Submicron Size Formulation of Linseed Oil Containing Omega-3 Fatty Acid for Topical Delivery

The aim of the present study was to design and develop topical submicron size gel formulation of linseed oil with enhanced permeation through the skin for the management of psoriasis. Linseed oil contains significant amount of α-linolenic acid (ALA) an omega-3 fatty acid, which is responsible for its pharmacological actions. In order to enhance permeation through skin, microemulsion based gel formulation was prepared and characterized. Microemulsions were prepared by aqueous phase titration method, using linseed oil, Unitop 100, PEG 400, and distilled water as the oil phase, surfactant, cosurfactant and aqueous phase, respectively. Selected formulations were subjected to physical stability studies and consequently in vitro skin permeation studies. Surface morphology studies of optimized formulation were done by transmission electron microscopy (TEM). The droplet size of microemulsions ranged from 70 to 500 nm with average particle size 186 nm. The optimized microemulsion was converted into hydrogel using carbopol 971 which had a viscosity of 498 ± 0.04 cps. During in vitro permeation study the flux of microemulsion formulation and gel was found to be 19.05 and 10.2 µg/cm²/hr, respectively, which indicated better penetration of linseed oil through the skin. These result indicated that the developed ME formulation may be a good approach for topical therapy for the management of psoriasis.     

Isobaric vapor–liquid equilibria of binary system ethyl acetate + ethyl benzene + lithium bromide

The isobaric vapor–liquid equilibrium (VLE) behaviors for binary system, ethyl acetate + ethyl benzene, and ethyl acetate + ethyl benzene + LiBr (at saturation) were studied at the local ambient pressure (707 ± 1 mmHg). Equilibrium still was used where both liquid and vapor were continuously circulated. The experimental results showed that salt-free ethyl acetate + ethyl benzene system does not form an azeotrope point. The experimental results for ethyl acetate + ethyl benzene system were in a very good agreement with the predicted results using UNIFAC, UNIQUAC, NRTL, and Wilson models. Adding LiBr, as a salt, did show slight effects on the VLE behavior of ethyl acetate + ethyl benzene system.     

Nonionic Surfactant Vesicles as a Carrier for Transdermal Delivery of Frusemide

The aim of the present study was to formulate and evaluate the nonionic surfactant vesicles of frusemide in order to enhance its skin permeation. The process variables which could affect the preparation and properties of the niosome formulation studied included type of spans, ratio of span and cholesterol, ratio of cholesterol and dicetylphosphate (DCP), concentration of drug, type of solvent, hydration media and time of hydration. The formulated niosomes thus were characterized for various parameters such as surface morphology, size, entrapment efficiency, skin permeation, etc. Stability of the niosomes in terms of drug holding capacity was assessed for a period of 30 days on storage under defined conditions. The maximum entrapment efficiency of 77.73±2.36% was obtained with niosomes formulated from Span 60∶Cholesterol∶DCP (47.5∶47.5∶5) using chloroform:methanol (4∶1) as the solvent system at the hydration time of 1 hr. A direct relationship was observed between the percentage leaching of the drug out of the vesicles and temperature. Higher transdermal flux was obtained with niosomal gel (9.2±0.5 μg/cm²/hour) in comparison to conventional gel (6.4±0.3 μg/cm²/hour).     

Ligase-Independent Cloning of Amylase Gene from a Local Bacillus subtilis Isolate and Biochemical Characterization of the Purified Enzyme

Five hundred ninety-seven bacterial isolates from Turkish hot spring water sources were screened for their ability to produce extracellular α-amylase. Among them, a high enzyme-producing Bacillus subtilis isolate, A28, was selected, and its α-amylase gene was cloned and expressed in Escherichia coli by a ligase-independent method. α-Amylase from the recombinant strain was purified to homogeneity by Q-Sepharose anion exchange and Sephacryl S-100 gel filtration chromatographies. The final yield of the enzyme was about 22.5 % of the initial activity, with a 16.4-fold increase in specific activity compared with the culture lysate. The optimum temperature and pH of the enzyme were 70 °C and 6.0, respectively. The enzyme was highly active at acidic-neutral pH range of 4.5–7.0. The amy28 α-amylase retained 100 % of its activity after incubation at 50 °C for 90 min. Co⁺², Cu²⁺, Fe²⁺, Fe³⁺, Ni⁺², and Zn⁺² caused significant inhibition in enzyme activity, which was not affected by Na⁺, Mg²⁺, Li⁺, and Ba²⁺. The activity was inhibited about 70 % upon treatment of the enzyme with 10 mM ethylenediaminetetraacetic acid. However, Ca²⁺ ions known as high temperature stabilizer for other amylases did not stimulate the activity of the enzyme. Due to pH stability and thermostability of the recombinant amylase, this enzyme may be suitable in starch processing, brewing, and food industries.     

