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Total Antioxidant Power and Free Radical

Total Antioxidant Power and Free Radical

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Published by: Dr. Varaprasad Bobbarala on Jul 17, 2009
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06/13/2013

 
INTRODUCTION
Reactive oxygen in particular, free radicalsare considered to induce oxidative damage inbiomolecules and to play an important role in aging,cardiovascular diseases, cancer, and inflammatorydiseases
1-3
. In addition, they are also well known tobe major causers of material degradation and fooddeterioration
4
. Consequently, antioxidants are nowknown to be prospective protective or therapeuticagents. In the past few years, addition of syntheticantioxidants has begun to be restricted because oftheir health risks and toxicity
5
. The importance ofexploiting natural antioxidants from various sourcesand replacing synthetic antioxidants with naturalingredients has attracted increasing attention.Several natural compounds from plants exhibitantioxidant / radical scavenger properties are
Biosciences, Biotechnology Research Asia 
Vol.
5
(2),
 
837-840 (2008)
Total antioxidant power and free radicalscavenging capacity of some medicinal plants
P. KOTESWARA RAO¹, VARAPRASAD BOBBARALA², D. BHASKAR RAO¹,CH. RAVI KIRAN¹, K.V. RAGHAVA RAO¹ and T. RAGHAVA RAO¹
¹Department of Biochemistry, College of Science & Technology,Andhra University Visakhapatnam - 03 (India).²For U Biosciences, A/4A, Park lane Residency, East point colony,Visakhapatnam, A. P - 530 017 (India).(Received: October 28, 2008; Accepted: December 05, 2008)
ABSTRACT
Current research is now directed towards finding naturally occurring antioxidants of plantorigin. The present study was aimed to assess the
In vitro 
antioxidant activity of methanolic leafextracts of some medicinal plants by carrying the determination of Total Antioxidant power (FRAPAssay) and Free radical scavenging capacity (DPPH Assay) and results were compared with wellestablished antioxidants like Ascorbic Acid and Butylated Hydroxy Toluene(BHT). Our results clearlyshowed that all the plants of present study showed potent Total Antioxidant power and Free radicalscavenging capacity. Of all the plants tested, the highest total antioxidant power and free radicalscavenging capacity was shown by
X. mekongensis, B. cylindrica 
and
E. agallocha 
with nearlyequal to antioxidant capacity to stable antioxidants like BHT and Ascorbic acid. This data will beuseful in estimating the total pharmacological and phytochemical properties of these plants.
Key words:
Reactive oxygen species, DPPH Radical,Ascorbic acid Equivalents, Free radical scavengerspreferred because they are safe and environmentalfriendly. It has become clear that the direct freeradical scavenging effect and membrane protectionplay an important role in the action mechanism ofseveral old established drugs. Several herbs andspices have been reported to exhibit antioxidantactivity, including rosemary, sage, thyme, nutmeg,turmeric, white pepper, chili pepper, ginger, andseveral Chinese medicinal plants extracts
6-9
. The aimof the present work was to prove thephytotherapeutical significance of some popularmedicinal plants on the basis of their antioxidantactivity due to their influence on pathological freeradical reactions. The medicinal plants of presentstudy includes
Borreria hispida 
(BH) belongsRubiaceae,
Bruguiera cylindrica 
(BC)
 
and
Ceriops decandra 
(CD) belongs to Rhizophoraceae
, Eugenia bracteata 
(EB) belongs to Myrtaceae,
Excoecaria 
 
agallocha 
(EA) belongs to Euphorbiaceae,
Glycyrrhiza glabra 
(GG) belongs to Fabaceae,
Picrorhiza kurrow 
(PK) belongs to Scrophulariaceae,
Trianthima decandra 
(TD) belongs to Aizoaceae,
Xylocarpus mekongensis 
(XM) belongs toMeliaceae. The reason for selection is that, theseplants have a wide range of applications in traditionalmedicines and are involved in the treatment ofvarious skin diseases, liver disorders, cancer, HIV,cough, asthma, diarrohea, dysentery, ulcers andcontrolling of blood pressure etc,. In addition, someof these plants were also reported to be the potentantioxidants. Hence, in the present study, we madean attempt to study the total antioxidant power andradical scavenging capacity of methanolic extractsof these plants. This data will be used to estimatethe phytochemical and antioxidant intake of the localpopulation and to understand the therapeutic usesof these plants.
MATERIAL AND METHODS
Diphenyl picryl hydrazyl (DPPH) wasobtained from Himedia laboratories Pvt. Ltd,Mumbai, India. Ferric Chloride, 2,4, 6-tri pyridyl-s-triazine, Butylated hydroxy toluene (BHT) wereobtained from Sisco research laboratories Pvt. Ltd.,Mumbai, India. All other chemicals used were ofanalytical grade obtained from commercial sources.
Plant collection and preparation of the extraction
The plants are collected from the near byhilly region, Paderu and coastal region ofVisakhapatnam, Andhra Pradesh, India andauthenticated by Dr. M. Venkayya, Associateprofessor, Dept. of Botany, Andhra University,Visakhapatnam, Andhrapradesh.
 
