Abstract  We attempted to integrate lipase-catalyzed ethanolysis into fermentative bioethanol production. To produce bioethanol, ethanol fermentation from brown rice was conducted using a tetraploid Saccharomyces cerevisiae expressing α-amylase and glucoamylase. The resultant ethanol was distilled and separated into three fractions with different concentrations of water and fusel alcohols. In ethanolysis using the first fraction with 89.3% ethanol, a recombinant Aspergillus oryzae whole-cell biocatalyst expressing Fusarium heterosporum lipase (r-FHL) afforded the highest ethyl ester content of 94.0% after 96h. Owing to a high concentration of water in the bioethanol solutions, r-FHL, which works best in the presence of water when processing ethanolysis, was found to be more suitable for the integrative process than a commercial immobilized Candida antarctica lipase. In addition, r-FHL was used for repeated-batch ethanolysis, resulting in an ethyl ester content of more than 80% even after the fifth batch. Fusel alcohols such as 1-butanol and isobutyl alcohol are thought to decrease the lipase activity of r-FHL. Using this process, a high ethyl ester content was obtained by simply mixing bioethanol, plant oil, and lipase with an appropriate adjustment of water concentration. The developed process model, therefore, would contribute to biodiesel production from only biomass-derived feedstocks.

Abstract  A Resin-linker-vector (RLV) strategy is described for the radiosynthesis of tracer molecules containing the radionuclide (18) F, which releases the labelled vector into solution upon nucleophilic substitution of a polystyrene-bound arylsulfonate linker with [(18) F]-fluoride ion. Three model linker-vector molecules 7 a-c containing different alkyl spacer groups were assembled in solution from (4-chlorosulfonylphenyl)alkanoate esters, exploiting a lipase-catalysed chemoselective carboxylic ester hydrolysis in the presence of the sulfonate ester as a key step. The linker-vector systems were attached to aminomethyl polystyrene resin through amide bond formation to give RLVs 8 a-c with acetate, butyrate and hexanoate spacers, which were characterised by using magic-angle spinning (MAS) NMR spectroscopy. On fluoridolysis, the RLVs 8 a, b containing the longer spacers were shown to be more effective in the release of the fluorinated model vector (4-fluorobutyl)phenylcarbamic acid tert-butyl ester (9) in NMR kinetic studies and gave superior radiochemical yields (RCY≈60 %) of the (18) F-labelled vector. The approach was applied to the synthesis of the radiopharmaceutical O-(2-[(18) F]-fluoroethyl)-L-tyrosine ([(18) F]-FET), delivering protected [(18) F]-FET in >90 % RCY. Acid deprotection gave [(18) F]-FET in an overall RCY of 41 % from the RLV.

Abstract  Stress induces long-lasting changes in neuronal gene expression and cholinergic neurotransmission, but the underlying mechanism(s) are incompletely understood. Here, we report that chromatin structure and histone modifications are causally involved in this transcriptional memory. Specifically, the AChE gene encoding the acetylcholine-hydrolyzing enzyme acetylcholinesterase is known to undergo long-lasting transcriptional and alternative splicing changes after stress. In mice subjected to stress, we identified two alternative 5' exons that were down-regulated after stress in the hippocampus, accompanied by reduced acetylation and elevated trimethylation of H3K9 at the corresponding promoter. These effects were reversed completely by daily administration of the histone deacetylase (HDAC) inhibitor sodium butyrate for 1 wk after stress. H3K9 hypoacetylation was associated with a selective, sodium butyrate-reversible promoter accumulation of HDAC4. Hippocampal suppression of HDAC4 in vivo completely abolished the long-lasting AChE-related and behavioral stress effects. Our findings demonstrate long-lasting stress-inducible changes in AChE's promoter choices, identify the chromatin changes that support this long-term transcriptional memory, and reveal HDAC4 as a mediator of these effects in the hippocampus.

