Food and Chemical Toxicology 48 (2010) 1734–1740

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Food and Chemical Toxicology

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Chemical profile, antifungal, antiaflatoxigenic and antioxidant activity ofCitrus maxima Burm. and Citrus sinensis (L.) Osbeck essential oils andtheir cyclic monoterpene, DL-limonene

Priyanka Singh a, Ravindra Shukla a, Bhanu Prakash a, Ashok Kumar a, Shubhra Singh b,Prashant Kumar Mishra a, Nawal Kishore Dubey a,*

a Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221 005, Indiab Allahabad Agricultural Institute, Deemed University, Allahabad 211 007, India

a r t i c l e i n f o

Article history:Received 13 February 2010Accepted 5 April 2010

Keywords:Aflatoxin B1

Aspergillus flavusCitrus maximaCitrus sinensisDL-LimoneneEssential oil

0278-6915/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.fct.2010.04.001

* Corresponding author. Tel.: +91 542 2313625; faxE-mail addresses: nkdubeybhu@gmail.com,

(N.K. Dubey).

a b s t r a c t

The study deals with antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima and Citrus sin-ensis essential oils (EOs) and their phytochemical composition. The EOs were obtained by hydrodistilla-tion and their chemical profile was determined through GC and GC–MS analysis. Both the EOs and their1:1 combination showed broad fungitoxic spectrum against different food contaminating moulds. TheEOs and their combination completely inhibited aflatoxin B1 (AFB1) production at 500 ppm, whereas,DL-limonene, the major component of EOs showed better antiaflatoxigenic efficacy even at 250 ppm. Boththe oils exhibited antioxidant activity as DPPH free radical scavenger in dose dependent manner. The IC50

for radical scavenging efficacy of C. maxima and C. sinensis oils were to be 8.84 and 9.45 ll ml�1, respec-tively. The EOs were found non-mammalian toxic showing high LD50 for mice (oral, acute). The oils maybe recommended as safe plant based antimicrobials as well as antioxidants for enhancement of shelf lifeof food commodities by checking their fungal infestation, aflatoxin production as well as lipidperoxidation.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Food safety is a fundamental concern of both consumers andthe food industry, especially as the number of reported cases offood-associated infections continues to increase (Alzoreky andNakahara, 2003). It has been estimated that as many as 30% of peo-ple in industrialized countries suffer from food borne diseases eachyear (WHO, 2002). Microorganisms play a major role in contamina-tion of stored foods deteriorating them quantitatively as well asqualitatively. Fungi are significant destroyer of foodstuffs duringstorage, rendering them unfit for human consumption by retardingtheir nutritive value and sometimes by producing mycotoxins.Approximately 25–40% of cereals worldwide are contaminatedwith mycotoxins produced by different storage fungi (Kumaret al., 2007). Nearly, 70% of the total production of food grains inmany tropical and sub-tropical countries is retained at farm levelwhere the unscientific and faulty storage conditions enhance thechances of fungal attack and thereby mycotoxin production.Amongst the mycotoxins, aflatoxins are carcinogenic, mutagenic,

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immunosuppressive agents, produced by Aspergillus flavus andAspergillus parasiticus on variety of food products.

Pathogenic and toxigenic fungi are mostly controlled by syn-thetic fungicides but their treatment can be often problematicdue to their residual nature and high toxicity to mammals (Chenet al., 2008). The use of synthetic chemicals as antimicrobial forthe management of plant pathogens has undoubtedly increasedcrop protection but with considerable deterioration of environ-mental quality and human health (Cutler and Cutler, 1999). Foodborne microorganisms and their toxigenic strains are showingresistance to synthetic fungicides day by day (El-Ghaouth, 1997).As a consequence, there is increasing interest to investigate saferand alternative means of food preservation to minimize microbialcontaminations and to improve the quality of foods. A shift fromsynthetic chemicals to botanical antimicrobials is gaining popular-ity because of their environment safety and biorational mode of ac-tion (Varma and Dubey, 1999). Amongst different groups of plantproducts, essential oils (EOs) have recently become a focal pointof research to flavour food stuff, to produce perfumes, hygienicitems and in many other manufacturing areas (Pavela, 2007;Kumar et al., 2008). Being volatile in nature, the essential oils arebeing recommended as fumigants for preservation of foodcommodities.

Table 2Chemical profile of the essential oil of C. sinensis.

