The attached table below shows the zone of inhibition (ZOI) in mm of:
1. Rutin
2. Heliomycin
3. AgNPs(silver nanoparticles)
4. AgNPs + Rutin
5. AgNPs + Heliomycin
6. Tetracycline (positive ctrl)
Hence, my question is how to know if there's an interaction effect (additive, synergistic, or antagonistic) antimicrobial effect of AgNPs + Rutin & AgNPs + Heliomycin when referring to the table:
Phil Geis
Synergism: When the effect (Diameter of zone of inhibition/mm) of combined agents (A + B) is greater than the sum of their individual effects
PREPARATION OF RUTIN (R):
- Rutin is a flavonoid glycosidic compound plant derivative.
- Extraction Source: Rutin, a natural flavonoid, extracted from dried ground leaves of Ruta graveneoles.
- Extraction Method: Dried leaves (1000 g) mixed with 70% ethanol, filtered, and extraction repeated five times.
- Evaporation: Followed by evaporation under reduced pressure.
- Precipitate Formation: Aqueous layer collected, left to stand for three days, resulting in a yellow precipitate.
- Filtration and Washing: Precipitate filtrated and washed three times with dichloromethane: ethyl acetate (2:1).
- Yield: The process yielded 500 mg of a yellow solid compound.
PREPARATION OF HELIOMYCIN (H):
- Heliomycin is a secondary metabolite marine actinomycetes derivative.
- Extraction Source: Heliomycin extracted from the culture broth of actinomycete AB5.
- Harvesting: Fermentation broth harvested after four days.
- Centrifugation: Centrifuged at 4000 rpm for 10 min to obtain a mycelial cake.
- Multiple Extractions: Mycelial cake extracted three times with methanol; aqueous phase extracted with ethyl acetate.
- Crude Extract: Brown crude extract formed, dissolved in methanol, and left to stand overnight.
- Precipitate Formation: Yellow precipitate formed, split apart, filtrated.
- Washing: Precipitate washed three times with methanol.
- Yield: Resulted in a yellow solid compound.
PREPARATION OF Silver nanoparticles (AgNPs):
- Silver Nanoparticle (AgNP) Synthesis: Achieved by reducing AgNO3 (silver nitrate) using extracellular polymeric substances (EPS) released by Bacillus nitratireducens.
- Biological Source: Bacillus nitratireducens isolated from soil.
- Reduction Mechanism: EPS from the bacterial source serves as a reducing agent for AgNO3, leading to the formation of silver nanoparticles.
AIM:
- Evaluation of Antimicrobial Effects: Testing potential synergistic or additive effects of silver nanoparticles (AgNPs) in combination with rutin (AgNPs + R) and heliomycin (AgNPs + H).
- Microorganism Target: Testing conducted against Pseudomonas fluorescens.
- Method Used: The disc diffusion method is employed to visualize and assess antimicrobial effects.
"Synergism" by ZOI is not a great idea as the metric reflects not only intrinsic efficacy but presence by diffusion - a consequence of solubility, binding to substrate, diffusion. Much like chromatography - components of a complex materials will be separated/diffuse at different rates.
The definition is sophomoric and shows to ZOI as not the best metric One has to anticipate A will diffuse as A will diffuse - whether alone of with another compound. Using both would not "add" to the diffusion or either. Synergy that might exist would be at the margins of both components where lower non-inhibitory concentrations of each are present to work in combination.
Could not find the solvent used for Heliomycin and Rutin in MIC. From extraction, appears they'd need some organic solvent that would require control.
The metric observed is very interesting. They performed MIC by classic methods - don't know why they used ZOI as when they could have performed synergy by that as well.
Yes, disk diffusion assay is not the std. method for testing synergism but it might give a preliminary idea of any interaction effect:
After reviewing the Quest. Table, I think that synergism were achieved:
I.e. Rutin = 9mm (ZOI)
AgNPs = 8 mm
R + AgNPs = 23 mm
And it support the definition of synergism.
The authors' definition. Not one of accepted methodology.
Please understand - this is a microbiological article published in a Marine journal where it would not be reviewed by microbiologists, much less critical review - witness the occasional misspellings of target bacterial names and failure to report solvent for Heliomycin and Rutin and most important absence of controls that would validate authors novel method for determining "synergy"
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