antifungal, aflatoxin inhibitory and free radical-scavenging activities of some medicinal plants...

ANTIFUNGAL, AFLATOXIN INHIBITORY AND FREERADICAL-SCAVENGING ACTIVITIES OF SOME MEDICINALPLANTS EXTRACTSjfq_441 1..8

RAVINDRA SHUKLA, PRIYANKA SINGH, BHANU PRAKASH, ANURADHA and N.K. DUBEY1

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

1Corresponding author. TEL: +91-9415295765;FAX: +91-5422368174; EMAIL:[email protected]

Received for Publication August 24, 2011Accepted for Publication January 25, 2012

doi:10.1111/j.1745-4557.2012.00441.x

ABSTRACT

The antifungal, aflatoxin inhibitory and antioxidant activity of methanol–aqueousextract (2:1) of 62 medicinal plants was explored. Based on the antifungal results, theextracts of 25 plants showed more than 50% antifungal activity and were furtherinvestigated for their aflatoxin inhibition and antioxidant properties. Methanol–aqueous extracts of Phyllanthus emblica and Terminalia chebula fruits caused 100%inhibition of aflatoxin production by the toxigenic strain of Aspergillus flavus insemisynthetic medium at 1 mg/mL. In addition, P. emblica (IC50 = 4.1 mg/mL) andT. chebula (IC50 = 6.9 mg/mL) fruits extracts exhibited strong antioxidant activityduring the 2,2-diphenyl-1-picrylhydrazyl radical-scavenging assay in comparisonwith butylated hydroxytoluene (IC50 = 8.1 mg/mL) and butylated hydroxyanisole(IC50 = 6 mg/mL).

PRACTICAL APPLICATIONS

Based on the results of the present study, methanol–aqueous extracts of Phyllanthusemblica and Terminalia chebula, being endowed with strong antifungal, aflatoxininhibitory and antioxidant activity, may be recommended as plant-based preserva-tives for the enhancement of shelf life of food items and their protection from theundesirable harmful effects of molds, aflatoxin and free radical-mediated damages.

INTRODUCTION

World agricultural industries suffer from severe losses of foodcommodities because of various pest infestations, and thesituation is alarming in tropical countries because of theirvaried climatic conditions, poor agricultural practices andsocioeconomic status providing a suitable platform to buildup and proliferate harmful microorganisms (Wagacha andMuthomi 2008). Among different storage pests, mold con-tamination has from time to time attracted the attention ofthe scientific community because of its ubiquitous nature andits secretion of toxigenic metabolites on food items underfavorable environmental conditions (Nguefack et al. 2009).According to the Food and Agriculture Organization, nearly25% of agricultural food products are affected by varioustypes of mycotoxin contamination throughout the world(WHO 1999). Aflatoxins produced by the toxigenic strain ofAspergillus flavus and Aspergillus parasiticus are regarded as

targeted mycotoxins by the scientific community because oftheir carcinogenic, teratogenic, mutagenic potential and ther-mostable nature along with the ability of bioaccumulation inthe mammalian systems (WHO 2006; Wu 2006; Wagacha andMuthomi 2008).

Reports regarding the negative effects of synthetic pesti-cides on treated food items, including enhancement of freeradicals resulting to oxidative damages of various cellularcomponents, off-flavors and changes in texture, adverse sideeffects on mammalian systems, and development of resis-tance by treated microorganisms, are currently an issue ofdiscussion among food scientists (Tolouee et al. 2010;Prakash et al. 2011a). Hence, the search for alternative preser-vatives is strongly felt by the food industry for postharvesttreatment of food items.

Plant products have long been regarded as a natural guardfrom the various harmful aspects of pest infestation. The bio-logical activity of different plant extracts and essential oils has

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Journal of Food Quality ISSN 1745-4557

1Journal of Food Quality •• (2012) ••–•• © 2012 Wiley Periodicals, Inc.

been investigated against bacteria, fungi, insects and lipid per-oxidation (Yi et al. 2008; Conte et al. 2009; Korukluoglu et al.2009; Rahman et al. 2009; Mahlo et al. 2010; Mehr et al. 2010;Prakash et al. 2010, 2011a; Silva et al. 2010; Belewa et al.2011). Recently, different plant products have gained specialsignificance as safe preservatives in the agri-food industriesand have been recognized as alternatives to synthetic chemi-cals in view of their endowed biologically active components,generally recognized as safe status for the environment as wellas consumers, availability of sufficient raw materials and theirefficacy at low concentrations in comparison with syntheticchemicals (Burt 2004; Bakkali et al. 2008; Prakash et al.2011a).

