Zorbax

Curcumin was dispersed in
soybean oil, and the mixture was stirred for 24 h for complete dissolution.
The supernatant was obtained by centrifugation, and the concentration
of curcumin in soybean oil was determined by HPLC (Agilent ZORBAX,
Agilent, USA) with an isocratic elution of methanol–water (88/12,
v/v) as the mobile phase. The flow rate was 1 mL/min, and the column
temperature was maintained at 40 °C. The detection wavelength
was set at 430 nm. Curcumin-encapsulated LPE (LPE@Curcumin) was prepared
by processing the oil phase (curcumin in soybean oil) and water phase
(0.1 wt % QAL, pH 7) in the Ultra-Turrax homogenizer for 2 min.
The in vitro release analysis was conducted according
to a previously reported method.^{31 (link)} Then,
0.5 mL of LPE@Curcumin was dispersed in 40 mL of phosphate-buffered
saline solution (pH 7.4) and acetate-buffered solution (pH 5.0) with
0.5 wt % SDS. The sample was placed in a shaking incubator at 37 °C
with shaking at 100 rpm. At specific time intervals, 1 mL of the mixture
was collected and an equivalent volume of buffer solution was added
to ensure the maintenance of the total volume. The curcumin concentration
was determined by HPLC. Three replicates were conducted for all of
the samples.

Vitamin C contents were determined as previously reported [17 ] with the utilization of high−performance liquid chromatography (HPLC). The samples were extracted with 10% (v/v) metaphosphoric acid before loading onto an ODS column (5 μm, 250 × 4.6 mm, Zorbax from Agilent Technologies, Santa Clara, CA, USA) attached to the HPLC system consisting of a Waters 515 pump (Waters Corporation, Milford, MA, USA) and a UV−975 UV/Vis detector (JASCO International Co., Ltd., Tokyo, Japan). Vitamin C was detected at 254 nm with an isocratic solvent system of 0.5% (v/v) KH₂PO₄ and a flow rate of 0.8 mL/min.

The vacuum-dried trypsin-digested peptides were labeled with an 8-plex iTRAQ (Sciex, USA) reagent, and the reaction was stopped by the addition of double-distilled H₂O for 30 minutes. All labeled samples were then placed in a test tube, centrifuged, and dissolved in 110 µL of mobile phase A (10 mM ammonium formate, 5% aqueous acetonitrile, pH, 10.0) in a high pH reverse column (Agilent, ZORBAX). One-dimensional fractionation was performed on a high-pH reverse column (Agilent, ZORBAX Extended-C18 2.1) using a separation gradient of buffer B (10 mM ammonium formate, 90% aqueous acetonitrile, pH, 10.0) for 40 minutes and increased from 5% to 30% linearly at a flow rate of 0.3 mL/min. Each tube was collected every 1 minute for a total of 40 tubes. Four tubes were combined into one component from the first tube, and 10 in total were obtained. The components were subjected to centrifugal drying for LC-MS analysis.