Natural occurrence of aflatoxin in green leafy vegetables

Food Chemistry 138 (2013) 1908–1913

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Natural occurrence of aflatoxin in green leafy vegetables P. Hariprasad, P. Durivadivel, M. Snigdha, G. Venkateswaran ⇑ Food Microbiology Department, Central Food Technological Research Institute, Mysore 570 020, Karnataka, India

a r t i c l e

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Article history: Received 3 October 2012 Received in revised form 20 November 2012 Accepted 23 November 2012 Available online 1 December 2012 Keywords: Aflatoxin Green leafy vegetables TLC ELISA Xylem sap Phloem sap

a b s t r a c t Natural occurrence of aflatoxin (AF) in agricultural soils, green leafy vegetables (GLVs) and persistence in processed foods was investigated. in total 33 soil samples and 81 GLVs which belonged to 9 groups collected from nine vegetable-growing regions were studied. Seventy percent of soils and 69.2% GLVs were contaminated with AF ranging from 0.0 to 88 ppb. Root samples frequently had higher concentration of AFB1 in comparison with shoot samples. Under greenhouse conditions all the tested plants were found to take up AF. From xylem and phloem sap experiments it was clear that AF was gaining entry into the plant system via water-conducting xylem tissue and was translocated to aerial plant parts, with subsequent entry into the phloem. Of the two cooking methods studied, pressure cooking of GLVs significantly reduced the AF level in comparison with ordinary boiling. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Green leafy vegetables (GLVs) are popular around the world, especially in Asia. The majority of the Indian population is vegetarian and daily intake of at least 100 g of fresh GLVs is recommended by nutrition experts (Reddy, 1999). India is endowed with an array of leafy vegetables suited for tropical, sub-tropical and temperate climates, which are grown throughout the year. Green leafy vegetables are valuable sources of vitamins A and C, iron, folic acid, dietary fibre, and minerals like calcium, phosphorus, sodium, potassium. It has been estimated that 100 g of tropical leafy vegetables can provide 60–140 mg of ascorbic acid, 100 lg of folic acid, 4–7 mg iron and 200–400 mg of calcium (Saxena, 1999). Some GLVs are known to be rich in lysine, an essential amino acid that is lacking in diets based on cereal and fibres, while others are medicinal (Maundu, 1995), anticarcinogenic and antiarteriosclerotic (Imungi, 2002). Green leafy vegetables also contain polyphenols which have beneficial physiological effects on humans as antioxidants. The commonly consumed GLVs in different parts of India are coriander, fenugreek, dill, amaranthus, mint, basella, lettuce, spinach, alternanthera, drumstick, etc. As GLVs happen to be to be a major part of India’s diet, it is important to analyse the various contaminations occurring in them, so that one can minimise the threat to consumer’s health. The distribution of aflatoxin (AF) in agricultural commodities has been fairly well characterised because of

⇑ Corresponding author. Tel.: +91 9739599259. E-mail address: [email protected] (G. Venkateswaran). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.11.093

its importance to the food supply. However little is known on the occurrence and fate of AF in GLVs. Aflatoxins are a group of mycotoxins produced mainly by Aspergillus flavus, A. parasiticus and A. nomius (Bayman & Cotty, 1993). These species are ubiquitous in nature and being a saprophyte grow on a wide variety of substrates, including decaying plant and animal debris under field conditions. Aflatoxins are carcinogenic contaminants of food and feeds that are frequently responsible for health and economic concerns in many countries. Aflatoxin B1 is the most potent toxic metabolite, that shows hepatotoxic, teratogenic and mutagenic properties, causing damage to mammals as toxic hepatitis, haemorrhage, oedema, immunosuppression and hepatic carcinoma. It has been classified as a Class 1 human carcinogen by the International Agency for Research on Cancer (IARC, 2002). The presence of AF and aflatoxigenic fungi in rhizospheric and non-rhizospheric environment could potentially result in a number of adverse environmental consequences. Predominance of these fungi in rhizospheric soil has been studied by Jofee (1969). Since both the fungi and their metabolites gain access to the plant under field conditions, the uptake of AF, its persistence in plants and effect on consumers is of interest. Absorption of AF by young germinating seedlings grown in contaminated soil could seriously affect the growth and development of plants (Ahmad & Sinha, 2002; Chatterjee, 1988; El-Naghy, Fadl-Allah, & Samhan, 1999). Until recently, research has focused mainly on the occurrence of aflatoxigenic fungi in stored grains, grain susceptibility to fungal infection and factors affecting postharvest contamination. But being a soil-borne organism Aspergillus spp. produce AF in rhizosphere and non-rhizosphere soil and may enter the plant via

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P. Hariprasad et al. / Food Chemistry 138 (2013) 1908–1913 Table 1 Natural occurrence of aflatoxin in soil samples from vegetable growing fields. Sl. No.

