10mM Phosphate Buffer Research Articles

Quantification of the Plasma Concentration of Vadadustat by High-Performance Liquid Chromatography with Ultraviolet Detection and Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

An inexpensive, simple, and accurate plasma concentration measurement system is needed to actively conduct pharmacokinetic and pharmacodynamic analyses of vadadustat, hypoxia-inducible factor-prolyl hydroxylase inhibitor, in clinical settings. In this study, the authors aimed to develop a method for measuring vadadustat in human plasma that could be applied for therapeutic drug monitoring using high-performance liquid chromatography with ultraviolet detection (HPLC-UV) in a clinical setting. Plasma samples (100 μL) were pretreated with acetonitrile using butyl paraoxybenzoate as an internal standard. Chromatographic separation was performed on a SunShell PFP C18 column (2.6 μm, 4.6 mm × 150 mm). The mobile phase consisted of (A) 20 mM of phosphate buffer (pH 2.4) and (B) acetonitrile (60:40, v/v), delivered isocratically at a flow rate of 1 mL/min. The analytes were detected by UV absorbance at a wavelength of 220 nm, and the column temperature was 40°C. To evaluate the applicability of HPLC-UV in a clinical setting, blood samples were collected at 19 time points from 7 patients who had been taking vadadustat. The calibration curve was linear over the concentration range of 0.2-150 mcg/mL (R2 > 0.99). Intra-assay and interassay accuracy, precision, and stability met the Food and Drug Administration recommendations. The vadadustat plasma concentrations of patients analyzed using the current HPLC-UV method were almost equal to those measured using ultra-performance liquid chromatography-tandem mass spectrometry (mean difference: 0.13 mcg/mL). Large variability in the dose-adjusted plasma concentrations of vadadustat at 12 hours after administration was observed between patients (coefficient of variation = 57.6%). This HPLC-UV method is a simple, accurate quantification method for evaluating plasma concentrations in patients taking vadadustat in a clinical setting.

Rapid Detection of the Anti-Tumor Drug Etoposide in Biological Samples by Using a Nanoporous-Gold-Based Electrochemical Sensor.

Monitoring etoposide is important due to its wide usage in anti-tumor therapy; however, the commonly used HPLC method is expensive and often requires complicated extraction and detection procedures. Electrochemical analysis has great application prospects because of its rapid response and high specificity, sensitivity, and efficiency with low cost and high convenience. In this study, we constructed a nanoporous gold (NPG)-modified GCE for the detection of etoposide. The electrochemical oxidation of etoposide by NPG caused a sensitive current peak at +0.27 V with good reproductivity in 50 mM of phosphate buffer (pH 7.4). The relationship between etoposide concentration and peak current was linear in the range between 0.1 and 20 μM and between 20 and 150 μM, with a detection sensitivity of 681.8 μA mM-1 cm-2 and 197.2 μA mM-1 cm-2, respectively, and a limit of detection (LOD) reaching 20 nM. The electrode had a good anti-interference ability to several common anions and cations. Spiked recovery tests in serum, urine, and fermentation broth verified the excellent performance of the sensor in terms of sensitivity, reproducibility, and specificity. This may provide a promising tool for the detection of etoposide in biological samples.

The antihypertensive action of C-phycocyanin is related to the prevention of angiotensin II-caused vascular dysfunction in chronic kidney disease.

