Oxygen is highly reactive atom that is capable of becoming part of potentially damaging molecule commonly called “free radical.” Free radicals are capable of attacking cells of the body, causing them to lose their structure and function. Free radicals have been implicated in the pathogenesis of at least 50 diseases. Free radial formation is controlled naturally by various compounds known as antioxidants. It is when the ability of antioxidant is limited that this damage can become cumulative and debilitating. Following criteria should be considered while selecting an antioxidant. It should be able to produce desire redox reaction. It should be physiologically and chemically compatible. It should be physiologically inert. It should be non-toxic both in the reduced and oxidized forms. It should be effective in low concentration. It should provide prolonged stability to the formulation.

1. Antioxidants
SUNIL KUMAR
ASSISTANT PROFESSOR
DEPT. OF PHARMACEUTICAL CHEMISTRY
ISF COLLEGE OF PHARMACY
WEBSITE: - WWW.ISFCP.ORG
EMAIL: sunil.medichem@gmail.com
ISF College of Pharmacy, Moga
Ghal Kalan,nGT Road, Moga- 142001, Punjab, INDIA
Internal Quality Assurance Cell - (IQAC)

2. 2
INTRODUCTION
REACTIVE OXYGEN SPECIES (ROS)
SOURCES OF FREE RADICALS
MECHANISM OF CELL DAMAGE
ANTIOXIDANT
NATURAL ANTIOXIDANTS
ANTIOXIDANT PROTECTION SYSTEM
OXIDATIVE STRESS AND HUMAN DISEASE
SCREENING MODEL OF ANTIOXIDANT ACTIVITY
IN VITRO METHODS
IN VIVO METHODS
LIST OFANTIOXIDANT PLANTS
REFERENCES
Content

3. 3
 Oxygen is highly reactive atom that is capable of becoming part
of potentially damaging molecule commonly called “free radical.”
 Free radicals are capable of attacking cells of the body, causing
them to lose their structure and function.
 Free radicals have been implicated in the pathogenesis of at
least 50 diseases.
 Free radial formation is controlled naturally by various compounds
known as antioxidants.
 It is when the ability of antioxidant is limited that this damage can
become cumulative and debilitating.
Introduction

4. 4
Following criteria should be considered while selecting an antioxidant.
1) It should be able to produce desire redox reaction.
2) It should be physiologically and chemically compatible.
3) It should be physiologically inert.
4) It should be non-toxic both in the reduced and oxidized forms.
5) It should be effective in low concentration.
6) It should provide prolonged stability to the formulation.
Selection criteria of antioxidants

5. 5
Free radicals are electrically charged molecules. i.e. they have an unpaired
electron, which cause them to seek out and capture electron form other substance
in order to neutralize themselves.
Although the initial attack cause the free radical to become neutralized, another
free radical is formed in this process, causing a chain reaction is occur.
And until subsequent free radicals are deactivated, thousand of free
radical reaction can occur within second of the initial reaction.
Antioxidants are capable of stabilizing or deactivating, free radical before they
attack cells.

6. 6Reactive oxygen species (ROS)
Reactive oxygen species (ROS) is define as all highly reactive, oxygen-containing
molecules, including free radicals.
All are capable of reacting with membrane lipids, nucleic acids, proteins, enzymes
and other small molecules, resulting in cellular damage.
ROS are generated by a number of pathways. Most of the oxidants produced by cells
occur as:
Normal aerobic metabolism: Approximately 90% of the oxygen utilized by the cell
Oxidative burst from phagocytes (white blood cells)
Xenobiotic metabolism, i.e., detoxification of toxic substances.

7. 7Various ROS and corresponding neutralizing antioxidants
REACTIVE OXYGEN SPECIES NEUTRALIZING ANTIOXIDANTS
Hydroxyl radical Vitamin C, Glutathione, Flavonoids, Lipoic
acid
Superoxide radical Vitamin C, Glutathione, Flavonoids, SOD
Hydrogen peroxide Vitamin C, Glutathione, Beta carotene,
Vitamin E, Flavonoids, Lipoic acid.
Lipid peroxides Beta carotene, Vitamin E,
Flavonoids, Glutathione peroxidase

8. 8Sources of free radicals
INTERNAL SOURCE:
 Mitochondria
 Phagocytes
Xanthine oxidase
Exercise
Inflammation
Ischemia and reperfusion.
EXTERNAL SOURCES:
Cigarette smoke
Radiation
U.V. light
Certain drugs
Reagents and industrial solvents pollution

