Evaluation of Nostoc commune for potential antibacterial activity and UV-HPLC analysis of methanol extract
P Kaushik, A Chauhan, G Chauhan, P Goyal
agar-well diffusion assay, antibacterial activity, minimum inhibitory concentration and uv-hplc
P Kaushik, A Chauhan, G Chauhan, P Goyal. Evaluation of Nostoc commune for potential antibacterial activity and UV-HPLC analysis of methanol extract. The Internet Journal of Microbiology. 2007 Volume 5 Number 1.
Cyanobacteria (blue-green algae) are a group of extraordinary diverse Gram-negative prokaryotes that originated 3.5 billion years ago. The medicinal and nutrient qualities of cyanobacteria were first appreciated as early as 1500 BC, when
Material and Methods
Organism and growth conditions
Preparation of extracts
At the stationary phase of growth (25 days), the culture was harvested and spent media was collected after filtering the biomass. The collected biomass was dried in hot air oven at 60 ° C for one h and then placed in solvent. Left the mixture for few hours at room temperature and then sonicated for 10 min followed by centrifugation at 4000 rpm for 10 min. After centrifugation, supernatant was collected in a preweighed test tube and then concentrated under nitrogen until completely dry, again weighed and then resuspended in the appropriate solvent to make the solution of known concentration for the antibacterial assay. Same procedure was adopted for the preparation of aqueous extract.
The microorganisms used in antibacterial assay were collected from Institute of Microbial Technology (IMTECH), Chandigarh, India .The species employed include four Gram-positive bacteria (
Evaluation of Antibacterial Activity
Determination of Minimum Inhibitory Concentration (MIC)
Minimum inhibitory concentration of active crude extract(s) was determined by broth microdilution method as recommended by National Committee for Clinical Laboratories Standards (NCCLS, 1997). The test was performed in 96 wells microtiter plates. Two fold serial dilutions of all active extracts were made in Cation-Adjusted Muller-Hinton Broth (CAMHB from Hi-Media) ranging from 1 to 512 µg/ml. Ciprofloxacin was used as standard antibiotic for the assay. Each inoculum was prepared in the same medium at a density adjusted to a 25% transmittance turbidity standard (10 8 cfu/ml) and diluted to 1:100 for the assay. The final volume in the wells was 200 µl. After 24 h of incubation at 37°C, the calculated amount of nitrogen dried cyanobacterial material present in the most diluted extract that produced a visible inhibition was defined as MIC.
UV-Vis spectroscopic analysis
The absorption spectrum of methanol extract of
High performance liquid chromatography of methanol extract of
Results and Discussion
The antibacterial activity of different extracts of
Aqueous extracts of the
No antibacterial activity was detected in dichloromethane extracts and culture supernatant and none of the methanol extracts and aqueous extract showed antibacterial activity against
Minimum inhibitory concentration of methanol extract of
UV-Vis spectrophotometric analysis of active methanol extract reveals the highest absorption spectra at 423 nm wave-length (Fig. 1-A). Several peaks were observed during HPLC analysis indicating the presence of active compounds in extract (Fig. 1-B). The antibacterial activity of these extracts might be due to the presence of one or more compounds. These extracts can further be subjected to various advanced techniques such as Nuclear Magnetic Resonance (NMR) and Mass Spectroscopy to determine the exact chemical molecule responsible for their bioactivity. This would definitely be turning point for pharmaceutical sciences in determining a novel antibacterial compound.
The above findings indicate the discovery of novel chemicals that can lead to the development of pharmaceuticals by cyanobacteria. The current scenario of antibiotics is very threatening with significant emergence of resistance among bacterial pathogens against available antibiotics. The present investigation reveals that cyanobacterium