No. Compounds % RTa

1 a-Pinene 0.36 9.522 Sabinine 0.37 10.873 b-Pinine 0.03 11.054 Methyl heptenone 0.01 11.175 b-Myrcene 1.71 11.356 Octanal 0.43 11.777 a-Phellandrene 0.04 12.05

P. Singh et al. / Food and Chemical Toxicology 48 (2010) 1734–1740 1735

The present paper reports on the antifungal, antiaflatoxigenicand antioxidant activity of EOs of leaves of Citrus maxima Burm.and peel of Citrus sinensis (L.) Osbeck and their combination. Thechemical profile of the EOs was studied for their standardizationand their LD50 were calculated in order to record their safety limits.It may be noted that in Indian herbal system of medicine, leafinfusion and dried peel of C. maxima and C. sinensis have been usedin epilepsy, convulsive cough, dyspepsia, diarrhea and ulcers(Prajapati et al., 2003).

8 (E)-b-ocimene 0.21 12.279 a-Terpinene 0.02 12.5010 DL-Limonene 90.66 12.92

11 cis-Ocimine 0.02 13.6512 b-Ocimine 0.04 14.2213 Caprylic alcohol 0.05 14.5514 Terpinolene 0.18 15.5215 a-Terpinoline 0.06 15.5516 Linalylacetate 2.80 15.9517 Nonanal 0.05 16.0718 2,6-Dimethyl-1,3,5,7-octatetraene 0.03 17.0019 Myrtenylacetate 0.06 17.6520 Artemiseole 0.18 17.8021 Isopulegol 0.26 18.3522 t-Sabinine hydrate 0.42 19.6723 3-Cyclohexene-1-methanol 0.02 20.2724 Decanal 0.02 20.7725 b-Citronellol 0.17 21.8526 Laevo-beta-pinene 0.46 21.9527 Z-Citral 0.09 22.5228 DL-Carvone 0.05 22.80

29 Geranyl formate 0.65 23.1230 Z-Citral 0.02 23.8731 3-Methyl-6-isopropenyl-2-cyclohexen-1-one 0.29 24.1232 Perillaldehyde 0.03 24.22

a RT – retention time.

2. Materials and methods

2.1. Test organisms

The toxigenic strain of A. flavus (NKD-116) was chosen as test fungus for thepresent study. The strain has earlier been isolated during mycological survey ofsome edibles in our laboratory. Some other moulds viz. Aspergillus fumigatus, Asper-gillus niger, Aspergillus terreus, Alternaria alternata, Cladosporium herbarum, Curvular-ia lunata, Fusarium oxysporum, Helminthosporium oryzae and Trichoderma viridewere procured from the Division of Mycology and Plant Pathology, IARI, New Delhiand their cultures were maintained on Potato Dextrose Agar (PDA; Potato infusion200 g, dextrose 20 g, agar 15 g and distilled water 1 L; pH 5.6 ± 0.2; Qualigens,Mumbai) at 28 ± 2 �C.

2.2. Plant material

C. maxima and C. sinensis grown in the premises of Banaras Hindu University,Varanasi were identified by morphological features with the help of Flora of BHUCampus (Dubey, 2004). The voucher specimens (LHP/Rut-21/2008 and LHP/Rut-22/2008) were deposited at the Laboratory of Herbal Pesticides, Banaras Hindu Uni-versity, Varanasi.

2.3. Extraction of essential oils

Fresh leaves of C. maxima and peels of C. sinensis (200 g) were collected in July2008 and subjected to hydrodistillation using a Clevenger-type apparatus for 4 h(Shukla et al., 2009). The water traces removed with the help of capillary tubeand anhydrous sodium sulphate. An oil combination was also prepared using 1:1(v/v) ratio of both the EOs. EOs were stored at 4 �C in airtight containers prior toanalysis by gas chromatography–mass spectrometry (GC–MS). Pure DL-limonenewas purchased from Genuine Chemical Co., Mumbai.

2.4. GC and GC–MS

The EOs and their combinations were analyzed on gas chromatography (Perkin-Elmer Auto XL GC, MA, USA) equipped with a flame ionization detector. The GC con-ditions were as follows: column, EQUITY-5 (60 m � 0.32 mm � 0.25 lm); H2 wasthe carrier gas; column Head 159 pressure 10 psi; oven temperature program iso-therm 2 min at 70 �C, 3 �C/min gradient to 250 �C, isotherm 10 min; injection tem-perature, 250 �C; detector temperature 280 �C.

