In-Vitro Models For Antioxidant Activity Evaluation: A Review

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Antioxidants are gaining a lot of importance as a panacea for a large number
of life-style diseases like aging, cancer, diabetes, cardiovascular and other
degenerative diseases etc. owing to our sedentary way of life and stressful
existence. Added to these are the deleterious effects of pollution and exposure
to harmful chemicals. All the above cause can accumulation of harmful free radicals.

Free radicals are types of Reactive oxygen species (ROS), which include all
highly reactive, oxygen-containing molecules. Types of ROS include the hydroxyl
radical, the super oxide anion radical, hydrogen peroxide, singlet oxygen, nitric
oxide radical, hypochlorite radical, and various lipid peroxides. All these
are capable of reacting with membrane lipids, nucleic acids, proteins and enzymes
and other small molecules, resulting in cellular damage.  In living organisms
various ROSs can be formed in different ways, including normal aerobic respiration,
stimulated polymorphonuclear leukocytes and macrophages and peroxisomes. These
appear to be the main endogenous sources of most of the oxidants produced by
cells. Exogenous sources of free radicals include tobacco smoke, ionizing radiation,
certain pollutants, organic solvents and pesticides.


Free radicals may be defined as chemical species associated with an odd or
unpaired electron. They are neutral, short lived, unstable and highly reactive
to pair up the odd electron and finally achieve stable configuration. They are
capable of attacking the healthy cells of the body, causing them to lose their
structure and function. Cell damage caused by free radicals appears to be a
major contributor to aging and degenerative diseases of aging such as cancer,
cardiovascular disease, cataracts, immune system decline, liver diseases, diabetes
mellitus, inflammation, renal failure, brain dysfunction and stress among others.   


To protect the cells and organ systems of the body against reactive oxygen
species, humans have evolved a highly sophisticated and complex antioxidant
protection system, that functions interactively and synergistically to neutralize
free radicals. Thus, antioxidants are capable of stabilizing or deactivating,
free radicals before they attack cells1. Antioxidants are absolutely
critical for maintaining optimal cellular and systemic health and well-being.


Naturally there is a dynamic balance between the amount of free radicals produced
in the body and antioxidants to scavenge or quench them to protect the body
against deleterious effects. The amount of antioxidant principles present under
normal physiological conditions may be insufficient to neutralize free radicals
generated. Therefore, it is obvious to enrich our diet with antioxidants to
protect against harmful diseases. Hence there has been an increased interest
in the food industry and in preventive medicine in the development of “Natural
antioxidants” from plant materials. That is why plants with antioxidant properties
are becoming more and more popular all over the world.


Considering the importance of this area, we have listed some important in-vitro
models for evaluating antioxidant activity.


{mospagebreak title=In-vitro models for evaluating antioxidant activity}


IN-VITRO MODELS FOR EVALUATING ANTIOXIDANT ACTIVITY


Conjugated diene assay


This method allows dynamic quantification of conjugated dienes as a result
of initial PUFA (Poly unsaturated fatty acids) oxidation by measuring UV absorbance
at 234 nm. The principle of this assay is that during linoleic acid oxidation,
the double bonds are converted into conjugated double bonds, which are characterized
by a strong UV absorption at 234 nm. The activity is expressed in terms of Inhibitory
concentration (IC50)2-4.


DPPH Method (1, 1 diphenyl 2, picryl hydrazyl)


This is the most widely reported method for screening of antioxidant activity of many plant drugs. DPPH assay method is based on the reduction of methanolic solution of colored free radical DPPH by free radical scavenger. The procedure involves measurement of decrease in absorbance of DPPH at its absorption maxima of 516 nm, which is proportional to concentration of free radical scavenger added to DPPH reagent solution. The activity is expressed as effective concentration EC50. 5-7


Super oxide radical scavenging activity


In-vitro super oxide radical scavenging activity is measured by riboflavin/light/NBT
(Nitro blue tetrazolium) reduction. Reduction of NBT is the most popular method.
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 formazon that can be easured at 560nm. The capacity of extracts to inhibit
the colour to 50% is measured in terms of EC50. Antioxidant activity
of Ailanthus, flavanoids and Triphala has been reported in terms of super oxide
radical scavenging activity. The super oxide radical can also be detected by
oxidation of hydroxylamine, yielding nitrite which is measured colorimetric
reaction.8-9


Hydroxyl radical scavenging activity


Hydroxyl radical scavenging capacity of an extract is directly related to its
antioxidant activity. This method involves in-vitro generation of hydroxyl
radicals using Fe3+ /ascorbate/EDTA/H2O2
system using Fenton reaction. Scavenging of this hydroxyl radical in presence
of antioxidant is measured. In one of the methods the hydroxyl radicals formed
by the oxidation is made to react with DMSO (dimethyl sulphoxide) to yield formaldehyde.
Formaldehyde formed produces intense yellow color with Nash reagent (2M ammonium
acetate with 0.05M acetic acid and 0.02M acetyl acetone in distilled water).
The intensity of yellow color formed is measured at 412nm spectrophotometrically
against reagent blank. The activity is expressed as % hydroxyl radical scavenging.8
 


