A Humanized Mouse Model Coupled with Computational Analysis Identifies Potent Glycolipid Agonist of Invariant NKT Cells.

Invariant natural killer T (iNKT) cells play an important role in many innate and adaptive immune responses, with potential applications in cancer immunotherapy. The glycolipid KRN7000, an α-galactosylceramide, potently activates iNKT cells but has shown limited anticancer effects in human clinical trials conducted so far. In spite of almost three decades of structure-activity relationship studies, no alternative glycolipid has yet emerged as a superior clinical candidate. One reason for the slow progress in this area is that standard mouse models do not accurately reflect the specific ligand recognition by human iNKT cells and their requirements for activation. Here we evaluated a series of KRN7000 analogues using a recently developed humanized mouse model that expresses a human αTCR chain sequence and human CD1d. In this process, a more stimulatory, previously reported but largely overlooked glycolipid was identified, and its activity was probed and rationalized via molecular simulations.

Influence of the extraction conditions on chia oil quality and partially defatted flour antioxidant properties.

Partially defatted chia flour (PDCF) is a by-product of oil extraction from chia seeds (Salvia hispanica L.). It may be used as an ingredient to improve food products, especially due to its antioxidant properties. In this work, we studied the best screw press extraction conditions that allow preserving the antioxidant properties of PDCF. A central composite design was applied to perform a response surface analysis in order to optimize the oil extraction. The variables considered for optimization were seed moisture content and pressing temperature. Besides the oil quality indicators, the study was focused on the assessment of PDCF properties, including total polyphenol content and antioxidant capacity determined by chemical methods. Our results show that, within the range of screw press conditions evaluated, the chemical quality of the oil and the antioxidant properties of PDCF are both preserved. The best results (highest oil yield and stability) were obtained under a seed moisture content of 10.2% and a pressing temperature of 58.5 °C. In general, our results indicate that screw press methodology can be applied to process chia seeds, using a wide range of conditions, to concurrently produce good quality oil and a PDCF with beneficial properties.

A favorable path to domain separation in the orange carotenoid protein.

The orange carotenoid protein (OCP) is responsible for nonphotochemical quenching (NPQ) in cyanobacteria, a defense mechanism against potentially damaging effects of excess light conditions. This soluble two-domain protein undergoes profound conformational changes upon photoactivation, involving translocation of the ketocarotenoid inside the cavity followed by domain separation. Domain separation is a critical step in the photocycle of OCP because it exposes the N-terminal domain (NTD) to perform quenching of the phycobilisomes. Many details regarding the mechanism and energetics of OCP domain separation remain unknown. In this work, we apply metadynamics to elucidate the protein rearrangements that lead to the active, domain-separated, form of OCP. We find that translocation of the ketocarotenoid canthaxanthin has a profound effect on the energetic landscape and that domain separation only becomes favorable following translocation. We further explore, characterize, and validate the free energy surface (FES) using equilibrium simulations initiated from different states on the FES. Through pathway optimization methods, we characterize the most probable path to domain separation and reveal the barriers along that pathway. We find that the free energy barriers are relatively small (<5 kcal/mol), but the overall estimated kinetic rate is consistent with experimental measurements (>1 ms). Overall, our results provide detailed information on the requirement for canthaxanthin translocation to precede domain separation and an energetically feasible pathway to dissociation.

Spectral Features of Canthaxanthin in HCP2. A QM/MM Approach.

The increased interest in sequencing cyanobacterial genomes has allowed the identification of new homologs to both the N-terminal domain (NTD) and C-terminal domain (CTD) of the Orange Carotenoid Protein (OCP). The N-terminal domain homologs are known as Helical Carotenoid Proteins (HCPs). Although some of these paralogs have been reported to act as singlet oxygen quenchers, their distinct functional roles remain unclear. One of these paralogs (HCP2) exclusively binds canthaxanthin (CAN) and its crystal structure has been recently characterized. Its absorption spectrum is significantly red-shifted, in comparison to the protein in solution, due to a dimerization where the two carotenoids are closely placed, favoring an electronic coupling interaction. Both the crystal and solution spectra are red-shifted by more than 50 nm when compared to canthaxanthin in solution. Using molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) studies of HCP2, we aim to simulate these shifts as well as obtain insight into the environmental and coupling effects of carotenoid-protein interactions.