C-phycocyanin (CPC) is a photosynthetic protein found in Arthrospira maxima with a nephroprotective and antihypertensive activity that can prevent the development of hemodynamic alterations caused by chronic kidney disease (CKD). However, the complete nutraceutical activities are still unknown. This study aims to determine if the antihypertensive effect of CPC is associated with preventing the impairment of hemodynamic variables through delaying vascular dysfunction. Twenty-four normotensive male Wistar rats were divided into four groups: (1) sham + 4 mL/kg/d vehicle (100 mM of phosphate buffer, PBS) administered by oral gavage (og), (2) sham + 100 mg/kg/d og of CPC, (3) CKD induced by 5/6 nephrectomy (CKD) + vehicle, (4) CKD + CPC. One week after surgery, the CPC treatment began and was administrated daily for four weeks. At the end treatment, animals were euthanized, and their thoracic aorta was used to determine the vascular function and expression of AT1, AT2, and Mas receptors. CKD-induced systemic arterial hypertension (SAH) and vascular dysfunction by reducing the vasorelaxant response of angiotensin 1-7 and increasing the contractile response to angiotensin II. Also, CKD increased the expression of the AT1 and AT2 receptors and reduced the Mas receptor expression. Remarkably, the treatment with CPC prevented SAH, renal function impairment, and vascular dysfunction in the angiotensin system. In conclusion, the antihypertensive activity of CPC is associated with avoiding changes in the expression of AT1, AT2, and Mas receptors, preventing vascular dysfunction development and SAH in rats with CKD.

Purification of thioredoxin reductase from Spirulina platensis by affinity chromatography and investigation of kinetic properties

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and nicotinamide adenine dinucleotide phosphate (NADPH). Spirulina platensis, which is one of the blue-green algae in the form of spiral rings, belongs to the cyanobacteria class. Spirulina platensis can produce Trx under stress conditions. If it can produce Trx, it also has TrxR activity. Therefore, in this study, the TrxR enzyme was purified for the first time from Spirulina platensis, an algae the most grown and also used as a nutritional supplement in the world. A two-step purification process was used: preparation of the homogenate and 2′,5′-ADP sepharose 4B affinity chromatography. The enzyme was purified with a purification fold of 1059.51, a recovery yield of 9.7 %, and a specific activity of 5.77 U/mg protein. The purified TrxR was tested for purity by SDS-PAGE. The molecular weight of its subunit was found to be about 45 kDa. Optimum pH, temperature and ionic strength of the enzyme were pH 7.0, 40 °C and 750 mM in phosphate buffer respectively. The Michaelis constant (Km) and maximum velocity of enzyme (Vmax) values for NADPH and 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) are 5 μM and 2.2 mM, and 0.0033 U/mL and 0.0044 U/mL, respectively. Storage stability of the purified enzyme was determined at several temperatures. The inhibition effects of Ag+, Cu2+, Al3+ and Se4+ metal ions on the purified TrxR activity were investigated in vitro. While Se4+ ion increased the enzyme activity, other tested metal ions showed different type of inhibitory effects on the Lineweaver–Burk graphs.

Structural Elucidation of Alkali Degradation Impurities of Favipiravir from the Oral Suspension: UPLC-TQ-ESI-MS/MS and NMR.

A novel stability-indicating, reversed-phase, high-performance liquid chromatography (RP-HPLC) method was developed and validated for the determination of favipiravir in an oral suspension. The effective separation of favipiravir and its degradation products was achieved on a Zorbax Eclipse Plus C18 column (5 μm particle size, 150 mm length × 4.6 mm diameter). The mobile phase was prepared by mixing 5 mM of phosphate buffer (pH 3.5) and methanol in a 75:25 v/v ratio delivered at a 1.0 mL/min flow rate. The eluents were monitored using a photodiode array detector at a wavelength of 322 nm. The stability-indicating nature of this method was evaluated by performing force degradation studies under various stress conditions, such as acidic, alkali, oxidative, thermal, and photolytic degradation. Significant degradation was observed during the alkali stress degradation condition. The degradation products generated during various stress conditions were well separated from the favipiravir peak. In addition, the major degradation product formed under alkali stress conditions was identified using UPLC-ESI-TQ-MS/MS and NMR. Method validation was performed according to the ICH Q2 (R1) guideline requirements. The developed method is simple, accurate, robust, and reliable for routine quality control analysis of favipiravir oral suspensions.

