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International Biodeterioration & Biodegradation 62 (2008) 348–351

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ib iod

Antimycotic and antiaflatoxigenic potency of Adenocalymma alliaceumMiers. on fungi causing biodeterioration of food commodities andraw herbal drugs

Ravindra Shukla, Ashok Kumar, Chandra Shekhar Prasad, Bhawana Srivastava, Nawal Kishore Dubey*

Laboratory of Herbal Pesticide, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi-221005, India

a r t i c l e i n f o

Article history:Received 21 July 2007Received in revised form 14 November 2007Accepted 14 November 2007Available online 16 June 2008

Keywords:Adenocalymma alliaceumAflatoxin B1

BiodeteriorationFood commoditiesHerbal raw materials

* Corresponding author. Tel.: þ91 542 2313625; faxE-mail addresses: nkdubey2@rediffmail.com, nkdu

0964-8305/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.ibiod.2007.11.006

a b s t r a c t

This study characterized the antifungal and antiaflatoxigenic efficacy of an aqueous extract ofAdenocalymma alliaceum against fungal isolates that cause biodeterioration of cereal grains, legumeseeds, dry fruits, fresh fruits, and raw herbal drugs during storage and transportation. Fungal species thatcause biodeterioration were isolated from stored food and drug commodities by serial dilutions. Apoisoned food technique was adopted to assess fungitoxicity of the plant extract against fungal isolates.Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of extractsagainst two dominant storage fungi, Aspergillus flavus and Aspergillus niger, were superior to those of twocommonly used synthetic fungicides. Aflatoxin B1 synthesis was inhibited at low extract concentrationsin SMKY medium. Germination of legume seeds was unaffected by the extract application but ratherseedling growth was enhanced. Hence the aqueous extract of A. alliaceum possessed a wide spectrum offungitoxicity against fungi associated with deterioration of food commodities and herbal drugs. In ad-dition, it possessed antiaflatoxigenic activity and was non-phytotoxic.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Most foods are prone to biodeterioration by moulds and otherfungi during post-harvest processing, transport, and storage(Chauhan, 2004). These pathogens are opportunistic and ubiqui-tous, causing significant deterioration of food products, mainlythrough hydrolytic enzymes; thus they are responsible for con-siderable economic loss (Mishra and Dubey, 1994; Kumar et al.,2007a). Mould infections alter the chemical and nutritional char-acteristics of grains and seeds and, more importantly, contaminatethe remaining food with mycotoxins (Singh et al., 1990; Halt, 1998;Candlish et al., 2001; Juglal et al., 2002; Rasooli and Abyaneh,2004). Deterioration of stored raw herbal drugs by moulds andtheir toxins is quite alarming and a challenge for the pharmaceu-tical industry (Efuntoye, 1996; Roy, 2003).

Aflatoxin is one of the most common and dangerous mycotoxinsproduced by Aspergillus flavus during biodeterioration. About 4.5billion people in developing countries are systematically exposed touncontrolled amounts of aflatoxin (Williams et al., 2004). Aflatox-icosis induces depressed feed efficiency, abnormal liver chemistry,depressed immune response, carcinogenesis, and even death (Pier,

: þ91 542 2368174.bey@bhu.ac.in (N.K. Dubey).

All rights reserved.

1992). In plants, aflatoxins inhibit seed germination, seedlinggrowth, root elongation, and chlorophyll and carotenoid synthesis,and retard protein, nucleic acid, and synthesis of some enzymes(Jones et al., 1980).

The widespread indiscriminate use of chemical preservativeshas resulted in the emergence of resistant microorganisms leadingto food-borne diseases (Gibbons, 1992; Kaur and Arora, 1999;Akimpelu, 2001). To reduce this problem, there is a need to adoptstrategies that are accessible, simple in application, and nontoxic tohumans and plants, and that have sustainable broad-spectrumfungitoxicity.

Aromatic herbs are widely claimed to have broad-spectrumantimicrobial activity (Shah et al., 1992; Parimelazhagan andFrancis, 1999; Ovolade et al., 2004). Adenocalymma allliaceumMiers. (family: Bignoniaceae), commonly known as ‘‘garliccreeper,’’ is native to the Amazon rain forests of South America. Theleaves and flowers are widely consumed by Brazilians as a sub-stitute for garlic (Zoghbi et al., 1984). The plant has a number oftraditional medicinal properties; research has documented its usefor analgesic, antiarthritic, anti-inflammatory, antipyretic, anti-rheumatic, antitussive, depurative, purgative, and vermifugeproperties (Taylor, 2006). The present study was an attempt toexplore the fungicidal and antiaflatoxigenic potential of garliccreeper.

