{"paper_id":"44ed24bf-ea02-414e-b612-9b97bec03875","body_text":"Role of Non-ionic surfactant on the interaction of drugs with Bovine serum albumin (BSA) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Role of Non-ionic surfactant on the interaction of drugs with Bovine serum albumin (BSA) Prakash Karunanithi, M. Senthilkumar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5323332/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Serum albumins are the soluble protein part in the circulatory system. They have a significant role in transport and delivery of drug in blood. Bovine serum albumin (BSA) is generally used as model protein which is structurally similar to human serum albumin (HSA). Interaction of drug at the protein binding level can induce many changes like distribution rate, elimination of drug or most importantly solubilization of a hydrophobic drug. Protein-surfactant interaction employed to cause conformational changes in protein by change of polarity and on many occasions help in stabilization of protein. Generally, nonionic surfactants are used for these purposes and hence are widely studied. The studies on drug binding with BSA in presence of nonionic surfactant are however, rare in literature. In this study, we have taken four drugs for analyzing their binding with BSA in presence of a nonionic, hydrophobic surfactant, pluronic L-81 and investigate their role in the system. The drugs are Tinidazole (TNZ), Piroxicam (PY), Methylparaben (MP) and Propylparaben (PP). The characterizations were done using UV-visible spectroscopy and fluorescence spectroscopy. The UV-spectroscopic measurements were conducted for BSA-drug mixtures without and with pluronic L-81. The absorbance values were used for determining the binding constant values using Benesi-Hildebrand (B-H) plot. Fluorimetry studies of the BSA + L-81 + drug mixtures were carried out at room temperature which showed that there was quenching effect on addition of drug to BSA + L-81 mixture. Further, the quenching was of dynamic nature. The fluorescence data were analyzed using Stern-Volmer and Lineweaver-Burk equations. With the data, association constant K A for all the systems were determined. The docking studies confirmed the binding of the drugs with BSA. Pluronic L-81 Tinidazole Piroxicam Methylparaben Propylparaben Solubilization Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 1. Introduction Serum albumins are the most abundant protein present in blood plasma. They are also referred as blood albumin which carries many compounds of blood like hormones and ions. They also transport the drugs by binding and release in the circulatory system 1 . They play an important role in transport and delivery of drug ligands in blood and on many occasions they increase the solubility of hydrophobic drugs [ 1 ]. Bovine serum albumin (BSA) is used extensively as a model protein in place of human serum albumin (HSA) because of its structural similarity, low cost and easy availability. BSA and HSA have about 86% similarity in amino acid sequence [ 2 ]. BSA consists of 583 amino acids in a single peptide chain and is made up of three homologous domains. It has two tryptophan residues which have a role to stabilize the structure through different kind of bonding [ 3 , 4 ].They are, (i) Trp-212 located in hydrophobic pocket of their protein and (ii) Trp-134 located on the surface of the molecule. The studies of drug-protein can provide information regarding structural features that decide the therapeutic effectiveness of the drug. They also provide useful information about the possibility and stability of drug-drug interaction, drug-protein interaction, side effects and dosage information [ 5 – 7 ]. So, the studies have been interesting in the research field of medicinal chemistry with different pharmacokinetic properties like distribution, transportation and excretion. The knowledge of the protein receptors (binding with BSA) is necessary for any new drug prior to their clinical trial [ 8 – 10 ]. The functions of proteins depend on its three dimensional structure. This structure can be restored or disturbed in different chemical environments. The marginal stability of protein when present in native globular confirmation depends on various interactions taking place in the proteins, viz., the temperature, pH of the medium, presence of coenzyme, type of inhibitors and substrates [ 11 , 12 ]. Surfactants act as denaturing agents for membranes in proteins and lipids. The study of interaction of surfactants with globular proteins has lot of importance and it is extensively studied and reported [ 13 , 14 ]. From this angle, surfactants are classified based on their reaction with protein. In the first type, there is denaturation or protein unfolding and in the second type, they can only bind where the ternary structure of the protein is left undisturbed. Generally, ionic surfactants are capable of denaturing protein, but nonionic surfactants do not [ 15 – 17 ]. Among the study of BSA-surfactant interaction it is observed that SDS is an efficient protein denaturant. Also, there are several reports on BSA + SDS interaction [ 18 , 19 ]. There are many reports on multifunctional binding nature of BSA that binds a wide range of molecules [ 20 , 21 ]. Also, there are many reports on the interaction of drug with BSA 22–24 .The binding function is a medium for transportation of soluble substances between different tissues and organs. Albumin has one important function (i.e.) transporting fatty acids. Binding also helps to protect against the toxic effects of the drug bound ligand. Hence, binding studies with BSA have significant applications in the rational designing of drug in many pharmaceuticals. The effect of additives or drugs on micellization behavior has been investigated before [ 25 , 26 ]. The presence of surfactants in the drug-protein complex can cause conformational changes in protein due to changes in polarity and can, in many instances, result in stability of the protein [ 27 – 31 ]. Protein-surfactant interaction has a wide variety of applications in the biological, cosmetic, pharmaceutical and industrial systems [ 32 ]. But, the role of nonionic surfactant in a drug-BSA system is rare in literature. In this context it is important to mention that though pluronic L-81 is a hydrophobic surfactant and directly it doesn’t find any application in the pharmaceutical industry, it has been used in mixed micellar formulation for better efficacy by earlier research groups [ 33 – 34 ]. Work reported here includes the incorporation of a nonionic hydrophobic surfactant, namely, pluronic L-81 into complex of BSA and drug and analyze the change in binding parameters due to the conformational changes induced by the surfactant. We have used four drugs in this study. They are Tinidazole (TNZ), Piroxicam (PY), Methylparaben (MP) and Propylparaben (PP). The characterization techniques employed are UV-visible spectroscopy and fluorescence spectroscopy. The docking studies were also carried out to assess the interactions. The following works on different drugs on BSA has been reported in the literature shown in Table 1 . Table 1 Literature survey of BSA with different drugs compared to our data Systems Binding Constant (M − 1 ) References BSA + Methylparaben 3.32×10 9 [ 1 ] BSA + Tinidazole BSA + Piroxicam BSA + Methylparaben BSA + Propylparaben 6.0×10 − 3 7.0×10 − 3 3.6×10 − 2 1.2×10 − 2 This work BSA + L-81 + Tinidazole BSA + L-81 + Piroxicam BSA + L-81 + Methylparaben BSA + L-81 + Propylparaben 8.39×10 − 3 7.9×10 − 3 5.60×10 − 2 3.11×10 − 2 This work 2. Experimental Section 2.1 Materials and methods All the reagents were of analytical grade. Double distilled water was used for the experiments. The drugs (98% purity) Tinidazole (TNZ), Piroxicam (PY), Methylparaben (MP) and Propylparaben (PP) were procured from MMC health care limited and Chemical Ltd chemical company, Chennai respectively. BSA (≥ 98% purity) was procured from Sigma Aldrich, supplied by Subra scientific company Pondicherry. Pluronic L-81 (≥ 99% purity) also was obtained from Sigma Aldrich. A stock solution of pluronic L-81 was prepared for 18mM after weighing the appropriate quantity and solubilizing in distilled water. This co-polymer is solubilized better in cold temperatures (around 5 o C). At room temperature it showed a somewhat turbid appearance which disappears on dilution. The stock drug solution of Tinidazole (TNZ) for 4.0×10 − 2 M, Piroxicam (PY) for 3.0×10 − 3 M, Methylparaben (MP) for 6.5×10 − 2 M and Propylparaben (PP) for 4.5×10 − 2 M were prepared. Bovine serum albumin solution was prepared by taking 1.002g of BSA and making upto 100 ml in a standard measuring flask using buffer pH 7.4. The buffer solution was prepared by weighing 1.37g buffer of potassium di-hydrogen phosphate in 100ml, double distilled water and 1.41g of disodium hydrogen phosphate in other two standard flasks. In the following step, 81 ml of disodium and 19 ml of potassium di-hydrogen phosphate were taken to prepare 100ml buffer solution. The pH was adjusted using a pH meter to 7.4 by using standard NaOH solution and then it was calibrated using a known buffer solution. Different concentrations of the drug solutions were used for calibration of the instrument and from among them, an optimum concentration was chosen for all the experiments which were kept constant. The optimized drug concentration was mixed with different concentrations of BSA. The mixture of drug and BSA were taken for UV-spectroscopic and fluorescence measurements with and without L-81. All the experiments were carried out at room temperature. 2.2. UV-Visible spectroscopy UV Spectroscopic measurements of the four different drugs with Pluronic L-81 in presence of BSA were carried out using a Shimadzu (UV-2600) spectrophotometer baseline in the range of 200–400 nm. All the readings were taken at room temperature. 2.3. Fluorescence spectroscopy Fluorescence intensity measurements were carried out with by F-4700 FL spectrophotometer. The excitation and emission wavelength were maintained at 250 nm and 550 nm respectively. BSA was used as a fluorescence probe. Scanning and slit range was 1200 nm/min and 5nm. In this study, we have taken four different drugs with Pluronic L-81 in presence of BSA in aqueous medium. 2.4. Docking study Docking study was carried out for Tinidazole, Piroxicam, Methylparaben, Propylparaben and surfactant pluronic L-81by using Autodock 4.0 software. 3. Results and discussion In this work, the surfactant pluronic L-81 is added to drug-BSA combination. Each drug has its specific interaction affinity with BSA. However, the presence of surfactant changes the interactivity between drug and BSA. The presence and absence of the surfactant in the drug-BSA medium was assessed through the following characterizations. Each drug has been described on separately. 3.1 UV spectroscopic measurements To investigate the nature of binding of BSA with drug, UV spectroscopic measurements were taken. There were four drugs chosen. Each drug was taken with BSA with and without the non-ionic surfactant pluronic L-81. The absorbance values of the aliquots were recorded and analyzed. Each drug is discussed separately. The calibration of each drug was done and the results showed correlation coefficient above 0.9. The molecular structures of four drugs viz., tinidazole, piroxicam, methylparaben and propylparaben, as well as pluronic L-81, are shown in Fig. 1 . 3.1.1. Tinidazole (TNZ) Tinidazole (TNZ) was taken in 0.008 mM concentration. It showed a maximum at 318 nm which was coincidence with literature value [ 35 , 36 ]. On addition of 0.8µM BSA in the drug solution, there was increase in the intensity of absorbance of 318 nm peak. Additionally, the peak for BSA appeared prominently at 279 nm [ 37 , 38 ]. There was no extra peak formed and there was no shifting of the λ max value of TNZ is shown in Fig. 2 . Subsequent aliquots were taken by using the same BSA concentration and varying (increase) concentration of drug. In all those samples, the same trend followed with a small red shift. The peak at 318nm for TNZ and 279nm for BSA were observed to increase in the absorbance intensity. The concentration of TNZ was varied from 0.008mM to 0.028mM. The increase of absorbance of the drug at λ max 318nm at higher concentrations and BSA peak at 279nm is suggestive of strong interaction between the TNZ molecule and BSA. Using the absorbance values and applying Benesi-Hildebrand (B-H) plot, the binding constant values were calculated. The absorbance of pure BSA is A 0 , the absorbance of formed complex after interaction with drug is A. Then we can do a baseline correction by calculating A-A 0 in the process of binding. The binding constant K has been calculated using formula, K = 1/slope* A-A 0 The slope is taken from a graph plotted between 1/concentration of drug and 1/A-A 0 . The A-A 0 value of the highest concentration of drug is taken for calculation. Thus, the binding constant of the system is calculated and shown in Table 2 . Table 2 Binding constant of BSA + TNZ Concentration TNZ mM Wavelength (nm) A 0 A A 0 -A 1/TNZ Concentration 1/ A 0 -A 0 0.008 0.012 0.016 0.02 0.024 0.028 318 279 279 279 279 279 279 0.255 0.255 0.255 0.255 0.255 0.255 0.255 0.255 0.693 0.752 0.788 0.792 0.905 0.1061 0 0.438 0.497 0.533 0.537 0.650 0.806 0 125 83.4 62.5 50 41.7 35.7 0 2.28 2.01 1.87 1.86 1.53 1.24 Binding constant k = 6.0×10 − 3 M − 1 To the above mixture of BSA + TNZ, a non-ionic surfactant pluronic L-81 was incorporated and the experiment was repeated as before. A set of readings with the same increased absorbance trend was obtained. The A, A 0 values were substituted and binding constant was found to be K = 128 M − 1 . It is shown in Fig. 3 and Table 3 . Table 3 Binding constant of BSA + L-81 + TNZ Concentration TNZ mM Wavelength (nm) A 0 A A 0 -A 1/TNZ Concentration 1/ A 0 -A 0 0.008 0.012 0.016 0.02 0.024 0.028 318 279 279 279 279 279 279 0.226 0.226 0.226 0.226 0.226 0.226 0.226 0.266 0.812 0.825 0.866 0.889 0.939 1.066 0 0.546 0.559 0.6 0.629 0.673 0.8 0 125 83.4 62.5 50 41.7 35.7 0 1.83 1.78 1.67 1.60 1.48 0.93 Binding constant k = 8.39×10 − 3 M − 1 The discrepancy of K values shows that the ternary system of BSA + L-81 + TNZ has a higher binding constant. Hence, the incorporation of the L-81 in the drug-BSA mixture is helping to strengthen the bonds in the complex. The binding constant value was calculated to be K = 173 M − 1 . The higher binding constant is the parameter to assess the strength of bond. It is shown in Table 3 . 