation of effective ingredients; and high-efficient liquid chromatographic determination combining the change of the spectrum. By acting traditional Chinese medicine on different types of smooth muscle cells in the vas deferens, the behavior of dissociation liquid of the traditional Chinese medicine on a high-efficient liquid chromatography column is examined, and the affinity of active ingredients of the traditional Chinese medicine and certain opium receptor subtype can be examined by using opium receptor excitant as a reference to opium receptor antagonist. The method takes an opium receptor on the vas deferens as a sieving model of key links of analgesics effect, can make an active ingredient sieving to the traditional Chinese medicine with abirritation, can also directly determine whether the active ingredients have the affinity with certain opium receptor subtype, and have good application prospect.

Description

technical field [0001] The invention belongs to the field of screening of active ingredients of traditional Chinese medicine and research on mechanism of action, and in particular relates to a method for establishing a cell membrane chromatographic model for screening active ingredients of traditional Chinese medicine for analgesia and determining opioid receptor subtypes bound thereto. Background technique [0002] Traditional TCM active ingredient screening models, such as whole animal, system, and organ models, have the disadvantages of long research cycle, high cost, and low efficiency; enzyme and receptor models cannot reflect the characteristics of multi-components and multi-targets of TCM. Among many models, cell membrane chromatography stands out as a new method for efficient screening of active ingredients in traditional Chinese medicine. [0003] Cell membrane chromatography (Cell Membrane Chromatography, CMC) is to immobilize the active tissue membrane of human or...

Claims

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Application Information

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IPC IPC(8): G01N30/02
Inventor 聂红尹震
Owner JINAN UNIVERSITY
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