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Antifungal, aflatoxin inhibition and antioxidant activity of Callistemon lanceolatus (Sm.) Sweet essential oil and its major component 1,8-cineole against fungal isolates from chickpea seeds
Food Control 25 (2012) 27e33
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Antifungal, aflatoxin inhibition and antioxidant activity of Callistemon lanceolatus (Sm.) Sweet essential oil and its major component 1,8-cineole against fungal isolates from chickpea seeds Ravindra Shukla, Priyanka Singh, Bhanu Prakash, N.K. Dubey* Laboratory of Herbal Pesticides, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2011 Received in revised form 28 September 2011 Accepted 4 October 2011
The present investigation reports the extent of molds and aflatoxin contamination to Avarodhi, Kabuli, Pusa 256, Radha and Samrat varieties of chickpea seeds. The study also examines the chemical composition of Callistemon lanceolatus (Sm.) Sweet essential oil and its antifungal, antiaflatoxin and antioxidant activity. During standardization of chemical profile, a total of 8 compounds constituting 0.862 mg/mL of oil composition were analyzed by GCeMS analysis where 1,8-cineole was recorded as a major component (0.56 mg/mL). The antifungal activity of EO and 1,8-cineole was evaluated by contact assay on Czapek’s dox agar. The EO (0.227e0.908 mg/mL) and 1,8-cineole (0.918 mg/mL) showed remarkable antifungal effect against all the fungal isolates of chickpea. Their minimal inhibitory (MIC) and fungicidal (MFC) concentrations for Aspergillus flavus were lower than those of the prevalent systemic fungicide Nystatin. Aflatoxin B1 (AFB1) production by NKD-208 isolates of A. flavus was strongly inhibited even at the lower fungistatic concentration of EO and 1,8-cineole.There was no adverse effect of EO treatment on chickpea seed germination suggesting its non-phytotoxic nature. Based on the findings of present investigation, C. lanceolatus essential oil may be recommended as botanical preservative for the enhancement of shelf life of food items in- view of the adverse effect of synthetic preservatives and its strong antifungal, aflatoxin inhibition and antioxidant activity. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Aflatoxin B1 Antifungal Antioxidant Callistemon lanceolatus Essential oil
1. Introduction Molds are ubiquitous microorganisms with a great capacity to colonize different kinds of substrates and to proliferate under extreme environmental conditions (Nguefack et al., 2009). They spoil various types of foods viz. cereals, legumes, spices, vegetables, fruits etc. and also produce mycotoxins that can be mutagenic, teratogenic, carcinogenic causing feed refusal and emesis in humans or animals (Frisvad, Skouboe, & Samson, 2005; Prakash et al., 2010; Prakash et al., 2011; Reddy, Raghavender, Salleh, Reddy, & Reddy, 2011). There are reports that hepatic carcinoma and other serious diseases may be induced by consuming food or using raw materials for food processing contaminated with aflatoxins. Nearly 5 billion people are exposed to aflatoxins in different developing countries and aflatoxicosis is ranked 6th among the 10 most severe health risks identified by WHO (Prakash et al., 2010). Moreover, aflatoxins are proved resistant to heat and have an ability to accumulate in the organism (Galvano, Ritieni, Piva, & Pietri, 2005). * Corresponding author. Tel.: þ91 9415295765; fax: þ91 5422368174. E-mail address: [email protected] (N.K. Dubey). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.10.010
Chickpea (Cicer arietinum L.) is the most nutritive among the food legumes and extensively used as protein adjunct to starchy diet (Sastri, 1950). Chickpea after dehulling is valued for its nutritive seeds with high protein content (12.3e31.5%) and 0.3% phosphorus. South-east Asia contributes about 80% to the global chickpea production, and India is the principal chickpea producing country in the region with 83% share (ICRISAT, 2011). Fungal contamination of chickpea seeds is the major problem in Indian sub-continent (Ahmad & Singh, 1991; Dawar, Syed, & Ghaffar, 2007). Although, different synthetic antimicrobials have been successfully commercialized in recent years, they encounter major problems not only due to their adverse side effects on consumers but also for the development of resistance by microorganisms (Tolouee et al., 2010). Hence, there must be optimization of alternative methods for pest and disease control that produce minimal damage to the environment and human health. With an expanding list of food-borne pathogens, there is an urgent need to explore the novel strategy to prevent food contamination. Essential oils as well as compounds derived from aromatic plants possess a wide range of activities of which the antimicrobial activity is most studied (Burt, 2004). Their applications
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as preservatives in food or antiseptics and disinfectants are widely studied (Holley & Patel, 2005). In this regard, plant essential oils and their compounds may offer a great potential. Some essential oils and their components viz. eugenol, carvone, carvacrol, citral, thymol and limonene are bioactive compounds of some essential oils which are in commercial use as food additives through encapsulation technologies (Burt, 2004; Prakash et al., 2010).Therefore, their composition and antimicrobial activities must be continued to be investigated for better alternatives. Callistemon lanceolatus (Sm.) Sweet (family: Myrtaceae) is native to Australia and is widely distributed in subtropical and tropical regions; commonly known as bottle brush because of their cylindrical brush like flowers resembling a traditional bottle. Phytochemical and antimicrobial activity of Callistemon rigidus, Callistemon citrinus, Callistemon viminalis, Callistemon linearis (Das, Zaman, & Singh, 2009; Dongmo et al., 2010; Jazet et al., 2009) have earlier been reported but little is known about biological activity of C. lanceolatus. In the present study, chickpea seeds were screened for incidence of mycoflora and aflatoxin contamination by the toxigenic strain of Aspergillus flavus. In addition the chemically characterized EO of C. lanceolatus was assessed for its antifungal, aflatoxin inhibition and antioxidant activity.
2. Materials and methods 2.1. Collection of chickpea seeds and mycological analysis Avarodhi, Kabuli, Pusa 256, Radha and Samrat varieties of chickpea (C. arietinum L.), stored from 6 to 8 months,were procured from local markets of Varanasi, Uttar Pradesh, India. The seed mycoflora was examined using the agar plate method (Embaby & Abdel-Galil, 2006) as recommended by International Seed Testing Association (ISTA, 1966). Seeds were surfacesterilized with 1% solution of sodium hypochlorite and rinsed in three changes of sterile distilled water. Four seeds of each variety were placed equidistantly in Petri plates containing potato dextrose agar (PDA) medium (Potato infusion, 200 g; Dextrose, 20 g; Agar, 15 g; Distilled water, 1 L, pH, 5.6 0.2; HiMedia Laboratories Pvt. Ltd., Mumbai) and incubated for 7 days (28 2 C). The developing fungal colonies were isolated, identified (Burnett & Hunter, 1999) and routinely maintained on PDA in the presence of antibiotic Streptomycin (300 mg/L). The incidence of fungi was determined based on the occurrence of a particular species in samples of 10 seeds.
2.2. Detection of aflatoxigenic isolates of A. flavus A. flavus isolates from each variety of chickpea seed were screened for the production of aflatoxin B1 (AFB1) following Kumar, Mishra, Dubey, and Tripathi (2007). The isolates were cultured separately in 25 mL SMKY broth (sucrose 200 g; MgSO4$7H2O, 0.5 g; KNO3, 0.3 g and yeast extract, 7 g; 1 L distilled water) in 100 mL flask for 10 days. The content of each flask was filtered and extracted with 20 mL chloroform in a separating funnel. The extract was evaporated to dryness on water bath and redissolved in 1 mL chloroform. AFB1 was detected by thin layer chromatography. Fifty micro liter chloroform extract was spotted on Thin Layer Chromatography (TLC) plates and developed in the solvent system comprising toluene/isoamyl alcohol/methanol (90:32:2; v/v/v). The plate was air dried and the intensity of AFB1 observed in UVtransilluminator (360 nm).
2.3. Plant materials and extraction of EO C. lanceolatus, planted in Botanical garden of Banaras Hindu University, Varanasi, India was identified by morphological features with the help of Flora of BHU Campus (Dubey, 2004, p. 112 and 113) and the voucher specimens (LHP/Ver-21/2008 and LHP/Are/17/ 2008) were deposited at the Laboratory of Herbal Pesticides, Banaras Hindu University, Varanasi, India. Fresh plant leaves (200 g) were collected in the month of June and subjected to hydro-distillation (4 h) using a Clevenger-type apparatus (Kumar et al., 2007). The yield (mL/kg) of EOs was averaged over four experiments and calculated on the basis of plant material fresh weight. 2.4. GC and GCeMS EO of C. lanceolatus was subjected to gas chromatography (PerkinElmer Auto XL GC, MA, USA) equipped with a flame ionization detector and the GC condition were: EQUITY-5 column (60 m 0.32 mm 0.25 mm); H2 was the carrier gas; column head pressure 10 psi; oven temperature program isotherm 2 min at 70 C, 3 C/min gradient to 250 C, isotherm 10 min; injection temperature, 250 C; detector temperature 280 C. GCeMS analysis was performed using Perkin Elmer Turbomass GCeMS. The column size and oven conditions of GCeMS were the same of the GC-FID. Helium was the carrier gas. The effluent of the GC column was introduced directly into the source of MS and spectra obtained in the EI mode with 70 eV ionization energy. The sector mass analyzer was set to scan from 40 to 500 amu for 2 s. The identification of individual compounds was based on their retention times relative to those of authentic samples and matching spectral peaks available with Wiley, NIST and NBS mass spectral libraries or with the published data (Adams, 2007). 2.5. Antifungal assay The antifungal activity of EO and 1,8-cineole, the major constituent, (procured from Genuine Chemical Co., Mumbai, India) was tested against the fungal isolates of chickpea by contact assay based on hyphal growth inhibition (Shukla, Kumar, Singh, & Dubey, 2009) using Czapek-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 distilled water, pH 6.8 0.2; Sisco Research Lab., Mumbai). The requisite amount of EO was dissolved in 0.5 mL acetone, and added to 9.5 mL molten CDA in different Petri plates to achieve final concentrations (0.227, 0.454, 0.681, 0.908 mg/mL). 1,8-cineole was tested at 0.918 mg/mL. CDA plates containing acetone (0.5 mL) only, served as negative control. In addition, CDA plates treated with reference antifungal nystatin (1.0 mg/mL) were used as positive control. A 5 mm disc of test fungus was placed upside down on the center of the plate with fungal species in contact with growth medium. Cultures were incubated in the dark at 28 2 C (7 days). Antifungal index was calculated as the following-
Antifungal indexð%Þ ¼
1
Da 100 Db
where Da: average diameter of fungal growth in the treatment; Db: average diameter of fungal growth in the control sets. 2.6. Determination of MIC and MFC The minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for A. flavus (the most prevalent fungus) were determined by broth dilution method as reported
R. Shukla et al. / Food Control 25 (2012) 27e33
earlier (Shukla et al., 2009). Different concentrations of the EO and 1,8-cineole were dissolved in 0.5 mL acetone and incorporated to 9.5 mL CD broth tubes containing 106 spores/mL. The tubes were incubated at 30 C for a week. The lowest concentration that did not permit any visible growth of fungus was taken as MIC. Cells from the tubes showing no growth were sub-cultured on treatment-free CDA plates to determine if the inhibition was reversible. MFC is the lowest concentration at which no growth occurred on the plates.