Place of collection

No. of samples

Aflatoxigenic fungi

Total aflatoxin (ppb)

1 2 3 4 5 6 7 8 9

Pandavapur Mysore T. Narasipura Mandya Nanjanagudu Bannur Srirangapatna Nagamangala H. D. Kote

5 5 3 4 2 3 5 2 4

+ + + + + + + + +

0.0–5.0 0.0–13.6 0.0–13.5 0.0–24.2 0.0–14.0 0.0–2.6 0.0–41.5 3.0–7.5 0.0–9.7

+ = Presence.

conducting tissues and ultimately may accumulate in aerial plant parts or in seeds. Aflatoxin plant uptake studies have been already reported in maize seedlings (Mertz, Lee, Zuber, & Lillehoj, 1980), mustard (Ahmad & Sinha, 2002), soybean (Jones, Chancey, Morton, Dashek, & Llewellyn, 1980) and lettuce (Crisan, 1973). In the case of GLVs whole plant or plant parts are consumed fresh. Hence the probability of exposure to AF from GLVs is high. There are not many reports regarding uptake of AF by GLVs. Hence the present study focused on (i) screening of agricultural soil samples and local GLVs for the presence of AFs, (ii) studying the ability of GLVs to take up AF under laboratory and greenhouse conditions, and (iii) investigating the effect of normal boiling and pressure cooking on the fate of AF in GLVs. 2. Material and methods 2.1. Collection of plant, soil and seed materials Thirty-three agricultural soil samples were collected from nine vegetable growing regions of Mandya and Mysore Districts of southern Karnataka during March–August, 2011 (Table 1). Soil samples were collected randomly from five selected sites in each field and pooled to get a composite sample. Samples were labelled and immediately brought to the laboratory for further analysis. In total 81 leafy vegetables belonging to nine species (Table 2), which are regularly used in local diets were collected from local vegetable markets of Pandavapur, Mysore, T. Narasipura, Mandya, Nanganagudu, Bannur, Srirangapatna, Nagamangala and H.D. Kote, in the Mysore and Mandya districts of Karnataka state during March–August, 2011 (Table 2). Seed samples of GLVs for greenhouse studies were procured from local private seed agencies, Mysore, Karnataka.

media (HiMedia, Mumbai) and incubated at 28 ± 2 °C. The characteristic colonies of Aspergillus were counted and expressed as mean colony-forming units per gram soil (cfu/g). All Aspergillus isolates were identified up to the generic level, and the isolates belonging to A. flavus were identified morphologically to species level by following the manual for the identification of the genus Aspergillus (Raper & Fennel, 1965). 2.3. Detection of toxigenicity of Aspergillus flavus Aspergillus flavus isolates were grown on 10 ml potato dextrose broth (PDB) for 7 days at 28 ± 2 °C. After the growth period AFs were extracted with three volumes of 100 ml chloroform and extract was evaporated to dryness. The extract was later analysed for the presence of toxin using thin layer chromatography (TLC) and competitive indirect enzyme linker immunosorbent assay (ciELISA). 2.4. Extraction and quantification of AF from soil samples Soil samples (25 g) were taken in a stoppered conical flask to which 100 ml of chloroform:distilled water (3:1) were added. The samples were incubated in a shaker at 100 rpm at room temperature for 12 h. The samples were then filtered through Whatman No. 1 filter paper and filtrates were concentrated using a rotary evaporator. The collected chloroform extract was again dried using a rotary evaporator and the residue was redissolved in 1 ml chloroform and dried under nitrogen gas. The concentrated extract was analysed for the presence of AF by TLC and ciELISA as explained below. 2.5. Extraction and quantification of aflatoxin from green leafy vegetables

2.2. Isolation of Aspergillus spp. from soil samples Fungi were isolated using standard plate method. One gram of soil sample was homogenised in 10 ml sterile saline (0.85%) and serially diluted, pour plated in triplicate on Aspergillus differential

Green leafy vegetables were partitioned into shoot and root, blot dried and subjected to AF analysis. Samples (500 g shoot or 100 g root) were ground in a mechanical blender into a fine paste. One hundred gram of shoot and 25 g of root paste were mixed with

Table 2 List of green leafy vegetables used in the present study. Sl. No.

