Antioxidant Tests

7/30/2019 Antioxidant Tests

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ANTIOXIDANT TEST

In biological systems potentially harmful reactive oxygen species (ROS) are produced during

the normal aerobic metabolism. Antioxidants are deployed to prevent generation of ROS or to

scavenge those formed. Deficiency of antioxidative defenses may lead to oxidative stress,

which might be associated with a variety of disorders such as coronary heart diseases, neural

disorders, diabetes, arthritis and cancers. Although organisms are bestowed with antioxidant

and repair systems that have evolved to protect them against oxidative damage, these systems

are insufficient to prevent the damage totally. Hence antioxidants in diet are of great

importance as possible protective agents to help human body to reduce oxidative damage.

Recently a large number of natural antioxidants have been isolated from different plants.

Human diet containing m e d i c i n a l herb possessing antioxidative properties would bepotentially useful to help human body to reduce oxidative damage. Much research has

concentrated on different plant extracts abilities to induce antioxidant effects.

Natural antioxidative compounds from plants have aroused great attention due to concerns

about the safety of synthetic antioxidants. Increasing efforts have been made to search for

plant-derived antioxidants. Many herbals contain antioxidant compounds which protect cells

against the damaging effects of reactive oxygen species, such as singlet oxygen, superoxide,

peroxyl radicals, hydroxyl radicals and peroxynitrite. Overproduction of such free radicals can

cause oxidative damage to biomolecules (e.g. lipids, proteins, DNA), eventually leading to

many chronic diseases, such as atherosclerosis, cancer, diabetes, aging, and other degenerative

diseases in humans. Plants (fruits, vegetables, medicinal herbs, etc.) may contain a wide

variety of free radical scavenging molecules, such as phenolic compounds (e.g. phenolic acids,

flavonoids, quinones, coumarins, lignans, stilbenes, tannins), nitrogen compounds (alkaloids,

amines, betalains), vitamins, terpenoids (including carotenoids), and some other endogenous

metabolites, which are rich in antioxidant.

Free radical formation by oxygen

Oxygen has double-edged properties, being essential for life; it can also aggravate the damage

within the cell by oxidative events. Free radicals and its adverse effects were discovered in

the last decade. These are dangerous substances produced in the body along with toxins and

wastes which are formed during the normal metabolic process of the body. Oxidative stress and

impaired antioxidant system have been implicated in the pathophysiology of diverse disease

states.


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The body obtained energy by the oxidation of carbohydrates, fats and proteins through both

aerobic and anaerobic process leads the generation of free radicals. Over production of the free

radicals can responsible for tissue injury. Cell membranes are made of unsaturated lipids

and these unsaturated lipid molecules of cell membranes are particularly susceptible to free

radicals. Oxidative damage can direct to a breakdown or even hardening of lipids, which

composition of all cell walls. Breakdown or hardening is due to lipid peroxidation leads to

death of cell or it becomes unfeasible for the cell to properly get its nutrients or get signals to

achieve another. In addition, other biological molecules including RNA, DNA and protein

enzymes are also susceptible to oxidative damage.

Reactive oxygen-free radicals (ROS) have been implicated in many diseases and in

aging process. These free radicals, which cause tissue damage via oxidative stress, aregenerated by aerobic respiration, inflammation, and lipid peroxidation. Antioxidant systems

minimize or prevent deleterious effects of the ROS. High concentration of hydrogen peroxide

is deleterious to cells, and its accumulation causes oxidation of cellular targets such as DNA,

proteins, and lipids, leading to mutagenesis and cell death.

Environmental agents also initiate free radical generation leads different complication in

body. The toxicity of lead, pesticides, cadmium, ionizing radiation, alcohol, cigarette smoke,

UV light and pollution may all be due to their free radical initiating capability. A free radical

may defined as a molecule or molecular fragments containing one or more unpaired electrons in

its outermost atomic or molecular orbital and are capable of independent existence.


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Problem of free radical

Free radical stress leads to a wide number of health problems which include tissue injury and

progression of disease conditions such as arthritis, hemorrhagic shock, atherosclerosis, diabetes,

hepatic injury, aging, and ischemia, reperfusion injury of many tissues, gastritis, tumor

promotion, neuron degenerative diseases and carcinogenesis. Safer antioxidants suitable for long

term use are needed to prevent or stop the progression of free radical mediated disorders. Free

radical damage and oxidative stress are the major reasons for liver tissue damage. Lipid

peroxidation is regarded as one of the basic mechanisms of tissue damage caused by free

radicals .The antioxidants are the first line defense against such damage and provide protection

against the deteriorating outcome.

