Changes in the Antioxidant Potential of Camellia sinensis Cultures under the Influence of Phenolic Precursors.

The viability, productivity and survival of higher plants under the adverse factors influence are largely determined by the functional activity of the antioxidant system. The aim of our work was to investigate changes in formation of high-molecular (superoxide dismutase and peroxidase) and low-molecular (phenolics, including flavanols and proanthocyanidins) antioxidants in callus culture of Camellia sinensis under influence of phenolic precursors (L-phenylalanine-3 mM, trans-cinnamic acid-1 mM, naringenin-0.5 mM). According to the data obtained, the effect of precursors on tea callus cultures did not lead to significant increasing of superoxide dismutase and peroxidase activity in most cases. However, it led to the increased accumulation of the total phenolics content, as well as flavanols and proanthocyanidins contents. For C. sinensis callus cultures, the most promising regulator of phenolic compounds was L-phenylalanine, in the presence of which its content increased almost twice. Thus, the exogenous effect of various precursors is possible to use for the targeted regulation of certain phenolics classes accumulation in plant cells.

Natural Deep Eutectic Solvents for the Extraction of Triterpene Saponins from Aralia elata var. mandshurica (Rupr. & Maxim.) J. Wen.

The roots of the medicinal plant Aralia elata are rich in biologically active natural products, with triterpene saponins constituting one of their major groups. These metabolites can be efficiently extracted by methanol and ethanol. Due to their low toxicity, natural deep eutectic solvents (NADES) were recently proposed as promising alternative extractants for the isolation of natural products from medicinal plants. However, although NADES-based extraction protocols are becoming common in routine phytochemical work, their application in the isolation of triterpene saponins has not yet been addressed. Therefore, here, we address the potential of NADES in the extraction of triterpene saponins from the roots of A. elata. For this purpose, the previously reported recoveries of Araliacea triterpene saponins in extraction experiments with seven different acid-based NADES were addressed by a targeted LC-MS-based quantitative approach for, to the best of our knowledge, the first time. Thereby, 20 triterpene saponins were annotated by their exact mass and characteristic fragmentation patterns in the total root material, root bark and root core of A. elata by RP-UHPLC-ESI-QqTOF-MS, with 9 of them being identified in the roots of this plant for the first time. Triterpene saponins were successfully extracted from all tested NADES, with the highest efficiency (both in terms of the numbers and recoveries of individual analytes) achieved using a 1:1 mixture of choline chloride and malic acid, as well as a 1:3 mixture of choline chloride and lactic acid. Thereby, for 13 metabolites, NADES were more efficient extractants in comparison with water and ethanol. Our results indicate that new, efficient NADES-based extraction protocols, giving access to high recoveries of triterpene saponins, might be efficiently employed in laboratory practice. Thus, our data open the prospect of replacing alcohols with NADES in the extraction of A. elata roots.

Challenging Structure Elucidation of Lumnitzeralactone, an Ellagic Acid Derivative from the Mangrove Lumnitzera racemosa.

The previously undescribed natural product lumnitzeralactone (1), which represents a derivative of ellagic acid, was isolated from the anti-bacterial extract of the Indonesian mangrove species Lumnitzera racemosa Willd. The structure of lumnitzeralactone (1), a proton-deficient and highly challenging condensed aromatic ring system, was unambiguously elucidated by extensive spectroscopic analyses involving high-resolution mass spectrometry (HRMS), 1D 1H and 13C nuclear magnetic resonance spectroscopy (NMR), and 2D NMR (including 1,1-ADEQUATE and 1,n-ADEQUATE). Determination of the structure was supported by computer-assisted structure elucidation (CASE system applying ACD-SE), density functional theory (DFT) calculations, and a two-step chemical synthesis. Possible biosynthetic pathways involving mangrove-associated fungi have been suggested.

Study of the Antioxidant Properties of Filipendula ulmaria and Alnus glutinosa.