Table 1Fungitoxic spectrum of Adenocalymma alliaceum extract against biodeterioratingfungal isolates from different commodities

Target fungus % Fungal inhibition at following concentration (mean� SE)

5.0 mg ml�1 7.5 mg ml�1 10.0 mg ml�1

Absidia ramosa 61.25� 0.10 100 100Alternaria alternata 67.56� 0.03 83.33� 0.15 100Aspergillus flavus 20.77� 0.12 31.55� 0.16 54.88� 0.29Aspergillus fumigatus 48.88� 1.00 81.55� 0.16 100Aspergillus niger 18.55� 0.08 20.00� 0.10 40.00� 0.23Aspergillus terreus 85.50� 0.08 100 100Botryodiplodia

theobromae74.11� 0.08 100 100

Cladosporiumcladosporioides

28.22� 0.06 31.88� 0.06 51.55� 0.13

Colletotrichumgloeosporioides

67.77� 0.06 86.66� 0.11 100

Curvularia lunata 62.66� 0.18 87.44� 0.06 100Dreschlera sp. 100 100 100Fusarium oxysporum 73.33� 0.11 100 100Fusarium roseum 100 100 100Humicola sp. 100 100 100Mucor sp. 100 100 100Penicillium

chrysogenum55.00� 1.00 70.23� 0.82 100

Penicillium italicum 71.05� 0.52 82.00� 0.03 100Pestalotia sp. 90.24� 0.00 100 100Rhizopus nigricans 43.77� 0.06 82.66� 0.23 100Rhizopus stolonifer 82.82� 0.62 100 100

Table 2MIC and MFC of A. alliaceum extract (mg ml�1) against A. flavus and A. niger

Treatment A. flavus A. niger

MIC MFC MIC MFC

A. alliaceum extract 15.0 20.0 17.5 20.0Micronised wettable

sulphur (Wettasul-80)27.5 >30.0 27.5 >30.0

Carbendazim (Bavistin) 16.5 20.0 20.0 20.0

Values are mean (n¼ 3).

R. Shukla et al. / International Biodeterioration & Biodegradation 62 (2008) 348–351 349

2. Material and methods

2.1. Isolation of microorganisms

The biodeteriorating fungi were isolated from cereal grains (Oryza sativa L.,Triticum aestivum L.), legume seeds (Cajanus cajun L., Cicer arietinum L., Glycine maxL., Phaseolus mungo L., Pisum sativum L.), dry fruits (Anacardium occidentale L.,Buchnania lanzan Spreng., Phoenix dactylifera L., Prunus amygdalus Batsch.), diseasedfruits (Citrus limon L., Mangifera indica L.) and stored raw herbal medicines (Acoruscalamus L., Boerhavia diffusa L., Rauwolfia serpentina L., Withania somnifera L.). Serialdilution technique was adopted for isolation of fungi according to Aziz et al. (1998).Fungal isolates were identified following Ainsworth et al. (1973) and Burnett andHunter (1999). Isolates were maintained on potato dextrose agar (PDA) medium at28� 2 �C, RH 80% in BOD incubator (Scintech, India). The aflatoxigenic strain ofA. flavus Navjot 4NSt was procured from the Indian Agriculture Research Institute,New Delhi, and used in the antiaflatoxigenic assay.

2.2. Preparation of leaf extracts

Fresh leaves of A. alliaceum collected from the botanical garden of Banaras HinduUniversity were washed thoroughly with 2% sodium hypochloride solution andsterile distilled water. Dried leaves were pulverized into a fine powder using anelectric blender. Powdered leaves (100 g) were soaked in 100 ml of water for 24 h.The homogenate was filtered through double-layered cheesecloth and then passedthrough Whatman no. 1 filter paper; the filtrate was freeze-dried at �33 �C(Lyophilizer, CHRIST alpha, Germany). Dried aqueous extract was dissolved in dis-tilled water to a concentration of 50 mg ml�1.

