Effect of Ascorbic Acid on the Degradation of Cyanocobalamin and Hydroxocobalamin in Aqueous Solution: A Kinetic Study

The degradation kinetics of 5 × 10⁻⁵ M cyanocobalamin (B₁₂) and hydroxocobalamin (B_12b) in the presence of ascorbic acid (AH₂) was studied in the pH range of 1.0–8.0. B₁₂ is degraded to B_12b which undergoes oxidation to corrin ring cleavage products. B_12b alone is directly oxidized to the ring cleavage products. B₁₂ and B_12b in degraded solutions were simultaneously assayed by a two-component spectrometric method at 525 and 550 nm without interference from AH₂. Both degrade by first-order kinetics and the values of the rate constants at pH 1.0–8.0 range from 0.08 to 1.05 × 10⁻⁵ s⁻¹ and 0.22–7.62 × 10⁻⁵ s⁻¹, respectively, in the presence of 0.25 × 10⁻³ M AH₂. The t_1/2 values of B₁₂ and B_12b range from 13.7 to 137.5 h and 2.5–87.5 h, respectively. The second-order rate constants for the interaction of AH₂ with B₁₂ and B_12b are 0.05–0.28 × 10⁻² and 1.10–30.08 × 10⁻² M⁻¹ s⁻¹, respectively, indicating a greater effect of AH₂ on B_12b compared to that of B₁₂. The k_obs–pH profiles for both B₁₂ and B_12b show the highest rates of degradation around pH 5. The degradation of B₁₂ and B_12b by AH₂ is affected by the catalytic effect of phosphate ions on the oxidation of AH₂ in the pH range 6.0–8.0.KEY WORDS: ascorbic acid, cyanocobalamin, degradation, hydroxocobalamin, kinetics, two-component spectrometry     

Optimization of Storage and Manufacture Conditions for Imidapril Hydrochloride in Solid State as a Way to Reduce Costs of Antihypertensive Therapy

The effect of temperature and relative humidity (RH) on the stability of imidapril hydrochloride (IMD) in solid state was investigated. The main aim of this study was to determine the most appropriate conditions of storage and manufacture of IMD so that the efficiency of the technological process could be improved and its costs could be minimized. A reversed-phase high-performance liquid chromatography was validated and applied for the determination of IMD degradation samples under the following operating conditions: stationary phase, LiChrospher 100 RP-18 (size 5 μm) 250 × 4 mm I.D., and mobile phase, acetonitrile–methanol–phosphate buffer, pH 2.0, 0.035 mol L⁻¹ (60:10:30 v/v/v). The effect of temperature on IMD degradation rate was analyzed under increased RH ∼76.4% (within temperature range of 70–90°C) and decreased RH ∼0% (within temperature range of 90–110°C). The influence of RH was investigated under 90°C within RH range of 25.0–76.4%. IMD degradation accords with autocatalytic reaction model, and RH has no influence on its mechanism yet it increases its rate. The reaction also accelerates under high temperatures and in the presence of IMD degradation product. Pure IMD is more stable than other structurally related angiotensin-converting enzyme inhibitors, such as enalapril maleate, but it still should be stored in tightly closed containers and protected from moisture and high temperatures.KEY WORDS: angiotensin-converting enzyme inhibitors, imidapril hydrochloride, RP-HPLC, stability, thermodynamics     

Photodegradation of Moxifloxacin in Aqueous and Organic Solvents: A Kinetic Study

The kinetics of photodegradation of moxifloxacin (MF) in aqueous solution (pH 2.0–12.0), and organic solvents has been studied. MF photodegradation is a specific acid-base catalyzed reaction and follows first-order kinetics. The apparent first-order rate constants (k_obs) for the photodegradation of MF range from 0.69 × 10⁻⁴ (pH 7.5) to 19.50 × 10⁻⁴ min⁻¹ (pH 12.0), and in organic solvents from 1.24 × 10⁻⁴ (1-butanol) to 2.04 × 10⁻⁴ min⁻¹ (acetonitrile). The second-order rate constant (k₂) for the [H⁺]-catalyzed and [OH⁻]-catalyzed reactions are 6.61 × 10⁻² and 19.20 × 10⁻² M⁻¹ min⁻¹, respectively. This indicates that the specific base-catalyzed reaction is about three-fold faster than that of the specific acid-catalyzed reaction probably as a result of the rapid cleavage of diazabicyclononane side chain in the molecule. The k_obs-pH profile for the degradation reactions is a V-shaped curve indicating specific acid-base catalysis. The minimum rate of photodegradation at pH 7–8 is due to the presence of zwitterionic species. There is a linear relation between k_obs and the dielectric constant and an inverse relation between k_obs and the viscosity of the solvent. Some photodegraded products of MF have been identified and pathways proposed for their formation in acid and alkaline solutions.KEY WORDS: acid-base catalysis, kinetics, moxifloxacin, photodegradation, rate–pH profile, solvent effect     

Binding of Sulfamethazine to β-cyclodextrin and Methyl-β-cyclodextrin

β-cyclodextrin (βCD) and methyl-β-cyclodextrin (MβCD) complexes with sulfamethazine (SMT) were prepared and characterized by different experimental techniques, and the effects of βCD and MβCD on drug solubility were assessed via phase-solubility analysis. The phase-solubility diagram for the drug showed an increase in water solubility, with the following affinity constants calculated: 40.4 ± 0.4 (pH 2.0) and 29.4 ± 0.4 (pH 8.0) M⁻¹ with βCD and 56 ± 1 (water), 39 ± 3 (pH 2.0) and 39 ± 5 (pH 8.0) M⁻¹ with MβCD. According to ¹H NMR and 2D NMR spectroscopy, the complexation mode involved the aromatic ring of SMT included in the MβCD cavity. The complexes obtained in solid state by freeze drying were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and thermal analysis. The amorphous complexes obtained in this study may be useful in the preparation of pharmaceutical dosage forms of SMT.     