Low-molecular weight hyaluronidase from the venom of Bungarus caeruleus (Indian common krait) snake: Isolation and partial characterization

Snake venom hyaluronidases known as “spreading factor” are not extensively studied. Recently, it is argued that beyond its role as a spreading factor, venom hyaluronidase (HYL) deserves to be explored as a possible therapeutic target for inhibiting the systemic distribution of venom/toxins and also for minimizing local tissue destruction. In this context, in the present study, a low-molecular weight HYL has been isolated from Bungarus caeruleus (Indian krait) venom by single step chromatography on HPLC system. The apparent molecular weight determined by SDS-PAGE is 14 ± 2 kDa, as confirmed by zymogen study and LC–MS as well. The enzyme had optimal pH 6 and temperature 37°C. The Michaelis–Menten constant (K_m) was found to be 8.48 µg/mL at 37°C. The activity of purified enzyme was completely inhibited by Ba²⁺ metal ion and N-acetyl imidazole group-specific agents. This work yielded a highly active HYL from B. caeruleus the first one to be isolated. Further studies on its pharmacological actions will be interesting to develop lead molecules for better management of snakebite.     

Effect of tretinoin inclusion in dimethyl-beta-cyclodextrins on release rate from a hydrogel formulation

The aim of this work was to study the release, permeation and skin retention profiles of 0.05% tretinoin hydrogel formulations in which tretinoin was in free form or complexed with dimethyl-beta-cyclodextrin in a stoichiometry of 1:4. Theoretically, this complexation will mainly allow to: overcome drug’s low water solubility and low stability; enhance the drug permeation by promoting skin absorption and alleviate drug inducing local irritation. In vitro release, permeation and skin retention tests were performed in both formulations in order to compare the main advantages of this complexation. The influence of the thermodynamic activity on the drug release profile was also investigated. This study proved that tretinoin inclusion complexes formulation with excess of cyclodextrins had better release profile than the free tretinoin formulation. It was concluded that in this study, thermodynamic activity was not the driving force for the release rate improvement observed with cyclodextrins. Probably, this improvement was due to the increased availability of tretinoin near the membrane surface. In fact, the percentage of total drug that had been retained in the skin was 0.41?±?0.08% for complexed tretinoin gel and 0.17?±?0.04% for the free tretinoin gel.     

Expression,Purification, and Characterization of NADP+-Dependent Malic Enzyme from the Oleaginous Fungus Mortierella Alpina

Malic enzymes are a class of oxidative decarboxylases that catalyze the oxidative decarboxylation of malate to pyruvate and carbon dioxide, with concomitant reduction of NAD(P)⁺ to NAD(P)H. The NADP⁺-dependent malic enzyme in oleaginous fungi plays a key role in fatty acid biosynthesis. In this study, the malic enzyme-encoding complementary DNA (cDNA) (malE1) from the oleaginous fungus Mortierella alpina was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant protein (MaME) was purified using Ni-NTA affinity chromatography. The purified enzyme used NADP⁺ as the cofactor. The K _m values for l-malate and NADP⁺ were 2.19?±?0.01 and 0.38?±?0.02 mM, respectively, while the V _max values were 147?±?2 and 302?±?14 U/mg, respectively, at the optimal condition of pH 7.5 and 33 °C. MaME is active in the presence of Mn²⁺, Mg²⁺, Co²⁺, Ni²⁺, and low concentrations of Zn²⁺ rather than Ca²⁺, Cu²⁺, or high concentrations of Zn²⁺. Oxaloacetic acid and glyoxylate inhibited the MaME activity by competing with malate, and their K _i values were 0.08 and 0.6 mM, respectively.     

