Research ArticleComposition In Vitro Antioxidant and AntimicrobialActivities of Essential Oil and Oleoresins Obtained from BlackCumin Seeds (Nigella sativa L)
Sunita Singh1 S S Das1 G Singh1 Carola Schuff2
Marina P de Lampasona2 and Ceacutesar A N Catalaacuten2
1 Chemistry Department DDU Gorakhpur University Gorakhpur Uttar Pradesh 273009 India2 INQUINOA-CONICET Instituto de Quımica Organica Facultad de Bioquımica Quımica y FarmaciaUniversidad Nacional de Tucuman T4000INI San Miguel de Tucuman Argentina
Correspondence should be addressed to G Singh gsingh4usyahoocom
Received 6 April 2013 Accepted 24 October 2013 Published 6 February 2014
Academic Editor Afaf K El-Ansary
Copyright copy 2014 Sunita Singh et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
Gas chromatography-mass spectrometry (GC-MS) analysis revealed the major components in black cumin essential oils whichwere thymoquinone (376) followed by p-cymene (312) 120572-thujene (56) thymohydroquinone (34) and longifolene (20)whereas the oleoresins extracted in different solvents contain linoleic acid as amajor componentThe antioxidant activity of essentialoil and oleoresins was evaluated against linseed oil system at 200 ppm concentration by peroxide value thiobarbituric acid valueferric thiocyanate ferrous ion chelating activity and 11-diphenyl-2-picrylhydrazyl radical scavenging methods The essential oiland ethyl acetate oleoresin were found to be better than synthetic antioxidants The total phenol contents (gallic acid equivalentsmgGAEper g) in black cumin essential oil ethyl acetate ethanol and n-hexane oleoresinswere calculated as 1147plusmn005 1088plusmn09968 plusmn 006 and 833 plusmn 001 respectively by Folin-Ciocalteau method The essential oil showed up to 90 zone inhibition againstFusarium moniliforme in inverted petri plate method Using agar well diffusion method for evaluating antibacterial activity theessential oil was found to be highly effective against Gram-positive bacteria
1 Introduction
Preservation of food degradation mainly by oxidation pro-cesses or by microorganism activity during productionstorage and marketing is an important issue in the foodindustry There is currently a large interest in substitutingsynthetic food preservatives and synthetic antioxidants forsubstance that can be marketed as natural Synthetic antioxi-dants such as gallates butylated hydroxytoluene (BHT) buty-lated hydroxyanisole (BHA) and tert-butyl hydroquinone(TBHQ) were the first preservatives designed for widespreadindustrial use However some physical properties of BHAand BHT such as their high volatility and instability atelevated temperatures strict legislation on the use of syn-thetic food additives and consumer preferences have shiftedthe attention of manufacturers from synthetic to naturalantioxidant [1] It is well known that most spices possess awide range of biological and pharmacological activities
Black cumin (Nigella sativa L) belonging to familyRanunculaceae is a spice that has been used for decadesfor both culinary and medicinal purposes It is also usedas a natural remedy for asthma hypertension diabetesinflammation cough bronchitis headache eczema feverdizziness and influenza [2] The seeds are known to becarminative stimulant and diuretic [3] The essential oilfrom the seeds of this herbaceous plant has been found tocontain high concentrations of thymoquinone and its relatedcompounds such as thymol and dithymoquinone whichhave been implicated in the prevention of inflammation [4]antioxidant activities [5] such as quenching reactive oxygenspecies antimicrobial activity [6] and anticarcinogenic andantiulcer activity [2]
Thepresent paper dealswith the chemistry and antioxida-tive and antimicrobial behavior of essential oil and oleoresins(extracted in ethanol ethyl acetate and n-hexane) of blackcumin seeds
Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 918209 10 pageshttpdxdoiorg1011552014918209
2 BioMed Research International
2 Materials and Methods
The seeds of black cumin were purchased from the localmarket of Gorakhpur Uttar Pradesh India A voucherspecimen was deposited at the herbarium of the Faculty ofScience DDU Gorakhpur University
21 Reagents Thiobarbituric acid (TBA) 111015840-diphenyl-2-picrylhydrazyl radical (DPPH) and linoleic acid are ofAcros (New Jersey USA) butylated hydroxytoluene (BHT)butylated hydroxyanisole (BHA) and propyl gallate (PG) areof S D Fine Chemicals Ltd Mumbai India Folin-Ciocalteureagent and gallic acid were from Qualigens Chemicals LtdMumbai India and Qualikems Chemicals Ltd New DelhiIndia respectively Tween 20 and ferrozine were fromMerckPvt Ltd Mumbai India Ampicillin was purchased fromRanbaxy Fine Chemicals (New Delhi) India Crude linseedoil was obtained from local oil mill in Gorakhpur All solventsused were of analytical grade
22 Sample Extraction Powdered seeds of black cumin(250 g) were subjected to hydrodistillation in Clevengerapparatus for 3 h according to the method recommended byEuropean Pharmacopoeia [7] A volatile oil with light orangecharacteristic odour was obtained with yield of 09 It wasdried over anhydrous sodium sulphate and the sample wasstored at 4∘C before use
Oleoresins were obtained by extracting 30 g of powderedspice with 300mL of various solvents (ethanol ethyl acetateand n-hexane) for 3 h in Soxhlet extractor Evaporation ofthe solvents at reduced pressure gave viscous extracts Theoleoresins were stored in freezer until further use
23 Chemical Characterization
231 Gas Chromatography-Mass Spectrometry (GC-MS)Analysis of the volatile oils and oleoresins was run on aHewlett Packard (6890) GC-Ms system coupled to a quadru-ple mass spectrometer (model HP 5973) with a capillarycolumn of HP-5MS (5 phenyl methylsiloxane length =30m inner diameter = 025mm and film thickness =025 120583m) GC-MS interphase ion source and selective massdetector temperatures were maintained at 280∘C 230∘C and150∘C respectively Carrier gas used was helium with a flowrate of 10mLminminus1The oven temperature was programmedas follows
For essential oil at 60∘C for 1min then increased from60 to 185∘C at the rate of 15∘Cminminus1 and held at the rate of9∘Cminminus1 and held at 275∘C for 2min
For oleoresin 60∘C for zero min then increased from 60to 300∘C at the rate of 15∘Cminminus1 and held at the rate of5∘Cminminus1 and held at 300∘C for 10min
24 Identification of Components Most of the componentswere identified on the basis of comparison of their retentionindices and mass spectra with published data [6 8 9]and computer matching was done with the Wiley 275 andNational Institute of Standards Technology libraries provided
Table 1 Chemical composition of essential oil obtained from blackcumin seeds analyzed by GC-MS
Compounds MS RI IdentificationΦ
120572-Thujene 56 919 MS RI co-GC120572-Pinene 14 928 MS RI co-GCSabinene 08 967 MS RI co-GC120573-Pinene 17 973 MS RI co-GC120572-Phellandrene 01 1003 MS RI120572-Terpinene 02 1012 MS RI co-GC119901-Cymene 314 1019 MS RI co-GCLimonene 10 1024 MS RI co-GC18-Cineole 01 1025 MS RI co-GC120574-Terpinene 02 1050 MS RI co-GCtrans-Sabinene hydrate 01 1101 MS RIUnidentified Blowast 68 1113 minus
Terpinen-4-ol 10 1172 MS RI co-GC119901-Cymen-8-ol Trace 1179 MS RI120572-Terpineol Trace 1189 MS RI co-GCCuminal Trace 1240 MS RICarvone Trace 1241 MS RIThymoquinone 376 1248 MS RItrans-Sabinene hydrate acetate 01 1258 MS RIBornyl acetate 02 1285 MS RIThymol 02 1289 MS RICarvacrol 14 1295 MS RI120572-Longipinene 05 1353 MS RILongifolene 20 1405 MS RIThymohydroquinone 34 1559 MS RI10-epi-120574-Eudesmol 03 1625 MS RI120573-Eudesmol 05 1652 MS RI120572-Eudesmol 04 1655 MS RITotal 902Trace lt 005 the retention index was calculated using a homologousseries of n-alkanes C8ndashC18 ΦCo-GC coinjection with an authentic samplePercentages were obtained from electronic integration Trace measurementsusing a selective mass detector
with the computer controllingGC-MS systemsThe retentionindices were calculated using a homologous series of n-alkanes C
8ndashC18
and C8ndashC22
for essential oil and oleoresinsrespectively which are reported in Tables 1 and 2
3 Antioxidant Activity
The antioxidant activity is system dependent and accordingto the method adopted and lipid system used as substrateHence different methods have been adopted in order toassess antioxidative potential of black cumin oil and itsoleoresins are as follows
31 Chelating Activity on Ferrous Ions The chelating activityof the aqueous and ethanolic extract on ferrous ions (Fe2+)was measured according to the method described by Deckerand Welch [10] Aliquots of 1mL of different concentrations
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Table 2 Chemical composition of oleoresins obtained from black cumin (Nigella sativa L) seeds in different solvents analysed by GC-MS
Compounds M1 M2 M3 RI IdentificationΦ
120572-Thujene Trace 04 06 919 MS RI co-GC119901-Cymene 09 28 22 1019 MS RI co-GCUnidentified A 05 05 04 1092 minus
Thymoquinone 57 61 37 1248 MS RICarvacrol 04 Trace Trace 1295 MS RI co-GC120572-Longipinene Trace Trace Trace 1353 MS RILongifolene 05 06 03 1405 MS RIThymohydroquinone 25 16 05 1559 MS RIPalmitic acid ethyl ester 28 Trace Trace 1979 MS co-GCLinoleic acid methyl ester 06 05 05 minus MS co-GCLinoleic acid ethyl ester 116 Trace 06 minus MS co-GCOleic acid ethyl ester 46 Trace 02 minus MS co-GCOleic acid 03 Trace 02 minus MS co-GCLinoleic acid 330 439 277 minus MSLinoleic acid butyl ester 09 57 160 minus MSOleic acid butyl ester 12 45 73 minus MSGlyceryl palmitate 37 16 23 minus MSGlyceryl linoleate 277 219 231 minus MSSitosterol Trace 13 Trace minus MS co-GCTotal 964 909 852Trace lt 005 the retention index was calculated using a homologous series of n-alkanes C8ndashC20 ΦCo-GC coinjection with an authentic samplePercentages were obtained from electronic integration measurements using selective mass detectorM1 ethanol oleoresin M2 ethyl acetate oleoresin M3 n-hexane oleoresin
of the samples were mixed with 37mL of deionized waterThe mixture was incubated with FeCl
2(2mM 01mL)
After incubation the reaction was initiated by addition offerrozine (5mM and 02mL) for 10min at room temperatureand then the absorbance was measured at 562 nm in aspectrophotometer A lower absorbance indicates a higherchelating power The chelating activity of the extract on Fe2+was compared with that of EDTA that was used as positivecontrol Chelating activity was calculated using the followingformula
Chelating activity ()
= [1 minus (
Absorbance of sampleAbsorbance of control
)] times 100
(1)
32 Scavenging Effect on DPPH TheDPPH assay constitutesa quick and low cost method which has frequently been usedfor the evaluation of the antioxidative potential of variousnatural products [11] Due to its odd electron DPPH givesa strong absorption band at 517 nm (deep violet colour) Inthe presence of a free radical scavenger this electron becomespaired resulting in the absorption loss and consecutivestoichiometric decolorization with respect to the number ofelectron acquired The absorbance change produced by thisreaction is assessed to evaluate the antioxidant potential ofthe test sample 5 10 15 and 20 120583L of the sample were addedto 5mLof 0004methanol solution ofDPPHAfter a 30minincubation period at room temperature the absorbance was
read against a blank at 515 nm All determination was per-formed in triplicate and results were performed in triplicateand results are reported as scavenging effect () versusconcentration in Figure 2
33 Estimation of Total Phenolic Content (TPC) TPC weredetermined using the Folin-Ciocalteu reagent methoddescribed by Singleton and Rossi [12] Gallic acid stocksolution (1000 120583gmLminus1) was prepared by dissolving 100mgof gallic acid in 100mL of ethanol Various dilutions ofstandard gallic acid were prepared from this stock solutionCalibration curve (Figure 3) was plotted by mixing 1mLaliquots of 10ndash100120583gmLminus1 of gallic acid solutionswith 50mLof Folin-Ciocalteu reagent (diluted tenfold) and 40mL ofsodium carbonate solution (75 g Lminus1) The absorbance wasmeasured after 30min at 20∘C at 765 nm
4 Evaluation of Antioxidant Activity forLinseed Oil System
For present investigation crude linseed oil having initialperoxide value 52meq kgminus1 was taken to assess the antiox-idant activity of black cumin oil and its oleoresins This oilis most frequently used edible oil in central Europe and israther unstable because of the presence of substantial amountof linoleic acid The antioxidant activity of volatile oil andextract was examined by comparing the activity of knownantioxidants such as PG BHT and BHA by the followingperoxide value and thiobarbituric acid value methods
4 BioMed Research International
41 Peroxide Value Method For measuring the peroxidevalue (PV) a modified oven test was used [13] Theantioxidant activity of black cumin oil and its oleoresinsin different solvents were compared with the syntheticantioxidants such as PG BHT and BHA For this purposecalculated quantities of each (200 ppm)were dissolved to 30 gof linseed oil in an open mouthed beaker The mixtures werethoroughly homogenized and placed in incubator at 80∘CThe peroxide values (meq of oxygen kgminus1) were measured inevery seven days and test was replicated for three times Acontrol sample was prepared under similar conditionwithoutany additive The effects of oil and oleoresins in term ofperoxidation at 90∘C are shown in Figure 4
42 Thiobarbituric Acid Value (TBA) TBA value of differentsamples was determined according to the method previouslyreported [13] About 100mg of oil sample was dissolved in25mL of 1-butanol A 25mL aliquot of the above solutionwas mixed thoroughly with 50mL of TBA reagent (200mgTBA in 100mL of 1-butanol) and incubated at 95∘C After 2 hthe reaction mixture was cooled to room temperature underrunning water and absorbance was measured at 530 nm withHitachi-U-2000 spectrophotometer (Tokyo Japan) At thesame time a reagent blank (without TBA reagent) was alsodoneThe thiobarbituric acid value (meq ofmalondialdehydeper g) was calculated as
TBA value = 50 times (119860 minus 119861)119872
(2)
where 119860 is absorbance of the test sample 119861 is absorbance ofthe reagent blank and119872 is mass of the sample
43 Determination of Antioxidant Activity in Linoleic AcidSystem Antioxidant activity of black cumin oil and itsoleoresins was compared to synthetic standards accordingto the ferric thiocyanate method in linoleic acid emulsion[14] The reaction medium contained black cumin oil andoleoresins at the concentration of 1mg100mL of absoluteethanol (2mL) an emulsion of 251 linoleic acid in ethanol(2mL) 4mL of 005M-phosphate buffer (pH = 70) and2mL of distilled water The solution (10mL) was mixed andincubated at 40∘C in the darkThe same solution without anyadditives was taken as control sample At regular intervalsduring incubation 01mL aliquot of the mixture was dilutedwith 97mL of 75 ethanol followed by the addition of02mL of 30 ammonium thiocyanate and 01mL of 20mMof FeCl
2in 35 HCl the absorbance of red colour was
measured at 500 nm in Hitachi-U-2000 spectrophotometer(Tokyo Japan) The control and standard were subjected tothe same procedure except for the control where there wasno addition of sample and for the standard 1mL of samplewere replaced with 1mg of PG BHT and BHA These stepsare repeated every 48 h until the control sample reached itsmaximum Low absorbance value indicates the efficiency ofthe test samples to inhibit lipid oxidation The results werereported as incubation time versus absorbance in Figure 6
5 Antimicrobial Investigations
51 Antifungal Assay The antifungal activity of the essentialoil and extract against various pathogenic fungi Aspergilluslavus (1884) Aspergillus niger (2479) Fusarium moniliforme(1893) Fusarium graminearum (2088) and Penicillium viridi-catum (2007) were tested by the inverted petri plate [15] andpoison food medium methods [16] All the fungi cultureswere procured from the Microbial Type Culture Collection(MTCC) and their reference numbers are given in theparentheses The cultures were maintained in Czapek agarmedium Each test was replicated three times and fungi toxi-city was measured in terms of percentage mycelial inhibitioncalculated with the following equation
colony of the control and treated sets respectively
52 Antibacterial Assay The essential oil and extract wereindividually tested against a panel of microorganisms usingagar well diffusion method [17] Three Gram-positive bacte-ria Staphylococcus aureus (3103) Bacillus cereus (430) andBacillus subtilis (1790) and two Gram negative bacteriaEscherichia coli (1672) and Pseudomonas aeruginosa (1942)All the bacterial strains were procured from the MicrobialType Culture Collection (MTCC) Institute of MicrobialTechnology (Chandigarh India) and their reference num-bers are given in parentheses The bacterial cultures weregrown on nutrient agar medium and stored at 4∘C In orderto prepare a bacterial strain for test initially one loopfulof bacterial culture was transferred from slant to nutrientbroth solution (10mL) and was stored at 37∘C for 24 hThe control plate without the addition of essential oil orextract containing DMSO was also maintained under thesame conditions After incubating for 24 h at 37∘C all plateswere examined for any zones of growth inhibition and thediameters of these zones were measured in millimeters
6 Statistical Analysis
For the essential oil or oleoresin three samples were preparedfor assays of every antioxidant and antimicrobial attributeThe data are presented as mean (standard deviation of threedeterminations (data are not shown) Statistical analyseswere performed using a one-way analysis of variance [18] Aprobability value of119875 lt 005was considered to be significant
7 Results and Discussions
71 Phytochemistry Careful and detailed interpretations ofthe experimental GC-MS data (EM fragmentation retentionindices) were carried out which permitted identification ofa large number of components in essential oil and oleo-resins (Tables 1 and 2) Table 1 shows identification of 33components in black cumin oil representing about 902 ofthe total amount The major component in black cumin oil
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0
25
50
75
100
0 5 10 15 20 25
Black cumin oilEtOH oleon-hexane oleo
Ethyl acetate oleoEDTA
Chel
atin
g eff
ect (
)
Concentration (120583g mLminus1)
Figure 1 Chelating effect of black cumin oil and its differentoleoresins
was thymoquinone (376) followed by p-cymene (314)120572-thujene (56) thymohydroquinone (34) longifolene(20) and carvacrol (14) Burits and Bucar [8] character-ized many components in black cumin essential oil such asthymoquinone (278ndash570) p-cymene (71ndash155) carvacrol(58ndash116) trans-anethole (025ndash23) 4-terpineol (20ndash66) and 10ndash80 of longifolene These results are slightdifferent from the work reported by Hajhashemi et al [19]who reported that p-cymene (373) and thymoquinone(137) were the major components of black cumin Singh etal [6] also reported p-cymene as the major component in theblack cumin essential oil
From Table 2 it is evident that in ethanol oleoresin19 components constitute 964 of the total weight in theethyl acetate oleoresin a total of 19 components making909 of the whole mass and in case of n-hexane oleoresin19 compounds constituting about 852 of the total weightwere identified The oleoresins were mainly comprised ofunsaturated fatty acids and their different esters The majorcomponents in all three oleoresins were linoleic acid (unsat-urated fatty acid) followed by glyceryl linoleate glycerylpalmitate oleic acid and other minor components Thepresence of unsaturated fatty acids in oleoresins was wellsupported by the various reported work [20ndash26]
72 Antioxidant Investigations
721 Chelating Activity on Ferrous Ions The ferrous ion(Fe2+) chelating effect of black cumin oil and its differentoleoresins is presented in Figure 1 The chelating activityof the extracts was concentration dependent Black cuminoil exhibited higher chelating activity in comparison to theoleoresins but was not effective chelator as EDTA Maximumchelating of metal ions at 200120583gmLminus1 for black cumin oiland EDTA was found to be 7456 and 8790 respectivelywhereas the oleoresins were less effective in metal chelationand their metal chelating activity ranges from 221 to 445
Figure 2 Scavenging effect () of black cumin oil and its oleoresinson DPPH radical
0
02
04
06
08
1
12
0 10 20 30 40 50 60 70 80 90 100
Abs
orba
nce a
t 765
nm
Gallic acid
Gallic acid (120583g mLminus1)
y = 00101x + 00178
R2 = 0982
Figure 3 Calibration curve of gallic acid
722 Scavenging Effect on DPPH Radical DPPH∙ is a stableradical showing amaximum absorbance at 515 nm InDPPH∙assay the antioxidant was able to reduce the stable radicalDPPH to the yellow-colored diphenylpicrylhydrazone Themethod is based on the reduction of DPPH∙ in alcoholicsolution in the presence of a hydrogen-donating antioxidantdue to formation of the nonradical form DPPH-H in thereaction DPPH∙ is usually used as a reagent to evaluatefree radical and accepts an electron or hydrogen radical tobecome a stable diamagnetic molecule The disappearanceof the DPPH radical absorption at 515 nm by the action ofantioxidants is taken as a measure of antioxidant activityThescavenging effects of black cumin oil and oleoresins onDPPHradical linearly increased as concentration increased from5 to 20120583gmLminus1 (Figure 2) At 20120583gmLminus1 the scavengingactivity of black cumin oil and ethyl acetate oleoresin was954 and 8975 respectively comparatively higher than
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0
100
200
300
400
500
0 7 14 21 28
PGBlack cumin oilEthyl acetate oleoBHT
EtOH oleo
BHAControl
Incubation time (days)
Pero
xide
val
ue (m
eq kg
minus1)
n-hexane oleo
Figure 4 Inhibitory effect of black cumin oil and its oleoresins onthe primary oxidation of linseed oil measured using peroxide valuemethod
BHT and BHA but lower than PG However the scavengingactivity of BHA BHT and PG was more effective at lowerconcentration and was 698 721 and 863 at 5 120583gmLminus1but as the concentration increases the differences in scav-enging activity between BHA BHT and oleoresins (speciallyethyl acetate) become less significant Ethanol and n-hexaneoleoresins showed moderate scavenging activity
723 Estimation of TPC The amount of total phenols wasdetermined with Folin-Ciocalteu reagent Gallic acid wasused as standard compound The absorbance for variousdilutions of gallic acid with Folin-Ciocalteu reagent andsodium carbonate was obtained and found standard curveequation 119910 = 00101119909 + 00178 1198772 = 0982 (Figure 3) Thetotal phenol contents (gallic acid equivalents mg GAE perg) in black cumin essential oil ethyl acetate ethanol and n-hexane oleoresins were calculated as 1147plusmn005 1088plusmn09968 plusmn 006 and 833 plusmn 001 respectively The value suggeststhat the black cumin oil and its oleoresins have lesser amountof total phenols [27] The differences in the total phenoliccontent among the samplesmight be due tomany differencessuch as the environmental conditions genetic background oragricultural techniques applied
73 Antioxidant Assays in Linseed Oil System The changes ofPV in linseed oil of all investigated samples are presented inFigure 4 The rate of oxidative reactions in linseed oil withadditives was almost similar to that of the blank sampleThe stability of the linseed oil samples to the formation ofperoxides can be ranked in the following descending order
PG gt Black cumin oil gt Ethyl acetate oleo gt BHT gtBHA gt EtOH oleo gt n-hexane oleo gt Control
Simultaneously with the measurements of PV changes insecondary product such as malondialdehyde the compound
0 7 14 21 28
PGBlack cumin oilEthyl acetate oleoBHA
BHTControl
Incubation time (days)
0
005
01
015
02
025
03
TBA
val
ue (m
eq O
2Kg
minus1)
EtOH oleo
n-hexane oleo
Figure 5 Inhibitory effect of black cumin oil and its oleoresinson the primary oxidation of linseed oil measured using TBA valuemethod
0
05
1
15
2
25
3
0 2 4 6 8 10 12
PGBlack cumin oilBHTEthyl acetate oleo
BHA
EtOH oleoControl
Abs
orba
nce a
t 500
nm
Incubation time (days)
n-hexane oleo
Figure 6 Inhibitory effect of black cumin oil and its oleoresins onthe primary oxidation of linoleic acid system measured using ferricthiocyanate method
used as an indicator of lipid peroxidation was measured byTBAvalues (Figure 5) were also determined after every sevendays Black cumin oil and its ethyl acetate oleoresin showedstrong inhibition at 002 concentration as compared toBHT and BHA (119875 lt 005) but lower than PG whereas theethanol and n-hexane oleoresin showed moderate inhibitionat 002 concentration as compared to the other additivesFrom the above results it should be confirmed that formationof primary oxidation species peroxides was quite similarwith the secondary oxidation products and the changes ofboth oxidation characteristics are in a good correlation
74 Antioxidant Activity in Linoleic Acid System The FTCmethod was used to measure the amount of peroxides at
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the primary stage of linoleic acid peroxidation (Figure 6)Since the concentration of the peroxide decreases as theantioxidant activity increases the intensity of the pigmentwill be reduced leading to lower absorbance Absorbancevalues of the control as well as black cumin oil and itsoleoresins increase until day 10 and then decreased on day12 due to the malondialdehyde formation from linoleic acidoxidation There was a significant difference (119875 lt 005)between the control and the tested essential oil and oleoresinsAs can be seen in Figure 6 both black cumin oil andoleoresins showed good antioxidative property in the linoleicacid system and were significantly (119875 lt 005) different fromthe control
Antioxidant activities of essential oils and oleoresinsmay be related to the diverse compounds present in themincluding terpenes sesquiterpenes and phenolic acids whichact in various ways such as inhibition of peroxidation scav-enging the radicals and chelating the metal ions The mainconstituents of black cumin oil were thymoquinone (376)and p-cymene (314) with minor amounts of longifolenecarvacrol and thymohydroquinone which were responsiblefor the antioxidant activity of black cumin oil [8 9 19 28ndash31] Furthermore free radical scavenging effects of thesecomponentswere studied on the reactions generating reactiveoxygen species such as superoxide anion radical hydroxylradical using spectrophotometric methods [5] The resultsobtained using different assays were well correlated withthe previous work reported be many workers [5 30] whofound thymoquinone as a main constituent responsible forthe activity It has been suggested that phenolic content iscorrelated with the antioxidant activity [32] It is consideredthat the antioxidant activity of phenolic compounds is dueto their high redox potentials which allow them to actas reducing agents hydrogen donors Thymoquinone wasalso present in small amount with higher percentage ofunsaturated fatty acid linoleic acid in different oleoresinsStudies [33] have shown that these unsaturated fatty acidshave anti-rather than pro-oxidant activity but still researchhas been going on for the exact role of unsaturated fatty acidsagainst the oxidative stress
75 Antimicrobial Investigations Using inverted petri platetechnique (Table 3) the volatile oil exhibited more than 90zone inhibition for F moniliforme and P viridicatum It wasalso found to be highly effective in controlling the growthof Aspergillus species and F graminearum where (50) and(65) zone inhibition was observed respectively For othertested fungi the essential oil exerted less activity Howeverusing the same method the oleoresins have revealed lessactivity except for F moniliforme in which only up to 40mycelial zone inhibition was obtained Moreover using foodpoison technique (Table 4) the volatile oil showed clear zoneof growth inhibition against F graminearum at 10 120583L Thevolatile oil showed strong antifungal activity against all testedAspergillus species in the food poison method Ethyl acetateand ethanol oleoresin showed up to 30 zone inhibitionat 10 120583L dose The n-hexane oleoresin showed very feeble
