New Anti-aflatoxin marine and Terrestrial extracts with assessment of their Antioxidants, Antimicrobial, Glycemic and Cholinergic properties bioscreening of new Anti-aflatoxin natural extracts

1. ~ 445 ~
_______________________________________
* Corresponding author: Doaa A. Ghareeb,
E-mail address: d.ghareeb@yahoo.com
Available online at www.ijrpp.com
Print ISSN: 2278 – 2648
Online ISSN: 2278 - 2656 IJRPP | Volume 2 | Issue 3 | 2013 Research article
New Anti-aflatoxin marine and Terrestrial extracts with assessment of
their Antioxidants, Antimicrobial, Glycemic and Cholinergic
properties bioscreening of new Anti-aflatoxin natural extracts
Nadia Fouad1
, Doaa A.Ghareeb1
*, El-Sayed E. Hafez2
, Mohameed A. El-Saadani1
Mohamed M. El –Sayed1
.
1
Biochemistry Department, Faculty of Science, Alexandria University, City for Scientific,
Egypt.
2
Research and Technology Applications, SRAT, New Borg El Arab, Alexandria, Egypt.
ABSTRACT
Aflatoxins are a group of closely related mycotoxins that are widely distributed in nature in different agricultural
communities. These toxins can produce a variety of diseases related to liver and brain damage. In this study, the
phytochemical constituents of different plants ethanolic extracts (Thymus vulgaris, Cinnamuom zeilanicum,
Menthe Pulegium, Adiantum capillus and Berberis vulgaris) and algae extracts (Ulva lactuca, Jania ruben and
Petrocladiella capillacea) were examined as antifungal, antibacterial, antioxidant beside cholinergic and
glycemic behaviours. The extracts antifungal activity was examined both on the Aspergillus flavus growth and
on aflatoxins production. Results showed that both tested C. zeilanicum concentrations gave undetectable
Aflatoxins B1 and B2 levels, while B. vulgaris (1%) decreased B1 concentrations than control. Furthermore, it
was found that B. vulgaris act as potent anti-AChE while the other tested terrestrial and marine extracts acted as
activators. Additionally, B. vulgaris and marine algae extracts showed inhibitory effects toward α-glucosidase
activity. As the growth of A.flavus affected by our tested extracts, differential display was performed on fungus
genome to study up-down regulation. Results showed alteration in A. flavus gene expression at different
concentrations of C. Zeilanicum and B. vulgaris (0.1%, 0.5% and 1%) that confirmed the alterations that took
place in aflatoxins production. In conclusion, this study showed the positive effect of our extracts as anti-
aflatoxin so they could be used as preservative during food storage and as aflatoxicosis therapeutic extracts.
Key words: Cinnamuom zeilanicum, Berberis vulgaris, Aspergillus flavus, mycotoxins, marine algae.
INTRODUCTION
Mycotoxins are secondary metabolites of moulds
that exert toxic effects on animals and humans [1].
General interest in mycotoxins rose in 1960 when a
feed-related mycotoxicosis called turkey X disease,
this was later proved to be caused by aflatoxins,
appeared in farm Animals in England [2],
subsequently, it was found that aflatoxins are
hepatocarcinogens for animals and humans, and
these stimulated researches on mycotoxins [3].
Aflatoxins are a group of closely related
mycotoxins that are widely distributed in nature in
different agricultural communities. It has been
demonstrated that Asperagillus flavus (A. flavus)
can infect corn and produce Aflatoxins specially
type B1 (AFB1) [4]. Aflatoxins have a very wide
range of biological activities particularly B1, which
causes great economic losses and poses health,
hazards both to human and farm animals.
Aflatoxicosis is poisoning resulting from ingestion
of aflatoxins in contaminated food or feed. Milk
and milk products- aflatoxin contamination is
International Journal of Research in
Pharmacology & Pharmacotherapeutics

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produced by two ways; either toxins pass to milk
with ingestion of feeds contaminated with
aflatoxin, or it results as subsequent contamination
of milk and milk products with fungi [5]. AFB1
was shown to be excreted in human urine [6].
Kidney damage induced by aflatoxicosis was
demonstrated in fish [7] and chicken [8]; however,
the effects of AFB1 contaminated diet on the rat
kidney has not been much elucidated histologically.
