Food Control 34 (2013) 763e769

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Food Control
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Biological activities of Boswellia sacra extracts on the growth and

aflatoxins secretion of two aflatoxigenic species of Aspergillus species
Saifeldin A.F. El-Nagerabi a, *, Abdulkadir E. Elshafie b, Suleiman S. AlKhanjari a,
Saif N. Al-Bahry b, Mohamed R. Elamin c
a
Department of Biological Sciences and Chemistry, College of Arts and Sciences, University of Nizwa, P.O. Box 33, PC 616 Birkat Al Mouz, Nizwa, Oman
b
Department of Biology, College of Science, Sultan Qaboos University, P.O. Box 36, PC 123 Al Khoud, Muscat, Oman
c
Department of Chemistry, Sudan Academy of Science, P.O. Box 268, PC 11111 Khartoum North, Sudan

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

Article history: Aflatoxins are the most serious carcinogenic, hepatotoxic, teratogenic and mutagenic secondary me-
Received 5 April 2013 tabolites which adversely affect human and animal health. This study was designed to evaluate the
Received in revised form in vitro inhibitory effect of different concentrations of Boswellia sacra resin (2.5, 5, 7.5 and 10 g/100 ml),
14 June 2013
leaf extract (5, 7.5, 10, 12.5 and 15 ml/100 ml), and essential oil (1, 2, 3, and 4 ml/100 ml) on the growth
Accepted 22 June 2013
and aflatoxins production by two species of Aspergilli, namely Aspergillus flavus (SQU21) and Aspergillus
parasiticus (CBS921.7). Resin of B. sacra caused 57.9e92.1% inhibition of aflatoxin secretion by A. flavus
Keywords:
and 43.6e95.7% for A. parasiticus. However, the mycelial dry weights were significantly increased by 20.9
Aflatoxins
Aspergillus flavus
e52.7% for A. flavus, and 8.9e68.5% for A. parasiticus. The leaf extract of B. sacra apparently enhanced
A. parasiticus aflatoxins production by 20e50%, and mycelial dry weight by 25.5e29.1% for A. flavus and A. parasiticus.
Detoxification The essential oil of B. sacra at different concentrations similarly inhibited the fungal growth and afla-
Frankincense toxins production by 45.8e83.7% for A. flavus and 41.3e83.5% for A. parasiticus which indicates the
antifungal activity of this oil. None of the B. sacra extracts detoxified pure aqueous aflatoxin B1. We have
concluded that B. sacra resin and essential oil possess biological activity against biochemical synthesis
and metabolic pathway of aflatoxin production of the two Aspergillus species. Therefore, the resin and
essential oil of B. sacra can be recommended as safe plant based bioreservatives to enhance shelf life of
food and feed products with reference to adverse effect of physical and synthetic chemical preservatives
and their antimicrobial and aflatoxins inhibition activity.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction (Mothana, Hasson, Schultz, Mowitz, & Lindequist, 2011). The

The genus Boswellia of the family Burseraceae includes eight
species that grow in the Arabian Peninsula, India and East Africa
(Gamarda, Dayton, Distefano, Pitonzo, & Schillaci, 2007; Hasson
et al., 2011; Miller & Morris, 2004). There are many varieties of
Boswellia serrata in India, Boswellia carteri in East Africa and China,
Boswellia frereana in Somalia, and Boswellia sacra in Oman, Island of
Soqotra (Yemen). They produce slightly different types of resin
known as frankincense (Olibanum, oleogum), “Luban” in Arabic
and “Levonah” in Hebrew (Hasson et al., 2011). Frankincense resin
has been important for civilizations of the Arabian Peninsula and
North Africa as precious commercial product and incense material
* Corresponding author. Tel.: þ968 96365051; fax: þ96825443191.
E-mail addresses: nagerabi@hotmail.com, nagerabi@unizwa.edu.om (S.A.F. El-
Nagerabi).
chemical nature of Boswellia species has been reported by many
researchers. These investigations led to identification of 67e72
chemical constituents in different Boswellia species such as essen-
tial oil with monoterpene hydrocarbon (32.8%), a-thujene (9.3%), a-
pinene (8.3%), diterpene (31.7%), incensol (14.8%), and oxygenated
monoterpenes (30.7%) with p-cymene (13%), 2-hydroxy-5-
methoxy-acetophenone (16.3%), and comphore (11.6%) (Al-Harrasi
& Al-Saidi, 2008; Mothana et al., 2011). In addition, frankincense
has been reported to be a rich source of non-volatile triterpenoic
constituents from the types of ursane, oleonane, and lupine, which
are in many cases responsible for various biological activities
(Büchele, Zugmaier, & Simmet, 2003; Safayhi & Sailer, 1997; Singh
et al., 2008). Boswellia species is among the most important me-
dicinal plants, which are used for relieving fever and pains as well
as disturbed stomach (Miller & Morris, 2004; Mothana et al., 2011).
