Cold-Pressed Pomegranate Seed Oil: Study of Punicic Acid Properties by Coupling of GC/FID and FTIR.

Over the last decades, we have witnessed an increasing interest in food-related products containing vegetable oils. These oils can be obtained either by extraction or by mechanical pressing of different parts of plants (e.g., seeds, fruit, and drupels). Producers of nutraceuticals have ceaselessly searched for unique and effective natural ingredients. The enormous success of argan oil has been followed by discoveries of other interesting vegetable oils (e.g., pomegranate oil) containing several bioactives. This work describes the pomegranate fruit extract and seed oil as a rich source of conjugated linolenic acid as a metabolite of punicic acid (PA), deriving from the omega-5 family (ω-5). Through the chemical characterization of PA, its nutritional and therapeutic properties are highlighted together with the physiological properties that encourage its use in human nutrition. We analyzed the composition of all fatty acids with beneficial properties occurring in pomegranate seed oil using gas chromatography (GC) with flame-ionization detection (FID) analysis combined with Fourier transform infrared spectroscopy (FTIR). Pomegranate seed oil mainly consists of 9,11,13-octadic-trienoic acid (18:3), corresponding to 73 wt % of the total fatty acids. Nine components were identified by GC in PSO, varying between 0.58 and 73.19 wt %. Using midinfrared (MIR) spectroscopy, we compared the composition of pomegranate seed oil with that of meadowfoam seed oil (MSO), which is also becoming increasingly popular in the food industry due to its high content of long chain fatty acids (C20-22), providing increased oil stability. From the results of FTIR and MIR spectroscopy, we found that punicic acid is unique in PSO (73.19 wt %) but not in MSO.

Microbial enrichment of blackcurrant press residue with conjugated linoleic and linolenic acids.

AIMS: The aim of the study was to investigate the isomerization of linoleic (LA) and linolenic acids (LNAs) into their conjugated isomers by Propionibacterium freudenreichii DSM 20270 and utilize this feature for microbial enrichment of blackcurrant press residue (BCPR) with health-beneficial conjugated fatty acids. METHODS AND RESULTS: First, the ability of P. freudenreichii to isomerize 0·4 mg ml-1 of LA and LNA was studied in lactate growth medium. Free LA and α-LNA were efficiently converted into conjugated linoleic (CLA) and α-linolenic acid (α-CLNA), being the predominant isomers c9,t11-CLA and c9,t11,c15-CLNA, respectively. The bioconversion of α-LNA by P. freudenreichii was more efficient in terms of formation rate, yield and isomer-specificity. Thereafter, free LA and LNAs obtained from hydrolysed BCPR neutral lipids, by lipolytically active oat flour, were subjected to microbial isomerization in BCPR slurries. In 10% (w/v) slurries, a simultaneous enrichment in c9,t11-CLA and c9,t11,c15-CLNA of up to 0·51 and 0·29 mg ml-1 was observed from starting levels of 0·96 mg LA ml-1 and 0·37 mg α-LNA ml-1 respectively. CONCLUSIONS: This study shows that growing cultures of P. freudenreichii DSM 20270 are able to simultaneously enrich BCPR with health-beneficial conjugated isomers of LA and α-LNA. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that microbial isomerization technique can be utilized to enrich lipid-containing plant materials with bioactive compounds and thereby enable valorization of low value plant-based side streams from food industry into value-added food ingredients.

Pinocembrin and its linolenoyl ester derivative induce wound healing activity in HaCaT cell line potentially involving a GPR120/FFA4 mediated pathway.

Wound healing represents an urgent need from the clinical point of view. Several diseases result in wound conditions which are difficult to treat, such as in the case of diabetic foot ulcer. Starting from there, the medicinal research has focused on various targets over the years, including GPCRs as new wound healing drug targets. In line with this, GPR120, known to be an attractive target in type 2 diabetes drug discovery, was studied to finalize the development of new wound healing agents. Pinocembrin (HW0) was evaluated as a suitable compound for interacting with GPR120, and was hybridized with fatty acids, which are known endogenous GPR120 ligands, to enhance the wound healing potential and GPR120 interactions. HW0 and its 7-linolenoyl derivative (HW3) were found to be innovative wound healing agents. Immunofluorescence and functional assays suggested that their activity was mediated by GPR120, and docking simulations showed that the compounds could share the same pocket occupied by the known GPR120 agonist, TUG-891.