3.1.2. Piroxicam (PY) Piroxicam has a global charge with zwitterions-anionic drug. The UV studies of piroxicam shows that the λ max is at 353nm. The literature value is 353nm [ 39 ]. Like the previous case, here also the BSA concentration was kept constant and PY concentration was raised from 0.0024 mM to 0.0084 mM. There was increased absorption intensity with a small red shift of the λ max . But, the 279nm peak of BSA was seen to only enhance the absorption. The Benesi–Hildebrand (B-H) plot calculations showed that the binding energy of the PY + BSA was K = 46 M − 1 in the complex which is a strong one indicated by red shift. The results are shown in Table 4 . Table 4 Binding constant of PY + BSA Concentration PY mM Wavelength (nm) A 0 A A 0 -A 1/PY Concentration 1/ A 0 -A 0 0.0024 0.0036 0.0048 0.006 0.0072 0.0084 353 353 353 353 353 353 353 0.158 0.158 0.158 0.158 0.158 0.158 0.158 0.158 0.213 0.264 0.343 0.403 0.485 0.561 0 0.055 0.106 0.185 0.245 0.327 0.403 0 416.7 277.8 208.4 166.7 138.9 119 0 18.18 9.43 5.40 4.08 3.05 2.48 Binding constant k = 7.0×10 − 3 M − 1 In the next step, to the BSA + PY mixture, pluronic L-81 was added. The results are shown in Fig. 4 a and Table 5 . There was only small increase of the absorbance λ max (353nm). But, BSA peak at 279nm was undisturbed. There was no intersection of the spectra and no new spectra appeared. Only the increase in absorbance could be due to complex formation by BSA + L-81 + PY. The absorbance values were used to calculate the Benesi-Hildebrand (B-H) plot to determine the binding constant which was found to be 617 M − 1 shown in Table 5 . In this system also, the binding constant shows that the presence of L-81 has worked towards strong binding of PY and BSA. Table 5 Binding constant of PY + BSA + L-81 Concentration PY mM Wavelength (nm) A 0 A A 0 -A 1/PY Concentration 1/ A 0 -A 0 0.0024 0.0036 0.0048 0.006 0.0072 0.0084 260 270 270 270 270 270 270 0.118 0.118 0.118 0.118 0.118 0118 0.118 0.118 0.635 0.666 0.735 0.805 0.811 0.889 0 0.517 0.548 0.617 0.687 0.693 0.771 0 416.7 277.8 208.4 166.7 138.9 119 0 1.93 1.82 1.62 1.45 1.44 1.29 Binding constant k = 7.9×10 − 3 M − 1 3.1.3. Methylparaben (MP) Parabens are commonly used for cosmetic products. The external application of paraben can be tuned depending on the requirement. Hence, it's interaction with different surfactants is a topic of research. The BSA + MP interaction has been reported before (Naik & Nandibewoor, 2013). Methylparaben showed spectra with λ max at 254nm. This is in good agreement in the literature values [ 40 ]. Addition of BSA causes red shift of the λ max to 278nm (Fig. 6a and Table 6 ). Red shifting by 24nm is an indication of the strong bonding developed between MP and BSA to form a complex. The (B-H) plot was used to determine the binding constant which was found to be K = 27 M − 1 . Table 6 Binding constant of BSA + MP Concentration MP mM Wavelength (nm) A 0 A A 0 -A 1/MP Concentration 1/ A 0 -A 0 0.032 0.038 0.044 0.052 0.057 0.064 254 259 259 259 258 259 259 0.327 0.327 0.327 0.327 0.327 0.327 0.327 0.327 0.866 0.916 0.956 1.014 1.105 1.177 0 0.539 0.589 0.629 0.687 0.778 0.850 0 31.25 26.31 22.72 19.23 17.54 15.62 0 1.85 1.69 1.58 1.45 1.28 1.17 Binding constant k = 3.6×10 − 2 M − 1 Incorporation of pluronic L-81 into the MP + BSA mixture, there was no significant change in the nature of spectra. The results are shown in Fig. 5 b and Table 7 . The red shift of λ max observed in Fig. 5 b, remains unaffected by addition of pluronic L-81. The absorbance values were taken to apply the B-H plot and determine the binding constant K = 24 M − 1 . Table 7 Binding constant of BSA + L-81 + MP Concentration MP mM Wavelength (nm) A 0 A A 0 -A 1/MP Concentration 1/ A 0 -A 0 0.032 0.038 0.044 0.052 0.057 0.064 254 259 259 259 259 259 259 0.256 0.256 0.256 0.256 0.256 0.256 0.256 0.256 0.659 0.703 0.734 0.751 0.798 0.972 0 0.403 0.447 0.478 0.495 0.542 0.716 0 31.25 26.21 22.72 19.23 17.54 15.62 0 2.48 2.23 2.09 2.02 1.84 1.39 Binding constant k = 5.60×10 − 2 M − 1 Here, the binding constant (K) was observed to be lower in presence of L-81 as compared to the BSA + MP mixture alone. Hence, it can be predicted that the non-ionic surfactant pluronic L-81 captured the drug MP + BSA complex with weaker binding became presence of pluronic L-81 in Fig. 7 . 3.1.4. Propylparaben (PP) Propylparaben displayed the absorption spectra at (λ max ) at 255nm. The evidence in literature is 255nm [ 41 , 42 ]. The results are shown in Fig. 6a and Table 8 . As observed, addition of BSA to PP there was a red shift to λ max = 275nm. The 24nm shifting was because of the strong bonding developed between PP and BSA to form a complex. With increase in concentration of drug to a fixed quantity of BSA, there was only progressive increase in absorbance without formation of new peaks or any intersecting point. Hence, no new compound is expected to have been formed. The (B-H) plot of the absorbance data showed that the value of K is 8.4 M − 1 . Table 8 Binding constant of BSA + PP Concentration PP mM Wavelength (nm) A 0 A A 0 -A 1/PP Concentration 1/ A 0 -A 0 0.13 0.16 0.19 0.21 0.24 0.27 254 259 259 259 259 259 259 0.267 0.267 0.267 0.267 0.267 0.267 0.267 0.267 0.802 0.869 0.979 0.989 1.009 1.086 0 0.563 0.602 0.712 0.722 0.742 0.819 0 7.69 6.25 5.26 4.76 4.17 3.70 0 1.80 1.66 1.40 1.38 1.34 1.22 Binding constant k = 1.2×10 − 2 M − 1 To know the effect of the pluronic L-81 in the PP-BSA system, the next part of the study was carried out. To fixed quantities of L-81 and BSA, different concentrations of drug, PP were added. Here, the drug concentration ranged from 0.13mM to 0.24mM. The results are shown in Figure 8 b and Table 9 . There was increase in absorbance with increase in concentration of drug. This shows that there is complex formation, between BSA and PP and that, in presence of pluronic L-81 the complex formation occurs with greater binding force. Like the previous case, there is a significant red shift 25nm which is indicative of strong bonding between PP and BSA when pluronic L-81 present (λ max = 280nm). The K value determined from the absorbance values was K = 4.5 M − 1 . Table 9 Binding constant of BSA + L-81 + PP Concentration PP mM Wavelength (nm) A 0 A A 0 -A 1/PP Concentration 1/ A 0 -A 0 0.13 0.16 0.19 0.21 0.24 0.27 254 259 259 259 259 259 259 0.197 0.197 0.197 0.197 0.197 0.197 0.197 0.197 0.531 0.586 0.598 0.6 0.789 0.839 0 0.334 0.389 0.401 0.403 0.592 0.642 0 7.69 6.25 5.26 4.76 4.17 3.70 0 2.99 2.57 2.49 2.48 1.68 1.55 Binding constant k = 3.11×10 − 2 M − 1 A high value of K in presence of L-81 in the PP-BSA system indicates that pluronic L-81 is not enhancing the binding factor to the PP + BSA complex. In Table 10 , all the four drugs with BSA with and without pluronic L-81 are shown. This reveals that there is a strong role of pluronic L-81 in the binding of drug with BSA in Tinidazole and Piroxicam, but with MP and PP the binding is weaker in presence of L-81. Table 10 Benesi-Hildebrand (B-H) binding constant values using UV data Ligand Docking Score Tinidazole -4.52 Piraxicam -4.37 Methylparaben -4.39 Pluronic L-81 -0.75 Propylparaben -4.03 Hence, it can be inferred from UV studies that out of the four drugs two had enhanced binding with BSA in presence of pluronic-L81. This may be due to specific nature of the drug and surfactant combination. 3.2. Spectro Fluorimetry The changes in confirmation of BSA were monitored by the intrinsic fluorescence intensity before and after addition of pluronic L-81 when the protein is bound with the drug [ 43 ]. Fluorescence measurements provide information about the molecular environment present in the vicinity of the fluorophores. We have used four drugs in this study. They are discussed separately. 3.2.1. Tinidazole (TNZ) When drug TNZ was added to BSA in presence of pluronic L-81 surfactant, the fluorescence intensity of BSA got reduced [ 44 ]. This is show in Fig. 10 a. In subsequent aliquots keeping the concentration of BSA and L-81 constant, the drug concentration was varied. The quantitative analysis of the binding of TNZ with BSA in presence of L-81 was carried out at 343nm. There was reduction in the intensity due to quenching effect. The maximum wavelength of BSA didn't shift from the original position. The remarkable lowering of fluorescence intensity indicated that there is positive interaction of TNZ with BSA and L-81. The hydrophobic surfactant L-81 was helpful in maintaining the environmental polarity and its micelles helped protecting the complex formed between the TNZ and BSA. There was possible quenching mechanism which is evident from the application Stern-Volmer plots as shown in Fig. 12 a. The quenching data were applied to Stern-Volmer equation. F/F 0 = 1 + K sv [Q] = 1 + K q τ a [Q] Where F 0 and F stand for the fluorescence intensity of BSA with and without TNZ in presence of pluronic L-81. The parameters K q , K sv , τ a and [Q] represent quenching rate constant, Stern- Volmer constant, the average lifetime of the drug and concentration of quencher respectively. The linear nature of Stern-Volmer regression curve suggests that the quenching interaction between BSA + TNZ is of dynamic nature. The Line-weaver bulk plot is displayed in Fig. 11 a and Table 11 . Table 11 Lineweaver-Burk calculation for association constant (K A ) Samples of different drugs Binding Constant of BSA (Without L-81) Binding Constant of BSA (With L-81) M − 1 M − 1 Tinidazole (TNZ) Piroxicam (PY) Methylparaben (MP) Propylparaben (MP) 128 46 27 8.4 173 617 24 4.5 3.2.2. Piroxicam (PY) Piroxicam addition in BSA solution in presence of pluronic L-81 was investigated by fluorescence studies. It is shown in Fig. 10 b. A fixed quantity of BSA + L-81 was added to varied concentration of the drug piroxicam. The confirmation of BSA in the molecular environment was assessed by fluorophore molecules [ 45 ]. The effect of piroxicam on the BSA + L-81 shows that there is significant lowering of fluorescence intensity of BSA, which points towards the interaction between BSA and PY. The micellar cavity of pluronic L-81 which was initially present near BSA, in the later stage it takes up the drug molecules of piroxicam. As the concentration of drug increases, there is gradual lowering of the fluorescence intensity. The intensity values were used to determine the Stern-Volmer constant by making use of the equation. The results showed linearity of Stern-Volmer curve shown in Fig. 12 b indicating dynamic nature of quenching. The association constant value of piroxicam was found to be 3.4×10 3 L Mol − 1 . The association factors of the four drugs and the Lineweaver-Burk are displayed in Table 11 . 