inhibition zone in both techniques The lower antimicrobialefficacy of the oleoresins is due to their low volatility [34]
The antibacterial investigations were undertaken usingagar well diffusion method (Table 4) Using this method theblack cumin oil has shown better activity than oleoresinsand commercial bactericide that is ampicillin The volatileoil was found to be highly effective against B subtilis Bcereus and S aureus and showed complete zone of inhibitionat 3000 ppm concentration whereas in oleoresins more than20mm inhibition was obtained for Gram-positive bacteriaIn addition more than 20 and 25mm zone inhibition wasobtained for P aeruginosa and E coli The results obtainedusing agar well diffusion method were well correlated withthe earlier reported work [6 30 35] where black cumin seedoil has been shown to be effective against a wide spectrumof organisms bacteria like B cereus B subtilis S aureus Sepidermis E coli and P aeruginosa
The results obtained in antimicrobial investigations ofblack cumin oil and oleoresins were in good agreementwith the previous reported work [36] Thymoquinone p-cymene (monoterpene) longifolene (sesquiterpene) andthymohydroquinone were responsible for strong antimicro-bial activity of black cumin oil [29] El Alfy et al [35]isolated thymohydroquinone as antimicrobial compoundfrom the volatile oil of Nigella sativa seeds Oleoresins havehigh concentration of unsaturated fatty acids along withthymohydroquinone in small amount which is responsiblefor its moderate antimicrobial effects Long chain fatty acidslike linoleic acid and oleic acid were previously reportedto possess antibacterial and antifungal activity [36ndash39] p-Cymene is not an efficient antimicrobial compound whenused alone but it potentiate the activity of compounds likecarvacrol [40] The antimicrobial activity of essential oils canoften be correlated to its content of phenolic constituentsThe type of bacteria also has an influence on the effectivenessof the volatile oil and oleoresins Gram-negative bacteriawere generally less susceptible than Gram-positive bacteria[41] The difference in the susceptibility of the bacteria arisesas a result of differences in their cell membrane structurewhich is more complex in case of Gram-negative bacteriaThe antimicrobial activity of a given essential oil may dependon only one or two of the major constituents that make upthe oil However increasing amounts of evidence indicatethat the inherent activity of essential oils may not only relyexclusively on the ratio in which the main active constituentsare present but also on interactions between these andminorconstituents in the oils and oleoresins
8 Conclusions
Seeds of black cumin seem to possess magical propertiesand have been worked out extensively This study revealedthat black cumin essential oil and its oleoresins constitutea good alternative source of essential fatty acids comparedwith common vegetable oil The present results showedthat essential oil and oleoresins of black cumin exhibitedhigher antioxidant activity than synthetic antioxidantsThesefindings could be used to prepare multipurpose products forpharmaceutical applications and its usage as dietary source of
8 BioMed Research International
Table 3 (a) Antifungal investigations of black cumin oil and its oleoresins ( zone inhibitiona) using inverted petriplate method (b)Antifungal investigations of black cumin oil and its oleoresins ( zone inhibition) using food poisoned method
(a)
Mycelial zone inhibition at different dosesa of sample ()Samples Doses (120583L) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
(b)
Mycelial zone inhibitiona at different doses of sample ()Samples Doses (ppm) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
Table 4 Antibacterial activity of black cumin oil and its oleoresinsagainst a few bacterial species using agar well diffusion method
Diameter of inhibition zone (mma)Samples Doses (ppmb) BS BC SA EC PA
Black cumin oil 1000 ++ 253 plusmn 14 ++ minus 189 plusmn 017
3000 ++ ++ ++ 203 plusmn 014 279 plusmn 015
Ethanol oleoresin 1000 164 plusmn 081 minus 156 plusmn 036 minus minus
3000 283 plusmn 020 minus 259 plusmn 042 minus minus
n-Hexane oleoresin 1000 115 plusmn 031 minus 91 plusmn 11 minus minus
3000 209 plusmn 19 minus 134 plusmn 26 minus minus
Ethyl acetate oleoresin1000 223 plusmn 08 minus 177 plusmn 23 minus minus
3000 443 plusmn 07 minus 409 plusmn 12 minus minus
Ampicillin 1000 156 plusmn 032 minus 91 plusmn 11 minus minus
3000 132 plusmn 02 minus 134 plusmn 26 minus minus
aAverage of three replicates ++ indicates complete inhibition and minus indicates no inhibitionbDMSO was used as solventBS Bacillus subtilis SA Staphylococcus aureus BC Bacillus cereus EC Escherichia coli PA Pseudomonas aeruginosa
BioMed Research International 9
antioxidant should be considered largely for alleviating andameliorating diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to theHead of Department of Chem-istry DDU Gorakhpur University Gorakhpur for providinglaboratory facilities The financial support from UGC toSunita Singh (JRF) and Emeritus Fellow to Dr Gurdip Singhis also acknowledged
References
[1] G Zengin A Aktumsek G O Guler Y S Cakmak andE Yildiztugay ldquoAntioxidant properties of methanolic extractand fatty acid composition of Centaurea urvillei DC subsphayekiana Wagenitzrdquo Records of Natural Products vol 5 no 2pp 123ndash132 2011
[2] H Lutterodt M Luther M Slavin et al ldquoFatty acid profilethymoquinone content oxidative stability and antioxidantproperties of cold-pressed black cumin seed oilsrdquo Food Scienceand Technology vol 43 no 9 pp 1409ndash1413 2010
[3] S Shah and K S Ray ldquoStudy on antioxidant and antimicrobialproperties of black cumin (Nigella sativa Linn)rdquo Journal of FoodScience and Technology vol 40 no 1 pp 70ndash73 2003
[4] I Tekeoglu A Dogan and L Demiralp ldquoEffects of thymo-quinone (volatile oil of black cumin) on rheumatoid arthritis inrat modelsrdquo Phytotherapy Research vol 20 no 10 pp 869ndash8712006
[5] I Kruk TMichalska K Lichszteld A Kladna andH Y Aboul-Enein ldquoThe effect of thymol and its derivatives on reactionsgenerating reactive oxygen speciesrdquo Chemosphere vol 41 no7 pp 1059ndash1064 2000
[6] G Singh P Marimuthu C S De Heluani and C Cata-lan ldquoChemical constituents and antimicrobial and antioxidantpotentials of essential oil and acetone extract of Nigella sativaseedsrdquo Journal of the Science of Food and Agriculture vol 85no 13 pp 2297ndash2306 2005
[7] S A Maisonneuve European Pharmacopoeia Part 1 Sainte-Ruffine 1983
[8] M Burits and F Bucar ldquoAntioxidant activity of Nigella sati-vaessential oilrdquo Phytotherapy Research vol 14 pp 323ndash3282000
[9] N Ilaiyaraja and F Khanum ldquoNigella sativa L a review of thera-peutic applicationsrdquo Journal of Herbal Medicine and Toxicologyvol 4 no 2 pp 1ndash8 2010
[10] E A Decker and B Welch ldquoRole of ferritin as a lipid oxidationcatalyst in muscle foodrdquo Journal of Agricultural and FoodChemistry vol 38 no 3 pp 674ndash677 1990
[11] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo Food Scienceand Technology vol 28 no 1 pp 25ndash30 1995
[12] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic as it reagentsrdquo TheAmerican Journal of Enology and Viticulture vol 16 pp 144ndash158 1965
[13] K D Economou V Oreopoulou and C D ThomopoulosldquoAntioxidant activity of some plant extracts of the familylabiataerdquo Journal of the American Oil Chemists Society vol 68no 2 pp 109ndash113 1991
[14] G Singh S Maurya M P de Lampasona and C A N CatalanldquoA comparison of chemical antioxidant and antimicrobialstudies of cinnamon leaf and bark volatile oils oleoresins andtheir constituentsrdquo Food and Chemical Toxicology vol 45 no 9pp 1650ndash1661 2007
[15] G Singh I P S Kapoor P Singh C S de Heluani M P deLampasona and C A N Catalan ldquoChemistry antioxidant andantimicrobial investigations on essential oil and oleoresins ofZingiber officinalerdquo Food and Chemical Toxicology vol 46 no10 pp 3295ndash3302 2008
[16] G P Rao and A K Shrivastava ldquoToxicity of essential oils ofhigher plants against fungal pathogens of sugarcanerdquo inCurrentTrend in Sugarcane Pathology G P Rao A G Gillasple PP Upadhaya A Bergamin V P Agnihotri and C T ChenEds pp 347ndash365 International Books and Periodicals SupplyService Delhi India 1994
[17] K Ramadas G Suresh N Janarthanan and S Masila-mani ldquoAntifungal activity of 13-disubstituted symmetrical andunsymmetrical thioureasrdquo Pesticide Science vol 52 no 2 pp145ndash151 1998
[18] NCCLS (National Committee for Clinical Laboratory Stan-dards) Performance Standards for Antimicrobial Disc Suscepti-bility Test 6th edition 1997
[19] V Hajhashemi A Ghannadi and H Jafarabadi ldquoBlack cuminseed essential oil as a potent analgesic and antiinflammatorydrugrdquo Phytotherapy Research vol 18 no 3 pp 195ndash199 2004
[20] P J Houghton R Zarka B De Las Heras and J R SHoult ldquoFixed oil of Nigella sativa and derived thymoquinoneinhibit eicosanoid generation in leukocytes andmembrane lipidperoxidationrdquo Planta Medica vol 61 no 1 pp 33ndash36 1995
[21] M T SultanM S Butt FMAnjumA Jamil S Akhtar andMNasir ldquoNutritional profile of indigenous cultivar of black cuminseeds and antioxidant potential of its fixed and essential oilrdquoPakistan Journal of Botany vol 41 no 3 pp 1321ndash1330 2009
[22] S Cheikh-Rouhou S Besbes G Lognay C Blecker CDeroanne and H Attia ldquoSterol composition of black cumin(Nigella sativa L) and Aleppo pine (Pinus halepensisMill) seedoilsrdquo Journal of Food Composition and Analysis vol 21 no 2 pp162ndash168 2008
[23] M F Ramadan ldquoNutritional value functional properties andnutraceutical applications of black cumin (Nigella sativa L) anoverviewrdquo International Journal of Food Science and Technologyvol 42 no 10 pp 1208ndash1218 2007
[24] T D Parker D A Adams K Zhou M Harris and L Yu ldquoFattyacid composition and oxidative stability of cold-pressed edibleseed oilsrdquo Journal of Food Science vol 68 no 4 pp 1240ndash12432003
[25] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAmerican Oil Chemical Society vol 83 pp 847ndash854 2006
[26] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAgricultural and Food Chemistry vol 53 no 3 pp 566ndash5732005
[27] R R Sokal Introduction to Biostatistics W H Freeman SanFrancisco Calif USA 1973
10 BioMed Research International
[28] H Hosseinzadeh S Parvardeh M N Asl H R Sadeghniaand T Ziaee ldquoEffect of thymoquinone and Nigella sativa seedsoil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampusrdquo Phytomedicine vol 14no 9 pp 621ndash627 2007
[29] S Bourgou A Pichette B Marzouk and J Legault ldquoBioactiv-ities of black cumin essential oil and its main terpenes fromTunisiardquo South African Journal of Botany vol 76 no 2 pp 210ndash216 2010
[30] H J Harzallah E Noumi K Bekir et al ldquoChemical composi-tion antibacterial and antifungal properties of Tunisian Nigellasativa fixed oilrdquoAfrican Journal of Microbiology Research vol 6no 22 pp 4675ndash4679 2012
[31] V S Deepa and P S Kumar ldquoPreliminary phytochemicalinvestigations and in vitro antioxidant activity in selected partsof Andrographis spprdquo Journal of Pharmacological Research vol3 no 9 pp 2206ndash2210 2010
[32] M Amensour E Sendra A Jamal S Bouhdid J A Perez-Alvarez and J Fernandez-Lopez ldquoTotal phenolic content andantioxidant activity of myrtle (Myrtus communis) extractsrdquoNatural Product Communications vol 4 no 6 pp 819ndash8242009
[33] M Di Nunzio V Valli and A Bordoni ldquoPro- and anti-oxidanteffects of polyunsaturated fatty acid supplementation in HepG2cellsrdquo Prostaglandins Leukotrienes and Essential Fatty Acids vol85 no 3-4 pp 121ndash127 2011
[34] G Singh SMaurya CCatalan andMP de Lampasona ldquoStud-ies on essential oils part 42 chemical antifungal antioxidantand sprout suppressant studies on ginger essential oil and itsoleoresinrdquo Flavour and Fragrance Journal vol 20 no 1 pp 1ndash62005
[35] T S El Alfy H M El Fatatry and M A Toama ldquoIsolationand structure assignment of an antimicrobial principle from thevolatile oil of Nigella sativa L seedsrdquo Pharmazie vol 30 no 2pp 109ndash111 1975
[36] M A Khan ldquoChemical composition and medicinal propertiesof Nigella sativa Linnrdquo Inflammopharmacology vol 7 no 1 pp15ndash35 1999
[37] L J McGaw A K Jager and J van Staden ldquoIsolation ofantibacterial fatty acids from Schotia brachypetalardquo Fitoterapiavol 73 no 5 pp 431ndash433 2002
[38] A R McCutcheon T E Roberts E Gibbons et al ldquoAntiviralscreening of British Columbian medicinal plantsrdquo Journal ofEthnopharmacology vol 49 no 2 pp 101ndash110 1995
[39] S Javed A A Shahid M S Haider et al ldquoNutritionalphytochemical potential and pharmacological evaluation ofNigella Sativa (Kalonji) and Trachyspermum Ammi (Ajwain)rdquoMedicinal Plants Research vol 6 no 5 pp 768ndash775 2012
[40] P Rattanachaikunsopon and P Phumkhachorn ldquoAssessmentof factors influencing antimicrobial activity of carvacrol andcymene against Vibrio cholerae in foodrdquo Journal of Bioscienceand Bioengineering vol 110 no 5 pp 614ndash619 2010
[41] M Gilles J Zhao M An and S Agboola ldquoChemical com-position and antimicrobial properties of essential oils of threeAustralian Eucalyptus speciesrdquo Food Chemistry vol 119 no 2pp 731ndash737 2010
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
The seeds of black cumin were purchased from the localmarket of Gorakhpur Uttar Pradesh India A voucherspecimen was deposited at the herbarium of the Faculty ofScience DDU Gorakhpur University
21 Reagents Thiobarbituric acid (TBA) 111015840-diphenyl-2-picrylhydrazyl radical (DPPH) and linoleic acid are ofAcros (New Jersey USA) butylated hydroxytoluene (BHT)butylated hydroxyanisole (BHA) and propyl gallate (PG) areof S D Fine Chemicals Ltd Mumbai India Folin-Ciocalteureagent and gallic acid were from Qualigens Chemicals LtdMumbai India and Qualikems Chemicals Ltd New DelhiIndia respectively Tween 20 and ferrozine were fromMerckPvt Ltd Mumbai India Ampicillin was purchased fromRanbaxy Fine Chemicals (New Delhi) India Crude linseedoil was obtained from local oil mill in Gorakhpur All solventsused were of analytical grade
22 Sample Extraction Powdered seeds of black cumin(250 g) were subjected to hydrodistillation in Clevengerapparatus for 3 h according to the method recommended byEuropean Pharmacopoeia [7] A volatile oil with light orangecharacteristic odour was obtained with yield of 09 It wasdried over anhydrous sodium sulphate and the sample wasstored at 4∘C before use
Oleoresins were obtained by extracting 30 g of powderedspice with 300mL of various solvents (ethanol ethyl acetateand n-hexane) for 3 h in Soxhlet extractor Evaporation ofthe solvents at reduced pressure gave viscous extracts Theoleoresins were stored in freezer until further use
23 Chemical Characterization
231 Gas Chromatography-Mass Spectrometry (GC-MS)Analysis of the volatile oils and oleoresins was run on aHewlett Packard (6890) GC-Ms system coupled to a quadru-ple mass spectrometer (model HP 5973) with a capillarycolumn of HP-5MS (5 phenyl methylsiloxane length =30m inner diameter = 025mm and film thickness =025 120583m) GC-MS interphase ion source and selective massdetector temperatures were maintained at 280∘C 230∘C and150∘C respectively Carrier gas used was helium with a flowrate of 10mLminminus1The oven temperature was programmedas follows
For essential oil at 60∘C for 1min then increased from60 to 185∘C at the rate of 15∘Cminminus1 and held at the rate of9∘Cminminus1 and held at 275∘C for 2min
For oleoresin 60∘C for zero min then increased from 60to 300∘C at the rate of 15∘Cminminus1 and held at the rate of5∘Cminminus1 and held at 300∘C for 10min
24 Identification of Components Most of the componentswere identified on the basis of comparison of their retentionindices and mass spectra with published data [6 8 9]and computer matching was done with the Wiley 275 andNational Institute of Standards Technology libraries provided
Table 1 Chemical composition of essential oil obtained from blackcumin seeds analyzed by GC-MS
Compounds MS RI IdentificationΦ
120572-Thujene 56 919 MS RI co-GC120572-Pinene 14 928 MS RI co-GCSabinene 08 967 MS RI co-GC120573-Pinene 17 973 MS RI co-GC120572-Phellandrene 01 1003 MS RI120572-Terpinene 02 1012 MS RI co-GC119901-Cymene 314 1019 MS RI co-GCLimonene 10 1024 MS RI co-GC18-Cineole 01 1025 MS RI co-GC120574-Terpinene 02 1050 MS RI co-GCtrans-Sabinene hydrate 01 1101 MS RIUnidentified Blowast 68 1113 minus
Terpinen-4-ol 10 1172 MS RI co-GC119901-Cymen-8-ol Trace 1179 MS RI120572-Terpineol Trace 1189 MS RI co-GCCuminal Trace 1240 MS RICarvone Trace 1241 MS RIThymoquinone 376 1248 MS RItrans-Sabinene hydrate acetate 01 1258 MS RIBornyl acetate 02 1285 MS RIThymol 02 1289 MS RICarvacrol 14 1295 MS RI120572-Longipinene 05 1353 MS RILongifolene 20 1405 MS RIThymohydroquinone 34 1559 MS RI10-epi-120574-Eudesmol 03 1625 MS RI120573-Eudesmol 05 1652 MS RI120572-Eudesmol 04 1655 MS RITotal 902Trace lt 005 the retention index was calculated using a homologousseries of n-alkanes C8ndashC18 ΦCo-GC coinjection with an authentic samplePercentages were obtained from electronic integration Trace measurementsusing a selective mass detector
with the computer controllingGC-MS systemsThe retentionindices were calculated using a homologous series of n-alkanes C
8ndashC18
and C8ndashC22
for essential oil and oleoresinsrespectively which are reported in Tables 1 and 2
3 Antioxidant Activity
The antioxidant activity is system dependent and accordingto the method adopted and lipid system used as substrateHence different methods have been adopted in order toassess antioxidative potential of black cumin oil and itsoleoresins are as follows
31 Chelating Activity on Ferrous Ions The chelating activityof the aqueous and ethanolic extract on ferrous ions (Fe2+)was measured according to the method described by Deckerand Welch [10] Aliquots of 1mL of different concentrations
BioMed Research International 3
Table 2 Chemical composition of oleoresins obtained from black cumin (Nigella sativa L) seeds in different solvents analysed by GC-MS
Compounds M1 M2 M3 RI IdentificationΦ
120572-Thujene Trace 04 06 919 MS RI co-GC119901-Cymene 09 28 22 1019 MS RI co-GCUnidentified A 05 05 04 1092 minus
Thymoquinone 57 61 37 1248 MS RICarvacrol 04 Trace Trace 1295 MS RI co-GC120572-Longipinene Trace Trace Trace 1353 MS RILongifolene 05 06 03 1405 MS RIThymohydroquinone 25 16 05 1559 MS RIPalmitic acid ethyl ester 28 Trace Trace 1979 MS co-GCLinoleic acid methyl ester 06 05 05 minus MS co-GCLinoleic acid ethyl ester 116 Trace 06 minus MS co-GCOleic acid ethyl ester 46 Trace 02 minus MS co-GCOleic acid 03 Trace 02 minus MS co-GCLinoleic acid 330 439 277 minus MSLinoleic acid butyl ester 09 57 160 minus MSOleic acid butyl ester 12 45 73 minus MSGlyceryl palmitate 37 16 23 minus MSGlyceryl linoleate 277 219 231 minus MSSitosterol Trace 13 Trace minus MS co-GCTotal 964 909 852Trace lt 005 the retention index was calculated using a homologous series of n-alkanes C8ndashC20 ΦCo-GC coinjection with an authentic samplePercentages were obtained from electronic integration measurements using selective mass detectorM1 ethanol oleoresin M2 ethyl acetate oleoresin M3 n-hexane oleoresin
of the samples were mixed with 37mL of deionized waterThe mixture was incubated with FeCl
2(2mM 01mL)
After incubation the reaction was initiated by addition offerrozine (5mM and 02mL) for 10min at room temperatureand then the absorbance was measured at 562 nm in aspectrophotometer A lower absorbance indicates a higherchelating power The chelating activity of the extract on Fe2+was compared with that of EDTA that was used as positivecontrol Chelating activity was calculated using the followingformula
Chelating activity ()
= [1 minus (
Absorbance of sampleAbsorbance of control
)] times 100
(1)
32 Scavenging Effect on DPPH TheDPPH assay constitutesa quick and low cost method which has frequently been usedfor the evaluation of the antioxidative potential of variousnatural products [11] Due to its odd electron DPPH givesa strong absorption band at 517 nm (deep violet colour) Inthe presence of a free radical scavenger this electron becomespaired resulting in the absorption loss and consecutivestoichiometric decolorization with respect to the number ofelectron acquired The absorbance change produced by thisreaction is assessed to evaluate the antioxidant potential ofthe test sample 5 10 15 and 20 120583L of the sample were addedto 5mLof 0004methanol solution ofDPPHAfter a 30minincubation period at room temperature the absorbance was
read against a blank at 515 nm All determination was per-formed in triplicate and results were performed in triplicateand results are reported as scavenging effect () versusconcentration in Figure 2
33 Estimation of Total Phenolic Content (TPC) TPC weredetermined using the Folin-Ciocalteu reagent methoddescribed by Singleton and Rossi [12] Gallic acid stocksolution (1000 120583gmLminus1) was prepared by dissolving 100mgof gallic acid in 100mL of ethanol Various dilutions ofstandard gallic acid were prepared from this stock solutionCalibration curve (Figure 3) was plotted by mixing 1mLaliquots of 10ndash100120583gmLminus1 of gallic acid solutionswith 50mLof Folin-Ciocalteu reagent (diluted tenfold) and 40mL ofsodium carbonate solution (75 g Lminus1) The absorbance wasmeasured after 30min at 20∘C at 765 nm
4 Evaluation of Antioxidant Activity forLinseed Oil System
For present investigation crude linseed oil having initialperoxide value 52meq kgminus1 was taken to assess the antiox-idant activity of black cumin oil and its oleoresins This oilis most frequently used edible oil in central Europe and israther unstable because of the presence of substantial amountof linoleic acid The antioxidant activity of volatile oil andextract was examined by comparing the activity of knownantioxidants such as PG BHT and BHA by the followingperoxide value and thiobarbituric acid value methods
4 BioMed Research International
41 Peroxide Value Method For measuring the peroxidevalue (PV) a modified oven test was used [13] Theantioxidant activity of black cumin oil and its oleoresinsin different solvents were compared with the syntheticantioxidants such as PG BHT and BHA For this purposecalculated quantities of each (200 ppm)were dissolved to 30 gof linseed oil in an open mouthed beaker The mixtures werethoroughly homogenized and placed in incubator at 80∘CThe peroxide values (meq of oxygen kgminus1) were measured inevery seven days and test was replicated for three times Acontrol sample was prepared under similar conditionwithoutany additive The effects of oil and oleoresins in term ofperoxidation at 90∘C are shown in Figure 4
42 Thiobarbituric Acid Value (TBA) TBA value of differentsamples was determined according to the method previouslyreported [13] About 100mg of oil sample was dissolved in25mL of 1-butanol A 25mL aliquot of the above solutionwas mixed thoroughly with 50mL of TBA reagent (200mgTBA in 100mL of 1-butanol) and incubated at 95∘C After 2 hthe reaction mixture was cooled to room temperature underrunning water and absorbance was measured at 530 nm withHitachi-U-2000 spectrophotometer (Tokyo Japan) At thesame time a reagent blank (without TBA reagent) was alsodoneThe thiobarbituric acid value (meq ofmalondialdehydeper g) was calculated as
TBA value = 50 times (119860 minus 119861)119872
(2)
where 119860 is absorbance of the test sample 119861 is absorbance ofthe reagent blank and119872 is mass of the sample
43 Determination of Antioxidant Activity in Linoleic AcidSystem Antioxidant activity of black cumin oil and itsoleoresins was compared to synthetic standards accordingto the ferric thiocyanate method in linoleic acid emulsion[14] The reaction medium contained black cumin oil andoleoresins at the concentration of 1mg100mL of absoluteethanol (2mL) an emulsion of 251 linoleic acid in ethanol(2mL) 4mL of 005M-phosphate buffer (pH = 70) and2mL of distilled water The solution (10mL) was mixed andincubated at 40∘C in the darkThe same solution without anyadditives was taken as control sample At regular intervalsduring incubation 01mL aliquot of the mixture was dilutedwith 97mL of 75 ethanol followed by the addition of02mL of 30 ammonium thiocyanate and 01mL of 20mMof FeCl
2in 35 HCl the absorbance of red colour was
measured at 500 nm in Hitachi-U-2000 spectrophotometer(Tokyo Japan) The control and standard were subjected tothe same procedure except for the control where there wasno addition of sample and for the standard 1mL of samplewere replaced with 1mg of PG BHT and BHA These stepsare repeated every 48 h until the control sample reached itsmaximum Low absorbance value indicates the efficiency ofthe test samples to inhibit lipid oxidation The results werereported as incubation time versus absorbance in Figure 6
5 Antimicrobial Investigations
51 Antifungal Assay The antifungal activity of the essentialoil and extract against various pathogenic fungi Aspergilluslavus (1884) Aspergillus niger (2479) Fusarium moniliforme(1893) Fusarium graminearum (2088) and Penicillium viridi-catum (2007) were tested by the inverted petri plate [15] andpoison food medium methods [16] All the fungi cultureswere procured from the Microbial Type Culture Collection(MTCC) and their reference numbers are given in theparentheses The cultures were maintained in Czapek agarmedium Each test was replicated three times and fungi toxi-city was measured in terms of percentage mycelial inhibitioncalculated with the following equation
colony of the control and treated sets respectively
52 Antibacterial Assay The essential oil and extract wereindividually tested against a panel of microorganisms usingagar well diffusion method [17] Three Gram-positive bacte-ria Staphylococcus aureus (3103) Bacillus cereus (430) andBacillus subtilis (1790) and two Gram negative bacteriaEscherichia coli (1672) and Pseudomonas aeruginosa (1942)All the bacterial strains were procured from the MicrobialType Culture Collection (MTCC) Institute of MicrobialTechnology (Chandigarh India) and their reference num-bers are given in parentheses The bacterial cultures weregrown on nutrient agar medium and stored at 4∘C In orderto prepare a bacterial strain for test initially one loopfulof bacterial culture was transferred from slant to nutrientbroth solution (10mL) and was stored at 37∘C for 24 hThe control plate without the addition of essential oil orextract containing DMSO was also maintained under thesame conditions After incubating for 24 h at 37∘C all plateswere examined for any zones of growth inhibition and thediameters of these zones were measured in millimeters
6 Statistical Analysis
For the essential oil or oleoresin three samples were preparedfor assays of every antioxidant and antimicrobial attributeThe data are presented as mean (standard deviation of threedeterminations (data are not shown) Statistical analyseswere performed using a one-way analysis of variance [18] Aprobability value of119875 lt 005was considered to be significant
7 Results and Discussions
71 Phytochemistry Careful and detailed interpretations ofthe experimental GC-MS data (EM fragmentation retentionindices) were carried out which permitted identification ofa large number of components in essential oil and oleo-resins (Tables 1 and 2) Table 1 shows identification of 33components in black cumin oil representing about 902 ofthe total amount The major component in black cumin oil
