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

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...

Examples

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...

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...

Examples

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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