Traditionally, the frankincense resin has been used for the treat-
ment of rheumatic and other inflammatory diseases (Banno et al.,

0956-7135/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodcont.2013.06.039
764 S.A.F. El-Nagerabi et al. / Food Control 34 (2013) 763e769
2006; Langmead & Rampton, 2006; Mothana et al., 2011). Toxicity
studies show that resin preparations could be effective in the
treatment of chronic colitis with minimal side effects (Gupta et al.,
2001). Supplementation of different plant extracts including resin
from Boswellia was beneficial in modulating the alteration induced
in kidney and heart of animals under toxic effect of AFB1
(Abulmajeed, 2011). Essential oil prepared from hydrodistillation
has tumor cell-specific cytotoxicity in multiple cancer cell types
(Suhailet al., 2011). Therefore, it has gained obvious attention of
researchers and pharmaceutical companies. Several studies have
reported on the anticancer, anti-inflammatory, immunomodula-
tory, antimicrobial, and antiviral activities of several Boswellia
species (Akihisa et al., 2006; Ammon, Safayhi, Mack, & Sabieraj,
1993; Badria, Mikhaeil, Maatooq, & Amer, 2003; Banno et al.,
2006; Mikhaeil, Moatooq, Badria, & Amer, 2003; Mothana &
Lindequist, 2005; Mothana, Mentel, Reiss, & Lindequist, 2006;
Mothana et al., 2011). This prompted us to evaluate the antifungal
properties and detoxification activities of these extracts on afla-
toxigenic Aspergillus species.
Moulds are significant decomposers that adversely affecting
human food and animal feed, by retarding their nutritive value and
producing toxigenic secondary metabolites (El-Nagerabi, Al-Bahry,
Elshafie, & AlHilali, 2012; El-Nagerabi, Elshafie, AlKhanjari, Al-
Bahry, & Elamin, 2013; Herzallah, 2009; Jouany, 2007; Mishra &
Dubey, 1994; Salim & Ahmad, 2010). These mycotoxins are mainly
produced by the fungal genera of Aspergillus, Fusarium and Peni-
cillium, which contaminate agricultural products before or after
harvest. They lowered growth performance, and cause sickness or
death of humans and animals (Kumar, Basu, & Rajendran, 2008;
Salim & Ahmad, 2010; Wagacha & Muthomi, 2008). Of these my-
cotoxins, aflatoxins (AFs), are the most toxic secondary metabolites
of the genus Aspergillus flavus, Aspergillus parasiticus, Aspergillus
nominus and Aspergillus pseudotamarii (El-Nagerabi, Al-Bahry, et al.,
2012; Elshafie, ElMubarak, El-Nagerabi, & Elshafie, 2010). Aflatoxins
in general (AFB1, AFB2, AFG1 and AFG2) and particularly Aflatoxin B1
are the most serious threat to public health due to their carcino-
genic, hepatotoxic, teratogenic and mutagenic effects for both hu-
man and his domestic animals (Banu & Muthumary, 2010; Joseph,
Jayaprakasha, Selvi, Jena, & Sakariah, 2005; Peraica, Domijan,
Ana-Marija, Jurjevic, & Cvjetkovic, 2002; Wild & Turner, 2002).
