Research Article Antioxidative Evaluation of -and from Crude Palm Oildownloads.hindawi.com/journals/jamc/2013/351671.pdf · 2019-07-31 · In Vitro Antioxidative Evaluation of -and

Hindawi Publishing CorporationJournal of Analytical Methods in ChemistryVolume 2013 Article ID 351671 10 pageshttpdxdoiorg1011552013351671

Research ArticleIn Vitro Antioxidative Evaluation of 120572- and 120573-Carotene Isolatedfrom Crude Palm Oil

Surashree Sen Gupta1 and Mahua Ghosh12

1 Department of Chemical Technology University of Calcutta Kolkata West Bengal 700 009 India2Department of Chemical Technology University College of Science amp Technology University of Calcutta 92 APC RoadKolkata 700009 India

Correspondence should be addressed to Mahua Ghosh mahuaggmailcom

Received 22 May 2013 Revised 6 September 2013 Accepted 24 September 2013

Academic Editor Jian Yang

Copyright copy 2013 S Sen Gupta and M Ghosh This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The present work describes the isolation of 120572- and 120573-carotene from crude palm oil and their antioxidant potential in an in vitromodel Pure product was isolated by the method adopted Antioxidant activities of the isolated 120572- and 120573-carotene were analyzed infive different concentrations of 0001 0005 001 005 and 01 (wv) From the several assays conducted an observation wasmadethat the antioxidant activity of the product shifted between antioxidant and prooxidant effects depending on the concentration andthe system analyzed The metal chelation DPPH radical scavenging and superoxide scavenging activities showed almost similarresults in terms of high activity at lowest concentrations ABTS-scavenging activity was displayed only by a particular antioxidantconcentration of 01 Lipid peroxidation assay pronounced the activity of 01 antioxidant in inhibiting oxidation of sensitivebioactive lipids In vitro antidenaturation test again specified the efficacy of low concentrations in preventing protein denaturationThrough this study a definite dosage formulation for consumption of carotenoids is being proposed which will enhance healthpromotion and prevent chronic diseases when taken as fortified foods or dietary supplements

1 Introduction

120572- and 120573-carotene are important members of the carotenoidfamily As a retinol precursor with a high conversion rate120572- and 120573-carotene provide a substantial proportion of thevitamin A in the human diet [1] Recently some researchershave shown that 120572- and 120573-carotene due to their antioxi-dant activities possess important health-promoting activitieswhich might be helpful in the prevention and protectionagainst a number of serious health disorders such as cancercardiovascular disease and colorectal adenomas [2] Forthese reasons there is a strong interest in using 120572- and 120573-carotene and other carotenoids as functional ingredients infood products

Crude palm oil has a significant amount of carotene thatcan be extracted and in recent years various methods forextracting carotenes from palm oil have been developedOf these the method involving the combination of trans-esterification and molecular distillation processes is the most

cost-effective one with ease of execution [3] and this wasfollowed in this study

A number of studies have shown that 120572- and 120573-caroteneand other carotenoids have lipid-soluble antioxidant activityIn homogeneous lipid solutions in membrane models andalso in intact cells 120572- and 120573-carotene have been mostlystudied [4 5] Recent findings suggest that the positionand orientation of the carotenoids in the membrane areimportant factors in determining their relative effectivenessin protecting against free radicals [6] 120572- and 120573-carotenepartially or completely protect intact cells (eg human livercell line HepG2) against oxidant-induced lipid peroxidationand the protective effect is independent of provitamin Aactivity [4] 120572- and 120573-carotene suppressed lipid peroxidationin mouse and rat tissues [5] The consumption of food-basedantioxidants like 120572- and 120573-carotene seems to be useful for theprevention ofmacular degeneration and cataracts [7] A well-known fact is that 120572- and 120573-carotene quench singlet oxygenwith higher efficiency than many other antioxidants [8]

2 Journal of Analytical Methods in Chemistry

In contrast to the physiologically relevant properties theknowledge on antioxidant potential of 120572- and 120573-carotene invitro is scarce as most studies conducted so far were in vivoHence the aim of the present study was to isolate 120572- and 120573-carotene from crude palm oil their characterization and invitro investigation of their antioxidative efficacy

2 Experimental

21 Materials Crude palm oil imported from Indonesiawas collected from local Vanaspati manufacturer All otherreagents were of analytical grade and procured from MerckIndia Ltd Mumbai India

22 Isolation of 120572- and 120573-Carotene from Crude Palm OilCrude palm oil was chemically transesterified with methanoland sodium hydroxide Oil and alcohol were taken in dif-ferent molar ratios and stirred while maintaining a hightemperature for a definite time interval [3] Syntheses of esterswere monitored by thin layer chromatography The effectsof different reaction parameters for this particular reactionsuch as temperature substratemolar ratio and concentrationof alkali were optimized with individual sets of reactionmixture Oil methanol ratio of 1 10 on weight basis stirringrate of 200 rpm temperature of 80∘C for 3 hours and aconcentration of 02 NaOH were optimised for maximumyield of 120572- and 120573-carotene A two-phase mixture comprisinga glycerol phase and an ester phase consisting of fatty estersand the carotenewas obtainedThe ester phasewas distilled at300∘C at 10minus01mbar pressure in a molecular distillation unit(SIBATA Japan serial no N40394) The residue was diluted5 times and saponified with ethanolic potassium hydroxide at80∘CThe carotene was extracted from the saponified residuewith a mixture of hexane and water where the extract phasewas rich in carotene Pure carotene was obtained on solvent(hexane) removal which was crystallized and recrystallizedwith acetone

23 Spectrophotometric Analysis For spectrophotometricanalysis 001 g measured amount of the crystallized caroteneproduct was taken in a calibrated test tube and dissolvedinto 25mL of petroleum ether The solution was scannedunder UV-visible spectrophotometer (UV-1700 PharmaSpecUV-VIS Spectrophotometer Shimadzu Japan) between 300ndash600 nm The 120582max value was noted and the molar extinctioncoefficient (120576max) using 1 cm cell was calculated for definiteconcentration of the sample Further spectral conformationwas carried out by scanning the solid carotene crystals underthe UV-visible range of 200ndash800 nm with UV spectropho-tometer (U-3501 Hitachi Japan)

24 Characterisation by Thin Layer Chromatography (TLC)Crystalline samples were dissolved in petroleum ether andspotted in glass TLC plates coated with silica gel stationaryphase The TLC plate was placed in a chamber saturatedwith solvent vapour The solvent system consisted of 60petroleum ether20 acetone20 dichloromethane [9]After development of the plates they were visualized by

subjecting them to iodine vapour The 119877119891values for the spot

on the TLC plate were calculated

119877119891=

distance from baseline to sample frontdistance from baseline to solvent front

(1)

25 Characterisation by High Performance Liquid Chromatog-raphy (HPLC) Analysis of the carotenoids was conductedusing Waters HPLC (Milford Massachusetts US) with aWaters 1525 binary HPLC pump consisting of a vacuumdegasser and a Waters 2487 dual 120582 absorbance detectorReverse-phase HPLC equipped with Nova-pak C

18column

39 times 150mm 4 120583m was used for the analysis of the antiox-idant The UV detector was set at 450 nm The mobilephase consisted of acetonitrile methanol tetrahydrofuran(50 45 5 by volume) [10] The crystallized carotenoids weredissolved in 5mL of the mobile phase The mobile phase wasisocratic solvent Injection volume of 200120583L and the flow ratewere set at 1mLmin Data acquisition was completed in 40minutes