2.7. Effect of EO and 1,8-cineole on aflatoxin B1 synthesis Requisite amounts of EO and 1,8-cineole were dissolved separately in 0.5 mL acetone, and added to 24.5 mL SMKY to achieve the different concentrations between (0.091e0.908) and (0.092e0.918) mg/mL respectively. The medium was inoculated with toxigenic isolate NKD-208 of A. flavus to give 106 spores/mL and incubated at 28 2 C (10 days). The medium was filtered and fungal mat was dried at 80 C (12 h) to determine the net mycelial dry weight. The filtrate was used for aflatoxin extraction as described above. For quantitative estimations, fluorescent spot of AFB1 on TLC plate was scrapped, dissolved in 5 mL cold methanol, and centrifuged (3000 rpm, 5 min). Optical density of the supernatant recorded at 360 nm and the AFB1 amount calculated according to Kumar et al. (2007):
Aflatoxin B1 contentðmg=LÞ ¼
DM 1000 El
where, D ¼ absorbance, M ¼ molecular weight of aflatoxin (312), E ¼ molar extinction coefficient of aflatoxin B1 (21,800) and l ¼ path length (1 cm cell was used).
2.8. Antioxidant activity 2.8.1. DPPH radical scavenging assay through TLC The antioxidant activity of C. lanceolatus EO was determined following Tepe, Daferera, Sokmen, Sokmen, and Polissiou (2005). 4.54 mg of the EO (1:10 dilution in methanol) was applied on TLC plate and developed in ethyl acetate and methanol (1:1) the plate was sprayed with 0.2% DPPH solution in methanol (2,2-diphenyl-1picrylhydrazil) and left at room temperature for 30 min. Yellow spot formed due to bleaching of purple color of DPPH reagent was recorded as positive antioxidant activity of EO. 2.8.2. Free radical scavenging activity Free radical scavenging activity of the C. lanceolatus EO was measured by recording the extent of bleaching of the purplecolored DPPH solution to yellow following Prakash et al. (2010). Different concentrations (1.816e9.08 mg/mL) of the essential oil were added to 0.004% DPPH solution in methanol (5 mL). After a 30 min of incubation at room temperature, the absorbance was taken against a blank at 517 nm using spectrophotometer. Butylated hydroxytoluene (BHT) and Butylated hydroxyanisole (BHA) (2.0e10 mg/mL) were used as positive control. Scavenging of DPPH free radical with reduction in absorbance of the sample was taken as a measure of their antioxidant activity IC50, which represented the concentration of the EO that caused 50% neutralization of DPPH radicals, was calculated from the graph plotting between percentage inhibition and concentration.
I% ¼
Ablank Asample ¼ Ablank 100
where, Ablank is the absorbance of the control (without test compound), and Asample is the absorbance of the test compound.
29
2.8.3. b-carotene/linoleic acid assay The b-carotene/linoleic acid bleaching test was performed by the method described by Ebrahimabadi et al. (2010). A stock solution of b-carotene and linoleic acid was prepared by dissolving 0.5 mg of b-carotene in 1 mL of chloroform, 25 mL of linoleic acid and 200 mL Tween 40. The chloroform was completely evaporated under vacuum in a rotatory evaporator at 40 C; then 100 mL of distilled water was added and the resulting mixture was vigorously stirred. The samples (2 g/L) were dissolved in DMSO and 350 mL of each sample solution were added to 2.5 mL of the above mixture in test tubes. The test tubes were incubated in a hot water bath at 50 C; for 2 h, together with blanks, BHT and BHA as a positive control and the other contained the same volume of DMSO instead of the samples as a negative control. The absorbance of each sample was measured at 470 nm. The test tube with BHT and BHA maintained its yellow color during the incubation period. The absorbance was measured at 470 nm on an ultraviolet spectrometer. Antioxidant activities (inhibition percentage, I%) of the samples was calculated using the following equation:
I% ¼
AbCarotene after 2 h assay =Ainitial bCarotene 100
where, Ab-Carotene after 2 h assay is the absorbance of b-Carotene after 2 h assay remaining in the samples and Ainitial b-Carotene is the absorbance of b-Carotene at the beginning of the experiments.
2.9. Phytotoxicity assay The phytotoxicity of the EO and 1,8-cineole in terms of seed germination and seedling growth of chickpea was assayed with respect to control sets following Kordali et al. (2008). Two layers of filter paper were placed on the bottom of each Petri plate (9 cm) and 5 seeds were placed equidistantly on the filter paper moistened with 10 mL of distilled water. 9.08 mg of the EO and 9.18 mg of EO compounds were dripped on Whatman No. 1 filter paper strip placed on the lid using a micropipette. Petri plates were sealed with parafilm to prevent escaping of volatile compounds and kept at 23 2 C in a growth chamber. Percent germination of seeds of control and treated sets was recorded. The length of radical and plumule was monitored at 24, 48, 72, 96, 120 and 144 h interval. 2.9.1. Statistical analysis Antifungal, antiaflatoxigenic and phytotoxicity experiments were performed in triplicate and data analyzed are mean SE subjected to one way ANOVA. Means are separated by Tukey’s multiple range tests when ANOVA was significant (p < 0.05) (SPSS 10.0; Chicago, IL, USA).
Table 1 Fungi isolated from chickpea seeds. Legumes
Fungi isolated
Cicer arietinum L. Var. Avarodhi
A. flavus (10), Aspergillus niger (4), Alternaria alternata (4), Fusarium oxysporum (4), Absidia ramosa (2) A. flavus (7), Mucor sp. (3), Trichoderma sp. (3), Fusarium nivale (4), Dreschlera sp. (2) A. flavus (5), Penicillium citrinum (4), Mucor sp. (2), Aspergillus fumigatus (4) A. flavus (10), Fusarium sp.(5)
Cicer arietinum L. Var. Kabuli Cicer arietinum L. Var. Pusa 256 Cicer arietinum L. Var. Radha Cicer arietinum L. Var. Samrat
A. flavus (6), Chaetomium sp. (3), P. citrinum (4), A. niger (4), Aspergillus oryzae (2)
Values in parentheses (*) are incidence of a particular species in samples of 10 seeds in direct plating method.
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Table 2 Aflatoxin B1 production potential of different Aspergillus flavus isolates in SMKY medium. Legumes Cicer Cicer Cicer Cicer Cicer
arietinum arietinum arietinum arietinum arietinum
Fungal isolates L. L. L. L. L.
var. var. var. var. var.
Avarodhi Kabuli Pusa 256 Radha Samrat
A. A. A. A. A.
flavus flavus flavus flavus flavus
NKD 188 NKD 189 NKD 197 NKD 208 NKD 209
AFB1 217.3 305.0 nf 365.3 nf
Values for aflatoxin B1 content is given in mg/l; nf ¼ not found.
3. Results During mycological analysis, A. flavus predominated in almost all the 5 varieties of chickpea seeds (Table 1). The Avarodhi and Radha varieties were heavily contaminated with A. flavus where all the 10 seeds of each variety were found positive with this fungus. Seven seeds out of 10 from var. Kabuli were found contaminated with A. flavus. Five different fungal species were isolated from Avarodhi, Kabuli and Samrat varieties whereas, only 2 species from Radha. Aspergillus niger and Penicillium citrinum were found on seeds of 2 varieties and recorded as the second dominant fungus after A. flavus. Out of 5 isolates of A. flavus procured from 5 varieties of chickpea seeds, only 3 isolates were found to be AFB1 producers. All the isolates were named on the basis of their culture collection number and deposited in our laboratory. A. flavus NKD-208, isolated from Radha variety of chickpea was recorded to be the most toxigenic isolate which produced 365.3 mg/L AFB1 in SMKY broth followed by A. flavus NKD 189 (305.0 mg/L) and A. flavus NKD 188 (217.3 mg/L) (Table 2). The hydro-distillation of C. lanceolatus leaves yielded colorless oil (yield: 6.9 g/kg). The EO composition of C. lanceolatus is presented in Table 3. Total 8 components were identified which constitutes 0.862 mg/mL of the essential oil. 1,8-cineole was found the major component with 0.56 mg/mL of the oil followed by apinene (0.18 mg/mL) and g-terpinene (0.11 mg/mL). b-pinene, isoamyl bromide, p-cymene, limonene and geraniol formate were the minor components of the oil. The EO of C. lanceolatus exhibited moderate to high antifungal activity (Table 4). Even at the lowest concentration of 0.227 mg/mL, EO caused more than 50% mycelial inhibition of most of the fungi except A. niger (39.7%). Aspergillus oryzae, Fusarium oxysporum, Fusarium sp. and Mucor sp. were found most susceptible fungi against the EO and their 100% growth inhibition was achieved at 0.681 mg/mL. However, remarkable antifungal index (71.3e96.0%) of EO was recorded against rest of the fungi at the same concentration. All the fungal species were completely inhibited at 0.908 mg/mL of EO. At 0.918 mg/mL, 1,8-cineole showed antifungal activity, ranging between 52.3 and 89.9%. The reference antifungal agent, nystatin showed the lowest antifungal activity among all the
Table 3 Chemical composition of C. lanceolatus essential oil. Compounds
Composition (mg/mL)
Retention time (Min.)