Sensitivity Enhancement of Pre-Capillary Chelation Method for the Separation of Metal Ions: Experimental and DFT Study

Background: Heavy metal toxicity has proven to be a major threat and there are several health risks associated with it. The toxic effects of these metals, even though they do not have any biological role, remain present in some or the other form harmful for the human body and its proper functioning. They sometimes act as a pseudo element of the body while at certain times they may even interfere with metabolic processes. Sensitivity enhancement and selective pre-capillary chelation and separation method was developed for the simultaneous determination of metal ions by capillary zone electrophoresis (CZE) with UV light as a detector. Method: This method was based on chelation of metal ions such as nickel(II), cobalt(II), lead(II) and zinc(II) with 2.0 mM Ammonium Morpholine-4-Carbodithioate (AMC) at pH 7.2 prior to analysis in 2.0 mM of phosphate buffer. Results: Different optimal conditions such as effect of pH, concentration of AMC, applied voltage, nature of the buffer solution and excipient ions were investigated to enhance the sensitivity of the method. Conclusion: The developed method separate nickel(II), cobalt(II), lead(II) & zinc(II) in less than 5 min with good reproducibility and recoveries ranging from 93.50 to 100.00 % in agricultural materials. Furthermore, the interaction and Density Functional Theory (DFT) based studies reveal that the metal ions form relatively stable complexes with AMC and follows the experimental trend performed with CZE.

Optimization of immobilization of Pseudomonas cepacia lipase on multiwalled carbon nanotubes functionalized with glycyrrhizin and Tween 80.

In the present study, multiwalled carbon nanotubes (MWCNTs) were functionalized with glycyrrhizin and Tween 80 and applied for immobilization of Pseudomonas cepacia lipase (PcL). Characterization of f-MWCNTs was performed through Fourier-transform infrared spectroscopy, thermal gravimetric, field emission scanning electron microscopy, and energy-dispersive X-ray spectroscopy analysis. The optimum specific activity of immobilized PcL (studied by Plackett-Burman statistical design) occurred at 0.3 mg/mL of f-MWCNTs, 25 mM of phosphate buffer (pH 6.0), 15 min sonication time, 8 U/mL of enzyme concentration, and 24 h immobilization time at 4 °C in the absence of glutaraldehyde. In these conditions, the specific activity was 16.57 ± 0.71 U/mg, which was very close to the predicted amount (16.62 ± 0.64 U/mg). The results of thermal and pH stability showed that the stability of immobilized PcL was higher than that of the free PcL. The activity of immobilized PcL on f-MWCNTs held 93% after being incubated for 60 min at 70 °C. Moreover, the immobilized PcL on f-MWCNTs retained about 65% of its initial activity after 30 days of storage at 25 °C. In addition, about 50% of initial activity of immobilized PcL retained after 10 cycles of uses. Therefore, f-MWCNTs could be introduced as suitable support for enzymes immobilization.

On-Site and Daily Stress-Measurement Using Gold-Linked Electrochemical Immuno Assay for IgA Determination

