Dietary plants and antioxidant enzymes in hydroperoxide-induced oxidative stress in U 937

1. Dietary plants and antioxidant enzymes in hydroperoxide-induced oxidative stress in U 937 Presenter Miss Wantana Phookongchana 4311075 Advisor Dr Roongsiri Shotepativatekul 1

2. Free radicals Introduction • Molecules have been chemically damaged (modified) by removing a single electron.This results in a molecule that has at least one of its electrons un-paired electron in atomic orbital. • This molecule is very reactive since it wants to have its electrons all paired up. To do this it goes looking for electrons from some other molecules. When it finds another electron it takes it from that molecule. 2

3. Introduction Alzheimerimer’s disease Heart disease Aging 3

4. Introduction Antioxidants Any substance that, when present in low concentration compaired to that of oxidisable substrate, significantly delay or inhibit the oxidation of that substrate 4

5. Introduction ชนิดของ antioxidants • 1. Primary antioxidants work by preventing the formation of new free radical species • - Catalase, superoxide dismutase, glutathione peroxidase, metal binding protein • 2. Secondary antioxidants trap radicals thereby preventing chain reactions • - Vitamin C,E,beta-carotene • 3. Tertiary antioxidants -repair biomolecules damaged by free radicals • - DNA-repaired enzymes 5

6. Introduction Superoxide dismutase (SOD) SOD • 2 O2- + 2H+ H2O2 + O2 Catalase Catalase H2O2 H2O + O2 6

7. Introduction ชื่อวิทยาศาสตร์Hibiscus sabdariffa Linn.ชื่อสามัญJamaican sorrel, Roselle of Rama Biological Activity: Antiatherosclerotic Effect,Antihepatotoxic Activity,Antihypertensive Activity ชื่อวิทยาศาสตร์Cassia siamea Britt. ชื่อสามัญCassod Tree, Thai Copper Pod,Siamese Cassia Biological Activity:AntiAlzheimer’s Activity,Vasodilator Activity 7

8. Introduction ชื่อวิทยาศาสตร์Apium graveolens ชื่อสามัญ Celery Biological Activity: Antihypercholesterolemia ActivityAntimutagenic Activity Anti-inflammatory Activity ชื่อวิทยาศาสตร์Ocimum basilicum Linn ชื่อสามัญSweet Basil Biological Activity: Anti-inflammation Activity Antihemorrhagic Activity, Antibacterial Activity 8

9. Introduction ชื่อวิทยาศาสตร์Averrhoa carambola Linn ชื่อสามัญ Carambola, star fruit Biological Activity: Antibacterial Activity Anti-inflammation Activity 9

10. Objective เพื่อศึกษาผลของสารสกัดจากพืชที่มีต่อ antioxidant enzymes ในเซลล์ U 937 ที่ถูกเหนี่ยวนำให้เกิด oxidative stress โดย hydroperoxide 10

11. Materials and Methods 11

12. Method Cell culture Treatment of cells Collect cells Cell lysate preparation Protein concentration assay Enzyme activity Superoxide dismutase Catalase 12

13. Preparation of plant extracts Washed Chopped Dried at 500C Extracted with 80% ethanol Shaken over night 13 Lyophilized

14. Cell culture Resuspend with RPMI +1%Fetal calf serum Centrifuge 1200 rpm, 5 min U 937 cells Incubate overnight Adjust cells 500,000 cells/ml Add to 6 well plate 2 ml/well Cell count 14

15. Treatment of cells 1.กระเจี๊ยบ 2.ขี้เหล็ก 3.คื่นฉ่าย 4.โหระพา 5.มะเฟือง 6.untreat 7.DMSO 8.t-BOOH Stock 10 mg/ml Conc : 3.0 1.2 0.6 0.12 0.03 Final conc : 500 200 100 20 5 mg/ml mg/ml 3mM 0.5 mM 15