Table 1Chemical profile of the essential oil of C. maxima.

No. Compounds % RTa

1 a-Pinene 0.40 9.552 Sabinine 0.93 10.903 b-Pinine 3.71 11.074 Methyl heptenone 1.25 11.205 b-Myrcene 0.90 11.406 Hexanal 0.12 11.807 Sabinine 0.42 12.308 DL-Limonine 31.83 13.00

9 t-Ocimine 1.19 13.7010 Linalool 0.16 15.9711 1-Hexene,4-methyl 15.22 16.1512 1-Hexene,3,3-dimethyl 0.67 18.4513 Geranyl formate 1.83 22.0214 Z-Citral 13.38 22.6515 Geranyl formate 2.43 23.2216 E-Citral 17.75 24.0217 Geranylacetate 0.82 29.2018 b-Farnesene 0.45 31.27

a RT – retention time.

GC–MS analysis was also performed using PerkinElmer Turbomass GC–MS. TheGC column was EQUITY-5 (60 m � 0.32 mm � 0.25 lm) fused silica capillary col-umn. The GC conditions were as follows: Injection temperature, 250 �C; columntemperature, isothermal at 70 �C for 2 min, then programmed to 250 �C at 37 �C/min and held at this temperature for 10 min; ion source temperature, 250 �C. He-lium was used as the carrier gas. The effluent of the GC column was introduced di-rectly into the source of MS and spectra were obtained in the EI mode with 70 eVionization energy. The sector mass analyzer was set to scan from 40 to 500 amufor 2 s. The identification of individual compounds was based on comparison oftheir relative retention times with those of authentic samples on capillary column,and by matching of their mass spectra of peaks with available Wiley, NIST and NBSmass spectral libraries or with published data in the literature (Adams, 2007). Theconstituents identified by GC and GC–MS analysis, their retention times and areapercentages (concentrations) are summarised in Tables 1–3.

2.5. Antifungal assay

The antifungal activity of EOs, their combination and DL-limonene was testedagainst fungal isolates by poisoned food assay which produce hyphal growth inhi-bition (Chang et al., 2008) using Czapek’s-dox agar (CDA) medium (NaNO3, 2 g;K2HPO4, 1 g; MgSO4�7H2O, 0.5 g; KCl, 0.5 g; FeSO4�7H2O, 0.01 g; sucrose, 30 g; agar,15 g; 1 L distilled water, pH 6.8 ± 0.2; Sisco Research Lab., Mumbai). Requisiteamount of EOs/combination and DL-limonene was dissolved separately in 0.5 mlacetone and then mixed with 9.5 ml CDA in Petri plates (9.0 cm) to achieve finalconcentrations 250, 500, 750 and 1000 ppm. The prepared plates were inoculatedaseptically with an assay disc (5 mm), cut from the periphery of 7 days old cultureof A. flavus. The control sets were prepared using sterilized distilled water in placeof EO and incubated at 28 ± 2 �C. The efficacy of treatments was evaluated 0–10 days by measuring the diameter of the fungus colonised. All the tests were per-formed in triplicate and percent inhibition of the radial growth of the test fungi bythe oils at day-10 was calculated as follows:

Percent mycelial inhibition ¼ dc � dtdc

� 100;

where dc = mean colony diameter of control sets, dt = mean colony diameter of treat-ment sets.

Table 3Chemical profile of the C. maxima and C. sinensis oil combination.

No. Compounds % RTa

1 a-Pinene 0.02 15.822 Sabinine 0.50 17.373 Methyl heptenone 0.65 17.604 b-Pinene 1.73 17.705 b-Myrcene 0.51 17.826 Cymol 1.27 19.527 Octanal 0.22 18.408 b-Ocimene 0.30 18.909 DL-Limonine 69.78 19.75

10 b-terpinene 8.69 22.4511 (�)-Carvyl acetate 0.09 23.6012 1,3,4-Trimethyl-3-cyclohexene-1-carbaldehyde 0.10 24.0713 L-Carveol 0.14 24.22

14 2-(3,3-Dimethylcyclohexylidene)ethanol 0.30 24.6015 c-Terpinene 0.16 26.1016 a-terpinene 0.30 26.6217 Decanal 0.06 26.7218 (�)-Carvyl acetate 0.12 26.8219 Neryl acetate 0.83 27.5020 Carveol 3.86 28.1221 Linalyl acetate 1.28 28.4722 L-Carvone 0.13 28.60

23 Z-citral 5.24 29.2224 Sabinol 0.41 32.3025 a-Fenchene 0.13 32.4526 Linalyl acetate 0.25 33.1227 t-caryophyllene 0.18 35.40

a RT – retention time.

