Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 489071 8 pageshttpdxdoiorg1011552013489071

Research ArticleAntioxidant Potential and Oil Composition ofCallistemon viminalis Leaves

Muhammad Zubair1 Sadia Hassan1 Komal Rizwan1 Nasir Rasool1

Muhammad Riaz1 M Zia-Ul-Haq2 and Vincenzo De Feo3

1 Department of Chemistry Government College University Faisalabad 38000 Pakistan2Department of Pharmacognosy University of Karachi Karachi 75270 Pakistan3 Department of Pharmaceutical and Biomedical Sciences University of Salerno 84100 Salerno Italy

Correspondence should be addressed to Vincenzo De Feo defeounisait

Received 10 December 2012 Accepted 15 January 2013

Academic Editors A Bosabalidis J C Parajo and B Vyskot

Copyright copy 2013 Muhammad Zubair et al 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 study was designed to investigate the antioxidant potential and oil composition of Callistemon viminalis leaves GC-MS analysis of the n-hexane extract revealed the presence of 40 compounds Leaves contained appreciable levels of total phenoliccontents (027ndash085GAEmgg) and total flavonoid contents (225ndash796CEmgg) DPPH radical scavenging IC

50and inhibition

of linoleic acid peroxidation were found to be in the ranges of 284ndash562 120583gml and 401ndash702 respectively The haemolytic effectof the plant leaves was found in the range of 179ndash495 The antioxidant activity of extracts was also studied using sunflower oilas an oxidative substrate and found that it stabilized the oil The correlation between the results of different antioxidant assays andoxidation parameters of oil indicated that leavesrsquo methanolic extract exhibiting higher TPC and TFC and scavenging power wasalso more potent for enhancing the oxidative stability of sunflower oil

1 Introduction

The genus Callistemon belongs to family Myrtaceae that hasa great medicinal importance The majority of Callistemonspecies are found in east and southeast of Australia Phy-tochemical studies of different callistemon species revealedthe presence of different monoterpenes sesquiterpenesflavonoids Callistemon species are used for forestry essentialoil production farm treewindbreak plantings degraded landreclamation and as bioindicators for environmental man-agement and ornamental horticulture among other applica-tions [1 2] Earlier phytochemical explorations of membersof this genus resulted in the identification of C-methylflavonoids triterpenoids and phloroglucinol derivatives [3ndash6] Moreover some medicinal properties like antimicrobialantistaphylococcal antithrombin and nematicidal activitiesand larvicidal and pupicidal values have been reported forthis genus [7] Callistemon viminalis (family Myrtaceae) isa small tree or shrub with pendulous foliage although someforms are more pendulous than othersThis is an ornamental

plant commonly known as bottlebrush that is found in severalareas with the exception of localities extremely cold anddry It is also found along the streets and in the botanicalgardens [1ndash3] C viminalis is edible and its leaves are atea substitute and have a delightfully refreshing flavour andfragrance Antihelminthic and antibacterial activities of Cviminalis various parts have been reported [8] It has beenused to prepare a hot drink locally referred to as ldquoteardquo for thetreatment of gastroenteritis diarrhea and skin infections [9]As part of our studies on exploringmedicinal flora of Pakistanfor their compositional nutritional and antioxidant potential[10ndash16] we studied the plant C viminalis leaves to explore itsantioxidant potential and oil composition

2 Materials and Methods

21 Materials The fresh leaves of the fully matured plant Cviminalis were collected on the basis of intensive review andethnopharmacological information from Botanical GardenUniversity of Agriculture Faisalabad Pakistan (A plane

2 The Scientific World Journal

Soaked in petroleum ether

Petroleum ether extractResidue

Soaked in chloroform

Chloroform extract Residue

Soaked in ethylacetate

Ethylacetate extract

Soaked in acetone

Acetone extract

Residue

Residue

Soaked in absolutemethanol

Absolute methanol extract

Residue

Soaked in 95 methanol

Residue

Residue

Residue

Soaked in methanolwater(91)

90 methanol extract

95 methanol extract

(23 g)

(19 g)

(25 g)

(22 g)

(215 g)

(235 g)

(195 g)

119899-Butanol extract(24 g)

Soaked in 119899-Butanol

Powdered plant material (1000 g)

Figure 1 Schematic diagram showing preparation of extracts of C viminalis leaves

region latitude 31∘-261015840 N longitude 73∘-061015840 E and altitude1844 meters above main sea level) and further identified bya Taxonomist Dr Mansoor Hameed from Department ofBotany University of Agriculture Faisalabad Pakistan

22 Sample Preparation The plant leaves were washed withdistilled water and then shade driedThe grinded fine powderof leaves was extracted with petroleum ether (2 times 2 L) for6 h at room temperature After filtering the extract was con-centrated through rotary vacuum evaporator (Eyela TokyoRikakikai Co Ltd Japan) This process was repeated thriceto obtain a sufficient quantity of petroleum ether extractTheremaining plant residue was further extracted with other dif-ferent polarity-based solvents and obtained successively chlo-roform ethylacetate acetone n-butanol absolute methanol95 methanol (95 5 methanol water vv) and 90methanol (90 10 methanol water vv) extracts (Figure 1)

All obtained extracts after drying were stored at minus4∘C tillfurther analysis

23 Preparation of n-Hexane Extract for GC-MS AnalysisThe plant material (10 g of powdered leaves) was extractedwith 300mL n-hexane by using a Soxhlet apparatus for 6 hThe obtained n-hexane extract was filtered and evaporatedby using a rotary evaporator and freeze dryer respectively togive the crude dried extract The dried extract was stored at4∘C until used

24 Evaluation of Antioxidant Activity The plant leaves totalphenolic contents (TPCs) total flavonoid contents (TFCs)DPPH radical scavenging IC

50 and inhibition of linoleic

acid peroxidation were determined by the followingmethodsdescribed by Rasool et al [17] Reducing the potential of theplant extracts was also determined [18]

The Scientific World Journal 3

25 Haemolytic Activity Haemolytic activity of the plant wasevaluated by following the already reported procedure [19]

26 Determination of Antioxidant Efficacy Using SunflowerOil as Oxidation Substrate

261 Stabilization of Sunflower Oil The crude concentratedvarious extracts of the plant were separately added into thepreheated (50∘C) refined bleached and deodorized sun-flower oil (SO) at concentration of 300 ppm (ww) The oilsamples were stirred for 30 minutes at 50∘C for uniform dis-persion All oil samples were separately stabilized and storedin 100mL airtight bottle A control sample was also prepared(without extract) under the same set of analytical conditionsSamples were stored at room temperature Synthetic antioxi-dant (BHT)was employed at its legal limit of 200 ppm to com-pare the efficacy of extracts Stabilized and control oil samples(100mL) were placed in dark brown airtight glass bottleswith narrow necks and subjected to accelerated storage in anelectric hot air oven (IM-30 Irmeco Gmbh amp Co Germany)at 60∘C for 28 days All oil samples were prepared in triplicateOil samples were taken after every 7-day intervals

262 Measurement of Oxidation Parameters of SunflowerOil The oxidative deterioration level was assessed by themeasurement of peroxide value (PV) free fatty acids (FFAs)conjugate diene (CD) conjugate triene (CT) and p-anisidinevalues Determination of the FFA and PV of stabilized andcontrol sunflower oil samples weremade following the AOCSOfficial methods Cd 8ndash53 and F 9a-44 respectively [20] Theoxidation products such as conjugated dienes and conjugatedtrienes were analyzed by following the IUPAC method IID23 [21] The absorbance was noted at 232 and 268 nmrespectively The determination of the p-anisidine value wasmade following an IUPAC method II D 26 [21]

27 Oil Analysis TheGC-MS analysis of the n-hexane extractwas performed using GC 6850 network gas chromatographicsystem equipped with 7683B series auto injector and 5973 iinert mass selective detector (Agilent Technologies USA)Compounds were separated on anHP-5MS capillary columnhaving 5 phenyl polysiloxane a stationary phase with thecolumn length 300m internal diameter 025mm and filmthickness 025120583m The temperature of injector port was300∘C and 10 120583L of sample was injected in the split modewith split ratio 30 1 The helium was used as the carrier gasat constant flow with the flow rate of 15mLmin The tem-perature program used for column oven was an initialtemperature 150∘Cand held for 1min then ramped at a rate of10∘Cmin up to 290∘C and finally held at this temperature for5min The temperature of MSD transfer line was 300∘C Formass spectra determination MSD was operated in electromionization (EI) mode with the ionization energy of 70 eVwhile the mass range scanned was 3ndash500mz The temper-ature of ion source was 230∘C and that of MS quadrupole150∘C The identification of components was based on thecomparison of their mass spectra with those of NIST massspectral library [22 23]

28 Statistical Analysis Each sample was analyzed individu-ally in triplicate and data were reported as mean (119899 = 3 times 3 times1) plusmn standard deviation (119899 = 3 times 3 times 1) Data were analyzedby analysis of variance (ANOVA) usingMinitab 2000 version132 statistical software (Minitab Inc Pennysylvania USA)

3 Results and Discussion

31 Antioxidant Analysis The yield (g100 g) of variousextracts from the plant C viminalis leaves using differentsolvents ranged from 190 to 25The amounts of TPC andTFC (Table 1) from plant leaves in different solvent systemswere found to be ranged from 027 to 085GAE (mgg ofleaves extracts) and 225ndash796CE (mgg of leaves extracts)respectively The ability of different solvents to extract TPCwas found asfollows absolute methanol gt 90 methanol gtchloroform gt acetone gt 95 methanol gt ethylacetate gt n-butanol gt petroleum ether The effect of different solventson TFC values was found in the following order absolutemethanol gt 95 methanol gt chloroform gt acetone gt 90methanol gt ethylacetate gt n-butanol gt petroleum etherThese differences in the amount of TPC and TFC may bedue to the varied efficiency of the extracting solvents to dis-solve endogenous compounds The leaves extracts exhibiteddifferent radical scavenging activity having IC