It has been stated, in fact, that the contamination of
milk and milk products with AFM1 displayed
variations according to geography, country and
season. The pollution level of AFM1 was
differentiated further by hot and cold seasons, due
to the fact that grass, pasture, weed, and rough
feeds were found more commonly in spring and
summer than in winter. At the end of summer,
greens were consumed more than concentrated
feed, causing a decreased level of AFM1 in milk [5]
(table 1).
Aflatoxin causes genetic damage in bacteria and
cultured cells in vitro and experimental animals,
exposed to aflatoxin in vivo. Types of genetic
damage observed include formation of DNA and
albumin adducts, gene mutations, micronucleus
formation, sister chromatid exchange, and mitotic
recombination. Metabolically activated aflatoxin
B1 specifically induced G to T transversion
mutations in bacteria. G to T transversion in codon
249 of the p53 tumor-suppressor gene have been
found in human liver tumors from geographic areas
with high risk of aflatoxin exposure and in
experimental animals [9]. In humans and
susceptible animal species, aflatoxin B1 is metab-
olized by cytochrome P450 enzymes to aflatoxin-8,
9-epoxide, a reactive form that binds to DNA and
to albumin in the blood serum, forming adducts.
Comparable levels of the major aflatoxin B1
adducts (the N7-guanine and serum albumin
adducts) have been detected in humans and
susceptible animal species [10]. The 8, 9-epoxide
metabolite can be detoxified through conjugation
with glutathione, mediated by the enzyme
glutathione S-transferase (GST). The activity of
GST is much higher in animal species that are re-
sistant to aflatoxin carcinogenicity, such as mice,
than in susceptible animal species such as rats.
Humans have lower GST activity than either mice
or rats, suggesting that humans are less capable of
detoxifying aflatoxin-8, 9-epoxide. In studies of
rats and trout, treatment with chemopreventive
agents reduced the formation of aflatoxin B1–
guanine adducts and the incidence of liver tumors
[11].
Plants have a long history of use in the treatment of
cancer. Hartwell [12], in his review of plants used
against cancer, listed more than 3000 plant species
that have reportedly been used in the treatment of
cancer . The genus Mentha (Lamiaceae) includes
aromatic herbs of difficult taxonomic classification
due to a great variability in their morphological
characters and frequent hybridization. Popularly
known as “Khalvash”, is consumed mainly for its
antiseptic, insect repellent, carminative,
antispasmodic, diaphoretic and anti-inflammatory
properties in Iran [13]. No well-documented
investigation the cytotoxicity of this herbal
medication in cancer cells has been reported.
Thyme (Thymus vulgaris L.) belonging to the
lamiaceae family, is a pleasant smelling perennial
shrub, which grows in several regions in the world
[14]. Thymus and its oil have been used as
fumigants, antiseptics, antioxidant, and mouth
washes. The main essential oil in thyme, thymol, is
active against Salmonella and Ataphylococcus
bacteria [15]. Cinnamuom zeilanicum is native to
India and Sri Lanka (Ceylon). It is now cultivated
in many tropical countries including Mexico. Most
of the original uses are still prevalent; mainly as a
treatment for diarrhea, stomach upset, against
respiratory ailments and externally as a skin
antiseptic and rubefacient.
Cinnamon bark may possess a potentiating effect
on insulin, and can be useful in the treatment of
type 2 diabetes; as well as lowering triglyceride
levels and serum cholesterol [16]. Cinnamon
constituents possess antioxidant action and may
prove beneficial against free radical damage to cell
membranes [17].
Adiantum capillus veneris belonging to the
Adiantaceae family is one of the most common and
widely distributed species [18]. Victor et al., [19]
reported the antimicrobial activity of leaves and
pinnae oils.
Berberis vulgaris is a shrub in the family
Berberidaceae, native to central and southern
Europe, northwest Africa and western Asia. The
presence of protoberberines and bisbenzyl-
isoquinoline alkaloids (berbamine, tetrandrine and
chondocurine) has been well established as anti-
inflammatory and immuno-suppressive activities
[20].
