Characterization of silver release from commercially available functional (nano)textiles.

Silver, both in the nano as well as in other forms, is used in many applications including antimicrobial textiles. Washing of such textiles has already been identified as an important process that results in the release of silver into wastewater. This study thus investigated the release of silver from eight different commercially available silver-textiles during a washing and rinsing cycle. The silver released was size-fractionated and characterized using electron microscopy. In addition, the antimicrobial functionality of the textiles was tested before and after washing. Three of the textiles contained nanosized silver (labeled or confirmed by manufacturers' information), another used a metallic silver wire and four contained silver in undeclared form. The initial silver content of the textiles was between 1.5 and 2925mg Ag/kg. Only four of the investigated textiles leached detectable amounts of silver, of which 34-80% was in the form of particles larger than 450nm. Microscopic analysis of the particles released in the washing solutions identified Ti/Si-AgCl nanocomposites, AgCl nanoparticles, large AgCl particles, nanosilver sulfide and metallic nano-Ag, respectively. The nanoparticles were mainly found in highly agglomerated form. The identified nanotextiles showed the highest antimicrobial activity, whereas some of the other textiles, e.g. the one with a silver wire and the one with the lowest silver content, did not reduce the growth of bacteria at all. The results show that different silver textiles release different forms of silver during washing and that among the textiles investigated AgCl was the most frequently observed chemical form in the washwater.

Metabolism of the major Echinacea alkylamide N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide by human recombinant cytochrome P450 enzymes and human liver microsomes.

Echinacea preparations are used for the treatment and prevention of upper respiratory tract infections. The phytochemicals believed responsible for the immunomodulatory properties are the alkylamides found in ethanolic extracts, with one of the most abundant being the N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide (1). In this study, we evaluated the human cytochrome P450 enzymes involved in the metabolism of this alkylamide using recombinant P450s, human liver microsomes and pure synthetic compound. Epoxidation, N-dealkylation and hydroxylation products were detected, with different relative amounts produced by recombinant P450s and microsomes. The major forms showing activity toward the metabolism of 1 were CYP1A1, CYP1A2 (both producing the same epoxide and N-dealkylation product), CYP2A13 (producing two epoxides), and CYP2D6 (producing two epoxides and an hydroxylated metabolite). Several other forms showed less activity. In incubations with human liver microsomes and selective inhibitors, CYP2E1 was found to be principally responsible for producing the dominant, hydroxylation product, whereas CYP2C9 was the principal source of the epoxides and CYP1A2 was responsible for the dealkylation product. In summary, in this study the relative impacts of the main human xenobiotic-metabolizing cytochrome P450s on the metabolism of a major Echinacea alkylamide have been established and the metabolites formed have been identified.

Permeability studies of Kavalactones using a Caco-2 cell monolayer model.

OBJECTIVE: To examine the bioavailability of kavalactones in vitro and the possible differences in their bioavailability because of variations in either chemical structure or the method of extraction used. RESEARCH DESIGN AND METHODS: Caco-2 cell monolayers were used to determine the potential bioavailability of kavalactones. Kavalactones were added to the apical layer and basolateral samples were taken over 150 min to examine the concentration diffusing across the cell monolayer. Kavalactone concentrations in these samples were determined by high pressure liquid chromatography. RESULTS: Kavalactones were found to be potentially bioavailable as they all readily crossed the Caco-2 monolayers with apparent permeabilities (P(app)) increasing from 42 x 10(-6) cm/s and most exhibiting more than 70% crossing within 90 min. Not all differences in their bioavailability can be related to kavalactone structural differences as it appears that bioavailability may also be affected by co-extracted compounds. For example, the P(app) for kawain from ethanol extracts was higher than the values obtained for the same compound from water extracts or for the kavalactone alone. CONCLUSIONS: While the extraction method used (ethanol or water) influences the total (but not the relative) concentrations of kavalactones, it does not markedly affect their bioavailability. Hence, any differences between an ethanolic or an aqueous extract in terms of the propensity of kava to cause liver damage is not because of differing kavalactone bioavailabilities.

