Food and Chemical Toxicology 48 (2010) 1734–1740

Contents lists available at ScienceDirect

Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Chemical profile, antifungal, antiaflatoxigenic and antioxidant activity of

Citrus maxima Burm. and Citrus sinensis (L.) Osbeck essential oils and
their 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, India
b
Allahabad Agricultural Institute, Deemed University, Allahabad 211 007, India

a r t i c l e i n f o a b s t r a c t

Article history: The study deals with antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima and Citrus sin-
Received 13 February 2010 ensis essential oils (EOs) and their phytochemical composition. The EOs were obtained by hydrodistilla-
Accepted 5 April 2010 tion and their chemical profile was determined through GC and GC–MS analysis. Both the EOs and their
1:1 combination showed broad fungitoxic spectrum against different food contaminating moulds. The
EOs and their combination completely inhibited aflatoxin B1 (AFB1) production at 500 ppm, whereas,
Keywords: DL-limonene, the major component of EOs showed better antiaflatoxigenic efficacy even at 250 ppm. Both
Aflatoxin B1
the oils exhibited antioxidant activity as DPPH free radical scavenger in dose dependent manner. The IC50
Aspergillus flavus
Citrus maxima
for radical scavenging efficacy of C. maxima and C. sinensis oils were to be 8.84 and 9.45 ll ml1, respec-
Citrus sinensis tively. The EOs were found non-mammalian toxic showing high LD50 for mice (oral, acute). The oils may
DL-Limonene be recommended as safe plant based antimicrobials as well as antioxidants for enhancement of shelf life
Essential oil of food commodities by checking their fungal infestation, aflatoxin production as well as lipid
peroxidation.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction immunosuppressive agents, produced by Aspergillus flavus and

Food safety is a fundamental concern of both consumers and
the food industry, especially as the number of reported cases of
food-associated infections continues to increase (Alzoreky and
Nakahara, 2003). It has been estimated that as many as 30% of peo-
ple in industrialized countries suffer from food borne diseases each
year (WHO, 2002). Microorganisms play a major role in contamina-
tion of stored foods deteriorating them quantitatively as well as
qualitatively. Fungi are significant destroyer of foodstuffs during
storage, rendering them unfit for human consumption by retarding
their nutritive value and sometimes by producing mycotoxins.
Approximately 25–40% of cereals worldwide are contaminated
with mycotoxins produced by different storage fungi (Kumar
et al., 2007). Nearly, 70% of the total production of food grains in
many tropical and sub-tropical countries is retained at farm level
where the unscientific and faulty storage conditions enhance the
chances of fungal attack and thereby mycotoxin production.
Amongst the mycotoxins, aflatoxins are carcinogenic, mutagenic,
* Corresponding author. Tel.: +91 542 2313625; fax: +91 542 2368174.
E-mail addresses: nkdubeybhu@gmail.com, nkdubey2@rediffmail.com
(N.K. Dubey).
Aspergillus parasiticus on variety of food products.
Pathogenic and toxigenic fungi are mostly controlled by syn-
thetic fungicides but their treatment can be often problematic
due to their residual nature and high toxicity to mammals (Chen
et al., 2008). The use of synthetic chemicals as antimicrobial for
the management of plant pathogens has undoubtedly increased
crop protection but with considerable deterioration of environ-
mental quality and human health (Cutler and Cutler, 1999). Food
borne microorganisms and their toxigenic strains are showing
resistance to synthetic fungicides day by day (El-Ghaouth, 1997).
As a consequence, there is increasing interest to investigate safer
and alternative means of food preservation to minimize microbial
contaminations and to improve the quality of foods. A shift from
synthetic 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 plant
products, essential oils (EOs) have recently become a focal point
of research to flavour food stuff, to produce perfumes, hygienic
items and in many other manufacturing areas (Pavela, 2007;
Kumar et al., 2008). Being volatile in nature, the essential oils are
being recommended as fumigants for preservation of food
commodities.