Densities and Viscosities of the Regular Quinary System: Benzene (1) + Toluene (2) + Ethylbenzene (3) + Heptane (4) + Cyclooctane (5) and Its Quaternary Sub-Systems at 293.15 and 298.15 K

The densities and viscosities of the regular quinary system: benzene (1) + toluene (2) + ethylbenzene (3) + heptane (4) + cyclooctane (5), and all its corresponding quaternary sub-systems were measured over the entire composition range at 293.15 and 298.15 K. Kinematic viscosity–composition data reported in the present study were utilized to test the predictive capability of some well-known viscosity models. The models subjected to testing were: the predictive version of the McAllister’s three-body model, a generalized corresponding states principle, the group contribution GC-UNIMOD method, and the Allan and Teja correlation. Results of testing these models led to an overall AAD (%) of 1.20 for the one quaternary system that did not contain cyclooctane, and an overall AAD (%) of 8.59 for the remaining cyclooctane-containing systems. For the quinary system, the overall AAD (%) was found to be 2.92; the last two values were calculated using the value of cyclooctane of 10.595.     

Effect of coexisting cations on the adsorption of cesium onto poly (β-cyclodextrin)/bentonite composite

A novel poly(β-cyclodextrin)/bentonite composite (β-CD/BNC) was successfully prepared through graft polymerisation by using ammonium persulphate–sodium bisulphate as initiators, and characterized by FT-IR and EDS. The equilibrium data fit Freundlich isotherm satisfactorily. Adsorption kinetic was fitted with pseudo-second-order. The maximum adsorption capacities for Cs⁺ by β-CD/BNC in absence and presence of Na⁺ and Mg²⁺ were 48.83 ± 0.35, 47.30 ± 0.28, and 42.52 ± 0.85 mg g^?1, respectively. Adsorption of Cs⁺ was suppressed by presence of Mg²⁺ more than Na⁺. β-CD/BNC had a higher affinity to Cs⁺ than Na⁺ and Mg²⁺. β-CD/BNC was an effective sorbent for the treatment cesium waste water.     

A Novel Process for Direct Production of Acetone–Butanol–Ethanol from Native Starches Using Granular Starch Hydrolyzing Enzyme by Clostridium saccharoperbutylacetonicum N1-4

In this work, a new approach for acetone–butanol–ethanol (ABE) production has been proposed. Direct fermentation of native starches (uncooked process) was investigated by using granular starch hydrolyzing enzyme (GSHE) and Clostridium saccharoperbutylacetonicum N1-4. Even the process was carried out under suboptimal condition for activity of GSHE, the production of ABE was similar with that observed in conventional process or cooked process in terms of final solvent concentration (21.3?±?0.4 to 22.4?±?0.4 g/L), butanol concentration (17.5?±?0.4 to 17.8?±?0.3 g/L) and butanol yield (0.33 to 0.37 g/g). The production of solvents was significantly dependent on the source of starches. Among investigated starches, corn starch was more susceptible to GSHE while cassava starch was the most resistant to this enzyme. Fermentation using native corn starch resulted in the solvent productivity of 0.47 g/L h, which was about 15 % higher than that achieved in cooked process. On the contrary, uncooked process using cassava and wheat starch resulted in the solvent productivity of 0.30 and 0.37 g/L h, which were respectively about 30 % lower than those obtained in cooked process. No contamination was observed during all trials even fermentation media were prepared without sterilization. During the fermentation using native starches, no formation of foam is observed. This uncooked process does not require cooking starchy material; therefore, the thermal energy consumption for solvent production would remarkably be reduced in comparison with cooked process.     

Composition and stability constants of copper(II) complexes with succinic acid determined by capillary electrophoresis

The advantage of capillary electrophoresis was demonstrated for studying a complicated system owing to the dependence of direction and velocity of the electrophoretic movement on the charge of complex species. The stability constants of copper(II) complexes with ions of succinic acid were determined by capillary electrophoresis, including the 1?:?2 metal to ligand complexes which are rarely mentioned. The measurements were carried out at 25 °C and ionic strength of 0.1, obtained by mixing the solutions of succinic acid and lithium hydroxide up to pH 4.2–6.2. It was shown that while pH was more than 4.5 the zone of copper(II) complexes with succinate moves as an anion. It is impossible to treat this fact using only the complexes with a metal-ligand ratio of 1?:?1 (CuL⁰, CuHL⁺). The following values of stability constants were obtained: log β(CuL) = 2.89 ± 0.02, log β(CuHL⁺) = 5.4 ± 0.5, log β(CuL₂^2?) = 3.88 ± 0.05, log β(CuHL₂^?) = 7.2 ± 0.3.     