[Introduction]Mental stress is closely connected with our physical and mental wellness. In particular, depression may be associated with stress. Therefore, stress measurement can contribute to assess our lifestyle and increase our quality of life. However, objective stress-measurement is not established. To establish objective stress-measurement, an individual must indicate the stress level by obtaining numerous data continuously throughout the day. Therefore, the measurement method which can be suitable for this condition is needed. In this paper, we detect the secretory Immunoglobulin A (sIgA), which is the candidate of salivary stress-markers, with original electrochemical immunoassay: Gold-Linked Electrochemical Immuno Assay (GLEIA). This biosensor is based on a sandwich-type immunosensor, and adopt the electrochemical method to detect the reduction peak from Au nanoparticles linked to the secondary antibody. GLEIA is convenient and cost-effective that only requires low sample volume (10 µL). In addition, all devices that GLEIA needs are minimal such as note PC and portable potentiostat and printed electrode. Therefore, GLEIA method shows various advantages suitable for stress measurement as compared with other sIgA detection methods. In this paper, we aim to the on-site and continuous measurement of salivary IgA in our daily life, to determine the various concentration of sIgA in standard solution and artificial saliva using GLEIA.[Experimental]Primary Anti-IgA Antibody solution was dropped onto the working electrode for immobilizing the antibody by physical adsorption. After, the Au nanoparticle solution was mixed with the phosphate buffer (50 mM, pH=8.3). The anti-IgA to dissolve the 5 mM of phosphate buffer add the Au nanoparticle solution. For the preparing of secondary antibody modified Au nanoparticle, the antibody was conjugated with Au nanoparticle under the pH=8.3 condition and kept for 10 min at room temperature. Then, 10% BSA in PBS buffer and 1% PEG in PBS buffer were added in preparing Au solution. Finally, the concentration was adjusted for OD520=6.0 by centrifuge and exclude the supernatant. The Au Anti-IgA conjugate was diluted by 3 times with 50 w/v of trehalose (OD520=2) and dripping 5µL of this solution in the immunomodule. Then the immunomodule was dried in vacuum condition for 5 minutes. Dried immunomodule was stored at 4 ℃ until use. Different concentrations of the IgA antigen solution (0, 10, 60, 150, 300 ng/mL) were made by diluting in PBS. For the detection of Antibody-Antigen reaction, the IgA solutions were mixed in prepared immunomodule, After the 1 minute, and 1.4 µL of the solution was placed on immunosensor to incubate for 1 hour, after rinsing with PBS, and eliminate the PBS solution with the air gun. The direct redox reaction was performed in 2M KCl solution (20 µL) covering the entire three-electrode zone at room temperature.[Results and Discussion]We obtained the linear response to relate the concentration of sIgA (10-300 ng/mL) in D-PBS buffer with the artificial-saliva which includes salivary inorganic salt and typically glycoprotein (mucin). Figure 1 shows the differential pulse voltammogram of GLEIA with IgA in artificial saliva (a) and calibration curve (b). In addition, the calibration curve shows the linear range within 10-300 ng/mL and the correlation coefficient (R2) of the calibration curve for this relationship was 0.963. The peak current intensity is decreased from in standard solution. It may prevent the Antigen-Antibody reaction from artificial saliva which pH is nearly 8.5. Generally, the antibody shows the best affinity in pH= 6.5~8.4 condition. However, these results show that GLEIA is suitable for salivary IgA detection because the salivary pH is mainly neutral. Furthermore, we evaluate the selectivity of the immunosensor by comparing it with the reduction peak between IgA= 300ng/mL or 0 ng/mL and interference protein such as α-amylase, human serum albumin, immunoglobulin G (IgG), lysozyme and mucin. We acknowledge GLEIA shows the signal increasing within the existence of interference proteins. This may be the affinity of antibody and non-specific reaction between antibody and the proteins. More importantly, we recognize that the reduction peak is increasing with the concentration of sIgA in the existence of interference proteins. Therefore, GLEIA can be applied for stress measurement using salivary sIgA that can be used in daily life anywhere and anytime whenever necessary. In the future, we try to detect the sIgA in real saliva for on-site stress measurement using GLEIA and to integrate the various immunosensors for stress-markers in saliva.nd anytime whenever necessary. In the future, we try to detect the sIgA in real saliva for on-site stress measurement using GLEIA and to integrate the various immunosensors for stress-markers in saliva. Figure 1

Metode mikrokoloni (slide culture) sebagai metode diagnostic alternative yang lebih cepat untuk diagnosis tuberculosis paru