Abstract  To compare the fermentation characteristics of d-lactate, l-lactate and dl-lactate mixture by fecal microbiota of pigs, in vitro fermentation test was conducted with d-lactate, l-lactate and dl-lactate mixture as substrates, feces of piglets as inocula. The concentrations of lactate and short chain fatty acid (SCFA) were quantified during the fermentation. The composition of bacterial communities in the inocula and 24 h fermentation fluids was analyzed by pyrosequencing. The results showed that, when 5 mmol/l lactate was used, there was no significant difference in utilizing efficiency among d-lactate, l-lactate and dl-lactate mixture, propionate and butyrate were the major end-products converted from lactate. However, when 25 mmol/l lactates were used, a higher utilizing efficiency of dl-lactate mixture and a slower utilizing rate of d-lactate were observed, acetate and propionate became the main end-products. The SCFA proportions were significantly affected by lactate source and its concentration. Pyrosequencing analysis showed that after 24 h fermentation, significant difference in the composition of bacterial communities (genus and OTU levels) was observed among different substrate groups. The results suggest that lactate accumulation in the hindgut is related to the cooperation between microbiota members, and regulating bacterial community may be a possible way to control lactate accumulation.

Abstract  This study was conducted to evaluate the effects of 2 different daily doses of short-chain fructooligosaccharides (scFOS), a prebiotic ingredient, added to a calf milk replacer on growth performance, carcass characteristics, and fecal concentrations of short-chain fatty acids of preruminant veal calves. In total, 112 male Prim'Holstein calves, between 8 and 10d of age, were randomized in this study according to their body weight and were bred until the age of 168 d. They were fed a calf milk replacer containing 5% soluble wheat proteins as well as cereal-based pellets, the composition of which was adapted to cover the needs of the animals throughout the study. After 2wk of adaptation, the calf milk replacer was supplemented or not supplemented with a daily dose of 3 or 6g of scFOS. Growth performance of calves, as measured by body weight, cold carcass weight, feed intake, average daily gain, and feed conversion ratio, was recorded and feces samples were taken to evaluate short-chain fatty acid concentrations. The inclusion of wheat proteins in milk replacer did not negatively affect the growth performance of calves in comparison with general standards. The addition of scFOS in the milk reduced the feed conversion ratio of veal calves in a dose-dependent manner and tended to increase the carcass weight. A general trend was observed for an increased production of total short-chain fatty acids in time, but scFOS decreased acetate proportion to the benefit of butyrate proportion. These data suggest that inclusion of scFOS in the calf milk replacer allowed the growth performance of preruminant calves to be enhanced, possibly via a modification of the activities of microbial fermentation.

Abstract  A new method of phase transfer hollow fiber liquid phase microextraction (PT-HF-LPME) combined with electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICP-MS) has been developed for the determination of trace Co, Pd, Cd and Bi in environmental and biological samples. In PT-HF-LPME, an intermediate solvent (1-butanol) was added into the sample solution to ensure the maximum contact area between the target metal ions and the chelating reagent (8-hydroxyquinoline, 8-HQ), which accelerated the formation of 8-HQ-metal complexes and their subsequent extraction by extraction solvent (toluene). The experimental parameters affecting the extraction efficiency of PT-HF-LPME for the target metals were studied by simplex optimization and orthogonal array design (OAD) experiments. Under the optimized conditions, the enrichment factors for Co, Pd, Cd and Bi were 110, 393, 121 and 111-fold, respectively, the limits of detection (LODs, 3σ) ranged from 3.7 to 8.3 ng L(-1). The relative standard deviations (RSDs, c=0.5 ng mL(-1), n=7) were 8.7, 6.2, 12.4 and 12.9% for Co, Pd, Cd and Bi, respectively. To validate the accuracy of the proposed method, two Certified Reference Materials of GSBZ50009-88 Environment Water and GBW09103 Human Urine were analyzed, and the results obtained for Cd were in good agreement with the certified values. Finally, the developed method was successfully applied to the analysis of Co, Pd, Cd and Bi in lake water and human urine samples.

Abstract  Abstract Integration-defective lentiviral vectors (IDLVs) are being increasingly deployed in both basic and preclinical gene transfer settings. Often, however, the IDLV transgene expression profile is muted when compared to that of their integration-proficient counterparts. We hypothesized that the episomal nature of IDLVs turns them into preferential targets for epigenetic silencing involving chromatin-remodeling histone deacetylation. Therefore, vectors carrying an array of cis-acting elements and transcriptional unit components were assembled with the aid of packaging constructs encoding either the wild-type or the class I mutant D116N integrase moieties. The transduction levels and transgene-product yields provided by each vector class were assessed in the presence and absence of the histone deacetylase (HDAC) inhibitors sodium butyrate and trichostatin A. To investigate the role of the target cell replication status, we performed experiments in growth-arrested human mesenchymal stem cells and in post-mitotic syncytial myotubes. We found that IDLVs are acutely affected by HDACs regardless of their genetic makeup or target cell replication rate. Interestingly, the magnitude of IDLV transgene expression rescue due to HDAC inhibition varied in a vector backbone- and cell type-dependent manner. Finally, investigation of histone modifications by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) revealed a paucity of euchromatin marks distributed along IDLV genomes when compared to those measured on isogenic integration-competent vector templates. These findings support the view that IDLVs constitute preferential targets for epigenetic silencing involving histone deacetylation, which contributes to dampening their full transcriptional potential. Our data provide leads on how to most optimally titrate and deploy these promising episomal gene delivery vehicles.