Development of Controlled-Release Matrix Tablet of Risperidone: Influence of Methocel®- and Ethocel®-Based Novel Polymeric Blend on <Emphasis Type="Italic">In Vitro</Emphasis> Drug Release and Bioavailability

Controlled-release (CR) matrix tablet of 4 mg risperidone was developed using flow bound dry granulation–slugging method to improve its safety profile and compliance. Model formulations F1, F2, and F3, consisting of distinct blends of Methocel® K100 LV-CR and Ethocel® standard 7FP premium, were slugged. Each batch of granules (250–1,000 μm), obtained by crushing the slugs, was divided into three portions after lubrication and then compressed to 9-, 12-, and 15-kg hard tablets. In vitro drug release studies were carried out in 0.1 N HCl (pH 1.2) and phosphate buffer (pH 6.8) using a paddle dissolution apparatus run at 50 rpm. The CR test tablet, containing 30% Methocel® and 60% Ethocel® (F3) with 12-kg hardness, exhibited pH-independent zero-order release kinetics for 24 h. The drug release rate was inversely proportional to the content of Ethocel®, while the gel layer formed of Methocel® helped in maintaining the integrity of the matrix. Changes in the hardness of tablet did not affect the release kinetics. The tablets were reproducible and stable for 6 months at 40 ± 2°C/75 ± 5% relative humidity. Risperidone and its active metabolite, 9-hydroxyrisperidone, present in the pooled rabbit’s serum, were analyzed with HPLC-UV at λ_max 280 nm. The CR test tablet exhibited bioequivalence to reference conventional tablet in addition to the significantly (p < 0.05) optimized peak concentration, C_max, and extended peak time, T_max, of the active moiety. There was a good association between drug absorption in vivo and drug release in vitro (R² = 0.7293). The successfully developed CR test tablet may be used for better therapeutic outcomes of risperidone.KEY WORDS: controlled release, dry granulation slugging method, risperidone     

Effect of Acetate and Carbonate Buffers on the Photolysis of Riboflavin in Aqueous Solution: A Kinetic Study

The photolysis of riboflavin (RF) in the presence of acetate buffer (pH 3.8–5.6) and carbonate buffer (pH 9.2–10.8) has been studied using a multicomponent spectrophotometric method for the simultaneous assay of RF and its photoproducts. Acetate and carbonate buffers have been found to catalyze the photolysis reaction of RF. The apparent first-order rate constants for the acetate-catalyzed reaction range from 0.20 to 2.86 × 10⁻⁴ s⁻¹ and for the carbonate-catalyzed reaction from 3.33 to 15.89 × 10⁻⁴ s⁻¹. The second-order rate constants for the interaction of RF with the acetate and the carbonate ions range from 2.04 to 4.33 × 10⁻⁴ M⁻¹ s⁻¹ and from 3.71 to 11.80 × 10⁻⁴ M⁻¹ s⁻¹, respectively. The k-pH profile for the acetate-catalyzed reaction is bell shaped and for the carbonate-catalyzed reaction a steep curve. Both HCO₃⁻ and CO₃^2 − ions are involved in the catalysis of the photolysis reaction in alkaline solution. The rate constants for the HCO₃⁻ and CO₃^2 − ions catalyzed reactions are 0.72 and 1.38 × 10⁻³ M⁻¹ s⁻¹, respectively, indicating a major role of CO₃^2 − ions in the catalysis reaction. The loss of RF fluorescence in acetate buffer suggests an interaction between RF and acetate ions to promote the photolysis reaction. The optimum stability of RF solutions is observed in the pH range 5–6, which is suitable for pharmaceutical preparations.KEY WORDS: acetate effect, carbonate effect, photolysis, riboflavin, spectrophotometric assay     

Adsorption and Degradation of Doxorubicin from Aqueous Solution in Polypropylene Containers

The purpose of this study was to examine doxorubicin adsorption in polypropylene containers as a function of pH and drug concentration based on anecdotal evidence of such adsorption. Doxorubicin loss was first examined in high-performance liquid chromatography (HPLC) glass inserts by UV absorbance to determine appropriate pH and time durations for subsequent analysis. Doxorubicin loss was then investigated in polypropylene microcentrifuge tubes at different pH values and starting drug concentration at 37°C over 48 h using HPLC with fluorescent detection. Doxorubicin concentrations was essentially constant in HPLC glass inserts at pH 4.8 up to 12 h but declined 5% at pH 7.4 by 3 h. The percent doxorubicin adsorption was calculated in polypropylene microcentrifuge tubes from extrapolations to zero time and was the least at pH 4.8, but increased with pH values 6.5 and 7.4, and decreased with drug concentration to reach a maximum adsorption of 45% in 2.0 μg/mL at pH 7.4 and 37°C. Degradation rate constants, ranging from 0.0021 to 0.019 h⁻¹, also increased with pH in these studies. Determinations of low amounts of doxorubicin in polypropylene containers at pH 7.4 may be underestimated if adsorption and degradation issues are not taken into account.KEY WORDS: adsorption, analysis, chemical stability, doxorubicin, glass, HPLC, polypropylene     

Simple Chromatographic Method for Simultaneous Analyses of Phosphatidylcholine, Lysophosphatidylcholine, and Free Fatty Acids