BioMed Research International 5
0
25
50
75
100
0 5 10 15 20 25
Black cumin oilEtOH oleon-hexane oleo
Ethyl acetate oleoEDTA
Chel
atin
g eff
ect (
)
Concentration (120583g mLminus1)
Figure 1 Chelating effect of black cumin oil and its differentoleoresins
was thymoquinone (376) followed by p-cymene (314)120572-thujene (56) thymohydroquinone (34) longifolene(20) and carvacrol (14) Burits and Bucar [8] character-ized many components in black cumin essential oil such asthymoquinone (278ndash570) p-cymene (71ndash155) carvacrol(58ndash116) trans-anethole (025ndash23) 4-terpineol (20ndash66) and 10ndash80 of longifolene These results are slightdifferent from the work reported by Hajhashemi et al [19]who reported that p-cymene (373) and thymoquinone(137) were the major components of black cumin Singh etal [6] also reported p-cymene as the major component in theblack cumin essential oil
From Table 2 it is evident that in ethanol oleoresin19 components constitute 964 of the total weight in theethyl acetate oleoresin a total of 19 components making909 of the whole mass and in case of n-hexane oleoresin19 compounds constituting about 852 of the total weightwere identified The oleoresins were mainly comprised ofunsaturated fatty acids and their different esters The majorcomponents in all three oleoresins were linoleic acid (unsat-urated fatty acid) followed by glyceryl linoleate glycerylpalmitate oleic acid and other minor components Thepresence of unsaturated fatty acids in oleoresins was wellsupported by the various reported work [20ndash26]
72 Antioxidant Investigations
721 Chelating Activity on Ferrous Ions The ferrous ion(Fe2+) chelating effect of black cumin oil and its differentoleoresins is presented in Figure 1 The chelating activityof the extracts was concentration dependent Black cuminoil exhibited higher chelating activity in comparison to theoleoresins but was not effective chelator as EDTA Maximumchelating of metal ions at 200120583gmLminus1 for black cumin oiland EDTA was found to be 7456 and 8790 respectivelywhereas the oleoresins were less effective in metal chelationand their metal chelating activity ranges from 221 to 445
Figure 2 Scavenging effect () of black cumin oil and its oleoresinson DPPH radical
0
02
04
06
08
1
12
0 10 20 30 40 50 60 70 80 90 100
Abs
orba
nce a
t 765
nm
Gallic acid
Gallic acid (120583g mLminus1)
y = 00101x + 00178
R2 = 0982
Figure 3 Calibration curve of gallic acid
722 Scavenging Effect on DPPH Radical DPPH∙ is a stableradical showing amaximum absorbance at 515 nm InDPPH∙assay the antioxidant was able to reduce the stable radicalDPPH to the yellow-colored diphenylpicrylhydrazone Themethod is based on the reduction of DPPH∙ in alcoholicsolution in the presence of a hydrogen-donating antioxidantdue to formation of the nonradical form DPPH-H in thereaction DPPH∙ is usually used as a reagent to evaluatefree radical and accepts an electron or hydrogen radical tobecome a stable diamagnetic molecule The disappearanceof the DPPH radical absorption at 515 nm by the action ofantioxidants is taken as a measure of antioxidant activityThescavenging effects of black cumin oil and oleoresins onDPPHradical linearly increased as concentration increased from5 to 20120583gmLminus1 (Figure 2) At 20120583gmLminus1 the scavengingactivity of black cumin oil and ethyl acetate oleoresin was954 and 8975 respectively comparatively higher than
6 BioMed Research International
0
100
200
300
400
500
0 7 14 21 28
PGBlack cumin oilEthyl acetate oleoBHT
EtOH oleo
BHAControl
Incubation time (days)
Pero
xide
val
ue (m
eq kg
minus1)
n-hexane oleo
Figure 4 Inhibitory effect of black cumin oil and its oleoresins onthe primary oxidation of linseed oil measured using peroxide valuemethod
BHT and BHA but lower than PG However the scavengingactivity of BHA BHT and PG was more effective at lowerconcentration and was 698 721 and 863 at 5 120583gmLminus1but as the concentration increases the differences in scav-enging activity between BHA BHT and oleoresins (speciallyethyl acetate) become less significant Ethanol and n-hexaneoleoresins showed moderate scavenging activity
723 Estimation of TPC The amount of total phenols wasdetermined with Folin-Ciocalteu reagent Gallic acid wasused as standard compound The absorbance for variousdilutions of gallic acid with Folin-Ciocalteu reagent andsodium carbonate was obtained and found standard curveequation 119910 = 00101119909 + 00178 1198772 = 0982 (Figure 3) Thetotal phenol contents (gallic acid equivalents mg GAE perg) in black cumin essential oil ethyl acetate ethanol and n-hexane oleoresins were calculated as 1147plusmn005 1088plusmn09968 plusmn 006 and 833 plusmn 001 respectively The value suggeststhat the black cumin oil and its oleoresins have lesser amountof total phenols [27] The differences in the total phenoliccontent among the samplesmight be due tomany differencessuch as the environmental conditions genetic background oragricultural techniques applied
73 Antioxidant Assays in Linseed Oil System The changes ofPV in linseed oil of all investigated samples are presented inFigure 4 The rate of oxidative reactions in linseed oil withadditives was almost similar to that of the blank sampleThe stability of the linseed oil samples to the formation ofperoxides can be ranked in the following descending order
PG gt Black cumin oil gt Ethyl acetate oleo gt BHT gtBHA gt EtOH oleo gt n-hexane oleo gt Control
Simultaneously with the measurements of PV changes insecondary product such as malondialdehyde the compound
0 7 14 21 28
PGBlack cumin oilEthyl acetate oleoBHA
BHTControl
Incubation time (days)
0
005
01
015
02
025
03
TBA
val
ue (m
eq O
2Kg
minus1)
EtOH oleo
n-hexane oleo
Figure 5 Inhibitory effect of black cumin oil and its oleoresinson the primary oxidation of linseed oil measured using TBA valuemethod
0
05
1
15
2
25
3
0 2 4 6 8 10 12
PGBlack cumin oilBHTEthyl acetate oleo
BHA
EtOH oleoControl
Abs
orba
nce a
t 500
nm
Incubation time (days)
n-hexane oleo
Figure 6 Inhibitory effect of black cumin oil and its oleoresins onthe primary oxidation of linoleic acid system measured using ferricthiocyanate method
used as an indicator of lipid peroxidation was measured byTBAvalues (Figure 5) were also determined after every sevendays Black cumin oil and its ethyl acetate oleoresin showedstrong inhibition at 002 concentration as compared toBHT and BHA (119875 lt 005) but lower than PG whereas theethanol and n-hexane oleoresin showed moderate inhibitionat 002 concentration as compared to the other additivesFrom the above results it should be confirmed that formationof primary oxidation species peroxides was quite similarwith the secondary oxidation products and the changes ofboth oxidation characteristics are in a good correlation
74 Antioxidant Activity in Linoleic Acid System The FTCmethod was used to measure the amount of peroxides at
BioMed Research International 7
the primary stage of linoleic acid peroxidation (Figure 6)Since the concentration of the peroxide decreases as theantioxidant activity increases the intensity of the pigmentwill be reduced leading to lower absorbance Absorbancevalues of the control as well as black cumin oil and itsoleoresins increase until day 10 and then decreased on day12 due to the malondialdehyde formation from linoleic acidoxidation There was a significant difference (119875 lt 005)between the control and the tested essential oil and oleoresinsAs can be seen in Figure 6 both black cumin oil andoleoresins showed good antioxidative property in the linoleicacid system and were significantly (119875 lt 005) different fromthe control
Antioxidant activities of essential oils and oleoresinsmay be related to the diverse compounds present in themincluding terpenes sesquiterpenes and phenolic acids whichact in various ways such as inhibition of peroxidation scav-enging the radicals and chelating the metal ions The mainconstituents of black cumin oil were thymoquinone (376)and p-cymene (314) with minor amounts of longifolenecarvacrol and thymohydroquinone which were responsiblefor the antioxidant activity of black cumin oil [8 9 19 28ndash31] Furthermore free radical scavenging effects of thesecomponentswere studied on the reactions generating reactiveoxygen species such as superoxide anion radical hydroxylradical using spectrophotometric methods [5] The resultsobtained using different assays were well correlated withthe previous work reported be many workers [5 30] whofound thymoquinone as a main constituent responsible forthe activity It has been suggested that phenolic content iscorrelated with the antioxidant activity [32] It is consideredthat the antioxidant activity of phenolic compounds is dueto their high redox potentials which allow them to actas reducing agents hydrogen donors Thymoquinone wasalso present in small amount with higher percentage ofunsaturated fatty acid linoleic acid in different oleoresinsStudies [33] have shown that these unsaturated fatty acidshave anti-rather than pro-oxidant activity but still researchhas been going on for the exact role of unsaturated fatty acidsagainst the oxidative stress
75 Antimicrobial Investigations Using inverted petri platetechnique (Table 3) the volatile oil exhibited more than 90zone inhibition for F moniliforme and P viridicatum It wasalso found to be highly effective in controlling the growthof Aspergillus species and F graminearum where (50) and(65) zone inhibition was observed respectively For othertested fungi the essential oil exerted less activity Howeverusing the same method the oleoresins have revealed lessactivity except for F moniliforme in which only up to 40mycelial zone inhibition was obtained Moreover using foodpoison technique (Table 4) the volatile oil showed clear zoneof growth inhibition against F graminearum at 10 120583L Thevolatile oil showed strong antifungal activity against all testedAspergillus species in the food poison method Ethyl acetateand ethanol oleoresin showed up to 30 zone inhibitionat 10 120583L dose The n-hexane oleoresin showed very feeble
inhibition zone in both techniques The lower antimicrobialefficacy of the oleoresins is due to their low volatility [34]
The antibacterial investigations were undertaken usingagar well diffusion method (Table 4) Using this method theblack cumin oil has shown better activity than oleoresinsand commercial bactericide that is ampicillin The volatileoil was found to be highly effective against B subtilis Bcereus and S aureus and showed complete zone of inhibitionat 3000 ppm concentration whereas in oleoresins more than20mm inhibition was obtained for Gram-positive bacteriaIn addition more than 20 and 25mm zone inhibition wasobtained for P aeruginosa and E coli The results obtainedusing agar well diffusion method were well correlated withthe earlier reported work [6 30 35] where black cumin seedoil has been shown to be effective against a wide spectrumof organisms bacteria like B cereus B subtilis S aureus Sepidermis E coli and P aeruginosa
The results obtained in antimicrobial investigations ofblack cumin oil and oleoresins were in good agreementwith the previous reported work [36] Thymoquinone p-cymene (monoterpene) longifolene (sesquiterpene) andthymohydroquinone were responsible for strong antimicro-bial activity of black cumin oil [29] El Alfy et al [35]isolated thymohydroquinone as antimicrobial compoundfrom the volatile oil of Nigella sativa seeds Oleoresins havehigh concentration of unsaturated fatty acids along withthymohydroquinone in small amount which is responsiblefor its moderate antimicrobial effects Long chain fatty acidslike linoleic acid and oleic acid were previously reportedto possess antibacterial and antifungal activity [36ndash39] p-Cymene is not an efficient antimicrobial compound whenused alone but it potentiate the activity of compounds likecarvacrol [40] The antimicrobial activity of essential oils canoften be correlated to its content of phenolic constituentsThe type of bacteria also has an influence on the effectivenessof the volatile oil and oleoresins Gram-negative bacteriawere generally less susceptible than Gram-positive bacteria[41] The difference in the susceptibility of the bacteria arisesas a result of differences in their cell membrane structurewhich is more complex in case of Gram-negative bacteriaThe antimicrobial activity of a given essential oil may dependon only one or two of the major constituents that make upthe oil However increasing amounts of evidence indicatethat the inherent activity of essential oils may not only relyexclusively on the ratio in which the main active constituentsare present but also on interactions between these andminorconstituents in the oils and oleoresins
8 Conclusions
Seeds of black cumin seem to possess magical propertiesand have been worked out extensively This study revealedthat black cumin essential oil and its oleoresins constitutea good alternative source of essential fatty acids comparedwith common vegetable oil The present results showedthat essential oil and oleoresins of black cumin exhibitedhigher antioxidant activity than synthetic antioxidantsThesefindings could be used to prepare multipurpose products forpharmaceutical applications and its usage as dietary source of
8 BioMed Research International
Table 3 (a) Antifungal investigations of black cumin oil and its oleoresins ( zone inhibitiona) using inverted petriplate method (b)Antifungal investigations of black cumin oil and its oleoresins ( zone inhibition) using food poisoned method
(a)
Mycelial zone inhibition at different dosesa of sample ()Samples Doses (120583L) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
(b)
Mycelial zone inhibitiona at different doses of sample ()Samples Doses (ppm) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
Table 4 Antibacterial activity of black cumin oil and its oleoresinsagainst a few bacterial species using agar well diffusion method
Diameter of inhibition zone (mma)Samples Doses (ppmb) BS BC SA EC PA
Black cumin oil 1000 ++ 253 plusmn 14 ++ minus 189 plusmn 017
3000 ++ ++ ++ 203 plusmn 014 279 plusmn 015
Ethanol oleoresin 1000 164 plusmn 081 minus 156 plusmn 036 minus minus
3000 283 plusmn 020 minus 259 plusmn 042 minus minus
n-Hexane oleoresin 1000 115 plusmn 031 minus 91 plusmn 11 minus minus
3000 209 plusmn 19 minus 134 plusmn 26 minus minus
Ethyl acetate oleoresin1000 223 plusmn 08 minus 177 plusmn 23 minus minus
3000 443 plusmn 07 minus 409 plusmn 12 minus minus
Ampicillin 1000 156 plusmn 032 minus 91 plusmn 11 minus minus
3000 132 plusmn 02 minus 134 plusmn 26 minus minus
aAverage of three replicates ++ indicates complete inhibition and minus indicates no inhibitionbDMSO was used as solventBS Bacillus subtilis SA Staphylococcus aureus BC Bacillus cereus EC Escherichia coli PA Pseudomonas aeruginosa
BioMed Research International 9
antioxidant should be considered largely for alleviating andameliorating diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to theHead of Department of Chem-istry DDU Gorakhpur University Gorakhpur for providinglaboratory facilities The financial support from UGC toSunita Singh (JRF) and Emeritus Fellow to Dr Gurdip Singhis also acknowledged
References
[1] G Zengin A Aktumsek G O Guler Y S Cakmak andE Yildiztugay ldquoAntioxidant properties of methanolic extractand fatty acid composition of Centaurea urvillei DC subsphayekiana Wagenitzrdquo Records of Natural Products vol 5 no 2pp 123ndash132 2011
[2] H Lutterodt M Luther M Slavin et al ldquoFatty acid profilethymoquinone content oxidative stability and antioxidantproperties of cold-pressed black cumin seed oilsrdquo Food Scienceand Technology vol 43 no 9 pp 1409ndash1413 2010
[3] S Shah and K S Ray ldquoStudy on antioxidant and antimicrobialproperties of black cumin (Nigella sativa Linn)rdquo Journal of FoodScience and Technology vol 40 no 1 pp 70ndash73 2003
[4] I Tekeoglu A Dogan and L Demiralp ldquoEffects of thymo-quinone (volatile oil of black cumin) on rheumatoid arthritis inrat modelsrdquo Phytotherapy Research vol 20 no 10 pp 869ndash8712006
[5] I Kruk TMichalska K Lichszteld A Kladna andH Y Aboul-Enein ldquoThe effect of thymol and its derivatives on reactionsgenerating reactive oxygen speciesrdquo Chemosphere vol 41 no7 pp 1059ndash1064 2000
[6] G Singh P Marimuthu C S De Heluani and C Cata-lan ldquoChemical constituents and antimicrobial and antioxidantpotentials of essential oil and acetone extract of Nigella sativaseedsrdquo Journal of the Science of Food and Agriculture vol 85no 13 pp 2297ndash2306 2005
[7] S A Maisonneuve European Pharmacopoeia Part 1 Sainte-Ruffine 1983
[8] M Burits and F Bucar ldquoAntioxidant activity of Nigella sati-vaessential oilrdquo Phytotherapy Research vol 14 pp 323ndash3282000
[9] N Ilaiyaraja and F Khanum ldquoNigella sativa L a review of thera-peutic applicationsrdquo Journal of Herbal Medicine and Toxicologyvol 4 no 2 pp 1ndash8 2010
[10] E A Decker and B Welch ldquoRole of ferritin as a lipid oxidationcatalyst in muscle foodrdquo Journal of Agricultural and FoodChemistry vol 38 no 3 pp 674ndash677 1990
[11] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo Food Scienceand Technology vol 28 no 1 pp 25ndash30 1995
[12] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic as it reagentsrdquo TheAmerican Journal of Enology and Viticulture vol 16 pp 144ndash158 1965
[13] K D Economou V Oreopoulou and C D ThomopoulosldquoAntioxidant activity of some plant extracts of the familylabiataerdquo Journal of the American Oil Chemists Society vol 68no 2 pp 109ndash113 1991
[14] G Singh S Maurya M P de Lampasona and C A N CatalanldquoA comparison of chemical antioxidant and antimicrobialstudies of cinnamon leaf and bark volatile oils oleoresins andtheir constituentsrdquo Food and Chemical Toxicology vol 45 no 9pp 1650ndash1661 2007
[15] G Singh I P S Kapoor P Singh C S de Heluani M P deLampasona and C A N Catalan ldquoChemistry antioxidant andantimicrobial investigations on essential oil and oleoresins ofZingiber officinalerdquo Food and Chemical Toxicology vol 46 no10 pp 3295ndash3302 2008
[16] G P Rao and A K Shrivastava ldquoToxicity of essential oils ofhigher plants against fungal pathogens of sugarcanerdquo inCurrentTrend in Sugarcane Pathology G P Rao A G Gillasple PP Upadhaya A Bergamin V P Agnihotri and C T ChenEds pp 347ndash365 International Books and Periodicals SupplyService Delhi India 1994
[17] K Ramadas G Suresh N Janarthanan and S Masila-mani ldquoAntifungal activity of 13-disubstituted symmetrical andunsymmetrical thioureasrdquo Pesticide Science vol 52 no 2 pp145ndash151 1998
[18] NCCLS (National Committee for Clinical Laboratory Stan-dards) Performance Standards for Antimicrobial Disc Suscepti-bility Test 6th edition 1997
[19] V Hajhashemi A Ghannadi and H Jafarabadi ldquoBlack cuminseed essential oil as a potent analgesic and antiinflammatorydrugrdquo Phytotherapy Research vol 18 no 3 pp 195ndash199 2004
[20] P J Houghton R Zarka B De Las Heras and J R SHoult ldquoFixed oil of Nigella sativa and derived thymoquinoneinhibit eicosanoid generation in leukocytes andmembrane lipidperoxidationrdquo Planta Medica vol 61 no 1 pp 33ndash36 1995
[21] M T SultanM S Butt FMAnjumA Jamil S Akhtar andMNasir ldquoNutritional profile of indigenous cultivar of black cuminseeds and antioxidant potential of its fixed and essential oilrdquoPakistan Journal of Botany vol 41 no 3 pp 1321ndash1330 2009
[22] S Cheikh-Rouhou S Besbes G Lognay C Blecker CDeroanne and H Attia ldquoSterol composition of black cumin(Nigella sativa L) and Aleppo pine (Pinus halepensisMill) seedoilsrdquo Journal of Food Composition and Analysis vol 21 no 2 pp162ndash168 2008
[23] M F Ramadan ldquoNutritional value functional properties andnutraceutical applications of black cumin (Nigella sativa L) anoverviewrdquo International Journal of Food Science and Technologyvol 42 no 10 pp 1208ndash1218 2007
[24] T D Parker D A Adams K Zhou M Harris and L Yu ldquoFattyacid composition and oxidative stability of cold-pressed edibleseed oilsrdquo Journal of Food Science vol 68 no 4 pp 1240ndash12432003
[25] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAmerican Oil Chemical Society vol 83 pp 847ndash854 2006
[26] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAgricultural and Food Chemistry vol 53 no 3 pp 566ndash5732005
[27] R R Sokal Introduction to Biostatistics W H Freeman SanFrancisco Calif USA 1973
10 BioMed Research International
[28] H Hosseinzadeh S Parvardeh M N Asl H R Sadeghniaand T Ziaee ldquoEffect of thymoquinone and Nigella sativa seedsoil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampusrdquo Phytomedicine vol 14no 9 pp 621ndash627 2007
[29] S Bourgou A Pichette B Marzouk and J Legault ldquoBioactiv-ities of black cumin essential oil and its main terpenes fromTunisiardquo South African Journal of Botany vol 76 no 2 pp 210ndash216 2010
[30] H J Harzallah E Noumi K Bekir et al ldquoChemical composi-tion antibacterial and antifungal properties of Tunisian Nigellasativa fixed oilrdquoAfrican Journal of Microbiology Research vol 6no 22 pp 4675ndash4679 2012
[31] V S Deepa and P S Kumar ldquoPreliminary phytochemicalinvestigations and in vitro antioxidant activity in selected partsof Andrographis spprdquo Journal of Pharmacological Research vol3 no 9 pp 2206ndash2210 2010
[32] M Amensour E Sendra A Jamal S Bouhdid J A Perez-Alvarez and J Fernandez-Lopez ldquoTotal phenolic content andantioxidant activity of myrtle (Myrtus communis) extractsrdquoNatural Product Communications vol 4 no 6 pp 819ndash8242009
[33] M Di Nunzio V Valli and A Bordoni ldquoPro- and anti-oxidanteffects of polyunsaturated fatty acid supplementation in HepG2cellsrdquo Prostaglandins Leukotrienes and Essential Fatty Acids vol85 no 3-4 pp 121ndash127 2011
[34] G Singh SMaurya CCatalan andMP de Lampasona ldquoStud-ies on essential oils part 42 chemical antifungal antioxidantand sprout suppressant studies on ginger essential oil and itsoleoresinrdquo Flavour and Fragrance Journal vol 20 no 1 pp 1ndash62005
[35] T S El Alfy H M El Fatatry and M A Toama ldquoIsolationand structure assignment of an antimicrobial principle from thevolatile oil of Nigella sativa L seedsrdquo Pharmazie vol 30 no 2pp 109ndash111 1975
[36] M A Khan ldquoChemical composition and medicinal propertiesof Nigella sativa Linnrdquo Inflammopharmacology vol 7 no 1 pp15ndash35 1999
[37] L J McGaw A K Jager and J van Staden ldquoIsolation ofantibacterial fatty acids from Schotia brachypetalardquo Fitoterapiavol 73 no 5 pp 431ndash433 2002
[38] A R McCutcheon T E Roberts E Gibbons et al ldquoAntiviralscreening of British Columbian medicinal plantsrdquo Journal ofEthnopharmacology vol 49 no 2 pp 101ndash110 1995
[39] S Javed A A Shahid M S Haider et al ldquoNutritionalphytochemical potential and pharmacological evaluation ofNigella Sativa (Kalonji) and Trachyspermum Ammi (Ajwain)rdquoMedicinal Plants Research vol 6 no 5 pp 768ndash775 2012
[40] P Rattanachaikunsopon and P Phumkhachorn ldquoAssessmentof factors influencing antimicrobial activity of carvacrol andcymene against Vibrio cholerae in foodrdquo Journal of Bioscienceand Bioengineering vol 110 no 5 pp 614ndash619 2010
[41] M Gilles J Zhao M An and S Agboola ldquoChemical com-position and antimicrobial properties of essential oils of threeAustralian Eucalyptus speciesrdquo Food Chemistry vol 119 no 2pp 731ndash737 2010
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Table 2 Chemical composition of oleoresins obtained from black cumin (Nigella sativa L) seeds in different solvents analysed by GC-MS
Compounds M1 M2 M3 RI IdentificationΦ
120572-Thujene Trace 04 06 919 MS RI co-GC119901-Cymene 09 28 22 1019 MS RI co-GCUnidentified A 05 05 04 1092 minus
Thymoquinone 57 61 37 1248 MS RICarvacrol 04 Trace Trace 1295 MS RI co-GC120572-Longipinene Trace Trace Trace 1353 MS RILongifolene 05 06 03 1405 MS RIThymohydroquinone 25 16 05 1559 MS RIPalmitic acid ethyl ester 28 Trace Trace 1979 MS co-GCLinoleic acid methyl ester 06 05 05 minus MS co-GCLinoleic acid ethyl ester 116 Trace 06 minus MS co-GCOleic acid ethyl ester 46 Trace 02 minus MS co-GCOleic acid 03 Trace 02 minus MS co-GCLinoleic acid 330 439 277 minus MSLinoleic acid butyl ester 09 57 160 minus MSOleic acid butyl ester 12 45 73 minus MSGlyceryl palmitate 37 16 23 minus MSGlyceryl linoleate 277 219 231 minus MSSitosterol Trace 13 Trace minus MS co-GCTotal 964 909 852Trace lt 005 the retention index was calculated using a homologous series of n-alkanes C8ndashC20 ΦCo-GC coinjection with an authentic samplePercentages were obtained from electronic integration measurements using selective mass detectorM1 ethanol oleoresin M2 ethyl acetate oleoresin M3 n-hexane oleoresin
of the samples were mixed with 37mL of deionized waterThe mixture was incubated with FeCl
2(2mM 01mL)
After incubation the reaction was initiated by addition offerrozine (5mM and 02mL) for 10min at room temperatureand then the absorbance was measured at 562 nm in aspectrophotometer A lower absorbance indicates a higherchelating power The chelating activity of the extract on Fe2+was compared with that of EDTA that was used as positivecontrol Chelating activity was calculated using the followingformula
Chelating activity ()
= [1 minus (
Absorbance of sampleAbsorbance of control
)] times 100
(1)
32 Scavenging Effect on DPPH TheDPPH assay constitutesa quick and low cost method which has frequently been usedfor the evaluation of the antioxidative potential of variousnatural products [11] Due to its odd electron DPPH givesa strong absorption band at 517 nm (deep violet colour) Inthe presence of a free radical scavenger this electron becomespaired resulting in the absorption loss and consecutivestoichiometric decolorization with respect to the number ofelectron acquired The absorbance change produced by thisreaction is assessed to evaluate the antioxidant potential ofthe test sample 5 10 15 and 20 120583L of the sample were addedto 5mLof 0004methanol solution ofDPPHAfter a 30minincubation period at room temperature the absorbance was
read against a blank at 515 nm All determination was per-formed in triplicate and results were performed in triplicateand results are reported as scavenging effect () versusconcentration in Figure 2
33 Estimation of Total Phenolic Content (TPC) TPC weredetermined using the Folin-Ciocalteu reagent methoddescribed by Singleton and Rossi [12] Gallic acid stocksolution (1000 120583gmLminus1) was prepared by dissolving 100mgof gallic acid in 100mL of ethanol Various dilutions ofstandard gallic acid were prepared from this stock solutionCalibration curve (Figure 3) was plotted by mixing 1mLaliquots of 10ndash100120583gmLminus1 of gallic acid solutionswith 50mLof Folin-Ciocalteu reagent (diluted tenfold) and 40mL ofsodium carbonate solution (75 g Lminus1) The absorbance wasmeasured after 30min at 20∘C at 765 nm
4 Evaluation of Antioxidant Activity forLinseed Oil System
For present investigation crude linseed oil having initialperoxide value 52meq kgminus1 was taken to assess the antiox-idant activity of black cumin oil and its oleoresins This oilis most frequently used edible oil in central Europe and israther unstable because of the presence of substantial amountof linoleic acid The antioxidant activity of volatile oil andextract was examined by comparing the activity of knownantioxidants such as PG BHT and BHA by the followingperoxide value and thiobarbituric acid value methods
4 BioMed Research International
