The proanthocyanin-related transcription factors MYBC1 and WRKY44 regulate branch points in the kiwifruit anthocyanin pathway.

The groups of plant flavonoid metabolites termed anthocyanins and proanthocyanins (PA) are responsible for pigmentation in seeds, flowers and fruits. Anthocyanins and PAs are produced by a pathway of enzymes which are transcriptionally regulated by transcription factors (TFs) that form the MYB-bHLH-WD40 (MBW) complex. In this study, transcriptomic analysis of purple-pigmented kiwifruit skin and flesh tissues identified MYBC1, from subgroup 5 of the R2R3 MYB family, and WRKY44 (highly similar to Arabidopsis TTG2) as candidate activators of the anthocyanin pathway. Transient over-expression of MYBC1 and WRKY44 induced anthocyanin accumulation in tobacco leaves. Dual luciferase promoter activation assays revealed that both MYBC1 and WRKY44 were able to strongly activate the promoters of the kiwifruit F3'H and F3'5'H genes. These enzymes are branch points of the pathway which specifies the type of anthocyanin accumulated. Stable over-expression of MYBC1 and WRKY44 in kiwifruit calli activated the expression of F3'5'H and PA-related biosynthetic genes as well as increasing levels of PAs. These results suggest that while previously characterised anthocyanin activator MYBs regulate the overall anthocyanin biosynthesis pathway, the PA-related TFs, MYBC1 and WRKY44, more specifically regulate key branch points. This adds a layer of regulatory control that potentially balances anthocyanin and PA levels.

Consumption of an Anthocyanin-Rich Extract Made From New Zealand Blackcurrants Prior to Exercise May Assist Recovery From Oxidative Stress and Maintains Circulating Neutrophil Function: A Pilot Study.

Aim: To evaluate blackcurrant anthocyanin-rich extract (BAE) consumption on time- and dose-dependent plasma anthocyanin bioavailability and conduct a pilot study to explore the potential effect of BAE in promoting recovery from exercise-induced oxidative stress, and maintenance of circulating neutrophil function. Methods: Time- and dose-dependent blackcurrant anthocyanin bioavailability was assessed using LC-MS in 12 participants over 6 h after the ingestion of a placebo or BAE containing 0.8, 1.6, or 3.2 mg/kg total anthocyanins. In a separate pilot intervention exercise trial, 32 participants consumed either a placebo or 0.8, 1.6, or 3.2 mg/kg BAE (8 individuals per group), and then 1 h later performed a 30 min row at 70% VO2max. Blood was collected during the trial for oxidative, antioxidant, inflammatory, and circulating neutrophil status. Results: Consumption of BAE caused a time- and dose-dependent increase in plasma anthocyanins, peaking at 2 h after ingestion of 3.2 mg/kg BAE (217 ± 69 nM). BAE consumed 1 h prior to a 30 min row had no effect on plasma antioxidant status but hastened the recovery from exercise-induced oxidative stress: By 2 h recovery, consumption of 1.6 mg/kg BAE prior to exercise caused a significant (P < 0.05) 34 and 32% decrease in post-exercise plasma oxidative capacity and protein carbonyl levels, respectively, compared to placebo. BAE consumption prior to exercise dose-dependently attenuated a small, yet significant (P < 0.01) transient 13 ± 2% decline in circulating neutrophils observed in the placebo group immediately post-exercise. Furthermore, the timed consumption of either 1.6 or 3.2 mg/kg BAE attenuated a 17 ± 2.4% (P < 0.05) decline in neutrophil phagocytic capability of opsonised FITC-Escherichia coli observed 6 h post-exercise in the placebo group. Similarly, a dose-dependent increase in neutrophil surface expression of complement receptor-3 complex (CR3, critical for effective phagocytosis of opsonised microbes), was observed 6 h post-exercise in both 1.6 and 3.2 mg/kg BAE intervention groups. Conclusions: Consumption of BAE (>1.6 mg/kg) 1 h prior to exercise facilitated recovery from exercise-induced oxidative stress and preserved circulating neutrophil function. This study provides data to underpin a larger study designed to evaluate the efficacy of timed BAE consumption on post-exercise recovery and innate immunity.

Sweet Poisons: Honeys Contaminated with Glycosides of the Neurotoxin Tutin.

