Improved methods of analysis for betaines in <Emphasis Type="Italic">Ascophyllum nodosum</Emphasis> and its commercial seaweed extracts

Beneficial effects of seaweeds and their extracts on crop performance have been attributed to a variety of compounds, including the betaines which are quaternary ammonium betaines. Methods of analysis of betaines published thus far suffer from low sensitivity, lack of baseline separation of individual betaines and from interference from other sample constituents. A rapid cleanup protocol and a sensitive LC-MS/MS method of analysis were developed to afford baseline separation of four betaines in the brown alga Ascophyllum nodosum and its commercial seaweed extract. Using this method, the presence of glycine betaine, δ-aminovaleric acid betaine, γ-aminobutyric acid betaine and laminine in A. nodosum, and commercial extracts derived from A. nodosum, were confirmed and quantified. The major betaine present was γ-aminobutyric acid betaine accounting for 0.008–0.014% of the dry weight of the seaweed and 0.014–0.027% of the dry weight of the commercial extracts. Seasonal variation in betaine content was observed. Differences in the total betaine content were observed between A. nodosum of the yellow (0.011–0.017% dry weight) and the olive green (0.017–0.021% dry weight) coloured morphologies.     

Osmoprotective activity, urea protection, and accumulation of hydrophilic betaines in Escherichia coli and Staphylococcus aureus

The hydrophilic betaines, deanol betaine, triethanol betaine, diethanolthetin and methylethanolthetin, and also thioxanium betaine and citrulline betaine, were accumulated by Escherichia coli. All betaines tested had significant osmoprotective activity for E. coli and, with the exception of citrulline betaine and diethanolthetin, also demonstrated urea protection. Staphylococcus aureus accumulated only methylethanolthetin, deanol betaine and thioxanium betaine: the first two had an osmoprotective effect but conferred no urea protection. Diethanolthetin and thioxanium betaine significantly decreased urea tolerance for S. aureus.     

Betaine distribution in Angiosperms

Over 140 Angiosperm species, included in 45 of the 62 orders listed by Engler, have been investigated for the presence of betaines, which were detected in 86% of the species examined and in 43 of the orders. Moreover, betaines were reported earlier in a further seven of the orders. Thus, it can be concluded that betaines are very widely distributed in Angiosperms. The most commonly detected betaines in the study were glycinebetaine and trigonelline, although others, such as prolinebetaine, trans-4-hydroxyprolinebetaine and pipecolatebetaine were found, although with a very restricted distribution. In the large majority of species tested, betaine levels were low (below 0.1%, dry weight).     

Relationship between osmoprotection and the structure and intracellular accumulation of betaines by Escherichia coli

Abstract Naturally occuring betaines, especially glycine betaine and proline betaine, were accumulated by Escherichia coli from urine. In synthetic hyperosmotic medium, with an homologous series of added betaines, (CH₃)₃N⁺-(CH₂) _n -COO⁻, osmoprotective activity and intracellular accumulation decreased monotonically as n increased from 1 to 5. In contrast, α -substituted glycine betaines were accumulated in a similar manner to glycine betaine, but with different osmoprotective activities. Arsenobetaine, with a quaternary arsonium group, was also accumulated but amino acids which can become negatively charged in a chemically basic environment were not.     

Enhanced leaf chlorophyll levels in plants treated with seaweed extract

Application to the soil of an aqueous alkaline extract ofAscophyllum nodosum resulted in higher concentrations of chlorophyll in the leaves of treated plants in comparison to control plants treated with an equivalent volume of water. Positive results were obtained with all species tested (tomato, dwarf French bean, wheat, barley, maize). When the seaweed extract was applied as a foliar spray, similar effects on leaf chlorophyll contents were obtained, except in the case of dwarf French bean plants, for which no significant difference was recorded between test and control plants. When the betaines present in the seaweed extract were applied as a mixture in the same concentrations as those in the diluted seaweed extract (-aminobutyric acid betaine 0.96 mg L^–1, -aminovaleric acid betaine 0.43 mg L^–1, glycinebetaine 0.34 mg L^–1), very similar leaf chlorophyll levels were recorded for the seaweed extract and betaine treated plants. This suggests strongly that the enhanced leaf chlorophyll content of plants treated with seaweed extract is dependent on the betaines present.     

