Detoxification of aflatoxin B1 by an aqueous extract

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ARTICLE IN PRESS Microbiological Research xxx (2013) xxx–xxx

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Detoxification of aflatoxin B1 by an aqueous extract from leaves of Adhatoda vasica Nees S. Vijayanandraj a , R. Brinda b , K. Kannan a , R. Adhithya a , S. Vinothini a , K. Senthil c , Ramakoteswara Rao. Chinta d , V. Paranidharan a , R. Velazhahan a,∗ a

Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore 641 003, Tamil Nadu, India Department of Food and Agricultural Processing Engineering, Agricultural Engineering College and Research Institute, Tamil Nadu Agricultural University, Coimbatore 641003, Tamil Nadu, India c Department of Agricultural Entomology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore 641 003, Tamil Nadu, India d Jawaharlal Nehru Technological University Hyderabad, Kukatpally, Hyderabad 500085, Andhra Pradesh, India b

a r t i c l e

i n f o

Article history: Received 11 April 2013 Received in revised form 28 June 2013 Accepted 1 July 2013 Available online xxx Keywords: Adhatoda vasica Aflatoxin B1 Detoxification Medicinal plant

a b s t r a c t The effectiveness of aqueous extracts of various medicinal plants in detoxification of aflatoxin B1 (AFB1) was tested in vitro by thin-layer chromatography and enzyme-linked immunosorbent assay (ELISA). Among the different plant extracts, the leaf extract of Vasaka (Adhatoda vasica Nees) showed the maximum degradation of AFB1 (≥98%) after incubation for 24 h at 37 ◦ C. The aflatoxin detoxifying activity of the A. vasica leaf extract was significantly reduced by heating to 100 ◦ C for 10 min or autoclaving at 121 ◦ C for 20 min. Dialysis had no effect on aflatoxin detoxifying ability of A. vasica extract and the dialyzed extract showed similar level of detoxification of AFB1 as that of the untreated extract. A time course study of aflatoxin detoxification by A. vasica extract showed that 69% of the toxin was degraded within 6 h and ≥95% degradation was observed after 24 h of incubation. Detoxification of AFB1 by A. vasica extract was further confirmed by liquid chromatography–mass spectrometry (LC–MS) analysis. Phytochemical analysis revealed the presence of alkaloids in methanolic extract of A. vasica leaves. A partially purified alkaloid from A. vasica leaves by preparative TLC exhibited strong AFB1 detoxification activity. © 2013 Published by Elsevier GmbH.

1. Introduction Aflatoxins are a group of carcinogenic secondary metabolites produced by certain strains of Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius (Diener et al. 1987; Kurtzman et al. 1987). These fungi are common contaminants of groundnut, maize, cottonseed and tree nuts. Aflatoxin B1 (AFB1) and three structurally similar compounds (AFB2, AFG1 and AFG2) have been detected as contaminants of crops in the field and during storage, transportation, and processing. Among the 18 different types of aflatoxins identified so far, AFB1 is considered the most toxic (Ong 1975). The International Agency for Research on Cancer (IARC), classified aflatoxins as class I human carcinogens (IARC 2002). Human beings are exposed to aflatoxins directly from consumption of contaminated food or, indirectly, from food stuffs originating from animals previously exposed to aflatoxins in feeds. Since aflatoxins are acutely toxic, carcinogenic, mutagenic, teratogenic and immunosuppressive to most mammalian species, their presence in food commodities greatly impacts the food and feed industries

∗ Corresponding author. Tel.: +91 422 6611226; fax: +91 422 6611437. E-mail address: [email protected] (R. Velazhahan).

(Williams et al. 2004). The aflatoxins are extremely durable under most conditions of storage, handling and processing of foods or feeds. Hence preventing the contamination of food by A. flavus and A. parasiticus, is the most rational and economic approach to avoid potential health hazards. However, prevention is not always possible under certain agronomic and storage practices. In that context, detoxification of aflatoxin is another option for commodities already contaminated with aflatoxin. Various physical, chemical and biological methods have been described for detoxification of aflatoxins in foods and feeds (Ciegler et al. 1966; Cole and Kirksey 1971; Mann and Rehm 1976; Basappa 1983; Harvey et al. 1989; Piva et al. 1995; Tejada-Castaneda et al. 2008; Oluwafemi et al. 2010). However, all these methods have their own shortcomings. Physical methods like pressure cooking and roasting are employed for detoxification of aflatoxins from food commodities, but certain nutrients are destroyed in this process (Yazdanpanah et al. 2005; Park and Kim 2006). A few chemical methods like ammoniation, treatment with formaldehyde and calcium hydroxide, and sodium bisulfite have been found to be effective in detoxification of aflatoxins (Piva et al. 1995). However, their use in food industry is restricted because of food safety issues that may arise due to chemical residues. Several researchers have demonstrated the biological detoxification of aflatoxins by employing microorganisms (Ciegler

