Microalgae as a New Source of Oxylipins: A Comprehensive LC-MS-Based Analysis Using Conventional and Green Extraction Methods.

Microalgae are promising sources of essential lipids, including omega-3 and omega-6 polyunsaturated fatty acids (n-3 and n-6 PUFA) and novel lipid metabolites like oxylipins. However, limited data exist on the oxylipin profile, its characterization, and the potential impact of the extraction process on these metabolites in microalgae. Thus, our study aimed to investigate the fatty acid and oxylipin profile of four microalgal species of interest (Microchloropsis gaditana, Tisochrysis lutea, Phaeodactylum tricornutum, and Porphyridium cruentum) while also examining the impact of the extraction method, with a focus on developing a greener process using ultrasound-assisted extraction (UAE) and ethanol. The UAE method showed similar oxylipin profiles, generally yielding concentrations comparable to those of the conventional Folch method. In total, 68 oxylipins derived from n-3 and n-6 PUFA were detected, with the highest concentrations of n-3 oxylipins found in P. tricornutum and T. lutea and of n-6 oxylipins in P. cruentum. This study provides the most extensive oxylipin characterization of these microalgae species to date, offering insights into alternative extraction methods and opening new avenues for further investigation of the significance of oxylipins in microalgae.

Anti-inflammatory discovery of sesquiterpenoids and a jasmonic acid derivative from Artemisia stolonifera.

Eleven previously undescribed sesquiterpenoids (8-18), one undescribed jasmonic acid derivative (35) and 28 known compounds were isolated from the leaves of Artemisia stolonifera. Undescribed compounds with their absolute configurations were determined by extensive spectroscopic analysis, single-crystal X-ray diffraction and ECD calculation. Compound 8 was identified as a rare sesquiterpenoid featuring a rearranged 5/8 bicyclic ring system, whereas compound 17 was found to be an unprecedented monocyclic sesquiterpenoid with methyl rearrangement. Evaluation of biological activity showed that compounds 1-5 and 7 displayed cytotoxicity against six tumor cells. In the meantime, compounds 11, 12, 18 and 35 exhibited inhibitory effects against LPS-stimulated NO production in RAW 264.7 macrophage cells and reduced the transcription of IL-6 and IL-1ß in a dose-dependent manner at 25, 50 and 100 µM. Moreover, the anti-inflammatory-based network pharmacology and molecular docking analyses revealed potential target proteins of 11, 12, 18 and 35.

Pronounced anti-neuroinflammatory jasmonates and terpenes isolated from lychee seeds.

Lychee is a favorite fruit of the Cantonese and native to Southeast Asia. In this study, the anti-neuroinflammatory bioactive compounds of lychee seeds have been carried out. Five new jasmonates (1, 2, 6-8) and seventeen known compounds were isolated using a series of chemical and chromatographic methods. Their chemical structures were identified through comprehensive spectroscopic analysis. Anti-neuroinflammatory activities were assayed and evaluated for the purified compounds. Most of the compounds exhibited pronounced anti-neuroinflammatory activities on nitric oxide (NO) induced by lipopolysaccharide (LPS) in BV-2 microglia cells. Moreover, compounds 1, 2 and 20 could reduce the expression of LPS-induced pro-inflammatory factors (iNOS and COX-2), inhibit the expression of mRNA levels of iNOS, COX-2, IL-6 and block NF-κB nuclear translocation in dose-dependent manners. This study suggested that lychee phytochemicals could be benefit to some neuroinflammatory-associated diseases, such as Alzheimer's disease.

Method optimization of oxylipin hydrolysis in nonprocessed bovine milk indicates that the majority of oxylipins are esterified.