50value 284ndash

562120583gmL Absolute methanolic extract exhibited lowestIC50

(284120583gmL) followed by 95 methanol (341 120583gmL)chloroform (382 120583gmL) acetone (414 120583gmL) 90 meth-anol (452 120583gmL) ethylacetate (458 120583gmL) n-butanol(521 120583gmL) and petroleum ether (562 120583gmL) extractsThe free radical scavenging activity of absolute methanoland 95 methanol extracts was superior to that of othersolvent extracts However All extracts offered slightly lessscavenging activity as compared to the synthetic antioxidantBHT (192 120583gmL) The nature and amount of secondarymetabolites of the plant cause the variation in free radicalscavenging ability [24] The free radical scavenging activitydepends upon the chemical composition of extracts Percentinhibition of linoleic acid oxidation ranged from 401 to702 The absolute methanolic extract exhibited the highestinhibition of linoleic acid oxidation (702) and petroleumether exhibited the lowest inhibition (401) When theresults of inhibition of linoleic acid oxidation were com-pared with standard BHT (928) all the samples showedsignificantly (119875 lt 005) less antioxidant activity (Table 1)The order of inhibition of linoleic acid oxidation offered byvarious extracts of leaves was as follows BHT gt absolutemethanol gt 95 methanol gt chloroform gt acetone gt 90methanol gt ethylacetate gt n-butanol gt petroleum ether

Results of the present study showed that among all thesolvent extracts absolute methanolic extract of plant leavesextracted the highest amount of TPC and TFC which alsodemonstrated the highest antioxidant activity as measuredby DPPH radical scavenging and inhibition of linoleic acidoxidation This may be due to the high polarity of methanolwhereas petroleum ether demonstrated the least antioxi-dant activity probably because of its low polarity Previousreports [25 26] also revealed that the methanolic extracts ofplant materials offer more effective antioxidants Antioxidant

4 The Scientific World Journal

Table 1 Antioxidant activity of C viminalis leavesa

Extractsfractionstandard Yield(g100 g)

Total phenolic contentsb(mgg)

Total flavonoid contentsc(mgg)

DPPH IC50(120583gmL)

Inhibition in linoleicacid system ()

Petroleum ether 230 plusmn 004 027 plusmn 0001 225 plusmn 004 562 plusmn 054 401 plusmn 052Chloroform 190 plusmn 002 071 plusmn 0007 653 plusmn 006 382 plusmn 045 664 plusmn 077Ethylacetate 215 plusmn 003 048 plusmn 0005 511 plusmn 004 458 plusmn 051 541 plusmn 052n-Butanol 240 plusmn 004 044 plusmn 0004 396 plusmn 004 521 plusmn 062 482 plusmn 052Acetone 195 plusmn 002 063 plusmn 0007 643 plusmn 006 414 plusmn 052 568 plusmn 065Absolute methanol 250 plusmn 004 085 plusmn 0009 796 plusmn 008 284 plusmn 019 702 plusmn 07795 methanol 220 plusmn 003 052 plusmn 0006 669 plusmn 007 341 plusmn 041 682 plusmn 07790 methanol 235 plusmn 003 075 plusmn 0007 595 plusmn 007 452 plusmn 051 564 plusmn 061BHT mdash mdash mdash 192 plusmn 022 928 plusmn 091aValues are mean plusmn SD of three separate experimentsbTotal phenolic contents expressed as gallic acid equivalentcTotal flavonoid contents are expressed as catechin equivalent

compounds were extracted from Catharanthus roseus shootsand found that methanol gave the maximum antioxidantyield [17] Similar results were observed in the present inves-tigations as methanol was most effective to extract antioxida-tive compounds

32 Haemolytic Activity Haemolytic activity was analyzedagainst human red blood cells (RBCs) using Triton X-100as positive control The lysis of RBCs caused by the plantextracts was observed Ethylacetate extract showed thehighest haemolytic effect (495) followed by petroleumether (448) 90 methanol (394) chloroform (261)95 methanol (249) acetone (233) absolute methanol(203) and n-butanol (179) extracts respectively Thehaemolytic effect of n-butanol and absolute methanol extractwas less then other extracts (Table 2) The mechanical stabil-ity of the erythrocytic membrane is a good indicator of theeffect of various in vitro studies by various compounds forthe screening of cytotoxicity The percentage lysis of humanerythrocytes was below 50 for all samples All these resultswere in safe range Though it can be expected that the plantextracts have a minor cytotoxicity [19 27] So pharmacolog-ically this kplant may be safe to use for human beings as asource of potential drug

33 Oil Analysis The chemical compounds identified byGC-MS analysis of n-hexane extract presented in Table 3The mass spectrum of each compound was compared withthat in the NIST 05 library Almost 40 different com-pounds were identified in n-hexane extract of plant leavesThe major compounds determined in the n-hexane extractwere 25568a-pentamethyl-trans-4a56788a-hexahydro-gamma-chromene (2760) (10E12E)-1012-tetradecadienylacetate (1162) Z-7-tetradecenal (498) 13-cyclohexadi-ene (397) respectively Some of the compounds werepresent in traces or less concentration as compared to otheridentified compounds The n-hexane extract may have somefatty acidsmethyl esters which may be implicated in someantioxidant and antimicrobial activities During the literaturereview it was found that the activities of some phytocompo-nents such as flavonoids palmitic acid (hexadecanoic acid

Table 2 Percentage haemolysis caused by C viminalis leaves differ-ent extractsa

Extracts Percentage ofhaemolysis

Petroleum ether 448 plusmn 001Chloroform 261 plusmn 002Ethylacetate 495 plusmn 005Acetone 233 plusmn 002n-Butanol 179 plusmn 001Absolute methanol 203 plusmn 00295 methanol 249 plusmn 00490 methanol 394 plusmn 003Phosphate Buffer Saline (PBS) 000Triton X-100 998 plusmn 101aValues are mean plusmn SD of three separate experiments

ethyl ester and n-hexadecanoic acid) unsaturated fatty aciddocosatetraenoic acid and octadecatrienoic acid as antimi-crobial antiinflammatory and antioxidant activities [28]Therefore the chemical constituents found in may C vimi-nalis leaves play major roles in the biological activities andpharmacological properties

34 Stabilization of Sunflower Oil Formation of free fattyacids (FFAs) might be an important measure of rancidityof foods FFAs are formed due to hydrolysis of triglyceridesand may get promoted by the reaction of oil with moisture[29] FFA content went on increasing with the increase instorage period for all the samples but no regular pattern ofincrease could be observed Control exhibited the highestFFA while sunflower oil stabilized with BHT exhibited least(Figure 2) Initially there was no increase in FFA of stabilizedoil samples but after seven days of storage an increase thatwas observed showed the free fatty acid (FFA) contents of oilsamples stabilized with leaves extract of C viminalis underambient storage conditions All the oil samples stabilized

The Scientific World Journal 5

Table 3 GC-MS analysis of n-hexane extract of C viminalis leaves

Peak number Retention time Compounds area1 5411 n-Octane 0872 6350 2-Methyloctane 0623 7754 o-Xylene 0564 7882 3-Methyloctane 0685 8964 n-Nonane 1446 13004 13-Cyclohexadiene 3977 16301 Undecane 1078 19590 n-Dodecane 1029 22354 2-Isopropyl-5-methylphenol 16410 23309 Exo-2-hydroxy cineole 12611 24266 Etyhl-59-dimethyl-24-decadienoate 39512 25413 3-Methyl-5-(26-dimethylheptyl)-15-pent-2-enolide 12013 25497 n-Tetradecane 12714 26880 4-Oxo-120573-isodamascol 07115 28026 n-Pentadecane 06216 28170 24-Di-tert-butylphenol 27517 28391 Durohydroquinone 09218 28867 (10E12E)-1012-Tetradecadienyl acetate 116219 29027 25568a-Pentamethyl-trans-4a56788a-hexahydro-gamma-chromene 276020 29783 (minus)-Spathulenol 20721 30117 Hexadecane 23422 31919 10-Methylicosane 12523 33537 Heneicosane 30924 33583 Origanene 05425 35008 n-Nonadecane 06226 35837 Eicosanoic acid 33727 36382 n-Tetracosane 18628 37672 154-Dibromotetrapentacontane 04929 37791 Trans-phytol 10530 38074 Cis-9cis-12-octadecadienoic acid 23831 38157 Z-7-Tetradecenal 49832 38411 Stearic acid 06033 38892 n-Tetratricoaconate 08134 40175 Ergost-722-dien-911-epoxy-3-ol acetate(ester) 07535 40558 Furostan-12-one 23736 42576 Mono(2-ethylhexyl)phthalate 22137 44934 Trans-squalene 17438 45506 n-Hexatriacontane 05039 47309 120572-Tocopherol-120573-D-mannoside 08240 49184 Gamma-sitosterol 242

with plant extracts were found to show a slow followed bya gradual increase in free fatty acid contents The lowervalues of free fatty acid contents of stabilized oil samples thancontrol indicated the effectiveness of leaves extracts as naturalantioxidant in retarding the free fatty acid contents Of allextracts methanol extract was most efficient after BHT toinhibit the formation of FFA contents

Peroxide value (PV) is usually used to evaluate the extentof primary oxidation products in oils The highest PV wasobserved for control sample followed by petroleum ether gtchloroform gt ethylacetate gt n-butanol gt acetone gt 90methanol gt 95 methanol gt absolute methanol gt BHTrespectively All used extracts of the plant controlled peroxide

value appreciably revealing good antioxidant efficacy ofextracts in stabilization of oil A regular increase in PV asa function of storage time was observed for all the samplesat all intervals Initially the difference in peroxide contentof control and stabilized oil samples was not noticeable itbecame significant (119875 lt 005) just after heating up to one day(Figure 3)

The results for para-anisidine values (PAVs) which usu-ally determine the amount of aldehyde in oils presented inFigure 4 The control sample showed the maximum increasein para-anisidine values indicating a higher rate of secondaryproduct formation A slow increase in PAV of stabilizedsunflower oil as compared with the control indicating the

6 The Scientific World Journal

0

02

04

06

08

1

12

0 7 14 21 28

Free

fatty

acid

s (

)

Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Figure 2 Free fatty acid contents () of sunflower oil stabilizedwithC viminalis leaves extracts

0

2

4

6

8

10

12

14

16

0 7 14 21 28

Pero

xide

val

ues (

meq

kg)

Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Figure 3 Peroxide values of sunflower oil stabilized with C vimi-nalis leaves extracts

antioxidant potential of the plant leaves Adecreasing order ofstability of oil treatedwith different extracts of plant regardingpara-anisidine values was found to be BHT gt absolutemethanol gt acetone gt 90 methanol gt 95 methanol gt n-butanol gt ethylacetate gt chloroform gt petroleum ether gtcontrol

The formation of conjugated diene (Figure 5) and triene(Figure 6) analyzed for the control and stabilized sunflower

0

2

4

6

8

10

12

14

16

18

0 7 14 21 28

Incr

ease

in P

AV

Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Figure 4 Relative increase in p-ansidine values of sunflower oilstabilized with C viminalis leaves extracts

0

2

4

6

8

10

12

14

16

18

0 7 14 21 28Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Con

juga

ted

dien

e (1205761

cm 120582232

nm)

Figure 5 Relative increase in conjugated dienes content (CD) ofsunflower oil stabilized with C viminalis leaves extracts

oil respectively Highest contents were observed for controlindicating greater intensity of oxidation

The determination of CD and CT is a good measure ofthe oxidative state of oils [30] and thus a good indicator ofeffectiveness of antioxidants CD and CT contents went onincreasing with the increase in storage time A slow increasein CD and CT of the stabilized sunflower oil as comparedwith those of the control indicated the antioxidant potential

The Scientific World Journal 7

0

1

2

3

4

5

6

0 7 14 21 28Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Con

juga

ted

trie

ne (1205761

cm 120582232

nm)

Figure 6 Relative increase in conjugated trienes content (CT) ofsunflower oil stabilized with C viminalis leaves extracts

of the C viminalis leaves Stabilized sunflower oil conjugateddiene and triene contens values were found to be in range of166ndash1354 and 12ndash389 (1205761 cm 232 nm) respectively Absolutemethanolic extract showed the lowest values and petroleumether exhibited highest All extracts played a prominent rolefor stabilization of sunflower oil but after standard BHTmethanol extract was most efficient to stabilize the oil

4 Conclusions

The results of the present study concluded that the plantpossessed considerable antioxidant potential and it may alsobe used to stabilize the edible sunflower oil Hence the plantleaves investigated can be explored as a potential antioxidantsource of natural origin Cytotoxicity of plant extracts againsthuman erythrocytes was checked and it was in safe range sothe investigated plant may be safe use for pharmaceutical andnatural therapies

Conflict of Interests

The authors do not have a direct financial relation with thecommercial identities mentioned in this paper that mightlead to a conflict of interests

References

[1] R D Spencer and P F Lumley ldquoCallistemonrdquo in Flora of NewSouth Wales G J Harden Ed vol 2 pp 168ndash173 New SouthWales University Press Sydney Australia 1991

[2] G S Wheeler ldquoMaintenance of a narrow host range by Oxyopsvitiosa a biological control agent of Melaleuca quinquenerviardquoBiochemical Systematics and Ecology vol 33 no 4 pp 365ndash3832005

[3] J W Wrigley and M Fagg Bottlebrushes Paperbarks and TeaTrees and All Other Plants in the Leptospermum Alliance Angusand Rovertson Sydney Australia 1993

[4] E Wollenweber R Wehde M Dorr G Lang and J F StevensldquoC-Methyl-flavonoids from the leaf waxes of some MyrtaceaerdquoPhytochemistry vol 55 no 8 pp 965ndash970 2000

[5] F Huq and L N Misra ldquoAn alkenol and C-methylated flavonesfrom Callistemon lanceolatus leavesrdquo Planta Medica vol 63 no4 pp 369ndash370 1997

[6] R S Varma andM R Parthasarathy ldquoTriterpenoids of Calliste-mon lanceolatus leavesrdquo Phytochemistry vol 14 no 7 pp 1675ndash1676 1975

[7] C Gomber and S Saxena ldquoAnti-staphylococcal potential ofCallistemon rigidusrdquo Central European Journal of Medicine vol2 no 1 pp 79ndash88 2007

[8] S K Srivastava A Ahmad K V Syamsunder K K Aggarwaland S P S Khanuja ldquoEssential oil composition of Callistemonviminalis leaves from Indiardquo Flavour and Fragrance Journal vol18 no 5 pp 361ndash363 2003

[9] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobial Research vol 12 pp 564ndash582 1999

[10] K Rizwan M Zubair N Rasool M Riaz M Zia-Ul-Haqand V de Feo ldquoPhytochemical and Biological Studies of Agaveattenuatardquo International Journal of Molecular Sciences vol 13pp 6440ndash6451 2012

[11] M N Bari M Zubair K Rizwan et al ldquoBiological activities ofOpuntia Monacantha cladodesrdquo Journal of Chemical SocietyPakistan vol 34 pp 990ndash995 2012

[12] M Zubair K Rizwan N Rasool N Afshan M Shahid andV Ahmed ldquoAntimicrobial potential of various extract and frac-tions of leaves of Solanum nigrumrdquo International Journal ofPhytomedicine vol 3 no 1 pp 63ndash67 2011

[13] N Rasool K Rizwan M Zubair et al ldquoAntioxidant activityof various extracts and organic fractions of Ziziphus jujubardquoInternational Journal of Phytomedicine vol 3 pp 346ndash352 2011

[14] M Zia-Ul-Haq S Cavar M Qayum I Imran and V DefeoldquoCompositional studies antioxidant and antidiabetic activitiesof Capparis decidua (Forsk) Edgewrdquo International Journal ofMolecular Sciences vol 12 pp 8846ndash8861 2011

[15] M Zia-Ul-Haq S Ahmad S Iqbal D L Luthria and RAmarowicz ldquoAntioxidant potential of lentil cultivars commonlyconsumed in Pakistanrdquo Oxidation Communication vol 34 pp819ndash831 2011

[16] M Zia-Ul-Haq S Ahmad L Calani et al ldquoCompositionalstudy and antioxidant potential of Ipomoea hederacea Jacq andLepidium sativum L sedesrdquo Molecules vol 17 pp 10306ndash103212012

[17] N Rasool K Rizwan M Zubair K U R Naveed I Imran andV U Ahmed ldquoAntioxidant potential of different extracts andfractions of Catharanthus roseus shootsrdquo International Journalof Phytomedicine vol 3 no 1 pp 108ndash114 2011

[18] B Sultana F Anwar and R Przybylski ldquoAntioxidant activityof phenolic components present in barks of Azadirachta indicaTerminalia arjunaAcacia nilotica andEugenia jambolana Lamtreesrdquo Food Chemistry vol 104 no 3 pp 1106ndash1114 2007

[19] W A Powell C M Catranis and C A Maynarrd ldquoDesign ofself processing antibacterial peptide of plant protectionrdquo Lettersin Applied Microbiology vol 31 pp 163ndash165 2000

[20] American Oil Chemists Society (AOCS) Official and Recom-mended Practices of the American Oil Chemists Society AOCSPress Champaign Ill USA 5th edition 1997

8 The Scientific World Journal

[21] International Union of Pure And Applied Chemistry (IUPAC)Standard Methods For the Analysis of Oils Fats and DerivativesC Paquot andA Hautfenne Eds Blackwell Scientific LondonUK 7th edition 1987

[22] Y Massada Analysis of Essential Oils by Gas ChromatographyandMass Spectrometry JohnWileyampSonsNewYorkNYUSA1976

[23] Mass Spectral Library NISTEPANIHUSA 2002 httpwwwnistgovsrdnistlahtm

[24] Y Sudjaroen R Haubner G Wurtele et al ldquoIsolation andstructure elucidation of phenolic antioxidants from Tamarind(Tamarindus indica L) seeds and pericarprdquo Food and ChemicalToxicology vol 43 no 11 pp 1673ndash1682 2005

[25] P Siddhuraju and K Becker ldquoAntioxidant properties of varioussolvent extracts of total phenolic constituents from three dif-ferent agroclimatic origins of drumstick tree (Moringa oleiferaLam) leavesrdquo Journal of Agricultural and Food Chemistry vol51 no 8 pp 2144ndash2155 2003

[26] S A S Chatha F AnwarMManzoor and J U R Bajwa ldquoEval-uation of the antioxidant activity of rice bran extracts usingdifferent antioxidant assaysrdquo Grasas y Aceites vol 57 no 3 pp328ndash335 2006

[27] P Sharma and J D Sharma ldquoIn vitro hemolysis of human eryth-rocytesmdashby plant extracts with antiplasmodial activityrdquo Journalof Ethnopharmacology vol 74 no 3 pp 239ndash243 2001

[28] P Kumar S Kumaravel and C Lalitha ldquoScreening of antioxi-dant activity total phenolics and GC-MS study of Vitex negun-dordquo African Journal of Biotechnology vol 4 pp 191ndash195 2010

[29] N Frega M Mozzon and G Lercker ldquoEffects of free fatty acidson oxidative stability of vegetable oilrdquo Journal of the AmericanOil Chemistsrsquo Society vol 76 no 3 pp 325ndash329 1999

[30] S H Yoon S K KimM G Shin and K H Kim ldquoComparativestudy of physical methods for lipid oxidation measurement inoilsrdquo Journal of the American Oil Chemistsrsquo Society vol 62 no10 pp 1487ndash1489 1985

4 The Scientific World Journal

Table 1 Antioxidant activity of C viminalis leavesa

Extractsfractionstandard Yield(g100 g)

Total phenolic contentsb(mgg)

Total flavonoid contentsc(mgg)

DPPH IC50(120583gmL)

Inhibition in linoleicacid system ()