The objective of the present investigation was to explorethe in vitro, antifungal, aflatoxin inhibition and antioxidantactivity of the methanol–aqueous extracts (2:1) of differentplant extracts so as to ascertain their possible application inprotection of food commodities from the harmful effects oftoxigenic strains of A. flavus, aflatoxin B1 contamination, aswell as undesirable changes generated by the free radical-mediated process during different storage conditions. Theselection of 62 plant species for testing in the present investi-gation is based on their application in the treatment ofvarious human diseases as astringent, laxative, carminativeand aphrodisiac agent in traditional system of medicine (Pra-japati et al. 2003).

MATERIALS AND METHODS

Plant Materials

The plant parts such as leaves, stems, barks and rhizomes of 62plant species were collected from their natural habitat fromdifferent regions of the Eastern Uttar Pradesh, India betweenAugust and December, 2008. The plants were cleaned, air-dried and stored under dark refrigerated conditions. Allsamples were identified and authenticated by Prof. N. K.Dubey. Their voucher specimens were prepared and depos-ited in the Herbarium of the Laboratory of Herbal Pesticides,Department of Botany, Banaras Hindu University and Vara-nasi, India. Botanical names, common names, family andplant parts used to obtain the extracts are listed in Table 1.

Preparation of Plant Extracts

Ten grams of ethnomedicinally used parts of the plant specieswere ground to a particle size of <1 mm and soaked separatelyin 100 mL methanol–water (2:1 v/v basis) for 24–48 h. Thehomogenate was filtered through a double-layered cheesecloth and then was passed through Whatman no.1 filter paper(Whatman, Brentford, U.K.). The solvent was evaporatednearly to dryness using a roto-evaporator. The resulting crys-talline or heavy syrup concentrate was subsequently lyo-

philized (Lyophilizer, Alpha, Christ, Osterode, Germany) toyield a dry residue. The stock solution was prepared by dis-solving dried extract in dimethyl sulfoxide (DMSO) to a con-centration of 50 mg/mL and kept at 4C in the dark.

Antifungal Assay

The poisoned food method, described by Singh et al. (2010)with slight modification, was used for determining the inhibi-tion of radial mycelial growth of the test fungus by the plantextracts. An aflatoxin B1-producing strain of A. flavus NKD-235 previously isolated from Arachis hypogaea L. (Shuklaet al. 2009) was chosen as test micoorganism. Czapek’s dox-agar (CDA) medium (NaNO3, 2 g; K2HPO4, 1 g; MgSO4, 0.5 g;KCl, 0.5 g; FeSO4, 0.01 g; sucrose, 30 g; agar, 15 g; 1 L distilledwater, pH 6.8 � 0.2; Sisco Research Lab., Mumbai, India) wasused for the assay. Streptomycin (300 mg/L) was added to themedium for controlling bacterial growth. Each plant extract(20 mg) in 0.4 mL DMSO was amended to 9.6 mL moltenCDA in Petri plates (90 mm). CDA plates containing DMSO(0.4 mL) only served as control. A 5-mm disc of test fungus (7days old) was placed upside down on the center of the platewith fungal species in contact with the growth medium. Cul-tures were incubated in the dark at 28 � 2C (7 days). Colonydiameter of the test fungus in treatment and control set wasmeasured, and antifungal activity was calculated in terms ofpercent mycelial inhibition following Kumar et al. (2007).