a-pinene b-pinene Isoamyl Bromide p-cymene Limonene 1,8-cineole g-terpinene Geraniol Formate
0.18 0.001 0.002 0.002 0.004 0.56 0.11 0.003
15.90 17.75 18.70 19.60 19.77 20.07 26.72 28.45
Total
0.862
treatments with only 34.9e58.8% inhibition of fungal isolates. None of the fungi was completely inhibited (100%) by 1,8-cineole and nystatin at 0.918 mg/mL and 1.0 mg/mL respectively. The MICs of C. lanceolatus EO and 1,8-cineole were 0.682 0.01 and 1.285 mg/mL 0.05 mg/mL, respectively against A. flavus. MFC of EO (1.135 mg/mL) was also lower than that of 1,8-cineole (1.83 mg/mL). However, MIC (1.78 mg/mL) and MFC (>2.0 mg/mL) of nystatin were comparatively higher than those of C. lanceolatus EO and 1,8-cineole. It is evident from Table 5 that EO and 1,8-cineole inhibited AFB1 production in a dose dependent manner. In control set, 325.3 mg/L AFB1 was secreted. However, at 0.455 mg/mL of C. lanceolatus EO, only 39.6 mg/L AFB1 was calculated. The oil caused complete prevention of AFB1 secretion in SMKY broth at 0.546 mg/mL, lower than the concentration at which no mycelium was found (i.e. 0.819 mg/mL). On the other hand, 1,8-cineole completely inhibited AFB1 at 0.918 mg/mL, whereas 121.7 mg mycelial weight was recorded at this concentration. The discoloration of the purple color of the DPPH on TLC plates confirmed the positive antioxidant activity of EO. The oil showed free radical scavenging activity in dose dependent manner and its IC50 value was 4.02 mg/mL. IC50 values of synthetic antioxidant BHT (7.28 mg/mL), BHA (4.44 mg/mL) were also determined as positive control. Oxidation of linoleic acid was moderately inhibited by the EO (30.16%) compare to positive control BHA (76.71%) BHT (64.23%) and a negative control (7.34%). Percent inhibition and IC50 values of EO and synthetic antioxidant are summarized in Table 6. A 100% germination of seeds was recorded following 48 h in all the three sets, including control. The size of radicles in EO treated seeds was somehow equal to the untreated control but higher than 1,8-cineole treated sets. The mean length of radicle was 128.5, 130.2 and 122.6 mm in EO, control and 1,8-cineole treated seeds. Similarly, no remarkable difference in the length of plumule was observed after 144 h in control (24.2 mm), EO (24.7 mm) and 1,8cineole (22.7 mm) treated seeds. 4. Discussion In the present investigation Avarodhi, Kabuli, Pusa 256, Radha and Samrat varieties of chickpea were screened for the first time to determine their inherent mycobiota.. All 5 varieties of chickpea were found contaminated with various fungi, where, 14 fungal species belonging to 9 genera were identified and isolated. Species of Aspergillus (56.3%) predominated in all the seeds followed by Fusarium sp. (14.0%). Only fragmentary work has been performed earlier regarding fungal association of chickpea seeds. These results confirm the earlier observations where Aspergillus species were one of the most predominant fungi and aflatoxin producers in some of the stored grains (Amadi & Adeniyi, 2009; Reddy, Reddy, & Muralidharan, 2009). Tabuc, Stroia, and Neacsu (2010) reported Aspergillus species in 45 of the 56 analyzed samples of corn, wheat, barley and oat. Reddy et al. (2011) stated that the genus Aspergillus was the most dominant among the prevalent genera of fungi isolated from some cereals and pulses. Similar results were obtained in a mycological study conducted with cowpea seeds in Africa (Houssou et al., 2009). The composition of EOs varies with respect to ecological and geographical conditions, age of plant and time of harvesting (Prakash et al., 2010). Such a variation in chemical composition of EOs would definitely alter their biological activity. Hence, the chemical profile of an EO must be standardized before its recommendation as food preservative. Most chemical components of EOs are terpenoids, including monoterpenes, sesquiterpenes, and their oxygenated derivatives. In the present investigation 1,8-cineole/ eucalyptol, an oxygenated monoterpene, was the major component
R. Shukla et al. / Food Control 25 (2012) 27e33
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Table 4 Antifungal index of C. lanceolatus essential oil and its major component against different fungal isolates. Fungal isolates
Antifungal Index (%) C. lanceolatus EO
Absidia ramosa Alternaria alternata, Aspergillus fumigatus Aspergillus niger Aspergillus oryzae Chetomium sp. Dreschelera sp. Fusarium nivale Fusarium oxysporum Fusarium sp. Mucor sp. Penicillium citrinum Trichoderma sp.
1,8-cineole
Nystatin
0.227 mg/mL
0.454 mg/mL
0.681 mg/mL
0.908 mg/mL
0.918 mg/mL
1.0 mg/mL
61.0 1.9 48.6 1.6 52.0 2.8 39.7 3.0 62.5 2.6 62.5 1.9 68.5 1.7 51.2 0.3 58.4 2.1 79.3 1.5 81.6 2.6 50.0 2.5 57.0 1.0
71.8 1.1 65.2 1.8 71.3 2.2 53.2 3.0 80.8 2.6 78.0 3.5 81.5 2.2 70.8 3.0 81.5 2.1 88.2 1.8 93.5 3.3 72.1 1.6 72.6 3.8
93.3 1.0 89.7 3.6 84.3 2.8 71.3 3.0 100 88.9 4.1 95.4 2.6 91.8 2.2 100 100 100 96.0 2.8 94.1 1.5
100 100 100 100 100 100 100 100 100 100 100 100 100
76.5 2.2 68.5 2.8 68.3 3.3 52.3 4.5 71.1 1.9 67.2 2.8 80.0 3.4 73.6 1.8 73.2 2.6 80.0 3.3 89.9 2.6 68.9 2.8 70.3 1.9
40.0 3.0 52.8 4.1 41.7 2.2 38.5 2.6 56.7 3.7 34.9 1.8 58.3 3.4 58.8 4.6 40.2 1.8 48.3 1.8 58.6 1.0 50.2 2.8 43.5 3.4
Concentrations are given in ml/mL, percent values are mean (n ¼ 3) SE.
of EO. a-pinene and g-terpinene, the monoterpene hydrocarbons, were second and third dominant components whereas; p-cymene and b-pinene were present in traces. 1,8-cineole-a-pinene has also been reported abundantly in several myrtaceous plants like Eucalyptus saligna, Melaleuca armillaris, C. rigidus, C. citrinus, Myrtus communis (Aidi, Mhamdi, & Marzouk, 2007; Dongmo et al., 2010; Farag et al., 2004). Amongst the toxigenic strains of A. flavus, the strain NKD 208 isolated from Radha variety of chickpea was found to be highly toxigenic and selected for detailed studies in the present investigation. Although, there are earlier reports on variation of antifungal activity of plant products against different species of a particular genus of a fungus (Prakash et al., 2010; Shukla et al., 2009) but literature is mostly silent on variation of their efficacy at strain level of a particular fungal species. The efficacy of C. lanceolatus EO has so far been not well-explored against storage fungi and mycotoxin contamination. However, there are earlier reports on antifungal activity of other species of Callistemon viz. C. citrinus, C. viminalis, C. rigidus against pathogenic yeasts and some filamentous fungi (Delahaye et al., 2009; Dongmo et al., 2010; Jazet et al., 2009). To the best of our knowledge, there has not been a relevant study on the effectiveness of any species of Callistemon oil against aflatoxin production by A. flavus. The biological activity of essential oils may be only due to their major components or synergistic/antagonistic interaction between different oil components Hence, 1,8-cineole, the major component along with the respective EO were tested in the present investigation to correlate whether the efficacy of the EO was due to
synergism between existing components or only due to the major component. The results indicate that none of the fungi was completely inhibited at 0.918 mg/mL of 1,8-cineole, whereas, 100% fungal inhibition was achieved at this concentration of EO. The MIC of 1,8-cineole (1.285 mg/mL) against A. flavus was approximately double to that of C. lanceolatus EO (0.682 mg/mL). Similarly, AFB1 production was inhibited at 0.918 mg/mL of 1,8-cineole and 0.546 mg/mL of EO. Hence, the enhanced antifungal and aflatoxin inhibitory activities C. lanceolatus EO may be attributed to some minor components that have a synergistic effect with the major components rather than 1,8-cineole, the major component. In addition, the better efficacy of the EO over the reference antifungal Nystatin and its broad fungitoxic spectrum inhibiting the growth of the all the fungal isolates strengthen its merits as a post harvest antimicrobial against food deteriorating fungi. Moreover, lower MIC of C. lanceolatus EO than that of some earlier reported oils viz., Lippia alba (Shukla et al., 2009) Satureja hortensis (Dikbas, Kotan, Dadasoglu, & Sahin, 2008), Daucus carota (Tavares et al., 2008) strengthens its high efficacy and possible economical exploitation. The antifungal mechanism of EO is speculated to involve membrane disruption by their lipophilic compounds (Cowan, 1999). The low-molecular weight and highly lipophilic components of EOs pass easily through cell membranes and cause disruption to the fungal cell organization (Chao et al., 2005). The EO inhibited aflatoxin production at concentrations lower than its fungitoxic concentration. Thus, the inhibition of AFB1 production cannot be completely attributed to reduced fungal
Table 5 Effect of C. lanceolatus EO and 1,8-cineole on mycelial biomass and Aflatoxin B1 production by A. flavus (NKD-208) in SMKY medium. Concentration (mg/mL)
C. lanceolatus EO MDW
0.0 (Control) 0.091 0.182 0.273 0.364 0.455 0.546 0.637 0.728 0.819 0.908
399.7 382.0 342.0 311.0 179.3 123.0 81.7 51.3 18.0 0.0 0.0
AFB1
2.9a 5.0b 2.0c 3.7d 2.3e 3.7f 3.1g 1.3h 2.3i 0.0j 0.0j
325.3 254.0 196.3 157.0 95.7 39.6 0.0 0.0 0.0 0.0 0.0
4.2a 4.0b 3.1c 5.6d 4.7e 0.8f 0.0g 0.0g 0.0g 0.0g 0.0g
Concentration
1,8-cineole
(mg/mL)
MDW
0.0 (control) 0.092 0.184 0.276 0.368 0.460 0.552 0.644 0.736 0.828 0.918
399.7 383.7 356.0 322.7 294.3 243.7 201.7 199.3 163.7 147.3 121.7
AFB1
2.9a 3.1b 4.0c 4.1d 3.3e 2.9f 1.7g 5.8g 3.8h 1.8i 3.2j
325.3 307.7 282.3 266.3 236.7 190.7 130.0 101.0 60.0 22.6 0.0
4.2a 5.3b 4.0c 2.7d 5.6e 3.7f 2.0g 2.0h 3.6i 2.6j 0.0k
MDW ¼ mycelial dry weight (mg); AFB1 ¼ aflatoxin B1 content (mg/l), values are mean (n ¼ 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.
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R. Shukla et al. / Food Control 25 (2012) 27e33
Table 6 Antioxidant activity of EO in DPPH free radical scavenging activity and b-carotene/ linoleic acid bleaching assay. Sample
DPPH (IC50)
b-carotene/linoleic acid inhibition (%)
C. lanceolatus EO BHT BHA Negative control
4.02 0.07a,y 7.28 0.03b,z 4.44 0.03c,z ND
30.16 64.23 76.75 7.34
0.16c 0.24b 0.21a 0.33d
ymg/mL; zmg/mL. The means followed by same letter in the same column are not significantly different according to ANOVA and tukey’s multiple comparison tests. Value are mean (n ¼ 3) SE.
growth, but may be because of inhibition of carbohydrate catabolism in A. flavus by acting on some key enzymes, reducing its ability to produce aflatoxins as has been reported by Tian et al. (2011). Determination of exact mechanism of AFB1 suppression by C. lanceolatus EO requires further investigations. The antioxidant activity of C. lanceolatus EO was confirmed by DPPH radical scavenging assay and b-carotene-linoleic acid bleaching assay. C. lanceolatus EO recorded better free-radical scavenger as compared to earlier reported EOs, of Ocimum gratissimum (Prakash et al., 2011), Citrus maxima, Citrus sinensis (Singh et al., 2010), Stachys inflata (Ebrahimabadi et al., 2010) Satureja montana, Satureja subspicata (Cavar, Maksimovic, Solic, Mujkic, & Besta, 2008) and Semenovia tragioides (Bamoniri et al., 2010). There was no adverse effect of EO treatment on seed germination, suggesting its non-phytotoxic nature. In addition, the nonmammalian toxicity of C. lanceolatus EO in terms of high LD50 has earlier been reported by our research group (Shukla, Singh, Prakash, Kumar, Mishra,& Dubey, 2011). To the best of our knowledge, there is no report on any toxic phytochemical from C. lanceolatus. Because of pleasant camphorlike spicy aroma and cooling taste, the cyclic ether 1,8-cineole is used in flavorings, fragrances, and cosmetics. It is an ingredient in many brands of mouthwash and cough suppressant. Hence, there would be no chance of their negative effects on sensory quality, although detailed investigations on organoleptic parameters are needed before final recommendation. In conclusion, the EO of C. lanceolatus may be recommended as a plant based food preservative for enhancement of shelf life of stored food commodities by checking their fungal contamination, aflatoxin secretion and lipid per oxidation because of antifungal, aflatoxin inhibition and antioxidant activity. The yield of EO (6.9 g/ kg) was relatively high in the present investigation, and sufficient amount of raw materials would be available as the plant grows luxuriantly in the area. The findings of the present investigation would draw the attention of food industries regarding large scale application of C. lanceolatus EO as a plant based food additive. Acknowledgments This work was financially supported by Council of Scientific and Industrial Research (CSIR), New Delhi, India. References Adams, R. P. (2007). Identification of essential oil components by gas chromatography/ mass spectrometry. Carol Stream, IL, USA: Allured Publishing Corporation. Ahmad, S. K., & Singh, P. L. (1991). Mycofloral changes and aflatoxin contamination in stored chickpea seeds. Food Additives & Contaminants, 8, 723e730. Aidi, W. W., Mhamdi, B., & Marzouk, B. (2007). Essential oil composition of two myrtus communis L. varieties grown in North Tunisia. Italian Journal of Biochemistry, 56, 180e186. Amadi, J. E., & Adeniyi, D. O. (2009). Mycotoxin production by fungi isolated from stored grains. African Journal of Biotechnology, 8, 1219e1221.
