Sodium taurocholate

Sodium taurocholate, cholesterol, sodium chloride (NaCl), sodium oleate, ammonium formate, formic acid, potassium hydroxide (KOH), hydrochloric acid (HCl), griseofulvin, furosemide, dipyridamole, and acyclovir were purchased from Merck Chemicals Ltd. Ibuprofen was obtained from BSAF chemical company, Paracetamol was from Mallinckrodt Pharmaceuticals and mefenamic acid from Sigma Aldrich. Phosphatidylcholine from soybean (PC S) was purchased from Lipoid company. See Table 1 for physicochemical properties and molecular structures. Chloroform was from Rathburn Chemical Company, FaSSIF media was purchased from Biorelevant.com, and sodium phosphate monobasic monohydrate (NaH₂PO₄·H₂O) was purchased from Fisher Scientific. All acetonitrile (ACN) and methanol (MeOH) solvents were HPLC gradient (VWR). All water is ultrapure Milli-Q water.Table 1

Physicochemical properties and molecular structures of drugs.

Compound	a/b/n	pKa	LogP	Structure
Ibuprofen	a	5.3	3.97
mefenamic acid	a	4.2	5.12
furosemide	a	3.9	2.03
dipyridamole	b	6.2	3.77
Paracetamol	n	–	0.46
griseofulvin	n	–	2.18
acyclovir	n	2.52/9.35	−1.56

This method was modified from (31 (link)). C. difficile was grown on BHI agar overnight at 37°C. A single colony from the BHI agar plate was inoculated in 10 ml of BHI broth with 0.5% yeast extract and 0.1% L-cysteine (Merck Millipore, Darmstadt, Germany) and incubated at 37°C overnight under anaerobic conditions, and 1 ml of the BHI culture was sub-cultured into BHI agar with 0.1% L-cysteine and incubated at 37°C in an anaerobic jar for 7 days. After 7 days of incubation at 37°C, the sporulation efficiency was confirmed by phase-contrast microscopy and measurement of heat-resistant CFU and spore crops harvested immediately or after overnight incubation at 4°C. The spores were washed in PBS two times; suspended in PBS containing 125 mM Tris, 200 mM EDTA, 0.3 mg/ml proteinase K (Amresco, USA), and 1% sarcosyl; and incubated with gentle shaking at 37°C for 2 h. The spores were centrifuged (6,500 × g, 10 min) and the pellet was resuspended in water and washed 10 times. After the final suspension in water, the spores were heat-treated (60°C, 20 min) to kill any residual cells. The spore supernatants were stored at 4°C until testing. To calculate the spore CFU, aliquots were serially diluted in PBS and plated onto BHI agar supplemented with 0.1% sodium taurocholate (Merck Millipore). The plates were incubated for 48 h before the enumeration of CFU.

Sodium taurocholate, cholesterol, sodium chloride (NaCl), sodium oleate, ammonium formate, potassium hydroxide (KOH), hydrochloric acid (HCl), naproxen, phenytoin, piroxicam, fenofibrate, probucol, griseofulvin, carvedilol, tadalafil, and indomethacin were purchased from Merck Chemicals Ltd. Aprepitant and felodipine were provided through OrBiTo by Dr. R. Holm, Head of Preformulation, Lundbeck, Denmark. Zafirlukast was purchased from Stratech Scientific Ltd. Phosphatidylcholine from soybean (lecithin) was purchased from Lipoid company. Chloroform from Rathburn Chemical Company. FaSSIF-v1 media was purchased from Biorelevant.com Ltd. Sodium phosphate monobasic monohydrate (NaH2PO4·H2O) and formic acid from Fisher Scientific. All acetonitrile (ACN) and methanol (MeOH) solvents were HPLC gradient (VWR). All water was ultrapure Milli-Q.

The cholesterol assimilation by L. casei with/without different treatments with laser was determined using MRS broth medium supplemented with water soluble cholesterol (Sigma-Aldrich; St. Louis, MO, USA) at final concentration of 2 mg/mL in addition to 0.2% sodium taurocholate (Merck; Darmstadt, Germany) as previously described by [10 ,25 ] where the supplemented medium was inoculated by a 2% activated L. casei culture and incubated for 18 h at 37 °C to mimic the intestinal conditions. The L. casei suspensions were centrifuged at 14,000 rpm at 4 °C for 10 min to collect the supernatant. A calorimetric method was used to investigate the cholesterol residues by measuring the absorbance at 550 nm [26 (link)].

Nimesulide was gifted from PharmEvo Pakistan. Microcrystalline cellulose (Avicel PH102) and carboxymethylcellulose (Ac-di-sol®) were purchased from FMC, Brussels, Belgium. Octadecanoic acid, magnesium stearate, and sodium lauryl sulphate were purchased from RDH Germany. Hydroxypropyl methylcellulose (Methocel K4M) was gifted from Colorcon, USA. Potassium dihydrogen phosphate, Hydrochloric acid, Sodium hydroxide, Sodium Chloride, sodium taurocholate, citric acid, lecithin, disodium hydrogen Phosphate, Triton X 100, sodium dihydrogen phosphate, glacial acetic acid, Sodium hydroxide pellets, citric acid, ammonium acetate and HPLC grade, all were purchased from Merck, Darmstadt, Germany.

To establish a rat model of SAP, experimental rats were treated with 5% sodium taurocholate (Sigma, United States) at a dose of 1 mL/kg body weight, via a slow biliopancreatic duct injection using a micro-infusion pump at a rate of 0.2 mL/min. Blood was taken from the abdominal aorta 12 h after treatment. Blood samples were placed at room temperature for 2 h, and the serum was obtained. The amylase (Amy) value was usually 3–5 times higher in the SAP rats compared to the normal rats. SAP rat serum was filtered through a 2 μm filter, followed by adding DMEM medium at a 10-fold dilution (i.e. 10% concentration).