Seeking faster, alternative methods for glycolipid biosurfactant characterization and purification.

Biosurfactants have been investigated as potential alternatives for synthetic surfactants in several areas, for example, in environmental and pharmaceutical fields. In that regard, extensive research has been carried out with sophorolipids and rhamnolipids that also present various biological properties with therapeutic significance. These biosurfactants are obtained as complex mixtures of slightly different molecules, and thus when studying these microbial glycolipids, the ability to identify and purify the produced compounds is of extreme importance. This study aimed to develop improved methodologies for the identification, separation, and purification of sophorolipids and rhamnolipids. Therefore, an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was modified to ensure faster characterization of both sophorolipids and rhamnolipids, enabling the identification and fragmentation pattern description of 10 and 13 congeners, respectively. The separation and purification of these biosurfactants was achieved with novel reversed-phase solid-phase extraction methods guaranteeing the isolation of different glycolipids, including those considered for their significant biological activity (e.g. antimicrobial, anticancer). It was possible to isolate sophorolipids and rhamnolipids with purity of 94% and 99%, respectively. The methods presented herein can be easily implemented and are expected to make purification of these biosurfactants easier, facilitating the study of their individual properties in further works.

Evaluation of Antioxidant and Antibacterial Activities, Cytotoxicity of Acacia seyal Del Bark Extracts and Isolated Compounds.

Water extract of Acacia seyal bark is used traditionally by the population in Djibouti for its anti-infectious activity. The evaluation of in vitro antibacterial, antioxidant activities and cytotoxicity as well as chemical characterization of Acacia seyal bark water and methanolic extracts were presented. The water extract has a toxicity against the MRC-5 cells at 256 µg/mL while the methanolic extract has a weak toxicity at the same concentration. The methanolic extract has a strong antioxidant activity with half maximal inhibitory concentration (IC50) of 150 ± 2.2 µg/mL using 1-diphenyl-2-picrylhydrazyl (DPPH) and IC50 of 27 ± 1.3 µg/mL using 2,2'-azino-bis 3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical methods. For ferric reducing/antioxidant power (FRAP) assay, the result is 45.74 ± 5.96 µg Vitamin C Equivalent (VCE)/g of dry weight (DW). The precipitation of tannins from methanol crude extract decreases the MIC from 64 µg/mL to 32 µg/mL against Staphylococcus aureus and Corynebacterium urealyticum. However, the antioxidant activity is higher before tannins precipitation than after (IC50 = 150 µg/mL for methanolic crude extract and 250 µg/mL after tannins precipitation determined by DPPH method). By matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, the results showed that the condensed tannins consist of two types of catechin and gallocatechin-based oligomers. The fractionation led to the identification of three pure compounds: two flavanols catechin and epicatechin; one triterpene as lupeol; and a mixture of three steroids and one fatty acid: campesterol, stigmasterol, clionasterol, and oleamide.

Structure and Potential Cellular Targets of HAMLET-like Anti-Cancer Compounds made from Milk Components.

The HAMLET family of compounds (Human Alpha-lactalbumin Made Lethal to Tumours) was discovered during studies on the properties of human milk, and is a class of protein-lipid complexes having broad spectrum anti-cancer, and some specific anti-bacterial properties. The structure of HAMLET-like compounds consists of an aggregation of partially unfolded protein making up the majority of the compound's mass, with fatty acid molecules bound in the hydrophobic core. This is a novel protein-lipid structure and has only recently been derived by small-angle X-ray scattering analysis. The structure is the basis of a novel cytotoxicity mechanism responsible for anti-cancer activity to all of the around 50 different cancer cell types for which the HAMLET family has been trialled. Multiple cytotoxic mechanisms have been hypothesised for the HAMLET-like compounds, but it is not yet clear which of those are the initiating cytotoxic mechanism(s) and which are subsequent activities triggered by the initiating mechanism(s). In addition to the studies into the structure of these compounds, this review presents the state of knowledge of the anti-cancer aspects of HAMLET-like compounds, the HAMLET-induced cytotoxic activities to cancer and non-cancer cells, and the several prospective cell membrane and intracellular targets of the HAMLET family. The emerging picture is that HAMLET-like compounds initiate their cytotoxic effects on what may be a cancer-specific target in the cell membrane that has yet to be identified. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Bioguided isolation of (9Z)-octadec-9-enoic acid from Phellodendron amurense Rupr. and identification of fatty acids as PTP1B inhibitors.