Common name

Local name

Family

Botanical name

Plant part used

1 2 3 4 5 6 7 8 9

Coriander Fenugreek Amaranthus Amaranthus Cabbage Spinach Mint Dill Bladderdock

Coriander Menthya Dhantu Keere Kosu Palak Pudina Sapsege Chukki

Apiaceae Fabaceae Amaranthaceae Amaranthaceae Brassicaceae Amaranthaceae Lamiaceae Apiaceae Polygonaceae

Coriandrum sativum Trigonella foenum-graecum Amaranthus bicolor Amaranthus sp. Brassica oleracea Spinacia oleracea Mentha arvensis Anethum graveolens Rumex vesicarius

Shoot Shoot Shoot Shoot Leaf Leaf Shoot Shoot Leaf

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500 ml and 125 ml of methanol:chloroform (1:1), respectively and extracted for 3 h on a rotary shaker at 150 rpm in the dark. The chloroform layer was separated and filtered through Whatman No. 1 filter paper. The organic phase was completely evaporated and crude AF was dissolved in 20 ml of 30% acetone followed by addition of 10 ml of 20% lead acetate to the acetone mixture to remove chlorophyll, lipids and fatty acid contaminants (Mertz et al., 1980). This step was repeated until chlorophyll was completely removed. Finally, the crude extract was dissolved in 10 ml chloroform and concentrated. The concentrated extract was analysed for the presence of AF by TLC and ciELISA as explained below.

approximately 1 cm2 pieces and combined with 500 ml of distilled water. One sample was cooked at 100 °C for 20 min in a steel vessel. The other was cooked at 121 °C for 20 min in a commercial pressure cooker at 15 lb/in2. After cooling, the cooked GLVs were drained in a sieve. Residues of AFB1 in cooked GLVs and drained water were extracted and quantified through ciELISA. A control was prepared where AF was extracted and quantified from plant material without cooking. The percent reduction in AF level from different cooking methods was calculated by considering the level of AF in control as 100%. The following experiments were done in two series of triplicates containing 50 plants each.

2.6. Aflatoxin uptake studies under greenhouse conditions

2.10. Thin layer chromatography

Seeds of selected GLVs were sown in sterilised coir pith and grown for 10 days under greenhouse conditions. To the 10-day old seedlings, AF (100 lg/l) spiked water was added for the next 10 days (10 ml/seedling). Distilled-water-treated seedlings served as a control. After treatment, seedlings were removed from coir pith without damaging the root system. Roots of all seedlings were washed with running water followed by 5% acetone in water. Seedlings were blot dried, separated root from shoot and used for the extraction of AF as explained earlier. For each treatment there were 50 plants of three replicates and the experiment was repeated twice.

Presence of different AF (AFB1, AFB2, AFG1 and AFG2) contamination in soil samples and GLVs was determined by TLC. Equal volumes were spotted and the plates were developed with mobile phase consisting of chloroform:ethyl acetate (8:2). Developed chromatograms were evaluated visually under UV illumination (365 nm). Aflatoxin B and G were visualised as blue and green spots, respectively. These spots were compared with the standard AFs obtained from Sigma.