Betaine distribution in the Malvaceae

Aerial parts of 26 taxa, distributed in 18 genera and all 5 tribes of the Malvaceae have been examined for the presence of betaines. Glycinebetaine was obtained in high yield (0.5-4.6%, dry weight) from all the plants studied, except Abelmoschus moschatus, in extracts of which glycinebetaine was not detected. Trigonelline was recorded for 16 of the plants tested, but the yields were low (0.005-0.07%, dry weight). Roots and flowers of a few of the species were also examined for betaines. The same compounds as those found in the aerial parts were usually detected, but the glycinebetaine contents of the roots and flowers were considerably lower.     

Significance of betaines in the increased chlorophyll content of plants treated with seaweed extract

Seaweed extract, prepared by alkaline extraction of Ascophyllum nodosum (L.) Le Jol., applied either to the soil or to the foliage of tomato plants, produced leaves with higher chlorophyll levels than those of control plants. The effects on leaf chlorophyll content were investigated using a cucumber bioassay procedure devised for cytokinins. The seaweed extract was shown to increase the chlorophyll levels of the cucumber cotyledons, but ‘peaks’ of activity were obtained when widely different concentrations were used. The possibility that these effects were the result of betaines present in the extract was considered. Glycinebetaine, γ-aminobutyric acid betaine and δ-aminovaleric acid betaine all produced significantly enhanced chlorophyll concentrations in the cotyledons. ‘Peaks’ of activity were observed for each betaine: for glycinebetaine at 10⁻⁶ and between 10⁻⁴ and 10¹ mg 1⁻¹, for γ-aminobutyric acid betaine at 10⁻⁶, between 10⁻⁴ and 10⁻¹, and 10¹ mg 1⁻¹, and for δ-aminovaleric acid betaine between 10⁻⁵ and 10¹ mg 1⁻¹. It was concluded that the effects of enhancing chlorophyll levels produced by the seaweed extract were due, at least in part, to betaines.     

Retention of enzyme activity following freeze-drying the mycelium of ectomycorrhizal isolates: part II. Enzymes acting upon carbon compounds

Freeze-dried ectomycorrhizal fungus cultures were tested for qualitative enzymatic activity and compared with that of the non-lyophilized culture. Enzymes involved in the utilization of starch, cellulose, lipid, lignin and urea were tested for their qualitative presence/activity. Expression of amylase and urease was stronger than that of lipase and lignin-degrading activity for the isolates tested. Variation among the species of Laccaria was low and prominently seen only for cellulase and urease. Amanita muscaria showed significant variation relative to other members of the Agaricales reported in the present study, except for β-glucosidase activity. All the enzymatic tests showed an unequivocal uniformity between the lyophilized vegetative mycelium (L) and the respective non-lyophilized mycelial cultures (NL), indicating that the lyophilization procedure maintained stable enzyme activity. This revised version was published online in November 2006 with corrections to the Cover Date.     

Antifungal activity of ovotransferrin towards genus Candida

The inhibiting activity of ovotransferrin was tested towards different species belonging to genus Candida.Of one hundred strains tested, only C. krusei showed a noticeable resistance, while the other species appeared to be more sensitive than bacteria to the action of ovotransferrin. The influence of anions, such as bicarbonate and citrate, on the inhibiting activity of ovotransferrin was also investigated. Moreover it was observed that iron saturated ovotransferrin retained its activity, thus suggesting an interaction between the protein and Candida cells.     

