Isolation and partial characterisation of acid phosphatase isozymes from dormant oilseed of Corylus avellana L.

The acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) complement from dormant hazel (Corylus avellana L.) seeds was found to exhibit significant electrophoretic heterogeneity partially attributable to the presence of distinct molecular forms. In axiferous tissue, total acid phosphatase activity increased in a biphasic fashion during chilling, a treatment necessary to alleviate seed dormancy. Three acid phosphatase isozymes were isolated from cotyledons of dormant hazel seeds by successive ammonium sulphate precipitation, size-exclusion, Concanavalin A affinity, cation- and anion-exchange chromatographies resulting in 75-, 389- and 191-fold purification (APase1, APase2, APase3, respectively). The three glycosylated isoforms were isolated to catalytic homogeneity as determined by electrophoretic, kinetic and heat-inactivation studies. The native acid phosphatase complement of hazel seeds had an apparent Mr of 81.5±3.5 kDa as estimated by size-exclusion chromatography, while the determined pI values were 5.1 (APase1), 6.9 (APase2) and 7.3 (APase3). The optimum pH for p-nitrophenyl phosphate hydrolysis was pH 3 (APase1), pH 5.6 (APase2) and pH 6 (APase3). The hazel isozymes hydrolysed a variety of phosphorylated substrates in a non-specific manner, exhibiting low Km and the highest specificity constant (Vmax/Km) for pyrophosphate. They were not primary phytases since they could not initiate phytic acid hydrolysis, while APase2 and APase3 had significant phospho-tyrosine phosphatase activity. Inorganic phosphate was a competitive inhibitor, while activity was significantly impaired in the presence of vanadate and fluoride.

Isolation and characterisation of phytase from dormant Corylus avellana seeds

Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26), which catalyses the step-wise hydrolysis of phytic acid, was purified from cotyledons of dormant Corylus avellana L. seeds. The enzyme was separated from the major soluble acid phosphatase by successive (NH4)2SO4 precipitation, gel filtration and cation exchange chromatography resulting in a 300-fold purification and yield of 7.5%. The native enzyme positively interacted with Concanavalin A suggesting that it is putatively glycosylated. After size exclusion chromatography and SDS–PAGE it was found to be a monomeric protein with molecular mass 72±2.5 kDa. The hazel enzyme exhibited optimum activity for phytic acid hydrolysis at pH 5 and, like other phytases, had broad substrate specificity. It exhibited the lowest Km (162 μM) and highest specificity constant (Vmax/Km) for phytic acid, indicating that this is the preferred in vivo substrate. It required no metal ion as a co-factor, while inorganic phosphate and fluoride competitively inhibited enzymic activity (Ki=407 μM and Ki=205 μM, respectively).

New bis(triazinyl) pyridines for selective extraction of americium(III)

New hydrophobic, tridentate nitrogen heterocyclic reagents (BATPs) such as 2,6-bis(5,5,8,8-tetramethyl-5,6,7,8-tetrahydrobenzo[1,2,4]triazin-3-yl) pyridine (1) and 2,6-bis(9,9,10,10-tetramethyl-9,10-dihydro-1,2,4-triaza-anthrane-3-yl) pyridine (2) have been studied. I is resistant to hydrolysis in 3 M nitric acid, whereas 2 is resistant to both acid hydrolysis and radiolysis. The molecules are able to give significantly enhanced separations of americium(III) from an excess of europium(III) in nitric acid. Typically, for 1 D-Am = 500 and SFAm,/Eu = 5000 compared with D-Am = 30 and SFAm /Eu = 400 with the reference molecule 2,6-bis(isopropyl[1,2,4]triazin-3-yl) pyridine (7). In order to increase the stability of 1 and 2, the labile alpha-benzylic hydrogens that are present in 7 have been replaced by alkyl groups. Three molecules of 1 are able to enclose completely the coordination sphere of the M(III) in the crystal structure of [Y(1)(3)][Y(NO3)(5)]center dot NO3 center dot 2.5H(2)O.

Extraction of polyphenols from processed black currant (Ribes nigrum L.) residues

The total phenol and anthocyanin contents of black currant pomace and black currant press residue (BPR) extracts, extracted with formic acid in methanol or with methanol/water/acetic acid, were studied. Anthocyanins and other phenols were identified by means of reversed phase HPLC, and differences between the two plant materials were monitored. In all BPR extracts, phenol levels, determined by the Folin-Ciocalteu method, were 8-9 times higher than in the pomace extracts. Acid hydrolysis liberated a much higher concentration of phenols from the pomace than from the black currant press residue. HPLC analysis revealed that delphinidin-3-O-glucoside, delphinidin-3-O-rutinoside, cyanidin-3-O-glucoside, and cyanidin-3-O-rutinoside were the major anthocyanins and constituted the main phenol class (approximate to 90%) in both types of black currant tissues tested. However, anthocyanins were present in considerably lower amounts in the pomace than in the BPR. In accordance with the total phenol content, the antioxidant activity determined by scavenging of 2,2'-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) radical cation, the ABTS(center dot+) assay, showed that BPR extracts prepared by solvent extraction exhibited significantly higher (7-10 times) radical scavenging activity than the pomace extracts, and BPR anthocyanins contributed significantly (74 and 77%) to the observed high radical scavenging capacity of the corresponding extracts.

