Taxol as chemical detoxificant of aflatoxin produced by aspergillus flavus isolated from sunflower seed

doi:10.4236/health.2010.27119

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Vol.2, No.7, 789-795 (2010)

doi:10.4236/health.2010.27119

Health

Taxol as chemical detoxificant of aflatoxin produced by

aspergillus flavus isolated from sunflower seed

Narasimhan Banu1*, Johnpaul Muthumary2

1Department of Biote h n ol o gy, Vels University, Pallavaram, Chennai, India; *Corresponding Author: [email protected]

2Centre for Advanced Studies in Botany, University of Madras, Chennai, India

Received 24 November 2009; revised 10 March 2010; accepted 12 March 2010.

ABSTRACT

Aflatoxins are the potent toxic, mutagenic, het-

erogenic and carcinogenic metabolites produc-

ed by species of A. flavus and A. parasiticus. In

the present study, an attempt has been made to

prevent aflatoxin production using an antican-

cerous drug taxol. Taxol (Paclitaxel) is a well

known drug for its anticancerous property mainly

to treat breast and ovarian cancers. It was ob-

tained from Taxus brevifolia and it was also ob-

tained from the endophytic fungi present in

Taxus brefivolia [1]. Therefore, this drug is spe-

cifically selected to screen its activity on the

control of A. flavus and AFB1 production at va-

rious concentrations. Among the 6 concentra-

tions used, 3 μg of taxol was found to be suitable

to control the growth and AFB1 production. The

content of AFB1 found at this concentration was

6 ppm by TLC and 6.3 ppm by HPTLC. The

complete elimination of AFB1 might require

higher concentrations of taxol.

Keywords: Aflatoxins; Taxol; Anticancerous Drug;

Liver Cancer

1. INTRODUCTION

Aflatoxins are the potent toxic, mutagenic, heterogenic

and carcinogenic metabolites produced by species of A.

flavus and A. parasiticus in food and feed, especially the

oil seeds and their products, both at pre and post harvest

conditions. Their occurrence in food and feed materials

has caused not only health hazards in animals and hu-

mans but also economic losses especially to the export-

ing countries. They are initially classified as human car-

cinogens by the International Agency on Research in

Cancer in 1993 and further epidemiological experimen-

tal research continues to show a strong link between

aflatoxin exposure and hepato cellular carcinom a (HCC).

Although the prevention of mycotoxin contamination

in the field is the main goal of agricultural and food in-

dustries, under certain environ mental conditions the con -

tamination of various commod ities with fungi like Fusa-

rium, Aspergillus, Alternaria and Penicillium and their

mycotoxins are unavoidable for producers. Decontami-

nation/detoxification procedure is useful in order to re-

cuperate mycotoxin contaminated commodities. The

ideal decontamination procedure should be easy to use,

inexpensive and should not lead to the formation of

compound that are still toxic, or may reverse to reform

the parent mycotoxin or alter the nutritional and palat-

ability properties of the grain or grain products. The

frequent occurrence of aflatoxins in food materials poses

a serious threat to the consumers. Therefore, a consider-

able concern has been shown for prevention of the my-

cotoxins [2]. Hepatocellular carcinoma (HCC) is one of

the most prevalent cancers worldwide with incidence

rates highest in geographical regions of Africa and Asia

exhibiting climatic similarities of high heat, humidity

and poor food storage conditions. Reports have shown

that aflatoxins causes primary liver cancer in humans on

a worldwide basis [3-5]. The International Agency for

Research on Cancer [6] has declared AFB1 to be class I

carcinogen, on the basis of animal assays. Hence ap-

proaches involving physical [7-9] chemical and biologi-

cal [10-15] methods have been made to detoxify afla-

toxins in food and feedstuffs in the recent past. In India,

mycotoxin contamination in food and its control were

extensively studied by Bilgrami and his associates [16].

Examination of physico-chemical and biochemical

characteristics of AFB1 molecule reveals two important

sites for toxicological activity [17]. The first site is the

double bond in position c-8, 9, of furo-furan ring. The

aflatoxin-DNA and protein interactions, which occur at

this site, alter the normal biochemical functions of these

macromolecules leading to deleterious effects at the cel-

lular level. The second reactive group is the lactone-ring

in the coumarin moiety. The lactone ring is easily hy-

N. Banu et al. / HEALTH 2 (2010) 789-795

790

drolyzed; it is therefore a vulnerable site for degrad ation.

Hence the degradation treatments should be aimed at

removing the double bond of the terminal furan ring or

in opening the lactone ring. Once the lactone ring is

opened, further reactions could occur to alter the binding

properties of the terminal furan ring to DNA and pro-

teins.