41 Peroxide Value Method For measuring the peroxidevalue (PV) a modified oven test was used [13] Theantioxidant activity of black cumin oil and its oleoresinsin different solvents were compared with the syntheticantioxidants such as PG BHT and BHA For this purposecalculated quantities of each (200 ppm)were dissolved to 30 gof linseed oil in an open mouthed beaker The mixtures werethoroughly homogenized and placed in incubator at 80∘CThe peroxide values (meq of oxygen kgminus1) were measured inevery seven days and test was replicated for three times Acontrol sample was prepared under similar conditionwithoutany additive The effects of oil and oleoresins in term ofperoxidation at 90∘C are shown in Figure 4
42 Thiobarbituric Acid Value (TBA) TBA value of differentsamples was determined according to the method previouslyreported [13] About 100mg of oil sample was dissolved in25mL of 1-butanol A 25mL aliquot of the above solutionwas mixed thoroughly with 50mL of TBA reagent (200mgTBA in 100mL of 1-butanol) and incubated at 95∘C After 2 hthe reaction mixture was cooled to room temperature underrunning water and absorbance was measured at 530 nm withHitachi-U-2000 spectrophotometer (Tokyo Japan) At thesame time a reagent blank (without TBA reagent) was alsodoneThe thiobarbituric acid value (meq ofmalondialdehydeper g) was calculated as
TBA value = 50 times (119860 minus 119861)119872
(2)
where 119860 is absorbance of the test sample 119861 is absorbance ofthe reagent blank and119872 is mass of the sample
43 Determination of Antioxidant Activity in Linoleic AcidSystem Antioxidant activity of black cumin oil and itsoleoresins was compared to synthetic standards accordingto the ferric thiocyanate method in linoleic acid emulsion[14] The reaction medium contained black cumin oil andoleoresins at the concentration of 1mg100mL of absoluteethanol (2mL) an emulsion of 251 linoleic acid in ethanol(2mL) 4mL of 005M-phosphate buffer (pH = 70) and2mL of distilled water The solution (10mL) was mixed andincubated at 40∘C in the darkThe same solution without anyadditives was taken as control sample At regular intervalsduring incubation 01mL aliquot of the mixture was dilutedwith 97mL of 75 ethanol followed by the addition of02mL of 30 ammonium thiocyanate and 01mL of 20mMof FeCl
2in 35 HCl the absorbance of red colour was
measured at 500 nm in Hitachi-U-2000 spectrophotometer(Tokyo Japan) The control and standard were subjected tothe same procedure except for the control where there wasno addition of sample and for the standard 1mL of samplewere replaced with 1mg of PG BHT and BHA These stepsare repeated every 48 h until the control sample reached itsmaximum Low absorbance value indicates the efficiency ofthe test samples to inhibit lipid oxidation The results werereported as incubation time versus absorbance in Figure 6
5 Antimicrobial Investigations
51 Antifungal Assay The antifungal activity of the essentialoil and extract against various pathogenic fungi Aspergilluslavus (1884) Aspergillus niger (2479) Fusarium moniliforme(1893) Fusarium graminearum (2088) and Penicillium viridi-catum (2007) were tested by the inverted petri plate [15] andpoison food medium methods [16] All the fungi cultureswere procured from the Microbial Type Culture Collection(MTCC) and their reference numbers are given in theparentheses The cultures were maintained in Czapek agarmedium Each test was replicated three times and fungi toxi-city was measured in terms of percentage mycelial inhibitioncalculated with the following equation
colony of the control and treated sets respectively
52 Antibacterial Assay The essential oil and extract wereindividually tested against a panel of microorganisms usingagar well diffusion method [17] Three Gram-positive bacte-ria Staphylococcus aureus (3103) Bacillus cereus (430) andBacillus subtilis (1790) and two Gram negative bacteriaEscherichia coli (1672) and Pseudomonas aeruginosa (1942)All the bacterial strains were procured from the MicrobialType Culture Collection (MTCC) Institute of MicrobialTechnology (Chandigarh India) and their reference num-bers are given in parentheses The bacterial cultures weregrown on nutrient agar medium and stored at 4∘C In orderto prepare a bacterial strain for test initially one loopfulof bacterial culture was transferred from slant to nutrientbroth solution (10mL) and was stored at 37∘C for 24 hThe control plate without the addition of essential oil orextract containing DMSO was also maintained under thesame conditions After incubating for 24 h at 37∘C all plateswere examined for any zones of growth inhibition and thediameters of these zones were measured in millimeters
6 Statistical Analysis
For the essential oil or oleoresin three samples were preparedfor assays of every antioxidant and antimicrobial attributeThe data are presented as mean (standard deviation of threedeterminations (data are not shown) Statistical analyseswere performed using a one-way analysis of variance [18] Aprobability value of119875 lt 005was considered to be significant
7 Results and Discussions
71 Phytochemistry Careful and detailed interpretations ofthe experimental GC-MS data (EM fragmentation retentionindices) were carried out which permitted identification ofa large number of components in essential oil and oleo-resins (Tables 1 and 2) Table 1 shows identification of 33components in black cumin oil representing about 902 ofthe total amount The major component in black cumin oil
BioMed Research International 5
0
25
50
75
100
0 5 10 15 20 25
Black cumin oilEtOH oleon-hexane oleo
Ethyl acetate oleoEDTA
Chel
atin
g eff
ect (
)
Concentration (120583g mLminus1)
Figure 1 Chelating effect of black cumin oil and its differentoleoresins
was thymoquinone (376) followed by p-cymene (314)120572-thujene (56) thymohydroquinone (34) longifolene(20) and carvacrol (14) Burits and Bucar [8] character-ized many components in black cumin essential oil such asthymoquinone (278ndash570) p-cymene (71ndash155) carvacrol(58ndash116) trans-anethole (025ndash23) 4-terpineol (20ndash66) and 10ndash80 of longifolene These results are slightdifferent from the work reported by Hajhashemi et al [19]who reported that p-cymene (373) and thymoquinone(137) were the major components of black cumin Singh etal [6] also reported p-cymene as the major component in theblack cumin essential oil
From Table 2 it is evident that in ethanol oleoresin19 components constitute 964 of the total weight in theethyl acetate oleoresin a total of 19 components making909 of the whole mass and in case of n-hexane oleoresin19 compounds constituting about 852 of the total weightwere identified The oleoresins were mainly comprised ofunsaturated fatty acids and their different esters The majorcomponents in all three oleoresins were linoleic acid (unsat-urated fatty acid) followed by glyceryl linoleate glycerylpalmitate oleic acid and other minor components Thepresence of unsaturated fatty acids in oleoresins was wellsupported by the various reported work [20ndash26]
72 Antioxidant Investigations
721 Chelating Activity on Ferrous Ions The ferrous ion(Fe2+) chelating effect of black cumin oil and its differentoleoresins is presented in Figure 1 The chelating activityof the extracts was concentration dependent Black cuminoil exhibited higher chelating activity in comparison to theoleoresins but was not effective chelator as EDTA Maximumchelating of metal ions at 200120583gmLminus1 for black cumin oiland EDTA was found to be 7456 and 8790 respectivelywhereas the oleoresins were less effective in metal chelationand their metal chelating activity ranges from 221 to 445
Figure 2 Scavenging effect () of black cumin oil and its oleoresinson DPPH radical
0
02
04
06
08
1
12
0 10 20 30 40 50 60 70 80 90 100
Abs
orba
nce a
t 765
nm
Gallic acid
Gallic acid (120583g mLminus1)
y = 00101x + 00178
R2 = 0982
Figure 3 Calibration curve of gallic acid
722 Scavenging Effect on DPPH Radical DPPH∙ is a stableradical showing amaximum absorbance at 515 nm InDPPH∙assay the antioxidant was able to reduce the stable radicalDPPH to the yellow-colored diphenylpicrylhydrazone Themethod is based on the reduction of DPPH∙ in alcoholicsolution in the presence of a hydrogen-donating antioxidantdue to formation of the nonradical form DPPH-H in thereaction DPPH∙ is usually used as a reagent to evaluatefree radical and accepts an electron or hydrogen radical tobecome a stable diamagnetic molecule The disappearanceof the DPPH radical absorption at 515 nm by the action ofantioxidants is taken as a measure of antioxidant activityThescavenging effects of black cumin oil and oleoresins onDPPHradical linearly increased as concentration increased from5 to 20120583gmLminus1 (Figure 2) At 20120583gmLminus1 the scavengingactivity of black cumin oil and ethyl acetate oleoresin was954 and 8975 respectively comparatively higher than
6 BioMed Research International
0
100
200
300
400
500
0 7 14 21 28
PGBlack cumin oilEthyl acetate oleoBHT
EtOH oleo
BHAControl
Incubation time (days)
Pero
xide
val
ue (m
eq kg
minus1)
n-hexane oleo
Figure 4 Inhibitory effect of black cumin oil and its oleoresins onthe primary oxidation of linseed oil measured using peroxide valuemethod
BHT and BHA but lower than PG However the scavengingactivity of BHA BHT and PG was more effective at lowerconcentration and was 698 721 and 863 at 5 120583gmLminus1but as the concentration increases the differences in scav-enging activity between BHA BHT and oleoresins (speciallyethyl acetate) become less significant Ethanol and n-hexaneoleoresins showed moderate scavenging activity
723 Estimation of TPC The amount of total phenols wasdetermined with Folin-Ciocalteu reagent Gallic acid wasused as standard compound The absorbance for variousdilutions of gallic acid with Folin-Ciocalteu reagent andsodium carbonate was obtained and found standard curveequation 119910 = 00101119909 + 00178 1198772 = 0982 (Figure 3) Thetotal phenol contents (gallic acid equivalents mg GAE perg) in black cumin essential oil ethyl acetate ethanol and n-hexane oleoresins were calculated as 1147plusmn005 1088plusmn09968 plusmn 006 and 833 plusmn 001 respectively The value suggeststhat the black cumin oil and its oleoresins have lesser amountof total phenols [27] The differences in the total phenoliccontent among the samplesmight be due tomany differencessuch as the environmental conditions genetic background oragricultural techniques applied
73 Antioxidant Assays in Linseed Oil System The changes ofPV in linseed oil of all investigated samples are presented inFigure 4 The rate of oxidative reactions in linseed oil withadditives was almost similar to that of the blank sampleThe stability of the linseed oil samples to the formation ofperoxides can be ranked in the following descending order
PG gt Black cumin oil gt Ethyl acetate oleo gt BHT gtBHA gt EtOH oleo gt n-hexane oleo gt Control
Simultaneously with the measurements of PV changes insecondary product such as malondialdehyde the compound
0 7 14 21 28
PGBlack cumin oilEthyl acetate oleoBHA
BHTControl
Incubation time (days)
0
005
01
015
02
025
03
TBA
val
ue (m
eq O
2Kg
minus1)
EtOH oleo
n-hexane oleo
Figure 5 Inhibitory effect of black cumin oil and its oleoresinson the primary oxidation of linseed oil measured using TBA valuemethod
0
05
1
15
2
25
3
0 2 4 6 8 10 12
PGBlack cumin oilBHTEthyl acetate oleo
BHA
EtOH oleoControl
Abs
orba
nce a
t 500
nm
Incubation time (days)
n-hexane oleo
Figure 6 Inhibitory effect of black cumin oil and its oleoresins onthe primary oxidation of linoleic acid system measured using ferricthiocyanate method
used as an indicator of lipid peroxidation was measured byTBAvalues (Figure 5) were also determined after every sevendays Black cumin oil and its ethyl acetate oleoresin showedstrong inhibition at 002 concentration as compared toBHT and BHA (119875 lt 005) but lower than PG whereas theethanol and n-hexane oleoresin showed moderate inhibitionat 002 concentration as compared to the other additivesFrom the above results it should be confirmed that formationof primary oxidation species peroxides was quite similarwith the secondary oxidation products and the changes ofboth oxidation characteristics are in a good correlation
74 Antioxidant Activity in Linoleic Acid System The FTCmethod was used to measure the amount of peroxides at
BioMed Research International 7
the primary stage of linoleic acid peroxidation (Figure 6)Since the concentration of the peroxide decreases as theantioxidant activity increases the intensity of the pigmentwill be reduced leading to lower absorbance Absorbancevalues of the control as well as black cumin oil and itsoleoresins increase until day 10 and then decreased on day12 due to the malondialdehyde formation from linoleic acidoxidation There was a significant difference (119875 lt 005)between the control and the tested essential oil and oleoresinsAs can be seen in Figure 6 both black cumin oil andoleoresins showed good antioxidative property in the linoleicacid system and were significantly (119875 lt 005) different fromthe control
Antioxidant activities of essential oils and oleoresinsmay be related to the diverse compounds present in themincluding terpenes sesquiterpenes and phenolic acids whichact in various ways such as inhibition of peroxidation scav-enging the radicals and chelating the metal ions The mainconstituents of black cumin oil were thymoquinone (376)and p-cymene (314) with minor amounts of longifolenecarvacrol and thymohydroquinone which were responsiblefor the antioxidant activity of black cumin oil [8 9 19 28ndash31] Furthermore free radical scavenging effects of thesecomponentswere studied on the reactions generating reactiveoxygen species such as superoxide anion radical hydroxylradical using spectrophotometric methods [5] The resultsobtained using different assays were well correlated withthe previous work reported be many workers [5 30] whofound thymoquinone as a main constituent responsible forthe activity It has been suggested that phenolic content iscorrelated with the antioxidant activity [32] It is consideredthat the antioxidant activity of phenolic compounds is dueto their high redox potentials which allow them to actas reducing agents hydrogen donors Thymoquinone wasalso present in small amount with higher percentage ofunsaturated fatty acid linoleic acid in different oleoresinsStudies [33] have shown that these unsaturated fatty acidshave anti-rather than pro-oxidant activity but still researchhas been going on for the exact role of unsaturated fatty acidsagainst the oxidative stress
75 Antimicrobial Investigations Using inverted petri platetechnique (Table 3) the volatile oil exhibited more than 90zone inhibition for F moniliforme and P viridicatum It wasalso found to be highly effective in controlling the growthof Aspergillus species and F graminearum where (50) and(65) zone inhibition was observed respectively For othertested fungi the essential oil exerted less activity Howeverusing the same method the oleoresins have revealed lessactivity except for F moniliforme in which only up to 40mycelial zone inhibition was obtained Moreover using foodpoison technique (Table 4) the volatile oil showed clear zoneof growth inhibition against F graminearum at 10 120583L Thevolatile oil showed strong antifungal activity against all testedAspergillus species in the food poison method Ethyl acetateand ethanol oleoresin showed up to 30 zone inhibitionat 10 120583L dose The n-hexane oleoresin showed very feeble
inhibition zone in both techniques The lower antimicrobialefficacy of the oleoresins is due to their low volatility [34]
The antibacterial investigations were undertaken usingagar well diffusion method (Table 4) Using this method theblack cumin oil has shown better activity than oleoresinsand commercial bactericide that is ampicillin The volatileoil was found to be highly effective against B subtilis Bcereus and S aureus and showed complete zone of inhibitionat 3000 ppm concentration whereas in oleoresins more than20mm inhibition was obtained for Gram-positive bacteriaIn addition more than 20 and 25mm zone inhibition wasobtained for P aeruginosa and E coli The results obtainedusing agar well diffusion method were well correlated withthe earlier reported work [6 30 35] where black cumin seedoil has been shown to be effective against a wide spectrumof organisms bacteria like B cereus B subtilis S aureus Sepidermis E coli and P aeruginosa
The results obtained in antimicrobial investigations ofblack cumin oil and oleoresins were in good agreementwith the previous reported work [36] Thymoquinone p-cymene (monoterpene) longifolene (sesquiterpene) andthymohydroquinone were responsible for strong antimicro-bial activity of black cumin oil [29] El Alfy et al [35]isolated thymohydroquinone as antimicrobial compoundfrom the volatile oil of Nigella sativa seeds Oleoresins havehigh concentration of unsaturated fatty acids along withthymohydroquinone in small amount which is responsiblefor its moderate antimicrobial effects Long chain fatty acidslike linoleic acid and oleic acid were previously reportedto possess antibacterial and antifungal activity [36ndash39] p-Cymene is not an efficient antimicrobial compound whenused alone but it potentiate the activity of compounds likecarvacrol [40] The antimicrobial activity of essential oils canoften be correlated to its content of phenolic constituentsThe type of bacteria also has an influence on the effectivenessof the volatile oil and oleoresins Gram-negative bacteriawere generally less susceptible than Gram-positive bacteria[41] The difference in the susceptibility of the bacteria arisesas a result of differences in their cell membrane structurewhich is more complex in case of Gram-negative bacteriaThe antimicrobial activity of a given essential oil may dependon only one or two of the major constituents that make upthe oil However increasing amounts of evidence indicatethat the inherent activity of essential oils may not only relyexclusively on the ratio in which the main active constituentsare present but also on interactions between these andminorconstituents in the oils and oleoresins
8 Conclusions
Seeds of black cumin seem to possess magical propertiesand have been worked out extensively This study revealedthat black cumin essential oil and its oleoresins constitutea good alternative source of essential fatty acids comparedwith common vegetable oil The present results showedthat essential oil and oleoresins of black cumin exhibitedhigher antioxidant activity than synthetic antioxidantsThesefindings could be used to prepare multipurpose products forpharmaceutical applications and its usage as dietary source of
8 BioMed Research International
Table 3 (a) Antifungal investigations of black cumin oil and its oleoresins ( zone inhibitiona) using inverted petriplate method (b)Antifungal investigations of black cumin oil and its oleoresins ( zone inhibition) using food poisoned method
(a)
Mycelial zone inhibition at different dosesa of sample ()Samples Doses (120583L) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
(b)
Mycelial zone inhibitiona at different doses of sample ()Samples Doses (ppm) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
Table 4 Antibacterial activity of black cumin oil and its oleoresinsagainst a few bacterial species using agar well diffusion method
Diameter of inhibition zone (mma)Samples Doses (ppmb) BS BC SA EC PA
Black cumin oil 1000 ++ 253 plusmn 14 ++ minus 189 plusmn 017
3000 ++ ++ ++ 203 plusmn 014 279 plusmn 015
Ethanol oleoresin 1000 164 plusmn 081 minus 156 plusmn 036 minus minus
3000 283 plusmn 020 minus 259 plusmn 042 minus minus
n-Hexane oleoresin 1000 115 plusmn 031 minus 91 plusmn 11 minus minus
3000 209 plusmn 19 minus 134 plusmn 26 minus minus
Ethyl acetate oleoresin1000 223 plusmn 08 minus 177 plusmn 23 minus minus
3000 443 plusmn 07 minus 409 plusmn 12 minus minus
Ampicillin 1000 156 plusmn 032 minus 91 plusmn 11 minus minus
3000 132 plusmn 02 minus 134 plusmn 26 minus minus
aAverage of three replicates ++ indicates complete inhibition and minus indicates no inhibitionbDMSO was used as solventBS Bacillus subtilis SA Staphylococcus aureus BC Bacillus cereus EC Escherichia coli PA Pseudomonas aeruginosa
BioMed Research International 9
antioxidant should be considered largely for alleviating andameliorating diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to theHead of Department of Chem-istry DDU Gorakhpur University Gorakhpur for providinglaboratory facilities The financial support from UGC toSunita Singh (JRF) and Emeritus Fellow to Dr Gurdip Singhis also acknowledged
References
[1] G Zengin A Aktumsek G O Guler Y S Cakmak andE Yildiztugay ldquoAntioxidant properties of methanolic extractand fatty acid composition of Centaurea urvillei DC subsphayekiana Wagenitzrdquo Records of Natural Products vol 5 no 2pp 123ndash132 2011
[2] H Lutterodt M Luther M Slavin et al ldquoFatty acid profilethymoquinone content oxidative stability and antioxidantproperties of cold-pressed black cumin seed oilsrdquo Food Scienceand Technology vol 43 no 9 pp 1409ndash1413 2010
[3] S Shah and K S Ray ldquoStudy on antioxidant and antimicrobialproperties of black cumin (Nigella sativa Linn)rdquo Journal of FoodScience and Technology vol 40 no 1 pp 70ndash73 2003
[4] I Tekeoglu A Dogan and L Demiralp ldquoEffects of thymo-quinone (volatile oil of black cumin) on rheumatoid arthritis inrat modelsrdquo Phytotherapy Research vol 20 no 10 pp 869ndash8712006
[5] I Kruk TMichalska K Lichszteld A Kladna andH Y Aboul-Enein ldquoThe effect of thymol and its derivatives on reactionsgenerating reactive oxygen speciesrdquo Chemosphere vol 41 no7 pp 1059ndash1064 2000
[6] G Singh P Marimuthu C S De Heluani and C Cata-lan ldquoChemical constituents and antimicrobial and antioxidantpotentials of essential oil and acetone extract of Nigella sativaseedsrdquo Journal of the Science of Food and Agriculture vol 85no 13 pp 2297ndash2306 2005
[7] S A Maisonneuve European Pharmacopoeia Part 1 Sainte-Ruffine 1983
[8] M Burits and F Bucar ldquoAntioxidant activity of Nigella sati-vaessential oilrdquo Phytotherapy Research vol 14 pp 323ndash3282000
[9] N Ilaiyaraja and F Khanum ldquoNigella sativa L a review of thera-peutic applicationsrdquo Journal of Herbal Medicine and Toxicologyvol 4 no 2 pp 1ndash8 2010
[10] E A Decker and B Welch ldquoRole of ferritin as a lipid oxidationcatalyst in muscle foodrdquo Journal of Agricultural and FoodChemistry vol 38 no 3 pp 674ndash677 1990
[11] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo Food Scienceand Technology vol 28 no 1 pp 25ndash30 1995
[12] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic as it reagentsrdquo TheAmerican Journal of Enology and Viticulture vol 16 pp 144ndash158 1965
[13] K D Economou V Oreopoulou and C D ThomopoulosldquoAntioxidant activity of some plant extracts of the familylabiataerdquo Journal of the American Oil Chemists Society vol 68no 2 pp 109ndash113 1991
[14] G Singh S Maurya M P de Lampasona and C A N CatalanldquoA comparison of chemical antioxidant and antimicrobialstudies of cinnamon leaf and bark volatile oils oleoresins andtheir constituentsrdquo Food and Chemical Toxicology vol 45 no 9pp 1650ndash1661 2007
[15] G Singh I P S Kapoor P Singh C S de Heluani M P deLampasona and C A N Catalan ldquoChemistry antioxidant andantimicrobial investigations on essential oil and oleoresins ofZingiber officinalerdquo Food and Chemical Toxicology vol 46 no10 pp 3295ndash3302 2008
[16] G P Rao and A K Shrivastava ldquoToxicity of essential oils ofhigher plants against fungal pathogens of sugarcanerdquo inCurrentTrend in Sugarcane Pathology G P Rao A G Gillasple PP Upadhaya A Bergamin V P Agnihotri and C T ChenEds pp 347ndash365 International Books and Periodicals SupplyService Delhi India 1994
[17] K Ramadas G Suresh N Janarthanan and S Masila-mani ldquoAntifungal activity of 13-disubstituted symmetrical andunsymmetrical thioureasrdquo Pesticide Science vol 52 no 2 pp145ndash151 1998
[18] NCCLS (National Committee for Clinical Laboratory Stan-dards) Performance Standards for Antimicrobial Disc Suscepti-bility Test 6th edition 1997
[19] V Hajhashemi A Ghannadi and H Jafarabadi ldquoBlack cuminseed essential oil as a potent analgesic and antiinflammatorydrugrdquo Phytotherapy Research vol 18 no 3 pp 195ndash199 2004
[20] P J Houghton R Zarka B De Las Heras and J R SHoult ldquoFixed oil of Nigella sativa and derived thymoquinoneinhibit eicosanoid generation in leukocytes andmembrane lipidperoxidationrdquo Planta Medica vol 61 no 1 pp 33ndash36 1995
[21] M T SultanM S Butt FMAnjumA Jamil S Akhtar andMNasir ldquoNutritional profile of indigenous cultivar of black cuminseeds and antioxidant potential of its fixed and essential oilrdquoPakistan Journal of Botany vol 41 no 3 pp 1321ndash1330 2009
[22] S Cheikh-Rouhou S Besbes G Lognay C Blecker CDeroanne and H Attia ldquoSterol composition of black cumin(Nigella sativa L) and Aleppo pine (Pinus halepensisMill) seedoilsrdquo Journal of Food Composition and Analysis vol 21 no 2 pp162ndash168 2008
[23] M F Ramadan ldquoNutritional value functional properties andnutraceutical applications of black cumin (Nigella sativa L) anoverviewrdquo International Journal of Food Science and Technologyvol 42 no 10 pp 1208ndash1218 2007
[24] T D Parker D A Adams K Zhou M Harris and L Yu ldquoFattyacid composition and oxidative stability of cold-pressed edibleseed oilsrdquo Journal of Food Science vol 68 no 4 pp 1240ndash12432003
[25] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAmerican Oil Chemical Society vol 83 pp 847ndash854 2006
[26] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAgricultural and Food Chemistry vol 53 no 3 pp 566ndash5732005
[27] R R Sokal Introduction to Biostatistics W H Freeman SanFrancisco Calif USA 1973
10 BioMed Research International
[28] H Hosseinzadeh S Parvardeh M N Asl H R Sadeghniaand T Ziaee ldquoEffect of thymoquinone and Nigella sativa seedsoil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampusrdquo Phytomedicine vol 14no 9 pp 621ndash627 2007
[29] S Bourgou A Pichette B Marzouk and J Legault ldquoBioactiv-ities of black cumin essential oil and its main terpenes fromTunisiardquo South African Journal of Botany vol 76 no 2 pp 210ndash216 2010
[30] H J Harzallah E Noumi K Bekir et al ldquoChemical composi-tion antibacterial and antifungal properties of Tunisian Nigellasativa fixed oilrdquoAfrican Journal of Microbiology Research vol 6no 22 pp 4675ndash4679 2012
[31] V S Deepa and P S Kumar ldquoPreliminary phytochemicalinvestigations and in vitro antioxidant activity in selected partsof Andrographis spprdquo Journal of Pharmacological Research vol3 no 9 pp 2206ndash2210 2010
[32] M Amensour E Sendra A Jamal S Bouhdid J A Perez-Alvarez and J Fernandez-Lopez ldquoTotal phenolic content andantioxidant activity of myrtle (Myrtus communis) extractsrdquoNatural Product Communications vol 4 no 6 pp 819ndash8242009
[33] M Di Nunzio V Valli and A Bordoni ldquoPro- and anti-oxidanteffects of polyunsaturated fatty acid supplementation in HepG2cellsrdquo Prostaglandins Leukotrienes and Essential Fatty Acids vol85 no 3-4 pp 121ndash127 2011
[34] G Singh SMaurya CCatalan andMP de Lampasona ldquoStud-ies on essential oils part 42 chemical antifungal antioxidantand sprout suppressant studies on ginger essential oil and itsoleoresinrdquo Flavour and Fragrance Journal vol 20 no 1 pp 1ndash62005
[35] T S El Alfy H M El Fatatry and M A Toama ldquoIsolationand structure assignment of an antimicrobial principle from thevolatile oil of Nigella sativa L seedsrdquo Pharmazie vol 30 no 2pp 109ndash111 1975
[36] M A Khan ldquoChemical composition and medicinal propertiesof Nigella sativa Linnrdquo Inflammopharmacology vol 7 no 1 pp15ndash35 1999
[37] L J McGaw A K Jager and J van Staden ldquoIsolation ofantibacterial fatty acids from Schotia brachypetalardquo Fitoterapiavol 73 no 5 pp 431ndash433 2002
[38] A R McCutcheon T E Roberts E Gibbons et al ldquoAntiviralscreening of British Columbian medicinal plantsrdquo Journal ofEthnopharmacology vol 49 no 2 pp 101ndash110 1995