Abstract  Neuroadaptations associated with behavioral sensitization induced by repeated exposure to methamphetamine (MA) appear to be involved in compulsive drug pursuit and use. Increased histone acetylation, an epigenetic effect resulting in altered gene expression, may promote sensitized responses to psychostimulants. The role of histone acetylation in the expression and acquisition of MA-induced locomotor sensitization was examined by measuring the effect of histone deacetylase inhibition by sodium butyrate (NaB). For the effect on expression, mice were treated repeatedly with MA (10 days of 2mg/kg MA) or saline (10 days), and then vehicle or NaB (630 mg/kg, intraperitoneally) was administered 30 min prior to MA challenge and locomotor response was measured. NaB treatment increased the locomotor response to MA in both acutely MA treated and sensitized animals. For acquisition, NaB was administered 30 min prior to each MA exposure (10 days of 1 or 2mg/kg), but not prior to the MA challenge test. Treatment with NaB during the sensitization acquisition period significantly increased locomotor activation by MA in sensitized mice only. NaB alone did not significantly alter locomotor activity. Acute NaB or MA, but not the combination, increased striatal acetylation at histone H4. Repeated treatment with MA, but not NaB or MA plus NaB, increased striatal acetylation at histone H3. Although increased histone acetylation may alter the expression of genes involved in acute locomotor response to MA and in the acquisition of MA-induced sensitization, results for acetylation at H3 and H4 showed little correspondence with behavior.

Abstract  ETHNOPHARMACOLOGICAL RELEVANCE: Local communities in Malaysia consume Pereskia bleo Kunth. (Cactaceae) leaves as raw vegetables or as a concoction and drink as a tea to treat diabetes, hypertension, rheumatism, cancer-related diseases, inflammation, gastric pain, ulcers, and for revitalizing the body.

AIM OF THE STUDY: To evaluate anti-nociceptive activity of the extracts and vitexin, isolated for the first time in this species, in two analgesic models; formalin-induced licking and acetic acid-induced abdominal writhing.

MATERIALS AND METHODS: Three and a half kilos of P. bleo leaves were extracted using Soxhlet apparatus with ethanol for 72 h. The crude ethanol extract was treated with activated charcoal overnight and subjected to a liquid-liquid partition yielding hexane, dichloromethane, ethyl acetate and butanol extracts. All extracts, including the crude ethanol and vitexin isolated from the ethyl acetate partition were tested for peripheral anti-nociceptive activity using formalin test and acetic acid-induced abdominal writhing, besides having their acute toxicity assays performed.

RESULTS: The phytochemical analyses resulted in the isolation of vitexin (1), β-sitosterol glucoside (2) and β-sitosterol (3) isolated from the ethyl acetate, dichloromethane and hexane extracts, respectively. This is the first time vitexin and β-sitosterol glucoside are isolated from this species. The anti-nociceptive activities for all extracts were only moderate. Vitexin, which was isolated from the ethyl acetate extract did not show any activity in all models tested when used alone at the same concentration as it appears in the extract.

CONCLUSION: This study showed that all the extracts possess moderate anti-nociceptive activity. Vitexin is not the compound responsible for the anti-nociceptive effect in the ethyl acetate extract. Further investigations are needed to identify the compound(s) that might be responsible for the anti-nociceptive activity in this plant.