Aflatoxins have received considerable attention due
to their hepatocarcinogenic nature [21]. The ability

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of some herbal drugs and condiments to inhibit
mould growth and aflatoxin production by
Aspergillus flavus have been reported [21].
However, the investigation of seaweeds for this
purpose has not been recorded. Ulva lactuca is a
common species of macroalgae found in green
tides [22]. The main aim of this study was to find
out a natural extract affecting the aflatoxin
production. Moreover, the second aim was to
assess the other biological activities of these
extracts which have anti-aflatoxin properties.
MATERIALS AND METHODS
Thymus vulgaris, Cinnamuom zeilanicum, Menthe
Pulegium, Adiantum Capillus and Berberis
Vulgaris were purchased from the markets; algae
were collected from the Abu Kir coast and
identified by Prof. Dr. Samy Shaalan,
Microbiology and Botany Department, Faculty of
Science, Alexandria University, Egypt. The algae
were classified as two red algae (Jania ruben and
Pterocladiella capillacea) and green algae, Ulva
lactuca. Corn seeds were collected under the
authorization and supervision of Dr. Gamal Fathy,
Department of Microbiology, Faculty of Science,
El-Mannsoura University, Egypt.
Isolation of fungi from corn seeds
Twenty gm agar dissolved in one liter distilled
water supplemented with 10% NaCl. The fungi
were isolated after 10 days. The Fungi purification
step was carried by transferring and storage on
PDA media (200 gm potato, 20 gm dextrose and 20
gm agar in one liter distilled water).
Preparations of plant and algae extractions
Both Plants and algal tissues were collected and
dried at room temperature, powdered and sieved.
Powered plants 250 grams were separately soaked
in absolute ethanol (500 ml) for 3 days at 25◦
C in
shaker incubator (600 r.p.m.). The supernatant was
collected by filtration using Buchner filter. The
collected supernatant was evaporated under
vacuum to sticky oil solution. The resident oily
solution was lyophilized and weighed.
Plant and algae phytochemical screening
Dried plants and algae were phytochemically
screened for alkaloids, phlobatannins, saponnins,
flavonoids, steroids, terpenoids and cardiac
glycosides (Ayoola et al, 2008). Extraction of
alkaloids was performed according to [23] and for
Tannins Edeoga et al method was used. Morover,
for saponins extraction a method of Harbone [24]
was performed and Sofowara [25] method was
used for phlobatannins extraction. Test of
flavonoids and steroids were performed according
to Hussain et al [26]. In case of terpenoids and
cardiac glycosides were extracted according to
Seniya et al. [27], respectively. For determination
of the total phenolic content the method of Anesini
et al. [28] was used. About 0.4 mL of the plant
extract aliquot was transferred into a test tube
containing 0.8 mL of the 10% Folin-Ciocalteu-
phenol reagent. After 3 min, 1.6 mL of the 10%
sodium carbonate solution was added. The contents
were mixed routinely, using glass rod and left to
stand at room temperature for 1h. Absorbance
measurements were recorded at 750 nm using a
spectrophotometer while Gallic acid was used for
the preparation of the standard curve.
Effect of plants and algae crude extracts on
fungi growth
The effect of the plants and algal extracts on the
fungal growth was preformed according to the
method of Huynh et al. [29]. Sabouraud’s dextrose
media was prepared by adding 20 gm glucose; 10
gm yeast extract-10gm peptone prepared on one
liter distilled water. Equally amount of
Asperagillus flavus (about 3 inoculums) was mixed
with 35 mL media that contained 351 µL DMSO
(+ve control), water (-ve control) or different
concentrations of plants or algae (0.01, 0.05, 0.1,
0.5, 1%) of each plant or algae and left for 10 days
until completely growth. The effect of different
plant and algae concentrations on fungus growth
was observed by photographic assay and on the
aflatoxin production (B1and B2 toxin) by HPLC
assay.
