reactive oxygen species DPPH 15g/ml 88.8 200g/ml 78 4g/ml DNA 240g/ml 1
; BHT C E (1) 2
DPPH DNA DNA NRK-52E 3
H 2 O 2 DMEM C (Vit. C) 2,2-dipheny1-1-picrylhydrazyl (DPPH) phenazine methosulfate PMS nitro blue tetrazoliumnbt dihydronicotinamide adenine dinucleotidenadh buty1ated hydroxytoluene (BHT) 4
(free radical) (oxygen-derived free radical) (reactive oxygen speciesros) (superoxide aniono - 2 ) (hydrogen peroxideh 2 O 2 ) (hydroxyl radical OH) (oxidative stress) (singlet oxygen O 2 ) (lipid hydroperoxidelooh) (nitric oxideno) 5
, DNA (2) (catalaseglutathione peroxidase) ( C Eβ- ) (BHT ) 6
C Eβ- (3,4) 7
( ) ( ) (Toona sinensis Roem.Cedrela s inensis A. Juss) (Meliaceae) ( ) (100g) (82.6g) (2.8g)(0.4) (2.0g) (1.5g) C(77.3mg) (0.93mg) (426.0mg) (1.0mg) (122.0mg) (5) C C C BHT BHT 8
1. 2. 45 3. ( ) DPPH Shimada (4) 2,2-dipheny1-1-picrylhydrazylDPPH (517nm ) DPPH ( ) DPPH DPPH AH DPPH 2 A DPPH 9
1. 1000l ( 1g/ml100g/ml C 1g/ml70g/mlBHT 1g/ml150 g/ml)0.4mmdpph 250l 10 3 2. 1000l 3. 517nm DPPH 4. DPPH ( 517nm 517nm ) 100% 517nm Robak Gryglewski (4) phenazine methosulfate PMS dihydronicotinamide adenine dinucleotide NADH nitro blue tetrazolium NBT 560nm 10
1.250l 50g/mL ~ 500g/mL 250l 300M NBT120 PMS936M NADH 5 250l 2. 560nm 3. ( 560nm 560nm ) 100% 560nm DNA 10-9 DNA DNA (6) DNA (supercoiled) DNA DNAplasmid DNA (7) C DNA H 2 O 2 11
1. DNA(p uc18 ) (E.coli) p uc18 20 ml LB Ampicillin (50 g/ml) 37 12-16 1.5 x 10 9 cells / ml 2. (E.coli) DNAplasmid DNAp uc18 1 1.5ml E.coli (12-14,000 rpm) 2 Solution I 200l5 Solution II 200l 5 Solution III 200l5 3 (12,000rpm) 5 DNA 4 spin column 1.5ml 3 spin column1 DNA spin column 5 washing solution 700l 1 63 washing solution 7 spin column 1.5ml 60 5-10 8 (60~70 ph7~8.5)50~100l spin column 12
12000rpm DNA DNA 3. 1% Agarose DNA 1 0.25g Agarose 25 ml TAE 1% 2 ethidium bromide 3 37 30 2µl 110V 20 ( ) p uc18 10µl 10µl 10µl 10µl 10µl 10µl 10µl H 2 O 2 1µl 1µl 1µl 1µl 1µl FeSO 4 1µl 1µl 1µl 1µl 1µl / C 1µl 1µl 1µl ddh 2 O 3µl 2µl 2µl 1µl p uc18 DNA DNA 4µg/ml20µg/ml60µg/ml DNA 13
NRK-52E (8) C NRK-52E MTS 酶 PMSphenazine methosulfate MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-(carboxymethoxyphenyl)-2-(4-sulfophenyl )-2H-tetrazolium) formazan 490nm 1.96-well plate well 5 10 3 100µl 37 5CO 2 24 2. 95µl Vit C 5 320µg/ml 30 3. H 2 O 2 0.50.81.01.5mM 16 4. 5. MTS ( :333µg/ml)PMS( :25µM) 6. 2 MTS formazan 7. 490nm 490nm 100% 490nm 14
ph 113.13g 6.53g ph 4.7 C ph 3.3 DPPH DPPH % 100 90 80 70 60 50 40 30 20 10 0 0 20 40 60 80 100 120 140 160 180 200 ( µ g/m l) Vit C BHT ( ) DPPH 15
(g/ml) ( ) DPPH DPPH ( 517nm 517nm ) 100% 517nm Vit C BHT 1.0 3.30 2.30 2.90 2.5 18.40 11.80 4.30 5.0 39.20 25.10 9.30 10.0 60.60 87.10 5.20 15.0 88.80 92.70 25.10 20.0 89.90 95.60 28.60 30.0 95.30 32.60 50.0 95.90 39.60 60.0 95.60 43.00 70.0 96.10 42.70 80.0 47.80 100.0 72.30 150.0 84.10 1 DPPH 2 C BHT 1. EC 50 9.4g/ml C EC 50 7g/ml 2. DPPH 3. C 1g/ml15g/ml DPPH DPPH 4.BHT 1g/ml80g/ml DPPH 100g/ml150g/ml DPPH C 5. C BHT 16