[39] S Javed A A Shahid M S Haider et al ldquoNutritionalphytochemical potential and pharmacological evaluation ofNigella Sativa (Kalonji) and Trachyspermum Ammi (Ajwain)rdquoMedicinal Plants Research vol 6 no 5 pp 768ndash775 2012
[40] P Rattanachaikunsopon and P Phumkhachorn ldquoAssessmentof factors influencing antimicrobial activity of carvacrol andcymene against Vibrio cholerae in foodrdquo Journal of Bioscienceand Bioengineering vol 110 no 5 pp 614ndash619 2010
[41] M Gilles J Zhao M An and S Agboola ldquoChemical com-position and antimicrobial properties of essential oils of threeAustralian Eucalyptus speciesrdquo Food Chemistry vol 119 no 2pp 731ndash737 2010
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
41 Peroxide Value Method For measuring the peroxidevalue (PV) a modified oven test was used [13] Theantioxidant activity of black cumin oil and its oleoresinsin different solvents were compared with the syntheticantioxidants such as PG BHT and BHA For this purposecalculated quantities of each (200 ppm)were dissolved to 30 gof linseed oil in an open mouthed beaker The mixtures werethoroughly homogenized and placed in incubator at 80∘CThe peroxide values (meq of oxygen kgminus1) were measured inevery seven days and test was replicated for three times Acontrol sample was prepared under similar conditionwithoutany additive The effects of oil and oleoresins in term ofperoxidation at 90∘C are shown in Figure 4
42 Thiobarbituric Acid Value (TBA) TBA value of differentsamples was determined according to the method previouslyreported [13] About 100mg of oil sample was dissolved in25mL of 1-butanol A 25mL aliquot of the above solutionwas mixed thoroughly with 50mL of TBA reagent (200mgTBA in 100mL of 1-butanol) and incubated at 95∘C After 2 hthe reaction mixture was cooled to room temperature underrunning water and absorbance was measured at 530 nm withHitachi-U-2000 spectrophotometer (Tokyo Japan) At thesame time a reagent blank (without TBA reagent) was alsodoneThe thiobarbituric acid value (meq ofmalondialdehydeper g) was calculated as
TBA value = 50 times (119860 minus 119861)119872
(2)
where 119860 is absorbance of the test sample 119861 is absorbance ofthe reagent blank and119872 is mass of the sample
43 Determination of Antioxidant Activity in Linoleic AcidSystem Antioxidant activity of black cumin oil and itsoleoresins was compared to synthetic standards accordingto the ferric thiocyanate method in linoleic acid emulsion[14] The reaction medium contained black cumin oil andoleoresins at the concentration of 1mg100mL of absoluteethanol (2mL) an emulsion of 251 linoleic acid in ethanol(2mL) 4mL of 005M-phosphate buffer (pH = 70) and2mL of distilled water The solution (10mL) was mixed andincubated at 40∘C in the darkThe same solution without anyadditives was taken as control sample At regular intervalsduring incubation 01mL aliquot of the mixture was dilutedwith 97mL of 75 ethanol followed by the addition of02mL of 30 ammonium thiocyanate and 01mL of 20mMof FeCl
2in 35 HCl the absorbance of red colour was
measured at 500 nm in Hitachi-U-2000 spectrophotometer(Tokyo Japan) The control and standard were subjected tothe same procedure except for the control where there wasno addition of sample and for the standard 1mL of samplewere replaced with 1mg of PG BHT and BHA These stepsare repeated every 48 h until the control sample reached itsmaximum Low absorbance value indicates the efficiency ofthe test samples to inhibit lipid oxidation The results werereported as incubation time versus absorbance in Figure 6
5 Antimicrobial Investigations
51 Antifungal Assay The antifungal activity of the essentialoil and extract against various pathogenic fungi Aspergilluslavus (1884) Aspergillus niger (2479) Fusarium moniliforme(1893) Fusarium graminearum (2088) and Penicillium viridi-catum (2007) were tested by the inverted petri plate [15] andpoison food medium methods [16] All the fungi cultureswere procured from the Microbial Type Culture Collection(MTCC) and their reference numbers are given in theparentheses The cultures were maintained in Czapek agarmedium Each test was replicated three times and fungi toxi-city was measured in terms of percentage mycelial inhibitioncalculated with the following equation
colony of the control and treated sets respectively
52 Antibacterial Assay The essential oil and extract wereindividually tested against a panel of microorganisms usingagar well diffusion method [17] Three Gram-positive bacte-ria Staphylococcus aureus (3103) Bacillus cereus (430) andBacillus subtilis (1790) and two Gram negative bacteriaEscherichia coli (1672) and Pseudomonas aeruginosa (1942)All the bacterial strains were procured from the MicrobialType Culture Collection (MTCC) Institute of MicrobialTechnology (Chandigarh India) and their reference num-bers are given in parentheses The bacterial cultures weregrown on nutrient agar medium and stored at 4∘C In orderto prepare a bacterial strain for test initially one loopfulof bacterial culture was transferred from slant to nutrientbroth solution (10mL) and was stored at 37∘C for 24 hThe control plate without the addition of essential oil orextract containing DMSO was also maintained under thesame conditions After incubating for 24 h at 37∘C all plateswere examined for any zones of growth inhibition and thediameters of these zones were measured in millimeters
6 Statistical Analysis
For the essential oil or oleoresin three samples were preparedfor assays of every antioxidant and antimicrobial attributeThe data are presented as mean (standard deviation of threedeterminations (data are not shown) Statistical analyseswere performed using a one-way analysis of variance [18] Aprobability value of119875 lt 005was considered to be significant
7 Results and Discussions
71 Phytochemistry Careful and detailed interpretations ofthe experimental GC-MS data (EM fragmentation retentionindices) were carried out which permitted identification ofa large number of components in essential oil and oleo-resins (Tables 1 and 2) Table 1 shows identification of 33components in black cumin oil representing about 902 ofthe total amount The major component in black cumin oil
BioMed Research International 5
0
25
50
75
100
0 5 10 15 20 25
Black cumin oilEtOH oleon-hexane oleo
Ethyl acetate oleoEDTA
Chel
atin
g eff
ect (
)
Concentration (120583g mLminus1)
Figure 1 Chelating effect of black cumin oil and its differentoleoresins
was thymoquinone (376) followed by p-cymene (314)120572-thujene (56) thymohydroquinone (34) longifolene(20) and carvacrol (14) Burits and Bucar [8] character-ized many components in black cumin essential oil such asthymoquinone (278ndash570) p-cymene (71ndash155) carvacrol(58ndash116) trans-anethole (025ndash23) 4-terpineol (20ndash66) and 10ndash80 of longifolene These results are slightdifferent from the work reported by Hajhashemi et al [19]who reported that p-cymene (373) and thymoquinone(137) were the major components of black cumin Singh etal [6] also reported p-cymene as the major component in theblack cumin essential oil
From Table 2 it is evident that in ethanol oleoresin19 components constitute 964 of the total weight in theethyl acetate oleoresin a total of 19 components making909 of the whole mass and in case of n-hexane oleoresin19 compounds constituting about 852 of the total weightwere identified The oleoresins were mainly comprised ofunsaturated fatty acids and their different esters The majorcomponents in all three oleoresins were linoleic acid (unsat-urated fatty acid) followed by glyceryl linoleate glycerylpalmitate oleic acid and other minor components Thepresence of unsaturated fatty acids in oleoresins was wellsupported by the various reported work [20ndash26]
72 Antioxidant Investigations
721 Chelating Activity on Ferrous Ions The ferrous ion(Fe2+) chelating effect of black cumin oil and its differentoleoresins is presented in Figure 1 The chelating activityof the extracts was concentration dependent Black cuminoil exhibited higher chelating activity in comparison to theoleoresins but was not effective chelator as EDTA Maximumchelating of metal ions at 200120583gmLminus1 for black cumin oiland EDTA was found to be 7456 and 8790 respectivelywhereas the oleoresins were less effective in metal chelationand their metal chelating activity ranges from 221 to 445
Figure 2 Scavenging effect () of black cumin oil and its oleoresinson DPPH radical
0
02
04
06
08
1
12
0 10 20 30 40 50 60 70 80 90 100
Abs
orba
nce a
t 765
nm
Gallic acid
Gallic acid (120583g mLminus1)
y = 00101x + 00178
R2 = 0982
Figure 3 Calibration curve of gallic acid
722 Scavenging Effect on DPPH Radical DPPH∙ is a stableradical showing amaximum absorbance at 515 nm InDPPH∙assay the antioxidant was able to reduce the stable radicalDPPH to the yellow-colored diphenylpicrylhydrazone Themethod is based on the reduction of DPPH∙ in alcoholicsolution in the presence of a hydrogen-donating antioxidantdue to formation of the nonradical form DPPH-H in thereaction DPPH∙ is usually used as a reagent to evaluatefree radical and accepts an electron or hydrogen radical tobecome a stable diamagnetic molecule The disappearanceof the DPPH radical absorption at 515 nm by the action ofantioxidants is taken as a measure of antioxidant activityThescavenging effects of black cumin oil and oleoresins onDPPHradical linearly increased as concentration increased from5 to 20120583gmLminus1 (Figure 2) At 20120583gmLminus1 the scavengingactivity of black cumin oil and ethyl acetate oleoresin was954 and 8975 respectively comparatively higher than
6 BioMed Research International
0
100
200
300
400
500
0 7 14 21 28
PGBlack cumin oilEthyl acetate oleoBHT
EtOH oleo
BHAControl
Incubation time (days)
Pero
xide
val
ue (m
eq kg
minus1)
n-hexane oleo
Figure 4 Inhibitory effect of black cumin oil and its oleoresins onthe primary oxidation of linseed oil measured using peroxide valuemethod
BHT and BHA but lower than PG However the scavengingactivity of BHA BHT and PG was more effective at lowerconcentration and was 698 721 and 863 at 5 120583gmLminus1but as the concentration increases the differences in scav-enging activity between BHA BHT and oleoresins (speciallyethyl acetate) become less significant Ethanol and n-hexaneoleoresins showed moderate scavenging activity
723 Estimation of TPC The amount of total phenols wasdetermined with Folin-Ciocalteu reagent Gallic acid wasused as standard compound The absorbance for variousdilutions of gallic acid with Folin-Ciocalteu reagent andsodium carbonate was obtained and found standard curveequation 119910 = 00101119909 + 00178 1198772 = 0982 (Figure 3) Thetotal phenol contents (gallic acid equivalents mg GAE perg) in black cumin essential oil ethyl acetate ethanol and n-hexane oleoresins were calculated as 1147plusmn005 1088plusmn09968 plusmn 006 and 833 plusmn 001 respectively The value suggeststhat the black cumin oil and its oleoresins have lesser amountof total phenols [27] The differences in the total phenoliccontent among the samplesmight be due tomany differencessuch as the environmental conditions genetic background oragricultural techniques applied
73 Antioxidant Assays in Linseed Oil System The changes ofPV in linseed oil of all investigated samples are presented inFigure 4 The rate of oxidative reactions in linseed oil withadditives was almost similar to that of the blank sampleThe stability of the linseed oil samples to the formation ofperoxides can be ranked in the following descending order
PG gt Black cumin oil gt Ethyl acetate oleo gt BHT gtBHA gt EtOH oleo gt n-hexane oleo gt Control
Simultaneously with the measurements of PV changes insecondary product such as malondialdehyde the compound
0 7 14 21 28
PGBlack cumin oilEthyl acetate oleoBHA
BHTControl
Incubation time (days)
0
005
01
015
02
025
03
TBA
val
ue (m
eq O
2Kg
minus1)
EtOH oleo
n-hexane oleo
Figure 5 Inhibitory effect of black cumin oil and its oleoresinson the primary oxidation of linseed oil measured using TBA valuemethod
0
05
1
15
2
25
3
0 2 4 6 8 10 12
PGBlack cumin oilBHTEthyl acetate oleo
BHA
EtOH oleoControl
Abs
orba
nce a
t 500
nm
Incubation time (days)
n-hexane oleo
Figure 6 Inhibitory effect of black cumin oil and its oleoresins onthe primary oxidation of linoleic acid system measured using ferricthiocyanate method
used as an indicator of lipid peroxidation was measured byTBAvalues (Figure 5) were also determined after every sevendays Black cumin oil and its ethyl acetate oleoresin showedstrong inhibition at 002 concentration as compared toBHT and BHA (119875 lt 005) but lower than PG whereas theethanol and n-hexane oleoresin showed moderate inhibitionat 002 concentration as compared to the other additivesFrom the above results it should be confirmed that formationof primary oxidation species peroxides was quite similarwith the secondary oxidation products and the changes ofboth oxidation characteristics are in a good correlation
74 Antioxidant Activity in Linoleic Acid System The FTCmethod was used to measure the amount of peroxides at
BioMed Research International 7
the primary stage of linoleic acid peroxidation (Figure 6)Since the concentration of the peroxide decreases as theantioxidant activity increases the intensity of the pigmentwill be reduced leading to lower absorbance Absorbancevalues of the control as well as black cumin oil and itsoleoresins increase until day 10 and then decreased on day12 due to the malondialdehyde formation from linoleic acidoxidation There was a significant difference (119875 lt 005)between the control and the tested essential oil and oleoresinsAs can be seen in Figure 6 both black cumin oil andoleoresins showed good antioxidative property in the linoleicacid system and were significantly (119875 lt 005) different fromthe control
Antioxidant activities of essential oils and oleoresinsmay be related to the diverse compounds present in themincluding terpenes sesquiterpenes and phenolic acids whichact in various ways such as inhibition of peroxidation scav-enging the radicals and chelating the metal ions The mainconstituents of black cumin oil were thymoquinone (376)and p-cymene (314) with minor amounts of longifolenecarvacrol and thymohydroquinone which were responsiblefor the antioxidant activity of black cumin oil [8 9 19 28ndash31] Furthermore free radical scavenging effects of thesecomponentswere studied on the reactions generating reactiveoxygen species such as superoxide anion radical hydroxylradical using spectrophotometric methods [5] The resultsobtained using different assays were well correlated withthe previous work reported be many workers [5 30] whofound thymoquinone as a main constituent responsible forthe activity It has been suggested that phenolic content iscorrelated with the antioxidant activity [32] It is consideredthat the antioxidant activity of phenolic compounds is dueto their high redox potentials which allow them to actas reducing agents hydrogen donors Thymoquinone wasalso present in small amount with higher percentage ofunsaturated fatty acid linoleic acid in different oleoresinsStudies [33] have shown that these unsaturated fatty acidshave anti-rather than pro-oxidant activity but still researchhas been going on for the exact role of unsaturated fatty acidsagainst the oxidative stress
75 Antimicrobial Investigations Using inverted petri platetechnique (Table 3) the volatile oil exhibited more than 90zone inhibition for F moniliforme and P viridicatum It wasalso found to be highly effective in controlling the growthof Aspergillus species and F graminearum where (50) and(65) zone inhibition was observed respectively For othertested fungi the essential oil exerted less activity Howeverusing the same method the oleoresins have revealed lessactivity except for F moniliforme in which only up to 40mycelial zone inhibition was obtained Moreover using foodpoison technique (Table 4) the volatile oil showed clear zoneof growth inhibition against F graminearum at 10 120583L Thevolatile oil showed strong antifungal activity against all testedAspergillus species in the food poison method Ethyl acetateand ethanol oleoresin showed up to 30 zone inhibitionat 10 120583L dose The n-hexane oleoresin showed very feeble
inhibition zone in both techniques The lower antimicrobialefficacy of the oleoresins is due to their low volatility [34]
The antibacterial investigations were undertaken usingagar well diffusion method (Table 4) Using this method theblack cumin oil has shown better activity than oleoresinsand commercial bactericide that is ampicillin The volatileoil was found to be highly effective against B subtilis Bcereus and S aureus and showed complete zone of inhibitionat 3000 ppm concentration whereas in oleoresins more than20mm inhibition was obtained for Gram-positive bacteriaIn addition more than 20 and 25mm zone inhibition wasobtained for P aeruginosa and E coli The results obtainedusing agar well diffusion method were well correlated withthe earlier reported work [6 30 35] where black cumin seedoil has been shown to be effective against a wide spectrumof organisms bacteria like B cereus B subtilis S aureus Sepidermis E coli and P aeruginosa
The results obtained in antimicrobial investigations ofblack cumin oil and oleoresins were in good agreementwith the previous reported work [36] Thymoquinone p-cymene (monoterpene) longifolene (sesquiterpene) andthymohydroquinone were responsible for strong antimicro-bial activity of black cumin oil [29] El Alfy et al [35]isolated thymohydroquinone as antimicrobial compoundfrom the volatile oil of Nigella sativa seeds Oleoresins havehigh concentration of unsaturated fatty acids along withthymohydroquinone in small amount which is responsiblefor its moderate antimicrobial effects Long chain fatty acidslike linoleic acid and oleic acid were previously reportedto possess antibacterial and antifungal activity [36ndash39] p-Cymene is not an efficient antimicrobial compound whenused alone but it potentiate the activity of compounds likecarvacrol [40] The antimicrobial activity of essential oils canoften be correlated to its content of phenolic constituentsThe type of bacteria also has an influence on the effectivenessof the volatile oil and oleoresins Gram-negative bacteriawere generally less susceptible than Gram-positive bacteria[41] The difference in the susceptibility of the bacteria arisesas a result of differences in their cell membrane structurewhich is more complex in case of Gram-negative bacteriaThe antimicrobial activity of a given essential oil may dependon only one or two of the major constituents that make upthe oil However increasing amounts of evidence indicatethat the inherent activity of essential oils may not only relyexclusively on the ratio in which the main active constituentsare present but also on interactions between these andminorconstituents in the oils and oleoresins
8 Conclusions
Seeds of black cumin seem to possess magical propertiesand have been worked out extensively This study revealedthat black cumin essential oil and its oleoresins constitutea good alternative source of essential fatty acids comparedwith common vegetable oil The present results showedthat essential oil and oleoresins of black cumin exhibitedhigher antioxidant activity than synthetic antioxidantsThesefindings could be used to prepare multipurpose products forpharmaceutical applications and its usage as dietary source of
8 BioMed Research International
Table 3 (a) Antifungal investigations of black cumin oil and its oleoresins ( zone inhibitiona) using inverted petriplate method (b)Antifungal investigations of black cumin oil and its oleoresins ( zone inhibition) using food poisoned method
(a)
Mycelial zone inhibition at different dosesa of sample ()Samples Doses (120583L) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
(b)
Mycelial zone inhibitiona at different doses of sample ()Samples Doses (ppm) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
Table 4 Antibacterial activity of black cumin oil and its oleoresinsagainst a few bacterial species using agar well diffusion method
Diameter of inhibition zone (mma)Samples Doses (ppmb) BS BC SA EC PA
Black cumin oil 1000 ++ 253 plusmn 14 ++ minus 189 plusmn 017
3000 ++ ++ ++ 203 plusmn 014 279 plusmn 015
Ethanol oleoresin 1000 164 plusmn 081 minus 156 plusmn 036 minus minus
3000 283 plusmn 020 minus 259 plusmn 042 minus minus
n-Hexane oleoresin 1000 115 plusmn 031 minus 91 plusmn 11 minus minus
3000 209 plusmn 19 minus 134 plusmn 26 minus minus
Ethyl acetate oleoresin1000 223 plusmn 08 minus 177 plusmn 23 minus minus
3000 443 plusmn 07 minus 409 plusmn 12 minus minus
Ampicillin 1000 156 plusmn 032 minus 91 plusmn 11 minus minus
3000 132 plusmn 02 minus 134 plusmn 26 minus minus
aAverage of three replicates ++ indicates complete inhibition and minus indicates no inhibitionbDMSO was used as solventBS Bacillus subtilis SA Staphylococcus aureus BC Bacillus cereus EC Escherichia coli PA Pseudomonas aeruginosa
BioMed Research International 9
antioxidant should be considered largely for alleviating andameliorating diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to theHead of Department of Chem-istry DDU Gorakhpur University Gorakhpur for providinglaboratory facilities The financial support from UGC toSunita Singh (JRF) and Emeritus Fellow to Dr Gurdip Singhis also acknowledged
References
[1] G Zengin A Aktumsek G O Guler Y S Cakmak andE Yildiztugay ldquoAntioxidant properties of methanolic extractand fatty acid composition of Centaurea urvillei DC subsphayekiana Wagenitzrdquo Records of Natural Products vol 5 no 2pp 123ndash132 2011
[2] H Lutterodt M Luther M Slavin et al ldquoFatty acid profilethymoquinone content oxidative stability and antioxidantproperties of cold-pressed black cumin seed oilsrdquo Food Scienceand Technology vol 43 no 9 pp 1409ndash1413 2010
[3] S Shah and K S Ray ldquoStudy on antioxidant and antimicrobialproperties of black cumin (Nigella sativa Linn)rdquo Journal of FoodScience and Technology vol 40 no 1 pp 70ndash73 2003
[4] I Tekeoglu A Dogan and L Demiralp ldquoEffects of thymo-quinone (volatile oil of black cumin) on rheumatoid arthritis inrat modelsrdquo Phytotherapy Research vol 20 no 10 pp 869ndash8712006
[5] I Kruk TMichalska K Lichszteld A Kladna andH Y Aboul-Enein ldquoThe effect of thymol and its derivatives on reactionsgenerating reactive oxygen speciesrdquo Chemosphere vol 41 no7 pp 1059ndash1064 2000
[6] G Singh P Marimuthu C S De Heluani and C Cata-lan ldquoChemical constituents and antimicrobial and antioxidantpotentials of essential oil and acetone extract of Nigella sativaseedsrdquo Journal of the Science of Food and Agriculture vol 85no 13 pp 2297ndash2306 2005
[7] S A Maisonneuve European Pharmacopoeia Part 1 Sainte-Ruffine 1983
[8] M Burits and F Bucar ldquoAntioxidant activity of Nigella sati-vaessential oilrdquo Phytotherapy Research vol 14 pp 323ndash3282000
[9] N Ilaiyaraja and F Khanum ldquoNigella sativa L a review of thera-peutic applicationsrdquo Journal of Herbal Medicine and Toxicologyvol 4 no 2 pp 1ndash8 2010
[10] E A Decker and B Welch ldquoRole of ferritin as a lipid oxidationcatalyst in muscle foodrdquo Journal of Agricultural and FoodChemistry vol 38 no 3 pp 674ndash677 1990
[11] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo Food Scienceand Technology vol 28 no 1 pp 25ndash30 1995
[12] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic as it reagentsrdquo TheAmerican Journal of Enology and Viticulture vol 16 pp 144ndash158 1965
[13] K D Economou V Oreopoulou and C D ThomopoulosldquoAntioxidant activity of some plant extracts of the familylabiataerdquo Journal of the American Oil Chemists Society vol 68no 2 pp 109ndash113 1991
[14] G Singh S Maurya M P de Lampasona and C A N CatalanldquoA comparison of chemical antioxidant and antimicrobialstudies of cinnamon leaf and bark volatile oils oleoresins andtheir constituentsrdquo Food and Chemical Toxicology vol 45 no 9pp 1650ndash1661 2007
[15] G Singh I P S Kapoor P Singh C S de Heluani M P deLampasona and C A N Catalan ldquoChemistry antioxidant andantimicrobial investigations on essential oil and oleoresins ofZingiber officinalerdquo Food and Chemical Toxicology vol 46 no10 pp 3295ndash3302 2008
[16] G P Rao and A K Shrivastava ldquoToxicity of essential oils ofhigher plants against fungal pathogens of sugarcanerdquo inCurrentTrend in Sugarcane Pathology G P Rao A G Gillasple PP Upadhaya A Bergamin V P Agnihotri and C T ChenEds pp 347ndash365 International Books and Periodicals SupplyService Delhi India 1994
[17] K Ramadas G Suresh N Janarthanan and S Masila-mani ldquoAntifungal activity of 13-disubstituted symmetrical andunsymmetrical thioureasrdquo Pesticide Science vol 52 no 2 pp145ndash151 1998
[18] NCCLS (National Committee for Clinical Laboratory Stan-dards) Performance Standards for Antimicrobial Disc Suscepti-bility Test 6th edition 1997
[19] V Hajhashemi A Ghannadi and H Jafarabadi ldquoBlack cuminseed essential oil as a potent analgesic and antiinflammatorydrugrdquo Phytotherapy Research vol 18 no 3 pp 195ndash199 2004
[20] P J Houghton R Zarka B De Las Heras and J R SHoult ldquoFixed oil of Nigella sativa and derived thymoquinoneinhibit eicosanoid generation in leukocytes andmembrane lipidperoxidationrdquo Planta Medica vol 61 no 1 pp 33ndash36 1995
[21] M T SultanM S Butt FMAnjumA Jamil S Akhtar andMNasir ldquoNutritional profile of indigenous cultivar of black cuminseeds and antioxidant potential of its fixed and essential oilrdquoPakistan Journal of Botany vol 41 no 3 pp 1321ndash1330 2009
[22] S Cheikh-Rouhou S Besbes G Lognay C Blecker CDeroanne and H Attia ldquoSterol composition of black cumin(Nigella sativa L) and Aleppo pine (Pinus halepensisMill) seedoilsrdquo Journal of Food Composition and Analysis vol 21 no 2 pp162ndash168 2008
[23] M F Ramadan ldquoNutritional value functional properties andnutraceutical applications of black cumin (Nigella sativa L) anoverviewrdquo International Journal of Food Science and Technologyvol 42 no 10 pp 1208ndash1218 2007
[24] T D Parker D A Adams K Zhou M Harris and L Yu ldquoFattyacid composition and oxidative stability of cold-pressed edibleseed oilsrdquo Journal of Food Science vol 68 no 4 pp 1240ndash12432003
[25] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAmerican Oil Chemical Society vol 83 pp 847ndash854 2006
[26] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAgricultural and Food Chemistry vol 53 no 3 pp 566ndash5732005
[27] R R Sokal Introduction to Biostatistics W H Freeman SanFrancisco Calif USA 1973
10 BioMed Research International
[28] H Hosseinzadeh S Parvardeh M N Asl H R Sadeghniaand T Ziaee ldquoEffect of thymoquinone and Nigella sativa seedsoil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampusrdquo Phytomedicine vol 14no 9 pp 621ndash627 2007
[29] S Bourgou A Pichette B Marzouk and J Legault ldquoBioactiv-ities of black cumin essential oil and its main terpenes fromTunisiardquo South African Journal of Botany vol 76 no 2 pp 210ndash216 2010
[30] H J Harzallah E Noumi K Bekir et al ldquoChemical composi-tion antibacterial and antifungal properties of Tunisian Nigellasativa fixed oilrdquoAfrican Journal of Microbiology Research vol 6no 22 pp 4675ndash4679 2012
[31] V S Deepa and P S Kumar ldquoPreliminary phytochemicalinvestigations and in vitro antioxidant activity in selected partsof Andrographis spprdquo Journal of Pharmacological Research vol3 no 9 pp 2206ndash2210 2010
[32] M Amensour E Sendra A Jamal S Bouhdid J A Perez-Alvarez and J Fernandez-Lopez ldquoTotal phenolic content andantioxidant activity of myrtle (Myrtus communis) extractsrdquoNatural Product Communications vol 4 no 6 pp 819ndash8242009
