2.3. Preparation and Characterization of CL Plant Extract

Rhizomes of CL collected from India were dried under shade and finely powdered. A volume of 50 g of biomass was extracted in 100% methanol solution with cycles of sonication at room temperature (RT), in the dark. Following filtration, the extract was concentrated using a rotary evaporator and stored at −80 °C. After lyophilization, CL was kept at RT in the dark and protected from moisture. For CL extract characterization, the liquid phase was filtered (Ø 20 µm), and the sample in the proportion 1:10 (extract: methanol) was analyzed by HPLC-DAD (HITACHI, LabChrom Elite, Tokyo, Japan) and monitored by the computer software EZChrome elite (Agilent Technologies, v 3.02, Santa Clara, CA, USA). The compounds separation was performed on a reversed phase LiChroCART 250-4 column (Phosursohere. RP-18e. 5 μm, Merck, Darmstadt, Germany) at RT, using acetonitrile containing 0.1% (v/v) formic acid (ACN–FA) and ultrapure water containing formic acid 0.1% (v/v) (upW-FA), as the mobile phases. The flow rate was 0.8 mL/min, and the elution gradient was 5% (v/v) of ACN-FA at time 0 min, 30% (v/v) of ACN-FA at time 30 min, 90% (v/v) of ACN-FA at time 40 min, and 95% (v/v) of upW-FA at time 60 min. Spectral data from all compounds were accumulated in the range of 230–550 nm, and chromatograms were recorded at 400 nm. CC (and other curcuminoids) were quantified at 400 nm by the external method, using a commercial curcumin standard (>65% purity).