Natural extracts Bioscreening:
The natural extract was used as antibacterial
according to Aboaba et al. [30]. Three bacterial
strains; Escherichia coli, bacillus and
pesudomonass were antigonstic using the prepared
discs. The plant sterile discs method were used to
detect the antibacterial activity of the plant extracts

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in which, sterilized filter paper (3MM) disc was
dipped in 1% of each plant extract for 24 hr at 37
ᵒ
C, another disc was dipped in 1% DMSO which
used as control. Duplicate plates were prepared for
each extract and controls, one bacteria strain was
sprayed on LB agar media for each plate then the
different extract discs were distributed separately at
constant distance. The plates were incubated at
28ᵒC -30ᵒC for 48 hr. The diameters of cleared
zones were observed. The transparently cleared
zones showed bactericidal activity while the un-
cleared zones containing micro colonies showed
bacteriostatic activity.
The plants extract were examined as Antifungal
according to yang et al. [31]. Three mold fungi
alternaria, fusaria solenai, risobous, and mucour
were used in this assay. Spore suspensions of
remaining test fungi were prepared by washing the
surface of each malt agar plate with 10–15mL of
sterile deionized water (DI) according to ASTM
standard D4445-91. In one set of tests, a mixture of
three mold spore suspensions was transferred to
spray bottle and diluted to 100mL with DI water to
yield 3x107
spores/ mL. The spray bottle was
adjusted to deliver 1mL in spray/plate. Sterilized
filter paper (3MM) disc was dipped in 1% of each
plant extract for 24 hr at 37 ᵒ
C; another disc was
dipped in 1% DMSO which used as control.
Duplicate plates were prepared for each extract and
controls, one fungi strain was sprayed on PDA agar
media plate then the different extract discs were
distributed separately at constant distance. The
Petri dishes were incubated at optimum
temperature 26ᵒ
C-28◦
C for 7-10 days and then were
examined to observe the inhibition zones.
Cholinergic effect of the extracted plants was
preformed according to Gearhart et al. [32].
Whenever, the effect of the plants extracts on
Alpha glucosidae, method of Han and Srinivasan
[33] was performed. A 100 μL of plant and algae
extract (test), organic solvents (control) or dH2O
(blank) were diluted with 2.5 mL 0.1 M phosphate
buffer pH 7.4. Then 100 μL of liver homogenate
was added, mixed well and incubated in a water
bath with the reaction mixture at 30°C for 5 min.
PNPG (para-nitrophenyl- α -D-glucoside) 500 μL,
5 mM, was added and the reaction was allowed to
proceed for 15 min. The reaction was stopped by
the addition of 2mL of 1M Na2CO3. The yellow p-
nitrophenol released was read at 400 nm. Under the
conditions of the assay the reaction was linear for
at least 10 min. A unit of enzyme activity was
defined as nmoles of p-nitrophenol released/min. A
standard curve was constructed using various
concentrations of p-nitrophenol.
Effect of the plant extracts on the
mycotoxin producing fungi using
differential display
Extraction of RNA from fungi was performed
using RNA extraction kit (QIAgene, Germany)
according to the manufacture procedures. The
extracted RNA was subjected to cDNA synthesis
using reverse transcriptase and the reaction
constituents was as follow; cDNA was synthesized
using reverse transcriptase (Fermentas, USA) and
its buffer (5X) [50 mM Tris-HCl (pH 8.3 at 25˚C),
250 mM KCl, 20 mM MgCl2 and 50 mM DTT] in
presence of random hexamer primer (Promega,
USA). 5µl of RNA was added to (10 µl (5x) RT-
Buffer, 5 µL (25 mM) dNTPs, 5 µL of primer, 0.5
µL (20 u/ µL) of RT-enzyme, 24.5 µL H2O). The
mixture was incubated at 37˚C for 60 minutes, then
at 70˚C for 10 minutes (for enzyme inactivation)
followed by storage at 4˚C until be used. For
second PCR; six arbitrary primers were used for
PCR amplification according to Williams,.et al [34]
with some modifications. All primers (Obp18,
CTG CTG GGA C; 16S (F), AGG AGG TGA TCC
AAC CGC; 16S (R), AAC TGG AGG AAG GTG
GGGAT; NAR48, CCTTTCCCTC; NAR49, GAC
GAC GAC GACGAC; NAR50, ACG GAG TTG
GAG GTC) were re-suspended in Milli-Q water at
a concentration of 100 pmol/ µL as stock solution.