3.2.3. Methylparaben (MP) Different concentrations of methylparaben were added to fixed quantity of BSA in presence and absence of L-81 and fluorescence spectra were recorded. As there was an increase in MP concentration, there was more lowering of the intensity of fluorescence spectra. There are previous reports of BSA + MP interaction. But, the above system in presence of surfactant has not been carried out [ 46 ]. In this study also, a similar trend was observed as the case of BSA + MP system. The lowering of intensity with increase in drug solution indicated that there is quenching reaction taking place. The results are displayed in Fig. 12 c and Table 10 . This intensity values were used to calculate the association constant which was found to be 3.49×10 3 L Mol − 1 . The Stern-Volmer plot displayed a straight line that suggested quenching to be of dynamic nature. Lineweaver-Burk was also plotted which is shown in Fig. 11 c. 3.2.4. Propylparaben (PP) The effect of the addition of propylparaben to BSA and pluronic L-81 was observed from the fluorescence studies. The molecular environment of the fluorophore molecule, here the BSA molecule, was changed due to the addition of PP and pluronic L-81. It is shown in Fig. 9 d. There was a gradual lowering of intensity values on the addition of more amounts PP to BSA and pluronic L-81. This was nothing but quenching action by the drug [ 47 ]. The quenching mechanism was evaluated by Stern-Volmer equation and the results are shown in Fig. 12 d. A near linear curve predicts the dynamic nature of quenching. The association constant K A for the system was evaluated which was found to be 3.3×10 3 L Mol − 1 . A straight line was obtained for Lineweaver-Burk plot as shown in Fig. 11 d. 3.2.5. Docking Analysis Molecular modeling technique is used to predict how a protein interacts with small molecules. In this study, molecular docking was performed using the Autodock 4.2 program. Since it was not possible to take three molecules simultaneously for docking study, using the above software, we have used the docking model of all the four drugs and the surfactant with BSA. From the binding energy values of five combinations, the comparative interaction is assessed. Molecular docking of compound Fig. 13 (a-e) was performed with the protein BSA. The ligand often connects to the protein through a variety of interactions, including pi-alkyl interactions, pi-pi interaction, pi-donor hydrogen bond interactions and conventional hydrogen bond interactions [ 48 – 50 ]. Our molecular docking studies showed the same kind of interactions. In compound (a), the BSA shows two hydrogen bonds Gln220 (B) and Lys 221(B) with bond lengths 3.21 Å and 2.57 Å respectively. This was observed to have a binding energy of -4.52 Kcal mol − 1 .The combination of compound (c) with BSA shows one hydrogen bond at Lys524 (B) site with bond length of 2.95 Å and the binding energy of this compound was seen to be -4.39 Kcal mol − 1 mentioned in Table 12 . The binding energy of the compound (d) was − 4.03 Kcal mol − 1 . The BSA shows three hydrogen bonds with Arg256 (A) of varying bond lengths 3.30 Å, 3.15 Å and 3.10 Å respectively. One hydrogen bond was observed with Tyr149(A) having a bond length of 3.33 Å. The binding energy of the compound (e) was − 0.75 Kcal mol − 1 . This value is the lowest in the table, which means that there is no interference if this molecule is present in between drug and surfactant. With this molecule, the BSA shows hydrogen bonds with Asn44 (B).The bond lengths noted were 2.40 Å and 3.25 Å. Yet another hydrogen bond with Lys 20 (B) of bond length 2.89 Å was also found in the structure. Table 12 Docking score of drugs and surfactants Quenchers BSA with L-81 with quenchers 1/Q F 0 -F 1/F 0 -F K A L mol − 1 Tinidazole (TNZ) 416.7 277.8 208.4 166.7 45.73 50.72 55.89 64.34 0.022 0.019 0.017 0.015 3.4×10 3 Piroxicam (PY) 1225 83.4 62.5 50 0.051 0.068 0.071 0.083 45.45 14.70 14.08 12.04 6.9×10 3 Methylparaben (MP) 31.25 26.21 22.72 19.23 251.4 329.5 337.2 353.4 0.0039 0.0030 0.0029 0.0028 3.49×10 3 Propylparaben (PP) 7.69 6.25 5.26 4.76 23.01 26.02 28.13 32.85 0.043 0.038 0.035 0.030 3.3×10 3 Comparing of the bonding in all the combinations from Fig. 13a to Fig. 13e, it can be said that compound (a), with TNZ has the lowest binding energy, hence, gives a more stable combination with BSA. 4. Conclusion Pluronic L-81, a non-ionic surfactant has been used earlier in binary combination in the pharmaceutical industry as polymeric drug delivery vehicles. In this work, the effect of presence of pluronic L-81 on the binding behavior of four drugs with BSA was investigated by UV-visible spectroscopy and fluorescence measurements. There was enhanced binding observed due to incorporation of L-81 in the complex of drug-BSA. Dynamic quenching is suggested from the quenching behavior of drugs. The results were compared with molecular docking studies. This study discloses use of a non-ionic surfactant to improve the drug-BSA binding which can be extended too many other drug formulations. Abbreviations TNZ-Tinidazole PY-Piroxicam MP- Methylparaben PP-Propylparaben BSA-Bovine serum albumin Pluronic L-81 Declarations Acknowledgement The medicine samples were provided by Applied Communication and Control, Dehradun, and MMC Health Care Limited, Chennai. Funding The authors declare that they have not received any funding from research work Conflict of Interest The authors declare that they have no conflict of interest Ethical Approval statement and Declaration It is not applicable Author Contribution Prakash Karunanithi: Investigation, Data curation M. Senthilkumar: Investigation, Formal analysis, Methodology, Review & Writing-original draft. References Naik KM, Nandibewoor ST (2013) Spectral characterization of the binding and conformational changes of bovine serum albumin upon interaction with an anti-fungal drug, methylparaben. Spectr Act A Mole Biomolecular Spect 105: 418–423. https://doi.org/10.1016/j.saa.2012.12.055 Fuhrman Kirk D, Allinson JM (1992) Atomic structure and chemistry of human serum albumin. Nature 358:710–713 Hu YJ, Ou-Yang Y, Bai AM, Li W, Liu Y (2010) Investigation of the interaction between ofloxacin and bovine serum albumin: Spectroscopic approach. 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International J Molecular Sci 24: (14), 11442. https://doi.org/10.3390/ijms241411442 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-5323332\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":374875084,\"identity\":\"779d41e1-1b4b-46d8-9da7-c1b60d4ac080\",\"order_by\":0,\"name\":\"Prakash Karunanithi\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Prakash\",\"middleName\":\"\",\"lastName\":\"Karunanithi\",\"suffix\":\"\"},{\"id\":374875085,\"identity\":\"4b8f5c2e-b234-4b26-9a18-bb3591537a6c\",\"order_by\":1,\"name\":\"M. 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L-81.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5323332/v1/5eb8d7d53e73b3ea6edd5d03.png\"},{\"id\":69552302,\"identity\":\"15eba7d7-a35a-4076-937f-37200fea1b1a\",\"added_by\":\"auto\",\"created_at\":\"2024-11-21 14:43:02\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":67128,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eBenesi-Hildebrand (B-H) plot of methylparaben and (BSA) protein (a) Without L-81 and (b) With L-81.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5323332/v1/e8e89a1e17f9b1b6c0a039d5.png\"},{\"id\":69551135,\"identity\":\"70fe9c93-52e5-4d51-8f17-ab434b2b36dd\",\"added_by\":\"auto\",\"created_at\":\"2024-11-21 14:35:02\",\"extension\":\"png\",\"order_by\":8,\"title\":\"Figure 8\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":219635,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eUV-spectra of propylparaben and (BSA) protein (a) Without L-81 and (b) With L-81.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"8.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5323332/v1/6462bbb44b1179b4913f35e0.png\"},{\"id\":69551136,\"identity\":\"06a3d0bb-fd44-4b21-8aac-8ee94d834fe0\",\"added_by\":\"auto\",\"created_at\":\"2024-11-21 14:35:02\",\"extension\":\"png\",\"order_by\":9,\"title\":\"Figure 9\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":60368,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eBenesi\\u003cem\\u003e-\\u003c/em\\u003eHildebrand (B-H) plot of propylparaben and (BSA) protein (a) Without L-81 and (b) With L-81.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"9.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5323332/v1/e97f42d21cdb36b24fa94850.png\"},{\"id\":69551143,\"identity\":\"6e01d718-a23a-45d5-bcb7-944ac71ea9b3\",\"added_by\":\"auto\",\"created_at\":\"2024-11-21 14:35:03\",\"extension\":\"png\",\"order_by\":10,\"title\":\"Figure 10\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":354515,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFluorescence spectra of four drugs with pluronic system in presence of BSA protein (a) Tinidazole (TNZ), (b) Piroxicam (PY), (c) Methylparaben (MP) and Propylparaben (PP).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"10.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5323332/v1/e7148577f069ca4ad53d2b25.png\"},{\"id\":69552304,\"identity\":\"d80cf8a4-b3b3-4d0c-97f8-33dd3b5fb8e3\",\"added_by\":\"auto\",\"created_at\":\"2024-11-21 14:43:02\",\"extension\":\"png\",\"order_by\":11,\"title\":\"Figure 11\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":49883,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eLineweaver\\u003cem\\u003e-\\u003c/em\\u003eBurkeplot of four drug and (BSA) protein with L-81 (a) Tinidazole (TNZ), (b) Piroxicam (PY), (c) Methylparaben (MP) and Propylparaben (PP).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"11.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5323332/v1/35c23eab3752871b7e74823b.png\"},{\"id\":69551139,\"identity\":\"f0306f9c-ebd8-41e6-8d9b-2e4749aa3bfc\",\"added_by\":\"auto\",\"created_at\":\"2024-11-21 14:35:02\",\"extension\":\"png\",\"order_by\":12,\"title\":\"Figure 12\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":57376,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eStern-volmer plot of four drug and (BSA) protein with L-81 (a) Tinidazole (TNZ), (b) Piroxicam (PY), (c) Methylparaben (MP) and Propylparaben (PP)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"12.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5323332/v1/ad2b7f868b263c9e72fb3f79.png\"},{\"id\":69551141,\"identity\":\"dc36dc79-aebd-4838-bd11-4484560eab0f\",\"added_by\":\"auto\",\"created_at\":\"2024-11-21 14:35:03\",\"extension\":\"png\",\"order_by\":13,\"title\":\"Figure 13\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":985284,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e2D and 3D docking image of drugs and surfactant (a) Tinidazole, (b) Piroxicam, (c) Methylparaben, (d) Propylparaben and (e) Pluroni L81.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"13.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5323332/v1/fb7f43ab1a677b13b92f69f9.png\"},{\"id\":69754661,\"identity\":\"b8f54a27-65d7-43e2-b4e9-2849a02d25fd\",\"added_by\":\"auto\",\"created_at\":\"2024-11-25 03:02:13\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":3170402,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-5323332/v1/a6cc2ef4-2c09-41ce-9dc3-6e5623272964.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Role of Non-ionic surfactant on the interaction of drugs with Bovine serum albumin (BSA)\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eSerum albumins are the most abundant protein present in blood plasma. They are also referred as blood albumin which carries many compounds of blood like hormones and ions. They also transport the drugs by binding and release in the circulatory system\\u003csup\\u003e1\\u003c/sup\\u003e. They play an important role in transport and delivery of drug ligands in blood and on many occasions they increase the solubility of hydrophobic drugs [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. Bovine serum albumin (BSA) is used extensively as a model protein in place of human serum albumin (HSA) because of its structural similarity, low cost and easy availability. BSA and HSA have about 86% similarity in amino acid sequence [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. BSA consists of 583 amino acids in a single peptide chain and is made up of three homologous domains. It has two tryptophan residues which have a role to stabilize the structure through different kind of bonding [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e].They are, (i) Trp-212 located in hydrophobic pocket of their protein and (ii) Trp-134 located on the surface of the molecule. The studies of drug-protein can provide information regarding structural features that decide the therapeutic effectiveness of the drug. They also provide useful information about the possibility and stability of drug-drug interaction, drug-protein interaction, side effects and dosage information [\\u003cspan additionalcitationids=\\\"CR6\\\" citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. So, the studies have been interesting in the research field of medicinal chemistry with different pharmacokinetic properties like distribution, transportation and excretion. The knowledge of the protein receptors (binding with BSA) is necessary for any new drug prior to their clinical trial [\\u003cspan additionalcitationids=\\\"CR9\\\" citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eThe functions of proteins depend on its three dimensional structure. This structure can be restored or disturbed in different chemical environments. The marginal stability of protein when present in native globular confirmation depends on various interactions taking place in the proteins, viz., the temperature, pH of the medium, presence of coenzyme, type of inhibitors and substrates [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eSurfactants act as denaturing agents for membranes in proteins and lipids. The study of interaction of surfactants with globular proteins has lot of importance and it is extensively studied and reported [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. From this angle, surfactants are classified based on their reaction with protein. In the first type, there is denaturation or protein unfolding and in the second type, they can only bind where the ternary structure of the protein is left undisturbed. Generally, ionic surfactants are capable of denaturing protein, but nonionic surfactants do not [\\u003cspan additionalcitationids=\\\"CR16\\\" citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eAmong the study of BSA-surfactant interaction it is observed that SDS is an efficient protein denaturant. Also, there