Enterococcus faecium sp. GR7 was enhanced via arginine deiminase pathway. Process parameters including fermentation media and environmental conditions were optimized using independent experiments and response surface methodology . Fermentation media (EAPM) were optimized using independent experiments which resulted in 4-fold increase in arginine deiminase specific activity as compared to basal medium. To further enhance arginine deiminase activity in E. faecium sp. GR7 and biomass production including a five-level central composite design (CCD) was employed to study the interactive effect of three-process variables. Response surface methodology suggested a quadratic model which was further validated experimentally where it showed approximately 15-fold increase in arginine metabolism (in terms of arginine deiminase specific activity) over basal medium. By solving the regression equation and analyzing the response surface cartons, optimal concentrations of the media components (g/L) were determined as arginine 20.0; tryptone 15.0; lactose 10.0; K2HPO4 3.0; NaCl 1.0, MnSO4 0.6 mM; Tween 80 1%; pH 6.0 for achieving specific arginine deiminase activity of 4.6 IU/mG with concomitant biomass production of 12.1 mg/L. The model is significant as the coefficient of determination (R2) was 0.87 to 0.90 for all responses. Enhanced arginine deiminase yield from E. faecium, a GRAS lactic acid bacterial strain, is desirable to explore in vitro therapeutic potential of the arginine metabolizing E. faecium sp. GR7.Arginine metabolism in  Enterococcus faecalis, Lactobacillus sp., Lactococcus, Leuconostoc, Oenococcus, Streptococcus, and Weissella [L-arginine is classified as conditionally essential amino acid for protein synthesis that is metabolized through citrulline, ornithine, creatine, proline, and polyamines in human body , 2. It ieissella , 5–10. Ieissella .Enterococcus sp. RSM, formerly known as Box-Wilson methodology, is the most widely used statistical technique to evaluate relationship between a set of controllable experimental factors and observed results [E. faecium sp. GR7 which was carried out in two steps. Independent experiments for the selection of most influential process parameters including fermentation media and environmental conditions were performed followed by application of CCD design for final optimization of media components to enhance ADI activity and biomass production using influential process variables.The present study was aimed to screen and optimize media components that increase ADI activity of  results . RSM is E. faecium sp. GR7 used in the study was cultured in MAM medium (g/L) consisting of tryptone 10.0; glucose 5.0; yeast extract 5.0; arginine 3.0; KH2PO4 0.5; MgSO4 0.2; MnSO4 0.05; Tween 80 1.0 mL/L; Agar 2.0; pH 6.0 [; pH 6.0 . OpticalμL of 50 mM arginine, 2.3 mL of 50 mM acetate buffer (pH 5.5), 50 μL of cell wall or cytoplasm preparation, and 3.6 μL of sodium azide (0.05% w/v). Controls without substrate and without enzyme were included. After incubation at 37°C for 1 h, the reaction was stopped by adding 0.5 mL solution of 2N HCl, and precipitated protein was removed by centrifugation. Citrulline content after enzyme assay in CFS was determined by Archibald's method [3PO4-H2SO4 (3/1 v/v) and 250 μL of diacetyl monoxime  in 10% (v/v) methanol, mixed and boiled in the dark for 30 min. After cooling for 10 min, absorbance was measured at 460 nm and one enzyme unit was calculated as the amount of enzyme required to catalyze formation of 1 μmol citrulline per min. Finally, specific activity was calculated as international enzyme units present per mg (IU/mG) of protein.To determine enzyme activity, 24 h-old cultures were centrifuged at 8,000 rpm for 10 min. Cell free supernatant (CFS) was assessed for extracellular protein and enzyme activity. For assaying intracellular ADI activity, cell pellet was resuspended in lysis buffer. Total protein was estimated by measuring absorption at 280 nm using standard curve of BSA. ADI activity was assayed using standard method of De Angelis et al. . Assay ms method . One milEnterococcus confirmatory broth [E. faecium sp. GR7. Inoculums were subcultured twice in MAM broth at 37°C for 24 h and used at 1% v/v for each experiment. ADI specific activity was determined following standard