This study describes a simple chromatographic method for the simultaneous analyses of phosphatidylcholine (PC) and its hydrolytic degradation products: lysophosphatidylcholine (LPC) and free fatty acids (FFA). Quantitative determination of PC, LPC, and FFA is essential in order to assure safety and to accurately assess the shelf life of phospholipid-containing products. A single-run normal-phase high-performance liquid chromatography (HPLC) with evaporative light scattering detector has been developed. The method utilizes an Allsphere silica analytical column and a gradient elution with mobile phases consisting of chloroform: chloroform–methanol (70:30%, v/v) and chloroform–methanol–water–ammonia (45:45:9.5:0.5%, v/v/v/v). The method adequately resolves PC, LPC, and FFA within a run time of 25 min. The quantitative analysis of PC and LPC has been achieved with external standard method. The free fatty acids were analyzed as a group using linoleic acid as representative standard. Linear calibration curves were obtained for PC (1.64–16.3 μg, r² = 0.9991) and LPC (0.6–5.0 μg, r² = 0.9966), while a logarithmic calibration curve was obtained for linoleic acid (1.1–5.8 μg, r² = 0.9967). The detection and quantification limits of LPC and FFA were 0.04 and 0.1 μg, respectively. As a means of validating the applicability of the assay to pharmaceutical products, PC liposome was subjected to alkaline hydrolytic degradation. Quantitative HPLC analysis showed that 97% of the total mass balance for PC could be accounted for in liposome formulation. The overall results show that the HPLC method could be a useful tool for chromatographic analysis, stability studies, and formulation characterization of phospholipid-based pharmaceuticals.KEY WORDS: evaporative light scattering detection, free fatty acid, lysophosphatidylcholine, phosphatidylcholine     

Development and Clinical Evaluation of Clotrimazole–β-Cyclodextrin Eyedrops for the Treatment of Fungal Keratitis

Fungal keratitis is a serious corneal disease that may result in loss of vision. There are limited treatment options available in Iraqi eye hospitals which might be the main reason behind the poor prognosis of many cases. The purpose of this study was to prepare and pharmaceutically evaluate clotrimazole–β-cyclodextrin (CTZ–β-CD) eyedrops then clinically assess its therapeutic efficacy on fungal keratitis compared with extemporaneous amphotericin B eyedrops (0.5% w/v). A CTZ–β-CD ophthalmic solution was prepared and evaluated by various physicochemical, microbiological, and biological tests. The prepared formula was stable in 0.05 M phosphate buffer pH 7.0 at 40 ± 2°C and 75 ± 5% RH for a period of 6 months. Light has no significant effect on the formula’s stability. The CTZ–β-CD eyedrops efficiently complied with the isotonicity, sterility, and antimicrobiological preservative effectiveness tests. Results of the clinical study revealed that 20 (80%) patients showed a favorable response to the CTZ–β-CD eyedrops, while 16 patients (64%) exhibited a favorable response to amphotericin B (P > 0.05). The mean course of treatment was significantly (P < 0.05) less in the CTZ treatment group than in the amphotericin group (21.5 ± 5.2 vs. 28.3 ± 6.4 days, respectively). The CTZ formulation was significantly (P < 0.05) more effective in the management of severe cases and also against Candida sp. than amphotericin B. There was no significant difference (P < 0.05) between both therapies against filamentous fungi. The CTZ–β-CD formulation can be used alternatively to other ophthalmic antimycotic treatment options in developing countries where stability, cost, or efficacy is a limiting factor.Key words: clotrimazole, β-cyclodextrin, eyedrops, fungal keratitis, Iraq     

Controlled Release Matrix Tablets of Olanzapine: Influence of Polymers on the <Emphasis Type="Italic">In Vitro</Emphasis> Release and Bioavailability

Controlled-release (CR) tablet formulation of olanzapine was developed using a binary mixture of Methocel® K100 LV-CR and Ethocel® standard 7FP premium by the dry granulation slugging method. Drug release kinetics of CR tablet formulations F1, F2, and F3, each one suitably compressed for 9-, 12-, and 15-kg hardness, were determined in a dissolution media of 0.1 N HCl (pH 1.5) and phosphate buffer (pH 6.8) using type II dissolution apparatus with paddles run at 50 rpm. Ethocel® was found to be distinctly controlling drug release, whereas the hardness of tablets and pH of the dissolution media did not significantly affect release kinetics. The CR test tablets containing 30% Methocel® and 60% Ethocel® (F3) with 12-kg hardness exhibited pH-independent zero-order release kinetics for 24 h. In vivo performance of the CR test tablet and conventional reference tablet were determined in rabbit serum using high-performance liquid chromatography coupled with electrochemical detector. Bioavailability parameters including C_max, T_max, and AUC_0–48 h of both tablets were compared. The CR test tablets produced optimized C_max and extended T_max (P < 0.05). A good correlation of drug absorption in vivo and drug release in vitro (R² = 0.9082) was observed. Relative bioavailability of the test tablet was calculated as 94%. The manufacturing process employed was reproducible and the CR test tablets were stable for 6 months at 40 ± 2°C/75 ± 5% relative humidity. It was concluded that the CR test tablet formulation successfully developed may improve tolerability and patient adherence by reducing adverse effects.Key words: bioavailability, controlled release, Ethocel®, olanzapine     

The Effect of Acid pH Modifiers on the Release Characteristics of Weakly Basic Drug from Hydrophlilic–Lipophilic Matrices

