Gx 271

LH20 F1 was subjected to semi-preparative HPLC, equipped with a Gilson 321 pump, a Gilson GX-271 liquid handler with a 2 mL sample loop and a Gilson Prep ELS™ II detector. Pinnatifidanoside D (9.1 mg) was isolated in an isocratic mode using CH₃CN:H₂O:FA (13:87:0.1, v/v), at 4 mL min⁻¹, on Atlantis Prep T3 at 40 °C.

Different fractions of the L. citriodora extract were recovered using a Gilson preparative HPLC system (Gilson Inc., Middleton, WI, USA) which equipped with automated liquid handling solutions (model GX-271), a binary pump (model 331/332) and UV-vis detector (model UV-Vis 156). The solvent of the collected fractions was evaporated in a Savant SpeedVac Concentrator SC250 EXP (Thermo Scientific, Waltham, MA, USA).
The composition of the whole extract and the fractions was analyzed through a HPLC-ESI-TOF-MS system. The chromatographic separation was carried out using an Agilent 1200 series rapid-solution LC equipment (Agilent Technologies, Palo Alto, CA, USA) with an autosampler, a binary pump and a diode-array detector (DAD).
Both HPLC systems were coupled to a time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) with an electrospray ionization (ESI) interface (model G1607A, Agilent Technologies, Palo Alto, CA, USA). Calibration solution was injected at the beginning of each analysis by a 74900-00-05 Cole-Palmer syringe pump (Cole-Palmer, Vernon Hills, IL, USA) directly connected to the interface.
Finally, absorbance and fluorescence measurements for the antioxidant assays were performed on a Synergy Mx Monochromator-Based Multi-Mode Micro plate reader (Bio-Tek instruments Inc., Winooski, VT, USA) in 96-well microplates.

Two cores (8 mm in diameter) of bacteria and agar were obtained from each plate, placed directly into MeOH (2 mL), and extracted for 30 min. The extract was transferred into a clean glass vial and evaporated using a SpeedVac concentrator; the extract was dissolved in MeOH (100 μL) and diluted with H₂O (1 mL). The solution was then placed on a Gilson GX-271 liquid handling system for automated SPE (Biotage: EVOLUTE ABN, 25 mg absorbent mass, 1 mL reservoir volume), washed using H₂O (1 mL) to remove media components, and eluted with MeOH (1 mL) directly into an LC-MS-certified vial.

Electrochemical bulk measurements were carried out in a homemade H-cell. Each compartment was filled with 28 ml 0.1 M H₂SO₄ (Merck Suprapur diluted with Merck Milli-Q) before the experiment. The working electrodes and reference electrodes (Basi, 3 M Ag/AgCl) were immersed in one compartment whereas the counter electrodes (glassy carbon, SIGRADUR G, HTW) were immersed in the other compartment. The compartments were covered with Parafilm to avoid evaporation of electrolytes. Convection in the system for equal distribution of dissolution product was enabled through Ar-purging of the anode compartments. Samples were taken by an automated liquid handler (Gilson GX-271). The electrochemical protocol (Gamry Interface1000 B) was started after the first sample was extracted. The total volume of electrolyte in both compartments was kept between the initial 28 ml and 24 ml at any time.

Optical rotations were measured at 20 °C in acetonitrile using an Anton Paar MCP 300 polarimeter in a 100-mm-long 350 μL cell. UV spectra were recorded at 20 °C in acetonitrile or methanol using a PerkinElmer Lambda 5 spectrophotometer. Electronic circular dichroism spectra were acquired at 20 °C in acetonitrile on a JASCO J-810 spectropolarimeter. NMR spectra were recorded on Bruker 300, 500, 600 and 700 MHz spectrometers (Bruker, Rheinstetten, Germany). The chemical shifts (δ) are reported as ppm based on the solvent signal, and coupling constants (J) are in hertz. Preparative HPLC was conducted with a Gilson system equipped with a 322 pumping device, a GX-271 fraction collector, a 171 diode array detector and a prep ELSII. All solvents were HPLC grade, purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France).

All reactions were carried out under argon atmosphere. Preparative high-performance liquid chromatography (HPLC) was performed using a Gilson 331 and 332 pumps with a UV/VIS-155 detector and GX-271 liquid handler. Column was Phenomenex Luna LC Column (5 µm C18 100 Å, 150 × 21.2 mm). ¹H NMR spectra were recorded on a 300 MHz INOVA VARIAN spectrometer. Chemical shift values are given in ppm and referred as the internal standard to TMS (tetramethylsilane). The coupling constants (J) are reported in Hertz (Hz). Mass Spectra were obtained on an Agilent 6120 mass spectrometer with electrospray ionization source (1200 Aligent LC-MS spectrometer, Positive). Mobile phase flow was 1.0 mL/min with a 3.0 min gradient from 20% aqueous media (0.1% formic acid) to 95% CH₃CN (0.1% formic acid) and a 9.0 min total acquisition time. All the tested compounds possess a purity of at least 95%, which was determined by LC/MS Data recorded using an Agilent 1200 liquid chromatography and Agilent 6120 mass spectrometer, and further supported by clean NMR spectra. Generation of these tested compounds was described in the Supplementary Method.

Evaluation of the SCFA concentrations in colonic digesta was performed adopting the procedure of Loo et al. [40 (link)] with minor changes. After colonic fermentation, supernatants (1.5 mL) collected after centrifugation were combined with four volumes of an internal standard mixture containing 1.59 mmol/L of 4-methyl valeric acid mixed with formic acid and orthophosphoric acid (1% both) and vortexed for 30 s. From the final mixture, 1 mL of each sample was dispensed into 1.5 mL flip capped tubes and centrifuged (10,000× g, 10 min at 4 °C). The supernatants were separated and stored at 4 °C until analysis. Acetic, butyric, propionic, iso-valeric, valeric, iso-butyric, and heptanoic acids were used as analytical standards to create the standard curves [44 (link)].
Aliquots (2 μL) of the sample and standards were injected into a gas chromatograph (7890B Agilent, CA, USA) that was fitted with a capillary column of 12 × 0.53 mm internal diameter (ID) and a film thickness of 0.5 µm (SGE BP21, SGE International, Ringwood, VIC, Australia, P/N 054473), a flame ionisation detector (FID), an autosampler (Gilson GX-271, Gilson Inc., Middleton, WI, USA), and an autoinjector. The FID and injection port were set at temperatures of 240 and 200 °C, respectively. Helium was applied as a carrier gas at a flow rate of 14.4 mL/min along with hydrogen, nitrogen, and air as makeup gases.