The oxidation of polyunsaturated fatty acids produces bioactive primary oxidation products known as oxylipins. In many biological matrices, the majority of oxylipins are bound (i.e. esterified), and a relatively small proportion (<10%) exists in the free form. The present study tested whether this extends to bovine milk following method evaluation of various extraction and base hydrolysis protocols for measuring bound oxylipins. Free (unbound) oxylipins were also measured. Folch extraction followed by sodium carbonate hydrolysis in the presence of methanol containing 0.1% of acetic acid and 0.1% of butylated hydroxytoluene resulted in greater oxylipin concentrations and better surrogate standard recoveries compared to other methods that did not involve Folch extraction or the addition of methanol with hydrolysis base. Sodium hydroxide was better than sodium carbonate in hydrolyzing bound oxylipins under the same conditions. Milk analysis of oxylipins with mass-spectrometry following Folch extraction and sodium hydroxide hydrolysis revealed that 95% of oxylipins in bovine milk were esterified. Most of the detected oxylipins were derived from linoleic acid, which accounted for 92 and 88% of oxylipins in the free and esterified pools, respectively. These results demonstrate that the majority of bovine milk oxylipins are bound, and that linoleic-acid derived metabolites are the most abundant oxylipin species in free and bound lipid pools. Additional studies are needed to understand the role of different oxylipin pools in both calf and human nutrition. PRACTICAL APPLICATION: A method involving Folch lipid extraction and sodium hydroxide hydrolysis was validated for esterified oxylipin measurements in bovine milk. Application of the method revealed that the majority (∼95%) of oxylipins in bovine milk were bound. Linoleic-acid derived oxylipins were the most abundant species in both bound and free milk fractions (88-92%). The results highlight the presence of a new pool of oxidized lipids in milk, potentially involved in modifying its sensory and nutritional properties.

Isolation and Antitrypanosomal Characterization of Furoquinoline and Oxylipin from Zanthoxylum zanthoxyloides.

In the absence of vaccines, there is a need for alternative sources of effective chemotherapy for African trypanosomiasis (AT). The increasing rate of resistance and toxicity of commercially available antitrypanosomal drugs also necessitates an investigation into the mode of action of new antitrypanosomals for AT. In this study, furoquinoline 4, 7, 8-trimethoxyfuro (2, 3-b) quinoline (compound 1) and oxylipin 9-oxo-10, 12-octadecadienoic acid (compound 2) were isolated from the plant species Zanthoxylum zanthoxyloides (Lam) Zepern and Timler (root), and their in vitro efficacy and mechanisms of action investigated in Trypanosomabrucei (T. brucei), the species responsible for AT. Both compounds resulted in a selectively significant growth inhibition of T. brucei (compound 1, half-maximal effective concentration EC50 = 1.7 µM, selectivity indices SI = 74.9; compound 2, EC50 = 1.2 µM, SI = 107.3). With regards to effect on the cell cycle phases of T. brucei, only compound 1 significantly arrested the second growth-mitotic (G2-M) phase progression even though G2-M and DNA replication (S) phase arrest resulted in the overall reduction of T. brucei cells in G0-G1 for both compounds. Moreover, both compounds resulted in the aggregation and distortion of the elongated slender morphology of T. brucei. Analysis of antioxidant potential revealed that at their minimum and maximum concentrations, the compounds exhibited significant oxidative activities in T. brucei (compound 1, 22.7 µM Trolox equivalent (TE), 221.2 µM TE; compound 2, 15.0 µM TE, 297.7 µM TE). Analysis of growth kinetics also showed that compound 1 exhibited a relatively consistent growth inhibition of T. brucei at different concentrations as compared to compound 2. The results suggest that compounds 1 and 2 are promising antitrypanosomals with the potential for further development into novel AT chemotherapy.

Simple liquid chromatography-electrospray ionization ion trap mass spectrometry method for the quantification of galacto-oxylipin arabidopsides in plant samples.

A simple and sensitive method to quantify five different arabidopsides by HPLC-ion trap mass spectrometry in complex plant samples was developed and validated. Arabidopsides are oxidized galactolipids first described in Arabidopsis thaliana but also produced by other plant species under stress conditions. External calibration was performed using arabidopsides purified from freeze-thawed Arabidopsis leaves. Lipids were extracted and pre-purified on an SPE silica column before HPLC-MS analysis. Arabidopsides were separated on a C18 column using a gradient of mQ water and acetonitrile:mQ water (85:15) supplemented with formic acid (0.2%) and ammonium formate (12 mM). The method was validated according to European commission decision 2002/657/CE. LOD, LOQ, linearity, intra-day and inter-day precision and accuracy, selectivity, matrix effects and recoveries were determined for the five metabolites. The established method is highly selective in a complex plant matrix. LOD and LOQ were, respectively, in the range 0.098-0.78 and 0.64-1.56 µM, allowing the arabidopside quantification from 25.6-62.4 nmol/g fresh weight. Calibration curve correlation coefficients were higher than 0.997. Matrix effects ranged from -2.09% to 6.10% and recoveries between 70.7% and 109%. The method was successfully applied to complex plant matrixes: Arabidopsis thaliana and Nasturtium officinale.