The solubility of weakly basic drugs within passage though GI tract leads to pH-dependent or even incomplete release of these drugs from extended release formulations and consequently to lower drug absorption and bioavailability. The aim of the study was to prepare and evaluate hydrophilic–lipophilic (hypromellose–montanglycol wax) matrix tablets ensuring the pH-independent delivery of the weakly basic drug verapamil-hydrochloride by an incorporation of three organic acidifiers (citric, fumaric, and itaconic acids) differing in their concentrations, pK_a, and solubility. The dissolution studies were performed by the method of changing pH values, which better corresponded to the real conditions in the GI tract (2 h at pH 1.2 and then 10 h at pH 6.8). Within the same conditions, pH of matrix microenvironment was measured. To determine relationships between the above mentioned properties of acidifiers and the monitored effects (the amount of released drug and surface pH of gel layer in selected time intervals—360 and 480 min), the full factorial design method and partial least squares PLS-2 regression were used. The incorporation of the tested pH modifiers significantly increased the drug release rate from matrices. PLS-components explained 75% and 73% variation in the X- and Y-data, respectively. The obtained results indicated that the main crucial points (p < 0.01) were the concentration and strength of acidifier incorporated into the matrix. Contrary, the acid solubility surprisingly did not influence the selected effects except for the surface pH of gel layer in time 480 min.Key words: gel layer, matrix tablets, pH-independent drug release, pH modifiers, statistical evaluation     

Characterization of the kinetic and thermodynamic landscape of RNA folding using a novel application of isothermal titration calorimetry

A novel isothermal titration calorimetry (ITC) method was applied to investigate RNA helical packing driven by the GAAA tetraloop–receptor interaction in magnesium and potassium solutions. Both the kinetics and thermodynamics were obtained in individual ITC experiments, and analysis of the kinetic data over a range of temperatures provided Arrhenius activation energies (ΔH^‡) and Eyring transition state entropies (ΔS^‡). The resulting rich dataset reveals strongly contrasting kinetic and thermodynamic profiles for this RNA folding system when stabilized by potassium versus magnesium. In potassium, association is highly exothermic (ΔH_25°C = −41.6 ± 1.2 kcal/mol in 150 mM KCl) and the transition state is enthalpically barrierless (ΔH^‡ = −0.6 ± 0.5). These parameters are sigificantly positively shifted in magnesium (ΔH_25°C = −20.5 ± 2.1 kcal/mol, ΔH^‡ = 7.3 ± 2.2 kcal/mol in 0.5 mM MgCl₂). Mixed salt solutions approximating physiological conditions exhibit an intermediate thermodynamic character. The cation-dependent thermodynamic landscape may reflect either a salt-dependent unbound receptor conformation, or alternatively and more generally, it may reflect a small per-cation enthalpic penalty associated with folding-coupled magnesium uptake.     

Biochemical studies on a versatile esterase that is most catalytically active with polyaromatic esters

