The influence of casein and urea as nitrogen sources on in vitro equine caecal fermentation.

To access the fermentative response of equine caecal microbial population to nitrogen availability, an in vitro study was conducted using caecal contents provided with adequate energy sources and nitrogen as limiting nutrient. Two nitrogen (N) sources were provided, protein (casein) and non-protein (urea). Caecal fluid, taken from three cannulated horses receiving a hay-concentrate diet, was mixed with a N-free buffer-mineral solution. The influence of four N levels (3.7, 6.3, 12.5 or 25 mg of N in casein or urea) was studied using the gas production technique. Total volatile fatty acids (VFA), NH3-N and gas production were measured after a 24-h incubation period. Microbial biomass was estimated using adenine and guanine bases as internal markers, and ATP production was estimated stoichiometrically. Microbial growth efficiency (YATP) and gas efficiency (Egas) were estimated. Fermentation with casein as the sole N source was generally characterized by lower total VFA, NH3-N, total gas production and higher acetate : propionate (A : P) ratio and YATP than with urea. Results herein presented indicate that, under these in vitro conditions, caecal microbial population does in fact use urea N, but less efficiently than casein in terms of microbial growth.

Initial in vitro evaluations of the antibacterial activities of glucosinolate enzymatic hydrolysis products against plant pathogenic bacteria.

AIMS: The aim of the study was to evaluate the in vitro antibacterial effects of glucosinolate hydrolysis products (GHP) against plant pathogenic micro-organisms namely Agrobacterium tumefaciens, Erwinia chrysanthemi, Pseudomonas cichorii, Pseudomonas tomato, Xanthomonas campestris and Xanthomonas juglandis. METHODS AND RESULTS: Using a disc diffusion assay, seven different doses of 10 GHP were tested against each bacteria. The results showed that the isothiocyanates were potent antibacterials, whilst the other GHP were much less efficient. Moreover, the antibacterial effects were dose-dependent, increasing with the dose applied; 2-phenylethylisothiocyanate and sulforaphane showed the strongest inhibitory effects. The overall results show a great potential for using the isothiocyanates as an alternative tool to control undesired bacterial growth in plants. CONCLUSIONS: Glucosinolate hydrolysis products and more specifically the isothiocyanates: benzylisothiocyanate, 2-phenylethylisothiocyanate, the isothiocyanate Mix and sulforaphane, were effective phytochemicals against the in vitro growth of the phytopathogenic bacteria. The antibacterial activity exhibited by these phytochemicals reinforces their potential as alternatives to the traditional chemical control of phytopathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This current in vitro study is the first providing comparative data on GHP as potential control agents for plant pathogenic bacteria. However, more studies are needed to determine their possible allelopathic impacts e.g. inhibition of plant growth and negative effects on beneficial soil bacteria and fungi (mycorrhizae).

The antimicrobial effects of glucosinolates and their respective enzymatic hydrolysis products on bacteria isolated from the human intestinal tract.

AIMS: The aim of the study was to evaluate the in vitro antibacterial activity of glucosinolates and their enzymatic hydrolysis product against bacteria isolated from the human intestinal tract. METHODS AND RESULTS: Using a disc diffusion bioassay, different doses of intact glucosinolates and their corresponding hydrolysis products were tested. There were clear structure-activity and concentration differences with respect to the in vitro growth inhibition effects as well as differences in the sensitivities of the individual bacteria. The most effective glucosinolate hydrolysis products were the isothiocyanates; sulforaphane and benzyl isothiocyanate were the best inhibitors of growth. Indole-3-carbinol had some inhibitory effects against the Gram-positive bacteria but had no effect, even at the highest dose, against the Gram-negative bacteria. Indole-3-acetonitrile had some inhibitory activity against the Gram-negative bacteria. Glucosinolates, nitriles and amines were ineffective at all the doses used. CONCLUSIONS: Glucosinolate hydrolysis products and specifically the isothiocyanates SFN and BITC have significant antimicrobial activity against Gram-positive and Gram-negative bacteria, and might be useful in controlling human pathogens through the diet. SIGNIFICANCE AND IMPACT OF THE STUDY: This the first major in vitro study demonstrating the potential of these natural dietary chemicals as an alternative to, or in combination with, current antibiotic-based therapies for treating infectious diseases.