procedures of De Angelis et al. [Six culture media including MAM ; MRS 1414; TGYE ry broth ; and M9 ry broth  each sups et al.  and Archs et al. .E. faecium sp. GR7 was grown in the selected medium (TGYE) and the effect of various parameters, that is, initial pH (5.0 to 7.0), inoculum size (1 to 5% v/v), subculturing period (1 to 4 days), incubation temperature (25°C to 50°C), and culture incubation conditions, that is, aerobic, anaerobic, shaking (200 rpm) on ADI production was studied individually, by varying one factor at a time. At each step, the selected factor was included in the basal medium (selected from the previous experiment) for getting a set of conditions that enhanced ADI activity in E. faecium sp. GR7. Rest of the conditions and enzyme assays were described previously.E. faecium sp. GR7 was investigated. In the production medium, carbon of the basal media was replaced with glucose, galactose, sucrose, maltose, lactose, and fructose, and nitrogen with yeast extract, peptone, tryptone, and beef extract which were tested at levels ranging from 5 to 20 g/L (w/v).The effect of various carbon and nitrogen sources on enzyme production in Effect of inducer concentration, that is, arginine on ADI production was studied by supplementing basal medium with different concentrations of arginine from 5 to 20 mM (w/v).2HPO4 (0.5–5 g/L) (w/v), MnSO4 (0.2–2 mM) (w/v), CuSO4 (0.025–0.075 mM) (w/v), ZnSO4 (2–6 mM) (w/v), and surfactants including CTAB, SDS, Tween 80, and Triton X-100 (0.1% w/v) to investigate their role in improving enzyme production and secretion by E. faecium sp. GR7.Basal medium was supplemented with various salts such as NaCl (0.5–5 g/L) (w/v), KE. faecium sp. GR7. RSM is a successive and exploratory tool for establishing the influence and interaction among variables on biological activities [E. faecium sp. GR7. A quadratic model obtained by a multiple regression technique for three factors, that is, tryptone, lactose, and arginine was studied at five different levels along with four constant variables, so that, interactions among these variables at different levels could be studied for two responses, that is, ADI activity and biomass  and incubated at 30°C for 24 h. The samples were collected after 4 h intervals to estimate specific ADI activity and growth A600 for optimized and unoptimized media basal medium.The response surface model was validated under the predicted conditions in triplicates. On the basis of results obtained in statistical RSM analysis, the optimized medium (g/L) was composed of tryptone 15.0; lactose 10.0; arginine 20.0; MnSOP value <0.05 was used as the criterion for statistical significance.One way Anova analysis was carried out, and the results are presented as mean ± standard deviation of three triplicate experiments. A probability value of Enterococcus confirmatory media, and M9 minimal salt media were selected on the basis of previous literature citations for studying ADI enzyme production in E. faecium sp. GR7. The highest specific ADI activity of 0.182 ± 0.001 IU/mG was observed in TGYE media when supplemented with 15 mM arginine , E. faecium sp. GR7 yielded maximum ADI activity of 0.187 IU/mG at pH 6.0 when incubated at 30°C for 24 h  inoculum size and after 2nd subculturing period  of 0.131 IU/mG . TryptonE. faecium sp. GR7 was obtained in production media supplemented 15 mM arginine as compared to unsupplemented basal media as shown in The ADI pathway is an arginine inducible system in most of the microorganisms , 27–29. 4, CuSO4, and ZnSO4, E. faecium sp. GR7 showed highest ADI activity of 0.186 ± 0.010 IU/mG at the concentration of 0.6 mM  as compared to TGYE basal media (0.182 ± 0.001 IU/mG).A new medium including the best source of carbon, nitrogen, inducer, salts, and surfactants for ADI production was improved, and bacterium 4, NaCl, K2HPO4, Tween 80, and pH-6 at 30°C for 24 h under aerobic conditions as constant factors was studied at five different levels along with other variables, and therefore, the interaction among the variables at their different levels could be studied. When grown in unoptimized TGYE broth, E. faecium sp. GR7 showed 0.182 IU/mG of specific ADI activity with a growth of 0.823 at A600 nm  after 24 h of incubation at 30°C. In order to enhance specific ADI activity