The therapy of type-2 diabetes mellitus is one of the major challenges of our age. A possible strategy to prevent the progression of this disease is the inhibition of protein tyrosine phosphatase 1B (PTP1B), a major negative regulator in the insulin and leptin signalling pathway. Phellodendri amurensis cortex is a well-known Asian herbal drug traditionally used as antiphlogistic, antibacterial, and anti-inflammatory agent, and its efficacy against diabetes-related symptoms is reported as well. However, information regarding active principle(s) or the molecular mode of action was scarce. By bioguided isolation using an IN VITRO enzyme assay with human recombinant PTP1B, (9 Z)-octadec-9-enoic acid (oleic acid) could be identified as a major PTP1B inhibitor in the bark of Phellodendron amurENSE Rupr. (Rutaceae); it showed an IC50 value of 6.2 µM. Consistent with this inhibition of PTP1B, oleic acid was capable of enhancing insulin signalling in wild-type, but not PTP1B knockout fibroblasts. By testing a series of other fatty acids of different chain length and degree of saturation, their general PTP1B-inhibitory potential in the micromolar range was observed. More pronounced effects were associated with a longer carbon backbone and saturation of the double bonds. Therefore, our work provides first scientific evidences for the antidiabetic properties of P. amurense via a new target, effects which seem to be explainable by oleic acid. The discovery of a PTP1B inhibition by many fatty acids also adds a novel facet to the pharmacological properties of a class of compounds that is found in many food items in considerable amount and triggers speculation over their possible involvement in the feedback regulation of cellular fatty acid synthesis.

Hydroxyl octadecenoic acids biosynthesized by crown galls of Panax quinquefolium induced by artermisinic acid.

OBJECTIVE: To investigate the effect of artermisinic acid on the secondary metabolites production of Panax quinquefolium crown galls. METHODS: Artemisinic acid was added into the suspended cells of Panax quinquefolium crown galls and co-culture for two days. Products were isolated with chromatographic method. RESULTS: Three hydroxyl octadecenoic acids [9,12,13-trihydroxy-10-octadecenoic acid (1), 11,12,13-trihydroxy-9-octadecenoic acid (2) and 11-hydroxy-12,13-epoxy-9-octadecenoic acid (3)] were isolated from crown galls of Panax quinquefolium. CONCLUSION: Artermisinic acid as one of the new type of phytohormones that might induce the production of 13-lipoxygenases in crown galls of Panax quinquefolium.

Identification of oleamide in Guatteria recurvisepala by LC/MS-based Plasmodium falciparum thioredoxin reductase ligand binding method.

Our current research on applications of mass spectrometry to natural product drug discovery against malaria aims to screen plant extracts for new ligands to Plasmodium falciparum thioredoxin reductase (PfTrxR) followed by their identification and structure elucidation. PfTrxR is involved in the antioxidant defense and redox regulation of the parasite and is validated as a promising target for therapeutic intervention against malaria. In the present study, detannified methanol extracts from Guatteria recurvisepala, Licania kallunkiae, and Topobea watsonii were screened for ligands to PfTrxR using ultrafiltration and liquid chromatography/mass spectrometry-based binding experiments. The PfTrxR ligand identified in the extract of Guatteria recurvisepala displayed a relative binding affinity of 3.5-fold when incubated with 1 µM PfTrxR. The ligand corresponding to the protonated molecule m/z 282.2792 [M+ H]+ was eluted at a retention time of 17.95 min in a 20-min gradient of 95% B consisting of (A) 0.1%formic acid in 95% H2O-5% ACN, and (B) 0.1% formic acid in 95% ACN-5% H2O in an LC-QTOF-MS.Tandem MS of the protonated molecule m/z 282.2792 [M + H]+, C18H36NO (DBE: 2; error: 1.13 ppm) resulted in two daughter ions m/z 265.2516[M + H-NH3]+ (DBE: 3; error: 0.35 ppm) and m/z 247.2405 [M + H-NH3-H2O] +, (DBE: 4; error:2.26 ppm). The PfTrxR ligand was identified as oleamide and confirmed by comparison of the retention time, molecular formula, accurate mass,and double bond equivalence with the standard oleamide. This is the first report on the identification of oleamide as a PfTrxR ligand from Guatteria recurvisepala R. E. Fr. and the corresponding in vitro activity against P. falciparum strain K1 (IC50 4.29 µg/mL).