Poisonings due to consumption of honeys containing plant toxins have been reported widely. One cause is the neurotoxin tutin, an oxygenated sesquiterpene picrotoxane, traced back to honeybees (Apis mellifera) collecting honeydew produced by passionvine hoppers (Scolypopa australis) feeding on sap of the poisonous shrub tutu (Coriaria spp.). However, a pharmacokinetic study suggested that unidentified conjugates of tutin were also present in such honeys. We now report the discovery, using ion trap LC-MS, of two tutin glycosides and their purification and structure determination as 2-(ß-d-glucopyranosyl)tutin (4) and 2-[6'-(α-d-glucopyranosyl)-ß-d-glucopyranosyl]tutin (5). These compounds were used to develop a quantitative triple quadrupole LC-MS method for honey analysis, which showed the presence of tutin (3.6 ± 0.1 µg/g honey), hyenanchin (19.3 ± 0.5), tutin glycoside (4) (4.9 ± 0.4), and tutin diglycoside (5) (4.9 ± 0.1) in one toxic honey. The ratios of 4 and 5 to tutin varied widely in other tutin-containing honeys. The glycosidation of tutin may represent detoxification by one or both of the insects involved in the food chain from plant to honey.

Unusual immuno-modulatory triterpene-caffeates in the skins of russeted varieties of apples and pears.

Three triterpene-caffeates have been isolated from skins of a russeted apple cultivar "Merton Russet" and identified by LC-MS and NMR as betulinic acid-3-cis-caffeate, betulinic acid-3-trans-caffeate, and oleanolic acid-3-trans-caffeate. Betulinic acid-3-trans-caffeate and oleanolic acid-3-trans-caffeate were also found in russeted pear skins. These compounds have not been previously reported in apples or pears, or in any other foods. Their presence was related to suberized tissue as they were only found in russet portions of the partially russeted apple cultivar "Cox's Orange Pippin" and were not detected in the waxy apple cultivar "Royal Gala". High concentrations of betulinic acid-3-trans-caffeate were found in the bark of both "Merton Russet" and "Royal Gala" trees. The three triterpene-caffeates showed anti-inflammatory activity in vitro, inhibiting NF-κB activation with IC50's of 6-9 µM. Betulinic acid-3-trans-caffeate, the predominant compound in the apples, was immuno-modulatory at around 10 µM in the in vitro and ex vivo bioassays, boosting production of the pro-inflammatory cytokine TNFα in cells stimulated with bacterial lipopolysaccharides.

Blackcurrant proanthocyanidins augment IFN-gamma-induced suppression of IL-4 stimulated CCL26 secretion in alveolar epithelial cells.

Epidemiological studies reveal that fruit consumption reduces the prevalence of airway inflammation and childhood asthma. In particular, blackcurrant polyphenolic extracts have been shown to alleviate lung inflammation. Since IL-4-stimulated eotaxin-3 (CCL26) secretion is a major factor in the continuous eosinophil recruitment observed in atopic asthma, our focus was to evaluate the effectiveness of blackcurrant polyphenolic compounds on CCL26 secretion in human alveolar epithelial cells. Our results indicate that a proanthocyanin-enriched blackcurrant extract (BC-P), but not anthocyanin-enriched blackcurrant extract suppressed both IL-4- and IL-13-stimulated CCL26 secretion in a dose-dependent manner. Furthermore pre-incubation of cells with BC-P caused a time-dependent suppression of IL-4-stimulated CCL26 secretion. Moreover, epigallocatechin (EGC), and to a lesser extent epicatechin, metabolites identified in the proanthocyanidin extract, suppressed IL-4-stimulated CCL26 secretion. EGC was also effective at reducing the cellular phosphorylated STAT-6/STAT-6 ratio. Furthermore, both BC-P and purified EGC potentiated the ability of IFN-gamma to suppress IL-4-stimulated CCL26 secretion. The progression of an allergic immune response is complex, identifying plant compounds that target specific cellular events and complement the body's own immune actions is important for the development of functional foods. Our findings support the potential for blackcurrant polyphenolic compounds to reduce eosinophil recruitment and alleviate eosinophilic-driven airway inflammation.

Blueberry fruit polyphenolics suppress oxidative stress-induced skeletal muscle cell damage in vitro.

