Controlled formation of mono- and dihydroxy-resolvins from EPA and DHA using soybean 15-lipoxygenase.

Resolvins and protectins are important anti-inflammatory and pro-resolution compounds derived from the enzymatic oxidation of omega-3 fatty acids all-cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and all-cis-4,7,10,13,16,19-docosahexaenoic acid (DHA). We have developed a simple, controlled method to synthesize an array of resolvin and protectin analogs from fatty acid starting materials using soybean 15-lipoxygenase. The conditions were optimized for the production of both mono- and dihydroxy derivatives, with enzyme concentration and pH found to have a significant effect on the reaction products. The methods were applied to five biologically important omega-3 and omega-6 fatty acid substrates. Mono- and dihydroxy compounds were successfully synthesized from all substrates and the products were characterized by normal phase (NP) HPLC, GC-MS, TOF-MS, UV-visible (UV-vis) spectroscopy, and NMR spectroscopy. The methods could be further applied to any polyunsaturated fatty acids containing the cis-1,4,7,10-undecatetraene moiety to produce a range of novel compounds with potential biological activity.

Quantitative determination of fatty acid compositions in micro-encapsulated fish-oil supplements using Fourier transform infrared (FTIR) spectroscopy.

The research describes a rapid method for the determination of fatty acid (FA) contents in a micro-encapsulated fish-oil (µEFO) supplement by using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic technique and partial least square regression (PLSR) analysis. Using the ATR-FTIR technique, the µEFO powder samples can be directly analysed without any pre-treatment required, and our developed PLSR strategic approach based on the acquired spectral data led to production of a good linear calibration with R(2)=0.99. In addition, the subsequent predictions acquired from an independent validation set for the target FA compositions (i.e., total oil, total omega-3 fatty acids, EPA and DHA) were highly accurate when compared to the actual values obtained from standard GC-based technique, with plots between predicted versus actual values resulting in excellent linear fitting (R(2)≥0.96) in all cases. The study therefore demonstrated not only the substantial advantage of the ATR-FTIR technique in terms of rapidness and cost effectiveness, but also its potential application as a rapid, potentially automated, online monitoring technique for the routine analysis of FA composition in industrial processes when used together with the multivariate data analysis modelling.

Isolation of phosphorylated polysaccharides from algae: the immunostimulatory principle of Chlorella pyrenoidosa.

The major immunostimulatory principle in the hot aqueous extract of Chlorella pyrenoidosa has been isolated by a sequence of ethanol precipitation, precipitation with a cationic surfactant (CTAB), size exclusion chromatography, and anion exchange chromatography. A series of phosphorylated polysaccharides were obtained having different molecular masses but with similar structures. The higher molecular mass fractions showed considerable activity in the stimulation of mouse peritoneal macrophages to synthesize nitric oxide. The structure of the major polysaccharide was established by sugar analysis, configurational analysis, and 1D and 2D NMR experiments at 500 and 800 MHz on the parent polysaccharide, the de-O-acetylated polysaccharide, and on the components obtained after hydrolysis of the phosphate diesters. It had a beta-D-Galp-(1-->3)-beta-D-Galp-(1-->3)-backbone with half of the Galp units substituted at O-6 by terminal beta-D-Glcp units. The remaining Galp units were substituted on O-6 by about equal amounts of alpha-D-Manp-1-phosphate and 3-O-Me-alpha-Manp-1-phosphate diesters. The substituents were not located in a regularly alternating fashion on the backbone. The O-acetyl groups were largely located on O-2 and O-4 of Galp and 35% of the Galp residues were O-acetylated. This is the second observation of a phosphorylated polysaccharide in an alga and the first where it is present to a significant extent.

First isolation and structural determination of cyclic beta-(1-->2)-glucans from an alga, Chlorella pyrenoidosa.

The aqueous extract of the edible green microalgae Chlorella pyrenoidosa is of interest because of its immunostimulatory activity. Some components in the extract have been identified previously, namely a unique type of arabinogalactan and a galactofuran. Further fractionation of this extract was accomplished by treating the aqueous solution of the fraction precipitated by addition of 1.5vol of 95% ethanol with cetyltrimethylammonium bromide. The residue obtained by concentration of the supernatant was fractionated further by anion-exchange chromatography and size-exclusion chromatography on Sephadex G-100. Two fractions from the latter column were retained, of which one was a starch-like alpha-(1-->4)-linked d-glucan with some alpha-(1-->6) branches, and the other contained a starch plus a mixture of beta-(1-->2)-d-glucans. ESI mass spectrometry was used to show that the mixture contained both cyclic and linear beta-(1-->2)-d-glucans in a cyclic:linear ratio of 64:36, based on intensities of mass spectral peaks. For the cyclic beta-(1-->2)-d-glucans, ring sizes ranged from 18 to 35 monosaccharides with the ring containing 21 glucose units (54% of the cyclic glucans) being greater than three times more abundant than the next most abundant component, the ring containing 22 glucose units (15%). No rings containing 20 glucose units were present. This is the first observation of cyclic beta-(1-->2)-d-glucans in algae, as far as we are aware. For the linear beta-(1-->2)-d-glucans, the component containing 20 glucoses was most abundant (35% of the linear glucans), while the component containing 21 glucose units was the next most abundant (17%). These relatively low-molecular-weight glucans had low immunostimulatory activity.