Plants of the 'Libellus de Medicinalibus Indorum Herbis' from Mexico, 1552. Zephyranthes fosteri (Amaryllidaceae) Alkaloids.

The Libellus de Medicinalibus Indorum Herbis (Booklet of Indian Medicinal Plants) is the first book of medicinal plants written in the American continent. It was first published in 1939 as 'An Aztec Herbal'. One of the depicted plants is Huetzcanixochitl (laughing flower) interpreted as Zephyranthes fosteri (Amaryllidaceae). No chemical or pharmacological studies are reported for this species; so, we decide to investigate it. The GC/MS of the bulbs and aerial parts extracts indicated that they contain Amaryllidaceae alkaloids, among them: lycorine, 3-O-acetylpowelline, and norlycoramine. An unknown major alkaloid was isolated and identified by 1 H, 13 C-NMR and MS, as 3'-demethoxy-6-epimesembranol (1). The methanolic extract, the alkaloid fraction, and compound 1 inhibited acetylcholinesterase in vitro. Mesembrine alkaloids are found in Sceletium species (Aizoaceae). Several are known as serotonin recapture inhibitors and have been proposed as potential antidepressant drugs. The presence of 1 suggests that Z. fosteri was probably used in pre-Columbian times in Mexico as a 'stimulant and euphoriant', alike Sceletium tortuosum by several ethnic groups in South Africa.

Spectral Signatures of Canthaxanthin Translocation in the Orange Carotenoid Protein.

The orange carotenoid protein (OCP) is involved in the photoprotective processes in cyanobacteria via nonphotochemical quenching. Triggered by blue-green light absorption, the carotenoid chromophore undergoes translocation, displacing around 12 Å from the C-terminal domain (CTD) to the N-terminal domain (NTD). The detailed molecular rearrangements that occur within the carotenoid and the protein during this process remain largely elusive. By using a combination of molecular dynamics, well-tempered metadynamics, and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations, we were able to mimic the translocation of the carotenoid from the inactive OCPO and obtain metastable red-shifted states in the photoactivation mechanism, replicating the λmax values of reference experimental spectra. In addition, our simulations give insight into the structure of the red-shifted form of the inactive state of OCP.

Targeted metabolomics to assess the authenticity of bakery products containing chia, sesame and flax seeds.

Food fraud is perpetrated with increasing frequency along the food chain, triggering the need for new and modern tools to detect food authenticity. Chia, flax and sesame seeds are well known for the good nutritional characteristics of their oils, but there is a lack of knowledge regarding the authenticity of these seeds and food products containing them as well. In the present work, we propose a method based on targeted metabolomics to identify the polyphenols present in seeds, which can be used as markers of authenticity. We tentatively identified 44 polyphenols in the different seeds by HPLC-DAD-ESI-qTOF (MS/MS). Chemometrics allowed the selection of 12 compounds, which are nominated as novel markers for seed authentication. Some of these compounds were also found in a lab-scale preparation of cookies supplemented with the studied seeds. The proposed chemical markers resisted the baking process, representing good candidates to be used in the authentication of raw material and bakery products containing these seeds.

Clinanthus microstephium, an Amaryllidaceae Species with Cholinesterase Inhibitor Alkaloids: Structure-Activity Analysis of Haemanthamine Skeleton Derivatives.

Plants of the Amaryllidaceae family are well-known (not only) for their ornamental value but also for the alkaloids that they produce. In this report, the first phytochemical study of Clinanthus genus was carried out. The chemical composition of alkaloid fractions from Clinanthus microstephium was analyzed by GC/MS and NMR. Seven known compounds belonging to three structural types of Amaryllidaceae alkaloids were identified. An epimeric mixture of a haemanthamine-type compound (6-hydroxymaritidine) was tested as an inhibitor against acetyl- and butyrylcholinesterase enzymes (AChE and BChE, respectively), two enzymes relevant in the treatment of Alzheimer's disease, with good results. Structure-activity relationships through molecular docking studies with this alkaloid and other structurally related compounds were discussed.