1736 P. Singh et al. / Food and Chemical Toxicology 48 (2010) 1734–1740

2.6. Efficacy of EOs, their combination and DL-limonene in suppression of aflatoxinproduction

SMKY broth medium (Sucrose, 200 g; MgSO4�7H2O, 0.5 g; KNO3, 0.3 g; Yeast ex-tract, 7.0 g; distilled water, 1000 ml; pH, 5.6 ± 0.2; Sisco Research Lab., Mumbai)was used to investigate antiaflatoxigenic activity of EOs, their combination andDL-limonene. The method of Sinha et al. (1993) was adopted for the estimation ofaflatoxin B1 (AFB1). Different concentrations of the oils, their combination and lim-onene (250, 500, 750 and 1000 ppm) were prepared separately by dissolving theirrequisite amount in 0.5 ml acetone and then mixing it with 24.5 ml SMKY mediumin 100 ml Erlenmeyer flask. The control set was prepared without EO or DL-limo-nene. Flasks were inoculated with 0.5 ml spore suspension (�106 spores ml�1) pre-pared in 0.1% tween-80 and incubated at 28 ± 2 �C for 10 days. The content of eachflask was filtered (Whatman filter paper No. 1) and mycelium was oven dried at100 �C to determine mycelial dry weight. The filtrate was extracted with 20 mlchloroform in a separating funnel. The chloroform extract was evaporated till dry-ness on water bath at 70 �C and redissolved in 1 ml chloroform. Fifty microliterchloroform extract spotted on TLC plate (20 � 20 cm of Silica gel-G) and developedin toluene: isoamyl alcohol: methanol (90:32:2;v/v/v). The intensity of AFB1 wasobserved in UV transilluminator (Zenith, Agra) at wavelength of 360 nm.

For quantitative estimation, spots of AFB1 on TLC were scraped and dissolved in5 ml methanol, centrifuged at 3000 rpm (5 min) and optical density of supernatantwas recorded at 360 nm using spectophotometer (Systronics, India Ltd., Mumbai).The amount of AFB1 in control and treatment sets was calculated following Singhet al. (2008):

Aflatoxin B1 content ðlg kg�1Þ ¼ D�ME� L

� 1000;

where D = optical density, M = mol. wt. of aflatoxin B1 (312), E = molar extinctioncoefficient (21,800) and L path length (1 cm cell was used).

2.7. Fungitoxic spectrum of the essential oils and their combination

The mycotoxic spectrum of the EOs and their combination was evaluated by thepoisoned food technique at 750 ppm against some Aspergillus spp. viz. A. fumigatus,A. niger, A. terreus, A. alternata and other filamentous fungi like C. herbarum, C. lunata,F. oxysporum, H. oryzae and T. viride.

2.8. Antioxidant activity of C. maxima and C. sinensis EOs

The antioxidant activity of the essential oils was observed by DPPH radical scav-enging assay on TLC and measuring the free radical scavenging activity throughspectrophotometer following Tepe et al. (2005).

To determine the antioxidant activity of EOs of C. maxima and C. sinensis, 5 ll(1:10 dilution in methanol) was spotted separately on TLC plate and developed inethyl acetate and methanol (1:1). The plate was sprayed with 0.2% DPPH solutionin methanol (2,2-diphenyl-1-picrylhydrazil) and left at room temperature for30 min. Yellow spot formed due to bleaching of purple color of DPPH reagent wasrecorded as positive antioxidant activity of EO.

Free radical scavenging activity of EOs of C. maxima and C. sinensis was mea-sured by recording the extent of bleaching of the purple-colored DPPH solutionto yellow. Different concentrations (2.0–10.00 ll ml�1) of the samples were addedseparately to 0.004% DPPH solution in methanol (5 ml). After 30 min of incubationat room temperature, the absorbance was taken against a blank at 517 nm usingspectrophotometer. Scavenging of DPPH free radical with reduction in absorbanceof the sample was taken as a measure of the antioxidant activity of the essential oilsas followed by Sharififar et al. (2007). IC50, which represented the concentration ofthe EO that caused 50% neutralization of DPPH radicals, was calculated from thegraph plotting between percentage inhibition and concentration

I % ¼ Ablank � Asample=Ablank� �

� 100;

where Ablank is the absorbance of the control (without test compound), and Asample isthe absorbance of the test compound.