The vegetative growth and associated aflatoxins produced by
A. flavus, and A. parasiticus have been proven to be sensitive to
different plant extracts and essential oils (EOs) extracted from
various medicinal and herbal plants (El-Nagerabi, Al-Bahry, et al.,
2012; Fiori et al., 2000; Prakash, Singh, Kedia, & Dubey, 2012;
Soliman & Badeaa, 2002). Aqueous leaf extracts obtained from
mature leaves of Vernonia amygdalina, Sena elata and Cymbopogon
citrutus apparently inhibited the fungal growth and reduced afla-
toxins production by Aspergillus species (Suleiman, Emua, & Taiga,
2008). Similar effects were obtained by plant extracts of Syzigium
aromaticum, Curcuma longa, Allium sativum and Ocimum sanctum
(Reddy, Reddy, & Muralidharan, 2009), and extracts from fruit rinds
of Garcinia cowa and Garcinia pendunculata (Joseph et al., 2005),
herbal compounds (Gowda, Malati, & Suganthi, 2004), dry leaves
and calyx extracts of Hibiscus sabdariffa (Al-Shayeb & Mabrook,
1984; El-Nagerabi, Al-Bahry, et al., 2012; El-Nagerabi, Elshafie,
et al., 2013), and fruit pulp of Adansonia digitata (Baobab) (El-
Nagerabi, Elshafie, et al., 2013). Many of the tested oils had
different effects on the fungal growth and aflatoxin production
(Gandomi et al., 2009; Shukla, Singh, Prakash, & Dubey, 2012;
Szczerbanik, Jolbing, Morris, & Holford, 2007). Oils extracted from
anise, baobab, black cumin, caraway, cinnamon, clove and fennel
revealed variable fungistatic effects against A. flavus and inhibition
of aflatoxins production by Aspergillus species (Bullerman, Lieu &
Seier, 1977; El-Nagerabi, Al-Bahry, et al., 2012; El-Nagerabi,
Elshafie, et al., 2013; Farag, Daw, & Abo-Raya, 1989; Hasan, 1994;
Patkar, Usha, Shetty, Poster, & Lacey, 1993; Soher, 1999).
Worldwide, various researchers are studying the antimicrobial
properties of various plant extracts and essential oils (Eos) on the
fungal growth and the subsequent aflatoxin production. Boswellia
extracts proved effective against a panel of bacteria (Weckesser
et al., 2007). However, neither water nor methanolic extracts
showed any activity against Candida albicans and Candida maltosa
(Hasson et al., 2011). All essential oils of Boswellia species possessed
antimicrobial activity, especially against Gram-positive bacteria
(Mothana et al., 2011). Frankincense oil at a concentration of 2%
showed the strongest inhibition of mycelial growth and spores of
pathogenic fungi (Udomsilp, Piyo, Khang-Khun, & Thobunluepop,
2009). Supplementation of aqueous extract from B. serrata resins
effectively ameliorated the deviation induced in both kidneys
and hearts of animals in response to AFB1 administration
(Abdulmajeed, 2011).
Numerous synthetic chemicals have been used for controlling
fungal invasion of agricultural crops and food commodities (Kumar,
Shukla, Singh, & Dubey, 2009). However, reduction and detoxifi-
cation of aflatoxins in food by physical and chemical methods have
not yet proven to be an effective or desirable practice. This is due to
their residual toxicities and adverse effects on the food chain
(Alberts, Gelderblom, Botha, & Vanzyl, 2009; Kumar et al., 2009).
Therefore, the biological detoxification offers promising alternative
for eliminating aflatoxin and safe guarding the quality of the food
and feed (Alberts et al., 2009; Oguz, 2011). The current debate on
the negative effects of synthetic preservatives has also renewed the
interest of consumers towards natural food additives for improving
the shelf life of food products and protecting them from toxic mi-
croorganisms (Prakash, Shukla, Singh, & Mishra, 2011). Since these
compounds are eco-friendly and harmless to humans, there is
increasing attention, both in industry and academic research, to
herbal, medicinal and aromatic plants for their antifungal activities
against food spoilage and aflatoxigenic fungi (El-Nagerabi, Al-
Bahry, et al., 2012; El-Nagerabi, Elshafie, et al., 2013; Gandomi
et al., 2009; Maraqa et al., 2007; Montes-Belmont & Carvajal, 1998;
Patkar et al., 1993; Soher, 1999; Soliman & Badeaa, 2002). Many
studies revealed the antimicrobial and antiviral properties of
different species of Boswellia plant (Mothana & Lindequist, 2005;
Mothana et al., 2006, 2011). Therefore, the present study was
designed to evaluate the in vitro biological effects of frankincense
resin, green leaf extract, and essential oil of B. sacra from Oman on
the mycelial growth and aflatoxins production by two Aspergillus
species, namely A. flavus (Strain SQU21) and A. parasiticus (Strain
CBS921.7). We anticipate these findings will lead to major devel-
opment in food production and preservation technology.
2. Materials and methods
2.1. A. flavus and A. parasiticus isolates
Two species of high aflatoxin-producers namely A. flavus
(SQU21) and A. parasiticus (CBS921.7) [NRR22999] were obtained
from the culture collection of Sultan Qaboos University, Oman. The
isolates were inoculated on Potato Dextrose Agar (PDA) and iden-
tified according to Raper and Fennell (1965).