4株羊瘤胃功能性细菌的筛选

通过DNS法测定羊瘤胃源功能性细菌产生的纤维素酶和淀粉酶的活力,福林酚法测定产生的蛋白酶的活力,检测细菌产生酶的特性。同时检测菌株的发酵液对大肠埃希菌(ATCC25922)、副溶血弧菌(ATCC17802)、藤黄八叠球菌(HY78)和产气杆菌(AS1489)等指示菌的抑制能力,分析它们的抑菌活性。结果表明,羊瘤胃源细菌C13产生的纤维素酶活力最高,产酶量也最高;而细菌C5产淀粉酶活力和蛋白酶活力最高,产生淀粉酶和蛋白酶的能力也最高。抑菌活性检测发现,细菌C9对副溶血弧菌(ATCC17802)有很高的抑制作用,而细菌C12对大肠埃希菌(ATCC25922)的抑制能力最明显。     

Molecular characterization of the cai operon necessary for carnitine metabolism in Escherichia coii

The sequence encompassing the cai genes of Escherichia coli, which encode the carnitine pathway, has been determined. Apart from the already identified caiB gene coding for the carnitine dehydratase, five additional open reading frames were identified. They belong to the caiTABCDE operon, which was shown to be located at the first minute on the chromosome and transcribed during anaerobic growth in the presence of carnitine. The activity of carnitine dehydratase was dependent on the CRP regulatory protein and strongly enhanced in the absence of a functional H-NS protein, in relation to the consensus sequences detected in the promoter region of the cai operon. In vivo expression studies led to the synthesis of five polypeptides in addition to CaiB, with predicted molecular masses of 56 613 Da (CaiT), 42 564 Da (CaiA), 59311 Da (CaiC), 32 329 Da (CaiD) and 21 930 Da (CaiE). Amino acid sequence similarity or enzymatic analysis supported the function assigned to each protein. CaiT was suggested to be the transport system for carnitine or betaines, CaiA an oxidoreduction enzyme, and CaiC a crotonobetaine/carnitine CoA ligase. CaiD bears strong homology with enoyl hydratases/isomerases. Overproduction of CaiE was shown to stimulate the carnitine racemase activity of the CaiD protein and to markedly increase the basal level of carnitine dehydratase activity. It is inferred that CaiE is an enzyme involved in the synthesis or the activation of the still unknown cofactor required for carnitine dehydratase and carnitine racemase activities. Taken together, these data suggest that the carnitine pathway in E. coli resembles that found in a strain situated between Agrobacterium and Rhizobium.     

Inhibitory Effects of Some Spice and Herb Extracts Against Arcobacter butzleri, A. cryaerophilus, and A. skirrowii

Seventeen spice and medicinal plant extracts (methanol and chloroform) were assayed for their antimicrobial activity against Arcobacter butzleri, A. cryaerophilus, and A. skirrowii. In general, all of the tested extracts were able, to a different extent, to inhibit the growth of the selected Arcobacter species. Cinnamon, bearberry, chamomile, sage and rosemary extracts showed strong antimicrobial activity toward arcobacter strains tested. Overall, the methanol extracts showed better activity than the chloroform extracts (P < 0.05); however, enhanced antibacterial activity of chloroform extracts of cinnamon and rosemary has been observed in comparison with their methanol counterparts. The inhibitory dose of the most active extracts (the diameter of zone of inhibition ≥ 20 mm) was determined using the disc-diffusion method as well.     

Inhibitory effect of the essential oil from <Emphasis Type="Italic">Chamaecyparis obtusa</Emphasis> on the growth of food-borne pathogens

In this study, the antibacterial activity of essential oil from Chamaecyparis obtusa (Sieb. et Zucc) leaves and twigs was investigated. The test strains were Klebsiella pneumoniae, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Escherichia coli, Legionella pneumophila, and Methicilline-resistant Staphylococcus aureus. Antibacterial activity was estimated by measuring bacterial growth inhibition. Histopathological examination was also performed. C. obtusa oil distinctly inhibited the growth of all test strains and exhibited the strongest antibacterial activity against L. monocytogenes. It was chromatographically divided into several fractions. The fractions were further tested against antibacterial activity and their chemical compositions were analyzed. The fraction containing terpinen-4-ol (TA) showed high antibacterial activity toward all strains tested. Tests with authentic samples showed that TA played a major role in the antibacterial activity of C. obtusa oil, and in a mice test, the oil actively minimized inflammation by S. aureus.     