2.9. Determination of safety profile of the oils

The safety profile of the essential oils of C. maxima and C. sinensis was deter-mined on mice (Mus musculus L., average weight 50.0 g, age 3 months) by recordingtheir LD50 values which represent the lethal dose of oil per unit weight for killing of50% population of test animals (Singh et al., 2009). A stock solution was prepared bymixing the EOs, tween-80 and distilled water in 2:1:1 ratio. Different dilutions ofthe stock solution of each essential oil were made separately with distilled water.Each dilution was orally given to each group of animal (12 mice) separately inwhich the dose of oil was 0.2–3.2 ml. In control set, equal dose of tween-80 and dis-tilled water was given. After 24 h, the mortality of the test animals was recordedand LD50 of EOs was calculated by probit analysis (Finney, 1971).

2.10. Statistical analysis

Antifungal and antiaflatoxigenic experiments were performed in triplicate anddata analyzed were mean ± SE. Data obtained were subjected to one way ANOVA.Means were separated by Tukey’s multiple range tests when ANOVA was significant(p < 0.05) (SPSS 10.0; 241 Chicago, IL, USA).

3. Results

The essential oils of C. maxima, C. sinensis and their combinationwere pale-yellow in color. The yield of C. maxima and C. sinensisEOs was 7.3 and 10.6 ml/kg, respectively. The GC–MS profile de-picted that DL-limonene (31.83%), E-citral (17.75%), 1-hexene-4-methyl (15.22%) and Z-citral (13.38%) were the major componentsin C. maxima oil (Table 1), whereas, DL-limonene represented90.66% in C. sinensis followed by lynalyl acetate (2.80) (Table 2).In oil combination DL-limonene was again the major component(69.78%) followed by b-terpinene (8.69%), Z-citral (5.24%) and car-veol (3.86%) (Table 3).

During antifungal assay, all the concentrations of EOs werefound significantly effective over control from day-2 to day-10according to ANOVA and tukey’s comparison tests (Table 4). A cor-responding decrease in mycelial growth was recorded with in-creased concentration of EOs. Complete inhibition of A. flavuswas found at 750 ppm of both the EOs and their combination. EOof C. maxima was found most efficacious when compared with thatof C. sinensis and oil combination. At 500 ppm, A. flavus was inhib-ited 48.1%, 46.2% and 44.0% against EO of C. maxima, C. sinensis andtheir combination, respectively. DL-Limonene, completely inhibitedthe growth of A. flavus at 500 ppm.

The EOs of C. maxima, C. sinensis and their combination com-pletely inhibited AFB1 production at 500 ppm in SMKY broth whileDL-limonene could inhibit at 250 ppm. At 500 ppm, mycelialgrowth was recorded in all the EOs and DL-limonene treated sets,but aflatoxin B1 production was completely inhibited (Table 5).

The oils and their combination also exhibited a broad fungitoxicspectrum against all the fungi tested and caused 100% inhibition of

Table 4Antifungal activity of C. maxima, C. sinensis EOs, their oil combination and DL-limonene on A. flavus.

Treatments (ppm) Diameter (mean ± SE) of mycelial growth (mm) including disc diameter of 5 mm

