Buffer Medium pH Research Articles

Multicomponent crystals: Solubility enhancement of simvastatin using arginine and glycine coformers

Context: Simvastatin can be modified by the formation of multicomponent crystals to increase its solubility. Aims: To compare the solubility of multicomponent simvastatin crystals to pure simvastatin. Methods: The in silico study of simvastatin and the coformers arginine and glycine revealed non-covalent interactions, so multicomponent preparations of simvastatin crystals were prepared by solvent evaporation using a mole ratio of 1:1; 1:2 and 2:1. Results: Each simvastatin-arginine and simvastatin-glycine ratio increased the solubility, with the highest increase observed for the 1:2 ratio compared to pure simvastatin. Conclusions: Simvastatin-arginine multicomponent crystals (1:2) showed the best dissolution profile in phosphate buffer medium pH 7.0 with 67.69% dissolution, while simvastatin-glycine multicomponent crystals (1:2) exhibited the best dissolution profile in buffer media pH 1.2 with 16.19% dissolution. Characterization of the multicomponent crystals revealed a shift in the peaks, a decreased melting point, and enthalpy, indicating decreased % crystallinity and the formation of a new solid phase.

SIMPLEX OPTIMIZATION FOR THE SPECTROPHOTOMETRIC DETERMINATION OF AZITHROMYCIN DRUG VIA ION-PAIR FORMATION

A spectrophotometric determination of azithromycin was optimized using the simplex model. The approach has been proven to be accurate and sensitive. The analyte has been reacted with bromothymol blue (BTB) to form a colored ion pair which has been extracted in chloroform in a buffer medium of pH=4 of potassium phthalate. The extracted colored product was assayed at 415 nm and exhibited a linear quantification range over (1 - 20) g/ml. The excipients did not exhibit any interferences with the proposed approach for assaying azithromycin in pharmaceutical formulations.

Endoplasmic reticulum-targeted fluorescent probes for metal-free tracking of carbon monoxide in living cells

Endoplasmic Reticulum (ER) targetable fluorescent probes, ER-NapNO2 and ER-NapAllyl, have been designed and synthesized based on 1,8-naphthalimide fluorophore, for the selective and targeted detection of carbon monoxide (CO) in aqueous buffer medium of pH 7.4 at 37 °C. ER-targetable moiety, methyl sulfonamide unit, was attached with the one part with the fluorophore and another part was connected with CO reactive zone i.e. 4-nitrobenzyle and allyl group. Very weak fluorescence signals were observed with both the probes due to the possible PET process operating from naphthalimide moiety to electron withdrawing units i.e. -NO2 and allyl group. Interestingly, both of the probes exhibited their selective detection behavior towards CO in aqueous reaction buffer in absence of any metal ions e.g., palladium, rhodium, iron etc. The possible expulsion of highly fluorescent naphthalimide-derivative was identified from the reaction mixture during the reaction of both the probes and CO. The detection technique is found to highly selective towards CO over various reactive oxygen, nitrogen, sulfur, and other biological species with low detection limit, 0.080 µM and 0.161 µM of ER-NapNO2 and ER-NapAllyl respectively. Theoretical study using time dependent density functional theory (TDDFT) optimized the end-products formed after the reaction with CO and probes which excellently corroborate with the experimental observation. Also, the sensing reactions are exothermic with computed free energy changes for ER-NapNO2 and ER-NapAllyl are − 158.74 kcal/mol and − 28.51 kcal/mol respectively. Cellular confocal fluorescence microscopic experiments showed that these probes are also efficient to detect CO in ER specific regions in living cells.

The effect of polysorbate 20 and polysorbate 80 on the solubility of quercetin.

