László Kursinszki

Characterization and identification of isoflavonoid glycosides in the root of Spiny restharrow (Ononis spinosa L.) by HPLC-QTOF-MS, HPLC-MS/MS and NMR

Restharrow root has been used in traditional medicine for thousands of years; however, the active ingredients responsible for the diuretic effect are still unknown. Previous studies have proved that the root extract contains isoflavonoids, however only few derivatives were identified, mostly relying on retention times or UV data. The aim of our work was to perform a detailed structural characterization of the complete isoflavonoid profile in the aqueous-methanolic extract of Ononis spinosa root by high-performance liquid chromatography coupled with electrospray ionization accurate-mass quadrupole time-of-flight and tandem mass spectrometry in positive ionization mode (HPLC-ESI-QTOF-MS, HPLC-ESI-MS/MS) and nuclear magnetic resonance spectroscopy (NMR). On the basis of the accurate masses and fragmentation patterns isoflavones (formononetin, calycosin and pseudobaptigenin) and pterocarpans (maackiain and medicarpin) were identified. Two further dihydroisoflavone aglycones, namely onogenin and sativanone and a unique glucoside were isolated and their structures were elucidated by NMR experiments. Calycosin, onogenin and sativanone were detected in this plant for the first time. In contrast to previous works, the presence of biochanin A could not be confirmed, however its regioisomer calycosin and its derivatives were identified. Similarly, neither tectorigenin derivatives could be detected, however the isobar compound sativanone and its various glucosides were elucidated. The presence of genistein and daidzein could not be confirmed in the extract. Fragmentation pathways for onogenin and sativanone are presented. In the aqueous-methanolic extract 9 glucosides, 6 minor and 8 major glucoside malonates, 4 glucoside acetates and 7 aglycones were found. In total, 34 compounds were successfully identified.

HPLC-ESI-MS/MS of brain neurotransmitter modulator lobeline and related piperidine alkaloids in Lobelia inflata L

There is a renewed interest in lobelia alkaloids because of their activity on the central nervous system. Lobeline, the most active of them, a nicotinic receptor ligand and neurotransmitter transporter inhibitor, is a candidate pharmacotherapy for metamphetamine abuse. In the present work, high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry in positive ion mode was used for investigating the alkaloid profile in Lobelia inflata L. Chromatographic separations were achieved on a Gemini C6-phenyl reversed-phase column providing good peak shape and improved selectivity. Being mostly 2,6-disubstituted piperidines, lobelia alkaloids presented abundant [M + H](+) ions with typical fragmentation. Identification was possible from a few specific ions, especially those resulting from excision of one of the substituents. Based on fragmentation pattern of lobeline as reference compound, 52 alkaloids were identified in the aqueous methanolic extract of L. inflata in contrast to the previously known some 20. Structural variability of these alkaloids identified arises basically from their substituents which can be phenyl-2-ketoethyl- or phenyl-2-hydroxyethyl units as well as their methyl-, ethyl- or propyl- homologues attached in different combinations. Several propyl homologue lobelia alkaloids and five hydroxypiperidine derivatives were found in the plant at the first time. In addition to 8-O-esters of 2-monosubstituted piperidine alkaloids previously reported by us in L. inflata, a 3-hydroxy-3-phenylpropanoic acid ester of hydroxyallosedamine ring-substituted was also identified as a new natural product. High-performance liquid chromatography-electrospray ionization tandem mass spectrometry can be successfully applied to Lobeliacae plant samples in the routine screening for new and known bioactive constituents, quality control of the crude drug, lobelia herba, alkaloid production studies, breeding and chemotaxonomy.

New Chemotypes of the Lichens Xanthoparmelia pulvinaris and X. subdiffluens (Parmeliaceae, Ascomycota)

Abstract: Farkas, E., Kursinszki, L., Szőke, É. & Molnár, K. 2015. New chemotypes of the lichens Xanthoparmelia pulvinaris and X. subdiffluens (Parmeliaceae, Ascomycota). — Herzogia 28: 679–689. New chemotypes of Xanthoparmelia pulvinaris and X. subdiffluens containing norstictic acid were identified among many of the Hungarian collections of these species using HPTLC and confirmed by LC-MS. The two species are characterised, the possible functional role of norstictic acid is briefly discussed and the importance of protecting populations with norstictic acid in nature is emphasized.