9. 9Mechanism of cell damage

10. 10
Antioxidant is substance that prevents or slows the breakdown of another
substance by oxygen.
Antioxidants are chemical substances that donate an electron to the free radical
and convert it to a harmless molecule.
Antioxidants are substances used by the body to protect itself from damage
caused by oxidation.
Oxidation is a process that causes damage in our tissues through the work of
free radicals.
An antioxidant is a chemical that prevents the oxidation of other chemicals.
Antioxidant

11. 11Natural antioxidants
Naturally occurring antioxidants differ in their composition, their physical and
chemical properties, their mechanism and site of action.
 Enzymes:- Reduced glutathione (GSH), Superoxide dismutase(SOD),
Catalase(CAT), Glutathione peroxidase(GPx)
 High molecular weight compounds(proteins):- Albumin, Ceruplasmin,
Transferin, Haptoglobin
 Low molecular weight compounds:
a) Lipid soluble antioxidants:- Tocopherol, Carotenoids, Quinine
and Some Polyphenols,
b) Water soluble antioxidants:- Ascorbic acid, Uric acid and Some
Polyphenols.
 Minerals:- Selenium, Manganese, Copper, Zinc
 Vitamins:-VitaminA, C, and E.
 Plants antioxidants:- Soyabean, Citrus peel, Sesame seed, Grapes and
wines, Tomato, Orange, Apple, Ashwagandha, Carrot,
Liquorice, Amla.

12. 12
Endogenous Antioxidants
 Bilirubin, Thiols, e.g., glutathione, lipoic acid, N-acetyl cysteine, NADPH and
NADH, Uric acid
 Enzymes:
– Copper/Zinc and manganese-dependent super oxide dismutase (SOD)
– Iron-dependent catalase
– Selenium-dependent glutathione peroxidase
Dietary Antioxidants
Vitamin C, Vitamin E, Beta carotene and other carotenoids and oxycarotenoids,
e.g., Lycopene and lutein
 Polyphenols, e.g. flavonoids, flavones, flavonols, and proanthocyanidins
Metal Binding Proteins
Albumin (copper), Ceruloplasmin (copper), Metallothionein (copper), Ferritin
(iron), Myoglobin (iron), Transferrin (iron)
Antioxidant protection system

13. 13Oxidative stress and human disease

14. 14Major groups of antioxidant 20 metabolites
Important groups of antioxidant activity are
Phenol
Phenolic acids
Anthocyanins
Flavones
Flavonoids
Flavonols
Tannins
Isoflavanoids
These group of compound show antioxidant activity & plant defense mechanisms
against pathogenic micro-oranism.

15. 15Antioxidant activity IN VITRO METHODS
In vitro methods divided into two major groups.
HYDROGEN ATOM TRANSFER REACTION :
Oxygen radical absorbance capacity (ORAC)
Total radical trapping antioxidant potential (TRAP)
β-carotene bleaching.
ELECTRON TRANSFER REACTION :
Trolox equivalent antioxidant capacity (TEAC)
Ferric reducing antioxidant power (FRAP)
α, α,-diphenyl -β-picryl-hydrazyl redical scavenging assay (DPPH)
Superoxide anion radical scavenging assay
Hydrogen radical Scavenging assay
Nitric oxide radical scavenging assay
Total phenol assay

16. 16(1-1-DIPHENYL-2-PICRYL-HYDRAZYL) DPPH
DPPH is a molecule containing a stable free radical.
This method is based on the reduction of DPPH in methanol solution in the presence
of a hydrogen-donating antioxidant due to the formation of the non radical from
DPPH-H.
This transformation results in color change from purple to yellow, which is
measured spectrophotometrically.
DPPH is reduced by donating hydrogen to the antioxidant molecule and resulting in
the absorbance decrease.
The decrease in the absorbance is directly proportional to the radical scavenging
activity

17. 17Mechanism
Free radical scavenging potentials of the extracts were tested against a methanolic
solution of 1, 1-diphenyl-2-picryl hydroxyl (DPPH).
 Antioxidants react with DPPH and convert it to 1-1-diphenyl -2-picryl hydrazine .
The degree of discoloration indicates the scavenging potential of the antioxidant
extract. The change in the absorbance produced at 517nm has been used as a measure
of antioxidant activity.

18. 18Procedure
The reaction mixture (3.0 ml) consist of 1.0ml of DPPH in methanol (0.3 mM), 1.0
ml of the extract and 1.0 ml of methanol.
It is incubated for 10 min in dark, then the absorbance is
measured at 517 nm.
Positive controls: Ascorbic acid, Gallic acid, BHA, α-tocopherol,
Quercetin, BHT, Rutin, Catechin.
The percentage of inhibition can be calculated using the formula:
Inhibition (%) = (A0 – A1 / A0) × 100
Where : A0 :- Absorbance of control
A1:- Absorbance of test.

