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Screening method of safety and efficacy of skin antioxidants through use of a plurality of normal human skin cells

An anti-oxidant and skin cell technology, which is applied in the field of anti-oxidation safety efficacy evaluation and analysis and detection of personal skin care products, can solve the problems of high variation, difficult operation, and long time consumption

Inactive Publication Date: 2013-09-25
GUANGZHOU HUADAI BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these experiments are time-consuming, expensive, difficult to operate, and have high inter-laboratory variability, which is not conducive to high-throughput screening and inter-laboratory promotion of suspected antioxidant substances.

Method used

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  • Screening method of safety and efficacy of skin antioxidants through use of a plurality of normal human skin cells
  • Screening method of safety and efficacy of skin antioxidants through use of a plurality of normal human skin cells
  • Screening method of safety and efficacy of skin antioxidants through use of a plurality of normal human skin cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] Example 1 Keratinocyte detection of vitamin C cytotoxicity and antioxidant efficacy

[0132] 1. Isolation, culture and identification of primary human keratinocytes:

[0133] 1) Thoroughly wash the fresh foreskin of the child with 0.01mol / L PBS buffer solution containing 100U / mL penicillin and 100ug / mL streptomycin for 2 to 3 times; then remove the subcutaneous fat under sterile conditions and connective tissue; cut the cleaned foreskin into skin pieces of about 2.0mm×3.0mm; unfold the skin piece, soak it in 0.25% lyase (Disspases II) with a mass concentration of 0.25% to separate the epidermis and dermis. Digest at 37°C for 2±0.5 hours or overnight at 4°C for 12-18 hours;

[0134] 2) Take out the separated skin piece from the lysing enzyme (Disspases II), gently separate the epidermis and dermis with tweezers, and collect the epidermis in 0.01mol / In PBS of L; wash the epidermis thoroughly with this PBS solution for 2 to 3 times, cut the epidermal tissue thoroughly w...

Embodiment 2

[0164] Example 2 Fibroblast detection of proanthocyanidin cytotoxicity and antioxidant efficacy

[0165] 1. Isolation, culture and identification of primary human fibroblasts

[0166] (1) Thoroughly wash the fresh foreskin of the child with 100 U / mL penicillin and 100 ug / mL streptomycin and 0.01 mol / L PBS buffer for 2 or 3 times; then remove the subcutaneous foreskin under aseptic conditions. Fat and connective tissue; cut the cleaned foreskin into skin pieces of about 2.0mm×3.0mm; unfold the skin piece, and soak the skin piece upwards in 0.25% lyase (Disspases II) to separate the epidermis and dermis. Digest at 37°C for 2±0.5 hours or overnight at 4°C for 12-18 hours;

[0167] (2) Take out the separated skin piece from the lyase (Disspases II), gently separate the epidermis and dermis with tweezers, and collect the dermis in 0.01mol of double antibody (100U / mL penicillin, 100ug / mL streptomycin) / L of PBS; thoroughly wash the dermis with this PBS solution for 2 to 3 times, a...

Embodiment 3

[0199] Example 3 Skin melanocytes detect the cytotoxicity and antioxidant efficacy of kojic acid

[0200] 1. Isolation and culture of primary human melanocytes

[0201] 1.1 Thoroughly wash the fresh foreskin of the child cut off by the surgical ring with 0.01mol / L PBS buffer solution containing 100U / mL penicillin and 100ug / mL streptomycin for 2-3 times; then remove the subcutaneous fat and Connective tissue; cut the cleaned foreskin into skin pieces of about 2.0mm×3.0mm; unfold the skin piece, soak it in 0.25% lyase (Disspases II) with the mass concentration to separate the epidermis and dermis, and there are 37 ways Digest for 2±0.5 hours at ℃ or overnight at 4℃ for 12-18 hours;

[0202] 1.2 Take out the separated skin piece from the lysing enzyme (Disspases II), gently separate the epidermis and dermis with tweezers, and collect the epidermis in 0.01mol / L containing double antibody (100U / mL penicillin, 100ug / mL streptomycin). Wash the epidermis thoroughly with this PBS sol...

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Abstract

The invention discloses a screening method of safety and efficacy of skin antioxidants through use of a plurality of normal human skin cells. The screening method includes the following steps: (1) separation, culture and identification of normal human primary cells; (2) toxicity test of substances for testing; (3) preparation of the substances for testing and concentration determination of the substances for testing; (4) determination of ultraviolet-light induced radiation dose; and (5) verification of antioxidant effects of the substances for testing. The normal human skin cells are prepared through standardized culture in vitro, so that the normal human skin cells has strong division propagation capability, high degree of standardization, less difference among batches, and the same activity and functions as that in vivo. Results which are obtained through use of the normal healthy human skin cells are more reliable than the results obtained through use of animal or human cell lines. Through use of the screening method, toxic effects and antioxidant efficacy of the substances for testing can be evaluated in two aspects including a qualitative aspect and a quantitative aspect. The method, through replacement of the living animal and human skin, can be directly used for toxicity and efficacy tests of antioxidant substances in products including chemicals, cosmetics, pharmaceuticals and the like.

Description

technical field [0001] The invention belongs to the technical field of evaluation, analysis and detection of antioxidant safety and efficacy of personal skin care products, and in particular relates to a method for screening the safety and efficacy of skin suspected antioxidants by using various skin cells of normal people. Background technique [0002] The skin is the largest organ of the human body and the main organ for the toxic effects of exogenous chemical factors (chemicals, drugs, cosmetics, etc.) or physical and mechanical factors (ultraviolet rays, microwaves, etc.). With the development of modern industry and the increasingly serious environmental pollution, the types and frequency of oxidative stress sources in daily life have increased significantly compared with before. Various sources of oxidative stress attack skin cells and their proteins, cell lipids and even DNA, causing oxidative damage to cells, directly or indirectly leading to various skin sub-health a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02
Inventor 程树军秦瑶步犁谈伟君
Owner GUANGZHOU HUADAI BIOLOGICAL TECH CO LTD
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