26 Antioxidant Assay of Isolated Carotene Crystals Theantioxidant activity of the isolated carotenoids was examinedby seven different in vitro assay systems Different concen-trations of the antioxidant were prepared in ethanol Theconcentrations were 0001 0005 001 005 and 01 (wv)Their antioxidant activities were measured consecutively

261 Assay of DPPH Radical-Scavenging Activity Antioxi-dant activity of 120572- and 120573-carotene was determined by thescavenging activity of the stable DPPH free radical Themethod was described by Katerere and Eloff [11] Differentconcentrations of the test samples were placed into test tubestaking 02mL from each of them with 4mL of 02mMof ethanolic solution of DPPH Absorbance at 517 nm wasdetermined after 40 minutes using a solution of ethanoland DPPH (3 1) as control Radical scavenging activity wasexpressed as the inhibition percentage and was calculatedusing the following formulae

Radical scavenging activity = [1198600minus 1198601

1198600

] times 100 (2)

where 1198600is the absorbance of the control and 119860

1is the

absorbance of the sample

262 Assay of Reductive Potential The reductive potential ofthe antioxidant was determined according to the method ofDorman and Hiltunen [12] The reaction mixture containingdifferent concentrations of the antioxidants (50ndash250120583gmL)in 1mL of ethanol phosphate buffer (25mL 02M pH66) and potassium ferricyanide (25mL 1 wtvol) wasincubated at 50∘C for 20 minutes A portion (25mL) oftrichloroacetic acid (10 wtvol) was added to the mixturewhich was then allowed to rest for 10 minutes From themixture 25mL was taken and mixed with distilled water(25mL) and FeCl

3(05mL 01 wtvol) and the absorbance

was measured at 700 nm in a spectrophotometer Higherabsorbance of the reaction mixture indicated greater reduc-tive potential

Journal of Analytical Methods in Chemistry 3

263 Assay of Metal Chelating Activity The Fe2+ chelatingability of antioxidants was estimated by the method ofDinis et al [13] Samples with concentrations of about 50ndash250120583gmL were added to 008mL of FeCl

2(25mmolL)

solution To the system 03mL of ferrozine (6mmolL) solu-tion was added and the mixture was shaken vigorously andleft to stand at room temperature for 10 minutes Absorbancewas thereby measured spectrophotometrically at 562 nmPercentage of inhibition of ferrozine-Fe2+ complex formationwas calculated as follows

inhibition = [1198600minus 1198601

1198600

] times 100 (3)


1is the


264 ABTS Radical Scavenging Activity ABTS radical cationscavenging activity of flaxseed lignan was assessed by themethod of Re et al [14] and Zhao et al [15] with someminor modifications Measured volume of 5mL of 7mMABTS was mixed with 88120583L of 140mM K

2S2O8and kept

overnight Next day the solution was diluted with 50ethanol and an initial absorbance of 07 was set at 734 nmThen 1mL of diluted ABTS was mixed with 20120583L of sampleand absorbance wasmeasured at 734 nm at 1-second intervalsfor 5 minutes

265 Lipid Oxidation in a Linoleic Acid Emulsion Model Sys-tem The ammonium thiocyanate method was used to assessthe antioxidant activity of flaxseed lignans with some minormodifications of the procedure of Gulcin [16] A linoleicacid preemulsion was made by vortexing 028 g of linoleicacid with 028 g of Tween 20 in 50mL of 005120583L phosphatebuffer (pH 74) Working solutions of SDG (02mL) wereadded and mixed with 25mL linoleic acid emulsion and23mL phosphate buffer (02 120583L) vortexed and incubatedat 37∘C overnight Aliquots (100 120583L) from the incubatedmixture were withdrawn at 1 24 48 72 96 and 120 hours ofincubation and tested for lipid peroxidation by adding 5mLof ethanol (75) 01mL of NH

4SCN (30 wv) and 01mL

of FeCl2(01 wv) The absorbance of the reaction mixture

was measured at 500 nm against ethanol


1198600

] times 100 (4)


1is the


266 Superoxide Anion Scavenging Activity Superoxideanion scavenging activity of flaxseed lignan was based onthe method described by Yaping et al [17] and Wang et al[18] with some minor modifications About 45mL of Tris-HCl buffer (50mmolL pH 82) and 10mL tested sampleswith various concentrations were mixed in tubes with lidsThen the mixture was incubated for 20min in the waterbath at 25∘C Meanwhile 04mL of 25mmolL pyrogallolpreheated at 25∘C was added immediately After 4min

the reactionwas terminated by 01mLHCl solution (8molL)and the mixture was centrifuged at 4000 rpm for 15minThe absorbance of sample and control was determined byUV spectrophotometer at 325 nm Scavenging activity wascalculated using the following equation

Superoxide anion scavenging activity () = (1198600 minus 119860119904)1198600

times 100

(5)

where 1198600 is the absorbance without sample and 119860119904 isabsorbance with sample

267 Assay of In Vitro Anti-denaturation Effects The assayhelps to assess the anti-denaturationanti-inflammatoryeffect of proteins The method is based on the works ofWilliams [19] An amount of 25mL 1 BSA was mixed with25mL tris acetate buffer (005M) and 25mL of the testsolutionsThemixtures were heated at 69∘C for 4minutes andcooled and then the absorbances of the turbidities were readat 660 nm

inhibition of denaturation = (1198600 minus 119860119904)1198600

times 100 (6)

where 1198600 is the absorbance of control and 119860119904 is absorbancewith sample

27 Statistical Analysis Statistical analysis was performedwith one-way analysis of variance (ANOVA) When ANOVAdetected significant differences between mean values meanswere compared using Tukeyrsquos test For statistical stud-ies OriginLab software (Origin7 OriginLab CorporationNorthampton UK) was used Statistical significance wasdesignated as 119875 lt 005 Three replications for each of theexperiments and assays were conducted (119899 = 3) A meanof the three values was reported in each case The values areexpressed as Mean plusmn SEM

3 Results and Discussions

31 Isolation Spectroscopic Analysis and TLC of 120573-CaroteneIn the present work the palm oil was initially transesterifiedand then concentrated by distillation for isolating 120572- and120573-carotene Amount of the carotene crystals obtained was12 Spectroscopic studies showed maximum absorptionat 440 nm (Figure 1(a)) and a minor absorbance peak at4666 nm (Figure 1(b)) The peak at 440 nm corresponds tothe absorbance by 120573-carotene as confirmed by calculation ofthe molar extinction coefficient (120576max)

120576max =119860

119862119871

(7)

where119860 is absorbance of the specifiedmolecule at maximumwavelength (120582max) 119862 is concentration of the active moleculeand 119871 is distance (1 cm)

The extinction coefficient was calculated to be 1 28300 Lmolminus1 cmminus1 which is in correlation with established


Spectrum150 A

(01

00d

iv)

100 A

4400 nm 1287 A

5000 nm3700 nm(20div)

(a)

Spectrum150 A

(01

00d

iv)

100 A

4666 nm 1033 A

5000 nm3700 nm(20div)

(b)

200 300 400 500 600 700 800(nm)

1110

9876543210

minus1

(Abs

)

(c)