% mycelial inhibition 1= − ×dc dt

dc00

dc = average diameter of fungal colony in control setdt = average diameter of fungal colony in treatment set

Efficacy of Plant Extracts in Inhibition ofAflatoxin B1 Production by the ToxigenicStrain of A. flavus NKD-235

The extracts of 25 plants that showed more than 50% antifun-gal activity were investigated for aflatoxin inhibition. Todetermine the antiaflatoxigenicity of the plant extracts,24.5 mL of semisynthetic SMKY broth medium (sucrose,200 g; MgSO4.7H2O, 0.5 g; KNO3, 0.3 g; yeast extract, 7 g in1 L distilled water; pH 5.6 � 0.2, Sisco Research Lab) was dis-pensed into 100-mL Erlenmeyer flasks. The requisite volumeof extract solution in DMSO was used in different flasks toachieve final concentrations of 1 mg/mL of growth medium.A fungal disc (5 mm in diameter) from the periphery of a7-day-old culture of toxigenic strain of A. flavus NKD-235was incorporated into each flask. Cultures were incubated ona rotary shaker at 28 � 2C for 10 days. The culture mediumwas filtered (Whatman no. 1) and mycelial dry weight(MDW) was determined using a hot air oven (80C, 12 h). The

ANTIFUNGAL ACTIVITY OF SOME PLANT EXTRACTS R. SHUKLA ET AL.

2 Journal of Food Quality •• (2012) ••–•• © 2012 Wiley Periodicals, Inc.

TABLE 1. ANTIFUNGAL ACTIVITY OF PLANT EXTRACTS AGAINST ASPERGILLUS FLAVUS NKD-235

Plants Vernacular/common name Family Part used

% mycelial inhibition

(2 mg/mL)

Abrus precatorius L. Ratti Papilionaceae L 65.4 � 2.2o

Aegle marmelos (L.) Correa Bael Rutaceae L 62.0 � 0.5n

Aloe barbadensis Mill. Ghikumari Liliaceae L 70.1 � 0.9q

Allamanda cathartica L. Golden trumpet Apocyanaceae L 12.2 � 1.2c

Alstonia scholaris (L.) R.Br. Saptaparni Apocyanaceae L 23.1 � 2.1e

Anagallis arvensis L. Krishna-neel Primulaceae L 22.0 � 0.0e

Andrographis paniculata (Burm.f.) Wall. Kaalmegh Acanthaceae S, L 38.2 � 1.4h

Anisomeles ovata R.Br. Indian catmint Lamiaceae L 32.6 � 1.7g

Artabotrys odoratissimus R.Br. Kantili champa Annonaceae L 72.4 � 1.0q

Bixa orellana L. Sindoori Bixaceae F 39.4 � 0.4i

Calotropis procera (Ait.) R.Br. Madar Asclepiadaceae L 70.3 � 1.3q

Carica papaya L. Papeeta Caricaceae L 62.0 � 0.6n

Catharanthus roseus (L.) G.Don Sadabahar Apocyanaceae L 28.5 � 1.4f

Clerodendrum viscosum Vent. Bhates Verbenaceae L 45.4 � 0.4k

Cordia dichotoma Frost. Lasorha Ehretiaceae L 40.4 � 0.4i

Diospyros ebenum J.Koenig Tendu Ebenaceae L 0.0 � 0.0a

Dracaena cancinna Kunth. Dragon tree Liliaceae L 49.7 � 2.7l

Duranta repens L. Pigeon berry Verbenaceae L 34.9 � 0.9g

Eclipta prostrate L. Bhringraj Asteraceae L 48.9 � 0.9l

Ehratia leavis Roxb. Datranga Ehretiaceae L 35.2 � 2.2g

Eupatorium cannabinum L. Hemp agrimony Asteraceae L 52.5 � 0.3m

Evolvulus alsinoides L. Shankhapushpi Convolvulaceae S, L 71.6 � 0.4q

Hamelia patens Jacq. Scarlet bush Rubiaceae L 8.0 � 0.6b

Ipomoea carnea Mart. Morning glory Convolvulaceae L 40.0 � 0.0i

Ixora arborea Roxb. Ixora Rubiaceae L 41.6 � 0.4j

Jatropha curcas L. Ratanjot Euphorbiaceae L 75.5 � 0.5r

Justicia adhatoda L. Vasak Acanthaceae L, B 39.1 � 0.6l

Lantana camara L. Ghaneri Verbenaceae L 43.0 � 0.0j

Launaea nudicularis Roxb. Muskani Asteraceae L 31.4 � 1.0f

Lawsonia inermis L. Mehndi Lythraceae L 51.3 � 1.8l

Melia azaderach L. Bakain Meliaceae L 81.4 � 1.4tMoringa oleifera Lamk. Sahijan Moringaceae L 85.5 � 0.5u