Quercetin acts as an antioxidant, anti-inflammatory, wound healing, and anti-aging so quercetin can be used as a topical preparation. However, it has low solubility in water at 0.01 mg/ml at 25oC. Increasing the solubility of quercetin in water was done by the addition of surfactants. This study compared the solubility of quercetin in Polysorbate 20 (P20) and Polysorbate 80 (P80) in a citrate buffer medium pH 4.5±0.2. The surfactants Polysorbate 80 and Polysorbate 20 differ in their alkyl chain length. Polysorbate 80 has an alkyl chain length of 18, while Polysorbate 20 has an alkyl chain length of 12. The concentrations of surfactant are above, below, and at the critical micelle concentration (CMC) values. The concentrations of quercetin were determined at the maximum wavelength by spectrophotometric method. The results of the quercetin solubility test without surfactant were 3.89±0.59 mg/L. The results of the quercetin solubility test by adding Polysorbate 20 at a concentration of 42.0 ppm; 57.5 ppm; and 73.0 ppm were 3.62±0.72, 4.04±0.23 and 8.35±1.97 mg/L, respectively. While the solubility of quercetin by adding Polysorbate 80 at a concentration of 4.0 ppm, 11.5 ppm, and 19.0 ppm was 11.15±0.72, 11.37±1.23 and 14.17±1.96 mg/L, respectively. The solubility of quercetin is greater after the addition of surfactant Polysorbate 20 only at the concentration above the CMC value and the solubility of quercetin is greater with the addition of surfactant Polysorbate 80 at all concentrations. Surfactant Polysorbate 20 increases the solubility of quercetin in citrate buffer pH 4.5±0.2 only at concentrations above the CMC value of 2.14 times. Polysorbate 80 can increase the solubility of quercetin in citrate buffer pH 4.5±0.2 at concentrations below, at, and above CMC by 2.87, 2.92, and 3.63 times, respectively. Polysorbate 80 can increase the solubility of quercetin in citrate buffer pH 4.5±0.2 higher than Polysorbate 20.

Development of a membraneless single-enzyme biofuel cell powered by glucose

This work presents the development of a membraneless single-enzyme biofuel cell powered by glucose (GBFC). The GBFC biocathode is based on a graphite rod electrode (GRE) coated with a layer of Prussian blue (PB) nanoparticles entrapped into poly(pyrrole-2-carboxylic acid) (PPCA) shell, an additional layer of PPCA, and covalently to polymer linked glucose oxidase (GOx). The bioanode is based on GRE modified with a nanobiocomposite composed of poly(1,10-phenanthroline-5,6-dione), gold nanoparticles entrapped in a PPCA shell, and GOx linked by an amide bond to polymer. The operation of the developed single-enzyme GBFC is based on GOx-catalysed oxidation of glucose on both the bioanode and biocathode and reduction of H2O2 electrocatalysed by PB on the biocathode. The GBFC operated in O2-saturated buffer medium pH 6.0 containing glucose. An open-circuit voltage (OCV) of 646 mV, a maximum power density of 10.94 μW/cm2 and a current density of 60.52 μA/cm2 at 40 mM glucose were determined. OCV and current density were directly proportional to glucose concentration in 0.01–10.00 mM and 0.05–124.00 mM concentration ranges, respectively. In addition, GBFC had good operational stability and retained more than 90% of the initial OCV after 36 days.

Microencapsulation of Ruellia tuberosa L. Aqueous Root Extracts Using Chitosan-Sodium Tripolyphosphate and Their In Vitro Biological Activities.

The current study aims to perform microencapsulation of R. tuberosa L. extracts using chitosan crosslinked to sodium tripolyphosphate (NaTPP) as wall materials by spray drying and to analyze their in vitro biological activities. The influence of manufacturing conditions, like pH, chitosan concentration, and stirrer time, was assessed. Results showed that microcapsules prepared in pH 4 with a concentration of 0.1% (w/v) chitosan, and 90 min stirring time had 51.80% encapsulation efficiency and high in vitro biological activity. These were shown by high in vitro alpha amylase inhibition and antioxidant activity with IC50 values of 50.65 μg/mL and 123.97 μg/mL, respectively. Releases of the bioactive compounds in microcapsules of R. tuberosa L. were carried out on phosphate buffer medium pH 2.2 and pH 7.4 with times release of 30, 60, 90, and 120 min. The bioactive compounds were released in pH 2.2 in 120 min at 2.48%. At pH 7.4, the active ingredients were more easily released, by 79.90% in 120 min. The microcapsules' morphology showed a rough surface with spherical forms and the average sizes were 53.41 μm. This study supports the essential role of microencapsulation in improving plant extracts with reserved biological activities.