Alkylamides from echinacea modulate induced immune responses in macrophages.

The ability of Echinacea and its components to alter the immune response was examined in vitro in a macrophage cell line under either basal or immunostimulated conditions. Potential immunostimulatory and inflammatory activity was determined using a nuclear transcription factor (NFkappaB) expression, tumour necrosis factor alpha (TNFalpha) and nitric oxide (NO) production as biomarkers. In the absence of alternate stimulation, the only significant effects seen were a decrease in NFkappaB expression by a 2-ene alkylamide ((2E)-N-isobutylundeca-2-ene-8,10-diynamide (1)) and a decrease in TNFalpha levels by cichoric acid and an Echinacea alkylamide fraction (EPL AA). When the cells were stimulated by lipopolysaccharide (LPS), inhibition of the increased NFkappaB expression levels was caused by cichoric acid, an Echinacea preparation (EPL), EPL AA and a 2,4-diene ((2E,4E,8Z,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamide (2)). Increases in TNFalpha levels were inhibited by cichoric acid, EPL and EPL AA but enhanced by 1 in the presence of LPS, while only EPL AA was able to inhibit the stimulated increases in NO. When using phorbol myristate acetate to stimulate the cells, NFkappaB and NO levels were unaffected by Echinacea or its components while only cichoric acid and 2 inhibited TNFalpha levels. Although cichoric acid was found to have an effect, it is probably not an important contributor to the Echinacea modulation of the immune response in vivo, as it is not bioavailable. Echinacea appears to attenuate the response of macrophages to an immune stimulus and its combination of phytochemicals exhibits different pharmacological properties to one or more of the isolated major individual components.

Comparison of Echinacea alkylamide pharmacokinetics between liquid and tablet preparations.

The relative oral bioavailability of alkylamides from two different Echinacea dosage forms (liquid and tablet) were compared in a small two-way crossover study in humans (n=3). The liquid preparation investigated contained a mixture of Echinacea purpurea root (300 mg/ml) and Echinacea angustifolia root (200 mg/ml) extracted in 60% ethanol. The tablet preparation investigated was also a mixture of E. purpurea root (675 mg/tablet) and E. angustifolia root (600 mg/tablet), but was prepared from the dried 60% ethanolic extracts of these two Echinacea species. Alkylamides were found to be rapidly absorbed and measurable in plasma from both preparations. No significant differences in the tetraene alkylamide pharmacokinetic parameters for T(1/2), AUC(t-lin) and C(max) in the two different preparations were found. T(max) increased from 20 min for the liquid to 30 min for the tablet, which is not unexpected as the tablet required time for disintegration before absorption could occur. These results suggested that there was no significant difference in the bioavailability of alkylamides from the liquid and tablet Echinacea formulations. Furthermore, the results also indicated that the absorption site and any alkylamide loss due to digestive processes were similar in both preparations.

Stereoselective synthesis, natural occurrence and CB(2) receptor binding affinities of alkylamides from herbal medicines such as Echinacea sp.

A divergent synthesis of (2E,4E,8E,10E)- and (2E,4E,8E,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamides from pent-4-yn-1-ol allowed identification of the (2E,4E,8E,10Z)-isomer for the first time in Echinacea species. A short, stereoselective synthesis of the (2E,4E,8E,10Z)-isomer is also described which allowed further biological evaluation of this material, and the demonstration that this isomer does not occur in Spilanthes mauritiana as previously reported.

Echinacea intake induces an immune response through altered expression of leucocyte hsp70, increased white cell counts and improved erythrocyte antioxidant defences.