[33] M Di Nunzio V Valli and A Bordoni ldquoPro- and anti-oxidanteffects of polyunsaturated fatty acid supplementation in HepG2cellsrdquo Prostaglandins Leukotrienes and Essential Fatty Acids vol85 no 3-4 pp 121ndash127 2011
[34] G Singh SMaurya CCatalan andMP de Lampasona ldquoStud-ies on essential oils part 42 chemical antifungal antioxidantand sprout suppressant studies on ginger essential oil and itsoleoresinrdquo Flavour and Fragrance Journal vol 20 no 1 pp 1ndash62005
[35] T S El Alfy H M El Fatatry and M A Toama ldquoIsolationand structure assignment of an antimicrobial principle from thevolatile oil of Nigella sativa L seedsrdquo Pharmazie vol 30 no 2pp 109ndash111 1975
[36] M A Khan ldquoChemical composition and medicinal propertiesof Nigella sativa Linnrdquo Inflammopharmacology vol 7 no 1 pp15ndash35 1999
[37] L J McGaw A K Jager and J van Staden ldquoIsolation ofantibacterial fatty acids from Schotia brachypetalardquo Fitoterapiavol 73 no 5 pp 431ndash433 2002
[38] A R McCutcheon T E Roberts E Gibbons et al ldquoAntiviralscreening of British Columbian medicinal plantsrdquo Journal ofEthnopharmacology vol 49 no 2 pp 101ndash110 1995
[39] S Javed A A Shahid M S Haider et al ldquoNutritionalphytochemical potential and pharmacological evaluation ofNigella Sativa (Kalonji) and Trachyspermum Ammi (Ajwain)rdquoMedicinal Plants Research vol 6 no 5 pp 768ndash775 2012
[40] P Rattanachaikunsopon and P Phumkhachorn ldquoAssessmentof factors influencing antimicrobial activity of carvacrol andcymene against Vibrio cholerae in foodrdquo Journal of Bioscienceand Bioengineering vol 110 no 5 pp 614ndash619 2010
[41] M Gilles J Zhao M An and S Agboola ldquoChemical com-position and antimicrobial properties of essential oils of threeAustralian Eucalyptus speciesrdquo Food Chemistry vol 119 no 2pp 731ndash737 2010
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Figure 1 Chelating effect of black cumin oil and its differentoleoresins
was thymoquinone (376) followed by p-cymene (314)120572-thujene (56) thymohydroquinone (34) longifolene(20) and carvacrol (14) Burits and Bucar [8] character-ized many components in black cumin essential oil such asthymoquinone (278ndash570) p-cymene (71ndash155) carvacrol(58ndash116) trans-anethole (025ndash23) 4-terpineol (20ndash66) and 10ndash80 of longifolene These results are slightdifferent from the work reported by Hajhashemi et al [19]who reported that p-cymene (373) and thymoquinone(137) were the major components of black cumin Singh etal [6] also reported p-cymene as the major component in theblack cumin essential oil
From Table 2 it is evident that in ethanol oleoresin19 components constitute 964 of the total weight in theethyl acetate oleoresin a total of 19 components making909 of the whole mass and in case of n-hexane oleoresin19 compounds constituting about 852 of the total weightwere identified The oleoresins were mainly comprised ofunsaturated fatty acids and their different esters The majorcomponents in all three oleoresins were linoleic acid (unsat-urated fatty acid) followed by glyceryl linoleate glycerylpalmitate oleic acid and other minor components Thepresence of unsaturated fatty acids in oleoresins was wellsupported by the various reported work [20ndash26]
72 Antioxidant Investigations
721 Chelating Activity on Ferrous Ions The ferrous ion(Fe2+) chelating effect of black cumin oil and its differentoleoresins is presented in Figure 1 The chelating activityof the extracts was concentration dependent Black cuminoil exhibited higher chelating activity in comparison to theoleoresins but was not effective chelator as EDTA Maximumchelating of metal ions at 200120583gmLminus1 for black cumin oiland EDTA was found to be 7456 and 8790 respectivelywhereas the oleoresins were less effective in metal chelationand their metal chelating activity ranges from 221 to 445
Figure 2 Scavenging effect () of black cumin oil and its oleoresinson DPPH radical
0
02
04
06
08
1
12
0 10 20 30 40 50 60 70 80 90 100
Abs
orba
nce a
t 765
nm
Gallic acid
Gallic acid (120583g mLminus1)
y = 00101x + 00178
R2 = 0982
Figure 3 Calibration curve of gallic acid
722 Scavenging Effect on DPPH Radical DPPH∙ is a stableradical showing amaximum absorbance at 515 nm InDPPH∙assay the antioxidant was able to reduce the stable radicalDPPH to the yellow-colored diphenylpicrylhydrazone Themethod is based on the reduction of DPPH∙ in alcoholicsolution in the presence of a hydrogen-donating antioxidantdue to formation of the nonradical form DPPH-H in thereaction DPPH∙ is usually used as a reagent to evaluatefree radical and accepts an electron or hydrogen radical tobecome a stable diamagnetic molecule The disappearanceof the DPPH radical absorption at 515 nm by the action ofantioxidants is taken as a measure of antioxidant activityThescavenging effects of black cumin oil and oleoresins onDPPHradical linearly increased as concentration increased from5 to 20120583gmLminus1 (Figure 2) At 20120583gmLminus1 the scavengingactivity of black cumin oil and ethyl acetate oleoresin was954 and 8975 respectively comparatively higher than
6 BioMed Research International
0
100
200
300
400
500
0 7 14 21 28
PGBlack cumin oilEthyl acetate oleoBHT
EtOH oleo
BHAControl
Incubation time (days)
Pero
xide
val
ue (m
eq kg
minus1)
n-hexane oleo
Figure 4 Inhibitory effect of black cumin oil and its oleoresins onthe primary oxidation of linseed oil measured using peroxide valuemethod
BHT and BHA but lower than PG However the scavengingactivity of BHA BHT and PG was more effective at lowerconcentration and was 698 721 and 863 at 5 120583gmLminus1but as the concentration increases the differences in scav-enging activity between BHA BHT and oleoresins (speciallyethyl acetate) become less significant Ethanol and n-hexaneoleoresins showed moderate scavenging activity
723 Estimation of TPC The amount of total phenols wasdetermined with Folin-Ciocalteu reagent Gallic acid wasused as standard compound The absorbance for variousdilutions of gallic acid with Folin-Ciocalteu reagent andsodium carbonate was obtained and found standard curveequation 119910 = 00101119909 + 00178 1198772 = 0982 (Figure 3) Thetotal phenol contents (gallic acid equivalents mg GAE perg) in black cumin essential oil ethyl acetate ethanol and n-hexane oleoresins were calculated as 1147plusmn005 1088plusmn09968 plusmn 006 and 833 plusmn 001 respectively The value suggeststhat the black cumin oil and its oleoresins have lesser amountof total phenols [27] The differences in the total phenoliccontent among the samplesmight be due tomany differencessuch as the environmental conditions genetic background oragricultural techniques applied
73 Antioxidant Assays in Linseed Oil System The changes ofPV in linseed oil of all investigated samples are presented inFigure 4 The rate of oxidative reactions in linseed oil withadditives was almost similar to that of the blank sampleThe stability of the linseed oil samples to the formation ofperoxides can be ranked in the following descending order
PG gt Black cumin oil gt Ethyl acetate oleo gt BHT gtBHA gt EtOH oleo gt n-hexane oleo gt Control
Simultaneously with the measurements of PV changes insecondary product such as malondialdehyde the compound
0 7 14 21 28
PGBlack cumin oilEthyl acetate oleoBHA
BHTControl
Incubation time (days)
0
005
01
015
02
025
03
TBA
val
ue (m
eq O
2Kg
minus1)
EtOH oleo
n-hexane oleo
Figure 5 Inhibitory effect of black cumin oil and its oleoresinson the primary oxidation of linseed oil measured using TBA valuemethod
0
05
1
15
2
25
3
0 2 4 6 8 10 12
PGBlack cumin oilBHTEthyl acetate oleo
BHA
EtOH oleoControl
Abs
orba
nce a
t 500
nm
Incubation time (days)
n-hexane oleo
Figure 6 Inhibitory effect of black cumin oil and its oleoresins onthe primary oxidation of linoleic acid system measured using ferricthiocyanate method
used as an indicator of lipid peroxidation was measured byTBAvalues (Figure 5) were also determined after every sevendays Black cumin oil and its ethyl acetate oleoresin showedstrong inhibition at 002 concentration as compared toBHT and BHA (119875 lt 005) but lower than PG whereas theethanol and n-hexane oleoresin showed moderate inhibitionat 002 concentration as compared to the other additivesFrom the above results it should be confirmed that formationof primary oxidation species peroxides was quite similarwith the secondary oxidation products and the changes ofboth oxidation characteristics are in a good correlation
74 Antioxidant Activity in Linoleic Acid System The FTCmethod was used to measure the amount of peroxides at
BioMed Research International 7
the primary stage of linoleic acid peroxidation (Figure 6)Since the concentration of the peroxide decreases as theantioxidant activity increases the intensity of the pigmentwill be reduced leading to lower absorbance Absorbancevalues of the control as well as black cumin oil and itsoleoresins increase until day 10 and then decreased on day12 due to the malondialdehyde formation from linoleic acidoxidation There was a significant difference (119875 lt 005)between the control and the tested essential oil and oleoresinsAs can be seen in Figure 6 both black cumin oil andoleoresins showed good antioxidative property in the linoleicacid system and were significantly (119875 lt 005) different fromthe control
Antioxidant activities of essential oils and oleoresinsmay be related to the diverse compounds present in themincluding terpenes sesquiterpenes and phenolic acids whichact in various ways such as inhibition of peroxidation scav-enging the radicals and chelating the metal ions The mainconstituents of black cumin oil were thymoquinone (376)and p-cymene (314) with minor amounts of longifolenecarvacrol and thymohydroquinone which were responsiblefor the antioxidant activity of black cumin oil [8 9 19 28ndash31] Furthermore free radical scavenging effects of thesecomponentswere studied on the reactions generating reactiveoxygen species such as superoxide anion radical hydroxylradical using spectrophotometric methods [5] The resultsobtained using different assays were well correlated withthe previous work reported be many workers [5 30] whofound thymoquinone as a main constituent responsible forthe activity It has been suggested that phenolic content iscorrelated with the antioxidant activity [32] It is consideredthat the antioxidant activity of phenolic compounds is dueto their high redox potentials which allow them to actas reducing agents hydrogen donors Thymoquinone wasalso present in small amount with higher percentage ofunsaturated fatty acid linoleic acid in different oleoresinsStudies [33] have shown that these unsaturated fatty acidshave anti-rather than pro-oxidant activity but still researchhas been going on for the exact role of unsaturated fatty acidsagainst the oxidative stress
75 Antimicrobial Investigations Using inverted petri platetechnique (Table 3) the volatile oil exhibited more than 90zone inhibition for F moniliforme and P viridicatum It wasalso found to be highly effective in controlling the growthof Aspergillus species and F graminearum where (50) and(65) zone inhibition was observed respectively For othertested fungi the essential oil exerted less activity Howeverusing the same method the oleoresins have revealed lessactivity except for F moniliforme in which only up to 40mycelial zone inhibition was obtained Moreover using foodpoison technique (Table 4) the volatile oil showed clear zoneof growth inhibition against F graminearum at 10 120583L Thevolatile oil showed strong antifungal activity against all testedAspergillus species in the food poison method Ethyl acetateand ethanol oleoresin showed up to 30 zone inhibitionat 10 120583L dose The n-hexane oleoresin showed very feeble
inhibition zone in both techniques The lower antimicrobialefficacy of the oleoresins is due to their low volatility [34]
The antibacterial investigations were undertaken usingagar well diffusion method (Table 4) Using this method theblack cumin oil has shown better activity than oleoresinsand commercial bactericide that is ampicillin The volatileoil was found to be highly effective against B subtilis Bcereus and S aureus and showed complete zone of inhibitionat 3000 ppm concentration whereas in oleoresins more than20mm inhibition was obtained for Gram-positive bacteriaIn addition more than 20 and 25mm zone inhibition wasobtained for P aeruginosa and E coli The results obtainedusing agar well diffusion method were well correlated withthe earlier reported work [6 30 35] where black cumin seedoil has been shown to be effective against a wide spectrumof organisms bacteria like B cereus B subtilis S aureus Sepidermis E coli and P aeruginosa
The results obtained in antimicrobial investigations ofblack cumin oil and oleoresins were in good agreementwith the previous reported work [36] Thymoquinone p-cymene (monoterpene) longifolene (sesquiterpene) andthymohydroquinone were responsible for strong antimicro-bial activity of black cumin oil [29] El Alfy et al [35]isolated thymohydroquinone as antimicrobial compoundfrom the volatile oil of Nigella sativa seeds Oleoresins havehigh concentration of unsaturated fatty acids along withthymohydroquinone in small amount which is responsiblefor its moderate antimicrobial effects Long chain fatty acidslike linoleic acid and oleic acid were previously reportedto possess antibacterial and antifungal activity [36ndash39] p-Cymene is not an efficient antimicrobial compound whenused alone but it potentiate the activity of compounds likecarvacrol [40] The antimicrobial activity of essential oils canoften be correlated to its content of phenolic constituentsThe type of bacteria also has an influence on the effectivenessof the volatile oil and oleoresins Gram-negative bacteriawere generally less susceptible than Gram-positive bacteria[41] The difference in the susceptibility of the bacteria arisesas a result of differences in their cell membrane structurewhich is more complex in case of Gram-negative bacteriaThe antimicrobial activity of a given essential oil may dependon only one or two of the major constituents that make upthe oil However increasing amounts of evidence indicatethat the inherent activity of essential oils may not only relyexclusively on the ratio in which the main active constituentsare present but also on interactions between these andminorconstituents in the oils and oleoresins
8 Conclusions
Seeds of black cumin seem to possess magical propertiesand have been worked out extensively This study revealedthat black cumin essential oil and its oleoresins constitutea good alternative source of essential fatty acids comparedwith common vegetable oil The present results showedthat essential oil and oleoresins of black cumin exhibitedhigher antioxidant activity than synthetic antioxidantsThesefindings could be used to prepare multipurpose products forpharmaceutical applications and its usage as dietary source of
8 BioMed Research International
Table 3 (a) Antifungal investigations of black cumin oil and its oleoresins ( zone inhibitiona) using inverted petriplate method (b)Antifungal investigations of black cumin oil and its oleoresins ( zone inhibition) using food poisoned method
(a)
Mycelial zone inhibition at different dosesa of sample ()Samples Doses (120583L) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
(b)
Mycelial zone inhibitiona at different doses of sample ()Samples Doses (ppm) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
Table 4 Antibacterial activity of black cumin oil and its oleoresinsagainst a few bacterial species using agar well diffusion method
Diameter of inhibition zone (mma)Samples Doses (ppmb) BS BC SA EC PA
Black cumin oil 1000 ++ 253 plusmn 14 ++ minus 189 plusmn 017
3000 ++ ++ ++ 203 plusmn 014 279 plusmn 015
Ethanol oleoresin 1000 164 plusmn 081 minus 156 plusmn 036 minus minus
3000 283 plusmn 020 minus 259 plusmn 042 minus minus
n-Hexane oleoresin 1000 115 plusmn 031 minus 91 plusmn 11 minus minus
3000 209 plusmn 19 minus 134 plusmn 26 minus minus
Ethyl acetate oleoresin1000 223 plusmn 08 minus 177 plusmn 23 minus minus
3000 443 plusmn 07 minus 409 plusmn 12 minus minus
Ampicillin 1000 156 plusmn 032 minus 91 plusmn 11 minus minus
3000 132 plusmn 02 minus 134 plusmn 26 minus minus
aAverage of three replicates ++ indicates complete inhibition and minus indicates no inhibitionbDMSO was used as solventBS Bacillus subtilis SA Staphylococcus aureus BC Bacillus cereus EC Escherichia coli PA Pseudomonas aeruginosa
BioMed Research International 9
antioxidant should be considered largely for alleviating andameliorating diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to theHead of Department of Chem-istry DDU Gorakhpur University Gorakhpur for providinglaboratory facilities The financial support from UGC toSunita Singh (JRF) and Emeritus Fellow to Dr Gurdip Singhis also acknowledged
References
[1] G Zengin A Aktumsek G O Guler Y S Cakmak andE Yildiztugay ldquoAntioxidant properties of methanolic extractand fatty acid composition of Centaurea urvillei DC subsphayekiana Wagenitzrdquo Records of Natural Products vol 5 no 2pp 123ndash132 2011
[2] H Lutterodt M Luther M Slavin et al ldquoFatty acid profilethymoquinone content oxidative stability and antioxidantproperties of cold-pressed black cumin seed oilsrdquo Food Scienceand Technology vol 43 no 9 pp 1409ndash1413 2010
[3] S Shah and K S Ray ldquoStudy on antioxidant and antimicrobialproperties of black cumin (Nigella sativa Linn)rdquo Journal of FoodScience and Technology vol 40 no 1 pp 70ndash73 2003
[4] I Tekeoglu A Dogan and L Demiralp ldquoEffects of thymo-quinone (volatile oil of black cumin) on rheumatoid arthritis inrat modelsrdquo Phytotherapy Research vol 20 no 10 pp 869ndash8712006
[5] I Kruk TMichalska K Lichszteld A Kladna andH Y Aboul-Enein ldquoThe effect of thymol and its derivatives on reactionsgenerating reactive oxygen speciesrdquo Chemosphere vol 41 no7 pp 1059ndash1064 2000
[6] G Singh P Marimuthu C S De Heluani and C Cata-lan ldquoChemical constituents and antimicrobial and antioxidantpotentials of essential oil and acetone extract of Nigella sativaseedsrdquo Journal of the Science of Food and Agriculture vol 85no 13 pp 2297ndash2306 2005
[7] S A Maisonneuve European Pharmacopoeia Part 1 Sainte-Ruffine 1983
[8] M Burits and F Bucar ldquoAntioxidant activity of Nigella sati-vaessential oilrdquo Phytotherapy Research vol 14 pp 323ndash3282000
[9] N Ilaiyaraja and F Khanum ldquoNigella sativa L a review of thera-peutic applicationsrdquo Journal of Herbal Medicine and Toxicologyvol 4 no 2 pp 1ndash8 2010
[10] E A Decker and B Welch ldquoRole of ferritin as a lipid oxidationcatalyst in muscle foodrdquo Journal of Agricultural and FoodChemistry vol 38 no 3 pp 674ndash677 1990
[11] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo Food Scienceand Technology vol 28 no 1 pp 25ndash30 1995
[12] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic as it reagentsrdquo TheAmerican Journal of Enology and Viticulture vol 16 pp 144ndash158 1965
[13] K D Economou V Oreopoulou and C D ThomopoulosldquoAntioxidant activity of some plant extracts of the familylabiataerdquo Journal of the American Oil Chemists Society vol 68no 2 pp 109ndash113 1991
[14] G Singh S Maurya M P de Lampasona and C A N CatalanldquoA comparison of chemical antioxidant and antimicrobialstudies of cinnamon leaf and bark volatile oils oleoresins andtheir constituentsrdquo Food and Chemical Toxicology vol 45 no 9pp 1650ndash1661 2007
[15] G Singh I P S Kapoor P Singh C S de Heluani M P deLampasona and C A N Catalan ldquoChemistry antioxidant andantimicrobial investigations on essential oil and oleoresins ofZingiber officinalerdquo Food and Chemical Toxicology vol 46 no10 pp 3295ndash3302 2008
[16] G P Rao and A K Shrivastava ldquoToxicity of essential oils ofhigher plants against fungal pathogens of sugarcanerdquo inCurrentTrend in Sugarcane Pathology G P Rao A G Gillasple PP Upadhaya A Bergamin V P Agnihotri and C T ChenEds pp 347ndash365 International Books and Periodicals SupplyService Delhi India 1994
[17] K Ramadas G Suresh N Janarthanan and S Masila-mani ldquoAntifungal activity of 13-disubstituted symmetrical andunsymmetrical thioureasrdquo Pesticide Science vol 52 no 2 pp145ndash151 1998
[18] NCCLS (National Committee for Clinical Laboratory Stan-dards) Performance Standards for Antimicrobial Disc Suscepti-bility Test 6th edition 1997
[19] V Hajhashemi A Ghannadi and H Jafarabadi ldquoBlack cuminseed essential oil as a potent analgesic and antiinflammatorydrugrdquo Phytotherapy Research vol 18 no 3 pp 195ndash199 2004
[20] P J Houghton R Zarka B De Las Heras and J R SHoult ldquoFixed oil of Nigella sativa and derived thymoquinoneinhibit eicosanoid generation in leukocytes andmembrane lipidperoxidationrdquo Planta Medica vol 61 no 1 pp 33ndash36 1995
[21] M T SultanM S Butt FMAnjumA Jamil S Akhtar andMNasir ldquoNutritional profile of indigenous cultivar of black cuminseeds and antioxidant potential of its fixed and essential oilrdquoPakistan Journal of Botany vol 41 no 3 pp 1321ndash1330 2009
[22] S Cheikh-Rouhou S Besbes G Lognay C Blecker CDeroanne and H Attia ldquoSterol composition of black cumin(Nigella sativa L) and Aleppo pine (Pinus halepensisMill) seedoilsrdquo Journal of Food Composition and Analysis vol 21 no 2 pp162ndash168 2008
[23] M F Ramadan ldquoNutritional value functional properties andnutraceutical applications of black cumin (Nigella sativa L) anoverviewrdquo International Journal of Food Science and Technologyvol 42 no 10 pp 1208ndash1218 2007
[24] T D Parker D A Adams K Zhou M Harris and L Yu ldquoFattyacid composition and oxidative stability of cold-pressed edibleseed oilsrdquo Journal of Food Science vol 68 no 4 pp 1240ndash12432003
[25] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAmerican Oil Chemical Society vol 83 pp 847ndash854 2006
[26] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAgricultural and Food Chemistry vol 53 no 3 pp 566ndash5732005
[27] R R Sokal Introduction to Biostatistics W H Freeman SanFrancisco Calif USA 1973
10 BioMed Research International
[28] H Hosseinzadeh S Parvardeh M N Asl H R Sadeghniaand T Ziaee ldquoEffect of thymoquinone and Nigella sativa seedsoil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampusrdquo Phytomedicine vol 14no 9 pp 621ndash627 2007
[29] S Bourgou A Pichette B Marzouk and J Legault ldquoBioactiv-ities of black cumin essential oil and its main terpenes fromTunisiardquo South African Journal of Botany vol 76 no 2 pp 210ndash216 2010
[30] H J Harzallah E Noumi K Bekir et al ldquoChemical composi-tion antibacterial and antifungal properties of Tunisian Nigellasativa fixed oilrdquoAfrican Journal of Microbiology Research vol 6no 22 pp 4675ndash4679 2012
[31] V S Deepa and P S Kumar ldquoPreliminary phytochemicalinvestigations and in vitro antioxidant activity in selected partsof Andrographis spprdquo Journal of Pharmacological Research vol3 no 9 pp 2206ndash2210 2010
[32] M Amensour E Sendra A Jamal S Bouhdid J A Perez-Alvarez and J Fernandez-Lopez ldquoTotal phenolic content andantioxidant activity of myrtle (Myrtus communis) extractsrdquoNatural Product Communications vol 4 no 6 pp 819ndash8242009
[33] M Di Nunzio V Valli and A Bordoni ldquoPro- and anti-oxidanteffects of polyunsaturated fatty acid supplementation in HepG2cellsrdquo Prostaglandins Leukotrienes and Essential Fatty Acids vol85 no 3-4 pp 121ndash127 2011
[34] G Singh SMaurya CCatalan andMP de Lampasona ldquoStud-ies on essential oils part 42 chemical antifungal antioxidantand sprout suppressant studies on ginger essential oil and itsoleoresinrdquo Flavour and Fragrance Journal vol 20 no 1 pp 1ndash62005
[35] T S El Alfy H M El Fatatry and M A Toama ldquoIsolationand structure assignment of an antimicrobial principle from thevolatile oil of Nigella sativa L seedsrdquo Pharmazie vol 30 no 2pp 109ndash111 1975
[36] M A Khan ldquoChemical composition and medicinal propertiesof Nigella sativa Linnrdquo Inflammopharmacology vol 7 no 1 pp15ndash35 1999
[37] L J McGaw A K Jager and J van Staden ldquoIsolation ofantibacterial fatty acids from Schotia brachypetalardquo Fitoterapiavol 73 no 5 pp 431ndash433 2002
[38] A R McCutcheon T E Roberts E Gibbons et al ldquoAntiviralscreening of British Columbian medicinal plantsrdquo Journal ofEthnopharmacology vol 49 no 2 pp 101ndash110 1995
[39] S Javed A A Shahid M S Haider et al ldquoNutritionalphytochemical potential and pharmacological evaluation ofNigella Sativa (Kalonji) and Trachyspermum Ammi (Ajwain)rdquoMedicinal Plants Research vol 6 no 5 pp 768ndash775 2012
[40] P Rattanachaikunsopon and P Phumkhachorn ldquoAssessmentof factors influencing antimicrobial activity of carvacrol andcymene against Vibrio cholerae in foodrdquo Journal of Bioscienceand Bioengineering vol 110 no 5 pp 614ndash619 2010
[41] M Gilles J Zhao M An and S Agboola ldquoChemical com-position and antimicrobial properties of essential oils of threeAustralian Eucalyptus speciesrdquo Food Chemistry vol 119 no 2pp 731ndash737 2010
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Figure 4 Inhibitory effect of black cumin oil and its oleoresins onthe primary oxidation of linseed oil measured using peroxide valuemethod
BHT and BHA but lower than PG However the scavengingactivity of BHA BHT and PG was more effective at lowerconcentration and was 698 721 and 863 at 5 120583gmLminus1but as the concentration increases the differences in scav-enging activity between BHA BHT and oleoresins (speciallyethyl acetate) become less significant Ethanol and n-hexaneoleoresins showed moderate scavenging activity
723 Estimation of TPC The amount of total phenols wasdetermined with Folin-Ciocalteu reagent Gallic acid wasused as standard compound The absorbance for variousdilutions of gallic acid with Folin-Ciocalteu reagent andsodium carbonate was obtained and found standard curveequation 119910 = 00101119909 + 00178 1198772 = 0982 (Figure 3) Thetotal phenol contents (gallic acid equivalents mg GAE perg) in black cumin essential oil ethyl acetate ethanol and n-hexane oleoresins were calculated as 1147plusmn005 1088plusmn09968 plusmn 006 and 833 plusmn 001 respectively The value suggeststhat the black cumin oil and its oleoresins have lesser amountof total phenols [27] The differences in the total phenoliccontent among the samplesmight be due tomany differencessuch as the environmental conditions genetic background oragricultural techniques applied
73 Antioxidant Assays in Linseed Oil System The changes ofPV in linseed oil of all investigated samples are presented inFigure 4 The rate of oxidative reactions in linseed oil withadditives was almost similar to that of the blank sampleThe stability of the linseed oil samples to the formation ofperoxides can be ranked in the following descending order
PG gt Black cumin oil gt Ethyl acetate oleo gt BHT gtBHA gt EtOH oleo gt n-hexane oleo gt Control
Simultaneously with the measurements of PV changes insecondary product such as malondialdehyde the compound
0 7 14 21 28
PGBlack cumin oilEthyl acetate oleoBHA
BHTControl
Incubation time (days)
0
005
01
015
02
025
03
TBA
val
ue (m
eq O
2Kg
minus1)
EtOH oleo
n-hexane oleo
Figure 5 Inhibitory effect of black cumin oil and its oleoresinson the primary oxidation of linseed oil measured using TBA valuemethod
0
05
1
15
2
25
3
0 2 4 6 8 10 12
PGBlack cumin oilBHTEthyl acetate oleo
BHA
EtOH oleoControl
Abs
orba
nce a
t 500
nm
Incubation time (days)
n-hexane oleo
Figure 6 Inhibitory effect of black cumin oil and its oleoresins onthe primary oxidation of linoleic acid system measured using ferricthiocyanate method