2.2. Sources and characteristics of B. sacra extracts
In this study, frankincense resin powder, green leaf, and oil were
used. The resin is obtained through incisions made in the trunks of
Boswellia trees and the resin oozes from the incisions, allowed to dry
for 3e4 days, manually collected, cleaned from plant debris, and sent
to the market without any other treatment. The resin granules were
 S.A.F. El-Nagerabi et al. / Food Control 34 (2013) 763e769
purchased from the local market of Nizwa, Oman. The resins were
ground into fine powder and stored in polyethylene bags at room
temperature (25e29  C). The oil was extracted from the dry resins
through hydrodistillation using a Quart Stove Still Unit with Cle-
venger’s apparatus (Department of Chemistry, Sudan Academy of
Science). Each one kilogram yields about 120e150 ml of oil. After
complete exhaustion, the collected oil was kept in glass vials and
stored at 4e5  C. The green leaves were collected from Dhofar re-
gion, Oman. The green leaf extract was prepared by mixing 50 g of
leaves with 200 ml distilled water and grinding this mixture using an
automatic blender at high speed for 2 min. The mixture was filtered
through Whatman filter paper No. 4. The filtrate was kept in a re-
agent bottle and stored in a refrigerator at 4e5  C.
2.3. Inoculation of Aspergillus species on media containing
frankincense extracts
A. flavus (SQU21) and A. parasiticus (CBS921.7) were inoculated
on Potato Dextrose Agar (PDA) and incubated at ambient temper-
ature (25e29  C) for 10 days. After the incubation period, sterile
thin glass tubes of 5 mm in diameter were used to cut several small
circular discs from the sporulating cultures. Two discs were added
aseptically to 200 ml of sterile yeast malt broth in 250 ml conical
flasks containing resin (0.0%, 2.5%, 5.0%, 7.5% and 10.0% w/v), leaf
extract (0.0%, 5%, 7.5%, 10%, 12.5% and 15% v/v), or oil (0.0%, 1%, 2%,
3% and 4% v/v) of frankincense (olibanum). As a negative control,
10% resin, 15% leaf extract and 4% oil were added to yeast malt broth
without any fungal inoculation. Triplicates of the inoculated flasks
were incubated at 25e29  C for 15 days. Another sets were pre-
pared and the mycelia of the fungi were filtered and the dry weight
was determined using the Oven method.
2.4. Effect of frankincense extracts on pure aflatoxin B1
Pure aflatoxin B1 of 870 ppb concentration was prepared in
100 ml sterile distilled water. The highest concentrations of resin
(10 g/100 ml) and leaf extract (15 ml/100 ml) and oil (4 ml/100 ml)
were selected and added separately to different flasks containing
pure aflatoxin B1. As a control, flask containing aflatoxin B1 and
without any extract was set. The flasks were incubated at 25e29  C
for 10 days. The concentration of the aflatoxin B1 was measured.
2.5. Extraction and determination of aflatoxin by Alfa Test-P Affinity
For aflatoxin extraction, the 200 ml fungal culture were mixed
with 5 g of NaCl salt and 100 ml extraction solution of meth-
anol:water (70:30 V/V) as described by El-Nagerabi, Al-Bahry, et al.
(2012). The content was then blended at high speed for 1 min and
filtered; 15 ml of the filtrate were diluted with 30 ml distilled water,
mixed thoroughly and filtered through glass microfilters. Ten
milliliter from the diluted filtrate (10 ml ¼ 1.0 g sample equivalent)
were passed through Afla-Test-P Affinity Column at a rate of 1e2
drops per second. The column was then cleaned using 10 ml
distilled water, and the aflatoxin was eluted with one ml methanol
(HPLC grade). Then, 1 ml of Afla-Test developer was added to the
elute in the cuvette, and mixed. The blank contains 1 ml methanol
and aflatoxin developer. The concentration of the aflatoxin was
measured using calibrated and adjusted Vicam fluorometer (Series-
4EX) with excitation wavelength of 360 nm and emission wave-
length of 440 nm (Elshafie & Al-Shally, 1998).
2.6. Statistical analysis
A one-way ANOVA test (correlation coefficient) was used to
determine the variation between the effect of different
765
concentrations of B. sacra extracts on aflatoxin secretion and
mycelial dry weight compared to the control. The analysis was
carried out using SPSS software (version 11.0).