0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.04.001
 P. Singh et al. / Food and Chemical Toxicology 48 (2010) 1734–1740 1735

The present paper reports on the antifungal, antiaflatoxigenic Table 2

and antioxidant activity of EOs of leaves of Citrus maxima Burm. Chemical profile of the essential oil of C. sinensis.

and peel of Citrus sinensis (L.) Osbeck and their combination. The No. Compounds % RTa
chemical profile of the EOs was studied for their standardization 1 a-Pinene 0.36 9.52
and their LD50 were calculated in order to record their safety limits. 2 Sabinine 0.37 10.87
It may be noted that in Indian herbal system of medicine, leaf 3 b-Pinine 0.03 11.05
infusion and dried peel of C. maxima and C. sinensis have been used 4 Methyl heptenone 0.01 11.17
5 b-Myrcene 1.71 11.35
in epilepsy, convulsive cough, dyspepsia, diarrhea and ulcers 6 Octanal 0.43 11.77
(Prajapati et al., 2003). 7 a-Phellandrene 0.04 12.05
8 (E)-b-ocimene 0.21 12.27
9 a-Terpinene 0.02 12.50
2. Materials and methods
10 DL-Limonene 90.66 12.92

2.1. Test organisms 11 cis-Ocimine 0.02 13.65

12 b-Ocimine 0.04 14.22
The toxigenic strain of A. flavus (NKD-116) was chosen as test fungus for the 13 Caprylic alcohol 0.05 14.55
present study. The strain has earlier been isolated during mycological survey of 14 Terpinolene 0.18 15.52
some edibles in our laboratory. Some other moulds viz. Aspergillus fumigatus, Asper- 15 a-Terpinoline 0.06 15.55
gillus niger, Aspergillus terreus, Alternaria alternata, Cladosporium herbarum, Curvular- 16 Linalylacetate 2.80 15.95
ia lunata, Fusarium oxysporum, Helminthosporium oryzae and Trichoderma viride 17 Nonanal 0.05 16.07
were procured from the Division of Mycology and Plant Pathology, IARI, New Delhi 18 2,6-Dimethyl-1,3,5,7-octatetraene 0.03 17.00
and their cultures were maintained on Potato Dextrose Agar (PDA; Potato infusion 19 Myrtenylacetate 0.06 17.65
200 g, dextrose 20 g, agar 15 g and distilled water 1 L; pH 5.6 ± 0.2; Qualigens, 20 Artemiseole 0.18 17.80
Mumbai) at 28 ± 2 °C. 21 Isopulegol 0.26 18.35
22 t-Sabinine hydrate 0.42 19.67
23 3-Cyclohexene-1-methanol 0.02 20.27
2.2. Plant material 24 Decanal 0.02 20.77
25 b-Citronellol 0.17 21.85
C. maxima and C. sinensis grown in the premises of Banaras Hindu University, 26 Laevo-beta-pinene 0.46 21.95
Varanasi were identified by morphological features with the help of Flora of BHU 27 Z-Citral 0.09 22.52
Campus (Dubey, 2004). The voucher specimens (LHP/Rut-21/2008 and LHP/Rut- 28 DL-Carvone 0.05 22.80
22/2008) were deposited at the Laboratory of Herbal Pesticides, Banaras Hindu Uni-
29 Geranyl formate 0.65 23.12
versity, Varanasi.
30 Z-Citral 0.02 23.87
31 3-Methyl-6-isopropenyl-2-cyclohexen-1-one 0.29 24.12
2.3. Extraction of essential oils 32 Perillaldehyde 0.03 24.22
a
RT – retention time.
Fresh leaves of C. maxima and peels of C. sinensis (200 g) were collected in July
2008 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 tube
and 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 to GC–MS analysis was also performed using PerkinElmer Turbomass GC–MS. The
analysis by gas chromatography–mass spectrometry (GC–MS). Pure DL-limonene GC column was EQUITY-5 (60 m  0.32 mm  0.25 lm) fused silica capillary col-