used as an indicator of lipid peroxidation was measured byTBAvalues (Figure 5) were also determined after every sevendays Black cumin oil and its ethyl acetate oleoresin showedstrong inhibition at 002 concentration as compared toBHT and BHA (119875 lt 005) but lower than PG whereas theethanol and n-hexane oleoresin showed moderate inhibitionat 002 concentration as compared to the other additivesFrom the above results it should be confirmed that formationof primary oxidation species peroxides was quite similarwith the secondary oxidation products and the changes ofboth oxidation characteristics are in a good correlation
74 Antioxidant Activity in Linoleic Acid System The FTCmethod was used to measure the amount of peroxides at
BioMed Research International 7
the primary stage of linoleic acid peroxidation (Figure 6)Since the concentration of the peroxide decreases as theantioxidant activity increases the intensity of the pigmentwill be reduced leading to lower absorbance Absorbancevalues of the control as well as black cumin oil and itsoleoresins increase until day 10 and then decreased on day12 due to the malondialdehyde formation from linoleic acidoxidation There was a significant difference (119875 lt 005)between the control and the tested essential oil and oleoresinsAs can be seen in Figure 6 both black cumin oil andoleoresins showed good antioxidative property in the linoleicacid system and were significantly (119875 lt 005) different fromthe control
Antioxidant activities of essential oils and oleoresinsmay be related to the diverse compounds present in themincluding terpenes sesquiterpenes and phenolic acids whichact in various ways such as inhibition of peroxidation scav-enging the radicals and chelating the metal ions The mainconstituents of black cumin oil were thymoquinone (376)and p-cymene (314) with minor amounts of longifolenecarvacrol and thymohydroquinone which were responsiblefor the antioxidant activity of black cumin oil [8 9 19 28ndash31] Furthermore free radical scavenging effects of thesecomponentswere studied on the reactions generating reactiveoxygen species such as superoxide anion radical hydroxylradical using spectrophotometric methods [5] The resultsobtained using different assays were well correlated withthe previous work reported be many workers [5 30] whofound thymoquinone as a main constituent responsible forthe activity It has been suggested that phenolic content iscorrelated with the antioxidant activity [32] It is consideredthat the antioxidant activity of phenolic compounds is dueto their high redox potentials which allow them to actas reducing agents hydrogen donors Thymoquinone wasalso present in small amount with higher percentage ofunsaturated fatty acid linoleic acid in different oleoresinsStudies [33] have shown that these unsaturated fatty acidshave anti-rather than pro-oxidant activity but still researchhas been going on for the exact role of unsaturated fatty acidsagainst the oxidative stress
75 Antimicrobial Investigations Using inverted petri platetechnique (Table 3) the volatile oil exhibited more than 90zone inhibition for F moniliforme and P viridicatum It wasalso found to be highly effective in controlling the growthof Aspergillus species and F graminearum where (50) and(65) zone inhibition was observed respectively For othertested fungi the essential oil exerted less activity Howeverusing the same method the oleoresins have revealed lessactivity except for F moniliforme in which only up to 40mycelial zone inhibition was obtained Moreover using foodpoison technique (Table 4) the volatile oil showed clear zoneof growth inhibition against F graminearum at 10 120583L Thevolatile oil showed strong antifungal activity against all testedAspergillus species in the food poison method Ethyl acetateand ethanol oleoresin showed up to 30 zone inhibitionat 10 120583L dose The n-hexane oleoresin showed very feeble
inhibition zone in both techniques The lower antimicrobialefficacy of the oleoresins is due to their low volatility [34]
The antibacterial investigations were undertaken usingagar well diffusion method (Table 4) Using this method theblack cumin oil has shown better activity than oleoresinsand commercial bactericide that is ampicillin The volatileoil was found to be highly effective against B subtilis Bcereus and S aureus and showed complete zone of inhibitionat 3000 ppm concentration whereas in oleoresins more than20mm inhibition was obtained for Gram-positive bacteriaIn addition more than 20 and 25mm zone inhibition wasobtained for P aeruginosa and E coli The results obtainedusing agar well diffusion method were well correlated withthe earlier reported work [6 30 35] where black cumin seedoil has been shown to be effective against a wide spectrumof organisms bacteria like B cereus B subtilis S aureus Sepidermis E coli and P aeruginosa
The results obtained in antimicrobial investigations ofblack cumin oil and oleoresins were in good agreementwith the previous reported work [36] Thymoquinone p-cymene (monoterpene) longifolene (sesquiterpene) andthymohydroquinone were responsible for strong antimicro-bial activity of black cumin oil [29] El Alfy et al [35]isolated thymohydroquinone as antimicrobial compoundfrom the volatile oil of Nigella sativa seeds Oleoresins havehigh concentration of unsaturated fatty acids along withthymohydroquinone in small amount which is responsiblefor its moderate antimicrobial effects Long chain fatty acidslike linoleic acid and oleic acid were previously reportedto possess antibacterial and antifungal activity [36ndash39] p-Cymene is not an efficient antimicrobial compound whenused alone but it potentiate the activity of compounds likecarvacrol [40] The antimicrobial activity of essential oils canoften be correlated to its content of phenolic constituentsThe type of bacteria also has an influence on the effectivenessof the volatile oil and oleoresins Gram-negative bacteriawere generally less susceptible than Gram-positive bacteria[41] The difference in the susceptibility of the bacteria arisesas a result of differences in their cell membrane structurewhich is more complex in case of Gram-negative bacteriaThe antimicrobial activity of a given essential oil may dependon only one or two of the major constituents that make upthe oil However increasing amounts of evidence indicatethat the inherent activity of essential oils may not only relyexclusively on the ratio in which the main active constituentsare present but also on interactions between these andminorconstituents in the oils and oleoresins
8 Conclusions
Seeds of black cumin seem to possess magical propertiesand have been worked out extensively This study revealedthat black cumin essential oil and its oleoresins constitutea good alternative source of essential fatty acids comparedwith common vegetable oil The present results showedthat essential oil and oleoresins of black cumin exhibitedhigher antioxidant activity than synthetic antioxidantsThesefindings could be used to prepare multipurpose products forpharmaceutical applications and its usage as dietary source of
8 BioMed Research International
Table 3 (a) Antifungal investigations of black cumin oil and its oleoresins ( zone inhibitiona) using inverted petriplate method (b)Antifungal investigations of black cumin oil and its oleoresins ( zone inhibition) using food poisoned method
(a)
Mycelial zone inhibition at different dosesa of sample ()Samples Doses (120583L) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
(b)
Mycelial zone inhibitiona at different doses of sample ()Samples Doses (ppm) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
Table 4 Antibacterial activity of black cumin oil and its oleoresinsagainst a few bacterial species using agar well diffusion method
Diameter of inhibition zone (mma)Samples Doses (ppmb) BS BC SA EC PA
Black cumin oil 1000 ++ 253 plusmn 14 ++ minus 189 plusmn 017
3000 ++ ++ ++ 203 plusmn 014 279 plusmn 015
Ethanol oleoresin 1000 164 plusmn 081 minus 156 plusmn 036 minus minus
3000 283 plusmn 020 minus 259 plusmn 042 minus minus
n-Hexane oleoresin 1000 115 plusmn 031 minus 91 plusmn 11 minus minus
3000 209 plusmn 19 minus 134 plusmn 26 minus minus
Ethyl acetate oleoresin1000 223 plusmn 08 minus 177 plusmn 23 minus minus
3000 443 plusmn 07 minus 409 plusmn 12 minus minus
Ampicillin 1000 156 plusmn 032 minus 91 plusmn 11 minus minus
3000 132 plusmn 02 minus 134 plusmn 26 minus minus
aAverage of three replicates ++ indicates complete inhibition and minus indicates no inhibitionbDMSO was used as solventBS Bacillus subtilis SA Staphylococcus aureus BC Bacillus cereus EC Escherichia coli PA Pseudomonas aeruginosa
BioMed Research International 9
antioxidant should be considered largely for alleviating andameliorating diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to theHead of Department of Chem-istry DDU Gorakhpur University Gorakhpur for providinglaboratory facilities The financial support from UGC toSunita Singh (JRF) and Emeritus Fellow to Dr Gurdip Singhis also acknowledged
References
[1] G Zengin A Aktumsek G O Guler Y S Cakmak andE Yildiztugay ldquoAntioxidant properties of methanolic extractand fatty acid composition of Centaurea urvillei DC subsphayekiana Wagenitzrdquo Records of Natural Products vol 5 no 2pp 123ndash132 2011
[2] H Lutterodt M Luther M Slavin et al ldquoFatty acid profilethymoquinone content oxidative stability and antioxidantproperties of cold-pressed black cumin seed oilsrdquo Food Scienceand Technology vol 43 no 9 pp 1409ndash1413 2010
[3] S Shah and K S Ray ldquoStudy on antioxidant and antimicrobialproperties of black cumin (Nigella sativa Linn)rdquo Journal of FoodScience and Technology vol 40 no 1 pp 70ndash73 2003
[4] I Tekeoglu A Dogan and L Demiralp ldquoEffects of thymo-quinone (volatile oil of black cumin) on rheumatoid arthritis inrat modelsrdquo Phytotherapy Research vol 20 no 10 pp 869ndash8712006
[5] I Kruk TMichalska K Lichszteld A Kladna andH Y Aboul-Enein ldquoThe effect of thymol and its derivatives on reactionsgenerating reactive oxygen speciesrdquo Chemosphere vol 41 no7 pp 1059ndash1064 2000
[6] G Singh P Marimuthu C S De Heluani and C Cata-lan ldquoChemical constituents and antimicrobial and antioxidantpotentials of essential oil and acetone extract of Nigella sativaseedsrdquo Journal of the Science of Food and Agriculture vol 85no 13 pp 2297ndash2306 2005
[7] S A Maisonneuve European Pharmacopoeia Part 1 Sainte-Ruffine 1983
[8] M Burits and F Bucar ldquoAntioxidant activity of Nigella sati-vaessential oilrdquo Phytotherapy Research vol 14 pp 323ndash3282000
[9] N Ilaiyaraja and F Khanum ldquoNigella sativa L a review of thera-peutic applicationsrdquo Journal of Herbal Medicine and Toxicologyvol 4 no 2 pp 1ndash8 2010
[10] E A Decker and B Welch ldquoRole of ferritin as a lipid oxidationcatalyst in muscle foodrdquo Journal of Agricultural and FoodChemistry vol 38 no 3 pp 674ndash677 1990
[11] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo Food Scienceand Technology vol 28 no 1 pp 25ndash30 1995
[12] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic as it reagentsrdquo TheAmerican Journal of Enology and Viticulture vol 16 pp 144ndash158 1965
[13] K D Economou V Oreopoulou and C D ThomopoulosldquoAntioxidant activity of some plant extracts of the familylabiataerdquo Journal of the American Oil Chemists Society vol 68no 2 pp 109ndash113 1991
[14] G Singh S Maurya M P de Lampasona and C A N CatalanldquoA comparison of chemical antioxidant and antimicrobialstudies of cinnamon leaf and bark volatile oils oleoresins andtheir constituentsrdquo Food and Chemical Toxicology vol 45 no 9pp 1650ndash1661 2007
[15] G Singh I P S Kapoor P Singh C S de Heluani M P deLampasona and C A N Catalan ldquoChemistry antioxidant andantimicrobial investigations on essential oil and oleoresins ofZingiber officinalerdquo Food and Chemical Toxicology vol 46 no10 pp 3295ndash3302 2008
[16] G P Rao and A K Shrivastava ldquoToxicity of essential oils ofhigher plants against fungal pathogens of sugarcanerdquo inCurrentTrend in Sugarcane Pathology G P Rao A G Gillasple PP Upadhaya A Bergamin V P Agnihotri and C T ChenEds pp 347ndash365 International Books and Periodicals SupplyService Delhi India 1994
[17] K Ramadas G Suresh N Janarthanan and S Masila-mani ldquoAntifungal activity of 13-disubstituted symmetrical andunsymmetrical thioureasrdquo Pesticide Science vol 52 no 2 pp145ndash151 1998
[18] NCCLS (National Committee for Clinical Laboratory Stan-dards) Performance Standards for Antimicrobial Disc Suscepti-bility Test 6th edition 1997
[19] V Hajhashemi A Ghannadi and H Jafarabadi ldquoBlack cuminseed essential oil as a potent analgesic and antiinflammatorydrugrdquo Phytotherapy Research vol 18 no 3 pp 195ndash199 2004
[20] P J Houghton R Zarka B De Las Heras and J R SHoult ldquoFixed oil of Nigella sativa and derived thymoquinoneinhibit eicosanoid generation in leukocytes andmembrane lipidperoxidationrdquo Planta Medica vol 61 no 1 pp 33ndash36 1995
[21] M T SultanM S Butt FMAnjumA Jamil S Akhtar andMNasir ldquoNutritional profile of indigenous cultivar of black cuminseeds and antioxidant potential of its fixed and essential oilrdquoPakistan Journal of Botany vol 41 no 3 pp 1321ndash1330 2009
[22] S Cheikh-Rouhou S Besbes G Lognay C Blecker CDeroanne and H Attia ldquoSterol composition of black cumin(Nigella sativa L) and Aleppo pine (Pinus halepensisMill) seedoilsrdquo Journal of Food Composition and Analysis vol 21 no 2 pp162ndash168 2008
[23] M F Ramadan ldquoNutritional value functional properties andnutraceutical applications of black cumin (Nigella sativa L) anoverviewrdquo International Journal of Food Science and Technologyvol 42 no 10 pp 1208ndash1218 2007
[24] T D Parker D A Adams K Zhou M Harris and L Yu ldquoFattyacid composition and oxidative stability of cold-pressed edibleseed oilsrdquo Journal of Food Science vol 68 no 4 pp 1240ndash12432003
[25] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAmerican Oil Chemical Society vol 83 pp 847ndash854 2006
[26] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAgricultural and Food Chemistry vol 53 no 3 pp 566ndash5732005
[27] R R Sokal Introduction to Biostatistics W H Freeman SanFrancisco Calif USA 1973
10 BioMed Research International
[28] H Hosseinzadeh S Parvardeh M N Asl H R Sadeghniaand T Ziaee ldquoEffect of thymoquinone and Nigella sativa seedsoil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampusrdquo Phytomedicine vol 14no 9 pp 621ndash627 2007
[29] S Bourgou A Pichette B Marzouk and J Legault ldquoBioactiv-ities of black cumin essential oil and its main terpenes fromTunisiardquo South African Journal of Botany vol 76 no 2 pp 210ndash216 2010
[30] H J Harzallah E Noumi K Bekir et al ldquoChemical composi-tion antibacterial and antifungal properties of Tunisian Nigellasativa fixed oilrdquoAfrican Journal of Microbiology Research vol 6no 22 pp 4675ndash4679 2012
[31] V S Deepa and P S Kumar ldquoPreliminary phytochemicalinvestigations and in vitro antioxidant activity in selected partsof Andrographis spprdquo Journal of Pharmacological Research vol3 no 9 pp 2206ndash2210 2010
[32] M Amensour E Sendra A Jamal S Bouhdid J A Perez-Alvarez and J Fernandez-Lopez ldquoTotal phenolic content andantioxidant activity of myrtle (Myrtus communis) extractsrdquoNatural Product Communications vol 4 no 6 pp 819ndash8242009
[33] M Di Nunzio V Valli and A Bordoni ldquoPro- and anti-oxidanteffects of polyunsaturated fatty acid supplementation in HepG2cellsrdquo Prostaglandins Leukotrienes and Essential Fatty Acids vol85 no 3-4 pp 121ndash127 2011
[34] G Singh SMaurya CCatalan andMP de Lampasona ldquoStud-ies on essential oils part 42 chemical antifungal antioxidantand sprout suppressant studies on ginger essential oil and itsoleoresinrdquo Flavour and Fragrance Journal vol 20 no 1 pp 1ndash62005
[35] T S El Alfy H M El Fatatry and M A Toama ldquoIsolationand structure assignment of an antimicrobial principle from thevolatile oil of Nigella sativa L seedsrdquo Pharmazie vol 30 no 2pp 109ndash111 1975
[36] M A Khan ldquoChemical composition and medicinal propertiesof Nigella sativa Linnrdquo Inflammopharmacology vol 7 no 1 pp15ndash35 1999
[37] L J McGaw A K Jager and J van Staden ldquoIsolation ofantibacterial fatty acids from Schotia brachypetalardquo Fitoterapiavol 73 no 5 pp 431ndash433 2002
[38] A R McCutcheon T E Roberts E Gibbons et al ldquoAntiviralscreening of British Columbian medicinal plantsrdquo Journal ofEthnopharmacology vol 49 no 2 pp 101ndash110 1995
[39] S Javed A A Shahid M S Haider et al ldquoNutritionalphytochemical potential and pharmacological evaluation ofNigella Sativa (Kalonji) and Trachyspermum Ammi (Ajwain)rdquoMedicinal Plants Research vol 6 no 5 pp 768ndash775 2012
[40] P Rattanachaikunsopon and P Phumkhachorn ldquoAssessmentof factors influencing antimicrobial activity of carvacrol andcymene against Vibrio cholerae in foodrdquo Journal of Bioscienceand Bioengineering vol 110 no 5 pp 614ndash619 2010
[41] M Gilles J Zhao M An and S Agboola ldquoChemical com-position and antimicrobial properties of essential oils of threeAustralian Eucalyptus speciesrdquo Food Chemistry vol 119 no 2pp 731ndash737 2010
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
the primary stage of linoleic acid peroxidation (Figure 6)Since the concentration of the peroxide decreases as theantioxidant activity increases the intensity of the pigmentwill be reduced leading to lower absorbance Absorbancevalues of the control as well as black cumin oil and itsoleoresins increase until day 10 and then decreased on day12 due to the malondialdehyde formation from linoleic acidoxidation There was a significant difference (119875 lt 005)between the control and the tested essential oil and oleoresinsAs can be seen in Figure 6 both black cumin oil andoleoresins showed good antioxidative property in the linoleicacid system and were significantly (119875 lt 005) different fromthe control
Antioxidant activities of essential oils and oleoresinsmay be related to the diverse compounds present in themincluding terpenes sesquiterpenes and phenolic acids whichact in various ways such as inhibition of peroxidation scav-enging the radicals and chelating the metal ions The mainconstituents of black cumin oil were thymoquinone (376)and p-cymene (314) with minor amounts of longifolenecarvacrol and thymohydroquinone which were responsiblefor the antioxidant activity of black cumin oil [8 9 19 28ndash31] Furthermore free radical scavenging effects of thesecomponentswere studied on the reactions generating reactiveoxygen species such as superoxide anion radical hydroxylradical using spectrophotometric methods [5] The resultsobtained using different assays were well correlated withthe previous work reported be many workers [5 30] whofound thymoquinone as a main constituent responsible forthe activity It has been suggested that phenolic content iscorrelated with the antioxidant activity [32] It is consideredthat the antioxidant activity of phenolic compounds is dueto their high redox potentials which allow them to actas reducing agents hydrogen donors Thymoquinone wasalso present in small amount with higher percentage ofunsaturated fatty acid linoleic acid in different oleoresinsStudies [33] have shown that these unsaturated fatty acidshave anti-rather than pro-oxidant activity but still researchhas been going on for the exact role of unsaturated fatty acidsagainst the oxidative stress
75 Antimicrobial Investigations Using inverted petri platetechnique (Table 3) the volatile oil exhibited more than 90zone inhibition for F moniliforme and P viridicatum It wasalso found to be highly effective in controlling the growthof Aspergillus species and F graminearum where (50) and(65) zone inhibition was observed respectively For othertested fungi the essential oil exerted less activity Howeverusing the same method the oleoresins have revealed lessactivity except for F moniliforme in which only up to 40mycelial zone inhibition was obtained Moreover using foodpoison technique (Table 4) the volatile oil showed clear zoneof growth inhibition against F graminearum at 10 120583L Thevolatile oil showed strong antifungal activity against all testedAspergillus species in the food poison method Ethyl acetateand ethanol oleoresin showed up to 30 zone inhibitionat 10 120583L dose The n-hexane oleoresin showed very feeble
inhibition zone in both techniques The lower antimicrobialefficacy of the oleoresins is due to their low volatility [34]
The antibacterial investigations were undertaken usingagar well diffusion method (Table 4) Using this method theblack cumin oil has shown better activity than oleoresinsand commercial bactericide that is ampicillin The volatileoil was found to be highly effective against B subtilis Bcereus and S aureus and showed complete zone of inhibitionat 3000 ppm concentration whereas in oleoresins more than20mm inhibition was obtained for Gram-positive bacteriaIn addition more than 20 and 25mm zone inhibition wasobtained for P aeruginosa and E coli The results obtainedusing agar well diffusion method were well correlated withthe earlier reported work [6 30 35] where black cumin seedoil has been shown to be effective against a wide spectrumof organisms bacteria like B cereus B subtilis S aureus Sepidermis E coli and P aeruginosa
The results obtained in antimicrobial investigations ofblack cumin oil and oleoresins were in good agreementwith the previous reported work [36] Thymoquinone p-cymene (monoterpene) longifolene (sesquiterpene) andthymohydroquinone were responsible for strong antimicro-bial activity of black cumin oil [29] El Alfy et al [35]isolated thymohydroquinone as antimicrobial compoundfrom the volatile oil of Nigella sativa seeds Oleoresins havehigh concentration of unsaturated fatty acids along withthymohydroquinone in small amount which is responsiblefor its moderate antimicrobial effects Long chain fatty acidslike linoleic acid and oleic acid were previously reportedto possess antibacterial and antifungal activity [36ndash39] p-Cymene is not an efficient antimicrobial compound whenused alone but it potentiate the activity of compounds likecarvacrol [40] The antimicrobial activity of essential oils canoften be correlated to its content of phenolic constituentsThe type of bacteria also has an influence on the effectivenessof the volatile oil and oleoresins Gram-negative bacteriawere generally less susceptible than Gram-positive bacteria[41] The difference in the susceptibility of the bacteria arisesas a result of differences in their cell membrane structurewhich is more complex in case of Gram-negative bacteriaThe antimicrobial activity of a given essential oil may dependon only one or two of the major constituents that make upthe oil However increasing amounts of evidence indicatethat the inherent activity of essential oils may not only relyexclusively on the ratio in which the main active constituentsare present but also on interactions between these andminorconstituents in the oils and oleoresins
8 Conclusions
Seeds of black cumin seem to possess magical propertiesand have been worked out extensively This study revealedthat black cumin essential oil and its oleoresins constitutea good alternative source of essential fatty acids comparedwith common vegetable oil The present results showedthat essential oil and oleoresins of black cumin exhibitedhigher antioxidant activity than synthetic antioxidantsThesefindings could be used to prepare multipurpose products forpharmaceutical applications and its usage as dietary source of
8 BioMed Research International
Table 3 (a) Antifungal investigations of black cumin oil and its oleoresins ( zone inhibitiona) using inverted petriplate method (b)Antifungal investigations of black cumin oil and its oleoresins ( zone inhibition) using food poisoned method
(a)
Mycelial zone inhibition at different dosesa of sample ()Samples Doses (120583L) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
(b)
Mycelial zone inhibitiona at different doses of sample ()Samples Doses (ppm) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
Table 4 Antibacterial activity of black cumin oil and its oleoresinsagainst a few bacterial species using agar well diffusion method
Diameter of inhibition zone (mma)Samples Doses (ppmb) BS BC SA EC PA
Black cumin oil 1000 ++ 253 plusmn 14 ++ minus 189 plusmn 017
3000 ++ ++ ++ 203 plusmn 014 279 plusmn 015
Ethanol oleoresin 1000 164 plusmn 081 minus 156 plusmn 036 minus minus
3000 283 plusmn 020 minus 259 plusmn 042 minus minus
n-Hexane oleoresin 1000 115 plusmn 031 minus 91 plusmn 11 minus minus
3000 209 plusmn 19 minus 134 plusmn 26 minus minus
Ethyl acetate oleoresin1000 223 plusmn 08 minus 177 plusmn 23 minus minus
3000 443 plusmn 07 minus 409 plusmn 12 minus minus
Ampicillin 1000 156 plusmn 032 minus 91 plusmn 11 minus minus
3000 132 plusmn 02 minus 134 plusmn 26 minus minus
aAverage of three replicates ++ indicates complete inhibition and minus indicates no inhibitionbDMSO was used as solventBS Bacillus subtilis SA Staphylococcus aureus BC Bacillus cereus EC Escherichia coli PA Pseudomonas aeruginosa
BioMed Research International 9
antioxidant should be considered largely for alleviating andameliorating diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to theHead of Department of Chem-istry DDU Gorakhpur University Gorakhpur for providinglaboratory facilities The financial support from UGC toSunita Singh (JRF) and Emeritus Fellow to Dr Gurdip Singhis also acknowledged
References
[1] G Zengin A Aktumsek G O Guler Y S Cakmak andE Yildiztugay ldquoAntioxidant properties of methanolic extractand fatty acid composition of Centaurea urvillei DC subsphayekiana Wagenitzrdquo Records of Natural Products vol 5 no 2pp 123ndash132 2011
[2] H Lutterodt M Luther M Slavin et al ldquoFatty acid profilethymoquinone content oxidative stability and antioxidantproperties of cold-pressed black cumin seed oilsrdquo Food Scienceand Technology vol 43 no 9 pp 1409ndash1413 2010
[3] S Shah and K S Ray ldquoStudy on antioxidant and antimicrobialproperties of black cumin (Nigella sativa Linn)rdquo Journal of FoodScience and Technology vol 40 no 1 pp 70ndash73 2003
[4] I Tekeoglu A Dogan and L Demiralp ldquoEffects of thymo-quinone (volatile oil of black cumin) on rheumatoid arthritis inrat modelsrdquo Phytotherapy Research vol 20 no 10 pp 869ndash8712006
[5] I Kruk TMichalska K Lichszteld A Kladna andH Y Aboul-Enein ldquoThe effect of thymol and its derivatives on reactionsgenerating reactive oxygen speciesrdquo Chemosphere vol 41 no7 pp 1059ndash1064 2000
[6] G Singh P Marimuthu C S De Heluani and C Cata-lan ldquoChemical constituents and antimicrobial and antioxidantpotentials of essential oil and acetone extract of Nigella sativaseedsrdquo Journal of the Science of Food and Agriculture vol 85no 13 pp 2297ndash2306 2005
[7] S A Maisonneuve European Pharmacopoeia Part 1 Sainte-Ruffine 1983
[8] M Burits and F Bucar ldquoAntioxidant activity of Nigella sati-vaessential oilrdquo Phytotherapy Research vol 14 pp 323ndash3282000
[9] N Ilaiyaraja and F Khanum ldquoNigella sativa L a review of thera-peutic applicationsrdquo Journal of Herbal Medicine and Toxicologyvol 4 no 2 pp 1ndash8 2010
[10] E A Decker and B Welch ldquoRole of ferritin as a lipid oxidationcatalyst in muscle foodrdquo Journal of Agricultural and FoodChemistry vol 38 no 3 pp 674ndash677 1990
[11] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo Food Scienceand Technology vol 28 no 1 pp 25ndash30 1995
[12] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic as it reagentsrdquo TheAmerican Journal of Enology and Viticulture vol 16 pp 144ndash158 1965
[13] K D Economou V Oreopoulou and C D ThomopoulosldquoAntioxidant activity of some plant extracts of the familylabiataerdquo Journal of the American Oil Chemists Society vol 68no 2 pp 109ndash113 1991
[14] G Singh S Maurya M P de Lampasona and C A N CatalanldquoA comparison of chemical antioxidant and antimicrobialstudies of cinnamon leaf and bark volatile oils oleoresins andtheir constituentsrdquo Food and Chemical Toxicology vol 45 no 9pp 1650ndash1661 2007