OBJECTIVE: To study the effect of Echinacea tablets on the expression of leucocyte heat shock protein 70 (hsp70), erythrocyte haemolysis, plasma antioxidant status, serum chemistry, haematological values and plasma alkylamide concentrations. METHOD: Eleven healthy individuals (26-61 years of age) were evaluated at baseline (day 1) and on day 15 after consuming two commercially blended Echinacea tablets daily for 14 days. RESULTS: Echinacea supplementation enhanced the fold increase in leucocyte hsp70 expression after a mild heat shock (P=0.029). White cell counts (WCC) were also increased (P=0.043). We also observed a preventative effect against free radical induced erythrocyte haemolysis (P=0.006) indicative of an antioxidant effect. CONCLUSION: The pilot study suggests that Echinacea may invoke an immune response through altered expression of hsp70 and increased WCC.

Cytochrome P450 enzyme-mediated degradation of Echinacea alkylamides in human liver microsomes.

Echinacea preparations are widely used herbal remedies for the prevention and treatment of colds. In this study we have investigated the metabolism by human liver microsomes of the alkylamide components from an Echinacea preparation as well as that of pure synthetic alkylamides. No significant degradation of alkylamides was evident in cytosolic fractions. Time- and NADPH-dependent degradation of alkylamides was observed in microsomal fractions suggesting they are metabolised by cytochrome P450 (P450) enzymes in human liver. There was a difference in the susceptibility of 2-ene and 2,4-diene pure synthetic alkylamides to microsomal degradation with (2E)-N-isobutylundeca-2-ene-8,10-diynamide (1) metabolised to only a tenth the extent of (2E,4E,8Z,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamide (3) under identical incubation conditions. Markedly less degradation of 3 was evident in the mixture of alkylamides present in an ethanolic Echinacea extract, suggesting that metabolism by liver P450s was dependent both on their chemistry and the combination present in the incubation. Co-incubation of 1 with 3 at equimolar concentrations resulted in a significant decrease in the metabolism of 3 by liver microsomes. This inhibition by 1, which has a terminal alkyne moiety, was found to be time- and concentration-dependent, and due to a mechanism-based inactivation of the P450s. Alkylamide metabolites were detected and found to be the predicted epoxidation, hydroxylation and dealkylation products. These findings suggest that Echinacea may effect the P450-mediated metabolism of other concurrently ingested pharmaceuticals.

Echinacea alkamide disposition and pharmacokinetics in humans after tablet ingestion.

Echinacea is a widely used herbal remedy for the treatment of colds and other infections. However, almost nothing is known about the disposition and pharmacokinetics of any of its components, particularly the alkamides and caffeic acid conjugates which are thought to be the active phytochemicals. In this investigation, we have examined serial plasma samples from 9 healthy volunteers who ingested echinacea tablets manufactured from ethanolic liquid extracts of Echinacea angustifolia and Echinacea purpurea immediately after a standard high fat breakfast. Caffeic acid conjugates could not be identified in any plasma sample at any time after tablet ingestion. Alkamides were rapidly absorbed and were measurable in plasma 20 min after tablet ingestion and remained detectable for up to 12 h. Concentration-time curves for 2,4-diene and 2-ene alkamides were determined. The maximal concentrations for the sum of alkamides in human plasma were reached within 2.3 h post ingestion and averaged 336+/-131 ng eq/mL plasma. No obvious differences were observed in the pharmacokinetics of individual or total alkamides in 2 additional fasted subjects who took the same dose of the echinacea preparation. This single dose study provides evidence that alkamides are orally available and that their pharmacokinetics are in agreement with the one dose three times daily regimen already recommended for echinacea.

Permeability studies of alkylamides and caffeic acid conjugates from echinacea using a Caco-2 cell monolayer model.

