Novel Nanocombinations of l-Tryptophan and l-Cysteine: Preparation, Characterization, and Their Applications for Antimicrobial and Anticancer Activities

Tungsten oxide WO3 nanoparticles (NPs) were prepared in a form of nanosheets with homogeneous size and dimensions in one step through acid precipitation using a cation exchange column. The resulting WO3nanosheet surface was decorated with one of the two amino acids (AAs) l-tryptophan (Trp) or l-cysteine (Cys) and evaluated for their dye removal, antimicrobial, and antitumor activities. A noticeable improvement in the biological activity of WO3 NPs was detected upon amino acid modification compared to the original WO3. The prepared WO3-Trp and WO3-Cys exhibited strong dye removal activity toward methylene blue and safranin dyes with complete dye removal (100%) after 6 h. WO3-Cys and WO3-Trp NPs revealed higher broad-spectrum antibacterial activity toward both Gram-negative and Gram-positive bacteria, with strong antifungal activity toward Candida albicans. Anticancer results of the modified WO3-Cys and WO3-Trp NPs against various kinds of cancer cells, including MCF-7, Caco-2, and HepG-2 cells, indicate that they have a potent effect in a dose-dependent manner with high selectivity to cancer cells and safety against normal cells. The expression levels of E2F2 and Bcl-2 genes were found to be suppressed after treatment with both WO3-Cys and WO3-Trp NPs more than 5-FU-treated cells. While expression level of the p53 gene in all tested cells was up-regulated after treatment 5–8 folds more as compared to untreated cells. The docking results confirmed the ability of both NPs to bind to the p53 gene with relevant potency in binding to other tested gens and participation of cysteine SH-functional group in such interaction.

Novel Nanocombinations of L-tryptophan and L-cysteine: Preparation, Characterization and Their Applications for Dye Decolorization, Antimicrobial and Anticancer Activities

Tungsten oxide WO3 nanoparticles (NPs) were prepared in a form of nanosheets with a homogeneous size and dimensions in one step through acid precipitation using a cation exchange column. The resulting WO3 nanosheets surface was decorated with one of the two amino acids (AAs) L-tryptophan (Trp) or L-cysteine (Cys) for their dye removal, antimicrobial, and antitumor activities. A noticeable improvement in the biological activity of WO3 NPs was detected upon amino acid modification compared to the original WO3. The prepared WO3-Trp and WO3-Cys exhibited strong dye removal activity toward methylene blue and safranin dyes with complete dye removal (100%) after 6 h. WO3-Cys NPs and WO3-Trp NPs revealed higher broad-spectrum antibacterial activity toward both G-ve and G+ve bacteria with strong antifungal activity toward Candida albicans. Anticancer results of the modified WO3-Cys and WO3-Trp NPs against various kinds of cancer cells including MCF-7, caco-2, and HepG-2 cells indicated that they have a potent effect in a dose-dependent manner with high selectivity to cancer cells and safety against normal cells. The expression levels of E2F2, Bcl-2 genes were found to be suppressed after treatment with both WO3-Cys and WO3-Trp NPs more than 5-FU-treated cells. While expression level of the p53 gene in all tested cells was evidently up-regulated after treatment by more than 5-8 folds as compared to untreated cells. The docking results confirmed the ability of both NPs to bind to P53 gene with relevant potency in binding to other tested gens and participation of cysteine SH-functional group in such interaction.

Quercetin as a precursor in the synthesis of analogues of fulvic acids and their antibacterial properties

A simple, fast and effective method for producing synthetic substances with properties similar to natural humic substances has been proposed. The synthesis method is based on the oxidation of quercetin by molecular oxygen in an alkaline medium, followed by conversion to the acid form by passing through a cation exchange column. Study of elemental and functional compositions, spectral properties (UV/Vis and IR range) and redox characteristics allowed qualifying the resulting product as a synthetic fulvic acid. The enhanced antibacterial properties of the obtained synthetic product were established. The minimum concentration of inhibition of synthetic fulvic acid derived from quercetin is 25 g mL–1, which is in 100 times less than for natural humic substances.