Figure 1 Absorbance spectrum of 120573-carotene120572-carotene pigments isolated from crude palm oil (a) absorbance maxima of 120573-carotene at440 nm (b) absorbance of 120572-carotene at 4666 nm (c) absorbance profile of 120573-carotene120572-carotene mixture in a crystalline form

values of extinction coefficient [20] The minor peak at4666 nm is that of120572-carotenewhich is in correlationwith theextinction coefficient (1 02 979 Lmolminus1 cmminus1) of 120572-carotene[21]

The absorption at given wavelength range can beexpressed as the sum of individual carotenoids as observedin Figure 1(c) where UVvisible spectrophotometric studyof the crystalline carotenoid pigment displays a uniformplateau-shaped absorption range representing an assemblageof 120572- and 120573-caroteneThe range was observed between 300 to550 nm which is in harmony with previously derived results

On thin layer chromatographic analysis119877119891factor was cal-

culated to be 095 which corresponds to previously reportedvalues [22] The single spot indicated that 120572- and 120573-carotenehad overlapped over the specified area resulting in theincurrence of an intermediate value of retention factor

32 Detection of 120573-Carotene Using High Performance Liq-uid Chromatography (HPLC) On HPLC two peaks wereobserved with retention times at 282 and 305 minutes 120573-carotene eluted as a single peak at 305minutes (Figure 2) and120572-carotene at 282minutes as deciphered by comparisonwithauthentic standards

33 Antioxidant Assay of Isolated 120573-Carotene The antiox-idant activity of the isolated carotene crystals were exam-ined by seven different in vitro assay systems Differentconcentrations of the antioxidant was prepared in ethanolThe concentrations prepared were 0001 0005 001 005and 01 (wv) Their antioxidant activities were measuredin terms of DPPH radical scavenging activity reducingactivity metal chelation ABTS radical scavenging activitylipid peroxidation superoxide scavenging activity and anti-denaturation effect

331 Assay of DPPH Radical-Scavenging Activity TheDPPHradical scavenging method was based on the reduction ofmethanolic DPPH solution in the presence of a hydrogendonating antioxidant due to the formation of the non-radical form DPPH-H [23] Monitoring the decrease of theradical in terms of decreasing absorbance values leads to theassessment of the antioxidant activity of the product Thecarotene crystals were able to reduce and decolourise 11-diphenyl-2-picrylhydrazyl efficiently at low concentrationsvia their hydrogen donating ability (Figure 3) This is anelectron transfer-based assay which measures the capacity ofan antioxidant in the reduction of an oxidant Here the degree


00012

00010

00008

00006

00004

00002

00000

000 500 1000 1500 2000 2500 3000 3500 4000Minutes

(AU

)

120572-carotene

120573-carotene

Figure 2 HPLC chromatogram of 120572- and 120573-carotene

000 002 004 006 008 01015

20

25

30

35

40

45

50

DPP

H ra

dica

l sca

veng

ing

activ

ity (

)

Concentration of antioxidants ()

a a

bc d

Figure 3 DPPH radical scavenging activity for 120572- and 120573-carotenecrystals at different concentrations Values are Mean plusmn SEM of 3observations At the 005 level difference in superscripts (a b c d)indicates significant difference in means

of colour change is correlated with the samplersquos antioxidantactivity

The order of antioxidant potency by DPPH method isconclusive of the behavior of the antioxidant at differentconcentration gradients Colour interference by 120572-carotenewith DPPH chromogen and 120573-carotene may result in a lowermeasured DPPH activity at high concentrations [24] More-over the presence of higher concentrations of antioxidantsresults in prooxidant effects which induce oxidative stressusually by inhibiting antioxidant systems Hence the lowestconcentration of the antioxidant (0001) that was analysedfor DPPH radical scavenging activity was found to be theoptimum concentration that showed efficacy in scavengingthe free radicals Furthermore on comparing the mean val-ues all the concentration means varied significantly amongthemselves except for 0001 and 0005 concentrations at119875 lt 005

332 Assay of Reductive Potential Again assay of reducingcapacity is an effective means to understand the antioxidant

000 002 004 006 008 010minus01

00

01

02

03

04

05

06

07

08

Abso

rban

ce (Aring

)


a bc

d

e

Figure 4 Reducing activity of120572- and120573-carotene crystals at differentconcentrations Values are Mean plusmn SEM of 3 observations At the005 level the difference in superscripts (a b c d e) indicatessignificant difference in means

activity of various antioxidants Reducing capacity serves asa significant indicator of the potential antioxidant activityof any bioactive species Here reduction potential bearsa proportional dependency on the absorbance measured(Figure 4) Here all the different concentration mean valueswere significantly different from each other at 119875 lt 005The measured absorbance serves to indicate the change inreduction potential of the tested species The reducing power(transformation of Fe3+ to Fe2+) of the antioxidant makesit an efficient electron donor which can react with freeradicals to convert them to more stable products therebyterminating radical chain reactionsThe antioxidant exerts anantioxidant effect by reducing Fe3+ to Fe2+ Such bioactiveproperty of the antioxidant leads to the development ofits chemoprotective potential too From this assay it wasdeduced that in terms of reducing activity 120572- and 120573-caroteneshowed the expected tendency of concentration variationWith increasing carotenoid concentrations the absorbanceincreased indicating an increase in the reducing activity ofthe antioxidant Hence higher amounts of the antioxidantwill serve as an efficient reducing agent though its radicalscavenging activity showed a contrary behaviour

333 Assay of Metal Chelating Activity Again metal chela-tion activity is an example of a complexation reactionwhere Ferrozine [disodium salt of 3-(2-pyridyl)-56-bis(4-phenylsulfonic acid)-124-triazine] is a complex-formingagent of Fe(II) and will form a magenta complex Fe(II)-(Ferrozine)(III) withmaximumabsorbance at 562 nmHencein the presence of a reducing agent the complex formation ishampered resulting in decrease in the colour of the complexand hence a decrease in the absorbance Measurement ofabsorbance therefore allows estimating the metal chelatingactivity of the coexisting chelator [25] In this case 120572- and120573-carotene interfered with the formation of ferrous-ferrozine


000 002 004 006 008 01012

14

16

18

20

22

24

26

28

30

Met

al ch

elat

ion

activ

ity (

)


b

a

c

d

e

Figure 5 Metal chelation activity of 120572- and 120573-carotene crystals atdifferent concentrations Values are Mean plusmn SEM of 3 observationsAt the 005 level the difference in superscripts (a b c d e) indicatessignificant difference in means

complexThe steric hindrance generated by the bulky carbonchains of120572- and120573-carotene prevented the unsaturated groupsfrom acting as chelating agents at higher concentrationsMoreover with increasing concentrations the prooxidanteffect is active and 120572- and 120573-carotene become the sourceof complexation resulting in higher absorbance values andhence reducedmetal chelation activities (Figure 5) In fine 120572-and 120573-carotene act as a better DPPH-radical scavenger than ametal chelatorThe presence of unsaturated groups could be acontributing factor towards their effective reducing propertyAll the different concentrationmean values were significantlydifferent from each other at 119875 lt 005

334 ABTS Radical Scavenging Activity The ABTS assay isbased on the ability of antioxidants to scavenge the long-liferadical cation ABTS+ The scavenging of radicals producesa decreased absorbance at 734 nm Here absorbance wasfound to increase at 734 nm for all concentrations except for01 concentration (Figure 6) In case of 01 concentrationcommencement of radical-scavenging activity was observedbeyond 240 seconds as indicated by lowering absorbancevalues