BACKGROUND: Echinacea is composed of three major groups of compounds that are thought to be responsible for stimulation of the immune system--the caffeic acid conjugates, alkylamides and polysaccharides. This study has focussed on the former two classes, as these are the constituents found in ethanolic liquid extracts. OBJECTIVE: To investigate the absorption of these two groups of compounds using Caco-2 monolayers, which are a model of the intestinal epithelial barrier. RESULTS: The caffeic acid conjugates (caftaric acid, echinacoside and cichoric acid) permeated poorly through the Caco-2 monolayers although one potential metabolite, cinnamic acid, diffused readily with an apparent permeability (Papp) of 1 x 10(-4) cm/s. Alkylamides were found to diffuse through Caco-2 monolayers with Papp ranging from 3 x 10(-6) to 3 x 10(-4) cm/s. This diversity in Papp for the different alkylamides correlates to structural variations, with saturation and N-terminal methylation contributing to decreases in Papp. The transport of the alkylamides is not affected by the presence of other constituents and the results for synthetic alkylamides were in line with those for the alkylamides in the echinacea preparation. CONCLUSION: Alkylamides but not caffeic acid conjugates are likely to cross the intestinal barrier.

Cytomegalovirus keratitis in acquired immunodeficiency syndrome.

A man with acquired immunodeficiency syndrome developed a generalized rash and bilateral dendritic epithelial keratitis without retinitis. Cytologic examination of superficial corneal scrapings showed many megalosyncytial giant cells that were highly characteristic of cytomegalovirus (CMV) infection. Viral cultures yielded CMV from 2 separate specimens obtained by corneal epithelial debridement from both eyes. The slightly elevated, opaque, branching, nonulcerative epitheliopathy recurred after corneal scrapings and persisted despite oral and topical antiviral therapy. Stromal keratouveitis subsequently developed. This case report confirms that CMV can produce corneal involvement and suggests that CMV keratitis may be an emergent complication of acquired immunodeficiency syndrome.

Short-term efficacy of apraclonidine hydrochloride added to maximum-tolerated medical therapy for glaucoma. Apraclonidine Maximum-Tolerated Medical Therapy Study Group.

PURPOSE: We determined whether the addition of topical apraclonidine hydrochloride to eyes that are receiving maximal medical therapy but still have inadequate intraocular pressure control and that are scheduled to undergo surgery could adequately decrease intraocular pressure, postponing the need for further intervention. METHODS: We performed a prospective, 90-day, multicentered, placebo-controlled, double-masked parallel study. We enrolled one eye each of 174 glaucoma patients with inadequate intraocular pressure control who were on maximally tolerated medical therapy. We continued to administer maximum medical therapy for glaucoma. Study medications were either apraclonidine hydrochloride 0.5% or placebo (apraclonidine's vehicle). Patients were instructed to take the study medication every eight hours. We measured intraocular pressure, change in intraocular pressure from baseline, and the number of eyes requiring surgery after the addition of study medication. RESULTS: Fifty-two (60%) of 86 patients treated with apraclonidine maintained adequate intraocular pressure control throughout the study and avoided surgery, compared with 28 (32%) of 88 patients treated with placebo (P < .001). Apraclonidine treatment resulted in significantly more patients attaining an additional 20% reduction or more in intraocular pressure from baseline and an intraocular pressure less than or equal to 20 mm Hg (P < .05). The most common ocular complication was conjunctival hyperemia (11 of 86 patients, 12.8%). The most frequent nonocular problem was dry mouth (four patients, 4.7%). CONCLUSION: Apraclonidine appeared to be safe in all eyes and efficacious in some eyes. It significantly lowered intraocular pressure when used in combination with maximally tolerated medical therapy, which delayed or prevented further glaucoma surgery for at least 90 days in 52 (60%) of 86 treated patients.

The efficacy of apraclonidine as an adjunct to timolol therapy. Apraclonidine Adjunctive Therapy Study Group.