Despite wide distribution of pulmonary tuberculosis in Indonesia, its diagnosis is still an important issue to be dealt with. Fourty seven sputums from pulmonary tuberculosis patients in Surabaya were examined to detect Mycobacterium tuberculosis using three methods, i.e. the acidfast stain Ziehl Neelsen, microcolony (slide culture) and Lowenstein Jensen. Sputums were collected spontaneously from the patients. All of them were decontaminated and centrifuged. After the supernatant fluids were carefully decanted, the sediments were resuspended in 1 ml of 10 mMphosphate buffer (pH 7.4) and the suspensions were then inoculated on to two 76 x 13 mm glass microscope slides. One of them was stained by Ziehl Neelsen method and the other was inoculated into microcolony media for seven days and the waste suspension was inoculated into LowensteinJensen media. The results of the microcolony method analysis were compared with the Ziehl Neelsen staining. Employing McNemar test, a significant difference was observed between the microcolony method and the Ziehl Neelsen staining (?² = 5,88). The sensitivity and spesificity ofmicrocolony were 100% and 89% while the Ziehl Neelsen were 60% and 84% respectively. In conclusions microcolony method was better compared with the Ziehl Neelsen staining in the detection of Mycobacterium tuberculosis. Microcolony method was able to reduce time required todetect Mycobacterium tuberculosis in patient suspected with pulmonary tuberculosis.

Enantioseparation of Penconazole, Myclobutanil and Triadimefon by Cyclodextrin-Modified Micellar Electrokinetic Chromatography

Simultaneous enantioseparation of three chiral triazole fungicides i.e. penconazole, myclobutanil and triadimefon bycyclodextrin-modified micellar electrokinetic chromatography (CD-MEKC) under acidic condition was investigated withdifferent HP--CD concentrations as chiral selector. The three triazole fungicides have an asymmetrically substitutedcarbon atom respectively, and they all contain two enantiomers. In order to achieve the best enantioresolution of thesetriazole fungicides, the HP--CD concentration range was optimized from 0 to 40 mM in 10 mM increment. The resultsindicate that separation of enantiomers was not achieved with concentration range of HP--CD from 0 to 20 mM.Simultaneous enantioseparation was successfully achived at 30 mM and 40 mM HP--CD concentration in 25 mMphosphate buffer (pH 3.0) solution that containing 50 mM SDS, 10% methanol and 5% acetonitrile. However,enantioresolution decreased for myclobutanil and triadimefon enantiomers with HP--CD concentration greater than 30mM. In addition, an increase in the concentration of HP--CD caused a significant increase in the analysis time. In light ofthese aspects, the optimal concentration for HP--CD was decided to be 30 mM for the simultaneous chiral separation ofthe three triazole fungicides with resolutions (Rs) between enantiomers for penconazole, Rs = 0.81; myclobutanil, Rs = 1.14;triadimefon, Rs = 2.09; peak efficiencies (N) greater than 108 000 for all stereoisomers and analysis time of less than 15min.

Graphene modified Palladium sensor for electrochemical analysis of norepinephrine in pharmaceuticals and biological fluids

A novel voltammetric sensor for the determination of norepinephrine (NE); an important neurotransmitter, has been developed using graphene modified palladium (Pd) sensor. Graphene was synthesized in the laboratory and was characterized by SEM, TEM, XRD and Raman spectroscopic measurements. The electrochemical response of modified sensor towards NE determination was studied by the means of electrochemical impedance spectroscopy, cyclic voltammetry and square wave voltammetry. The modified sensor exhibited excellent electrocatalytic activity for the oxidation of NE, leading to a remarkable enhancement in the peak current and lowering of peak potential. The anodic peak current was found to increase linearly with increasing the concentration of NE using graphene modified Pd sensor in the range 0.0005–0.5mM in phosphate buffer of pH 7.2 with limit of detection and limit of quantification as 67.44nM and 224.8nM, respectively. The detection sensitivities of bare and modified Pd sensors were 0.851μAmM−1 and 17.428μAmM−1, respectively, revealing the good electrocatalytic properties of graphene modified sensor. The proposed method was successfully applied for the determination of NE in pharmaceutical dosage forms and human urine samples. It was observed that using developed sensor NE can be detected in urine samples even in the presence of high concentration of uric acid.