[Studies on the chemical constituents of Codonopsis pilosula].

OBJECTIVE: To study the chemical constituents of Codonopsis pilosula. METHODS: The compounds were isolated and purified by column chromatography and their structures were elucidated through spectroscopic techniques (NMR) and physicochemical properties. RESULTS: The compounds were isolated as hesperidin(I), n-hexyl beta-sophoroside(II), atractylenolide III (III), lobetyolin(IV), lobetyolinin(V), taraxerol(VI), taraxeryl acetate(VII), alpha-spinasterol(VIII),9,10,13-trihydroxy-(E)-11-octadecenoic acid (IX),beta-sitosterol(X),beta-daucosterol(XI)and sugar(XII). CONCLUSION: Compounds 1 -2 and 9 are isolated from this plant for the first time.

Oleamide in Ipomoea and Dillenia Species and Inflammatory Activity Investigated through Ion Channel Inhibition.

BACKGROUND: Oleamide is an essential substance for human health. So, the plants with high oleamide content are great sources for health care products. OBJECTIVE: This study is conducted to investigate the quality of oleamide in plants and test the bioactivity in the selected two studied species. METHODS: The three Ipomoea and five Dillenia species including Ipomoea alba, Ipomoea aquatica and Ipomoea pes-caprae, and Dillenia indica, Dillenia obovata, Dillenia ovata, Dillenia parviflora and Dillenia pentagyna were investigated for the quantity of oleamide by high-performance liquid chromatography. The biological activity test was conducted on the powder formulation of the chosen plants, Dillenia ovata and Dillenia parviflora at a ratio of 30:70, for anti-inflammatory activity ex vivo on a panel of molecular targets through ion channel inhibition including voltage-gated sodium channel, voltage-gated potassium channel, and the cardiac ion as human ether-a-go-go related gene. RESULTS: The results showed that the leaf extracts of I. aquatica and D. ovata gave the highest and subsequent oleamide quantity i.e. 7.52 and 5.17 mg/g, respectively. Out of the Dillenia formulation which contained various compounds, oleamide showed the highest percentages of inhibition at 8.0-20.0%, and 6.2-14.2% in voltage-gated sodium channel, and voltage-gated potassium channel which had slightly lower values than the oleamide standard, and no effect as 0.0% value inhibition in the cardiac ion channel. CONCLUSION: The Dillenia formulation exhibits anti-inflammatory activity without affecting the heart. Accordingly, the three studied Ipomoea and three studied Dillenia species may be used for the same activity as a single component or formulation with effective solvent for disease treatments.

Exploring the potential of chitosan-based particles as delivery-carriers for promising antimicrobial glycolipid biosurfactants.

Driven by the need to find alternatives to control Staphylococcus aureus infections, this work describes the development of chitosan-based particulate systems as carriers for antimicrobial glycolipids. By using a simple ionic gelation method stable nanoparticles were obtained showing an encapsulation efficiency of 41.1 ± 8.8 % and 74.2 ± 1.3 % and an average size of 210.0 ± 15.7 nm and 329.6 ± 8.0 nm for sophorolipids and rhamnolipids chitosan-nanoparticles, respectively. Glycolipids incorporation and particle size was correspondingly corroborated by FTIR-ATR and TEM analysis. Rhamnolipids chitosan nanoparticles (RLs-CSp) presented the highest antimicrobial effect towards S. aureus (ATCC 25923) exhibiting a minimal inhibitory concentration of 130 µg/mL and a biofilm inhibition ability of 99 %. Additionally, RLs-CSp did not interfere with human dermal fibroblasts (AG22719) viability and proliferation under the tested conditions. The results revealed that the RLs-CSp were able to inhibit bacterial growth showing adequate cytocompatibility and might become, after additional studies, a valuable approach to prevent S. aureus related infections.

Direct Observation of Sophorolipid Micelle Docking in Model Membranes and Cells by Single Particle Studies Reveals Optimal Fusion Conditions.

