Analytical and active markers for quality assessment of Leonurus cardiaca and L. japonicus: RP-HPLC, HPTLC, and 1H-qNMR approaches for the determination of defined phenolic and N-containing constituents

Officinal European L. cardiaca, East Asian L. japonicus, and South African Leonotis leonurus are traditionally used for cardiovascular, gynecological, and neurological disorders. Nevertheless, a phytochemical assessment as a basis for their quality control and comparison amongst them has not yet been reported. A RP-HPLC method was newly developed for the quantification of leonurine. Only the Nucleodur C18 Pyramid column (RP-phase, polar endcapping) yielded stable retention times. No leonurine could be detected in any sample of L. leonurus or L. cardiaca despite numerous literature claims including official assessment reports (EMA/HMPC 2010) that name it as an active constituent. Surprisingly, L. japonicus fruits (Chin. Ph.) did not contain any leonurine, either, which was thus identified as a specific taxonomic marker with known pharmacological activity as it was detected in every sample of L. japonicus aerial parts. Furthermore, a novel RP-HPLC method for the simultaneous quantification of twelve phenolics was developed. Ferulic acid was found in every sample of every drug. Lavandulifolioside and verbascoside were not present in any sample of L. japonicus, but in every sample of the aerial parts of L. cardiaca. Lavandulifolioside was firstly found in L. leonurus, just as isoquercitrin, which was also detected in L. cardiaca but not in L. japonicus. In contrast to literature data, hyperoside could not be detected in L. cardiaca but in both L. japonicus and L. leonurus. An instrumental HPTLC method for stachydrine was newly developed, using postchromatographic derivatization by Vágújfalvi reagent, an automatic TLC setup, and winCATS data analysis software. The results of an equally novel ¹H-qNMR procedure using its N-CH₃ singlet δ 3.03 ppm in comparison with the singlet of the two vinylic protons of the internal standard maleic acid at δ 6.18 ppm were always within the standard deviation of the HPTLC data, thus stachydrine could be quantified in every examined sample.