INHIBITORY EFFECT OF THE ACETONE EXTRACT OF MAIZE LEAVES ON THE MYCELIAL GROWTH OF ASPERGILLUS PARASITICUS ALONG WITH ITS AFLATOXINS PRODUCTION

Maneesh Kumar 1,2* , Harish Kumar 1 , Roshan Kamal Topno 3 , Ganesh Chandra Sahoo 2 , Subham Kumar 1 , Kundan Kumar 1 , Siddharth Jha 1 and Sushil Kumar 4 . 1. Department of Biotechnology, College of Commerce, Arts and Science (Magadh University, Bodh Gaya), Patna-800020, Bihar, India. 2. Department of Virology, Rajendra Memorial Research Institute of Medical Sciences (ICMR), Agamkuan, Patna-800007, Bihar, India. 3. Department of Epidemiology, Rajendra Memorial Research Institute of Medical Sciences (ICMR), Agamkuan, Patna-800007, Bihar, India. 4. Department of Pathology, NIMS University, Jaipur, Rajasthan, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5 (11), 751-757 752 as well as aflatoxins contamination most commonly due to their hot and humid climatic conditions. These climatic conditions are favourable for fungal growth and aflatoxins production (Kumar et al., 2017b). Some time food items are destroyed by oxidative stress due to free radical generation. The free radicals damage many cellular molecules such as nucleic acid, proteins and lipids (Prakash et al., 2015). Aflatoxins production, oxidative stress and free radical generation are directly proportional to each other (Jayashree and Subramanyam, 2000). Contamination of A. parasiticus and aflatoxins in cereal crops are a matter of serious concern around the world, and there is an urgent need to solve this problem.
Many synthetic food preservatives are used to prevent the contamination of fungus and mycotoxins, but these preservatives some time show harmful effects in human life. Synthetic food preservatives such as hexachlorobenzene (HCB) cause prophyria or poisoning in human (Jones et al., 1980). Other preservatives like captan, captafol and folpet cause irritation in skin, dermal sensitization and many respiratory problems (Royce et al., 1993;Vllaplana and Romaguera, 1993). It has been reported that synthetic preservatives like formaldehyde, nitrates, sorbates, butylatedhydroxyanisole and butylatedhydroxytoulene give the symptoms of hypersensitivity, hyperactivity, allergy, cancer and asthma (Anand and Sati, 2013).
Plants contain many types of secondary metabolites, which possess anti-fungal and anti-bacterial properties (Sharma and Sharma, 2012). Extracts of many plants showed antifungal properties against the growth of Aspergillus species (Thanaboripat et al., 2004;Krishnamurthy and Shashikala, 2006). Present study was designed to reveal the antifungal and anti-aflatoxigenic properties of the acetonic extract of Maize leaves against the infection A. parasiticus and aflatoxins production.

Pathogenic organism used:-
The toxigenic strain of A. parasiticus was procured from Department of Food Science & Technology, Pondicherry University, Pondicherry, India. The culture was revived and maintained on fresh Czapek-Dox Agar (CDA) medium. The CDA culture plates were incubated for next 10 days at 28ºC to ensure purity and viability.
Preparation of acetonic extract:-Fresh leaves of maize (Malviya Hybrid Makka) were collected from agricultural regions of Dr. Rajendra Prasad Central Agriculture University at Pusa, in Samastipur district and kept in sterilized collecting polybag. These maize leaves were properly cleaned with running tap water followed by distilled water trice. Thereafter, leaves were dried in an oven for 72 hours. The leaves were crushed entirely by using mortar and pestle. About 60 grams of crushed leaves were weighed, and kept back into a sterilized conical flask. 300 ml of acetone was added into the flask and left for 72 hours. Afterword, the solution was filtered through Whatman filter paper no. 1, and filtrate was collected into a flat bottom dish. Evaporation of the acetonic extract was performed at standard room temperature till dryness. The dry residue was again dissolved in small amount of acetone and water in the ratio of 50:50 (v/v), and kept at 4ºC for the experimental work.
Antifungal activity:-Agar plate diffusion method was employed to analyze the antifungal activity of the acetonic extract (Kumar, 2017). Different acetonic extract concentrations (50µl, 100µl, 150µl, 200µl and 250µl/ml) were added into 20 ml of molten CzapekDox Agar (CDA) medium in different Petri plates. Freshly prepared fungal disc (5 mm diameter) of A. parasiticus was spotted at the centre of each petri plate containing different concentrations of the acetonic extract. One Petri plate had taken as control, containing only 20 ml of CDA medium and a fungal disc (5 mm diameter) of A. parasiticus. In next step, all petri plates were incubated at 28±2ºC for next 10 days under controlled condition. After the incubation period, the mycelial development of the A. parasiticus was measured in diameter (in cm) on the medium of each petri plate. Percentage of inhibition of the growth of the A. parasiticus was calculated by the following formula: Where, DC = Average increase in mycelial growth in the control sample. 753 DT = Average increase in mycelial growth of the test sample.