Petroleum ether 230 plusmn 004 027 plusmn 0001 225 plusmn 004 562 plusmn 054 401 plusmn 052Chloroform 190 plusmn 002 071 plusmn 0007 653 plusmn 006 382 plusmn 045 664 plusmn 077Ethylacetate 215 plusmn 003 048 plusmn 0005 511 plusmn 004 458 plusmn 051 541 plusmn 052n-Butanol 240 plusmn 004 044 plusmn 0004 396 plusmn 004 521 plusmn 062 482 plusmn 052Acetone 195 plusmn 002 063 plusmn 0007 643 plusmn 006 414 plusmn 052 568 plusmn 065Absolute methanol 250 plusmn 004 085 plusmn 0009 796 plusmn 008 284 plusmn 019 702 plusmn 07795 methanol 220 plusmn 003 052 plusmn 0006 669 plusmn 007 341 plusmn 041 682 plusmn 07790 methanol 235 plusmn 003 075 plusmn 0007 595 plusmn 007 452 plusmn 051 564 plusmn 061BHT mdash mdash mdash 192 plusmn 022 928 plusmn 091aValues are mean plusmn SD of three separate experimentsbTotal phenolic contents expressed as gallic acid equivalentcTotal flavonoid contents are expressed as catechin equivalent

compounds were extracted from Catharanthus roseus shootsand found that methanol gave the maximum antioxidantyield [17] Similar results were observed in the present inves-tigations as methanol was most effective to extract antioxida-tive compounds

32 Haemolytic Activity Haemolytic activity was analyzedagainst human red blood cells (RBCs) using Triton X-100as positive control The lysis of RBCs caused by the plantextracts was observed Ethylacetate extract showed thehighest haemolytic effect (495) followed by petroleumether (448) 90 methanol (394) chloroform (261)95 methanol (249) acetone (233) absolute methanol(203) and n-butanol (179) extracts respectively Thehaemolytic effect of n-butanol and absolute methanol extractwas less then other extracts (Table 2) The mechanical stabil-ity of the erythrocytic membrane is a good indicator of theeffect of various in vitro studies by various compounds forthe screening of cytotoxicity The percentage lysis of humanerythrocytes was below 50 for all samples All these resultswere in safe range Though it can be expected that the plantextracts have a minor cytotoxicity [19 27] So pharmacolog-ically this kplant may be safe to use for human beings as asource of potential drug

33 Oil Analysis The chemical compounds identified byGC-MS analysis of n-hexane extract presented in Table 3The mass spectrum of each compound was compared withthat in the NIST 05 library Almost 40 different com-pounds were identified in n-hexane extract of plant leavesThe major compounds determined in the n-hexane extractwere 25568a-pentamethyl-trans-4a56788a-hexahydro-gamma-chromene (2760) (10E12E)-1012-tetradecadienylacetate (1162) Z-7-tetradecenal (498) 13-cyclohexadi-ene (397) respectively Some of the compounds werepresent in traces or less concentration as compared to otheridentified compounds The n-hexane extract may have somefatty acidsmethyl esters which may be implicated in someantioxidant and antimicrobial activities During the literaturereview it was found that the activities of some phytocompo-nents such as flavonoids palmitic acid (hexadecanoic acid

Table 2 Percentage haemolysis caused by C viminalis leaves differ-ent extractsa

Extracts Percentage ofhaemolysis

Petroleum ether 448 plusmn 001Chloroform 261 plusmn 002Ethylacetate 495 plusmn 005Acetone 233 plusmn 002n-Butanol 179 plusmn 001Absolute methanol 203 plusmn 00295 methanol 249 plusmn 00490 methanol 394 plusmn 003Phosphate Buffer Saline (PBS) 000Triton X-100 998 plusmn 101aValues are mean plusmn SD of three separate experiments

ethyl ester and n-hexadecanoic acid) unsaturated fatty aciddocosatetraenoic acid and octadecatrienoic acid as antimi-crobial antiinflammatory and antioxidant activities [28]Therefore the chemical constituents found in may C vimi-nalis leaves play major roles in the biological activities andpharmacological properties

34 Stabilization of Sunflower Oil Formation of free fattyacids (FFAs) might be an important measure of rancidityof foods FFAs are formed due to hydrolysis of triglyceridesand may get promoted by the reaction of oil with moisture[29] FFA content went on increasing with the increase instorage period for all the samples but no regular pattern ofincrease could be observed Control exhibited the highestFFA while sunflower oil stabilized with BHT exhibited least(Figure 2) Initially there was no increase in FFA of stabilizedoil samples but after seven days of storage an increase thatwas observed showed the free fatty acid (FFA) contents of oilsamples stabilized with leaves extract of C viminalis underambient storage conditions All the oil samples stabilized

The Scientific World Journal 5

Table 3 GC-MS analysis of n-hexane extract of C viminalis leaves

Peak number Retention time Compounds area1 5411 n-Octane 0872 6350 2-Methyloctane 0623 7754 o-Xylene 0564 7882 3-Methyloctane 0685 8964 n-Nonane 1446 13004 13-Cyclohexadiene 3977 16301 Undecane 1078 19590 n-Dodecane 1029 22354 2-Isopropyl-5-methylphenol 16410 23309 Exo-2-hydroxy cineole 12611 24266 Etyhl-59-dimethyl-24-decadienoate 39512 25413 3-Methyl-5-(26-dimethylheptyl)-15-pent-2-enolide 12013 25497 n-Tetradecane 12714 26880 4-Oxo-120573-isodamascol 07115 28026 n-Pentadecane 06216 28170 24-Di-tert-butylphenol 27517 28391 Durohydroquinone 09218 28867 (10E12E)-1012-Tetradecadienyl acetate 116219 29027 25568a-Pentamethyl-trans-4a56788a-hexahydro-gamma-chromene 276020 29783 (minus)-Spathulenol 20721 30117 Hexadecane 23422 31919 10-Methylicosane 12523 33537 Heneicosane 30924 33583 Origanene 05425 35008 n-Nonadecane 06226 35837 Eicosanoic acid 33727 36382 n-Tetracosane 18628 37672 154-Dibromotetrapentacontane 04929 37791 Trans-phytol 10530 38074 Cis-9cis-12-octadecadienoic acid 23831 38157 Z-7-Tetradecenal 49832 38411 Stearic acid 06033 38892 n-Tetratricoaconate 08134 40175 Ergost-722-dien-911-epoxy-3-ol acetate(ester) 07535 40558 Furostan-12-one 23736 42576 Mono(2-ethylhexyl)phthalate 22137 44934 Trans-squalene 17438 45506 n-Hexatriacontane 05039 47309 120572-Tocopherol-120573-D-mannoside 08240 49184 Gamma-sitosterol 242

with plant extracts were found to show a slow followed bya gradual increase in free fatty acid contents The lowervalues of free fatty acid contents of stabilized oil samples thancontrol indicated the effectiveness of leaves extracts as naturalantioxidant in retarding the free fatty acid contents Of allextracts methanol extract was most efficient after BHT toinhibit the formation of FFA contents

Peroxide value (PV) is usually used to evaluate the extentof primary oxidation products in oils The highest PV wasobserved for control sample followed by petroleum ether gtchloroform gt ethylacetate gt n-butanol gt acetone gt 90methanol gt 95 methanol gt absolute methanol gt BHTrespectively All used extracts of the plant controlled peroxide

value appreciably revealing good antioxidant efficacy ofextracts in stabilization of oil A regular increase in PV asa function of storage time was observed for all the samplesat all intervals Initially the difference in peroxide contentof control and stabilized oil samples was not noticeable itbecame significant (119875 lt 005) just after heating up to one day(Figure 3)

The results for para-anisidine values (PAVs) which usu-ally determine the amount of aldehyde in oils presented inFigure 4 The control sample showed the maximum increasein para-anisidine values indicating a higher rate of secondaryproduct formation A slow increase in PAV of stabilizedsunflower oil as compared with the control indicating the

6 The Scientific World Journal

0

02

04

06

08

1

12

0 7 14 21 28

Free

fatty

acid

s (

)

Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Figure 2 Free fatty acid contents () of sunflower oil stabilizedwithC viminalis leaves extracts

0

2

4

6

8

10

12

14

16

0 7 14 21 28

Pero

xide

val

ues (

meq

kg)

Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Figure 3 Peroxide values of sunflower oil stabilized with C vimi-nalis leaves extracts

antioxidant potential of the plant leaves Adecreasing order ofstability of oil treatedwith different extracts of plant regardingpara-anisidine values was found to be BHT gt absolutemethanol gt acetone gt 90 methanol gt 95 methanol gt n-butanol gt ethylacetate gt chloroform gt petroleum ether gtcontrol

The formation of conjugated diene (Figure 5) and triene(Figure 6) analyzed for the control and stabilized sunflower

0

2

4

6

8

10

12

14

16

18

0 7 14 21 28

Incr

ease

in P

AV

Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Figure 4 Relative increase in p-ansidine values of sunflower oilstabilized with C viminalis leaves extracts

0

2

4

6

8

10

12

14

16

18

0 7 14 21 28Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Con

juga

ted

dien

e (1205761

cm 120582232

nm)

Figure 5 Relative increase in conjugated dienes content (CD) ofsunflower oil stabilized with C viminalis leaves extracts

oil respectively Highest contents were observed for controlindicating greater intensity of oxidation

The determination of CD and CT is a good measure ofthe oxidative state of oils [30] and thus a good indicator ofeffectiveness of antioxidants CD and CT contents went onincreasing with the increase in storage time A slow increasein CD and CT of the stabilized sunflower oil as comparedwith those of the control indicated the antioxidant potential

The Scientific World Journal 7

0

1

2

3

4

5

6

0 7 14 21 28Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Con

juga

ted

trie

ne (1205761

cm 120582232

nm)

Figure 6 Relative increase in conjugated trienes content (CT) ofsunflower oil stabilized with C viminalis leaves extracts

of the C viminalis leaves Stabilized sunflower oil conjugateddiene and triene contens values were found to be in range of166ndash1354 and 12ndash389 (1205761 cm 232 nm) respectively Absolutemethanolic extract showed the lowest values and petroleumether exhibited highest All extracts played a prominent rolefor stabilization of sunflower oil but after standard BHTmethanol extract was most efficient to stabilize the oil