0 day 2 days 4 days 6 days 8 days 10 days % Growth inhibitionat day-10

Control 5.00 ± 0.00a 13.6 ± 0.66a 26.2 ± 0.25a 37.4 ± 0.75a 53.2 ± 0.41a 82.8 ± 0.92a –C. maxima (250) 5.00 ± 0.00a 8.07 ± 0.56cd 13.9 ± 0.52cd 32.3 ± 0.47c 41.7 ± 0.61c 63.4 ± 0.43c 23.4C. maxima (500) 5.00 ± 0.00a 6.95 ± 0.49d 8.82 ± 0.29e 25.6 ± 0.67d 32.4 ± 0.81e 42.9 ± 0.51e 48.1C. maxima (750) 5.00 ± 0.00a 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00f 100C. maxima (1000) 5.00 ± 0.00a 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00f 100C. sinensis (250) 5.00 ± 0.00a 9.06 ± 0.08c 15.1 ± 0.32bc 34.6 ± 0.53bc 43.3 ± 0.71bc 65.7 ± 0.36bc 18.2C. sinensis (500) 5.00 ± 0.00a 8.26 ± 0.33cd 12.2 ± 0.61d 26.2 ± 0.64d 33.6 ± 0.53de 44.5 ± 0.67de 46.2C. sinensis (750) 5.00 ± 0.00a 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00f 100C. sinensis (1000) 5.00 ± 0.00a 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00f 100Oil combination (250) 5.00 ± 0.00a 10.8 ± 0.49b 16.9 ± 0.52b 35.2 ± 0.66ab 44.8 ± 0.54b 66.4 ± 0.47b 19.8Oil combination (500) 5.00 ± 0.00a 9.48 ± 0.28c 14.2 ± 0.75c 27.8 ± 0.43d 35.7 ± 0.71d 46.3 ± 0.77d 44.0Oil combination (750) 5.00 ± 0.00a 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00f 100Oil combination (1000) 5.00 ± 0.00a 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00f 100

DL-Limonene (500) 5.00 ± 0.00a 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00f 100

DL-Limonene (1000) 5.00 ± 0.00a 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00e 5.00 ± 0.00f 5.00 ± 0.00f 100

Values are mean (n = 3) ± SE.The means followed by same letter in the same column are not significantly different according to ANOVA and Tukey’s multiple comparison tests.

Table 5Efficacy of essential oils, their combination and DL-limonene on mycelial biomass (g) and aflatoxin B1 elaboration (lg kg�1) of a toxigenic strain of A. flavus in SMKY medium.

Conc. (ppm) Dry weight of mycelium (g) ± SE Aflatoxin B1 (lg kg�1)

C. maxima C. sinensis Oil combination DL-Limonene C. maxima C. sinensis Oil combination DL-Limonene

Control 0.778 ± 0.003a 0.778 ± 0.003a 0.778 ± 0.003a 0.778 ± 0.003a 572.2 ± 2.17a 572.2 ± 2.17a 572.2 ± 2.17a 572.2 ± 2.17a

250 0.653 ± 0.006b 0.520 ± 0.005b 0.674 ± 0.003b 0.366 ± 0.02b 187.6 ± 1.10b 181.2 ± 0.99b 193.9 ± 1.30b 0.00 ± 0.00b

500 0.293 ± 0.008c 0.241 ± 0.008c 0.331 ± 0.005c 0.240 ± 0.01c 0.00 ± 0.00c 0.00 ± 0.00c 0.00 ± 0.00c 0.00 ± 0.00b

750 0.00 ± 0.00d 0.00 ± 0.00d 0.00 ± 0.00d 0.00 ± 0.00d 0.00 ± 0.00c 0.00 ± 0.00c 0.00 ± 0.00c 0.00 ± 0.00b

1000 0.00 ± 0.00d 0.00 ± 0.00d 0.00 ± 0.00d 0.00 ± 0.00d 0.00 ± 0.00c 0.00 ± 0.00c 0.00 ± 0.00c 0.00 ± 0.00b

Values are mean (n = 3) ± SE.The means followed by same letter in the same column are not significantly different according to ANOVA and Tukey’s multiple comparison tests.

P. Singh et al. / Food and Chemical Toxicology 48 (2010) 1734–1740 1737

the mycelial growth of A. fumigatus, A. terreus, A. alternata, F. oxy-sporum, H. oryzae and T. viride at 750 ppm.

Both the EOs exhibited DPPH radical scavenging activity in dosedependent manner (Fig. 1). The IC50 of C. maxima and C. sinensis oilswere calculated to be 8.84 and 9.45 ll/ml, respectively indicatingtheir strong antioxidant efficacy.

The LD50 of the C. maxima and C. sinensis oils, determinedthrough oral administration on mice, was calculated to be36680.04 and 19938.54 ll/kg body weight, respectively (Table 6

Concentration (µl/ml)

1 2 3 4 5 6 7 8 9 10 11

% ra

dica

l sca

veng

ing

activ

ity

0

10

20

30

40

50

60

70

Citrus maxima oil Citrus sinensis oil

Fig. 1. Radical scavenging activity of C. maxima and C. sinensis oils.

and 7). The high LD50 value indicates their favourable safety profileand non-mammalian toxicity.

