GC MS, hoạt tính kháng nấm va kháng oxy hóa

Efficacy of chemically characterized Piper betle L. essential oil against fungal and
aflatoxin contamination of some edible commodities and its antioxidant activity
Bhanu Prakash, Ravindra Shukla, Priyanka Singh, Ashok Kumar,
Prashant Kumar Mishra, Nawal Kishore Dubey
⁎
Laboratory of Herbal Pesticides, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, India
abstractarticle info
Article history:
Received 12 January 2010
Received in revised form 20 May 2010
Accepted 15 June 2010
Keywords:
Aflatoxin B
1
Antifungal
Antioxidant
Essential oil
Piper betle
The study investigates fungal contamination in some dry fruits, spices and areca nut and evaluation of the
essential oil (EO) of Piper betle var. magahi for its antifungal, antiaflatoxigenic and antioxidant properties. A
total of 1651 fungal isolates belonging to 14 species were isolated from the samples and Aspergillus was
recorded as the dominant genus with 6 species. Eleven aflatoxin B
1
(AFB
1
) producing strains of A. flavus were
recorded from the samples. Eugenol (63.39%) and acetyleugenol (14.05%) were the major components of 32
constituents identified from the Piper betle EO through GC and GC–MS analysis. The minimum inhibitory
concentration (MIC) of P. betle EO was found 0.7 μl/ml against A.flavus. The EO reduced AFB
1
production in a

dose dependent manner and completely inhibited at 0.6 μl/ml. This is the first report on efficacy of P. betle EO
as aflatoxin suppressor. EO also exhibited strong antioxidant potent ial as its IC
50
value (3.6 μg/ml) was close
to that of ascorbic acid (3.2 μg/ml) and lower than that of the synthetic antioxidants such as butylated
hydroxytouene (BHT) (7.4 μg/ml) and butylated hydroxyanisole (BHA) (4.5 μg/ml). P. betle EO thus exhibited
special merits possessing antifungal, aflatoxin suppressive and antioxidant characters which are desirable for
an ideal preservative. Hence, its application as a plant based food additive in protection and enhancement of
shelf life of edible commodities during storage and processing is strongly recommended in view of the
toxicological implications by synthetic preservatives.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
Microbial contamination is a major problem of food and feedstuffs
during storage. Among microorganisms, moulds have potent capability
to spoil the food items by producing hydrolytic enzymes. Different types
of mycotoxins have been reported in mould contaminated edible
commodities from diverse meteorological regions of the world
(Tatsadjieu et al., 2009). However, in tropical and sub-tropical countries,
improper and traditional storage conditions provide conducive condi-
tions for the growth and proliferation of moulds. There are reports of
severe cases of mycotoxicoses in humans and livestock due to
consumption of such contaminated commodities (Bhatnagar and
Garcia, 2001). Aflatoxins produced by toxigenic strains of A. flavus,
have received significant attention throughout the world because of
their hepatocarcinogenic, teratogenic, mutagenic and immunosuppres-
sive properties (Leontopoulos et al., 2003). About 5 billion people are
exposed to aflatoxins in developing countries and aflatoxicosis is ranked
6th among the 10 most important health risks identified by WHO
(Williams et al.,2004). Despite such a high level of toxigenicity, aflatoxin
contamination in edible commodities has attracted less attention than

the bacterial contamination.
Several synthetic additives and preservatives are effectively used
in management of post harvest losses but their continuous application
may cause the development of fungal resistance as well as residual
toxicity (Brent and Hollomon, 1998). Synthetic preservatives are also
responsible for the origin of partially reduced form of oxygen such as
superoxide (O
2
−
) hydrogen peroxide (H
2
O
2
) and hydroxyl radicals
(OH
−
) which are highly reactive molecules causing oxidative diseases
by damaging the proteins, lipids and DNA (Halliwell, 1997) and also
responsible for the stimulation of aflatoxin biosynthesis (Jayashree
and Subramanyam 2000).
To overcome these problems some plant based preservatives such as
azadirachtin, carvone, allyl isothiocynate from Azadirachta indica, Carum
carvi and mustard oil, respectively have been developed as safe and
novel antimicrobials and are used on large scale as food additives
(Chacon et al., 2006; de Carvalho and da Fonseca, 2006; Gopal et al.,
2007). Among natural products, essential oils (EOs) of higher plants and
their components are gaining interest as food additives and widely
accepted by consumers because of their relatively high volatility,
ephemeral nature and bi odegradability. Carvacrol, cinnamaldehyde,
citral, thymol and limonene are some major bioactive compounds of

