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

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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

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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

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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

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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

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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

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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

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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

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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

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• 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