Antioxidant Presentation

8/3/2019 Antioxidant Presentation

1/32


2/32

ANTIOXIDANT An antioxidant is a molecule capable of inhibiting the oxidation of

other molecules.

SIGNIFICANCE---Exposure of biological systems to xenobiotics, pollutants, ionizing radiation orU.V. light

and development of certain pathological conditions lead to oxidative stress, consequently increase production of oxy radicals[2]. Cell damage caused by free radicals appears to be a major contributor in aging and degenerative diseases of aging such as cancer, cardiovascular disease, cataracts, compromised immune system, rheumatoid arthritis and brain dysfunction.


3/32

Free radicals have been implicated in thepathogenesis of at least 50 diseases.Fortunately, free radical formation iscontrolled naturally by various beneficialcompounds known as antioxidants.

Antioxidants are capable of stabilize,deactivate or scavenge free radicals beforethey attack cells.


4/32

MECHANISM OF ACTION

Antioxidant action

1-Includes free radical scavenging capacity

2-Inhibition of lipid peroxidation

3-Metal ion chelating ability and reducingcapacity.


5/32

NATURAL ANTIOXIDANTS

a) Enzymes-Antioxidant enzymes such as

reduced glutathione (GSH), superoxide

dismutase(SOD), catalase(CAT), glutathionePeroxidase (GPx)

b)High molecular weight compounds

(proteins): Albumin, ceruplasmin, transferin,

haptoglobin bind to redox active metals and limitsthe production of metal catalyzed free radicals.


6/32

c)Low molecular weight compounds: These aresubdivided into lipid soluble,carotenoids, quinine

and some polyphenols) and water soluble antioxidants(ascorbic acid, uric acid and

some polyphenols) d)Minerals: Selenium, manganese, copper, and zinc are recognized as versatile antioxidants. e)Vitamins: Vitamin A, C, and E are

proved to have significant role in preventing or minimizing peroxidation damage in biologicalsystem

f)Plant antioxidants


7/32

IN-VIVO ANTIOXIDANT SCREENING

METHODS

Antioxidant activity can be measured using in-vitro as well as in-vivo methods. The chemistryresponsible for these effects is ready donation ofelectrons to reactive oxygen species (ROS) by

antioxidants. METHODS

Lipid peroxidation (LPO) : LPO is an autocatalytic process, which is a common consequence of cell death. MDA (Malondialdehyde) is one of the end

products in the lipid per-oxidation process whichis accepted as an indicator of lipid peroxidation.


8/32

Method-I-The tissues are homogenized in 0.1 M

buffer pH 7.4 with a Teflon-glass homogenizer. LPO in this homogenate was determined by measuring the

amounts of Malondialdehyde (MDA) produced.

0.2 ml of tissue homogenate, 0.2 ml of 8.1%sodium

dodecyl sulphate (SDS), 1.5 ml of 20% acetic acid

and 1.5 ml of 8% TBA were added. The volume of

the mixture was made up to 4 ml with distilled water

and then heated at 95C on a water bath for 60 min using glass balls as condenser.


9/32

After incubation the

tubes were cooled to room temperature and final

volume was made to 5 ml in each tube. 5.0 ml of

butanol: pyridine (15:1) mixture was added and the

contents were vortexed thoroughly for 2 min. After

centrifugation at 3000rpm for 10 min, the upper organic layer was taken and its OD read at 532 nm

against an appropriate blank without the sample. The

levels of lipid peroxides were expressed as n moles

of thiobarbituric acid reactive substances (TBARS)/

mg protein using an extinction coefficient of 1.56

105 M1cm-1


10/32

Method-II-Three sets of tubes were used

for the assay. The first set had 22ml of bufferand 02ml of FeSO4 (07 mM finalconcentration); another set had 22 ml of

buffer and02 ml of H2O2 (075 mM finalconcentration); the third set contained 22mlof buffer and 02 ml of distilled water. Each setwas divided into two batches. To each tube ofone batch 06 ml of the tissue homogenatewas added.


11/32

The tubes were incubated on a mechanical shaker at100120 oscillation/min (for aeration) for 60 min. Afterthe incubation 05 ml of 40%trichloroacetic acid (TCA)

was added. To each tube of the second batch 05 ml

of 40 % TCA was added at zero time immediately

after the addition of 06ml of the homogenate .To all the tubes 025 ml of 5 HCl was added and the

Contents mixed thoroughly. This was followed by the

addition of 05ml of 2%thiobarbituric acid. The tubes

were shaken and incubated in a water bath at 90C for 20 min.


12/32

They were then cooled and 3 ml of

chloroform was added. After thorough mixingthey were centrifuged for 15 min at 3000 g.The supernatant was aspirated and itsabsorbance at 532 nm determined using

water blank. Standard malonaldehyde wastreated in a similar fashion and the colourdeveloped was measured.

Method-III: one volume of homogenatewas mixed with 0.5 volume of trichloroaceticacid (15%w/v) and centrifuged at 200 rev for10 min.