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Please cite this article in press as: Vijayanandraj S, et al. Detoxification of aflatoxin B1 by an aqueous extract from leaves of Adhatoda vasica Nees. Microbiol Res (2013), http://dx.doi.org/10.1016/j.micres.2013.07.008


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Table 1 Detoxification of AFB1 by aqueous extracts from various medicinal plants. Medicinal plants

Family

AFB1 recovered (ng)a

% reduction over control

A. moschatus Medik A. precatorius (L.) A. vasica Nees A. paronychioides A.St.-Hil Aloe vera (L.) Burm A. paniculata (Burm. f.) Wall. ex Nees Artemesia nilagirica (Clark) Pamp B. monnieri (L.) Pennell C. sappan (L.) C. gigantea (L.) W.T.Aiton Clerodendrum inerme (L.) Gaertn Catharanthus roseus (L.) G. Don Cassia fistula (L.) Cassia occidentalis (L.) Cassia angustifolia Vahl Coleus aromaticus Bentham Coleus forskohlii Briq Cuminum cyminum (L.) Curcuma longa (L.) Eclipta alba (L.) Hassk Lippia nodiflora (L.) Michx Murraya koenigii (L.) Spreng Ocimum basilicum (L.) Ocimum sanctum (L.) Rhinacanthus nasuta (L.) Kurz Syzygium aromaticum (L.) Merrill & Perry T. ammi (L.) Sprague ex Turrill Tinospora cardifolia (Wild) Miers Trigonella foenum-graecum (L.) Wedelia chinensis (Osbeck) Merr Withania somnifera (L.) Dunal

Malvaceae Fabaceae Acanthaceae Amaranthaceae Liliaceae Acanthaceae Asteraceae Scrophulariaceae Caesalpiniaceae Asclepiadaceae Verbenaceae Apocynaceae Caesalpiniaceae Caesalpiniaceae Caesalpiniaceae Laminaceae Laminaceae Apiaceae Zingiberaceae Compositae Verbenaceae Rutaceae Lamiaceae Lamiaceae Acanthaceae Myrtaceae Apiaceae Meninspermeaceae Fabaceae Asteraceae Solanaceae

97.5 58.4 1.2 63.6 49.7 13.9 66.4 46.1 51.3 50.4 58.4 53.6 63.6 46.8 55.4 50.2 60.9 11.0 44.2 43.5 55.9 54.7 66.4 50.2 59.4 63.6 39.0 43.3 41.9 62.2 87.6

2.5 41.6 98.3 36.4 50.3 86.1 33.6 53.9 48.7 49.6 41.6 46.4 36.4 53.2 44.6 49.8 39.1 89.0 55.8 56.5 44.1 45.3 33.6 49.8 40.6 36.4 61.0 56.7 58.1 37.3 12.4

AFB1 (100 ng) was mixed with 500 ␮l of aqueous extract from medicinal plants and incubated at 37 ◦ C. After 24 h the AFB1 was recovered from the mixture and analyzed by ELISA. a Mean of three replications.

et al. 1966; Cole and Kirksey 1971; El-Nezami et al. 1998; Shantha 1999). The major drawbacks in using microorganisms are their utilization of nutrients from foods for their own growth and multiplication and release of undesirable compounds. One of the most practical approaches is the mixing of non-nutritive adsorbents with aflatoxin-contaminated feeds, which bind the toxins and inhibit their absorption from the gastrointestinal tract, thus minimizing the toxic effects to livestock and the carryover of these fungal metabolites into animal products (Ramos and Hernandez 1997). Several adsorbents have been shown to impair nutrient utilization by poultry. Hence, there is a need to identify biologically safe and cost-effective aflatoxin detoxifying compounds for use in food and feed industries. Natural phytochemicals may be an alternative to synthetic chemicals for detoxification of aflatoxins. Several medicinal plants that are known to combat microbial infections have been reported to have aflatoxin detoxification potential (El-Mofty et al. 1994; Gyamfi and Aniya 1998; De Boer et al. 2005; Farombi et al. 2005; Hajare et al. 2005; Sandosskumar et al. 2007; Velazhahan et al. 2010). Recently we demonstrated that pre-feeding of Wistar rats with a spray-dried formulation of Adhatoda vasica (L.) Nees leaf extract (500 mg kg−1 body weight consecutively for 7 days) counteracted the hepatic dysfunction induced by subsequent treatment with AFB1 (Brinda et al. 2013). This paper reports the potential of aqueous extracts from A. vasica (Nees) leaves in the detoxification of aflatoxin B1.