Role of Glutamate Decarboxylase-like Protein 1 (GADL1) in Taurine Biosynthesis

This manuscript concerns the tissue-specific transcription of mouse and cattle glutamate decarboxylase-like protein 1 (GADL1) and the biochemical activities of human GADL1 recombinant protein. Bioinformatic analysis suggested that GADL1 appears late in evolution, only being found in reptiles, birds, and mammals. RT-PCR determined that GADL1 mRNA is transcribed at high levels in mouse and cattle skeletal muscles and also in mouse kidneys. Substrate screening determined that GADL1, unlike its name implies, has no detectable GAD activity, but it is able to efficiently catalyze decarboxylation of aspartate, cysteine sulfinic acid, and cysteic acid to β-alanine, hypotaurine, and taurine, respectively. Western blot analysis verified the presence of GADL1 in mouse muscles, kidneys, C2C12 myoblasts, and C2C12 myotubes. Incubation of the supernatant of fresh muscle or kidney extracts with cysteine sulfinic acid resulted in the detection of hypotaurine or taurine in the reaction mixtures, suggesting the possible involvement of GADL1 in taurine biosynthesis. However, when the tissue samples were incubated with aspartate, no β-alanine production was observed. We proposed several possibilities that might explain the inactivation of ADC activity of GADL1 in tissue protein extracts. Although β-alanine-producing activity was not detected in the supernatant of tissue protein extracts, its potential role in β-alanine synthesis cannot be excluded. There are several inhibitors of the ADC activity of GADL1 identified. The discovery of GADL1 biochemical activities, in conjunction with its expression and activities in muscles and kidneys, provides some tangible insight toward establishing its physiological function(s).

Chiral discrimination of sibutramine enantiomers by capillary electrophoresis and proton nuclear magnetic resonance spectroscopy

Capillary electrophoresis (CE) and proton nuclear magnetic resonance spectroscopy ((1)H-NMR) have been used to discriminate the enantiomers of sibutramine using cyclodextrin derivatives. Possible correlation between CE and (1)H-NMR was examined. Good correlation between the (1)H-NMR shift non-equivalence data for sibutramine and the degree of enantioseparation in CE was observed. In CE study, a method of enantiomeric separation and quantitation of sibutramine was developed using enantiomeric standards. The method was based on the use of 50 mM of phosphate buffer of pH 3.0 with 10 mM of methyl-beta-cyclodextrin (M-β-CD). 0.05% of LOD, 0.2% of LOQ for S-sibutramine enantiomer was achieved, and the method was validated and applied to the quantitative determination of sibutramine enantiomers in commercial drugs. On a 600 MHz (1)H-NMR analysis, enantiomer signal separation of sibutramine was obtained by fast diastereomeric interaction with a chiral selector M-β-CD. For chiral separation and quantification, N-methyl proton peaks (at 2.18 ppm) were selected because of its being singlet and simple for understanding of diastereomeric interaction. Effects of temperature and concentration of chiral selector on enantiomer signal separation were investigated. The optimum condition was 0.5 mg/mL of sibutramine and 10 mg/mL of M-β-CD at 10°C. Distinguishment of 0.5% of S-sibutramine in R-sibutramine was found to be possible by (1)H-NMR with M-β-CD as chiral selector. Host-guest interaction between sibutramine and M-β-CD was confirmed by (1)H-NMR studies and CE studies. A Structure of the inclusion complex was proposed considering (1)H-NMR and 2D ROESY studies.

Studies on the Electrochemical Behavior of Thiazolidine and Its Applications Using a Flow–Through Chronoamperometric Sensor Based on a Gold Electrode

The electrochemical behaviors of thiazolidine (tetrahydrothiazole) on gold and platinum electrodes were investigated in a Britton-Robinson buffer (pH 2.77–11.61), acetate buffer (pH 4.31), phosphate buffer solutions (pH 2.11 and 6.38) and methanol or acetonitrile containing various supporting electrolytes. Detection was based on a gold wire electrochemical signal obtained with a supporting electrolyte containing 20% methanol-1.0 mM of phosphate buffer (pH 6.87, potassium dihydrogen phosphate and dipotassium hydrogen phosphate) as the mobile phase. Comparison with results obtained with a commercial amperometric detector shows good agreement. Using the chronoamperometric sensor with the current at a constant potential, and measurements with suitable experimental parameters, a linear concentration from 0.05 to 16 mg L−1 was found. The limit of quantification (LOQ) of the method for thiazolidine was found to be 1 ng.

