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Transcript of Dietary plants and antioxidant enzymes in hydroperoxide-induced oxidative stress in U 937 Presenter...
Dietary plants and antioxidant enzymes in hydroperoxide-induced
oxidative stress in U 937Presenter Miss Wantana
Phookongchana 4311075Advisor Dr Roongsiri Shotepativatekul
1
IntroductionFree 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.
Introduction
3
Alzheimerime r’s disease
Heart diseaseAging
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
ชนิ�ดของ antioxidants Introduction
5
1. Primary antioxidants work by preventing the formation of new free radical species - Catalase, superoxide dismutase, glutathione peroxidase, metal binding protein2. Secondary antioxidants trap radicals thereby preventing chain reactions - Vitamin C,E,beta-carotene3. Tertiary antioxidants -repair biomolecules damaged by free radicals - DNA-repaired enzymes
IntroductionSuperoxide dismutase (SOD)
2 O2- + 2H+
H2O2 + O2
Catalase H2O2 H2O + O2
6
SOD
Catalase
ชื่��อวิ�ทยาศาสตร์� Hibiscus sabdariffa Linn. ชื่��อสามั�ญ Jamaican sorrel, Roselle of Rama
ชื่��อวิ�ทยาศาสตร์� Cassia siamea Britt .
ชื่��อสามั�ญ Cassod Tree, Thai Copp er Pod,Siamese Cassia
Introduction
Biological Activity: Antiatherosclerotic Effect,Antihepatotoxic
Activity,Antihypertensive Activity
Biological Activity:AntiAlzheimer ’s Activity,Vasodilator
Activity 7
ชื่��อวิ�ทยาศาสตร์� Apium graveolensชื่��อสามั�ญ Celery
ชื่��อวิ�ทยาศาสตร์� Ocimum basilicum Linn
ชื่��อสามั�ญ Sweet Basil
Biological Activity: -Anti inflam mation Activity
Antihemorrha gic Activity,
Antibacteria l Activity
Introduction
Biological Activity: Antihypercholesterolemia Activity Antimutagenic Activity Anti-inflammatory Activity
8
ชื่��อวิ�ทยาศาสตร์� Averrhoa ca rambola Linn
ชื่��อสามั�ญ Carambola, star fruit
Introduction
Biological Activity: Antibacterial Activity Anti-inflammation Activity
9
Objective
เพื่� อศึ�กษาผลของสารสก�ดจากพื่�ชที่� มี�ต่�อ antioxidant enzymes ในิเซลล U
937 ที่� ถู"กเหนิ� ยวนิ&าให'เก�ด oxidative stress โดย
hydroperoxide
10
Materials and Methods
11
Cell culture
Treatment of cells
Collect cells
Cell lysate preparation
Enzyme activity
Protein concentration assay
Method
CatalaseSuperoxide dismutase 12
Extracted with 80% ethanol
Lyophilized
Preparation of plant extracts
Washed
Dried at 500C
Chopped
Shaken over night 13
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
Treatment of cells
15
1.กระเจ�*ยบ2 .ข�,เหล-ก3 .คื่� นิฉ่�าย4 .โหระพื่า5 .มีะเฟื1อง6 .unt r eat7 .DMSO8 .
-t BOOH
Stock 10 mg/mlConc : 3.0 1.2 0.6 0.12 0.03Final conc : 500 200 100 20 5
3mM 0.5 mM
mg/mlg/ml
Duplicate
5 20100200500 g/ml
สาร์สกั�ด0.5 ml/well UntreatDM
SOT-BOOH
1 hr,370C,5%CO2
Final conc :
1 hr,370C,5%CO2
Collect cell
Cell :2.0 mlสารสก�ด:0.5
ml
0.5 mM t-BOOH: 0.5 ml/well
3 ml
16
Centrifuge
1,200 rpm, 5
min
Discard supernatant
Resuspend with cold 1x PBSDiscard supernatant-700C
Collect cells
17
Centrifuge 1,200 rpm, 5 min
RIPA lysis buffer
Protease inhibitor
1ml : 10 l
Cell
Cell homogenate
Homogenizer
Centrifuge 1000 rpm, 5 min
Collect supernatant
Transfer
- 70 0C
Cell lysate preparation
18
Principle
Catalase Reaction: 2 H2O2 => 2 H2O + O2
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 activity
19
Method
+ 12 l 3% H2O2
888 l 50 mM Phosphate buffer pH 7.4
100 l Cell lysate
Incubate 370C
Absorbance monitored for 5 min at 240 nm
20
2O2-+ 2H+
H2O2 + O2
Principle
Xanthine + NBT Blue formazan Xanthine oxidase
- 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
SOD
Superoxide dismutase activity
NBT
Blue formazan
21
Mixture- 0.05 M Sodium carbonate buffer pH 10.2- 3.0 mM Xanthine- 0.75 mM Nitro blue tetrazolium(NBT)- Cell lysate
Method
Xanthine oxidase
6 mM copper(II) chloride
Incubate RT , 30 min
Centrifuge1,500
rpm,10 min
Supernatant
Abs 560 nm
22
Protein assay
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
Bicinchoninic acid (BCA kit)
Reagent A : Reagent B50 : 1
- Cell lysate- Protein Std.(BSA):0.2,0.4,0.6,0.8,1.0 mg/ml200 l/well 25 l/well
96 well plate370C, 30 min
Microplate reader 562 nm
method
24Reagent A: Bicinchoninic acid , 0.1 NaOHReagent B: Copper sulfate pentahydrate
Protein assayResult
Protein standard curve
y = 0.0016x
R2 = 0.98410.000
0.500
1.000
1.500
2.000
0 200 400 600 800 1000Concentration(g/ml)
Abso
rban
ce
25
Result
Catalase activity
0
200
400
600
U D T กระเจ�*ยบ ข�,เหล-ก คื่� นิฉ่�าย มีะเฟื1อง โหระพื่า
CAT ac
tivity
(U/m
g prot
ein)
520100200500
Concentration(g/ml)
26Figure 2 Plant extracts did not affect catalase activity in hydroperoxide-inducedoxidative stress in U937
Result
27
0.00
0.50
1.00
1.50
2.00 SOD
activity (U
/mg p
rotein
) 520100200500
U D T กระเจ�*ยบ ข�,เหล-ก คื่� นิฉ่�าย โหระพื่า มีะเฟื1อง
Superoxide dismutase
Concentration(g/ml)
Figure 3 Plant extracts did not affect superoxide dismutase activity in hydroperoxide-induced oxidative stress in U937
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
• 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
ConclusionDietary plants; Roselle, Cassod three,Cerely,Carambola
did not have any effect on antioxidant enzyme in hydroperoxide-induced oxidative stress in U937