Biological activity of roots and aerial parts of Zinnia peruviana on pathogenic micro-organisms in planktonic state and biofilm forming

Microbial resistance to antibiotics affects the control of clinical infections and is a growing concern in global public health. One important mechanism whereby micro-organisms acquire resistance is biofilm formation. This context has led to the investigation of new antimicrobial substances from plants popularly used in folk medicine. In this work, we studied the antimicrobial and antibiofilm activity of Zinnia peruviana roots, ziniolide (major root metabolite) and aerial parts against Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. The minimum inhibitory and minimum microbicidal concentration and inhibition of biofilm production was determined. All Z. peruviana extracts showed antimicrobial activity, but that corresponding to the roots was the most active one. The best inhibitory and microbicidal activity was detected against Gram-positive bacteria (0·039–0·078 mg ml⁻¹). The acetonic extract from Z. peruviana leaves showed moderate activity against Gram-positive bacteria (0·625 mg ml⁻¹). Acetonic extract of Z. peruviana flowers showed weak activity (1·25–5 mg ml⁻¹). All the extracts tested showed inhibition of biofilm formation, as well as the ziniolide, however, roots and flowers extracts showed higher antibiofilm activity particularly against Staphylococcus, Listeria and Candida. The extracts tested may be a promising natural alternative for the control of microbial infections.     

Reconfirmation of antimicrobial activity in the coelomic fluid of the earthwormEisenia fetida andrei by colorimetric assay

A novel tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) was used in the assessment of antimicrobial activity in earthworm in the presence of phenazine methosulphate (PMS) as an electron coupling reagent. This activity was purified from the coelomic fluid of the earthworm (ECF),Eisenia fetida andrei (Oligochaeta, Lumbricidae, annelids) using a series of column chromatography techniques and was tested against three Gram-negative strains ofEscherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and three Gram-positive strains ofStaphylococcus aureus, Bacillus megaterium, Arthrobacter sp., respectively. Only the pigment-free eluate of coelomic fluid of the earthworm (ECFPE) showed activity againstB. megaterium amongst three isolated active fractions. The anion (DEAE-52) exchange effluent of the ECFPE was reported to have the strongest activity againstP. aeruginosa amongst the three active fractions. The 20% acetonitrile eluate (AE) by Sep-Pak C₁₈ cartridge was also tested and showed fair resistance againstE. coli, P. aeruginosa andArthrobacter sp., respectively.     

Cephalosporin acetylesterase activity of Fusaria

Summary Cephalosporin acetylesterase activity was detected in a number of fusaria tested. Of these Fusarium oxysporum AY F-298 was found to be the most active. Deacetyl derivatives of cephalothin and phenoxymethyl cephalosporin prepared by hydrolysis of the parent compounds with AY F-298 showed low antimicrobial activity.     

Biological activity of new aza analogues of quinolones

A series of novel derivatives of 4H-pyrido[1,2-a]pyrimidine, 1,4-dihydro-4-oxo-1,5-naphthyridine and 1,4-dihydro-4-oxo-1,6-naphthyridine were prepared and their biological activity was compared with that of nalidixic acid. Thein vitro antibacterial activity of the tested compounds was lower than that of nalidixic acid except for two agents,1b and2c, with a higher activity againstEnterococcus faecalis. The compounds were tested for their ability to cure four plasmids from two species ofEnterobacteriaceae. The derivatives eliminated three plasmids (pKM101, pBR322, F'lac) at one-half or one-quarter of the minimal inhibitory concentration. Plasmid RP4 was unaffected by the treatment. None of these compounds showed better antichloroplast activity than nalidixic acid.     

