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
Introduction
Free radicals

• 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
Alzheimerimers 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
??????? 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
??????????????? Hibiscus sabdariffa Linn.
????????? Jamaican sorrel, Roselle of Rama
Biological Activity Antiatherosclerotic
Effect,Antihepatotoxic
Activity,Antihypertensive Activity
??????????????? Cassia siamea Britt. ?????????
Cassod Tree, Thai Copper Pod,Siamese Cassia
Biological ActivityAntiAlzheimers
Activity,Vasodilator
Activity
7
8
Introduction
??????????????? Apium graveolens ?????????
Celery
Biological Activity Antihypercholesterolemia
Activity
Antimutagenic Activity
Anti-inflammatory Activity
??????????????? Ocimum basilicum Linn ?????????
Sweet Basil Biological Activity
Anti-inflammation Activity
Antihemorrhagic Activity,
Antibacterial Activity
8
9
Introduction
??????????????? Averrhoa carambola Linn ?????????
Carambola, star fruit
Biological Activity Antibacterial Activity
Anti-inflammation
Activity
9
10
Objective
???????????????????????????????????? antioxidant
enzymes ??????? U 937 ??????????????????????
oxidative stress ??? hydroperoxide
10
11
Materials and Methods
11
12
Method
Cell culture
Treatment of cells
Collect cells
Cell lysate preparation
Protein concentration assay
Enzyme activity
Catalase
Superoxide dismutase
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 1Fetal 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.????????? 2.???????? 3.???????? 4.?????? 5.?????
?? 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
??????? 0.5 ml/well
5
20
100
200
500 mg/ml
Untreat
T-BOOH
Final conc
DMSO
Duplicate
1 hr,370C,5CO2
0.5 mM t-BOOH 0.5 ml/well
3 ml
???????0.5 ml
Cell 2.0 ml
1 hr,370C,5CO2
Collect cell
16
17
Collect cells
Centrifuge 1,200 rpm, 5 min
Discard supernatant
Resuspend with cold 1x PBS
Discard supernatant
-700C
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 gt 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 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

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
32
(No Transcript)
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