was purchased from Genuine Chemical Co., Mumbai. umn. The GC conditions were as follows: Injection temperature, 250 °C; column
temperature, isothermal at 70 °C for 2 min, then programmed to 250 °C at 37 °C/
2.4. GC and GC–MS 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-
The EOs and their combinations were analyzed on gas chromatography (Perkin- rectly into the source of MS and spectra were obtained in the EI mode with 70 eV
Elmer Auto XL GC, MA, USA) equipped with a flame ionization detector. The GC con- ionization energy. The sector mass analyzer was set to scan from 40 to 500 amu
ditions were as follows: column, EQUITY-5 (60 m  0.32 mm  0.25 lm); H2 was for 2 s. The identification of individual compounds was based on comparison of
the carrier gas; column Head 159 pressure 10 psi; oven temperature program iso- their relative retention times with those of authentic samples on capillary column,
therm 2 min at 70 °C, 3 °C/min gradient to 250 °C, isotherm 10 min; injection tem- and by matching of their mass spectra of peaks with available Wiley, NIST and NBS
perature, 250 °C; detector temperature 280 °C. mass spectral libraries or with published data in the literature (Adams, 2007). The
constituents identified by GC and GC–MS analysis, their retention times and area
percentages (concentrations) are summarised in Tables 1–3.
Table 1
Chemical profile of the essential oil of C. maxima. 2.5. Antifungal assay
No. Compounds % RTa
The antifungal activity of EOs, their combination and DL-limonene was tested
1 a-Pinene 0.40 9.55 against fungal isolates by poisoned food assay which produce hyphal growth inhi-
2 Sabinine 0.93 10.90 bition (Chang et al., 2008) using Czapek’s-dox agar (CDA) medium (NaNO3, 2 g;
3 b-Pinine 3.71 11.07 K2HPO4, 1 g; MgSO47H2O, 0.5 g; KCl, 0.5 g; FeSO47H2O, 0.01 g; sucrose, 30 g; agar,
4 Methyl heptenone 1.25 11.20 15 g; 1 L distilled water, pH 6.8 ± 0.2; Sisco Research Lab., Mumbai). Requisite
5 b-Myrcene 0.90 11.40 amount of EOs/combination and DL-limonene was dissolved separately in 0.5 ml
6 Hexanal 0.12 11.80 acetone and then mixed with 9.5 ml CDA in Petri plates (9.0 cm) to achieve final
7 Sabinine 0.42 12.30 concentrations 250, 500, 750 and 1000 ppm. The prepared plates were inoculated
8 DL-Limonine 31.83 13.00 aseptically with an assay disc (5 mm), cut from the periphery of 7 days old culture
9 t-Ocimine 1.19 13.70 of A. flavus. The control sets were prepared using sterilized distilled water in place
10 Linalool 0.16 15.97 of EO and incubated at 28 ± 2 °C. The efficacy of treatments was evaluated 0–
11 1-Hexene,4-methyl 15.22 16.15 10 days by measuring the diameter of the fungus colonised. All the tests were per-
12 1-Hexene,3,3-dimethyl 0.67 18.45 formed in triplicate and percent inhibition of the radial growth of the test fungi by
13 Geranyl formate 1.83 22.02 the oils at day-10 was calculated as follows:
14 Z-Citral 13.38 22.65
15 Geranyl formate 2.43 23.22 dc  dt
16 E-Citral 17.75 24.02 Percent mycelial inhibition ¼  100;
dc
17 Geranylacetate 0.82 29.20
18 b-Farnesene 0.45 31.27
where dc = mean colony diameter of control sets, dt = mean colony diameter of treat-
a
RT – retention time. ment sets.
1736 P. Singh et al. / Food and Chemical Toxicology 48 (2010) 1734–1740

Table 3 To determine the antioxidant activity of EOs of C. maxima and C. sinensis, 5 ll

