Synergistic Effect of Quercetin with Allicin from the Ethanolic …
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2.9 Fluorescence Confocal Microscopy
The biofilm formation in the presence and absence of extract of A. cepa and
Azithromycin were observed by fluorescence microscopy. The biofilm was formed on
glass slides for a period of 72 h, and then the samples were challenged with the extract
for 2 h keeping the control untreated. Then the samples were treated with 10 mg/mL
of acridine orange solution and were incubated at 37 °C for 1 h. Stained cells were
observed using an Axioplant Fluorecence Microscope equipped with digital camera
(d × m 1200, Nikon, Japan).
2.10 FTIR Analysis of Biofilm Formation
Biofilm was formed upon Chitin flakes for a period of 72 h at 37 °C, and challenged with plant extract and kept for drying at 37 °C for 48 h. The FTIR
spectra were recorded in the range of 450–4000 cm
−1 using Spectrum 100 FTIR
Spectrophotometer.
2.11 GC–MS Analysis
The study of Gas Chromatography–Mass Spectrometry is the process of optimization
that includes the process of introducing the plant extract into the column followed by
the separation of the constituents being present and the target molecules are analyzed
including the process of Mass Spectrometry. The chromatographic analysis of the
methanolic extract of the A. cepa was done using GC Model—Trace GC Ultra,
MS model—POLARISQ (Thermoscientific). The data obtained were searched using
NIST Library. The spectra formed as a result of GC-MS were linked with their
existing library.
2.12 Protein Structure Preparation and Ligand Search
from Online Database
Bioactive compounds from A. cepa, i.e. allicin and quercitin were found from
PubChem Database (Morris et al. 1998). Molecular docking studies were carried
out to elucidate the interaction of allicin and quercitin with biofilm-forming protein.
3-D atomic co-ordinate of the protein is not available in the protein data bank (PDB)
so homology modelling and fold recognition was employed to model the protein.
Protein intimin from E coli. was found to have maximum sequence identity. So, the
73
2.9 Fluorescence Confocal Microscopy
The biofilm formation in the presence and absence of extract of A. cepa and
Azithromycin were observed by fluorescence microscopy. The biofilm was formed on
glass slides for a period of 72 h, and then the samples were challenged with the extract
for 2 h keeping the control untreated. Then the samples were treated with 10 mg/mL
of acridine orange solution and were incubated at 37 °C for 1 h. Stained cells were
observed using an Axioplant Fluorecence Microscope equipped with digital camera
(d × m 1200, Nikon, Japan).
2.10 FTIR Analysis of Biofilm Formation
Biofilm was formed upon Chitin flakes for a period of 72 h at 37 °C, and challenged with plant extract and kept for drying at 37 °C for 48 h. The FTIR
spectra were recorded in the range of 450–4000 cm
−1 using Spectrum 100 FTIR
Spectrophotometer.
2.11 GC–MS Analysis
The study of Gas Chromatography–Mass Spectrometry is the process of optimization
that includes the process of introducing the plant extract into the column followed by
the separation of the constituents being present and the target molecules are analyzed
including the process of Mass Spectrometry. The chromatographic analysis of the
methanolic extract of the A. cepa was done using GC Model—Trace GC Ultra,
MS model—POLARISQ (Thermoscientific). The data obtained were searched using
NIST Library. The spectra formed as a result of GC-MS were linked with their
existing library.
2.12 Protein Structure Preparation and Ligand Search
from Online Database
Bioactive compounds from A. cepa, i.e. allicin and quercitin were found from
PubChem Database (Morris et al. 1998). Molecular docking studies were carried
out to elucidate the interaction of allicin and quercitin with biofilm-forming protein.
3-D atomic co-ordinate of the protein is not available in the protein data bank (PDB)
so homology modelling and fold recognition was employed to model the protein.
Protein intimin from E coli. was found to have maximum sequence identity. So, the