Synthesis of EPA- and DHA-Enriched Structured Acylglycerols at the sn-2 Position Starting from Commercial Salmon Oil by Enzymatic Lipase Catalysis under Supercritical Conditions.

There is consistent evidence that long-chain polyunsaturated fatty acids (LCPUFA) belonging to the n-3 series, i.e., eicosapentaenoic (20:5n-3, EPA) and docosahexaenoic (22:6n-3, DHA) acids, decrease the risk of heart, circulatory and inflammatory diseases. Furthermore, the bioavailability of such fatty acids has been shown to depend on their location in triacylglycerol (TG) molecules at the sn-2 position. Consequently, great attention has been accorded to the synthesis of structured acylglycerols (sAG), which include EPA or DHA at the sn-2 position. The aim of this work was to synthesize sAG starting from deodorized refined commercial salmon oil. For this, immobilized lipase B from Candida antarctica (nonspecific) was used as a catalyst for the intra-interesterification process under CO2 supercritical conditions (CO2SC). According to the CO2SC reaction time, three different fractions including sAG compounds were obtained. The location of EPA and DHA at the sn-2 position in the resulting glycerol backbone was identified by mass spectrometry (MALDI-TOF) analysis. In all fractions obtained, a marked decrease in the starting TG content was observed, while an increase in the DHA content at the sn-2 position was detected. The fraction obtained after the longest reaction time period (2 h) led to the highest yield of sn-2 position DHA in the resulting sAG molecule.

Preparation, Physicochemical Characterisation and DoE Optimisation of a Spray-Dried Dry Emulsion Platform for Delivery of a Poorly Soluble Drug, Simvastatin.

In the presented study, insight into the development and optimisation of the dry emulsion formulation and spray drying process is provided. The aim was to facilitate the dissolution of the poorly soluble, highly lipophilic drug, simvastatin, by forming spray-dried dry emulsion particles having adequate powder flow properties, while assuring sufficient drug content. Simvastatin and a mixture of caprylic, capric triglyceride and 1-oleoyl-rac-glycerol were employed as a model drug and solubilising oils, respectively. A matrix of the dry emulsions was composed at a fixed ratio mixture of mannitol and HPMC. Tween 20 was used in low amounts as the primary emulsion stabiliser. To facilitate process optimisation, a DoE surface response design was used to study the influence of formulation and process parameters on the particle size distribution, powder bulk properties, emulsion reconstitution ability, drug stability and process yield of spray-dried products. Two-fluid nozzle geometry was identified, studied and confirmed to be important for most product critical quality attributes. Models obtained after the study showed acceptable coefficients of determination and provided good insight in the relationship governing the process and product characteristics. Five model optimised products showed adequate process yield, suitable particle size distribution, good reconstitution ability and improved dissolution profile, when compared to a non-lipid-based tablet and the pure drug. However, the obtained dry emulsion powders exhibited poor flow character according to the Carr index. The optimised product was further analysed with NMR during lipolysis to gain insight into the species formed during digestion and the kinetics of their formation.

Syntheses of L-Rhamnose-Linked Amino Glycerolipids and Their Cytotoxic Activities against Human Cancer Cells.

A major impediment to successful cancer treatment is the inability of clinically available drugs to kill drug-resistant cancer cells. We recently identified metabolically stable L-glucosamine-based glycosylated antitumor ether lipids (GAELs) that were cytotoxic to chemotherapy-resistant cancer cells. In the absence of commercially available L-glucosamine, many steps were needed to synthesize the compound and the overall yield was poor. To overcome this limitation, a facile synthetic procedure using commercially available L-sugars including L-rhamnose and L-glucose were developed and the L-GAELs tested for anticancer activity. The most potent analog synthesized, 3-amino-1-O-hexadecyloxy-2R-(O-α-L-rhamnopyranosyl)-sn- glycerol 3, demonstrated a potent antitumor effect against human cancer cell lines derived from breast, prostate, and pancreas. The activity observed was superior to that observed with clinical anticancer agents including cisplatin and chlorambucil. Moreover, like other GAELs, 3 induced cell death by a non-membranolytic caspase-independent pathway.

