While the review provides an extensive summary of antimicrobial effects observed with natural-product-based metallic nanoparticles (NP-MNPs), one major concern arises regarding the claimed synergistic antimicrobial mechanisms—particularly those illustrated in Figures 3 and the associated mechanistic discussion.
The paper suggests that combining phytochemicals with metallic nanoparticles enhances antimicrobial action through well-established mechanisms such as efflux pump inhibition, ROS generation, and membrane destabilization. However, these mechanistic conclusions are primarily drawn from scattered literature reports without presenting any direct or comparative mechanistic data, nor are distinctions made between effects caused by the phytochemical, the nanoparticle, or their conjugate.
For instance, many of the cited studies (e.g., Table 2 and Table 3) report zone of inhibition values or MICs from agar diffusion tests, which do not allow for mechanistic attribution, let alone synergy quantification. In the absence of mechanistic assays (e.g., membrane permeability, DNA-binding kinetics, efflux modulation), quantification of ROS generation using probes (e.g., DCFDA), or time-kill kinetics to differentiate between additive and synergistic bactericidal effects, the central assertion that MNPs and plant extracts act “synergistically” through multiple, simultaneous mechanisms remains speculative.
Furthermore, the antimicrobial enhancement is often described as “synergistic” merely because the MNP formulation has higher inhibition than the free extract or metal ion alone. But synergy implies a greater-than-additive effect, which requires combination index (CI) analysis, isobolograms, or Loewe additivity models, none of which are applied or cited.
How do the authors justify the mechanistic synergy claims in the absence of standard synergy evaluation protocols (e.g., FIC index, time-kill synergy studies, mechanistic validation assays)? Could some of the reported enhancements in antimicrobial effects be due to additive or even concentration-dependent effects rather than true synergistic mechanisms?