OBJECTIVE: To compare the intraocular pressure (IOP) lowering efficacy of 0.5% and 1.0% apraclonidine hydrochloride when used adjunctively with 0.5% timolol maleate in 129 patients. DESIGN: A multicenter, randomized, double-masked clinical trial. Adult patients of either sex diagnosed as having either open-angle glaucoma or ocular hypertension were enrolled in the study. Patients using only 0.5% timolol maleate twice daily for at least 4 weeks and who had 8 AM IOPs of at least 22 mm Hg and no greater than 30 mm Hg 12 hours after dosing were eligible for the study. After 8 AM baseline IOPs were obtained while patients were taking timolol only, they were then randomized to receive either 0.5% or 1.0% apraclonidine twice daily in addition to their timolol. Intraocular pressures were measured at 8 AM (before morning dosing) and at 11 AM (3 hours after dosing) on days 14 and 90 and at 8 AM only on day 45. RESULTS: Both concentrations of apraclonidine produced significant IOP reductions from baseline at all visits (P < .001). At 8 AM, after the nighttime dose, the additional mean IOP reduction from the timolol baseline ranged from 2.5 to 3.3 mm Hg (10.3% to 13.6% reduction, respectively). At 11 AM, 3 hours after the morning dose, the additional IOP reduction from the timolol baseline ranged from 4.7 to 5.2 mm Hg (20.0% to 21.7%, respectively). No difference in IOP reduction was observed between the 0.5% and 1.0% apraclonidine concentrations and no loss of IOP efficacy was observed for either concentration for the duration of the study. Sensitivity to 0.5% and 1.0% apraclonidine was observed in nine (13.8%) and 13 (20.3%) patients, respectively. Overall, therapy was discontinued owing to ocular or nonocular side effects with 0.5% and 1.0% apraclonidine in 14 (21.5%) and 16 (25%) patients, respectively. CONCLUSIONS: We believe that 0.5% apraclonidine is equally effective as 1.0% apraclonidine when used twice daily as the first adjunctive drug to timolol. The drug effect is maintained for at least 90 days.

Topical apraclonidine hydrochloride in eyes with poorly controlled glaucoma. The Apraclonidine Maximum Tolerated Medical Therapy Study Group.

OBJECT: We determined whether the addition of topical apraclonidine hydrochloride to eyes receiving maximal medical therapy, with inadequate intraocular pressure (IOP) control, and scheduled to undergo surgery, could adequately lower IOP, postponing the need for surgical intervention. DESIGN: A prospective 90 day, multi-centered, placebo-controlled, doublemasked parallel study. PATIENTS: We enrolled 174 glaucoma patients with inadequate IOP control on maximally tolerated medical therapy. All were candidates for either laser trabeculoplasty or invasive surgical intervention. We enrolled only one eye per patient. INTERVENTIONS: We continued to administer maximum-tolerated medical therapy for glaucoma. Patients took the study medication every eight hours. Study medications were either apraclonidine hydrochloride 0.5% or placebo (apraclonidine's vehicle). MAJOR OUTCOME MEASURES: We evaluated IOP, IOP change from baseline, and the number of eyes requiring surgery after the addition of study medication. RESULTS: Sixty one percent of patients treated with apraclonidine maintained adequate IOP control throughout the study, avoiding additional surgery compared to 33.9% patients treated with placebo (P < .001). Apraclonidine treatment resulted in significantly more patients achieving either an additional > or = 20% reduction in IOP from baseline (resulting in an IOP < or = 20 mm Hg) (P < 0.05). The most common ocular complications were conjunctival hyperemia (12.6%), itching and foreign body sensation (6.8%), and tearing (4.5%). The most frequent non-ocular adverse events related to apraclonidine were dry mouth (4.5%) and unusual taste perception (2.2%). CONCLUSIONS: Apraclonidine appears safe and efficacious. It significantly lowered IOP when used in combination with a patient's maximum tolerated medical therapy. This delayed or prevented further glaucoma surgery for at least 90 days in approximately 60% of treated patients.

Structural differences in chlorosomes from Chloroflexus aurantiacus grown under different conditions support the BChl c-binding function of the 5.7 kDa polypeptide.