are several reports on BSA\\u0026thinsp;+\\u0026thinsp;SDS interaction [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. There are many reports on multifunctional binding nature of BSA that binds a wide range of molecules [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. Also, there are many reports on the interaction of drug with BSA \\u003csup\\u003e22\\u0026ndash;24\\u003c/sup\\u003e.The binding function is a medium for transportation of soluble substances between different tissues and organs. Albumin has one important function (i.e.) transporting fatty acids. Binding also helps to protect against the toxic effects of the drug bound ligand. Hence, binding studies with BSA have significant applications in the rational designing of drug in many pharmaceuticals. The effect of additives or drugs on micellization behavior has been investigated before [\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. The presence of surfactants in the drug-protein complex can cause conformational changes in protein due to changes in polarity and can, in many instances, result in stability of the protein [\\u003cspan additionalcitationids=\\\"CR28 CR29 CR30\\\" citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. Protein-surfactant interaction has a wide variety of applications in the biological, cosmetic, pharmaceutical and industrial systems [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. But, the role of nonionic surfactant in a drug-BSA system is rare in literature. In this context it is important to mention that though pluronic L-81 is a hydrophobic surfactant and directly it doesn\\u0026rsquo;t find any application in the pharmaceutical industry, it has been used in mixed micellar formulation for better efficacy by earlier research groups [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e]. Work reported here includes the incorporation of a nonionic hydrophobic surfactant, namely, pluronic L-81 into complex of BSA and drug and analyze the change in binding parameters due to the conformational changes induced by the surfactant. We have used four drugs in this study. They are Tinidazole (TNZ), Piroxicam (PY), Methylparaben (MP) and Propylparaben (PP). The characterization techniques employed are UV-visible spectroscopy and fluorescence spectroscopy. The docking studies were also carried out to assess the interactions. The following works on different drugs on BSA has been reported in the literature shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eLiterature survey of BSA with different drugs compared to our data\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"3\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"char\\\" char=\\\"\\u0026times;\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eSystems\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eBinding\\u003c/p\\u003e \\u003cp\\u003eConstant (M\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eReferences\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eBSA\\u0026thinsp;+\\u0026thinsp;Methylparaben\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026times;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.32\\u0026times;10\\u003csup\\u003e9\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e[\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eBSA\\u0026thinsp;+\\u0026thinsp;Tinidazole\\u003c/p\\u003e \\u003cp\\u003eBSA\\u0026thinsp;+\\u0026thinsp;Piroxicam\\u003c/p\\u003e \\u003cp\\u003eBSA\\u0026thinsp;+\\u0026thinsp;Methylparaben\\u003c/p\\u003e \\u003cp\\u003eBSA\\u0026thinsp;+\\u0026thinsp;Propylparaben\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026times;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e6.0\\u0026times;10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003e7.0\\u0026times;10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003e3.6\\u0026times;10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003e1.2\\u0026times;10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eThis work\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eBSA\\u0026thinsp;+\\u0026thinsp;L-81\\u0026thinsp;+\\u0026thinsp;Tinidazole\\u003c/p\\u003e \\u003cp\\u003eBSA\\u0026thinsp;+\\u0026thinsp;L-81\\u0026thinsp;+\\u0026thinsp;Piroxicam\\u003c/p\\u003e \\u003cp\\u003eBSA\\u0026thinsp;+\\u0026thinsp;L-81\\u0026thinsp;+\\u0026thinsp;Methylparaben\\u003c/p\\u003e \\u003cp\\u003eBSA\\u0026thinsp;+\\u0026thinsp;L-81\\u0026thinsp;+\\u0026thinsp;Propylparaben\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"char\\\" char=\\\"\\u0026times;\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e8.39\\u0026times;10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003e7.9\\u0026times;10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003e5.60\\u0026times;10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/sup\\u003e\\u003c/p\\u003e \\u003cp\\u003e3.11\\u0026times;10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eThis work\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e\"},{\"header\":\"2. Experimental Section\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.1 Materials and methods\\u003c/h2\\u003e \\u003cp\\u003eAll the reagents were of analytical grade. Double distilled water was used for the experiments. The drugs (98% purity) Tinidazole (TNZ), Piroxicam (PY), Methylparaben (MP) and Propylparaben (PP) were procured from MMC health care limited and Chemical Ltd chemical company, Chennai respectively. BSA (\\u0026ge;\\u0026thinsp;98% purity) was procured from Sigma Aldrich, supplied by Subra scientific company Pondicherry. Pluronic L-81 (\\u0026ge;\\u0026thinsp;99% purity) also was obtained from Sigma Aldrich.\\u003c/p\\u003e \\u003cp\\u003eA stock solution of pluronic L-81 was prepared for 18mM after weighing the appropriate quantity and solubilizing in distilled water. This co-polymer is solubilized better in cold temperatures (around 5\\u003csup\\u003eo\\u003c/sup\\u003eC). At room temperature it showed a somewhat turbid appearance which disappears on dilution. The stock drug solution of Tinidazole (TNZ) for 4.0\\u0026times;10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/sup\\u003e M, Piroxicam (PY) for 3.0\\u0026times;10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/sup\\u003e M, Methylparaben (MP) for 6.5\\u0026times;10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/sup\\u003e M and Propylparaben (PP) for 4.5\\u0026times;10\\u003csup\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/sup\\u003e M were prepared.\\u003c/p\\u003e \\u003cp\\u003eBovine serum albumin solution was prepared by taking 1.002g of BSA and making upto 100 ml in a standard measuring flask using buffer pH 7.4. The buffer solution was prepared by weighing 1.37g buffer of potassium di-hydrogen phosphate in 100ml, double distilled water and 1.41g of disodium hydrogen phosphate in other two standard flasks. In the following step, 81 ml of disodium and 19 ml of potassium di-hydrogen phosphate were taken to prepare 100ml buffer solution. The pH was adjusted using a pH meter to 7.4 by using standard NaOH solution and then it was calibrated using a known buffer solution.\\u003c/p\\u003e \\u003cp\\u003eDifferent concentrations of the drug solutions were used for calibration of the instrument and from among them, an optimum concentration was chosen for all the experiments which were kept constant. The optimized drug concentration was mixed with different concentrations of BSA. The mixture of drug and BSA were taken for UV-spectroscopic and fluorescence measurements with and without L-81. All the experiments were carried out at room temperature.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.2. UV-Visible spectroscopy\\u003c/h2\\u003e \\u003cp\\u003eUV Spectroscopic measurements of the four different drugs with Pluronic L-81 in presence of BSA were carried out using a Shimadzu (UV-2600) spectrophotometer baseline in the range of 200\\u0026ndash;400 nm. All the readings were taken at room temperature.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.3. Fluorescence spectroscopy\\u003c/h2\\u003e \\u003cp\\u003eFluorescence intensity measurements were carried out with by F-4700 FL spectrophotometer. The excitation and emission wavelength were maintained at 250 nm and 550 nm respectively. BSA was used as a fluorescence probe. Scanning and slit range was 1200 nm/min and 5nm. In this study, we have taken four different drugs with Pluronic L-81 in presence of BSA in aqueous medium.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e2.4. Docking study\\u003c/h2\\u003e \\u003cp\\u003eDocking study was carried out for Tinidazole, Piroxicam, Methylparaben, Propylparaben and surfactant pluronic L-81by using Autodock 4.0 software.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"3. Results and discussion\",\"content\":\"\\u003cp\\u003eIn this work, the surfactant pluronic L-81 is added to drug-BSA combination. Each drug has its specific interaction affinity with BSA. However, the presence of surfactant changes the interactivity between drug and BSA. The presence and absence of the surfactant in the drug-BSA medium was assessed through the following characterizations. Each drug has been described on separately.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.1 UV spectroscopic measurements\\u003c/h2\\u003e \\u003cp\\u003eTo investigate the nature of binding of BSA with drug, UV spectroscopic measurements were taken. There were four drugs chosen. Each drug was taken with BSA with and without the non-ionic surfactant pluronic L-81. The absorbance values of the aliquots were recorded and analyzed. Each drug is discussed separately. The calibration of each drug was done and the results showed correlation coefficient above 0.9. The molecular structures of four drugs viz., tinidazole, piroxicam, methylparaben and propylparaben, as well as pluronic L-81, are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec9\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.1.1. Tinidazole (TNZ)\\u003c/h2\\u003e \\u003cp\\u003eTinidazole (TNZ) was taken in 0.008 mM concentration. It showed a maximum at 318 nm which was coincidence with literature value [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e]. On addition of 0.8\\u0026micro;M BSA in the drug solution, there was increase in the intensity of absorbance of 318 nm peak. Additionally, the peak for BSA appeared prominently at 279 nm [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]. There was no extra peak formed and there was no shifting of the λ\\u003csub\\u003emax\\u003c/sub\\u003e value of TNZ is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e. Subsequent aliquots were taken by using the same BSA concentration and varying (increase) concentration of drug. In all those samples, the same trend followed with a small red shift. The peak at 318nm for TNZ and 279nm for BSA were observed to increase in the absorbance intensity. The concentration of TNZ was varied from 0.008mM to 0.028mM. The increase of absorbance of the drug at λ\\u003csub\\u003emax\\u003c/sub\\u003e 318nm at higher concentrations and BSA peak at 279nm is suggestive of strong interaction between the TNZ molecule and BSA. Using the absorbance values and applying Benesi-Hildebrand (B-H) plot, the binding constant values were calculated. The absorbance of pure BSA is A\\u003csub\\u003e0\\u003c/sub\\u003e, the absorbance of formed complex after interaction with drug is A. Then we can do a baseline correction by calculating A-A\\u003csub\\u003e0\\u003c/sub\\u003e in the process of binding. The binding constant K has been calculated using formula,\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eK\\u0026thinsp;=\\u0026thinsp;1/slope* A-A\\u003csub\\u003e0\\u003c/sub\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe slope is taken from a graph plotted between 1/concentration of drug and 1/A-A\\u003csub\\u003e0\\u003c/sub\\u003e. The A-A\\u003csub\\u003e0\\u003c/sub\\u003e value of the highest concentration of drug is taken for calculation. Thus, the binding constant of the system is calculated and shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eBinding constant of BSA\\u0026thinsp;+\\u0026thinsp;TNZ\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"9\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c9\\\" colnum=\\\"9\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c2\\\" namest=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eConcentration\\u003c/p\\u003e \\u003cp\\u003eTNZ mM\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eWavelength\\u003c/p\\u003e \\u003cp\\u003e(nm)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eA\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e1/TNZ\\u003c/p\\u003e \\u003cp\\u003eConcentration\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e1/ A\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c2\\\" namest=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.008\\u003c/p\\u003e \\u003cp\\u003e0.012\\u003c/p\\u003e \\u003cp\\u003e0.016\\u003c/p\\u003e \\u003cp\\u003e0.02\\u003c/p\\u003e \\u003cp\\u003e0.024\\u003c/p\\u003e \\u003cp\\u003e0.028\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e318\\u003c/p\\u003e \\u003cp\\u003e279\\u003c/p\\u003e \\u003cp\\u003e279\\u003c/p\\u003e \\u003cp\\u003e279\\u003c/p\\u003e \\u003cp\\u003e279\\u003c/p\\u003e \\u003cp\\u003e279\\u003c/p\\u003e \\u003cp\\u003e279\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.255\\u003c/p\\u003e \\u003cp\\u003e0.255\\u003c/p\\u003e \\u003cp\\u003e0.255\\u003c/p\\u003e \\u003cp\\u003e0.255\\u003c/p\\u003e \\u003cp\\u003e0.255\\u003c/p\\u003e \\u003cp\\u003e0.255\\u003c/p\\u003e \\u003cp\\u003e0.255\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0.255\\u003c/p\\u003e \\u003cp\\u003e0.693\\u003c/p\\u003e \\u003cp\\u003e0.752\\u003c/p\\u003e \\u003cp\\u003e0.788\\u003c/p\\u003e \\u003cp\\u003e0.792\\u003c/p\\u003e \\u003cp\\u003e0.905\\u003c/p\\u003e \\u003cp\\u003e0.1061\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.438\\u003c/p\\u003e \\u003cp\\u003e0.497\\u003c/p\\u003e \\u003cp\\u003e0.533\\u003c/p\\u003e \\u003cp\\u003e0.537\\u003c/p\\u003e \\u003cp\\u003e0.650\\u003c/p\\u003e \\u003cp\\u003e0.806\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e125\\u003c/p\\u003e \\u003cp\\u003e83.4\\u003c/p\\u003e \\u003cp\\u003e62.5\\u003c/p\\u003e \\u003cp\\u003e50\\u003c/p\\u003e \\u003cp\\u003e41.7\\u003c/p\\u003e \\u003cp\\u003e35.7\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c9\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e2.28\\u003c/p\\u003e \\u003cp\\u003e2.01\\u003c/p\\u003e \\u003cp\\u003e1.87\\u003c/p\\u003e \\u003cp\\u003e1.86\\u003c/p\\u003e \\u003cp\\u003e1.53\\u003c/p\\u003e \\u003cp\\u003e1.24\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"8\\\" nameend=\\\"c8\\\" namest=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eBinding constant k\\u0026thinsp;=\\u0026thinsp;6.0\\u0026times;10\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/b\\u003e\\u003c/sup\\u003e \\u003cb\\u003eM\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/b\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"1\\\" nameend=\\\"c9\\\" namest=\\\"c9\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo the above mixture of BSA\\u0026thinsp;+\\u0026thinsp;TNZ, a non-ionic surfactant pluronic L-81 was incorporated and the experiment was repeated as before. A set of readings with the same increased absorbance trend was obtained. The A, A\\u003csub\\u003e0\\u003c/sub\\u003e values were substituted and binding constant was found to be K\\u0026thinsp;=\\u0026thinsp;128 M\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. It is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e and Table\\u0026nbsp;\\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 3\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eBinding constant of BSA\\u0026thinsp;+\\u0026thinsp;L-81\\u0026thinsp;+\\u0026thinsp;TNZ\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"8\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eConcentration TNZ mM\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eWavelength\\u003c/p\\u003e \\u003cp\\u003e(nm)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eA\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1/TNZ Concentration\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c8\\\" namest=\\\"c7\\\"\\u003e \\u003cp\\u003e1/ A\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.008\\u003c/p\\u003e \\u003cp\\u003e0.012\\u003c/p\\u003e \\u003cp\\u003e0.016\\u003c/p\\u003e \\u003cp\\u003e0.02\\u003c/p\\u003e \\u003cp\\u003e0.024\\u003c/p\\u003e \\u003cp\\u003e0.028\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e318\\u003c/p\\u003e \\u003cp\\u003e279\\u003c/p\\u003e \\u003cp\\u003e279\\u003c/p\\u003e \\u003cp\\u003e279\\u003c/p\\u003e \\u003cp\\u003e279\\u003c/p\\u003e \\u003cp\\u003e279\\u003c/p\\u003e \\u003cp\\u003e279\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.226\\u003c/p\\u003e \\u003cp\\u003e0.226\\u003c/p\\u003e \\u003cp\\u003e0.226\\u003c/p\\u003e \\u003cp\\u003e0.226\\u003c/p\\u003e \\u003cp\\u003e0.226\\u003c/p\\u003e \\u003cp\\u003e0.226\\u003c/p\\u003e \\u003cp\\u003e0.226\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.266\\u003c/p\\u003e \\u003cp\\u003e0.812\\u003c/p\\u003e \\u003cp\\u003e0.825\\u003c/p\\u003e \\u003cp\\u003e0.866\\u003c/p\\u003e \\u003cp\\u003e0.889\\u003c/p\\u003e \\u003cp\\u003e0.939\\u003c/p\\u003e \\u003cp\\u003e1.066\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.546\\u003c/p\\u003e \\u003cp\\u003e0.559\\u003c/p\\u003e \\u003cp\\u003e0.6\\u003c/p\\u003e \\u003cp\\u003e0.629\\u003c/p\\u003e \\u003cp\\u003e0.673\\u003c/p\\u003e \\u003cp\\u003e0.8\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e125\\u003c/p\\u003e \\u003cp\\u003e83.4\\u003c/p\\u003e \\u003cp\\u003e62.5\\u003c/p\\u003e \\u003cp\\u003e50\\u003c/p\\u003e \\u003cp\\u003e41.7\\u003c/p\\u003e \\u003cp\\u003e35.7\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e1.83\\u003c/p\\u003e \\u003cp\\u003e1.78\\u003c/p\\u003e \\u003cp\\u003e1.67\\u003c/p\\u003e \\u003cp\\u003e1.60\\u003c/p\\u003e \\u003cp\\u003e1.48\\u003c/p\\u003e \\u003cp\\u003e0.93\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"1\\\" nameend=\\\"c8\\\" namest=\\\"c8\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"4\\\" nameend=\\\"c4\\\" namest=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eBinding constant k\\u0026thinsp;=\\u0026thinsp;8.39\\u0026times;10\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/b\\u003e\\u003c/sup\\u003e \\u003cb\\u003eM\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/b\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"4\\\" nameend=\\\"c8\\\" namest=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe discrepancy of K values shows that the ternary system of BSA\\u0026thinsp;+\\u0026thinsp;L-81\\u0026thinsp;+\\u0026thinsp;TNZ has a higher binding constant. Hence, the incorporation of the L-81 in the drug-BSA mixture is helping to strengthen the bonds in the complex. The binding constant value was calculated to be\\u003c/p\\u003e \\u003cp\\u003eK\\u0026thinsp;=\\u0026thinsp;173 M\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. The higher binding constant is the parameter to assess the strength of bond. It is shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec10\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.1.2. Piroxicam (PY)\\u003c/h2\\u003e \\u003cp\\u003ePiroxicam has a global charge with zwitterions-anionic drug. The UV studies of piroxicam shows that the λ\\u003csub\\u003emax\\u003c/sub\\u003e is at 353nm. The literature value is 353nm [\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e]. Like the previous case, here also the BSA concentration was kept constant and PY concentration was raised from 0.0024 mM to 0.0084 mM. There was increased absorption intensity with a small red shift of the λ\\u003csub\\u003emax\\u003c/sub\\u003e. But, the 279nm peak of BSA was seen to only enhance the absorption. The Benesi\\u0026ndash;Hildebrand (B-H) plot calculations showed that the binding energy of the PY\\u0026thinsp;+\\u0026thinsp;BSA was K\\u0026thinsp;=\\u0026thinsp;46 M\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e in the complex which is a strong one indicated by red shift. The results are shown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab4\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 4\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eBinding constant of PY\\u0026thinsp;+\\u0026thinsp;BSA\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"8\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eConcentration\\u003c/p\\u003e \\u003cp\\u003ePY mM\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eWavelength\\u003c/p\\u003e \\u003cp\\u003e(nm)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eA\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e1/PY Concentration\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e1/ A\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.0024\\u003c/p\\u003e \\u003cp\\u003e0.0036\\u003c/p\\u003e \\u003cp\\u003e0.0048\\u003c/p\\u003e \\u003cp\\u003e0.006\\u003c/p\\u003e \\u003cp\\u003e0.0072\\u003c/p\\u003e \\u003cp\\u003e0.0084\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e353\\u003c/p\\u003e \\u003cp\\u003e353\\u003c/p\\u003e \\u003cp\\u003e353\\u003c/p\\u003e \\u003cp\\u003e353\\u003c/p\\u003e \\u003cp\\u003e353\\u003c/p\\u003e \\u003cp\\u003e353\\u003c/p\\u003e \\u003cp\\u003e353\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.158\\u003c/p\\u003e \\u003cp\\u003e0.158\\u003c/p\\u003e \\u003cp\\u003e0.158\\u003c/p\\u003e \\u003cp\\u003e0.158\\u003c/p\\u003e \\u003cp\\u003e0.158\\u003c/p\\u003e \\u003cp\\u003e0.158\\u003c/p\\u003e \\u003cp\\u003e0.158\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.158\\u003c/p\\u003e \\u003cp\\u003e0.213\\u003c/p\\u003e \\u003cp\\u003e0.264\\u003c/p\\u003e \\u003cp\\u003e0.343\\u003c/p\\u003e \\u003cp\\u003e0.403\\u003c/p\\u003e \\u003cp\\u003e0.485\\u003c/p\\u003e \\u003cp\\u003e0.561\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.055\\u003c/p\\u003e \\u003cp\\u003e0.106\\u003c/p\\u003e \\u003cp\\u003e0.185\\u003c/p\\u003e \\u003cp\\u003e0.245\\u003c/p\\u003e \\u003cp\\u003e0.327\\u003c/p\\u003e \\u003cp\\u003e0.403\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e416.7\\u003c/p\\u003e \\u003cp\\u003e277.8\\u003c/p\\u003e \\u003cp\\u003e208.4\\u003c/p\\u003e \\u003cp\\u003e166.7\\u003c/p\\u003e \\u003cp\\u003e138.9\\u003c/p\\u003e \\u003cp\\u003e119\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e18.18\\u003c/p\\u003e \\u003cp\\u003e9.43\\u003c/p\\u003e \\u003cp\\u003e5.40\\u003c/p\\u003e \\u003cp\\u003e4.08\\u003c/p\\u003e \\u003cp\\u003e3.05\\u003c/p\\u003e \\u003cp\\u003e2.48\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"8\\\" nameend=\\\"c8\\\" namest=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eBinding constant k\\u0026thinsp;=\\u0026thinsp;7.0\\u0026times;10\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/b\\u003e\\u003c/sup\\u003e \\u003cb\\u003eM\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/b\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eIn the next step, to the BSA\\u0026thinsp;+\\u0026thinsp;PY mixture, pluronic L-81 was added. The results are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea and Table\\u0026nbsp;\\u003cspan refid=\\\"Tab5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e. There was only small increase of the absorbance λ\\u003csub\\u003emax\\u003c/sub\\u003e (353nm). But, BSA peak at 279nm was undisturbed. There was no intersection of the spectra and no new spectra appeared. Only the increase in absorbance could be due to complex formation by BSA\\u0026thinsp;+\\u0026thinsp;L-81\\u0026thinsp;+\\u0026thinsp;PY. The absorbance values were used to calculate the Benesi-Hildebrand (B-H) plot to determine the binding constant which was found to be 617 M\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003eshown in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003e. In this system also, the binding constant shows that the presence of L-81 has worked towards strong binding of PY and BSA.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab5\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 5\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eBinding constant of PY\\u0026thinsp;+\\u0026thinsp;BSA\\u0026thinsp;+\\u0026thinsp;L-81\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"9\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c9\\\" colnum=\\\"9\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eConcentration\\u003c/p\\u003e \\u003cp\\u003ePY mM\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eWavelength\\u003c/p\\u003e \\u003cp\\u003e(nm)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eA\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e1/PY Concentration\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c9\\\" namest=\\\"c8\\\"\\u003e \\u003cp\\u003e1/ A\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.0024\\u003c/p\\u003e \\u003cp\\u003e0.0036\\u003c/p\\u003e \\u003cp\\u003e0.0048\\u003c/p\\u003e \\u003cp\\u003e0.006\\u003c/p\\u003e \\u003cp\\u003e0.0072\\u003c/p\\u003e \\u003cp\\u003e0.0084\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e260\\u003c/p\\u003e \\u003cp\\u003e270\\u003c/p\\u003e \\u003cp\\u003e270\\u003c/p\\u003e \\u003cp\\u003e270\\u003c/p\\u003e \\u003cp\\u003e270\\u003c/p\\u003e \\u003cp\\u003e270\\u003c/p\\u003e \\u003cp\\u003e270\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.118\\u003c/p\\u003e \\u003cp\\u003e0.118\\u003c/p\\u003e \\u003cp\\u003e0.118\\u003c/p\\u003e \\u003cp\\u003e0.118\\u003c/p\\u003e \\u003cp\\u003e0.118\\u003c/p\\u003e \\u003cp\\u003e0118\\u003c/p\\u003e \\u003cp\\u003e0.118\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.118\\u003c/p\\u003e \\u003cp\\u003e0.635\\u003c/p\\u003e \\u003cp\\u003e0.666\\u003c/p\\u003e \\u003cp\\u003e0.735\\u003c/p\\u003e \\u003cp\\u003e0.805\\u003c/p\\u003e \\u003cp\\u003e0.811\\u003c/p\\u003e \\u003cp\\u003e0.889\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.517\\u003c/p\\u003e \\u003cp\\u003e0.548\\u003c/p\\u003e \\u003cp\\u003e0.617\\u003c/p\\u003e \\u003cp\\u003e0.687\\u003c/p\\u003e \\u003cp\\u003e0.693\\u003c/p\\u003e \\u003cp\\u003e0.771\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e416.7\\u003c/p\\u003e \\u003cp\\u003e277.8\\u003c/p\\u003e \\u003cp\\u003e208.4\\u003c/p\\u003e \\u003cp\\u003e166.7\\u003c/p\\u003e \\u003cp\\u003e138.9\\u003c/p\\u003e \\u003cp\\u003e119\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e1.93\\u003c/p\\u003e \\u003cp\\u003e1.82\\u003c/p\\u003e \\u003cp\\u003e1.62\\u003c/p\\u003e \\u003cp\\u003e1.45\\u003c/p\\u003e \\u003cp\\u003e1.44\\u003c/p\\u003e \\u003cp\\u003e1.29\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"1\\\" nameend=\\\"c9\\\" namest=\\\"c9\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"8\\\" nameend=\\\"c8\\\" namest=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eBinding constant k\\u0026thinsp;=\\u0026thinsp;7.9\\u0026times;10\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;3\\u003c/b\\u003e\\u003c/sup\\u003e \\u003cb\\u003eM\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/b\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"1\\\" nameend=\\\"c9\\\" namest=\\\"c9\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.1.3. Methylparaben (MP)\\u003c/h2\\u003e \\u003cp\\u003eParabens are commonly used for cosmetic products. The external application of paraben can be tuned depending on the requirement. Hence, it's interaction with different surfactants is a topic of research. The BSA\\u0026thinsp;+\\u0026thinsp;MP interaction has been reported before (Naik \\u0026amp; Nandibewoor, 2013).