2. Materials and methods 2.1. Plant materials The medicinal plants viz., Abelmoschus moschatus Medik, Abrus precatorius (L.) A. vasica Nees, Alternanthera paronychioides

A.St.-Hil, Aloe vera (L.) Burm, Andrographis paniculata (Burm. f.) Wall. ex Nees, Artemisia nilagirica (Clark) Pamp, Bacopa monnieri (L.) Pennell, Caesalpinia sappan (L.), Calotropis gigantea (L.) W.T.Aiton, Clerodendrum inerme (L.) Gaertn, Catharanthus roseus (L.) G. Don, Cassia fistula (L.), Cassia occidentalis (L.), Cassia angustifolia Vahl, Coleus aromaticus Bentham, Coleus forskohlii Briq, Cuminum cyminum (L.), Curcuma longa (L.), Eclipta alba (L.) Hassk, Lippia nodiflora (L.) Michx, Murraya koenigii (L.) Spreng, Ocimum basilicum (L.), Ocimum sanctum (L.), Rhinacanthus nasuta (L.) Kurz, Syzygium aromaticum (L.) Merrill and Perry, Trachyspermum ammi (L.) Sprague ex Turrill, Tinospora cardifolia (Wild) Miers, Trigonella foenum-graecum (L.), Wedelia chinensis (Osbeck) Merr and Withania somnifera (L.) Dunal were collected from the Department of Medicinal Plants, Horticulture College and Research Institute, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. 2.2. Preparation of plant extracts Aqueous extracts of medicinal plants were prepared by homogenizing one gram of leaves/seeds with 3 ml of sterile distilled water and the homogenate was centrifuged at 14,000 × g for 15 min and the supernatant was used for further studies. 2.3. Test for detoxification of aflatoxins by plant extracts Five hundred microliters of plant extract was mixed with 100 ng of aflatoxin B1 (Sigma Chemical Co., St. Louis, USA) in a micro centrifuge tube and incubated at 37 ◦ C for 24 h in an incubator. After incubation, the aflatoxin in the mixture was extracted with 500 ␮l of chloroform. The chloroform fraction was evaporated on a heat block at 60 ◦ C and the residue was dissolved in 10 ␮l of methanol and analyzed by thin layer chromatography (TLC)/enzyme-linked




Table 2 Detoxification of AFB1 by boiled, autoclaved and dialyzed extract of A. vasica as assessed by ELISA. Treatment


% degradation

Untreated extract Autoclaving (121 ◦ C for 20 min) Boiling (100 ◦ C for 10 min) Dialysis (dialyzed with a membrane having molecular cut off 12-14 kDa)

4.41 a 58.82 c 47.05 b 5.88 a

95.59 41.18 52.95 94.12

AFB1 (100 ng) was mixed with 500 ␮l of A. vasica extract and incubated at 37 ◦ C. After 24 h the AFB1 was recovered from the mixture by extraction with chloroform and analyzed by ELISA. Means within the column followed by same letter are not significantly different (P = 0.05) by Duncan’s multiple range test. a Mean of five replications.

Fig. 1. Effect of heat treatment on AFB1 detoxification ability of A. vasica leaf extract as assessed by TLC. Aqueous extract of A. vasica was subjected to boiling at 100 ◦ C for 10 min or autoclaving at 121 ◦ C for 20 min. The treated extract (500 ␮l) was incubated with AFB1 (100 ng) at 37 ◦ C for 24 h. Following incubation, aflatoxin in the solution was extracted with chloroform and analyzed by TLC. Lane 1, AFB1 (100 ng); Lane 2, boiled extract; Lane 3, autoclaved extract; Lane 4, untreated extract.

