ORIGINAL PAPER

A novel combination of the essential oils of Cinnamomumcamphora and Alpinia galanga in checking aflatoxin B1 productionby a toxigenic strain of Aspergillus flavus

Bhawana Srivastava Æ Priyanka Singh ÆRavindra Shukla Æ Nawal Kishore Dubey

Received: 29 March 2007 / Accepted: 31 July 2007 / Published online: 15 August 2007

� Springer Science+Business Media B.V. 2007

Abstract The growth of a toxigenic strain (Saktiman

3Nst) of Aspergillus flavus decreased progressively with

increasing concentration of essential oils from leaves of

Cinnamomum camphora and rhizome of Alpinia galanga

incorporated into SMKY liquid medium. The oils signifi-

cantly arrested aflatoxin B1 elaboration by A. flavus. The

oil of C. camphora completely checked aflatoxin B1 elab-

oration at 750 ppm (mg/L) while that of A. galanga

showed complete inhibition at 500 ppm only. The oil

combination of C. camphora and A. galanga showed more

efficacy than the individual oils showing complete inhibi-

tion of AFB1 production even at 250 ppm.

Keywords Antimicrobial � Antiaflatoxigenic � Aflatoxin �Fungitoxicity � Aspergillus flavus

Introduction

Many agricultural commodities are vulnerable to attack by a

group of fungi that are able to produce toxic metabolites

called mycotoxins. During the last few decades increasing

interest has been shown in mycotoxins in food commodities

because of their carcinogenic nature. According to FAO

estimates, 25% of the world food crops are affected by

mycotoxins each year (Pittet 1998). About 4.5 billion people

living in developing countries are chronically exposed to

uncontrolled amounts of toxins. Aflatoxicosis is recently

recognized as the sixth amongst the 10 most important

health risks identified by WHO for developing countries

(Williams et al. 2004). Among various mycotoxins,

aflatoxins have currently assumed significance due to their

deleterious effects on human beings, poultry and livestock.

A variety of tissues and organs such as the liver, kidney,

nervous system and gastrointestinal system have been

reported to be affected by aflatoxin. There are also reports on

adverse effects of aflatoxin on germination of seeds (Sinha

and Sinha 1993). Use of synthetic fungicides is the most

frequently encountered storage technology for protection of

food commodities from fungal deterioration as well as

mycotoxin contaminations. However, most of the synthetic

fungicides cause residual toxicity on grains and often disturb

the food chain. Such residues have often contributed to the

development of fungal resistance, which occurs when the

fungi are exposed to sub-lethal concentrations.

Plant products, especially essential oils, are one of the

most promising groups of natural compounds for the

development of safer antifungal agents and their employ-

ment for the control of different storage pests is also well

documented (Don-Pedro 1985; Varma and Dubey 2001).

During the past decade, interest in the use of these sub-

stances has increased due to their advantages over synthetic

fungicides, indigenous nature and non-mammalian toxicity

(Olivier et al. 1998; Varma and Dubey 1999). However,

little attention has been paid on efficacy of essential oils in

checking aflatoxin production. Hence, the present investi-

gation has been undertaken to find out the efficacy of

essential oils of Cinnamomum camphora (leaves) and

Alpinia galanga (rhizomes) in checking aflatoxin elabora-

tion by a toxigenic strain of Aspergillus flavus. The leaves

of C. camphora and rhizomes of A. galanga have been in

use since long in Indian system of medicine and the plants

grow luxuriously in the sub-tropical region of the Hima-

layan region of the country.

B. Srivastava � P. Singh � R. Shukla � N. K. Dubey (&)

Centre of Advanced Study in Botany, Banaras Hindu University,

Varanasi 221005, India

e-mail: nkdubey2@rediffmail.com;

bhawana_bhu53@rediffmail.com

123

World J Microbiol Biotechnol (2008) 24:693–697

DOI 10.1007/s11274-007-9526-0

Materials and methods

Isolation of essential oils from plants

The plants viz; C. camphora (L.) Presl. and A. galanga (L.)

Willd. were identified with the help of different floras (Bailey

1958; Duthie 1960; Maheshwari 1963; Santapau 1967;

Dubey 2004). Leaves of C. camphora (F. Lauraceae) were

collected from the botanical garden, Banaras Hindu Uni-

versity. Fresh rhizomes of A. galanga (F. Zingiberaceae)

were collected from the local market. These plant parts were

cut separately into small pieces with the help of scissors and

mortar and pestle after washing with sterilized distilled

water. The volatile fraction i.e. essential oil was isolated

through hydro distillation by Clavenger’s apparatus. The

isolated fractions of plant parts exhibited two distinct lay-

ers—an upper oily layer and the lower aqueous layer. Both

the layers were separated and the essential oils were stored in

clear glass vials separately after removing water traces with

the help of capillary tube and anhydrous sodium sulphate

(Tripathi et al. 2004).