2.7. Xylem sap experiment Seedlings were grown as explained earlier, with regular water spiked with AFB1 (100 lg/l) for about 12 days. After treatment period, using sterile blade, stem of seedlings were cut 0.5 cm above the cotyledonary leaf and seedlings were kept in a moist chamber for 15 min. xylem sap collected on the cut surface under root pressure was drawn and pooled (Schurr & Schulze, 1995). Detection of AF in xylem sap was done by ciELISA. For each treatment there were 15 plants of two replicates and the experiment was repeated twice. 2.8. Phloem sap experiment Aphids, which are known to feed on GLVs (T. foenum-graecum and A. bicolor) were collected along with the branches of plants from vegetable growing fields. Immediately these aphids were brought to the laboratory, and removed from plants using a Camlin brush without damaging their stylets and maintained on selected GLVs under greenhouse conditions. The aphid technique was used to determine the presence of AF in phloem (Kawabe, Fukomorita, & Chino, 1980). The plants were grown under greenhouse conditions for 20 days as explained earlier with regular watering with water spiked with AFB1 (100 lg/ l). Aphids were released (approximately 25 aphids/plant) onto each plant and allowed to feed for 2 days. At the end of the feeding period, aphids from treated and control seedlings were collected separately. Aphids were pooled in an Eppendorf tube and crushed with water:chloroform (1:3); the chloroform layer was separated and evaporated to get the concentrate. Complete dried extract was dissolved in methanol:water (1:1) and used for ciELISA. For each plant group there were three replicates of two plants and the experiment was repeated twice.

2.11. Indirect competitive enzyme linked immunosorbent assay The total AF content was estimated from the soil samples and GLVs by the modified method of Reddy, Reddy, and Muralidharan (2009). AFB1-oxime and AFB1-OVA conjugate were prepared following the standard procedures (Kolosova, Shim, Yang, Eremin, & Chung, 2006). To perform ciELISA, the wells of microtitre plates (Maxi-sorp F96, Nalge Nunc International, Roskilde, Denmark) were coated overnight at 4 °C with 100 ll of AFB1-OVA conjugate in carbonate buffer (1 mg/ml), at pH 9.6, and then washed thrice with PBS containing 0.05% Tween 20 (PBST). AFB1 standards (Sigma) in 10% (v/v) methanol-PBS (50 ll), or different dilutions of samples (50 ll), were added to the wells. After 15 min incubation at 37 °C, 100 ll of anti-AFB1 antibody (Sigma) (1:10000) in PBS were added and incubated at 37 °C for 45 min. The plates were washed with PBST 4–5 times. Subsequently, 100 ll of secondary antibody conjugated with horseradish peroxidase (HRP) (1:10000) (Bangalore Genei) in PBS was added and incubated at 37 °C for 45 min. At the end of the incubation period the plates were washed 4–5 times with PBST. One hundred microlitres of substrate (TMB-H2O2) (Bangalore Genei) were added and incubated at room temperature for 10–15 min. The reaction was stopped by adding 100 ll of stop solution (2 M H2SO4) and the colour developed was read at 450 nm. Standard curves using absorbance (A) vs. logarithm of analyte concentration were plotted. AF in the samples were determined from the standard curve and expressed in ppb. 2.12. Method validation Validation of the ciELISA method was done by determination of recoveries of uncontaminated spiked GLVs (C. sativum and A. bicolor) at 50 and 100 ppb of AFB1. The repeatability and reproducibility were also ascerrtained at spiking levels as mentioned above. The repeatability was determined under repetitive conditions on the same day while reproducibility was estimated with time intervals (at 5 different days of the month).

2.9. Preparation of cooked green leafy vegetables 2.13. Statistical analysis GLVs grown under greenhouse conditions in coir pith with regular watering with AF spiked water (100 lg/l) as explained earlier were used. GLVs shoots (100 g) were rinsed thrice with distilled water, drained and blot dried. Washed GLVs were chopped into

All data collected from laboratory and greenhouse experiments were analysed separately for each experiment and were subjected to arcsine transformation and analysis of variance (ANOVA) (SPSS,

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version 16; SPSS Inc., Chicago, IL). Significant effects of treatments were determined by F-values (p 6 0.05). Treatment means were separated using Tukey’s HSD test.

Table 4 Greenhouse studies on uptake of AF in some selected Green leafy vegetables. Sample name

Coriandrum sativum Trigonella foenum-graecum Amaranthus bicolor Amaranthus sp. Brassica oleracea Spinacia oleracea Mentha arvensi Anethum graveolens Rumex vesicarius

3. Results 3.1. Aflatoxigenic fungi and aflatoxin in soil Table 1 shows the levels of natural AF in 33 soil samples collected from agricultural fields. Thin-layer chromatography analysis showed that only 31% of soil samples were contaminated and AFB1 was found to be predominant. But ciELISA results revealed 70% soil samples were contaminated with AF, the highest being 41.5 ppb recorded in samples collected from Srirangapatna. All the A. flavus strains isolated from 9 different agricultural fields were found to be aflatoxigenic.

