*3.4. Analysis of Flavonoids in the Extracts by HPLC*

Without any doubt, pharmacological activity of *P. cretica* depends on its chemical constituents. Therefore, flavonoid profiles in the *P. cretica* extracts were analyzed by HPLC. Figure 3 indicated that the HPLC chromatograms of extracts obtained by UAE and HRE showed highly similar characteristics. A total of five flavonoids, including luteolin-7-*O*-glucoside, rutin, quercetin, luteolin, and apigenin, were identified, and their elution times were 37.340, 39.021, 43.031, 50.568, and 54.306 min, respectively. Based on the results of HPLC quantitative analysis, the contents of the five flavonoids (mg/g plant material, dry weight) were in the following descending order: Luteolin-7-*O*-glucoside (3.237 ± 0.015 mg/g) > rutin (1.483 ± 0.009 mg/g) > apigenin (0.803 ± 0.013 mg/g) > luteolin (0.679 ± 0.007 mg/g) > quercitrin (0.582 ± 0.011 mg/g). However, the contents of five flavonoids extracted by HRE were all lower compared to the UAE: Luteolin-7-*O*-glucoside (2.862 ± 0.005 mg/g) > rutin (1.133 ± 0.007 mg/g) > apigenin (0.659 ± 0.011 mg/g) > luteolin (0.486 ± 0.009 mg/g) > quercitrin (0.574 ± 0.013 mg/g). Thus, it could be concluded that the UAE was a promising extraction method for the separation of bioactive flavonoids from *P. cretica* in the future. To our knowledge, this work was the first quantitative analysis of these five flavonoids in *P. cretica*.

**Figure 3.** HPLC profiles of standards mixture (**a**) and *P. cretica* extract obtained by optimized ultrasonic-assisted extraction (UAE) (**b**). *P. cretica* extract obtained by heat reflux extraction (HRE) (**c**). Peaks (1): Luteolin-7-*O*-glucoside, (2) rutin, (3) quercitrin, (4) luteolin, and (5) apigenin.