Murraya koenigii (L.) spreng. Curry patta Rutaceae L 78.1 � 0.8s

Murraya paniculata (L.) Jacq. Kaamini Rutaceae L 64.5 � 0.4n

Nepeta hindostana (Roth.) Haines Jatamansi Lamiaceae L 33.3 � 2.3g

Ocimum basilicum L. Kaali tulsi Lamiaceae L 95.8 � 0.4w

Oxalis corniculata L. Tinpatiya Oxalidaceae L 36.3 � 1.3h

Paederia foetida Gandhaprasarini Rubiaceae L 14.2 � 2.2c

Peristrophe bicalyculata (Retz.) Nees Kakajangha Acanthaceae L, S 28.2 � 0.2f

Phyllanthus emblica L. Amla Phyllanthaceae F 98.4 � 0.9x

Plumeria rubra L. Temple tree Apocyanaceae B 28.5 � 0.5f

Polyalthea longifolia Sonn. Asoka Annonaceae L 51.0 � 0.0l

Pongamia pinnata (L.) Pierre Karanj Fabaceae L 95.7 � 0.7w

Putranjiva roxburghii Wall. Putranjiva Madhumalti Putranjivaceae L 41.5 � 1.3i

Quisqualis indica L Sarpgandha Combretaceae L 38.6 � 0.3h

Rauvolfia serpentina (L.) Benth. ex Kurz Bara chandrika Apocyanaceae R 25.7 � 2.0e

Rauvolfia tetraphylla L. Rendi Apocyanaceae R 43.5 � 0.3j

Ricinus communis L. Toothed dock Euphorbiaceae L 79.4 � 0.6s

Rumex dentatus L. Kamarkas polygonaceae L 17.0 � 2.0d

Salvia plebia R.Br. Reetha Lamiaceae L 42.1 � 0.9j

Sapindus mucorossi Gaertn. Akarkara Sapindaceae B 68.0 � 0.0p

Spilanthes acmella Murr. Seeta ashok Asteraceae L 34.8 � 0.8g

Saraca asoca (Roxb.) Wilde Arjun Fabaceae B 41.2 � 1.3i

Terminalia arjuna Roxb. Bahera Combretaceae B 82.7 � 1.6t

Terminalia bellerica (Gaertn.) Roxb. Harrah Combretaceae F 95.2 � 0.6w

Terminalia chebula Retz. Giloy Combretaceae F 90.1 � 1.1v

Tinospora cordifolia (Thunb.) Miers Sahadevi Menispermaceae S 34.4 � 1.6g

Vernonia cinerea (L.) Less. Nirgundi Asteraceae L 40.3 � 0.3i

Vitex negundo L. Ashwagandha Lamiaceae L, S 64.7 � 1.1n

Withania somnifera (L.) Dunal Dhataki Solanaceae L 45.6 � 0.9k

Woodfordia fructicosa (L.) Kurz. Adarakh Lythraceae L 31.7 � 1.7f

Zingiber officinale Roscoe Ratti Zingiberaceae Rh 92.1 � 1.5v

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

ANOVA, analysis of variance; B, bark; F, fruit; L, leaves; R, root; Rh, rhizome; S, stem; SE, standard error.

R. SHUKLA ET AL. ANTIFUNGAL ACTIVITY OF SOME PLANT EXTRACTS


filtrate was extracted with 20 mL CHCl3 in a separatingfunnel and the extract was passed through anhydrous Na2SO4

and evaporated to dryness. The residue was then re-dissolvedin 1 mL CHCl3. Fifty microliter of the extract was spotted onthin-layer chromatography (TLC) plates and developed intoluene : isoamylalcohol : methanol (90:32:2 v/v; QualigensIndia Ltd., Mumbai, India). Plates were air-dried and viewedunder ultraviolet-visible transilluminator (365 nm). Thefluorescent spots were scrapped off the plates, dissolved in5 mL cold CH3OH and centrifuged (3,000 rpm, 5 min).Absorbance (265 nm) of the supernatant was taken using aspectrophotometer (Systronics India Ltd., Mumbai, India).

The amount of aflatoxin B1 (AFB1) was calculated accord-ing to the following formula as described by Kumar et al.(2007)

AFB content g 11 ( )μ LD M

E L= ×

×× 000

where D = absorbance; M = molecular weight of AFB1 (312);E = molar extinction coefficient of AFB1 (21,800); andL = path length (1 cm cell was used).