Abstract  Hwangryun-haedok-tang (HRT) is the common recipe in traditional Asian medicine, and microbial fermentation is used for the conventional methods for processing traditional medicine. We investigated the inhibitory effect of the n-butanol fraction of HRT (HRT-BU) and fHRT (fHRT-BU) on the RANKL-induced osteoclastogenesis in bone-marrow-derived macrophages. mRNA expression of osteoclastogenesis-related genes were evaluated by real-time QPCR. The activation of signaling pathways was determined by western blot analysis. The marker compounds of HRT-BU and fHRT-BU were analyzed by HPLC. The inhibitory effect of HRT or fHRT on ovariectomy-induced bone loss were evaluated using OVX rats with orally administered HRT, fHRT (300, 1000 mg/kg), or its vehicle for 12 weeks. fHRT-BU significantly inhibited RANKL-induced osteoclastogenesis, and phosphorylation of p38, IKKα/β, and NF-κBp65 compared to HRT-BU. In addition, fHRT-BU also significantly inhibited the mRNA expression of Nfκb2, TNF-α, NFATc1, TRAP, ATPv0d2, and cathepsin K. Furthermore, administration of fHRT had a greater effect on the increase of BMD, and greater improved bone microstructure of the femora than that of HRT in ovariectomy rats. This study demonstrated that bacterial fermentation enhances the inhibitory effect of HRT on osteoclastogenesis and bone loss. These results suggest that fermented HRT might have the beneficial effects on bone disease by inhibiting osteoclastogenesis.

Abstract  The larvicidal activity of crude petroleum ether, toluene, n-butanol, ethyl acetate, acetone, and methanol extracts of the seeds of Clausena lansium was assayed for their toxicities against the early fourth instar larvae of Aedes albopictus. The larval mortality was observed after 24-h exposure. The LC(50) value of petroleum ether extract was 22.99 ppm, showing the best larvicidal activity among all six solvent extracts. A cinnamon amide compound lansiumamide B (N-methyl-N-cis-styrylcinnamamide) was isolated from the petroleum ether extract by column chromatographic method, which exhibited a strong larvicidal activity against the early fourth instar larvae of A. albopictus with LC(50) and LC(90) values of 0.45 and 2.19 ppm, respectively. The structure was elucidated by (1)H NMR, (13)C NMR spectral data. The larvicidal activity against mosquito of lansiumamide B from the seed of C. lansium was evaluated for the first time.

Abstract  The present study describes the phytochemical investigations of the crude extracts of rhizomes and leaves of Geranium wallichianum. The crude extracts were fractionated to obtain n-hexane, ethyl acetate, and n-butanol fractions, which were subjected to different biological activities and enzyme inhibition assays to explore the therapeutic potential of this medicinally important herb. The results indicated that the crude extracts and different fractions of rhizomes and leaves showed varied degree of antimicrobial activities and enzyme inhibitions in different assays. Overall, the rhizome extract and its different fractions showed comparatively better activities in various assays. Furthermore, the purified constituents from the repeated chromatographic separations were also subjected to enzyme inhibition studies against three different enzymes. The results of these studies showed that lipoxygenase enzyme was significantly inhibited as compared to urease. In case of chemical constituents, the sterols (2-4) showed no inhibition, while ursolic acid (1) and benzoic ester (6) showed significant inhibition of urease enzymes.

Abstract  AIMS: The objective of this study was to comprehensively evaluate quillaja (QSP) and yucca saponin (YSP) products with respect to their effects on diversity of rumen bacteria and archaea, abundance of selected microbes, and feed degradability and fermentation.

METHODS AND RESULTS: Both QSP and YSP at doses 0-0.6 g l(-1) tended to increase degradability of feed substrate in in vitro rumen cultures, but to different extents. Neither one of the saponins affected the concentrations of ammonia, total volatile fatty acids, or molar proportion of acetate. However, QSP increased molar proportion of propionate and decreased that of butyrate, whereas YSP tended to decrease that of butyrate. As determined by qPCR, QSP and YSP did not affect the abundance of total bacteria or Ruminococcus albus. The QSP did not affect the abundances of Fibrobacter succinogenes or genus Prevotella, but tended to decrease that of Ruminococcus flavefaciens, whereas YSP significantly increased the abundance of R. flavefaciens and Prevotella, and numerically increased that of F. succinogenes. Both saponins increased archaeal abundance, although to small magnitudes (0.3-0.4 log). The protozoal populations were decreased significantly by QSP, but not by YSP. Based on DGGE and T-RFLP analysis, both saponins altered the bacterial community and species organization, but less so the archaeal community.