Skeletal muscle damage can result from disease and unaccustomed or excessive exercise. Muscle dysfunction occurs via an increased level of reactive oxygen species and hence there is potential in antioxidants as amelioration strategies. We explored the putative benefit of fruit polyphenolic extracts in reducing the susceptibility of skeletal muscle cells to oxidative stress. Muscle myotubes were simultaneously challenged with fruit extracts (1-50 microg/mL) and calcium ionophore (A23187), hydrogen peroxide, or 2,4-dinitrophenol and damage monitored by release of cytosolic enzymes. A blueberry fruit extract displayed a potent and significant dose-dependent protective capacity. Evaluation of the protective capacity of anthocyanin sub-extracts of blueberry fruit and pure individual glycosides, with identification of extract polyphenolic components using MS, suggested that malvidin galactoside and/or glucoside were the active compounds. These in vitro data support the concept that blueberry fruits or derived foods rich in malvidin glycosides may be beneficial in alleviating muscle damage caused by oxidative stress. More research on the benefits of blueberry fruit consumption in human intervention studies is warranted.

Chemical composition and in vitro anti-inflammatory activity of apple phenolic extracts and of their sub-fractions.

Apple extract powders from three different manufacturers were investigated for their anti-inflammatory activity, their total phenolic content, and their chemical composition. The samples represented two production batches for two products and a single batch of a third. The samples showed similar, but clearly different, anti-inflammatory activities, and had substantially different total phenolic contents, and different chemical compositions. Differences in chemical composition for batches of the same product were significant, although not as great as differences between products. The samples were fractionated into chemical classes. The most active fractions were those that contained epicatechin, catechin with phloridzin and quercetin glycosides, or those that contained procyanidin polymers. It was not possible to link activity to the presence of individual components or combinations of these. If fruit extracts are to be reliably linked to validated health benefits, then the source materials, the extraction processes, and the final composition of such products need to be more clearly defined than at present.

Environmental regulation of leaf colour in red 35S:PAP1 Arabidopsis thaliana.

* High-temperature, low-light (HTLL) treatment of 35S:PAP1 Arabidopsis thaliana over-expressing the PAP1 (Production of Anthocyanin Pigment 1) gene results in reversible reduction of red colouration, suggesting the action of additional anthocyanin regulators. High-performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS) and Affimetrix-based microarrays were used to measure changes in anthocyanin, flavonoids, and gene expression in response to HTLL. * HTLL treatment of control and 35S:PAP1 A. thaliana resulted in a reversible reduction in the concentrations of major anthocyanins despite ongoing over-expression of the PAP1 MYB transcription factor. Twenty-one anthocyanins including eight cis-coumaryl esters were identified by LCMS. The concentrations of nine anthocyanins were reduced and those of three were increased, consistent with a sequential process of anthocyanin degradation. Analysis of gene expression showed down-regulation of flavonol and anthocyanin biosynthesis and of transport-related genes within 24 h of HTLL treatment. No catabolic genes up-regulated by HTLL were found. * Reductions in the concentrations of anthocyanins and down-regulation of the genes of anthocyanin biosynthesis were achieved by environmental manipulation, despite ongoing over-expression of PAP1. Quantitative PCR showed reduced expression of three genes (TT8, TTG1 and EGL3) of the PAP1 transcriptional complex, and increased expression of the potential transcriptional repressors AtMYB3, AtMYB6 and AtMYBL2 coincided with HTLL-induced down-regulation of anthocyanin biosynthesis. * HTLL treatment offers a model system with which to explore anthocyanin catabolism and to discover novel genes involved in the environmental control of anthocyanins.

Antifungal saponins from Paris polyphylla Smith.

Three steroidal saponins, including one new and two known compounds, were isolated from the rhizomes of Paris polyphylla Smith. One- and two-dimensional NMR, LC-MS, and interpretation of hydrolytic cleavage experiments led to the identification of the structure of the new saponin as ( 25R)-spirost-5-ene-3 beta,17 alpha-diol (pennogenin) 3- O-{ O- alpha- L-rhamnopyranosyl-(1-->2)- O-[ O- beta-xylopyranosyl-(1-->5)- alpha- L-arabinofuranosyl-(1-->4)]- beta- D-glucopyranoside}. The isolated saponins were evaluated for their antifungal activity against Cladosporium cladosporioides and Candida species and showed comparable activity to chemicals used in some commercial products.