Culture of fungus and medium

A toxigenic strain of A. flavus (Saktiman 3Nst) was used for

aflatoxin estimation. The strain was procured from Labo-

ratory of Mycotoxins , Department of Botany, University of

Bhagalpur, India. The method followed by Sinha and Sinha

(1993) was adopted for the estimation of the aflatoxin.

200 g of Sucrose, 0.5 g MgSO4 � 7H2O; 0.3 G KNO3 and

7 g yeast extract were mixed in 1 L of distilled water

(Dienner and Davis 1966) to make 1,000 mL of SMKY

medium.

Analysis of fungitoxic and anti-aflatoxigenic properties

of individual oils

An aliquot of 25 mL of SKMY medium was taken in

100 mL conical flask to which requisite amounts of the

essential oils of C. camphora and A. galanga were added

separately after taking two drops of Tween-80 so as to get

the concentration of 250 ppm (mg/L), 500, 750 and

1,000 ppm. The flasks were aseptically inoculated sepa-

rately with 5.0 mm disc of 7 days old culture of toxigenic

strain of A. flavus. The control set comprised the medium

without oil. The flasks were incubated for 10 days at

28 � 2 �C. Each control and treated flask was kept in

triplicates. After incubation, the content of each flask was

filtered through Whatman filter paper No. 1. The mycelia

were allowed to dry at 100 �C for 12 h. The mycelial dry

weight (biomass) was used to compare fungal growth in

treated and control sets. The filtrate was extracted with

20 mL chloroform in a separating funnel. The chloroform

extract was passed through anhydrous sodium sulphate

kept on a Whatman filter paper No. 42. The extract was

evaporated to dryness on water bath and the residue was

dissolved in 1 mL chloroform. Aflatoxins were detected by

thin layer chromatography (TLC) (Soares and Rodriguez

1989). Silica Gel-G (with 13% CaSO4 as binder) was used

as stationary phase for the TLC. The chloroform extract

obtained for aflatoxin screening was spotted on TLC plates.

The spotted plates were developed in the solvent system

comprising toluene: iso amyl alcohol: methanol (90:32:2 v/

v). The developed plates were air dried and the intensity of

aflatoxins was observed in ultraviolet fluorescence analysis

cabinet at 360 nm (AOAC 1984). The presence of afla-

toxins was confirmed chemically by derivatization with

trifluoroacetic acid and by spraying the developed plates

with aqueous solution of 50% sulphuric acid. For quanti-

tative estimation spots of aflatoxin B1 on TLC plates were

scrapped out and dissolved in 5 mL cold methanol, shaken

and centrifuged at 604.8g for 5 min. Optical density of

supernatants were taken at the wave length of 360 nm and

the amount of aflatoxin B1 was calculated (AOAC 1984).

Quantitative estimation of aflatoxin

The quantity of aflatoxin in control and treatment sets was

estimated by Spectrophotometer at 360 nm (AOAC 1984).

The amount of aflatoxin present in sample was calculated

according to the formula—

aflatoxin content (lg/kg) ¼ D�M

E � l� 1000

where, D = Optical density, M = Mol. wt. of aflatoxin (=

312), l = Path length (1 cm cell was used), E = Constant

(= 21,800)

Fungitoxic and antiaflatoxigenic effect of oil

combination

The oils of C. camphora and A. galanga were mixed in

equal amounts and a combination was prepared. The fun-

gitoxic and anti-aflatoxigenic activities of the oil

combination were recorded at 250, 500, 750 and

1,000 ppm following the method as earlier described in

case of analysis of individual oils.

GC-MS analysis

GC-MS analysis of oil samples and their combination was

done at Central Institute of Medicinal and Aromatic Plants,

694 World J Microbiol Biotechnol (2008) 24:693–697

123

Lucknow, India. The analysis was carried out on Perkin-

Elmer Turbomass/Auto XL system using a PE-5

(50 · 0.32 M, 0.25 l film thickness) capillary column with

oven temperature programmed from 100 to 28 �C at 3 �C/

min initial temperature holder of 20 min. Helium was

employed as carrier gas at 10 psi inlet pressure and spectra

generated at 70 eV. Identification of compounds was car-

ried out by comparing the MS of each peak with Wiley and

NIST libraries research program.

