Potassium dihydrogen phosphate

Raloxifene HCl (RLX), USP 99.7% purity, batch number CFR018007 was purchased from BDR Pharmaceuticals International (Mumbai, India). Cremophor^® RH40 (polyoxyl 40 hydrogenated castor oil) was kindly provided by BASF SE (Ludwigshafen, Germany). Transcutol^® HP was a kind gift from Gattefosse (Lyon, France). Miglyol^® 812 (caprylic/capric triglyceride) was a kind gift from EIPICO for pharmaceutical industries (Cairo, Egypt). Hydroxypropyl methyl cellulose (HPMC), methocel™ K100LV (100 cP at a 2% addition rate in water), was purchased from Chempoint (Bellevue, WA, USA). Poly vinyl alcohol (PVA) (average Mw 146,000–186,000), chitosan (CS) high molecular weight (800–2000 cP), and acetonitrile were purchased from Sigma–Aldrich, Co. (St. Louis, MO, USA). Normal saline was purchased from Otsuka Pharmaceutical Co., S.A.E (Cairo, Egypt). Xylazine (XYLA-JECT^®) was purchased from ADWIA Pharmaceuticals Co. (Cairo, Egypt). Ethyl acetate and formic acid were bought from El-Nasr Pharmaceutical Chemicals Co. (Cairo, Egypt). Olmesartan obtained from AstraZeneca (Cairo, Egypt). Potassium dihydrogen phosphate and disodium hydrogen phosphate were purchased from Sisco Research Laboratories (Mumbai, India). HPLC-grade methanol was purchased from Research-lab Fine Chem Industries (Mumbai, India). All other chemicals and solvents were of analytical grade and used as received.

The chemicals utilized during study: ammonium sulphate, 2,4-dinitro-phenyl-hydrazine, 1-chloro-2,4-dinitrobenzene, ethylenediaminetetraacetic acid, 5,5-di-thio-bi(2-nitrobenzoic acid), N-ethylmaleimide, nitro blue tetrazolium, reduced nicotinamide adenine dinucleotide, potassium dihydrogen phosphate, phenazinemethosulphate, sodium pyrophosphate, reduced glutathione, sodium azide, thiobarbituric acid, 5-thio-2-nitrobenzoic acid and trichloro acetic acid were procured from Sisco Research Laboratory, India. Bovine serum albumin, Bradford reagent and Pb-acetate were purchased from Sigma-Aldrich, St. Louis, USA. All primary antibodies (produced in rabbit) viz. anti-Bcl-2 (dilution 1:1000), anti-Bad (dilution 1:3000), anti-Cyt C (dilution 1:1000), anti-Apaf-1 (dilution 1:1000), anti-caspase 3 (dilution 1:1000), anti-caspase 8 (dilution 1:1000), anti-caspase 9 (dilution 1:1000), anti-Fas (dilution 1:2000), anti-Bid (dilution 1:1000) and anti-actin (dilution 1:3000) for immunoblotting were purchased from Sigma-Aldrich Chemical Company St. Louis, USA. Appropriate HRP conjugated secondary antibody (dilution 1:3000) produced in goat was also purchased from Sigma-Aldrich Chemical Company St. Louis, USA. Methanol, formic acid, acetic acid and acetonitrile were obtained from Merck, India.

Bovine serum albumin (BSA), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Bradford reagent, Collagenase type I, antibodies for immunoblotting were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO), USA. Solvents (HPLC grade) were obtained from Merck, Mumbai, India. Kits for measurement of serum biochemical parameters were purchased from Span diagnostic Ltd., India. 1-Chloro-2,4-dinitrobenzene, ammonium sulphate, sodium arsenite (NaAsO₂), 2,4-dinitrophenylhydrazine, ethylene diaminetetraacetic acid, 5,5-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, nitro blue tetrazolium, reduced nicotinamide adenine dinucleotide, potassium dihydrogen phosphate, phenazinemethosulphate, sodium pyrophosphate, reduced glutathione, sodium azide, thiobarbituric acid, 5-thio-2-nitrobenzoic acid and trichloro acetic acid were purchased from Sisco Research Laboratory, Mumbai, India.

The composition of Klebsiella blue agar media per litre of distilled water is as follows: solution A — 3 g potassium dihydrogen phosphate (Sisco Research Laboratories Pvt. Ltd.), 6 g dipotassium phosphate (EMPLURA, Merck Life Science Pvt. Ltd.), 6 g sodium chloride (Sigma Aldrich), 64 mg methylene blue (Spectro Chem Pvt. Ltd.); solution B — 100 mg magnesium sulphate dihydrate (MERCK Specialties Pvt. Ltd.) and 17 g agar (Hi Media Laboratories Pvt. Ltd.). Solutions A and B were separately autoclaved at 121 °C for 20 min. On cooling, filter sterilised bile salt, tryptophan, and glycerol 1.5 g/L, 2 g/L, and 0.2%, respectively, were added to solution A. KBA was constituted by mixing solutions A and B and poured onto sterile Petri dishes. The Luria Bertani broth (LB) (Hi Media Laboratories Pvt. Ltd.) and HiCrome™ Klebsiella Selective agar Base (HiMedia Laboratories Pvt. Ltd.) were used for comparison.

Etodolac was kindly gifted from European Egyptian Pharmaceutical Industries, Egypt and manufactured by Polpharma Pharmaceutical Works, Warsaw, Poland. Poloxamer 407 (Plx 407; Pluronic acid F127), phosphotungstic acid and MO were obtained from Sigma–Aldrich Chemie GmbH, Germany. Dialysis tubing cellulose membrane (molecular weight cutoff 12,000 g/mole) was purchased from Sigma Chemical Company (Sigma-Aldrich Corp., St. Louis, MO, USA). Poly(vinyl alcohol) (PVA; M.Wt. 22000 Da) was purchased from Sigma-Aldrich (Sigma-Aldrich Co., St. Louis, MO, USA). Potassium dihydrogen phosphate and disodium hydrogen phosphate were purchased from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. HPLC grade of acetonitrile and methyl-t-butyl-ether were purchased from SDFCL – S. D. Fine Chemical Limited, Mumbai, India.