Antimicrobial activity of flavonoids extracted from bergamot (Citrus bergamia Risso) peel, a byproduct of the essential oil industry.

AIMS: To evaluate the antimicrobial properties of flavonoid-rich fractions derived from bergamot peel, a byproduct from the Citrus fruit processing industry and the influence of enzymatic deglycosylation on their activity against different bacteria and yeast. METHODS AND RESULTS: Bergamot ethanolic fractions were tested against Gram-negative bacteria (Escherichia coli, Pseudomonas putida, Salmonella enterica), Gram-positive bacteria (Listeria innocua, Bacillus subtilis, Staphylococcus aureus, Lactococcus lactis) and the yeast Saccharomyces cerevisiae. Bergamot fractions were found to be active against all the Gram-negative bacteria tested, and their antimicrobial potency increased after enzymatic deglycosylation. The minimum inhibitory concentrations of the fractions and the pure flavonoids, neohesperidin, hesperetin (aglycone), neoeriocitrin, eriodictyol (aglycone), naringin and naringenin (aglycone), were found to be in the range 200 to 800 microg ml(-1). The interactions between three bergamot flavonoids were also evaluated. CONCLUSION: The enzyme preparation Pectinase 62L efficiently converted common glycosides into their aglycones from bergamot extracts, and this deglycosylation increased the antimicrobial potency of Citrus flavonoids. Pairwise combinations of eriodictyol, naringenin and hesperetin showed both synergistic and indifferent interactions that were dependent on the test indicator organism. SIGNIFICANCE AND IMPACT OF THE STUDY: Bergamot peel is a potential source of natural antimicrobials that are active against Gram-negative bacteria.

In vitro evaluation of the prebiotic activity of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel.

The prebiotic effect of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel was studied using pure and mixed cultures of human faecal bacteria. This was compared to the prebiotic effect of fructo-oligosaccharides (FOS). Individual species of bifidobacteria and lactobacilli responded positively to the addition of the bergamot extract, which contained oligosaccharides in the range of three to seven. Fermentation studies were also carried out in controlled pH batch mixed human faecal cultures and changes in gut bacterial groups were monitored over 24 h by fluorescent in situ hybridisation, a culture-independent microbial assessment. Addition of the bergamot oligosaccharides (BOS) resulted in a high increase in the number of bifidobacteria and lactobacilli, whereas the clostridial population decreased. A prebiotic index (PI) was calculated for both FOS and BOS after 10 and 24 h incubation. Generally, higher PI scores were obtained after 10 h incubation, with BOS showing a greater value (6.90) than FOS (6.12).

The tu8 mutation of Arabidopsis thaliana encoding a heterochromatin protein 1 homolog causes defects in the induction of secondary metabolite biosynthesis.

The tu8 mutant of Arabidopsis thaliana (L.) Heynh. was previously described as deficient in pathogen-induced auxin and glucosinolate (GSL) accumulation, as well as in heat-induced accumulation of cytosolic Hsp90, the latter feature was accom"panied by reduced thermotolerance at higher temperatures. The mutated gene was shown to be a novel allele of TERMINAL FLOWER2, encoding the only Arabidopsis homolog for heterochromatin protein 1 (Kim et al., 2004). In this report, we investigated the influence of heat stress on auxin and GSL content, as well as the accumulation of several secondary metabolites derived from the phenylpropanoid pathway, including anthocyanins and sinapine derivatives, in the mutant tu8. tu8 had less sinapine and sinapoyl esters compared to the wild type. In addition, the induction of sinapine by heat shock in Columbia was not found in tu8. Anthocyanins were also induced by heat stress in wild type plants, whereas tu8 showed only slight induction of these compounds and only at higher temperatures. GSLs were induced at higher temperatures in the wild type, but induction was absent in tu8. Transcript levels known to be involved in IAA/glucosinolate synthesis and metabolism (nitrilase and myrosinase) were examined and both showed developmental regulation, while only nitrilase mRNA levels differed between wild type and mutant seedlings. Treatment of Columbia and tu8 with jasmonic acid (JA), a known inducer of glucosinolates, showed differences between wild type and tu8 with respect to induction of individual GSLs and anthocyanins. However, the transcript level of the TU8/TFL2 gene after heat shock and jasmonate treatment did not change. Loss of function or altered function in the heterochromatin protein most likely lead to the pleiotropic phenotype observed for the tu8 mutant.