2.3. Antifungal assay

The fungitoxic activity of the aqueous extract was tested against bio-deteriorating fungal isolates by the poisoned food technique (Perrucci et al., 1994)using Czapek Dox (CD) agar medium (NaNO3, 2 g; K2HPO4, 1 g; MgSO4$7H2O, 0.5 g;KCl, 0.5 g; FeSO4$7H2O, 0.01 g; sucrose, 30 g; agar, 15 g; Himedia, Mumbai in 1 ldistilled water, pH 6.8� 0.2). The requisite amount of aqueous extract in distilledwater was mixed with 8 ml sterilized CD agar in different petri plates to achieve finalconcentrations of 5, 7.5, and 10 mg ml�1 of growth medium. The control set had only2 ml distilled water in place of extract. The extract-amended medium wasinoculated individually at the center with a 5-mm inoculum disc of each test fungusand incubated at 28� 2 �C (7 days). The inhibition of fungal growth was calculatedby the formula

I ¼ ½ðCFc � CFtÞ=CFc�100

where I¼ percentage of inhibition; CFc¼ growth in the control; and CFt¼ growth inthe treatment (Albuquerque et al., 2006).

2.4. Determination of minimum inhibitory concentration (MIC) and minimumfungicidal concentration (MFC)

The MIC and MFC values of extract for A. flavus and Aspergillus niger (thetwo most prevalent biodeteriorating pathogens of cereals and legumes) weredetermined according to the method described earlier (Irobi and Daramola, 1993;Irkin and Korukluoglu, 2007). Different extract concentrations (5–20 mg ml�1) wereincorporated in CD broth. Fungal spore suspension (1 ml) in 0.1% Tween-80 wasadded to each tube and incubated at 30 �C for a week. CD broth with 1 ml inoculumserved as control. The lowest extract concentration that did not permit any visiblefungal growth was taken as the MIC. The tubes that did not show visible fungalgrowth were sub-cultured on extract-free CDA plates to determine if the inhibitionwas reversible. Minimum fungicidal concentration (MFC) was the lowest concen-tration that did not permit growth on the plates.

The MIC of Adenocalymma extract was also compared with the MICs of twocommonly used fungicides – Wettasul-80 (micronised wettable sulphur, SulphurMills Ltd., Mumbai) and Bavistin (Carbendazim-50, BASF India Ltd., Mumbai) by thetechnique described above.

2.5. Efficacy of the extract on aflatoxin B1 (AFB1) synthesis

To determine antiaflatoxigenicity of the aqueous extract, 25 ml of semi-syn-thetic SMKY (Ranjan and Sinha, 1991) liquid medium (sucrose, 200 g; MgSO4$7H2O,0.5 g in 1 l distilled water; KNO3, 0.3 g; yeast extract, 7 g; pH 5.6� 0.2) was dis-pensed into 100-ml Erlenmeyer flasks. The requisite volume of extract solution wasused in different flasks to achieve final concentrations of 5, 7.5, 10, 12.5, 15, 17.5, and20 mg ml�1 of growth medium. A fungal disc (5 mm diameter) from the periphery ofa 7-day-old culture of toxigenic strain A. flavus Navjot 4NSt was incorporated intoeach flask. Cultures were incubated on a rotary shaker at 28� 2 �C for 10 days. Theculture medium was filtered (Whatman no. 1) and mycelial dry weight wasdetermined using a hot air oven (110 �C, 12 h).

The filtrate was extracted with 20 ml CHCl3 in a separating funnel and the ex-tract was passed through anhydrous Na2SO4 and evaporated to dryness; the residuewas then re-dissolved in 1 ml CHCl3. Fifty microliters of the extract was spotted on

TLC plates and on AFB1; plates were developed in toluene:isoamyl alcohol:methanol(90:32:2 v/v; Qualigens India Ltd., Mumbai). Plates were air-dried and viewed underUV transilluminator (365 nm). The fluorescent spots were scrapped off the plates,dissolved in 5 ml cold CH3OH, and centrifuged (3000 rpm, 5 min) and absorbance(265 nm) of supernatant was taken using a spectrophotometer (Systronics, India).The amount of AFB1 was calculated according to the following formula as given byKumar et al. (2007b)

½D�M=E � L�1000;

where D¼ absorbance; M¼molecular weight of AFB1 (312); E¼molar extinctioncoefficient of AFB1 (21,800); and L¼ path length (a 1-cm cell was used).