Unlike DPPH radical scavenging activity the ABTS-scavenging power was exhibited only at 01 antioxidantconcentration Apparently here the antioxidant activity of120572- and 120573-carotene cannot be well differentiated based ontheir concentrations as can be established in the case ofDPPH method or metal chelation Generation of oxidativestress was evident from the increase in absorbance valuesat 005 concentration The lowest concentration of 0001showed higher absorbance readings than the ones at 0005concentration This could be due to the inability of theantioxidant to scavenge the long-life free radicals at theminimal concentration At still higher value of 001 con-centration the absorbance value reaches an almost constant

0 50 100 150 200 250 300

092

093

094

095

096

097

Abso

rban

ce (Aring

)

Time (s)

00010005001

00501

d ef f f fa

b b

c cc

jg

h hi i i

k l l

m m

n

o o p op

Figure 6 ABTS-radical scavenging activity of 120572- and 120573-carotenecrystals at different concentrations Values are Mean plusmn SEM of 3observations At the 005 level at time intervals 0 60 120 180 240and 300 seconds the difference in superscripts indicates significantdifference in means Superscripts (1) (a b c) for 0001 (2) (d e f)for 0005 (3) (g h i) for 001 (4) (j k l m) for 005 and (5)(n o p) for 01 are used

value beyond 4 minutes indicating a possibility of ABTS-scavenging capacity at higher time periods At 119875 lt 005 forconcentrations 0001 0005 001 005 and 01 thepopulationmeanswere significantly different fromeach otherthroughout the time range tested except for 60 120 secondsand 180 240 300 seconds among themselves for 0001concentration 120 180 240 300 seconds among themselvesfor 0005 concentration 60 120 seconds and 180 240 300seconds among themselves for 001 concentration 120 180seconds and 240 300 seconds among themselves for 005concentration 60 120 300 seconds and 180 240 secondsamong themselves for 01 concentration

335 Lipid Oxidation in a Linoleic Acid Emulsion ModelSystem The relative inhibitory effect of 120572- and 120573-carotene onlipid peroxidation using a model linoleic acid peroxidationassay is shown in Figure 7 The basis of lipid peroxidationmethod involves the use of heat as the free radical initiatorwhich instigates the oxidation of Fe2+ to Fe3+ (8) and is conse-quentlymeasured by assessing the amount of peroxyl radicalsthat is generated in the prepared linoleic acid emulsion dueto the lipid oxidation reaction of the linoleic acid [26]

Fe2+ + LOOH = Fe3+ + LO∙ +OHminus (8)

where LOOH denotes a lipid derived hydroperoxide and LO∙denotes a lipid peroxyl radical The Fe3+ subsequently reactswith ammonium thiocyanate to form ferric thiocyanateThe absorbance at 500 nm measures the amount of ferricthiocyanate formed in the medium as a result of the above


0 20 40 60 80 100 120

0

10

20

30

40

50

60

Prev

entio

n of

per

oxid

atio

n (

)

Time (hours)

00010005001

00501

a

b

c

d

eo

e

f

g

mh

ni

k

ql

p

r

s

t

j

Figure 7 Prevention of lipid peroxidation by 120572- and 120573-carotenecrystals at different concentrations Values are Mean plusmn SEM of3 observations At the 005 level for the time span 0 hours 24hours 48 hours 72 hours 96 hours and 120 hours the difference insuperscripts indicates significant difference in means Superscripts(1) (a b c) for 0001 (2) (d e f) for 0005 (3) (g h i j) for 001(4) (k l m n o) for 005 and (5) (p q r s t) for 01 are used

reaction Higher absorbance readings (ie lower preventionof lipid peroxidation) are associated with increased con-centration of peroxyl radicals in the solution which occurssimultaneously with the formation of the colouring complex120572- and 120573-carotene served to scavenge the Fe2+ ion fromthe medium where the scavenging efficacy increased withincreasing concentration of the antioxidants A variety ofbeta carotene isomers and metabolites were used to test forvarious in vitro assays like FRAP assay lipid peroxidationand so forth No ferric reducing activity (FRAP assay) wasobserved for the isomers Between the major isomers no sig-nificant differences in bleaching the ABTS∙+ or in scavengingperoxyl radicals (ROO∙) generated by thermal degradationof AAPH (using a chemiluminescence assay) were detected[27]The carotenoids displayed lipophilic antioxidant activityby dissolving completely in the linoleic acid emulsion andserving as an efficient radical scavenger A mixture of 120572- and120573-carotene in association increased the activity of the productcrystals against lipid peroxidation as indicated by high levelof inhibition of lipid peroxidation and therefore sufficientantioxidant activity at higher concentrations

In case of 0001 concentration prevention of peroxida-tion was not detected before 72 hours unlike 01 concen-tration where 097 prevention was observed only after 24hours Beyond 4 days the inhibition of lipid peroxidationreduced for all concentrations Thus antioxidant activity ofthe products showed potencymaximumup to 4 days beyondwhich the effectiveness diminished gradually and predomi-nance of the peroxyl radicals was observed as indicated by

000 002 004 006 008 010

0

20

40

60

80

100

Supe

roxi

de sc

aven

ging

activ

ity (

)

Concentration of the antioxidants ()

aa

b

c

d

Figure 8 Superoxide scavenging activity by 120572- and 120573-carotenecrystals at different concentrations Values are Mean plusmn SEM of 3observations At the 005 level the difference in superscripts (a bc d) indicates significant difference in means

higher absorbance values At 119875 lt 005 for the time span 0hours 24 hours 48 hours 72 hours 96 hours and 120 hoursthe population means are significantly different from eachother for all the five different carotene concentrations exceptfor 72 from 120 hours for 0005 concentration

336 Superoxide Anion Scavenging Assay Again 120572- and 120573-carotene crystals exhibited a concentration-dependent super-oxide scavenging activity similar to DPPH radical scavengingactivity The losses of linearity in the dose response curve atlow concentrations of 120572- and 120573-carotene were a reflectionof not only their superoxide scavenging efficiency but alsothe prooxidant effect of the products As both 120572- and 120573-carotene can interchange between a reduced form and anoxidised form they display antioxidant and pro-oxidantproperties related to their dosage and half-life [28] Necessarypreponderance must be given to the antioxidant activity ofcarotenoids at low concentrations especially while consid-ering their scavenging activities (Figure 8) Here pyrogallolacted as the free radical initiatorwhich absorbed oxygen fromthe air turning purple from a colourless solution by formingsuperoxide anions Superoxide anion is an oxygen-centeredreactive oxygen species (superoxide anion and hydrogenperoxide are the main reactive oxygen species causing theoxidation of cells and tissues) They react with protons inwater solution to form hydrogen peroxide (9) which serve asa substrate for the generation of hydroxyl radicals and singletoxygen [29]

2O2

minus

+ 2H+ 997888rarr H2O2+O2 (9)

Superoxide radical is a very harmful species to cellularcomponents as a precursor of more reactive oxygen speciesand is known to be produced in vivo and can result inthe formation of H