Nitric oxide radical inhibition activity


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 anti oxidant 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.8


Reducing Power Method

This method is based on the principle of increase in the absorbance of the reaction
mixture. Increase in the absorbance indicates increase in the antioxidant activity.
In this method antioxidant compound forms a colored complex with potassium ferricyanide,
trichloro acetic acid and ferric chloride, which is measured at 700nm. Increase
in absorbance of the reaction mixture indicates the reducing power of the samples.10
     


Phospho molybdenum Method

It is a spectroscopic method for the quantitative determination of antioxidant capacity, through the formation of phospho molybdenum complex. The assay is based on the reduction of Mo (VI) to Mo (V) by the sample analyte and subsequent formation of a green phosphate Mo (V) complex at acidic pH. 11


Peroxynitrite radical scavenging activity


Peroxynitrite is now recognized by researchers as the culprit in many toxic reactions that were previously ascribed to its chemical precursors, superoxide and nitric oxide. Hence, an in vitro method for scavenging of peroxy radical has been developed to measure antioxidant activity. The scavenging activity is measured by monitoring the oxidation of dihydrorhodamine on a microplate fluorescence spectrophotometer at 485nm12


ABTS (2,2-azinobis(3-ethyl benzothiazoline-6-sulfonicacid) diamonium salt
)Method


This is a measure of antioxidant activity as opposed to antioxidant concentration
which might include a proportion of biologically inactive antioxidants. It also
permits the measurement of antioxidant activity of mixtures of substances and
hence helps to distinguish between additive and synergistic effects. The antioxidant
activity of wines was measured by using this method. The assay is based on interaction
between antioxidant and ABTS+ radical cation which has a characteristic color
showing maxima at 645, 734 and 815nm.13-16


DMPD (N, N-dimethyl-p-phenylene diamine dihydrochloride) Method


This assay is based on the reduction of buffered solution of colored DMPD in
acetate buffer and ferric chloride. The procedure involves measurement of decrease
in absorbance of DMPD in presence of scavengers at its absorption maxima of
505nm. The antioxidant activity of wines was measured by using this method.
The activity was expressed as percentage reduction of DMPD. 13-16


Oxygen Radical Absorbance Capacity (ORAC)


ORAC is an exciting and revolutionary new test tube analysis that can be utilized to test "Antioxidant Power" of foods and other chemical substances. It calculates the ability of a product or chemical to protect against potentially damaging free radicals. This analytical procedure measures the ability of a food, vitamin, nutritional supplement, or other chemicals to protect against the attack by free radicals, or to act as an Antioxidant. The test is performed using Trolox (a water-soluble analog of Vitamin E) as a standard to determine the Trolox Equivalent (TE). The ORAC value is then calculated from the Trolox Equivalent and expressed as ORAC units or value. The higher the ORAC value, the greater the "Antioxidant Power".


This assay is based on generation of free radical using AAPH (2,2-azobis 2-amido
propane dihydrochloride) and measurement of decrease in fluorescence in presence
of free radical scavengers. Hong et.al., (1996) have reported automated ORAC
assay. In this assay b-phycoerythrin (b-PE) was used as target free radical
damage, AAPH as a peroxy radical generator and Trolox as a standard control.
After addition of AAPH to the test solution, the fluorescence is recorded and
the antioxidant activity is expressed as trolox equivalent. 17-19


b-Carotene Linoleate model
This is one of the rapid method to screen antioxidants, which is mainly based on the principle that Linoleic acid, which is an unsaturated fatty acid, gets oxidized by "Reactive Oxygen Species” (ROS) produced by oxygenated water. 


The products formed will initiate the b-carotene oxidation, which will lead to discoloration. Antioxidants decrease the extent of discoloration, which is measured at 434nm and the activity is measured. 20


Xanthine oxidase Method
This is one of the recent methods for evaluation of anti oxidant activity.21, 22. The percentage inhibition in the xanthine oxidase activity in presence of anti oxidants is measured. Xanthine oxidase enzyme produces uric acid together with super oxide radicals from xanthine and the amount of uric acid is measured at 292nm.  


FRAP Method
FRAP (Ferric Reducing Ability of Plasma) is one of the most rapid test and very useful for routine analysis. The antioxidative activity is estimated by measuring the increase in absorbance caused by the formation of ferrous ions from FRAP reagent containing TPTZ (2,4,6 – tri (2 – pyridyl) – s – triazine) and FeCl36H2O. The absorbance is measured spectrophotometrically at 595nm23.  