Optimized Chromatographic Determination of Naphazoline and Chlorpheniramine in Eye/Nose Drops Using Cation-exchange Column

A simple and efficient liquid chromatographic method has been developed and validated for the simultaneous determination of naphazoline and chlorpheniramine in eye/nose drops, in presence of naphazoline degradation product and naphthalene acetic acid (NAPD). The separation was achieved within 6.0 min, employing a mixture of 53.5% v/v water-acetonitrile containing 78.70 mM/L sodium dihydrogen orthophosphate adjusted to pH 5.30 as isocratic mobile phase, pumped at 1.0 mL/min through a strong cation-exchange column (10 μm particle size), the analytes were monitored at 230 nm. Statistical experimental designs and graphic representations (response surface methodology) were used for optimizing the chromatographic separation. The linearity plots were linear over concentration range up to 125% of the analytes nominal concentrations (100%) with regression coefficients (r) > 0.99, method’s accuracy (RSD < 2.0%), repeatability and intermediate precision (RSD < 2.0%) were verified. System suitability parameters were also within the acceptable range. The validated method was successfully employed for the routine analysis of eye/nose drops.

LC–MS/MS Analysis of Choline Compounds in Japanese-Cultivated Vegetables and Fruits

Choline is an essential nutrient and choline esters are potential functional food ingredients. We aimed to analyze the choline compound content in 19 cultivated fruits and vegetables and identify those with high acetylcholine content. We utilized liquid chromatography with tandem mass spectrometry to quantify choline compounds according to the standard addition method. Choline compounds were extracted from lyophilized fruit/vegetable powders and passed through a weakly acidic cation exchange column, resulting in a concentrated solution of choline compounds. The compounds were separated on a pentafluorophenyl column and then analyzed using positive mode electrospray ionization. Results showed that acetylcholine and choline were the primary choline compounds in all agricultural products; propionylcholine and butyrylcholine were minor compounds in 17 and 12 agricultural products, respectively. The acetylcholine concentration was 2900-fold higher in eggplants (6.12 mg/100 g fresh weight [FW]) than in other agricultural products (average: 2.11 × 10−3 mg/100 g FW). The concentration of acetylcholine differed only 2-fold between eggplant cultivars with the highest (′Higomurasaki′: 5.53 mg/100 g FW) and lowest (′Onaga nasu′: 2.79 mg/100 g FW) concentrations. The half-life of acetylcholine in eggplants was approximately 16 days, which is longer the shelf life of eggplants. Thus, eggplants can be a good source of acetylcholine.

Molecular Cloning and Chartacterization DNA Polymerase I from thermophilic Geobacillus SBS 4S strain

Thermostable DNA polymerases are extensively used in biotechnology and life science applications. DNA Polymerase I was isolated from a hyperthermophile bacteria Geobacillus SBS 4S. Primers were designed using the template sequence of DNA Polymerase I gene of Geobacillus kaustophilus HTA26 strain. Nco 1 and Hind III sites were introduced on the forward and reverse primers respectively. Polymerase I gene of 2.6 Kb was cloned in pTZ57/ RT vector. Cloned gene of Polymerase I was restricted with Nco 1 and Bam H1, and ligated to pET 22b vector. Nco 1 site was used to insert twenty two N-terminal aminoacids (pelB) leader sequence at the start of the gene, which lead the recombinant protein in the periplasmic space, which increases the half life of recombinant protein. pelB fused with DNA Polymerase I produces soluble protein, which was detected after sonication. Sequencing shows that DNA polymerase I consists of 2499 bp with encodes for 832 amino acids, showed 99 % similarity with Geobacillus Kaustophillus . The expression of pelB fused DNA Polymerase I was optimized at different concentrations of IPTG and lactose. Highest expression was observed with 0.5mM IPTG and 20mM lactose. After Harvesting and sonication of BL21 codon plus cells, Polymerase I was produced in the soluble fraction. The supernatant containing the protein of interest, was separated after centrifugation at 10,000 rpm for 20 min. The protein was purified by ammonium sulphate precipitation and cation exchange column. The activity of purified DNA Polymerase I was checked by PCR reaction.