some essential oils which are recommended as food additives by
European commission with no harm to human health (Burt, 2004).
International Journal of Food Microbiology 142 (2010) 114–119
⁎ Corresponding author. Tel.: +91 9415295765.
E-mail address: (N.K. Dubey).
0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2010.06.011
Contents lists available at ScienceDirect
International Journal of Food Microbiology
journal homepage: www.elsevier.com/locate/ijfoodmicro
Piper betle L. (family: Piperaceae) is an indigenous climber of the
Indo-Malaya region. Its ethno-medicinal application has been well
known for a long time. It is used traditionally in skin and eye diseases
(Farnsworth and Bunyapraphatsara, 1992). Carminative, aphrodisiac
and anticancerous properties of P. betle EO have also been reported
(Manosroi et al., 2006; Bissa et al., 2007).
The present study was performed to investigate fungal contamina-
tion in some dry fruits, spices and areca nut. In addition, the EO of P.
betle var. magahi was evaluated for its antifungal, antiaflatoxigenic
and antioxidant properties in order to assess its efficacy as a food
additive.
2. Materials and methods
2.1. Chemicals and equipments
Chemicals and equipment viz. chloroform, methanol, sodium
sulphate, tween-80, toluene, isoamyl alcohol, PDA (potato, 200 g;
dextrose, 20 g; agar, 18 g and distilled water 1000 ml) and SMKY
medium (sucrose, 200 g; MgSO4·7H2O, 0.5 g; KNO3, 0.3 g; yeast
extract, 7.0 g; distilled water, 1000 ml), ascorbic acid, butylated
hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and, 2,2-
diphenyl-1-picrylhydrazil (DPPH) were procured from HiMedia Labo-

ratories Pvt. Ltd., Mumbai, India. Eugenol and Nystatin were procured
from Genuine Chemical Company, Mumbai and Wettasul-80 from
Sulphur Mills Ltd., Mumbai, India. The major equipment used were
hydro-distillation apparatus (Merck Specialities Pvt. Ltd., Mumbai,
India), centrifuge, UV transilluminator (Zenith Engineers, Agra, India)
and spectrophotometer (Systronics India Ltd., Mumbai, India).
2.2. Edible commodities
A total of 70 samples of edible commodities viz. dry fruits (Anacardium
occidentale L., Prunus amygdalus Batsch., Arachis hypogea L.), spices ( Piper
nigrum L., Piper longum L., C. sativum L.)andanut(Areca catechu L.) were
collected fro m retail outlets located i n Varanasi, India. The collected
samples w ere stored in sterilized polythene b ags to prevent further
contaminat ion a nd wer e store d at 10 °C until an alysis.
2.3. Moisture content and pH
Fifty grams of each sample was dried at 100 °C in hot air oven for
24 h and moisture content was calculated based on difference with
the fresh weight (Mandeel 2005). One gram of each material was
finely ground using mortar-pestle and 1:10 (sample: distilled water)
suspension of each sample was prepared and stirred for 24 h. The pH
of the suspension was recorded using electronic pH meter.
2.4. Mycological analysis
Mycological analysis of selected edible commodities was carried
out according to Aziz et al., (1998). Ten grams of each powdered sample
was homogenized in 90 ml sterile distilled water in an Erlenmeyer
flask (250 ml). Five fold serial dilutions were prepared and 1 ml of
aliquot (10
−4
) of each sample was inoculated on a Petri dish containing
10 ml freshly prepared PDA medium. Three replicates of each sample
were prepared and incubated (27±2 °C) for seven days. Different