Chemical profile of the C. maxima and C. sinensis oil combination. (1:10 dilution in methanol) was spotted separately on TLC plate and developed in
ethyl acetate and methanol (1:1). The plate was sprayed with 0.2% DPPH solution
No. Compounds % RTa in methanol (2,2-diphenyl-1-picrylhydrazil) and left at room temperature for
1 a-Pinene 0.02 15.82 30 min. Yellow spot formed due to bleaching of purple color of DPPH reagent was
2 Sabinine 0.50 17.37 recorded as positive antioxidant activity of EO.
3 Methyl heptenone 0.65 17.60 Free radical scavenging activity of EOs of C. maxima and C. sinensis was mea-
4 b-Pinene 1.73 17.70 sured by recording the extent of bleaching of the purple-colored DPPH solution
5 b-Myrcene 0.51 17.82 to yellow. Different concentrations (2.0–10.00 ll ml1) of the samples were added
6 Cymol 1.27 19.52 separately to 0.004% DPPH solution in methanol (5 ml). After 30 min of incubation
7 Octanal 0.22 18.40 at room temperature, the absorbance was taken against a blank at 517 nm using
8 b-Ocimene 0.30 18.90 spectrophotometer. Scavenging of DPPH free radical with reduction in absorbance
9 DL-Limonine 69.78 19.75 of the sample was taken as a measure of the antioxidant activity of the essential oils
as followed by Sharififar et al. (2007). IC50, which represented the concentration of
10 b-terpinene 8.69 22.45
the EO that caused 50% neutralization of DPPH radicals, was calculated from the
11 ()-Carvyl acetate 0.09 23.60
graph plotting between percentage inhibition and concentration
12 1,3,4-Trimethyl-3-cyclohexene-1-carbaldehyde 0.10 24.07
13 L-Carveol 0.14 24.22

14 2-(3,3-Dimethylcyclohexylidene)ethanol 0.30 24.60 I % ¼ Ablank  Asample =Ablank  100;
15 c-Terpinene 0.16 26.10
16 a-terpinene 0.30 26.62 where Ablank is the absorbance of the control (without test compound), and Asample is
17 Decanal 0.06 26.72 the absorbance of the test compound.
18 ()-Carvyl acetate 0.12 26.82
19 Neryl acetate 0.83 27.50
2.9. Determination of safety profile of the oils
20 Carveol 3.86 28.12
21 Linalyl acetate 1.28 28.47 The safety profile of the essential oils of C. maxima and C. sinensis was deter-
22 L-Carvone 0.13 28.60 mined on mice (Mus musculus L., average weight 50.0 g, age 3 months) by recording
23 Z-citral 5.24 29.22 their LD50 values which represent the lethal dose of oil per unit weight for killing of
24 Sabinol 0.41 32.30 50% population of test animals (Singh et al., 2009). A stock solution was prepared by
25 a-Fenchene 0.13 32.45 mixing the EOs, tween-80 and distilled water in 2:1:1 ratio. Different dilutions of
26 Linalyl acetate 0.25 33.12 the stock solution of each essential oil were made separately with distilled water.
27 t-caryophyllene 0.18 35.40 Each dilution was orally given to each group of animal (12 mice) separately in
a which the dose of oil was 0.2–3.2 ml. In control set, equal dose of tween-80 and dis-
RT – retention time.
tilled water was given. After 24 h, the mortality of the test animals was recorded
and LD50 of EOs was calculated by probit analysis (Finney, 1971).