Structurally different chlorosomes were isolated from the green photosynthetic bacterium Chloroflexus aurantiacus grown under different conditions. They were analysed with respect to variable pigment-protein stoichiometries in view of the presumed BChl c-binding function of the 5.7 kDa chlorosome polypeptide. Under high-light conditions on substrate-limited growth medium the pigment-protein ratio of isolated chlorosomes was several times lower than under low-light conditions on complex medium. Proteolytic degradation of the 5.7 kDa polypeptide in high-light chlorosomes led to a 60% decrease of the absorbance at 740 nm. The CD spectrum of high-light chlorosomes exhibited a sixfold lower relative intensity at 740 nm (delta A/A740) than low-light chlorosomes, but it showed a fivefold increase in intensity upon degradation of the 5.7 kDa polypeptide compared to a twofold increase in low-light chlorosomes. It seems probable that BChl c in the chlorosomes is present as oligomers bound to the 5.7 kDa polypeptide. Our data suggest further that compared to low-light chlorosomes smaller oligomers or single BChl c molecules are bound to the 5.7 kDa polypeptide in high-light chlorosomes resulting in lower rotational strength.

Giant circular dichroism of chlorosomes fromChloroflexus aurantiacus treated with 1-hexanol and proteolytic enzymes.

The circular dichroism (CD) spectrum of isolated chlorosomes fromChloroflexus aurantiacus showed a conservative, S-shaped signal with a negative maximum at 723 nm, a positive maximum at 750 nm and a zero-crossing at 740 nm. Proteolytic treatment of chlorosomes with trypsin at 37°C did not change the CD signal or the absorption spectrum in contrast to treatment with proteinase K, where a twofold increase in rotational strength and a slight decrease of the absorption band at 740 nm were observed. Treatment with saturating 1-hexanol concentrations resulted in a blue shift of the absorption band at 740 nm as well as in changes of the CD spectrum. These changes reversed when the sample was diluted to half the saturating 1-hexanol concentration. In contrast to that, we observed an irreversible formation of a giant CD signal using the combination of 1-hexanol and proteinase K treatment. Electron micrographs of chlorosomes treated with both 1-hexanol and proteinase K showed large aggregates of multiple chlorosome size. By comparison of proteinase K induced effects with trypsin effects it appeared that the 5.7 kDa polypeptide has a structural role in the organisation of BChlc in the chlorosome.

Paired comparison of contrast sensitivity in diffractive multifocal IOLs and conventional monofocal IOLs.

Multifocal intraocular lens (IOL) optics produce a retinal image of reduced contrast, which results from splitting incoming light between multiple focal points. This study sought to determine whether the reduced-contrast image results in functional loss of contrast sensitivity. Contrast sensitivity was measured in patients in the US Food and Drug Administration (FDA) study of the 3M Diffractive IOL, using the Pelli-Robson Letter Chart. Fifty-eight of these patients, including six from our clinic, were 'best-case' patients with no pathology, and also had paired multifocal/monofocal implants. Data from these 'best-care' patients with paired eyes indicate no functional difference in contrast sensitivity. Additional data obtained on our six patients with the Regan Low Contrast Charts suggest small predictable differences may exist for some patients at very low contrast levels. Further study is needed to determine whether these differences have functional significance.

Cilioretinal arteries. A study based on review of stereo fundus photographs and fluorescein angiographic findings.

The incidence of cilioretinal arteries in 2,000 eyes of 1,000 consecutive patients was determined by review of stereo color fundus photographs and fluorescein angiographs. One or more cilioretinal arteries were present in 49.5% of all patients or in 32.1% of the eyes. The arteries occurred bilaterally in 14.6% and contributed to some portion of the macular circulation in 18.7% of the patients. A great deal of variability of size, number, and distribution of cilioretinal vessels was observed. Prior studies that were based on direct visualization of the fundus have reported cilioretinal arteries in 7% to 29.6% of patients examined. Careful review of stereo fundus photographs and early phase fluorescein angiographs aided in the observation of these vessels that might otherwise have been unrecognized. We believe the method of review accounts for the high incidence of cilioretinal arteries in our series.