16. สารสกัด 0.5 ml/well 5 20 100 200 500 mg/ml Untreat T-BOOH Final conc : DMSO Duplicate 1 hr,370C,5%CO2 0.5 mM t-BOOH: 0.5 ml/well 3 ml สารสกัด:0.5 ml Cell :2.0 ml 1 hr,370C,5%CO2 Collect cell 16

17. Collect cells Centrifuge 1,200 rpm, 5 min Discard supernatant Resuspend with cold 1x PBS -700C Discard supernatant Centrifuge 1,200 rpm, 5 min 17

18. Cell lysate preparation RIPA lysis buffer Protease inhibitor 1ml : 10 ml Homogenizer Cell homogenate Cell Transfer - 70 0C Collect supernatant Centrifuge 1000 rpm, 5 min 18

19. Catalase activity Principle Catalase catalyses the breakdown of hydrogen peroxide (H2O2) with the release free Oxygen.. Changes in absorbance were taken to proportional to breakdown of H2O2 catalase Catalase Reaction: 2 H2O2 => 2 H2O+ O2 19

20. Method 888 ml 50 mM Phosphate buffer pH 7.4 + 12 ml 3% H2O2 Incubate 370C 100 ml Cell lysate Absorbance monitored for 5 min at 240 nm 20

21. Superoxide dismutase activity Principle - Xanthine is oxidized by oxygen and xanthine oxidase that produce H2O2 and O2- - O2- reduces nitro blue tetrazolium (NBT) that produces blue formazan - Superoxide dismutase inhibit Blue formazan formation Blue formazan Xanthine oxidase Xanthine + NBT Blue formazan NBT SOD 2O2-+ 2H+ H2O2 + O2 21

22. Method 6 mM copper(II) chloride Mixture - 0.05 M Sodium carbonate buffer pH 10.2 - 3.0 mM Xanthine - 0.75 mM Nitro blue tetrazolium(NBT) - Cell lysate Xanthine oxidase Incubate RT , 30 min Centrifuge 1,500 rpm,10 min Supernatant Abs 560 nm 22

23. Protein assay Bicinchoninic acid (BCA kit) Principle Proteins reduce alkaline Cu(II) to Cu(I) . Bicinchoninic acid is highly specific chromogenic reagent for Cu(I), forming a purple complex with absorbance maximum at 562 nm. Absorbance is directly proportional to protein concentration 23

24. method Reagent A : Reagent B 50 : 1 - Cell lysate - Protein Std.(BSA):0.2,0.4,0.6,0.8,1.0 mg/ml 200 ml/well 25 ml/well 96 well plate 370C, 30 min Microplate reader 562 nm Reagent A: Bicinchoninic acid , 0.1 NaOH Reagent B: Copper sulfate pentahydrate 24

25. Result Protein assay 25

26. Result Figure 2 Plant extracts did not affect catalase activity in hydroperoxide-induced oxidative stress in U937 26

27. Result Figure 3 Plant extracts did not affect superoxide dismutase activity in hydroperoxide-induced oxidative stress in U937 27

28. Discussion • I. Levels of superoxide dismutase (SOD) and catalase in t-BOOH treated cells were notsignificantly altered as compared to untreated cells • t-BOOH may not exert its effect through the antioxidant enzymes mechanism since the antioxidant enzymes were not increased in response to t-BOOH treatment • Antioxidant enzymes may take time to respond to t-BOOH treatment so any change in activity was not observed 28

29. Discussion II. Dietary plants did not have any effect on catalase and superoxide dismutase • Penetration of some active ingredients in the crude extracts into cells may require longer incubation time for the effect to take place • The active ingredients in the plant extracts used in this study did not have any effect on catalase and superoxide dismutase activity 29

30. Conclusion Dietary plants; Roselle, Cassod three,Cerely,Carambola did not have any effect on antioxidant enzyme in hydroperoxide-induced oxidative stress in U937

31. Thank you for your attention