The demographic situation of the last few decades is characterized by the increased numbers of elderly and senile people, i.e., by the aging of the population. In humans, ageing is closely associated with the enhanced production of reactive oxygen species (ROS), development of systemic inflammation and related vascular atherosclerotic alterations and metabolic disorders, like obesity, diabetes mellitus and neurodegenerative diseases. As these age-related alterations are directly associated with up-regulation of ROS production and development of chronic oxidative stress, their onset can be essentially delayed by continuous daily consumption of dietary antioxidants-natural products of plant origin. Such antioxidants (in the form of plant extracts, biologically active complexes or individual compounds) can be supplemented to functional foods, i.e., dietary supplementations for daily diet aiming prolongation of active life and delay of the senescence onset. Thereby, use of widely spread medicinal plants might essentially improve cost efficiency of this strategy and availability of antioxidant-rich functional foods. Therefore, here we addressed, to the best of our knowledge for the first time, the antioxidant activity of the extracts prepared from the aerial parts of Filipendula ulmaria and Alnus glutinosa growing in the Kaliningrad region of Russia, and assessed the contents of the biologically active substances underlying these properties. It was found that the extract prepared with the leaves of Filipendula ulmaria and female catkins of Alnus glutinosa demonstrated high antioxidant activity, although the former plant was featured with a higher antioxidant potential. The highest antioxidant activity detected in the methanol extracts of Alnus glutinosa reached 1094.02 ± 14.53 µmol TE/g, radical scavenging of activity was 584.45 ± 35.3 µmol TE/g, reducing capacity at interaction with iron complex-471.63 ± 7.06 µmol TE/g. For the methanol extracts of Filipendula ulmaria the antioxidant activity reached 759.78 ± 19.08 µmol TE/g, antioxidant activity for free radical removal was 451.08 ± 24.45 µmol TE/g and antioxidant activity for restorative ability with iron complex was 332.28 ± 10.93 µmol TE/g. These values are consistent with the total yields of the extracts and their content of ellagic acid. The ethyl acetate extracts of the both plants showed just minimal antioxidant activity. Thus, the considered extracts have an essential potential. This creates good prospects for the further use of herbal extracts of Filipendula ulmaria and Alnus glutinosa as a source of natural antioxidants.

Authentication of saffron spice accessions from its common substitutes via a multiplex approach of UV/VIS fingerprints and UPLC/MS using molecular networking and chemometrics.

Saffron is a spice revered for its unique flavor and health attributes often subjected to fraudulence. In this study, molecular networking as a visualization tool for UPLC/MS dataset of saffron and its common substitutes i.e. safflower and calendula (n = 21) was employed for determining genuineness of saffron and detecting its common substitutes i.e. safflower and calendula. Saffron was abundant in flavonol-O-glycosides and crocetin esters versus richness of flavanones/chalcones glycosides in safflower and cinnamates/terpenes in calendula. OPLS-DA identified differences in UPLC/MS profiles of different saffron accessions where oxo-hydroxy-undecenoic acid-O-hexoside was posed as saffron authentication marker and aided in discrimination between Spanish saffron of high quality from its inferior grade i.e. Iranian saffron along with crocetin di-O-gentiobiosyl ester and kaempferol-O-sophoroside. Kaempferol-O-neohesperidoside and N,N,N,-p-coumaroyl spermidine were characteristic safflower metabolites, whereas, calendulaglycoside C and di-O-caffeoyl quinic acid were unique to calendula. UV/VIS fingerprint spectral regions of picrocrocin (230-260 nm) and crocin derivatives (400-470 nm) were posed as being discriminatory of saffron authenticity and suggestive it can replace UPLC/MS in saffrom quality determination.

Unraveling the metabolome composition and its implication for Salvadora persica L. use as dental brush via a multiplex approach of NMR and LC-MS metabolomics.

Salvadora persica L. (toothbrush tree, Miswak) is well recognized in most Middle Eastern and African countries for its potential role in dental care, albeit the underlying mechanism for its effectiveness is still not fully understood. A comparative MS and NMR metabolomics approach was employed to investigate the major primary and secondary metabolites composition of S. persica in context of its organ type viz., root or stem to rationalize for its use as a tooth brush. NMR metabolomics revealed its enrichment in nitrogenous compounds including proline-betaines i.e., 4-hydroxy-stachydrine and stachydrine reported for the first time in S. persica. LC/MS metabolomics identified flavonoids (8), benzylurea derivatives (5), butanediamides (3), phenolic acids (8) and 5 sulfur compounds, with 21 constituents reported for the first time in S. persica. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) of either NMR or LC/MS dataset clearly separated stem from root specimens based on nitrogenous compounds abundance in roots and is justifying for its preference as toothbrush versus stems. The presence of betaines at high levels in S. persica (9-12 µg/mg dry weight) offers novel insights into its functioning as an osmoprotectant that maintains the hydration of oral mucosa. Additionally, the previously described anti-inflammatory activity of stachydrine along with the antimicrobial effects of sulfonated flavonoids, benzylisothiocynate and ellagic acid derivatives are likely contributors to S. persica oral hygiene health benefits. Among root samples, variation in sugars and organic acids levels were the main discriminatory criterion. This study provides the first standardization of S. persica extract using qNMR for further inclusion in nutraceuticals.

Biological activity and stability analyses of knipholone anthrone, a phenyl anthraquinone derivative isolated from Kniphofia foliosa Hochst.

