Virucidal and Immunostimulating Activities of Monogalactosyl Diacylglyceride from Coccomyxa sp. KJ, a Green Microalga, against Murine Norovirus and Feline Calicivirus.

Human noroviruses are the most common pathogens causing acute gastroenteritis and may lead to more severe illnesses among immunosuppressed people, including elderly and organ transplant recipients. To date, there are no safe and effective vaccines or antiviral agents for norovirus infections. In the present study, we aimed to demonstrate the antiviral activity of monogalactosyl diacylglyceride (MGDG) isolated from a microalga, Coccomyxa sp. KJ, against murine norovirus (MNV) and feline calicivirus (FCV), the surrogates for human norovirus. MGDG showed virucidal activities against these viruses in a dose- and time-dependent manner-MGDG at 100 µg/mL reduced the infectivity of MNV and FCV to approximately 10% after 60 min incubation. In the animal experiments of MNV infection, intraoral administration of MGDG (1 mg/day) exerted a therapeutic effect by suppressing viral shedding in the feces and produced high neutralizing antibody titers in sera and feces. When MGDG was orally administered to immunocompromised mice treated with 5-fluorouracil, the compound exhibited earlier stopping of viral shedding and higher neutralizing antibody titers of sera than those in the control mice administered with distilled water. Thus, MGDG may offer a new therapeutic and prophylactic alternative against norovirus infections.

Isolation and compositional analysis of galactoglycerolipids from perilla [Perilla frutescens (L.) Britton] leaves and comparison to the galactoglycerolipids from spinach and parsley.

The aim of this study was to isolate monogalactosyldiacylglycerols (MGDGs) and digalactosyldiacylglycerols (DGDGs) from perilla [Perilla frutescens (L.) Britton] and to investigate their fatty acid profiles. Perilla displayed the greatest total MGDG and DGDG content among the three types of leaf vegetables tested, that is, spinach, parsley, and perilla, containing 0.16 g/100 g MGDG and 0.04 g/100 g DGDG (on wet weight basis). High purity MGDG (approximately 97 g/100 g) and DGDG (approximately 86 g/100 g) were isolated from perilla chloroform/methanol (2:1, v/v) extracts by two-step silica gel column chromatography. MGDGs were primarily composed of 18:3n-3 and 16:3n-3, predominantly located at the sn-1 and sn-2 positions, respectively. In DGDG, 18:3n-3 and 16:0 were the most abundant fatty acids and were primarily found at the sn-1 and sn-2 positions, respectively. PRACTICAL APPLICATION: MGDGs and DGDGs are the most prevalent forms of galactoglycerolipids found in leaf vegetables including perilla and have been shown to exert health-beneficial effects, such as antitumor, anti-inflammatory, anticancer, and appetite-suppressing activities. Both MGDGs and DGDGs possess emulsifying properties. The present study may help better understand the health-beneficial effects of MGDG and DGDG from perilla, by providing total composition and positional distribution of the fatty acids. The present study also successfully established a protocol to isolate high purity MGDG and DGDG from perilla, thereby increasing their possible use as an ingredient in foods and nutraceuticals.

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

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

Molecular Characterization and Anti-inflammatory Activity of Galactosylglycerides and Galactosylceramides from the Microalga Isochrysis galbana.

Isochrysis galbana is a marine microalga rich in PUFAs that is widely used as feed in aquaculture and more recently investigated for its potential in food applications and as source of bioactive compounds. In this study, the biomass obtained from cultures of I. galbana has been investigated to determine its content in glycosylglycerides and glycosylceramides. By using NMR, UPLC-MS/MS, and fatty acid profiles, the structures of ten monogalactosyldiacylglycerols (MGDGs 1-10) and nine digalactosyldiacylglycerols (DGDGs 11-19) have been established. Two distinctive features of the galactosylglycerides from I. galbana are the wide presence of highly unsaturated acyl chains derived from stearidonic acid (18:4Δ6Z,9Z,12Z,15Z) and octadecapentaenoic acid (18:5Δ3Z,6Z,9Z,12Z,15Z), as well as the unusual coexistence of αß-DGDGs and ßß-DGDGs. Three new galactosylceramides, isogalbamides A-C (20-22), have also been isolated and characterized by NMR and MS/MS. These metabolites, which are the first galactosylceramides described from microalgae, derive from unprecedented tetraolefinic sphingoid bases. In anti-inflammatory assays, the MGDG and DGDG mixtures and the isolated DGDGs 11 and 12 showed significant activity as inhibitors of the production of the pro-inflammatory cytokine TNF-α in lipopolysaccharide-stimulated human THP-1 macrophages, while the galactosylceramides showed moderated activity.