In addition to the antioxidant defense system of our body, antioxidants that are mainly supplied

as dietary consumptions can also impede carcinogenesis by scavenging oxygen radicals or

interfering with the binding of carcinogens to DNA, include vitamin C, vitamin E (-tocopherol,

-tocopherol), -carotenoids (-carotene, -carotene, - cryptoxanthin, lutein, zeaxanthin,

lycopene) and several polyphenolic compounds including flavonoids (catechins, flavonols,

flavones, isoflavonoids).

Several potential plant-derived antioxidants such as quercetin, carnosol, thymol, carnosic acid,

hydroxytyrosol, gallic acid derivatives, tannins, catechins, rutin, morin, ellagic acid, eugenol, and

rosemarinic acids have come to attention because of their extensive use in dietary

supplementation, food preservation, and treating various free radical mediated diseases.

Methods of evaluating antioxidant activity

Antioxidant property of the various fraction of the plant was determined by following methods-

Determination of DPPH radical scavenging assay (Quantitative analysis) Determination of total phenolic content Determination of total antioxidant capacity by phosphomolybdenum method Determination of total flavonoids content

DPPH radical scavenging assay


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DPPH free radical scavenging activity of the plant fractions was determined following the

method described by Braca et al. (2001).

Principle

DPPH (1,1-diphenyl-2-picrylhydrazyl) radical has been widely used to evaluate the free radical

scavenging capacity of antioxidants. DPPH free radical is reduced to the corresponding

hydrazine when it reacts with hydrogen donors. DPPH can generate stable free radicals in

aqueous or methanol solution. With this method it was possible to determine the antiradical

power of an antioxidant by measuring the decrease in the absorbance of DPPH at 517 nm.

Resulting from a color change from purple to yellow the absorbance decreased when the DPPH

was scavenged by an antioxidant, through donation of hydrogen to form a stable DPPH

molecule. In the radical form, this molecule has an absorbance at 517 nm which disappears after

acceptance of an electron or hydrogen radical from an antioxidant compound to become a stable

diamagnetic molecule. When the odd electron of DPPH radical becomes paired with hydrogen

from a free radical scavenging antioxidant to form the reduced DPPH-H, then the color turns

from purple to yellow as the molar absorptive of the DPPH radical reduces from 9660 to 1640 at

517 nm. Scavenging of DPPH free radicals by antioxidants decreases the absorbance.

The DPPH assay was used to measure the level of antioxidants in a substance. DPPH is a wellknown radical and a trap ("scavenger") for other radicals. Therefore, rate reduction of a chemical

reaction upon addition of DPPH is used as an indicator of the radical nature of that reaction.


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Materials & Reagents

1,1-diphenyl-2-picrylhydrazyl

UV- visible spectrophotometer

L-Ascorbic acid

Beaker (100 & 200ml)

Distilled water

Test tube

Methanol

Aluminum foil

Pipette (5ml)

Spatula

Analytical balance

6.1.3. Methods

2.0 ml of a methanol solution of the extract at different concentration (400,200,100, 50,25, 12.5,6.25,3.125,1.5625,0.78125}g/ml were made.

After this all the testtubes were filled up by to 5 ml by mixing 3 ml DPPHsolution

A blank solution was made which contain only DPPH solution of 5 ml(20g/ml).

After 30 min reaction period at room temperature in dark place theabsorbance was measured against at 517 nm against methanol as blank by using

a UV- visible spetrophotometer.

Inhibition free radical DPPH in percent (I%) was calculated as follows: (I%) = (1Asample/Ablank) X 100

Where Ablank is the absorbance of the control reaction (containing all reagents except the


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test material).

Extract concentration providing 50% inhibition (IC50) was calculated fromthe graph plotted inhibition percentage against extract concentration.

L-Ascorbic acid was used as positive control.


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Estimation of Total Phenolic Content

The total phenolic concentration of the extract ofSpondias pinnata skin was determined by

the modified Folin-Ciocalteu method. The process of measuring total phenolic content of the

crude extract of Spondias pinnata fruit involves the use of Folin-Ciocalteu reagent. The

Folin-Ciocalteu reagent is a mixture of phosphomolybdate and phosphotungstate used for

the colorimetric assay of phenolic and polyphenolic antioxidants. The FCR actually

measures a samples reducing capacity. The exact chemical nature of the FC reagent is not

known, but it is believed to contain heteropolyphosphotunstates - molybdates. Sequences of

reversible one- or two-electron reduction reactions lead to blue species, possibly

(PMoW11O40)4-

. It is believed that the molybdenum is easier to be reduced in the complex

and electron- transfer reaction occurs between reductants and Mo (VI):

Mo (VI) + e Mo (V)

It measures the amount of substance being tested needed to inhibit the oxidation of the

Folin-Ciocalteu reagent. The reagent does not contain phenol. Rather, the reagent will react

with phenols and nonphenolic reducing substances to form chromogens that can be detected

spectrophotometrically. The generated chromogens give a strong absorption maximum at

760 nm.

