Comparisons between the in vitro and in vivo efficacies of potential fungal antagonists of Armillaria mellea

Seventeen fungal isolates were tested in vitro as potential antagonists of two isolates of the root rot pathogen, Armillaria mellea. Some of the isolates were also added on mushroom composts with living mycelia to the roots of Armillaria-inoculated potted strawberry plants in the glasshouse to find out if they had the same degree of efficacy against the disease. Dactylium dendroides isolate SP was the most effective in reducing mycelial growth of A. mellea isolate 1 (Am1), followed by Trichoderma harzianum isolate Th2 and T. viride isolate Tv4. Th2, Th22, Tv3 and SP grew extensively over Am1 colonies, disintegrating the rhizomorphs. Isolate Tham1 of T hamatum was the most effective in reducing mycelial growth of A. mellea isolate 2 (Am2), followed by Tv3. Th12, Th22, Tv1, Tv3 and SP inhibited the initiation and growth of rhizomorphs of Am2. Regeneration tests showed that both Am1 and Am2 attacked by Trichoderma isolates and SP were no longer viable. Th23 and SP were almost as effective in vivo as in vitro. But isolate Co of Chaetomium olivaceum, which was ineffective in vitro, was found effective in vivo. Conversely, Th2, which exhibited good antagonistic activity in vitro, performed poorly in vivo. These results show that the in vitro and in vivo efficacies of potential antagonists may not necessarily be closely correlated. Hence, there is a danger that potentially effective isolates may be discarded if decisions are made only on the basis of preliminary screening tests carried out under laboratory conditions.

13C-2H correlation NMR spectroscopy studies of the in vivo transformations of natural products from Artemisia annua

13C-2H correlation NMR spectroscopy (13C-2H COSY) permits the identification of 13C and 2H nuclei which are connected to one another by a single chemical bond via the sizeable 1JCD coupling constant. The practical development of this technique is described using a 13C-2H COSY pulse sequence which is derived from the classical 13C-1H correlation experiment. An example is given of the application of 13C-2H COSY to the study of the biogenesis of natural products from the anti-malarial plant Artemisia annua, using a doubly-labelled precursor molecule. Although the biogenesis of artemisinin, the anti-malarial principle from this species, has been extensively studied over the past twenty years there is still no consensus as to the true biosynthetic route to this important natural product – indeed, some published experimental results are directly contradictory. One possible reason for this confusion may be the ease with which some of the metabolites from A. annua undergo spontaneous autoxidation, as exemplified by our recent in vitro studies of the spontaneous autoxidation of dihydroartemisinic acid, and the application of 13C-2H COSY to this biosynthetic problem has been important in helping to mitigate against such processes. In this in vivo application of 13C-2H COSY, [15-13C2H3]-dihydroartemisinic acid (the doubly-labelled analogue of the natural product from this species which was obtained through synthesis) was fed to A. annua plants and was shown to be converted into several natural products which have been described previously, including artemisinin. It is proposed that all of these transformations occurred via a tertiary hydroperoxide intermediate, which is derived from dihyroartemisinic acid. This intermediate was observed directly in this feeding experiment by the 13C-2H COSY technique; its observation by more traditional procedures (e.g., chromatographic separation, followed by spectroscopic analysis of the purified product) would have been difficult owing to the instability of the hydroperoxide group (as had been established previously by our in vitro studies of the spontaneous autoxidation of dihydroartemisinic acid). This same hydroperoxide has been reported as the initial product of the spontaneous autoxidation of dihydroartemisinic acid in our previous in vitro studies. Its observation in this feeding experiment by the 13C-2H COSY technique, a procedure which requires the minimum of sample manipulation in order to achieve a reliable identification of metabolites (based on both 13C and 2H chemical shifts at the 15-position), provides the best possible evidence for its status as a genuine biosynthetic intermediate, rather than merely as an artifact of the experimental procedure.

