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 β-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 β- and γ-secretases, as well as to accumulation of cellular AβPP. When tested on the neuronally differentiated PC12-htau cells, both compounds proved effective in suppressing the active forms of GSK3β 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

Alzheimer's disease: Exploring nature's 'medicinal chest' for new therapeutic agents

Natural products have served humanity as a valuable source for the discovery and development of therapeutic agents. In addition, these phytochemicals can function as lead compounds for the development of synthetic analogs aimed at treating human diseases. In our aging society, Alzheimer's disease (AD) is the most common cause of dementia, which is characterized by a significant and progressive loss of memory and other cognitive functions. As society demographics change, the predominance of AD and other age-related dementias is increasing, with concurrent financial and societal costs. AD represents one of the most remarkable scientific challenges for drug discovery as the search for effective disease-modifying agents has been unsuccessful. Medicinal plants have been used for their “anti-aging” properties, and cognitive enhancing properties. In the past decades, natural products have been studied for their anti-AD properties, and their potential for developing therapeutic agents against several molecular targets has been evaluated. This insight evaluates the prospects of medicinal plants for providing disease-modifying, as well as disease-preventing, agents for AD. © 2020 Anthony Tsarbopoulos, De Gruyter

Determination of carbofuran, carbaryl and their main metabolites in plasma samples of agricultural populations using gas chromatography-tandem mass spectrometry

A gas chromatography-tandem mass spectrometric (GC-MS/MS) method has been developed for the determination of carbofuran (2,3-dihydro-2,2- dimethylbenzofuran-7-yl methylcarbamate), carbaryl (1-naphthyl-N- methylcarbamate) and their main metabolites in human blood plasma. Optimization of the isolation of the compounds from plasma matrix included the precipitation, denaturation and digestion of plasma proteins. Derivatization was achieved by the use of trifluoroacetic acid anhydride and was optimized for temperature, time and volume of derivatization agent. In the proposed method, a mild precipitation technique was applied using β-mercaptoethanol and ascorbic acid in combination with solid-phase extraction technique using Oasis HLB (Hydrophobic Lipophilic Balance) cartridges for further clean up of samples. Carbamate linkage was not hydrolyzed to its phenol product, but both carbamate phenol and ketones were transformed into trifluoroacetyl derivatives in order to become volatile compounds and were determined using tandem mass spectrometry. The linearity of the method was shown for nine concentrations in the range of 0.50-250 ng mL-1 in fortified plasma aliquots. Limits of detection (LODs) for all compounds ranged from 0.015-0.151 ng mL-1. Inter-day and intra-day assays (RSD) for all compounds, at three concentration levels of 2.5, 25 and 100 ng mL-1 (n=3) in fortified plasma samples were less than 18%. Accuracy (%E r) was calculated at three concentration levels, 8, 80 and 160 ng mL-1 (n=3), and ranged from -12.0 to 15.0%. Matrix effect was evaluated so mean recoveries were calculated for all compounds and ranged from 81-107%. Specificity for the use of this method to biological monitoring studies was achieved including four main metabolites of CF, 1-naphthol and 2-naphthol from the naphthalene metabolism pathways, and both the parent compound of carbofuran and carbaryl. The proposed method was applied to plasma samples of pesticide users

Development and validation of an ultra performance liquid chromatography-tandem mass spectrometry method for the quantification of daptomycin in human plasma

A rapid, simple and accurate analytical method based on ultra performance liquid chromatography (UPLC) combined with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a hybrid q TOF instrument has been developed and fully validated for the quantification of daptomycin (DPT) in human plasma. The samples were analyzed after simple pretreatment involving protein precipitation, while chromatographic separation of DPT and the internal standard (reserpine) was achieved on an Acquity BEH C18 column (100mm×2.1mm, 1.7μm) using gradient elution with 0.1% aqueous formic acid (FA) and acetonitrile with 0.1% FA (with DPT eluting at 2.60min). The method presented good fit (r>0.999) over the quantification range of 0.01-10μgmL-1 with the lower limit of quantitation (LLOQ) being 0.01μgmL-1 of human plasma for DPT. The intra- and inter-day precision, measured as % relative standard deviation, was less than 11% for DPT. The validation results showed that the developed method demonstrated adequate selectivity, sensitivity, precision and accuracy and therefore was successfully applied to the analysis of clinical samples following intravenous (iv) administration of 5.4mgkg-1 DPT to patients suffering from post-traumatic osteomyelitis induced by methicillin-resistant Staphylococcus aureus (MRSA). The developed methodology is the first report of an accurate mass tandem MS method for the analysis of this potent antibiotic in human plasma and can be used to further study pharmacokinetic, bioequivalence and even metabolic aspects related to this drug. © 2011 Elsevier B.V

In-depth analysis of crocetin ester glycosides from dried/processed stigmas of Crocus sativus L. by HPLC-ESI-MS n (n = 2, 3)

Introduction: Saffron stigmas from Crocus sativus L. (Iridaceae) are used as a drug in folk medicine, as a food additive and as a dying agent for at least 3500 years. Despite this long-term use the chemical composition of saffron seems still to be not fully known. Objective: An analytical strategy for detailed investigations of aqueous saffron extract is developed based on reverse-phase high-performance liquid chromatography electrospray ionisation (HPLC-ESI) multistage mass spectrometry (MS n ) for crocins. Methods: Commercially available stigmas are analysed by reverse-phase HPLC in combination with ESI/three-dimensional (3D)-ion trap mass spectrometry (MS) and MS n (n = 2 and 3). Sodium chloride is added to the analyte solution ready for injection to promote abundant [M + Na] + adduct ions of crocins, being ideal precursor ions for low-energy collision-induced dissociation (CID)-MS 2/3 . Results: This strategy allows the detailed structural elucidation of known as well as previously unknown crocin derivatives (molecular mass of the aglycon, oligosaccharide chain length and linkage determination). The two isomeric trisaccharide substituents neapolitanose and gentiotriose are distinguished based on linkage-specific cross-ring cleavage for the first time. Furthermore, crocins containing up to six hexose units are also observed. Five novel crocin ester glycosides shifted by a mass difference of −40 Da indicate the presence of the here newly described C 17 -aglycon, termed norcrocetin (crocetin = C 20 ). Conclusions: These findings indicate the action of at least two different carotenoid cleavage dioxygenases (CCD2 and tentatively CCD4) during biosynthesis of this new bis-apocarotenoid aglycon (norcrocetin) and the existence of even higher glycosylated crocin derivatives at trace level. © 2019 John Wiley & Sons, Ltd

In-chain neutral hydrocarbon loss from crocin apocarotenoid ester glycosides and the crocetin aglycon (Crocus sativus L.) by ESI-MSn (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 (MSn, 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 MS2-experiments. All sodiated precursor ions and major product ions were accompanied by a loss of 92 Da, which was elucidated as C7H8-loss from the aglycon by skeletal rearrangement via an eight-membered transition state as previously described for intact C40-carotenoids. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd

Publisher Correction: Crocus-derived compounds alter the aggregation pathway of Alzheimer’s Disease - associated beta amyloid protein (Scientific Reports, (2020), 10, 1, (18150), 10.1038/s41598-020-74770-x)

The original version of this Article contained an error in the title of the paper, where the phrase “Disease -associated” was incorrectly given as “Disease: associated”. This has now been corrected in the PDF and HTML versions of the Article. © 2021, The Author(s)