Statistical treatment of the results

The analysis of data was performed with the SPSS program

version 11.0. Mean and standard error of data were cal-

culated using SPSS software. The statistical level of

significance was fixed at P \ 0.05 (Hussaini et al. 2006).

Results

A corresponding decrease in fungal mycelial growth and

aflatoxin elaboration with increasing concentration of oils

was observed in the present study. It is evident from

Table 1 that the both the oils showed complete inhibition

of growth of the toxigenic strain of A. flavus at 1,000 ppm.

At decreasing concentrations, both the oils showed a

similar trend of reduction in growth of the toxigenic strain.

However, both the oils showed antiaflatoxigenic properties

at a concentration lower than the fungitoxic concentra-

tion.(Table 2) The C. camphora oil showed complete

inhibition of aflatoxin production at 750 ppm while that of

A. galanga at 500 ppm. Thus, A. galanga oil was found to

be more efficacious in inhibiting aflatoxin production than

the C. camphora oil. The oil combination of C. camphora

and A. galanga was found superior in efficacy than the

individual oils. It showed complete inhibition of the fungal

growth at 750 ppm and inhibition of aflatoxin production

even at 250 ppm the lowest concentration tested. The

inhibition of the growth of A. flavus and the aflatoxin B1 by

the oils and the combination is shown in Figs. 1 and 2.

The major components of C. camphora oil as deter-

mined by GC-MS were fenchone (34.82%), camphene

(23.77%), a-thujene (17.45%), L-limolene (7.54%) and cis-

p-menthane (5.81%). In case of A. galanga oil bicyclo

(4.2.0) oct-1-ene, 7-exoethenyl (58.46%), trans-caryo-

phyllene (7.05%), a-pinene (14.94%) with camphene

(2.15%), germacrene (1.78%) and citronellyl acetate

(1.41%) were recorded as major components (Table 3).

The combination of the essential oils of C. camphora

and A. galanga showed a totally different chemical profile.

The major components of combination as determined by

GC-MS are a-pinene (28.22%), fenchone (17.87%),

Table 1 Efficacy of essential oils and their combination on mycelial

biomass (g) � SE of a toxigenic strain of A. flavus in SMKY medium

Concentration

mg/L (ppm)

Biomass of mycelium (g) � SE

C. camphora A. galanga Oil combination

Control 0.54 � 0.01 0.54 � 0.01 0.54 � 0.01

250 0.46 � 0.00 0.42 � 0.02 0.32 � 0.01

500 0.34 � 0.01 0.30 � 0.01 0.26 � 0.01

750 0.28 � 0.01 0.22 � 0.01 0.00 � 0.00

1,000 0.00 � 0.00 0.00 � 0.00 0.00 � 0.00

Table 2 Efficacy of essential oils and their combination on aflatoxin

B1 elaboration (ppb) � SE of a toxigenic strain of A. flavus in SMKY

medium

Concentration

mg/L (ppm)

Aflatoxin elaboration (ppb) � SE

C. camphora A. galanga Oil combination

Control 561.60 � 4.16 561.60 � 4.16 561.60 � 4.16

250 436.80 � 4.16 418.40 � 4.16 0.00 � 0.00

500 187.20 � 1.98 0.00 � 0.00 0.00 � 0.00

750 0.00 � 0.00 0.00 � 0.00 0.00 � 0.00

1,000 0.00 � 0.00 0.00 � 0.00 0.00 � 0.00

Fig. 1 Photograph showing inhibition of A. flavus growth by essen-

tial oils. (a) Inhibition by C. camphora oil, (b) Inhibition by

A. galanga oil, (c) Inhibition by combination of C. camphora and

A. galanga oils

World J Microbiol Biotechnol (2008) 24:693–697 695

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camphene (15.42%), pentadecanol (10.44%), g-terpinene

(4.22%), b-asarone (3.39%), b-terpinene (3.16%), a-phel-

landrene (1.35%) and trans-caryophyllene (1.00%).

Discussion

The present study has thus shown that the essential oils

may have potential use in protecting foodstuffs against

A. flavus growth and aflatoxin contamination as well. Since

these plants have long been valued for their medicinal

properties, their oils may be recommended as antimicrobial

for protection of food commodities from storage fungi and

aflatoxins.