2O2via dismutation reaction H

2O2is a

nonradical reactive oxygen species which acts as a strong


000 002 004 006 008 01020

30

40

50

60

70

80

90

100

Inhi

bitio

n of

den

atur

atio

n (

)


a

b

c

d d

Figure 9 Inhibition of denaturation in vitro by 120572- and 120573-carotenecrystals at different concentrations Values are Mean plusmn SEM of 3observations At the 005 level the difference in superscripts (a bc d) indicates significant difference in means

oxidant leading to harmful reactions Here 120572- and 120573-caroteneat a concentration of 0001 scavenged superoxide anionswith utmost efficiency thereby reducing the probability ofperoxide formation Radical scavengers can be prooxidantunless linked to a radical sink and superoxides are suchradical sinks and hence efficiently scavenge radicals at lowconcentrations Increasing concentrations of the antioxidantslead to the appearance of prooxidant activity resulting in thedecrease in scavenging capacity Here the antioxidant itselfbehaved as a radical that bigoted another radical with higherefficiency at lower concentrations and it promoted oxidationat higher concentrations At 119875 lt 005 all the populationmeans are significantly different from each other except for0001 and 0005 concentrations

337 Assay of In Vitro Antidenaturation Effects The presentstudy is the first of its kind to report the efficacy of 120573-carotenein protecting cells from denaturationinflammatory effect invitro (Figure 9)

When BSA is heated it undergoes denaturation andexpresses antigens associated to Type III hypersensitive reac-tion which are related to diseases such as serum sicknessglomerulonephritis rheumatoid arthritis and systemic lupuserythematosus An in-depth study of the antidenaturationeffect of 120572- and 120573-carotene can be utilised for the treatmentof such diseases by their specific dosage formulations Anefficient antioxidant is one which is able to compete withendogenous scavengers and interact with endogenous path-ways by localizing themselves in the appropriate area One ofthe features of several nonsteroidal anti-inflammatory drugsis their ability to stabilize (prevent denaturation) heat treatedBSA at pathological pH [pH 62ndash65] [19] The increasedsynthesis of heat shock results in a ubiquitous physiologicalresponse of cells to environmental stress and hence promotesdegradation of proteins In this study an attempt was made in

vitro to study the course of action of 120572- and120573-carotene in pre-venting thermal denaturation and aggregation of BSAproteinat increased temperatures A variety of cellular internal andexternal stress are generated due to environmental imbalancewhich leads to the formation of reactive oxygen speciesresulting in the origin of several disturbances in the normalredox conditions of the cells leading to their deteriorationin the course of time These stresses denature proteinsenhancing the probability of the formation of reactive oxygenspecies in the medium leading to inflammation and otherphysiological malfunctionsThese aggregates if not disposedof or their formation being prevented can lead to cell deathIn response to the appearance of damaged proteins cellsinduce the expression of such harmful species However asan effective antioxidant 120572- and 120573-carotene prevented suchdenaturation which functioned as molecular chaperone andprevented protein aggregation or degradation Carotenoids(including 120573-carotene) were found to promote health whentaken at dietary levels but showed adverse effects whenconsumed at a high dose by subjects who smoke or wereexposed to asbestos [30]The effectiveness was the greatest atlow concentrations which can be utilized in drug designingThe high apparent antioxidant capacity of 120572- and 120573-caroteneat low concentrations also has an added advantage in terms ofcost-effectiveness of the developed drug product At119875 lt 005the concentration means are significantly different from eachother except for the concentrations 005 and 01

4 Conclusion

From the results obtained both 120572- and 120573-carotene wereisolated effectively and their antioxidant study gave inter-esting observations Table 1 summarizes the efficacy of thecarotenoids in responding to the different antioxidant assaysWhile 0001 concentration was effective DPPHsuperoxideradical scavenger metal chelator and antidenaturant 01concentration showed effective reducing activity and lipidperoxidation Moreover 01 was the only concentrationwhich showed any ABTS radical scavenging activity Com-pounds often enhance the antioxidant capacity of cells butare ineffective in test tube assays The antioxidant activityof 120572- and 120573-carotene can shift into a prooxidant phasedepending on such factors as oxygen tension or carotenoidconcentration Good correlations observed among the dif-ferent hydrophilic antioxidant assays namely metal chela-tion DPPH radical and superoxide scavenging activitiessuggested that these methods have almost similar predictivecapacity for antioxidant activities of various concentrations of120572- and 120573-carotene ABTS-scavenging activity was displayedat a particular antioxidant concentration of 01 In case oflipophilic antioxidant assay it was observed that almost 51inhibition of lipid peroxidationwas displayed by 01 activityThe right dose essentially differentiates a potent remedyfrom a subsequent harmful intake Hence in hydrophilicenvironment mostly lower concentrations of antioxidantswere effective whereas in lipophilic medium higher con-centrations were effective This could be due to the higherlipid affinity of the carotenoids being nonpolar in nature


Table 1 Relative efficacy of each antioxidant assay for individual antioxidant concentration

Concentrations()

Antioxidant assay

DPPHradical

scavengingactivity ()

Reducingactivity (A)

Metalchelation

()

ABTSradical

scavengingactivity()

Lipidperoxidation ()

Superoxide radicalscavenging activity

()

Anti-denaturationassay ()

0001 +++++ + +++++ minus + +++++ +++++0005 ++++ ++ ++++ minus ++ ++++ ++++001 +++ +++ +++ minus +++ +++ +++005 ++ ++++ ++ minus ++++ ++ ++01 + +++++ + + +++++ + +

In consequence they can act as substantial chemoprotectantsand prevent harmful physiological activities if consumed ina proper dose

Acknowledgment

The authors would like to acknowledge the ldquoCouncil ofScientific and Industrial Researchrdquo (CSIR) for the financialsupport received

References

[1] Y Yuan Y Gao L Mao and J Zhao ldquoOptimisation ofconditions for the preparation of 120573-carotene nanoemulsionsusing response surface methodologyrdquo Food Chemistry vol 107no 3 pp 1300ndash1306 2008

[2] D Albanes ldquo120573-Carotene and lung cancer a case studyrdquo Ameri-can Journal of Clinical Nutrition vol 69 no 6 pp 1345Sndash1350S1999

[3] M Nitsche W Johannisbauer and V Jordan ldquoProcess forObtaining Carotene fromPalmOilrdquo US Patent 5 902 890 1999

[4] A A Woodall G Britton and M J Jackson ldquoCarotenoids andprotection of phospholipids in solution or in liposomes againstoxidation by peroxyl radicals relationship between carotenoidstructure and protective abilityrdquo Biochimica et Biophysica Actavol 1336 no 3 pp 575ndash586 1997

[5] K R Martin M L Failla and J C Smith Jr ldquo120573-Carotene andlutein protectHepG2human liver cells against oxidant- induceddamagerdquo Journal of Nutrition vol 126 no 9 pp 2098ndash21061996

[6] T Iyama A Takasuga and M Azuma ldquo120573-carotene accumu-lation in mouse tissues and a protective role against lipidperoxidationrdquo International Journal for Vitamin and NutritionResearch vol 66 no 4 pp 301ndash305 1996

[7] V Agte and K Tarwadi ldquoThe importance of nutrition in theprevention of ocular disease with special reference to cataractrdquoOphthalmic Research vol 44 no 3 pp 166ndash172 2010

[8] P Di Mascio S Kaiser and H Sies ldquoLycopene as the most effi-cient biological carotenoid singlet oxygen quencherrdquoArchives ofBiochemistry and Biophysics vol 274 no 2 pp 532ndash538 1989

[9] P F Guo Preparation antioxidant activity and stability oflycopene and 120573-carotene from Papaya [PhD thesis] 2009

[10] J G Jackson E L Lien S J White N J Bruns and C FKuhlman ldquoMajor carotenoids in mature human milk longitu-dinal and diurnal patternsrdquo Journal of Nutritional Biochemistryvol 9 no 1 pp 2ndash7 1998