19. 19
Plant polyphenols, a diverse group of phenolic compounds (flavanols, flavonols,
anthocyanins, phenolic acids, etc.) possess an ideal structural chemistry for free
radical scavenging activity.
Antioxidative properties of polyphenols arise from their high reactivity as hydrogen
or electron donors from the ability of the polyphenol derived radical to stabilize and
delocalize the unpaired electron (chain-breaking function) and from their potential to
chelate metal ions (termination of the Fenton reaction)
The amount of total phenol content can be determined by Folin-Ciocateu reagent
method
Total phenolic content

20. 20Procedure
0.5 ml of extract and 0.1 ml of Folin-Ciocalteu reagent (0.5 N) are mixed and
incubated at room temperature for 15 min.
Then 2.5 ml saturated sodium carbonate(2%) is added and further incubated for 30
min at room temperature and absorbance measured at 760 nm.
Positive control:- Gallic acid , Tannic acid, Quercetin,
Chlorogenic acid, Pyrocatechol.
The total phenolic content is expressed in terms of standard
equivalent (mgg -1 of extracted compound).

21. 21Superoxide anion radical scavenging (so)
Superoxide anion is a weak oxidant, it gives rise to generation of
powerful and dangerous hydroxyl radicals
In-vitro super oxide radical scavenging activity is measured by
riboflavin/NBT (Nitro blue tetrazolium) reduction.
The method is based on generation of super oxide radical by auto oxidation of
riboflavin in presence of light.
The super oxide radical reduces NBT to a blue colored formazan that can be measured
at 560nm.
The capacity of extracts to inhibit the colour to 50% is measured
in terms of EC50

22. 22Procedure
The superoxide anion radicals are generated in 3.0 ml of Tris-HCl buffer (0.02 M, pH
8.3), containing 0.5 ml of NBT (0.3mM), 0.5 ml NADH (0.936 mM) solution, 1.0 ml
extract and 0.5 ml Tris-HCl buffer (0.02 M, pH 8.3).
The reactionis started by adding 0.5 ml phenazine methosulfate-nicotinamide adenine
dinucleotide (PMS-NADH) solution (0.12 mM) to the mixture, incubated at 25°C for 5
min and then the absorbance is measured at 560 nm against a blank sample.
Positive control: Gallic acid, BHA, Ascorbic acid,
α-tocopherol, Curcumin,
Quercetin or Trolox.

23. 23Hydrogen peroxide radical scavenging
Hydrogen peroxide occurs naturally at low concentration levels in the air, water,
human body, plants, microorganisms, food and beverages.
Human beings exposed to H2O2 indirectly via the environment are estimated as
0.28 mg/kg/day with intake from leaf crops contributing most to this exposure.
 Hydrogen peroxide enters the human body through inhalation of vapour through
eye or skin contact.
In the body, H2O2 is rapidly decomposed into oxygen and water and this may
produce hydroxyl radicals (OH˙) that can initiate lipid peroxidation and cause DNA
damage.

24. 24Procedure
A solution of hydrogen peroxide (40 mM) is prepared in phosphate buffer (50mM, pH
7.4).
The concentration of hydrogen peroxide is determined by absorption at 230 nm using
a spectrophotometer.
Extract(20 - 60 µg/ml) in distilled water is added to hydrogen peroxide and absorbance
at 230 nm is determined after10 min against a blank solution containing phosphate
buffer without hydrogen peroxide.
The percentage of hydrogen peroxide scavenging is calculated as follows:
% Scavenged (H2O2) = (A0 – A1 / A0) X 100
 Where: A0 is the absorbance of control and
A1 is the absorbance of test.
Positive control: Ascorbic acid, Rutin, BHA, α–tocopherol,
Quercetin.

25. 25Nitric oxide radical scavenging (no)
Nitric oxide, because of its unpaired electron, is classified as a free radical and
displays important reactivity’s with certain types of proteins and other free radicals.
 In vitro inhibition of nitric oxide radical is also a measure of antioxidant activity.
This method is based on the inhibition of nitric oxide radical generated from
sodium nitroprusside in buffer saline and measured by Griess reagent.
 In presence of scavengers, the absorbance of the chromophore is evaluated at
546nm.
The activity is expressed as % reduction of nitric oxide.