In vivo transformations of dihydroartemisinic acid in Artemisia annua plants

[15-(CH3)-C-13-H-2]-dihydroartemisinic acid (2a) and [15-(CH3)-H-2]-dihydroartemisinic acid (2b) have been fed via the root to intact Artemisia annua plants and their transformations studied in vivo by one-dimensional H-2 NMR spectroscopy and two-dimensional, C-13-H-2 correlation NMR spectroscopy (C-13-(2) H COSY). Labelled dihydroartemisinic acid was transformed into 16 12-carboxy-amorphane and cadinane sesquiterpenes within a few days in the aerial parts of A. annua, although transformations in the root were much slower and more limited. Fifteen of these 16 metabolites have been reported previously as natural products from A. annua. Evidence is presented that the first step in the transformation of dihydroartemisinic acid in vivo is the formation of allylic hydroperoxides by the reaction of molecular oxygen with the Delta(4,5)-double bond in this compound. The origin of all 16 secondary metabolites might then be explained by the known further reactions of such hydroperoxides. The qualitative pattern for the transformations of dihydroartemisinic acid in vivo was essentially unaltered when a comparison was made between plants, which had been kept alive and plants which were allowed to die after feeding of the labelled precursor. This, coupled with the observation that the pattern of transformations of 2 in vivo demonstrated very close parallels with the spontaneous autoxidation chemistry for 2, which we have recently demonstrated in vitro, has lead us to conclude that the main 'metabolic route' for dihydroartemisinic acid in A. annua involves its spontaneous autoxidation and the subsequent spontaneous reactions of allylic hydroperoxides which are derived from 2. There may be no need to invoke the participation of enzymes in any of the later biogenetic steps leading to all 16 of the labelled 11,13-dihydro-amorphane sesquiterpenes which are found in A. annua as natural products. (C) 2003 Elsevier Ltd. All rights reserved.

In vivo transformations of artemisinic acid in Artemisia annua plants

Artemisinic acid labeled with both C-13 and H-2 at the 15-position has been fed to intact plants of Artemisia annua via the cut stem, and its in vivo transformations studied by 1D- and 2D-NMR spectroscopy. Seven labeled metabolites have been isolated, all of which are known as natural products from this species. The transformations of artemisinic acid-as observed both for a group of plants, which was kept alive by hydroponic administration of water and for a group, which was allowed to die by desiccation-closely paralleled those, which have been recently described for its 11,13-dihydro analog, dihydroartemisinic acid. It seems likely therefore that similar mechanisms, involving spontaneous autoxidation of the Delta(4,5) double bond in both artemisinic acid and dihydroartemisinic acid and subsequent rearrangements of the resultant allylic hydroperoxides, may be involved in the biological transformations, which are undergone by both compounds. All of the sesquiterpene metabolites, which were obtained from in vivo transformations of artemisinic acid retained their unsaturation at the 11,13-position, and there was no evidence for conversion into any 11,13-dihydro metabolite, including artemisinin, the antimalarial drug, which is produced by A. annua. This observation led to the proposal of a unified biosynthetic scheme, which accounts for the biogenesis of many of the amorphane and cadinane sesquiterpenes that have been isolated as natural products from A. annua. In this scheme, there is a bifurcation in the biosynthetic pathway starting from amorpha-4,11-diene leading to either artemisinic acid or dihydroartemisinic acid; these two committed precursors are then, respectively, the parents for the two large families of highly oxygenated 11,13-dehydro and 11,13-dihydro sesquiterpene metabolites, which are known from this species. (C) 2007 Elsevier Ltd. All rights reserved.

In vivo transformations of dihydro-epi-deoxyarteannuin B in Artemisia annua plants

[15-(CH3)-C-13-H-2]-dihydro-epi-deoxyarteannuin B (4a) has been fed to intact Artemisia annua plants via the root and three labeled metabolites (17a-19a) have been identified by 1D- and 2D-NMR spectroscopies. The in vivo transformations of 4a in A. annua are proposed to involve enzymatically-mediated processes in addition to possible spontaneous autoxidation. In the hypothetical spontaneous autoxidation pathway, the tri-substituted double bond in 4a appears to have undergone 'ene-type' reaction with oxygen to form an allylic hydroperoxide, which subsequently rearranges to the allylic hydroxyl group in the metabolite 3 alpha-hydroxy-dihydro-epi-deoxyarteannuin B (17a). In the enzymatically-mediated pathways, compound 17a has then been converted to its acetyl derivative, 3 alpha-acetoxy-dihydro-epi-deoxyarteannuin B (18a), while oxidation of 4a at the 'unactivated' 9-position has yielded 9 beta-hydroxy-dihydro-epi-deoxyarteannuin B (19a). Although all of the natural products artemisinin ( 1), arteannuin K ( 7), arteannuin L ( 8), and arteannuin M ( 9) have been suggested previously as hypothetical metabolites from dihydro-epi-deoxyarteannuin B in A. annua, none were isolated in labeled form in this study. It is argued that the nature of the transformations undergone by compound 4a are more consistent with a degradative metabolism, designed to eliminate this compound from the plant, rather than with a role as a late precursor in the biosynthesis of artemisinin or other natural products from A. annua. (C) 2007 Elsevier Ltd. All rights reserved.