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

production
SMKY broth medium (Sucrose, 200 g; MgSO47H2O, 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 and
DL-limonene. The method of Sinha et al. (1993) was adopted for the estimation of
aflatoxin B1 (AFB1). Different concentrations of the oils, their combination and lim-
onene (250, 500, 750 and 1000 ppm) were prepared separately by dissolving their
requisite amount in 0.5 ml acetone and then mixing it with 24.5 ml SMKY medium
in 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 ml1) pre-
pared in 0.1% tween-80 and incubated at 28 ± 2 °C for 10 days. The content of each
flask was filtered (Whatman filter paper No. 1) and mycelium was oven dried at
100 °C to determine mycelial dry weight. The filtrate was extracted with 20 ml
chloroform 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 microliter
chloroform extract spotted on TLC plate (20  20 cm of Silica gel-G) and developed
in toluene: isoamyl alcohol: methanol (90:32:2;v/v/v). The intensity of AFB1 was
observed in UV transilluminator (Zenith, Agra) at wavelength of 360 nm.
For quantitative estimation, spots of AFB1 on TLC were scraped and dissolved in
5 ml methanol, centrifuged at 3000 rpm (5 min) and optical density of supernatant
was recorded at 360 nm using spectophotometer (Systronics, India Ltd., Mumbai).
The amount of AFB1 in control and treatment sets was calculated following Singh
et al. (2008):
1 DM
Aflatoxin B1 content ðlg kg Þ ¼  1000;
EL
where D = optical density, M = mol. wt. of aflatoxin B1 (312), E = molar extinction
coefficient (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 the
poisoned 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 through
spectrophotometer following Tepe et al. (2005).
2.10. Statistical analysis
Antifungal and antiaflatoxigenic experiments were performed in triplicate and
data 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 combination
were pale-yellow in color. The yield of C. maxima and C. sinensis
EOs 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 components
in C. maxima oil (Table 1), whereas, DL-limonene represented
90.66% 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 were
found significantly effective over control from day-2 to day-10
according 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. flavus
was found at 750 ppm of both the EOs and their combination. EO
of C. maxima was found most efficacious when compared with that
of 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 and
their combination, respectively. DL-Limonene, completely inhibited
the 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 while
DL-limonene could inhibit at 250 ppm. At 500 ppm, mycelial
growth 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 fungitoxic
spectrum against all the fungi tested and caused 100% inhibition of
 P. Singh et al. / Food and Chemical Toxicology 48 (2010) 1734–1740 1737

Table 4
Antifungal 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 inhibition
at 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 23.4
C. 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 48.1
C. 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 100
C. 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 100
C. 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 18.2
C. 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 46.2
C. 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 100
C. 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 100
Oil 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 19.8
Oil 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 44.0
Oil 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 100
Oil 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 5
Efficacy of essential oils, their combination and DL-limonene on mycelial biomass (g) and aflatoxin B1 elaboration (lg kg1) of a toxigenic strain of A. flavus in SMKY medium.

Conc. (ppm) Dry weight of mycelium (g) ± SE Aflatoxin B1 (lg kg1)

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

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

the mycelial growth of A. fumigatus, A. terreus, A. alternata, F. oxy- and 7). The high LD50 value indicates their favourable safety profile
sporum, H. oryzae and T. viride at 750 ppm. and non-mammalian toxicity.
Both the EOs exhibited DPPH radical scavenging activity in dose
dependent manner (Fig. 1). The IC50 of C. maxima and C. sinensis oils 4. Discussion
were calculated to be 8.84 and 9.45 ll/ml, respectively indicating
their strong antioxidant efficacy. The findings of present investigation revealed the antimycotic
The LD50 of the C. maxima and C. sinensis oils, determined as well aflatoxin inhibitory efficacy of the essential oils of C. max-
through oral administration on mice, was calculated to be ima, C. sinensis and their combination. The oils and the combination
36680.04 and 19938.54 ll/kg body weight, respectively (Table 6 were standardised by their chemical profile. The composition of
essential oils varies with respect to ecological and geographical
condition, age of plant and time of harvesting (Bagamboula et al.,
70 2004) and thus different chemotypes of a particular essential oil
have been reported. Such a variation in chemical composition of
Citrus maxima oil
% radical scavenging activity