Bamoniri, A., Ebrahimabadi, A. H., Mazoochi, A., Behpour, M., Kashi, F. J., & Batooli, H. (2010). Antioxidant and antimicrobial activity evaluation and essential oil analysis of Semenovia tragioides Boiss. from Iran. Food Chemistry, 122, 553e558. Burnett, W. L., & Hunter, B. B. (1999). Illustrated genera of imperfect fungi (4th ed.). St. Paul, Minnesota: APS Press. Burt, S. (2004). Essential oils: their antibacterial properties and potential applications in foodsda review. International Journal of Food Microbiology, 94, 223e253. Cavar, S., Maksimovic, M., Solic, M. E., Mujkic, A. J., & Besta, R. (2008). Chemical composition and antioxidant and antimicrobial activity of two Satureja essential oils. Food Chemistry, 111, 648e653. Chao, L. K., Hua, K. F., Hsu, H. Y., Cheng, S. S., Lin, J. Y., & Chang, S. T. (2005). Study on the anti-inflammatory activity of essential oil from leaves of Cinnamomum osmophloeum. Journal of Agricultural and Food Chemistry, 53, 7274e7278. Cowan, M. M. (1999). Plant products as antimicrobial agents. Clinical Microbiology Reviews, 12, 564e582. Das, A., Zaman, K., & Singh, A. V. (2009). Phytochemical and chemical composition evaluation of volatile oil of Callistemon linearis DC Leaf. Advances in Natural and Applied Sciences, 3, 56e59. Dawar, S., Syed, F., & Ghaffar, A. (2007). Seed borne fungi associated with chickpea in pakistan. Pakistan Journal of Botany, 39, 637e643. Delahaye, C., Rainford, L., Nicholson, A., Mitchell, S., Lindo, J., & Ahmad, M. (2009). Antibacterial and antifungal analysis of crude extracts from the leaves of Callistemon viminalis. Journal of Medical and Biological Sciences, 3, 1e7. Dikbas, N., Kotan, R., Dadasoglu, F., & Sahin, F. (2008). Control of Aspergillus flavus with essential oil and methanol extract of Satureja hortensis. International Journal of Food Microbiology, 124, 179e182. Dongmo, B. N., Dongmo, P. M. J., Ngoune, L. T., Kwazou, N. L., Zollo, P. H. A., & Menut, C. (2010). Antifungal activities of essential oils of some Cameroonian Myrtaceae on Aspergillus flavus Link ex. Fries. Asian Journal of Experimental Biological Sciences, 1, 907e914. Dubey, N. K. (2004). Flora of BHU campus. Varanasi: Banaras Hindu University Press. Ebrahimabadi, A. H., Ebrahimabadi, E. H., Djafari-Bidgoli, Z., Kashi, F. J., Mazoochi, A., & Batooli, H. (2010). Composition and antioxidant and antimicrobial activity of the essential oil and extracts of Stachys inflata Benth from Iran. Food Chemistry, 119, 452e458. Embaby, E. M., & Abdel-Galil, M. M. (2006). Seed borne fungi and mycotoxins associated with some legume seeds in Egypt. Journal of Applied Sciences Research, 2, 1064e1071. Farag, R. S., Shalaby, A. S., El-Baroty, G. A., Ibrahim, N. A., Ali, M. A., & Hassan, E. M. (2004). Chemical and biological evaluation of the essential oils of different Melaleuca species. Phytotherapy Research, 18, 30e35. Frisvad, C. J., Skouboe, P., & Samson, A. R. (2005). Taxonomic comparison of three different groups of aflatoxin producers and new efficient producer of aflatoxin B1, sterigmatocystine and 3-Omethylsterigmatocystine Aspergillus rumbelli sp. nov. Systematic and Applied Microbiology, 28, 442e453. Galvano, F., Ritieni, A., Piva, G., & Pietri, A. (2005). Mycotoxins in the human food chain. In Duarate Diaz (Ed.), The mycotoxin blue book (pp. 187e225). England: Nottingham University Press. Holley, R. A., & Patel, D. (2005). Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiology, 22, 273e292. Houssou, P. A., Ahohuendo, B. C., Fandohan, P., Kpodo, K., Hounhouigan, D. J., & Jakobsen, M. (2009). Natural infection of cowpea (Vigna unguiculata (L.) Walp.) by toxigenic fungi and mycotoxin contamination in Benin, West Africa. Journal of Stored Products Research, 45, 40e44. ICRISAT. (2011). Chickpea (Internet). International Crop Research Institute for the Semi Arid Tropics. Available from www.icrisat.org. ISTA. (1966). International rules for seed testing. Proceeding of International Seed Testing Association (Moisture and Aeration), 31, 54e55. Jazet, P. M. D., Tatsadjieu, L. N., Ndongson, B. D., Kuate, J., Zollo, P. H. A., & Menut, C. (2009). Correlation between chemical composition and antifungal properties of essential oils of Callistemon rigidus and Callistemon citrinus of Cameroon against Phaeoramularia angolensis. Journal of Medicinal Plants Research, 3, 9e15. Kordali, S., Cakir, A., Ozer, H., Cakmakci, R., Kesdek, M., & Mete, E. (2008). Antifungal, phytotoxic and insecticidal properties of essential oil isolated from Turkish Origanum acutidens and its three components, catvacrol, thymol and p-cymene. Bioresource Technology, 99, 8788e8795. Kumar, R., Mishra, A. K., Dubey, N. K., & Tripathi, Y. B. (2007). Evaluation of Chenopodium ambrosioides oil as a potential source of antifungal, antiaflatoxigenic and antioxidant activity. International Journal of Food Microbiology, 115, 159e164. Nguefack, J., Dongmo, J. B. L., Dakole, C. D., Leth, V., Vismer, H. F., Torp, J., et al. (2009). Food preservative potential of essential oils and fractions from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris against mycotoxigenic fungi. International Journal of Food Microbiology, 131, 151e156. Prakash, B., Shukla, R., Singh, P., Kumar, A., Mishra, P. K., & Dubey, N. K. (2010). Efficacy of chemically characterized Piper betle L. essential oil against fungal and aflatoxin contamination of some edible commodities and its antioxidant activity. International Journal of Food Microbiology, 142, 114e119. Prakash, B., Shukla, R., Singh, P., Mishra, P. K., Dubey, N. K., & Kharwar, R. N. (2011). Efficacy of chemically characterized Ocimum gratissimum L. essential oil as an antioxidant and a safe plant based antimicrobial against fungal and aflatoxin B1 contamination of spices. Food Research International, 44, 385e390.
R. Shukla et al. / Food Control 25 (2012) 27e33 Reddy, K. R. N., Raghavender, C. R., Salleh, B., Reddy, C. S., & Reddy, B. N. (2011). Potential of aflatoxin B1 production by Aspergillus flavus strains on commercially important food grains. International Journal of Food Science and Technology, 46, 161e165. Reddy, K. R. N., Reddy, C. S., & Muralidharan, K. (2009). Detection of Aspergillus spp. and aflatoxin B1 in rice in India. Food Microbiology, 26, 27e31. Sastri, B. N. (1950). The Wealth of India, a dictionary of Indian raw materials and industrial products. Council of Scientific and Industrial Research, 2, 427. Shukla, R., Kumar, A., Singh, P., & Dubey, N. K. (2009). Efficacy of Lippia alba (Mill.) N.E. Brown essential oil and its monoterpene aldehyde constituents against fungi isolated from some edible legume seeds and aflatoxin B1 production. International Journal of Food Microbiology, 135, 165e170. Shukla, R., Singh, P., Prakash, B., Kumar, A., Mishra, P. K., & Dubey, N. K. (2011). Efficacy of essential oils of Lippia alba (Mill.)N.E. Brown and Callistemon lanceolatus (Sm.)Sweet and their major constituents on mortality, oviposition and feeding behavior of pulse beetle, Callosobruchus chinensis L. Journal of the Science of Food and Agriculture, . doi:10.1002/ jsfa.4453.
33
Singh, P., Shukla, R., Prakash, B., Kumar, A., Singh, S., Mishra, P. K., et al. (2010). Chemical profile, antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima Burm. and Citrus sinensis (L.) Osbeck essential oils and their cyclic monoterpene, dl-limonene. Food and Chemical Toxicology, 48, 1734e1740. Tabuc, C., Stroia, C., & Neacsu, A. (2010). Incidence of Aspergillus strains and of aflatoxin B1 in cereals in south-western Romania. Research Journal of Agricultural Science, 42, 322e327. Tavares, A. C., Goncalves, M. J., Cavaleiro, C., Cruz, M. T., Lopes, M. C., Canhoto, J., et al. (2008). Essential oil of Daucus carota subsp. halophilus: composition, antifungal activity and cytotoxicity. Journal of Ethnopharmacology, 119, 129e134. Tepe, B., Daferera, D., Sokmen, A., Sokmen, M., & Polissiou, M. (2005). Antimicrobial and antioxidant activities of the essential oil and various extracts of Salvia tomentosa Miller (Lamiaceae). Food Chemistry, 90, 333e340. Tian, J., Ban, X., Zeng, H., He, J., Huang, B., & Youwei, W. (2011). Chemical composition and antifungal activity of essential oil from Cicuta virosa L. var. latisecta Celak. International Journal of Food Microbiology, 145, 464e470. Tolouee, M., Alinezhad, S., Saberi, R., Eslamifar, A., Zad, S. J., Jaimand, K., et al. (2010). Effect of Matricaria chamomilla L. flower essential oil on the growth and ultrastructure of Aspergillus niger van Tieghem. International Journal of Food Microbiology,139,127e133.