Preparative enzymatic synthesis of glucuronides of zearalenone and five of its metabolites.

The resorcylic acid lactones zearalenone ( 1), alpha-zearalenol ( 2), beta-zearalenol ( 3), alpha-zearalanol (zeranol) ( 4), beta-zearalanol (taleranol) ( 5), and zearalanone ( 6) were converted to their glucuronides on a preparative scale in good yields. Reactions were conducted with bovine uridine 5'-diphosphoglucuronyl transferase (UDPGT) as catalyst and uridine 5'-diphosphoglucuronic acid (UDPGA) as cofactor. The glucuronides were isolated by column chromatography and characterized by NMR spectroscopy and mass spectrometry. Although the principal products were 4- O-glucuronides (i.e., linkage through a phenolic hydroxyl), significant quantities of the 6'- O-glucuronides (i.e., linkage through the aliphatic hydroxyl) of alcohols 2, 4, and 5 were also isolated. In the case of 3, the 2- O-glucuronide was isolated as the minor product. Overall isolated yields of glucuronides, performed on a 20-50 mg scale, were typically ca. 80% based on the resorcylic acid lactone starting material. LC-UV-MS (2) analysis of purified specimens revealed MS (2) fragmentations useful for defining the point of attachment of the glucuronide moiety to the zearalenone nucleus.

Semisynthesis of S-desoxybrevetoxin-B2 and brevetoxin-B2, and assessment of their acute toxicities.

Brevetoxins are neurotoxins associated with blooms of marine algae such as Karenia brevis and can accumulate in the marine food chain, causing intoxication of marine animals and people consuming seafood. Brevetoxin-B2 ( 5) is a toxic metabolite produced in shellfish exposed to algae that contain brevetoxin-B ( 1). S-Desoxybrevetoxin-B2 ( 4) has been proposed as a cometabolite produced during this transformation, and while LC-MS analyses suggest its presence in shellfish, it has not yet been isolated and characterized. Studies on these materials are severely constrained by the difficulty of obtaining and purifying them from natural sources. We have developed a convenient one-pot conversion of commercially available brevetoxin-B ( 1) into S-desoxybrevetoxin-B2 ( 4), and a simple method for converting 4 into brevetoxin-B2 ( 5). Full NMR and mass-spectral characterization of 4 and 5 confirmed their structures and showed that the ratio of diastereoisomers in the synthetic 4 and 5 was similar to that observed in naturally contaminated shellfish. The LD 50 values for 4, 5, and dihydrobrevetoxin-B ( 6) by ip injection in mice were 211, 400, and 250 microg/kg, respectively. The methodology for synthesis of brevetoxin metabolites should greatly facilitate toxicological, biochemical and immunochemical studies of these substances, as well as the production of analytical standards.

Comparison of enzymically glucuronidated flavonoids with flavonoid aglycones in an in vitro cellular model of oxidative stress protection.

This study modeled, in vitro, the potential effect of conjugative (phase II) metabolism on the cytoprotective capacity of fruit flavonoids against oxidative stress. Flavonoid aglycones were compared with their corresponding isomeric mixtures of glucuronides for their ability to enhance the survival of cultured human Jurkat T and neuroblastoma cells stressed with hydrogen peroxide. Various polyphenolic compounds were tested as substrates in vitro for an ovine liver glucuronyl transferase preparation. Flavonoids and their glycoside derivatives were found to be good substrates, whereas phenolic acids were either poor or nonsubstrates. Five common flavonoids were glucuronidated to prepare mixtures for bioassay testing. Glucuronidation generally weakened the cytoprotective capacities of flavonoids (in the presence of H(2)O(2)), but some compounds were weakened much more than others. The concentration that halved cell death was well below 0.5 microM for most flavonoids tested, but glucuronidation increased median effective concentration values to a range of 1-16 microM. This compares with the generally accepted physiological range (0.1-10 microM) for circulating dietary polyphenolics detected in the body. Therefore, some flavonoids may retain a reduced cytoprotective capacity in vitro, after glucuronidation, whereas others may be effectively inactivated.

