C18 solid phase extraction column

Fresh plant parts of Hibiscus sabdariffa were extracted with water for 15 min at room temperature in 1:10 ratio. The aqueous extract was vortexed vigorously and centrifuged at 16,000 × g for 5 min at 4 °C and subjected to flash chromatography by C18 solid phase extraction (SPE) columns (Waters, USA). The fractions were eluted with 60% ethanol/0.01% trifluoroacetic acid (TFA) and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) (AB SCIEX 4700 MALDI-TOF/TOF).

Fresh Pereskia bleo plant parts were extracted with water for 15 min at room temperature at a 1:10 ratio. The aqueous extracts were vortexed vigorously and centrifuged at 16000 × g for 5 min at 4°C before being subjected to flash chromatography using C18 solid phase extraction (SPE) columns (Waters, United States). The fractions were eluted with 60% ethanol and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS; AB SCIEX 4700 MALDI-TOF/TOF). The MALDI spectra were acquired in the m/z range of 500–6000, with a focus m/z 3500. Total laser shots were 2250 with a laser intensity of 3500.

All reagents for liquid chromatography (LC) and mass spectrometry (MS) analysis were of LC-MS grade and purchased from Merck (Darmstadt, Germany). Trypsin Gold was purchased from Promega (Madison, WI, USA). C18 solid phase extraction columns were purchased from Waters Corporation (Milford, MA, USA). Millipore Protease Inhibitor Cocktail Set I was purchased from Merck (Darmstadt, Germany). Invitrogen SuperScript™ IV First-Strand Synthesis System and Applied Biosystems PowerUp SYBR Green PCR Master Mix were purchased from Thermo Fisher Scientific (Vilnius, Lithuania). Ambion TRIzol Reagent was purchased from Sigma-Aldrich (St. Louis, MO, USA). Invitrogen nucleophosmin monoclonal antibody (NA24), Invitrogen natriuretic peptides A polyclonal antibody, Pierce BCA Protein Assay Kit and Pierce ECL chemiluminescence kit were purchased from Thermo Scientific (Rockford, IL, USA). The moesin polyclonal antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). The S100A8/A9 inhibitor (ABR-238901) was a gift from Active Biotech AB (Lund, Sweden).

ACQUITY UPLC XEVO TQ-S liquid chromatography-tandem mass spectrometer (with electrospray ion source) was from Waters Corporation (Milford, MA, USA). MS2 vortex mixer was from IKA (Staufen, Germany). Centrifuge 5810 high-speed centrifuge was from Eppendorf Corporation (Hamburg, Germany). R-201 rotary evaporator was from Shanghai Kexing Instrument Co., Ltd. (Shanghai, China); N-EVAP 112 nitrogen blowing instrument was from Organomation Company (Berlin, MA, USA); 12-channel solid-phase extraction device was from Supelco company (Bellefonte, PA, USA); Ultrasonic Cleaner was from Shanghai Kedao ultrasound Instrument Co., Ltd. (Shanghai, China); Organic microporous membrane (0.22 μm) was from Tianjin Jinteng test equipment Co., Ltd. (Tianjin, China); AL 204 electronic balance with sensitivity of 0.1 mg, SL-502N desktop balance with sensitivity of 0.01 g; Amino solid phase extraction column (60 mg/3 mL), C18 solid phase extraction column (1 g/6 mL), MAX (60 mg/3 mL), HLB (200 mg/6 mL) and PRIME HLB SPE solid phase extraction column (60 mg/3 mL) were from Waters Corporation (Milford, MA, USA).