Preparation of Monoacylglycerol Derivatives from Indonesian Edible Oil and Their Antimicrobial Assay against Staphylococcus aureus and Escherichia coli.

In the present work, linoleic acid and oleic acid were isolated from Indonesian corn oil and palm oil and they were used to prepare monoacylglycerol derivatives as the antibacterial agent. Indonesian corn oil contains 57.74% linoleic acid, 19.88% palmitic acid, 11.84% oleic acid and 3.02% stearic acid. While Indonesian palm oil contains 44.72% oleic acid, 39.28% palmitic acid, 4.56% stearic acid and 1.54% myristic acid. The oleic acid was purified by using Urea Inclusion Complex (UIC) method and its purity was significantly increased from 44.72% to 94.71%. Meanwhile, with the UIC method, the purity of ethyl linoleate was increased from 57.74% to 72.14%. 1-Monolinolein and 2-monoolein compounds were synthesized via two-step process from the isolated linoleic acid and oleic acid, respectively. The preliminary antibacterial assay shows that the 1-monolinolein did not give any antibacterial activity against Staphylococcus aureus and Escherichia coli, while 2-monoolein showed weak antibacterial activity against Staphylococcus aureus.

Molecular simulation approach to the rational design of self-assembled nanoparticles for enhanced peroral delivery of doxorubicin.

Oral chemotherapy is preferred but challenged by low bioavailability of anticancer drugs. Self-assembled nanoparticles are promising in solving this problem but the design of appropriate nanocarrier remains a difficulty. Here, using doxorubicin (DOX) as a drug model, the objective of this study was to illustrate how to design an efficient drug delivery system using molecular simulation and elucidate the influence of fatty glyceride chain length in hyaluronic acid (HA) copolymers on peroral absorption of DOX. The compatibility between DOX and HA-g-glyceryl monocaprylate (HGC), HA-g-glyceryl monolaurate (HGL) and HA-g-glyceryl monostearate (HGS) was assessed by molecular simulation using solubility parameters and Flory-Huggins interaction parameters (χFH). Thereafter, HA copolymers were synthesized to verify the prediction. Among the copolymers, HGS showed the best compatibility with DOX followed by HGC and HGL. The physicochemical properties and stability of all the nanoparticles were copolymers structure dependent, with HGS-DOX nanoparticles showing the superior properties followed by HGC-DOX, HGL-DOX nanoparticles. The same order was found in cellular uptake, epithelial transport and in vivo absorption studies in rats, with HGS-DOX nanoparticles showing 7 times higher absorption after peroral administration than intravenous injection of DOX solution. In conclusion, molecular simulation is an effective strategy to the rational nanoparticle design for oral doxorubicin delivery.

Monomyristin and Monopalmitin Derivatives: Synthesis and Evaluation as Potential Antibacterial and Antifungal Agents.

In the present work, monoacylglycerol derivatives, i.e., 1-monomyristin, 2-monomyristin, and 2-monopalmitin were successfully prepared from commercially available myristic acid and palmitic acid. The 1-monomyristin compound was prepared through a transesterification reaction between ethyl myristate and 1,2-O-isopropylidene glycerol, which was obtained from the protection of glycerol with acetone, then followed by deprotection using Amberlyst-15. On the other hand, 2-monoacylglycerol derivatives were prepared through enzymatic hydrolysis of triglycerides in the presence of Thermomyces lanuginosa lipase enzymes. The synthesized products were analyzed using fourier transform infrared (FTIR) spectrophotometer, gas or liquid chromatography-mass spectrometer (GC-MS or LC-MS), and proton and carbon nuclear magnetic resonance (¹H- and 13C-NMR) spectrometers. It was found that monomyristin showed high antibacterial and antifungal activities, while 2-monopalmitin did not show any activity at all. The 1-monomyristin compound showed higher antibacterial activity against Staphylococcus aureus and Aggregatibacter actinomycetemcomitans and also higher antifungal activity against Candida albicans compared to the positive control. Meanwhile, 2-monomyristin showed high antibacterial activity against Escherichia coli. The effect of the acyl position and carbon chains towards antibacterial and antifungal activities was discussed.