Inhibition of aflatoxins production:-
To analyze the anti-aflatoxigenic properties of acetonic extract from Maize leaves, Czapek Dox Broth (CDB) medium was arranged. The sterilized fresh CDB media were poured into different conical flasks (25 ml). The requisite amount of acetonic extract of different concentrations (50µl/ml, 100µl/ml, 150µl/ml, 200µl/ml and 250µl/ml) was added into each flask containing CDB medium. The fungal discs of 5 mm diameter were inoculated into each flask containing different concentrations of the acetonic extract. A flask containing only 25 ml of CDB medium and a fungal disc (5 mm diameter) was used as control. Subsequently, all flasks were incubated at 28±2ºC for 10 days. After 10 days, the mycelium of A. parasiticus of each flask was filtered through Whatman filter paper no. 1. All the filtered mycelium of A. parasiticus was autoclaved to kill the spore pathogenicity. Then, the fungal mycelium were dehydrated entirely in an oven (at 80ºC) and weighed. The dried remnants were extracted by using 20 ml chloroform, and chloroform extract was stored at room temperature to evaporate till complete dryness. Again, 1ml of chloroform was added into residue. 60 µl of the chloroform extract was loaded on the thin layer chromatography (TLC) plates following the method of Turner and his colleagues (Turner et al., 2009) for confirmation of aflatoxin presence. Solvent system for TLC plate was prepared by the mixture of chloroform and acetone in the ratio of 9: 1 (v/v). After running the test sample, the TLC plate was dried and then transferred into a UV transilluminator (360 nm) to authenticate the presence of aflatoxins. The blue and green colour bands were observed on TLC plate under UV transilluminator. The blue and green colour bands indicated the presence of aflatoxins in the mold. All bands were scratched from the TLC plate, and dissolved in 5ml methanol followed by centrifugation at 3000 rpm for 5 minutes. Absorbance of supernatant was noticed at 360 nm, and the quantification of aflatoxins was done by using following formula: Where, D = Absorbance, E = Molar extinction coefficient of aflatoxins, M = molecular weight of aflatoxins, L = Path length.
Phytochemical Screening:-Test for different types of phytoconstituents such as proteins, carbohydrates, terpenes and flavonoids were carried out of the acetonic extract of Maize leaves according to the method of Harbone (Harborne, 1993).  The graph3 is presenting the anti-aflatoxigenic effect of the acetonic extract of Maize leaves. The result of antiaflatoxigenic activity revealed that this extract has capacity to inhibit the aflatoxins synthesis by A. parasiticus. The graph4 presents that the extract showed 100 % inhibition of aflatoxins production at the concentration of 200 µl/ml. The mycelium growth and aflatoxins production were analysed to decrease on increasing the concentration of the acetonic extract. It was also analysed that decrease in mycelial biomass leads to decrease in aflatoxins production. Therefore, mycelial growth must be controlled below the limit to arrest the synthesis of aflatoxins.
Qualitative phytochemical screening of the acetonic extract of Maize leaves showed the presence of proteins, carbohydrates, terpenes and flavoinds. It has been reported that flavonoids have potential to inhibit the production of aflatoxins (Mallozi et al., 1996;Patel et al., 2010). Flavonoids, singly or in combination with other phytochemical compounds might be responsible for the antifungal and anti-aflatoxigenic properties of the acetonic extract of Maize leaves.