3. Results and discussion
The potential effects of certain plant extracts and biocontrol
agents on the fungal growth and aflatoxins production have been
under investigation by many authors (Gowda et al., 2004; Joseph
et al., 2005; Reddy, Reddy, & Muralidharan, 2009; Suleiman,
Emua, & Taiga, 2008). Much emphasis was given to herbal, me-
dicinal and aromatic plants for their antifungal activities against
food spoilage and aflatoxigenic fungi (El-Nagerabi, Al-Bahry, et al.,
2012; El-Nagerabi, Elshafie, et al., 2013; Gandomi et al., 2009;
Maraqa et al., 2007; Montes-Belmont & Carvajal, 1998; Patkar et al.,
1993; Soher, 1999; Soliman & Badeaa, 2002). In the present study,
the effects of different concentrations of frankincense resin, green
leaf extract, and essential oil of B. sacra on the growth and aflatoxins
production of A. flavus (SQU21) and A. parasiticus (CBS921.7) were
investigated. The results showed that the total aflatoxins produced
by the two species were significantly (p < 0.05) inhibited by all
tested concentrations of frankincense resin (2.5, 5, 7.5, and 10 g/
100 ml) in comparison with the control. The percentages of afla-
toxins inhibition ranged between 57.9e92.1% and 43.6e95.7% for
A. flavus (SQU21) and A. parasiticus (CBS921.7), respectively (Fig. 1).
On the contrary, the mycelial dry weights of the tested species were
significantly (p < 0.05) increased with concentrations of resin
(Fig. 2). The dry weight was enhanced by 20.9e52.7% for A. flavus
(SQU21) and 8.9e68.5% for A. parasiticus (CBS921.7).
It is commonly known that not all isolates of Aspergillus are able
to produce aflatoxin; however, more than 50% of these isolates are
aflatoxin-producers secreting different types of aflatoxins (El-
Nagerabi, Al-Bahry, et al., 2012; El-Nagerabi, Elshafie, et al., 2013;
Lisker, Michaeli, & Frank, 1993). The two species used in the present
study produce high concentrations of aflatoxins and were recently
used in similar investigations (El-Nagerabi, Al-Bahry, et al., 2012;
El-Nagerabi, Elshafie, et al., 2013). Numerous studies have been
conducted on anticancer, anti-inflammatory, immunomodulatory,
antimicrobial, and antiviral activities of many species of Boswellia
(Akihisa et al., 2006; Ammon et al., 1993; Banno et al., 2006; Badria
et al., 2003; Mikhaeil et al., 2003; Mothana & Lindequist, 2005;
Mothana et al., 2006, 2011). To our knowledge, the detoxification
properties of resin, leaf and oil extract of B. sacra on aflatoxigenic
fungi had not been evaluated. This enforces the need for investi-
gating the inhibitory effects and detoxification properties of B. sacra
extracts and further identification of the biologically active chem-
ical ingredients. In the present investigations, the concentrations of
frankincense resin of between 2.5 and 10% resulted in 57.9e92.1%
Fig. 1. Aflatoxin production of A. flavus strain SQU21 and A. parasiticus strain CBS921.7
at different concentrations of B. sacra resin (identical letters and numbers indicate no
significant difference, p < 0.05).
766 S.A.F. El-Nagerabi et al. / Food Control 34 (2013) 763e769

Fig. 2. Mycelial dry weights of A. flavus strain SQU21 and A. parasiticus strain CBS921.7
at different concentrations of B. sacra resin (identical letters and numbers indicate no
significant difference, p < 0.05).
inhibition of aflatoxins production by A. flavus (SQU21) and 43.6e
95.7% for A. parasiticus (CBS921.7). In a similar study, although
Boswellia extracts were effective against different bacterial species
(Weckesser et al., 2007), neither water nor methanolic extracts
revealed any activity against C. albicans and C. matosa yeasts
(Hasson et al., 2011). Other studies using different plant extracts
showed apparent inhibition of the fungal growth and aflatoxin
production. Plant extracts of S. aromaticum, C. longa, A. sativum and
O. sanctum (5 g/kg) effectively inhibit the growth of A. flavus (65e
78%) and AFB1 production (72.2e85.7%) (Reddy et al., 2009). Extract
from fruit rinds of G. cowa and G. pendunculata at 2000e4000 ppb
concentration completely inhibited the growth of A. flavus and
aflatoxin B1 production up to 100% (Joseph et al., 2005). Fruit
extract of A. digitata (baobab) (1.5, 3, 5, and 7%) apparently inhibited
the total aflatoxin secretion up to 20.4e68.5% for A. flavus and 11.9e
69.1% for A. parasiticus, whereas the inhibition of aflatoxin B1 pro-
duction ranged between 29.9e79.2% and 13e68% for the two
strains, respectively (El-Nagerabi, Elshafie, et al., 2013). Bullerman
et al. (1977) stated that 0.02e20% cinnamon extracts inhibit afla-
toxin production by 25e100%, and 2% of cinnamon led to 97% in-
hibition of aflatoxin secretion. The leaves and calyx extracts of
H. sabdariffa (5e12.5%) caused 91.5e97.9% reduction in aflatoxin B1
production by A. flavus and A. parasiticus (Al-Shayeb & Mabrook,
1984; El-Nagerabi, Al-Bahry, et al., 2012). Our findings showed
that the highest inhibition (92.1e95.7%) was obtained at 10%
frankincense resin. These findings suggest the possibility of the
presence of different aflatoxin inhibitors in B. sacra extracts which
interfere with the biochemical synthesis pathways of aflatoxin.