Total aflatoxin (ppb) Root

Shoot

74.0 58.1 81.9 66.7 ND 98.8 ND 74.6 89.8

23.0 31.7 22.5 12.1 ND 23.6 ND 21.5 33.2

ND = not determined.

Table 5 Presence of aflatoxin in xylem and phloem sap of some green leafy vegetables. Sl. No.

Green leafy vegetables

1 2 3 4 5 6 7 8 9

Coriandrum sativum Trigonella foenum-graecum Amaranthus bicolor Amaranthus sp. Brassica oleracea Spinacia oleracea Mentha arvensis Anethum graveolens Rumex vesicarius

Aflatoxin

3.2. Aflatoxin in green leafy vegetables From 81 samples tested by TLC and ciELISA, 69.2% were found to be contaminated with AF. Highest totals of 88 ppb and 47.6 ppb AF were recorded in roots of A. bicolor and shoots of R. vesicarius, respectively. Roots of all tested GLVs showed higher levels of AF in comparison with shoots and AFB1 was the predominant AF (Table 3).

Xylem sap

Phloem sap

+ + + + ND + ND + ND

+ + + + ND ND ND ND ND

+ = positive for AFB1; ND = not determined.

3.3. Aflatoxin uptake studies under greenhouse conditions All tested leafy vegetables under greenhouse conditions took up AF when grown in coir pith spiked with AFB1. Among these, highest AFB1 of 98.8 ppb in S. oleraceae root and least AFB1 of 58.1 ppb in the roots of A. graveolens were recorded. In the case of shoots, R. vesicarius showed highest uptake of 33.2 ppb and Amaranthus sp. showed least uptake of 12.1 ppb of AFB1 (Table 4). Xylem and phloem sap experiment revealed the presence of AF in the saps of all tested plants (Table 5).

Cs

Tf

a

a

b

b

b

3.4. Effect of ordinary and pressure cooking of green leafy vegetables on aflatoxin B1 Of all the selected GLVs used, loss of AFB1 was significantly higher (p 6 0.05) when cooked under pressure at 121 °C for 20 min (15 lb/in2) than by ordinary cooking without pressure under similar conditions (Fig. 1). An average of 90.5% and 46.5% reduction of AF levels was recorded for pressure cooking and ordinary boiling, respectively.

a

a

Ab

a

a

b

b

b

b

A. sp.

So

a

Ag

Rv

Fig. 1. Reduction of AFB1 residues in GLVs before and after boiling and pressure cooking, as determined by ciELISA. Data were generated by considering AFB1 residue as 100% in controls. Cs: Coriandrum sativum, Tf: Trigonella foenum-graecum, Ab: Amaranthus bicolor, Asp: Amaranthus sp., Bo: Brassica oleracea, So: Spinacia oleracea, Ma: Mentha arvensis, Ag: Anethum graveolens, Rv: Rumex vesicarius.

Table 3 Natural occurrence of aflatoxin (ppb) in green leafy vegetables. Sl. No.

Place of collection

Cs

Tf

Ab

Asp.

Bo

So

Ma

Ag

Rv

Shoot

Root

Shoot

Root

Shoot

Root

Shoot

Root

Shoot

Root

Shoot

Root

Shoot

Root

Shoot

Root

Shoot

Root

1 2 3 4 5 6 7 8 9

Pandavapur Mysore T. Narasipura Mandya Nanjanagudu Bannur Srirangapatna Nagamangala H. D. Kote