Optimized small-molecule pull-downs define MLBP1 as an acyl-lipid-binding protein.

Abscisic acid (ABA) receptors belong to the START domain superfamily, which encompasses ligand-binding proteins present in all kingdoms of life. START domain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterized START domain proteins are the 14 PYR/PYL/RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently in Nicotiana benthamiana coupled to untargeted LC-MS to identify candidate binding ligands. We optimized this method using ABA-PYL interactions and show that ABA co-purifies with wild-type PYL5 but not a binding site mutant. The Kd of PYL5 for ABA is 1.1 µm, which suggests that the method has sufficient sensitivity for many ligand-protein interactions. Using this method, we surveyed a set of 37 START domain-related proteins, which resulted in the identification of ligands that co-purified with MLBP1 (At4G01883) or MLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed that MLBP1 binds to monolinolenin, which we confirmed using recombinant MLBP1. Monolinolenin also co-purified with MLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein-metabolite interaction and better understand protein-ligand interactions in plants.

NMR and Computational Studies as Analytical and High-Resolution Structural Tool for Complex Hydroperoxides and Diastereomeric Endo-Hydroperoxides of Fatty Acids in Solution-Exemplified by Methyl Linolenate.

A combination of selective 1D Total Correlation Spectroscopy (TOCSY) and 1H-13C Heteronuclear Multiple Bond Correlation (HMBC) NMR techniques has been employed for the identification of methyl linolenate primary oxidation products without the need for laborious isolation of the individual compounds. Complex hydroperoxides and diastereomeric endo-hydroperoxides were identified and quantified. Strongly deshielded C-O-O-H 1H-NMR resonances of diastereomeric endo-hydroperoxides in the region of 8.8 to 9.6 ppm were shown to be due to intramolecular hydrogen bonding interactions of the hydroperoxide proton with an oxygen atom of the five-member endo-peroxide ring. These strongly deshielded resonances were utilized as a new method to derive, for the first time, three-dimensional structures with an assignment of pairs of diastereomers in solution with the combined use of 1H-NMR chemical shifts, Density Functional Theory (DFT), and Our N-layered Integrated molecular Orbital and molecular Mechanics (ONIOM) calculations.

Effect of Blanching Pomegranate Seeds on Physicochemical Attributes, Bioactive Compounds and Antioxidant Activity of Extracted Oil.

This study investigated the effect of blanching pomegranate seeds (PS) on oil yield, refractive index (RI), yellowness index (YI), conjugated dienes (K232), conjugated trienes (K270), total carotenoid content (TCC), total phenolic compounds (TPC) and DPPH radical scavenging of the extracted oil. Furthermore, phytosterol and fatty acid compositions of the oil extracted under optimum blanching conditions were compared with those from the oil extracted from unblanched PS. Three different blanching temperature levels (80, 90, and 100 °C) were studied at a constant blanching time of 3 min. The blanching time was then increased to 5 min at the established optimum blanching temperature (90 °C). Blanching PS increased oil yield, K232, K270, stigmasterol, punicic acid, TPC and DPPH radical scavenging, whereas YI, ß-sitosterol, palmitic acid and linoleic acid were decreased. The RI, TCC, brassicasterol, stearic acid, oleic acid and arachidic acid of the extracted oil were not significantly (p > 0.05) affected by blanching. Blanching PS at 90 °C for 3 to 5 min was associated with oil yield, TPC and DPPH. Blanching PS at 90 °C for 3 to 5 min will not only increase oil yield but could also improve functional properties such as antioxidant activity, which are desirable in the cosmetic, pharmaceutical, nutraceutical and food industries.

Punicic acid: A potential compound of pomegranate seed oil in Type 2 diabetes mellitus management.

