2.1. Collection of Plant Material Azadirachta indica (neem) leaves were collected from Botanical Pesticide Garden of the Institute of Environmental Science and identified by the professional taxonomist of the Department of Botany of University of Rajshahi and a voucher specimen was deposited at the herbarium of the department.
2.2. Extraction Procedure Fresh and shade dried leaves of A. indica were used for the study (Cheenickal and Mendez, 2017). Neem leaves were washed properly by running tap water followed by distilled water and placed in a shade for drying out the surface water (Francine et al., 2015). After 6 hours, 100 gm fresh leaves were taken and divided into two parts, 50 gm in each. Part-A: fresh leaves were used immediately for UAE aqueous extraction and Part-B: fresh leaves were allowed for week long drying and grinding for methanol extraction. After drying and grinding 50 gm fresh leaves reduced to 18 gm dried powder.
2.2.1. Processing of Part-A Fresh leaves (50 gm) of A. indica were blended in a conventional juice machine with 250 ml distilled water (material solvent ratio 1:5) for better extraction (Toma et al., 2001). The juice was transferred to a 500 ml conical flask and placed in an ultrasonic bath for 30 minutes treatments at 40o C bath temperature. Power Sonic 405 (Microprocess controlled Bench Top Ultrasonic Cleaner) was used for ultrasound treatment. The plant extract were then collected and filtered through three layers of polyester cloth and liquid extracts were dried at 60o C in a conventional water bath. Dried crude UAE aqueous extracts stored in an air tight bottle and preserved in cold chamber for further use.
2.2.2. Processing of Part-B Dried leaves powder (18 gm) of A. indica were dissolved in 54 ml of methanol (material solvent ratio 1:3) in a conical flask for 72 hours with intermittent shaking as per standard method (Latha et al., 2015). The plant extract was then collected and filtered through filter paper (Whatman No.1) and the obtained liquid extracts were subjected to rotary evaporator and subsequently concentrated under reduced pressure (in vacuum at 40o C). The dried leaves methanol extract was stored in an air tight bottle and preserved in cold chamber for further use.
2.4. Phytochemical Screening Test Phytochemical screening tests are the qualitative study of the extract to identify the presence of different types of pharmacologically active compounds in the extract. These studies were helpful to compare the efficiency of the existing extraction procedure with conventional extraction procedure. The extract was subjected to phytochemical tests for plant secondary metabolites, alkaloids, anthraquinones, flavonoids, glycosides, saponins, steroids, 2.4. Phytochemical Screening Test Phytochemical screening tests are the qualitative study of the extract to identify the presence of different types of pharmacologically active compounds in the extract. These studies were helpful to compare the efficiency of the existing extraction procedure with conventional extraction procedure. The extract was subjected to phytochemical tests for plant secondary metabolites, alkaloids, anthraquinones, flavonoids, glycosides, saponins, steroids.
2.5. Antimicrobial Activity: Antimicrobial activity was done by disc diffusion method (Baker et al., 1993; Mukhtar and Tukur, 2000) on 5 pathogenic bacteria and 3 pathogenic fungi. Five human pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Shigella boydii, Shigella dysenteriae and Salmonella typhi) and three pathogenic fungi (Curvularia lunata, Fusarium chlamydosporum and Fusarium oxysporum) were collected from the Microbiology Lab, Department of Biochemistry and Molecular Biology, University of Rajshahi, Bangladesh. Nutrient agar media was used for sub-culturing bacteria at 37o C and potato dextrose agar (PDA) media was used for sub-culturing fungus at 25oC.