Lutein standard

Lutein content was determined using reverse phase High Performance Liquid chromatography (RP-HPLC-Shimadzu-LC2010 with UV detector, Japan) following the method of Leema et al., [35 (link)]. For sample preparation, a pre-weighed amount of lyophilized (-52 °C, Virtis, USA) C. vulgaris biomass (20 mg) was suitably disrupted and saponified at 40 °C for 30 min by the addition 2 ml of 10 M KOH with 2.5 % ascorbic acid. The reaction was stopped by cooling in ice and Lutein was extracted by the addition of 18 ml of methanol: dichloromethane (3:1 v/v). The mixture was centrifuged at 2500 rpm for 15 min for removing the supernatant, rotary evaporated (Buchi, Switzerland) and reconstituted with 2 ml of HPLC mobile phase. The samples from different treatments and standard Lutein (Sigma, USA) were filtered through a 0.22 μM syringe filter (acrodisc) prior to injection. The Lutein content from different treatments were analyzed using reverse phase C-18 column (Phenomenex, Luna, USA, 4.6 × 25 × 250 mm, 5 μm particle size) with isocratic solvent system methanol: dichloromethane:acetonitrile:water (67.5:22.5:9.5:0.5, v/v) at a flow rate of 1 mL/min at 450 nm. The calibration curve for standard Lutein (Sigma, USA) was prepared for identification and quantification of Lutein present in the extract.