Phytochemical Differentiation of Saffron (Crocus sativus L.) by High Resolution Mass Spectrometry Metabolomic Studies.

The metabolite profiling of saffron (Crocus sativus L.) from several countries was measured by using ultra-performance liquid chromatography combined with high resolution mass spectrometry (UPLC-HR MS). Multivariate statistical analysis was employed to distinguish among the several samples of C. sativus L. from Greece, Italy, Morocco, Iran, India, Afghanistan and Kashmir. The results of this study showed that the phytochemical content in the samples of C. sativus L. were obviously diverse in the different countries of origin. The metabolomics approach was deemed to be the most suitable in order to evaluate the enormous array of putative metabolites among the saffron samples studied, and was able to provide a comparative phytochemical screening of these samples. Several markers have been identified that aided the differentiation of a group from its counterparts. This can be important for the selection of the appropriate saffron sample, in view of its health-promoting effect which occurs through the modulation of various biological and physiological processes.

Crocus-derived compounds alter the aggregation pathway of Alzheimer's Disease: associated beta amyloid protein.

Natural products have played a dominant role in the discovery of lead compounds for the development of drugs aimed at the treatment of human diseases. This electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)-based study demonstrates that dietary antioxidants, isolated components from the stigmas of saffron (Crocus sativus L.) may be effective in inhibiting Aß fibrillogenesis, a neuropathological hallmark of Alzheimer's Disease (AD). This study reveals a substantial alteration in the monomer/oligomer distribution of Aß1-40, concomitant with re-direction of fibril formation, induced by the natural product interaction. These alterations on the Aß1-40 aggregation pathway are most prominent for trans-crocin-4 (TC4). Use of ESI-IMS-MS, electron microscopy alongside Thioflavin-T kinetics, and the interpretation of 3-dimensional Driftscope plots indicate a correlation of these monomer/oligomer distribution changes with alterations to Aß1-40 amyloid formation. The latter could prove instrumental in the development of novel aggregation inhibitors for the prevention, or treatment of AD.

Metabolomic fingerprinting and genetic discrimination of four Hypericum taxa from Greece.

Hypericum perforatum has plenty of uses in traditional medicine and is the source of top-selling herbal drugs and food supplements. The secondary metabolite chemistry for most of the nearly 500 Hypericum taxa is still unknown, even though they are used interchangeably. In the present study, we characterized four Hypericum populations from Achaia, Greece, belonging to H. perforatum ssp. veronense, H. perfoliatum, H. triquetrifolium, and an uninvestigated taxon, H. empetrifolium ssp. empetrifolium, in terms of their essential oils and polar bioactives in methanolic extracts via GC-MS, LC-HRMS, LC-DAD-MS, and HPLC-DAD. We also performed sequence analysis of nrITS to explore the genetic profile of these taxa and to examine whether their genotype is correlated to the metabolome. Sixty-three non-volatile compounds, phloroglucinols in their majority, and over one hundred (113) volatiles, mostly sesqui- and mono- terpenes, were detected. The concentration of the major polar constituents varied greatly among samples. In particular, phloroglucinols' diversity and abundance in H. empetrifolium ssp. empetrifolium was remarkable. The PCA and Biplot analysis revealed the contribution of each compound to the total chemodiversity and also revealed certain compounds that contribute to the discrimination of the samples. Sequence analysis of nrITS revealed different genetic profiles and markers which can be used for the identification of the four Hypericum taxa. The Mantel test showed a relatively strong correlation between the genetic profile and the volatile compounds and low with the main polar metabolites.

The Crocus sativus Compounds trans-Crocin 4 and trans-Crocetin Modulate the Amyloidogenic Pathway and Tau Misprocessing in Alzheimer Disease Neuronal Cell Culture Models.

Crocus sativus L. natural compounds have been extensively used in traditional medicine for thousands of years. Recent research evidence is now emerging in support of its therapeutic potential for different pathologies including neurodegenerative diseases. Herein, the C. sativus L. natural compounds trans-crocin 4 and trans-crocetin were selected for in depth molecular characterization of their potentially protective effects against Alzheimer's Disease (AD), utilizing two AD neuronal cell culture models (SH-SY5Y overexpressing APP and PC12 expressing hyperphosphorylated tau). Biologically relevant concentrations, ranging from 0.1 µM to 1 mM, applied for 24 h or 72 h, were well tolerated by differentiated wild type SH-SY5Y and PC12 cells. When tested on neuronally differentiated SH-SY5Y-APP both trans-crocin 4 and trans-crocetin had significant effects against amyloidogenic pathways. Trans-crocin 4 significantly decreased of ß-secretase, a key enzyme of the amyloidogenic pathway, and APP-C99, while it decreased γ-secretases that generate toxic beta-amyloid peptides. Similarly, trans-crocetin treatment led to a reduction in ß- and γ-secretases, as well as to accumulation of cellular AßPP. When tested on the neuronally differentiated PC12-htau cells, both compounds proved effective in suppressing the active forms of GSK3ß and ERK1/2 kinases, as well as significantly reducing total tau and tau phosphorylation. Collectively, our data demonstrate a potent effect of trans-crocin 4 and trans-crocetin in suppressing key molecular pathways of AD pathogenesis, rendering them a promising tool in the prevention and potentially the treatment of AD.