Structural degradation or inactivation of aflatoxins has

been found to be possible by the use of chemicals such

as chlorinating agents (sodium hypochlorite, chlorine

dioxide and gaseous chlorine), oxidizing agents (H2O2,

ozone, sodium bisulfite) and hydrolytic agents-acids

(organic and inorganic) and alkalies (sodium hydroxide,

ammonium hydroxide, potassium hydroxide, etc.). Some

of these chemicals are already being used in food indus-

try and are less prone to consumer resistance [18]. How-

ever, according to Park and Liang [8], most of these

chemicals are impractical and are particularly unsafe be-

cause they form toxic residues or dam age nutrient content,

flavour, odour, color, texture or functional properties of

the product.

Among different processes, ammoniation process has

been extensively worked out and largely accepted in

spite of certain resulting nutritional losses. Liquor am-

monia, gaseous ammonia, in situ liberation of ammonia

by reaction of urea and urease as well as reacting amino

methylamine with lime has been used .

The noteworthy losses affecting the nutritional quality

of ammoniated animal feeds include 10% reduction of

protein quality [19], an irreversible reduction in the de-

gree of unsaturation in lipids [20], a significant drip in

lysine and methionine content [21,22]. The presence of

residual toxicity arising from hydrolyzed products [23]

along with the potential for covalent bonding of AFB1 to

proteins [24] and the loss in nu tritional quality, mak e the

ammoniation treatment processes seem less acceptable.

However, under defined conditions the treatment of

grains (peanuts, cotton seed, corn) and their meals with

ammonia appears to be a commercially viable approach

to detoxification of aflatoxins to the extent of 99% [8]

particularly for feed purposes.

Ammonia degradation proceeds through hydrolysis of

lactone ring, and is followed by decarboxylation to pro-

duce non-toxic compounds. Due to severity of aflatoxins

contamination in selected agricultural commodities, in

various locales, specific decontamination processes have

been approved and put into use [8,25]. Ammoniation of

feeds is authorized by Food and Drug Administration of

the U.S.A. In the U.S.A., Arizona, California and Texas

permit the ammoniation of cotton seed products, and

Texas, North Carolina, Georgia and Alabama have ap-

proved the use in aflatoxin-contaminated corn. Mexico

and South Africa have approved the procedure for use on

corn. Treated peanut meal is widely used in animal feeds

in Europe and elsewhere; consequently the process is

routinely used in France, Senegal, Sudan and Brazil.

Several member countries of the European Community

import ammonia treated peanut meal on a regular basis.

Ammonia treatment processes for feed mill and at farm

level have been worked out intensively by Park et al.

[25]. Low ammonia concentration (0.2 to 2.0%) at high

pressure (35-50 psi) and high temperature (80-120ºC

would require less time (20-60 minutes) as compared to

high ammonia concentration (1-5%) at atmospheric

pressure, ambient conditions needed more time (14-21

days) for treating feed materials containing 12-16%

moisture [25] at feed mill level and form level, respec-

tively. Shannaz and Ghaffar [26] studied the use of am-

monia gas in the reduction of aflatoxin and aflatoxin

producing fung i in sunflow er seeds. Us e of ammonia g as

reduced the seed germination but infection of A. flavus

decreased with consequent reduction in aflatoxin pro-

duction. Feeding lactating cows with ammoniated peanut

meal can result in reduced levels of AFM1 in milk of

cows [27]. Namazi et al. [28] demonstrated that 0.9-

1.0% ammonia inhibited fungal growth together with

aflatoxin producti on.

Pathological and histo-pathological examinations made

with experimental and farm animals fed with ammoni-

ated meals did not show any signs of aflatoxicosis. Also

there were no differences in egg production and immu-

nological responses in poultry. Sodium bisulfite can re-

act with aflatoxin B1, G1, M1 and aflatoxicol at various

temperatures and concentrations at various times to form

water-soluble products [29]. Potassium bisulphite is a

common food preservative and does not pose any con-

sumer resistance problem [30]. Aflatoxin containing

copra at moisture contents of 24% and 7% was effec-

tively detoxified by ammonium hydroxide (> 97% and

89% reduction, respectively) [31]. Sharma et al. [32]

prevented aflatoxin formation in the commodities like

peanut and corn samples by the treatment with an aque-

ous solution of 2-chloroethylphosphoric acids. Buller-

man [33] studied the effects of cinnamon on growth and

aflatoxin production by known toxigenic strains of A.

parasiticus. It was observed that the cinnamon is an ef-

fective inhibitor of aflatoxin production even though

mycelium growth may be permitted.

Aflatoxin production by Aspergillus parasiticus was

markedly checked by O-vanillin on the cereals and oil

seeds by Bilgrami et al. [34]. Maximum inhibition was

recorded on rice (85.6%) foll owed by gro und nut (76.2 5%),

wheat (54.2%), maize (52.3%) and mustard (51.1%).