TRAP Method
This method is defined as total radical trapping antioxidant parameter. The fluorescence of R-Phycoerythrin is quenched by ABAP (2,2’-azo–bis (2-amidino-propane) hydrochloride) as a radical generator. This quenching reaction is measured in presence of antioxidants. The antioxidative potential is evaluated by measuring the delay in decoloration.24  


Cytochrome C test


Superoxide anions were assayed spectrophotometrically by a cytochrome reduction method described by McCord and Fridovich (1969). Xanthine oxidase converts xanthine to uric acid and yields superoxide anions and these radicals directly reduce ferri-cytochrome C to ferro-cytochrme C, having an absorbance change at 550 nm. When test compounds showed superoxide scavenger activity, there was a decrease in the reduction of ferri-cytochrome C. 25


Erythrocyte ghost system


This method involves isolation of erythrocytes ghost cells and the induction of lipid peroxidation using erythrocyte ghosts and the induction of tetra-butyl hydroxy peroxide (t-BHP). TBARS (thio barbituric acid reactive substance) produced during the reaction is measured at 535 nm.26  


Microsomal lipid peroxidation or Thiobarbituric acid (TBA) assay


TBA test is one of the most frequently used tests for measuring the peroxidation
of lipids. Method involves isolation of microsomes from rat liver and induction
of lipid peroxides with ferric ions leading to the production of small amount
of Malonaldehyde (MDA). TBA reacts with MDA to form a pink chromagen, which
can be detected spectrophotometricaly at 532 nm. 27-28



{mospagebreak title=References}


REFERENCES


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1991, 569-605.


2. Jacob,V. and Michael, A., Nutritional antioxidants: mechanism of action,
analyses of activities and medical applications, Nutrition, 49, 1999,
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3. Ashok, K.J., Imbalance in antioxidant defence and human diseases:Multiple
approach of natural antioxidant therapy, Current Science, 2001, 1179-1186.


4. David, G.B., Erik, E.A., Rohini, S. and Alfins, Antioxidant enzyme expression
and ROS damage in prostatic intraepithelial neoplasia and cancer, Cancer,
89, 2000, 124-134.


5. Vani, T., Rajani, M., Sarkar, S. and Shishoo, C.J., Antioxidant properties
of the ayurvedic formulation triphala and its constituents, Inter. J. Pharmacognosy,
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6. Sanchez-Moreno, C., Larrauri, J. and Saura-Calixto, F., Free radical scavenging
capacity of selected red and white wine, J. Sci.Food. Agric., 79, 1999, 1301-1304.


7. Navarro, M.C…. et al., Free radical scavenger and anti hepatotoxic activity
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8. Babu, B.H., Shylesh, B.S. and Padikkala, J., Antioxidant and hepatoprotective
effect of Alanthus icicifocus, Fitoterapia, 72, 2001, 272-277.


9. Robak, J. and Gryglewski, R.J, Flavonoids are scavengers of superoxide anions,
Biochem. Pharmcol., 37, 1998, 837-841.


10. Jayaprakash, G.K., Singh, R.P. and Sakariah, K.K., Antioxidant activity of grape seed extracts on peroxidation models in-vitro, J.Agric Food Chem., 55, 2001, 1018-1022.


11. Kanner, J…. et al., Natural antioxidants in grapes and wines, J. Agric. Food. Chem., 42, 1994, 64-69.


12. Hye Rhi Choi… et al., Peroxynitrite Scavenging Activity of Herb extracts, Phytother. Res., 16, 2002, 364-367.


13. Rice-Evans, C and Miller, N.J., Total antioxidant status in plasma and body fluids, Methods Enzymol, 243, 1994, 279-293.


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15. Simonetti, P., Pietta, P. and Testolin, G., Polyphenol content and total antioxidant potential selected Italian wines, J. Agric. Food.Chem., 45, 1997, 1152-1155.


16. Vinson, J.A and Hontz, B,A., Phenol antioxidant index: comparative antioxidant effectiveness of red and white wines, J. Agri. Food. Chem., 43, 1995, 401-403.


17. Frei, B., Stocker, R., England, L. and Ames, B.N., Ascorbate the most effective antioxidant in human blood plasma, Advances in medicine experiment and biology, 264, 1990, 155-63.


18. Cao, G., Alessio, H.M. and Culter, R.G., Oxygen radical absorbance capacity assay for antioxidant free radicals, Bio. Med.,14, 1993, 303-311.


19. Ronald, L.P…. et al., Anti oxidant capacity as influenced by total phenolic & anthocyanin content maturity and variety of vaccinum species, J. Agri Food Chem., 46, 1998, 2686-2693.