4. Discussion

The findings of present investigation revealed the antimycoticas well aflatoxin inhibitory efficacy of the essential oils of C. max-ima, C. sinensis and their combination. The oils and the combinationwere standardised by their chemical profile. The composition ofessential oils varies with respect to ecological and geographicalcondition, age of plant and time of harvesting (Bagamboula et al.,2004) and thus different chemotypes of a particular essential oilhave been reported. Such a variation in chemical composition ofEOs would definitely alter their biological activity. Hence, determi-nation of chemical profile of essential oil is important beforerecommending it for antimicrobial activity. In the present investi-gation, the GC–MS analysis revealed that monoterpenes constitutemore than 90% of both the EOs and their combinations. The majorcomponent of the EOs is a cyclic monoterpene, DL-limonene fol-lowed by E-citral, Z-citral (monoterpene aldehydes) and carveol,the hydroxylated derivatives of DL-limonene. EOs, rich in monoter-penes are recognized as food preservatives (Ruberto and Baratta,2000). In addition, monoterpenic EOs are considered as naturalantioxidants (Yanishlieva et al., 1999) and are reported as antican-cerous (Sharma et al., 2009). DL-Limonene, which was found to bein appreciable amounts in the EOs of this study, showed aflatoxininhibitory efficacy at a concentration lower than the individual oilsand the combination. Hence, it may be concluded that othercomponents present in the oil would be masking the efficacy of

Table 6LD50 of the essential oil of C. maxima.

Dose Log of doseconc. (x)

Total numberof animals (n)

Number ofdead animal

Percentmortality

Percent Correctedmortality

Empericalprobit

Expectedprobit (Y)

Weighingcoefficient (w)

Workingprobit (y)

Weight (nw) nwx nwy nwx2 nwy2 nwxy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 160.2 0.3010 12.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 – – – – –0.4 0.6021 12.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 – – – – –0.8 0.9030 12.0 1.0 8.33 8.33 3.61 3.90 0.405 3.66 58.32 52.668 213.5678 47.5632 78.2064 192.871.2 1.0791 12.0 3.0 25.0 25.0 4.33 4.30 0.532 4.33 76.608 82.6728 331.7124 89.2188 1436.3136 357.97321.6 1.2041 12.0 4.0 33.33 33.33 4.57 5.10 0.634 4.57 91.296 109.9308 418.044 109.9308 1914.2232 503.37242.0 1.3010 12.0 6.0 50.0 50.0 5.0 5.50 0.581 4.96 83.664 108.8484 414.9732 141.6144 2058.2664 539.88722.4 1.3802 12.0 9.0 75.0 75.0 5.67 5.75 0.532 5.67 76.608 105.7344 434.3664 145.9356 2462.8572 599.5142.8 1.4471 12.0 10.0 83.33 83.33 5.97 5.80 0.503 5.95 72.432 104.8188 431.2596 151.6884 2567.7192 624.093.2 1.5051 12.0 12.0 100.00 100.00 8.72 6.20 0.370 6.79 53.28 80.1936 361.7712 120.7032 2456.4264 544.5144Control – 12.0 0.0 0.0 0.0 0.0 – – – – – – – – –

LD50 36680.04 ll/kg body weight.

Table 7LD50 of the essential oil of C. sinensis.

Dose Log of doseconc. (x)

Total numberof animals (n)

Number ofdead animal

Percentmortality (y)

Percent correctedmortality

Empericalprobit

Expectedprobit (Y)

Weighingcoefficient (w)

Workingprobit (y)

Weight (nw) nwx nwy nwx2 nwy2 nwxy

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 160.2 0.3010 12.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 – – – – –0.4 0.6021 12.0 1.0 8.33 8.33 3.61 3.5 0.269 3.623 38.736 23.3208 140.34 14.052 508.4508 84.49080.8 0.9030 12.0 4.0 33.33 33.33 4.57 5.15 0.627 4.573 90.288 81.5376 412.8864 73.6356 1888.1292 372.87121.0 1.0 12.0 6.0 50.0 50.0 5.0 5.7 0.532 4.87 76.608 76.608 373.08 76.608 1816.8996 373.08121.2 1.0791 12.0 9.0 75.0 75.0 5.67 6.0 0.439 5.617 63.216 68.2212 355.0836 73.6224 1994.5044 383.19981.4 1.1461 12.0 11.0 91.67 91.67 6.39 6.2 0.370 6.361 53.28 61.0644 338.9136 69.9864 2155.8288 388.43041.6 1.2041 12.0 12.0 100.00 100.00 8.72 6.4 0.302 6.94 43.488 52.3644 301.806 63.0528 2094.5328 363.408Control – 12.0 0.0 0.0 0.0 0.0 – – – – – – – – –

LD50 19938.54 ll/kg body weight.