CONCLUSIONS: This study demonstrated that saponins, although not effective in mitigating methane emission, may improve feed utilization at low doses, and modulate ruminal microbial communities in a dose-dependent manner.

SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study suggest that saponins at low doses may directly stimulate the growth of some rumen bacteria including cellulolytic bacteria, thus improving digestibility of feeds, independent of their defaunation activity. In contrast, saponins at high doses modulate rumen fermentation characteristically similar to defaunation.

Abstract  ETHNOPHARMACOLOGICAL RELEVANCE: The dried flowers of Lonicera japonica Thunb and dried roots of Anemarrhena asphodeloides BUNGE have been used for the treatment of a variety of inflammatory diseases in traditional Korean medicine.

OBJECTIVE: The aim of the study is to evaluate the anti-inflammatory effects of WIN-34B, a new herbal medicine, in fibroblast-like synoviocytes (FLS) obtained from patients with osteoarthritis (OA).

MATERIALS AND METHODS: WIN-34B is isolated from the n-butanol fraction of dried flowers of L. japonica and dried roots of A. asphodeloides. The anti-inflammatory effects of WIN-34B on cell viability, the production and release of inflammatory mediators, matrix metalloproteinases (MMPs), aggrecanases, tissue inhibitor of matrix proteinases (TIMP) is compared with celecoxib in IL-1β-stimulated human OA FLS. Furthermore, the effect of WIN-34B on inhibitory kappa B-α (IκB-α) phosphorylation and mitogen-activated protein kinases (MAPK) in the IL-1β-stimulated OA FLS was also evaluated.

RESULTS: WIN-34B significantly inhibited the IL-1β-induced cell viability in human OA FLS without cytotoxicity. Compared to celecoxib, WIN-34B exhibited similar or better anti-inflammatory effects through significant suppression of inflammatory mediators (IL-1β, TNF-α, PGE2 and NO), MMPs (MMP-1, MMP-3 and MMP-13) and aggrecanases (ADAMTS-4 and ADAMTS-5), and enhancement of TIMPs (TIMP-1 and TIMP-3). Moreover, WIN-34B reduced the phosphorylation of IκB-α, ERK1/2, p38 and JNK1/2 in IL-1β-stimulated OA FLS.

CONCLUSIONS: WIN-34B exhibited similar or better anti-inflammatory properties in IL-1β-stimulated OA FLS compared to celecoxib. The anti-inflammatory effects of WIN-34B are due to inhibition of inflammatory mediators (IL-1β, TNF-α, PGE2 and NO) and regulation of MMPs, ADAMTSs and TIMPs via the inhibition of IκB-α and MAPK phosphorylation in IL-1β-stimulated OA FLS.

Abstract  The present study evaluated the potential of affecting amino acid metabolism through intestinal fermentation in domestic cats, using dietary guar gum as a model. Apparent protein digestibility, plasma fermentation metabolites, faecal fermentation end products and fermentation kinetics (exhaled breath hydrogen concentrations) were evaluated. Ten cats were randomly assigned to either guar gum- or cellulose-supplemented diets, that were fed in two periods of 5 weeks in a crossover design. No treatment effect was seen on fermentation kinetics. The apparent protein digestibility (P = 0·07) tended to be lower in guar gum-supplemented cats. As a consequence of impaired small-intestinal protein digestion and amino acid absorption, fermentation of these molecules in the large intestine was stimulated. Amino acid fermentation has been shown to produce high concentrations of acetic and butyric acids. Therefore, no treatment effect on faecal propionic acid or plasma propionylcarnitine was observed in the present study. The ratio of faecal butyric acid:total SCFA tended to be higher in guar gum-supplemented cats (P = 0·05). The majority of large-intestinal butyric acid is absorbed by colonocytes and metabolised to 3-hydroxy-butyrylcoenzyme A, which is then absorbed into the bloodstream. This metabolite was analysed in plasma as 3-hydroxy-butyrylcarnitine, which was higher (P = 0·02) in guar gum-supplemented cats. In all probability, the high viscosity of the guar gum supplement was responsible for the impaired protein digestion and amino acid absorption. Further research is warranted to investigate whether partially hydrolysed guar gum is useful to potentiate the desirable in vivo effects of this fibre supplement.

