Plant material
The Lucknow local market was visited to obtain the plant leaves used in this investigation. With the assistance of Central Institute of Medicine and Aromatic plants, Lucknow, and other literature survey comparisons, the plant leaf was recognized and verified.
Drying and Grinding of Plant
The plants were cleaned and then chopped in to little bits with scissors and knives. They were kept for drying in a room without any exposure to light for about two weeks, however they may also be dried in an oven (at 40 to 50 0C) if necessary. After the plants have dried fully, make sure the powder is uniform in size and that the surface area is increased for improved extraction. To keep materials dry until extraction, they were kept in tightly closed plastic containers (Hassan et al. 2019; Kezetas et al. 2021).
Fungal strain:
A. fumigatus and F. solani were acquired from Chandigarh’s Microbial Type Culture Collection and Gene Bank. On potato dextrose agar, the fungus was cultivated at 25 0C. after 10 days, the fungus spores were harvested from cultures on agar plates (Poala et al. 2011; Naveen et al. 2012). The fungus suspension was kept -40 0C in 20% glycerol.
Dtermination of antifungal activity:
Agar well diffusion was used to test plant extracts for their ability to inhibit the growth of fungus (Hassan et al. 2019; Eman et al. 2021).The Potato Dextrose Agar (PDA) medium was made in accordance with the standard composition provided by Himedia. 40 gm pf the medium was suspended in 1L of water, and the medium was autoclaved at 121 0C and 15 pressure for 15 minutes. Each plate was filled with 20ml of theculture media after the sterilising media had been added using aseptic procedures into sterile glass petri dishes. After allowing thre plates to properly solidify, the media was inoculated using the spread plate technique with the appropriate fungus isolates, A. fumigatus and F. solani on PDA media. For this, 100 ml of the culture broth of each isolated was added over the media and evenlly spread using a sterile glass rod. Ten minutes after spreading, sterile microtips were used to pierce wells into the media plates, and each well was subsequently filled with 20 ml of the appropriate extract on separate plates. The samples were allowed to diffuse into the media through the well before the plates were parafilm sealed and incubated for 48 hrs at 27 0C (Fungus).
The plates had two well one of the positive controls that was filled with fluconazole (Sanam et al. 2019) of 1200ppm concentration and the negative control that was filled with pure solvent in which samples were prepared. After 5-7 days of incubation, the plates were examined for the zone of inhibition, a clear area surrounding the well whse diameter was measured in millimeters and noted.
Minimum Inhibitory Concentration (MIC)
MIC was calculated using various extracts concentrations (Gayoso et al. 2004; eman et al. 2021; Jeyasakthy et al. 2017). For MIC test, potato dextrose broth was used, the media was prepared as per the standard composition and autoclaved for sterilization. A series of six tubes was used, and one blank test tube starting from the highest concentration of antifungal agent followed by the lower ones. After the respective concentration of the antifungal agents were prepared in each tubes, each tubes containing the media and antifungal agentwas loaded with 100ml of 24hr mold fungus that is A. fumigatus and F. solani. After addition of fungus culture the tubes were plugged and incubated at 35 0C for 24-48 hrs. the tubes were observed for the turbidity in the medium, which is an indication of fungal growth. The lowest concentration of the antifungul agent at which no turbidity was seen is taken as the MIC value for set of analysis.