These chemicals in many cases are responsible for various biolog-
ical activities of frankincense (Büchele et al., 2003; Safayhi & Sailer,
1997; Singh et al., 2008). On the contrary, the addition of different
concentrations of frankincense resin to the yeast malt broth and
inoculation with the two Aspergillus species significantly (p < 0.05)
enhanced the growth and vigour of the two species (Fig. 2).
Nonetheless, different concentrations of calyx extract (5e12.5%)
from H. sabdariffa did not inhibit or enhance the mycelial growth of
Aspergillus species (El-Nagerabi, Al-Bahry, et al., 2012). However,
extract from the dry leaves of H. sabdariffa apparently inhibited the
growth vigour of Aspergillus fumigatus, Rhizopus stolonifer and Tri-
chophyton mentagrophyte (Guerin & Reveillere, 1984). The growth
of A. flavus was completely inhibited by the hexane and chloroform
extracts from fruit rinds of G. cowa and G. penduculata (2000e
4000 ppb), whereas extracts from G. cowa completely inhibited
aflatoxin B1 production (Joseph et al., 2005). Among the plant ex-
tracts tested, S. aromaticum (5 g/kg) showed complete inhibition of
A. flavus and AFB1 production, whereas C. longa, A. sativum, and
O. sanctum (5 g/kg) effectively reduced the fungal growth (65e78%)
and AFB1 production (72.2e85.7%) (Reddy et al., 2009). Cinnamon
at the concentrations of between 0.02 and 20% inhibited the growth
of A. parasiticus by 16e100% as well as inhibition of aflatoxin
Fig. 3. Aflatoxin production of A. flavus strain SQU21 and A. parasiticus strain CBS921.7
at different concentrations of B. sacra leaf extract (identical letters and numbers
indicate no significant difference, p < 0.05).
biosynthesis (Bullerman et al., 1977). Therefore, it is apparent that
frankincense resin contains inhibitors which interfere with the
biochemical sequences of aflatoxin biosynthesis as shown in
similar studies (Da Costa, Geraldo, Arrotéia & Kemmelmeier, 2010;
El-Nagerabi, Al-Bahry, et al., 2012; El-Nagerabi, Elshafie, et al.,
2013). These chemicals in many cases are responsible for various
biological activities of frankincense (Büchele et al., 2003; Safayhi &
Sailer, 1997; Singh et al., 2008).
In the present study, inoculation of the two species of A. flavus
(SQU21) and A. parasiticus (CBS921.7) in yeast malt broth supple-
mented with different concentrations of frankincense leaf extracts
(5, 7.5, 10, 12.5, and 15% v/v) significantly (p < 0.05) increased both
aflatoxin production (Fig. 3) as well as the mycelial dry weights of
the two Aspergillus species (Fig. 4). The highest concentration of the
leaf extracts caused 20e50% increase in aflatoxin production, and
25.3e29.1% mycelial dry weight for A. flavus and A. parasiticus. This
enhancement of aflatoxin production and the fungal vigour may be
due to the high nutrient contents of the green leaves. Although, it is
evident that the frankincense resin of B. sacra contains aflatoxin
inhibitors, the leaf extracts enhanced the mycelial growth. This
indicates the interference of these inhibitors with the biochemical
events of aflatoxin biosynthesis as concluded by many researchers
(Da Costa et al., 2010; El-Nagerabi, Al-Bahry, et al., 2012; El-
Nagerabi, Elshafie, et al., 2013).
The inhibition of the vegetative growth and aflatoxin production
of A. flavus and A. parasiticus by essential oils (Eos) extracted from
herbal, medicinal and aromatic plants have been recommended by
many authors (ex. El-Nagerabi, Al-Bahry, et al., 2012; El-Nagerabi,
Fig. 4. Mycelial dry weights of A. flavus strain SQU21 and A. parasiticus strain CBS921.7
at different concentrations of B. sacra leaves extract (identical letters and numbers
indicate no significant difference, p < 0.05).