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Antifungal, aflatoxin inhibition and antioxidant activity of Callistemon lanceolatus (Sm.) Sweet essential oil and its major component 1,8-cineole against fungal isolates from chickpea seeds Ravindra Shukla, Priyanka Singh, Bhanu Prakash, N.K. Dubey* Laboratory of Herbal Pesticides, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2011 Received in revised form 28 September 2011 Accepted 4 October 2011
The present investigation reports the extent of molds and aflatoxin contamination to Avarodhi, Kabuli, Pusa 256, Radha and Samrat varieties of chickpea seeds. The study also examines the chemical composition of Callistemon lanceolatus (Sm.) Sweet essential oil and its antifungal, antiaflatoxin and antioxidant activity. During standardization of chemical profile, a total of 8 compounds constituting 0.862 mg/mL of oil composition were analyzed by GCeMS analysis where 1,8-cineole was recorded as a major component (0.56 mg/mL). The antifungal activity of EO and 1,8-cineole was evaluated by contact assay on Czapek’s dox agar. The EO (0.227e0.908 mg/mL) and 1,8-cineole (0.918 mg/mL) showed remarkable antifungal effect against all the fungal isolates of chickpea. Their minimal inhibitory (MIC) and fungicidal (MFC) concentrations for Aspergillus flavus were lower than those of the prevalent systemic fungicide Nystatin. Aflatoxin B1 (AFB1) production by NKD-208 isolates of A. flavus was strongly inhibited even at the lower fungistatic concentration of EO and 1,8-cineole.There was no adverse effect of EO treatment on chickpea seed germination suggesting its non-phytotoxic nature. Based on the findings of present investigation, C. lanceolatus essential oil may be recommended as botanical preservative for the enhancement of shelf life of food items in- view of the adverse effect of synthetic preservatives and its strong antifungal, aflatoxin inhibition and antioxidant activity. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Aflatoxin B1 Antifungal Antioxidant Callistemon lanceolatus Essential oil
1. Introduction Molds are ubiquitous microorganisms with a great capacity to colonize different kinds of substrates and to proliferate under extreme environmental conditions (Nguefack et al., 2009). They spoil various types of foods viz. cereals, legumes, spices, vegetables, fruits etc. and also produce mycotoxins that can be mutagenic, teratogenic, carcinogenic causing feed refusal and emesis in humans or animals (Frisvad, Skouboe, & Samson, 2005; Prakash et al., 2010; Prakash et al., 2011; Reddy, Raghavender, Salleh, Reddy, & Reddy, 2011). There are reports that hepatic carcinoma and other serious diseases may be induced by consuming food or using raw materials for food processing contaminated with aflatoxins. Nearly 5 billion people are exposed to aflatoxins in different developing countries and aflatoxicosis is ranked 6th among the 10 most severe health risks identified by WHO (Prakash et al., 2010). Moreover, aflatoxins are proved resistant to heat and have an ability to accumulate in the organism (Galvano, Ritieni, Piva, & Pietri, 2005). * Corresponding author. Tel.: þ91 9415295765; fax: þ91 5422368174. E-mail address: [email protected] (N.K. Dubey). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.10.010
Chickpea (Cicer arietinum L.) is the most nutritive among the food legumes and extensively used as protein adjunct to starchy diet (Sastri, 1950). Chickpea after dehulling is valued for its nutritive seeds with high protein content (12.3e31.5%) and 0.3% phosphorus. South-east Asia contributes about 80% to the global chickpea production, and India is the principal chickpea producing country in the region with 83% share (ICRISAT, 2011). Fungal contamination of chickpea seeds is the major problem in Indian sub-continent (Ahmad & Singh, 1991; Dawar, Syed, & Ghaffar, 2007). Although, different synthetic antimicrobials have been successfully commercialized in recent years, they encounter major problems not only due to their adverse side effects on consumers but also for the development of resistance by microorganisms (Tolouee et al., 2010). Hence, there must be optimization of alternative methods for pest and disease control that produce minimal damage to the environment and human health. With an expanding list of food-borne pathogens, there is an urgent need to explore the novel strategy to prevent food contamination. Essential oils as well as compounds derived from aromatic plants possess a wide range of activities of which the antimicrobial activity is most studied (Burt, 2004). Their applications
28
R. Shukla et al. / Food Control 25 (2012) 27e33
as preservatives in food or antiseptics and disinfectants are widely studied (Holley & Patel, 2005). In this regard, plant essential oils and their compounds may offer a great potential. Some essential oils and their components viz. eugenol, carvone, carvacrol, citral, thymol and limonene are bioactive compounds of some essential oils which are in commercial use as food additives through encapsulation technologies (Burt, 2004; Prakash et al., 2010).Therefore, their composition and antimicrobial activities must be continued to be investigated for better alternatives. Callistemon lanceolatus (Sm.) Sweet (family: Myrtaceae) is native to Australia and is widely distributed in subtropical and tropical regions; commonly known as bottle brush because of their cylindrical brush like flowers resembling a traditional bottle. Phytochemical and antimicrobial activity of Callistemon rigidus, Callistemon citrinus, Callistemon viminalis, Callistemon linearis (Das, Zaman, & Singh, 2009; Dongmo et al., 2010; Jazet et al., 2009) have earlier been reported but little is known about biological activity of C. lanceolatus. In the present study, chickpea seeds were screened for incidence of mycoflora and aflatoxin contamination by the toxigenic strain of Aspergillus flavus. In addition the chemically characterized EO of C. lanceolatus was assessed for its antifungal, aflatoxin inhibition and antioxidant activity.
2. Materials and methods 2.1. Collection of chickpea seeds and mycological analysis Avarodhi, Kabuli, Pusa 256, Radha and Samrat varieties of chickpea (C. arietinum L.), stored from 6 to 8 months,were procured from local markets of Varanasi, Uttar Pradesh, India. The seed mycoflora was examined using the agar plate method (Embaby & Abdel-Galil, 2006) as recommended by International Seed Testing Association (ISTA, 1966). Seeds were surfacesterilized with 1% solution of sodium hypochlorite and rinsed in three changes of sterile distilled water. Four seeds of each variety were placed equidistantly in Petri plates containing potato dextrose agar (PDA) medium (Potato infusion, 200 g; Dextrose, 20 g; Agar, 15 g; Distilled water, 1 L, pH, 5.6 0.2; HiMedia Laboratories Pvt. Ltd., Mumbai) and incubated for 7 days (28 2 C). The developing fungal colonies were isolated, identified (Burnett & Hunter, 1999) and routinely maintained on PDA in the presence of antibiotic Streptomycin (300 mg/L). The incidence of fungi was determined based on the occurrence of a particular species in samples of 10 seeds.
2.2. Detection of aflatoxigenic isolates of A. flavus A. flavus isolates from each variety of chickpea seed were screened for the production of aflatoxin B1 (AFB1) following Kumar, Mishra, Dubey, and Tripathi (2007). The isolates were cultured separately in 25 mL SMKY broth (sucrose 200 g; MgSO4$7H2O, 0.5 g; KNO3, 0.3 g and yeast extract, 7 g; 1 L distilled water) in 100 mL flask for 10 days. The content of each flask was filtered and extracted with 20 mL chloroform in a separating funnel. The extract was evaporated to dryness on water bath and redissolved in 1 mL chloroform. AFB1 was detected by thin layer chromatography. Fifty micro liter chloroform extract was spotted on Thin Layer Chromatography (TLC) plates and developed in the solvent system comprising toluene/isoamyl alcohol/methanol (90:32:2; v/v/v). The plate was air dried and the intensity of AFB1 observed in UVtransilluminator (360 nm).
2.3. Plant materials and extraction of EO C. lanceolatus, planted in Botanical garden of Banaras Hindu University, Varanasi, India was identified by morphological features with the help of Flora of BHU Campus (Dubey, 2004, p. 112 and 113) and the voucher specimens (LHP/Ver-21/2008 and LHP/Are/17/ 2008) were deposited at the Laboratory of Herbal Pesticides, Banaras Hindu University, Varanasi, India. Fresh plant leaves (200 g) were collected in the month of June and subjected to hydro-distillation (4 h) using a Clevenger-type apparatus (Kumar et al., 2007). The yield (mL/kg) of EOs was averaged over four experiments and calculated on the basis of plant material fresh weight. 2.4. GC and GCeMS EO of C. lanceolatus was subjected to gas chromatography (PerkinElmer Auto XL GC, MA, USA) equipped with a flame ionization detector and the GC condition were: EQUITY-5 column (60 m 0.32 mm 0.25 mm); H2 was the carrier gas; column head pressure 10 psi; oven temperature program isotherm 2 min at 70 C, 3 C/min gradient to 250 C, isotherm 10 min; injection temperature, 250 C; detector temperature 280 C. GCeMS analysis was performed using Perkin Elmer Turbomass GCeMS. The column size and oven conditions of GCeMS were the same of the GC-FID. Helium was the carrier gas. The effluent of the GC column was introduced directly into the source of MS and spectra obtained in the EI mode with 70 eV ionization energy. The sector mass analyzer was set to scan from 40 to 500 amu for 2 s. The identification of individual compounds was based on their retention times relative to those of authentic samples and matching spectral peaks available with Wiley, NIST and NBS mass spectral libraries or with the published data (Adams, 2007). 2.5. Antifungal assay The antifungal activity of EO and 1,8-cineole, the major constituent, (procured from Genuine Chemical Co., Mumbai, India) was tested against the fungal isolates of chickpea by contact assay based on hyphal growth inhibition (Shukla, Kumar, Singh, & Dubey, 2009) using Czapek-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 distilled water, pH 6.8 0.2; Sisco Research Lab., Mumbai). The requisite amount of EO was dissolved in 0.5 mL acetone, and added to 9.5 mL molten CDA in different Petri plates to achieve final concentrations (0.227, 0.454, 0.681, 0.908 mg/mL). 1,8-cineole was tested at 0.918 mg/mL. CDA plates containing acetone (0.5 mL) only, served as negative control. In addition, CDA plates treated with reference antifungal nystatin (1.0 mg/mL) were used as positive control. A 5 mm disc of test fungus was placed upside down on the center of the plate with fungal species in contact with growth medium. Cultures were incubated in the dark at 28 2 C (7 days). Antifungal index was calculated as the following-
Antifungal indexð%Þ ¼
1
Da 100 Db
where Da: average diameter of fungal growth in the treatment; Db: average diameter of fungal growth in the control sets. 2.6. Determination of MIC and MFC The minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for A. flavus (the most prevalent fungus) were determined by broth dilution method as reported
R. Shukla et al. / Food Control 25 (2012) 27e33
earlier (Shukla et al., 2009). Different concentrations of the EO and 1,8-cineole were dissolved in 0.5 mL acetone and incorporated to 9.5 mL CD broth tubes containing 106 spores/mL. The tubes were incubated at 30 C for a week. The lowest concentration that did not permit any visible growth of fungus was taken as MIC. Cells from the tubes showing no growth were sub-cultured on treatment-free CDA plates to determine if the inhibition was reversible. MFC is the lowest concentration at which no growth occurred on the plates.