2.6. Phytotoxicity assay

The phytotoxicity of aqueous extract with respect to seed germination andseedling growth of the chick pea (C. arietinum L. var. Radha) was assayed followingthe method of Nwachukwu and Umechuruba (2001). Seeds were soaked in toxic andhyper toxic concentrations of extract for 3 h and kept on moistened double-layeredWhatman no. 1 filter paper in sterilized petri plates (20 cm) at 27 �C. After two days,the number of germinated seeds was recorded and radicles and plumules lengthswere measured every 24 h up to 144 h (6 days).

2.7. Statistical analysis

All the treatments were in triplicate and the data are mean� SE. The data wereanalyzed with one-way ANOVA and means were separated by Tukey’s multiplerange tests using software (SPSS 10.0; Chicago, IL, USA).

3. Results

The fungi isolated from different stored commodities are listedin Table 1. Aspergillus spp. were most prevalent on stored legumes,cereals, dry fruits, and raw herbal drugs. A. niger and A. flavus were

Table 3Effect of A. alliaceum extract on A. flavus biomass and Aflatoxin B1 production

Extract (mg ml�1) Mycelial dry weight (g) Mycotoxin content(mg kg�1)

0 (control) 0.444� 0.006a 244.22� 20.19a

5.0 0.300� 0.040b 141.19� 16.63b

7.5 0.214� 0.008bc 61.05� 13.75c

10.0 0.146� 0.004cd 8.10� 4.58cd

12.5 0.107� 0.012de 0.00� 0.00d

15.0 0.034� 0.018e 0.00� 0.00d

17.5 0.000� 0.000f 0.00� 0.00d

20.0 0.000� 0.000f 0.00� 0.00d

Values are mean (n¼ 3)� SE.The means followed by same letter in the same column are not significantly differentaccording to ANOVA and Tukey’s multiple comparison tests.

R. Shukla et al. / International Biodeterioration & Biodegradation 62 (2008) 348–351350

isolated from 12 out of 17 commodities examined. Stored rawherbal drugs were found to be heavily infested with different fungalspecies and a maximum of six fungal species were isolated from R.serpentina; A. calamus followed with five. In all four dried fruitsexamined Zygomycetes genera (Rhizopus and Mucor spp.) weremost frequent, followed by Aspergillus.

The present study reports on the broad-spectrum fungitoxicityof Adenocalymma extract (Table 1). The inhibitory effect of theextract was concentration-dependent. Most of the fungi treatedwere susceptible to 10 mg ml�1 concentration of the extract withcomplete inhibition of spore germination. Among all of them, A.niger, A. flavus, and Cladosporium cladosporioides were found to bemost resistant, with their spore germination inhibited only 40%,55%, and 52%, respectively, against Adenocalymma extract. The mostsensitive fungal strains were Mucor sp., Dreschlera sp., Fusariumroseum, and Humicola sp. had absolute inhibition of spore germi-nation at a low concentration (5 mg ml�1).

The MIC and MFC values of A. alliaceum extract against the twomost prevalent fungi are presented in Table 2. The extract preventedspore germination of A. flavus completely from a concentration15 mg ml�1 upwards, whereas the MIC for A. niger was 17.5 mg ml�1.A lethal concentration of extract (20 mg ml�1) was common for bothspecies. Wettasul-80, the commonly used synthetic fungicide, hada high MIC (27.5 mg ml�1) and MFC (30 mg ml�1) relative to those ofA. alliaceum. The other fungicide, Bavistin, with fungicidal activity at20 mg ml�1, was similar to that of the extract. To sum up, the extractremained fungistatic at a concentration (15 mg ml�1) lower thanthat of Bavistin (16.5 mg ml�1).

Table 3 summarizes the effect of extract on aflatoxin B1 pro-duction by toxigenic A. flavus Navjot 4NSt strain. The extractinhibited AFB1 synthesis completely at 12.5 mg ml�1 despite themycelial growth upto 17.5 mg ml�1. AFB1 content declined signi-ficantly in SMKY medium in a dose-dependent manner. Bothmycelial growth and AFB1 production followed a declining trendwith increase in extract concentrations.

Table 4Effect of Adenocalymma alliaceum extract on seed germination and seedling growth of c

Germination period (h) Mean length of seedlings (cm.)� SE

Control To

R P R

24 0.25� 0.02 – 0.48 1.36� 0.07 – 172 3.40� 0.10 – 3.96 4.60� 0.10 – 4.120 5.66� 0.66 1.00� 1.00 6.144 7.73� 0.39 2.70� 0.20 9.