and studying growth, experiments were designed to optimize media constituents.A new medium was formed by selecting most influencing variables from independent experiments, which was further optimized using RSM to enhance enzyme production. In statistical tool CCD, each selected variable, that is, lactose, tryptone, and arginine while keeping MnSOY) and growth (G) as a function of these variables. Multiple regression analysis of the experimental data was carried out, and statistical equation was generated which gives ADI enzyme activity and growth according to  as appropriate to the experimental design used. Based on the CCD, the experimental levels of specific ADI activity under each set of condition were determined and compared with the corresponding predicted levels suggested by Design-Expert 8.0.2 . The maxrding to  and (2),F-value when compared to other models  indicated the “P-value” to be 0.0018, which implies the model to be significant. The “lack of fit F-value” of 99.43 with P-value <0.0001 implies that the lack of fit is significant. There is only a 0.01% chance that a “lack of fit F-value” this large could occur due to noise. ANOVA indicated the R2 value of 0.8745 for response Y. This ensures a good correlation between observed and expected values of the quadratic model and indicates that this model could explain 87% response variability. The adequate precision which measures the signal-to-noise ratio of 9.227 indicates an adequate signal.Quadratic model was found to be the “best fit model” for the specific enzyme activity also for growth with the highest r models . ANOVA fA600 with the highest F-value when compared to other models  indicated the “P-value” to be 0.0004, which implies the model to be significant. The “lack of fit P-value” of <0.0002 implies the lack of fit is significant. There is only a 0.02% chance that a “lack of fit F-value” this large could occur due to noise. A 91% response variability showed good correlation between observed and expected values of the quadratic model as ANOVA indicated the R2 value of 0.9099 for response Y. The adequate precision which measures the signal-to-noise ratio of 9.58 indicates an adequate signal.Quadratic model is also found to be the “best fit model” for the growth at r models . ANOVA fE. faecium sp. GR7 shows very poor specific ADI activity (0.182 ± 0.001 IU/mG) in unoptimized TGYE broth, which was enhanced to 0.732 ± 0.006 IU/mG in new media and finally raised to 15 folds (4.27 IU/mG) in RSM optimized media (run 16) as shown in x and y-axis while keeping the other third variable at their central value, that is, lactose 10 g/L, 15 g/L tryptone, and arginine 10 g/L. CCD employed has revealed that tryptone at concentration of 15 g/L was optimal for maximum enzyme activity. This nitrogen source plays important role in ADI specific activity and biomass production, as growth increases with the concomitant increase in tryptone concentration  was mostly influenced by an increase in the concentration of tryptone and lactose  and optimal media. Biomass concentration of the bacteria cultivated in optimal medium in batch culture conditions reached 0.751 at A600 (12.1 mg/L) after 20 h of incubation with concomitant production of 4.27 IU mG−1 IU/mG specific ADI activity. This being higher in comparison with bacteria in TGYE medium with 0.32 at A600 (5.17 mg/L) biomass after 20 h of incubation with specific ADI activity of 0.20 IU/mG. Though the biomass yield is lesser in optimized medium but higher specific ADI activity is having more significance from industrial perspective. lactose . Presenc Figures , althoug Figures . Based oClostridium [Enterococcus [Halobacterium [Lactobacillus [Lactococcus [Leuconostoc [Mycoplasma [Pseudomonas [Streptococcus [Weissella [E. faecium sp. GR7 was enhanced to 4 folds in fermentation media (EAPM) using independent experiments which was further raised by 15 folds in RSM optimized media supplemented with 15 mM arginine in comparison to basal medium. Specific ADI activities reported in GRAS LAB range from 0.10 to 2.80 IU/mG  [Lactobacillus sake (above 0.6 mM glucose) [Lactobacillus sanfranciscensis CB1 (above 54 mM glucose) [Streptococcus lactis  [Leuconostoc oenos, Streptococcus mitis, and Weissella confusa GR7 [E. faecium sp. GR7 at (10 g/L) which was approximately 