20. Joseph, K… et al, Natural antioxidants in grapes & wines, J. Agri food chem., 42, 1994.


21. Pieroni, A… et al, In vitro anti oxidant activity of ethnic Albanians in southern Italy, Phytother. Res., 16, 2002, 467-473.


22. Opoku, A.R., Maseko, N.F. and Terblanche, S.E., The in vitro antioxidative activity of some traditional Zulu medicinal plants, Phytother. Res., 16, 2002, S51-S56.


23. Benzie, I.F.F. and Strain, J.J.,The ferric reducing ability of plasma (FRAP) as a measure of ‘antioxidant power’: The FRAP Assay, Anal. Biochem., 239, 1996, 70-76.


24. Ghiselli, A…. et al., A Fluorescence based method for measuring total plasma antioxidant capability, Free Radic. Biol. Med., 18, 1995, 29-36.


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1075-78


26.Chiaki, S and Naomi, O (1998) “Antioxidative polyphenols isolated from Theobroma
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27.Kimura, Y., Okuda, H and Okuda, T (1984) “Studies on activities of tannins
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Acta
: 754: 38-41



{mospagebreak title=About authors}





ABOUT AUTHORS

H.N.Shivaprasad*1, S.Mohan2, M.D.Kharya 3,
Mahendra R Shiradkar4 and K. Lakshman5    

H..N.Shivaprasad*1 is a lecturer in Pharmacognosy &
Phytochemistry at
PES College of Pharmacy
, Bangalore. He has completed his graduation and
post-graduation from Rajiv Gandhi University of Health Sciences, India. He is
the university rank holder at graduate and postgraduate levels. He has worked
as Executive, R & D in Himalaya Drugs, Bangalore. He is doing his Doctoral
[Ph.D.] research at Dr. Hari Singh Gour Vishwavidyalaya, Sagar [M.P]. 
He has several National and International publications to his credit. He has
written many health articles in Health and Fitness section of reputed daily
Vijay Times. He has also written many scientific articles to reputed herbal
websites. He has been awarded as Best presenter in 55th Indian Pharmaceutical
Congress, Chennai. He is the Editor for “Continuing Health Awareness”
a fortnightly bulletin which is circulated among all the PES Group of institutions. 
He is the reviewer for the prestigious PHARMACOGNOSY
JOURNAL
.  His research interest extends from screening of herbal drugs
for various pharmacological activities, Standardization of crude drugs, Phytochemistry
to tissue culture. 

Corresponding author address: Dr. H.N.Shivaprasad  
M.Pharm., [Ph.D.] Department of Pharmacognosy  PES College of Pharmacy,
50 Feet Road,Hanumanthanagar, Bangalore –560 050, IndiaPhone: 09448517387 [M],
080-26507428 [O], 080-26520671 [R]Fax: 080-26507428, E-mail: shivaprasad_hn@rediffmail.com



Dr. S.Mohan2 is a Principal and Professor in Pharmaceutical
chemistry at PES
College of Pharmacy
, Bangalore. He has a vast teaching experience of 30
years.   He has several National and International publications to
his credit. He has guided several M.Pharm and Ph.D. students till date. He had
the privilege to present scientific papers in International Conferences. He
has delivered invited lectures and chaired many scientific sessions in India
and abroad.He was the President of the Association of Pharmaceutical teachers
of India, Karnataka State branch and is a life member of several reputed professional
and academic organizations. He is the Editor-in-chief for “Continuing Health
Awareness”
a fortnightly bulletin which is circulated among all the PES
Group of institutions.   

  


Dr. M.D.Kharya 3 is a professor [Pharmacognosy] and Dean
at Dept. of Pharmaceutical Sciences, Dr. Hari singh Gour Vishwavidyalaya, Sagar
[M.P], India. He is a member of Senate and Chairman, board of studies, Dr. Hari
singh Gour Vishwavidyalaya, Sagar [M.P], India. He has several National and
International publications to his credit. He has more than thirty years of academic
experience. He is a life member of several reputed professional and academic
organizations. He is the President of the Association of Pharmaceutical teachers
of India, Madhya Pradesh. He is the Associate editor for Indian Journal of
Natural Products
.





Mr. Mahendra R Shiradkar 4 is an Asst. Professor
at Dept. of Pharmaceutical Chemistry, AISSMS College of Pharmacy, Pune. He has
several National and International publications to his credit. He is doing his
Doctoral [Ph.D.] research at Bhavnagar University, Gujarat.  


 





Dr. K. Lakshman 5 is an Asst. Professor & HOD, Dept.
of Pharmacognosy, PES College of Pharmacy, Bangalore. He has several National
and International publications to his credit. His research areas includes tissue
culture and screening of herbal drugs for various pharmacological activities
.

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