fungal colonies were counted and species were identified following
Raper and Fennel (1977), Pitt (1979) and Domsch et al., (1980).The
percent relative density of different fungi and their occurrence
frequency on each sample was determined following Singh et al .,
(2008). The cultures of fungal isolates were maintained on PDA.
Relative density %ðÞ=
No: of colony of fungus
total no: of colony of all fungal species
×100
The occurrence frequency of isolated fungi was determine
following Mandeel (2005)
Occurrence frequency %ðÞ=
No: of fungal isolates on each sample
total no: of fungal isolate on all samples
× 100
2.5. Detection of aflatoxigenic isolates
Randomly selected isolates of A. flavus from each sample were
screened for their aflatoxin B
1
(AFB
1
) producing potential by thin layer
chromatography (TLC) following Kumar et al., (2007). A. flavus isolates
were aseptically inoculated in 25 ml SMKY medium and incubated for
10 days (27±2 °C). The content of each flask was filtered and extracted
with 20 ml chloroform using separating funnel. The extract was
evaporated to dryness on water bath and was redissolved in 1 ml
chloroform. Fifty microliter of chloroform extract was spotted on TLC
plates and developed in toluene:isoamyl alcohol:methanol (90:32:2; v/
v/v). The plate was air dried and AFB

1
was observed in UV-
transilluminator (360 nm). The intensity of the blue fluorescent spot
in the UV transilluminator varies with different aflatoxigenic strains
from light blue to deep blue. The toxigenic A. flavus (LHPac-3), isolated
from A. catechu produced maximum blue fluorescence under UV light
and was, therefore, selected for further investigations.
2.6. Isolation of essential oil
Leaves of P. betle L.var. magahi were purchased from the local
market of Varanasi and subjected to hydro-distillation using Cleven-
ger's apparatus (Prasad et al., 2009). The essential oil (EO) was
separated and collected in sterilized glass vial. Water traces were
removed using anhydrous sodium sulphate and EO was stored at 4 °C
for the experimental processes.
2.7. GC and GC–MS analysis of P. betle EO
P. betle EO was subjected to gas chromatography (PerkinElmer Auto
XL GC, MA, USA) equipped with a flame ionization detector and the GC
condition were: EQUITY-5 column (60 m ×0.32mm×0.25 μm); H
2
was
the carrier gas; column head pressure 10 psi; oven temperature
prog ram isother m 2 min at 70 °C, 3 °C/min gradient to 250 °C,
isotherm10 min; injection temperature, 250 °C; detector temperature
280 °C. GC–MS analysis was performed using PerkinElmer Turbomass
GC–MS. The GC column was EQUITY-5 (60 m×0.32 mm×0.25 μm)
fused silica capillary column. The GC conditions were: injection
temperature, 250 °C; column temperature, 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. Helium was
the carrier gas. The effluent of the GC column was introduced directly

into the source of MS and spectra obtained in the EI mode with 70 eV
ionization energy. The sector mass analyzer was set to scan from 40 to
500 amu for 2 s. The identification of individual compounds is based on
their retention times relative to those of authentic samples and
matching spectral peaks available with Wiley, NIST and NBS mass
spectral libraries or with the published data (Adams, 2007).
2.8. Fungitoxic investigation of P. betle EO
The mini mum inhibitory concentrations ( MICs) of EO agains t
dif ferent fungal isolates were determined using Potato dextrose
bro th (PDB) medium following Shukla et al., (2008). Different
concentrat ions of e ssential oil (0.1 to 2.0 μl/ml) were dissolved in
0.5 ml acetone and then incorporated with 9.5 ml PDB in test t ubes.
Fun gal spore suspension (10
6
spores/ml) in 0.1 % Tween-80 was
inoculated to each tube and incubated for a week. PDB without
essential oil was served as control. The lowes t concentration of the
oil that did not permit any visible fungal growt h was recorded as
115B. Prakash et al. / International Journal of Food Microbiology 142 (2010) 114–119
MIC. The tubes showing no visible fungal growth were sub-cultured
on EO-free PDA p lates to determine if the inhibition was reversible.
The fungitoxic spectrum of P. betle EO against different fungal
isolates was observed at its MIC in PDB. The MI Cs of two prevale nt
fun gicides viz. Nystatin and Wettasul-80 were also determined
against A. flavus.
2.9. Efficacy of P. betle EO and eugenol in checking
aflatoxin B
1
production
Requisite amount of P. betle EO and eugenol were dissolved