Abstract  BACKGROUND: Steaming and roasting treatments are widely used enzyme deactivation methods in the oat food industry in China. Whether or not the enzyme deactivation treatments affect the nutritional function of oat foods is unknown. In the current study, we examined the effects of 4-week ingestion of steamed or roasted oat foods on the intestinal bacteria and short-chain fatty acids of rats.

RESULTS: Compared with rats taking no oat foods, rats taking normal oat foods or enzyme-deactivated oat foods showed significantly higher (P < 0.05) counts of Lactobacillus spp. and Bifidobacterium spp. in colon, significantly lower (P < 0.05) counts of Enterococcus spp. and coliforms in colon, and significantly higher (P < 0.05) levels of butyrate and acetate in colonic digesta. In addition, rats taking infrared roasting (IR)-treated oat foods also demonstrated significantly higher (P < 0.05) fecal Lactobacillus spp. counts and significantly lower (P < 0.05) cecal and fecal counts of E. coli, Enterococcus spp. and coliforms than rats taking no oat foods. As for the comparison between the enzyme-undeactivated oat group and the three enzyme-deactivated oat groups, there were no significant differences in most of the parameters (P > 0.05), though a few exceptions did exist.

CONCLUSION: Enzyme deactivation treatments did not decrease the beneficial role of oat food in the intestinal microbes and short-chain fatty acids of rats. © 2012 Society of Chemical Industry.

Abstract  Anaerobic ammonium-oxidizing bacteria were recently shown to use short-chain organic acids as additional energy source. The AMP-forming acetyl-CoA synthetase gene (acs) of Kuenenia stuttgartiensis, encoding an important enzyme involved in the conversion of these organic acids, was identified and heterologously expressed in Escherichia coli to investigate the activation of several substrates, that is, acetate, propionate and butyrate. The heterologously expressed ACS enzyme could complement an E. coli triple mutant deficient in all pathways of acetate activation. Activity was observed toward several short-chain organic acids, but was highest with acetate. These properties are in line with a mixotrophic growth of anammox bacteria. In addition to acs, the genome of K. stuttgartiensis contained the essential genes of an acetyl-CoA synthase/CO dehydrogenase complex and genes putatively encoding two isoenzymes of archaeal-like ADP-forming acetyl-CoA synthetase underlining the importance of acetyl-CoA as intermediate in the carbon assimilation metabolism of anammox bacteria.

Abstract  The World Anti-Doping Agency (WADA) has recently added desmopressin, a synthetic analogue of the endogenous peptide hormone arginine vasopressin, to the Prohibited List, owing to the potential masking effects of this drug on hematic parameters useful to detect blood doping. A qualitative method for detection of desmopressin in human urine by high-performance liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) has been developed and validated. Desmopressin purification from urine was achieved by means of delipidation with a 60:40 di-isopropyl ether/n-butanol and solid-phase extraction with WCX cartridges. The lower limit of detection was 25 pg/mL. Extraction recovery was determined as 59.3% (SD 29.4), and signal reduction owing to ion suppression was estimated to be 42.7% (SD 12.9). The applicability of the method was proven by the analysis of real urine samples obtained after intravenous, oral and intranasal administration of desmopressin, achieving unambiguous detection of the peptide in all the cases. Copyright © 2012 John Wiley & Sons, Ltd.

Abstract  1,4-Disubstituted bis-chromenyl triazole hybrids 5a-m have been synthesized in a three-step reaction sequence from 4-(bromomethyl)-2H-chromen-2-ones 3a-m. The intermediate azides 4a-m underwent a regioselective 1,3-dipolar cycloaddition with a 2H-chromen-2-one linked acetylenic dipolarophile in the presence of Cu (II)/ascorbate/water/n-butanol reaction medium. Three compounds 5h-j exhibited 6.25 μg/mL MIC against M. tuberculosis. Among the compounds screened for antifungal activity, lowest MIC of 6.25 μg/mL was observed for 5c against A. niger that also exhibited DNA cleavage observed by agarose gel electrophoresis. All the compounds were moderately active against both Gram-positive and Gram-negative bacterial strains. The cytotoxic effect of potent compounds on normal cells (V79 and HBL100) was assessed by MTT assay.

Abstract  INTRODUCTION: Phytochemical analyses of weeds, many of which have been used in traditional medicine worldwide, could lead to the identification of secondary metabolites with significant biological activity.