Herein, we applied a community genomic approach using a naphthalene-enriched community (CN1) to isolate a versatile esterase (CN1E1) from the α/β-hydrolase family. The protein shares low-to-medium identity (≤ 57%) with known esterase/lipase-like proteins. The enzyme is most active at 25–30°C and pH 8.5; it retains approximately 55% of its activity at 4°C and less than 8% at ≥ 55°C, which indicates that it is a cold-adapted enzyme. CN1E1 has a distinct substrate preference compared with other α/β-hydrolases because it is catalytically most active for hydrolysing polyaromatic hydrocarbon (phenanthrene, anthracene, naphthalene, benzoyl, protocatechuate and phthalate) esters (7200–21 000 units g⁻¹ protein at 40°C and pH 8.0). The enzyme also accepts 44 structurally different common esters with different levels of enantio-selectivity (1.0–55 000 units g⁻¹ protein), including (±)-menthyl-acetate, (±)-neomenthyl acetate, (±)-pantolactone, (±)-methyl-mandelate, (±)-methyl-lactate and (±)-glycidyl 4-nitrobenzoate (in that order). The results provide the first biochemical evidence suggesting that such broad-spectrum esterases may be an ecological advantage for bacteria that mineralize recalcitrant pollutants (including oil refinery products, plasticizers and pesticides) as carbon sources under pollution pressure. They also offer a new tool for the stereo-assembly (i.e. through ester bonds) of multi-aromatic molecules with benzene rings that are useful for biology, chemistry and materials sciences for cases in which enzyme methods are not yet available.Microorganisms play a crucial role in soil genesis by facilitating mineralization not only for soil organic matter (Leigh Mascarelli, 2009) but also for prevalent and persistent pollutants, such as polyaromatic hydrocarbons (PAH) and heterocyclic aromatic compounds (Lu et al., 2011). Such compounds are common additives in crude oil and industrial chemical products, such as dyes, flavouring compounds, plasticizers, perfumes, pesticides and insect repellent as well as, more recently, microelectronics, printed circuit boards, silk screen printing devices, optical disks and black colour tube matrices (Kästner, 2000; Chae et al., 2002). Multiple microorganisms can obtain energy from such hydrophobic aromatics (Lu et al., 2011), which are primarily released into the environment through anthropogenic activities. When bacteria are confronted with aromatic compounds, the cells encounter an interesting contradiction. On the one hand, such chemical species can be mineralized to yield carbon and energy for growth, which allows microorganisms to colonize niches that are refractory to other microbes (Dominguez-Cuevas et al., 2006; Lu et al., 2011). On the other hand, aromatic compounds over a certain threshold are toxic for bacteria because they partition and disorganize the cell membrane by removing lipids and proteins, which leads to cell death (Sikkema et al., 1995; von Wallbrunn et al., 2003). To cope with such activities, bacteria have developed multiple chemical tolerance mechanisms and an extensive enzyme arsenal (e.g. oxygenases, O-demetylases, CoA synthases and ligases, aldolases, alcohol dehydrogenases and α/β hydrolases) for mineralizing aromatic compounds by submitting them to the central metabolism (Pérez-Pantoja et al., 2008; 2009; Vilchez-Vargas et al., 2013).Recent studies suggest that multifunctional esterase/lipase-like proteins from the α/β hydrolase family that can hydrolyse both C-C and C-O bonds may exist in nature at much higher levels than previously thought (Alcaide et al., 2013). From an ecological perspective, such proteins may contribute to global carbon cycling processes for complex substrates, including recalcitrant organic pollutants. From a biotechnological perspective, such proteins may open unexpected research avenues for biotechnology applications. Esterases/lipases from the α/β hydrolase family have demonstrated such activities for catechol and biphenyl derivatives (Alcaide et al., 2013). However, the potential implication for the degradation of and biotechnology of complex organic molecules with two or more aromatic rings, specifically benzene rings (polyaromatic hydrocarbons – PAH), has not been described, even though the PAH degradation phenomenon is relatively well known (Pérez-Pantoja et al., 2008; 2009; Seo et al., 2009). Such activities are especially significant because the chemistry for PAH esters has been a subject of considerable interest due to their chemical and physical properties (e.g. polymers produced with versatile photo-reactivities) (Noh et al., 2001; Chae et al., 2002; Zhu et al., 2011). Heterogeneous catalysts have successfully been used to produce PAH-like (naphthalene, anthracene, phenanthrene, benzyl and phthalate) derivatives (Noh et al., 2001; Zhu et al., 2011; Maruyama et al., 2012). However, to the best of our knowledge, no study in the specialized literature has involved identification of potential applications for esterase/lipase-like proteins as catalysts for modifying PAHs.Because contaminated environments and microbial communities derived therefrom are an excellent source for enzymes that act on aromatics (Pérez-Pantoja et al., 2008; 2009; Guazzaroni et al., 2013), we now ask whether such α/β hydrolases can be identified therein. We generated a subset of 5500 clones (in Escherichia coli EPI300-T1^R; Epicentre Biotechnologies; Madison, WI, USA; Alcaide et al., 2013) from a naphthalene-enriched community (CN1) derived from PAH-contaminated soil (Guazzaroni et al., 2013), which included nearly 0.17 Gbp of community genomes, that were evaluated for their ability to hydrolyse α-naphthyl acetate (Reyes-Duarte et al., 2012). Four unique positive clones (hit rate 1:1375) were identified as active, and one (herein, CN1E1) was selected for in-depth analysis due to its high activity (halo/colour formation) and capacity for hydrolysis in agar-based assays using the model PAH ester naphthalene carboxylic acid methyl ester (Fig. 1). The insert [30 164 bp; nucleotide sequence available at the National Center for Biotechnology Information (NCBI) under accession number SRP030024] was sequenced [Roche 454 GS FLX Ti sequencer (454 Life Sciences, Branford, CT, USA) at LifeSequencing S.L., Valencia, Spain], analysed and compared with the sequences in the NCBI non-redundant public database (Altschul et al., 1997). Thirty-one predicted open reading frames were identified (Lukashin and Borodovsky, 1998); one encoded a putative esterase (CN1E1) with the α/β hydrolase fold. The protein [311 amino acids (AA); molecular weight (MW) 33 146 Da; isoelectric point (pI) 6.34] was produced in a soluble form (Fig. 2) upon expression in the pET-46 Ek/LIC vector (using the forward and reverse primers 5′-GACGACGACAAGATGGCGGTAGATCCG-3′ and 5′-GAGGAGAAGCCCGGTTATCTCGGTCCGGC-3′ respectively) and E. coli BL21 (DE3) (conditions described in Alcaide et al., 2013). The sequence was analysed, which indicated that it belonged to the α/β hydrolase superfamily; the esterase/lipase-like protein from Paenibacillus mucilaginosus 3016 ({"type":"entrez-protein","attrs":{"text":"YP_005313749.1","term_id":"379721618","term_text":"YP_005313749.1"}}YP_005313749.1) is the most similar enzyme (AA sequence identity: 54%; similarity: 68%). It was also homologous (≤ 56% identity) to predicted esterases/lipases from uncultured microorganisms derived from soil and marine sediment samples (Fig. 3). This enzyme can be categorized in the microbial family IV described by Arpigny and Jaeger (1999); it includes a typical -GlyxSerxGly-motif and presumptive Ser-Asp-His catalytic triad (Ser155, Asp250 and His280). CN1E1 was structurally most similar to a lipase/esterase-like protein from Alicyclobacillus acidocaldarius [identity: 45%; Protein Data Bank (PDB) code 2HM7_A].Open in a separate windowFigure 1Hydrolytic phenotype for E. coli BL21 (DE3) that either expressed or did not express CN1E1 using the pET-46 Ek/LIC vector. The cells were plated on fresh Luria Bertani (LB) plates with ampicillin (50 μg ml⁻¹). The plates [with 1.0 mM isopropyl-β-D-galactopyranoside (IPTG)] were incubated for 12 h at 37°C and then covered with a second layer that included the substrate [20 ml 5 mM N-(2-hydroxyethyl) piperazine-N′-(3-propanesulfonic acid) (EPPS) buffer, pH 8.0, 0.4% agarose and 320 μl of a naphthalene carboxylic acid methyl ester solution in acetonitrile (100 mg ml⁻¹)]. The active phenotype was apparent due to the colour change resulting from substrate hydrolysis and acetic acid formation, which changes the pH indicator from red to yellow.Open in a separate windowFigure 2A Coomassie-stained SDS-PAGE gel showed that the active form of the protein CN1E1 was over-expressed in E. coli at 16°C. The expression and purification conditions were reported in Martínez-Martínez and colleagues (2013). As shown, a high percentage