separately in 0.5 ml acetone and added to 24.5 ml SMKY to achieve the
various concentrations from 0.1 to 0.7 μl/ml. The medium inoculated
with 1 ml spore suspension (10
6
spores) of toxigenic isolate of A. flavus
(LHPac-3) was incubated for ten days at (27±2 °C). The medium was
filtered and mycelium was dried at 80 °C (12 h). AFB
1
was detected by
thin layer chromatography as mentioned in Section 2.5. The developed
blue spots on TLC plate were scratched, dissolved in methanol (5 ml)
and centrifuged at 3000 rpm (5 min). Absorbance of the supernatant
was recorded at 360 nm and AFB
1
was calculated following AOAC
(1984) and Kumar et al., (2007).
AFB
1
content μg = lðÞ
D×M
E×l
×1000:
D = absorbance, M = molecular weight (312), E = molar
extinction coefficient AFB
1
(21800), l = path length (1 cm).
2.10. Antioxidant activity of P. betle EO
The antioxidant activity of the EO was measured by DPPH radical
scavenging assay on TLC and measuring the free radical scavenging
activity through spectrophotometer following Tepe et al., (2005).

2.10.1. DPPH radical scavenging assay on TLC
To determine the antioxidant activity of EO, 5 μl (1:10 dilution in
methanol) was applied on TLC plate and developed in ethyl acetate
and methanol (1:1). The plate was sprayed with 0.2% DPPH solution in
methanol (2, 2-diphenyl-1-picrylhydrazil) and left at room temper-
ature for 30 min. Yellow spot formed due to bleaching of purple color
of DPPH reagent was recorded as positive antioxidant activity of EO.
2.10.2. Free radical scavenging activity
Free radical scavenging activity of the P. betle EO was measured by
recording the extent of bleaching of the purple-colored DPPH solution
to yellow. Different concentrations (1.25 to 10.00 μg/ml) of the
samples were added to 0.004% DPPH solution in methanol (5 ml).
After a 30 min of incubation at room temperature, the absorbance was
taken against a blank at 517 nm using spectrophotometer. Scavenging
of DPPH free radical with reduction in absorbance of the sample was
taken as a measure of their antioxidant activity following Sharififar
et al., (2007). Butylated hydroxytoluene (BHT), Butylated hydro-
xyanisole (BHA) and ascorbic acid were used as positive control. IC
50,
which represented the concentration of the essential oil that caused
50% neutralization of DPPH radicals, was calculated from the graph
plotting between percentage inhibition and concentration.
I% = A
blank
–A
sample
= A
blank

