Metabolic fingerprinting of Leontopodium species (Asteraceae) by means of ¹H NMR and HPLC-ESI-MS.

The genus Leontopodium, mainly distributed in Central and Eastern Asia, consists of ca. 34-58 different species. The European Leontopodium alpinum, commonly known as Edelweiss, has a long tradition in folk medicine. Recent research has resulted in the identification of prior unknown secondary metabolites, some of them with interesting biological activities. Despite this, nearly nothing is known about the Asian species of the genus. In this study, we applied proton nuclear magnetic resonance (¹H NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) metabolic fingerprinting to reveal insights into the metabolic patterns of 11 different Leontopodium species, and to conclude on their taxonomic relationship. Principal component analysis (PCA) of ¹H NMR fingerprints revealed two species groups. Discriminators for these groups were identified as fatty acids and sucrose for group A, and ent-kaurenoic acid and derivatives thereof for group B. Five diterpenes together with one sesquiterpene were isolated from Leontopodium franchetii roots; the compounds were described for the first time for L. franchetii: ent-kaur-16-en-19-oic acid, methyl-15α-angeloyloxy-ent-kaur-16-en-19-oate, methyl-ent-kaur-16-en-19-oate, 8-acetoxymodhephene, 19-acetoxy-ent-kaur-16-ene, methyl-15ß-angeloyloxy-16,17-epoxy-ent-kauran-19-oate. In addition, differences in the metabolic profile between collected and cultivated species could be observed using a partial least squares-discriminant analysis (PLS-DA). PCA of the LC-MS fingerprints revealed three groups. Discriminating signals were compared to literature data and identified as two bisabolane derivatives responsible for discrimination of group A and C, and one ent-kaurenoic acid derivative, discriminating group B. A taxonomic relationship between a previously unidentified species and L. franchetii and Leontopodium sinense could be determined by comparing NMR fingerprints. This finding supports recent molecular data. Furthermore, Leontopodium dedekensii and L. sinense, two closely related species in terms of morphology and DNA-fingerprints, could be distinguished clearly using ¹H NMR and LC-MS metabolic fingerprinting.

Quantification of cynaropicrin in artichoke leaf extracts by ¹H NMR spectroscopy.

The quantification of cynaropicrin in artichoke leaf extracts (ALE) by means of quantitative ¹H NMR was performed. The method relies on solid phase extraction for sample preparation and spectra acquisition in DMSO- D6 containing anthracene as the internal standard. The obtained ¹H NMR spectra revealed excellent signal purity for H-13(b) of cynaropicrin, which was thus chosen for quantification. Validation was performed in terms of selectivity, linearity, precision, accuracy, and robustness. The content of cynaropicrin in the analyzed samples showed considerable variation, ranging from non detectable to 1.6 %. The NMR approach does not rely on external calibration, which represents the major advantage compared to traditional approaches based on HPLC.

Identification and quantification of major steviol glycosides in Stevia rebaudiana purified extracts by 1H NMR spectroscopy.

The use of (1)H NMR spectroscopy for the characterization of Stevia rebaudiana extracts is presented. The developed method allows qualitative and quantitative determination of the major steviol glycosides in purified extracts and fractions obtained from various stages of the purification process. Moreover, it proved to be a powerful tool to differentiate between glycosides which are naturally occurring in the stevia plant and artifacts formed in the course of the manufacturing process. Identification of steviol glycosides was achieved by the use of 2D NMR techniques, whereas quantification is based on qHNMR using anthracene as internal standard. The solvent mixture pyridine-d(5)-DMSO-d(6) (6:1) enabled satisfactory separation of the signals to be integrated. Validation of the method was performed in terms of specificity, precision, accuracy, linearity, robustness, and stability. Quantitative results were compared to those obtained with the JECFA HPLC-UV method and were found to be in reasonable agreement. NMR analysis does not rely on the use of reference compounds and enables significantly faster analysis compared to HPLC-UV. Thus, NMR represents a feasible alternative to HPLC-based methods for the quality control of Stevia rebaudiana extracts.

Identification and pharmacological characterization of the anti-inflammatory principal of the leaves of dwarf elder (Sambucus ebulus L.).