Evaluation of cyclooxygenase oxylipins as potential biomarker for obesity-associated adipose tissue inflammation and type 2 diabetes using targeted multiple reaction monitoring mass spectrometry.

INTRODUCTION: Obesity is associated with adipose tissue inflammation which in turn drives insulin resistance and the development of type 2 diabetes. Oxylipins are a collection of lipid metabolites, subdivided in different classes, which are involved in inflammatory cascades. They play important roles in regulating adipose tissue homeostasis and inflammation and are therefore putative biomarkers for obesity-associated adipose tissue inflammation and the subsequent risk of type 2 diabetes onset. The objective for this study is to design an assay for a specific oxylipin class and evaluate these as potential prognostic biomarker for obesity-associated adipose tissue inflammation and type 2 diabetes. METHODS: An optimized workflow was developed to extract oxylipins from plasma using solid-phase extraction followed by analysis using ultra-high performance liquid chromatography coupled to a triple quadrupole mass spectrometer in multiple reaction monitoring mode. This workflow was applied to clinical plasma samples obtained from obese-type 2 diabetes patients and from lean and obese control subjects. RESULTS: The assay was analytically validated and enabled reproducible analyses of oxylipins extracted from plasma with acceptable sensitivities. Analysis of clinical samples revealed discriminative values for four oxylipins between the type 2 diabetes patients and the lean and obese control subjects, viz. PGF2α, PGE2, 15-keto-PGE2 and 13,14-dihydro-15-keto-PGE2. The combination of PGF2α and 15-keto-PGE2 had the most predictive value to discriminate type 2 diabetic patients from lean and obese controls. CONCLUSIONS: This proof-of-principle study demonstrates the potential value of oxylipins as biomarkers to discriminate obese individuals from obese-type 2 diabetes patients.

New oxylipins from Siegesbeckia glabrescens as potential antibacterial agents.

Seven rare oxylipins, siegesbeckins A-G (1-7) representing further bioactive constituents different from the general terpenyl compounds found in Siegesbeckia species, have been obtained from the aerial parts of Siegesbeckia glabrescens. These isolates were identified to be a series of methyl 4-methylpentanoates incorporating fatty acid moieties of different chain lengths, based on spectroscopic techniques, and their absolute configurations were determined via chemical degradation and comparison of experimental and theoretically calculated ECD spectra. With respect to bioactivity, antibacterial, anti-inflammatory and cytotoxic properties of selected compounds were evaluated. Compounds 1 and 5 showed moderate antibacterial activity against two Gram-positive bacteria with MIC values of 4.3 µg/mL, while 3 showed no pronounced activity in these assays.

Simultaneous detection of plant growth regulators jasmonic acid and methyl jasmonate in plant samples by a monoclonal antibody-based ELISA.

Methyl jasmonate (MeJA) and its free-acid form, jasmonic acid (JA), collectively referred to as jasmonates (JAs), are natural plant growth regulators that are widely present in higher plants. Simultaneous detection of JA and MeJA in plant samples is of significance and is a great challenging issue. In this study, coupling with two extraction methods, a sensitive monoclonal antibody (mAb) based enzyme-linked immunosorbent assay (ELISA) for simultaneous detection of JA and MeJA in plant samples was developed. The JA-bovine serum albumin (BSA) conjugate was used as an immunogen for the production of mAb. As the produced mAb exhibited higher recognition ability towards MeJA than towards JA, ELISA was established using MeJA as the standard. Under optimal experimental conditions, the IC50 and LOD values of ELISA for MeJA were 2.02 ng mL-1 and 0.20 ng mL-1, respectively. In the first extraction method, MeJA in plant samples was evaporated and only JA was extracted. In the second extraction method, both JA and MeJA were extracted. After methylation, JA in the extracts was converted into MeJA, and the whole MeJA in the extracts was measured by ELISA. Plant samples including the leaves of Salvia splendens, the flowers of Salvia splendens and the fruit of grapes were collected. JA and MeJA in these samples were detected by the proposed ELISA. It was found that the concentrations of JA in these three plant samples were about 3-5 times higher than those of MeJA in those samples. ELISA was also confirmed by HPLC. There was a good correlation between ELISA and HPLC.