2.7. Effect of EO and 1,8-cineole on aflatoxin B1 synthesis Requisite amounts of EO and 1,8-cineole were dissolved separately in 0.5 mL acetone, and added to 24.5 mL SMKY to achieve the different concentrations between (0.091e0.908) and (0.092e0.918) mg/mL respectively. The medium was inoculated with toxigenic isolate NKD-208 of A. flavus to give 106 spores/mL and incubated at 28 2 C (10 days). The medium was filtered and fungal mat was dried at 80 C (12 h) to determine the net mycelial dry weight. The filtrate was used for aflatoxin extraction as described above. For quantitative estimations, fluorescent spot of AFB1 on TLC plate was scrapped, dissolved in 5 mL cold methanol, and centrifuged (3000 rpm, 5 min). Optical density of the supernatant recorded at 360 nm and the AFB1 amount calculated according to Kumar et al. (2007):
Aflatoxin B1 contentðmg=LÞ ¼
DM 1000 El
where, D ¼ absorbance, M ¼ molecular weight of aflatoxin (312), E ¼ molar extinction coefficient of aflatoxin B1 (21,800) and l ¼ path length (1 cm cell was used).
2.8. Antioxidant activity 2.8.1. DPPH radical scavenging assay through TLC The antioxidant activity of C. lanceolatus EO was determined following Tepe, Daferera, Sokmen, Sokmen, and Polissiou (2005). 4.54 mg of the EO (1:10 dilution in methanol) was applied on TLC plate and developed in ethyl acetate and methanol (1:1) the plate was sprayed with 0.2% DPPH solution in methanol (2,2-diphenyl-1picrylhydrazil) and left at room temperature for 30 min. Yellow spot formed due to bleaching of purple color of DPPH reagent was recorded as positive antioxidant activity of EO. 2.8.2. Free radical scavenging activity Free radical scavenging activity of the C. lanceolatus EO was measured by recording the extent of bleaching of the purplecolored DPPH solution to yellow following Prakash et al. (2010). Different concentrations (1.816e9.08 mg/mL) of the essential oil were added to 0.004% DPPH solution in methanol (5 mL). After a 30 min of incubation at room temperature, the absorbance was taken against a blank at 517 nm using spectrophotometer. Butylated hydroxytoluene (BHT) and Butylated hydroxyanisole (BHA) (2.0e10 mg/mL) were used as positive control. Scavenging of DPPH free radical with reduction in absorbance of the sample was taken as a measure of their antioxidant activity IC50, which represented the concentration of the EO that caused 50% neutralization of DPPH radicals, was calculated from the graph plotting between percentage inhibition and concentration.
I% ¼
Ablank Asample ¼ Ablank 100
where, Ablank is the absorbance of the control (without test compound), and Asample is the absorbance of the test compound.
29
2.8.3. b-carotene/linoleic acid assay The b-carotene/linoleic acid bleaching test was performed by the method described by Ebrahimabadi et al. (2010). A stock solution of b-carotene and linoleic acid was prepared by dissolving 0.5 mg of b-carotene in 1 mL of chloroform, 25 mL of linoleic acid and 200 mL Tween 40. The chloroform was completely evaporated under vacuum in a rotatory evaporator at 40 C; then 100 mL of distilled water was added and the resulting mixture was vigorously stirred. The samples (2 g/L) were dissolved in DMSO and 350 mL of each sample solution were added to 2.5 mL of the above mixture in test tubes. The test tubes were incubated in a hot water bath at 50 C; for 2 h, together with blanks, BHT and BHA as a positive control and the other contained the same volume of DMSO instead of the samples as a negative control. The absorbance of each sample was measured at 470 nm. The test tube with BHT and BHA maintained its yellow color during the incubation period. The absorbance was measured at 470 nm on an ultraviolet spectrometer. Antioxidant activities (inhibition percentage, I%) of the samples was calculated using the following equation:
I% ¼
AbCarotene after 2 h assay =Ainitial bCarotene 100
where, Ab-Carotene after 2 h assay is the absorbance of b-Carotene after 2 h assay remaining in the samples and Ainitial b-Carotene is the absorbance of b-Carotene at the beginning of the experiments.
2.9. Phytotoxicity assay The phytotoxicity of the EO and 1,8-cineole in terms of seed germination and seedling growth of chickpea was assayed with respect to control sets following Kordali et al. (2008). Two layers of filter paper were placed on the bottom of each Petri plate (9 cm) and 5 seeds were placed equidistantly on the filter paper moistened with 10 mL of distilled water. 9.08 mg of the EO and 9.18 mg of EO compounds were dripped on Whatman No. 1 filter paper strip placed on the lid using a micropipette. Petri plates were sealed with parafilm to prevent escaping of volatile compounds and kept at 23 2 C in a growth chamber. Percent germination of seeds of control and treated sets was recorded. The length of radical and plumule was monitored at 24, 48, 72, 96, 120 and 144 h interval. 2.9.1. Statistical analysis Antifungal, antiaflatoxigenic and phytotoxicity experiments were performed in triplicate and data analyzed are mean SE subjected to one way ANOVA. Means are separated by Tukey’s multiple range tests when ANOVA was significant (p < 0.05) (SPSS 10.0; Chicago, IL, USA).
Table 1 Fungi isolated from chickpea seeds. Legumes
Fungi isolated
Cicer arietinum L. Var. Avarodhi
A. flavus (10), Aspergillus niger (4), Alternaria alternata (4), Fusarium oxysporum (4), Absidia ramosa (2) A. flavus (7), Mucor sp. (3), Trichoderma sp. (3), Fusarium nivale (4), Dreschlera sp. (2) A. flavus (5), Penicillium citrinum (4), Mucor sp. (2), Aspergillus fumigatus (4) A. flavus (10), Fusarium sp.(5)
Cicer arietinum L. Var. Kabuli Cicer arietinum L. Var. Pusa 256 Cicer arietinum L. Var. Radha Cicer arietinum L. Var. Samrat
A. flavus (6), Chaetomium sp. (3), P. citrinum (4), A. niger (4), Aspergillus oryzae (2)
Values in parentheses (*) are incidence of a particular species in samples of 10 seeds in direct plating method.
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R. Shukla et al. / Food Control 25 (2012) 27e33
Table 2 Aflatoxin B1 production potential of different Aspergillus flavus isolates in SMKY medium. Legumes Cicer Cicer Cicer Cicer Cicer
arietinum arietinum arietinum arietinum arietinum
Fungal isolates L. L. L. L. L.
var. var. var. var. var.
Avarodhi Kabuli Pusa 256 Radha Samrat
A. A. A. A. A.
flavus flavus flavus flavus flavus
NKD 188 NKD 189 NKD 197 NKD 208 NKD 209
AFB1 217.3 305.0 nf 365.3 nf
Values for aflatoxin B1 content is given in mg/l; nf ¼ not found.
3. Results During mycological analysis, A. flavus predominated in almost all the 5 varieties of chickpea seeds (Table 1). The Avarodhi and Radha varieties were heavily contaminated with A. flavus where all the 10 seeds of each variety were found positive with this fungus. Seven seeds out of 10 from var. Kabuli were found contaminated with A. flavus. Five different fungal species were isolated from Avarodhi, Kabuli and Samrat varieties whereas, only 2 species from Radha. Aspergillus niger and Penicillium citrinum were found on seeds of 2 varieties and recorded as the second dominant fungus after A. flavus. Out of 5 isolates of A. flavus procured from 5 varieties of chickpea seeds, only 3 isolates were found to be AFB1 producers. All the isolates were named on the basis of their culture collection number and deposited in our laboratory. A. flavus NKD-208, isolated from Radha variety of chickpea was recorded to be the most toxigenic isolate which produced 365.3 mg/L AFB1 in SMKY broth followed by A. flavus NKD 189 (305.0 mg/L) and A. flavus NKD 188 (217.3 mg/L) (Table 2). The hydro-distillation of C. lanceolatus leaves yielded colorless oil (yield: 6.9 g/kg). The EO composition of C. lanceolatus is presented in Table 3. Total 8 components were identified which constitutes 0.862 mg/mL of the essential oil. 1,8-cineole was found the major component with 0.56 mg/mL of the oil followed by apinene (0.18 mg/mL) and g-terpinene (0.11 mg/mL). b-pinene, isoamyl bromide, p-cymene, limonene and geraniol formate were the minor components of the oil. The EO of C. lanceolatus exhibited moderate to high antifungal activity (Table 4). Even at the lowest concentration of 0.227 mg/mL, EO caused more than 50% mycelial inhibition of most of the fungi except A. niger (39.7%). Aspergillus oryzae, Fusarium oxysporum, Fusarium sp. and Mucor sp. were found most susceptible fungi against the EO and their 100% growth inhibition was achieved at 0.681 mg/mL. However, remarkable antifungal index (71.3e96.0%) of EO was recorded against rest of the fungi at the same concentration. All the fungal species were completely inhibited at 0.908 mg/mL of EO. At 0.918 mg/mL, 1,8-cineole showed antifungal activity, ranging between 52.3 and 89.9%. The reference antifungal agent, nystatin showed the lowest antifungal activity among all the
Table 3 Chemical composition of C. lanceolatus essential oil. Compounds
Composition (mg/mL)
Retention time (Min.)