 S.A.F. El-Nagerabi et al. / Food Control 34 (2013) 763e769
Elshafie, et al., 2013; Maraqa et al., 2007; Soliman & Badeaa, 2002).
Many of these oils had different fungistatic activities and mode of
action (Gandomi et al., 2009; Shukla et al., 2012; Szczerbanik et al.,
2007). They have variable effects on mycelial growth and aflatoxin
production of A. flavus and A. parasiticus and associated aflatoxin
secretions. It was reported that 3% of Nigella sativa oil completely
inhibited aflatoxins B1, B2, G1 and G2 when using TLC for aflatoxin
detection and 50% inhibition when using sensitive monoclonal
antibody method (Maraqa et al., 2007). At concentration of 1e3%,
this oil caused 47.9e58.3% inhibition of aflatoxin B1 for A. flavus and
32e48% for A. parasiticus (El-Nagerabi, Al-Bahry, et al., 2012). The
highest inhibition levels of total aflatoxin and aflatoxin B1 secretion
by A. flavus (47.2e95.7%; 28.1e89.7%) and A. parasiticus (42.7e
93.3%; 25.9e80.2%) were obtained with essential oil extracted from
A. digitata seeds (El-Nagerabi, Elshafie, et al., 2013). Clove oil at
200e250 ppm inhibits the growth of A. parasiticus (Bullerman et al.,
1977). Essential oil of Cymbopogon flexuosus absolutely inhibits the
growth of A. flavus and aflatoxin B1 production at 1.3 mml1 and
1.0 mml1, respectively. Aflatoxin B1 production by NKD-208 iso-
lates of A. flavus was strongly inhibited at lower fungistatic con-
centrations of essential oil of Callistemon lanceolatus (Shukla et al.,
2012). The EOs of Thymus eriocalyx and Thymus x-porlock at 1/2e1/
16 dilutions were fungicidal and strongly inhibited aflatoxin pro-
duction (Rasooli & Abyaneh, 2004). C. citratus exhibits fungitoxicity
at 1000 ppm (Mishra & Dubey, 1994). Frankincense of B. carteri at
2% (v/v) showed the strongest mycelium inhibition against A. flavus
(11.1%), Fusarium moniliform (61.1%), Fusarium proliferratum (16.7%),
Pyrincularia grisea (33.3%), Bipolaris oryzae (33.3%), Rhizoctonia
solani (44.4%), and Alternaria brassicicola (71.3%) (Udomsilp et al.,
2009). In the present investigations, the effects of different con-
centrations of oil extracted from resin of B. sacra (1, 2, 3, and 4 ml/
100 ml) on aflatoxin production (Fig. 5) and the mycelial growth
rates (Fig. 6) of A. flavus (SQU21) and A. parasiticus (CBS921.7) were
recorded. The results showed that the oil of B. sacra significantly
(p < 0.05) reduced aflatoxin secretion for A. flavus (45.8e83.7%) and
A. parasiticus (41.3e83.5%). Similarly, the mycelial dry weights of
the two Aspergillus species were significantly (p < 0.05) inhibited
by the tested concentrations of B. sacra oil compared to the control.
This indicates the antifungal activity of B. sacra essential oil against
the growth of the two species of A. flavus (SQU21) and A. parasiticus
(CBS921.7). Similar conclusions were drawn by many authors using
different essential oils extracted from various plants such as
N. sativa and A. digitata (El-Nagerabi, Al-Bahry, et al., 2012; El-
Nagerabi, Elshafie, et al., 2013; Maraqa et al., 2007).
Detoxification of aflatoxins by plant extracts is safer and more
eco-friendly than detoxification by physical, chemical or microbi-
ological means (Alberts et al., 2009; Kumar et al., 2009). The
Fig. 5. Aflatoxin production of A. flavus strain SQU21 and A. parasiticus strain CBS921.7
at different concentrations of B. sacra oil (identical letters and numbers indicate no
significant difference, p < 0.05).