Preparation of emulsifying wax/glyceryl monooleate nanoparticles and evaluation as a delivery system for repurposing simvastatin in bone regeneration.

Simvastatin (Sim) is a widely known drug in the treatment of hyperlipidemia, which has attracted so much attention in bone regeneration due to its potential osteoanabolic effect. However, repurposing of Sim in bone regeneration will require suitable delivery systems that can negate undesirable off-target/side effects. In this study, we have investigated a new lipid nanoparticle (NP) platform that was fabricated using a binary blend of emulsifying wax (Ewax) and glyceryl monooleate (GMO). Using the binary matrix materials, NPs loaded with Sim (0-500 µg/mL) were prepared and showed an average particle size of about 150 nm. NP size stability was dependent on Sim concentration loaded in NPs. The suitability of NPs prepared with the binary matrix materials in Sim delivery for potential application in bone regeneration was supported by biocompatibility in pre-osteoclastic and pre-osteoblastic cells. Additional data demonstrated that biofunctional Sim was released from NPs that facilitated differentiation of osteoblasts (cells that form bones) while inhibiting differentiation of osteoclasts (cells that resorb bones). The overall work demonstrated the preparation of NPs from Ewax/GMO blends and characterization to ascertain potential suitability in Sim delivery for bone regeneration. Additional studies on osteoblast and osteoclast functions are warranted to fully evaluate the efficacy of Sim-loaded Ewax/GMO NPs using in-vitro and in-vivo approaches.

2-Arachidonoylglycerol: A signaling lipid with manifold actions in the brain.

2-Arachidonoylglycerol (2-AG) is a signaling lipid in the central nervous system that is a key regulator of neurotransmitter release. 2-AG is an endocannabinoid that activates the cannabinoid CB1 receptor. It is involved in a wide array of (patho)physiological functions, such as emotion, cognition, energy balance, pain sensation and neuroinflammation. In this review, we describe the biosynthetic and metabolic pathways of 2-AG and how chemical and genetic perturbation of these pathways has led to insight in the biological role of this signaling lipid. Finally, we discuss the potential therapeutic benefits of modulating 2-AG levels in the brain.

Synthesis of Alkyl-Glycerolipids Standards for Gas Chromatography Analysis: Application for Chimera and Shark Liver Oils.

Natural O-alkyl-glycerolipids, also known as alkyl-ether-lipids (AEL), feature a long fatty alkyl chain linked to the glycerol unit by an ether bond. AEL are ubiquitously found in different tissues but, are abundant in shark liver oil, breast milk, red blood cells, blood plasma, and bone marrow. Only a few AEL are commercially available, while many others with saturated or mono-unsaturated alkyl chains of variable length are not available. These compounds are, however, necessary as standards for analytical methods. Here, we investigated different reported procedures and we adapted some of them to prepare a series of 1-O-alkyl-glycerols featuring mainly saturated alkyl chains of various lengths (14:0, 16:0, 17:0, 19:0, 20:0, 22:0) and two monounsaturated chains (16:1, 18:1). All of these standards were fully characterized by NMR and GC-MS. Finally, we used these standards to identify the AEL subtypes in shark and chimera liver oils. The distribution of the identified AEL were: 14:0 (20-24%), 16:0 (42-54%) and 18:1 (6-16%) and, to a lesser extent, (0.2-2%) for each of the following: 16:1, 17:0, 18:0, and 20:0. These standards open the possibilities to identify AEL subtypes in tumours and compare their composition to those of non-tumour tissues.

Selective synthesis of partial glycerides of conjugated linoleic acids via modulation of the catalytic properties of lipases by immobilization on different supports.