OBJECTIVE: To perform an assessment of the chemical composition and exploitation potential of the invasive weed Oxalis pes-caprae. To evaluate the anti-oxidant activity of its extracts and isolate and characterise polyphenolic metabolites using LC-DAD-MS (ESI+) and NMR methods.

METHODOLOGY: Aerial parts of the invasive weed O. pes-caprae were extracted with solvents of increasing polarity and their major polyphenolic metabolites were identified by LC-DAD-MS (ESI+). The total phenolic content of the extracts was determined using the Folin-Ciocalteu method, while their anti-oxidant activity was evaluated on the basis of their ability to scavenge the stable free radical 1,1-diphenyl-2-picrylhydrazyl and hydrogen peroxide.

RESULTS: The major polyphenolic constituents of the extracts were tentatively characterised as chlorogenic acid, quinic ferulate, luteolin glucoside and cernuoside according to their MS and UV spectroscopic data. Cernuoside, an aureusidin glucoside, was isolated from the methanolic extract of the weed's flowers and its structure was unambiguously identified by 1D- and 2D-NMR spectroscopy. The butanol extract of O. pes-caprae displayed the highest anti-oxidant activity.

CONCLUSION: The metabolic profile of O. pes-caprae was studied and the structures of the major polyphenolic metabolites based on their MS and UV-vis spectra were tentatively assigned. The aureusidin glucoside cernuoside was isolated and characterised for the first time from O. pes-caprae. The extracts exhibited high levels of anti-oxidant activity.

Abstract  This work aimed to study the response of the growing rabbit caecal ecosystem (bacterial community and caecal environmental parameters) after a switch from a control to a low-fibre diet (LFD). A group of 160 rabbits were fed ad libitum a control diet (ADF: 20.4%) from weaning (36 days). At 49 days of age (day 0), 75 rabbits were switched to a LFD group (ADF: 10.7%), whereas 85 others (control group) remained on the control diet, for 39 days. Caecal contents were regularly sampled throughout the trial (60 rabbits per group). The bacterial community structure was characterized using CE-SSCP (capillary electrophoresis single strand conformation polymorphism) and total bacteria were quantified using real-time PCR. Redox potential (Eh), pH, NH(3)-N, volatile fatty acid (VFA) were measured in the caecum to characterize environmental parameters. The reduction of fibre in the diet modified the CE-SSCP profiles (P < 0.001) but not the diversity index (5.6 ± 0.8, ns). The number of 16S rRNA gene copies of total bacteria decreased (P < 0.01) in LFD rabbits compared with controls. In LFD rabbits, the caecal environment was less acid (+0.2 units; P < 0.01), more reductive (-11 mV; P < 0.05) and drier (+3.4 g 100 per g; P < 0.001), with an increase in NH3-N (+77%; P < 0.001) and a decrease in total VFA concentration (-17%; P < 0.001). We found significant correlations between the bacterial community, the quantity of bacteria and the caecal traits of the caecal ecosystem. Indeed, in both groups, the caecal traits barely constrained the total inertia of the CE-SSCP profile set (less than 14%), whereas total bacteria were positively related to total VFA, acetic acid and butyric acid levels, and Eh, and negatively related to pH. All the microbial and environmental modifications had occurred by day 2 and remained stable thereafter. These results suggest that the bacterial community in the growing rabbit caecum is able to adapt quickly after a change to in the dietary fibre supply to reach a new steady-state equilibrium.