Cholinesterase-inhibitory effect and in silico analysis of alkaloids from bulbs of Hieronymiella species.

BACKGROUND: In Argentina, the Amaryllidaceae family (59 species) comprises a wide variety of genera, only a few species have been investigated as a potential source of cholinesterases inhibitors to treat Alzheimer disease (AD). PURPOSE: To study the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities of the basic dichloromethane extracts (E) from Hieronymiella aurea, H. caletensis, H. clidanthoides, H. marginata, and H. speciosa species, as well as the isolated compounds from these plant extracts. STUDY DESIGN AND METHODS: AChE and BChE inhibitory activities were evaluated with the Ellman's spectrophotometric method. The alkaloids composition from the E was obtained by gas chromatography-mass spectrometry (GC-MS). The E were successively chromatographed on a silica gel column and permeated on Sephadex LH-20 column to afford the main alkaloids identified by means of spectroscopic data. Additionally, an in silico study was carried out. RESULTS: Nine known alkaloids were isolated from the E of five Hieronymiella species. Galanthamine was identified in all the species by GC-MS standing out H. caletensis with a relative abundance of 9.79% of the total ion current. Strong AChE (IC50 = 1.84 - 15.40 µg/ml) and moderate BChE (IC50 = 23.74 - 136.40 µg/ml) inhibitory activities were displayed by the extracts. Among the isolated alkaloids, only sanguinine and chlidanthine (galanthamine-type alkaloids) demonstrated inhibitory activity toward both enzymes. The QTAIM study suggests that sanguinine has the strongest affinity towards AChE, attributed to an additional interaction with Ser200 as well as stronger molecular interactions Glu199 and His440.These results allowed us to differentiate the molecular behavior in the active site among alkaloids possessing different in vitro inhibitory activities. CONCLUSION: Hieronymiella species growing in Argentina represent a rich and widespread source of galanthamine and others AChE and BChE inhibitors alkaloids. Additionally, the new trend towards the use of natural extracts as pharmaceuticals rather than pure drugs opens a pathway for the development of a phytomedicine derived from extracts of Hieronymiella spp.

Alkaloids from Hippeastrum argentinum and Their Cholinesterase-Inhibitory Activities: An in Vitro and in Silico Study.

Two new alkaloids, 4-O-methylnangustine (1) and 7-hydroxyclivonine (2) (montanine and homolycorine types, respectively), and four known alkaloids were isolated from the bulbs of Hippeastrum argentinum, and their cholinesterase-inhibitory activities were evaluated. These compounds were identified using GC-MS, and their structures were defined by physical data analysis. Compound 2 showed weak butyrylcholinesterase (BuChE)-inhibitory activity, with a half-maximal inhibitory concentration (IC50) value of 67.3 ± 0.09 µM. To better understand the experimental results, a molecular modeling study was also performed. The combination of a docking study, molecular dynamics simulations, and quantum theory of atoms in molecules calculations provides new insight into the molecular interactions of compound 2 with BuChE, which were compared to those of galantamine.

HPTLC and GC/MS Study of Amaryllidaceae Alkaloids of Two Narcissus Species.

In this article, we report on the alkaloid profile and dynamic of alkaloid content and diversity in two Narcissus plants at different stages of development. The alkaloid profile of the two Narcissus species was investigated by GC/MS and HPTLC. Fifty eight Amaryllidaceae alkaloids were detected, and 25 of them were identified in the different organs of N. tazetta and N. papyraceus. The alkaloid 3-O-methyl-9-O-demethylmaritidine is tentatively identified here for the first time from the Amaryllidaceae family, and four alkaloids (tazettamide, sternbergine, 1-O-acetyllycorine, 2,11-didehydro-2-dehydroxylycorine) are tentatively identified for the first time in the genus Narcissus. The different organs of the two species analyzed showed remarkable differences in their alkaloid pattern, type of biosynthesis, main alkaloid and number of alkaloids. Lycorine-type alkaloids dominated the alkaloid, metabolism in N. papyraceus, while alkaloids of narciclasine-, galanthamine- and homolycorine-types were found only in the species N. tazetta L.

Analysis of bioactive Amaryllidaceae alkaloid profiles in Lycoris species by GC-MS.