AIM OF THE STUDY: The performed investigations aimed on the identification of the anti-inflammatory principal of extracts of leaves of Sambucus ebulus L. (dwarf elder) in order to rationalize the traditional use of this plant for the treatment of chronically inflammatory diseases. MATERIALS AND METHODS: Dwarf elder leaf extract was subjected to activity guided fractionation using inhibition of TNFα induced expression of vascular cell adhesion molecule 1 (VCAM-1) on the surface of human umbilical vein endothelial cells (HUVECs) as monitoring tool (positive control: parthenolide 10µM, VCAM-1 expression (% of control): 5.35±0.38%). RESULTS: Bio-guided isolation resulted in identification of ursolic acid as anti-inflammatory principal. Besides its inhibitory effects against TNFα induced expression of VCAM-1 (IC(50) 6.25 µM), ursolic acid inhibits also TNFα induced expression of ICAM-1 (IC(50) value between 3.13 and 6.25 µM) (positive control: parthenolide 10 µM, ICAM-1 expression (% of control): 38.89±16.6%). Toxic effects of ursolic acid on HUVECs can be drastically reduced using an enriched extract instead of the pure compound. CONCLUSIONS: Our findings suggest an additional mechanism of the anti-inflammatory activity of ursolic acid by demonstrating its ability to inhibit TNFα-stimulated expression of VCAM-1 and ICAM-1 and support the traditional use of extracts and preparations of Sambucus ebulus L., rich in ursolic acid, for the treatment of chronically inflammatory processes.

Analysis of iridoids, secoiridoids and xanthones in Centaurium erythraea, Frasera caroliniensis and Gentiana lutea using LC-MS and RP-HPLC.

This study presents a new and validated HPLC method for the simultaneous determination of bioactive compounds in Centaurium erythraea, Frasera caroliniensis and Gentiana lutea. The iridoid loganic acid, four secoiridoids and 29 xanthones were separated on a RP-18 column, using aqueous o-phosphoric acid (0.085%, v/v) and acetonitrile as mobile phase. Phytochemical investigation of C. erythraea herb and F. caroliniensis roots resulted into isolation of 25 xanthones and three secoiridoids the structure of which was elucidated by spectroscopic means (NMR, MS and UV). 1,3,8-Trihydroxy-5,6-dimethoxyxanthone, isolated from C. erythraea, turned out to be a novel xanthone. The stability of the analytes was tested by subjecting samples to light, moisture and different temperatures. After six months of storage, decomposition of gentiopicroside and sweroside was observed. The swertiamarin content was nearly unchanged when stored at room temperature or in the refrigerator, but high temperature conditions reduced the content to 85%. In contrast, xanthones were stable under long-term, refrigerated and accelerated conditions. The established chromatographic method has been successfully applied for the quantification of the bioactive compounds in the three plants. The presence and distribution of polyoxygenated xanthones within the three members of the Gentianaceae family and their significance as analytical markers are discussed.

Iridoid glycosides from the leaves of Sambucus ebulus.

Six new iridoid glycosides (1-6) of the "Valeriana type" were isolated from leaves of Sambucus ebulus. The structures were elucidated by 1D- and 2D-NMR spectroscopy, mass spectrometry, and chemical degradation methods as 10-O-acetylpatrinoside-aglycone-11-O-[4''-O-acetyl-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-ribohexo-3-ulopyranoside] (1), 7-O-acetylpatrinoside-aglycone-11-O-[4''-O-acetyl-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-ribohexo-3-ulopyranoside] (2), 10-O-acetylpatrinoside-aglycone-11-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-ribohexo-3-ulopyranoside] (3), patrinoside-aglycone-11-O-[4''-O-acetyl-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-ribohexo-3-ulopyranoside] (4), 10-O-acetylpatrinoside-aglycone-11-O-[4''-O-acetyl-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranoside] (5), and patrinoside-aglycone-11-O-2'-deoxy-beta-D-glucopyranoside (6). Compounds 1-4 represent the first examples of acylated iridoid diglycosides bearing the uncommon D-ribohexo-3-ulopyranosyl sugar moiety. Compound 6 is the first iridoid glycoside with a 2-deoxy-D-glucopyranosyl sugar moiety.