Determination of Antioxidant Activity

Scavenging Activity of2,2-Diphenyl-1-Picrylhydrazyl Free Radical throughTLC. The antioxidant activity of extracts of 25 plant specieswas measured through 2,2-diphenyl-1-picrylhydrazyl(DPPH) free radical-scavenging assay as previously describedby Salazar et al. (2008) with slight modification. Briefly, 1 mLof each extract (1 mg/mL in methanol) was applied to a chro-matographic plate. Chromatography was conducted usingethyl acetate : methanol (1:1) as running solvent. The platewas developed using a DPPH solution (2 mg/mL in metha-nol) as spaying agent. The TLC was observed on ordinarylight after 30 min. The yellow spots from reduced DPPH wereobserved against a purple background.

Scavenging Activity of DPPH Free Radical throughSpectrophotometry. Assay of DPPH scavenging activityby spectrophotometry was conducted according to Prakashet al. (2010), with some modifications. Different concentra-tions (up to 200 mg/mL) of the extracts were added to 0.004%DPPH solution in methanol (5 mL). After 30 min of incuba-tion at room temperature (25 � 2C), the absorbance wastaken against a blank at 517 nm using a spectrophotometer.Scavenging of DPPH free radical with reduction in absor-bance of the sample was taken as a measure of their antioxi-dant activity following Sharififar et al. (2007). Butylatedhydroxytoluene (BHT) and butylated hydroxyanisole (BHA)were used as positive control. IC50, which represented the con-

centration of the extract that caused 50% neutralization ofDPPH radicals, was calculated from the graph plottingbetween percentage inhibition and concentration.

Percentage inhibition I A A A 1blank sample blank%( ) = −( ) × 00

where, A blank is the absorbance of the control (withoutextract), and A sample is the absorbance of the extract.

Statistical Analysis

All the experiments were performed in triplicate and dataanalysis was done on mean � standard error subjected toone-way analysis of variance (ANOVA). Means are separatedby the Tukey’s multiple-range test when ANOVA was signifi-cant (<0.05) (Statistical Package for the Social Sciences[SPSS] 10.0; SPSS Inc., Chicago, IL).

RESULTS AND DISCUSSION

The antifungal activity of plant extracts in terms of percentmycelial inhibition calculated with respect to control againstthe toxigenic strain of A. flavus (NKD-235) is presented inTable 1. All the plant extracts showed promising activityagainst the test fungus, except that of Diospyros ebenumshowing no activity. Twenty-five plant extracts showed morethan 50% antifungal activity. The antifungal activity ofextracts of Ocimum basilicum, Phyllanthus emblica, Pongamiapinnata, Terminalia bellerica, Terminalia chebula and Zingiberofficinale were recorded to be 95.8, 98.4, 95.7, 95.2, 90.1 and92.1%, respectively. However, extracts of Aloe barbadensis,Artabotrys odoratissimus, Calotropis procera, Evolvulus alsi-noides, Jatropha curcas, Melia azaderach, Moringa oleifera,Murraya koenigii, Ricinus communis and Terminalia arjunashowed moderate to high fungitoxicity ranging between 70and 85%. Allamanda cathartica, Hamelia patens, Paederiafoetida and Rumex dentatus showed poor antifungal activity.

MDW of the test fungus, AFB1 produced in SMKYmedium and AFB1 produced with respect to fungal growthare presented in Table 2. Extracts of P. emblica and T. chebulacaused complete inhibition of AFB1 production at 1 mg/mL,whereas at this concentration, 29.6 and 50.6 mg mycelialweight was found. Similarily, M. koenigii (3.0 mg/g), O. basi-licum (3.1 mg/g), Polyalthea longifolia (5.5 mg/g), P. pinnata(4.2 mg/g), T. arjuna (5.7 mg/g), T. bellerica (8.6 mg/g), Vitexnegundo (6.8 mg/g) and Z. officinale (2.4 mg/g) showed pro-nounced antiaflatoxigenic activity as compared with control(19.8 mg/g). Abrus precatorius, Aegle marmelos, A. barbaden-sis, A. odoratissimus,C. procera, Carica papaya, Eupatoriumcannabinum, E. alsinoides, J. curcas, Lawsonia inermis,M. azaderach, M. oleifera, R. communis, Sapindus mucorossiand Murraya paniculata extracts were found to be least effec-tive against the aflatoxin production.