[11] D R Katerere and J N Eloff ldquoAntibacterial and antioxidantactivity of Sutherlandia frutescens (Fabaceae) a reputed anti-HIVAIDS phytomedicinerdquo Phytotherapy Research vol 19 no9 pp 779ndash781 2005

[12] H J D Dorman and R Hiltunen ldquoFe(III) reductive andfree radical-scavenging properties of summer savory (Saturejahortensis L) extract and subfractionsrdquo Food Chemistry vol 88no 2 pp 193ndash199 2004

[13] T C P Dinis V M C Madeira and L M Almeida ldquoActionof phenolic derivatives (acetaminophen salicylate and 5-aminosalicylate) as inhibitors of membrane lipid peroxidationand as peroxyl radical scavengersrdquo Archives of Biochemistry andBiophysics vol 315 no 1 pp 161ndash169 1994

[14] R Re N Pellegrini A Proteggente A PannalaM Yang andCRice-Evans ldquoAntioxidant activity applying an improved ABTSradical cation decolorization assayrdquo Free Radical Biology andMedicine vol 26 no 9-10 pp 1231ndash1237 1999

[15] X Zhao H Sun A Hou Q Zhao T Wei and W XinldquoAntioxidant properties of two gallotannins isolated from theleaves of Pistacia weinmannifoliardquo Biochimica et BiophysicaActa vol 1725 no 1 pp 103ndash110 2005

[16] I Gulcin I G Sat S Beydemir M Elmastas and O IKufrevioglu ldquoComparison of antioxidant activity of clove(Eugenia caryophylata Thunb) buds and lavender (Lavandulastoechas L)rdquo Food Chemistry vol 87 no 3 pp 393ndash400 2004

[17] Z Yaping Y Wenli W Dapu L Xiaofeng and H TianxildquoChemiluminescence determination of free radical scavengingabilities of rsquotea pigmentsrsquo and comparison with rsquotea polyphe-nolsrsquordquo Food Chemistry vol 80 no 1 pp 115ndash118 2003

[18] J Wang Q Zhang Z Zhang and Z Li ldquoAntioxidant activityof sulfated polysaccharide fractions extracted from Laminariajaponicardquo International Journal of Biological Macromoleculesvol 42 no 2 pp 127ndash132 2008

[19] L A D Williams A OrsquoConnar L Latore et al ldquoThe invitro anti-denaturation effects induced by natural productsand non-steroidal compounds in heat treated (Immunogenic)bovine serum albumin is proposed as a screening assay for thedetection of anti-inflammatory compounds without the use ofanimals in the early stages of the drug discovery processrdquoWestIndian Medical Journal vol 57 no 4 pp 327ndash331 2008

[20] E Biehler F Mayer L Hoffmann E Krause and T BohnldquoComparison of 3 spectrophotometric methods for carotenoid


determination in frequently consumed fruits and vegetablesrdquoJournal of Food Science vol 75 no 1 pp C55ndashC61 2010

[21] R J Bushway A Yang and A M Yamani ldquoComparison ofalpha- and beta-carotene content of supermarket vs Roadsidestand produced in Mainerdquo Technical Bulletin vol 122 pp 1ndash161986

[22] B Fried K Beers and J Sherma ldquoThin-layer chromatographicanalysis of beta-carotene and lutein in Echinostoma trivolvis(Trematoda) rediaerdquo Journal of Parasitology vol 79 no 1 pp113ndash114 1993

[23] X L Li and A G Zhou ldquoEvaluation of the antioxidant effects ofpolysaccharides extracted from Lycium barbarumrdquo MedicinalChemistry Research vol 15 no 9 pp 471ndash482 2007

[24] T Yamaguchi H Takamura T Matoba and J Terao ldquoHPLCmethod for evaluation of the free radical-scavenging activityof foods by using 11-diphenyl-2-picrylhydrazylrdquo BioscienceBiotechnology and Biochemistry vol 62 no 6 pp 1201ndash12041998

[25] F Yamaguchi M Saito T Ariga Y Yoshimura and HNakazawa ldquoFree radical scavenging activity and antiulceractivity of garcinol from Garcinia indica fruit rindrdquo Journal ofAgricultural and Food Chemistry vol 48 no 6 pp 2320ndash23252000

[26] D D Kitts Y V Yuan A N Wijewickreme and L UThompson ldquoAntioxidant activity of the flaxseed lignan secoiso-lariciresinol diglycoside and its mammalian lignan metabolitesenterodiol and enterolactonerdquoMolecular and Cellular Biochem-istry vol 202 no 1-2 pp 91ndash100 1999

[27] L Mueller and V Boehm ldquoAntioxidant activity of 120573-carotenecompounds in different in vitro assaysrdquoMolecules vol 16 no 2pp 1055ndash1069 2011

[28] C C Teow V-D Truong R F McFeeters R L Thompson KV Pecota and G C Yencho ldquoAntioxidant activities phenolicand120573-carotene contents of sweet potato genotypes with varyingflesh coloursrdquo Food Chemistry vol 103 no 3 pp 829ndash838 2007

[29] B Halliwell J M C Gutteridge and C E Cross ldquoFree radicalsantioxidants and human disease where are we nowrdquo Journalof Laboratory and Clinical Medicine vol 119 no 6 pp 598ndash6201992

[30] S A R Paiva R M Russell and S K Dutta ldquo120573-caroteneand other carotenoids as antioxidantsrdquo Journal of the AmericanCollege of Nutrition vol 18 no 5 pp 426ndash433 1999

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Inorganic ChemistryInternational Journal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

International Journal ofPhotoenergy


Carbohydrate Chemistry

International Journal of


Journal of

Chemistry


Advances in

Physical Chemistry

Hindawi Publishing Corporationhttpwwwhindawicom

Analytical Methods in Chemistry

Journal of

Volume 2014

Bioinorganic Chemistry and ApplicationsHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

SpectroscopyInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Medicinal ChemistryInternational Journal of


Chromatography Research International


Applied ChemistryJournal of



Theoretical ChemistryJournal of


Journal of

Spectroscopy

Analytical ChemistryInternational Journal of


Journal of


Quantum Chemistry


Organic Chemistry International

ElectrochemistryInternational Journal of



CatalystsJournal of


In contrast to the physiologically relevant properties theknowledge on antioxidant potential of 120572- and 120573-carotene invitro is scarce as most studies conducted so far were in vivoHence the aim of the present study was to isolate 120572- and 120573-carotene from crude palm oil their characterization and invitro investigation of their antioxidative efficacy

2 Experimental

21 Materials Crude palm oil imported from Indonesiawas collected from local Vanaspati manufacturer All otherreagents were of analytical grade and procured from MerckIndia Ltd Mumbai India

22 Isolation of 120572- and 120573-Carotene from Crude Palm OilCrude palm oil was chemically transesterified with methanoland sodium hydroxide Oil and alcohol were taken in dif-ferent molar ratios and stirred while maintaining a hightemperature for a definite time interval [3] Syntheses of esterswere monitored by thin layer chromatography The effectsof different reaction parameters for this particular reactionsuch as temperature substratemolar ratio and concentrationof alkali were optimized with individual sets of reactionmixture Oil methanol ratio of 1 10 on weight basis stirringrate of 200 rpm temperature of 80∘C for 3 hours and aconcentration of 02 NaOH were optimised for maximumyield of 120572- and 120573-carotene A two-phase mixture comprisinga glycerol phase and an ester phase consisting of fatty estersand the carotenewas obtainedThe ester phasewas distilled at300∘C at 10minus01mbar pressure in a molecular distillation unit(SIBATA Japan serial no N40394) The residue was diluted5 times and saponified with ethanolic potassium hydroxide at80∘CThe carotene was extracted from the saponified residuewith a mixture of hexane and water where the extract phasewas rich in carotene Pure carotene was obtained on solvent(hexane) removal which was crystallized and recrystallizedwith acetone

