New Anti-aflatoxin marine and Terrestrial extracts with assessment of their Antioxidants,...

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* Corresponding author: Doaa A. Ghareeb, E-mail address: [email protected]

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.Ghareeb

1*, El-Sayed E. Hafez

2, Mohameed A. El-Saadani

1

Mohamed M. El –Sayed1.

1Biochemistry Department, Faculty of Science, Alexandria University, City for Scientific,

Egypt. 2Research 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

Doaa A. Ghareeb, et al / Int. J. of Res. in Pharmacology and Pharmacotherapeutics Vol-2(3)2013 [445- 458]

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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



in which, sterilized filter paper (3MM) disc was

dipped in 1% of each plant extract for 24 hr at 37 ᵒC, 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 ᵒC; 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 26ᵒC-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 950C for 1 min, followed by

annealing at 300C for 1 min and extension at 72

0C

for 1 min with initial delay for 5 min at 950C at the

beginning of the first cycle and post extension step

for 10 min at 720C 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



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].


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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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


Cinnamuom

zeilanicum

Adiantum

capillus

Mentha

pulegium

Pterocladiella

capillacea

Ulva lactuca

Jania ruben


454


456

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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