Peningkatan Stabilitas Termal Enzim α-Amilase dari Bacillus subtilis ITBCCB148 dengan Amobilisasi Menggunakan Zeolit

The objective of the research is to increase the thermal stability of -amylase from Bacillus subtilis ITBCCB148 by immobilization using zeolite. For that reason, firstly we need to produce, isolate, and purify the enzyme. The purification of the enzyme was conducted by the following steps: fractionation with ammonium sulphate, dialysis, and CM-cellulose cation exchange column chromatography. The purified enzyme was immobilized using zeolite. The success in immobilization of the enzyme was evaluated by comparing the thermal stability of the enzyme before and after immobilization. Activity of α-amylase was determined by the Mandels and Fuwa method. The protein content was determined based on the method by Lowry. The results showed that the specific activity of purified enzyme was 2473.7 U / mg, increased 19 times compared to crude extract of enzyme having specific activity of 1285.9 U / mg. The purified enzyme has the optimum temperature at 65ºC, while the immobilized enzyme has the optimum temperature at 75ºC. The thermal stability test of the purified enzyme at 65ºC for 100 minutes showed the purified enzyme having residual activity of 20%; t 1 / 2 = 30 min, k i = 0.023 min -1 and ΔGi = 103.65 kJ mol -1 . The thermal stability test of the immobilized enzyme at 65ºC for 100 minutes showed that the immobilized enzyme had residual activity of 40%; t 1/ 2 = 49 min, k i = 0.014 min -1 and ΔGi = 105.03 kJ mol -1 . Immobilization using zeolite has succeeded in increasing the thermal stability of enzyme by 1.64 times compared to the purified enzyme, which is indicated by the decreasing of k i value, the increase of half-life and denaturation energy change (ΔGi).

Ion Chromatographic Method for the Simultaneous Determination of Anions and Cations in Firecrackers and Matches Samples as Known Potential Explosives

<p class="Abstract">A reference ion chromatography method based on column switching has been presented for the simultaneous determining anions (Cl⁻, ClO₃⁻, NO₂⁻, SO₄²⁻, and NO₃⁻) and/or cations (NH₄⁺, Na⁺, K⁺, Ca²⁺, and Mg²⁺) using one pump, one type of mobile phase, and one detector. The reference method performed anion-exchange column and cation-exchange column and arranged serially via one 10-port valve. The determination of either anions or cations in one determination system could be made by switching the valve. When the use of 1.25 mM trimellitic acid as a mobile phase and the instrument was operated at a flow rate of 0.6 mL/min, five anions and five cations were determined on the anion-exchange column and the cation-exchange column, respectively. All anions target could be determined completely within 55 minutes, whereas the cations target could be determined within 35 minutes. The calculation of limit of detection using S/N=3 was 3.85 − 14.10 µM for anions and 2.95 − 10.58 µM for cations. The relative standard deviations of all ions were less than 3.82%, 3.29%, and 3.21% for retention time, peak area, and peak height, respectively. The reference method was then applied for the simultaneous determining anions and/or cations contained in firecrackers and matches samples as known potentially explosives.</p>

DEVELOPMENT OF ANALYTICAL METHOD FOR THE DETERMINATION OF NINHYDRIN-POSITIVE SUBSTANCES IN AMINO ACIDS BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Aim and Objectives: The aim of the work is focused on the optimization of the high-performance liquid chromatography (HPLC) method for the determination of ninhydrin-positive substances in amino acids using HPLC technique in a single method. Since, most of the amino acids are widely used in the determination of Renal and Nutrition drug products exist independently in the monograph for each amino acid either by TLC or HPLC.Methods: The chromatographic separation was performed using sodium amino acid analysis cation exchange column using Sodium Eluent Na 315, Sodium eluent Na 425, and Sodium Eluent Na 640 as Mobile phase, performed by gradient program with detection of wavelength 570 nm using flow rate as 0.4 mL/min. The method has been evaluated using post-column derivatization technique (HPLC/Pinnacle PCX).Results: All the amino acids were eluted correspondingly at the individual retention time and the method shall be validated as per the ICH Q2R1 Guideline.Conclusion: The method has been successfully evaluated and developed for the analytical applications.