1H-qNMR for direct quantification of stachydrine in Leonurus japonicus and L. cardiaca

Abstract

(1)H-qNMR-spectroscopy was successfully applied to quantify the pharmacologically active alkaloid stachydrine ((2S)-1,1-dimethylpyrrolidinium-2-carboxylic-acid) in aerial parts of Leonurus japonicus (Leonuri herba, yimucao; Chin.Ph.2010, DAB2012) which are used in TCM and Kampo for the treatment of various gynaecological and cardiovascular disorders. Pharmacological publications on this betaine describe cardiovascular, hypotensive, and tissue-protective effects. However, its pharmacopeial analytics poses severe difficulties as it does not contain any chromophore suitable for HPLC-UV-detection. Nine samples from three countries were prepared as decoctions and freeze-dried. (1)H-NMR-spectra were recorded in D2O. The direct-quantitative (1)H-qNMR-procedure was carried out using the N-CH3-singlet at δ 3.03 ppm in comparison to the δ 6.18 ppm singlet of the two vinylic protons of maleic-acid, which was identified as a most favourable internal standard. The quantification limit of stachydrine was 0.44 mg/g drug material. Neither reference-compounds for calibration-curves nor sample-pre-purification was necessary. This protocol revealed stachydrine contents in the range from 0.09 up to 1.01% (w/w) for the tested yimucao samples. Furthermore, between 0.18 and 0.21% of stachydrine was found in the L. japonicus fruit-drug (Leonuri fructus, chongweizi; Chin.Ph.2010) which was examined for this constituent for the first time. In four co-investigated samples of the closely related and similarly used European herb Leonurus cardiaca Ph.Eur., even higher contents up to 1.55% were attested. The presented quantitative (1)H-qNMR-method was shown to be precise with respect to concentration, and yielded highly reproducible data in a series of inter-day repetitions. Methodically, (1)H-qNMR may be a powerful tool for quality assurance of stachydrine containing plants and herbal drugs, especially for industrial routine protocols.