4 Conclusions

The results of the present study concluded that the plantpossessed considerable antioxidant potential and it may alsobe used to stabilize the edible sunflower oil Hence the plantleaves investigated can be explored as a potential antioxidantsource of natural origin Cytotoxicity of plant extracts againsthuman erythrocytes was checked and it was in safe range sothe investigated plant may be safe use for pharmaceutical andnatural therapies

Conflict of Interests

The authors do not have a direct financial relation with thecommercial identities mentioned in this paper that mightlead to a conflict of interests

References

[1] R D Spencer and P F Lumley ldquoCallistemonrdquo in Flora of NewSouth Wales G J Harden Ed vol 2 pp 168ndash173 New SouthWales University Press Sydney Australia 1991

[2] G S Wheeler ldquoMaintenance of a narrow host range by Oxyopsvitiosa a biological control agent of Melaleuca quinquenerviardquoBiochemical Systematics and Ecology vol 33 no 4 pp 365ndash3832005

[3] J W Wrigley and M Fagg Bottlebrushes Paperbarks and TeaTrees and All Other Plants in the Leptospermum Alliance Angusand Rovertson Sydney Australia 1993

[4] E Wollenweber R Wehde M Dorr G Lang and J F StevensldquoC-Methyl-flavonoids from the leaf waxes of some MyrtaceaerdquoPhytochemistry vol 55 no 8 pp 965ndash970 2000

[5] F Huq and L N Misra ldquoAn alkenol and C-methylated flavonesfrom Callistemon lanceolatus leavesrdquo Planta Medica vol 63 no4 pp 369ndash370 1997

[6] R S Varma andM R Parthasarathy ldquoTriterpenoids of Calliste-mon lanceolatus leavesrdquo Phytochemistry vol 14 no 7 pp 1675ndash1676 1975

[7] C Gomber and S Saxena ldquoAnti-staphylococcal potential ofCallistemon rigidusrdquo Central European Journal of Medicine vol2 no 1 pp 79ndash88 2007

[8] S K Srivastava A Ahmad K V Syamsunder K K Aggarwaland S P S Khanuja ldquoEssential oil composition of Callistemonviminalis leaves from Indiardquo Flavour and Fragrance Journal vol18 no 5 pp 361ndash363 2003

[9] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobial Research vol 12 pp 564ndash582 1999

[10] K Rizwan M Zubair N Rasool M Riaz M Zia-Ul-Haqand V de Feo ldquoPhytochemical and Biological Studies of Agaveattenuatardquo International Journal of Molecular Sciences vol 13pp 6440ndash6451 2012

[11] M N Bari M Zubair K Rizwan et al ldquoBiological activities ofOpuntia Monacantha cladodesrdquo Journal of Chemical SocietyPakistan vol 34 pp 990ndash995 2012

[12] M Zubair K Rizwan N Rasool N Afshan M Shahid andV Ahmed ldquoAntimicrobial potential of various extract and frac-tions of leaves of Solanum nigrumrdquo International Journal ofPhytomedicine vol 3 no 1 pp 63ndash67 2011

[13] N Rasool K Rizwan M Zubair et al ldquoAntioxidant activityof various extracts and organic fractions of Ziziphus jujubardquoInternational Journal of Phytomedicine vol 3 pp 346ndash352 2011

[14] M Zia-Ul-Haq S Cavar M Qayum I Imran and V DefeoldquoCompositional studies antioxidant and antidiabetic activitiesof Capparis decidua (Forsk) Edgewrdquo International Journal ofMolecular Sciences vol 12 pp 8846ndash8861 2011

[15] M Zia-Ul-Haq S Ahmad S Iqbal D L Luthria and RAmarowicz ldquoAntioxidant potential of lentil cultivars commonlyconsumed in Pakistanrdquo Oxidation Communication vol 34 pp819ndash831 2011

[16] M Zia-Ul-Haq S Ahmad L Calani et al ldquoCompositionalstudy and antioxidant potential of Ipomoea hederacea Jacq andLepidium sativum L sedesrdquo Molecules vol 17 pp 10306ndash103212012

[17] N Rasool K Rizwan M Zubair K U R Naveed I Imran andV U Ahmed ldquoAntioxidant potential of different extracts andfractions of Catharanthus roseus shootsrdquo International Journalof Phytomedicine vol 3 no 1 pp 108ndash114 2011

[18] B Sultana F Anwar and R Przybylski ldquoAntioxidant activityof phenolic components present in barks of Azadirachta indicaTerminalia arjunaAcacia nilotica andEugenia jambolana Lamtreesrdquo Food Chemistry vol 104 no 3 pp 1106ndash1114 2007

[19] W A Powell C M Catranis and C A Maynarrd ldquoDesign ofself processing antibacterial peptide of plant protectionrdquo Lettersin Applied Microbiology vol 31 pp 163ndash165 2000

[20] American Oil Chemists Society (AOCS) Official and Recom-mended Practices of the American Oil Chemists Society AOCSPress Champaign Ill USA 5th edition 1997

8 The Scientific World Journal

[21] International Union of Pure And Applied Chemistry (IUPAC)Standard Methods For the Analysis of Oils Fats and DerivativesC Paquot andA Hautfenne Eds Blackwell Scientific LondonUK 7th edition 1987

[22] Y Massada Analysis of Essential Oils by Gas ChromatographyandMass Spectrometry JohnWileyampSonsNewYorkNYUSA1976

[23] Mass Spectral Library NISTEPANIHUSA 2002 httpwwwnistgovsrdnistlahtm

[24] Y Sudjaroen R Haubner G Wurtele et al ldquoIsolation andstructure elucidation of phenolic antioxidants from Tamarind(Tamarindus indica L) seeds and pericarprdquo Food and ChemicalToxicology vol 43 no 11 pp 1673ndash1682 2005

[25] P Siddhuraju and K Becker ldquoAntioxidant properties of varioussolvent extracts of total phenolic constituents from three dif-ferent agroclimatic origins of drumstick tree (Moringa oleiferaLam) leavesrdquo Journal of Agricultural and Food Chemistry vol51 no 8 pp 2144ndash2155 2003

[26] S A S Chatha F AnwarMManzoor and J U R Bajwa ldquoEval-uation of the antioxidant activity of rice bran extracts usingdifferent antioxidant assaysrdquo Grasas y Aceites vol 57 no 3 pp328ndash335 2006

[27] P Sharma and J D Sharma ldquoIn vitro hemolysis of human eryth-rocytesmdashby plant extracts with antiplasmodial activityrdquo Journalof Ethnopharmacology vol 74 no 3 pp 239ndash243 2001

[28] P Kumar S Kumaravel and C Lalitha ldquoScreening of antioxi-dant activity total phenolics and GC-MS study of Vitex negun-dordquo African Journal of Biotechnology vol 4 pp 191ndash195 2010

[29] N Frega M Mozzon and G Lercker ldquoEffects of free fatty acidson oxidative stability of vegetable oilrdquo Journal of the AmericanOil Chemistsrsquo Society vol 76 no 3 pp 325ndash329 1999

[30] S H Yoon S K KimM G Shin and K H Kim ldquoComparativestudy of physical methods for lipid oxidation measurement inoilsrdquo Journal of the American Oil Chemistsrsquo Society vol 62 no10 pp 1487ndash1489 1985

The Scientific World Journal 5

Table 3 GC-MS analysis of n-hexane extract of C viminalis leaves

Peak number Retention time Compounds area1 5411 n-Octane 0872 6350 2-Methyloctane 0623 7754 o-Xylene 0564 7882 3-Methyloctane 0685 8964 n-Nonane 1446 13004 13-Cyclohexadiene 3977 16301 Undecane 1078 19590 n-Dodecane 1029 22354 2-Isopropyl-5-methylphenol 16410 23309 Exo-2-hydroxy cineole 12611 24266 Etyhl-59-dimethyl-24-decadienoate 39512 25413 3-Methyl-5-(26-dimethylheptyl)-15-pent-2-enolide 12013 25497 n-Tetradecane 12714 26880 4-Oxo-120573-isodamascol 07115 28026 n-Pentadecane 06216 28170 24-Di-tert-butylphenol 27517 28391 Durohydroquinone 09218 28867 (10E12E)-1012-Tetradecadienyl acetate 116219 29027 25568a-Pentamethyl-trans-4a56788a-hexahydro-gamma-chromene 276020 29783 (minus)-Spathulenol 20721 30117 Hexadecane 23422 31919 10-Methylicosane 12523 33537 Heneicosane 30924 33583 Origanene 05425 35008 n-Nonadecane 06226 35837 Eicosanoic acid 33727 36382 n-Tetracosane 18628 37672 154-Dibromotetrapentacontane 04929 37791 Trans-phytol 10530 38074 Cis-9cis-12-octadecadienoic acid 23831 38157 Z-7-Tetradecenal 49832 38411 Stearic acid 06033 38892 n-Tetratricoaconate 08134 40175 Ergost-722-dien-911-epoxy-3-ol acetate(ester) 07535 40558 Furostan-12-one 23736 42576 Mono(2-ethylhexyl)phthalate 22137 44934 Trans-squalene 17438 45506 n-Hexatriacontane 05039 47309 120572-Tocopherol-120573-D-mannoside 08240 49184 Gamma-sitosterol 242

with plant extracts were found to show a slow followed bya gradual increase in free fatty acid contents The lowervalues of free fatty acid contents of stabilized oil samples thancontrol indicated the effectiveness of leaves extracts as naturalantioxidant in retarding the free fatty acid contents Of allextracts methanol extract was most efficient after BHT toinhibit the formation of FFA contents

Peroxide value (PV) is usually used to evaluate the extentof primary oxidation products in oils The highest PV wasobserved for control sample followed by petroleum ether gtchloroform gt ethylacetate gt n-butanol gt acetone gt 90methanol gt 95 methanol gt absolute methanol gt BHTrespectively All used extracts of the plant controlled peroxide

value appreciably revealing good antioxidant efficacy ofextracts in stabilization of oil A regular increase in PV asa function of storage time was observed for all the samplesat all intervals Initially the difference in peroxide contentof control and stabilized oil samples was not noticeable itbecame significant (119875 lt 005) just after heating up to one day(Figure 3)