Sophorolipids (SLs) are naturally produced glycolipids that acts as drug delivery for a spectrum of biomedical applications, including as an antibacterial antifungal and anticancer agent, where they induce apoptosis selectively in cancerous cells. Despite their utility, the mechanisms underlying their membrane interactions, and consequently cell entry, remains unknown. Here, we combined a single liposome assay to observe directly and quantify the kinetics of interaction of SL micelles with model membrane systems, and single particle studies on live cells to record their interaction with cell membranes and their cytotoxicity. Our single particle readouts revealed several repetitive docking events on individual liposomes and quantified how pH and membrane charges, which are known to vary in cancer cells, affect the docking of SL micelles on model membranes. Docking of sophorolipids micelles was found to be optimal at pH 6.5 and for membranes with -5% negatively charge lipids. Single particle studies on mammalian cells reveled a two-fold increased interaction on Hela cells as compared to HEK-293 cells. This is in line with our cell viability readouts recording an approximate two-fold increased cytotoxicity by SLs interactions for Hela cells as compared to HEK-293 cells. The combined in vitro and cell assays thus support the increased cytotoxicity of SLs on cancer cells to originate from optimal charge and pH interactions between membranes and SL assemblies. We anticipate studies combining quantitative single particle studies on model membranes and live cell may reveal hitherto unknown molecular insights on the interactions of sophorolipid and additional nanocarriers mechanism.

[Study on hydrophobic constituents of Arnebia euchroma].

OBJECTIVE: To investigate the massive hydrophobic constituents of Arnebia euchroma, and compare the similarities and differences of AE and AEZ from different sources. METHODS: The hydrophobic constituents, AE01-1 and AEZ01-1, were obtained by the chromatograph of silica gel with elution of chloroform used in the chloroform extracts of Arnebia euchroma, AE01 and AEZ01. GC-MS was used to identify the main components of the hydrophobic constituents. RESULTS: 11 and 22 compounds were identified in the hydrophobic constituenty, AE01-1 and AEZ01-1, including 5 common compounds that were the main components. AE01-1, the hydrophobic constituents of AE, had 2 naphthoquinones, while AEZ01-1, the hydrophobic constituents of AEZ, had only lipid compounds. CONCLUSION: Compared to the AE, AEZ has fewer naphthoquinones with low polarity.

Oral shea nut oil triterpene concentrate supplement ameliorates pain and histological assessment of articular cartilage deterioration in an ACLT injured rat knee osteoarthritis model.

Osteoarthritis (OA) is a multifactorial joint disease and a common disabling condition in the elderly population. The associated pain and pathohistological changes in cartilage are common features of OA in both humans and animal models. Shea nut oil extract (SheaFlex75) contains a high triterpenoid concentration and has demonstrated anti-inflammatory and antiarthritic effects in both human and animal studies. In this study, we aim to investigate the potential of SheaFlex75 to prevent articular cartilage deterioration in a rat model of chronic OA progression. By employing anterior cruciate ligament transection (ACLT) with medial meniscectomy (MMx)-induced OA, we found attenuation of both early and chronic onset OA pain and cartilage degeneration in ACLT+MMx rats receiving SheaFlex75 dietary supplementation. Under long-term oral administration, the rats with induced OA presented sustained protection of both pain and OA cartilage integrity compared to the OA-control rats. Moreover, rats subjected to long-term SheaFlex75 ingestion showed normal biochemical profiles (AST, BUN and total cholesterol) and presented relatively lower triglycerides (TGs) and body weights than the OA-control rats, which suggested the safety of prolonged use of this oil extract. Based on the present evidence, preventive management is advised to delay/prevent onset and progression in OA patients. Therefore, we suggest that SheaFlex75 may be an effective management strategy for symptom relief and cartilage protection in patients with both acute and chronic OA.

Synthesis of aromatic triols and triacids from oleic and erucic acid: separation and characterization of the asymmetric and symmetric isomers.

Hexasubstituted benzene derivatives, aromatic triols 4, 9 and triacids 5, 10 have been synthesized from oleic and erucic acid derivatives followed by a cyclotrimerization step catalyzed by palladium-on-carbon (Pd/C) and chlorotrimethylsilane (TMSCl). The asymmetric isomer, which is the main product, was isolated from its symmetric isomer by flash chromatography. All the products were fully characterized by IR, (1)H NMR, (13)C NMR and mass spectrometry. The products are suitable monomers for the production of polyurethanes, polyesters and polyamides.