Diabetes is one of the most prevalent diseases in the worldwide. Type 2 diabetes mellitus (T2DM), the most common form of the disease, has become a serious threat to public health and is a growing burden on global economies. Due to the unexpected adverse effects of antidiabetic medicines, the use of nutraceuticals as a complementary therapy has drawn extensive attention by investigators. In this issue, a novel nutraceutical, Punicic acid (PA)-the main ingredient of pomegranate seed oil (PSO) that has potential therapeutic effects in T2DM-has been investigated. PA is a peroxisome proliferator-activated receptor gamma agonist, and unlike synthetic ligands, such as thiazolidinediones, it has no side effects. PA exerts antidiabetic effects via various mechanisms, such as reducing inflammatory cytokines, modulating glucose homeostasis, and antioxidant properties. In this review, we discussed the potential therapeutic effects of PSO and PA and represented the related mechanisms involved in the management of T2DM.

Preparation, optimization, and pharmacokinetic study of nanoliposomes loaded with triacylglycerol-bound punicic acid for increased antihepatotoxic activity.

Punicic acid of pomegranate oil (PAP) has gained heightened interest due to several health benefits, such as anticarcinogenic, antidiabetic, and antiatherosclerotic properties. However, these bioactivities have been hampered by chemical instability, poor water solubility, rapid metabolism, and low bioavailability of PAP. Therefore, this study was aimed at optimizing the liposomal formulation of Triacylglycerol-bound punicic acid with its regioisomers (TPAR) for improved oral bioavailability and increased hepatoprotection through antioxidation and anti-inflammation. Herein, the optimized TPAR nanoliposome (TPAR-NL) was developed using thin-film dispersion method and subsequently characterized with appropriate indices. The optimized TPAR-NL produced fairly stable spherical nanoparticles (˂ 200 nm) with encapsulation efficiency (%EE) of 85.77%, as well as enhanced in vitro release and improved oral bioavailability. The TPAR-NL exhibited profound antihepatotoxic effect in mice pretreated with carbon tetrachloride (CCl4 ) via reduction of serum alanine aminotransferase, aspartate aminotransferase, and total bilirubin levels compared with free TPAR. The TPAR-loaded liposome also significantly reduced oxidative stress by increasing superoxide dismutase and glutathione levels while lowering malonaldehyde concentration compared with the free TPAR. The TPAR-LNF further exhibited remarkable anti-inflammatory activity compared with the free drug via inhibition of interleukin-6 and tumor necrosis factor-alpha generation. Thus, the developed nanoliposomes potentiated the antihepatotoxic activity of TPAR via antioxidation and anti-inflammation.

Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis.

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(S)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST (Solanum tuberosum), CYP74C2 (Cucumis melo), CYP74C1_CS and CYP74C31 (both of Cucumis sativus), and CYP74C13_GM (Glycine max). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the "hydroperoxide-binding domain" (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs.

Galactolipase activity of Talaromyces thermophilus lipase on galactolipid micelles, monomolecular films and UV-absorbing surface-coated substrate.

Talaromyces thermophilus lipase (TTL) was found to hydrolyze monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) substrates presented in various forms to the enzyme. Different assay techniques were used for each substrate: pHstat with dioctanoyl galactolipid-bile salt mixed micelles, barostat with dilauroyl galactolipid monomolecular films spread at the air-water interface, and UV absorption using a novel MGDG substrate containing α-eleostearic acid as chromophore and coated on microtiter plates. The kinetic properties of TTL were compared to those of the homologous lipase from Thermomyces lanuginosus (TLL), guinea pig pancreatic lipase-related protein 2 and Fusarium solani cutinase. TTL was found to be the most active galactolipase, with a higher activity on micelles than on monomolecular films or surface-coated MGDG. Nevertheless, the UV absorption assay with coated MGDG was highly sensitive and allowed measuring significant activities with about 10 ng of enzymes, against 100 ng to 10 µg with the pHstat. TTL showed longer lag times than TLL for reaching steady state kinetics of hydrolysis with monomolecular films or surface-coated MGDG. These findings and 3D-modelling of TTL based on the known structure of TLL pointed out to two phenylalanine to leucine substitutions in TTL, that could be responsible for its slower adsorption at lipid-water interface. TTL was found to be more active on MGDG than on DGDG using both galactolipid-bile salt mixed micelles and galactolipid monomolecular films. These later experiments suggest that the second galactose on galactolipid polar head impairs the enzyme adsorption on its aggregated substrate.