13/32

One milliliter of the supernatant was

Mixed with 0.5ml TBA(0.7%w/v) and boiled for 10min.After cooling, the absorbance was recorded at 535 byspectrophotometer (Shimadzu UV-3100).MDAconcentration was calculated using extinctioncoefficient of 1.56 105M1 cm1.

Reduced Glutathione (GSH):

Method -I: To measure the GSH level, the tissue homogenate was taken. The homogenate was

Added with equal volume of 20%trichloroacetic acid

(TBA) containing 1 mM EDTA.


14/32

The mixture was allowed to stand for

5 min prior to centrifugation for 10 min at 200rpm.The supernatant (200 l) was thentransferred to a newest of test tubes and

added 1.8ml of the Ellmans reagent(0.1mM).Then all the test tubes make upto thevolume of 2ml.After completion of the totalreaction, solutions were measured at 412 nmagainst blank. Absorbance values werecompared with a standard curve generatedfrom standard curve from known GSH.


15/32

Glutathione Peroxidase (GSHPx): Cytosolic

GPx was assayed via a 3-ml cuvette containing 2.0 ml of 75 mmol/L phosphate buffer, pH 7.0. The following

solutions were then added : 50l of 60mmol/L

glutathione reductase solution (30 U/mL) , 50L OF

0.12 mol/L NaN3, 0.10 OF 0.15mmol/L Na2EDTA , 100Lof 3.0mmol/L NADPH, and 100Lof cytosolic

fraction obtained after centrifugation at 20,000 g for

25 minutes.


16/32

Glutathione-S-transferase ( GSt): The

reaction mixture (1mL) consisted of 0.1 Npotassium phosphate (pH 6.5), 1 nmol/LGSH,1mol/L l-chloro-2, 4-dinitrobenzene as

substrate and a suitable amount of cytosol (6mg protein/mL). The reaction mixture wasincubated at 370C for 5 min and the reactionwas initiated by the addition of the substrate.The increase in absorbance at 340 nm wasmeasured spectrophotometrically.


17/32

Glutathione Reductase: Method:Preparation of Liver Supernatant- Livers

(about 400 g) were obtained from 40 male Sprague- Dawley rats (200 to 250 g), which were killed by decapitation. The livers were cut into small pieces and homogenized in 9 ml of 0.25Mice-cold sucrose per g of rat liver in a Turmix blender.The homogenate was centrifuged for 45min at 14,000 rpm. The pellets were suspended in a small volume of 0.25Msucrose and centrifuged. The supernatants were combined with the previous centrifugate. The pooled material was adjusted to pH 5.5 with cold 0.2M acetic acid and centrifuged .


18/32

The rate of oxidation of NADPH by GSSG at 300 was

used as a standard measure of enzymatic activity.

The reaction system of 1ml contained: 1.0mM GSSG,

0.1 mM NADPH, 0.5 mM EDTA, 0.10 M sodium

phosphate buffer (pH 7.6), and a suitable amount of

the glutathione reductase sample to give a change in absorbance of 0.05 to 0.03/min. The oxidation of

1 mol of NADPH/min under these conditions is

used as a unit of glutathione reductase activity.

The specific activity is expressed as units per mg

of protein.


19/32

Superoxide dismutase (SOD):

Method-I: Assay mixture contained 0.1ml ofsample,

1.2ml of sodiumpyrophosphate buffer (pH8.3, 0.052 M), 0.1 ml phenazine methosulphate (186 M), 0.3 ml of 300 M nitroblue tetrazolium, 0.2 ml NADH (750 M). Reaction was started by addition of NADH.After incubation at 30Cfor 90 s, the reaction was stopped by the addition of 0.1ml glacial acetic acid. Reaction mixture was stirred vigorously with 4.0ml of n-butanol. Mixture was allowed to stand for 10min, centrifuged and butanol layer was separated


20/32

Color intensity of the chromogen in the butanollayer was measured at 560 nm

spectrophotometrically and concentration of SODwas expressed as units/mg protein.

Method-II : Supernatant was assayed for SOD

activity by following inhibition of pyrogallolautooxidation. Pyrogallol (24mmol/l)wasprepared in 10mmole/l HCl and kept at 40 Cbefore use.Catalase (30 mol/l stock solution)was

prepared in an alkaline buffer pH 9. Aliquots ofsupernatant (150g protein) were added to thisHCL buffer containing 25l pyrogallol and 10 l ofcatalase.


21/32

The final volume of 3ml was made up of the

same buffer.Changes in absorbance at 420 nmwere recorded at 1min interval for 5min. SODactivity was determined from a std. curve of %

inhibition of pyrogallol autooxidation withknown SOD activity. This assay was highlyreproducible, and the standard curve waslinear upto 250 g protein with a correlationcoefficient of 0.998. Data are expressed asSOD units/mg proteins compared withstandard.