The results for para-anisidine values (PAVs) which usu-ally determine the amount of aldehyde in oils presented inFigure 4 The control sample showed the maximum increasein para-anisidine values indicating a higher rate of secondaryproduct formation A slow increase in PAV of stabilizedsunflower oil as compared with the control indicating the

6 The Scientific World Journal

0

02

04

06

08

1

12

0 7 14 21 28

Free

fatty

acid

s (

)

Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Figure 2 Free fatty acid contents () of sunflower oil stabilizedwithC viminalis leaves extracts

0

2

4

6

8

10

12

14

16

0 7 14 21 28

Pero

xide

val

ues (

meq

kg)

Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Figure 3 Peroxide values of sunflower oil stabilized with C vimi-nalis leaves extracts

antioxidant potential of the plant leaves Adecreasing order ofstability of oil treatedwith different extracts of plant regardingpara-anisidine values was found to be BHT gt absolutemethanol gt acetone gt 90 methanol gt 95 methanol gt n-butanol gt ethylacetate gt chloroform gt petroleum ether gtcontrol

The formation of conjugated diene (Figure 5) and triene(Figure 6) analyzed for the control and stabilized sunflower

0

2

4

6

8

10

12

14

16

18

0 7 14 21 28

Incr

ease

in P

AV

Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Figure 4 Relative increase in p-ansidine values of sunflower oilstabilized with C viminalis leaves extracts

0

2

4

6

8

10

12

14

16

18

0 7 14 21 28Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Con

juga

ted

dien

e (1205761

cm 120582232

nm)

Figure 5 Relative increase in conjugated dienes content (CD) ofsunflower oil stabilized with C viminalis leaves extracts

oil respectively Highest contents were observed for controlindicating greater intensity of oxidation

The determination of CD and CT is a good measure ofthe oxidative state of oils [30] and thus a good indicator ofeffectiveness of antioxidants CD and CT contents went onincreasing with the increase in storage time A slow increasein CD and CT of the stabilized sunflower oil as comparedwith those of the control indicated the antioxidant potential

The Scientific World Journal 7

0

1

2

3

4

5

6

0 7 14 21 28Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Con

juga

ted

trie

ne (1205761

cm 120582232

nm)

Figure 6 Relative increase in conjugated trienes content (CT) ofsunflower oil stabilized with C viminalis leaves extracts

of the C viminalis leaves Stabilized sunflower oil conjugateddiene and triene contens values were found to be in range of166ndash1354 and 12ndash389 (1205761 cm 232 nm) respectively Absolutemethanolic extract showed the lowest values and petroleumether exhibited highest All extracts played a prominent rolefor stabilization of sunflower oil but after standard BHTmethanol extract was most efficient to stabilize the oil

4 Conclusions

The results of the present study concluded that the plantpossessed considerable antioxidant potential and it may alsobe used to stabilize the edible sunflower oil Hence the plantleaves investigated can be explored as a potential antioxidantsource of natural origin Cytotoxicity of plant extracts againsthuman erythrocytes was checked and it was in safe range sothe investigated plant may be safe use for pharmaceutical andnatural therapies

Conflict of Interests

The authors do not have a direct financial relation with thecommercial identities mentioned in this paper that mightlead to a conflict of interests

References

[1] R D Spencer and P F Lumley ldquoCallistemonrdquo in Flora of NewSouth Wales G J Harden Ed vol 2 pp 168ndash173 New SouthWales University Press Sydney Australia 1991

[2] G S Wheeler ldquoMaintenance of a narrow host range by Oxyopsvitiosa a biological control agent of Melaleuca quinquenerviardquoBiochemical Systematics and Ecology vol 33 no 4 pp 365ndash3832005

[3] J W Wrigley and M Fagg Bottlebrushes Paperbarks and TeaTrees and All Other Plants in the Leptospermum Alliance Angusand Rovertson Sydney Australia 1993

[4] E Wollenweber R Wehde M Dorr G Lang and J F StevensldquoC-Methyl-flavonoids from the leaf waxes of some MyrtaceaerdquoPhytochemistry vol 55 no 8 pp 965ndash970 2000

[5] F Huq and L N Misra ldquoAn alkenol and C-methylated flavonesfrom Callistemon lanceolatus leavesrdquo Planta Medica vol 63 no4 pp 369ndash370 1997

[6] R S Varma andM R Parthasarathy ldquoTriterpenoids of Calliste-mon lanceolatus leavesrdquo Phytochemistry vol 14 no 7 pp 1675ndash1676 1975

[7] C Gomber and S Saxena ldquoAnti-staphylococcal potential ofCallistemon rigidusrdquo Central European Journal of Medicine vol2 no 1 pp 79ndash88 2007

[8] S K Srivastava A Ahmad K V Syamsunder K K Aggarwaland S P S Khanuja ldquoEssential oil composition of Callistemonviminalis leaves from Indiardquo Flavour and Fragrance Journal vol18 no 5 pp 361ndash363 2003

[9] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobial Research vol 12 pp 564ndash582 1999

[10] K Rizwan M Zubair N Rasool M Riaz M Zia-Ul-Haqand V de Feo ldquoPhytochemical and Biological Studies of Agaveattenuatardquo International Journal of Molecular Sciences vol 13pp 6440ndash6451 2012

[11] M N Bari M Zubair K Rizwan et al ldquoBiological activities ofOpuntia Monacantha cladodesrdquo Journal of Chemical SocietyPakistan vol 34 pp 990ndash995 2012

[12] M Zubair K Rizwan N Rasool N Afshan M Shahid andV Ahmed ldquoAntimicrobial potential of various extract and frac-tions of leaves of Solanum nigrumrdquo International Journal ofPhytomedicine vol 3 no 1 pp 63ndash67 2011

[13] N Rasool K Rizwan M Zubair et al ldquoAntioxidant activityof various extracts and organic fractions of Ziziphus jujubardquoInternational Journal of Phytomedicine vol 3 pp 346ndash352 2011

[14] M Zia-Ul-Haq S Cavar M Qayum I Imran and V DefeoldquoCompositional studies antioxidant and antidiabetic activitiesof Capparis decidua (Forsk) Edgewrdquo International Journal ofMolecular Sciences vol 12 pp 8846ndash8861 2011

[15] M Zia-Ul-Haq S Ahmad S Iqbal D L Luthria and RAmarowicz ldquoAntioxidant potential of lentil cultivars commonlyconsumed in Pakistanrdquo Oxidation Communication vol 34 pp819ndash831 2011

[16] M Zia-Ul-Haq S Ahmad L Calani et al ldquoCompositionalstudy and antioxidant potential of Ipomoea hederacea Jacq andLepidium sativum L sedesrdquo Molecules vol 17 pp 10306ndash103212012

[17] N Rasool K Rizwan M Zubair K U R Naveed I Imran andV U Ahmed ldquoAntioxidant potential of different extracts andfractions of Catharanthus roseus shootsrdquo International Journalof Phytomedicine vol 3 no 1 pp 108ndash114 2011

[18] B Sultana F Anwar and R Przybylski ldquoAntioxidant activityof phenolic components present in barks of Azadirachta indicaTerminalia arjunaAcacia nilotica andEugenia jambolana Lamtreesrdquo Food Chemistry vol 104 no 3 pp 1106ndash1114 2007

[19] W A Powell C M Catranis and C A Maynarrd ldquoDesign ofself processing antibacterial peptide of plant protectionrdquo Lettersin Applied Microbiology vol 31 pp 163ndash165 2000

[20] American Oil Chemists Society (AOCS) Official and Recom-mended Practices of the American Oil Chemists Society AOCSPress Champaign Ill USA 5th edition 1997

8 The Scientific World Journal

[21] International Union of Pure And Applied Chemistry (IUPAC)Standard Methods For the Analysis of Oils Fats and DerivativesC Paquot andA Hautfenne Eds Blackwell Scientific LondonUK 7th edition 1987

[22] Y Massada Analysis of Essential Oils by Gas ChromatographyandMass Spectrometry JohnWileyampSonsNewYorkNYUSA1976

[23] Mass Spectral Library NISTEPANIHUSA 2002 httpwwwnistgovsrdnistlahtm

[24] Y Sudjaroen R Haubner G Wurtele et al ldquoIsolation andstructure elucidation of phenolic antioxidants from Tamarind(Tamarindus indica L) seeds and pericarprdquo Food and ChemicalToxicology vol 43 no 11 pp 1673ndash1682 2005

[25] P Siddhuraju and K Becker ldquoAntioxidant properties of varioussolvent extracts of total phenolic constituents from three dif-ferent agroclimatic origins of drumstick tree (Moringa oleiferaLam) leavesrdquo Journal of Agricultural and Food Chemistry vol51 no 8 pp 2144ndash2155 2003

[26] S A S Chatha F AnwarMManzoor and J U R Bajwa ldquoEval-uation of the antioxidant activity of rice bran extracts usingdifferent antioxidant assaysrdquo Grasas y Aceites vol 57 no 3 pp328ndash335 2006

[27] P Sharma and J D Sharma ldquoIn vitro hemolysis of human eryth-rocytesmdashby plant extracts with antiplasmodial activityrdquo Journalof Ethnopharmacology vol 74 no 3 pp 239ndash243 2001

[28] P Kumar S Kumaravel and C Lalitha ldquoScreening of antioxi-dant activity total phenolics and GC-MS study of Vitex negun-dordquo African Journal of Biotechnology vol 4 pp 191ndash195 2010

[29] N Frega M Mozzon and G Lercker ldquoEffects of free fatty acidson oxidative stability of vegetable oilrdquo Journal of the AmericanOil Chemistsrsquo Society vol 76 no 3 pp 325ndash329 1999

[30] S H Yoon S K KimM G Shin and K H Kim ldquoComparativestudy of physical methods for lipid oxidation measurement inoilsrdquo Journal of the American Oil Chemistsrsquo Society vol 62 no10 pp 1487ndash1489 1985