Working solution of each primer was prepared at
10 pmol/µL of PCR reaction. Amplification
reactions were performed in a 25 µL reaction
mixture containing; 2.5 µL Taq DNA polymerase
buffer , 2.5 µL 50 mmol/L MgCl2, 2 µL from each
primer (40 pmol/ µL ) (table 2), and 0.25 µL of taq
polymerase (AmpliTaq, Perkin-Elmer, 5 U/ µL),
2.5 µL from the cDNA, 2.5 µL dNTPase 4 mmol/L
and 12.75 µL of dH2O.
The amplification was performed for 40 cycles in a
Thermal-cycler. Each cycle consisted of
denaturation at 950
C for 1 min, followed by
annealing at 300
C for 1 min and extension at 720
C
for 1 min with initial delay for 5 min at 950
C at the
beginning of the first cycle and post extension step
for 10 min at 720
C after the end of the last cycle.
PCR products were separated on agarose gel
electrophoresis using 1.5% (w/v) agarose dissolved
in 0.5 X TBE buffer. The size of each band was
estimated by using DNA molecular marker.

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Finally, the gel was photographed by using gel
documentation system.
Statistical analysis
Data were analyzed by one-way analysis of
variance (ANOVA) using Primer of Biostatistics
(Version 5) software program. Significance of
means ± SD was detected groups by the multiple
comparisons Student-Newman-keuls test at p <
0.05.
Results and discussion
Table (2) shows that all tested plants and algae
contained cardiac glyceroids and flavonoids. All
tested plants and algae expect P. capillacea
contained saponins. C. zeilanicum and T. vulgaries
contained alkaloids, tannins, phobataninns steroids
and terpenoids. B. vulgaris, A. capillus, J. ruben, P.
capillacea and U. lactuca contained tannins and
phobataninns. Table (3) shows that Jania ruben
contained the highest concentration of flavonoids
(9.25%) followed by Mentha pulegium (7.43%).the
lowest flavonoids concentration was found in
Pterocladiella capillacea (1.32%). Substances
isolated from plants such as flavonoids,
isoflavonoids and biflavonoids, besides other
activities, have shown activity against some aspects
of fungal metabolism [35]. Our results shows that
the tested plants and had different amounts of
alkaloids, tannins, saponins, phlobatannins and
flavonoids. Table 4 represents that B. vulgaris
acted as antibacterial and antifungal toward E. coli,
Pesudomonass and Mucour. M. pulegium had
antibacterial effect toward Pesudomonass while A.
diantum showed antifungal effect toward Mucour.
On the other hand, other plants and tested algae
extracts did not showed anti microbial effect. Such
observation was supported by Irobi and Adedayo
[36], who found that polar solvent extract has high
antifungal activity against a wide range of fungal
isolates including Aspergillus niger and Candida
albicans. Antimicrobial activity may involve
complex mechanisms, like the inhibition of the
synthesis of cell walls and cell membranes, nucleic
acids and proteins, as well as the inhibition of the
metabolism of nuclide acids [37]. The ethanolic
extracts of the tested plants and algae were
evaluated for their effect on two important
enzymes, AChE enzyme (E.C.3.1.1.7) and α-
glucosidase enzymes (E.C.3.2.1.20).
In this study we found that the extracts of T.
vulgaris, A. capillus, M. pulegium and C.
zeilanicum significantly increased the activity of
AChE enzyme (figure 1) so act as activators.
Certain neurotoxins work by inhibiting
acetylcholinesterase, thus leading to excess
acetylcholine at the neuromuscular junction, thus
causing paralysis of the muscles needed for
breathing and stopping the beating of the heart.
[38]. On the other hand the extracts of B. vulgaris,
J. rupin, P. capillacea and U. lactuca were
significantly inhibited AChE activity. Therefore,
these extracts could be used as AD treatment.
Our results emphasize that the plant and algae
extracts expect B. vulgaris extracts (figure 1) acted
as glucosidase activators. It is well known that
glucosidase activators are effective in
hypoglycemic conditions and glycoprotein
formation [39] cellulose biosynthesis, tissue culture
[40], and in the treatment of genetic disorders like
pomp’s disease [41]. On the other side, B. vulgaris
extract worked as glucosidase inhibitor therefore is
considered as anti-diabetic agent. Matching with
our results, berberine the active alkaloid in B.
vulgaris is used as anti-diabetic agent [42].