a-pinene b-pinene Isoamyl Bromide p-cymene Limonene 1,8-cineole g-terpinene Geraniol Formate
0.18 0.001 0.002 0.002 0.004 0.56 0.11 0.003
15.90 17.75 18.70 19.60 19.77 20.07 26.72 28.45
Total
0.862
treatments with only 34.9e58.8% inhibition of fungal isolates. None of the fungi was completely inhibited (100%) by 1,8-cineole and nystatin at 0.918 mg/mL and 1.0 mg/mL respectively. The MICs of C. lanceolatus EO and 1,8-cineole were 0.682 0.01 and 1.285 mg/mL 0.05 mg/mL, respectively against A. flavus. MFC of EO (1.135 mg/mL) was also lower than that of 1,8-cineole (1.83 mg/mL). However, MIC (1.78 mg/mL) and MFC (>2.0 mg/mL) of nystatin were comparatively higher than those of C. lanceolatus EO and 1,8-cineole. It is evident from Table 5 that EO and 1,8-cineole inhibited AFB1 production in a dose dependent manner. In control set, 325.3 mg/L AFB1 was secreted. However, at 0.455 mg/mL of C. lanceolatus EO, only 39.6 mg/L AFB1 was calculated. The oil caused complete prevention of AFB1 secretion in SMKY broth at 0.546 mg/mL, lower than the concentration at which no mycelium was found (i.e. 0.819 mg/mL). On the other hand, 1,8-cineole completely inhibited AFB1 at 0.918 mg/mL, whereas 121.7 mg mycelial weight was recorded at this concentration. The discoloration of the purple color of the DPPH on TLC plates confirmed the positive antioxidant activity of EO. The oil showed free radical scavenging activity in dose dependent manner and its IC50 value was 4.02 mg/mL. IC50 values of synthetic antioxidant BHT (7.28 mg/mL), BHA (4.44 mg/mL) were also determined as positive control. Oxidation of linoleic acid was moderately inhibited by the EO (30.16%) compare to positive control BHA (76.71%) BHT (64.23%) and a negative control (7.34%). Percent inhibition and IC50 values of EO and synthetic antioxidant are summarized in Table 6. A 100% germination of seeds was recorded following 48 h in all the three sets, including control. The size of radicles in EO treated seeds was somehow equal to the untreated control but higher than 1,8-cineole treated sets. The mean length of radicle was 128.5, 130.2 and 122.6 mm in EO, control and 1,8-cineole treated seeds. Similarly, no remarkable difference in the length of plumule was observed after 144 h in control (24.2 mm), EO (24.7 mm) and 1,8cineole (22.7 mm) treated seeds. 4. Discussion In the present investigation Avarodhi, Kabuli, Pusa 256, Radha and Samrat varieties of chickpea were screened for the first time to determine their inherent mycobiota.. All 5 varieties of chickpea were found contaminated with various fungi, where, 14 fungal species belonging to 9 genera were identified and isolated. Species of Aspergillus (56.3%) predominated in all the seeds followed by Fusarium sp. (14.0%). Only fragmentary work has been performed earlier regarding fungal association of chickpea seeds. These results confirm the earlier observations where Aspergillus species were one of the most predominant fungi and aflatoxin producers in some of the stored grains (Amadi & Adeniyi, 2009; Reddy, Reddy, & Muralidharan, 2009). Tabuc, Stroia, and Neacsu (2010) reported Aspergillus species in 45 of the 56 analyzed samples of corn, wheat, barley and oat. Reddy et al. (2011) stated that the genus Aspergillus was the most dominant among the prevalent genera of fungi isolated from some cereals and pulses. Similar results were obtained in a mycological study conducted with cowpea seeds in Africa (Houssou et al., 2009). The composition of EOs varies with respect to ecological and geographical conditions, age of plant and time of harvesting (Prakash et al., 2010). Such a variation in chemical composition of EOs would definitely alter their biological activity. Hence, the chemical profile of an EO must be standardized before its recommendation as food preservative. Most chemical components of EOs are terpenoids, including monoterpenes, sesquiterpenes, and their oxygenated derivatives. In the present investigation 1,8-cineole/ eucalyptol, an oxygenated monoterpene, was the major component
R. Shukla et al. / Food Control 25 (2012) 27e33
31
Table 4 Antifungal index of C. lanceolatus essential oil and its major component against different fungal isolates. Fungal isolates
Antifungal Index (%) C. lanceolatus EO
Absidia ramosa Alternaria alternata, Aspergillus fumigatus Aspergillus niger Aspergillus oryzae Chetomium sp. Dreschelera sp. Fusarium nivale Fusarium oxysporum Fusarium sp. Mucor sp. Penicillium citrinum Trichoderma sp.
1,8-cineole
Nystatin
0.227 mg/mL
0.454 mg/mL
0.681 mg/mL
0.908 mg/mL
0.918 mg/mL
1.0 mg/mL
61.0 1.9 48.6 1.6 52.0 2.8 39.7 3.0 62.5 2.6 62.5 1.9 68.5 1.7 51.2 0.3 58.4 2.1 79.3 1.5 81.6 2.6 50.0 2.5 57.0 1.0
71.8 1.1 65.2 1.8 71.3 2.2 53.2 3.0 80.8 2.6 78.0 3.5 81.5 2.2 70.8 3.0 81.5 2.1 88.2 1.8 93.5 3.3 72.1 1.6 72.6 3.8
93.3 1.0 89.7 3.6 84.3 2.8 71.3 3.0 100 88.9 4.1 95.4 2.6 91.8 2.2 100 100 100 96.0 2.8 94.1 1.5
100 100 100 100 100 100 100 100 100 100 100 100 100
76.5 2.2 68.5 2.8 68.3 3.3 52.3 4.5 71.1 1.9 67.2 2.8 80.0 3.4 73.6 1.8 73.2 2.6 80.0 3.3 89.9 2.6 68.9 2.8 70.3 1.9
40.0 3.0 52.8 4.1 41.7 2.2 38.5 2.6 56.7 3.7 34.9 1.8 58.3 3.4 58.8 4.6 40.2 1.8 48.3 1.8 58.6 1.0 50.2 2.8 43.5 3.4
Concentrations are given in ml/mL, percent values are mean (n ¼ 3) SE.
of EO. a-pinene and g-terpinene, the monoterpene hydrocarbons, were second and third dominant components whereas; p-cymene and b-pinene were present in traces. 1,8-cineole-a-pinene has also been reported abundantly in several myrtaceous plants like Eucalyptus saligna, Melaleuca armillaris, C. rigidus, C. citrinus, Myrtus communis (Aidi, Mhamdi, & Marzouk, 2007; Dongmo et al., 2010; Farag et al., 2004). Amongst the toxigenic strains of A. flavus, the strain NKD 208 isolated from Radha variety of chickpea was found to be highly toxigenic and selected for detailed studies in the present investigation. Although, there are earlier reports on variation of antifungal activity of plant products against different species of a particular genus of a fungus (Prakash et al., 2010; Shukla et al., 2009) but literature is mostly silent on variation of their efficacy at strain level of a particular fungal species. The efficacy of C. lanceolatus EO has so far been not well-explored against storage fungi and mycotoxin contamination. However, there are earlier reports on antifungal activity of other species of Callistemon viz. C. citrinus, C. viminalis, C. rigidus against pathogenic yeasts and some filamentous fungi (Delahaye et al., 2009; Dongmo et al., 2010; Jazet et al., 2009). To the best of our knowledge, there has not been a relevant study on the effectiveness of any species of Callistemon oil against aflatoxin production by A. flavus. The biological activity of essential oils may be only due to their major components or synergistic/antagonistic interaction between different oil components Hence, 1,8-cineole, the major component along with the respective EO were tested in the present investigation to correlate whether the efficacy of the EO was due to
synergism between existing components or only due to the major component. The results indicate that none of the fungi was completely inhibited at 0.918 mg/mL of 1,8-cineole, whereas, 100% fungal inhibition was achieved at this concentration of EO. The MIC of 1,8-cineole (1.285 mg/mL) against A. flavus was approximately double to that of C. lanceolatus EO (0.682 mg/mL). Similarly, AFB1 production was inhibited at 0.918 mg/mL of 1,8-cineole and 0.546 mg/mL of EO. Hence, the enhanced antifungal and aflatoxin inhibitory activities C. lanceolatus EO may be attributed to some minor components that have a synergistic effect with the major components rather than 1,8-cineole, the major component. In addition, the better efficacy of the EO over the reference antifungal Nystatin and its broad fungitoxic spectrum inhibiting the growth of the all the fungal isolates strengthen its merits as a post harvest antimicrobial against food deteriorating fungi. Moreover, lower MIC of C. lanceolatus EO than that of some earlier reported oils viz., Lippia alba (Shukla et al., 2009) Satureja hortensis (Dikbas, Kotan, Dadasoglu, & Sahin, 2008), Daucus carota (Tavares et al., 2008) strengthens its high efficacy and possible economical exploitation. The antifungal mechanism of EO is speculated to involve membrane disruption by their lipophilic compounds (Cowan, 1999). The low-molecular weight and highly lipophilic components of EOs pass easily through cell membranes and cause disruption to the fungal cell organization (Chao et al., 2005). The EO inhibited aflatoxin production at concentrations lower than its fungitoxic concentration. Thus, the inhibition of AFB1 production cannot be completely attributed to reduced fungal
Table 5 Effect of C. lanceolatus EO and 1,8-cineole on mycelial biomass and Aflatoxin B1 production by A. flavus (NKD-208) in SMKY medium. Concentration (mg/mL)
C. lanceolatus EO MDW
0.0 (Control) 0.091 0.182 0.273 0.364 0.455 0.546 0.637 0.728 0.819 0.908
399.7 382.0 342.0 311.0 179.3 123.0 81.7 51.3 18.0 0.0 0.0
AFB1
2.9a 5.0b 2.0c 3.7d 2.3e 3.7f 3.1g 1.3h 2.3i 0.0j 0.0j
325.3 254.0 196.3 157.0 95.7 39.6 0.0 0.0 0.0 0.0 0.0
4.2a 4.0b 3.1c 5.6d 4.7e 0.8f 0.0g 0.0g 0.0g 0.0g 0.0g
Concentration
1,8-cineole
(mg/mL)
MDW
0.0 (control) 0.092 0.184 0.276 0.368 0.460 0.552 0.644 0.736 0.828 0.918
399.7 383.7 356.0 322.7 294.3 243.7 201.7 199.3 163.7 147.3 121.7
AFB1
2.9a 3.1b 4.0c 4.1d 3.3e 2.9f 1.7g 5.8g 3.8h 1.8i 3.2j
325.3 307.7 282.3 266.3 236.7 190.7 130.0 101.0 60.0 22.6 0.0
4.2a 5.3b 4.0c 2.7d 5.6e 3.7f 2.0g 2.0h 3.6i 2.6j 0.0k
MDW ¼ mycelial dry weight (mg); AFB1 ¼ aflatoxin B1 content (mg/l), values are mean (n ¼ 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.
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R. Shukla et al. / Food Control 25 (2012) 27e33
Table 6 Antioxidant activity of EO in DPPH free radical scavenging activity and b-carotene/ linoleic acid bleaching assay. Sample
DPPH (IC50)
b-carotene/linoleic acid inhibition (%)
C. lanceolatus EO BHT BHA Negative control
4.02 0.07a,y 7.28 0.03b,z 4.44 0.03c,z ND
30.16 64.23 76.75 7.34
0.16c 0.24b 0.21a 0.33d
ymg/mL; zmg/mL. The means followed by same letter in the same column are not significantly different according to ANOVA and tukey’s multiple comparison tests. Value are mean (n ¼ 3) SE.
growth, but may be because of inhibition of carbohydrate catabolism in A. flavus by acting on some key enzymes, reducing its ability to produce aflatoxins as has been reported by Tian et al. (2011). Determination of exact mechanism of AFB1 suppression by C. lanceolatus EO requires further investigations. The antioxidant activity of C. lanceolatus EO was confirmed by DPPH radical scavenging assay and b-carotene-linoleic acid bleaching assay. C. lanceolatus EO recorded better free-radical scavenger as compared to earlier reported EOs, of Ocimum gratissimum (Prakash et al., 2011), Citrus maxima, Citrus sinensis (Singh et al., 2010), Stachys inflata (Ebrahimabadi et al., 2010) Satureja montana, Satureja subspicata (Cavar, Maksimovic, Solic, Mujkic, & Besta, 2008) and Semenovia tragioides (Bamoniri et al., 2010). There was no adverse effect of EO treatment on seed germination, suggesting its non-phytotoxic nature. In addition, the nonmammalian toxicity of C. lanceolatus EO in terms of high LD50 has earlier been reported by our research group (Shukla, Singh, Prakash, Kumar, Mishra,& Dubey, 2011). To the best of our knowledge, there is no report on any toxic phytochemical from C. lanceolatus. Because of pleasant camphorlike spicy aroma and cooling taste, the cyclic ether 1,8-cineole is used in flavorings, fragrances, and cosmetics. It is an ingredient in many brands of mouthwash and cough suppressant. Hence, there would be no chance of their negative effects on sensory quality, although detailed investigations on organoleptic parameters are needed before final recommendation. In conclusion, the EO of C. lanceolatus may be recommended as a plant based food preservative for enhancement of shelf life of stored food commodities by checking their fungal contamination, aflatoxin secretion and lipid per oxidation because of antifungal, aflatoxin inhibition and antioxidant activity. The yield of EO (6.9 g/ kg) was relatively high in the present investigation, and sufficient amount of raw materials would be available as the plant grows luxuriantly in the area. The findings of the present investigation would draw the attention of food industries regarding large scale application of C. lanceolatus EO as a plant based food additive. Acknowledgments This work was financially supported by Council of Scientific and Industrial Research (CSIR), New Delhi, India. References Adams, R. P. (2007). Identification of essential oil components by gas chromatography/ mass spectrometry. Carol Stream, IL, USA: Allured Publishing Corporation. Ahmad, S. K., & Singh, P. L. (1991). Mycofloral changes and aflatoxin contamination in stored chickpea seeds. Food Additives & Contaminants, 8, 723e730. Aidi, W. W., Mhamdi, B., & Marzouk, B. (2007). Essential oil composition of two myrtus communis L. varieties grown in North Tunisia. Italian Journal of Biochemistry, 56, 180e186. Amadi, J. E., & Adeniyi, D. O. (2009). Mycotoxin production by fungi isolated from stored grains. African Journal of Biotechnology, 8, 1219e1221.