Rapid isolation and characterization of crocins, picrocrocin, and crocetin from saffron using centrifugal partition chromatography and LC-MS.

This study demonstrates a simple method for one-step isolation of the main secondary metabolites of a hydroalcoholic extract of Crocus sativus stigmas (saffron) using step-gradient centrifugal partition chromatography. The analysis was performed in dual and elution-extrusion mode, using five biphasic systems of the solvents heptane/ethyl acetate/butanol/ethanol/water in ratios of 4:10:0:4:10, 1:13:0:4:10, 1:12:1:4:10, 1:10:3:4:10, and 1:7:6:4:10. Five major crocins, picrocrocin, and crocetin were directly isolated in one step. Scaling up to preparative level, allowed the recovery of significantly high quantities of pure compounds, especially trans-crocin-4, saffron's principal crocin. Comparing dual-mode and elution-extrusion, in dual-mode, the trans-crocin-4 containing fractions were co-eluted with a high amount of free ß-d-glucose. In contrast, absence of free ß-d-glucose was observed in the corresponding trans-crocin-4 fractions obtained by the second method denoting its superiority against dual-mode. Initiating analysis with the 4th solvent-system afforded selective isolation of trans-crocin-4, with reduction in experimental time and solvent consumption. Structure elucidation was performed by nuclear magnetic resonance spectroscopy, liquid chromatography with mass spectrometry, and high-resolution tandem mass spectrometry. The proposed methodology comprises an integrated approach for the purification and characterization of biologically active saffron components in a fast, selective, and environmentally friendly manner.

Protein aggregation and neurodegeneration in prototypical neurodegenerative diseases: Examples of amyloidopathies, tauopathies and synucleinopathies.

Alzheimer's and Parkinson's diseases are the most prevalent neurodegenerative diseases that generate important health-related direct and indirect socio-economic costs. They are characterized by severe neuronal losses in several disease-specific brain regions associated with deposits of aggregated proteins. In Alzheimer's disease, ß-amyloid peptide-containing plaques and intraneuronal neurofibrillary tangles composed of hyperphosphorylated microtubule-associated protein tau are the two main neuropathological lesions, while Parkinson's disease is defined by the presence of Lewy Bodies that are intraneuronal proteinaceous cytoplasmic inclusions. α-Synuclein has been identified as a major protein component of Lewy Bodies and heavily implicated in the pathogenesis of Parkinson's disease. In the past few years, evidence has emerged to explain how these aggregate-prone proteins can undergo spontaneous self-aggregation, propagate from cell to cell, and mediate neurotoxicity. Current research now indicates that oligomeric forms are probably the toxic species. This article discusses recent progress in the understanding of the pathogenesis of these diseases, with a focus on the underlying mechanisms of protein aggregation, and emphasizes the pathophysiological molecular mechanisms leading to cellular toxicity. Finally, we present the putative direct link between ß-amyloid peptide and tau in causing toxicity in Alzheimer's disease as well as α-synuclein in Parkinson's disease, along with some of the most promising therapeutic strategies currently in development for those incurable neurodegenerative disorders.

Processed stigmas of Crocus sativus L. imaged by MALDI-based MS.

The processed, i.e. dried under certain conditions, stigmas of Crocus sativus L. are one of the most expensive plant parts used commercially. For the color, aroma and biological activity a very complex mixture of glycolipids termed crocins are responsible. Therefore studying structural composition and distribution in the commercial plant material is of great interest. We showed successfully the application of a MALDI-based mass spectrometric imaging (MSI) approach for stigmas towards different crocin species. MSI opens up the investigation of processed plant materials in various fields allowing studying the processing in detail as well as adulteration attempts (which are quite frequent due to the price of the material). Furthermore, we could demonstrate that a similar number of crocins present in stigmas could be detected by MALDI MSI compared to the classical approach of analyzing the solvent-extract of stigmas by MALDI-MS.