x100

where, A
blank
is the abso rbance of the control (without test
compound), and A
sample
is the absorbance of the test compound.
2.11. Statistical analysis
Antifungal and antioxidant experiments were performed in
triplicate and data analyzed are mean ±SE subjected to one way
ANOVA. Means are separated by the Tukey's multiple range test when
ANOVA was significant (p b 0.05) (SPSS 10.0; Chicago, IL, USA).
3. Results
Themoisturecontentofthecommoditiesvariedsignificantly. The
highest moisture content (25.90%) was recorded in A. hypogea followed by
P. amygdalus (21. 11%) and the lowest ( 11.36%) was in the case of
C. sati vum followed by the A. catechu (13.45%) . The magnitude of pH was
found i n acidic range. The lowest pH (4.7) was recorded in A. catechu while
the highest (6.45) in case of P. nigrum (Table 1). A total of 1651 fungal
isolates bel ongi ng to 14 species were recorded from the samples.
Aspergillus was recorded as the dominant genus. Aspergillus flavus and
Aspergillus niger were found in all the investigated samples. Some fungi
viz. Nigrospora sp., Mycelia sterilia, Aspergillus terreus were found only in P.
amygdalus, Anacardium occidentale, A. hypogea, respectively. The highest
percent relative density was recorded with A. flavus (40.69%) followed by
A. niger (24.10%) and C. cladosporioides (11.81%). The lowest relative
density was recorded with mucorales (0.72%) followed by Nigrospora sp.
(0.90%). Highest frequency of occurrence was recorded in A. catechu
(20.65%), whereas, minimum (9. 87%) in C. sativum and Piper longum.
During the investigations on toxigenicity of A. flavus isolates from
the selected commodities, 11 isolates out of 24 were found

aflatoxigenic with blue spots on TLC plates. The toxigenic A. flavus
(LHPac-3), isolated from A. catechu was used for antiaflatoxigenic
bioassay as it produced maximum blue fluorescence under UV light.
The yield of EO was 4.0 ml/kg through hydro-distillation. Chemical
compositions of EO were identified by the GC–MS analysis and 32
different components were identified. Their retention time and area
percentage are summarized in Table 2. Major components of EO were
eugenol (63.39%) and acetyleugenol (14.05%).
MIC of P. betle EO against A. flavus was found at 0.7 μl/ml. The oil
exhibited pronounced fungitoxicity against all the fungal isolates. The
lowest MIC (0.3 μl/ml) of the oil was recorded against M. sterilia and
the highest was observed against A. niger as 0.73 μl/ml (Table 3). The
fungicides viz. Nystatin and Wettasul-80 inhibited A. flavus at 1.85 μl/
ml and 2.78 mg/ml, respectively, and thus found to be less efficacious
than the P. betle EO.
The P. betle EO inhibited AFB
1
production in a dose dependent
manner. At the lowest concentration of 0.1 μl/ml, enhanced AFB
1
production (1165.93 μg/l) was recorded even higher than the control
set (978.93 μg/l). However, the P. betle EO inhibited AFB
1
production
on higher concentrations and completely inhibited at 0.6 μl/ml
(Table 4 ). Eugenol, the major component of the P. betle EO was
found to be more efficacious than the oil. It inhibited the growth of the
toxigenic strain LHPac-3 of A. flavus and the aflatoxin production at
0.4 μl/ml and 0.1 μl/ml, respectively.
The appearance of yellow spot due to bleaching the purple color of

the DPPH confirmed the positive antioxidant activity of EO. Percent
inhibition and IC
50
values of EO and synthetic antioxidant are
summarized in Fig 1. The oil showed strong free radical scavenging
activity as its IC
50
value (3.6 μg/ml) was found close to ascorbic acid
(3.2 μg/ml) and lower than BHT (7.4 μg/ml), BHA (4.5 μg/ml).
4. Discussion
The results of the present investigation indicate that all the selected
edible commodities were heavily contaminated with the different
mould species. The samples were also found associated with toxigenic
strains of A. flavus. Hence, the biodeterioration of the samples was
qualitative as well as quantitative in nature.
Moisture content and pH are two main abiotic factors responsible for
the growth and proliferation of moulds. In all the samples, pH and
moisture content ranged between 4.7 to 6.4 and 11 to 25%, respectively,
which are favorable limits for the growth of moulds. High moisture
content of most of the samples may be one of the factors for their
116 B. Prakash et al. / International Journal of Food Microbiology 142 (2010) 114–119
biodeterioration. However, a critical observation on the mycological
analysis of the samples clearly showed that neither moisture content
nor pH of the samples individually influenced the fungal distribution. A.
catechu having comparatively lower moisture content (13.45%) showed
the highest occurrence frequency and diversity of moulds as well as
aflatoxin content indicating that chemical profile of substrate may also
be a deciding factor for the growth of moulds strengthening the earlier
hypothesis of Singh et al., (2008).
GC and GC–MS analysis of EO revealed 32 different components