Oxylipin vanillyl acetals from Solanum lyratum.

Four undescribed oxylipin vanillyl acetals with four stereogenic carbons were isolated from the herbs of Solanum lyratum. A comprehensive set of spectroscopic methods were used to elucidate the structures and relative configurations of 1-4. The absolute configurations of the naturally occurring compounds are assigned as 7S, 9'S, 10'S, 11'R at the site of six-membered cyclic acetal attachment by electronic circular dichroism (ECD) calculations and the modified Mosher's method. Compounds 1 and 3 displayed moderate selective inhibition against Hep3B and HepG2 cells, respectively. Further Annexin V-FITC/PI staining assay revealed that 1 and 3 might have inhibitory effects on hepatoma cells through induction of apoptosis.

Inhibition of Biofilm Formation by Modified Oxylipins from the Shipworm Symbiont Teredinibacter turnerae.

The bioactivity-guided purification of the culture broth of the shipworm endosymbiont Teredinibacter turnerae strain 991H.S.0a.06 yielded a new fatty acid, turneroic acid (1), and two previously described oxylipins (2-3). Turneroic acid (1) is an 18-carbon fatty acid decorated by a hydroxy group and an epoxide ring. Compounds 1-3 inhibited bacterial biofilm formation in Staphylococcus epidermidis, while only 3 showed antimicrobial activity against planktonic S. epidermidis. Comparison of the bioactivity of 1-3 with structurally related compounds indicated the importance of the epoxide moiety for selective and potent biofilm inhibition.

In†Vivo Solid-Phase Microextraction for Sampling of Oxylipins in Brain of Awake, Moving Rats.

Oxylipins are key lipid mediators of important brain processes, including pain, sleep, oxidative stress, and inflammation. For the first time, an in-depth profile of up to 52 oxylipins can be obtained from the brains of awake moving animals using in vivo solid-phase microextraction (SPME) chemical biopsy tool in combination with liquid chromatography-high resolution mass spectrometry. Among these, 23 oxylipins are detectable in the majority of healthy wildtype samples. This new approach successfully eliminates the changes in oxylipin concentrations routinely observed during the analysis of post-mortem samples, allows time-course monitoring of their concentrations with high spatial resolution in specific brain regions of interest, and can be performed using the same experimental set-up as in vivo microdialysis (MD) thus providing a new and exciting tool in neuroscience and drug discovery.

Role of Jasmonate in Modulation of Mycorrhizae-Induced Resistance Against Fungal Pathogens.

Symbiotic association of plants with arbuscular mycorrhizal (AM) fungi brings about changes in levels of the phytohormone jasmonate (JA) in root and shoot tissues of a plant. The enhanced JA levels not only play a role in controlling the extent of AM colonization but are also involved in the expression of mycorrhizal-induced resistance (MIR) against pathogens. We describe a method used to study the levels of a volatile jasmonate derivative, methyl jasmonate (MeJA), in tomato plants colonized by AM fungi and in response to subsequent attack by the foliar pathogen Alternaria alternata.

Quantitative Jasmonate Profiling Using a High-Throughput UPLC-NanoESI-MS/MS Method.

Jasmonic acid (JA) and its many derivatives-collectively referred as jasmonates-occur ubiquitously in land plants and regulate a wide range of stress-responses and development. Measuring these signaling compounds is complicated by the large number of jasmonate derivatives and the comparatively low concentration of these metabolites in plant tissues. We, here, present a selective and sensitive method consisting of a two-phase extraction coupled with liquid chromatography, nanoelectrospray ionization, and mass spectrometry to determine jasmonate levels in tissues and fluids of various plant species. The application of stable deuterium-labelled standards in combination with authentic standards allows the absolute quantification of a multitude of jasmonates and, additionally, the semi-quantitative analysis of further metabolites from the jasmonate pathway.