Diffusion rate of quercetin from chitosan-TPP nanoparticles dispersion of onion (Allium cepa L.) ethanol extract in medium phosphate buffer pH 7.4

Onion extract contains quercetin, which has anti-inflammatory properties. The absorption of quercetin in the extract can be improved by using the ionic gelation method to composition the extract into a nanoparticle system. Chitosan is a polymer that is used to make nanoparticles that impact medicinal drug absorption. Although many studies of nanoparticle coatings with chitosan have been performed, the effect of the chitosan concentration used remains an intriguing research issue, especially as a natural compound carrier. The goal of this study was to examine how varying chitosan polymer concentrations affected the rate of quercetin diffusion from onion ( Allium cepa L.) ethanol extract nanoparticles. With 0.1% tripolyphosphate (TPP) as a crosslinker, the concentrations of chitosan used were 0.1% (F1), 0.2% (F2), 0.3% (F3), and 0.4% (F4). Organoleptic test, particle size measurement, zeta potential, polydisperse index, entrapment efficiency, density, and determination of quercetin diffusion rate using a phosphate buffer medium pH 7.4 were all used to analyze each composition. Transparent yellow nanoparticles with particle sizes ranging from 199.89 nm to 514.97 nm, a zeta potential of 47.73 mV to 51.36 mV, a polydispersity index of 0.57, an entrapment efficiency of 54.78 % to 59.06 %, and a density of 1.012 g/mL to 1.042 g/mL are the result of this system. In each composition, the rate of diffusion follows the Higuchi reaction kinetics. Increased chitosan concentration decreases the diffusion rate of onion ethanol extract nanoparticles ( Allium cepa L). The fastest diffusion rate value with requirements-meeting physical properties was obtained in nanoparticle systems containing a 0.1 % chitosan solution. Â

Characteristics and Release Profile : Formula of Insulin Nanoparticles using Medium Molecular Weight Chitosan and Pectin Polymers

Insulin is a macromolecular polypeptide hormone with low drug stability and permeability along the digestive tract. The nanoparticle delivery system has been proven to be able to increase the bioavailability of per-oral insulin. However, the formulation of insulin nanoparticles using chitosan and pectin polymers has not been widely studied. The purpose of this research is to figure out the physical characteristics and profile of insulin release from nanoparticle formulas made with ionic gelation techniques using chitosan and pectin polymers. The 0.1% insulin nanoparticle formula is made with variations of 2 levels of medium molecular chitosan and pectin concentrations to obtain 4 formulas, i.e. F1 (0.01%; 0.1%), F2 (0.03%; 0.1%), F3 (0.01%; 0.2%), and F4 (0.03%; 0.2%). The optimum formula is determined by the factorial design method contained in the Design Expert program using response characteristics in the form of percentage of the entrapment efficiency and zeta potential value. The selected formula is then tested for particle size and shape, and insulin release profile in vitro. The particle size and morphology are observed with TEM (Transmission Electron Microscope), while the insulin release profile is determined on HCl buffer media pH 1.2 and PBS pH 6.8. The optimization results of the formula show that F1 is the optimum formula with a desirability value of 0.786. The selected formula shows that the entrapment efficiency is 57.66%, the zeta potential is 12.0 mV, the shape of particles is spherical, and the size is <500 nm. In vitro studies show the profile of insulin release from the matrix following the Weibull kinetics model on HCl and Korsmeyer-Peppas media on PBS media, using the Fickian diffusion method. Overall, the insulin nanoparticles obtained have met the expected characteristic of the nanoparticles.

Spectrophotometric and Spectrofluorimetric Determination of Terazosin in Tablets by Eosin Y

Simple, sensitive and accurate two methods were described for the determination of terazosin. The spectrophotometric method (A) is based on measuring the spectral absorption of the ion-pair complex formed between terazosin with eosin Y in the acetate buffer medium pH 3 at 545 nm. Method (B) is based on the quantitative quenching effect of terazosin on the native fluorescence of Eosin Y at the pH 3. The quenching of the fluorescence of Eosin Y was measured at 556 nm after excitation at 345 nm. The two methods obeyed Beer’s law over the concentration ranges of 0.1-8 and 0.05-7 µg/mL for method A and B respectively. Both methods succeeded in the determination of terazosin in its tablets

Evaluation of the interaction of a guanylhydrazone derivative with cobalt ferrite nanoparticles and PAMAM electrochemical and UV/visible spectroscopic techniques