Plant leaves werecleaned with deionized water and dried at 50ºC for24 hours. The dried plants were ground and thensieved 80mesh. The dried leaves were ground to apowder using a milling machine. The dried powderwas then weighed and extracted with methanol, withreflux on a water bath at 40°C for three consecutivedays. The extracts were filtered and evaporatedunder vacuum to dryness with a rotary evaporatorand then placed in an oven at 60°C until constantweight was obtained and the solutions wereprepared with a concentration 1mg/ml usingmethanol.
Total Antioxidant powerFerric reducing ability of Plasma (FRAP) Assay
The total antioxidant potential of a samplewas determined using the ferric reducing ability ofplasma i.e., FRAP assay as a measure of antioxidantpower by Benzie and Strain
10
. The assay was basedon the reducing power of a compound (antioxidant).A potential antioxidant will reduce the ferric ion(Fe3+) to the ferrous ion (Fe2+); the latter forms ablue complex (Fe2+/TPTZ), which increases theabsorption at 593 nm. Briefly, the FRAP reagent wasprepared by mixing acetate buffer (300µM, pH 3.6),a solution of 10 µM TPTZ in 40 µM HCl, and 20µMFeCl3 at 10:1:1 (v/v/v). The reagent (300 µM) andsample solutions (10 µL) were added to each welland mixed thoroughly. The absorbance was takenat 593 nm after 10 min. Standard curve was preparedusing different concentrations of FeCl
3
. All solutionswere used on the day of preparation. The resultswere corrected for dilution (e.g.1000ml) andexpressed as ascorbic acid equivalents (µmoles/ml)or FRAP units. All determinations were performedin triplicates.
Free radical scavenging capacityDi Phenyl Picryl Hydrazyl radical scavengingassay (DPPH Assay)
The DPPH assay was carried out asdescribed by Cuendet
et al 
., (11). 5.0 ml of DPPHsolution (0.004%) in methanol was added to 50 µlof plant extract. After 30 min of incubation at 37
0
C,the absorbance was read against control at 517 nm.Ascorbic acid and BHT were used as positivecontrols. Percentage of inhibition = (absorbance ofcontrol –absorbance of test / absorbance of control)x100. All determinations were performed in triplicate.
RESULTS AND DISCUSSION
Recently much attention has been focusedon reactive oxygen species and free radicals, whichplay an important role in the genesis of variousdiseases such as inflammation, cataract, livercirrhosis and ischemia/reperfusion injury (12).Herbal drugs containing radical scavengers aregaining importance in the prevention and treatmentof such diseases¹³. Hence the present study isfocused on the determination of Total antioxidantpower by FRAP Assay and DPPH Assay.838Rao
et al.
,
Biosci., Biotech. Res. Asia,
Vol.
5
(2), 837-840 (2008)
 
Fig. 2: Showing the Free radical scavenging capacity (DPPH Assay)Total Antioxidant powerFerric reducing ability of Plasma (FRAP) Assay
The FRAP assay measures the reductionof Fe3+
 
(ferric iron) to Fe2+ (ferrous iron) in thepresence of antioxidants. Because the ferric-to-ferrous ion reduction occurs rapidly with allreductants with half reaction reduction potentialsabove that of Fe3+/Fe2+, the values in the FRAPassay will express the corresponding concentrationof electron-donating antioxidants (10). We electedto use the FRAP analysis for several reasons. TheFRAP assay is the only assay that directly measuresantioxidants or reductants in a sample. The otherassays are indirect because they measure theinhibition of reactive species (free radicals)generated in the reaction mixture, and these resultsalso depend strongly on the type of reactive speciesused. The FRAP assay, in contrast, uses antioxidantsas reductants in a redox-linked colorimetric reaction.Furthermore, the other assays, but not the FRAPassay, use a lag phase type of measurement. Thishas been difficult to standardize in previous
Fig.1: Showing the Total Antioxidant power (FRAP Assay)
Rao
et al.
,
Biosci., Biotech. Res. Asia,
Vol.
5
(2), 837-840 (2008)839

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