which constitute 97% of the oil. In the present investigation, eugenol
(63.39%) and its ester derivative acetyleugenol (14.05%) were recorded
as major components of oil. However, some earlier workers have
reported phenolics like chavibetol (53.1%) and chavibetol acetate
(15.5%) (Rimando et al., 1986), safrol (48.69%) (Arambewela et al.,
2005) and 4-allyl-2-methoxy-phenolacetate (31.47%), 3-allyl-6-meth-
oxyphenol (25.96%) (Apiwat et al., 2006) as prime components of P.
betel EO. Such chemotypic variations have been reported in most of the
EOs due to ecological and geographical conditions, age of the plant and
time of harvesting (Bagamboula et al., 2004). The apparent variation in
the chemical profile the oils may influence their antimicrobial activity.
Hence, it is advisable that the percentage of the major components of the
EOs should be mentioned if applied as food additive.
The literature is so far silent about the antifungal efficacy of P. betle
EO against storage fungi. Hence, detailed investigations were performed
to record its efficacy as fungitoxicant, aflatoxin suppressor and
antioxidant to evaluate it as a novel plant based antimicrobial and
food additive. The efficacy of EO against the moulds is either due to the
effect of major component or by the synergistic effect of overall
components (Burt, 2004). However, in the present investigation,
eugenol, the major component of the P. betle EO was more efficacious
as fungal growth inhibitor and aflatoxin suppressor than the EO. It
appears that the remaining components of the oil synergistically acted
in negative direction and reduce the activity of eugenol. It is also
Table 1
Mycoflora analysis of selected edible commodities.
Commodities
name
Fungal species pH Moisture
content

Total
isolates
Total
species
Occurence
frequency
A.f. A.n. A.fu. A.s. A.c. A.t. P.i. F.o. C.c. C.l. A.a. M.s. N.i. M.
Anacardium
occidentale
110 49 32 8 10 – 18 – 40 – 20 – 4 5.93±
0.23
ab
16.06±
1.11
b
291 9 17.62
Prunus
amygdalus
40 30 10 – 40 ––10 20 –––15 3 5.56 ±
0.18
bc
21.11±
1.13
c
168 8 10.18
Arachis
hypogea
140 39 12 ––18 7 – 35 – 12 –––6.44±
0.08
a

25.90±
1.09
d
263 7 15.93
Piper
nigrum
125 55 9 9 30 – 4 – 30 –––––6.45±
0.03
a
15.11±
0.93
ab
262 7 15.86
Piper
longum
70 40 – 10 ––17 – 20 – 6 –––5.93±
0.04
ab
14.87±
0.60
ab
163 6 9.87
Coriandrum
sativum
68 75 –– 8 ––10 – ––––2 5.12±
0.05
cd
11.36±
0.39
a

163 5 9.87
Areca
catechu
118 110 16 –––10 – 50 17 17 ––3 4.70 ±
0.06
d
13.45±
0.63
ab
341 8 20.65
Total
isolates
671 398 79 27 88 18 56 20 195 17 35 20 15 12 1651
Relative
density
40.69 24.10 4.78 1.60 5.33 1.09 3.39 1.21 11.81 1.03 2.12 1.21 0.90 0.72
A.f. Aspergillus flavus, A.n. Aspergillus niger, A.fu. Aspergillus fumigatus, A.s. Aspergillus sydowi, A.c. Aspergillus candidus, A.t. Aspergillus terreus, P.i. Penicillium italicum, F.o. Fusarium
oxysporum, C.c. Cladosporium cladosporoides, C.l. Curvularia lunata, A.a. Alternaria alternata, M.s. Mycelia sterlia, N.i. Nigrospora sp., M. Mucor sp.
The means followed by same letter in the same column are not significantly different according to ANOVA and Tukey's multiple comparison tests.
Table 2
Chemical composition of P. betle essential oil.
Sn. Compound Rt. (min.) %
1 α-pinene 9.6 0.09
2 Camphene 10.150 0.09
3 β-myrcene 11.425 0.12
4 L-limonene 13.100 0.28
5 Cis-ocimene 13.300 0.20
6 Phenyl acetylaldehyde 13.650 0.13
7 t-ocimene 13.800 0.66
8 Linalyl acetate 16.050 0.20