O-vanillin did not have any pronounced effect on seed

germination.

The prevention of aflatoxin producing fungi and afla-

N. Banu et al. / HEALTH 2 (2010) 789-795

791

toxin through some known anticarcinogenic compounds

viz., Redoxon (Ascorbic acid 0.1 g/ml) [35] and Serpasil

(Reserpine 2.5 mg/ml) [36] at different concentrations

[37]. It is evident that both these drugs had shown in-

hibitory effects on aflatoxin production at all concentra-

tions though in varying degrees.

Ozone effectively degraded AFB1 and AFG1 in 4%

dimethyl sulfoxide at room temperature within a few

minutes. The treated products were confirmed to be

non-toxic by various methods. It is reported to reduce

AFB1 levels by 91% in cottonseed meal containing 22%

moisture after treatment at 100ºC for 2 h; however, with

peanut meal (30% moisture) the reduction was only 78%

after exposure to ozone for 1 h. [38]. The destruction and

detoxification of aflatoxin B1, B2, G1 and G2 (50 µg/ml

in 4% dimethyl sulfoxide) with ozone were confirmed

by Maeba et al. [39].

2. MATERIALS AND METHODS

2.1. Chemical Method

2.1.1. Decontamination/Detoxification by Taxol

Taxol is an anti cancerous drug obtained from Taxus

brevifolia. Authentic sample of taxol (paclitaxel) was

obtained from Sigma chemicals. A sample of 0.02 mg

was dissolved in 1ml of 100% methanol. From this stock

different concentration of taxol viz., 0.5, 1.0, 1.5, 2.0,

2.5 and 3.0 µg was taken and added to 100 ml of Yeast

Extract Sucrose medium (2% yeast, 20% sucrose) [40]

separately. Then the medium was inoculated with a disc

of Aspergillus flavus isolate from the surface sterilized

sundried sunflower seed used for oil crushing and incu-

bated for 8 days at 30ºC as a stationary culture.

After 8 days, the cultures were killed and the culture

filtrate was filtered through Wh atman’s No.1 filter paper.

One hundred milliliter of culture filtrate was extracted

thrice with equal volume of chloroform. The chloroform

extract was dried over rotary evaporator. The final resi-

due was dissolved in 0.2 ml of chloroform. The same

procedure was fol l o wed for control wi t hout adding taxol.

2.1.2. Quantification of Aflatoxin B1 by TLC

Five, 10, 20 and 40 µl of the above extracts were applied

to pre-coated TLC plates (Merck) along with the stan-

dard aflatoxin B1. The plates were developed in a tank

containing chloroform: acetone in the ratio of 88:12.

After the development, the plates were viewed under

long UV light at 365 nm. Blue-fluorescence similar to

standard aflatoxin B1 indicated the presence of aflatoxin

B1. Quantification of aflatoxin B1 was made by evalu-

ating on the plate itself using long UV light. The role of

taxol on detoxification was determined by quantifying

the intensity of blue fluorescence of aflatoxin B1.

2.1.3. High Performance Thin Layer

Chromatography

Twenty micro liter of the above sample extracts were

loaded onto pre-coated silica gel plate. The plate was

developed in a saturated tank containing tertiary butyl

methyl ether: methanol: water in a ratio of 9.6: 0.3: 0.1.

The developing distance of the plate was up to 80mm.

The developed plates were scanned in a Camag TLC

Scanner 3 at 366 nm. The presence of blue-fluorescence

indicated the presence of aflatoxin and confirmed with

authentic sample.

3. RESULTS AND DISCUSSION

An attempt has been made in the present investig ation to

prevent af latoxin pro duction using an anticancerou s drug

taxol. Taxol (Paclitaxel) is a well known drug for it anti-

cancerous property. It was obtained from Taxus brevifo-

lia and it was also obtained from the endophytic fungi

present in Taxus brefivolia [1], Pestalotiopsis termina-

liae, an endophyte o f Termina lia a rjuna [41], and Pesta-

lotiopsis versicolor and Phyllosticta murrayicola, a

pathogenic fungi [42]. It is mainly used to treat breast

and ovarian cancers. Therefore, this drug is specifically

selected to screen its activity on the control of A. flavus

and AFB1 production at various concentrations.

From authentic taxol (0.02 mg) (Sigma chemicals),

0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 µg concentrations were

selected and amended with YES medium containing A.

flavus isolate. This isolate was used as control, its dry

weight was 3.9 g and its AFB1 content was 36 ppm

(Figure 2, Table 1). Except 2.5 µg concentration of

taxol, all the other concentrations showed marked reduc-

tion in the mycelial dry weight and AFB1 production.