Mapping antimalarial pharmacophores as a useful tool for the rapid discovery of drugs effective in vivo: design, construction, characterization, and pharmacology of metaquine

Resistant strains of Plasmodium falciparum and the unavailability of useful antimalarial vaccines reinforce the need to develop new efficacious antimalarials. This study details a pharmacophore model that has been used to identify a potent, soluble, orally bioavailable antimalarial bisquinoline, metaquine (N,N'-bis(7-chloroquinolin-4-yl)benzene-1,3-diamine) (dihydrochloride), which is active against Plasmodium berghei in vivo (oral ID50 of 25 mu mol/kg) and multidrug-resistant Plasmodium falciparum K1 in vitro (0.17 mu M). Metaquine shows strong affinity for the putative antimalarial receptor, heme at pH 7.4 in aqueous DMSO. Both crystallographic analyses and quantum mechanical calculations (HF/6-31+G*) reveal important regions of protonation and bonding thought to persist at parasitic vacuolar pH concordant with our receptor model. Formation of drug-heme adduct in solution was confirmed using high-resolution positive ion electrospray mass spectrometry. Metaquine showed strong binding with the receptor in a 1: 1 ratio (log K = 5.7 +/- 0.1) that was predicted by molecular mechanics calculations. This study illustrates a rational multidisciplinary approach for the development of new 4-aminoquinoline antimalarials, with efficacy superior to chloroquine, based on the use of a pharmacophore model.

Loss of 3-chlorotyrosine by inflammatory oxidants: Implications for the use of 3-chlorotyrosine as a bio-marker in vivo

Activated neutrophils generate the potent oxidant hypochlorous acid (HOCl) from the enzyme myeloperoxidase (MPO). A proposed bio-marker for MPO-derived HOCl in vivo is 3-chlorotyrosine, elevated levels of which have been measured in several human inflammatory pathologies. However, it is unlikely that HOCl is produced as the sole oxidant at sites of chronic inflammation as other reactive species are also produced during the inflammatory response. The work presented shows that free and protein bound 3-chlorotyrosine is lost upon addition of the pro-inflammatory oxidants, HOCl, peroxynitrite, and acidified nitrite. Furthermore, incubation of 3-chlorotyrosine with activated RAW264.7 macrophages or neutrophil-like HL-60 cells resulted in significant loss of 3-chlorotyrosine. Therefore, at sites of chronic inflammation where there is concomitant ONOO- and HOCl formation, it is possible measurement of 3-chlorotyrosine may represent an underestimate of the true extent of tyrosine chlorination. This finding could account for some of the discrepancies reported between 3-chlorotyrosine levels in tissues in the literature. (c) 2008 Elsevier Inc. All rights reserved.

Characterisation of cell death in bagged baby salad leaves

Baby leaf salads are gaining in popularity over traditional whole head lettuce salads in response to consumer demand for greater variety and convenience in their diet. Baby lettuce leaves are mixed, washed and packaged as whole leaves, with a shelf-life of approximately 10 days post-processing. End of shelf-life, as determined by the consumer, is typified by bruising, water-logging and blackening of the leaves, but the biological events causing this phenotype have not been studied to date. We investigated the physiological and ultrastructural characteristics during postharvest shelf-life of two lettuce varieties with very different leaf morphologies. Membrane disruption was an important determinant of cell death in both varieties. although the timing and characteristics of breakdown was different in each with Lollo rossa showing signs of aging such as thylakoid disruption and plastoglobuli accumulation earlier than Cos. Membranes in Lollo rossa showed a later, but more distinct increase in permeability than in Cos. as indicated by electrolyte leakage and the presence of cytoplasmic fragments in the vacuole, but Cos membranes show distinct fractures towards the end of shelf-life. The tissue lost less than 25% fresh weight during shelf-life and there was little protein loss compared to developmentally aging leaves in an ambient environment. Biophysical measurements showed that breakstrength was significantly reduced in Lollo rossa, whereas irreversible leaf plasticity was significantly reduced in Cos leaves. The reversible elastic properties of both varieties changed throughout shelf-life. We compared the characteristics of shelf-life in both varieties of bagged lettuce leaves with other leafy salad crops and discuss the potential targets for future work to improve postharvest quality of baby leaf lettuce. (C) 2007 Elsevier B.V. All rights reserved.