Phytochemical Study of Phenolic Compounds from Helichrysi flos by LC-DAD-MS

Helichrysum arenarium is a medicinal plant which has been long known in European traditional medicine as a cholagogue, choleretic and hepatoprotective agent. High-performance liquid chromatography coupled to a UV photodiode-array detector (DAD) and an electrospray ionization mass spectrometer (ESI-MS) was used to analyze the phytochemical constituents of its inflorescence extracts (crude methanolic extract and ethyl acetate fraction of hydrolyzed methanolic extract). Eight flavonoids, namely kaempferol, kaempferol-3-O-glucoside, apigenin, apigenin-7-O-glucoside, naringenin, naringenin-5-O-glucoside, naringenin-4’-O-glucoside, quercetin-3-O-glucoside (isoquercitrin), the chalcone derivative isosalipurposide (2’,4,4’,6’-tetrahydroxychalcone-6’-O-glucoside) and chlorogenic acid were identified in H. flos extracts. The characterization of the above constituents was based on the comparison of their retention times, UV and ESI-MS data recorded on-line with those of reference compounds and literature data.

Separation and characterization of homopipecolic acid isoflavonoid ester derivatives isolated from Ononis spinosa L. root

Spiny restharrow root (Ononis spinosa L.) and its preparations are mainly used for the treatment of urinary infections or bladder stones in numerous countries. Spiny restharrow root is rich in isoflavonoids (formononetin, calycosin and pseudobaptigenin), pterocarpans (medicarpin and maackiain) and dihydroisoflavonoids (onogenin and sativanone), which metabolites are present as glucosides, glucoside malonates, glucoside acetates and free aglycones in the root. The in-depth analysis of tandem mass spectrometric (MS) and high-resolution MS (HR-MS) data revealed the presence of nitrogen-containing compounds in the root extracts. An ion-exchange-based purification and a preparative-scale reversed phase chromatographic isolation procedure was developed for the characterization of these new natural products. For the unambiguous identification of the isolated compounds NMR experiments were carried out. The thorough characterization confirmed the presence of six piperidin-2-yl-acetic acid (homopipecolic acid) esters of isoflavonoid glucosides. This is the first report of homopipecolic acid esters isolated from higher plants.

Increasing the Anti-Addictive Piperidine Alkaloid Production of In VitroMicropropagated Indian Tobacco by Nitrate Treatments

Background: Lobelia inflata L. (Indian tobacco) is a traditional medicinal plant native to North America. It contains several piperidine alkaloids. Interest in Lobelia alkaloids, and in particular (-)-lobeline, the most active component, has increased in recent years due to their effect on the central nervous system. Thus, lobeline is currently the subject of renewed interest for its anti-addictive activity in the treatment of drug abuse, and neurological disorders. Our studies were aimed at introducing this species into cultivation in Hungary. Results: For direct characterization of di-substituted and mono-substituted piperidine alkaloids in extracts of L. inflata, a tandem mass spectrometric method was developed using electrospray ionization. The compounds (-) lobeline, norlobeline, lobelanidine, norlobelanine and other related structures were identified by HPLC-MS/MS. With the aim of increasing the alkaloid production, we have investigated the effect of changing the ammonium and potassium nitrate levels of the basic Murashige-Skoog medium. The highest dry mass, total alkaloid and lobeline content were measured in the herbs and roots cultured at 570 mg L-1 KNO3 content. Conclusions: The highest values for lobeline derivatives norlobeline and lobelidine were also recorded in the herba and roots of Lobelia inflata cultured on reduced KNO3 containing MS medium. The most sensitive response to media modification was observed in the case of lobelidine. Double-concentration of NH4NO3 had an inhibitory effect on plant growth, total alkaloid and lobeline content.