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Method for evaluating protective action of Chinese herbal medicine to DNA oxidation damnify

A technology of oxidative damage and protection, applied in the direction of biochemical equipment and methods, microbial determination/inspection, measurement devices, etc., can solve the problems of lung fibroblast DNA oxidative damage and so on

Active Publication Date: 2009-03-11
CHINA TOBACCO ZHEJIANG IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Liu Bin and others found that both extracts of Akebia officinalis can directly cause DNA oxidative damage in Chinese hamster lung fibroblasts

Method used

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  • Method for evaluating protective action of Chinese herbal medicine to DNA oxidation damnify
  • Method for evaluating protective action of Chinese herbal medicine to DNA oxidation damnify
  • Method for evaluating protective action of Chinese herbal medicine to DNA oxidation damnify

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Determination of DNA Oxidative Damage Caused by Fenton's Reagent

[0031] 1. Reagents and instruments:

[0032] All reagents were of analytical grade, and the experimental water was deionized ultrapure water. Imidazole buffer at pH 6.0; 0.1M PBS, pH=7.2-7.4; washing solution is PBS containing 0.1% Tween 20; blocking solution I is 40mM glycine, 1% BSA imidazole buffer; blocking solution II 10% BSA in PBS.

[0033] N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride (EDC) was purchased from Sigma; HRP-labeled rabbit anti-goat IgG was purchased from Beijing Boaosen Biotechnology Co., Ltd. Goat anti-8-OH-dG was purchased from abcam company; HRP chemiluminescence kit was purchased from Milipore company; carboxy-modified magnetic microspheres (1.5 μm, Bangs Laboratories, Inc.); DNA (5'-GCC AAC AGC CAG TGG GAA ACA AAAAAAAAA-NH 2 -3'), synthesized by Shanghai Yingjun Biotechnology Co., Ltd. Oxidizing agent: FeCl 2 Aqueous solution (containing 0.04% HCl...

Embodiment 2

[0040] Example 2 Selection of Goat Anti-8-OH-dG Dilution

[0041] Since the goat anti-8-OH-dG used in the experiment has a strong activity and has a great influence on the detection signal, it is necessary to select an appropriate dilution concentration to obtain the optimal signal-to-noise ratio. figure 2 The effect of different dilutions on the detected signal intensity is shown. The experimental conditions are: Fe 2+ The concentration is 0.288mM, H 2 o 2 The dilution of HRP-labeled rabbit anti-goat IgG is 1 / 20000. Such as Figure 4 It can be seen that as the dilution of goat anti-8-OH-dG increases, the detection signal shows a trend of first increasing and then decreasing, and reaches the maximum value near the dilution of 100000 times. If the dilution is too large and the concentration is too high, the probability of non-specific adsorption of the antibody in the control group on the magnetic beads will increase; if the dilution concentration is too small, the concen...

Embodiment 3

[0042] Example 3 Different Fe 2+ The effect of concentration on the intensity of chemiluminescence

[0043] Fenton's reagent that can generate hydroxyl radicals (ie Fe 2+ Mediated H 2 o 2 ) is a relatively classic DNA damage oxidant, where Fe 2+ The concentration has a significant effect on the amount of 8-OH-dG produced, image 3 show different Fe 2+ The influence of concentration on the luminous intensity can be seen from the figure, with Fe 2+ As the concentration increases, the amount of 8-OH-dG produced also increases, and then decreases. It may be that excessive oxidants can cause DNA strand breaks, thereby reducing the amount of 8-OH-dG produced. Our detection Methodological studies of this effect have also shown results similar to those in the literature. In the experiment, I represents the 20,000-fold dilution of HRP-labeled rabbit anti-goat IgG, II represents the 40,000-fold dilution; a represents the 100,000-fold dilution of the goat anti-8-OH-dG, and b repre...

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Abstract

The invention relates to an assessment method on the protective action of Chinese medicinal herb to the DNA oxidative damage, which comprises the steps that: DNA is fixed on the surface of a magnetic bead microsphere by covalent bonds; Chinese medicinal herb solution is added; the fixed DNA infects toxicity in vitro; magnetic bead separation is carried out to oxidize the components but reserve the oxidation-damaged DNA; 8-OH-dG antibodies are added to carry out immunoreaction with the 8-OH-dG generated by oxidative damage; enzyme-labeled second antibodies are added to react with the obtained product; and a luminous substrate is added, the high-sensitivity chemiluminescence method is adopted to detect the generated 8-OH-dG by DNA damage; the detection result is analyzed according to the detection result of the control group so as to judge whether the Chinese medicinal herb has the function of inhibiting the DNA oxidative damage to generate 8-OH-dG. The invention can detect and assess the protective action of Chinese medicinal herb to the DNA oxidative damage, and can be used for screening and developing high-efficiency and safe Chinese herbal preparations to protect the organism DNA from suffering oxidative damage.

Description

technical field [0001] The invention relates to a method for evaluating the protective effect of Chinese herbal medicine on DNA oxidative damage. Background technique [0002] Free radicals, reactive oxygen species (ROS), X-rays, γ-rays, asbestos, polycyclic aromatic hydrocarbons and other substances can attack DNA through different channels, forming covalent DNA adducts, causing DNA base mismatching, and DNA single strands Or double-strand breaks, resulting in DNA damage and ultimately cancerous changes (Poulsen, HE., Oxidative DNA modifications, Exp. Toxicol. Pathol., 2005, 57, 161-169.). DNA damage is associated with all changes in humans such as cancer, Alzheimer's disease, and the normal aging process. DNA oxidative damage products refer to DNA adducts produced by ROS damage to DNA. Among the more than twenty kinds of DNA oxidative damage products that have been discovered, guanine is most likely to be oxidatively damaged due to its molecular orbital having a higher en...

Claims

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Application Information

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IPC IPC(8): G01N33/543C12Q1/68
Inventor 储国海周国俊黄芳芳陈红君卢建忠
Owner CHINA TOBACCO ZHEJIANG IND
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