Absorption of kaempferol from endive, a source of kaempferol-3-glucuronide, in humans.

OBJECTIVE: To determine the absorption, excretion and metabolism of kaempferol in humans. DESIGN: A pharmacokinetic study of kaempferol from endive over 24 h. SUBJECTS: Four healthy males and four healthy females. RESULTS: Kaempferol, from a relatively low dose (9 mg), was absorbed from endive with a mean maximum plasma concentration of 0.1 microM, at a time of 5.8 h, indicating absorption from the distal section of the small intestine and/or the colon. Although a 7.5-fold interindividual variation between the highest and lowest maximum plasma concentration was observed, most individuals showed remarkably consistent pharmacokinetic profiles. This contrasts with profiles for other flavonoids that are absorbed predominantly from the large intestine (eg rutin). An average of 1.9% of the kaempferol dose was excreted in 24 h. Most subjects also showed an early absorption peak, probably corresponding to kaempferol-3-glucoside, present at a level of 14% in the endive. Kaempferol-3-glucuronide was the major compound detected in plasma and urine. Quercetin was not detected in plasma or urine indicating a lack of phase I hydroxylation of kaempferol. CONCLUSIONS: Kaempferol is absorbed more efficiently than quercetin in humans even at low oral doses. The predominant form in plasma is a 3-glucuronide conjugate, and interindividual variation in absorption and excretion is low, suggesting that urinary kaempferol could be used as a biomarker for exposure.

High-performance liquid chromatographic separation of natural and synthetic desulphoglucosinolates and their chemical validation by UV, NMR and chemical ionisation-MS methods.

Methods are described for the optimised extraction, desulphation and HPLC separation of desulphoglucosinolates. These methods provide rapid separation, identification and quantitative measurements of glucosinolates extracted from Brassica napus L and related crops, of unusual glucosinolates found in crucifer weed species, and also of synthetic alkylglucosinolates. The desulphoglucosinolates used in these studies were either chemically synthesised (at least one example from each major structural class), or purified from various plant sources. Validation of the identities of the desulphoglucosinolates was by comparison of retention times with standards, and by UV, 1H- and 13C-NMR and chemical ionisation MS analysis. A list of useful species, and the specific tissues, from which high concentrations of standards can be extracted is included.

Induction of Systemic Resistance to Albugo candida in Brassica juncea by Pre- or Coinoculation with an Incompatible Isolate.

ABSTRACT In an investigation of the interaction between two isolates of Albugo candida that were compatible (CO) and incompatible (IN) on a Brassica juncea accession, the IN isolate induced both local and systemic protection of cotyledons and true leaves against the CO isolate. The extent of the protection was proportional to the zoosporangia concentration used in the inducing (IN) inoculation. Protection was greatest locally on cotyledons and least on true leaves (the most remote tissue from the point of the inducing inoculation). Protection occurred when the two isolates were inoculated together but was greater when the interval between the IN and CO isolate inoculations was longer. The IN isolate induced only slight protection when it was inoculated after the CO isolate. No induced susceptibility to the IN isolate occurred with any treatment. There was some evidence of competition between CO and IN zoospores for infection sites (stomata). The occurrence of systemic protection and changes detected in phenylalanine ammonia lyase and total soluble peroxidase activities in inoculated cotyledons, particularly after the inducing (IN) inoculation, suggested that host-mediated factors also may be involved in the interaction between the two isolates.

Involvement of Cytochrome P450 in Glucosinolate Biosynthesis in White Mustard (A Biochemical Anomaly).