Cytotoxic activity of an octadecenoic acid extract from Euphorbia kansui (Euphorbiaceae) on human tumour cell strains.

We have investigated the cytotoxic and antitumour activity of an octadecenoic acid extract, mainly containing oleic and linoleic acids, from Euphorbia kansui on human gastric (SGC-7901), hepatocellular carcinoma (BEL-7402), and leukaemia (HL-60) tumour cell strains. Significant and dose-dependent antiproliferation effects were observed on tumour cells from the dose of 3.2 microg mL(-1), which were comparable with or better than those of the common antitumour agent 5-fluorouracil. Results from the clone formation assay and flow cytometry indicated that the mixture of octadecenoic acids resulted in a dose-dependent reduction in the number of tumour cells and significantly inhibited cell proliferation, with induced apoptosis and G(0)/G(1) phase cell cycle arrest. Also, the octadecenoic acids could not only cause cell apoptosis/necrosis but also functionally and structurally damage the tumour cell membrane and cell ultra-structures. These observations encourage further clinical evaluation of the inhibitory effects of octadecenoic acids on various forms of cancer.

Multi-objective Optimization of Molecular Distillation Conditions for Oleic Acid from a Rich-in-Fatty Acid Model Mixture.

Oleic acid is a commercially valuable compound and has many positive health effects. Determining optimum conditions in a physical separation process is an industrially significant point due to environmental and health related concerns. Molecular distillation avoids the use of chemicals and adverse effects of high temperature application. The objective of this study was to determine the molecular distillation conditions for oleic acid to increase its purity and distillation yield in a model fatty acid mixture. For this purpose, a short-path evaporator column was used. Evaporation temperature ranged from 110 to 190℃, while absolute pressure was from 0.05 to 5 mmHg. Results showed that elevating temperature generally increased distillation yield until a maximum evaporation temperature. Vacuum application also affected the yield at a given temperature, and amount of distillate increased at higher vacuums except the case applied at 190℃. A multi-objective optimization procedure was then used for maximizing both yield and oleic acid amounts in distillate simultaneously, and an optimum point of 177.36℃ and 0.051 mmHg was determined for this purpose. Results also demonstrated that evaporation of oleic acid was also suppressed by a secondary dominant fatty acid of olive oil - palmitic acid, which tended to evaporate easier than oleic acid at lower evaporation temperatures, and increasing temperature achieved to transfer more oleic acid to distillate. At 110℃ and 0.05 mmHg, oleic and palmitic acid concentrations in distillate were 63.67% and 24.32%, respectively. Outcomes of this study are expected to be useful for industrial process conditions.

The Effect of Solvent Type and Roasting Processes on Physico-Chemical Properties of Tigernut (Cyperus esculentus L.) Tuber Oil.

In this study, physico-chemical properties of raw and roasted tigernut oils extracted by two different solvents were determined. Peroxide values of raw and roasted tigernut oils extracted by petroleum ether and n-hexane solvents changed between 0.83 and 0.91 meqO2/100g to 1.57 and 1.63 meqO2/100g, respectively. While oleic acid contents of raw tigernut oils extracted by petroleum ether and n-hexane are determined as 66.83 and 67.47%, oleic acid contents of roasted tigernut oils extracted by petroleum ether and n-hexane were determined as 67.08 and 68.16%, respectively. The highest δ-tocopherol content was found in raw tigernut oil extracted by petroleum ether (54.91 mg/100g), while the lowest level is determined in roasted tigernut oil by n-hexane (50.77 mg/100g). As a result, the fatty acid profiles of roasted tigernut oil extracted by n-hexane were higher compared to results of raw tigernut oils extracted by petroleum ether (p < 0.05).

Fast Biodegradation of Diesel Hydrocarbons at High Concentration by the Sophorolipid-Producing Yeast Candida catenulata KP324968.