The utilization of these natural compounds as
substitutes for conventional fungicides in order to
prevent contamination by aflatoxins has been
considered because some flavonoids are
biologically active against A. flavus and A.
parasiticus [43]. As observed in the table (5), C.
zeilanicum was extremely potent inhibitor for
A.flavus growth because at high concentrations
(0.5% and 1%), it was completely inhibited the
fungi growth. Adiantum capillus had no effect on
growth of fungi either on low or high
concentrations. Finally, Mentha pulegium had
slightly effected fungi growth especially on
concentrations of 0.1% and 0.5%. Table (5) shows
that there was no differences detected in fungi
growth either in (–ve) control or (+ve) control (in
which different concentration of DMSO were
used). Comparing with control (-ve and +ve). P.
capillacea, U. lactuca and J. rupin had no
inhibitory effect toward fungi growth either at low
or high concentrations.
Figure 2 (A) shows that T. vulgaris at
concentrations of 0.01% and 1% increased the

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aflatoxin B1 concentration comparing with control
level. B. vulgaris showed an inhibitory effect
toward aflatoxin B1 production. On the other side,
low and high concentrations of C. zeilanicum
completely inhibited aflatoxin B1 production.
Moreover, figure 2 (B) shows that T. vulgaris
decreased the aflatoxin B2 production by 83 and 91
% at extract concentration of 0.01 and 1%. On the
same pattern, B. vulgaris inhibited B2 production
by 67% at concentration of 0.01 and by 90 % at
concentration of 1%. As usual, C. zeilanicum
concentrations completely arrested aflatoxin
production as it inhibited A. flavus growth.
Furthermore, figure 2 C shows that all algae extract
increased B1 than control especially P. capillacea
(156.6 ppm) followed by Jania rupin (127.60 ppm)
and Ulva lactuca (104.83 ppm). But they
completely inhibited the B2 production.
The inhibitory effect of C. zeilanicum could be
contributed to the presences of alkaloids and
flavonoids. The flavonoids that appear to be
primarily responsible for cinnamon’s antidiabetic
action are called procyanidins (type A). These
compounds are oligomers, a term used to denote
polymers consisting of just a few molecular units,
rather than many, as in most polymeric compounds.
Proanthocyanidins, a type of flavonoids in
cinnamon, have potent antioxidant capability and
may be able to inhibit tumor growth by starving the
cancer cells [44]. These special flavonoids may
also block the formation of nitrosamines, a
carcinogen that can damage the DNA in breast
tissue.
Several aflatoxin production inhibitors may act at
three levels: (1) modulate environmental and
physiological factors affecting aflatoxin
biosynthesis, (2) inhibit signaling circuits upstream
of the biosynthetic pathway, or (3) directly inhibit
gene expression or enzyme activity in the aflatoxin
production pathway as shown in figure (3). Till
now, the mode of action of most inhibitory
compounds is still unknown [45].
Figure (4 A) shows that when we used (16s) primer
with comparison to control and Bp marker, a new
up regulated band appeared on cinnamon
concentration (0.1%) between bp 30 and 40 and
disappeared on other concentrations. on the other
hand down regulated band disappeared on
cinnamon at concentration (1%) at 80 bp. When
using opb18 starting from 20 bp and ends on 40 bp
some band had low concentrations on cinnamon
concentrations (0.1%, 0.5%) and some disappeared,
but on concentration 1% they all appeared again
and also a new band on 56 bp appeared on both
control and cinnamon on concentration 1%.there
was a down regulated band at cinnamon
concentration (0.5%) at 120 bp. A new band
appeared at 100 bp on control concentrations
(0.1%, 1%) and on cinnamon concentrations (0.1%,
1%).
figure (4B) shows that for primer NAR48 with
comparing to control, band 24 bp in control had
concentrated band which disappeared on B3 (1%)
but appeared obviously on B2 (0.5%). about 3
bands between 30 bp and 50 bp disappeared from
concentration B1 and had slightly appearance on
concentrations B2 and B3. Band 50, 60 bp
appeared at all control concentrations but with
different concentration levels and those bands had
low concentration on samples treated with Berberis
vulgaris for all concentrations. For primer NAR49
all samples showed the same bands concentration
as 2 bands appeared between 10 and 20 bp and
band at 22 bp and another 2 bands at 37 bp and 80
bp. Finally for primer NAR50 sample B2 which
treated with concentration (0.5%) of Berberis
vulgaris extract had the same bands as control on
its different concentrations but with a slightly
decrease on its intensity. But for concentration
B1and B2 down regulated bands from 20 to 30 bp.
down regulated band at 50 bp disappeared at
Berberis vulgaris treated samples. Bands from 40
bp till 100 bp were disappeared from B3 (1%)
concentration.