22/32


23/32

The rate of the nucleotide oxidation (in thecontrol) and inhibition of the same by the

supernatant was calculated. One unit of the SODactivity was defined as the amount of the enzymerequired to inhibit the rate of NADH oxidation ofthe control by 50%.

Method-IV: The supernatant (50 l) wasadded to 0.75ml of carbonate buffer (100mM, pH10.2)AND10 l OF epinephrine (3mM). Thechange in absorbance of each sample was then

recorded at 480 nm in spectrophotometer for 2min at an interval of 15 sec. Parallel blank andstandard were run for determination SOD activity.


24/32

One unit of SOD is defined as the amount of enzyme required toproduce 50% inhibition of epinephrine autooxidation.

Catalase (CAT):

Method-I: 0.1 ml of supernatant was added to cuvette containing 1.9ml of 50mMphosphate buffer (pH 7.0).Reaction was started by the addition of 1.0

ml of freshly prepared 30 mM H2O2. The rate of decomposition ofH2O2 was measured spectrophotometrically from

changes in absorbance at 240 nm.Activity of catalase was expressed as units/ mg protein.


25/32

Method-II: The estimation was done spectrophotometrically following the decrease in absorbance at 230 nm. The tissue was homogenized in M/150 phosphate buffer (pH 7.0) AT 40C and centrifuged at 50000 rpm . The reaction mixture

contained 0.01 M phosphate buffer (pH 7.0), 2Mm H2O2 and the enzyme extract. The specific activity of catalase is expressed in terms of units/mg protein. A unit is defined as the velocity constant per

Second.


26/32

Glutamyl transpeptidase activity (GGT):

Method: The serum sample was added to asubstrate solution containing glycylglycine, MgCl2and Glutamyl-p-nitroanilide in 0.05Mtris (freebase), pH 8.2. The mixture was incubated at 370Cfor 1 min and the absorbance read at 405 nm at 1min interval for 5min. The activity of GGT wascalculated from the absorbance values.

Lactate Dehydrogenase (LDH): 10% (w/v)homogenates of the myocardium was prepared indistilled water using glass homogenizer.


27/32

(0.1 M sodium lactate), 1.0 ml of 0.66M phosphate

buffer pH 7.4, and 0.5 ml of 0.3 % triphenyl tetrazoniumchloride and 1.0ml of 10%homogenate.

The incubation was carried out at 370C for 45 min in

a water bath. After incubation, the reaction was

stopped by the addition of 6 ml glacial acetic acid. The formazon liberated was extracted in 6 ml of

toluene by keeping mixture over night in a refrigerator.

The toluene layer was then separated and its optical density was read at 505 nm in DU-2 Beckmanns

spectrophotometer. Blood was collected by cardiac

puncture and serum is separated.


28/32

LDH activity of the serum was estimated calorimetrically.

Method -II: The assay mixture consisted of 1.84ml

0f water, 1.o ml of phosphate buffer (Ph 7.4), NADPH to give an absorbance value of 0.5, 0.05 ml

0f supernatant ,and 0.1 ml of pyruate (0.33 nM final concentration). The total volume was 3.o ml; the Reaction was started by adding pyruate last and noting the decrease in A340 at intervals of 30 sec in a BeackmanDBmodel spectrophotometer.The activity of lactate dehydrogenase of each tissue was expressed as units per g of tissue, wet weight .

hi il i i i i


29/32

In this compilation various in vivo

antioxidant

screening methods have beendescribed which are

simple and require only use of

spectrophotometer and

chemicals. The procedures are very

popular and havebeen widely used to evaluate

antioxidant properties.


30/32

REFERENCES

1]. Tappel,A.L.: Fed. Proc. Fed.Am. Soc. Exp. Biol., 32:

1870-1874(1973).

[2]. Sies,H.:Ann.NY.Acad. Sci., 669: 7-20 (1992).

[3]. Langseth, L.:AntioxidantVita.Newslett. 4: 3 (1993). [4]. Halliwell,B.:Lancet., 344: 721-724 (1994).

[5]. Beris,H.:Drugs., 42: 569-605 (1991).

[6]. Jacob,V. andMicaael,A.:Nutrition., 49: 1-747 (1999).

[7]. Ashok,K.J.:Current Science., 1179-1186 (2001). [8]. David,G.B.,Erik,E.A.,Rohini, S. andAlfins :Cancer.,


31/32

89,124-134(2000). [9]. Schrauzer,G.N.:Biological trace element research,

33: 51-62(1992). [10]. Frankel, E.N. and Kanner, J.: Lancet., 341: 455-457 (1993). [11]. Frei, B., Stocker, R., England, L. and Ames, B.N.: Advances inmedicine experiment and biology., 264: 155-163(1990). [12]. Wilhelmina,L.,Charles, F.F.,Antonio,M. andRonald, L.P.: J.Agree. food. chem., 47: 4638-4644(1999). [13]. Hong, W., Guohua, C. and Ronald, L.P.: J. Agree.food.chem., 44: 701-705 (1996).


32/32

THANKS

Documents

Antioxidant Presentation