In the last decades, biodegradation as an environmentally friendly approach has raised interest in connection with the removal of hydrocarbon pollutants. Its capacity for removing pollutants strongly depends on the type of living cell and environmental conditions. The degradative activity of a new sophorolipid-producing yeast, Candida catenulata KP324968, in the removal of high concentrations of diesel from effluents was statistically evaluated considering the initial pH, the agitation speed, and the initial diesel concentration. The optimal setting of the operational variables at an initial pH of 4.7, an agitation speed of 204 rpm, and an initial diesel concentration of 93.4 g L-1 resulted in the highest total petroleum hydrocarbon removal efficiency: about 82.1% after 6 days (biodegradation rate: 0.378 g gcell-1 h-1). During the cell growth phase, the emulsification index in the medium increased and reached its highest level at 64.6% after 48 h. Further tests indicated that the emulsification capacity was obtained by in situ production of two sophorolipid molecules with an m/z of 533 and 583. In summary, its effective diesel removal and high emulsification capacity makes C. catenulata KP324968 an attractive candidate yeast for the degradation of hydrocarbons from aqueous environments.

A novel microbial technique for producing high-quality sophorolipids from horse oil suitable for cosmetic applications.

Horse oil contains linoleic, palmitoleic and unsaturated fatty acids that are similar to those in human skin, and may therefore be an ideal substance from which to isolate biosurfactants for cosmetic products to improve human skin quality. Herein, an innovative approach was developed to synthesise sophorolipids from horse oil by hydrolysis, followed by fermentation using the yeast Candida bombicola. The yield of sophorolipids from direct fermentation of horse oil and hydrolysed horse oil was 40.6 ± 1.3 g l-1 and 58.4 ± 1.8 g l-1 respectively. To further increase the yield, 30-40 g l-1 glucose was added in a fed-batch fermentation process to maintain the pH between 4.0 and 4.5, resulting in a conversion yield of 71.7 ± 0.8 g l-1 . The purity and structure of the synthesised sophorolipids were analysed by ultra-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. An in vitro human dermal fibroblast model was used as a surrogate for human skin to measure elastase inhibition activity. Antiwrinkle properties of isolated sophorolipids were better than those of horse oil or hydrolysed horse oil in several in vitro assays. Furthermore, no cytotoxicity was observed at a concentration of 50 µg ml-1 , and wound-healing capacity was evident in a cell culture model. Additionally, the synthesised sophorolipids attenuated lipopolysaccharide-induced expression of inflammatory cytokines in macrophages, and efficiently inhibited several strains of bacteria and yeast. In conclusion, fed-batch fermentation of hydrolysed horse oil is a novel and efficient approach for producing high-quality and high-yield sophorolipids that exhibit great potential as cosmetic ingredients.

A direct and fast method to monitor lipid oxidation progress in model fatty acid methyl esters by high-performance size-exclusion chromatography.

A new method based on high-performance size-exclusion chromatography (HPSEC) is proposed to quantitate primary and secondary oxidation compounds in model fatty acid methyl esters (FAMEs). The method consists on simply injecting an aliquot sample in HPSEC, without preliminary isolation procedures neither addition of standard internal. Four groups of compounds can be quantified, namely, unoxidised FAME, oxidised FAME monomers including hydroperoxides, FAME dimers and FAME polymers. Results showed high repeatability and sensitivity, and substantial advantages versus determination of residual substrate by gas-liquid chromatography. Applicability of the method is shown through selected data obtained by numerous oxidation experiments on pure FAME, mainly methyl linoleate, at ambient and moderate temperatures.

Protective effect of Mediterranean fish oil extracts on heat-induced denaturation of albumin.

Three oily extracts, obtained by acetone extraction from the entrails of different varieties of Mediterranean fishes, such as mackerel (Scomber scombrus), sardine (Sardina pilchardus) and horse mackerel (Trachurus mediterraneus), were characterized to determine their unsaturated fatty acid content. In an in-vitro model, their inhibitory effect was then evaluated against protein aggregation and their protective efficacy against heat-induced albumin denaturation assessed. The fish oil extracts tested in this study presented a significant amount of unsaturated fatty acids; in particular the extract obtained from the entrails of horse mackerel proved to have higher concentrations of DHA (docosahexaenoic acid) and oleic acid compared with the other two oils. The in-vitro study revealed an interesting protective effect of the oil extracts (particularly the horse mackerel extract) against heat-induced denaturation of albumin.