Sodium chloride (nacl)

Bovine face pieces were purchased from a local tannery. Scyliorhinus canicula fish skin waste was collected from the fish market, Monastir, Tunisia, and stored at −20°C until used. Sodium hydroxide (NaOH), sodium chloride (NaCl), and butanol (99%) were purchased from Loba Chemie, and acetic acid was purchased from Merck India Ltd. Acrylamide, bis-Acrylamide, Tris-HCl, SDS (sodium dodecyl sulfate), ammonium persulfate, TEMED (tetraacetylethylenediamine), glycerol, β-mercaptoethanol, bromophenol blue, and Coomassie R-250 were purchased from Sigma-Aldrich.

Bimatoprost was obtained from Dr. Pradeep Reddy Laboratories (Hyderabad, India). Glyceryl monostearate (GMS), chloroform, dimethyl sulfoxide (DMSO), sodium chloride, dibasic sodium phosphate and benzalkonium chloride were procured from Loba Chemie (Mumbai, India).

The β‐carotene was determined as described by Perez‐Lopez (2010 (link)) and Association of Official Agricultural Chemists (AOAC) (1980 ) with some modifications. Five grams (5 g) of the sample (raw, pickle, and relish products) in 1% butylated hydroxytoluene (BHT) (LOBA Chemie, India) was homogenized using a blender (Moulinex, China) for 5 min and placed into the falcon tube (BR Biochem, India). The homogenate was treated with 30 ml of solvent (50% hexane:25% acetone:25% ethanol) (LOBA Chemie, India) and then centrifuged at 4000 rpm at ambient temperature for 10 min. The supernatant was collected and filtered using Whatman No. 1 filter paper (Whatman International Ltd., India), and then was re‐extracted two times until the residue was colorless. The supernatant was combined, and the volume was recorded. The supernatant was saponified with 40% potassium hydroxide (LOBA Chemie, India) and left at ambient temperature for 12 h. The mixture was then transferred into the separating funnel and treated with the same volume of 10% w/v sodium chloride (LOBA Chemie, India) for removing moisture. The mixture was then shaken vigorously, and the upper phase was collected and dried over anhydrous sodium sulfate (LOBA Chemie, India). The β‐carotene content was estimated based on the standard curve of β‐carotene, and the result was expressed as mg/100 g (wet basis).