40 SKIN CARE


Inhibition of P.aeruginosa AHL (3-oxo-C12-HSL) secretion


140 120 100 80 60 40 20 0


Untreated control


-42% (***) -72% (***)


120 100 80 60 40 20 0


0.2% Litchi extract 0.4%


Untreated control


Inhibition of P.aeruginosa AHL (C4-HSL) secretion


-17% -19% (***)


120 100 80 60 40 20 0


0.2% Litchi extract 0.4%


Untreated control


Statistics: n=6


Mean ± SD Figure 1: Litchi sinensis extract decreases secretion of quorum sensing molecules AHL and PQS of P. aeruginosa


AHL and PQS quantification in P. aeruginosa culture medium AHL (3-oxo-C12-Homoserine Lactone and C4Butanoyl-HomoserineLactone) and PQS extraction: 500µL of supernatants were extracted with a 500µL of a mixture ethyl acetate 99.5%/ acetic acid 0.05%. After centrifugation at 15,000g for five minutes, the organic layer was collected and evaporated. The dry matter was then dissolved in 100µL of a mixture acetonitrile (10%) and formic acid (0.1%) and the samples were diluted to the 10th before injection. LC/MS-MS analysis conditions: 5µL of each


sample and standard were injected in the LC/ MS-MS equipment Ultimate 3000 (Dionex). The chromatographic column used was a C18 column. Compound elution was made using a gradient of 10 to 50% of acetonitrile followed by a gradient of 50 to 99% of acetonitrile with a debit of 300nl/min. Data acquisition was made with Q-Exactive Plus (Thermo Scientific).


AIP quantification in S. aureus culture medium AIP extraction: 60 µl of acetonitrile was added to 500 µl of sample, the mixture was stirred and loaded onto a 30 kDa ultrafilter and centrifuged at 14,000 g for ten minutes. The filtrate was collected and evaporated on Speed Vac for 30 minutes to remove acetonitrile and then acidified with Trifluoroacetic acid (TFA) to a final concentration of 1%. The samples were then desalted and


purified using a Targa C18 cartridge and evaporated to dryness by Speed Vac. The samples were taken up in 100 µL of a mixture of 3% acetonitrile/0.1% formic acid, then diluted to the 10th before injection in LC-MS. LC/MS-MS analysis conditions: For each


sample, 5 µl of extract or standard solution were injected. Chromatography was performed with Ultimate 3000 (Dionex). The chromatographic column used was a C18 column. After a period of three minutes of trapping on the meadow column, a gradient of 2.5 to 60% acetonitrile was applied for 30 minutes, followed by a gradient of 60 at 80% acetonitrile for five minutes with a flow rate of 300nl/minute. Data acquisition was made with Q-Exactive Plus (Thermo Scientific).


Mass spectrometry analysis for AHL, PQS and AIP quantification ■ Voltage source: 2500V ■ Sweeping gas: 0psi ■ Temperature of the transferred tube: 275°C ■ Collision energy: 28 normalized collision energy


Data analysis for AH, PQS and AIP Data and integration of XIC ‘eXtract Ion Chromatogram’ was made using Xcalibur 4.0 (Thermo Scientific). The XICs (or eXtract Ion Chromatogram) are


generated by plotting a curve whose ordinate is the intensity signal, intensity for a given m/z value or for a set of values on a continuous MS


Statistics: n=4 (**) P<0.01


0.5 0.4 0.3 0.2 0.1 0


Untreated control


-32% (**) -67% (***)


One Way ANOVA (***) p<0.001


spectra, and the abscissa is the retention time. For AIP analysis the chosen value was


961.3794 Da (AIP-I) corresponding to the monoprotonated and monoisotopic mass of the molecule of interest. The chosen extraction window was +/- 4ppm. AIP concentration in fmol/µL of S. aureus supernatant culture was deducted from standard curve obtained with synthetized AIP. For P. aeruginosa QS molecules, the


values chosen was 172.0968 Da (C4-HSL); 298.2011 Da (3-oxo-C12-HSL) and 260.1646 (PQS) corresponding to the monoprotonated monoisotopic mass of the molecules of interest. The chosen extraction window was +/- 3 ppm. QS molecules concentration in P.


aeruginosa supernatant culture were deducted from standard curve obtained with commercially available 3-oxo-C12-HSL, C4-HSL and PQS in pmol/µL


Results and statistics Results were expressed as mean percentage of six replicate ± Standard Deviation SD; untreated control was standardized to 100%. The statistical analysis was done using Student t test versus untreated control following Sigmaplot software recommendation (Systat Software Inc, USA). The threshold of significance was set to 5% (p<0.05).


Pseudomonas aeruginosa motility evaluation Bacterial culture P. aeruginosa ATCC15602 was cultured in Luria Bertani (LB) medium, at 37°C for 24 hours. Bacterial cells were rinsed with PBS and 5µl of bacteria suspension were placed on soft agar plates (Minimal Medium M63, Glucose 2 g/L, 0.3% agar) in the centre of the plate without any additional product (untreated control) and with Litchi sinensis at 0.2% and 0.4%. Furanone at 0.1µg/mL was used as positive inhibition control. Each condition was tested in quadruplicate.


0.2% Litchi extract Figure 2: Litchi sinensis extract decreases P. aeruginosa swarming PERSONAL CARE February 2024 0.4%


Bacterial swarming evaluation After 24 hours of culture, bacterial swarming was measured by image analysis which allowed to measure area of bacteria spreading on agar plate in square centimetre (cm2


).


Results & Statistics Results are expressed as mean area spreading ± standard deviation of quadruplicate


www.personalcaremagazine.com 0.2% Litchi extract


Student t test vs untreated control (*) p<0.05


(***) p<0.001


Inhibition of P.aeruginosa PQS secretion


-31% (***)


0.4%


Mean bacteria surface (cm2


)


3-oxo-C 12 HSL mean release (%)


C4-HSL mean release (%)


PQS mean release (%)


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