Abundant betaines in reef-building corals and ecological indicators of a photoprotective role

Betaines are well known as compatible solutes that exert protein- and membrane-stabilizing effects, including protective effects on photosynthesis in plants and free-living algae stressed by high irradiance or unusual temperatures. Betaines, however, have received minimal attention in reef-building corals. One goal of this research was to identify and quantify the betaines of reef-building corals with chemically definitive methods. Metabolite profiling was conducted on 10 species (6 genera) of Curaçao corals by liquid chromatography/time-of-flight mass spectrometry calibrated using six stable-isotope-labeled internal standards. Glycine betaine (GlyB), proline betaine (ProB), alanine betaine (AlaB), β-alanine betaine, hydroxyproline betaine (HProB), taurine betaine (TauB), trigonelline (Trig) and the chemically related sulfonium compound dimethylsulfoniopropionate were found in all species. Relative levels of betaines varied across species, with GlyB and ProB being most prominent. Betaines were collectively abundant; estimated total concentrations were 12 to 204 (mean = 75) mmol per liter of tissue. A second goal was to examine ecological patterns in betaine concentrations in field populations of Curaçao corals. Betaine concentrations exhibited intraspecific patterns that matched a priori predictions for molecules that defend photosynthesis against negative effects of high irradiance. In Madracis mirabilis—which occupies unshaded locations—GlyB, ProB, AlaB, HProB, and Trig were 37–94% more abundant in colonies at 5 m depth (high irradiance) than 20 m. In M. pharensis—which occupies exposed and shaded locations—GlyB, ProB, and AlaB were 30–44% more abundant in unshaded than shaded colonies at one depth. M. senaria exhibited 45–93% increases in concentrations of betaines—GlyB, AlaB, HProB, TauB, and Trig—between early and late in the day, although M. mirabilis and pharensis did not. The results indicate that multiple betaines occur commonly in reef-building coral species, and betaine concentrations are modulated in response to growth light conditions in ways consistent with betaines acting as agents of photoprotection of coral photosynthesis.     

Antimicrobial activity of LFchimera synthetic peptide against plant pathogenic bacteria

The present study was designed to evaluate potential antibacterial activities of synthetic LFchimera against five plant pathogenic bacteria such as Ralstonia solanacearum, Erwinia amylovora, Xanthomonas campestris, Pseudomonas syringae and Pectobacterium carotovorum. The agar disc-diffusion method with different concentrations (0.2, 0.4, 0.6 and 0.8 μM) of peptide was used to study the antibacterial activity of LFchimera against bacteria. The Minimum Inhibitory Concentration (MIC) of the LFchimera peptide were tested using serial dilution method at concentration ranging from 0 to 10 μM. The Results from agar disc-diffusion method revealed that LFchimera was effective against all bacterial strain in a dose-dependent manner. LFchimera showed highest activity in 0.8 μM which was significant compared to the standard antibiotic. LFchimera pepetide showed low MIC values (4 μM) against all tested bacteria. LFchimera peptide was found to show antibacterial activity against important phytopathogenic bacteria and can improve the potential of an antimicrobial peptide in plant disease management.     

Chemical Composition and Antibacterial Activity of Essential Oils from Different Parts of Litsea cubeba

The composition of the essential oils obtained by hydrodistillation of different parts of Litsea cubeba, including roots, stems, leaves, alabastra (flower buds), flowers, and fruits, were investigated by GC (RI) and GC/MS. The antimicrobial activity of the oils was assessed with disc diffusion and microbroth dilution assays. The results showed large variations in the composition among the different oils. The major components in the oils from roots and fruits, from stems, leaves, and alabastra, and from flowers were citral B (neral), β‐phellandrene, and β‐terpinene, respectively. The inhibition zone (DD) and MIC values for the bacterial strains tested, which were all sensitive to the essential oil of L. cubeba, were in the range of 10.1–35.0 mm and 100–1000 μg/ml, respectively. Hence, the oils of the various parts showed moderate activity against the tested bacteria. This investigation showed that the antibacterial activity of L. cubeba was attributed to the essential oils, thus they can be a potential medicinal resource.