Physicochemical Characterization of Berberine Chloride: A Perspective in the Development of a Solution Dosage Form for Oral Delivery

The objective of the present research was to evaluate the physicochemical characteristics of berberine chloride and to assess the complexation of drug with 2-hydroxypropyl-β-cyclodextrin (HPβCD), a first step towards solution dosage form development. The parameters such as log P value were determined experimentally and compared with predicted values. The pH-dependent aqueous solubility and stability were investigated following standard protocols at 25°C and 37°C. Drug solubility enhancement was attempted utilizing both surfactants and cyclodextrins (CDs), and the drug/CD complexation was studied employing various techniques such as differential scanning calorimetry, Fourier transform infrared, nuclear magnetic resonance, and scanning electron microscopy. The experimental log P value suggested that the compound is fairly hydrophilic. Berberine chloride was found to be very stable up to 6 months at all pH and temperature conditions tested. Aqueous solubility of the drug was temperature dependent and exhibited highest solubility of 4.05 ± 0.09 mM in phosphate buffer (pH 7.0) at 25°C, demonstrating the effect of buffer salts on drug solubility. Decreased drug solubility was observed with increasing concentrations of ionic surfactants such as sodium lauryl sulfate and cetyl trimethyl ammonium bromide. Phase solubility studies demonstrated the formation of berberine chloride-HPβCD inclusion complex with 1:1 stoichiometry, and the aqueous solubility of the drug improved almost 4.5-fold in the presence of 20% HPβCD. The complexation efficiency values indicated that the drug has at least threefold greater affinity for hydroxypropyl-β-CD compared to randomly methylated-β-CD. The characterization techniques confirmed inclusion complex formation between berberine chloride and HPβCD and demonstrated the feasibility of developing an oral solution dosage form of the drug.

Regioselective hydroxylation of daidzein using P450 (CYP105D7) from Streptomyces avermitilis MA4680

Regiospecific 3'-hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent K(m) and k(cat) values of CYP105D7 for daidzein were 21.83 +/- 6.3 microM and 15.01 +/- 0.6 min(-1) in the presence of 1 microM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO-difference spectra at 450 nm using the whole cell extract. When the whole-cell reaction for the 3'-hydroxylation reaction of daidzein was carried out with 100 microM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) showed a 3.6-fold higher conversion yield (24%) than the corresponding wild type cell (6.7%). In a 7 L (working volume 3 L) jar fermentor, the recombinants S. avermitilis grown in R2YE media produced 112.5 mg of 7,3',4'-trihydroxyisoflavone (i.e., 29.5% conversion yield) from 381 mg of daidzein in 15 h.

Direct pattern formation of bacterial cells using micro-droplets generated by electrohydrodynamic forces

Bacterial cells were patterned efficiently on a nutrient-coated membrane in a direct, rapid, and cost-effective manner using electro-hydrodynamically generated micro-droplets. By varying the viscosity and electrical conductivity of the bacterial printing solution, uniform droplets were obtained. Escherichia coli cells were directly printed onto a membrane filter, followed by overnight incubation on an agar plate which created line patterns of bacterial colonies with a width of approximately 160 μm. Optimization of the concentration of the chemical components [i.e. the ethylene glycol (EG), phosphate buffer (PB), and sodium chloride (NaCl)] in the processing bacterial solution allowed successful growth and patterning of the cells. The optimal conditions to achieve the most effective cell growth and patterning on these printed surfaces were an EG concentration of 40 vol% and a concentration as low as 10 mM of PB or NaCl in the printing solution. Cells passing through the electric nozzle during the printing process remained viable.