\\u003c/p\\u003e \\u003cp\\u003eMethylparaben showed spectra with λ\\u003csub\\u003emax\\u003c/sub\\u003e at 254nm. This is in good agreement in the literature values [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e]. Addition of BSA causes red shift of the λ\\u003csub\\u003emax\\u003c/sub\\u003e to 278nm (Fig.\\u0026nbsp;6a and Table\\u0026nbsp;\\u003cspan refid=\\\"Tab6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e). Red shifting by 24nm is an indication of the strong bonding developed between MP and BSA to form a complex. The (B-H) plot was used to determine the binding constant which was found to be K\\u0026thinsp;=\\u0026thinsp;27 M\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab6\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 6\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eBinding constant of BSA\\u0026thinsp;+\\u0026thinsp;MP\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"7\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eConcentration\\u003c/p\\u003e \\u003cp\\u003eMP mM\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eWavelength\\u003c/p\\u003e \\u003cp\\u003e(nm)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eA\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1/MP Concentration\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e1/ A\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.032\\u003c/p\\u003e \\u003cp\\u003e0.038\\u003c/p\\u003e \\u003cp\\u003e0.044\\u003c/p\\u003e \\u003cp\\u003e0.052\\u003c/p\\u003e \\u003cp\\u003e0.057\\u003c/p\\u003e \\u003cp\\u003e0.064\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e254\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e258\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.327\\u003c/p\\u003e \\u003cp\\u003e0.327\\u003c/p\\u003e \\u003cp\\u003e0.327\\u003c/p\\u003e \\u003cp\\u003e0.327\\u003c/p\\u003e \\u003cp\\u003e0.327\\u003c/p\\u003e \\u003cp\\u003e0.327\\u003c/p\\u003e \\u003cp\\u003e0.327\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.327\\u003c/p\\u003e \\u003cp\\u003e0.866\\u003c/p\\u003e \\u003cp\\u003e0.916\\u003c/p\\u003e \\u003cp\\u003e0.956\\u003c/p\\u003e \\u003cp\\u003e1.014\\u003c/p\\u003e \\u003cp\\u003e1.105\\u003c/p\\u003e \\u003cp\\u003e1.177\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.539\\u003c/p\\u003e \\u003cp\\u003e0.589\\u003c/p\\u003e \\u003cp\\u003e0.629\\u003c/p\\u003e \\u003cp\\u003e0.687\\u003c/p\\u003e \\u003cp\\u003e0.778\\u003c/p\\u003e \\u003cp\\u003e0.850\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e31.25\\u003c/p\\u003e \\u003cp\\u003e26.31\\u003c/p\\u003e \\u003cp\\u003e22.72\\u003c/p\\u003e \\u003cp\\u003e19.23\\u003c/p\\u003e \\u003cp\\u003e17.54\\u003c/p\\u003e \\u003cp\\u003e15.62\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e1.85\\u003c/p\\u003e \\u003cp\\u003e1.69\\u003c/p\\u003e \\u003cp\\u003e1.58\\u003c/p\\u003e \\u003cp\\u003e1.45\\u003c/p\\u003e \\u003cp\\u003e1.28\\u003c/p\\u003e \\u003cp\\u003e1.17\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"4\\\" nameend=\\\"c4\\\" namest=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eBinding constant k\\u0026thinsp;=\\u0026thinsp;3.6\\u0026times;10\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/b\\u003e\\u003c/sup\\u003e\\u003cb\\u003eM\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/b\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c7\\\" namest=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eIncorporation of pluronic L-81 into the MP\\u0026thinsp;+\\u0026thinsp;BSA mixture, there was no significant change in the nature of spectra. The results are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb and Table\\u0026nbsp;\\u003cspan refid=\\\"Tab7\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e. The red shift of λ\\u003csub\\u003emax\\u003c/sub\\u003e observed in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb, remains unaffected by addition of pluronic L-81. The absorbance values were taken to apply the B-H plot and determine the binding constant K\\u0026thinsp;=\\u0026thinsp;24 M\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab7\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 7\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eBinding constant of BSA\\u0026thinsp;+\\u0026thinsp;L-81\\u0026thinsp;+\\u0026thinsp;MP\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"7\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eConcentration\\u003c/p\\u003e \\u003cp\\u003eMP mM\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eWavelength\\u003c/p\\u003e \\u003cp\\u003e(nm)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eA\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e1/MP Concentration\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e1/ A\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.032\\u003c/p\\u003e \\u003cp\\u003e0.038\\u003c/p\\u003e \\u003cp\\u003e0.044\\u003c/p\\u003e \\u003cp\\u003e0.052\\u003c/p\\u003e \\u003cp\\u003e0.057\\u003c/p\\u003e \\u003cp\\u003e0.064\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e254\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.256\\u003c/p\\u003e \\u003cp\\u003e0.256\\u003c/p\\u003e \\u003cp\\u003e0.256\\u003c/p\\u003e \\u003cp\\u003e0.256\\u003c/p\\u003e \\u003cp\\u003e0.256\\u003c/p\\u003e \\u003cp\\u003e0.256\\u003c/p\\u003e \\u003cp\\u003e0.256\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.256\\u003c/p\\u003e \\u003cp\\u003e0.659\\u003c/p\\u003e \\u003cp\\u003e0.703\\u003c/p\\u003e \\u003cp\\u003e0.734\\u003c/p\\u003e \\u003cp\\u003e0.751\\u003c/p\\u003e \\u003cp\\u003e0.798\\u003c/p\\u003e \\u003cp\\u003e0.972\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.403\\u003c/p\\u003e \\u003cp\\u003e0.447\\u003c/p\\u003e \\u003cp\\u003e0.478\\u003c/p\\u003e \\u003cp\\u003e0.495\\u003c/p\\u003e \\u003cp\\u003e0.542\\u003c/p\\u003e \\u003cp\\u003e0.716\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e31.25\\u003c/p\\u003e \\u003cp\\u003e26.21\\u003c/p\\u003e \\u003cp\\u003e22.72\\u003c/p\\u003e \\u003cp\\u003e19.23\\u003c/p\\u003e \\u003cp\\u003e17.54\\u003c/p\\u003e \\u003cp\\u003e15.62\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e2.48\\u003c/p\\u003e \\u003cp\\u003e2.23\\u003c/p\\u003e \\u003cp\\u003e2.09\\u003c/p\\u003e \\u003cp\\u003e2.02\\u003c/p\\u003e \\u003cp\\u003e1.84\\u003c/p\\u003e \\u003cp\\u003e1.39\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"4\\\" nameend=\\\"c4\\\" namest=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eBinding constant k\\u0026thinsp;=\\u0026thinsp;5.60\\u0026times;10\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/b\\u003e\\u003c/sup\\u003e\\u003cb\\u003eM\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/b\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"3\\\" nameend=\\\"c7\\\" namest=\\\"c5\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eHere, the binding constant (K) was observed to be lower in presence of L-81 as compared to the BSA\\u0026thinsp;+\\u0026thinsp;MP mixture alone. Hence, it can be predicted that the non-ionic surfactant\\u003c/p\\u003e \\u003cp\\u003epluronic L-81 captured the drug MP\\u0026thinsp;+\\u0026thinsp;BSA complex with weaker binding became presence of pluronic L-81 in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e7\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.1.4. Propylparaben (PP)\\u003c/h2\\u003e \\u003cp\\u003ePropylparaben displayed the absorption spectra at (λ\\u003csub\\u003emax\\u003c/sub\\u003e) at 255nm. The evidence in literature is 255nm [\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e]. The results are shown in Fig.\\u0026nbsp;6a and Table\\u0026nbsp;\\u003cspan refid=\\\"Tab8\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003e. As observed, addition of BSA to PP there was a red shift to λ\\u003csub\\u003emax\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;275nm. The 24nm shifting was because of the strong bonding developed between PP and BSA to form a complex. With increase in concentration of drug to a fixed quantity of BSA, there was only progressive increase in absorbance without formation of new peaks or any intersecting point. Hence, no new compound is expected to have been formed. The (B-H) plot of the absorbance data showed that the value of K is 8.4 M\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab8\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 8\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eBinding constant of BSA\\u0026thinsp;+\\u0026thinsp;PP\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"8\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eConcentration PP mM\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eWavelength\\u003c/p\\u003e \\u003cp\\u003e(nm)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eA\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e1/PP Concentration\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e1/ A\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.13\\u003c/p\\u003e \\u003cp\\u003e0.16\\u003c/p\\u003e \\u003cp\\u003e0.19\\u003c/p\\u003e \\u003cp\\u003e0.21\\u003c/p\\u003e \\u003cp\\u003e0.24\\u003c/p\\u003e \\u003cp\\u003e0.27\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e254\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.267\\u003c/p\\u003e \\u003cp\\u003e0.267\\u003c/p\\u003e \\u003cp\\u003e0.267\\u003c/p\\u003e \\u003cp\\u003e0.267\\u003c/p\\u003e \\u003cp\\u003e0.267\\u003c/p\\u003e \\u003cp\\u003e0.267\\u003c/p\\u003e \\u003cp\\u003e0.267\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.267\\u003c/p\\u003e \\u003cp\\u003e0.802\\u003c/p\\u003e \\u003cp\\u003e0.869\\u003c/p\\u003e \\u003cp\\u003e0.979\\u003c/p\\u003e \\u003cp\\u003e0.989\\u003c/p\\u003e \\u003cp\\u003e1.009\\u003c/p\\u003e \\u003cp\\u003e1.086\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.563\\u003c/p\\u003e \\u003cp\\u003e0.602\\u003c/p\\u003e \\u003cp\\u003e0.712\\u003c/p\\u003e \\u003cp\\u003e0.722\\u003c/p\\u003e \\u003cp\\u003e0.742\\u003c/p\\u003e \\u003cp\\u003e0.819\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e7.69\\u003c/p\\u003e \\u003cp\\u003e6.25\\u003c/p\\u003e \\u003cp\\u003e5.26\\u003c/p\\u003e \\u003cp\\u003e4.76\\u003c/p\\u003e \\u003cp\\u003e4.17\\u003c/p\\u003e \\u003cp\\u003e3.70\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e1.80\\u003c/p\\u003e \\u003cp\\u003e1.66\\u003c/p\\u003e \\u003cp\\u003e1.40\\u003c/p\\u003e \\u003cp\\u003e1.38\\u003c/p\\u003e \\u003cp\\u003e1.34\\u003c/p\\u003e \\u003cp\\u003e1.22\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"8\\\" nameend=\\\"c8\\\" namest=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eBinding constant k\\u0026thinsp;=\\u0026thinsp;1.2\\u0026times;10\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/b\\u003e\\u003c/sup\\u003e\\u003cb\\u003eM\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/b\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo know the effect of the pluronic L-81 in the PP-BSA system, the next part of the study was carried out. To fixed quantities of L-81 and BSA, different concentrations of drug, PP were added. Here, the drug concentration ranged from 0.13mM to 0.24mM. The results are shown in\\u003c/p\\u003e \\u003cp\\u003eFigure\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e8\\u003c/span\\u003eb and Table\\u0026nbsp;\\u003cspan refid=\\\"Tab9\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003e. There was increase in absorbance with increase in concentration of drug. This shows that there is complex formation, between BSA and PP and that, in presence of pluronic L-81 the complex formation occurs with greater binding force. Like the previous case, there is a significant red shift 25nm which is indicative of strong bonding between PP and BSA when pluronic L-81 present (λ\\u003csub\\u003emax =\\u003c/sub\\u003e 280nm). The K value determined from the absorbance values was K\\u0026thinsp;=\\u0026thinsp;4.5 M\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab9\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 9\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eBinding constant of BSA\\u0026thinsp;+\\u0026thinsp;L-81\\u0026thinsp;+\\u0026thinsp;PP\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"9\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c8\\\" colnum=\\\"8\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c9\\\" colnum=\\\"9\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eConcentration