Although, many essential oils have been tested for

antifungal activity and their minimum inhibitory concen-

tration have been recorded. Dubey et al. (1983)

demonstrated the efficacy of essential oils of Ocimum ca-

num and Citrus medica as volatile fungitoxicant in

protection of some species against their post-harvest fungal

deterioration. The essential oils of Cymbopogon citratus,

Caesulia axillaris and Mentha arvensis have shown in vivo

fumigant activity in the management of storage fungi and

insects of some cereals without exhibiting mammalian

toxicity (Mishra et al. 1992; Varma and Dubey 2001).

Mallic and Nandi (1982), Varma and Dubey (1999), Tri-

pathi and Dubey (2004) and Holley and Patel (2005) have

recommended the use of volatile compounds in the control

of mould infestations during storage and enhanced shelf

life of food commodities. However, very insignificant

attention has been given to the efficacy of these products as

antiaflatoxigenic agents. In the present study, the oils

showed antiaflatoxigenic properties at concentrations lower

than their fungitoxic concentration. Thus the inhibition of

fungal mycelia by these oils may be through a mode other

than the aflatoxin inhibition. The difference in antifungal

and aflatoxin inhibition efficacy of essential oils may be

attributed to the oil composition, as has been reported by

Rasooli and Abyaneh (2004). The components of the oils

may be acting by different mode of action for antifungal

activity and aflatoxin inhibition.

It is also advisable that during screening programs, the

minimum inhibitory concentration of a product against

growth of fungal mycelium as well as aflatoxin elaboration

should be recorded so as to recommend it in the mainte-

nance of the quality of stored food commodities. Both the

oils are being reported for the first time in checking afla-

toxin production by the toxigenic strain of A. flavus. The

A. galanga oil was found to be more efficacious than the

C. camphora oil showing antiaflatoxigenic properties even

at lower concentrations.

In general, the inhibitory action of natural products on

fungal cells involves cytoplasm granulation, cytoplasmic

membrane rupture and inactivation and/or inhibition of

synthesis of intracellular and extracellular enzymes. These

actions can occur in an isolated or in a concomitant manner

and culminate with mycelium germination inhibition

(Cowan 1999). Phenolic compounds in the essential oils

Fig. 2 Chromatogram showing inhibition of aflatoxin B1 by essential

oils. (a) Inhibition by C. camphora oil, (b) Inhibition by A. galangaoil, (c) Inhibition by combination of C. camphora and A. galanga oils

Table 3 GC-MS based chemical profile of individual oils and their

combination

Compounds C. camphora(%)

A. galanga(%)

Combination

(%)

Fenchone 34.82 0.26 17.87

Camphene 23.77 2.15 15.42

a-Thujene 17.45 0.25 –

Germacrene – 1.78 –

trans-Caryophyllene – 7.05 1.00

L-Limolene 7.54 – –

Bicyclo(4.2.0)

oct-1-ene,7-exo ethenyl

– 58.46 –

a-Pinene – 14.94 28.22

cis-p-menthane 5.81 – –

Citronellyl acetate – 1.41 –

Pentadecanol – – 10.44

c-Terpinene – – 4.22

b-Asarone – – 3.39

b-Terpinene – – 3.16

a-Phellandrene – – 1.35

696 World J Microbiol Biotechnol (2008) 24:693–697

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have been mostly reported to be responsible for their bio-

logical properties (Deans et al. 1995; Dorman and Deans

2000), however some non-phenolic constituents of oils are

more effective. The aldehyde group is also believed to be

responsible for antimicrobial activity. Among the alcohols,

longer chain (C6–C10) molecules in the oils have been

reported to be more effective (Ultee et al. 2002; Holley and

Patel 2005). Such compounds present in the oils may be

held responsible for such biological activities. The inter-

esting finding of the present study is the better efficacy of

the oil combination of C. camphora and A. galanga in

checking the mycelial growth as well as aflatoxin elabo-

ration at a concentration lower than with the individual

oils. The GC-MS of the oil combination indicates its

altogether different chemical profile from both the indi-

vidual oils. The fungitoxic components of the individual

oils synergistically met together in the oil combination.

Hence, the oil combination was more efficacious than the

individual oil.

The results obtained justify future researches empha-

sizing the antimicrobial properties of plant products and

their possible use as viable alternatives to control the

microbial growth in stored food commodities. In addition,

the therapeutic use of essential oils and their combinations

comprising more than one fungitoxic ingredients may also

provide a solution for the rapid development of fungal

resistance which is currently noticed in case of different

prevalent antifungal therapeutics.

Acknowledgements Authors are thankful to UGC New Delhi, India

for financial assistance in the form of CAS Junior Research

Fellowship.

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