*2.6. HPLC Analysis*

The analysis of all the extracts, both from solvent extraction and belonged from SFE, was carried out by means of HPLC using a Smartline HPLC system (Knauer, Germany). Chromatographic separation was carried out using a 2.0 mm ID × 150 mL, with precolumn, C-18 TSKgel ODS-100 V, 21810 (TOSOH BIOSCIENCE), both thermostatically at 40 ◦C. The operative conditions: mobile phase, flow rate and gradient of elution utilized are reported in Table 2. For the mobile phase, methanol and water with trifluoroacetic acid (TFA) were used.


**Table 2.** The mobile phase, flow rate and gradient of elution utilized.

Absorbance spectra were recorded every 2 s, between 200 and 450 nm, with a bandwidth of 4 nm, and chromatograms were acquired at 254 and 280 nm. HPLC analysis was performed in duplicate.

A wavelength of 280 nm was used for quantification [18], while the calibration line was obtained from the integration of the absorption peaks obtained from a series of dilutions of Rutin, Isoquercitrin, Nicotiflorine and Narcissin.

### **3. Results and Discussion**
