Dwayne J. Jensen

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.

Short-term blackcurrant extract consumption modulates exercise-induced oxidative stress and lipopolysaccharide-stimulated inflammatory responses.

Exercise-induced oxidative stress is instrumental in achieving the health benefits from regular exercise. Therefore, inappropriate use of fruit-derived products (commonly applied as prophalytic antioxidants) may counteract the positive effects of exercise. Using human exercise and cellular models we found that 1) blackcurrant supplementation suppressed exercise-induced oxidative stress, e.g., plasma carbonyls (0.9 +/- 0.1 vs. 0.6 +/- 0.1 nmol/mg protein, placebo vs. blackcurrant), and 2) preincubation of THP-1 cells with an anthocyanin-rich blackcurrant extract inhibited LPS-stimulated cytokine secretion [TNF-alpha (16,453 +/- 322 vs. 10,941 +/- 82 pg/ml, control vs. extract, P < 0.05) and IL-6 (476 +/- 14 vs. 326 +/- 32 pg/ml, control vs. extract, P < 0.05)] and NF-kappaB activation. In addition to its antioxidant and anti-inflammatory properties, we found that postexercise plasma collected after blackcurrant supplementation enhanced the differential temporal LPS-stimulated inflammatory response in THP-1 cells, resulting in an early suppression of TNF-alpha (1,741 +/- 32 vs. 1,312 +/- 42 pg/ml, placebo vs. blackcurrant, P < 0.05) and IL-6 (44 +/- 5 vs. 36 +/- 3 pg/ml, placebo vs. blackcurrant, P < 0.05) secretion after 24 h. Furthermore, by using an oxidative stress cell model, we found that preincubation of THP-1 cells with hydrogen peroxide (H(2)O(2)) prior to extract exposure caused a greater suppression of LPS-stimulated cytokine secretion after 24 h, which was not evident when cells were simultaneously incubated with H(2)O(2) and the extract. In summary, our findings support the concept that consumption of blackcurrant anthocyanins alleviate oxidative stress, and may, if given at the appropriate amount and time, complement the ability of exercise to enhance immune responsiveness to potential pathogens.

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.

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.

Blackcurrant proanthocyanidins augment IFN-γ-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 bodys 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.

Analysis of taxol, 10-deacetylbaccatin III and related compounds in Taxus baccata

Abstract A method is described for the analysis of taxol and related taxanes in the foliage of Taxus spp. such as T. baccata. The method has been optimised for recovery and analysis of a range of taxanes of differing polarity, namely: taxol, cephalomannine, baccatin III and 10-deacetylbaccatin III (10-DAB-III). Measured recoveries for all these compounds generally exceeded 84%. The reversed-phase high-performance liquid chromatography system developed uses a “sterically protected” C8 column, and allowed analysis of the components of interest with minimum interference from co-extracted compounds. Typical detection limits for routine operation were in the range of 1–10 mg/kg depending on the component and the particular sample. Analysis of test samples of T. baccata have shown variations in taxol concentrations in the range 7–510 mg/kg and 10-deacetylbaccatin III in the range 52–1185 mg/kg.

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.

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.

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.

Degradation rate of sodium fluoroacetate in three New Zealand soils.

The degradation rate of sodium fluoroacetate (SFA) was assessed in a laboratory microcosm study incorporating 3 New Zealand soil types under different temperature (5 °C, 10 °C, or 20 °C) and soil moisture (35% or 60% water holding capacity) conditions using guideline 307 from the Organisation for Economic Co-operation and Development. A combination of nonlabeled and radiolabeled (14) C-SFA was added to soil microcosms, with sampling and analysis protocols for soil, soil extracts, and evolved CO(2) established using liquid scintillation counting and liquid chromatography-mass spectrometry. Degradation products of SFA and their rates of formation were similar in the 3 soil types. The major degradation pathway for SFA was through microbial degradation to the hydroxyl metabolite, hydroxyacetic acid, and microbial mineralization to CO(2), which constituted the major transformation product. Temperature, rather than soil type or moisture content, was the dominant factor affecting the rate of degradation. Soil treatments incubated at 20 °C displayed a more rapid loss of (14)C-SFA residues than lower temperature treatments. The transformation half-life (DT50) of SFA in the 3 soils increased with decreasing temperature, varying from 6 d to 8 d at 20 °C, 10 d to 21 d at 10 °C, and 22 d to 43 d at 5 °C.