The genus Lycoris, a group of Amaryllidaceae plants distributed in temperate regions of Eastern Asia, is already known for containing representative alkaloids typical of this botanical family with a wide range of biological activities (for example, lycorine and galanthamine). In the present work, the alkaloid profiles of nine species, L. albiflora, L. aurea, L. chinensis, L. haywardii, L. incarnata, L. longituba, L. radiata, L. sprengeri, and L. squamigera, and one variety (L. radiata var. pumila) have been evaluated by GC-MS. Structures belonging to the lycorine-, homolycorine-, haemanthamine-, narciclasine-, tazettine-, montanine- and galanthamine-series were identified and quantified, with galanthamine- and lycorine-type alkaloids predominating and usually showing a high relative abundance in comparison with other alkaloids of the extracts. Interestingly, L. longituba revealed itself to be a potential commercial source of bioactive alkaloids. In general terms, our results are consistent with the alkaloid profiles reported in the literature for previously studied species.

Wild daffodils of the section Ganymedes from the Iberian Peninsula as a source of mesembrane alkaloids.

The aim of this work was to perform a detailed study of the alkaloid content of Narcissus triandrus, as well as a complete analysis of the alkaloid profile of 18 wild populations, comprising all the taxa of the section Ganymedes. Through the application of a combination of spectroscopic and chromatographic methods, the isolation and structural elucidation of 3 compounds are reported for the first time from a natural source (2-oxomesembrenone, 7,7a-dehydromesembrenone and 2-oxoepimesembranol), together with the identification of 5 major common mesembrane alkaloids. Additionally, the GC-MS analysis of the alkaloid profile demonstrated the regular presence of mesembranes in all the studied plants, showing mesembrenone as the predominant compound without any typical Amaryllidaceae alkaloid being detected.

Wild Argentinian Amaryllidaceae, a new renewable source of the acetylcholinesterase inhibitor galanthamine and other alkaloids.

The Amaryllidaceae family is well known for its pharmacologically active alkaloids. An important approach to treat Alzheimer's disease involves the inhibition of the enzyme acetylcholinesterase (AChE). Galanthamine, an Amaryllidaceae alkaloid, is an effective, selective, reversible, and competitive AchE inhibitor. This work was aimed at studying the alkaloid composition of four wild Argentinian Amarillydaceae species for the first time, as well as analyzing their inhibitory activity on acetylcholinesterase. Alkaloid content was characterized by means of GC-MS analysis. Chloroform basic extracts from Habranthus jamesonii, Phycella herbertiana, Rhodophiala mendocina and Zephyranthes filifolia collected in the Argentinian Andean region all contained galanthamine, and showed a strong AChE inhibitory activity (IC50 between 1.2 and 2 µg/mL). To our knowledge, no previous reports on alkaloid profiles and AChEIs activity of wild Argentinian Amarillydaceae species have been publisihed. The demand for renewable sources of industrial products like galanthamine and the need to protect plant biodiversity creates an opportunity for Argentinian farmers to produce such crops.

Alkaloids from Narcissus serotinus.

Narcissus serotinus belongs to the Amaryllidaceae family, a group well known for an exclusive variety of alkaloids with interesting biological activities. This study was aimed at identifying the alkaloid constituents of N. serotinus collected in the Spanish region of Valencia, using a combination of chromatographic, spectroscopic, and spectrometric methods, including GC-MS and 2D NMR techniques. GC-MS analysis allowed for the direct identification of five known compounds. In addition, the isolation and structure elucidation of six new Amaryllidaceae alkaloids are described.

Two new alkaloids from Narcissus serotinus L.

The Amaryllidaceae family is well known for the presence of an exclusive group of alkaloids with a wide range of biological activities. Narcissus serotinus L. is a plant belonging to this family and its geographical distribution is mainly located along the Mediterranean coast. In the present work, specimens collected near Casablanca (Morocco) were used to study the alkaloid content of this species. Starting with 350 g of the whole plant we used standard extraction and purification procedures to obtain fractions and compounds for GC-MS and NMR analysis. As well as five known alkaloids, we isolated two new compounds: 1-O-(3´-acetoxybutanoyl)lycorine and narseronine. The latter has been previously published, but with an erroneous structure.