60 EOs would definitely alter their biological activity. Hence, determi-

Citrus sinensis oil
nation of chemical profile of essential oil is important before
50
recommending it for antimicrobial activity. In the present investi-
gation, the GC–MS analysis revealed that monoterpenes constitute
40
more than 90% of both the EOs and their combinations. The major
component of the EOs is a cyclic monoterpene, DL-limonene fol-
30
lowed by E-citral, Z-citral (monoterpene aldehydes) and carveol,
20 the hydroxylated derivatives of DL-limonene. EOs, rich in monoter-
penes are recognized as food preservatives (Ruberto and Baratta,
10 2000). In addition, monoterpenic EOs are considered as natural
antioxidants (Yanishlieva et al., 1999) and are reported as antican-
0 cerous (Sharma et al., 2009). DL-Limonene, which was found to be
1 2 3 4 5 6 7 8 9 10 11 in appreciable amounts in the EOs of this study, showed aflatoxin
Concentration (µl/ml) inhibitory efficacy at a concentration lower than the individual oils
and the combination. Hence, it may be concluded that other
Fig. 1. Radical scavenging activity of C. maxima and C. sinensis oils. components present in the oil would be masking the efficacy of
 1738
Table 6
LD50 of the essential oil of C. maxima.

Dose Log of dose Total number Number of Percent Percent Corrected Emperical Expected Weighing Working Weight (nw) nwx nwy nwx2 nwy2 nwxy
conc. (x) of animals (n) dead animal mortality mortality probit probit (Y) coefficient (w) probit (y)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
0.2 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 192.87
1.2 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 357.9732
1.6 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 503.3724
2.0 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 539.8872
2.4 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 599.514
2.8 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 624.09

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

3.2 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 544.5144
Control – 12.0 0.0 0.0 0.0 0.0 – – – – – – – – –

LD50 36680.04 ll/kg body weight.

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

Dose Log of dose Total number Number of Percent Percent corrected Emperical Expected Weighing Working Weight (nw) nwx nwy nwx2 nwy2 nwxy
conc. (x) of animals (n) dead animal mortality (y) mortality probit probit (Y) coefficient (w) probit (y)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
0.2 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 84.4908
0.8 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 372.8712
1.0 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 373.0812
1.2 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 383.1998
1.4 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 388.4304
1.6 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 363.408
Control – 12.0 0.0 0.0 0.0 0.0 – – – – – – – – –

LD50 19938.54 ll/kg body weight.