9 Decanal 20.975 0.18
10 Chavicol 23.275 0.55
11 Cyclohexene,4-methyl- 27.476 0.15
12 Chavicol 27.701 0.55
13 Eugenol 28.851 63.39
14 β-elemene 30.176 0.24
15 Methyl-eugenol 30.426 0.21
16 Undecanal 30.576 0.43
17 t-caryophyllene 31.501 4.22
18 Bicyclo(4.1.0)hept-3-en- 31.876 0.12
19 α -humulene 33.01 0.68
20 γ-muurolene 33.926 1.27
21 Germacrene D 34.251 2.85
22 Germacrene B 34.876 0.81
23 Acetyleugenol 35.826 14.05
24 Aluminum sulphate 38.651 0.34
25 Ledene 39.001 0.18
26 Globulol 40.126 0.12
27 4-allyl-1,2-diacetoxybenzene 40.676 0.13
28 γ-cadinene 40.926 3.85
29 γ-muurolene 41.426 0.15
30 t-caryophyllene 41.551 0.53
31 Aluminum sulphate 42.151 0.10
32 γ-ionene 42.751 0.13
Rt: retention time.
Table 3
Minimum inhibitory concentration (MIC) of P. betle essential oil against fungal isolates.
Fungal isolates MIC (μl/ml)
Aspergillus flavus 0.70± 0.000
ab

Aspergillus niger 0.73± 0.016
a
Aspergillus fumigatus 0.40± 0.000
fg
Aspergillus terreus 0.60± 0.000
bcde
Aspergillus sydowi 0.63± 0.033
abcd
Apergillus candidus 0.57± 0.033
cde
Penicillium italicum 0.40± 0.000
fg
Fusarium oxysporum 0.50± 0.000
ef
Alternaria alternata 0.53 ±0.033
de
Cladosporium cladosporoides 0.67± 0.033
abc
Curvularia lunata 0.50± 0.000
ef
Mucor sp. 0.37± 0.033
g
Nigrospora sp 0.53± 0.033
de
Mycelia sterilia 0.30± 0.000
g
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.
117B. Prakash et al. / International Journal of Food Microbiology 142 (2010) 114–119

apparent from the present investigation that the eugenol has tremen-
dous capacity as aflatoxin inhibitor than the growth suppressor. The
presence of OH group in eugenol may be able to form hydrogen bonds
with the active site of the target enzymes and increases the activity by
denaturing the enzyme, responsible for toxin secretion as emphasized
by Bluma et al., (2008).The oil exhibited remarkable fungitoxicity
against all the fungal isolates infesting different edible commodities. The
EO was also found more efficacious than the two commonly used
fungicides viz. Nystatin and Wettasul-80. The MIC of EO against A. flavus
was found to be lower than some earlier reported EOs viz. Ocimum
grattissimum, Ocimum basilicum, Cymbopoga citrates, Thymus vulgare,
and Monodora myristica (Nguefack et al., 2004). Hence, the EO of the P.
betle may be recommended for complete protection of food commod-
ities from the fungal infestation at low concentration.
At low concentration of EO (0.1 μl/ml), AFB
1
production by the
toxigenic strain of A. flavus was increased than the control. However, the
aflatoxin inhibitory efficacy of the oil enhanced with higher concentra-
tions and at 0.6 μl/ml, it completely checked aflatoxin production by the
toxigenic isolate. It shows that the low fungicide doses create some
stress condition which was responsible for the production of more
secondary metabolites as a defense mechanism by the fungus. Some
earlier workers have also reported that low fungicide doses to stimulate
the toxin production (Magan et al., 2002).
Free radical scavenging activity of the P. betle EO was found to be
concentration dependent. The IC
50
value of the EO was very close to that
of ascorbic acid and lower than that of BHT and BHA, thus, reflecting its