767
Fig. 6. Mycelial dry weights of A. flavus strain SQU21 and A. parasiticus strain CBS921.7
at different concentrations of B. sacra oil (identical letters and numbers indicate no
significant difference, p < 0.05).
biological detoxifications offer promising alternatives for aflatoxin
elimination and maintaining the quality and safety of food and feed
(Alberts et al., 2009; Oguz, 2011; Prakash et al., 2011). The potential
of some herbal, medicinal and aromatic plants to degrade aflatoxins
has been reported by many researchers (El-Nagerabi, Al-Bahry,
et al., 2012; El-Nagerabi, Elshafie, et al., 2013; Sandosskumar
et al., 2007). Garlic (A. sativum L. x) and onion (Allium cepa L.) roots
incubated in water containing 70 mg of aflatoxin B1 for 5 days
degrade aflatoxin and cause 58.5% reduction in aflatoxin level
(Velazhahan et al., 2010). The dialyzed seed extract of Trachy-
spermum ammi was reported to degrade 90% of aflatoxin G1 by
modification of its lactone ring structure (Velazhahan et al., 2010).
The aqueous extract of T. ammi seeds was reported to cause
approximately 80% reduction in aflatoxin content due to the pres-
ence of aflatoxin inactivation factors (Hajare, Haijare, & Sharma,
2005). In the present study, the species of the two selected Asper-
gillus species are aflatoxin-producers and secreting different pro-
files of aflatoxins. To our knowledge, the detoxification properties
of B. sacra resin, leaf and oil extracts had not been examined before.
Nonetheless, the antimicrobial property of B. serrata resin was
investigated. It ameliorates the deviation induced in both kidneys
and hearts of animal in response to AFB1 administration
(Abdulmajeed, 2011). All essential oils of Boswellia species revealed
antibacterial activity against Gram-positive bacteria (Mothana
et al., 2011). Oil concentration of 2% evidently inhibits mycelial
growth and sporulation (Udomsilp et al., 2009). Therefore, it is not
astonishing that the resin and oil extracts of B. sacra display enor-
mous inhibitory effects on aflatoxin production by these aflatoxi-
genic fungi. Thus, it is very important to evaluate the detoxification
properties of resin and oil extracted from B. sacra to aflatoxin
production and further isolation and identification of the active
chemical compounds which are associated with detoxification
phenomenon. In the present study, we investigated the effect of
10% (w/v) resin and 4% (v/v) oil extract of B. sacra on 870 ppb pure
aqueous aflatoxin B1. The results showed that the two extracts
slightly reduced aflatoxin B1 concentrations to 860 ppb and to
867 ppb in comparison with the control (870 ppb) which suggests
the lack of the detoxification properties of these extracts on afla-
toxin B1. Therefore, it is obvious that both resin and oil extracts had
inhibitory effects on aflatoxin secretion, whereas oil possesses
antifungal activity. On the other hand, resin enhanced the fungal
growth of the selected Aspergillus species (A. flavus SQU21 and
A. parasiticus CBS921.7). Nonetheless, none of the tested extracts
(resin and oil) showed detoxification or inactivation activities
against pure aflatoxin B1. However, they inhibit the biological
synthesis of aflatoxin by A. flavus (SQU21) and A. parasiticus
(CBS921.7), besides, the antifungal activity of oil extract.
768 S.A.F. El-Nagerabi et al. / Food Control 34 (2013) 763e769
4. Conclusion
This research investigates the biological activities of resin,
essential oil, and leaf extract of B. sacra on the growth and aflatoxin
secretion of A. flavus (SQU21) and A. parasiticus (CBS921.7). Our
findings proved that the resin powder and essential oil of B. sacra
evidently reduce aflatoxin production, whereas resin enhanced the
fungal growth which indicates the inhibitory effect to aflatoxin
biosynthesis and secretion pathway. The green leaf extract in-
creases both the fungal growth and aflatoxin secretion by the two
Aspergillus species. None of the extracts detoxify pure aflatoxin B1.
Therefore, resin powder and essential oil can be used as natural
plant additives to food and feed products based on their toxicity
studies (Abulmajeed, 2011; Banno et al., 2006; Gupta et al., 2001;
Langmead & Rampton, 2006; Miller & Morris, 2004; Mothana
et al., 2011; Suhail et al., 2011). However, safety and toxicity of
biologically active chemical ingredients which affect the fungal
growth and aflatoxin production need further investigations. The
identified chemical ingredients should be tested for their antifungal
activity and interference with the biosynthesis pathway of afla-
toxins. This will broaden our knowledge on their uses and future
development in food industry and pharmaceutical applications.
Acknowledgement
This research was funded by University of Nizwa. We thank the
Department of Biological Sciences and Chemistry, College of Arts
and Sciences, University of Nizwa and the Department of Biology,
College of Science, Sultan Qaboos University for providing space
and facilities. We thank the University of Nizwa Writing Center for
proof reading the English of this article.
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