One of the first steps in glucosinolate biosynthesis is the conversion of amino acids to their aldoximes. The biochemistry of this process is controversial, and several very different enzyme systems have been described. The major glucosinolate in white mustard (Sinapis alba) is sinalbin, which is derived from tyrosine via its aldoxime, and this conversion is catalyzed by a cytochrome P450 (Cyt P450) monooxygenase. Phenylethyl- and alkenylglucosinolates are also present in white mustard leaves, as are the enzymes catalyzing the relevant aldoxime formation from homophenylalanine and methionine homologs, respectively. These enzymes are similar to those found in Brassica sp. and are distinct from the tyrosine-dependent enzyme in that they contain no heme and are unaffected by Cyt P450 inhibitors. They are instead inhibited by the flavoprotein inhibitor diphenylene iodonium and by Cu2+. In both white mustard and oilseed rape (Brassica napus) methyl jasmonate specifically stimulates indolylglucosinolate biosynthesis and yet has no effect on sinalbin accumulation in either cotyledons or leaves of white mustard. White mustard appears to be unique among crucifers in having a Cyt P450 aldoxime-forming enzyme for biosynthesis of one glucosinolate, although it also contains all of the non-Cyt P450 enzyme systems found in other members of the family. Sinalbin biosynthesis in white mustard is therefore an inappropriate model system for the synthesis of other glucosinolates in crucifers, including canola and oilseed rape.

Glucosinolate Biosynthesis (Further Characterization of the Aldoxime-Forming Microsomal Monooxygenases in Oilseed Rape Leaves).

The initial steps in glucosinolate biosynthesis are thought to proceed from amino acids, via N-hydroxy amino acids, to aldoximes. We showed previously that microsomes from green leaves of oilseed rape (Brassica napus cv Bienvenu) contain two distinct monooxygenases that catalyze the conversion of homophenylalanine and dihomomethionine to their respective aldoximes. Further characterization of these enzymes has now demonstrated that the latter enzyme catalyzes the NADPH-dependent oxidative decarboxylation of two higher homologs of methionine, in addition to dihomomethionine. No activity was found for either enzyme with L-methionine, DL-homomethionine, L-phenylalanine, L-tyrosine, or L-tryptophan. Both of these rape monooxygenase activities are dependent on O2, not requiring any other O2 species or radical. The presence of an unoxidized sulfur atom and its relative position in the side chain of the aliphatic substrates are important for binding to the active site of the methionine-homolog enzyme. Neither enzyme has any characteristics of a cytochrome P450-type enzyme, and antiserum raised against cytochrome P450 reductase did not significantly inhibit monooxygenase activity.

Synthesis of glucosinolate precursors and investigations into the biosynthesis of phenylalkyl- and methylthioalkylglucosinolates.

The alkenyl and aromatic glucosinolates in oilseed rape (Brassica napus) are biosynthesized from chain-extended homologues of protein amino acids, including methionine and phenylalanine. Homologues of these two amino acids, homophenylalanine (2-amino-4-phenylbutyric acid) and dihomomethionine (2-amino-6-methylthiohexanoic acid) were synthesized both with and without a 1-14C label. Microsomal preparations from oilseed rape leaves were shown to contain enzyme systems which metabolize these compounds, with loss of 14CO2, and produce the aldoxime intermediates possible in the biosynthetic pathway utilizing homophenylalanine. These were characterized by comparison with authenticated synthetic compounds. Potential intermediates on the pathway between homophenylalanine and its corresponding aldoxime, the N-hydroxyamino- and the oximino acids, were synthesized and their possible role in the pathway investigated.

Phenylethylamine and piperidine alkaloids in aloe species.

Of the approximately 300 species of ALOE (Liliaceae) native to Africa and Arabia, leaf extracts of 224 species have been examined chromatographically for alkaloids using ninhydrin, Dragendorff's reagent, nitroprusside, and iodoplatinate as revealing agents. From these, 48 (21%) species contained compounds giving a strong colorimetric reaction with at least one of these reagents. Tyramine derivatives were identified in 18 species and piperidine derivatives in a further 6 species. The other coloured zones remain unidentified but often appeared to represent common amino acids. Some taxonomic correlations are suggested. The presence of the toxic hemlock alkaloids in aloes readily available for potential medicinal use by local inhabitants sounds a note of caution against the unthinking use of these otherwise useful plants.