3.1 0.3 0.8 0.4 1.9 1.4 0.5 – 0.9

21.5 21.0 16.5 3.5 15.0 16.5 12.1 – 4.1

1.1 5.9 8.0 2.0 4.7 1.4 – – 4.0

26.4 13.3 13.0 3.5 18.4 6.7 – – 10.8

– 20.2 11.2 – 1.0 3.0 0.4 11.0 –

– 88.0 29.0 – 8.0 4.2 11.3 31.2 –

– 0.8 – 0.2 0.6 – 9.6 0.4 0.6

– 22.0 – NA NA NA 38.3 3.5 4.9

– 10.1 – 0.5 26.0 – – 0.4 20.3

NA NA NA NA NA NA NA NA NA

– 4.1 15.3 – 1.0 0.9 11.2 2.5 –

– 27.0 NA – 6.0 3.4 35.6 7.3 –

1.6 2.3 22.0 – 30.9 – 11.0 3.6 –

3.25 NA NA NA NA NA NA NA NA

– 20.2 – 2.0 11.7 11.3 2.2 25.6 3.5

– NA – 3.9 35.2 22.3 3.3 41.2 –

– 14.3 – – 10.4 0.5 47.6 33.0 –

– NA NA NA NA NA NA NA NA

Cs: Coriandrum sativum, Tf: Trigonella foenum-graecum, Ab: Amaranthus bicolor, Asp: Amaranthus sp., Bo: Brassica oleracea, So: Spinacia oleracea, Ma: Mentha arvensis, Ag: Anethum graveolens, Rv: Rumex vesicarius, NA: Not available, – = aflatoxin not present.

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3.5. Recovery of aflatoxin B1 from green leafy vegetables The effectiveness of the extraction procedure adopted was tested by adding pure AFB1 to chopped GLVs followed by extraction and quantification as explained earlier. Recoveries from root and shoot samples of GLVs estimated by ciELISA were 87–91% and 76–78%, respectively. The detection limit of AFB1 was 0.01 ppb and quantification limit was 1000 ppb.

4. Discussion Many studies have been conducted to assess the occurrence and to describe the ecology of AF-producing fungi and AF in natural and agricultural environments. Soil and crop management practices and also a number of environmental factors influence the population size and the spatial distribution of A. flavus in cultivated soil (Abbas, Zablotowics, & Locke, 2004; Orum, Bigelow, Nelson, Howell, & Cotty, 1997). The present investigation revealed the occurrence of aflatoxigenic Aspergillus spp. in all the soil samples tested and 31% soil samples were found contaminated with AFs. Our observations are in accordance with that of Accinelli, Abbas, Zablotowicz, and Wilkinson (2008) who reported the presence of AFB1 in agricultural soil which ranged from 0.6 to 5.5 ng/g. Based on cultural methods they reported, soil contained from log10 3.1 to 4.5 A. flavus cfu/g with about 60% of isolates being aflatoxigenic. From the above studies it is clear that aflatoxigenic fungi in soil cannot only produce these AF but they may persist in soil up to the succeeding cropping season. As these toxins are very low in molecular weight, they can easily be taken up by the plants from contaminated soils and accumulate in roots and aerial plant parts. A similar observation was made by Mantle (2000), where ochratoxin A in some green coffees comes directly from fungal activity in soil rather than from fungal infection of cherries or processed green coffee. Our study showed that 69.2% of GLVs were naturally contaminated with AF, with AF in roots higher than in shoots. Among the four AF (AFB1, AFB2, AFG1 and AFG2) studied, AFB1 was frequently recorded in most of the GLVs analysed followed by AFB2 (data not shown). Uptake of AFB1 by these plants under natural conditions is supported by our greenhouse studies. The experiment confirmed that the absorption of AF is through root and is translocated to the aerial plant parts through conducting tissue. The concentration of AFB1 was found higher in roots in comparison with shoots. It may be due to the higher extraction efficiency in case of root when compared to shoot, and also root has direct contact with AF in soil or growth medium. Published reports show that not much work apparently has been conducted to examine the possibility of plants taking up AFs from contaminated soils. Early researchers reported lettuce seedlings taking up AFBl from soil and transferring the same to above-ground portions of plant (Mertz, Edward, Lee, & Zuber, 1981; Mertz et al., 1980). In addition, fumonisin was taken up by maize seedlings and concluded that fungus–plant interaction is necessary for FB1 translocation in maize seedlings (Zitomer et al., 2010). The mycotoxins citrinin, patulin and terreic acid were absorbed by rice seedling roots and translocated to shoots. 10-day analysis of toxin-treated rice plants showed uptake and persistence of citrinin, patulin and terreic acid (Rao, Govindaraju, Sivasithamparam, & Shanmugasundaram, 1982), and this indicates the potential for uptake from soils. This is the first report on the uptake of AF by GLVs which are major dietary constituents. The AF uptake was further supported by the analysis of sap from xylem and phloem which revealed the presence of AFB1. This result conclusively proves that initially, AFB1 was absorbed by roots, and then translocated to shoot via