Lipases B from Candida antarctica (CALB), Rhizomucor miehei (RML) and Thermomyces lanuginosus (TLL) were immobilized on octadecyl methacylate (OM) or octadecyl methacrylate (OMC) beads. Their specific activity and regioselectivity were studied in the synthesis of conjugated linoleic acid (CLA) partial glycerides, which presented nutraceutical properties. TLL derivatives were poor catalysts. Novozym® 435 was much better than Lipozyme® RM IM. RML activity (a GRAS enzyme) was modulated via immobilization. After only 3 h, OM-RML gave the highest CLA conversion (54% at 40 °C with 1:3 M ratio of glycerol to CLA). OM-RML reduced by a factor of 3.12 and 1.16 the activation energy of the reaction with Lipozyme® RM IM and Novozym® 435, respectively. The new GRAS preparation OM-RML brings forth an optimal regioselective preparation of sn-1 mono and sn-1,3 diacylglycerols rich in CLA, with a ratio of sn-1,3/sn-1,2 regioisomers of 21.8, compared to 2.3 for Novozym® 435.

Neem oil nanoemulsions: characterisation and antioxidant activity.

The aim of the present work is to develop nanoemulsions (NEs), nanosized emulsions, manufactured for improving the delivery of active pharmaceutical ingredients. In particular, nanoemulsions composed of Neem seed oil, contain rich bioactive components, and Tween 20 as nonionic surfactant were prepared. A mean droplet size ranging from 10 to 100 nm was obtained by modulating the oil/surfactant ratio. Physicochemical characterisation was carried out evaluating size, ζ-potential, microviscosity, polarity and turbidity of the external shell and morphology, along with stability in simulated cerebrospinal fluid (CSF), activity of Neem oil alone and in NEs, HEp-2 cell interaction and cytotoxicity studies. This study confirms the formation of NEs by Tween 20 and Neem oil at different weight ratios with small and homogenous dimensions. The antioxidant activity of Neem oil alone and in NEs was comparable, whereas its cytotoxicity was strongly reduced when loaded in NEs after interaction with HEp-2 cells.

Structured Mono- and Diacylglycerols with a High Content of Medium Chain Fatty Acids.

In the present work, direct enzyme-catalysed esterification of medium chain fatty acids (MCFA) from three different sources (Medium chain triacylglycerols, MCT; saponified MCT and a mixture of free MCFA) was evaluated to obtain structured mono- and diacylglycerols. The esterifications were carried out mixing the different sources of MCFA with glycerol at two weight ratios (1:1 and 4:1, w/w), using three immobilized lipases: Novozym 435, Lipozyme RM IM and Lipozyme TL IM; different reaction times (t = 0, 15, 30, 60, 120 min); enzyme loadings (5, 10 or 15% with respect to the total weight of substrates). The extent of esterification was determined by gas chromatography (GC) analysis of the acylglycerols produced. The highest incorporation of free MCFA into glycerol was obtained for a 1:1 (w/w) glycerol to free MCFA ratio, 5% of Novozym 435, at 50°C, 300 rpm, 10% of molecular sieves and a commercial MCFA mixture as starting material. Under these conditions, incorporation of at least 90% of MCFA into glycerol was achieved after 30 min of reaction.

Production of Biodiesel from Acid Oil via a Two-Step Enzymatic Transesterification.

A two-step enzymatic transesterification process in a solvent-free system has been developed as a novel approach to the production of biodiesel using acid oil from rice bran oil soapstock. The acid oil consisted of 53.7 wt% fatty acids, 2.4 wt% monoacylglycerols, 9.1 wt% diacylglycerols, 28.8 wt% triacylglycerols, and 6.0 wt% others. Three immobilized lipases were evaluated as potential biocatalysts, including Novozym 435 from Candida antarctica, Lipozyme RM IM from Rhizomucor miehei, and Lipozyme TL IM from Thermomyces lanuginosus. The effects of molar ratio of acid oil to ethanol, temperature, and enzyme loading were investigated to determine the optimum conditions for the transesterification with the three immobilized lipases. The optimum conditions of the three immobilized lipases were a molar ratio of 1:5 (acid oil to ethanol), the temperature range of 30-40°C, and the enzyme loading range of 5-10%. The two-step transesterification was then conducted under the optimum conditions of each lipase. The stepwise use of Novozym 435 and Lipozyme TL IM or Lipozyme RM IM and Lipozyme TL IM resulted in similar or higher levels of yield to the individual lipases. The maximum yields obtained in both stepwise uses were ca. 92%.