Enteric coated spheres produced by extrusion/spheronization provide effective gastric protection and efficient release of live therapeutic bacteria

We present a novel but simple enteric coated sphere formulation containing probiotic bacteria (Lactobacillus casei). Oral delivery of live bacterial cells (LBC) requires live cells to survive firstly manufacturing processes and secondly GI microbicidal defenses including gastric acid. We incorporated live L. casei directly in the granulation liquid, followed by granulation, extrusion, spheronization, drying and spray coating to produce dried live probiotic spheres. A blend of MCC, calcium-crosslinked alginate, and lactose was developed that gave improved live cell survival during manufacturing, and gave excellent protection from gastric acid plus rapid release in intestinal conditions. No significant loss of viability was observed in all steps except drying, which resulted in approximately 1 log loss of viable cells. Eudragit coating was used to protect dried live cells from acid, and microcrystalline cellulose (MCC) was combined with sodium alginate to achieve efficient sphere disintegration leading to rapid and complete bacterial cell release in intestinal conditions. Viability and release of L. casei was evaluated in vitro in simulated GI conditions. Uncoated spheres gave partial acid protection, but enteric coated spheres effectively protected dried probiotic LBC from acid for 2 h, and subsequently released all viable cells within 1h of transfer into simulated intestinal fluid.

Hydrolysis of N2O5 on submicron sulfuric acid aerosols

The kinetics of reactive uptake of gaseous N2O5 on submicron sulfuric acid aerosol particles has been investigated using a laminar flow reactor coupled with a differential mobility analyzer (DMA) to characterize the aerosol. The particles were generated by homogeneous nucleation of SO3/H2O mixtures. In the H2SO4 concentration range 26.3−64.5 wt % the uptake coefficient was γ = 0.033 ± 0.004, independent of acid strength. For an acid strength of 45 wt % γ was found to decrease with increasing temperature over the range 263−298 K. From this, temperature dependence values of −115 ± 30 kJ/mol and −25.5 ± 8.4 J/K mol were determined for the changes in enthalpy and entropy of the uptake process, respectively. The results are consistent with a previous model of N2O5 hydrolysis involving both a direct and an acid catalyzed mechanism, with uptake under the experimental conditions limited by mass accommodation.

Micellar catalysis for partial oxidation of toluene to benzoic acid in supercritical CO2: effects of fluorinated surfactants

One of the key hindrances on development of solid catalysts containing cobalt species for partial oxidation of organic molecules at mild conditions in conventional liquid phase is the severe metal leaching. The leached soluble Co species with a higher degree of freedom always out-performs those of solid supported Co species in oxidation catalysis. However, the homogeneous Co species concomitantly introduces separation problems. We have recently reponed for the first time, a new oxidation catalyst system for the oxidation of organic molecules in supercritical CO2 using the principle of micellar catalysis. [CF3(CF2)(8)COO](2)Co.xH(2)O (the fluorinated anionic moiety forms aqueous reverse micelles carrying water-soluble Co2+ cations in scCO(2)) was previously shown to be extremely active for the oxidation of toluene in the presence of sodium bromide in water-CO2 mixture, giving 98% conversion and 99% selectivity to benzoic acid at 120 degreesC. In this study, we show that the effects of varying the type of surfactant counterions and the length of the surfactant chains on catalysis. It is found that the use of [CF3(CF2)(8)COO](2)Mg.yH(2)O/Co(II) acetate is as effective as the [CF3(CF2)(8)COO](2)Co.xH(2)O and the fluorinated chain length used has a subtle effect on the catalytic rate measured. It is also demonstrated that this new type of micellar catalyst in scCO(2) can be easily separated via CO2 depressurisation and be reused without noticeable deactivation. (C) 2003 Elsevier B.V. All rights reserved.

