Inhibition of growth and aflatoxin biosynthesis of Aspergillus flavus by extracts of some Egyptian plants
A.-L.E. Mahmoud, Botany Department, Faculty of Science, Assiut University, Assiut 71516, Egypt.
The effect of five different concentrations (2, 4, 6, 8 and 10 mg ml−1) of an aqueous extracts of Lupinus albus, Ammi visnaga and Xanthium pungens were tested on growth and aflatoxin production by Aspergillus flavus in a chemically defined medium. All the plants inhibited mycelial growth and aflatoxin formation. The inhibitory effect was proportional with the applied concentration. Growth and aflatoxin production appeared to be correlated processes. The nature of the plant extract also affected the ratio of B1 to B2, and there was no correlation between the inhibition of aflatoxins or growth of the fungus and the resultant B1: B2 ratio.
Aflatoxins are a group of hepatocarinogenic secondary metabolites produced by Aspergillus flavus Link ex. Fries and A. parasiticus Spear ( Bennet & Christensen 1983). These have been reported to contaminate a variety of foods and feeds ( Bilgrami 1984; Mahmoud 1993).
Plant-produced compounds are of interest as a source of safer or more effective substitutes for synthetically produced antimicrobial agents. Some of the natural products, such as cinnamon and clove oil ( Bullerman et al. 1977 ), phenols ( Singh 1983), some spices ( Sahay & Ranjan 1990; Hasan & Mahmoud 1993) and many essential oils ( Mahmoud 1994), have been reported as effective inhibitors of fungal growth and aflatoxin production.
The extracts of several wild and medicinal plants have also been tested against aflatoxin-producing fungi ( Bilgrami et al. 1980 ). Although hundreds of plant species have been tested for antimicrobial properties ( Sharma et al. 1979 ; Ross et al. 1980 ; Masood & Ranjan 1991; Heisey & Gorham 1992; Prasad et al. 1994 ; Arora & Ohlan 1997), the vast majority have not yet been adequately evaluated ( Balandrin et al. 1985 ).
The present study was initiated with 15 plant species collected from the Aswan mountains and Botanical Garden of Aswan, Upper Egypt. After preliminary screening, 3 plants were finally selected for a detailed study and the fungitoxic role of their aqueous extracts against aflatoxigenic strain of A. flavus is discussed.
MATERIALS and METHODS
Plant extract preparation
Three plants, viz. Ammi visnaga (Umbelliferae), Lupinus albus (Leguminosae), and Xanthium pungens (Compositae) were collected from the Aswan mountains and Botanical Garden of Aswan, Upper Egypt. Leaves of each plant were thoroughly washed with 2% aqueous sodium hypochloride solution and sterile distilled water. An aqueous extract was prepared by blending 20 g of plant part in 100 ml sterile distilled water. The filtrate was used as the test extract.
Mould and media
A toxigenic strain of A. flavus isolated from poultry feeds ( Mahmoud 1993) was used in this study. This mould was maintained on slopes of Czapek Dox agar. Growth and aflatoxin production were studied in the defined medium of Reddy et al. (1971) . The medium, 50 ml in each 250 ml Erlenmeyer flask, was sterilized by autoclaving at 121 °C for 15 min. The aqueous extracts were prepared with different strengths of 2, 4, 6, 8 and 10 mg ml−1. 2 ml extracts of each strength were added separately to each flask and were replaced by 2 ml of sterile distilled water in case of control. Each flask was inoculated with 0·5 ml spore suspension (Ca 106 spores. ml−1), prepared from 7 d old culture of the toxigenic strain and incubated as stationary cultures at 28 + 1 °C. Three replicates were prepared for each treatment.
Growth and aflatoxin production
After 8 d of incubation, cultures were filtered, washed with distilled water, and weighed after drying to constant weight at 70 °C. The filtrate and washings were extracted with chloroflorm (50 ml), the extract reduced to dryness on a rotary evaporator and redissolved in 2 ml of chloroform. Aflatoxins were analysed by TLC on Silica Gel 60-coated plates with chloroform-acetone (9:1) as the developing solvent. The spots of aflatoxins B1 and B2 were removed from the plates, eluted with methanol and estimated spectrophotometrically according to A.O.A.C. (1984).
L.S.D at 5 and 1% of all data were calculated by (PC-state) computer program.
RESULTS and DISCUSSION
Data presented in Table 1 clearly show that both growth and aflatoxin biosynthesis by A. flavus were suppressed by aqueous extracts of the tested plants. The inhibitory effect of these extracts was proportional with their concentrations.
Effect of some plant extracts on growth and aflatoxin production by Aspergillus flavus
Among the plants tested, Lupinus albus appeared to be the most effective. The application of its extract at concentration of 10 mg ml−1 reduced both fungal growth and aflatoxin production by 45·3 and 60%, respectively.
No or slightly effect on fungal growth and aflatoxin production was observed in treatment with low concentration of Ammi visnaga; whereas high concentrations of its extract inhibited fungal growth and, consequently, aflatoxin formation. Maximum inhibitory effect was recorded at 10 mg ml−1 concentration of extract ( Table 1).
The extract of Xanthium pungens was comparatively less effective against growth and aflatoxin production of A. flavus. Although extract of this plant at concentration 2 mg ml−1 slightly enhanced fungal growth (3·5%), high concentration decreased both mycelial growth and toxin yield. Maximum inhibitory effect against growth and aflatoxin formation was recorded as 26·7 and 32·7%, respectively ( Table 1). Reduction of both fungal growth and aflatoxin production by treatment with Lupinus albus, Ammi visnaga, and Xanthium pungens was possibly due to interference by active principles of their extracts. Such interference may be at the biosynthetic levels. In this respect, Kumar & Prasad (1992) suggested that growth and aflatoxin production by A. flavus are proportionate processes. However, Bhatnagar & McCromick (1987) reported that growth and aflatoxin production by A. parasiticus are independent phenomena.
It was also observed that specific aflatoxin production (expressed as μg total aflatoxin per mg dry weight) was slightly decreased in cultures treated with A. visnaga and X. pungens. However, L. albus was comparatively more effective ( Table 1). This trend shows that extracts of these plants inhibited aflatoxin production by inhibiting the growth of A. flavus. In a similar study, Masood & Ranjan (1991) found that extracts of Argemone mexicana and Cyperus rotundus inhibited aflatoxin production by inhibiting the growth of A. flavus.
The nature of the plant extract also affected the ratio of B1 to B2, and there was no correlation between the inhibition of aflatoxins or growth of the fungus and the resultant B1:B2 ratio. It is widely accepted that aflatoxin B1 is the precursor of the other related congeners ( Badii & Moss 1988). A detailed aflatoxin biosynthetic study with common medicinal and wild plants may be helpful in elucidating the regulatory system of biosynthesis of the aflatoxins.
In conclusion, L. albus, A. visnaga, and X. pungens are a significant inhibitors against A. flavus growth and resultant aflatoxins. If inhibitory factor(s) could be examined at biosynthetic level, these plants could be used in controlling aflatoxin formation in food and feed.