Leonurus japonicus, Leonurus cardiaca, Leonotis leonurus: a novel HPLC study on the occurrence and content of the pharmacologically active guanidino derivative leonurine.
Leonurine is a prominent pharmacologically active guanidine alkaloid (4-{[amino(imino)methyl]amino} butyl-4-hydroxy-3,5-dimethoxybenzoate), mainly exerting cardiovascular, hypotensive, uterotonic, and neuroprotective effects. It is commonly regarded as the predominant active principle of Leonurus and Leonotis drugs (subfamily Lamioideae), though its presence has only been unambiguously proven for the aerial parts of Leonurus japonicus Houtt. (yimucao/Chin.Ph.,DAB), used in TCM/Kampo for the treatment of various gynaecological and cardiovascular disorders. Although a series of claims concerning the occurrence of leonurine in European Leonurus cardiaca L. (Ph.Eur.) can be found describing it as an important active principle, this has never been conclusively demonstrated. The same holds true for the officinal Leonurus japonicus fruits (chongweizi/Chin.Ph.) and the closely related South African herb Leonotis leonurus (L.) R.Br. Since no reliable HPLC determination and quantification method for leonurine has been published up to now, in the present study, a highly reproducible RP-HPLC method was newly developed using a special octadecyl-bonded stationary phase and an acetonitrile/water gradient (adjusted to pH 2.5 by phosphoric acid) as mobile phase (DAD/277nm). In particular, this use of reversed phase packing with hydrophilic endcapping clearly contributes to an improved peak shape for leonurine, to our knowledge the first application of this technique on a natural zwitterionic analyte, and clearly enhances the selectivity of separation compared to classical RP-phases. The method was shown to be precise with respect to concentration, exhibiting a linear response in the range of 2.5-12.5 microg/ml leonurine, detection limit well below 0.5 microg/ml, and correlation coefficients constantly higher than 0.99 (5 levels, n = 3) over numerous inter day repetitions, demonstrating the robustness of the newly developed HPLC protocol. Thus, nine samples of L. japonicus aerial parts, two of L. japonicus fruits, four of L. cardiaca aerial parts, as well as one sample each of L. cardiaca fruits, and Leonotis leonurus aerial parts were examined. No leonurine could be detected in any sample of L. cardiaca in contrast to newly published official drug assessments, which consequently have to be revised. Leonotis leonurus and surprisingly, seeds of L. japonicus did not contain leonurine, either. However, in aerial parts of L. japonicus drug samples, obtained from China and Japan, leonurine contents between 0.001 and 0.049% were determined, while L. japonicus from domestic cultivation displayed significantly higher amounts of at least 0.1%. Thus, the HPLC method described above could be used for quality control of leonurine contained in TCM/Kampo medicines and in pharmacopeial analytics for the differentiation of L. japonicus and L. cardiaca samples.
Department of Pharmaceutical Biology, Institute of Pharmacy, University of Leipzig, Germany.
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Kuchta
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Ortwein
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HW
Rauwald
D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D005638:Fruit / Q000737:chemistry; D005707:Gallic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000302:isolation & purification; D031332:Leonurus / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D010936:Plant Extracts / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry; D012015:Reference Standards; D015203:Reproducibility of Results; D013045:Species Specificity; D021241:Spectrometry, Mass, Electrospray Ionization
D010936:Plant Extracts; C013587:leonurine; D005707:Gallic Acid