In vitro anti-denaturation and anti-hyaluronidase activities of extracts and galactolipids from leaves of Impatiens parviflora DC.

The in vitro anti-denaturation and anti-hyaluronidase activities of Impatiens parviflora extracts and isolated galactolipids (MGDG-1, DGDG-1) were investigated. This is the first report on these compounds in I. parviflora. All extracts showed anti-hyaluronidase activity, but only methanolic extract from fresh leaves exhibited significant activity against heat-induced denaturation of BSA in a dose-dependent manner. At 500 µg/mL, the extract and the reference drug showed 79.05% and 99.81% inhibition of protein denaturation, respectively. These results indicate that fresh leaves of I. parviflora may be beneficial in inflammatory conditions, especially those associated with protein denaturation, such as rheumatoid arthritis. The study revealed that only MGDG-1 showed weak activity in anti-denaturation assay but both galactolipids were potent inhibitors of hyaluronidase. MGDG-1 completely inhibited the enzyme activity at the concentration of 127.9 µg/mL. These results indicate the potential of galactolipids in the treatment of diseases associated with the loss of hyaluronic acid.

Monogalactosyldiacylglycerols as Host Recognition Cues for Western Corn Rootworm Larvae (Coleoptera: Chrysomelidae).

Monogalactosyldiacylglycerol (MGDG) was identified as a host recognition cue for larvae of the western corn rootworm Diabrotica virgifera virgifera LeConte. An active glycolipid fraction obtained from an extract of germinating maize roots was isolated with thin layer chromatography using a bioassay-driven approach. When analyzed with LC-MS (positive ion scanning), the assay-active spot was found to contain four different MGDG species: 18:3-18:3 (1,2-dilinolenoyl), 18:2-18:3 (1-linoleoyl, 2-linolenoyl), 18:2-18:2 (1,2-dilinoleoyl), and 18:2-16:0 (1-linoleoyl, 2-palmitoyl). A polar fraction was also needed for activity. When combined with a polar fraction containing a blend of sugars (glucose:fructose:sucrose:myoinositol), the isolated MGDG elicited a unique tight-turning behavior by neonate western corn rootworm larvae that is indicative of host recognition. In behavioral bioassays where disks treated with the active blend were exposed to successive sets of rootworm larvae, the activity of MGDG increased over four exposures, suggesting that larvae may be responding to compounds produced after enzymatic breakdown of MGDG. In subsequent tests with synthetic blends composed of theoretical MGDG-breakdown products, larval responses to four synthetic blends were not significantly different (P<0.5) than the response to isolated MGDG. GC-MS analysis showed modest increases in the amounts of the 16:0, 18:0, and 18:3 free fatty acids released from MGDG after a 30-min exposure to rootworm larvae, which is consistent with the enzymatic breakdown hypothesis.

UPLC-MSE profiling of Phytoplankton metabolites: application to the identification of pigments and structural analysis of metabolites in Porphyridium purpureum.

A fast and high-resolution UPLC-MSE analysis was used to identify phytoplankton pigments in an ethanol extract of Porphyridium purpureum (Pp) devoid of phycobiliproteins. In a first step, 22 standard pigments were analyzed by UPLC-MSE to build a database including retention time and accurate masses of parent and fragment ions. Using this database, seven pigments or derivatives previously reported in Pp were unequivocally identified: ß,ß-carotene, chlorophyll a, zeaxanthin, chlorophyllide a, pheophorbide a, pheophytin a, and cryptoxanthin. Minor amounts of Divinyl chlorophyll a, a chemotaxonomic pigment marker for prochlorophytes, were also unequivocally identified using the database. Additional analysis of ionization and fragmentation patterns indicated the presence of ions that could correspond to hydroxylated derivatives of chlorophyll a and pheophytin a, produced during the ethanolic extraction, as well as previously described galactosyldiacylglycerols, the thylakoid coenzyme plastoquinone, and gracilamide B, a molecule previously reported in the red seaweed Gracillaria asiatica. These data point to UPLC-MSE as an efficient technique to identify phytoplankton pigments for which standards are available, and demonstrate its major interest as a complementary method for the structural elucidation of ionizable marine molecules.