Comparative Study of Soybean Oil and the Mixed Fatty Acids as Acyl Donors for Enzymatic Preparation of Feruloylated Acylglycerols in Ionic Liquids.

Feruloylated acylglycerols (FAGs) are the lipophilic derivatives of ferulic acid. In this work, soybean oil (SBO) and the mixed fatty acids (MFA) were selected as fatty acyl donors, and reacted with glyceryl monoferulate (GMF) to prepare FAGs in ionic liquids (ILs). Effect of various reaction parameters (time, temperature, enzyme concentration, and substrate ratio) and ILs on the GMF conversion and the reaction selectivity for FAGs formation were investigated. Response surface methodology (RSM) based on a 3-level-4-factor Box-Behnken experimental design was employed to evaluate the inactive effect of reaction parameters. For the esterification of GMF with MFA, the maximum GMF conversion (98.9 ± 0.9%) and FAG yield (88.9 ± 0.6%) were achieved in [C10mim]PF6. However, for the transesterification of GMF with SBO, the maximum GMF conversion (94.3 ± 0.7%) and FAG yield (83.8 ± 1.0%) were obtained in [C12mim]PF6. High FAG selectivities (∼0.90) were also obtained using SBO or MFA as acyl donors.

Enzymatic preparation of human milk fat substitutes and their oxidation stability.

Human milk fat substitutes (HMFSs), rich in palmitic acid (P) at the sn-2 position of triacylglycerol (TAG), were prepared from lard via Novozym435(®)- and Lipozyme RM-IM(®)-mediated two-step reactions. First, 2-palmitoyl monoacylglycerol (2-P-MAG, 90% purity) was prepared via Novozym435(®)mediated ethanolysis of lard. Then, 2-P-MAG, oleic acid (O), linoleic acid (L), and lard were dissolved in hexane and subjected to a Lipozyme RM-IM(®)-mediated reaction for HMFS preparation. The effect of the amount of 2-P-MAG and fatty acids (O and L) in HMFS preparation were investigated. Under the optimum reaction conditions: 7 mmol of lard, 3.0 mmol of 2-P-MAG, 5.2 mmol of O, 3.5 mmol of L, 10 mL of hexane, 10 wt% of Lipozyme RM-IMR(®) (against the total weight of substrates), 550 rpm, 37°C and 6 h, a HMFS with total fatty acid composition and P content at the sn-2 position of TAG similar to that of human milk fat was prepared. In the same way, a HMFS having polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (PUFA-HMFS) was prepared. The HMFS and PUFA-HMFS prepared in this study, as well as fats extracted from commercially available powdered milk for infants (FPM) were evaluated for their oxidation stability by an auto-oxidation test. The test showed HMFS and PUFA-HMFS to possess greater oxidation stability than that the FPMs. These results indicated that the HMFS and PUFA-HMFS prepared in this study have value as potential ingredients for powdered milk.

Ultrasound assisted enzyme catalyzed synthesis of glycerol carbonate from glycerol and dimethyl carbonate.

The present work illustrates the transesterification of glycerol to glycerol carbonate (GlyC) from dimethyl carbonate (DMC) using commercial immobilized lipase (Novozym 435) under ultrasonic irradiation. The experiments were performed in a batch reactor placed in an ultrasonic water bath using a sequence of experimental protocol to evaluate the effects of temperature, molar ratios of substrates, enzyme loading, duty cycle and ultrasound power on the conversion of glycerol to GlyC. It has been found that ultrasound-assisted lipase-catalyzed transesterification of glycerol would be a potential alternative to conventional alkali-catalyzed method, as high conversion (99.75%) was obtained at mild operating conditions: molar ratio of DMC to glycerol 3:1, catalyst amount of 13% (w/w), lower power input (100W), duty cycle 50% and temperature (60°C) in a relatively short reaction time (4h) using Novozym 435 as catalyst. Ultrasound reduces the reaction time up to 4h as compared to conventional stirring method (14h) catalyzed by Novozym 435. The repeated use of the catalyst under the optimum experimental condition resulted in decay in both enzyme activity and product conversion.