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P. Singh et al. / Food and Chemical Toxicology 48 (2010) 1734–1740 1739

DL-limonene and they may be working in negative direction. Therewas no synergism between the oil components when the two oilswere mixed together as their aflatoxin inhibitory concentrationwas the same (500 ppm). The efficacy of the C. maxima, C. sinen-sis, their combination oil and DL-limonene as aflatoxin inhibitor isbeing reported for the first time in the present investigation. Theantioxidant nature of the EOs in terms of free radical scavengermay be due to antioxidant activity of DL-limonene, the major con-stituent of the oils (Junior et al., 2009).

The LD50 of the C. maxima and C. sinensis oils on mice wasrecorded to be very high even in comparison to some of the wellknown botanical products (azadirachtin, >5000 mg kg�1; pyre-thrum, 350–500 mg kg�1) which are applied on large scale ascontrol of pests (Isman, 2006; Singh et al., 2009). The EOs arethus non-mammalian toxic satisfying acceptability aspect ofconsumer’s requirements and may be recommended as a safefungitoxicant in place of some commercial antifungals like zincdimethyl di thiocarbamate (LD50: 1889 mg kg�1) and mancozeb(LD50: 5000 mg kg�1) (available at http://www.rtvanderbilt.com/documents/MSDS/US/46603.pdf, http://www.epa.gov/espp/litsta-tus/effects/redleg-frog/mancozeb-maneb/appendix-a.pdf). Beingused in traditional medicine against different human diseasesand because of appreciable LD50 value on test animal, therewould be no chance of any oral toxicity by exploiting these oilsas fungitoxicant.

In conclusion, the EOs of C. maxima, C. sinensis and DL-limonenemay be recommended as a plant based antimicrobial as well asantioxidant food preservative for enhancement of shelf life ofstored food commodities by checking their fungal growth, afla-toxin production and possessing antioxidant activity. In general,the plant based antimicrobials are recognized to be cheaper overthe synthetics because of short term toxicological testing beforeformulation as antimicrobial (Varma and Dubey, 1999). The yieldof the Citrus oils in the present investigation was comparativelyhigh and sufficient amount of raw materials would be availableas the plants grow luxuriantly in the area. In addition, the peelsof C. sinensis remain mostly unused and are dumped as waste.Hence, exploitation of their oil as food preservative would behelpful in their utilization in right direction. In addition, in recentyears there has been considerable pressure by consumers to re-duce or eliminate chemically synthesized additives in foods(Lanciotti et al., 2004). On the other hand, interest in the possibleuse of plant based additives in foods to prevent microbial growthis considerably increased. Numerous EOs like lemon grass, basil,thyme, rosemary, sage, oregano, clove, etc. are proven potentiallyuseful additives for food preservation against mycotoxigenic fungiand bacteria likely to prolong shelf life and improve the quality ofstored food products (Nguefack et al., 2004). Formulation of manyplant volatiles viz. carvone (under the trade name Talent) andDMC base naturals are widely used as food additives and mostof them are generally recognized as safe (GRAS) (Singh et al.,2009). There would be no chance of alteration in the organolepticproperties of food commodities when citrus EOs or their compo-nents used as preservatives because the monoterpenes presentin these oils are widely used as natural ingredients in many foodproducts, soaps, soft drinks, cosmetics and perfumes for theirlemon-like flavour and odor (Shukla et al., 2009). Thus, Citrusessential oils would be compatible with the organoleptic param-eters with treated food commodities and their exploitation wouldbe safe to the consumers and may add appreciable flavour to thetreated foods.

The findings of the present investigation would draw the atten-tion of food industries regarding large scale exploitation of theseEOs as food additives for enhancement of the shelf life. On the basisof unique virtues possessing antifungal as well as antiaflatoxigenicactivity, broad fungitoxic spectrum, antioxidant activity and

favourable safety profile, the oils may be recommended as safeplant based preservatives during post-harvest processing of foods.

Conflict of interest

The authors declared that there are no conflicts of interest.

Funding sources

Council of scientific and Industrial Reserach (CSIR), New Delhi.

Acknowledgement

This work was financially supported by Council of Scientific andIndustrial Research (CSIR) New Delhi, India.

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