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Statistics: Mean ± SD n=6 One Way ANOVA (*) p<0.05


140 120 100 80 60 40 20 0


Untreated control


-39% (*) 0.50% P. lobata extract


Figure 3: P. lobata extract inhibits secretion by S. aureus of AIP QS molecule


measurements. The statistical analysis was done using a one-way ANOVA test following Sigmaplot software recommendation (Systat Software Inc, USA). The threshold of significance was set to 5% (p<0.05).


S. aureus secreted lipase activity Bacterial culture S. aureus ATCC35556 was cultivated in tryptic soy broth (TSB) medium with and without (untreated control), Pueraria lobata extract at 0.5% and 1% for 24 hours at 37°C in aerobiosis. Glycyrrhizic ammonium salt (Sigma 50531) at 0.2% was used as positive control for the inhibition of lipase release. After incubation, the cultures were centrifugated at 1000g for 10 minutes and the supernatants were filtered.


Lipase secretion quantification The secretion of triacylglycerol lipase in culture supernatants was determined by incubation of 100µL of supernatant with 100µL lipase substrate 1,2-Di-O-lauryl-rac-glycero-3- (glutaric acid 6-methylresorufin ester) at 0.1M (Sigma Aldrich 30058) in a phosphate buffer for one hour at 40°C. The lipase activity was evaluated by


recording fluorescence emission at 600 nm with an excitation at 520 nm. A decrease in fluorescence reflects a decrease in substrate transformation, thus a decrease in lipase release. Optical density (OD) at 600nm was


measured before and after the incubation to check bacterial population density after incubation with active ingredient and controls.


Statistics The results of lipase release are expressed as a mean percentage (%) of triplicate measurement ± standard deviation (SD), compared to the untreated control standardized to 100%. The statistical analysis was done using a one-way ANOVA Holm Sidack method test following Sigmaplot software recommendation (Systat Software Inc, USA). The threshold of significance was set to 5% (p<0.05).


Results and discussion P. aeruginosa QS molecule and swarming inhibition We first evidenced in the case of P. aeruginosa,


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140 120 100 80 60 40 20 0


Untreated control


Statistics: Mean ± SD n=6 One Way ANOVA (*) p<0.05


41


-94% (*) 0.25% 0.50% P. lobata extract


Figure 4: Pueraria lobata extract decreases secretion of lipase from S. aureus


that furanone at 0.2%, the reference inhibitor of QS molecules secretion8


significantly decreased


by 87% (p<0.01) the acyl homoserine lactone (3-oxo-C12-HSL) secretion (data not shown). Furthermore, furanone also inhibited the secretion of Pseudomonas Quinolone Signal (PQS) secretion by 97% (data not shown); these results validated the experiment. The Litchi sinensis extract showed an


inhibition capacity on the release of these three QS molecules (Figure 1). It strongly and significantly inhibited secretion of 3-oxo-C12 HSL compared to the untreated control with a dose related efficiency. 42% (p<0.001) of inhibition was obtained at 0.2% and 72% (p<0.001) was obtained at 0.4%. C4-HSL secretion was inhibited significantly at the same level with the two doses of Litchi sinensis extract (-17% and -19%, p<0.05). Regarding PQS, its secretion was inhibited significantly by Litchi sinensis extract at 0.4% (-24%, p<0.001). Some natural and synthetic quorum sensing


interfering molecules have been already identified. Among natural products, inhibitory activity by some flavonoids have been already demonstrated.9


We can thus hypothesize that


the effect demonstrated with Litchi sinensis extract can be linked to its flavonoids content.10 Swarming is a virulence trait of P.


aeruginosa. During the swarming process, the up-regulated expression of a variety of


virulence factors would benefit the population of bacterial cells in the process of colonizing new environments.11 As swarming is under Quorum Sensing controlled gene expression,12


we sought to see


if QS inhibition was associated with a decrease in P. aeruginosa swarming. After one day of incubation of P. aeruginosa with furanone at 0.1µg/mL, we observed an inhibition of bacteria swarming; swarming inhibition by furanone have already been reported by Muner Otton and coworkers13


experiment. Interestingly, the Litchi sinensis extract


also inhibited significantly the swarming of P. aeruginosa on agar plate, with a dose-related efficacy. The mean area spreading of the bacteria was respectively decreased by 32% (p<0.01) and 67% (p<0.001) with 0.2% and 0.4% of L. sinensis extract (Figure 2). For the first time, we evidenced the


inhibition of P.aeruginosa QS and swarming of a Litchi sinensis extract. Potential of purified flavonoids in inhibition of P. aeruginosa swarming have been already reported14


; this


reinforces our hypothesis that the activity of this extract is partly related to its flavonoid content.


S. aureus AIP and lipase inhibition Staphylococcus aureus relies on quorum sensing to exert its harmfulness. After one day of incubation with S. aureus suspension, P. lobata extract at 1% decreased significantly by 39% the secretion of S. aureus AIP (Figure 3) in culture medium. AIP are secreted out of the bacterial cell


and detected by the surrounding bacteria. AIP binding to their receptor on the detecting bacteria triggers the expression of genes related to virulence factors, as amongst which is lipase. S. aureus lipase, which belongs to the glycerol ester hydrolases, contributes to skin colonization by hydrolyzing human sebum as a nutrient source.15 In addition, when adherent to the skin, S.


aureus strongly reduces the barrier function of the skin using lipase.16


Licorice extract,


which is known to contain high amount of Glycyrrhizic acid, has been reported to inhibit expression of virulence genes in S. aureus.17 In this experiment, Glycyrrhizic ammonium salt at 0.2% was used as positive control; it


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; these results thus validated the


Secreted AIP I (% vs control)


Lipase activity (% vs untreated control)


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