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Table 3 Time course of AFB1 detoxification by A. vasica leaf extract as assessed by ELISA. Time intervals (h)


% degradation

6 12 24 48

30.74 c 18.42 b 4.50 a 4.36 a

69.26 81.58 95.50 95.64

AFB1 (100 ng) was mixed with 500 ␮l of A. vasica extract and incubated at 37 ◦ C. At different time intervals the AFB1 in the mixture was recovered by extraction with chloroform and analyzed by ELISA. Means within the column followed by same letter are not significantly different (P = 0.05) by Duncan’s multiple range test. a Mean of five replications.

Fig. 2. Time course of AFB1 detoxification by A. vasica leaf extract as assessed by TLC. AFB1 (100 ng) was mixed with 500 ␮l of A. vasica extract and incubated at 37 ◦ C. At different time intervals the AFB1 in the mixture was recovered by extraction with chloroform and analyzed by TLC. Lane 1, AFB1 (100 ng); Lane 2, 3 h; Lane 3, 6 h; Lane 4, 12 h; Lane 5, 24 h.

Fig. 3. Chromatogram of AFB1 standard and AFB1 after treatment with A. vasica extract. AFB1 (50 ng or 100 ng) was mixed with 500 ␮l of A. vasica extract and incubated at 37 ◦ C for 24 h. Following incubation, the AFB1 in the mixture was recovered by extraction with chloroform and analyzed by LC–MS.


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Fig. 4. Mass spectrum showing detoxification of AFB1 by A. vasica extract.

immunosorbent assay (ELISA). Ten microliters of the chloroform extract was spotted on 0.25 mm silica gel G TLC plate (Merck) and developed in chloroform:acetone:water (88:12:1; v:v:v). The chromatogram was viewed under UV light (365 nm). For quantitative estimation of AFB1, RIDA SCREEN FAST Aflatoxin Test kit (R-Biopharm AG, Darmstadt, Germany) was used according to the manufacturer’s manual. Since the highest aflatoxin detoxification activity was detected in A. vasica leaf extract, further detailed studies were performed with A. vasica leaf extract only.

2.4. Effect of dialysis and heat on aflatoxin detoxification properties of A. vasica leaf extract An aqueous extract of A. vasica was prepared by grinding 10 g of leaves with 30 ml of sterile water. The extract was centrifuged at 14,000 × g for 20 min and the supernatant was dialyzed against distilled water using dialysis membrane having molecular weight cut off 12,000–14,000 Da (Spectra/Por, Spectrum Laboratories, CA,

USA). The aflatoxin detoxification activity of the dialyzed extract was tested by ELISA as described earlier. In order to assess the effect of heat on aflatoxin detoxification properties of A. vasica extract, 1 ml of the aqueous extract of A. vasica in a 1.5 ml micro centrifuge tube was subjected to boiling at 100 ◦ C for 10 min or autoclaving (121 ◦ C for 20 min), cooled to room temperature and then assayed for aflatoxin B1 detoxification activity and quantitatively estimated by ELISA as described earlier.

2.5. Effect of incubation time on detoxification of AFB1 by A. vasica leaf extract To study the time course of aflatoxin detoxification, 500 ␮l of A. vasica leaf extract was mixed with 100 ng of aflatoxin B1 and incubated at 37 ◦ C for 3, 6, 12, 24 and 48 h. The aflatoxin content in the reaction mixture at the end of the reaction was determined by TLC method and quantitatively estimated by ELISA as described earlier.




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Fig. 5. TLC of methanolic extract of A. vasica after spraying with Dragendorff reagent.

2.6. Testing aflatoxin detoxification by mass spectrometric analysis To confirm detoxification of aflatoxin B1 by A. vasica extract, 500 ␮l of aqueous extract of A. vasica was mixed with 50 ng or 100 ng of AFB1 and incubated at 37 ◦ C for 24 h. After incubation, the aflatoxin in the mixture was extracted with an equal volume of chloroform. The chloroform fraction was then evaporated on a heat block at 60 ◦ C and the residue was dissolved in 10 ␮l of methanol and analyzed by liquid chromatography tandem mass spectrometry (LC–MS) using Agilent 1200 HPLC and Q-TOF 6500 Series. LC analysis was carried out using 4.6 mm × 150 mm Waters C18 5 ␮m column and the mobile phase employed was acetonitrile:water (50:50, v/v) at a flow rate of 0.5 ml/min. AFB1 was detected using an electrospray ionization interface (ESI) and tandem MS in the positive ion mode. 2.7. Partial purification of aflatoxin detoxifying principle from A. vasica leaves solvent partitioning of crude extract A. vasica leaves were homogenized using different solvents viz., methanol, hexane, ethyl acetate, petroleum ether and chloroform at 1:3 ratio. The leaf homogenates were filtered through two layers of muslin cloth and the filtrates were centrifuged at 12,000 × g for 10 min and the clear supernatants were removed and analyzed by TLC for phenols, terpenoids and alkaloids as described by Sadasivam and Manickam (1992). 2.8. Preparative TLC A. vasica leaves (100 g) were homogenized in 300 ml of methanol using a blender. The leaf homogenate was filtered through two layers of muslin cloth and the filtrate was centrifuged at 12,000 × g for 10 min. The supernatant was evaporated