Differential display showed that at different
concentrations of berberis vulgaris and
Cinnamuom zeilanicum there were up regulated
and down regulated bands which was an indication
on the effect of those plants on A. flavus growth or
even inhibit aflatoxin production (figure 5). The
present results come in agreement with Reib [46]
when he postulated that in A. parasiticus certain
chemicals that inhibit sporulation have also been
shown to inhibit the production of aflatoxin. And
also In A. parasiticus and A. nidulans chemical
inhibition of polyamine biosynthesis inhibits
sporulation and aflatoxin and sterigmatocystin
production [47]. Several papers have shown that
Aspergillus mutants deficient in sporulation are
also unable to produce aflatoxin [48].

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Table:1 Conditions favoring Aspergillus flavus Development
Factor optimum Range
Temperature 86ºF 80-110ºF
Relative humidity 85% 62-99%
Kernel moisture 18% 13-20%
Table 2: Qualitative phytochemical screening
Presence of phytochemical constituents: +; Absence of phytochemical constituents: -.
Table: 3 Concentration of flavonoids and saponins in tested plants and algae
Sample Flavonoids conc.% Saponins g/Kg
Berberis vulgaris 2.02 0.3
Cinnamuom zeilanicum 1.79 0.14
Thymus vulgaris 2.38 0.11
Mentha pulegium 7.43 0.43
Adiantum capillus ------ 0.04
Ulva lactuca 2.36 0.08
Jania ruben 9.25 0.12
Pterocladiella capillacea 1.32 -------
Alkaloi
ds
Tanin
s
Phobatanin
ns
Saponni
ns
Flavonoi
ds
Steroid
s
Terpenoi
ds
Cardiac
glyceroi
ds
Berberis
vulgaris
+ - - ++ + - + ++
Cinnamuom
zeilanicum
+ + + + + - + ++
Thymus
vulgaries
+ ++ + + + - + ++
Mentha
pulegium
+ ++ + + + - - ++
Adiantum
capillus
+ -
-
+ + - - +
Jania ruben + - - + + - + +
Pterocladiel
la
capillacea
+ - - - + - + +
Ulva lactuca + - - + + - + +

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452
Table 4: Antibacterial and anti fungal effect of plant and algae extracts.
Extracts
Escherichia
coli
Pesudomonass
Bacillus Rizopus Mucour
Fusaria
solenai
Thymus vulgaris - - - - - -
Adiantum capillus - - - - + -
Menthe pulegium - + - - - -
Cinnamuom zeilanicum - - - - - -
Berberis vulgaris + + - - + -
Jania rupin - - - - - -
Pterocladiella capillacea - - - - - -
Ulva lactuca - - - - - -
+ indicates antifungal or antibacterial effect
-Indicates bacterial or fungal resistance.
Table 5: Effect of plants and algae crude extracts on fungi growth.
Concentrations 0.01% 0.05% 0.1% 0.5% 1%
Control
(+ve)
Control
(-ve)
Thymus vulgaris
Berberis
vulgaris

9. Doaa A. Ghareeb, et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(3) 2013 [445-458]
Cinnamuom
zeilanicum
Adiantum
capillus
Mentha
pulegium
Pterocladiella
capillacea
Ulva lactuca
Jania ruben

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454

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CONCLUSION
We concluded that those results supporting the
hypothesis that we can use B.s vulgaris or C.
zeilanicum as protecting agents from A. flavus
infection or particularly from aflatoxin toxicity
because of the bioactive ingredients which they
contain.
Conflict of interest statement
We declare that there is no disclose any financial
and personal relationships with other people or
organizations that could inappropriately influence
(bias) our work.
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