The galactolipase activity of Fusarium solani (phospho)lipase.

The purified (phospho)lipase of Fusarium solani (FSL), was known to be active on both triglycerides and phospholipids. This study aimed at assessing the potential of this enzyme in hydrolyzing galactolipids. FSL was found to hydrolyze at high rates of synthetic medium chains monogalactosyldiacylglycerol (4658±146U/mg on DiC8-MGDG) and digalactosyldiacylglycerol (3785±83U/mg on DiC8-DGDG) and natural long chain monogalactosyldiacylglycerol extracted from leek leaves (991±85U/mg). It is the microbial enzyme with the highest activity on galactolipids identified so far with a level of activity comparable to that of pancreatic lipase-related protein 2. FSL maximum activity on galactolipids was measured at pH8. The analysis of the hydrolysis product of natural MGDG from leek showed that FSL hydrolyzes preferentially the ester bond at the sn-1 position of galactolipids. To investigate the structure-activity relationships of FSL, a 3D model of this enzyme was built. In silico docking of medium chains MGDG and DGDG and phospholipid in the active site of FSL reveals structural solutions which are in concordance with in vitro tests.

Isolation of the molecular species of monogalactosyldiacylglycerols from brown edible seaweed Sargassum horneri and their inhibitory effects on triglyceride accumulation in 3T3-L1 adipocytes.

The chemical composition of monogalactosyldiacylglycerols (MGDGs) from brown alga Sargassum horneri and their inhibitory effects on lipid accumulation were investigated in this study. A total of 10 molecular species of MGDGs were identified using nuclear magnetic resonance, alkaline hydrolysis, gas chromatography-flame ionization detector, and high-performance liquid chromatography-tandem mass spectrometry methods. Individual molecular species of MGDGs, including (2S)-1-O-myristoyl-2-O-palmitoleoyl-3-O-ß-D-galactopyranosyl-sn-glycerol (1), (2S)-1-O-myristoyl-2-O-linoleyl-3-O-ß-D-galactopyranosyl-sn-glycerol (3), (2S)-1-O-palmitoyl-2-O-linolenoyl-3-O-ß-D-galactopyranosyl-sn-glycerol (5), (2S)-1-O-myristoyl-2-O-oleyl-3-O-ß-D-galactopyranosyl-sn-glycerol (7), (2S)-1-O-palmitoyl-2-O-palmitoleoyl-3-O-ß-D-galactopyranosyl-sn-glycerol (8), (2S)-1-O-palmitoyl-2-O-linoleyl-3-O-ß-D-galactopyranosyl-sn-glycerol (9), and (2S)-1-O-palmitoyl-2-O-oleyl-3-O-ß-D-galactopyranosyl-sn-glycerol (10), were then furnished using semi-preparative high-performance liquid chromatography, and their inhibitory effects on triglyceride (TG) accumulation and free fatty acid (FFA) levels in 3T3-L1 adipocytes were evaluated. Compounds 3 and 9 showed inhibitory effects on TG and FFA accumulation, with TG levels of 1.568 ± 0.2808 and 1.701 ± 0.1460 µmol/L and FFA levels of 0.149 ± 0.0258 and 0.198 ± 0.0229 mequiv/L, respectively, which were more effective than other compounds. The primary structure-activity relationship suggested that linoleyl [18:2(ω-6)] in the sn-2 position played an important role on triglyceride accumulation inhibition.

A novel ether-linked phytol-containing digalactosylglycerolipid in the marine green alga, Ulva pertusa.

Galactosylglycerolipids (GGLs) and chlorophyll are characteristic components of chloroplast in photosynthetic organisms. Although chlorophyll is anchored to the thylakoid membrane by phytol (tetramethylhexadecenol), this isoprenoid alcohol has never been found as a constituent of GGLs. We here described a novel GGL, in which phytol was linked to the glycerol backbone via an ether linkage. This unique GGL was identified as an Alkaline-resistant and Endogalactosylceramidase (EGALC)-sensitive GlycoLipid (AEGL) in the marine green alga, Ulva pertusa. EGALC is an enzyme that is specific to the R-Galα/ß1-6Galß1-structure of galactolipids. The structure of U. pertusa AEGL was determined following its purification to 1-O-phytyl-3-O-Galα1-6Galß1-sn-glycerol by mass spectrometric and nuclear magnetic resonance analyses. AEGLs were ubiquitously distributed in not only green, but also red and brown marine algae; however, they were rarely detected in terrestrial plants, eukaryotic phytoplankton, or cyanobacteria.