R¼ radicle, P¼ plumule.The means in the same row are not significantly different according to ANOVA and Tuke

The results obtained during phytotoxicity testing of extract of A.alliaceum are shown in Table 4. A 100% germination of seeds wasrecorded following 72 h in all three sets, including controls. Seed-ling emergence was almost similar in all the sets until 96 h ofincubation, but the size of radicles in treated seeds was greater thanthat in the controls for the rest of the incubations. Plumules orig-inated after 96 h and mean length was a maximum of 5.66 cm in20 mg ml�1 seeds soaked followed by 3.86 cm in 10 mg ml�1

compared to controls at 2.70 cm.

4. Discussion

An attempt was made to evaluate the efficacy of leaf extract of A.alliaceum as a fungitoxicant and its possible applicability in thebiodeterioration control of wheat, rice, grain, peas, and dried fruits,as well as raw herbal drugs, and, in turn, enhancing their marketvalue. The superiority of extract over commonly used syntheticfungicides at the lowest levels of MICs further supports its exploi-tation as an alternative fungitoxicant.

Perusal of the literature reveals that Garcinia, Xanthium, Ammi,and Polymnia sonchifolia have also been exploited to control growthof moulds and aflatoxin production in a manner similar to that ofAdenocalymma (Mahmoud, 1999; Pinto et al., 2001; Joseph et al.,2005). The antiaflatoxigenic efficacy of the leaves of A. alliaceum isthe first report of this type. The fumigant activity and characteristicodour of the plant are due to the presence of diallyl, di-, tri-, andtetrasulphide (Gracas et al., 1984; Zoghbi et al., 2002). The diallylsulphides are known to be toxic to different microorganisms (Chenet al., 1999; Tsao and Yin, 2001). In addition, naphthoquinones inleaves of this plant also showed toxicity against other microbes(Itokawa et al., 1992). The garlicky substance Aliin, found in leavesof garlic creeper, may also be the antimicrobial component(Apparao et al., 1981). The volatile fungitoxicity of garlic creeperleaves against Drecshlera oryzae has been reported to persist forseven days at room temperature and at temperatures up to 50 �C(Chaturvedi et al., 1987). Hence the fresh leaves should be consid-ered for extraction and isolation of fungicides.

The biodeteriorating fungi can easily develop resistant traitsagainst a single active component, but the plant extract containingdifferent antimicrobial ingredients may be exploited for fungitoxicpotency owing to the synergistic effects of different components.Interestingly, no phytotoxicity was found in the current study onseed germination tests and, rather, enhanced seedling growth wasrecorded in extract-treated seeds. It was also significant to note thatfor a slight increase in extract concentration there was an abruptdecline in AFB1 synthesis in spite of a gradual decrease in mycelialbiomass.

Plant extracts are potentially useful additives for food preser-vation as they are likely to prolong shelf life and improve thequality of stored food products (Guynot et al., 2003; Suhr and

hick pea

xic concentration (10 mg ml�1) Hypertoxic concentration(20 mg ml�1)

P R P

25� 0.02 – 0.26� 0.01 –.41� 0.02 – 1.53� 0.14 –60� 0.10 – 3.46� 0.26 –73� 0.23 – 4.56� 0.23 –73� 0.93 0.83� 0.83 6.46� 0.81 2.06� 1.0350� 0.76 3.86� 0.69 8.33� 1.01 5.66� 1.33

y’s multiple comparison test.

R. Shukla et al. / International Biodeterioration & Biodegradation 62 (2008) 348–351 351

Nielsen, 2003; Tripathi et al., 2004). They may also be considered asadditives to control fungal contamination of raw herbal medicines.Garlic creeper is an abundant and easily available plant. Our datashowed that extracts from this plant have broad antifungal activityagainst moulds associated with biodeterioration of food and rawherbal medicines and that they possess antiaflatoxigenic proper-ties. Further research is warranted on garlic creeper extracts’ modeof action and on their practical applicability for food and drugpreservation.

Acknowledgements

The authors thank the Department of Science and Technology,New Delhi, for financial assistance. We also extend our thanks toProf. S. P. Singh for critical suggestions, and to Mr. S. K. Gond and Mr.V. C. Verma of the Mycopathology Laboratory, Banaras Hindu Uni-versity, Varanasi, India, for their guidance in fungal identification.

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