6.2.2.2. Materials & Reagents

Test tube

Analytical balance

Pipette

UV- visible spectrophotometer

Spatula

Vortex

Mixer

Folin-Ciocalteu reagent

Distilledwater


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Sodium carbonate (Na2CO3)

Methanol

Gallic acid

Aluminum foil

Composition of Folin-Ciocalteu Reagent

SL. No. Component Quantity (%)

1 Water 57.5

2 Lithium Sulfate 15.0

3 Sodium Tungstate Dihydrate 10.0

4 Hydrochloric Acid>=25% 10.0

5 Phosphoric Acid 85% solution in water 5.0

6 Molybdic Acid Sodium Dihydrate 2.5

6.2.2.3. Methods

0.5ml of a methanol solution of the crude extract of concentration of 1mg/mlwas mixed with 5ml Folin ciocalteu reagent (1:10 v/v distilled water) and 4 ml

(75g/L) of Sodium carbonate.

The mixture was vortexed for and allowed to stand for 30min in dark place forcolor development and the absorbance was measured at 760 nm against

methanol as blank by using a UV- visible spetrophotometer.

The total phenolics was expressed as gm of GAE (gallic acidequivalent)/100gm of the dried extract using the following equation

obtained from a standard Gallic acid calibration curve

Y=0.0162x+0.0215;R=0.9972.


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Total antioxidant capacity

Principle

The phosphomolybdenum method usually detects antioxidants such as ascorbic acid, some

phenolics, -tocopherol, and carotenoids. The phosphomolybdenum method is based on the

reduction of Mo(VI) to Mo(V) by the antioxidant compound and subsequent formation of a

green phosphate/Mo(V) complex at acid pH. In essence, it is believed that the molybdenum

is easier to reduce in the complex and electron-transfer reaction occurs between

reductants and Mo(VI) and the formation of a greenphosphate/Mo(V) complex with

a maximal absorption at 695 nm.

Mo (VI) + e Mo (V)

Sometimes a correlation analysis is performed between the total phenolic content and total

antioxidant capacity to reveal the correlation. It is obvious that the plant phenolic

compounds contribute to the major antioxidant activity. Based on the absorbance values of

the extract solution, reacted with reagent solution (0.6 M Sulfuric acid,28mM Sodium

Phosphate and 4mM Ammonium molybdate) and compared with the standard solutions of

L-ascorbic acid equivalents, result of the colorimetric analysis of the total antioxidant

capacity is given in table 6.9. Total antioxidant capacity of the sample is expressed as mg of

L-ascorbic acid per gm of dried extract.

Method

1. 300l of each fraction (200 g/ml) was taken in test tubes.

2. 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM

ammonium molybdate) was added into the test tubes.

3. The test tubes were incubated at 950 C for 90 minutes to complete reaction.

4. Then the absorbance of the solution was measured at 695 nm using a

spectrophotometer against blank after cooling to room temperature.


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Assay for total flavonoids concentration

Aluminium chloride colorimetric method was used for determination of total flavonoids

concentration in the samples of S. pinnata fruit skin. Each extract and fraction(0.5 ml,

1:10 gml-1) ) in methanol were separately mixed with 1.5 ml of methanol, 0.1 ml of

10%aluminum chloride, 0.1 ml of 1M potassium acetate and 2.8 ml of distilled water It was

allowed to stand for 30 min at room temperature and the absorbance of the reaction

mixture was measured at 415 nm. Total flavonoids content was determined as mg of

Quercetin equivalent per gram using the equation obtained from a standard Quercetin

calibration curve

Materials and Equipments

Test tubes

UV-2450 spectrophotometer

Test tubeholder

Beaker

Electronic balance

6.5.2. Reagents

Methanol

Aluminumchloride

Potassium acetate

Quercetin

Distilled water

Method

1. 1.0 ml of each fraction (200 g/ml) and standard (quercetin) in different

concentrations were taken in test tubes.

2. 3 ml of methanol was added into the test tubes.


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3. 200 l of 10% aluminium chloride solution was added.

4. 200 l of 1 M potassium acetate solution was added to the mixtures in the test

tubes.

5. Then 5.6 ml of distilled water was added into the test tubes.

6. The test tubes were incubated for 30 minutes at room temperature to complete

reaction.

7. Then the absorbance of the solution was measured at 415 nm using a

spectrophotometer (Shimadzu UV PC-1600) against blank.

8. Methanol was used as the blank.

9. Total flavanoid concentration o f the fractions was expressed as quercetin

equivalents (QE) after calculation using the following equation:

C = (c x V)/m

Where:

C = total flavonoid contents, mg/g plant extract in QE,

c = concentration of quercetin obtained from calibration curve (mg/ml),

V = the volume of the sample solution (ml)

m=weight of the sample(g)

Antioxidant Tests

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