Adc 2 developing chamber

Phospholipids were isolated and quantified as described recently (20 (link), 21 (link)). Bacterial overnight cultures grown in MHB to an optical density at 600 nm (OD₆₀₀) of 0.05 were incubated in 100 ml fresh MHB until the exponential-growth phase (OD₆₀₀ of 0.5 to 1) was reached. After adjusting S. aureus strains to equal optical densities, the Bligh-Dyer method (37 (link)) was used to extract lipids with a chloroform-methanol-sodium acetate buffer (20 mM, pH 4.6) mixture (1:1:1 [vol/vol/vol]). Isolated lipids were vacuum dried, resuspended in chloroform-methanol (2:1 [vol/vol), and spotted onto silica gel 60 F254 high-performance thin-layer chromatography (HPTLC) plates (Merck, Darmstadt, Germany) with a Linomat 5 sample application unit (Camag, Berlin, Germany). Polar lipids were separated in an ADC 2 developing chamber (Camag, Berlin, Germany) with a chloroform-methanol-water (65:25:4 [vol/vol/vol]) running solvent. Phospholipids were detected by staining of phosphate groups with molybdenum blue, and the LysPG content was determined in relation to the total phospholipid content by densitometry analysis performed with ImageJ (http://rsbweb.nih.gov/ij/docs/guide/index.html) as described recently (20 (link), 21 (link)).

Lipase from porcine pancreas (type VI-S L0382-100KU), orlistat (04139-25 mg) and 4-methylumbelliferyl oleate (4-MUO 75164-25 mg) were obtained from Sigma-Aldrich Australia. Chemical references EGCG, ECG, EC, caffeine, rutin, crocin, solvents used for high-performance thin layer chromatography (HPTLC) mobile phase (i.e. toluene, acetone, formic acid, ethyl acetate, glacial acetic acid, chloroform, methanol and ethanol), derivatization reagents: Fast Blue Salt B, Natural Products Reagent (NP reagent), Polyethylene glycol (PEG solution), 10% sulphuric acid reagent and p-anisaldehyde-sulfuric acid reagent were also purchased from Sigma-Aldrich, Australia. The RCM-107 capsules (AUST L 285569) were obtained from Tong Kang Lee Chinese medicine clinic and single herbal granules (extracted products) of Fu ling/Poria, He Ye/lotus leaf, Ze Xie/Alismatis rhizoma, Che Qian Zi/Plantaginis semen, Jue Ming Zi/Cassiae semen, Huai Hua/Sophorae flos, Zhi Zi/Gardeniae fructus, green tea (matcha) were supplied by GL natural health care Chinese medicine clinic. HPTLC glass plates 20 × 10 cm coated with silica gel 60 F₂₅₄ were produced by Merck. Chromatographic equipment (Linomat 5 sample applicator; ADC 2 developing chamber; automated derivatizer; visualizer; VisionCATS 2.4 software) were supplied by CAMAG. Molecular docking was conducted via virtual screening software PyRx (Version 0.8).