Nowadays, scientific research grows a lot around nanotechnology, which connects many areas of knowledge such as electrochemistry and magnetic nanoparticles (MNPs). The study of bioactive substances by electrochemical methods is already very conceptual, and the association of magnetic nanoparticles has emerged as a new bias of this technique. Here, we report the results of the interaction between the molecule LQM10, a derivative of guanylhydrazone, with nanocarriers: cobalt ferrite magnetic nanoparticles (CoFe2O4 MNPs) and polyamidoamine dendrimer (PAMAM), seeking to unite the characteristics of each nanocarrier to benefit LQM10. The electrochemical system was composed of 3 electrodes, Ag/AgCl/Cl− saturated (reference), platinum (auxiliary), and modified glassy carbon (GCE) (work) with CoFe2O4 MNPs alone and with PAMAM G3, in buffered medium pH 7.03 with or without a co-solvent (analytical grade ethanol). UV-visible spectroscopic studies were carried out in aqueous-ethanolic medium in different concentrations of PAMAM G3 and CoFe2O4 MNPs. The magnetic properties of CoFe2O4 MNPs and each nanocarriers were confirmed by vibrating sample magnetometer and by field effect calorimetry. LQM10, which has a redox profile in oxidative potentials, showed good interaction with all tested electrodes and revealed considerable values of formation constant (KF), highlighting the GCE modified with CoFe2O4 MNPs (KF = 1.92 × 106 L/mol) and PAMAM G3, corroborating with the results of UV-visible data (Kb = 7.60 × 105 L/mol). The generation of heat by magnetic hyperthermia of CoFe2O4 MNPs in the presence of PAMAM G3 and LQM10 was determined (SAR = 0.72 W/g). Therefore, we demonstrated the association of promising nanocarriers (PAMAM G3 and CoFe2O4 MNPs) with anticancer substances and their applicability as magnetic hyperthermia.

A spectroscopic deciphering of the differential interaction behavior of alkaloid drugs with native B-DNA and protonated DNA

The present study demonstrates a detailed spectroscopic investigation on the differential extent and modes of interaction of two alkaloid drugs, Sanguinarine (SG) and Berberine (BR), with biologically significant protonated and B-forms of herring sperm DNA in aqueous buffer medium of pH 3.5 and 7.4 respectively. The steady-state spectroscopic results substantiate the binding interaction of either drug molecule with protonated as well as B-form of DNA. The drug:DNA binding strength obtained employing the Benesi-Hildebrand equation reveals a significantly higher binding affinity of BR with B-DNA at pH 7.4 compared to that with protonated DNA at pH 3.5. However, the binding constant obtained for interaction of iminium form of SG with protonated DNA is found to be an order of magnitude higher compared to that in case of interaction with B-DNA. Moreover, the steady-state emission results validate the role of preferential electrostatic interaction between the cationic forms of the drugs (cationic BR and the iminium form of SG) and the negatively charged phosphate backbone of B-DNA under physiological conditions at pH 7.4 which has been further corroborated by monitoring the effect of a strong electrolyte on drug:DNA interaction under the various experimental conditions. The circular dichroic (CD) spectroscopic studies reveal the probable intercalative binding of the drug molecules within DNA at either pH. The time-resolved fluorescence decay measurements along with the time-resolved fluorescence anisotropy studies further substantiate the binding interaction of the drugs with protonated as well as B-form of herring sperm DNA.

Linum usitatissimum seed mucilage-alginate mucoadhesive microspheres of metformin HCl: Fabrication, characterization and evaluation

This study investigates the fabrication, characterization and optimization of Linum usitatissimum mucilage (LSM) with sodium alginate mucoadhesive microspheres loaded metformin HCl, a sustained release dosage form prepared by ionic gelation technique. The effect of changing the polymer ratio with drug was studied for drug encapsulation efficiency and in vitro drug release for 12 h. Drug entrapment efficiency of all formulations was in the range of 77.93 ± 3.67 to 92.25 ± 4.08% with sustained release of 80 ± 0.12% to 88 ± 0.33% for 12 h. It followed Korsmeyers Peppas model (R2 = 0.9786–0.9964) with super case II transport mechanism. Average microsphere size of all formulations was within the range of 817–911 μm. SEM, FTIR were also characterized and swelling behavior of microspheres was affected by pH of buffer medium. These microspheres also showed good mucoadhesivity in wash off test. The optimized LSM-alginate mucoadhesive microspheres containing metformin HCl demonstrated significant hypoglycemic effect in alloxan-induced diabetic rats for prolonged period of time.