Quantitation of Crocins and picrocrocin in saffron by HPLC: application to quality control and phytochemical differentiation from other crocus taxa.

A chromatographic method was developed and fully validated for the determination of the major saffron constituents, i.e., picrocrocin and five major crocins. Dried samples (styles of Crocus sativus and other Crocus taxa) were extracted with MeOH : water (1 : 1, v/v), and chromatographic separation of the analytes was achieved by reversed-phase chromatography using a gradient elution. Full validation was performed using spiked samples with analytes, which were isolated, purified, and characterized by MS due to a lack of commercial standards. The method showed a good fit (r2 > 0.999) for all analytes with limit of quantitation values in the range of 1-15 µg/mL, and demonstrated adequate intra- and inter-precision (< 15 % RSD) and accuracy (< 7 % RE). The method was applied to the analysis of various commercial saffron samples and of indigenous Crocus taxa and allowed for the first time the absolute quantitation of several Crocus components.

In-chain neutral hydrocarbon loss from crocin apocarotenoid ester glycosides and the crocetin aglycon (Crocus sativus L.) by ESI-MS(n) (n=2, 3).

The stigmas of Crocus sativus L. have been used as spice and colorant agent (i.e. saffron) for more than 4000 years. For an updated structural investigation of the aglycon present in the glycosylated crocetin apocarotenoids (i.e. crocins), seven representative derivatives ranging from one up to five glucosyl-residues with a maximum number of three monosaccharides per glycosylation site (glucose, gentiobiose, gentiotriose and neapolitanose) were isolated and purified by high-performance liquid chromatography. The compounds selected for further mass spectrometric investigation include glucosyl-, bis-glucosyl-, gentiobiosyl-, gentiobiosyl-glucosyl-, bis-gentiobiosyl-, gentiobiosyl-gentiotriosyl- and gentiobiosyl-neapolitanosyl-crocetin. Electrospray ionization in combination with low-energy collision-induced dissociation/tandem mass spectrometry of sodiated crocin precursor ions utilizing either a 3D-ion trap (MS(n) , n = 2, 3) or a QqTOF instrument, with the latter providing accurate mass determination with an accuracy of ±1-3 ppm or better at a resolution of 10,000 (full width at half maximum), was used. Major fragmentation pathways included loss of either one or two carbohydrate substituents leading to the sodiated aglycon without interglycosidic bond cleavage during in MS(2) -experiments. All sodiated precursor ions and major product ions were accompanied by a loss of 92 Da, which was elucidated as C7 H8 -loss from the aglycon by skeletal rearrangement via an eight-membered transition state as previously described for intact C40-carotenoids.

Comparison of different tandem mass spectrometric techniques (ESI-IT, ESI- and IP-MALDI-QRTOF and vMALDI-TOF/RTOF) for the analysis of crocins and picrocrocin from the stigmas of Crocus sativus L.

RATIONALE: The expensive spice saffron originating from the stigmas of Crocus sativus L. and also applied in traditional Chinese medicine (TCM) constitutes a complex mixture of glycoconjugates varying not only in the aglycon structure, but also in glycosylation pattern. Therefore, various tandem mass spectrometric techniques were evaluated for their usefulness in structural elucidation. METHODS: Three selected constituents of the stigmas of Crocus sativus L., trans- and cis-crocin-4 as well as picrocrocin, were isolated and purified by HPLC and finally analyzed by ESI-MS (ion trap, QqRTOF), IP-MALDI-MS (QqRTOF) and vMALDI-MS (TOF/RTOF) in combination with tandem mass spectrometry in collision energy regimes ranging from a few eV (LE) to 20 keV (HE) collisions for the first time. These data aid in structurally elucidating minor, unknown glycoconjugates originating from this plant-derived spice. RESULTS: LE-CID of isomeric crocins on either an ion trap with ESI or a QqRTOF-instrument with ESI or IP-MALDI as desorption/ionization technique only yielded a limited number of structurally diagnostic sodiated product ions related to the carbohydrate moiety as well as to the intact aglycon in contrast to true HE-CID. The low MW constituent picrocrocin did not yield useful LE-CID spectra, but showed a high number of structurally diagnostic product ions by HE-CID utilizing a vMALDI TOF/RTOF-instrument. CONCLUSIONS: The highest number of structurally diagnostic product ions allowing also determination of the carbohydrate linkage of the gentiobiose-moiety of isomeric crocins ((0,4)A(2), (3,5)A(2) product ions indicating a 1→6 carbohydrate linkage) was only achievable by HE-CID. Fragmentation of the aglycon was not observed by any collision energy regime applied.