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

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Presentation on theme: "Dietary plants and antioxidant enzymes in hydroperoxide-induced oxidative stress in U 937 Presenter Miss Wantana Phookongchana 4311075 Advisor Dr Roongsiri."— Presentation transcript:

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

2 Introduction Free radicals 2 - 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.

3 Introduction 3 Alzheimerimer ’ s disease Heart disease Aging

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

5 ชนิดของ antioxidants Introduction 5 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

6 Introduction Superoxide dismutase (SOD) 2 O H + H 2 O 2 + O 2 Catalase H 2 O 2 H 2 O + O 2 6 SOD Catalase

7 ชื่อวิทยาศาสตร์ Hibiscus sabdariffa Linn. ชื่อสามัญ Jamaican sorrel, Roselle of Rama ชื่อวิทยาศาสตร์ Cassia siamea Britt. ชื่อสามัญ Cassod Tree, Thai Copper Pod,Siamese Cassia Introduction Biological Activity: Antiatherosclerotic Effect,Antihepatotoxic Activity,Antihypertensive Activity Biological Activity:AntiAlzheimer ’ s Activity,Vasodilator Activity 7

8 ชื่อวิทยาศาสตร์ Apium graveolens ชื่อสามัญ Celery ชื่อวิทยาศาสตร์ Ocimum basilicum Linn ชื่อสามัญ Sweet Basil Biological Activity: Anti- inflammation Activity Antihemorrhagic Activity, Antibacterial Activity Introduction Biological Activity: Antihypercholesterolemia Activity Antimutagenic Activity Anti- inflammatory Activity 8

9 ชื่อวิทยาศาสตร์ Averrhoa carambola Linn ชื่อสามัญ Carambola, star fruit Introduction 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 Cell culture Treatment of cells Collect cells Cell lysate preparation Enzyme activity Protein concentration assay Method CatalaseSuperoxide dismutase 12

13 Extracted with 80% ethanol Lyophilized Preparation of plant extracts Washed Dried at 50 0 C Chop ped Shaken over night 13

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

15 Treatment of cells กระเจี๊ ยบ 2. ขี้เหล็ก 3. คื่น ฉ่าย 4. โหระพ า 5. มะเฟือ ง 6.untr eat 7.DMS O 8.t- BOOH Stock 10 mg/ml Conc : Final conc : mM 0.5 mM mg/ml  g/ml

16 Duplicate  g/ml สารสกัด 0.5 ml/well Untreat DM SO T-BOOH 1 hr,37 0 C,5%CO 2 Final conc : 1 hr,37 0 C,5%CO 2 Collect cell Cell :2.0 ml สารสกัด :0.5 ml 0.5 mM t-BOOH: 0.5 ml/well 3 ml 16

17 Centrifuge 1,200 rpm, 5 min Discard supernatant Resuspend with cold 1x PBS Discard supernatant-70 0 C Collect cells 17 Centrifuge 1,200 rpm, 5 min

18 RIPA lysis buffer Protease inhibitor 1ml : 10  l C ell homog enate Homogenizer Centrifuge 1000 rpm, 5 min Collect supernatant Transfer C Cell lysate preparation 18

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

20 Meth od + 12  l 3% H 2 O  l 50 mM Phosphate buffer pH  l Cell lysate Incubate 37 0 C Absorbance monitored for 5 min at 240 nm 20

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

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

23 Protein assay Princip le 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 Bicinchoninic acid (BCA kit)

24 Reagent A : Reagent B 50 : 1 - Cell lysate - Protein Std.(BSA):0.2,0.4,0.6,0.8,1.0 mg/ml 200  l/well25  l/well 96 well plate 37 0 C, 30 min Microplate reader 562 nm method 24 Reagent A: Bicinchoninic acid, 0.1 NaOH Reagent B: Copper sulfate pentahydrate

25 Protein assay Result 25

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

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

28 28 I. Levels of superoxide dismutase (SOD) and catalase in t-BOOH treated cells were not significantly 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 Discussion

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

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

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33 RIPA buffer (Radioimmunoprecipritation) - Tris-HCl - buffering agent prevents protein denaturatiom - NaCl - salt prevents non-specific aggregation - Triton-X100- non-ionic detergent to extract protein Protese inhibitor -Phenylmethylsulfonyl fluoride(PMSF) -EDTA-calcium chelator -Leupeptin -Aprotini -Pepstatin


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