Non-targeted chromatographic analyses of cuticular wax flavonoids from Physalis alkekengi L.

Since Chinese lantern (Physalis alkekengi L.) represents a rich source of various bioactive secondary metabolites, there is an urge for its detailed characterization. Non-polar flavonoid aglycones represent one of the few bioactive species found in plant's cuticular waxes. The separation of flavonoids is already extensively covered in the literature, but methods dedicated to separation and identification of methylated flavonoids are rather scarce. In the present study a non-targeted approach for the separation, isolation and identification of methylated flavonoids present in P. alkekengi L. var. franchetii cuticular waxes was established. A rapid and simple separation on HPTLC silica gel was developed for preliminary screening of flavonoids. Fast HPLC-UV-MS(n) and HPLC-UV methods using a C6-Phenyl and a C18 stationary phase were also developed, respectively. In both cases, the right combination of temperature and tetrahydrofuran, as a mobile phase modifier, were shown to be crucial for a baseline separation of all studied compounds. By employing a semi-preparative analog of the C18 column, a simultaneous isolation of pure unknown analytes was achieved. Using these developed methods in combination with NMR, four 3-O-methylated flavonols were detected and identified in P. alkekengi L. var. franchetii cuticular waxes: myricetin 3,7,3'-trimethyl ether, quercetin 3,7-dimethyl ether, myricetin 3,7,3',5'-tetramethyl ether and quercetin 3,7,3'-trimethyl ether. Moreover, the simple and fast isocratic HPLC-UV-MS(n) method (under 8min) should prove useful in quality control of P. alkekengi L. var. franchetii by enabling chromatographic fingerprinting of external methylated flavonols. Finally, a rationale for the mechanism of separation of these metabolites by HPLC is also given, which establishes a foundation for future development of chromatographic methods for methylated flavonols and related compounds.