11.57 and 8.05 times higher than yeast extract, and peptone respectively, as reported earlier in E. faecalis NJ402 [4, NaCl, and K2HPO4 are reported to magnify their activity [ADI pathway of bacteria provides number of functions such as sole energy provider (ATP) for the growth of a variety of microorganisms in various environmental conditions, especially arginine as main catabolite for nonfermenting bacteria, allowing anaerobic growth of strict aerobic bacteria, growth of fermentative bacteria when sugars are not available or at low sugar concentration, to supply carbamoyl phosphate for biosynthesis of citrulline or pyrimidines and to protect bacteria against acid or starvation environmental stress conditions , 30. Thestridium , Enterocrococcus , Halobacacterium , Lactobabacillus , Lactocotococcus , Leuconoconostoc , Mycoplacoplasma , Pseudomudomonas , Streptotococcus , and Weieissella . Arginineissella . Few repeissella , 24, 27.80 IU/mG . Previouri CUC-3 , Lactoba NCDO110 , Lactobansis CB1 , Streptos lactis , Weisselglucose) , Lactobaglucose) , Lactobaglucose) , and Strlactose) . Fructosfusa GR7 , 34. Tryis NJ402 . ADI patactivity .Conventional processes of optimization are usually time consuming and expensive. The single factor optimization cannot explain the actual interactions of the parameters of the experimental data because the interaction between different factors is overlooked, leading to a misinterpretation of the results. In this study, optimization of multiple factors yielded more accurate results from which one could choose the correct value of each response factor individually with a high certainty. Fermentation media (EAPM) optimized using single factor based independent experiments resulted in 4-fold increase in ADI specific activity; however, a 15-fold increase was observed in RSM optimized medium when compared with basal medium. The optimized media components could be selected as the best condition for further studies on the development of a cost effective and viable industrial fermentation process. Thus, this work demonstrated the advantage of statistical media optimization methods for enzyme production with a minimum number of experimental trials, simultaneously with a reduction in laborious and time-consuming media composition analysis strategies.Enterococcus was found to be a potential producer of ADI enzyme. In this study, result of bioprocess was optimized for future scaleup of ADI production process in E. faecium sp. GR7. A statistical strategy of CCD was used successfully to find out optimum values of significant response factors, which resulted in 15-fold increase in ADI production in RSM optimized media over basal media in E. faecium sp. GR7. It is interesting to report a novel lactic acid bacterial strain; that is, E. faecium having arginine metabolizing capacity can be ideally explored for production as a class of therapeutic enzyme against arginine auxotrophic cancers at low cost where the industrial needs are satisfied. The progress in the ADI research will undoubtedly facilitate the use of amino acid depriving enzymes in an innovative strategy in therapy of specific auxotrophic tumors and have profound influence on human health.A new strain of an


Purpose. To compare the efficacy of intravitreal bevacizumab versus ranibizumab in the treatment of neovascular age-related macular degeneration (nAMD). Methods. Retrospective, comparative study. The newly diagnosed nAMD patients who were treated with intravitreal bevacizumab or ranibizumab on an as-needed treatment regimen were included in the study. Main outcome measures were the change in best corrected visual acuity (BCVA), and central retinal thickness (CRT). Secondary outcome measures were the number of injections, and complications. Results. A total of 154 patients were included in the study. Bevacizumab group consisted of 79 patients, and ranibizumab group consisted of 74 patients. Mean follow-up time was 18.9 months, and 18.3 months in the bevacizumab and ranibizumab groups, respectively. There was not a significant difference between the two groups regarding the change in BCVA and CRT at all time points . The mean number of injections at month 12 was 4.8 and 4.7 in bevacizumab and ranibizumab groups, respectively (P > 0.05). No serious complications