Changes in the flavonoid and phenolic acid contents and antioxidant activity of red leaf lettuce (Lollo Rosso) due to cultivation under plastic films varying in ultraviolet transparency

Red leaf lettuce (Lollo Rosso) was grown under three types of plastic films that varied in transparency to UV radiation (designated as UV block, UV low, and UV window). Flavonoid composition was determined by high-performance liquid chromatography (HPLC), total phenolics by the Folin-Ciocalteu assay, and antioxiclant capacity by the oxygen radical absorbance capacity (ORAC) assay. Exposure to increased levels of UV radiation during cultivation caused the leaves to redden and increased concentrations of total phenols and the main flavonoids, quercetin and cyanidin glycosides, as well as luteolin conjugates and phenolic acids. The total phenol content increased from 1.6 mg of gallic acid equivalents (GAE)/g of fresh weight (FW) for lettuce grown under UV block film to 2.9 and 3.5 mg of GAE/g of FW for lettuce grown under the UV low and UV window films. The antioxiclant activity was also higher in lettuce exposed to higher levels of UV radiation with ORAC values of 25.4 and 55.1 mu mol of Trolox equivalents/g of FW for lettuce grown under the UV block and UV window films, respectively. The content of phenolic acids, quantified as caffeic acid, was also different, ranging from 6.2 to 11.1 mu mol/g of FW for lettuce cultivated under the lowest and highest UV exposure plastic films, respectively. Higher concentrations of the flavonoid glycosides were observed with increased exposure to UV radiation, as demonstrated by the concentrations of aglycones after hydrolysis, which were cyanidin (ranging from 165 to 793 mu g/g), quercetin (ranging from 196 to 880,mu g/g), and luteolin (ranging from 19 to 152 mu g/g). The results demonstrate the potential of the use of UV-transparent plastic as a means of increasing beneficial flavonoid content of red leaf lettuce when the crop is grown in polytunnels.

Changes in the flavonoid and phenolic acid contents and antioxidant activity of red leaf lettuce (Lollo Rosso) due to cultivation under plastic films varying in ultraviolet transparency

Red leaf lettuce (Lollo Rosso) was grown under three types of plastic films that varied in transparency to UV radiation (designated as UV block, UV low, and UV window). Flavonoid composition was determined by high-performance liquid chromatography (HPLC), total phenolics by the Folin-Ciocalteu assay, and antioxiclant capacity by the oxygen radical absorbance capacity (ORAC) assay. Exposure to increased levels of UV radiation during cultivation caused the leaves to redden and increased concentrations of total phenols and the main flavonoids, quercetin and cyanidin glycosides, as well as luteolin conjugates and phenolic acids. The total phenol content increased from 1.6 mg of gallic acid equivalents (GAE)/g of fresh weight (FW) for lettuce grown under UV block film to 2.9 and 3.5 mg of GAE/g of FW for lettuce grown under the UV low and UV window films. The antioxiclant activity was also higher in lettuce exposed to higher levels of UV radiation with ORAC values of 25.4 and 55.1 mu mol of Trolox equivalents/g of FW for lettuce grown under the UV block and UV window films, respectively. The content of phenolic acids, quantified as caffeic acid, was also different, ranging from 6.2 to 11.1 mu mol/g of FW for lettuce cultivated under the lowest and highest UV exposure plastic films, respectively. Higher concentrations of the flavonoid glycosides were observed with increased exposure to UV radiation, as demonstrated by the concentrations of aglycones after hydrolysis, which were cyanidin (ranging from 165 to 793 mu g/g), quercetin (ranging from 196 to 880,mu g/g), and luteolin (ranging from 19 to 152 mu g/g). The results demonstrate the potential of the use of UV-transparent plastic as a means of increasing beneficial flavonoid content of red leaf lettuce when the crop is grown in polytunnels.

Reactions of artemisinin and arteether with acid: Implications for stability and mode of antimalarial action

The currently accepted mechanism of trioxane antimalarial action involves generation of free radicals within or near susceptible sites probably arising from the production of distonic radical anions. An alternative mechanistic proposal involving the ionic scission of the peroxide group and consequent generation of a carbocation at C-4 has been suggested to account for antimalarial activity. We have investigated this latter mechanism using DFT (B3LYP/6-31+G* level) and established the preferred Lewis acid protonation sites (artemisinin O5a >> O4a approximate to O3a > O2a > O1a; arteether O4a >= O3a > O5b >> O2a > O1a; Figure 3) and the consequent decomposition pathways and hydrolysis sites. In neither molecule is protonation likely to occur on the peroxide bond O1-O2 and therefore lead to scission. Therefore, the alternative radical pathway remains the likeliest explanation for antimalarial action.