Regioisomeric distribution of 9- and 13-hydroperoxy linoleic acid in vegetable oils during storage and heating.

BACKGROUND: The oxidative deterioration of vegetable oils is commonly measured by the peroxide value, thereby not considering the contribution of individual lipid hydroperoxide isomers, which might have different bioactive effects. Thus, the formation of 9- and 13-hydroperoxy octadecadienoic acid (9-HpODE and 13- HpODE), was quantified after short-term heating and conditions representative of long-term domestic storage in samples of linoleic acid, canola, sunflower and soybean oil, by means of stable isotope dilution analysis-liquid chromatography-mass spectroscopy. RESULTS: Although heating of pure linoleic acid at 180 °C for 30 min led to an almost complete loss of 9-HpODE and 13-HpODE, heating of canola, sunflower and soybean oil resulted in the formation of 5.74 ± 3.32, 2.00 ± 1.09, 16.0 ± 2.44 mmol L-1 13-HpODE and 13.8 ± 8.21, 10.0 ± 6.74 and 45.2 ± 6.23 mmol L-1 9-HpODE. An almost equimolar distribution of the 9- and 13-HpODE was obtained during household-representative storage conditions after 56 days, whereas, under heating conditions, an approximately 2.4-, 2.8- and 5.0-fold (P ≤ 0.001) higher concentration of 9-HpODE than 13-HpODE was detected in canola, soybean and sunflower oil, respectively. CONCLUSION: A temperature-dependent distribution of HpODE regioisomers could be shown in vegetable oils, suggesting their application as markers of lipid oxidation in oils used for short-term heating. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Effect of α-linolenic acid-modified low molecular weight chondroitin sulfate on atherosclerosis in apoE-deficient mice.

METHODS: 8-week-age male ApoE(-/-) mice were fed with the atherogenic diet together with or without tested compounds (rosuvastatin calcium, α-LNA-LMWCS, LMWCS and α-LNA) for 16 weeks. When the animals were killed, blood plasma was isolated to test the level of TC, LDL-C, TNF-α, IL-6 and CRP by biochemistry analysis and ELISA method. The whole aorta and aortic root sections were also collected to study atherogenesis level and reveal the possible mechanism by histological examination, real-time PCR and Western blot analysis. RESULTS: The level of TC, LDL-C, TNF-α, IL-6 and CRP in plasma in H-LNA-LMWCS group were significantly lower than those of the control group (rosuvastatin calcium). Plaques in H-LNA-LMWCS group showed higher content of smooth muscle cells, lower content of lipid and macrophages, and lower mRNA levels of TNF-α, IL-6, CRP, MCP-1, VCAM-1 and ICAM-1 than those in the control group. In addition, α-LNA-LMWCS could reduce the nuclear translocation of NF-κB, inhibit expressions of p-ERK1/2, p-p38, MCP-1, VCAM-1 and ICAM-1 in mice aorta. CONCLUSION: α-LNA-LMWCS exhibited anti-atherosclerosis effect through regulating the lipid metabolism and diminishing the synthesis of pro-inflammatory cytokines. The possible mechanism may be that α-LNA-LMWCS could influence MAPK/ NF-κB related signal pathway. GENERAL SIGNIFICANCE: The results may provide significant suggestions for the application of α-LNA-LMWCS in anti-atherosclerosis.

A continuous spectrophotometric assay that distinguishes between phospholipase A1 and A2 activities.

A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase(®), guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement.

Robust inhibitory effects of conjugated linolenic acids on a cyclooxygenase-related linoleate 10S-dioxygenase: Comparison with COX-1 and COX-2.