Antifungal activity of plant extract clearly showed that outof 62 plant extract tested against the toxigenic strain ofA. flavus, none could check completely the growth of testfungus at 2 mg/mL. However, most of them showed pro-nounced efficacy in checking aflatoxin production. Extractsof P. emblica and T. chebula caused complete inhibition ofAFB1 production at 1 mg/mL. Thus, the results showed thatthe plant extracts exhibited more efficacy as aflatoxin inhibi-tion than the fungal growth. The results showed similaritywith the earlier hypothesis of some worker that plant-products extract/essential oils have two different mode ofaction against the fungal growth and toxin production(Rasooli and Razzaghi-Abyaneh 2004; Prakash et al. 2010,2011b).

Most of the plant extracts showed DPPH radical-scavenging activity in dose-dependent manner. The percentinhibition (IC50) value of methanolic extract of all the testedplant extracts was found between 4.1 and 160.5 mg/mL. Cal-culated IC50 of 25 plant extracts are presented in (Fig. 1). TheIC50 of P. emblica and T. chebula extracts was 4.1 and 6.9 mg/mL, respectively, which was close to that of synthetic antioxi-dants BHA (6 mg/mL) and BHT (8.1 mg/mL). Similarly, theextracts of M. oleifera and M. koenigii, O. basilicum, T. arjuna

and T. bellerica also exhibited remarkable free radical-scavenger activity with of IC50 19.6, 21.2, 23.4, 13.7 and13.3 mg/mL, respectively. These extracts showed bleaching ofviolet color of DPPH to yellow on TLC plates. The extract ofV. negundo, which showed significant antiaflatoxigenic activ-ity, could not exhibit radical-scavenger activity because ofpoor IC50 (160.5 mg/mL).

Among all plant extracts, the radical-scavenging activity ofextracts of P. emblica was found superior to that of BHA andBHT, while T. chebula was found close to BHT but more thanthe BHA. The high radical-scavenging activity of these plantextacts suggests their possible application as a plant-basedfood additive to overcome the harmful effects of reactiveoxygen species viz.; damages in lipid, protein, nucleic acidcomponents of food items, thus, protecting them from theharmful effects generated by the free radicals.

It is clear from the present investigation that the extracts ofP. emblica and T. chebula were found efficacious as antifungal,aflatoxin inhibitory as well as antioxidant. Hence, the findingsof present investigation are supportive to the earlier view ofsome workers that the plant extract have pronounced efficacyas an antimicrobial and antimycotoxin because of the pres-ence of antioxidant compounds viz. polyphenols, phenols,

TABLE 2. EFFECT OF PLANT EXTRACTS ONINHIBITION OF MYCELIA DRY WEIGHT ANDAFLATOXIN B1 PRODUCTION BY THETOXIGENIC STRAIN OF ASPERGILLUSFLAVUS-235

Plants MDW (mg) AFB1 (mg/L) AFB1 (mg/g)

Concentration (1 mg/mL) (1 mg/mL) (1 mg/mL)

Control 389.6 � 5.5x 308.6 � 6.8s 19.8n

Abrus precatorius L. 194.3 � 8.2o 146.3 � 2.6n 18.8l

Aegle marmelos (L.) Correa 186.6 � 4.6n 127.0 � 5.5l 17.0k

Aloe barbadensis Mill. 232.6 � 6.6s 184.6 � 3.6q 19.8n

Artabotrys odoratissimus R.Br. 126.6 � 3.5g 89.6 � 9.3i 17.5k

Calotropis procera (Ait.) R.Br. 261.0 � 5.5w 195.0 � 12.5r 18.6l

Carica papaya L. 170.0 � 4.3l 134.0 � 4.0m 19.7n

Eupatorium cannabinum L. 207.0 � 4.7p 154.6 � 12.3o 18.6l

Evolvulus alsinoides L. 146.6 � 4.6h 101.6 � 1.6j 17.2k

Jatropha curcas L. 251.6 � 7.7v 196.0 � 3.0r 19.4m

Lawsonia inermis L. 237.0 � 2.0t 182.0 � 4.0q 19.1m

Melia azaderach L. 182.6 � 2.6m 110.3 � 0.3k 15.1j

Moringa oleifera Lamk. 209.3 � 5.9p 155.0 � 5.0p 18.5lMurraya koenigii (L.) spreng. 91.3 � 7.8f 11.3 � 2.9c 3.0c