of the protein was produced in its soluble form, which yielded a purity greater than 98% after a single His₆-tag purification step. Abbreviation: MW, molecular weight marker. The lanes include the following: lane 1, soluble cell fraction after induction with 1.0 mM IPTG; lane 2, soluble cell fraction without IPTG; lane 3, molecular weight marker (from top to bottom: 225, 150, 100, 75, 50, 35, 25 and 15 kDa); and lane 4, pure protein after His₆-tag purification.Open in a separate windowFigure 3The unrooted circular neighbour-joining tree indicates the polypeptide sequence phylogenetic positions for the CN1E1 enzyme (in boldface) and reference hydrolases. The tree was constructed using an aligned 297 AA-long sequence. The GenBank and PDB accession numbers are indicated in brackets. For the dendrogram construction details, see Martínez-Martínez and colleagues (2013) and Tamura and colleagues (2007). The Family V cluster includes the sequences with the following accession numbers: YP960710.1, {"type":"entrez-protein","attrs":{"text":"ADP98993.1","term_id":"311696120","term_text":"ADP98993.1"}}ADP98993.1, ZP01735705.1, ZP01735705.1, {"type":"entrez-protein","attrs":{"text":"CAE54381.1","term_id":"50871368","term_text":"CAE54381.1"}}CAE54381.1, ZP01307774.1, {"type":"entrez-nucleotide","attrs":{"text":"X53869","term_id":"44522","term_text":"X53869"}}X53869, {"type":"entrez-protein","attrs":{"text":"AEO74498.1","term_id":"347304384","term_text":"AEO74498.1"}}AEO74498.1, YP001347584.1, NP251639.1, YP790224.1, YP0029804424.1, YP002909304.2, YP001810250.1, ZP03570306.1, YP299126.1, YP583166.1, YP002005156.1, 725653.1 and YP006884923.1. The Family VI cluster includes YP007541785, WP007625024, WP008294645, S78600 and PDB 3CN7. The scale bar represents 0.2 substitutions per position. The lipase/esterase families are depicted based on the Arpigny and Jaeger (1999) classification system. The abbreviations are as follows: EST, esterase; LIP, lipase; HYP, hypothetical protein; ACTIN, Actinobacteria; FIR, Firmicutes; PLANCT, Planctomycetes; PROT, Proteobacteria; UNCB, uncultured bacterium.The pure enzyme was most active at 25–30°C and pH 8.5 (Fig. 4); it retained approximately 55% of its activity at 4°C and less than 8% at ≥ 55°C, which indicates that it is a cold-adapted enzyme. The substrates used herein included 12 model esters [6 p-nitrophenol (pNP) and 6 triacylglycerol] and 86 structurally different esters (Martínez-Martínez et al., 2013). Based on the specific activities determined (units g⁻¹; one unit is the amount of enzyme that hydrolyses 1 μmol of substrate per min under the assay conditions, as reported by Martínez-Martínez et al., 2013), CN1E1 showed a capacity for accepting 61 esters with different alcohol and acid moieties (Fig. 5). Considering the acyl chain length and p-nitrophenyl ester hydrolysis, CN1E1 was most active with pNP-acetate (∼ 55 000 units g⁻¹ at 40°C and pH 8.0); it also hydrolysed pNP-dodecanoate, albeit at a rate of four orders of magnitude lower. As shown in Fig. 5, pNP esters were the preferred substrates among the examined esters. This enzyme hydrolysed short-chain triacylglycerols that ranged from triacetin (which was preferred; ∼ 23 000 units g⁻¹ at 40°C and pH 8.0) to tricaproin (∼ 21 units g⁻¹) and short-chain halogenated and non-halogenated alkyl and aryl esters; methyl bromoacetate (∼ 31 000 units g⁻¹) and butyl acetate (∼ 11 700 units g⁻¹) were preferred respectively (Fig. 5). Because the enzyme preferred short-chain triacylglycerols and short-to-medium size alkyl and aryl esters, the α/β-hydrolase is likely an esterase. It also showed a capacity for accepting tri-O-acetyl-glucal (∼ 12 500 units g⁻¹), the carbohydrate ester α-D-glucose pentaacetate (∼ 373 units g⁻¹) and hydroxycinnamic acid-like esters, such as 2,5-dihydroxycinnamic acid methyl ester (∼ 10 700 units g⁻¹), methyl cinnamate (∼ 28 units g⁻¹) and caffeic acid phenethyl ester (∼ 19 units g⁻¹) (Fig. 5). Such characteristics suggest that the enzyme can support polysaccharide degradation. Amino acid esters, such as L-proline and L-serine esters, were also hydrolysed (∼ 24–1.2 units g⁻¹); however, they were among the non-preferred substrates (Fig. 5). Under our assay conditions, the esterase was also active (from ∼ 609 to 5.2 units g⁻¹) and enantio-selective for (±)-menthyl-acetate, (±)-methyl lactate, (±)-neomenthyl-acetate, (±)-methyl-mandelate and (±)-glycidyl 4-nitrobenzoate with an (S)-preference (Fig. 5). γ-Butyrolactone (∼ 141 units g⁻¹), γ-valerolactone (∼ 81 units g⁻¹) and (±)-pantolactone [the (R) enantiomer was preferred; ∼ 606 units g⁻¹] were also accepted as substrates (Fig. 5).Open in a separate windowFigure 4The wild-type CN1E1 α/β hydrolase pH (A) and temperature (B) profiles. Esterase activity using pNP-propionate (at 410 nm) was determined as described previously (Martínez-Martínez et al., 2013). The standard deviation (SD) for the triplicate assays is shown. We examined pH values between 4.0 and 9.5 and temperatures between 4 and 80°C to determine the optimal parameters. The following buffers were examined at 40 mM: sodium citrate (pH 4.0–4.5), sodium acetate (pH 5.0–6.0), 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.5–6.0), piperazine-N,N′-bis(ethanesulfonic acid) (PIPES) (pH 6.0–7.0), 4-(2-hydroxyethylpiperazine-1-ethanesulfonic acid (HEPES) (pH 7.0–8.0), K/Na-phosphate (pH 7.5), Tris-HCl (pH 8.5) and glycine (pH 9.0–9.5). The pH was adjusted at 25°C. The pH and temperature profiles were collected at 40°C (panel A) and pH 7.0 (using 40 mM HEPES; panel B) respectively. In panels (A) and (B), 100% of the activity refers to 27.08 ± 1.80 and 21.17 ± 1.41 units mg⁻¹ respectively.Open in a separate windowFigure 5Substrate profile for the wild-type CN1E1 α/β hydrolase using a set of structurally diverse esters. The specific activities were calculated in triplicate as described by Martínez-Martínez and colleagues (2013) at 40°C in 20 mM HEPES buffer pH 7.0 (for pNP esters) or 5 mM EPPS buffer pH 8.0 (for the remaining esters). The standard deviation (SD) for the triplicate assays is shown. The chemicals used for the enzymatic experiments were the purest grade available and were purchased from Fluka-Aldrich-Sigma Chemical Co. (St. Louis, MO, USA). The hydroxycinnamic-like esters were supplied by Apin Chemicals (Oxon, UK), the methyl phenanthrene-3-carboxylate and methyl phenanthrene-9-carboxylate were supplied by Wuhan Farthest Chemical (Mainland, China), and the anthracene-9-carboxylic acid methyl ester and anthracene-3-carboxylic acid methyl ester were obtained from Alfa Aesar (Karlsruhe, Germany). Insets (A) to (C) in the figure represent a zoom for substrates hydrolysed at low rates.Because the active clone with CN1E1 actively hydrolysed naphthalene carboxylic acid methyl ester on an agar plate (Fig. 1), we further tested and quantified hydrolysis for the pure enzyme using this and other model PAH esters; the pH-indicator protocol (at 40°C and pH 8.0) used to examine hydrolysis for the above esters was also used for this experiment (Martínez-Martínez et al., 2013). CN1E1 showed broad reactivity towards PAH-like esters, including phthalate alkyl and aryl (benzene) esters (from ∼ 21 000 to 6100 units g⁻¹), alkyl esters of phenanthrene (from ∼ 20 500 to 7600 units g⁻¹), anthracene (from ∼ 18 400 to 11 000 units g⁻¹), naphthalene (from ∼ 10 000 to 7800 units g⁻¹) and protocatechuate (∼ 8800 units g⁻¹) and benzoate esters with substituent phenyl/benzene rings (from ∼ 7600 to 7200 units g⁻¹). The majority of such compounds were among the best substrates for CN1E1 (Fig. 5); this enzyme showed regio-selectivity for phenanthrene-3-carboxylate over methyl phenanthrene-9-carboxylate (2.7-fold) and, to a lesser extent, for anthracene-2-carboxylate over anthracene-9-carboxylate (1.7-fold). Although previous reports have shown that other esterases can degrade phthalate esters (Maruyama et al., 2005; Saito et al., 2010; Wu et al., 2013), to the best of our knowledge, the specialized literature has not reported an esterase with the capacity to degrade PAH esters with multiple aromatic (e.g. benzene) rings.The half-saturation (Michaelis) coefficient (K_m), catalytic rate constant (k_cat) and catalytic efficiency (k_cat/K_m) values were determined for the 14 best and structurally distinct substrates (2013) at pH 8.0 and 40°C (except for pNP-acetate were pH 7.0 was used to ensure substrate stability). The substrates analysed include pNP-acetate (the model p-nitrophenyl ester), triacetin (the model triacylglycerol), methyl bromoacetate (the model halogenated ester), tri-O-acetyl-glucal (the model polysaccharide-like ester), butyl acetate (the model alkyl ester), phenyl acetate (the model aryl ester), 2,5-dihydroxycinnamic acid methyl ester (the model hydroxycinnamic-like ester) and seven chemically distinct model PAH esters. As shown in 2013)