Bamoniri, A., Ebrahimabadi, A. H., Mazoochi, A., Behpour, M., Kashi, F. J., & Batooli, H. (2010). Antioxidant and antimicrobial activity evaluation and essential oil analysis of Semenovia tragioides Boiss. from Iran. Food Chemistry, 122, 553e558. Burnett, W. L., & Hunter, B. B. (1999). Illustrated genera of imperfect fungi (4th ed.). St. Paul, Minnesota: APS Press. Burt, S. (2004). Essential oils: their antibacterial properties and potential applications in foodsda review. International Journal of Food Microbiology, 94, 223e253. Cavar, S., Maksimovic, M., Solic, M. E., Mujkic, A. J., & Besta, R. (2008). Chemical composition and antioxidant and antimicrobial activity of two Satureja essential oils. Food Chemistry, 111, 648e653. Chao, L. K., Hua, K. F., Hsu, H. Y., Cheng, S. S., Lin, J. Y., & Chang, S. T. (2005). Study on the anti-inflammatory activity of essential oil from leaves of Cinnamomum osmophloeum. Journal of Agricultural and Food Chemistry, 53, 7274e7278. Cowan, M. M. (1999). Plant products as antimicrobial agents. Clinical Microbiology Reviews, 12, 564e582. Das, A., Zaman, K., & Singh, A. V. (2009). Phytochemical and chemical composition evaluation of volatile oil of Callistemon linearis DC Leaf. Advances in Natural and Applied Sciences, 3, 56e59. Dawar, S., Syed, F., & Ghaffar, A. (2007). Seed borne fungi associated with chickpea in pakistan. Pakistan Journal of Botany, 39, 637e643. Delahaye, C., Rainford, L., Nicholson, A., Mitchell, S., Lindo, J., & Ahmad, M. (2009). Antibacterial and antifungal analysis of crude extracts from the leaves of Callistemon viminalis. Journal of Medical and Biological Sciences, 3, 1e7. Dikbas, N., Kotan, R., Dadasoglu, F., & Sahin, F. (2008). Control of Aspergillus flavus with essential oil and methanol extract of Satureja hortensis. International Journal of Food Microbiology, 124, 179e182. Dongmo, B. N., Dongmo, P. M. J., Ngoune, L. T., Kwazou, N. L., Zollo, P. H. A., & Menut, C. (2010). Antifungal activities of essential oils of some Cameroonian Myrtaceae on Aspergillus flavus Link ex. Fries. Asian Journal of Experimental Biological Sciences, 1, 907e914. Dubey, N. K. (2004). Flora of BHU campus. Varanasi: Banaras Hindu University Press. Ebrahimabadi, A. H., Ebrahimabadi, E. H., Djafari-Bidgoli, Z., Kashi, F. J., Mazoochi, A., & Batooli, H. (2010). Composition and antioxidant and antimicrobial activity of the essential oil and extracts of Stachys inflata Benth from Iran. Food Chemistry, 119, 452e458. Embaby, E. M., & Abdel-Galil, M. M. (2006). Seed borne fungi and mycotoxins associated with some legume seeds in Egypt. Journal of Applied Sciences Research, 2, 1064e1071. Farag, R. S., Shalaby, A. S., El-Baroty, G. A., Ibrahim, N. A., Ali, M. A., & Hassan, E. M. (2004). Chemical and biological evaluation of the essential oils of different Melaleuca species. Phytotherapy Research, 18, 30e35. Frisvad, C. J., Skouboe, P., & Samson, A. R. (2005). Taxonomic comparison of three different groups of aflatoxin producers and new efficient producer of aflatoxin B1, sterigmatocystine and 3-Omethylsterigmatocystine Aspergillus rumbelli sp. nov. Systematic and Applied Microbiology, 28, 442e453. Galvano, F., Ritieni, A., Piva, G., & Pietri, A. (2005). Mycotoxins in the human food chain. In Duarate Diaz (Ed.), The mycotoxin blue book (pp. 187e225). England: Nottingham University Press. Holley, R. A., & Patel, D. (2005). Improvement in shelf-life and safety of perishable foods by plant essential oils and smoke antimicrobials. Food Microbiology, 22, 273e292. Houssou, P. A., Ahohuendo, B. C., Fandohan, P., Kpodo, K., Hounhouigan, D. J., & Jakobsen, M. (2009). Natural infection of cowpea (Vigna unguiculata (L.) Walp.) by toxigenic fungi and mycotoxin contamination in Benin, West Africa. Journal of Stored Products Research, 45, 40e44. ICRISAT. (2011). Chickpea (Internet). International Crop Research Institute for the Semi Arid Tropics. Available from www.icrisat.org. ISTA. (1966). International rules for seed testing. Proceeding of International Seed Testing Association (Moisture and Aeration), 31, 54e55. Jazet, P. M. D., Tatsadjieu, L. N., Ndongson, B. D., Kuate, J., Zollo, P. H. A., & Menut, C. (2009). Correlation between chemical composition and antifungal properties of essential oils of Callistemon rigidus and Callistemon citrinus of Cameroon against Phaeoramularia angolensis. Journal of Medicinal Plants Research, 3, 9e15. Kordali, S., Cakir, A., Ozer, H., Cakmakci, R., Kesdek, M., & Mete, E. (2008). Antifungal, phytotoxic and insecticidal properties of essential oil isolated from Turkish Origanum acutidens and its three components, catvacrol, thymol and p-cymene. Bioresource Technology, 99, 8788e8795. Kumar, R., Mishra, A. K., Dubey, N. K., & Tripathi, Y. B. (2007). Evaluation of Chenopodium ambrosioides oil as a potential source of antifungal, antiaflatoxigenic and antioxidant activity. International Journal of Food Microbiology, 115, 159e164. Nguefack, J., Dongmo, J. B. L., Dakole, C. D., Leth, V., Vismer, H. F., Torp, J., et al. (2009). Food preservative potential of essential oils and fractions from Cymbopogon citratus, Ocimum gratissimum and Thymus vulgaris against mycotoxigenic fungi. International Journal of Food Microbiology, 131, 151e156. Prakash, B., Shukla, R., Singh, P., Kumar, A., Mishra, P. K., & Dubey, N. K. (2010). Efficacy of chemically characterized Piper betle L. essential oil against fungal and aflatoxin contamination of some edible commodities and its antioxidant activity. International Journal of Food Microbiology, 142, 114e119. Prakash, B., Shukla, R., Singh, P., Mishra, P. K., Dubey, N. K., & Kharwar, R. N. (2011). Efficacy of chemically characterized Ocimum gratissimum L. essential oil as an antioxidant and a safe plant based antimicrobial against fungal and aflatoxin B1 contamination of spices. Food Research International, 44, 385e390.
R. Shukla et al. / Food Control 25 (2012) 27e33 Reddy, K. R. N., Raghavender, C. R., Salleh, B., Reddy, C. S., & Reddy, B. N. (2011). Potential of aflatoxin B1 production by Aspergillus flavus strains on commercially important food grains. International Journal of Food Science and Technology, 46, 161e165. Reddy, K. R. N., Reddy, C. S., & Muralidharan, K. (2009). Detection of Aspergillus spp. and aflatoxin B1 in rice in India. Food Microbiology, 26, 27e31. Sastri, B. N. (1950). The Wealth of India, a dictionary of Indian raw materials and industrial products. Council of Scientific and Industrial Research, 2, 427. Shukla, R., Kumar, A., Singh, P., & Dubey, N. K. (2009). Efficacy of Lippia alba (Mill.) N.E. Brown essential oil and its monoterpene aldehyde constituents against fungi isolated from some edible legume seeds and aflatoxin B1 production. International Journal of Food Microbiology, 135, 165e170. Shukla, R., Singh, P., Prakash, B., Kumar, A., Mishra, P. K., & Dubey, N. K. (2011). Efficacy of essential oils of Lippia alba (Mill.)N.E. Brown and Callistemon lanceolatus (Sm.)Sweet and their major constituents on mortality, oviposition and feeding behavior of pulse beetle, Callosobruchus chinensis L. Journal of the Science of Food and Agriculture, . doi:10.1002/ jsfa.4453.
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Singh, P., Shukla, R., Prakash, B., Kumar, A., Singh, S., Mishra, P. K., et al. (2010). Chemical profile, antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima Burm. and Citrus sinensis (L.) Osbeck essential oils and their cyclic monoterpene, dl-limonene. Food and Chemical Toxicology, 48, 1734e1740. Tabuc, C., Stroia, C., & Neacsu, A. (2010). Incidence of Aspergillus strains and of aflatoxin B1 in cereals in south-western Romania. Research Journal of Agricultural Science, 42, 322e327. Tavares, A. C., Goncalves, M. J., Cavaleiro, C., Cruz, M. T., Lopes, M. C., Canhoto, J., et al. (2008). Essential oil of Daucus carota subsp. halophilus: composition, antifungal activity and cytotoxicity. Journal of Ethnopharmacology, 119, 129e134. Tepe, B., Daferera, D., Sokmen, A., Sokmen, M., & Polissiou, M. (2005). Antimicrobial and antioxidant activities of the essential oil and various extracts of Salvia tomentosa Miller (Lamiaceae). Food Chemistry, 90, 333e340. Tian, J., Ban, X., Zeng, H., He, J., Huang, B., & Youwei, W. (2011). Chemical composition and antifungal activity of essential oil from Cicuta virosa L. var. latisecta Celak. International Journal of Food Microbiology, 145, 464e470. Tolouee, M., Alinezhad, S., Saberi, R., Eslamifar, A., Zad, S. J., Jaimand, K., et al. (2010). Effect of Matricaria chamomilla L. flower essential oil on the growth and ultrastructure of Aspergillus niger van Tieghem. International Journal of Food Microbiology,139,127e133.