23 Spectrophotometric Analysis For spectrophotometricanalysis 001 g measured amount of the crystallized caroteneproduct was taken in a calibrated test tube and dissolvedinto 25mL of petroleum ether The solution was scannedunder UV-visible spectrophotometer (UV-1700 PharmaSpecUV-VIS Spectrophotometer Shimadzu Japan) between 300ndash600 nm The 120582max value was noted and the molar extinctioncoefficient (120576max) using 1 cm cell was calculated for definiteconcentration of the sample Further spectral conformationwas carried out by scanning the solid carotene crystals underthe UV-visible range of 200ndash800 nm with UV spectropho-tometer (U-3501 Hitachi Japan)

24 Characterisation by Thin Layer Chromatography (TLC)Crystalline samples were dissolved in petroleum ether andspotted in glass TLC plates coated with silica gel stationaryphase The TLC plate was placed in a chamber saturatedwith solvent vapour The solvent system consisted of 60petroleum ether20 acetone20 dichloromethane [9]After development of the plates they were visualized by

subjecting them to iodine vapour The 119877119891values for the spot

on the TLC plate were calculated

119877119891=

distance from baseline to sample frontdistance from baseline to solvent front

(1)

25 Characterisation by High Performance Liquid Chromatog-raphy (HPLC) Analysis of the carotenoids was conductedusing Waters HPLC (Milford Massachusetts US) with aWaters 1525 binary HPLC pump consisting of a vacuumdegasser and a Waters 2487 dual 120582 absorbance detectorReverse-phase HPLC equipped with Nova-pak C

18column

39 times 150mm 4 120583m was used for the analysis of the antiox-idant The UV detector was set at 450 nm The mobilephase consisted of acetonitrile methanol tetrahydrofuran(50 45 5 by volume) [10] The crystallized carotenoids weredissolved in 5mL of the mobile phase The mobile phase wasisocratic solvent Injection volume of 200120583L and the flow ratewere set at 1mLmin Data acquisition was completed in 40minutes

26 Antioxidant Assay of Isolated Carotene Crystals Theantioxidant activity of the isolated carotenoids was examinedby seven different in vitro assay systems Different concen-trations of the antioxidant were prepared in ethanol Theconcentrations were 0001 0005 001 005 and 01 (wv)Their antioxidant activities were measured consecutively

261 Assay of DPPH Radical-Scavenging Activity Antioxi-dant activity of 120572- and 120573-carotene was determined by thescavenging activity of the stable DPPH free radical Themethod was described by Katerere and Eloff [11] Differentconcentrations of the test samples were placed into test tubestaking 02mL from each of them with 4mL of 02mMof ethanolic solution of DPPH Absorbance at 517 nm wasdetermined after 40 minutes using a solution of ethanoland DPPH (3 1) as control Radical scavenging activity wasexpressed as the inhibition percentage and was calculatedusing the following formulae

Radical scavenging activity = [1198600minus 1198601

1198600

] times 100 (2)


1is the


262 Assay of Reductive Potential The reductive potential ofthe antioxidant was determined according to the method ofDorman and Hiltunen [12] The reaction mixture containingdifferent concentrations of the antioxidants (50ndash250120583gmL)in 1mL of ethanol phosphate buffer (25mL 02M pH66) and potassium ferricyanide (25mL 1 wtvol) wasincubated at 50∘C for 20 minutes A portion (25mL) oftrichloroacetic acid (10 wtvol) was added to the mixturewhich was then allowed to rest for 10 minutes From themixture 25mL was taken and mixed with distilled water(25mL) and FeCl

3(05mL 01 wtvol) and the absorbance

was measured at 700 nm in a spectrophotometer Higherabsorbance of the reaction mixture indicated greater reduc-tive potential



2(25mmolL)



1198600

] times 100 (3)


1is the



2S2O8and kept







1198600

] times 100 (4)


1is the





times 100

(5)




times 100 (6)





120576max =119860

119862119871

(7)




Spectrum150 A

(01

00d

iv)

100 A

4400 nm 1287 A

5000 nm3700 nm(20div)

(a)

Spectrum150 A

(01

00d

iv)

100 A

4666 nm 1033 A

5000 nm3700 nm(20div)

(b)

200 300 400 500 600 700 800(nm)

1110

9876543210

minus1

(Abs

)

(c)










00012

00010

00008

00006

00004

00002

00000

000 500 1000 1500 2000 2500 3000 3500 4000Minutes

(AU

)

120572-carotene

120573-carotene


000 002 004 006 008 01015

20

25

30

35

40

45

50

DPP

H ra

dica

l sca

veng

ing

activ

ity (

)


a a

bc d





000 002 004 006 008 010minus01

00

01

02

03

04

05

06

07

08

Abso

rban

ce (Aring

)


a bc

d

e





000 002 004 006 008 01012

14

16

18

20

22

24

26

28

30

Met

al ch

elat

ion

activ

ity (

)


b

a

c

d

e





0 50 100 150 200 250 300

092

093

094

095

096

097

Abso

rban

ce (Aring

)

Time (s)

00010005001

00501

d ef f f fa

b b

c cc

jg

h hi i i

k l l

m m

n

o o p op







0 20 40 60 80 100 120

0

10

20

30

40

50

60

Prev

entio

n of

per

oxid

atio

n (

)

Time (hours)

00010005001

00501

a

b

c

d

eo

e

f

g

mh

ni

k

ql

p

r

s

t

j




000 002 004 006 008 010

0

20

40

60

80

100

Supe

roxi

de sc

aven

ging

activ

ity (

)


aa

b

c

d




2O2

minus

+ 2H+ 997888rarr H2O2+O2 (9)



2O2is a



000 002 004 006 008 01020

30

40

50

60

70

80

90

100

Inhi

bitio

n of

den

atur

atio

n (

)


a

b

c

d d






4 Conclusion




Concentrations()

Antioxidant assay

DPPHradical



Metalchelation

()

ABTSradical




()




Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



2(25mmolL)



1198600

] times 100 (3)


1is the



2S2O8and kept







1198600

] times 100 (4)


1is the





times 100

(5)




times 100 (6)





120576max =119860

119862119871

(7)




Spectrum150 A

(01

00d

iv)

100 A

4400 nm 1287 A

5000 nm3700 nm(20div)

(a)

Spectrum150 A

(01

00d

iv)

100 A

4666 nm 1033 A

5000 nm3700 nm(20div)

(b)

200 300 400 500 600 700 800(nm)

1110

9876543210

minus1

(Abs

)

(c)










00012

00010

00008

00006

00004

00002

00000

000 500 1000 1500 2000 2500 3000 3500 4000Minutes

(AU

)

120572-carotene

120573-carotene


000 002 004 006 008 01015

20

25

30

35

40

45

50

DPP

H ra

dica

l sca

veng

ing

activ

ity (

)


a a

bc d





000 002 004 006 008 010minus01

00

01

02

03

04

05

06

07

08

Abso

rban

ce (Aring

)


a bc

d

e





000 002 004 006 008 01012

14

16

18

20

22

24

26

28

30

Met

al ch

elat

ion

activ

ity (

)


b

a

c

d

e





0 50 100 150 200 250 300

092

093

094

095

096

097

Abso

rban

ce (Aring

)

