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Method for quantitatively evaluating antioxidant activity of antioxidant based on Caco-2 cell model

An anti-oxidant activity and antioxidant technology, which is applied in material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of complex operation process, can not reflect the absorption characteristics of antioxidants, limit practical value, etc., and achieve the effect of high biological relevance.

Inactive Publication Date: 2014-04-30
SHENZHEN POLYTECHNIC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The problem with this method is that the selected cell model cannot reflect the absorption characteristics of antioxidants in the intestinal tract, and there is no biological correlation study report of this method so far
Therefore, it remains highly questionable whether the assay results are predictive of the actual efficacy of antioxidants in vivo
In 2008, Canadian NIS laboratory Jensen (Jensen, G.S. Cell-based antioxidant protection assay. U.S. Provisional patent application. 60 / 985, 166) established an antioxidant activity evaluation method based on human blood red blood cell model. Preparation of human red blood cells, the operation process is complicated, which limits its practical value
In 2009, Quebec University Girard-Lalancette et al. (Karl Girard-Lalancette; André Pichette; Jean Legault. Sensitive cell-based assay using DCFH oxidation for the determination of pro-and antioxidant properties of compounds and mixtures: Analysis of fruit and vegetable juices. Food Chem .2009,115,720–726) established an antioxidant analysis method based on the mouse fibroblastoma (L929) cell model, and set the measurement point at the time point when free radicals acted on the cells for 90 minutes, so the repeatability of the method was poor. large error
At present, there is no research report on the establishment of an in vitro quantitative analysis method for antioxidant activity using this cell model.

Method used

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  • Method for quantitatively evaluating antioxidant activity of antioxidant based on Caco-2 cell model
  • Method for quantitatively evaluating antioxidant activity of antioxidant based on Caco-2 cell model
  • Method for quantitatively evaluating antioxidant activity of antioxidant based on Caco-2 cell model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033]Example 1 Determination of Antioxidant Pure Product Cytological Antioxidative Activity

[0034] 1) Dilute 100L of Caco-2 single cell suspension by 5×10 4 The concentration of cells / well was inoculated into a transparent flat-bottomed black 96 microwell plate at 37°C in 5% CO 2 Cultured for 24 hours; human colon adenocarcinoma cell Caco-2 was purchased from the American Type Culture Collection (ATCC) in the United States as a cell line for evaluating the antioxidant activity of antioxidants;

[0035] 2) Remove the growth medium from the 96-well plate in step 1), and wash the adherent cells once with 150L of 1×PBS;

[0036] 3) Prepare different concentrations of quercetin, (-)-epigallocatechin gallate, resveratrol, galangin, kaempferol, and kaempferol in the antioxidant-treated medium containing 60M DCFH-DA Rehmannia flavonoids, phloretin, quercetin-3-G, dihydroquercetin, L-ascorbic acid, rutin, caffeic acid, gallic acid, L-glutathione, (+)-catechin, ( -)- 18 pure antio...

Embodiment 2

[0045] Example 2 Determination of fruit cytology antioxidant activity

[0046] Similar to the steps in Example 1, the cytological antioxidant activities of blueberries and apples were determined to be 6.8±0.4mol QE / 100g fresh fruit and 1.8±0.1mol QE / 100g fresh fruit, respectively.

Embodiment 3

[0047] Example 3 Biocorrelation Analysis of Antioxidant Antioxidant Activity Quantitative Evaluation Method Based on Caco-2 Monolayer Cell Model

[0048] 1) SPF grade male SD rats, weighing 200-250g (about 8-9 weeks old), temperature 22-25°C, relative humidity 65%-75%, adapted to feeding for one week, randomly divided into sample group and control group according to body weight , 6 rats in each group, fasted for 12 hours before the experiment, and had free access to water;

[0049] 2) For the sample group, 0.2mmol / kg·b·w (b·w is the abbreviation of body weight) antioxidant pure product (dissolved in corn oil) was administered to SD rats at one time, with a volume of 1mL / 100g . The control group was gavaged with solvent corn oil;

[0050] 3) Before gavage and 1, 2, 4, 6, 8, and 10 hours after gavage, blood was collected through the tip of the tail, placed in a heparin-anticoagulated test tube, centrifuged at 3500r / min for 10min, and plasma was separated;

[0051] 4) The ORAC...

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Abstract

The invention discloses a method for quantitatively evaluating the antioxidant activity of an antioxidant based on a Caco-2 cell model, relating to an antioxidant. The method for cytologically and quantitatively evaluating the antioxidant activity of an antioxidant based on a Caco-2 single-layer cell model is established by using the Caco-2 cell model and optimizing the inoculation concentration and culture time of cells as well as the incubation conditions of a fluorescent probe (2',7'-dichlorodihydrofluorescein diacetate, DCFH-DA for short), a free radical initiator (2,2'-azobis(2-methylpropionamidine)dihydrochloride, AAPH for short), a pure antioxidant and Caco-2 cells. The method has the advantages of high biological relevance, easiness and high speed, and an effective analysis and evaluation tool is provided for the research of biological antioxidants.

Description

technical field [0001] The invention relates to antioxidants, in particular to a method for quantitatively evaluating the antioxidant activity of antioxidants based on a Caco-2 cell model. Background technique [0002] Epidemiological and clinical experimental studies have proved that the excess of free radicals in the human body is an important factor causing chronic degenerative diseases such as cardiovascular disease, cancer and aging, and dietary intake and supplementing antioxidants are effective ways to prevent these diseases. Therefore, evaluating the antioxidant activity of antioxidants has always been a hot field in the research of nutraceuticals. So far, the antioxidant activity evaluation methods mainly include three categories: in vitro chemical analysis, animal experiment and human food test. The in vitro chemical method is simple, fast, and low in analysis cost, but the measurement results cannot truly reflect the antioxidant capacity of antioxidants in the bo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 刘冬孙海燕万红霞李艳从彦丽唐旭蔚
Owner SHENZHEN POLYTECHNIC
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