Biosynthetic production of universally (13)C-labelled polyunsaturated fatty acids as reference materials for natural health product research.

Long-chain polyunsaturated fatty acids (LCPUFA) including eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have become important natural health products with numerous proven benefits related to brain function and cardiovascular health. Not only are omega-3 fatty acids available in a plethora of dietary supplements, but they are also increasingly being incorporated as triglycerides into conventional foods, including bread, milk, yoghurt and confectionaries. Recently, transgenic oil seed crops and livestock have been developed that enhance omega-3 fatty acid content. This diverse array of matrices presents a difficult analytical challenge and is compounded further by samples generated through clinical research. Stable isotope (13)C-labelled LCPUFA standards offer many advantages as research tools because they may be distinguished from their naturally abundant counterparts by mass spectrometry and directly incorporated as internal standards into analytical procedures. Further, (13)C-labelled LCPUFAs are safe to use as metabolic tracers to study uptake and metabolism in humans. Currently, (13)C-labelled LCPUFAs are expensive, available in limited supply and not in triglyceride form. To resolve these issues, marine heterotrophic microorganisms are being isolated and screened for LCPUFA production with a view to the efficient biosynthetic production of U-(13)C-labelled fatty acids using U-(13)C glucose as a carbon source. Of 37 isolates obtained, most were thraustochytrids, and either DHA or omega-6 docosapentaenoic acid (22:5n-6) were produced as the major LCPUFA. The marine protist Hyalochlorella marina was identified as a novel source of EPA and omega-3 docosapentaenoic acid (22:5n-3). As proof of principle, gram-level production of (13)C-labelled DHA has been achieved with high chemical purity ( >99%) and high (13)C incorporation levels (>90%), as confirmed by NMR and MS analyses. Finally, U-(13)C-DHA was enzymatically re-esterified to glycerol to yield a (13)C-labelled tridocosahexaenoin.

Immunostimulatory polysaccharides from Chlorella pyrenoidosa. A new galactofuranan. measurement of molecular weight and molecular weight dispersion by DOSY NMR.

Fractionation of the hot water extract of Chlorella pyrenoidosa was performed using a combination of ethanol precipitation, size exclusion chromatography, and anion exchange chromatography. One fraction contained a new polysaccharide, and this compound was shown to be a 1-->2-linked beta-d-galactofuranan from its 1D and 2D (1)H and (13)C NMR spectra, with a molecular weight of 15 kDa from DOSY NMR measurements. A number of other fractions were shown to have the same repeating unit as the previously identified arabinogalactan. However, arabinogalactans from different fractions were shown by DOSY NMR to have different molecular weights, which ranged from 27 to 1020 kDa. Agreement with molecular weights measured for some of these fractions by SEC-MALS was very good, further confirming the relationship established by Viel et al. between molecular weights of neutral polysaccharides and self-diffusion coefficients. The smaller molecular weight polysaccharides, the galactofuranan and the 27 and 50 kDa arabinogalactans, were shown to be close to monodisperse by analysis of the distributions of the self-diffusion coefficients for the polymers. The larger arabinogalactans had considerable variation in their molecular weights (188 +/- 109 kDa and 1020 +/- 370 kDa). Only the two larger arabinogalactans showed immunostimulatory activity.

Isolation, characterization and structural determination of a unique type of arabinogalactan from an immunostimulatory extract of Chlorella pyrenoidosa.

An arabinogalactan was isolated from a hot water extract of freeze-dried cells of the green microalga, Chlorella pyrenoidosa. This hot water extract is a proprietary immunomodulator, with the trademark Respondintrade mark (ONC-107). The arabinogalactan was recovered from the ethanol-soluble fraction of the supernatant resulting from a process that involved controlled ethanol precipitation followed by size exclusion chromatography on Sephadex G-100, then Cetavlon precipitation. Sugar analyses, GC-MS data for (S)-2-octyl glycosides, and 1D and 2D NMR experiments established unambiguously that the repeating unit was -->2)-alpha-L-Araf-(1-->3)-[alpha-L-Araf-(1-->4)]-beta-D-Galp-(1-->. This structure does not fit into any of the known classes of arabinogalactans. SEC/MALS experiments gave a molecular mass for the arabinogalactan isolated as 47 +/- 4 kDa but the original structure was probably larger.