(12g) Enzymatic Oligomerization of Resveratrol to Generate Novel Anti-Microbial Analogs

Enzymatic oligomerization of Resveratrol to generate novel
anti-microbial analogs

Plant
polyphenols such as flavonoids and stilbenoids have
been associated with several health benefits and thus have been widely studied in
recent years. Despite the current controversy regarding their potential for
pharmacological applications, recent studies suggest that natural phenols can
be converted into active metabolites in
vivo, and more attention is required on the in vitro synthesis of analogs to enhance their efficacy. Also, a
constant growth of anti-microbial resistance and rapid microbial evolution,
underscores the importance of identification of alternatives to generate new
and novel antimicrobial compounds.  We
propose that the antimicrobial activity can be significantly enhanced by
generating oligomers of these compounds. In particular, we produced, isolated
and characterized a dimer from resveratrol (d-viniferin, the tri-dehydrodimer of resveratrol) from an in vitro enzymatic oligomerization reaction
mediated by soybean peroxidase (SBP) in aqueous buffer. Dose response studies in
gram- positive
and gram- negative
bacteria show great antimicrobial potential of d-viniferin as compared to resveratrol.
For example, d-viniferin showed MIC and IC₅₀ values of 125 µM
and 59 µM respectively against E. coli
(in the presence of efflux pump inhibitor Phe-Arg-ß-naphthylamide, PABN, 25 µg/mL),
while resveratrol shows MIC and IC₅₀ values of 2000 µM and 250 µM
respectively. Significant improvement was observed against B. cereus with d-viniferin giving  MIC and IC₅₀ values of 5 µM and 15
µM, while for resveratrol MIC and IC₅₀ values were >150 µM, all
in presence of the efflux pump inhibitor 1-(1-Naphthylmethyl) piperazine (NMP).

The
significant increase in anti-microbial activity in presence of efflux pump
inhibitor PABN in the case of E. coli
indicated that efflux pumps are responsible for the extrusion of resveratrol
and its analogs from the cell. Thus, d-viniferin was tested against an E. coli strain with a deletion of tolC gene (part of AcrAB-tolC
efflux pump) in the presence and absence of PABN. In both cases, significant but
similar growth inhibition was observed ([+]PABN, MIC
and IC₅₀ values were 1.2 and 3 µM respectively; [-]PABN, MIC and IC₅₀
values were 2.5 and 7 µM respectively). Moreover, in the absence of PABN both
the dimer and monomer had no effect on E.
coli at these concentrations. The
effect of efflux pump inhibitor on B.
cereus was less significant; MIC and IC₅₀ values in the absence
of NMP were 12 and 30 µM respectively.

Visual
evidence discarded the possibility of d-viniferin causing cellular
aggregation leading to a lower cell density or count. Also, morphological
changes in B. cereus were observed
before and after incubation with d-viniferin; there was a decrease in the
cell size while cell wall was found to be unaltered and intact.  

In
summary, it was demonstrated that novel and active antimicrobials from plant
polyphenols can be generated in vitro.
In particular, d-viniferin has shown great antimicrobial activity and its
low toxicity against mammalian cells provides a new insight in the development
of antimicrobial with potential therapeutic applications.