water-conducting xylem tissue. In addition, AFB1 from xylem enters into food-conducting phloem tissue. Aflatoxins are relatively stable under dry conditions, but in the presence of moisture, variable losses can occur (Scott, 1991). Cooking loss of AFB1 has been reported to be 6–88% depending on rice-to-water ratio used and whether the rice was cooked under pressure (Rehana, Basappa, & Murthy, 1979). Park and Kim (2006) reported AF loss of 78–88% after pressure cooking contaminated rice. They concluded that pressure cooking significantly reduces AF levels, as compared to simple boiling. These findings support our results where AFB1 content in selected GLVs significantly reduced after pressure cooking compared to ordinary boiling. Even though natural occurrence of AF in leafy vegetables is clear from the present study, its low level of persistence after processing the food may lower the exposure limit to human beings. Chemoprevention of carcinogenic effect of AF by chlorophyll was investigated in rat multi-organ carcinogenesis model by Simonich et al. (2007). According to their observations chlorophyll and chlorphyllin provide potent chemoprotection against early biochemical and late pathophysiological biomarkers of AFB1 carcinogenesis. Faecal elimination and urinary metabolite studies provide supporting evidence that both agents protect by inhibiting carcinogen uptake from the gut, thus reducing the availability of AFB1 to the target organ. Their results support the fact that irrespective of source of AF contamination, consumption of GLVs may substantially lower carcinogenic effect of AF on animals and livestock. Even though the above findings suggest the effect of purified chlorophyll on reduced effect of AF-induced carcinogenesis, the maximum level of GLVs contaminated with AF that can be consumed in the regular diet has yet to be standardised. Therefore more studies are needed to relate soil content and uptake of AF by GLVs and the processing methods practiced in various Indian traditional foods prepared using GLVs on AF residual level. Acknowledgements We thank Sharnya, A., Niya poulose, Poorna Chandra Rao, K., and Krithika, R.J., for their valuable help in conducting this work. The authors are thankful to Director, CFTRI, for providing necessary facilities to carry out this study and Department of Biotechnology, India for financial support. References Abbas, H. K., Zablotowics, R. M., & Locke, M. A. (2004). Spatial variability of Aspergillus flavus soil populations under different crops and corn grain colonization and aflatoxins. Botany, 82, 1768–1775. Accinelli, C., Abbas, H. K., Zablotowicz, R. M., & Wilkinson, J. R. (2008). Aspergillus flavus aflatoxin occurrence and expression of aflatoxin biosynthesis genes in soil. Canadian Journal of Microbiology, 54, 371–379. Ahmad, M. S., & Sinha, K. K. (2002). Influence of aflatoxin B1 on seed germination, seedling growth, chlorophyll and carotenoid contents of mustard (Brassica juncea L.var.pusa bold) seeds. Mycotoxin Research, 18, 2–6. Bayman, P., & Cotty, P. J. (1993). Genetic diversity in Aspergillus flavus: Association with aflatoxin production and morphology. Canadian Journal of Botany, 71, 23–31. Chatterjee, D. (1988). Inhibitory effects of aflatoxin Bl on amylase of maize seed. Letters in Applied Microbiology, 7, 9–11. Crisan, E. V. (1973). Effects of aflatoxin on germination and growth of lettuce. Applied Microbiology, 25, 342–345. El-Naghy, M. A., Fadl-Allah, E. M., & Samhan, M. (1999). Effect of aflatoxin G1 on germination growth and metabolic activities of some crop plants. Cytobios, 97, 87–93. IARC (2002). Monographs on the evaluation of carcinogenic risks to humans world health organization. International agency for research on cancer. Some traditional herbal medicines, some mycotoxins, naphthalene and styrene (Vol. 82). France: IARC Press Lyon. Imungi, J. K. (2002). The brighter side of phenoliccompounds abundance in African leafy vegetables. Nairobi, IPGRI Newsletter for sub-Saharan Africa. Issue No. 17. Jofee, A. Z. (1969). The mycoflora of groundnut rhizosphere, soil and geocarposphere on light, medium and heavy soils and its relations to Aspergillus flavus. Mycopathologia, 37, 113–192.

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