PP mM\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eWavelength\\u003c/p\\u003e \\u003cp\\u003e(nm)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eA\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003eA\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e1/PP\\u003c/p\\u003e \\u003cp\\u003eConcentration\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"2\\\" nameend=\\\"c9\\\" namest=\\\"c8\\\"\\u003e \\u003cp\\u003e1/ A\\u003csub\\u003e0\\u003c/sub\\u003e-A\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.13\\u003c/p\\u003e \\u003cp\\u003e0.16\\u003c/p\\u003e \\u003cp\\u003e0.19\\u003c/p\\u003e \\u003cp\\u003e0.21\\u003c/p\\u003e \\u003cp\\u003e0.24\\u003c/p\\u003e \\u003cp\\u003e0.27\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e254\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003cp\\u003e259\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.197\\u003c/p\\u003e \\u003cp\\u003e0.197\\u003c/p\\u003e \\u003cp\\u003e0.197\\u003c/p\\u003e \\u003cp\\u003e0.197\\u003c/p\\u003e \\u003cp\\u003e0.197\\u003c/p\\u003e \\u003cp\\u003e0.197\\u003c/p\\u003e \\u003cp\\u003e0.197\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e0.197\\u003c/p\\u003e \\u003cp\\u003e0.531\\u003c/p\\u003e \\u003cp\\u003e0.586\\u003c/p\\u003e \\u003cp\\u003e0.598\\u003c/p\\u003e \\u003cp\\u003e0.6\\u003c/p\\u003e \\u003cp\\u003e0.789\\u003c/p\\u003e \\u003cp\\u003e0.839\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e0.334\\u003c/p\\u003e \\u003cp\\u003e0.389\\u003c/p\\u003e \\u003cp\\u003e0.401\\u003c/p\\u003e \\u003cp\\u003e0.403\\u003c/p\\u003e \\u003cp\\u003e0.592\\u003c/p\\u003e \\u003cp\\u003e0.642\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e7.69\\u003c/p\\u003e \\u003cp\\u003e6.25\\u003c/p\\u003e \\u003cp\\u003e5.26\\u003c/p\\u003e \\u003cp\\u003e4.76\\u003c/p\\u003e \\u003cp\\u003e4.17\\u003c/p\\u003e \\u003cp\\u003e3.70\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c8\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003cp\\u003e2.99\\u003c/p\\u003e \\u003cp\\u003e2.57\\u003c/p\\u003e \\u003cp\\u003e2.49\\u003c/p\\u003e \\u003cp\\u003e2.48\\u003c/p\\u003e \\u003cp\\u003e1.68\\u003c/p\\u003e \\u003cp\\u003e1.55\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"1\\\" nameend=\\\"c9\\\" namest=\\\"c9\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"8\\\" nameend=\\\"c8\\\" namest=\\\"c1\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eBinding constant k\\u0026thinsp;=\\u0026thinsp;3.11\\u0026times;10\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;2\\u003c/b\\u003e\\u003c/sup\\u003e \\u003cb\\u003eM\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/b\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colspan=\\\"1\\\" nameend=\\\"c9\\\" namest=\\\"c9\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eA high value of K in presence of L-81 in the PP-BSA system indicates that pluronic L-81 is not enhancing the binding factor to the PP\\u0026thinsp;+\\u0026thinsp;BSA complex. In Table\\u0026nbsp;\\u003cspan refid=\\\"Tab10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003e, all the four drugs with BSA with and without pluronic L-81 are shown. This reveals that there is a strong role of pluronic L-81 in the binding of drug with BSA in Tinidazole and Piroxicam, but with MP and PP the binding is weaker in presence of L-81.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab10\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 10\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eBenesi-Hildebrand (B-H) binding constant values using UV data\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"2\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eLigand\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eDocking Score\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTinidazole\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e-4.52\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePiraxicam\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e-4.37\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eMethylparaben\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e-4.39\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePluronic L-81\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e-0.75\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePropylparaben\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e-4.03\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eHence, it can be inferred from UV studies that out of the four drugs two had enhanced binding with BSA in presence of pluronic-L81. This may be due to specific nature of the drug and surfactant combination.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003e3.2. Spectro Fluorimetry\\u003c/h2\\u003e \\u003cp\\u003eThe changes in confirmation of BSA were monitored by the intrinsic fluorescence intensity before and after addition of pluronic L-81 when the protein is bound with the drug [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. Fluorescence measurements provide information about the molecular environment present in the vicinity of the fluorophores. We have used four drugs in this study. They are discussed separately.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.2.1. Tinidazole (TNZ)\\u003c/h2\\u003e \\u003cp\\u003eWhen drug TNZ was added to BSA in presence of pluronic L-81 surfactant, the fluorescence intensity of BSA got reduced [\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e]. This is show in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003ea. In subsequent aliquots keeping the concentration of BSA and L-81 constant, the drug concentration was varied. The quantitative analysis of the binding of TNZ with BSA in presence of L-81 was carried out at 343nm. There was reduction in the intensity due to quenching effect. The maximum wavelength of BSA didn't shift from the original position. The remarkable lowering of fluorescence intensity indicated that there is positive interaction of TNZ with BSA and L-81. The hydrophobic surfactant L-81 was helpful in maintaining the environmental polarity and its micelles helped protecting the complex formed between the TNZ and BSA. There was possible quenching mechanism which is evident from the application Stern-Volmer plots as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e12\\u003c/span\\u003ea. The quenching data were applied to Stern-Volmer equation.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eF/F\\u003csub\\u003e0\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;1\\u0026thinsp;+\\u0026thinsp;K\\u003csub\\u003esv\\u003c/sub\\u003e [Q]\\u0026thinsp;=\\u0026thinsp;1\\u0026thinsp;+\\u0026thinsp;K\\u003csub\\u003eq\\u003c/sub\\u003e τ\\u003csub\\u003ea\\u003c/sub\\u003e [Q]\\u003c/p\\u003e \\u003cp\\u003eWhere F\\u003csub\\u003e0\\u003c/sub\\u003e and F stand for the fluorescence intensity of BSA with and without TNZ in\\u003c/p\\u003e \\u003cp\\u003epresence of pluronic L-81. The parameters K\\u003csub\\u003eq\\u003c/sub\\u003e, K\\u003csub\\u003esv\\u003c/sub\\u003e, τ\\u003csub\\u003ea\\u003c/sub\\u003e and [Q] represent quenching rate constant, Stern- Volmer constant, the average lifetime of the drug and concentration of quencher respectively. The linear nature of Stern-Volmer regression curve suggests that the quenching interaction between BSA\\u0026thinsp;+\\u0026thinsp;TNZ is of dynamic nature. The Line-weaver bulk plot is displayed in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003ea and Table\\u0026nbsp;\\u003cspan refid=\\\"Tab11\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003e.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab11\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 11\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eLineweaver-Burk calculation for association constant (K\\u003csub\\u003eA\\u003c/sub\\u003e)\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"3\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eSamples of different drugs\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eBinding Constant of BSA (Without L-81)\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eBinding Constant of \\u003c/p\\u003e \\u003cp\\u003eBSA (With L-81)\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eM\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/b\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e\\u003cb\\u003eM\\u003c/b\\u003e\\u003csup\\u003e\\u003cb\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/b\\u003e\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTinidazole (TNZ)\\u003c/p\\u003e \\u003cp\\u003ePiroxicam (PY)\\u003c/p\\u003e \\u003cp\\u003eMethylparaben (MP)\\u003c/p\\u003e \\u003cp\\u003ePropylparaben (MP)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e128\\u003c/p\\u003e \\u003cp\\u003e46\\u003c/p\\u003e \\u003cp\\u003e27\\u003c/p\\u003e \\u003cp\\u003e8.4\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e173\\u003c/p\\u003e \\u003cp\\u003e617\\u003c/p\\u003e \\u003cp\\u003e24\\u003c/p\\u003e \\u003cp\\u003e4.5\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec15\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.2.2. Piroxicam (PY)\\u003c/h2\\u003e \\u003cp\\u003ePiroxicam addition in BSA solution in presence of pluronic L-81 was investigated by fluorescence studies. It is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003eb. A fixed quantity of BSA\\u0026thinsp;+\\u0026thinsp;L-81 was added to varied concentration of the drug piroxicam. The confirmation of BSA in the molecular environment was assessed by fluorophore molecules [\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e]. The effect of piroxicam on the BSA\\u0026thinsp;+\\u0026thinsp;L-81 shows that there is significant lowering of fluorescence intensity of BSA, which points towards the interaction between BSA and PY. The micellar cavity of pluronic L-81 which was initially present near BSA, in the later stage it takes up the drug molecules of piroxicam. As the concentration of drug increases, there is gradual lowering of the fluorescence intensity. The intensity values were used to determine the Stern-Volmer constant by making use of the equation.\\u003c/p\\u003e \\u003cp\\u003eThe results showed linearity of Stern-Volmer curve shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e12\\u003c/span\\u003eb indicating dynamic nature of quenching. The association constant value of piroxicam was found to be 3.4\\u0026times;10\\u003csup\\u003e3\\u003c/sup\\u003e L Mol\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. The association factors of the four drugs and the Lineweaver-Burk are displayed in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab11\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec16\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.2.3. Methylparaben (MP)\\u003c/h2\\u003e \\u003cp\\u003eDifferent concentrations of methylparaben were added to fixed quantity of BSA in presence and absence of L-81 and fluorescence spectra were recorded. As there was an increase in MP concentration, there was more lowering of the intensity of fluorescence spectra. There are previous reports of BSA\\u0026thinsp;+\\u0026thinsp;MP interaction. But, the above system in presence of surfactant has not been carried out [\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e]. In this study also, a similar trend was observed as the case of BSA\\u0026thinsp;+\\u0026thinsp;MP system. The lowering of intensity with increase in drug solution indicated that there is quenching reaction taking place. The results are displayed in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e12\\u003c/span\\u003ec and Table\\u0026nbsp;\\u003cspan refid=\\\"Tab10\\\" class=\\\"InternalRef\\\"\\u003e10\\u003c/span\\u003e. This intensity values were used to calculate the association constant which was found to be 3.49\\u0026times;10\\u003csup\\u003e3\\u003c/sup\\u003e L Mol\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. The Stern-Volmer plot displayed a straight line that suggested quenching to be of dynamic nature.\\u003c/p\\u003e \\u003cp\\u003eLineweaver-Burk was also plotted which is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003ec.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.2.4. Propylparaben (PP)\\u003c/h2\\u003e \\u003cp\\u003eThe effect of the addition of propylparaben to BSA and pluronic L-81 was observed from the fluorescence studies. The molecular environment of the fluorophore molecule, here the BSA molecule, was changed due to the addition of PP and pluronic L-81. It is shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig11\\\" class=\\\"InternalRef\\\"\\u003e9\\u003c/span\\u003ed. There was a gradual lowering of intensity values on the addition of more amounts PP to BSA and pluronic L-81. This was nothing but quenching action by the drug [\\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e47\\u003c/span\\u003e]. The quenching mechanism was evaluated by\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eStern-Volmer equation and the results are shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig9\\\" class=\\\"InternalRef\\\"\\u003e12\\u003c/span\\u003ed. A near linear curve predicts the dynamic nature of quenching. The association constant K\\u003csub\\u003eA\\u003c/sub\\u003e for the system was evaluated which was found to be 3.3\\u0026times;10\\u003csup\\u003e3\\u003c/sup\\u003e L Mol\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. A straight line was obtained for Lineweaver-Burk plot as shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e11\\u003c/span\\u003ed.