 P. Singh et al. / Food and Chemical Toxicology 48 (2010) 1734–1740
DL-limonene and they may be working in negative direction. There
was no synergism between the oil components when the two oils
were mixed together as their aflatoxin inhibitory concentration
was the same (500 ppm). The efficacy of the C. maxima, C. sinen-
sis, their combination oil and DL-limonene as aflatoxin inhibitor is
being reported for the first time in the present investigation. The
antioxidant nature of the EOs in terms of free radical scavenger
may 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 was
recorded to be very high even in comparison to some of the well
known botanical products (azadirachtin, >5000 mg kg1; pyre-
thrum, 350–500 mg kg1) which are applied on large scale as
control of pests (Isman, 2006; Singh et al., 2009). The EOs are
thus non-mammalian toxic satisfying acceptability aspect of
consumer’s requirements and may be recommended as a safe
fungitoxicant in place of some commercial antifungals like zinc
dimethyl di thiocarbamate (LD50: 1889 mg kg1) and mancozeb
(LD50: 5000 mg kg1) (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). Being
used in traditional medicine against different human diseases
and because of appreciable LD50 value on test animal, there
would be no chance of any oral toxicity by exploiting these oils
as fungitoxicant.
In conclusion, the EOs of C. maxima, C. sinensis and DL-limonene
may be recommended as a plant based antimicrobial as well as
antioxidant food preservative for enhancement of shelf life of
stored 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 over
the synthetics because of short term toxicological testing before
formulation as antimicrobial (Varma and Dubey, 1999). The yield
of the Citrus oils in the present investigation was comparatively
high and sufficient amount of raw materials would be available
as the plants grow luxuriantly in the area. In addition, the peels
of C. sinensis remain mostly unused and are dumped as waste.
Hence, exploitation of their oil as food preservative would be
helpful in their utilization in right direction. In addition, in recent
years 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 possible
use of plant based additives in foods to prevent microbial growth
is considerably increased. Numerous EOs like lemon grass, basil,
thyme, rosemary, sage, oregano, clove, etc. are proven potentially
useful additives for food preservation against mycotoxigenic fungi
and bacteria likely to prolong shelf life and improve the quality of
stored food products (Nguefack et al., 2004). Formulation of many
plant volatiles viz. carvone (under the trade name Talent) and
DMC base naturals are widely used as food additives and most
of them are generally recognized as safe (GRAS) (Singh et al.,
2009). There would be no chance of alteration in the organoleptic
properties of food commodities when citrus EOs or their compo-
nents used as preservatives because the monoterpenes present
in these oils are widely used as natural ingredients in many food
products, soaps, soft drinks, cosmetics and perfumes for their
lemon-like flavour and odor (Shukla et al., 2009). Thus, Citrus
essential oils would be compatible with the organoleptic param-
eters with treated food commodities and their exploitation would
be safe to the consumers and may add appreciable flavour to the
treated foods.
The findings of the present investigation would draw the atten-
tion of food industries regarding large scale exploitation of these
EOs as food additives for enhancement of the shelf life. On the basis
of unique virtues possessing antifungal as well as antiaflatoxigenic
activity, broad fungitoxic spectrum, antioxidant activity and
1739
favourable safety profile, the oils may be recommended as safe
plant 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 and
Industrial Research (CSIR) New Delhi, India.
References
Adams, R.P., 2007. Identification of Essential Oil Components by Gas
Chromatography/Mass Spectrometry. Allured Publishing Corporation, Carol
Stream, IL.
Alzoreky, N.S., Nakahara, K., 2003. Antimicrobial activity of extracts from
some edible plants commonly consumed in Asia. Int. J. Food Microbiol. 80,
223–230.
Bagamboula, C.F., Uyttendaele, M., Debevere, J., 2004. Inhibitory effect of thyme and
basil essential oils, carvacrol, thymol, estragol, linalool and p-cymene towards
Shigella sonnei and S. Flexneri. Food Microbiol. 21, 33–42.
Chang, H.T., Cheng, Y.H., Wu, C.L., Chang, S.T., Chang, T.T., Su, Y.C., 2008. Antifungal
activity of essential oil and its constituents from Calocedrus macrolepis var.
