Screening for Triterpenoid Saponins in Plants Using Hyphenated Analytical Platforms.

Recently the number of studies investigating triterpenoid saponins has drastically increased due to their diverse and potentially attractive biological activities. Currently the literature contains chemical structures of few hundreds of triterpenoid saponins of plant and animal origin. Triterpenoid saponins consist of a triterpene aglycone with one or more sugar moieties attached to it. However, due to similar physico-chemical properties, isolation and identification of a large diversity of triterpenoid saponins remain challenging. This study demonstrates a methodology to screen saponins using hyphenated analytical platforms, GC-MS, LC-MS/MS, and LC-SPE-NMR/MS, in the example of two different phenotypes of the model plant Barbarea vulgaris (winter cress), glabrous (G) and pubescent (P) type that are known to differ by their insect resistance. The proposed methodology allows for detailed comparison of saponin profiles from intact plant extracts as well as saponin aglycone profiles from hydrolysed samples. Continuously measured 1D proton NMR data during LC separation along with mass spectrometry data revealed significant differences, including contents of saponins, types of aglycones and numbers of sugar moieties attached to the aglycone. A total of 49 peaks were tentatively identified as saponins from both plants; they are derived from eight types of aglycones and with 2-5 sugar moieties. Identification of two previously known insect-deterrent saponins, hederagenin cellobioside and oleanolic acid cellobioside, demonstrated the applicability of the methodology for relatively rapid screening of bioactive compounds.

Industrial Scale Isolation, Structural and Spectroscopic Characterization of Epiisopiloturine from Pilocarpus microphyllus Stapf Leaves: A Promising Alkaloid against Schistosomiasis.

This paper presents an industrial scale process for extraction, purification, and isolation of epiisopiloturine (EPI) (2(3H)-Furanone,dihydro-3-(hydroxyphenylmethyl)-4-[(1-methyl-1H-imidazol-4-yl)methyl]-, [3S-[3a(R*),4b]]), which is an alkaloid from jaborandi leaves (Pilocarpus microphyllus Stapf). Additionally for the first time a set of structural and spectroscopic techniques were used to characterize this alkaloid. EPI has shown schistomicidal activity against adults and young forms, as well as the reduction of the egg laying adult worms and low toxicity to mammalian cells (in vitro). At first, the extraction of EPI was done with toluene and methylene chloride to obtain a solution that was alkalinized with ammonium carbonate. The remaining solution was treated in sequence by acidification, filtration and alkalinization. These industrial procedures are necessary in order to remove impurities and subsequent application of the high performance liquid chromatography (HPLC). The HPLC was employed also to remove other alkaloids, to obtain EPI purity higher than 98%. The viability of the method was confirmed through HPLC and electrospray mass spectrometry, that yielded a pseudo molecular ion of m/z equal to 287.1 Da. EPI structure was characterized by single crystal X-ray diffraction (XRD), (1)H and (13)C nuclear magnetic resonance (NMR) in deuterated methanol/chloroform solution, vibrational spectroscopy and mass coupled thermal analyses. EPI molecule presents a parallel alignment of the benzene and the methyl imidazol ring separated by an interplanar spacing of 3.758 Å indicating a π-π bond interaction. The imidazole alkaloid melts at 225°C and decomposes above 230°C under air. EPI structure was used in theoretical Density Functional Theory calculations, considering the single crystal XRD data in order to simulate the NMR, infrared and Raman spectra of the molecule, and performs the signals attribution.

Alkaloid analysis by high-performance liquid chromatography-solid phase extraction-nuclear magnetic resonance: new strategies going beyond the standard.

The hyphenated technique HPLC-SPE-NMR is an important tool for rapid dereplication of complex mixtures of in particular small molecules and has been successfully employed in natural product research. However, positively charged alkaloids at low pH are often poorly trapped on the generally used SPE cartridge limiting the general application of the procedure. In this work, two new approaches for efficient SPE trapping of alkaloids and elution efficiencies were evaluated using 24 model alkaloids. Use of a 0.1 M NaOH solution as the post-column dilution greatly enhanced trapping of alkaloids on the commonly used cartridge containing divinylbenzene polymer (GP resin). This procedure, however, was unsuitable for trapping phenolic alkaloids. Severe line broadening and immiscibility with water made chloroform-d(1) unsuited as eluent. None of these problems occurred when methanol-d(4) was used as eluent. Previously, mixed mode cation exchange sorbents have not been used in HPLC-SPE-NMR analysis of natural products. In contrast to GP resin this material showed good retention and elution characteristics for retention and elution of alkaloids. As well the use of methanol-d(4) containing 1% aqueous NaOD (40%) as methanol-d(4) containing 5% aqueous NH(4)OH (30%) as eluents were successful, even though elution of alkaloids with pK(a) of the corresponding acid above 10 proved difficult. Alkaloid extracts of Huperzia selago containing complex aliphatic alkaloids and Triclisia patens containing bisbenzylisoquinoline alkaloids were used for validation of the protocols for analysis of a diverse collection of alkaloids. Mixed mode cation exchange sorbent was efficient for trapping and elution of both types of alkaloids as evidenced by acquisition of 2D NMR data for all trapped compounds. In contrast, GP resin proved only viable for all the H. selago alkaloids whereas trapping and elution of bisbenzylisoquinoline alkaloids were dubious.

LC-DAD-MS/SPE-NMR hyphenation. A tool for the analysis of pharmaceutically used plant extracts: identification of isobaric iridoid glycoside regioisomers from Harpagophytum procumbens.

LC-DAD-MS monitored fractionation of a Harpagophytum procumbens DC. (Pedaliaceae) root extract was combined with a hyphenated LC-DAD-MS/SPE-NMR technique, thus providing the spectral data needed for structure elucidation. This approach allowed the characterization of isobaric iridoid glycoside regioisomers present only as minor constituents. The analytes were identified as the (E/Z) pairs of 6'-O-(p-coumaroyl)harpagide (6'-PCHG) and 8-O-(p-coumaroyl)-harpagide (8-PCHG). The fact that 8-(Z)-PCHG constitutes a new natural product underlines the analytical power of this combined approach. Furthermore, derivatives 6'-(Z)- and 6'-(E)-PCHG are new constituents for H. procumbens.