A method of synthesis of silicious inorganic ordered materials (MCM-41-SBA-1) employing polyacrylic acid-C(n)TAB-TEOS nanoassemblies

In this work we describe the synthesis of a variety of MCM-41 type hexagonal and SBA-1 type cubic mesostructures and mesoporous silicious materials employing a novel synthesis concept based on polyacrylic acid (Pac)-C(n)TAB complexes as backbones of the developing structures. The ordered porosity of the solids was established by XRD and TEM techniques. The synthesis concept makes use of Pac-C(n)TAB nanoassemblies as a preformed scaffold, formed by the gradual increase of pH. On this starting matrix the inorganic precursor species SiO2 precipitate via hydrolysis of TEOS under the influence of increasing pH. The molecular weight (MW) of Pac, as well as the length of carbon chain in C,TAB, determine the physical and structural characteristics of the obtained materials. Longer chain surfactants (C(16)TAB) lead to the formation of hexagonal phase, while shorter chain surfactants (C(14)TAB, C(12)TAB) favor the SBA-1 phase. Lower MW of Pac (approximate to2000) leads to better-organized structures compared to higher MW ( 450,000), which leads to worm-like mesostructures. Cell parameters and pore size increase with increasing polyelectrolyte and/or surfactant chain, while at the same time SEM photography reveals that the particle size decreases. Conductivity experiments provide some insight into the proposed self-assembling pathway. (C) 2003 Elsevier Inc. All rights reserved.

One-step catalytic cyclohexane oxidation to adipic acid using molecular oxygen

Using combination of Mn-Co transition metal species with N-hydroxyphthalimide as a catalyst for one-step oxidation of cyclohexane with molecular oxygen in acetic acid at 353 K can give more than 95% selectivity towards oxygenated products with adipic acid as a major product at a high conversion (ca. 78%). A turnover number of 74 for this partial oxidation are also recorded.

Extraction and determination of ellagic acid content in chestnut bark and fruit

Chestnuts are an important economic resource in the chestnut growing regions, not only for the fruit, but also for the wood. The content of ellagic acid (EA), a naturally occurring inhibitor of carcinogenesis, was determined in chestnut fruits and bark. EA was extracted with methanol and free ellagic acid was determined by HPLC with UV detection, both in the crude extract and after hydrolysis. The concentration of EA was generally increased after hydrolysis due to the presence of ellagitannins in the crude extract. The concentration varied between 0.71 and 21.6 ing g(-1) (d.w.) in un-hydrolyzed samples, and between 2.83 and 18.4 mg g(-1) (d.w.) ill hydrolyzed samples. In chestnut fruits, traces of EA were present in the seed, with higher concentrations in the pellicle and pericarp. However, all fruit tissues had lower concentrations of EA than had the bark. The concentration of EA in the hydrolyzed samples showed a non-linear correlation with the concentration in the unhydrolyzed extracts. (C) 2008 Elsevier Ltd. All rights reserved.

Biodiversity of human faecal bacteria isolated from phytic acid enriched chemostat fermenters

Background: Myo-inositol hexaphosphate (IP6) or phytic acid is found mostly in cereals and legumes and is thought to possess anti-carcinogenic properties. Aim: To isolate and identify faecal bacteria capable of phytic acid metabolism and to assess the effectiveness of prebiotics (dietary oligosaccharides, metabolised by selective colonic bacteria) in preserving the integrity of phytic acid. Methods: Faecal samples from three volunteers were used in continuous culture experiments under varying conditions of pH, substrate concentration and dilution rates, seventy three different isolates cultured at steady state were then screened for phytic acid metabolism and identified through partial sequencing of their 16S rRNA genes (16S ribosomal ribonucleic acid). Utilisation of phytic acid was also assessed in a continuous culture system enriched with prebiotic fructooligosaccharides (FOS). Results: Bacteroides spp., Clostridium spp. and facultatively anaerobic bacteria generally appeared to maintain viable counts in the presence of phytic acid. Bifidobacterium spp. and Lactobacillus spp. appeared less able to maintain viable counts in the presence of phytic acid. These results were confirmed by an increase in viable counts of Bacteroides spp., Clostridium spp. and a decrease in viable counts of Bifidobacterium spp. and Lactobacillus spp. once phytic acid was introduced to a FOS enriched continuous culture. Conclusions: The phytate metabolising biodiversity from the human large intestine does not appear to encompass major bacterial genera associated with beneficial or benign health effects (e.g. Lactobacillus spp. and Bifidobacterium spp).

Modelling the uptake and growth kinetics of Penicillium glabrum in a tannic acid-containing solid-state fermentation for tannase production

A mathematical growth model for the batch solid-state fermentation process for fungal tannase production was developed and tested experimentally. The unstructured model describes the uptake and growth kinetics of Penicillium glabrum in an impregnated polyurethane foam substrate system. In general, good agreement between the experimental data and model simulations was obtained. Biomass, tannase and spore production are described by logistic kinetics with a time delay between biomass production and tannase and spore formation. Possible induction mechanisms for the latter are proposed. Hydrolysis of tannic acid, the main carbon source in the substrate system, is reasonably well described with Michaelis-Menten kinetics with time-varying enzyme concentration but a more complex reaction mechanism is suspected. The metabolism of gallic acid, a tannase-hydrolysis product of tannic acid, was shown to be growth limiting during the main growth phase. (c) 2004 Elsevier Ltd. All rights reserved.