Formosana Florin leaf against plant pathogenic fungi. Bioresour. Technol. 99,
6266–6270.
Chen, P.J., Moore, T., Nesnow, S., 2008. Cytotoxic effects of propiconazole and its
metabolites in mouse and human hepatoma cells and primary mouse
hepatocytes. Toxicol. in Vitro. 22, 1476–1483.
Cutler, H.G., Cutler, S.J., 1999. Biological Active Natural products: Agrochemicals.
CRS Press, Boca Raton, USA. p. 299.
Dubey, N.K., 2004. Flora of BHU Campus. BHU Press, Varanasi, India.
El-Ghaouth, A., 1997. Biologically-based alternatives to synthetic fungicides for the
control of postharvest diseases. J. Ind. Microbiol. Biotechnol. 19, 160–162.
Finney, J.D., 1971. Probit Analysis. Cambridge University Press, London. p. 333.
Isman, M.B., 2006. Botanical insecticides, deterrents, and repellents in modern
agriculture and an increasingly regulated world. Annu. Rev. Entomol. 51,
45–66.
Junior, M.R.M., Rocha e Silva, T.A.A., Franchi, G.C., Nowill, A., Pastore, G.M., Hyslop,
S., 2009. Antioxidant potential of aroma compounds obtained by limonene
biotransformation of orange essential oil. Food Chem. 116, 8–12.
Kumar, A., Shukla, R., Singh, P., Prasad, C.S., Dubey, N.K., 2008. Assessment of
Thymus vulgaris L. essential oil as a safe botanical preservative against post-
harvest fungal infestation of food commodities. Innovat. Food Sci. Emerg.
Technol. 9, 575–580.
Kumar, R., Dubey, N.K., Tiwari, O.P., Tripathi, Y.B., Sinha, K.K., 2007. Evaluation of
some essential oils as botanical fungi toxicants for the protection of stored food
commodities from fungal infestation. J. Sci. Food Agric. 87, 1737–1742.
Lanciotti, R., Gianotti, A., Patrignani, F., Belletti, N., Guerzoni, M.E., Gardini, F., 2004.
Use of natural aroma compounds to improve shelf life and safety of minimally
processed fruits. Trends Food Sci. Technol. 15, 201–208.
Nguefack, J., Leth, V., Amvam-Zollo, P.H., Mathur, S.B., 2004. Evaluation of five
essential oils from aromatic plants of Cameroon for controlling food spoilage
and mycotoxin producing fungi. Int. J. Food Microbiol. 94, 329–334.
Pavela, R., 2007. Possibilities of botanical insecticide exploitation in plant
protection. Pest Technol. 1, 47–52.
Prajapati, N.D., Purohit, S.S., Sharma, A.K., Kumar, T., 2003. A Handbook of Medicinal
Plants: A Complete Source. Agrobios Publisher, Jodhpur, India.
Ruberto, G., Baratta, M.T., 2000. Antioxidant activity of selected essential oil
components in two lipid model systems. Food Chem. 69, 167–174.
Sharififar, F., Moshafi, M.H., Mansouri, S.H., Khodashenas, M., Khoshnoodi, M., 2007.
In vitro evaluation of antibacterial and antioxidant activities of the essential oil
and methanol extract of endemic Zataria multiflora Boiss. Food Control 18, 800–
805.
Sharma, P.R., Dilip, M.M., Muthiah, S., Pal, H.C., Shahi, A.K., Saxena, A.K., Qazi, G.N.,
2009. Anticancer activity of an essential oil from Cymbopogon flexuosus. Chem.
Biol. Interact. 179, 160–168.
Shukla, R., Kumar, A., Singh, P., Dubey, N.K., 2009. Efficacy of Lippia alba (Mill.) N.E.
Brown essential oil and its monoterpene aldehyde constituents against fungi
isolated from some edible legume seeds and aflatoxin B1 production. Int. J. Food
Microbiol. 135, 165–170.
Singh, P., Kumar, A., Dubey, N.K., Gupta, R., 2009. Essential oil of Aegle marmelos as a
safe plant-based antimicrobial against postharvest microbial infestations and
aflatoxin contamination of food commodities. J. Food Sci. 74, 302–307.
1740 P. Singh et al. / Food and Chemical Toxicology 48 (2010) 1734–1740
Singh, P., Srivastava, B., Kumar, A., Dubey, N.K., 2008. Fungal contamination of raw
materials of some herbal drugs and recommendation of Cinnamomum camphora
oil as herbal fungitoxicant. Microbial Ecol. 56, 555–560.
Sinha, K.K., Sinha, A.K., Prasad, G., 1993. The effect of Clove and Cinnamon oils on
the growth of and aflatoxin productions by Aspergillus flavus. Lett. Appl.
Microbiol. 16, 114–117.
Tepe, B., Daferera, D., Sokmen, A., Sokmen, M., Polissiou, M., 2005. Antimicrobial and
antioxidant activities of the essential oil and various extracts of Salvia tomentosa
Miller (Lamiaceae). Food chem. 90, 333–340.
Varma, J., Dubey, N.K., 1999. Perspective of botanical and microbial products as
pesticides of tomorrow. Curr. Sci. 76, 172–179.
WHO (World Health Organization), 2002. World health reports 2002: reducing
risks, promoting healthy life Geneva: World Health Organization, 30 October
2002. ISBN 92 4 156207 2 ISSN 1020-3311, p. 248.
Yanishlieva, N.V., Marinova, E.M., Gordon, M.H., Raneva, V.G., 1999. Antioxidant
activity and mechanism of action of thymol and carvacrol in two lipid systems.
Food Chem. 64, 59–66.