6 The Scientific World Journal

0

02

04

06

08

1

12

0 7 14 21 28

Free

fatty

acid

s (

)

Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Figure 2 Free fatty acid contents () of sunflower oil stabilizedwithC viminalis leaves extracts

0

2

4

6

8

10

12

14

16

0 7 14 21 28

Pero

xide

val

ues (

meq

kg)

Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Figure 3 Peroxide values of sunflower oil stabilized with C vimi-nalis leaves extracts

antioxidant potential of the plant leaves Adecreasing order ofstability of oil treatedwith different extracts of plant regardingpara-anisidine values was found to be BHT gt absolutemethanol gt acetone gt 90 methanol gt 95 methanol gt n-butanol gt ethylacetate gt chloroform gt petroleum ether gtcontrol

The formation of conjugated diene (Figure 5) and triene(Figure 6) analyzed for the control and stabilized sunflower

0

2

4

6

8

10

12

14

16

18

0 7 14 21 28

Incr

ease

in P

AV

Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Figure 4 Relative increase in p-ansidine values of sunflower oilstabilized with C viminalis leaves extracts

0

2

4

6

8

10

12

14

16

18

0 7 14 21 28Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Con

juga

ted

dien

e (1205761

cm 120582232

nm)

Figure 5 Relative increase in conjugated dienes content (CD) ofsunflower oil stabilized with C viminalis leaves extracts

oil respectively Highest contents were observed for controlindicating greater intensity of oxidation

The determination of CD and CT is a good measure ofthe oxidative state of oils [30] and thus a good indicator ofeffectiveness of antioxidants CD and CT contents went onincreasing with the increase in storage time A slow increasein CD and CT of the stabilized sunflower oil as comparedwith those of the control indicated the antioxidant potential

The Scientific World Journal 7

0

1

2

3

4

5

6

0 7 14 21 28Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Con

juga

ted

trie

ne (1205761

cm 120582232

nm)

Figure 6 Relative increase in conjugated trienes content (CT) ofsunflower oil stabilized with C viminalis leaves extracts

of the C viminalis leaves Stabilized sunflower oil conjugateddiene and triene contens values were found to be in range of166ndash1354 and 12ndash389 (1205761 cm 232 nm) respectively Absolutemethanolic extract showed the lowest values and petroleumether exhibited highest All extracts played a prominent rolefor stabilization of sunflower oil but after standard BHTmethanol extract was most efficient to stabilize the oil

4 Conclusions

The results of the present study concluded that the plantpossessed considerable antioxidant potential and it may alsobe used to stabilize the edible sunflower oil Hence the plantleaves investigated can be explored as a potential antioxidantsource of natural origin Cytotoxicity of plant extracts againsthuman erythrocytes was checked and it was in safe range sothe investigated plant may be safe use for pharmaceutical andnatural therapies

Conflict of Interests

The authors do not have a direct financial relation with thecommercial identities mentioned in this paper that mightlead to a conflict of interests

References

[1] R D Spencer and P F Lumley ldquoCallistemonrdquo in Flora of NewSouth Wales G J Harden Ed vol 2 pp 168ndash173 New SouthWales University Press Sydney Australia 1991

[2] G S Wheeler ldquoMaintenance of a narrow host range by Oxyopsvitiosa a biological control agent of Melaleuca quinquenerviardquoBiochemical Systematics and Ecology vol 33 no 4 pp 365ndash3832005

[3] J W Wrigley and M Fagg Bottlebrushes Paperbarks and TeaTrees and All Other Plants in the Leptospermum Alliance Angusand Rovertson Sydney Australia 1993

[4] E Wollenweber R Wehde M Dorr G Lang and J F StevensldquoC-Methyl-flavonoids from the leaf waxes of some MyrtaceaerdquoPhytochemistry vol 55 no 8 pp 965ndash970 2000

[5] F Huq and L N Misra ldquoAn alkenol and C-methylated flavonesfrom Callistemon lanceolatus leavesrdquo Planta Medica vol 63 no4 pp 369ndash370 1997

[6] R S Varma andM R Parthasarathy ldquoTriterpenoids of Calliste-mon lanceolatus leavesrdquo Phytochemistry vol 14 no 7 pp 1675ndash1676 1975

[7] C Gomber and S Saxena ldquoAnti-staphylococcal potential ofCallistemon rigidusrdquo Central European Journal of Medicine vol2 no 1 pp 79ndash88 2007

[8] S K Srivastava A Ahmad K V Syamsunder K K Aggarwaland S P S Khanuja ldquoEssential oil composition of Callistemonviminalis leaves from Indiardquo Flavour and Fragrance Journal vol18 no 5 pp 361ndash363 2003

[9] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobial Research vol 12 pp 564ndash582 1999

[10] K Rizwan M Zubair N Rasool M Riaz M Zia-Ul-Haqand V de Feo ldquoPhytochemical and Biological Studies of Agaveattenuatardquo International Journal of Molecular Sciences vol 13pp 6440ndash6451 2012

[11] M N Bari M Zubair K Rizwan et al ldquoBiological activities ofOpuntia Monacantha cladodesrdquo Journal of Chemical SocietyPakistan vol 34 pp 990ndash995 2012

[12] M Zubair K Rizwan N Rasool N Afshan M Shahid andV Ahmed ldquoAntimicrobial potential of various extract and frac-tions of leaves of Solanum nigrumrdquo International Journal ofPhytomedicine vol 3 no 1 pp 63ndash67 2011

[13] N Rasool K Rizwan M Zubair et al ldquoAntioxidant activityof various extracts and organic fractions of Ziziphus jujubardquoInternational Journal of Phytomedicine vol 3 pp 346ndash352 2011

[14] M Zia-Ul-Haq S Cavar M Qayum I Imran and V DefeoldquoCompositional studies antioxidant and antidiabetic activitiesof Capparis decidua (Forsk) Edgewrdquo International Journal ofMolecular Sciences vol 12 pp 8846ndash8861 2011

[15] M Zia-Ul-Haq S Ahmad S Iqbal D L Luthria and RAmarowicz ldquoAntioxidant potential of lentil cultivars commonlyconsumed in Pakistanrdquo Oxidation Communication vol 34 pp819ndash831 2011

[16] M Zia-Ul-Haq S Ahmad L Calani et al ldquoCompositionalstudy and antioxidant potential of Ipomoea hederacea Jacq andLepidium sativum L sedesrdquo Molecules vol 17 pp 10306ndash103212012

[17] N Rasool K Rizwan M Zubair K U R Naveed I Imran andV U Ahmed ldquoAntioxidant potential of different extracts andfractions of Catharanthus roseus shootsrdquo International Journalof Phytomedicine vol 3 no 1 pp 108ndash114 2011

[18] B Sultana F Anwar and R Przybylski ldquoAntioxidant activityof phenolic components present in barks of Azadirachta indicaTerminalia arjunaAcacia nilotica andEugenia jambolana Lamtreesrdquo Food Chemistry vol 104 no 3 pp 1106ndash1114 2007

[19] W A Powell C M Catranis and C A Maynarrd ldquoDesign ofself processing antibacterial peptide of plant protectionrdquo Lettersin Applied Microbiology vol 31 pp 163ndash165 2000

[20] American Oil Chemists Society (AOCS) Official and Recom-mended Practices of the American Oil Chemists Society AOCSPress Champaign Ill USA 5th edition 1997

8 The Scientific World Journal

[21] International Union of Pure And Applied Chemistry (IUPAC)Standard Methods For the Analysis of Oils Fats and DerivativesC Paquot andA Hautfenne Eds Blackwell Scientific LondonUK 7th edition 1987

[22] Y Massada Analysis of Essential Oils by Gas ChromatographyandMass Spectrometry JohnWileyampSonsNewYorkNYUSA1976

[23] Mass Spectral Library NISTEPANIHUSA 2002 httpwwwnistgovsrdnistlahtm

[24] Y Sudjaroen R Haubner G Wurtele et al ldquoIsolation andstructure elucidation of phenolic antioxidants from Tamarind(Tamarindus indica L) seeds and pericarprdquo Food and ChemicalToxicology vol 43 no 11 pp 1673ndash1682 2005

[25] P Siddhuraju and K Becker ldquoAntioxidant properties of varioussolvent extracts of total phenolic constituents from three dif-ferent agroclimatic origins of drumstick tree (Moringa oleiferaLam) leavesrdquo Journal of Agricultural and Food Chemistry vol51 no 8 pp 2144ndash2155 2003

[26] S A S Chatha F AnwarMManzoor and J U R Bajwa ldquoEval-uation of the antioxidant activity of rice bran extracts usingdifferent antioxidant assaysrdquo Grasas y Aceites vol 57 no 3 pp328ndash335 2006

[27] P Sharma and J D Sharma ldquoIn vitro hemolysis of human eryth-rocytesmdashby plant extracts with antiplasmodial activityrdquo Journalof Ethnopharmacology vol 74 no 3 pp 239ndash243 2001

[28] P Kumar S Kumaravel and C Lalitha ldquoScreening of antioxi-dant activity total phenolics and GC-MS study of Vitex negun-dordquo African Journal of Biotechnology vol 4 pp 191ndash195 2010

[29] N Frega M Mozzon and G Lercker ldquoEffects of free fatty acidson oxidative stability of vegetable oilrdquo Journal of the AmericanOil Chemistsrsquo Society vol 76 no 3 pp 325ndash329 1999

[30] S H Yoon S K KimM G Shin and K H Kim ldquoComparativestudy of physical methods for lipid oxidation measurement inoilsrdquo Journal of the American Oil Chemistsrsquo Society vol 62 no10 pp 1487ndash1489 1985

The Scientific World Journal 7

0

1

2

3

4

5

6

0 7 14 21 28Storage period (days)

ControlBHTPetroleum etherChloroformEthylacetate

Acetone119899-ButanolAbsolute methanol95 methanol90 methanol

Con

juga

ted

trie

ne (1205761

cm 120582232

nm)