Studies of total, organic and inorganic iodine in Canadian bovine milk samples with varying milk fat content using ion-exchange chromatography and neutron activation analysis

Chemical separation methods in conjunction with instrumental neutron activation analysis (INAA) were developed for measuring iodine levels in commercially available bovine milk with varying milk fat (MF) content. Samples of homogenized (3.25 % MF), partly skimmed (2 % MF), partly skimmed (1 % MF), partly skimmed calcium enriched (1 % MF + Ca), and skim (<0.05 %) milk were purchased from local supermarkets. Ion exchange chromatography, solvent extraction, and ammonium sulfate precipitation methods were applied to the separation of the inorganic, lipidic and proteic fractions of iodine in milk. The levels of iodine were measured by INAA in total reactor and epi-cadmium (EINAA) neutron flux in conjunction with conventional gamma-ray and Compton suppression spectrometry (CSS). A pseudo-cyclic INAA method coupled with CSS (PC-INAA-CSS) was also explored as an instrumental option to further lower the detection limit of iodine. The detection limits of 0.06, 0.06 and 0.02 μg mL^?1 for iodine were obtained using INAA-CSS, EINAA-CSS, and PC-INAA-CSS methods, respectively. Although the PC-INAA-CSS method provided the lowest detection limit, the INAA-CSS method was sufficient for the determination of total iodine in almost all samples analyzed in this work. The total iodine concentrations (μg mL^?1) were: 0.40 ± 0.01 (in 3.25 % MF), 0.40 ± 0.01 (2 % MF), 0.42 ± 0.01 (1 % MF), 0.42 ± 0.01 (<0.05 %), and 0.96 ± 0.01 (1 % MF + Ca) milk samples. Iodine bound to various fractions of the milk samples analyzed, in percent of total iodine content, ranged: (0.05–1.8), (1.9–4.8), (90–95) for the lipidic, proteic and anionic inorganic fractions respectively. Iodine recovery in all cases was higher than 96 %.     

A Validated Quantification of Benzocaine in Lozenges Using TLC and a Flatbed Scanner

We present a video-densitometric quantification method for benzocaine in lozenges. The quantification is based on a derivatisation reaction with 4-dimethylaminobenzaldehyde. Measurements were carried out using a 16-bit flatbed scanner. Benzocaine was separated to a distance of 50 mm in a vertical developing chamber without vapour saturation. We present an RP-18 phase separation on a cyanopropyl plate (Merck, Darmstadt, Germany) using water, CH₃CN, dioxane, ethanol, and NH₃ (25 %) (8 + 2 + 1 + 1 + 0.05, v/v) as the mobile phase. We also separated benzocaine in a normal phase system on silica gel 60 LiChrospere^® plates (Merck, Darmstadt, Germany) with the mobile phase MTBE/cyclohexane (1 + 1, v/v). The calibration functions for benzocaine in both separations were linear in the range from 1 to 1,000 ng per spot. The range of linearity covers two magnitudes of power because the Kubelka–Munk expression was used for data transformation. In the cyanopropyl-system, the benzocaine amount was quantified as 242.5 ± 18.2 ng in a spot or 6.86 ± 0.52 mg in a single lozenge. The amount of 7.0 mg benzocaine per lozenge was labelled. The combined uncertainty of sample and calibration measurements was statistically calculated using a significance level of α = 0.05 to a total relative uncertainty of 7.49 %. The separation method is inexpensive, fast and reliable.     