A novel galactooligosaccharide mixture increases the bifidobacterial population numbers in a continuous in vitro fermentation system and in the proximal colonic contents of pigs in vivo

Prebiotics are nondigestible food ingredients that encourage proliferation of selected groups of the colonic microflora, thereby altering the composition toward a more beneficial community. In the present study, the prebiotic potential of a novel galactooligosaccharide (GOS) mixture, produced by the activity of galactosyltransferases from Bifidobacterium bifidum 41171 on lactose, was assessed in vitro and in a parallel continuous randomized pig trial. In situ fluorescent hybridization with 16S rRNA-targeted probes was used to investigate changes in total bacteria, bifidobacteria, lactobacilli, bacteroides, and Clostridium histolyticum group in response to supplementing the novel GOS mixture. In a 3-stage continuous culture system, the bifidobacterial numbers for the first 2 vessels, which represented the proximal and traverse colon, increased (P < 0.05) after the addition of the oligosaccharide mixture. In addition, the oligosaccharide mixture strongly inhibited the attachment of enterohepatic Escherichia coli (P < 0.01) and Salmonella enterica serotype Typhimurium (P < 0.01) to HT29 cells. Addition of the novel mixture at 4% (wt:wt) to a commercial diet increased the density of bificlobacteria (P < 0.001) and the acetate concentration (P < 0.001), and decreased the pH (P < 0.001) compared with the control diet and the control diet supplemented with inulin, suggesting a great prebiotic potential for the novel oligosaccharide mixture. J. Nutr. 135: 1726-1731, 2005.

Antioxidant properties of carotenoids in vitro and in vivo

Carotenoids are a class of natural pigments familiar to all through the orange-red to yellow colors of many fruits, vegetables, and flowers, as well as for the provitamin A activity that some of them possess. A body of scientific evidence suggests that carotenoids may scavenge and deactivate free radicals, acting thereby as antioxidants both in food systems (in vitro) and in the human organism (in vivo). Overall, epidemiological evidence links higher carotenoid intakes and tissue concentrations with reduced cancer and cardiovascular disease risk. However, research has also shown that the antioxidant activity of carotenoids may shift to a prooxidant character depending mainly on the biological environment in which they act. A summary of the antioxidant potential of natural carotenoids both in oil model systems and in vivo is presented in this article.

Modulation of detoxification enzymes by watercress: in vitro and in vivo investigations in human peripheral blood cells

Epidemiological studies indicate that consumption of cruciferous vegetables (CV) can reduce the risk of cancer. Supposed mechanisms are partly the inhibition of phase I and the induction of phase II enzymes. The aim of this study was to investigate in vitro and in vivo effects of watercress (WC), a member of the CV family, on chemopreventive parameters using human peripheral blood mononuclear cells (PBMC) as surrogate cells. We investigated the hypothesis that WC reduces cancer risk by inducing detoxification enzymes in a genotype-dependent manner. In vitro gene expression and enzyme activity experiments used PBMC incubated with a crude extract from fresh watercress (WCE, 0.1-10 mu L/mL with 8.2 g WC per 1 mL extract) or with one main key compound phenethyl isothiocyanate (PEITC, 1-10 mu M). From an in vivo perspective, gene expression and glutathione S-transferase (GST) polymorphisms were determined in PBMC obtained from a human intervention study in which subjects consumed 85 g WC per day for 8 weeks. The influence of WC consumption on gene expression was determined for detoxification enzymes such as superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPX1), whilst the SOD and GPX activities in red blood cells were also analysed with respect to GST genotypes. In vitro exposure of PBMC to WCE or PEITC (24 h) increased gene expression for both detoxification enzymes GPX1 (5.5-fold, 1 mu L/mL WCE, 3.7-fold 1 mu M PEITC) and SOD2 (12.1-fold, 10 mu L/mL WCE, 7.3-fold, 10 mu M PEITC), and increased SOD2 activity (1.9-fold, 10 mu L/mL WCE). The WC intervention had no significant effect on in vivo PBMC gene expression, as high individual variations were observed. However, a small but significant increase in GPX (p = 0.025) and SOD enzyme activity (p = 0.054) in red blood cells was observed in GSTM1*0, but not in GSTM1*1 individuals, whilst the GSTT1 genotype had no impact. The results indicate that WC is able to modulate the enzymes SOD and GPX in blood cells in vitro and in vivo, and suggest that the capacity of moderate intake of CV to induce detoxification is dependent in part on the GSTM1 genotype.