Murraya paniculata (L.) Jacq. 163.3 � 2.4k 84.3 � 8.4h 12.0h

Ocimum basilicum L. 80.6 � 3.4e 10.3 � 3.8c 3.1c

Phyllanthus emblica L. 29.6 � 2.7a 0.0 � 0.0a 0.0a

Polyalthea longifolia Sonn. 215.0 � 5.0r 47.6 � 4.9g 5.5e

Pongamia pinnata (L.) Pierre 148.3 � 3.3h 25.3 � 6.4d 4.2d

Ricinus communis L. 210.3 � 1.6q 126.3 � 10.6l 14.9i

Sapindus mucorossi Gaertn. 240.6 � 1.6u 153.6 � 13.1o 15.9j

Terminalia arjuna Roxb. 158.0 � 6.9j 36.6 � 10.4e 5.7e

Terminalia bellerica (Gaertn.) Roxb. 35.6 � 3.9b 12.3 � 2.9c 8.6g

Terminalia chebula Retz. 50.6 � 5.4c 0.0 � 0.0a 0.0a

Vitex negundo L. 154.3 � 3.8i 42.3 � 8.6f 6.8f

Zingiber officinale Roscoe 77.6 � 4.3d 7.6 � 4.6b 2.4b

Values are mean (n = 3) � SE. The means followed by same letter in the same column are not signifi-cantly different according to ANOVA and Tukey’s multiple-comparison tests.ANOVA, analysis of variance; SE, standard error.



flavonoids (Ebana and Madunagu 1993; Mahoney and Moly-neux 2004; Radulovic et al. 2006; Milanovic et al. 2007;Palumbo et al. 2007; Samapundo et al. 2007). Recently, Tianet al. (2011) have reported the efficacy of some plant chemi-cals in inhibition of carbohydrate catabolism and some keyenzymes responsible for inhibition of aflatoxin production bysome food-infesting fungi. However, most of the authors havesuggested that high-phenolic content of plant extracts isresponsible for their antioxidant and aflatoxin-inhibitoryactivity (Selvi et al. 2003; Prakash et al. 2010, 2011b; Garciaet al. 2011).

Antimutagenic, anticancer, antiulcerogenic and immuno-stimulatory properties of P. emblica is well explored by theearlier workers (Suresh and Vasudevan 1994; Bandyopadhyayet al. 2000; Khan et al. 2002; Lambertini et al. 2004). T. che-bula is frequently used as an antiviral, antibacterial, antican-cer, expectorant, aphrodisiac, diuretic and growth inhibitoryof several cancer cell lines (Saleem et al. 2002; Prajapati et al.2003; Kamaraj and Rahuman 2010).The fruits of P. emblicaand T. chebula are two essential ingredients of triphala, anAyurvedic formulation used as a safe tonic in the Indiansystem of medicine for hundreds of years providing immu-nity to human body (Prajapati et al. 2003). Hence, it isexpected that there would be less chance for any mammaliantoxicity if these are used as food additives. These are commontrees of Indian flora and their fruits are easily available in bulkas raw materials.

Thus, the extracts of P. emblica and T. chebula showingpromising antifungal, aflatoxin inhibitory and free radical-scavenging activity should be further tested on food systems soas to recommend them as plant-based food additives in pro-tecting the food items from mold and aflatoxin contaminationas well as lipid peroxidation. To the best of our knowledge,

there is no previous report on aflatoxin inhibition activity ofmethanol–aqueous extract of P. emblica and T. chebula.

ACKNOWLEDGMENT

This work was financially supported by Council of Scientificand Industrial Research, New Delhi, India.

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FIG. 1. IC50 OF PLANT EXTRACTS SHOWINGDPPH RADICAL-SCAVENGING ACTIVITYValues are mean (n = 3) � SE. The meansfollowed by same letter are not significantlydifferent according to ANOVA and Tukey’smultiple-comparison tests.ANOVA, analysis of variance; DPPH,2,2-diphenyl-1-picrylhydrazyl; SE, standarderror.



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antifungal, aflatoxin inhibitory and free radical-scavenging activities of some medicinal plants...

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