Convenient large-scale purification of yessotoxin from Protoceratium reticulatum culture and isolation of a novel furanoyessotoxin.

Yessotoxins from a large-scale culture (226 L) of Protoceratium reticulatum strain CAWD129 were harvested by filtration followed by solid-phase extraction. The extract was purified by column chromatography over basic alumina and reverse-phase flash chromatography to afford pure yessotoxin (193 mg). Isolation of yessotoxin was greatly facilitated by selection of a strain which did not produce analogues that interfered with yessotoxin isolation. In addition to yessotoxin, numerous minor yessotoxins were detected by LC-MS in other fractions. From one of these, an early eluting minor analogue with the same molecular weight as yessotoxin and a similar mass spectrometric fragmentation pattern was isolated. This analogue was identified by NMR and mass spectrometry as a novel yessotoxin analogue containing a furan ring in the side chain. This finding reveals biosynthetic flexibility of the yessotoxin pathway in P. reticulatum and confirms earlier findings of production of many minor yessotoxin analogues by this alga. Production of these analogues appeared to be a constitutive trait of P. reticulatum CAWD129.

Comparison of the relative recovery of polyphenolics in two fruit extracts from a model of degradation during digestion and metabolism.

To simulate the effects of digestion and metabolism on the survival of different polyphenolic compounds, extracts of blueberry and apple were deglycosylated by acid hydrolysis, followed by enzymic glucuronidation under neutral conditions, yielding approximately 5% overall recovery of polyphenolics. The major polyphenolics before and after the treatment were compared, to estimate which species are likely to be present in the intestinal lumen, undegraded and available for absorption, after consumption of the fruit. Whereas blueberry extract consisted predominantly of anthocyanins, epicatechin and caffeoyl quinate esters, the major components of the treated extract were quercetin glucuronides and (unglucuronidated) caffeoyl quinates, with only traces of anthocyanidin derivatives. In apple extract, compositional changes were less marked, but caffeoyl quinates, procyanidins and quercetin were enriched at the expense of caffeic acid, epicatechin and catechin. Hydrophobic compounds like phloretin and quercetin were extensively glucuronidated, whereas caffeic acid and caffeoyl quinate were not. These results suggest that the major polyphenolic components of a fruit are not necessarily the most important contributors to any health benefits because the polyphenolic composition in the intestinal lumen and consequently, in the circulation, may be considerably different.

Methylated polyphenols are poor "chemical" antioxidants but can still effectively protect cells from hydrogen peroxide-induced cytotoxicity.

Several polyphenolic compounds, including flavonoids and phenolic acids, were compared with their per-methylated forms in both chemical and cell-based assays for antioxidant capacity. Methylation largely eliminated "chemical" antioxidant capacity, according to ferric reducing antioxidant power and oxygen radical absorbance capacity assays. Methylation, however, only moderately reduced protection of human Jurkat cells in culture, from hydrogen peroxide-mediated cytotoxicity, at physiologically relevant concentrations. Neither methylated nor un-methylated compounds were detectably metabolized by the cells. It appears that the protective mechanism of polyphenolic antioxidants against high concentrations of hydrogen peroxide in human cells may be largely unrelated to chemical antioxidant capacity.

Isolation and identification of pectenotoxins-13 and -14 from Dinophysis acuta in New Zealand.

Two novel pectenotoxins (PTXs), PTX-13 and -14, were isolated from extracts of Dinophysis acuta collected from the west coast of South Island, New Zealand. The compounds were identified as oxidized analogues of PTX-2 by NMR spectroscopic and LC-MS studies. PTX-13 (32R-hydroxyPTX-2) corresponds to the unidentified analogue PTX-11x reported by [Suzuki et al., 2003. Liquid chromatography-mass spectrometry of spiroketal stereoisomers of pectenotoxins and the analysis of novel pectenotoxin isomers in the toxic dinoflagellate Dinophysis acuta from New Zealand. J. Chromatogr. A 992, 141-150]. PTX-13 underwent slow deuteration at the 13beta-position during NMR analysis. PTX-14 corresponds to the 32,36-dehydration product of PTX-13, and may be an artifact.

Peripherally administered desacetyl alpha-MSH and alpha-MSH both influence postnatal rat growth and associated rat hypothalamic protein expression.