[15] G Singh I P S Kapoor P Singh C S de Heluani M P deLampasona and C A N Catalan ldquoChemistry antioxidant andantimicrobial investigations on essential oil and oleoresins ofZingiber officinalerdquo Food and Chemical Toxicology vol 46 no10 pp 3295ndash3302 2008
[16] G P Rao and A K Shrivastava ldquoToxicity of essential oils ofhigher plants against fungal pathogens of sugarcanerdquo inCurrentTrend in Sugarcane Pathology G P Rao A G Gillasple PP Upadhaya A Bergamin V P Agnihotri and C T ChenEds pp 347ndash365 International Books and Periodicals SupplyService Delhi India 1994
[17] K Ramadas G Suresh N Janarthanan and S Masila-mani ldquoAntifungal activity of 13-disubstituted symmetrical andunsymmetrical thioureasrdquo Pesticide Science vol 52 no 2 pp145ndash151 1998
[18] NCCLS (National Committee for Clinical Laboratory Stan-dards) Performance Standards for Antimicrobial Disc Suscepti-bility Test 6th edition 1997
[19] V Hajhashemi A Ghannadi and H Jafarabadi ldquoBlack cuminseed essential oil as a potent analgesic and antiinflammatorydrugrdquo Phytotherapy Research vol 18 no 3 pp 195ndash199 2004
[20] P J Houghton R Zarka B De Las Heras and J R SHoult ldquoFixed oil of Nigella sativa and derived thymoquinoneinhibit eicosanoid generation in leukocytes andmembrane lipidperoxidationrdquo Planta Medica vol 61 no 1 pp 33ndash36 1995
[21] M T SultanM S Butt FMAnjumA Jamil S Akhtar andMNasir ldquoNutritional profile of indigenous cultivar of black cuminseeds and antioxidant potential of its fixed and essential oilrdquoPakistan Journal of Botany vol 41 no 3 pp 1321ndash1330 2009
[22] S Cheikh-Rouhou S Besbes G Lognay C Blecker CDeroanne and H Attia ldquoSterol composition of black cumin(Nigella sativa L) and Aleppo pine (Pinus halepensisMill) seedoilsrdquo Journal of Food Composition and Analysis vol 21 no 2 pp162ndash168 2008
[23] M F Ramadan ldquoNutritional value functional properties andnutraceutical applications of black cumin (Nigella sativa L) anoverviewrdquo International Journal of Food Science and Technologyvol 42 no 10 pp 1208ndash1218 2007
[24] T D Parker D A Adams K Zhou M Harris and L Yu ldquoFattyacid composition and oxidative stability of cold-pressed edibleseed oilsrdquo Journal of Food Science vol 68 no 4 pp 1240ndash12432003
[25] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAmerican Oil Chemical Society vol 83 pp 847ndash854 2006
[26] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAgricultural and Food Chemistry vol 53 no 3 pp 566ndash5732005
[27] R R Sokal Introduction to Biostatistics W H Freeman SanFrancisco Calif USA 1973
10 BioMed Research International
[28] H Hosseinzadeh S Parvardeh M N Asl H R Sadeghniaand T Ziaee ldquoEffect of thymoquinone and Nigella sativa seedsoil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampusrdquo Phytomedicine vol 14no 9 pp 621ndash627 2007
[29] S Bourgou A Pichette B Marzouk and J Legault ldquoBioactiv-ities of black cumin essential oil and its main terpenes fromTunisiardquo South African Journal of Botany vol 76 no 2 pp 210ndash216 2010
[30] H J Harzallah E Noumi K Bekir et al ldquoChemical composi-tion antibacterial and antifungal properties of Tunisian Nigellasativa fixed oilrdquoAfrican Journal of Microbiology Research vol 6no 22 pp 4675ndash4679 2012
[31] V S Deepa and P S Kumar ldquoPreliminary phytochemicalinvestigations and in vitro antioxidant activity in selected partsof Andrographis spprdquo Journal of Pharmacological Research vol3 no 9 pp 2206ndash2210 2010
[32] M Amensour E Sendra A Jamal S Bouhdid J A Perez-Alvarez and J Fernandez-Lopez ldquoTotal phenolic content andantioxidant activity of myrtle (Myrtus communis) extractsrdquoNatural Product Communications vol 4 no 6 pp 819ndash8242009
[33] M Di Nunzio V Valli and A Bordoni ldquoPro- and anti-oxidanteffects of polyunsaturated fatty acid supplementation in HepG2cellsrdquo Prostaglandins Leukotrienes and Essential Fatty Acids vol85 no 3-4 pp 121ndash127 2011
[34] G Singh SMaurya CCatalan andMP de Lampasona ldquoStud-ies on essential oils part 42 chemical antifungal antioxidantand sprout suppressant studies on ginger essential oil and itsoleoresinrdquo Flavour and Fragrance Journal vol 20 no 1 pp 1ndash62005
[35] T S El Alfy H M El Fatatry and M A Toama ldquoIsolationand structure assignment of an antimicrobial principle from thevolatile oil of Nigella sativa L seedsrdquo Pharmazie vol 30 no 2pp 109ndash111 1975
[36] M A Khan ldquoChemical composition and medicinal propertiesof Nigella sativa Linnrdquo Inflammopharmacology vol 7 no 1 pp15ndash35 1999
[37] L J McGaw A K Jager and J van Staden ldquoIsolation ofantibacterial fatty acids from Schotia brachypetalardquo Fitoterapiavol 73 no 5 pp 431ndash433 2002
[38] A R McCutcheon T E Roberts E Gibbons et al ldquoAntiviralscreening of British Columbian medicinal plantsrdquo Journal ofEthnopharmacology vol 49 no 2 pp 101ndash110 1995
[39] S Javed A A Shahid M S Haider et al ldquoNutritionalphytochemical potential and pharmacological evaluation ofNigella Sativa (Kalonji) and Trachyspermum Ammi (Ajwain)rdquoMedicinal Plants Research vol 6 no 5 pp 768ndash775 2012
[40] P Rattanachaikunsopon and P Phumkhachorn ldquoAssessmentof factors influencing antimicrobial activity of carvacrol andcymene against Vibrio cholerae in foodrdquo Journal of Bioscienceand Bioengineering vol 110 no 5 pp 614ndash619 2010
[41] M Gilles J Zhao M An and S Agboola ldquoChemical com-position and antimicrobial properties of essential oils of threeAustralian Eucalyptus speciesrdquo Food Chemistry vol 119 no 2pp 731ndash737 2010
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
Table 3 (a) Antifungal investigations of black cumin oil and its oleoresins ( zone inhibitiona) using inverted petriplate method (b)Antifungal investigations of black cumin oil and its oleoresins ( zone inhibition) using food poisoned method
(a)
Mycelial zone inhibition at different dosesa of sample ()Samples Doses (120583L) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
(b)
Mycelial zone inhibitiona at different doses of sample ()Samples Doses (ppm) AN AF FM FG PV
aAverage of three replicatesAN Aspergillus niger AF Aspergillus flavus FM Fusarium moniliforme FG Fusarium graminearum and PV Penicillium viridicatum(minus) no inhibition
Table 4 Antibacterial activity of black cumin oil and its oleoresinsagainst a few bacterial species using agar well diffusion method
Diameter of inhibition zone (mma)Samples Doses (ppmb) BS BC SA EC PA
Black cumin oil 1000 ++ 253 plusmn 14 ++ minus 189 plusmn 017
3000 ++ ++ ++ 203 plusmn 014 279 plusmn 015
Ethanol oleoresin 1000 164 plusmn 081 minus 156 plusmn 036 minus minus
3000 283 plusmn 020 minus 259 plusmn 042 minus minus
n-Hexane oleoresin 1000 115 plusmn 031 minus 91 plusmn 11 minus minus
3000 209 plusmn 19 minus 134 plusmn 26 minus minus
Ethyl acetate oleoresin1000 223 plusmn 08 minus 177 plusmn 23 minus minus
3000 443 plusmn 07 minus 409 plusmn 12 minus minus
Ampicillin 1000 156 plusmn 032 minus 91 plusmn 11 minus minus
3000 132 plusmn 02 minus 134 plusmn 26 minus minus
aAverage of three replicates ++ indicates complete inhibition and minus indicates no inhibitionbDMSO was used as solventBS Bacillus subtilis SA Staphylococcus aureus BC Bacillus cereus EC Escherichia coli PA Pseudomonas aeruginosa
BioMed Research International 9
antioxidant should be considered largely for alleviating andameliorating diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to theHead of Department of Chem-istry DDU Gorakhpur University Gorakhpur for providinglaboratory facilities The financial support from UGC toSunita Singh (JRF) and Emeritus Fellow to Dr Gurdip Singhis also acknowledged
References
[1] G Zengin A Aktumsek G O Guler Y S Cakmak andE Yildiztugay ldquoAntioxidant properties of methanolic extractand fatty acid composition of Centaurea urvillei DC subsphayekiana Wagenitzrdquo Records of Natural Products vol 5 no 2pp 123ndash132 2011
[2] H Lutterodt M Luther M Slavin et al ldquoFatty acid profilethymoquinone content oxidative stability and antioxidantproperties of cold-pressed black cumin seed oilsrdquo Food Scienceand Technology vol 43 no 9 pp 1409ndash1413 2010
[3] S Shah and K S Ray ldquoStudy on antioxidant and antimicrobialproperties of black cumin (Nigella sativa Linn)rdquo Journal of FoodScience and Technology vol 40 no 1 pp 70ndash73 2003
[4] I Tekeoglu A Dogan and L Demiralp ldquoEffects of thymo-quinone (volatile oil of black cumin) on rheumatoid arthritis inrat modelsrdquo Phytotherapy Research vol 20 no 10 pp 869ndash8712006
[5] I Kruk TMichalska K Lichszteld A Kladna andH Y Aboul-Enein ldquoThe effect of thymol and its derivatives on reactionsgenerating reactive oxygen speciesrdquo Chemosphere vol 41 no7 pp 1059ndash1064 2000
[6] G Singh P Marimuthu C S De Heluani and C Cata-lan ldquoChemical constituents and antimicrobial and antioxidantpotentials of essential oil and acetone extract of Nigella sativaseedsrdquo Journal of the Science of Food and Agriculture vol 85no 13 pp 2297ndash2306 2005
[7] S A Maisonneuve European Pharmacopoeia Part 1 Sainte-Ruffine 1983
[8] M Burits and F Bucar ldquoAntioxidant activity of Nigella sati-vaessential oilrdquo Phytotherapy Research vol 14 pp 323ndash3282000
[9] N Ilaiyaraja and F Khanum ldquoNigella sativa L a review of thera-peutic applicationsrdquo Journal of Herbal Medicine and Toxicologyvol 4 no 2 pp 1ndash8 2010
[10] E A Decker and B Welch ldquoRole of ferritin as a lipid oxidationcatalyst in muscle foodrdquo Journal of Agricultural and FoodChemistry vol 38 no 3 pp 674ndash677 1990
[11] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo Food Scienceand Technology vol 28 no 1 pp 25ndash30 1995
[12] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic as it reagentsrdquo TheAmerican Journal of Enology and Viticulture vol 16 pp 144ndash158 1965
[13] K D Economou V Oreopoulou and C D ThomopoulosldquoAntioxidant activity of some plant extracts of the familylabiataerdquo Journal of the American Oil Chemists Society vol 68no 2 pp 109ndash113 1991
[14] G Singh S Maurya M P de Lampasona and C A N CatalanldquoA comparison of chemical antioxidant and antimicrobialstudies of cinnamon leaf and bark volatile oils oleoresins andtheir constituentsrdquo Food and Chemical Toxicology vol 45 no 9pp 1650ndash1661 2007
[15] G Singh I P S Kapoor P Singh C S de Heluani M P deLampasona and C A N Catalan ldquoChemistry antioxidant andantimicrobial investigations on essential oil and oleoresins ofZingiber officinalerdquo Food and Chemical Toxicology vol 46 no10 pp 3295ndash3302 2008
[16] G P Rao and A K Shrivastava ldquoToxicity of essential oils ofhigher plants against fungal pathogens of sugarcanerdquo inCurrentTrend in Sugarcane Pathology G P Rao A G Gillasple PP Upadhaya A Bergamin V P Agnihotri and C T ChenEds pp 347ndash365 International Books and Periodicals SupplyService Delhi India 1994
[17] K Ramadas G Suresh N Janarthanan and S Masila-mani ldquoAntifungal activity of 13-disubstituted symmetrical andunsymmetrical thioureasrdquo Pesticide Science vol 52 no 2 pp145ndash151 1998
[18] NCCLS (National Committee for Clinical Laboratory Stan-dards) Performance Standards for Antimicrobial Disc Suscepti-bility Test 6th edition 1997
[19] V Hajhashemi A Ghannadi and H Jafarabadi ldquoBlack cuminseed essential oil as a potent analgesic and antiinflammatorydrugrdquo Phytotherapy Research vol 18 no 3 pp 195ndash199 2004
[20] P J Houghton R Zarka B De Las Heras and J R SHoult ldquoFixed oil of Nigella sativa and derived thymoquinoneinhibit eicosanoid generation in leukocytes andmembrane lipidperoxidationrdquo Planta Medica vol 61 no 1 pp 33ndash36 1995
[21] M T SultanM S Butt FMAnjumA Jamil S Akhtar andMNasir ldquoNutritional profile of indigenous cultivar of black cuminseeds and antioxidant potential of its fixed and essential oilrdquoPakistan Journal of Botany vol 41 no 3 pp 1321ndash1330 2009
[22] S Cheikh-Rouhou S Besbes G Lognay C Blecker CDeroanne and H Attia ldquoSterol composition of black cumin(Nigella sativa L) and Aleppo pine (Pinus halepensisMill) seedoilsrdquo Journal of Food Composition and Analysis vol 21 no 2 pp162ndash168 2008
[23] M F Ramadan ldquoNutritional value functional properties andnutraceutical applications of black cumin (Nigella sativa L) anoverviewrdquo International Journal of Food Science and Technologyvol 42 no 10 pp 1208ndash1218 2007
[24] T D Parker D A Adams K Zhou M Harris and L Yu ldquoFattyacid composition and oxidative stability of cold-pressed edibleseed oilsrdquo Journal of Food Science vol 68 no 4 pp 1240ndash12432003
[25] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAmerican Oil Chemical Society vol 83 pp 847ndash854 2006
[26] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAgricultural and Food Chemistry vol 53 no 3 pp 566ndash5732005
[27] R R Sokal Introduction to Biostatistics W H Freeman SanFrancisco Calif USA 1973
10 BioMed Research International
[28] H Hosseinzadeh S Parvardeh M N Asl H R Sadeghniaand T Ziaee ldquoEffect of thymoquinone and Nigella sativa seedsoil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampusrdquo Phytomedicine vol 14no 9 pp 621ndash627 2007
[29] S Bourgou A Pichette B Marzouk and J Legault ldquoBioactiv-ities of black cumin essential oil and its main terpenes fromTunisiardquo South African Journal of Botany vol 76 no 2 pp 210ndash216 2010
[30] H J Harzallah E Noumi K Bekir et al ldquoChemical composi-tion antibacterial and antifungal properties of Tunisian Nigellasativa fixed oilrdquoAfrican Journal of Microbiology Research vol 6no 22 pp 4675ndash4679 2012
[31] V S Deepa and P S Kumar ldquoPreliminary phytochemicalinvestigations and in vitro antioxidant activity in selected partsof Andrographis spprdquo Journal of Pharmacological Research vol3 no 9 pp 2206ndash2210 2010
[32] M Amensour E Sendra A Jamal S Bouhdid J A Perez-Alvarez and J Fernandez-Lopez ldquoTotal phenolic content andantioxidant activity of myrtle (Myrtus communis) extractsrdquoNatural Product Communications vol 4 no 6 pp 819ndash8242009
[33] M Di Nunzio V Valli and A Bordoni ldquoPro- and anti-oxidanteffects of polyunsaturated fatty acid supplementation in HepG2cellsrdquo Prostaglandins Leukotrienes and Essential Fatty Acids vol85 no 3-4 pp 121ndash127 2011
[34] G Singh SMaurya CCatalan andMP de Lampasona ldquoStud-ies on essential oils part 42 chemical antifungal antioxidantand sprout suppressant studies on ginger essential oil and itsoleoresinrdquo Flavour and Fragrance Journal vol 20 no 1 pp 1ndash62005
[35] T S El Alfy H M El Fatatry and M A Toama ldquoIsolationand structure assignment of an antimicrobial principle from thevolatile oil of Nigella sativa L seedsrdquo Pharmazie vol 30 no 2pp 109ndash111 1975
[36] M A Khan ldquoChemical composition and medicinal propertiesof Nigella sativa Linnrdquo Inflammopharmacology vol 7 no 1 pp15ndash35 1999
[37] L J McGaw A K Jager and J van Staden ldquoIsolation ofantibacterial fatty acids from Schotia brachypetalardquo Fitoterapiavol 73 no 5 pp 431ndash433 2002
[38] A R McCutcheon T E Roberts E Gibbons et al ldquoAntiviralscreening of British Columbian medicinal plantsrdquo Journal ofEthnopharmacology vol 49 no 2 pp 101ndash110 1995
[39] S Javed A A Shahid M S Haider et al ldquoNutritionalphytochemical potential and pharmacological evaluation ofNigella Sativa (Kalonji) and Trachyspermum Ammi (Ajwain)rdquoMedicinal Plants Research vol 6 no 5 pp 768ndash775 2012
[40] P Rattanachaikunsopon and P Phumkhachorn ldquoAssessmentof factors influencing antimicrobial activity of carvacrol andcymene against Vibrio cholerae in foodrdquo Journal of Bioscienceand Bioengineering vol 110 no 5 pp 614ndash619 2010
[41] M Gilles J Zhao M An and S Agboola ldquoChemical com-position and antimicrobial properties of essential oils of threeAustralian Eucalyptus speciesrdquo Food Chemistry vol 119 no 2pp 731ndash737 2010
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
antioxidant should be considered largely for alleviating andameliorating diseases
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
Acknowledgments
The authors are grateful to theHead of Department of Chem-istry DDU Gorakhpur University Gorakhpur for providinglaboratory facilities The financial support from UGC toSunita Singh (JRF) and Emeritus Fellow to Dr Gurdip Singhis also acknowledged
References
[1] G Zengin A Aktumsek G O Guler Y S Cakmak andE Yildiztugay ldquoAntioxidant properties of methanolic extractand fatty acid composition of Centaurea urvillei DC subsphayekiana Wagenitzrdquo Records of Natural Products vol 5 no 2pp 123ndash132 2011
[2] H Lutterodt M Luther M Slavin et al ldquoFatty acid profilethymoquinone content oxidative stability and antioxidantproperties of cold-pressed black cumin seed oilsrdquo Food Scienceand Technology vol 43 no 9 pp 1409ndash1413 2010
[3] S Shah and K S Ray ldquoStudy on antioxidant and antimicrobialproperties of black cumin (Nigella sativa Linn)rdquo Journal of FoodScience and Technology vol 40 no 1 pp 70ndash73 2003
[4] I Tekeoglu A Dogan and L Demiralp ldquoEffects of thymo-quinone (volatile oil of black cumin) on rheumatoid arthritis inrat modelsrdquo Phytotherapy Research vol 20 no 10 pp 869ndash8712006
[5] I Kruk TMichalska K Lichszteld A Kladna andH Y Aboul-Enein ldquoThe effect of thymol and its derivatives on reactionsgenerating reactive oxygen speciesrdquo Chemosphere vol 41 no7 pp 1059ndash1064 2000
[6] G Singh P Marimuthu C S De Heluani and C Cata-lan ldquoChemical constituents and antimicrobial and antioxidantpotentials of essential oil and acetone extract of Nigella sativaseedsrdquo Journal of the Science of Food and Agriculture vol 85no 13 pp 2297ndash2306 2005
[7] S A Maisonneuve European Pharmacopoeia Part 1 Sainte-Ruffine 1983
[8] M Burits and F Bucar ldquoAntioxidant activity of Nigella sati-vaessential oilrdquo Phytotherapy Research vol 14 pp 323ndash3282000
[9] N Ilaiyaraja and F Khanum ldquoNigella sativa L a review of thera-peutic applicationsrdquo Journal of Herbal Medicine and Toxicologyvol 4 no 2 pp 1ndash8 2010
[10] E A Decker and B Welch ldquoRole of ferritin as a lipid oxidationcatalyst in muscle foodrdquo Journal of Agricultural and FoodChemistry vol 38 no 3 pp 674ndash677 1990
[11] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo Food Scienceand Technology vol 28 no 1 pp 25ndash30 1995
[12] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic as it reagentsrdquo TheAmerican Journal of Enology and Viticulture vol 16 pp 144ndash158 1965
[13] K D Economou V Oreopoulou and C D ThomopoulosldquoAntioxidant activity of some plant extracts of the familylabiataerdquo Journal of the American Oil Chemists Society vol 68no 2 pp 109ndash113 1991
[14] G Singh S Maurya M P de Lampasona and C A N CatalanldquoA comparison of chemical antioxidant and antimicrobialstudies of cinnamon leaf and bark volatile oils oleoresins andtheir constituentsrdquo Food and Chemical Toxicology vol 45 no 9pp 1650ndash1661 2007
[15] G Singh I P S Kapoor P Singh C S de Heluani M P deLampasona and C A N Catalan ldquoChemistry antioxidant andantimicrobial investigations on essential oil and oleoresins ofZingiber officinalerdquo Food and Chemical Toxicology vol 46 no10 pp 3295ndash3302 2008
[16] G P Rao and A K Shrivastava ldquoToxicity of essential oils ofhigher plants against fungal pathogens of sugarcanerdquo inCurrentTrend in Sugarcane Pathology G P Rao A G Gillasple PP Upadhaya A Bergamin V P Agnihotri and C T ChenEds pp 347ndash365 International Books and Periodicals SupplyService Delhi India 1994
[17] K Ramadas G Suresh N Janarthanan and S Masila-mani ldquoAntifungal activity of 13-disubstituted symmetrical andunsymmetrical thioureasrdquo Pesticide Science vol 52 no 2 pp145ndash151 1998
[18] NCCLS (National Committee for Clinical Laboratory Stan-dards) Performance Standards for Antimicrobial Disc Suscepti-bility Test 6th edition 1997
[19] V Hajhashemi A Ghannadi and H Jafarabadi ldquoBlack cuminseed essential oil as a potent analgesic and antiinflammatorydrugrdquo Phytotherapy Research vol 18 no 3 pp 195ndash199 2004
[20] P J Houghton R Zarka B De Las Heras and J R SHoult ldquoFixed oil of Nigella sativa and derived thymoquinoneinhibit eicosanoid generation in leukocytes andmembrane lipidperoxidationrdquo Planta Medica vol 61 no 1 pp 33ndash36 1995
[21] M T SultanM S Butt FMAnjumA Jamil S Akhtar andMNasir ldquoNutritional profile of indigenous cultivar of black cuminseeds and antioxidant potential of its fixed and essential oilrdquoPakistan Journal of Botany vol 41 no 3 pp 1321ndash1330 2009
[22] S Cheikh-Rouhou S Besbes G Lognay C Blecker CDeroanne and H Attia ldquoSterol composition of black cumin(Nigella sativa L) and Aleppo pine (Pinus halepensisMill) seedoilsrdquo Journal of Food Composition and Analysis vol 21 no 2 pp162ndash168 2008
[23] M F Ramadan ldquoNutritional value functional properties andnutraceutical applications of black cumin (Nigella sativa L) anoverviewrdquo International Journal of Food Science and Technologyvol 42 no 10 pp 1208ndash1218 2007
[24] T D Parker D A Adams K Zhou M Harris and L Yu ldquoFattyacid composition and oxidative stability of cold-pressed edibleseed oilsrdquo Journal of Food Science vol 68 no 4 pp 1240ndash12432003
[25] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAmerican Oil Chemical Society vol 83 pp 847ndash854 2006
[26] J Parry L Su M Luther et al ldquoFatty acid compositionand antioxidant properties of cold-pressed marionberry boy-senberry red raspberry and blueberry seed oilsrdquo Journal ofAgricultural and Food Chemistry vol 53 no 3 pp 566ndash5732005
[27] R R Sokal Introduction to Biostatistics W H Freeman SanFrancisco Calif USA 1973
10 BioMed Research International
[28] H Hosseinzadeh S Parvardeh M N Asl H R Sadeghniaand T Ziaee ldquoEffect of thymoquinone and Nigella sativa seedsoil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampusrdquo Phytomedicine vol 14no 9 pp 621ndash627 2007
[29] S Bourgou A Pichette B Marzouk and J Legault ldquoBioactiv-ities of black cumin essential oil and its main terpenes fromTunisiardquo South African Journal of Botany vol 76 no 2 pp 210ndash216 2010
[30] H J Harzallah E Noumi K Bekir et al ldquoChemical composi-tion antibacterial and antifungal properties of Tunisian Nigellasativa fixed oilrdquoAfrican Journal of Microbiology Research vol 6no 22 pp 4675ndash4679 2012
[31] V S Deepa and P S Kumar ldquoPreliminary phytochemicalinvestigations and in vitro antioxidant activity in selected partsof Andrographis spprdquo Journal of Pharmacological Research vol3 no 9 pp 2206ndash2210 2010
[32] M Amensour E Sendra A Jamal S Bouhdid J A Perez-Alvarez and J Fernandez-Lopez ldquoTotal phenolic content andantioxidant activity of myrtle (Myrtus communis) extractsrdquoNatural Product Communications vol 4 no 6 pp 819ndash8242009
[33] M Di Nunzio V Valli and A Bordoni ldquoPro- and anti-oxidanteffects of polyunsaturated fatty acid supplementation in HepG2cellsrdquo Prostaglandins Leukotrienes and Essential Fatty Acids vol85 no 3-4 pp 121ndash127 2011
[34] G Singh SMaurya CCatalan andMP de Lampasona ldquoStud-ies on essential oils part 42 chemical antifungal antioxidantand sprout suppressant studies on ginger essential oil and itsoleoresinrdquo Flavour and Fragrance Journal vol 20 no 1 pp 1ndash62005
[35] T S El Alfy H M El Fatatry and M A Toama ldquoIsolationand structure assignment of an antimicrobial principle from thevolatile oil of Nigella sativa L seedsrdquo Pharmazie vol 30 no 2pp 109ndash111 1975
[36] M A Khan ldquoChemical composition and medicinal propertiesof Nigella sativa Linnrdquo Inflammopharmacology vol 7 no 1 pp15ndash35 1999
[37] L J McGaw A K Jager and J van Staden ldquoIsolation ofantibacterial fatty acids from Schotia brachypetalardquo Fitoterapiavol 73 no 5 pp 431ndash433 2002
[38] A R McCutcheon T E Roberts E Gibbons et al ldquoAntiviralscreening of British Columbian medicinal plantsrdquo Journal ofEthnopharmacology vol 49 no 2 pp 101ndash110 1995
[39] S Javed A A Shahid M S Haider et al ldquoNutritionalphytochemical potential and pharmacological evaluation ofNigella Sativa (Kalonji) and Trachyspermum Ammi (Ajwain)rdquoMedicinal Plants Research vol 6 no 5 pp 768ndash775 2012
[40] P Rattanachaikunsopon and P Phumkhachorn ldquoAssessmentof factors influencing antimicrobial activity of carvacrol andcymene against Vibrio cholerae in foodrdquo Journal of Bioscienceand Bioengineering vol 110 no 5 pp 614ndash619 2010
[41] M Gilles J Zhao M An and S Agboola ldquoChemical com-position and antimicrobial properties of essential oils of threeAustralian Eucalyptus speciesrdquo Food Chemistry vol 119 no 2pp 731ndash737 2010
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
[28] H Hosseinzadeh S Parvardeh M N Asl H R Sadeghniaand T Ziaee ldquoEffect of thymoquinone and Nigella sativa seedsoil on lipid peroxidation level during global cerebral ischemia-reperfusion injury in rat hippocampusrdquo Phytomedicine vol 14no 9 pp 621ndash627 2007
[29] S Bourgou A Pichette B Marzouk and J Legault ldquoBioactiv-ities of black cumin essential oil and its main terpenes fromTunisiardquo South African Journal of Botany vol 76 no 2 pp 210ndash216 2010
[30] H J Harzallah E Noumi K Bekir et al ldquoChemical composi-tion antibacterial and antifungal properties of Tunisian Nigellasativa fixed oilrdquoAfrican Journal of Microbiology Research vol 6no 22 pp 4675ndash4679 2012
[31] V S Deepa and P S Kumar ldquoPreliminary phytochemicalinvestigations and in vitro antioxidant activity in selected partsof Andrographis spprdquo Journal of Pharmacological Research vol3 no 9 pp 2206ndash2210 2010
[32] M Amensour E Sendra A Jamal S Bouhdid J A Perez-Alvarez and J Fernandez-Lopez ldquoTotal phenolic content andantioxidant activity of myrtle (Myrtus communis) extractsrdquoNatural Product Communications vol 4 no 6 pp 819ndash8242009
[33] M Di Nunzio V Valli and A Bordoni ldquoPro- and anti-oxidanteffects of polyunsaturated fatty acid supplementation in HepG2cellsrdquo Prostaglandins Leukotrienes and Essential Fatty Acids vol85 no 3-4 pp 121ndash127 2011
[34] G Singh SMaurya CCatalan andMP de Lampasona ldquoStud-ies on essential oils part 42 chemical antifungal antioxidantand sprout suppressant studies on ginger essential oil and itsoleoresinrdquo Flavour and Fragrance Journal vol 20 no 1 pp 1ndash62005
[35] T S El Alfy H M El Fatatry and M A Toama ldquoIsolationand structure assignment of an antimicrobial principle from thevolatile oil of Nigella sativa L seedsrdquo Pharmazie vol 30 no 2pp 109ndash111 1975
[36] M A Khan ldquoChemical composition and medicinal propertiesof Nigella sativa Linnrdquo Inflammopharmacology vol 7 no 1 pp15ndash35 1999
[37] L J McGaw A K Jager and J van Staden ldquoIsolation ofantibacterial fatty acids from Schotia brachypetalardquo Fitoterapiavol 73 no 5 pp 431ndash433 2002
[38] A R McCutcheon T E Roberts E Gibbons et al ldquoAntiviralscreening of British Columbian medicinal plantsrdquo Journal ofEthnopharmacology vol 49 no 2 pp 101ndash110 1995
[39] S Javed A A Shahid M S Haider et al ldquoNutritionalphytochemical potential and pharmacological evaluation ofNigella Sativa (Kalonji) and Trachyspermum Ammi (Ajwain)rdquoMedicinal Plants Research vol 6 no 5 pp 768ndash775 2012
[40] P Rattanachaikunsopon and P Phumkhachorn ldquoAssessmentof factors influencing antimicrobial activity of carvacrol andcymene against Vibrio cholerae in foodrdquo Journal of Bioscienceand Bioengineering vol 110 no 5 pp 614ndash619 2010
[41] M Gilles J Zhao M An and S Agboola ldquoChemical com-position and antimicrobial properties of essential oils of threeAustralian Eucalyptus speciesrdquo Food Chemistry vol 119 no 2pp 731ndash737 2010
Submit your manuscripts athttpwwwhindawicom
PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014
The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014