But there was no correlation between the mycelial dry

weight and the AFB1 production. The dry weight of the

mycelium ranged from 2.6-4 . 6 g ( Table 1).

Among the 6 concentrations used, 3 µg of taxol was

found to be suitable to control the growth and AFB1

production (Table 1, Figure 8). The content of AFB1

found at this concentration was 6 ppm by TLC and 6.3

ppm by HPTLC. The complete elimination of AFB1

might require higher concentrations of taxol (Figures

1-8). This study indicate that the chemical taxol was a

effective inhibitor of aflatoxin production even though

mycelial growth may be permitted. Taxo l is quite expen-

sive drug and we could not take this drug as food addi-

tives but the availability of Sargassum wightii is inex-

pensive and safe [15].

The U.S. FDA has currently established action levels

(max) of aflatoxin to be 20 ppb for human foods (except

milk), 0.5 ppb for milk, 20 ppb for animal feeds, except

some cases of feeds meant for maturing and finishing

N. Banu et al. / HEALTH 2 (2010) 789-795

792

Table 1. Quantification by TLC and HPTLC.

Level of Aflatoxin B1 (ppm)

S.No. Concentration

of Taxol (μg)

Mycelial dry

weight (g)Quantifica-

tion by TLC

Quantification

by HPTLC

1 0.5 2.9 8 9.2

2 1.0 2.7 12 11

3 1.5 2.6 16 15

4 2.0 3.6 12 12

5 2.5 4.6 12 11

6 3.0 2.9 6 6.3

Control - 3.9 30 36

Figure 1. Authentic Afl atoxin B1.

Figure 2. Control.

Figure 3. Aspergillus flavus with 0.5 µg of Taxol.

Figure 4. Aspergillus flavus with 1µg of Taxol.

Figure 5. Aspergillus flavus with 1.5µg of Taxol.

Figure 6. Aspergillus flavus with 2.0µg of Taxol.

Figure 7. Aspergillus flavus with 2.5 µg of Taxol.

N. Banu et al. / HEALTH 2 (2010) 789-795

793

Figure 8. Asper.gillus flavus with 3.0 µg of Taxol.

of meat animals, which varied from 100 to 300 ppb [8].

From the viewpoint of health and economics, it is im-

perative that such low levels of aflatoxin are prescribed

to follow up. To achieve such low levels, decontamina-

tion/ detoxification procedures are useful in order to re-

cuperate mycotoxin contaminated commodities. The ideal

decontamination procedure should be easy to use, inex-

pensive and should not lead to the formation of com-

pounds that are still toxic or may reverse to reform the

parent mycotoxin or alter the nutritional and palatability

properties of the product. Hence approaches involving

physical and biological methods have been made to de-

toxify aflatoxin in food and feedstuffs by many workers.

It was reported by Dollear [38] that in the case of

groundnut oil, the oils were alkali refined, the soap stock

removed and the oils subjected to two washing. No afla-

toxin could be detected in 100 ml of the refined, washed

oils. It is evident that conventional processing practices

remove completely any aflatoxin that may be found in

crude oils. Banu and Muthumary [43] were also found

the absence of fungal spores and aflatoxin in refined oil

collected from Tamil Nadu Agro Industries Corporation.

Chlorophyllin (CHL) has been found to be a safe and

effective agent for chemoprevention in humans exposed

to aflatoxin [44]. Substitutio n of antifungal and aflatox in

inhibitory chemicals by natural compounds such as

thyme oils is recommended [45]. Examination of various

concentrations of thyme essential oils on the grow th of A.

parasiticus showed promising prospectus on the utiliza-

tion of natural plant oi l s and extract s.

Prevention of aflatoxin elaboration has received con-

siderable attention. Various fungicides, fumigants and

chemicals [46,47], plant extracts [48], antibiotics [49]

have been suggested for controlling the growth of afla-

toxin producing fungi as well as aflatoxin production.

Ranjan and Sinha [37] used two anticarcinogenic com-

pound viz., Redoxon [35] and Serpasil [36] on AFB1

production and A. flavus control. Complete inhibition of

AFB1 was noticed at higher concentration of redoxon.

Combination of serpaisl and redoxon also significantly

inhibited aflatoxin prod ucti on as well as myceli al growt h.

4. CONCLUSIONS

By this study, it is evident that the taxol had inhibitory

effects on aflatoxin production at all the concentrations

mentioned though in varying degrees. It is apparent from

this study that the anticarcinogenic drug taxol can also

be exploited for the prevention of aflatoxin production

by A. flavus.

5. ACKNOWLEDGEMENTS

We greatly acknowledge the Department of Science and Technology,

Govt. of India for providing the financial support to carryout the re-

search work.

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