There are many reports of the anti-inflammatory, anti-cancer, and anti-atherosclerotic activities of conjugated linolenic acids (cLNA). They constitute a small percentage of fatty acids in the typical human diet, although up to 80% of the fatty acids in certain fruits such as pomegranate. In the course of studying a bacterial fatty acid dioxygenase (Nostoc linoleate 10S-DOX, an ancient relative of mammalian cyclooxygenases), we detected strong inhibitory activity in a commercial sample of linoleic acid. We identified two cLNA isomers, ß-eleostearic (9E,11E,13E-18:3) and ß-calendic acid (8E,10E,12E-18:3), as responsible for that striking inhibition with a Ki of ~49nM and ~125nM, respectively, the most potent among eight cLNA tested. We also examined the effects of all eight cLNA on the activity of COX-1 and COX-2. Jacaric acid (8Z,10E,12Z-18:3) and its 12E isomer, 8Z,10E,12E-18:3, strongly inhibit the activity of COX-1 with a Ki of ~1.7 and ~1.1µM, respectively. By contrast, COX-2 was ≤30% inhibited at 10µM concentrations of the cLNA. Identifying the activities of the naturally occurring fatty acids is of interest in terms of understanding their interaction with the enzymes, and for explaining the mechanistic basis of their biological effects. The study also highlights the potential presence of inhibitory fatty acids in commercial lipids prepared from natural sources. Analysis of seven commercial samples of linoleic acid by HPLC and UV spectroscopy is illustrated as supplementary data.

Nitro-linolenic acid is a nitric oxide donor.

Nitro-fatty acids (NO2-FAs), which are the result of the interaction between reactive nitrogen species (RNS) and non-saturated fatty acids, constitute a new research area in plant systems, and their study has significantly increased. Very recently, the endogenous presence of nitro-linolenic acid (NO2-Ln) has been reported in the model plant Arabidopsis thaliana. In this regard, the signaling role of this molecule has been shown to be key in setting up a defense mechanism by inducing the chaperone network in plants. Here, we report on the ability of NO2-Ln to release nitric oxide (NO) in an aqueous medium with several approaches, such as by a spectrofluorometric probe with DAF-2, the oxyhemoglobin oxidation method, ozone chemiluminescence, and also by confocal laser scanning microscopy in Arabidopsis cell cultures. Jointly, this ability gives NO2-Ln the potential to act as a signaling molecule by the direct release of NO, due to its capacity to induce different changes mediated by NO or NO-related molecules such as nitration and S-nitrosylation or by the electrophilic capacity of these molecules through a nitroalkylation mechanism.

Gram scale synthesis of specialized pro-resolving mediator 17(S)-HDHA using lipoxygenase enhanced by water-soluble reducing agent TCEP.

17(S)-Hydroxy docosahexaenoic acid (17(S)-HDHA) is a specialized pro-resolving mediator. The oxidation of docosahexaenoic acid (DHA) to 17(S)-HDHA using soybean lipoxygenase was accomplished in the presence of the reducing agent TCEP in high yield and high enantio excess. We demonstrated application of this strategy to the synthesis of other fatty acids and to gram scale synthesis of 17(S)-HDHA.

Physiological function and ecological aspects of fatty acid-amino acid conjugates in insects.

In tritrophic interactions, plants recognize herbivore-produced elicitors and release a blend of volatile compounds (VOCs), which work as chemical cues for parasitoids or predators to locate their hosts. From detection of elicitors to VOC emissions, plants utilize sophisticated systems that resemble the plant-microbe interaction system. Fatty acid-amino acid conjugates (FACs), a class of insect elicitors, resemble compounds synthesized by microbes in nature. Recent evidence suggests that the recognition of insect elicitors by an ancestral microbe-associated defense system may be the origin of tritrophic interactions mediated by FACs. Here we discuss our findings in light of how plants have customized this defense to be effective against insect herbivores, and how some insects have successfully adapted to these defenses.

Lipoxygenase-catalyzed transformation of epoxy fatty acids to hydroxy-endoperoxides: a potential P450 and lipoxygenase interaction.