Comparative Physiological Analysis of Methyl Jasmonate in the Delay of Postharvest Physiological Deterioration and Cell Oxidative Damage in Cassava.

The short postharvest life of cassava is mainly due to its rapid postharvest physiological deterioration (PPD) and cell oxidative damage, however, how to effectively control this remains elusive. In this study, South China 5 cassava slices were sprayed with water and methyl jasmonate (MeJA) to study the effects of MeJA on reactive oxygen species, antioxidant enzymes, quality, endogenous hormone levels, and melatonin biosynthesis genes. We found that exogenous MeJA could delay the deterioration rate for at least 36 h and alleviate cell oxidative damage through activation of superoxide dismutase, catalase, and peroxidase. Moreover, MeJA increased the concentrations of melatonin and gibberellin during PPD, which had a significant effect on regulating PPD. Notably, exogenous MeJA had a significant effect on maintaining cassava quality, as evidenced by increased ascorbic acid content and carotenoid content. Taken together, MeJA treatment is an effective and promising way to maintain a long postharvest life, alleviate cell oxidative damage, and regulate storage quality in cassava.

Directed Non-targeted Mass Spectrometry and Chemical Networking for Discovery of Eicosanoids and Related Oxylipins.

Eicosanoids and related oxylipins are critical, small bioactive mediators of human physiology and inflammation. While ∼1,100 distinct species have been predicted to exist, to date, less than 150 of these molecules have been measured in humans, limiting our understanding of their role in human biology. Using a directed non-targeted mass spectrometry approach in conjunction with chemical networking of spectral fragmentation patterns, we find over 500 discrete chemical signals highly consistent with known and putative eicosanoids and related oxylipins in human plasma including 46 putative molecules not previously described. In plasma samples from 1,500 individuals, we find members of this expanded oxylipin library hold close association with markers of inflammation, as well as clinical characteristics linked with inflammation, including advancing age and obesity. These experimental and computational approaches enable discovery of new chemical entities and will shed important insight into the role of bioactive molecules in human health and disease.

Brain oxylipin concentrations following hypercapnia/ischemia: effects of brain dissection and dissection time.

PUFAs are precursors to bioactive oxylipin metabolites that increase in the brain following CO2-induced hypercapnia/ischemia. It is not known whether the brain-dissection process and its duration also alter these metabolites. We applied CO2 with or without head-focused microwave fixation for 2 min to evaluate the effects of CO2-induced asphyxiation, dissection, and dissection time on brain oxylipin concentrations. Compared with head-focused microwave fixation (control), CO2 followed by microwave fixation prior to dissection increased oxylipins derived from lipoxygenase (LOX), 15-hydroxyprostaglandin dehydrogenase (PGDH), cytochrome P450 (CYP), and soluble epoxide hydrolase (sEH) enzymatic pathways. This effect was enhanced when the duration of postmortem ischemia was prolonged by 6.4 min prior to microwave fixation. Brains dissected from rats subjected to CO2 without microwave fixation showed greater increases in LOX, PGDH, CYP and sEH metabolites compared with all other groups, as well as increased cyclooxygenase metabolites. In nonmicrowave-irradiated brains, sEH metabolites and one CYP metabolite correlated positively and negatively with dissection time, respectively. This study presents new evidence that the dissection process and its duration increase brain oxylipin concentrations, and that this is preventable by microwave fixation. When microwave fixation is not available, lipidomic studies should account for dissection time to reduce these artifacts.

Detection and identification of complex oxylipins in meadow buttercup (Ranunculus acris) leaves.