Figure 6 Relative increase in conjugated trienes content (CT) ofsunflower oil stabilized with C viminalis leaves extracts

of the C viminalis leaves Stabilized sunflower oil conjugateddiene and triene contens values were found to be in range of166ndash1354 and 12ndash389 (1205761 cm 232 nm) respectively Absolutemethanolic extract showed the lowest values and petroleumether exhibited highest All extracts played a prominent rolefor stabilization of sunflower oil but after standard BHTmethanol extract was most efficient to stabilize the oil

4 Conclusions

The results of the present study concluded that the plantpossessed considerable antioxidant potential and it may alsobe used to stabilize the edible sunflower oil Hence the plantleaves investigated can be explored as a potential antioxidantsource of natural origin Cytotoxicity of plant extracts againsthuman erythrocytes was checked and it was in safe range sothe investigated plant may be safe use for pharmaceutical andnatural therapies

Conflict of Interests

The authors do not have a direct financial relation with thecommercial identities mentioned in this paper that mightlead to a conflict of interests

References

[1] R D Spencer and P F Lumley ldquoCallistemonrdquo in Flora of NewSouth Wales G J Harden Ed vol 2 pp 168ndash173 New SouthWales University Press Sydney Australia 1991

[2] G S Wheeler ldquoMaintenance of a narrow host range by Oxyopsvitiosa a biological control agent of Melaleuca quinquenerviardquoBiochemical Systematics and Ecology vol 33 no 4 pp 365ndash3832005

[3] J W Wrigley and M Fagg Bottlebrushes Paperbarks and TeaTrees and All Other Plants in the Leptospermum Alliance Angusand Rovertson Sydney Australia 1993

[4] E Wollenweber R Wehde M Dorr G Lang and J F StevensldquoC-Methyl-flavonoids from the leaf waxes of some MyrtaceaerdquoPhytochemistry vol 55 no 8 pp 965ndash970 2000

[5] F Huq and L N Misra ldquoAn alkenol and C-methylated flavonesfrom Callistemon lanceolatus leavesrdquo Planta Medica vol 63 no4 pp 369ndash370 1997

[6] R S Varma andM R Parthasarathy ldquoTriterpenoids of Calliste-mon lanceolatus leavesrdquo Phytochemistry vol 14 no 7 pp 1675ndash1676 1975

[7] C Gomber and S Saxena ldquoAnti-staphylococcal potential ofCallistemon rigidusrdquo Central European Journal of Medicine vol2 no 1 pp 79ndash88 2007

[8] S K Srivastava A Ahmad K V Syamsunder K K Aggarwaland S P S Khanuja ldquoEssential oil composition of Callistemonviminalis leaves from Indiardquo Flavour and Fragrance Journal vol18 no 5 pp 361ndash363 2003

[9] MM Cowan ldquoPlant products as antimicrobial agentsrdquoClinicalMicrobial Research vol 12 pp 564ndash582 1999

[10] K Rizwan M Zubair N Rasool M Riaz M Zia-Ul-Haqand V de Feo ldquoPhytochemical and Biological Studies of Agaveattenuatardquo International Journal of Molecular Sciences vol 13pp 6440ndash6451 2012

[11] M N Bari M Zubair K Rizwan et al ldquoBiological activities ofOpuntia Monacantha cladodesrdquo Journal of Chemical SocietyPakistan vol 34 pp 990ndash995 2012

[12] M Zubair K Rizwan N Rasool N Afshan M Shahid andV Ahmed ldquoAntimicrobial potential of various extract and frac-tions of leaves of Solanum nigrumrdquo International Journal ofPhytomedicine vol 3 no 1 pp 63ndash67 2011

[13] N Rasool K Rizwan M Zubair et al ldquoAntioxidant activityof various extracts and organic fractions of Ziziphus jujubardquoInternational Journal of Phytomedicine vol 3 pp 346ndash352 2011

[14] M Zia-Ul-Haq S Cavar M Qayum I Imran and V DefeoldquoCompositional studies antioxidant and antidiabetic activitiesof Capparis decidua (Forsk) Edgewrdquo International Journal ofMolecular Sciences vol 12 pp 8846ndash8861 2011

[15] M Zia-Ul-Haq S Ahmad S Iqbal D L Luthria and RAmarowicz ldquoAntioxidant potential of lentil cultivars commonlyconsumed in Pakistanrdquo Oxidation Communication vol 34 pp819ndash831 2011

[16] M Zia-Ul-Haq S Ahmad L Calani et al ldquoCompositionalstudy and antioxidant potential of Ipomoea hederacea Jacq andLepidium sativum L sedesrdquo Molecules vol 17 pp 10306ndash103212012

[17] N Rasool K Rizwan M Zubair K U R Naveed I Imran andV U Ahmed ldquoAntioxidant potential of different extracts andfractions of Catharanthus roseus shootsrdquo International Journalof Phytomedicine vol 3 no 1 pp 108ndash114 2011

[18] B Sultana F Anwar and R Przybylski ldquoAntioxidant activityof phenolic components present in barks of Azadirachta indicaTerminalia arjunaAcacia nilotica andEugenia jambolana Lamtreesrdquo Food Chemistry vol 104 no 3 pp 1106ndash1114 2007

[19] W A Powell C M Catranis and C A Maynarrd ldquoDesign ofself processing antibacterial peptide of plant protectionrdquo Lettersin Applied Microbiology vol 31 pp 163ndash165 2000

[20] American Oil Chemists Society (AOCS) Official and Recom-mended Practices of the American Oil Chemists Society AOCSPress Champaign Ill USA 5th edition 1997

8 The Scientific World Journal

[21] International Union of Pure And Applied Chemistry (IUPAC)Standard Methods For the Analysis of Oils Fats and DerivativesC Paquot andA Hautfenne Eds Blackwell Scientific LondonUK 7th edition 1987

[22] Y Massada Analysis of Essential Oils by Gas ChromatographyandMass Spectrometry JohnWileyampSonsNewYorkNYUSA1976

[23] Mass Spectral Library NISTEPANIHUSA 2002 httpwwwnistgovsrdnistlahtm

[24] Y Sudjaroen R Haubner G Wurtele et al ldquoIsolation andstructure elucidation of phenolic antioxidants from Tamarind(Tamarindus indica L) seeds and pericarprdquo Food and ChemicalToxicology vol 43 no 11 pp 1673ndash1682 2005

[25] P Siddhuraju and K Becker ldquoAntioxidant properties of varioussolvent extracts of total phenolic constituents from three dif-ferent agroclimatic origins of drumstick tree (Moringa oleiferaLam) leavesrdquo Journal of Agricultural and Food Chemistry vol51 no 8 pp 2144ndash2155 2003

[26] S A S Chatha F AnwarMManzoor and J U R Bajwa ldquoEval-uation of the antioxidant activity of rice bran extracts usingdifferent antioxidant assaysrdquo Grasas y Aceites vol 57 no 3 pp328ndash335 2006

[27] P Sharma and J D Sharma ldquoIn vitro hemolysis of human eryth-rocytesmdashby plant extracts with antiplasmodial activityrdquo Journalof Ethnopharmacology vol 74 no 3 pp 239ndash243 2001

[28] P Kumar S Kumaravel and C Lalitha ldquoScreening of antioxi-dant activity total phenolics and GC-MS study of Vitex negun-dordquo African Journal of Biotechnology vol 4 pp 191ndash195 2010

[29] N Frega M Mozzon and G Lercker ldquoEffects of free fatty acidson oxidative stability of vegetable oilrdquo Journal of the AmericanOil Chemistsrsquo Society vol 76 no 3 pp 325ndash329 1999

[30] S H Yoon S K KimM G Shin and K H Kim ldquoComparativestudy of physical methods for lipid oxidation measurement inoilsrdquo Journal of the American Oil Chemistsrsquo Society vol 62 no10 pp 1487ndash1489 1985

8 The Scientific World Journal

[21] International Union of Pure And Applied Chemistry (IUPAC)Standard Methods For the Analysis of Oils Fats and DerivativesC Paquot andA Hautfenne Eds Blackwell Scientific LondonUK 7th edition 1987

[22] Y Massada Analysis of Essential Oils by Gas ChromatographyandMass Spectrometry JohnWileyampSonsNewYorkNYUSA1976

[23] Mass Spectral Library NISTEPANIHUSA 2002 httpwwwnistgovsrdnistlahtm

[24] Y Sudjaroen R Haubner G Wurtele et al ldquoIsolation andstructure elucidation of phenolic antioxidants from Tamarind(Tamarindus indica L) seeds and pericarprdquo Food and ChemicalToxicology vol 43 no 11 pp 1673ndash1682 2005

[25] P Siddhuraju and K Becker ldquoAntioxidant properties of varioussolvent extracts of total phenolic constituents from three dif-ferent agroclimatic origins of drumstick tree (Moringa oleiferaLam) leavesrdquo Journal of Agricultural and Food Chemistry vol51 no 8 pp 2144ndash2155 2003

[26] S A S Chatha F AnwarMManzoor and J U R Bajwa ldquoEval-uation of the antioxidant activity of rice bran extracts usingdifferent antioxidant assaysrdquo Grasas y Aceites vol 57 no 3 pp328ndash335 2006

[27] P Sharma and J D Sharma ldquoIn vitro hemolysis of human eryth-rocytesmdashby plant extracts with antiplasmodial activityrdquo Journalof Ethnopharmacology vol 74 no 3 pp 239ndash243 2001

[28] P Kumar S Kumaravel and C Lalitha ldquoScreening of antioxi-dant activity total phenolics and GC-MS study of Vitex negun-dordquo African Journal of Biotechnology vol 4 pp 191ndash195 2010

[29] N Frega M Mozzon and G Lercker ldquoEffects of free fatty acidson oxidative stability of vegetable oilrdquo Journal of the AmericanOil Chemistsrsquo Society vol 76 no 3 pp 325ndash329 1999

[30] S H Yoon S K KimM G Shin and K H Kim ldquoComparativestudy of physical methods for lipid oxidation measurement inoilsrdquo Journal of the American Oil Chemistsrsquo Society vol 62 no10 pp 1487ndash1489 1985