superiority as better preservative over the synthetic antioxidants. The
IC
50
of P. betle EO was also found quite lower than that of some earlier
reported EOs viz. Zataria multiflora and Thymus caramanicus whose IC
50
values were 22.4 and 263.09 μg/ml, respectively (Sharififar et al., 2007;
Safaei-Ghomi et al., 2009). Free Radical scavenging activity of EOs may
be due to the presence of thephenolic compounds or synergistic effect of
overall compounds (Sharififar et al., 2007). Because of free radical
scavenging activity, the oil may be recommended as a plant based
antioxidant in enhancement of shelf life of food commodities, thus,
retarding oxidative rancidity of lipids. In addition, its use as preservative
of edible commodities would protect the human being from oxidative
diseases.
EOs, being plant based product and biodegradable in nature may be
used as alternatives of synthetic preservatives and fumigants against
biodeterioration of food items. Many of the antimicrobial formulations
containing the EOs and their constituents are actually exempted from
toxicity data requirements by the EPA (Burt, 2004; Holley and Patel,
2005). Essential oils of many edible and medicinal plants are used in
different pharmaceutical preparations which minimize questions
regarding their safe use. Essential oils from aromatic and medicinal
plants are potentially useful as antimicrobial agents and their uses as
medicines have long been recognized (Kim et al., 1995). The attraction
of modern society towards herbal products (Smid and Gorris, 1999)
desiring fewer synthetic ingredients in foods and recommendation of
herbal products as ‘generally recognized as safe’ (GRAS) as food
additives may lead scientific interest in the exploitation of essential
oils as plant based food additives. A few EO based preservatives are

already commercially available (Mendoza-Yepes et al., 1997).
In conclusion, the present study explores the effica cy of P. betle EO as
antifungal, antiaflat oxigenic and antioxidan t agent. Our study is the first
report on antiaflatoxigenic activity of P. betle EO to the b est of o ur
knowledge. The leaves of the plant are chewed by most of the Indians
because of its stimulating qualities (Bissa et al., 2007). Hence, t her e would
be no chance of off-flavour and adverse of organoleptic taste of the treated
edibles if the P. betle EO is recommended as plant based antimicrobial.
Moreover, the P. betle EO oil would be cheaper in f ormula tion because of
availability of sufficient amount of raw material and high yield of the oil
during hydro-distillation. Based on the findings of the present investiga-
tion, it appears that P. betle EO has special merit possessing antifungal,
aflatox in suppressive and antioxidant characters which are de sirabl e of an
ideal preservative. Therefore, its application in protection and enhance-
ment of shelf life of edible commodities during the storage and processing
is strongly recommended as a botanical food additive.
Acknowledgement
Authors are thankful to Council of Scientific and Industrial
Research (CSIR), New Delhi, India for financial assistance.
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Table 4
Effect of different concentrations of P. betle essential oil and eugenol on mycelial weight
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Conc. Piper betle EO Eugenol
(μl/ml) MDW AfB
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content MDW AfB
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a
978.93 ±11.91
b
532.33 ±08.95
a
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a
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ab
1165.93 ± 24.37
a
494.33 ±07.31
ab
0.0
b
0.2 480.00± 10.39
bc
832.00 ±28.87
c
466.33 ±06.00
b
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0.3 433.67± 13.02
cd
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d
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c
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b
0.4 390.00± 12.58
de
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e
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d
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e
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e
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f
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