Fig. 6. Detoxification of AFB1 by alkaloid (Rf = 0.6) purified from the leaf extract of A. vasica by preparative TLC. Aflatoxin (100 ng) was mixed with 200 ␮l of A. vasica alkaloid and incubated at 37 ◦ C. After 24 h the aflatoxin in the mixture was recovered by extraction with chloroform and analyzed by TLC. Lane 1, AFB1 treated with alkaloid; Lane 2, AFB1 (100 ng).

to dryness in a flash evaporator and re-dissolved in 1 ml of methanol. Preparative TLC was used to purify the alkaloids from leaf extract of A. vasica. Aliquots (10 ␮l) of methanolic extract of A. vasica were spotted on a horizontal line on preparative TLC (20 cm × 20 cm, 3 mm thick silica gel, E. Merck, USA) plates. Plates were developed with a solvent system of methanol:chloroform (1:9). A portion of the TLC plate was sprayed with Dragendorff’s reagent using an atomizer and the retention factor (Rf ) of the orange colored spot developed on the TLC plate was recorded. The silica gel at Rf value 0.6 from the other non-sprayed portion of the TLC plate was gently scraped off and re-suspended in 1 ml of methanol in 1.5 ml micro centrifuge tubes, vortexed and centrifuged at 5000 × g for 5 min. The supernatant was collected and tested for its ability to detoxify aflatoxins by TLC method as described earlier.




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3. Results and discussion Detoxification of aflatoxin by plant products have been reported by several workers (Hajare et al. 2005; Sapcota et al. 2005; Sandosskumar et al. 2007; Velazhahan et al. 2010). Furthermore, several plant compounds have been shown to have ameliorative effects against AFB1 -induced toxicity in animals (Liu et al. 1995; Gyamfi and Aniya 1998; Oluwafemi and Taiwo 2004; Farombi et al. 2005; Choi et al. 2011). In the present study, aqueous extracts obtained from leaves or seeds of 31 medicinal plants were evaluated for their ability to detoxify the AFB1 . Of the various plant extracts, a leaf extract of A. vasica showed the maximum degradation of AFB1 after incubation for 24 h at 37 ◦ C. Quantification of AFB1 in the mixture revealed a reduction of over 98% of AFB1 suggesting that the aqueous extract of A. vasica degraded the toxin (Table 1). The effect of heat treatment and dialysis on aflatoxin detoxification potential of A. vasica leaf extract was tested. The aflatoxin detoxifying activity of the A. vasica leaf extract was significantly decreased upon boiling at 100 ◦ C for 10 min or autoclaving at 121 ◦ C for 20 min (Fig. 1). The boiled and dialyzed A. vasica extract recorded 52.9% and 41.1% degradation of AFB1, respectively (Table 2). It was found that dialysis has no effect on aflatoxin detoxifying ability of A. vasica extract as the dialyzed extract showed a level of detoxification of AFB1 similar to that of the untreated extract. A time course study of aflatoxin detoxification by A. vasica extract showed that 69% degradation had occurred within 6 h and more than 80% degradation occurred by 12 h after incubation (Fig. 2 and Table 3). By 24 h after incubation 95% of AFB1 was degraded. These results suggest the existence of high molecular weight (non-dialyzable), water soluble and thermolabile aflatoxin detoxification principle(s) in A. vasica extract. To confirm detoxification of aflatoxin B1 by A. vasica extract, the reaction mixture after incubation of AFB1 for 24 h with A. vasica extract was analyzed using liquid chromatography tandem mass spectrometry (LC–MS) in the positive ion mode. Fig. 3 shows the total ion chromatogram of AFB1. In the AFB1 mass spectrum, the most abundant fragment eluting at 6.37 min in positive ionization mode was the protonated molecule [M+H]+ at m/z 313 specific to AFB1 (Ventura et al. 2006) (Fig. 4). When AFB1 was incubated with A. vasica extract, the molecular base ion at m/z 313 specific for AFB1 had disappeared confirming almost complete degradation of AFB1 by A. vasica extract. Investigations on the aflatoxin detoxification using microorganisms, physical and chemical agents revealed structural alterations in aflatoxin molecules after detoxification (Ciegler et al. 1966; Lee et al. 1974; Cucullu et al. 1976). Ciegler et al. (1966) observed formation of a new fluorescing compound with concomitant disappearance of aflatoxin B1, when aflatoxin B1 was added to acid-producing fungal cultures. The new compound was further identified as hydroxydihydro-aflatoxin B1 (Ciegler and Peterson 1968). Cucullu et al. (1976) identified the ammoniation product of aflatoxin B1 as dihydro-4-hydroxy-6methoxyfuro [2,3-b] benzofuran, a non-fluorescent phenol similar to aflatoxin D1 that lacks the cyclopentenone ring with a molecular weight of 206. Experiments by Lee et al. (1974) revealed that the major product formed from aflatoxin B1 after treatment with ammonium hydroxide had a molecular weight of 286 and exhibited ultraviolet absorption, Ymax MeOH 227, 324 nm, non-fluorescent and lacked the lactone group characteristics of aflatoxin B1. The new product C16 H14 O5 arises from opening of the lactone ring of AFB1 during ammoniation, formation of the ammonium salt of the resultant hydroxyl acid and loss of carbon-dioxide from this ␤-keto acid. Velazhahan et al. (2010) reported detoxification of aflatoxin G1 by seed extract of Ajowan (T. ammi) and suggested the modification of lactone ring structure of AFG1 as the mechanism of detoxification. The disappearance of AFB1 in response to the treatment with A. vasica extract in the present study indicates detoxification of AFB1.