Herein, we characterize a generally applicable transformation of fatty acid epoxides by lipoxygenase (LOX) enzymes that results in the formation of a five-membered endoperoxide ring in the end product. We demonstrated this transformation using soybean LOX-1 in the metabolism of 15,16-epoxy-α-linolenic acid, and murine platelet-type 12-LOX and human 15-LOX-1 in the metabolism of 14,15-epoxyeicosatrienoic acid (14,15-EET). A detailed examination of the transformation of the two enantiomers of 15,16-epoxy-α-linolenic acid by soybean LOX-1 revealed that the expected primary product, a 13S-hydroperoxy-15,16-epoxide, underwent a nonenzymatic transformation in buffer into a new derivative that was purified by HPLC and identified by UV, LC-MS, and ¹H-NMR as a 13,15-endoperoxy-16-hydroxy-octadeca-9,11-dienoic acid. The configuration of the endoperoxide (cis or trans side chains) depended on the steric relationship of the new hydroperoxy moiety to the enantiomeric configuration of the fatty acid epoxide. The reaction mechanism involves intramolecular nucleophilic substitution (SNi) between the hydroperoxy (nucleophile) and epoxy group (electrophile). Equivalent transformations were documented in metabolism of the enantiomers of 14,15-EET by the two mammalian LOX enzymes, 15-LOX-1 and platelet-type 12-LOX. We conclude that this type of transformation could occur naturally with the co-occurrence of LOX and cytochrome P450 or peroxygenase enzymes, and it could also contribute to the complexity of products formed in the autoxidation reactions of polyunsaturated fatty acids.

Development of a high-throughput assay for measuring phospholipase A activity using synthetic 1,2-α-eleostearoyl-sn-glycero-3-phosphocholine coated on microtiter plates.

To date, several sensitive methods, based on radiolabeled elements or sterically hindered fluorochrome groups, are usually employed to screen phospholipase A (PLA) activities. With the aim of developing a convenient, specific, sensitive, and continuous new ultraviolet (UV) spectrophotometric assay for PLA, we have synthesized a specific glycerophosphatidylcholine (PC) esterified at the sn-1 and sn-2 positions, with α-eleostearic acid (9Z, 11E, 13E-octadecatrienoic acid) purified from Aleurites fordii seed oil. The conjugated triene present in α-eleostearic acid constitutes an intrinsic chromophore and, consequently, confers the strong UV absorption properties of this free fatty acid as well as of the glycerophospholipids harboring it. This coated PC film cannot be desorbed by the various buffers used during PLA assays. Following the action of PLA at the oil-water interface, α-eleostearic acid is freed and desorbed from the film and then solubilized with ß-cyclodextrin. The UV absorbance of the α-eleostearic acid is considerably enhanced due to the transformation from an adsorbed to a water-soluble state. The PLA activity can be measured continuously by recording the variations with time of the UV absorption spectra. The rate of lipolysis was monitored by measuring the increase of absorption at 272 nm, which was found to be linear with time and proportional to the amount of added PLA. This continuous high-throughput PLA assay could be used to screen new PLA and/or PLA inhibitors present in various biological samples.

Epoxy monomers derived from tung oil fatty acids and its regulable thermosets cured in two synergistic ways.

A novel biobased epoxy monomer with conjugated double bonds, glycidyl ester of eleostearic acid (GEEA) was synthesized from tung oil fatty acids and characterized by (1)H and (13)C NMR. Differential scanning calorimeter analysis (DSC) and Fourier transform infrared spectroscopy (FT-IR) were utilized to investigate the curing process of GEEA with dienophiles and anhydrides. DSC indicated that GEEA could cross-link with both dienophiles and anhydrides through Diels-Alder reaction and epoxy/anhydride ring-opening reaction. Furthermore, Diels-Alder cross-link was much more active than the ring-opening of epoxy and anhydride in the curing process. FT-IR also revealed that GEEA successively reacted with dienophiles and anhydrides in both cross-linking methods. Dynamic mechanical analysis and mechanical tensile testing were used to study the thermal and mechanical properties of GEEA cured by maleic anhydride, nadic methyl anhydride and 1,1'-(methylenedi-4,1-phenylene)bismaleimide. Due to the independence between the curing agents, dienophile and anhydride, a series of thermosetting polymers with various properties could be obtained by adjusting the composition of these two curing agents.