Development of UV-spectrophotometric method and its validation for estimating contents of Prulifloxacin in Simulated Intestinal Fluid

This study was performed with an objective of developing and validating an UV-spectroscopic method for estimating contents of prulifloxacin in simulated intestinal fluid (SIF) i.e. phosphate- buffer media with a pH of 6.8 as per ICH guidelines. The λmax for prulifloxacin in phosphate- buffer media pH 6.8 was found to be 272 nanometer. The calibration curve of drug followed linearity in-between 1-9 μg/ml concentration range and correlation co-efficient value was found equal to 0.9995. We tested this proposed method onto the bulk and marketed pharmaceutical formulation (tablets) also in order to find out contents of drug. Using developed method for estimation of prulifloxacin in SIF, drug was found to be in-between 101.91 and 104.02 % in marketed tablets which shows a good agreement with that of the claimed level. Accuracy of developed method was established through recovery experimentation, performed for three spiked percent concentrations- 75%, 100%, and 125%. The % recovery was found to be in between 97.27 and 101.82%. Low values of % RSD supported accuracy as well as the reproducibility of developed method. Precision of developed method was established by good in-limit intraday and interday experimental variations and through repeatability tests. Values of % RSD less than 2 confirmed about precision of developed method. The ruggedness of the developed method was validated by performing drug estimation by two different performers. This proposed spectroscopic method has proved to be a rapid and successful method for routine analysis of prulifloxacin in simulated intestinal fluid.

Modulation of Semiconducting Behavior and a Change in Morphology upon Gelation of a Peptide Appended Naphthalenediimide

A dipeptide-appended naphthalenediimide (NDI) is self-assembled in an aqueous phosphate buffer medium of pH 7.5 to form nanovesicular structures. Interestingly, these molecules are assembled to for...

Ca(II)+Ba(II) ions crosslinked alginate gels prepared by a novel diffusion through dialysis tube (DTDT) approach and preliminary BSA release study

In this work, plain and model therapeutic protein Bovine Serum Albumin(BSA) loaded alginate hydrogels, crosslinked with Ca(II)+Ba(II) ions, were prepared by ‘Diffusion Through Dialysis Tube’ (DTDT) technique which involved permeation of crosslinking ions into alginate solution via a dialysis tube with a molecular weight cutoff of 8 k Da. The model protein BSA loaded hydrogels were characterized by Fourier transform infrared (FTIR) and X-ray diffraction (XRD) analysis. The dynamic water uptake behavior of plain hydrogels was investigated in phosphate buffer saline (PBS) of pH 7.4 at 37 °C. Finally, the controlled release of therapeutic protein BSA from these hydrogels was studied in the phosphate buffer medium of pH 7.4 at 37 °C.The release data were interpreted in the terms of Power function model and First order kinetic model respectively.

Sensitive determination and electro-oxidative polymerization of azodyes on a carbon paste electrode modified with bentonite

A new composite carbon paste electrode incorporating bentonite (BentMCPE) was developed and used for extremely sensitive determination and electro-oxidative polymerization of azodyes using cyclic voltammetry. Lanaset Red 2B (LR2B) was selected as a model molecule for optimization of operational parameters such as amount of modifier, the pH of buffer medium, scan rate and dye concentration. Performance analyses showed that BentMCPE allows determination of LR2B, Acid Blue 113 (AB113) and Acid Yellow 17 (AY17) dyes as low as (6–8)×10−10mol/L at pH5.5. The peak intensities increased with the repetitive scans due to electro-catalytic anodic polymerization of the dyes. The results of multiple scans in single binary and ternary dye solutions showed that all of the dyes whether hydroxyl substituted (LR2B and AY17) or not (AB113) can be polymerized on the BentMCPE surface. Two possible electro-polymerization pathways were proposed by assuming formation of different radical intermediates produced from azo/hydrazone tautomers of the dyes. A comparison of FT-IR spectra recorded after the first and tenth repetitive scans confirmed that the electro-polymerization process proceeds by head to tail interactions of the radicals with dye monomers deposited on the electrode surface.