Keratinocyte wound healing activity of galactoglycerolipids from the fern Ophioglossum vulgatum L.

The genus Ophioglossum consists of ferns with different therapeutic properties, including vulnerary virtues. The species Ophioglossum vulgatum L. is traditionally used on wounds and burns as an ointment, suggesting the occurrence of lipophilic compounds with tissue repair properties. We isolated and characterized a galactosyldiacylglycerol mixture (1), composed mainly of 1,2-di-O-linolenoyl-3-O-ß-D-galactopyranosyl-glycerol, from the frond dichloromethane extract. The wound healing properties of 1 were assessed in vitro on keratinocytes. Scratch wound assays showed increased wound closure rates in keratinocyte monolayers exposed to subtoxic doses, previously determined in cytotoxicity assays. The strongest effect, obtained at a dose of 5 µg/mL, approached that of a platelet lysate used in clinical settings. The use of inhibitors of the main cellular pathways involved in wound repair, revealed important roles for intracellular calcium and the ERK1/2 MAP kinase. Conversely, a PCR array of genes involved in wound healing showed an almost total absence of gene modulation. Taken together, the data suggest that 1 acts through a Ca(2+)-dependent, nongenomic mechanism involving the activation of ERK1/2 MAP kinase. Hence, 1 is a main candidate to explain the wound healing virtues of O. vulgatum ointment, and is proposed as a possible new drug in tissue repair and regenerative medicine.

Isolation of fucoxanthin and highly unsaturated monogalactosyldiacylglycerol from brown alga Fucus evanescens C Agardh and in vitro investigation of their antitumor activity.

Fucoxanthin (FX) and highly unsaturated monogalactosyldiacylglycerol (MGDG) were isolated from the ethanol extract of brown alga Fucus evanescens. Their structures were identified by nuclear magnetic resonance, complemented by electrospray ionization mass spectrometry (ESIMS). MGDG was identified as 1-O-(5Z,8Z,11Z,14Z,17Z-eicosapentanoyl)-2-O-(6Z,9Z,12Z,15Z-octadecatetraenoyl)-3-O-ß-D-galactopiranosyl-sn-glycerol. Antitumor activity of these compounds was tested on human melanoma (SK-MEL-28) cells. MGDG and FX inhibited the growth of human melanoma cells in a dose-dependent manner. IC50 values for growth inhibition were 104 and 114 µM, correspondently.

Monogalactosyl diacylglycerol, a replicative DNA polymerase inhibitor, from spinach enhances the anti-cell proliferation effect of gemcitabine in human pancreatic cancer cells.

BACKGROUND: Gemcitabine (GEM) is used to treat various carcinomas and represents an advance in pancreatic cancer treatment. In the screening for DNA polymerase (pol) inhibitors, a glycoglycerolipid, monogalactosyl diacylglycerol (MGDG), was isolated from spinach. METHODS: Phosphorylated GEM derivatives were chemically synthesized. In vitro pol assay was performed according to our established methods. Cell viability was measured using MTT assay. RESULTS: Phosphorylated GEMs inhibition of mammalian pol activities assessed, with the order of their effect ranked as: GEM-5'-triphosphate (GEM-TP) > GEM-5'-diphosphate > GEM-5'-monophosphate > GEM. GEM suppressed growth in the human pancreatic cancer cell lines BxPC-3, MIAPaCa2 and PANC-1 although phospholylated GEMs showed no effect MGDG suppressed growth in these cell lines based on its selective inhibition of replicative pol species. Kinetic analysis showed that GEM-TP was a competitive inhibitor of pol alpha activity with nucleotide substrates, and MGDG was a noncompetitive inhibitor with nucleotide substrates. GEM combined with MGDG treatments revealed synergistic effects on the inhibition of DNA replicative pols alpha and gamma activities compared with GEM or MGDG alone. In cell growth suppression by GEM, pre-addition of MGDG significantly enhanced cell proliferation suppression, and the combination of these compounds was found to induce apoptosis. In contrast, GEM-treated cells followed by MGDG addition did not influence cell growth. CONCLUSIONS: GEM/MGDG enhanced the growth suppression of cells based on the inhibition of pol activities. GENERAL SIGNIFICANCE: Spinach MGDG has great potential for development as an anticancer food compound and could be an effective clinical anticancer chemotherapy in combination with GEM.