The leaf extract of A. vasica was analyzed for phenolics, terpenoids and alkaoids by TLC. Phenolics or terpenoids could not be detected in the leaf extract of A. vasica (data not shown). However, TLC analysis of the leaf extract of A. vasica for alkaloids using Dragendorff’s reagent showed the development of an orange colored spot at Rf = 0.6 (Fig. 5). The compounds eluted at Rf = 0.6 by preparative TLC exhibited strong AFB1 detoxification activity (Fig. 6) suggesting that the aflatoxin detoxification principle in A. vasica extract may be alkaloid(s). A. vasica commonly known as Vasaka is an indigenous medicinal plant and is commonly found throughout Tamil Nadu, India. It is frequently used as an ingredient in Ayurvedic medicine to treat cough, asthma and bronchitis (Claeson et al. 2000; Srivastava et al. 2001). The plant is a rich source of the quinazoline alkaloids, vasicine, vasicinone, deoxyvasicinone, vasicol, adhavasicinone and some minor compounds in the same series (Claeson et al. 2000). Both vasicine and vasicinone, formed by oxidation of vasicine at C-8 position, are the two major alkaloids of A. vasica (Srivastava et al. 2001) are known to possess interesting biological activities including respiratory stimulant, bronchodilator and hypersensitive activities. Anti-feedant and toxic activity of A. vasica leaf extract against Spodoptera littoralis (Sadek 2003) and larvicidal activities of methanolic fractions from A. vasica leaf extract against the bancroftian filariasis vector Culex quinquefasciatus and dengue vector Aedes aegypti (Thanigaivel et al. 2012) have been reported. Khodjaniyazov et al. (2012) reported that Anabasine, an alkaloid isolated from the plant Anabasis aphylla exhibited detoxification of pesticide DDT. However, aflatoxin detoxifying ability of A. vasica has not been reported earlier. In conclusion, our findings suggest that aqueous extract of A. vasica leaves is capable of detoxifying AFB1. The active component in A. vasica is water soluble, heat sensitive and high molecular weight (non-dialyzable). Alkaloids have been detected in the leaf extract of A. vasica and partially purified alkaloids from the leaves of A. vasica had aflatoxin detoxifying ability suggesting that the aflatoxin detoxification principle may be alkaloids. To the best of our knowledge, this is the first report of detoxification of aflatoxin by A. vasica. Further studies need to be undertaken for structural elucidation of the aflatoxin detoxifying principle in A. vasica and to determine the structure of degradation products of AFB1.

Acknowledgements We thank Prof. S. Muthukrishnan, Department of Biochemistry, Kansas State University, Manhattan, Kansas, USA for critical review of this manuscript. This work was supported in part by the Department of Science and Technology, Government of India.

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