A Novel β-Glucosidase from Humicola insolens with High Potential for Untreated Waste Paper Conversion to Sugars

Humicola insolens produced a new β-glucosidase (BglHi2) under solid-state fermentation. The purified enzyme showed apparent molecular masses of 116 kDa (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) and 404 kDa (gel-filtration), suggesting that it is a homotetramer. Mass spectrometry analysis showed amino acid sequence similarity with a β-glucosidase from Chaetomium thermophilum. Optima of pH and temperature were 5.0 and 65 °C, respectively, and the enzyme was stable for 60 min at 50 °C, maintaining 71 % residual activity after 60 min at 55 °C. BglHi2 hydrolyzed p-nitrophenyl-β-d-glucopyranoside and cellobiose. Cellobiose hydrolysis occurred with high apparent affinity (K _M?=?0.24?±?0.01 mmol L^?1) and catalytic efficiency (k _cat/K _M?=?1,304.92?±?53.32 L mmol^?1 s^?1). The activity was insensitive to Fe⁺³, Cr⁺², Mn⁺², Co⁺², and Ni²⁺, and 50–60 % residual activities were retained in the presence of Pb²⁺, Hg²⁺, and Cu²⁺. Mixtures of pure BglHi2 or H. insolens crude extract (CE) with crude extracts from Trichoderma reesei fully hydrolyzed Whatman no. 1 paper. Mixtures of H. insolens CE with T. reesei CE or Celluclast 1.5 L fully hydrolyzed untreated printed office paper, napkin, and magazine papers after 24–48 h, and untreated cardboard was hydrolyzed by a H. insolens CE/T. reesei CE mixture with 100 % glucose yield. Data revealed the good potential of BglHi2 for the hydrolysis of waste papers, promising feedstocks for cellulosic ethanol production.     

Development and validation of an analytical method for the quantification of cytochrome c in skin transport studies

A simple isocratic HPLC method for the quantification of Cytochrome c in skin permeation samples was developed and validated. The mobile phase comprised a 41 : 59 mixture of an organic phase A (0.1% trifluoroacetic acid in a 90 : 10 mixture of MeCN–H₂O) and an aqueous phase B (0.1% trifluoroacetic acid in H₂O). The Cytochrome c retention and run times were 2.62 and 8.0 min, respectively—much shorter than those for existing gradient methods. The response was accurate, precise and linear from 2.5 to 25 μg/mL. The mean recoveries for intra‐day and inter‐day analysis ranged from 88.5 to 103.8% and the RSD varied from 0.05 to 1.55%. The assay was used to quantify transport of Cytochrome c across intact and laser‐microporated porcine skin in vitro. Cytochrome c permeation and the amount of protein retained within the membrane over 24 h were quantified as a function of the number of micropores. Although no Cytochrome c permeation was observed across intact skin, laser microporation enabled delivery of 22.9 ± 3.3 and 56.0 ± 15.9 μg/cm² of the protein across skin samples with 300 and 1800 micropores, respectively. In conclusion, the HPLC method provided a fast, efficient means to quantify Cytochrome c in samples from skin transport studies. Copyright © 2009 John Wiley & Sons, Ltd.     

A New Amperometric Carbon Paste Enzyme Electrode for Ethanol Determination

Abstract

In this study, a new amperometric carbon paste enzyme electrode for determination of ethanol was developed. The carbon paste was prepared by mixing alcohol dehydrogenase, its coenzyme nicotinamide adenine dinucleotide (oxidized form, NAD⁺), poly(vinylferrocene) (PVF) that was used as a mediator, graphite powder and paraffin oil, then the paste was placed into cavity of a glass electrode body. Determination of ethanol was performed by oxidation of nicotinamide adenine dinucleotide (reduced form, NADH) generated enzymatically at +0.7 V. The effects of enzyme, coenzyme and PVF amounts; pH; buffer concentration and temperature were investigated. The linear working range of the enzyme electrode was 4.0×10^?4–4.5×10^?3 M, determination limit was 3.9×10^?4 M and response time was 50 s. The optimum pH, buffer concentration, temperature, and amounts of enzyme, NAD⁺ and PVF for enzyme electrode were found to be 8.5, 0.10 M, 37°C, 2.0, 6.0, and 12.0 mg, respectively. The storage stability of enzyme electrode at +4°C was 7 days. Enzyme electrode was used for determination of ethanol in two different wine samples and results were in good agreement with those obtained by gas chromatography.     