Effect of Counterion on the Solid State Photodegradation Behavior of Prazosin Salts

The effect of counterion was evaluated on the photodegradation behavior of six prazosin salts, viz., prazosin hydrochloride anhydrous, prazosin hydrochloride polyhydrate, prazosin tosylate anhydrous, prazosin tosylate monohydrate, prazosin oxalate dihydrate, and prazosin camsylate anhydrous. The salts were subjected to UV–Visible irradiation in a photostability test chamber for 10 days. The samples were analyzed for chemical changes by a specific stability-indicating high-performance liquid chromatography method. pH of the microenvironment was determined in 10% w/v aqueous slurry of the salts. The observed order of photostability was: prazosin hydrochloride anhydrous > prazosin camsylate anhydrous ∼ prazosin-free base > prazosin hydrochloride polyhydrate > prazosin tosylate anhydrous > prazosin oxalate dihydrate ∼ prazosin tosylate monohydrate. Multivariate analysis of the photodegradation behavior suggested predominant contribution of the state of hydration and also intrinsic photosensitivity of the counterion. Overall, hydrated salts showed higher photodegradation compared to their anhydrous counterparts. Within the anhydrous salts, aromatic and carbonyl counterion-containing salts showed higher susceptibility to light. The pH of microenvironment furthermore contributed to photodegradation of prazosin salts, especially for drug counterions with inherent higher pH. The study reveals importance of selection of a suitable drug salt form for photosensitive drugs during preformulation stage of drug development.     

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l⁻¹ of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l⁻¹ ammonium sulphate {(NH₄) ₂SO₄} (MMS3) + BA (0.25 mg l⁻¹), Kinetin (Kin; 0.25 mg l⁻¹), Indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l⁻¹ of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.     