Abstract  At weaning (33 days of age), 246 hybrid rabbits (782 ± 53 g live weight) were divided into six experimental groups and fed ad libitum six iso-ADF diets formulated according to a bifactorial arrangement with two protein levels (152 and 162 g/kg) and three soluble fibre-to-starch ratios (0.2, 0.6 and 1.5), the latter obtained by replacing starch (from 209 to 91 g/kg) with soluble fibre (from 48 to 136 g/kg). The trial lasted for 42 days until slaughter. The rabbits that were fed the diet with the highest protein level and the lowest soluble fibre-to-starch ratio showed the highest mortality rate (17.1% v. 1.7% on average; P < 0.001) and sanitary risk (mortality + morbidity: 20.0% v. 8.1%; P = 0.04) compared with the rabbits fed the other diets. With increasing dietary crude protein level, the digestibility of dry matter (DM; 0.615 to 0.626) and gross energy (0.620 to 0.630) as well as aNDF (without sodium sulphite; 0.298 to 0.323) and hemicelluloses (0.417 to 0.461) significantly (0.001 < P < 0.10) improved. Moreover, total volatile fatty acids (VFAs) in the caecal content increased (59.0 to 68.4 mmol/l; P = 0.01) and ileum crypt depth tended to reduce (P = 0.07). Neither growth performance nor slaughter results were affected by the protein level. When increasing soluble fibre-to-starch ratio, the digestibility of DM and gross energy did not change, whereas the digestibility of aNDF (0.264 to 0.352), ADF (0.167 to 0.267) and hemicelluloses (0.400 to 0.470) linearly increased (P < 0.001). At caecum, N-ammonia tended to decrease linearly (P = 0.08), total VFA concentration (56.0 to 67.3 mmol/l) and acetate proportion (80.4 to 83.3 mmol/100 mmol VFA) linearly increased (P < 0.01), whereas butyrate and valerate proportions decreased (0.01 < P < 0.05). Growth performance was similar among groups, whereas at slaughter the proportion of the gastrointestinal tract linearly increased (177 to 184 g/kg; P < 0.01) without effect on dressing percentage, however. As soluble fibre-to-starch ratio increased, meat pH linearly decreased and lightness (L*), redness (a*) and yellowness (b*) colour indexes increased (0.01 < P < 0.05).

Abstract  Combined with medium-pressure liquid chromatography (MPLC) and preparative high-performance liquid chromatography (perp-HPLC), high-speed countercurrent chromatography (HSCCC) was applied for separation and purification of flavone C-glycosides from the crude extract of leaves of Ficus microcarpae L. f. HSCCC separation was performed on a two-phase solvent system composed of methyl tert- butyl ether - ethyl acetate - 1-butanol - acetonitrile - 0.1% aqueous trifluoroacetic acid at a volume ratio of 1:3:1:1:5. Partially resolved peak fractions from HSCCC separation were further purified by preparative HPLC. Four well-separated compounds were obtained and their purities were determined by HPLC. The purities of these peaks were 97.28%, 97.20%, 92.23%, and 98.40%.. These peaks were characterized by ESI-MS(n). According to the reference, they were identified as orientin (peak I), isovitexin-3″-O-glucopyranoside (peak II), isovitexin (peak III), and vitexin (peak IV), yielded 1.2 mg, 4.5 mg, 3.3 mg, and 1.8 mg, respectively.

Abstract  The interfacial partitioning behavior of ampicillin and phenylglycine crystals in different two-phase systems has been investigated. The two-phase systems employed are water/dodecane, water/1-butanol, and water/pentane/methanol. By means of partition experiments and microscopic imaging, it has been shown that the mechanism of separation strongly depends on the choice of the two-phase system. While water/dodecane features a mechanism of sheer competitive adsorption at the interface, separation in water/1-butanol is mainly due to partitioning into both liquid phases, leading to a higher degree of separation. Experiments with water/pentane/methanol have illustrated the large potential of three-component systems, as slight variations in the composition can have large effects on the separation.

Abstract  The present study is to investigate which kinds of solvent extracts of Inulae Flos inhibit the chemokine productions in HaCaT cell and whether the inhibitory capacity of Inulae Flos is related with constitutional compounds. The 70% methanol extract showed comparatively higher inhibition of thymus and activation-regulated chemokine (TARC/CCL17) in HaCaT cells, therefore this extract was further partitioned with n-hexane, chloroform, ethyl acetate, butanol, and water. The ethyl acetate fraction inhibited TARC, macrophage-derived chemokine (MDC/CCL22), and regulated on activation of normal T-cell-expressed and -secreted (RANTES/CCL5) production in HaCaT cells better than the other fractions. The compounds of Inulae Flos, such as 1,5-dicaffeoylquinic acid and luteolin, inhibited TARC, MDC, and RANTES production in HaCaT cells. 1,5-Dicaffeoylquinic acid was contained at the highest concentrations both in the 70% methanol extract and ethyl acetate fraction and inhibited the secretion of chemokines dose-dependently more than the other compounds. Luteolin also represented dose-dependent inhibition on chemokine productions although it was contained at lower levels in 70% methanol extract and solvent fractions. These results suggest that the inhibitory effects of Inulae Flos on chemokine production in HaCaT cell could be related with constituent compounds contained, especially 1,5-dicaffeoylquinic acid and luteolin.