Identification and Mobility Study of Three Main Flavonoids from "Passiflora ligularis" leaves by Capillary Electrophoresis

Passiflora species are widely distributed throughout Latin America. Previously studies in our research group showed evidences of a hypoglycemic and antidiabetic effects of leaf extract from P. ligularis. The aim of this study was to develop and to optimize a Capillary Electrophoresis (CE) method for the analysis of Passiflora ligularis flavonoids.
Dried leaves were extract by infusion (plant:water, 1:10w/v, 95°C) for 10min, filtered and then lyophilized. All the experiments were carried out in Agilent 7100 CE equipped with DAD detector (diode array detector), control temperature and auto-sampler devise. The data were processed in the software Agilent ChemStation®. For the first use, the capillary was pretreated with a pressure flush with 1M NaOH solution (30 min). Daily the capillary was conditioned: 0.1M NaOH (5 min), waiting time (1 min), Milli-Q water (5 min) and running buffer (5 min). In between runs, the capillary was flushed with running buffer (2 min). The DAD spectra were obtained in a range of 200-500 nm, and the detection of flavonoids was set at 270 nm. The samples were introduced to the system by hydrodynamic injection (50 mbar/10 s).
The method for separation of flavonoids from leaves of P. ligularis was developed by trying different buffer compositions: 1) varying the concentration of sodium tetraborate (STB) 2) varying methanol percentage and 3) by adding a surfactant. Moreover, different electrophoretic conditions were tested (voltage and temperature). For all trials a fused silica capillary was used with a total length of 48.5 cm and an effective length 40 (50μm I.D). It was observed that high concentrations of STB reduce resolution (Wu et al 2007) and high percentage of MeOH increase migration times (Watson, 2012). Afterwards, the identification of chrysin, quercetin-3-O-glucoside and kaempferol-3-O-glucoside was accomplished by co-injecting references sample of those flavonoids with the aqueous extract.
Moreover, we evaluated the electrophoretic mobility (EM) of chrysin, quercetin-3-O-glucoside and kaempferol-3-O-glucoside. For this, we varied the pH value of the running buffer (7.03, 8.32, 9.32, 10.33 and 11.08). The EM was evaluated with the equation: V= μ_e*E (Watson 2012). The results showed that because of the weak acidic nature of the flavonoids, at high pH their mobility increases (Jiang et al 2004). However, the behavior of each flavonoid was different depending on its size, nature and shape (Unger 2009). In the case of quercetin-3-O-glucoside it was also seen that its mobility was altered in function of the formation of borate complexes with hydroxyl groups in position 3’ and ‘4 of the flavonoid skeleton (Voirin et al 2000). These results evidenced the complex behavior of analytes from plant extracts when analyzed by CE technique.

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