Desacetyl alpha-MSH predominates over alpha-MSH during development, but whether it is biologically active and has a physiological role is unclear. We compared the effects of 0.3 microg.g(-1).day(-1) desacetyl alpha-MSH with that of 0.3 microg.g(-1).day(-1) alpha-MSH on postnatal body growth by administering the peptides subcutaneously daily for postnatal days 0-14 and also used a two-dimensional gel electrophoresis gel-based proteomic approach to analyze protein changes in hypothalami, the relay center for body weight and growth regulation, after 14 days of treatment. We found that the growth rate between days 1 and 10 was significantly decreased by desacetyl alpha-MSH but not by alpha-MSH, but by day 14, a time reported for development of a mature pattern of hypothalamic innervation, both peptides had significantly increased neonatal growth compared with PBS-treated control rats. Desacetyl alpha-MSH significantly increased spleen weight, but alpha-MSH had no effect. alpha-MSH significantly decreased kidney weight, but desacetyl alpha-MSH had no effect. Both desacetyl alpha-MSH and alpha-MSH significantly decreased brain weight. By 14 days, both peptides significantly changed expression of a number of hypothalamic proteins, specifically metabolic enzymes, cytoskeleton, signaling, and stress response proteins. We show that peripherally administered desacetyl alpha-MSH is biologically active and induces responses that can differ from those for alpha-MSH. In conclusion, desacetyl alpha-MSH appears to be an important regulator of neonatal rat growth.

Isolation and identification of a cis-C8-diol-ester of okadaic acid from Dinophysis acuta in New Zealand.

A cis-isomer of a C(8)-diol ester of okadaic acid (1) was isolated during large-scale purification of pectenotoxins (PTXs) from extracts of Dinophysis acuta collected from the west coast of South Island, New Zealand. The compound was identified by NMR spectroscopic and liquid chromatography-mass spectrometry (LC-MS) studies, and is the first reported cis-isomer of an okadaic acid C(8)-diol-ester identified in Dinophysis. The more abundant trans-C(8)-diol ester of okadaic acid (2) isolated from the same Dinophysis extract was rapidly hydrolyzed to okadaic acid in vitro by the supernatant from green-lipped mussel hepatopancreas.

Isolation of yessotoxin 32-O-[beta-L-arabinofuranosyl-(5'-->1'')-beta-L-arabinofuranoside] from Protoceratium reticulatum.

Yessotoxin 32-O-[beta-L-arabinofuranosyl-(5'-->1'')-beta-L-arabinofuranoside] (3) was isolated from extracts of Protoceratium reticulatum during a large scale isolation of yessotoxin (1). The structure was characterized by mass spectrometry and NMR spectroscopy. Di-glycoside-3, along with the corresponding mono-glycoside (2) were detected in cultures of P. reticulatum originating from Europe and New Zealand, suggesting that production of arabinosides of 1 is a normal feature of this alga. Formation of multiply charged anions and fragmentation of 3 occurred much more readily than for 1 and 2 under the LC-MS conditions used in this study.

Identification of pectenotoxin-11 as 34S-hydroxypectenotoxin-2, a new pectenotoxin analogue in the toxic dinoflagellate Dinophysis acuta from New Zealand.

A new pectenotoxin, which has been named pectenotoxin-11 (PTX11), was isolated from the dinoflagellate Dinophysis acuta collected from the west coast of New Zealand. The structure of PTX11 was determined as 34S-hydroxypectenotoxin-2 by tandem mass spectrometry and UV and NMR spectroscopy. PTX11 appears to be only the third pectenotoxin identified as a natural biosynthetic product from algae after pectenotoxin-2 and pectenotoxin-12. The LD50 of PTX11 determined by mouse intraperitoneal injection was 244 microg/kg. The LD(min) of PTX11 in these experiments was 250 microg/kg. No signs of toxicity were recorded in mice following an oral dose of PTX11 at 5000 microg/kg. No diarrhea was observed in any of the animals administered with the test substance by either route of administration. Unlike pectenotoxin-2 (PTX2), PTX11 was not readily hydrolyzed to its corresponding seco acid by enzymes from homogenized green-lipped mussel (Perna canaliculus) hepatopancreas.