An MEC-MFC-Coupled System for Biohydrogen Production from Acetate

Microbial fuel cells (MFCs) are devices that use bacteria as the catalysts to oxidize organic and inorganic matter and generate current whereas microbial electrolysis cells (MECs) are a reactor for biohydrogen production by combining MFC and electrolysis. In an MEC, an external voltage must be applied to overcome the thermodynamic barrier. Here we report an MEC-MFC-coupled system for biohydrogen production from acetate, in which hydrogen was produced in an MEC and the extra power was supplied by an MFC. In this coupled system, hydrogen was produced from acetate without external electric power supply. At 10 mM of phosphate buffer, the hydrogen production rate reached 2.2 +/- 0.2 mL L(-1) d(-1), the cathodic hydrogen recovery (RH2) and overall systemic Coulombic efficiency (CEsys) were 88 to approximately 96% and 28 to approximately 33%, respectively, and the overall systemic hydrogen yield (Y(sysH2)) peaked at 1.21 mol-H2 mol-acetate(-1). The hydrogen production was elevated by increasing the phosphate buffer concentration, and the highest hydrogen production rate of 14.9 +/- 0.4 mL L(-1) d(-1) and Y(sysH2) of 1.60 +/- 0.08 mol-H2 mol-acetate(-1) were achieved at 100 mM of phosphate buffer. The performance of the MEC and the MFC was influenced by each other. This MEC-MFC-coupled system has a potential for biohydrogen production from wastes, and provides an effective way for in situ utilization of the power generated from MFCs.

Superoxide Dismutase Enhanced the Formation of Hydroxyl Radicals in a Reaction Mixture Containing Xanthone under UVA Irradiation

To clarify the effect of superoxide dismutase (SOD) on the formation of hydroxyl radical in a standard reaction mixture containing 15 microM of xanthone, 0.1 M of 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and 45 mM of phosphate buffer (pH 7.4) under UVA irradiation, electron paramagnetic resonance (EPR) measurements were performed. SOD enhanced the formation of hydroxyl radicals. The formation of hydroxyl radicals was inhibited on the addition of catalase. The rate of hydroxyl radical formation also slowed down under a reduced oxygen concentration, whereas it was stimulated by disodium ethylenediaminetetraacetate (EDTA) and diethyleneaminepentaacetic acid (DETAPAC). Above findings suggest that O(2), H(2)O(2), and iron ions participate in the reaction. SOD possibly enhances the formation of the hydroxyl radical in reaction mixtures of photosensitizers that can produce O(2)(-.).

Simple and Rapid Liquid Chromatographic Method for Determination of Histidine HCl in Pharmaceutical Preparations

Histidine (HID) is frequently compounded in pharmaceutical preparations for the treatment of gastrointestinal diseases. A simple and rapid liquid chromatographic method was developed and validated for the routine determination of HID in pharmaceutical preparations. The high-performance liquid chromatographic (HPLC) system consisted of an Purospher STAR RP18 endcapped column (4.6×250 mmi. d.) using amobile phase of 50 mMphosphate buffer with 3 mM sodium octanesulfonate: acetonitrile (95:5, v/v) with an ultraviolet/visible (UV/VIS) detector at 210 nm. The developed method satisfies the system suitability criteria and peak integrity for HID. The square of the correlation coefficient of the linear regression analysis was 0.9995. The relative standard deviations (RSDs) for intra- and inter-day tests were less than 2%. The recovery for 5 concentrations expressed as the closeness of the observed mean to the spiked concentration was 98.20%~101.02%. The limits of detection (LOD) and quantitation (LOQ) for HID were 2.2 and 7.3 μg/mL, respectively. The method was applied to quantitatively determine HID in 8 brands of HID-containing tablets on the Taiwanese market, and these were found to contain 100.59%±1.01% of the percentage claimed on the label. The results indicate that the established assay method is suitable for measuring HID in pharmaceutical preparations.