Time (s)

00010005001

00501

d ef f f fa

b b

c cc

jg

h hi i i

k l l

m m

n

o o p op







0 20 40 60 80 100 120

0

10

20

30

40

50

60

Prev

entio

n of

per

oxid

atio

n (

)

Time (hours)

00010005001

00501

a

b

c

d

eo

e

f

g

mh

ni

k

ql

p

r

s

t

j




000 002 004 006 008 010

0

20

40

60

80

100

Supe

roxi

de sc

aven

ging

activ

ity (

)


aa

b

c

d




2O2

minus

+ 2H+ 997888rarr H2O2+O2 (9)



2O2is a



000 002 004 006 008 01020

30

40

50

60

70

80

90

100

Inhi

bitio

n of

den

atur

atio

n (

)


a

b

c

d d






4 Conclusion




Concentrations()

Antioxidant assay

DPPHradical



Metalchelation

()

ABTSradical




()




Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


Spectrum150 A

(01

00d

iv)

100 A

4400 nm 1287 A

5000 nm3700 nm(20div)

(a)

Spectrum150 A

(01

00d

iv)

100 A

4666 nm 1033 A

5000 nm3700 nm(20div)

(b)

200 300 400 500 600 700 800(nm)

1110

9876543210

minus1

(Abs

)

(c)










00012

00010

00008

00006

00004

00002

00000

000 500 1000 1500 2000 2500 3000 3500 4000Minutes

(AU

)

120572-carotene

120573-carotene


000 002 004 006 008 01015

20

25

30

35

40

45

50

DPP

H ra

dica

l sca

veng

ing

activ

ity (

)


a a

bc d





000 002 004 006 008 010minus01

00

01

02

03

04

05

06

07

08

Abso

rban

ce (Aring

)


a bc

d

e





000 002 004 006 008 01012

14

16

18

20

22

24

26

28

30

Met

al ch

elat

ion

activ

ity (

)


b

a

c

d

e





0 50 100 150 200 250 300

092

093

094

095

096

097

Abso

rban

ce (Aring

)

Time (s)

00010005001

00501

d ef f f fa

b b

c cc

jg

h hi i i

k l l

m m

n

o o p op







0 20 40 60 80 100 120

0

10

20

30

40

50

60

Prev

entio

n of

per

oxid

atio

n (

)

Time (hours)

00010005001

00501

a

b

c

d

eo

e

f

g

mh

ni

k

ql

p

r

s

t

j




000 002 004 006 008 010

0

20

40

60

80

100

Supe

roxi

de sc

aven

ging

activ

ity (

)


aa

b

c

d




2O2

minus

+ 2H+ 997888rarr H2O2+O2 (9)



2O2is a



000 002 004 006 008 01020

30

40

50

60

70

80

90

100

Inhi

bitio

n of

den

atur

atio

n (

)


a

b

c

d d






4 Conclusion




Concentrations()

Antioxidant assay

DPPHradical



Metalchelation

()

ABTSradical




()




Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


00012

00010

00008

00006

00004

00002

00000

000 500 1000 1500 2000 2500 3000 3500 4000Minutes

(AU

)

120572-carotene

120573-carotene


000 002 004 006 008 01015

20

25

30

35

40

45

50

DPP

H ra

dica

l sca

veng

ing

activ

ity (

)


a a

bc d





000 002 004 006 008 010minus01

00

01

02

03

04

05

06

07

08

Abso

rban

ce (Aring

)


a bc

d

e





000 002 004 006 008 01012

14

16

18

20

22

24

26

28

30

Met

al ch

elat

ion

activ

ity (

)


b

a

c

d

e





0 50 100 150 200 250 300

092

093

094

095

096

097

Abso

rban

ce (Aring

)

Time (s)

00010005001

00501

d ef f f fa

b b

c cc

jg

h hi i i

k l l

m m

n

o o p op







0 20 40 60 80 100 120

0

10

20

30

40

50

60

Prev

entio

n of

per

oxid

atio

n (

)

Time (hours)

00010005001

00501

a

b

c

d

eo

e

f

g

mh

ni

k

ql

p

r

s

t

j




000 002 004 006 008 010

0

20

40

60

80

100

Supe

roxi

de sc

aven

ging

activ

ity (

)


aa

b

c

d




2O2

minus

+ 2H+ 997888rarr H2O2+O2 (9)



2O2is a



000 002 004 006 008 01020

30

40

50

60

70

80

90

100

Inhi

bitio

n of

den

atur

atio

n (

)


a

b

c

d d






4 Conclusion




Concentrations()

Antioxidant assay

DPPHradical



Metalchelation

()

ABTSradical




()




Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


000 002 004 006 008 01012

14

16

18

20

22

24

26

28

30

Met

al ch

elat

ion

activ

ity (

)


b

a

c

d

e





0 50 100 150 200 250 300

092

093

094

095

096

097

Abso

rban

ce (Aring

)

Time (s)

00010005001

00501

d ef f f fa

b b

c cc

jg

h hi i i

k l l

m m

n

o o p op







0 20 40 60 80 100 120

0

10

20

30

40

50

60

Prev

entio

n of

per

oxid

atio

n (

)

Time (hours)

00010005001

00501

a

b

c

d

eo

e

f

g

mh

ni

k

ql

p

r

s

t

j




000 002 004 006 008 010

0

20

40

60

80

100

Supe

roxi

de sc

aven

ging

activ

ity (

)


aa

b

c

d




2O2

minus

+ 2H+ 997888rarr H2O2+O2 (9)



2O2is a



000 002 004 006 008 01020

30

40

50

60

70

80

90

100

Inhi

bitio

n of

den

atur

atio

n (

)


a

b

c

d d






4 Conclusion




Concentrations()

Antioxidant assay

DPPHradical



Metalchelation

()

ABTSradical




()




Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


0 20 40 60 80 100 120

0

10

20

30

40

50

60

Prev

entio

n of

per

oxid

atio

n (

)

Time (hours)

00010005001

00501

a

b

c

d

eo

e

f

g

mh

ni

k

ql

p

r

s

t

j




000 002 004 006 008 010

0

20

40

60

80

100

Supe

roxi

de sc

aven

ging

activ

ity (

)


aa

b

c

d




2O2

minus

+ 2H+ 997888rarr H2O2+O2 (9)



2O2is a



000 002 004 006 008 01020

30

40

50

60

70

80

90

100

Inhi

bitio

n of

den

atur

atio

n (

)


a

b

c

d d






4 Conclusion




Concentrations()

Antioxidant assay

DPPHradical



Metalchelation

()

ABTSradical




()




Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


000 002 004 006 008 01020

30

40

50

60

70

80

90

100

Inhi

bitio

n of

den

atur

atio

n (

)


a

b

c

d d






4 Conclusion




Concentrations()

Antioxidant assay

DPPHradical



Metalchelation

()

ABTSradical




()




Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



Concentrations()

Antioxidant assay

DPPHradical



Metalchelation

()

ABTSradical




()




Acknowledgment


References










































Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






















Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of










Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Documents

Research Article Antioxidative Evaluation of -and from Crude Palm Oildownloads.hindawi.com/journals/jamc/2013/351671.pdf · 2019-07-31 · In Vitro Antioxidative Evaluation of -and