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section3\\\"\\u003e \\u003ch2\\u003e3.2.5. Docking Analysis\\u003c/h2\\u003e \\u003cp\\u003eMolecular modeling technique is used to predict how a protein interacts with small molecules. In this study, molecular docking was performed using the Autodock 4.2 program. Since it was not possible to take three molecules simultaneously for docking study, using the above software, we have used the docking model of all the four drugs and the surfactant with BSA. From the binding energy values of five combinations, the comparative interaction is assessed. Molecular docking of compound Fig.\\u0026nbsp;13 (a-e) was performed with the protein BSA. The ligand often connects to the protein through a variety of interactions, including pi-alkyl interactions, pi-pi interaction, pi-donor hydrogen bond interactions and conventional hydrogen bond interactions [\\u003cspan additionalcitationids=\\\"CR49\\\" citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e48\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e50\\u003c/span\\u003e]. Our molecular docking studies showed the same kind of interactions. In compound (a), the BSA shows two hydrogen bonds Gln220 (B) and Lys 221(B) with bond lengths 3.21 \\u0026Aring; and 2.57 \\u0026Aring; respectively. This was observed to have a binding energy of -4.52 Kcal mol\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e.The combination of compound (c) with BSA shows one hydrogen bond at Lys524 (B) site with bond length of 2.95 \\u0026Aring; and the binding energy of this compound was seen to be -4.39 Kcal mol\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e mentioned in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab12\\\" class=\\\"InternalRef\\\"\\u003e12\\u003c/span\\u003e. The binding energy of the compound (d) was \\u0026minus;\\u0026thinsp;4.03 Kcal mol\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. The BSA shows three hydrogen bonds with Arg256 (A) of varying bond lengths 3.30 \\u0026Aring;, 3.15 \\u0026Aring; and 3.10 \\u0026Aring; respectively. One hydrogen bond was observed with Tyr149(A) having a bond length of 3.33 \\u0026Aring;. The binding energy of the compound (e) was \\u0026minus;\\u0026thinsp;0.75 Kcal mol\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e. This value is the lowest in the table, which means that there is no interference if this molecule is present in between drug and surfactant. With this molecule, the BSA shows hydrogen bonds with Asn44 (B).The bond lengths noted were 2.40 \\u0026Aring; and 3.25 \\u0026Aring;. Yet another hydrogen bond with Lys 20 (B) of bond length 2.89 \\u0026Aring; was also found in the structure.\\u003c/p\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab12\\\" border=\\\"1\\\"\\u003e \\u003ccaption language=\\\"En\\\"\\u003e \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 12\\u003c/div\\u003e \\u003cdiv class=\\\"CaptionContent\\\"\\u003e \\u003cp\\u003eDocking score of drugs and surfactants\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"5\\\"\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e \\u003cp\\u003eQuenchers\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colspan=\\\"4\\\" nameend=\\\"c5\\\" namest=\\\"c2\\\"\\u003e \\u003cp\\u003eBSA with L-81 with quenchers\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1/Q\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eF\\u003csub\\u003e0\\u003c/sub\\u003e-F\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e1/F\\u003csub\\u003e0\\u003c/sub\\u003e-F\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003eK\\u003csub\\u003eA\\u003c/sub\\u003e L mol\\u003csup\\u003e\\u0026minus;\\u0026thinsp;1\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eTinidazole (TNZ)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e416.7\\u003c/p\\u003e \\u003cp\\u003e277.8\\u003c/p\\u003e \\u003cp\\u003e208.4\\u003c/p\\u003e \\u003cp\\u003e166.7\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e45.73\\u003c/p\\u003e \\u003cp\\u003e50.72\\u003c/p\\u003e \\u003cp\\u003e55.89\\u003c/p\\u003e \\u003cp\\u003e64.34\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.022\\u003c/p\\u003e \\u003cp\\u003e0.019\\u003c/p\\u003e \\u003cp\\u003e0.017\\u003c/p\\u003e \\u003cp\\u003e0.015\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3.4\\u0026times;10\\u003csup\\u003e3\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePiroxicam\\u003c/p\\u003e \\u003cp\\u003e(PY)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e1225\\u003c/p\\u003e \\u003cp\\u003e83.4\\u003c/p\\u003e \\u003cp\\u003e62.5\\u003c/p\\u003e \\u003cp\\u003e50\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0.051\\u003c/p\\u003e \\u003cp\\u003e0.068\\u003c/p\\u003e \\u003cp\\u003e0.071\\u003c/p\\u003e \\u003cp\\u003e0.083\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e45.45\\u003c/p\\u003e \\u003cp\\u003e14.70\\u003c/p\\u003e \\u003cp\\u003e14.08\\u003c/p\\u003e \\u003cp\\u003e12.04\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e6.9\\u0026times;10\\u003csup\\u003e3\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eMethylparaben\\u003c/p\\u003e \\u003cp\\u003e(MP)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e31.25\\u003c/p\\u003e \\u003cp\\u003e26.21\\u003c/p\\u003e \\u003cp\\u003e22.72\\u003c/p\\u003e \\u003cp\\u003e19.23\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e251.4\\u003c/p\\u003e \\u003cp\\u003e329.5\\u003c/p\\u003e \\u003cp\\u003e337.2\\u003c/p\\u003e \\u003cp\\u003e353.4\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.0039\\u003c/p\\u003e \\u003cp\\u003e0.0030\\u003c/p\\u003e \\u003cp\\u003e0.0029\\u003c/p\\u003e \\u003cp\\u003e0.0028\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3.49\\u0026times;10\\u003csup\\u003e3\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003ePropylparaben\\u003c/p\\u003e \\u003cp\\u003e(PP)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e7.69\\u003c/p\\u003e \\u003cp\\u003e6.25\\u003c/p\\u003e \\u003cp\\u003e5.26\\u003c/p\\u003e \\u003cp\\u003e4.76\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e23.01\\u003c/p\\u003e \\u003cp\\u003e26.02\\u003c/p\\u003e \\u003cp\\u003e28.13\\u003c/p\\u003e \\u003cp\\u003e32.85\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e \\u003cp\\u003e0.043\\u003c/p\\u003e \\u003cp\\u003e0.038\\u003c/p\\u003e \\u003cp\\u003e0.035\\u003c/p\\u003e \\u003cp\\u003e0.030\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e \\u003cp\\u003e3.3\\u0026times;10\\u003csup\\u003e3\\u003c/sup\\u003e\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eComparing of the bonding in all the combinations from Fig.\\u0026nbsp;13a to Fig.\\u0026nbsp;13e, it can be said that compound (a), with TNZ has the lowest binding energy, hence, gives a more stable combination with BSA.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/div\\u003e\"},{\"header\":\"4. Conclusion\",\"content\":\"\\u003cp\\u003ePluronic L-81, a non-ionic surfactant has been used earlier in binary combination in the pharmaceutical industry as polymeric drug delivery vehicles. In this work, the effect of presence of pluronic L-81 on the binding behavior of four drugs with BSA was investigated by UV-visible spectroscopy and fluorescence measurements. There was enhanced binding observed due to incorporation of L-81 in the complex of drug-BSA. Dynamic quenching is suggested from the quenching behavior of drugs. The results were compared with molecular docking studies. This study discloses use of a non-ionic surfactant to improve the drug-BSA binding which can be extended too many other drug formulations.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003eTNZ-Tinidazole\\u003c/p\\u003e\\n\\u003cp\\u003ePY-Piroxicam\\u003c/p\\u003e\\n\\u003cp\\u003eMP- Methylparaben\\u003c/p\\u003e\\n\\u003cp\\u003ePP-Propylparaben\\u003c/p\\u003e\\n\\u003cp\\u003eBSA-Bovine serum albumin\\u003c/p\\u003e\\n\\u003cp\\u003ePluronic L-81\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgement\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe medicine samples were provided by Applied Communication and Control, Dehradun, and MMC Health Care Limited, Chennai.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have not received any funding from research work\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of Interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no conflict of interest\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eEthical Approval statement and\\u0026nbsp;\\u003c/strong\\u003e\\u003cstrong\\u003eDeclaration\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u0026nbsp;It is not applicable\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contribution\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePrakash Karunanithi:\\u003c/strong\\u003e Investigation, Data curation\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eM.\\u003c/strong\\u003e \\u003cstrong\\u003eSenthilkumar: \\u0026nbsp;\\u003c/strong\\u003eInvestigation, Formal analysis, Methodology, Review \\u0026amp; Writing-original draft.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eNaik KM, Nandibewoor ST (2013) Spectral characterization of the binding and conformational changes of bovine serum albumin upon interaction with an anti-fungal drug, methylparaben. 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J Mol Liquid 383: 121921\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eCarolane M, Almeida \\u0026Eacute;ricaCM, Nascimento Jo\\u0026atilde;oBL, Martins, Tales HA da, Mota Di\\u0026ecirc;goM, de Oliveira CC, Gatto (2023) Crystal Design, Antitumor Activity and Molecular Docking of Novel Palladium(II) and Gold(III) Complexes with a Thiosemicarbazone Ligand. International J Molecular Sci 24: (14), 11442. \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.3390/ijms241411442\\u003c/span\\u003e\\u003cspan address=\\\"10.3390/ijms241411442\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Pluronic L-81, Tinidazole, Piroxicam, Methylparaben, Propylparaben, Solubilization\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-5323332/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-5323332/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eSerum albumins are the soluble protein part in the circulatory system. They have a significant role in transport and delivery of drug in blood. Bovine serum albumin (BSA) is generally used as model protein which is structurally similar to human serum albumin (HSA). Interaction of drug at the protein binding level can induce many changes like distribution rate, elimination of drug or most importantly solubilization of a hydrophobic drug. Protein-surfactant interaction employed to cause conformational changes in protein by change of polarity and on many occasions help in stabilization of protein. Generally, nonionic surfactants are used for these purposes and hence are widely studied. The studies on drug binding with BSA in presence of nonionic surfactant are however, rare in literature. In this study, we have taken four drugs for analyzing their binding with BSA in presence of a nonionic, hydrophobic surfactant, pluronic L-81 and investigate their role in the system. The drugs are Tinidazole (TNZ), Piroxicam (PY), Methylparaben (MP) and Propylparaben (PP). The characterizations were done using UV-visible spectroscopy and fluorescence spectroscopy. The UV-spectroscopic measurements were conducted for BSA-drug mixtures without and with pluronic L-81. The absorbance values were used for determining the binding constant values using Benesi-Hildebrand (B-H) plot. Fluorimetry studies of the\\u003c/p\\u003e \\u003cp\\u003eBSA\\u0026thinsp;+\\u0026thinsp;L-81\\u0026thinsp;+\\u0026thinsp;drug mixtures were carried out at room temperature which showed that there was quenching effect on addition of drug to BSA\\u0026thinsp;+\\u0026thinsp;L-81 mixture. Further, the quenching was of dynamic nature. The fluorescence data were analyzed using Stern-Volmer and Lineweaver-Burk equations. With the data, association constant K\\u003csub\\u003eA\\u003c/sub\\u003e for all the systems were determined. The docking studies confirmed the binding of the drugs with BSA.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Role of Non-ionic surfactant on the interaction of drugs with Bovine serum albumin (BSA)\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-11-21 14:34:58\",\"doi\":\"10.21203/rs.3.rs-5323332/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":3}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"e71e5124-644b-4357-bc79-8082f5329b86\",\"owner\":[],\"postedDate\":\"November 21st, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-11-25T02:53:46+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-11-21 14:34:58\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-5323332\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-5323332\",\"identity\":\"rs-5323332\",\"version\":[\"v1\"]},\"buildId\":\"qtupq5eGEP_6zYnWcrvyt\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}