Studies on antimicrobial, biochemical and image analysis in Mirabilis jalapa

Text Sample:
Anti microbial activity:
Sample extraction:
The determined Fresh plant leaves (200g) were ground, extracted with Diethyl ether, ethyl acetate and methanol separately and filtered. The plant residue was re-extracted by adding above solvents and filtered again after 48hs. Such procedure was repeated every 72hs, completing three filtration processes. The filtrate was concentrated on a rotary evaporator at 45ºC for solvent elimination, and the extracts were kept in sterile bottles under refrigerated conditions until use. The final volume is adjusted to a concentration of 1 mg/ml with the above solvents separately.
Test microorganisms:
The microbial strains are identified strains and were obtained from the MTCC, IMTECH, Chandigarh, India. The bacterial strains studied are Eschericia coli MTCC 739, Staphylococcus aureus MTCC 737, Klebsiella pneumoniae MTCC 109, Bacillus subtilis MTCC 441 and Aspergillus niger MTCC 282.
Antimicrobial assay:
The antibacterial assays were performed by the agar well diffusion method. Petri dishes (200 mm) were poured with nutrient agar (HI-Media) and allowed to solidify to make base layers.
The seed layers were prepared by inoculating 10mL of test organism suspension in 100 mL Mueller-Hinton agar(for bacteria) and Sabouraud Dextrose agar (for fungi) and wells, 6 mm in diameter, were made in the agar medium with the help of a sterile steel borer. About 100 L of each extract was added aseptically in wells. All the plates were incubated at 37 ± 1°C for 24 hours in the upright position. At the end of the incubation times, the diameters of the inhibition zones were measured in millimeters. Ampicillin (20 g/mL) and solvent (diethyl ether, ethyl acetate, and methanol 20 g/mL) were used as antimicrobial compounds against text microorganisms. The tests were conducted in triplicate.
Thin layer chromatography (TLC):
Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures.
The present study is performed on a sheet of glass, which is coated with a thin layer of adsorbent material, usually silica gel. This layer of adsorbent is known as the stationary phase.
After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.
The TLC analysis was performed on glass slides pre-coated with silica gel G/GF (E-Merck grade). Before use, glass slides were pre-washed with methanol, and dried in an oven at 105°C for 1 hour.
Plates are prepared with 5g Silica Gel G and GF in a ratio of 8:2 by using 5ml ethyl acetate as a solvent.
A small spot of solution containing the sample is applied to a plate, about one centimetre from the base. The plate is then dipped in to a suitable solvent, such as hexane or ethyl acetate, and placed in a sealed container. The extracts (10 L) were applied on the plates as bands of 7-mm width with the help of a linomat-5 sample applicator. The solvent moves up the plate by capillary action and meets the sample mixture, which is dissolved and is carried up the plate by the solvent. Different compounds in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase, and because of differences in solubility in the solvent. By changing the solvent, or perhaps using a mixture, the separation of components (measured by the Rf value) can be adjusted. TLC plate is visualized under UV radiation.
TLC runned in ethylacetate and hexane in the ratio of 1:9, 3:7 and 5:5 for 10%,30% and 50% mobile phase.