Incorporation of cis-9, trans-11 or trans-10, cis-12 conjugated linoleic acid into human erythrocytes in vivo

The purpose of this study was to determine the incorporation into erythrocytes of cis (c)-9,trans (t)-11 conjugated linoleic acid (CLA) and t10,c12 CLA consumed as supplements highly enriched in these isomers. Healthy men (31 8 years) consumed 1, 2, and 4 capsules containing approximately 80 g/100 g of either c9,t11 CLA or t10,c12 CLA for sequential 8-week periods. Fatty acid concentrations in erythrocyte total lipids were determined at baseline and after consumption of the highest dose. The increase in c9,t11 CLA concentration (0.31 g/100 g) was significantly greater than that in t10,c12 CLA (0.19 g/100 g). This was associated with minor changes in concentrations of some fatty acids of chain length greater than 20 carbons. These data suggest selective assimilation of individual CLA isomers into erythrocyte lipids and partial substitution for specific saturated and polyunsaturated fatty acids. (C) 2005 Elsevier Inc. All rights reserved.

Role of quercetin and its in vivo metabolites in protecting H9c2 cells against oxidative stress

The aim of this study was to investigate the potential of quercetin and two of its "in vivo" metabolites, 3'-O-methyl quercetin and 4'-O-methyl quercetin, to protect H9c2 cardiomyoblasts against H2O2-induced oxidative stress. As limited data are available regarding the potential uptake and cellular effects of quercetin and its metabolites in cardiac cells, we have evaluated the cellular association/uptake of the three compounds and their involvement in the modulation of two pro-survival signalling pathways: ERK1/2 signalling cascade and PI3K/Akt pathway. The three flavonols associated with cells to differing extents. Quercetin and its two O-methylated metabolites were able to reduce intracellular ROS production but only quercetin was able to counteract H2O2 cell damage, as measured by MTT reduction assay, caspase-3 activity and DNA fragmentation assays. Furthermore, only quercetin was observed to modulate pro-survival signalling through ERK1/2 and PI3K/Akt pathway. In conclusion we have demonstrated that quercetin, but not its O-methylated metabolites, exerts protective effects against H2O2 cardiotoxicity and that the mechanism of its action involves the modulation of PI3K/Akt and ERK1/2 signalling pathways. (c) 2006 Elsevier Masson SAS. All rights reserved.

Effects of biologically active tumour-promoting and non-promoting phorbol esters on in vivo growth of melanocytic cells

Sapintoxin A (SAP A), a naturally occurring biologically active but non-promoting phorbol ester, acts as an effective in vitro mitogen for freshly derived human melanocytes. Seven days after addition of 50 nM SAP A there was a four to fivefold increase in melanocyte number over that observed in untreated control cultures comparable to that achieved with a 50 nM concentration of 12-0-tetradecanoylphorbol 13-acetate (TPA). The fluorescent stage 2 promoter sapintoxin D (SAP D) also supported the growth of these cells, with a 50 nM dose producing an increase in cell number comparable to that observed with 200 nM TPA. Similar results were obtained with an established, but non-tumorigenic, line of murine melanocytes. The same compounds exerted a potent anti-proliferative effect against transformed melanocyte lines of murine and human origin associated with morphological alterations and an increase in melanin production consistent with induced cytodifferentiation.