26. 26Procedure
3.0 ml of 10mM sodium nitroprusside in phosphate buffer (pH-7.5) is added to 2.0
ml of extract and reference compound in different concentrations
(20 – 100 µg/ml).
The resulting solutions are then incubated at 25°C for 60 min
A similar procedure is repeated with methanol as blank, which serves as control.
To 5.0 ml of the incubated sample, 5.0ml of Griess reagent (1% sulphanilamide,
0.1% naphthyethylene diamine dihydrochloride (NED) in 2% H3PO3) is added and
pink chromophore generate during diazotization of nitrite ions with sulphanilamide
and subsequent coupling with NED was measured spectrophotometrically at 540nm.
Percent inhibition of the nitrite oxide generated is measured by comparing the
absorbance values of control and test preparations.
POSITIVE CONTROL: Curcumin, Caffeic acid, Sodium nitrite, BHA,
Ascorbic acid, Rutin, BHT or α-tocopherol

27. 27In vivo screening methods of antioxidant activity
Various in-vivo methods are used to evaluate the ability of antioxidant to reduce
the radical. Antioxidant activity can be measured using both serum sample as well
as tissue homogenate.
The different in vivo antioxidant screening methods
Lipid peroxidation (LPO)
Reduced glutathione (GSH)
Glutathione peroxidase (GPx)
Glutathione reductase (GR)
Glutathione-s-transferase (GST)
Superoxide dismutase (SOD)
Catalase (CAT)
Total protein
Lactate dehydrogenase (LDH)
Ornithine decarboxylase
Glucose 6 phosphatase (G6P)
Creatinine phosphokinase(CPK)

28. 28Lipid peroxidation
LPO is an autocatalytic process, which is a common cause of cell death.
This process may cause peroxidative tissue damage in inflammation, cancer and
toxicity of xenobiotics and aging.
MDA is one of the end products in the lipid per-oxidation process .
Malondialdehyde (MDA) is formed during oxidative degeneration as a product of
free oxygen radicals. Which is accepted as an indicator of lipid peroxidation.

29. 29Procedure
The reaction mixture contained 0.1 ml of sample, 0.2 ml of 8.1% sodium
dodecyl sulfate (SDS), 1.5 ml of 20% acetic acid solution of various pHs, and 1.5 ml
of 0.8% aqueous solution of thiobarbituric acid (TBA).
The pH of 20% acetic acid solution was adjusted with NaOH above pH 3.0, and in
the pH range of 1.0-3.0, 20% acetic acid containing 0.27 M HCl was
adjusted to the specified pH with NaOH.
The mixture was finally made up to 4.0 ml with distilled water, and heated at 95°C
for 60 min.
After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of the mixture of n-
butanol and pyridine (15: 1, v/v) were added, and the mixture was shaken vigorously.
After centrifugation at 4000 rpm for 10 min, the absorbance of the organic
layer (upper layer) was measured at 532 nm against blank without the sample.
The level of lipid peroxidase were expressed as thiobarbituric
acid reactive substance (TBARS)/mg protein.

30. 30Reduce glutathiol
If deficiency of GSH within living organisms can lead to tissue disorder and injury.
GSH is an intra-cellular reductant and plays major role in catalysis, metabolism and
transport.
It protects cells against free radicals, peroxides and other toxic compounds.
Glutathione was also implicated in diseases like cataract and aminoaciduria.
Deficiency of GSH leads to cataract formation in mice and rats.
Glutathione also plays an important role in the kidney and takes part in a transport
system involved in the reabsorption of amino acids.
 Thus GSH monoesters are also useful in the treatment of aminoacidouria.

31. 31Procedure
To measure the GSH level, the tissue homogenate (in 0.1 M phosphate buffer pH 7.4)
was taken.
The homogenate was added with equal volume of 20% trichloroacetic acid (TCA)
containing 1 mM EDTA to precipitate the tissue proteins.
The mixture was centrifugation for 10 min at 200 rpm allowed to stand for 5 min.
The supernatant (200 μl) was then transferred to a new set of test tubes and added
1.8ml of the 5, 5'-dithiobis-2-nitrobenzoic acid (0.1mM) was prepared in 0.3 M
phosphate buffer with 1% of sodium citrate solution).
Then all the test tubes make upto the volume of 2ml with double distilled water.
After completion of the total reaction, solutions were measured at 412 nm against
blank.
Absorbance values were compared with a standard curve generated from standard
curve from known GSH.