Simultaneous extraction and preconcentration of copper, silver and palladium with modified alumina and their determination by electrothermal atomic absorption spectrometry

Abstract In the present work, an easy solid phase extraction method using alumina modified with polyethylenimine as a new adsorbent was applied to the simultaneous extraction of copper, silver, and palladium ions prior to their determination with electrothermal atomic absorption spectrometry. The analytical procedure involved the complex formation of these cations with polyethylenimine as a chelating agent in buffer media of pH 7.0. Under the optimum conditions, a preconcentration factor of 200, 150, and 200, precision of ±5.4%, ±4.7%, and ±5.2% and linear calibration ranges of 15.0–140, 4.0–93, and 7.5–125 ng/L (in original solution) for Cu, Ag, and Pd were obtained, respectively. Also detection limits of 3.9, 1.1, and 2.0 ng/L were obtained for Cu, Ag, and Pd, respectively. The proposed method was applied to the determination of copper, silver, and palladium in some real samples with satisfactory results.

Spectrophotometric Determination of Al(III) in Environment Water with <i>p</i>-Carboxyarsenazo

Spectrophotometry determination of Al (III) with new color reagent p-Carboxyarsenazo(CAA) was reported, and the chromomeric reaction between CAA and Al (III) was studied. In a buffer medium of pH=4.6 HAc-NaAc, CAA reacts with Al (III) to form a red complex and exhibits an absorption maxima at 614nm; Beer,s law is obeyed for 0.10~0.80 μg/mL of Alumium in color solution, the color reaction exhibited high sensitivity (the apparent molar absorptivity obtained is 1.61×104 L/(mol.cm)), and the absorbance fit linear regression equation is A=0.0105+0.561X(μg/mL), the correlation coefficient is 0.9998, the detection limit of the method is 0.36 μg/L. The results obtained by this method in the determination of environment water samples were rapid, accurate and satisfactorily.

Catʼs Claw Oxindole Alkaloid Isomerization Induced by Cell Incubation and Cytotoxic Activity against T24 and RT4 Human Bladder Cancer Cell Lines

The antitumor activity of Uncaria tomentosa, a native vine from the Amazonian rainforest, has been ascribed to pentacyclic oxindole alkaloids occurring in its bark. Former studies have shown that this activity, as well as its intensity, depends on whether cat's claw alkaloids occur as original compounds or isomerized derivatives. This work addresses this aspect, using T24 and RT4 human bladder cancer cell lines for that purpose. Bark samples were extracted by dynamic maceration, prepurified with cross-linked polyvinylpyrrolidone and properly fractioned by an ion exchange process to obtain an oxindole alkaloid purified fraction. Alkaloid isomerization was induced by heating it under reflux at 85 °C. Samples collected after 5, 15, and 45 min of heating were analyzed by HPLC-PDA, freeze-dried at once, and separately assayed using the non-isomerized purified fraction for comparison purposes. The latter showed significant and dose-dependent cytotoxic activity against both T24 and RT4 cancer cell lines (IC50: 164.13 and 137.23 µg/mL, respectively). However, results for both cell lines were equivalent to those observed for isomerized samples (p > 0.05). The alkaloid isomerization induced by the incubation conditions (buffered medium pH 7.4 and temperature 37 °C) helps to explain the similar results obtained from non-isomerized and isomerized samples. Mitraphylline, speciophylline, uncarine F, and, to a lesser degree, pteropodine were more susceptible to isomerization under the incubation conditions. Thus, the alkaloid profile of all fractions and their cytotoxic activities against T24 and RT4 human bladder cancer cell lines are determined to a large extent by the incubation conditions.

The effect of serum albumin, surfactant and their mixture on the reduction of a cobalt(III) complex by ascorbic acid

Kinetic investigations on the reduction of CoIII-complex by a biologically active antioxidant molecule, ascorbic acid in a buffer medium of pH 9.0 have been performed using spectroscopic techniques. The reaction has been carried out in presence of a protein, bovine serum albumin (BSA), n-cetyltrimethylammonium bromide (CTAB) and their mixtures. The reaction rate changes remarkably on addition of protein. Unlike the complex, experimental results depict extensive binding of detergent and ascorbate anions with BSA. Both native and cooperative binding have been observed for CTAB–BSA interaction. But only native binding at specific binding sites have been observed for ascorbic acid–BSA interaction. The reactant molecules prefer to bind with domain I and III over II of the BSA molecules. Denaturing action of urea releases the protein bound ascorbates into bulk. The experimental findings have been further verified from the conductometric and circular dichroism studies.