% 100 75 50 25 0 0 100 200 300 400 500 600 700 800 900 1000 ( µg/ml) VIT C (g/ml) ( 517nm 517nm ) 100% 517nm Vit. C 50.0 6.00 93.00 100.0 5.00 94.00 200.0 78.00 95.00 400.0 80.00 95.00 800.0 74.00 1000.0 77.00 1 2 C 17
1 EC 50 139g/ml C EC 50 50g/ml 2 50g/ml400g/ml C 3 50g/ml100g/ml 100g/ml200g/ml 200g/ml 4 C 50g/ml 18
DNA DNA C DNA 7 1 4 1 5 7 : C 4µg/ml20µg/ml60µg/ml 1. DNA (supercoiled) band 2 1 2. H 2 O 2 DNA H 2 O 2 band 1 band 2 2 3. FeSO 4 H 2 O 2 3 4. H 2 O 2 FeSO 4 H 2 O 2 FeSO 4 Fe 2 DNA OH - DNA band 1 4 5. 5 7 4 DNA DNA 4µg/ml 6. 5 7 CDNA C DNA 19
100 75 50 25 P<0.001 0 0 0.5 1 1.5 1.8 H 2 O 2 (mm) H 2 O 2 125 100 75 50 25 0 0 80 160 240 320 µg/ml) P<0.001 vs. H 2 O 2 100 75 50 25 0 CTL H 2 O 2 80 160 240 320 H 2 0 2 + (µg/ml) 1.5mM H 2 O 2 20
150 125 100 75 50 P<0.001 25 0 CTL 80 160 240 320 Vit. C (µg/ml) C 100 75 50 25 0 CTL H 2 O 2 80 160 240 320 H 2 O 2 + Vit. C (µg/ml) 1.5mM H 2 O 2 C 1. H 2 O 2 2. 3. 80g/ml 160g/ml 240g/ml 320g/ml 4. C 240g/ml 320 g/ml 5. C 21
reactive oxygen speciesros, X DNA (6) (9) (10,11) DPPH reactive oxygen species, ROS DPPH DPPH DPPH C BHT 1. 15g/ml 88.80% 22
EC 50 9.4g/ml 2. C 10g/ml 87.10% 3.BHT 100g/ml 72.30% C DPPH BHT C C ( C) 1. 200g/ml 78 EC 50 139g/ml 2. C 50g/ml 93 C DNA H 2 O 2 Fe 2 (Fenton reaction) DNA 23
DNA DNA 4g/ml () C C DNA H 2 O 2 H 2 O 2 H 2 O 2 (0.50.81.01.51.8mM) H 2 O 2 H 2 O 2 C H 2 O 2 0.81.5mM C 24
240320µg/ml C C C DPPH C DNA C C 100g C 77.3mg C 45 C DPPH C phenolics polyphenolics (12) 25
ph 113.13g 6.53g ph 4.7 DPPH EC 50 9.4g/ml 1g/ml15g/ml DPPH 89.90 DPPH EC 50 139g/ml 50g/ml100g/ml 100g/ml200g/ml 200g/ml DNA DNA 4g/ml 240320µg/ml 26
C C C C BHT CE C 27
1 (2000) 7-10 2 (2002) 49: 123-9 3 4 5 9011 20 6Hydroxyl Radical Free Radical and Radiation Biology Program 8. Feb 2001 7Ai-Hsiang Lo, Yu-chih Liang, Shoei-Yn Lin-Shiau, Chi-Tang Ho and Jen-Kun Lin (2002) Carnosol, an antioxidant inn rosemary, suppreses inducible nitric oxide synthase through down-regulating nuclear factor-kb in mouse macrophage Carcinogenesis 23(6):983-991 8Masahiro Sakai, Toshiaki Tsukada and Raymond C. Harris (2001) Experimental Nephrology 928-39 9Sabina S. Peters (2001) Superoxide: An introductory Glimpse from the chemical/biochemical perspective Free Radical and Radiation Biology Program 77:222 10Iman Ahmad (2001) Hydrogen Peroxide Free Radical and Radiation Biology Program 77:222 11Sherry A. Rovinsky Hydrogen Peroxide:An ubiquitous reactive oxygen species Free Radical and Radiation Biology Program 77:222 12Fereidoon Shahidi (2000) Natural antioxidants : sources, effects and applications Singapore institute of food science & technology 28
29