Screening of linolipins, i.e. galactolipids containing esterified residues of divinyl ether oxylipins, in the leaves of several higher plants revealed the presence of these complex oxylipins in the meadow buttercup leaves. The rapid accumulation of linolipins occurred in the injured leaves of meadow buttercup, while intact leaves possessed no linolipins. These oxylipins were isolated from the injured leaves, separated and purified by HPLC. The structural analyses of linolipins by UV, mass-spectroscopy and NMR spectroscopy resulted in the identification of eight molecular species. Three of them were identical to linolipins B-D found earlier in the leaves of flax (Linum usitatissimum L.). Other molecular species were identified as 1-O-(ω5Z)-etherolenoyl-2-O-dinor-(ω5Z)-etherolenoyl-3-O-ß-D-galactopyranosyl-sn-glycerol, 1-O-(ω5Z)-etherolenoyl-2-O-(7Z,10Z,13Z)-hexadecatrienoyl-3-O-ß-D-galactopyranosyl-sn-glycerol, 1-O-(ω5Z)-etherolenoyl-2-O-(7Z,10Z)-hexadecadienoyl-3-O-ß-D-galactopyranosyl-sn-glycerol, 1-O-(ω5Z)-etherolenoyl-2-O-α-linolenoyl-3-O-ß-D-galactopyranosyl-sn-glycerol, and 1-O-(ω5Z)-etherolenoyl-2-O-palmitoyl-3-O-(α-galactopyranosyl-1-6-ß-D-galactopyranosyl)-sn-glycerol. The trivial names "linolipins E, F, G, H and I," respectively, have been ascribed to these novel complex oxylipins.

Potential of Physalis peruviana calyces as a low-cost valuable resource of phytoprostanes and phenolic compounds.

BACKGROUND: In Colombia, agro-industrial residues represent an enormous economic and environmental problem, which could be reduced if different techniques for the addition of value to such residues were implemented by this industrial sector. One of the fruits with the highest export rates is Physalis peruviana (goldenberry); however, this fruit is generally marketed without its calyx, generating a large amount of residues. To develop a strategy to add value to these residues, it is essential to know their chemical composition. RESULTS: In the present work, phytoprostanes (PhytoPs) - new active oxylipins - have been detected for the first time in Physalis peruviana calyces by ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS), F1t -phytoprostanes and D1t -phytoprostanes being the predominant and minor classes, respectively. In addition, we were able to characterize the phenolic compounds profile of this matrix using LC-IT-DAD-MS/MS, describing six phenolic derivatives for the first time therein. CONCLUSIONS: This study increases our knowledge of the chemical composition of the calyces of this fruit and thereby supports the recycling of this class of residue. Consequently, goldenberry calyces could be used as phytotherapeutic, nutraceutic, or cosmetic ingredients for the development of diverse natural products. © 2018 Society of Chemical Industry.

A stable method for routine analysis of oxylipins from dried blood spots using ultra-high performance liquid chromatography-tandem mass spectrometry.

Oxylipins are biologically important lipid mediators that are derived enzymatically from polyunsaturated fatty acids (PUFA) and have a major role in regulating inflammatory processes. The currently available methods for measuring oxylipins from human biological samples have limitations, which restricts their use in large studies. We have developed a novel method for measuring 21 oxylipins from dried blood spot (DBS) using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and stable isotope dilution analysis. Our new method is reproducible and precise and enables the high throughput analysis and quantitation of bioactive oxylipins in small volumes of blood. In the future, this new method can be readily applied to measure oxylipins in large studies. Abstract Oxylipins are downstream lipid mediators enzymatically-produced from polyunsaturated fatty acids (PUFA) that are implicated as the biological effectors of these fatty acids. Recently reported methods for the quantitation of oxylipins require complex extraction procedures. In this study, we report the development and validation of a novel system for the quantitation of 21 individual oxylipins from a dried blood spot (DBS) using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and stable isotope dilution analysis. Linearity and precision of the method were determined and the stabilities of the 12 most abundant oxylipins were tested during 2 months of storage at room temperature, after being spiked into blood and prepared as DBS on PUFAcoat™ paper. Responses were linear across the concentration range analysed for all oxylipins (r2 values ranged from 0.953 to 0.998). Intra-day and inter-day variations were ≤16% for all oxylipins. Recovery of oxylipins from the DBS ranged from 80 - 115%. The 12 spiked oxylipins were stable for 2 months when stored as DBS at room temperature. Our method is reproducible and precise, and provides the opportunity to accurately quantitate these oxylipins in a small sample volume.