Syringopicroside is a natural drug with antibacterial activity, which is the main ingredient of Syringa oblata Lindl (S. oblata). In order to further develop the application of S. oblata and evaluate the ability of syringopicroside against Streptococcus suis (S. suis), this investigation first applied an ultrasonic-assisted method to extract syringopicroside, and then response surface methodology (RSM) was performed to get the optimum condition. Based on RSM analysis, a second-order polynomial equation about the syringopicroside yield and four variables, including ultrasonic power, time, temperature, and liquid-to-solid ratio, was purposed. Through RSM prediction and model verification experiments, the optimum conditions were determined, as follows: ultrasonic time was 63 min, temperature was 60 °C, a liquid-to-solid ratio was set to 63 mL/g, and ultrasonic power was 835 W. Under this condition, a high syringopicroside yield was obtained (3.07 ± 0.13 mg/g), which was not significantly different with a predicated value. After separation and purification by HPD 500 microporous resin, then mass spectrum was applied to identify the main ingredient in aqueous extract. A minimal inhibitory concentration (MIC) assay revealed the value against S. suis of syringopicroside was 2.56 µg/µL and syringopicroside with sub-inhibitory concentrations that could effectively inhibit biofilm formation of S. suis. Besides, scanning electron microscopy analysis indicated syringopicroside could destroy the multi-layered aggregation structure of S. suis. Finally, molecular docking analysis confirmed that syringopicroside was combined with Orfy protein of S. suis through hydrogen bonds, hydrophobic interaction, and π-π stacking.
Background
Streptococcus suis is a zoonotic pathogen that causes serious systemic infections in pigs and humans. It is a great threat to pig breeding and public health safety. The formation of biofilm was one of the main reasons that it’s difficult to cure. Rhubarb water extract can inhibit the formation of biofilm of Streptococcus suis, but the main functional ingredient was not clear. And what’s the potential mechanism of emodin intervene the biofilm formation? Importantly, the gene expression of luxS of Streptococcus suis was significantly decreased with emodin treatment, if there is a possibility that emodin could combine with LuxS protein which in turn inhibit the formation of biofilm?
Methods
Optimization of green ultrasonic extraction of emodin from R. officinale by Response Surface Methodology. The activity of antibacterial was evaluated by MIC assay. And the ability that intervene biofilm formation was evaluated through Crystal violet staining and SEM. The combination mode between emodin and LuxS protein was identified through molecular docking.
Results
The optimum extraction conditions (ethanol concentration of 80%, extraction time of 21 min, and liquid to solid ratio of 12.5:1 mL/g) were determined through the interaction analysis of different influencing factors and model verification experiment. The minimal inhibitory concentration (MIC) of RUEP against S.suis was 15.625 µg·mL-1, and sub-inhibitory concentrations of RUEP (1/2, 1/4, 1/8 and 1/16 MIC) significantly inhibited the biofilm formation of S. suis. Moreover, scanning electron microscopy analysis confirmed that RUEP can damage the layered colony community structure in biofilm and effectively intervene in the biofilm formation of Streptococcus suis. Through molecular docking analyzed, the inhibition of biofilm formation could be realized through the formation of hydrogen bond with residues His14 and Glu 60, π-π staking with His61、Phe80 and hydrophobic interaction with Cys127.
Conclusion
Through our study, we found emodin have good antibacterial and antibiofilm formation properties and the potential mechanism of emodin intervention on biofilm formation of Streptococcus suis was inferred.
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