Antioxidants and ROS scavenging ability in ten Darjeeling tea clones may serve as markers for selection of potentially adapted clones against abiotic stress

Ten Darjeeling tea clones (BT15/263, RR17/144, B777, T253, B157, Sundaram, HV39, AV2, K1/1 and TTV1) were collected from the experimental garden of Darjeeling Tea Research and Development Centre, Kurseong. Total phenol, flavonoids and two antioxidating enzymes (peroxidase and superoxide dismutase) were estimated. The total phenol ranged between 241 and 28 GAE mg g⁻¹ of leaf dry weight. The highest amount obtained in four clones, B15/263 (241.47), RR17/144 (221.2), B777 (154.54) and B157 (140.23 mg g⁻¹). Flavonoids were estimated as Catechin equivalent (CE) and ranged between 56.88 and 20.81 CE mg g⁻¹ leaf dry weight. Higher amounts occurred in BT15/263 (56.88 mg g⁻¹), B777 (56.69) and RR17/144 (48.63). Antioxidant activities were measured following DPPH and ABTS free radicle scavenging procedures and the results were well according to total polyphenol content among the clones (in total phenols, ranges of correlation in DPPH assay were r² = 0.990–0.989, p ≤ 0.05; in flavonoids r² = 0.954, p ≤ 0.01–0.987, p ≤ 0.05). Similarly, ABTS percent scavenging results were quiet significant. The IC₅₀ values were determined for both DPPH and ABTS assay. PAGE expressions of isoforms in two antioxidative enzymes and quantification of them also varied much among the investigated clones. The incidence of total phenols, flavonoids, PRX and SOD and ROS scavenging assay in in-situ condition, might be used as biochemical markers towards the superior adaptability against abiotic stress. In the present work, four clones (B15/263, B777, RR17/144 and B157) would be designated as comparatively better suited to the predicted abiotic stress.     

Microscopic Modeling of País Grape Seed Extract Absorption in the Small Intestine

The concentration profiles and the absorbed fraction (F) of the País grape seed extract in the human small intestine were obtained using a microscopic model simulation that accounts for the extracts'' dissolution and absorption. To apply this model, the physical and chemical parameters of the grape seed extract solubility (C_s), density (ρ), global mass transfer coefficient between the intestinal and blood content (k) (effective permeability), and diffusion coefficient (D) were experimentally evaluated. The diffusion coefficient (D = 3.45 × 10⁻⁶ ± 5 × 10⁻⁸ cm²/s) was approximately on the same order of magnitude as the coefficients of the relevant constituents. These results were chemically validated to discover that only the compounds with low molecular weights diffused across the membrane (mainly the (+)-catechin and (−)-epicatechin compounds). The model demonstrated that for the País grape seed extract, the dissolution process would proceed at a faster rate than the convective process. In addition, the absorbed fraction was elevated (F = 85.3%). The global mass transfer coefficient (k = 1.53 × 10⁻⁴ ± 5 × 10⁻⁶ cm/s) was a critical parameter in the absorption process, and minor changes drastically modified the prediction of the extract absorption. The simulation and experimental results show that the grape seed extract possesses the qualities of a potential phytodrug.KEY WORDS: dose absorption, mathematical modeling, País grape seed extract, simulation     

Design and In Vitro Evaluation of Finasteride-Loaded Liquid Crystalline Nanoparticles for Topical Delivery

In this study, liquid crystalline nanoparticles (LCN) have been proposed as new carrier for topical delivery of finasteride (FNS) in the treatment of androgenetic alopecia. To evaluate the potential of this nanocarrier, FNS-loaded LCN was prepared by ultrasonication method and characterized for size, shape, in vitro release, and skin permeation–retention properties. The particle size ranged from 153.8 to 170.2 nm with a cubical shape and exhibited controlled release profile with less than 20% of the drug released in the first 24 h. The release profile was significantly altered with addition of different additives. Formulation with lower monoolein exhibited higher skin permeation with a flux rate of 0.061 ± 0.005 μg cm⁻² h⁻¹ in 24 h. The permeation however, significantly increased with glycerol, propylene glycol, and polyethylene glycol 400, while it declined for the addition of oleic acid. A similar trend was observed with skin retention study. In conclusion, FNS-loaded LCN could be advocated as a viable alternative for oral administration of the drug.Key words: androgenetic alopecia, finasteride, liquid crystalline nanoparticles, release, skin permeation–retention     

Development of bioelectrocatalytic activity stimulates mixed‐culture reduction of glycerol in a bioelectrochemical system

In a microbial bioelectrochemical system (BES), organic substrate such as glycerol can be reductively converted to 1,3-propanediol (1,3-PDO) by a mixed population biofilm growing on the cathode. Here, we show that 1,3-PDO yields positively correlated to the electrons supplied, increasing from 0.27 ± 0.13 to 0.57 ± 0.09 mol PDO mol⁻¹ glycerol when the cathodic current switched from 1 A m⁻² to 10 A m⁻². Electrochemical measurements with linear sweep voltammetry (LSV) demonstrated that the biofilm was bioelectrocatalytically active and that the cathodic current was greatly enhanced only in the presence of both biofilm and glycerol, with an onset potential of −0.46 V. This indicates that glycerol or its degradation products effectively served as cathodic electron acceptor. During long-term operation (> 150 days), however, the yield decreased gradually to 0.13 ± 0.02 mol PDO mol⁻¹ glycerol, and the current–product correlation disappeared. The onset potentials for cathodic current decreased to −0.58 V in the LSV tests at this stage, irrespective of the presence or absence of glycerol, with electrons from the cathode almost exclusively used for hydrogen evolution (accounted for 99.9% and 89.5% of the electrons transferred at glycerol and glycerol-free conditions respectively). Community analysis evidenced a decreasing relative abundance of Citrobacter in the biofilm, indicating a community succession leading to cathode independent processes relative to the glycerol. It is thus shown here that in processes where substrate conversion can occur independently of the electrode, electroactive microorganisms can be outcompeted and effectively disconnected from the substrate.