In vivo antitumor effect of liposomes with sialyl Lewis X including monogalactosyl diacylglycerol, a replicative DNA polymerase inhibitor, from spinach.

The glycoglycerolipid monogalactosyl diacylglycerol (MGDG) isolated from spinach selectively inhibits the activities of replicative DNA polymerase species and suppresses the growth of human cancer cell lines, while not affecting normal human cells. Liposomes, carrying surface-bound sialyl Lewis X (SLX) and containing MGDG (SLX-Lipo-MGDG) and the fluorescent dye Cy5.5, were administered intravenously to mice bearing HT-29 human colon adenocarcinoma tumors and liposome distribution observed using fluorescence imaging equipment in vivo. In an in vivo antitumor assay on nude mice bearing HT-29 solid tumors, SLX-Lipo-MGDG was shown to be a stronger and more promising suppressor of solid tumors than MGDG alone. These results suggest that spinach MGDG could be developed into an anticancer compound, SLX-Lipo-MGDG could serve as an effective clinical anticancer drug and that these liposomes may be useful tools as the basis for active targeting drug delivery systems.

Structural characterization and anti-HSV-1 and HSV-2 activity of glycolipids from the marine algae Osmundaria obtusiloba isolated from Southeastern Brazilian coast.

Glycolipids were extracted from the red alga Osmundaria obtusiloba from Southeastern Brazilian coast. The acetone insoluble material was extracted with chloroform/methanol and the lipids, enriched in glycolipids, were fractionated on a silica gel column eluted with chloroform, acetone and then methanol. Three major orcinol-positive bands were found in the acetone and methanol fractions, being detected by thin layer chromatography. The structures of the corresponding glycolipids were elucidated by ESI-MS and (1)H/(13)C NMR analysis, on the basis of their tandem-MS behavior and HSQC, TOCSY fingerprints. For the first time, the structure of sulfoquinovosyldiacylglycerol from the red alga Osmundaria obtusiloba was characterized. This molecule exhibited potent antiviral activity against HSV-1 and HSV-2 with EC(50) values of 42 µg/mL to HSV-1 and 12 µg/mL to HSV-2, respectively. Two other glycolipids, mono- and digalactosyldiacylglycerol, were also found in the alga, being characterized by ESI-MS/MS. The structural elucidation of algae glycolipids is a first step for a better understanding of the relation between these structures and their biological activities.

Galactolipids from Bauhinia racemosa as a new class of antifilarial agents against human lymphatic filarial parasite, Brugia malayi.

Bioassay guided fractionation of ethanolic extract of the leaves of Bauhinia racemosa led to the isolation of galactolipid and catechin class of the compounds (1-7) from the most active n-butanol fraction (F4). Among the active galactolipids, 1 emerged as the lead molecule which was active on both forms of lymphatic filarial parasite, Brugia malayi. It was found to be better than the standard drug ivermectin and diethylcarbamazine (DEC) in terms of dose and efficacy.

The influence of monogalactosyldiacylglycerols from different marine macrophytes on immunogenicity and conformation of protein antigen of tubular immunostimulating complex.

The tubular immunostimulating complex (TI-complex) is a novel nanoparticulate antigen delivery system consisting of cholesterol, triterpene glycoside cucumarioside A(2)-2, and glycolipid monogalactosyldiacylglycerol (MGDG) isolated from marine macrophytes. MGDG is crucial for the formation of a lipid matrix for the protein antigen incorporated in TI-complexes. Fatty acid composition and the physical state of this glycolipid depend on the taxonomic position of marine macrophytes. Therefore, the aim of the present work was to study the capacity of MGDGs, isolated from five species of marine macrophytes, to influence conformation and to enhance immunogenicity of porin from Yersinia pseudotuberculosis (YOmpF) as a model antigen of subunit vaccine based on TI-complexes. The trimeric porin was chosen for these experiments, because it was approximately two times more immunogenic than monomeric porin incorporated in TI-complexes. Immunization of mice with YOmpF within TI-complexes, comprised of different MGDGs, revealed a dependence of the immunostimulating effect of TI-complexes on the microvicosity of this glycolipid. TI-complexes comprising MGDGs from Sargassum pallidum and Ulva fenestrata with medium microviscosity induced maximal levels of anti-porin antibodies (four times higher when compared with those induced by pure porin). The adjuvant effect of TI-complexes based on other MGDGs varied by 2.8, 2.3 and 1.3 times for TI-complexes comprised of MGDGs from Zostera marina, Ahnfeltia tobuchiensis, and Laminaria japonica, respectively. MGDGs are also able to influence cytokine mechanisms of immunological regulation. DSC and spectroscopic studies showed that maximal immunostimulating effect of TI-complexes correlated with a moderate stabilizing influence of MGDGs from S. pallidum and U. fenestrata on the conformation of porin. The results obtained suggest lipid "nanofluidics" as a novel strategy for optimizing the immune response to protein antigens within lipid particulate systems.