Chemoenzymatic solvent-free synthesis of 1-monopalmitin using a microwave reactor.

An environmentally friendly method for the synthesis of 1-monopalmitin has been developed. The procedure consists of a two-step, solvent-free chemoenzymatic reaction. In the first step, palmitic acid is esterified with solketal (4-hydroxymethyl-2,2-dimethyl-1,3-dioxolane) using Novozym 435 by both conventional heating and microwave irradiation. The use of a microwave reactor allows the enzymatic synthesis of the intermediate compound with a similar yield as that achieved using conventional heating. In the second step, 1,2-acetonide-3-palmitoyl glycerol is cleaved to yield 1-monopalmitin by means of a cation-exchange resin and water or aliphatic alcohols as hydrolytic reagent in solvent-free conditions. The hydrolysis was accomplished in 15 min at 85 degrees C. The best yield was obtained using 1-pentanol. We conclude that the yield achieved depends on the batch and nature of the cation-exchange resin used as catalyst.

A highly versatile convergent/divergent "onion peel" synthetic strategy toward potent multivalent glycodendrimers.

Both convergent and divergent strategies for the synthesis of "onion peel" glycodendrimers are reported which resulted in one of the best multivalent ligands known against the virulent factor from a bacterial lectin isolated from Pseudomonas aeruginosa.

Synthesis and tribological investigation of lipoyl glycerides.

Lipoyl glycerides were synthesized by enzymatic transesterification of lipoic acid with high-oleic sunflower oil in 2-methyl-2-butanol solvent. The synthesis gave a crude product mixture comprising unreacted lipoic acid, free fatty acids, and several lipoyl glyceride structures of varying lipoic acid substitution. A more purified product mixture, devoid of unreacted lipoic acid and free fatty acids, was obtained in 61% yield. The crude and purified product mixtures were thoroughly characterized and their components positively identified. The tribological properties of the product mixtures were further investigated using a variety of methods. The product mixtures displayed significantly improved oxidation stability, cold-flow, and extreme pressure properties over those of the parent high-oleic sunflower oil. The extreme pressure results for the neat products showed a higher weld point for the crude than for the purified mixture. This was attributed to differences in the chemical properties of the components in the two product mixtures.

Synthesis, structural elucidation, and biochemical analysis of immunoactive glucuronosyl diacylglycerides of mycobacteria and corynebacteria.

Glucuronosyl diacylglycerides (GlcAGroAc2) are functionally important glycolipids and membrane anchors for cell wall lipoglycans in the Corynebacteria. Here we describe the complete synthesis of distinct acyl-isoforms of GlcAGroAc2 bearing both acylation patterns of (R)-tuberculostearic acid (C19:0) and palmitic acid (C16:0) and their mass spectral characterization. Collision-induced fragmentation mass spectrometry identified characteristic fragment ions that were used to develop "rules" allowing the assignment of the acylation pattern as C19:0 (sn-1), C16:0 (sn-2) in the natural product from Mycobacterium smegmatis, and the structural assignment of related C18:1 (sn-1), C16:0 (sn-2) GlcAGroAc2 glycolipids from M. smegmatis and Corynebacterium glutamicum. A synthetic hydrophobic octyl glucuronoside was used to characterize the GDP-mannose-dependent mannosyltransferase MgtA from C. glutamicum that extends GlcAGroAc2. This enzyme is an Mg(2+)/Mn(2+)-dependent metalloenzyme that undergoes dramatic activation upon reduction with dithiothreitol.