Pistagremic acid,a novel β-secretase enzyme (BACE1) inhibitor from Pistacia integerrima Stewart

A new triterpenic compound named pistagremic acid (PA) was once again isolated from Pistaciaintegerrima. The β-secretase inhibition study was carried out. Compound PA was found significantly active against β-secretase enzyme (BACE1) with IC₅₀ value of 350 ± 2 nM in comparison to the standard inhibitors [Asn670, Sta671, Val672]-amyloid-β/A4 precursor protein 770 fragment 662–675 (IC₅₀ = 290.71 ± 1 nM). The selectivity of this compound was also evaluated against the acetylcholinesterase and butyrylcholinesterase enzymes. Interestingly compound PA was found to be inactive against them and showed selectivity towards β-secretase enzyme (BACE1).     

Modelling and Optimization of Stability Constants of Cadmium or Zinc with Biological Buffers (DIPSO or TAPS) in Aqueous Solutions by Electrochemical Techniques

The complexation behavior of four systems involving cadmium(II) or zinc(II) in aqueous solutions with the biological buffers 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), and [(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid (TAPS) was studied by direct current polarography (DCP) and glass electrode potentiometry (GEP), at 25.0 ± 0.1 °C and ionic strength 0.1 mol·dm^?3 KNO₃. Except for the Cd–TAPS system, for which full characterization of the system was possible either by DCP or GEP, full characterization of the other metal-buffer systems (Zn–DIPSO, Zn–TAPS and Cd–DIPSO) was only possible using DCP. For Zn-buffers systems, ZnL⁺ and $ {\text{ZnL(OH)}}_{2}^{ - } $ ZnL(OH) 2 ? (where L stands for buffer) were identified. For the Zn–DIPSO system, the overall stability constant values (as log₁₀ β) are 2.1 ± 0.2 and 13.4 ± 0.2, respectively. For the Zn–TAPS system, the overall stability constants values (as log₁₀ β) are 2.4 ± 0.1 and 12.9 ± 0.3, respectively. For the Cd–DIPSO system, the overall stability constants values (as log₁₀ β) of CdL⁺ and CdL(OH) are 2.9 ± 0.1 and 6.9 ± 0.3, respectively. For the Cd–TAPS system, only the species CdL⁺ was identified with log₁₀ β = 2.5 ± 0.1.     

ZnO nanoparticles and poly(acrylic) acid-based polymer gel electrolyte for photo electrochemical cell

This work presents a photo electrochemical cell based on zinc oxide (ZnO) nanoparticles and poly(acrylic) acid (PAA) doped with sodium iodide (NaI) and iodine (I₂) polymer gel electrolyte. The ZnO powders were synthesized by sol–gel storage and sol–gel centrifugation. The ZnO powder synthesized via sol–gel centrifugation showed the optimal structural properties, with largest crystallite sizes of 58 nm, average particles size between 20 and 80 nm and indirect band gap energy of 3.20 eV. The highest conductivity [(8.0 ± 0.1) × 10^?2 S cm^?1] was obtained for PAA + 0.8 M NaI + 0.02 M I₂. This sample achieved the lowest activation energy (0.029 eV) and electrochemical stability at 1.6 V. The ZnO powder synthesized via sol–gel centrifugation and PAA + 0.8 M NaI + 0.02 M I₂ was fabricated as a Cu–ZnO/PAA + 0.8 M NaI + 0.02 M I₂/C-ITO photo electrochemical cell.     

Complexation of the cesium cation with 1,3-alternate-25,27-bis(1-octyloxy)calix[4]arene-crown-6

From extraction experiments and γ-activity measurements, the extraction constant corresponding to the equilibrium Cs⁺(aq) + I^?(aq) + 1(nb) ? 1·Cs⁺(nb) + I^?(nb) taking place in the two–phase water–nitrobenzene system (1 = 1,3-alternate-25,27-bis(1-octyloxy)calix[4]arene-crown-6; aq = aqueous phase, nb = nitrobenzene phase) was evaluated as log K _ex (1·Cs⁺, I^?) = 2.9 ± 0.1. Further, the stability constant of the 1·Cs⁺ complex in nitrobenzene saturated with water was calculated for a temperature of 25 °C: log β_nb (1·Cs⁺) = 8.8 ± 0.1. Finally, by using quantum–mechanical DFT calculations, the most probable structure of the resulting cationic complex species 1·Cs⁺ was derived.