32. 32Superoxide dismutase
SOD is a metalloprotein and is the first enzyme involved in the antioxidant defence
by lowering the steady-state level of O2.
Procedure :
Assay mixture contained 0.1ml of sample, 1.2ml of sodium pyrophosphate buffer
(pH 8.3, 0.052 M), 0.1 ml phenazine methosulphate (186 μM), 0.3 ml of 300 μM
nitroblue tetrazolium, 0.2 ml NADH (750 μM).
 Reaction was started by addition of NADH.
After incubation at 30°C for 90 s, then addition of 0.1ml glacial acetic acid to stop
reaction.
Reaction mixture was stirred vigorously with 4.0ml of n-butanol.
Mixture was allowed to stand for 10min, centrifuged and butanol layer was
separated.
Colour intensity of the chromogen in the butanol layer was measured at 560 nm
spectrophotometrically and concentration of SOD was expressed as units/mg.

33. 33Catalase
CAT is a hemeprotein, localized in the peroxisomes or the microperoxisomes.
This enzyme catalyses the decomposition of H2O2 to water and oxygen and thus
protecting the cell from oxidative damage by H2O2 and OH.
CAT is a key component of the antioxidant defense system.
Inhibition of these protective mechanisms results in enhanced sensitivity to free
radical-induced cellular damage

34. 34Procedure
0.1 ml of supernatant was added to cuvette containing 1.9 ml of 50 mM phosphate
buffer (pH 7.0)
Reaction was started by the addition of 1.0 ml of freshly prepared 30 mM H2O2
The rate of decomposition of H2O2 was measured spectrophotometrically from
changes in absorbance at 240 nm.
Absorbance is calculated was expressed as units/mg protein.

35. 35
 To counter the harmful effects of free radicals, antioxidant defense mechanism
operates to detoxify or scavenge these free radicals.
 By destroying free radicals and reducing cellular damage antioxidant as a group
can: Promote eye health and prevent macular degeneration, cataract and other
degenerative eye diseases.
 Boost up immune system.
 Prevent neurodegenerative disorders.
 Prevent DNA damage and there for have anticarcinogenic effect.
 Promotes cardiovascular health.
 Antioxidants can decrease LDL and cholesterol, increase high density lipoprotein
HDL, and lower blood pressure
Biological role of antioxidants

36. 36
ALZHEIMER'S
CATARACT
DIABETES
 THROMBOSIS
 ASTHMA
EXERCISE AND ANTIOXIDANTS
PREVENTING SKIN AGEING
PARKINSON’S DISEASE
Therapeutic uses of antioxidants

37. 37
Reactive O2
+
O2-Derived
Free Radicals
Oxidized bases
in DNA
Normal cell
Lipid peroxidation
in cell membranes
Cell repair
Exposure of DNA
Cell Death
Lipid Peroxides
DNA Repair
Normal Cell
Mutations Cell Death
No Repair
Compensatory
Cellular
Hypoproliferation
Less regulation of cell
growth & differentiation
Cell Hyperproliferation
Exposure of DNA
Further Mutations
Antioxidant
Scavengers &
Enzyme Systems
Role of antioxidants in carcinogenesis

38. 38
The imbalance between free radicals and defense systems may increase the
oxidative burden and lead to the damage of macromolecules. Such processes are
thought to play a role in pathological processes of various diseases.
 Antioxidants have an important role in biological system and their use is
implicated in the prevention of various diseases.
 When the normal level of antioxidants defense mechanism is not sufficient for the
eradication of free radical induced injury, administration of antioxidants have a
potential role to play.
It has shown that proper intake of antioxidant will help quench all these inevitably
free radicals in the body thus, improving the health by lowering the risk of various
diseases .
 The FDA has been previewed therefore, people will have lower health risks and
tend to live longer and have fewer disabilities.
Conclusion

39. 39References
 S. Chanda and R. Dave. In vitro models for antioxidant activity evaluation and some
medicinal plants possessing antioxidant properties: An overview. African Journal of
Microbiology Research 2009 Dec 3(13):981-996.
 Rice-Evans C, Miller N, Paganga G. Antioxidant properties of phenolic compounds.
Trends Plant Sci. 1997 2:152-159.
 McDonald S, Prenzler PD, Antolovich M, Robards K. Phenolic content and antioxidant
activity of olive extracts. Food Chem. 2001 73:73-84.
 Athukorala Y, Kim KN, Jeon YJ. Antiproliferative and antioxidant properties of an
enzymatic hydrolysate from brown alga Ecklonia cava. Food Chem. Toxicol. 2006
44:1065-1074.

40. 40
1. Discussion session
2. Questions and feedback
3. Assignment

41. 41
Thank you