Knipholone (1) and knipholone anthrone (2), isolated from the Ethiopian medicinal plant Kniphofia foliosa Hochst. are two phenyl anthraquinone derivatives, a compound class known for biological activity. In the present study, we describe the activity of both 1 and 2 in several biological assays including cytotoxicity against four human cell lines (Jurkat, HEK293, SH-SY5Y and HT-29), antiplasmodial activity against Plasmodium falciparum 3D7 strain, anthelmintic activity against the model organism Caenorhabditis elegans, antibacterial activity against Aliivibrio fischeri and Mycobacterium tuberculosis and anti-HIV-1 activity in peripheral blood mononuclear cells (PBMCs) infected with HIV-1c. In parallel, we investigated the stability of knipholone (2) in solution and in culture media. Compound 1 displays strong cytotoxicity against Jurkat, HEK293 and SH-SY5Y cells with growth inhibition ranging from approximately 62-95% when added to cells at 50 µM, whereas KA (2) exhibits weak to strong activity with 26, 48 and 70% inhibition of cell growth, respectively. Both 1 and 2 possess significant antiplasmodial activity against Plasmodium falciparum 3D7 strain with IC50 values of 1.9 and 0.7 µM, respectively. These results complement previously reported data on the cytotoxicity and antiplasmodial activity of 1 and 2. Furthermore, compound 2 showed HIV-1c replication inhibition (growth inhibition higher than 60% at tested concentrations 0.5, 5, 15 and 50 µg/ml and an EC50 value of 4.3 µM) associated with cytotoxicity against uninfected PBMCs. The stability study based on preincubation, HPLC and APCI-MS (atmospheric-pressure chemical ionization mass spectrometry) analysis indicates that compound 2 is unstable in culture media and readily oxidizes to form compound 1. Therefore, the biological activity attributed to 2 might be influenced by its degradation products in media including 1 and other possible dimers. Hence, bioactivity results previously reported from this compound should be taken with caution and checked if they differ from those of its degradation products. To the best of our knowledge, this is the first report on the anti-HIV activity and stability analysis of compound 2.

Thin Film Chemical Deposition Techniques as a Tool for Fingerprinting of Free Fatty Acids by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Metabolic fingerprinting is a powerful analytical technique, giving access to high-throughput identification and relative quantification of multiple metabolites. Because of short analysis times, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is the preferred instrumental platform for fingerprinting, although its power in analysis of free fatty acids (FFAs) is limited. However, these metabolites are the biomarkers of human pathologies and indicators of food quality. Hence, a high-throughput method for their fingerprinting is required. Therefore, here we propose a MALDI-TOF-MS method for identification and relative quantification of FFAs in biological samples of different origins. Our approach relies on formation of monomolecular Langmuir films (LFs) at the interphase of aqueous barium acetate solution, supplemented with low amounts of 2,5-dihydroxybenzoic acid, and hexane extracts of biological samples. This resulted in detection limits of 10-13-10-14 mol and overall method linear dynamic range of at least 4 orders of magnitude with accuracy and precision within 2 and 17%, respectively. The method precision was verified with eight sample series of different taxonomies, which indicates a universal applicability of our approach. Thereby, 31 and 22 FFA signals were annotated by exact mass and identified by tandem MS, respectively. Among 20 FFAs identified in Fucus algae, 14 could be confirmed by gas chromatography-mass spectrometry.

An UPLC-MS/MS method for the simultaneous identification and quantitation of cell wall phenolics in Brassica napus seeds.

The seed residues left after pressing of rapeseed oil are rich in proteins and could be used for human nutrition and animal feeding. These press cakes contain, however, antinutritives, with fiber being the most abundant one. The analysis of fiber phenolic component (localized to seed coat cell walls) is, therefore, important in breeding and food quality control. However, correct structure and content assignments of cell wall-bound phenolics are challenging due to their low stability during sample preparation. Here, a novel LC-MS/MS-based method for the simultaneous identification and quantitation of 66 cell wall-bound phenolics and their derivatives is described. The method was internally standardized, corrected for degradation effects during sample preparation, and cross-validated with a well-established UV-based procedure. This approach was successfully applied to the analysis of cell wall phenolic patterns in different B. napus cultivars and proved to be suitable for marker compound search as well as assay development.

Profiling of hydroxycinnamic acid amides in Arabidopsis thaliana pollen by tandem mass spectrometry.

Phenylpropanoid polyamine conjugates are widespread in plant species. Their presence has been established in seeds, flower buds, and pollen grains. A biosynthetic pathway proposed for hydroxycinnamoyl spermidine conjugates has been suggested for the model plant Arabidopsis thaliana with a central acyl transfer reaction performed by a BAHD-like hydroxycinnamoyl transferase. A detailed liquid chromatography (LC)-electrospray ionization-mass spectrometry- and tandem-mass-spectrometry (MS/MS)-based survey of wild-type and spermidine hydroxycinnamoyl transferase (SHT) mutants identified more than 30 different bis- and tris-substituted spermidine conjugates, five of which were glycosylated, in the methanol-soluble fraction of the pollen exine. On the basis of characterized fragmentation patterns, a high-throughput LC-MS/MS method for highly sensitive HCAA relative quantification (targeted profiling) was developed. Only minor qualitative and quantitative differences in the pattern of bis-acyl spermidine conjugates in the SHT mutant compared to wild-type plants provide strong evidence for the presence of multiple BAHD-like acyl transferases and suggest a much more complex array of enzymatic steps in the biosynthesis of these conjugates than previously anticipated.