Glycolipid composition of Hevea brasiliensis latex.

Glycolipids of fresh latex from three clones of Hevea brasiliensis were characterized and quantified by HPLC/ESI-MS. Their fatty acyl and sterol components were further confirmed by GC/MS after saponification. The four detected glycolipid classes were steryl glucosides (SG), esterified steryl glucosides (ESG), monogalactosyl diacylglycerols (MGDG) and digalactosyl diacylglycerols (DGDG). Sterols in SG, ESG and total latex unsaponifiable were stigmasterol, ß-sitosterol and Δ5-avenasterol. The latter was found instead of fucosterol formerly described. Galactolipids were mainly DGDG and had a fatty acid composition different from that of plant leaves as they contained less than 5% C18:3. Glycolipids, which represented 27-37% of total lipids, displayed important clonal variations in the proportions of the different fatty acids. ESG, MGDG and DGDG from clone PB235 differed notably by their higher content in furan fatty acid, which accounted for more than 40% of total fatty acids. Clonal variation was also observed in the relative proportions of glycolipid classes except MGDG (8%), with 43-51% DGDG, 30-34% SG and 7-19% ESG. When compared with other plant cell content, the unusual glycolipid composition of H. brasiliensis latex may be linked to the peculiar nature of this specialized cytoplasm expelled from laticiferous system, especially in terms of functional and structural properties.

Constituents and secondary metabolite natural products in fresh and deteriorated cassava roots.

A phytochemical analysis of cassava (Manihot esculenta Crantz) fresh roots and roots suffering from post-harvest physiological deterioration (PPD) has been carried out. The first isolation and identification of galactosyl diacylglycerides from fresh cassava roots is reported, as well as beta-carotene, linamarin, and beta-sitosterol glucopyranoside. The hydroxycoumarin scopoletin and its glucoside scopolin were identified from cassava roots during PPD, as well as trace quantities of esculetin and its glucoside esculin. There is no isoscopoletin in cassava roots during PPD.

Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2.

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the most abundant lipids in nature, mainly as important components of plant leaves and chloroplast membranes. Pancreatic lipase-related protein 2 (PLRP2) was previously found to express galactolipase activity, and it is assumed to be the main enzyme involved in the digestion of these common vegetable lipids in the gastrointestinal tract. Most of the previous in vitro studies were however performed with medium chain synthetic galactolipids as substrates. It was shown here that recombinant guinea pig (Cavia porcellus) as well as human PLRP2 hydrolyzed at high rates natural DGDG and MGDG extracted from spinach leaves. Their specific activities were estimated by combining the pH-stat technique, thin layer chromatography coupled to scanning densitometry and gas chromatography. The optimum assay conditions for hydrolysis of these natural long chain galactolipids were investigated and the optimum bile salt to substrate ratio was found to be different from that established with synthetic medium chains MGDG and DGDG. Nevertheless the length of acyl chains and the nature of the galactosyl polar head of the galactolipid did not have major effects on the specific activities of PLRP2, which were found to be very high on both medium chain [1786+/-100 to 5420+/-85U/mg] and long chain [1756+/-208 to 4167+/-167U/mg] galactolipids. Fatty acid composition analysis of natural MGDG, DGDG and their lipolysis products revealed that PLRP2 only hydrolyzed one ester bond at the sn-1 position of galactolipids. PLRP2 might be used to produce lipid and free fatty acid fractions enriched in either 16:3 n-3 or 18:3 n-3 fatty acids, both found at high levels in galactolipids.