Vit. C

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3 reactive oxygen species DPPH 15g/ml g/ml 78 4g/ml DNA 240g/ml 1

4 ; BHT C E (1) 2

5 DPPH DNA DNA NRK-52E 3

6 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

7 (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

8 , DNA (2) (catalaseglutathione peroxidase) ( C Eβ- ) (BHT ) 6

9 C Eβ- (3,4) 7

10 ( ) ( ) (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

11 ( ) DPPH Shimada (4) 2,2-dipheny1-1-picrylhydrazylDPPH (517nm ) DPPH ( ) DPPH DPPH AH DPPH 2 A DPPH 9

12 l ( 1g/ml100g/ml C 1g/ml70g/mlBHT 1g/ml150 g/ml)0.4mmdpph 250l l nm DPPH 4. DPPH ( 517nm 517nm ) 100% 517nm Robak Gryglewski (4) phenazine methosulfate PMS dihydronicotinamide adenine dinucleotide NADH nitro blue tetrazolium NBT 560nm 10

13 1.250l 50g/mL ~ 500g/mL 250l 300M NBT120 PMS936M NADH 5 250l nm 3. ( 560nm 560nm ) 100% 560nm DNA 10-9 DNA DNA (6) DNA (supercoiled) DNA DNAplasmid DNA (7) C DNA H 2 O 2 11

14 1. DNA(p uc18 ) (E.coli) p uc18 20 ml LB Ampicillin (50 g/ml) x 10 9 cells / ml 2. (E.coli) DNAplasmid DNAp uc ml 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 (60~70 ph7~8.5)50~100l spin column 12

15 12000rpm DNA DNA 3. 1% Agarose DNA g Agarose 25 ml TAE 1% 2 ethidium bromide µl 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

16 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 µl 37 5CO µl Vit C 5 320µg/ml H 2 O mM MTS ( :333µg/ml)PMS( :25µM) 6. 2 MTS formazan nm 490nm 100% 490nm 14

17 ph g 6.53g ph 4.7 C ph 3.3 DPPH DPPH % ( µ g/m l) Vit C BHT ( ) DPPH 15

18 (g/ml) ( ) DPPH DPPH ( 517nm 517nm ) 100% 517nm Vit C BHT DPPH 2 C BHT 1. EC g/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

19 % ( µg/ml) VIT C (g/ml) ( 517nm 517nm ) 100% 517nm Vit. C C 17

20 1 EC g/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

21 DNA DNA C DNA : C 4µg/ml20µg/ml60µg/ml 1. DNA (supercoiled) band H 2 O 2 DNA H 2 O 2 band 1 band FeSO 4 H 2 O H 2 O 2 FeSO 4 H 2 O 2 FeSO 4 Fe 2 DNA OH - DNA band DNA DNA 4µg/ml CDNA C DNA 19

22 P< H 2 O 2 (mm) H 2 O µg/ml) P<0.001 vs. H 2 O CTL H 2 O H (µg/ml) 1.5mM H 2 O 2 20

23 P< CTL Vit. C (µg/ml) C CTL H 2 O H 2 O 2 + Vit. C (µg/ml) 1.5mM H 2 O 2 C 1. H 2 O g/ml 160g/ml 240g/ml 320g/ml 4. C 240g/ml 320 g/ml 5. C 21

24 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

25 EC g/ml 2. C 10g/ml 87.10% 3.BHT 100g/ml 72.30% C DPPH BHT C C ( C) g/ml 78 EC g/ml 2. C 50g/ml 93 C DNA H 2 O 2 Fe 2 (Fenton reaction) DNA 23

26 DNA DNA 4g/ml () C C DNA H 2 O 2 H 2 O 2 H 2 O 2 ( mM) H 2 O 2 H 2 O 2 C H 2 O mM C 24

27 240320µg/ml C C C DPPH C DNA C C 100g C 77.3mg C 45 C DPPH C phenolics polyphenolics (12) 25

28 ph g 6.53g ph 4.7 DPPH EC g/ml 1g/ml15g/ml DPPH DPPH EC g/ml 50g/ml100g/ml 100g/ml200g/ml 200g/ml DNA DNA 4g/ml µg/ml 26

29 C C C C BHT CE C 27

30 1 (2000) (2002) 49: Hydroxyl Radical Free Radical and Radiation Biology Program 8. Feb Ai-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): Masahiro Sakai, Toshiaki Tsukada and Raymond C. Harris (2001) Experimental Nephrology Sabina 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

31 29

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