were detected in any of the groups. Conclusion. Both of the bevacizumab and ranibizumab found to be effective in the treatment of nAMD in regards of functional and anatomical outcomes with similar number of treatments and similar side effects. Neovascular age-related macular degeneration (nAMD) is a leading cause of visual loss among elderly population , 2. BefoIn this study, we aimed to compare the functional and anatomical outcomes of bevacizumab therapy to ranibizumab therapy on as-needed treatment regimen for nAMD in Turkish patients.The clinical data of the patients who were treated with either bevacizumab or ranibizumab between 2009 and 2011 were retrospectively evaluated for this study. Inclusion criteria were age of 50 years or more, newly diagnosed nAMD, no treatments received other than bevacizumab alone or ranibizumab alone, and a minimum follow-up period of 12 months. Patients were not included if they had any of the following criteria: retinal diseases other than nAMD, previous intravitreal injections, or a history of photodynamic therapy or laser photocoagulation. The tenets of the Declaration of Helsinki were followed throughout the study, and written informed consent was obtained from all patients before the treatments were started.Data collected from the patients' records included age, gender, type of choroidal neovascularization (CNV) , BCVA, and central retinal thickness (CRT) before treatment, at months 3, 6, 9, and 12 during treatment, and at the most recent follow-up examination. Cumulative numbers of injections were recorded for each patient.All patients underwent a standardized examination including measurement of BCVA via the Early Treatment Diabetic Retinopathy Study (ETDRS) chart at 4 meters, slit-lamp biomicroscopy and fundus examination and measurement of intraocular pressure (IOP) via applanation tonometry. Fundus photography, fluorescein angiography , and optical coherence tomography (OCT) imaging  were performed before treatment. All examinations were repeated monthly, except fluorescein angiography, which was repeated only when the cause of visual acuity deterioration could not be clarified with the clinical examination and other imaging methods. OCT was used for detecting subretinal fluid and measurement of CRT, the latter being defined as the mean thickness of the neurosensory retina in the central 1 mm diameter region, computed via OCT mapping software provided with the device.All injections were performed under sterile conditions after topical anesthesia, and 10% povidone-iodine scrub  was used on the lids and lashes, and then 5% povidone-iodine was administered on the conjunctival sac. Intravitreal bevacizumab  or ranibizumab  was injected with a 30-gauge needle through the pars plana at 3.5 mm to 4 mm posterior to the limbus. Patients were then instructed to consult the hospital if they experienced decreased vision, eye pain, or any new symptoms.visual loss of 1 or more lines,newly developed macular hemorrhage, evidence of CNV enlargement on examination or fluorescein angiography,any amount of persistent subretinal fluid one month after an injection.For the first three months of treatment, all patients received monthly doses of bevacizumab, or ranibizumab . The patients were then examined monthly and were retreated if they met any of the following criteria:t-test. Chi-square test was used to compare nominal parameters between the groups, and independent sample t-test was used for continuous parameters. The statistical analysis was performed using SPSS version . A P value of less than 0.05 was considered to be statistically significant.Visual acuity was converted to logarithm of Minimum Angle of Resolution (logMAR) for statistical analysis. The changes in BCVA and CRT over time were analyzed with paired sample A total of 154 patients met the inclusion criteria for the study. The bevacizumab group consisted of 79 patients, and the ranibizumab group consisted of 74 patients. The general characteristics of the patients were similar between the two groups . P < 0.05, for all). However, there was not a statistically significant difference between the two groups in regards of change in BCVA at all of the time points  , 0.86 ± 0.46 LogMAR (range 0.2–2.1 LogMAR), 0.83 ± 0.43 LogMAR (range 0.2–2.1 LogMAR), 0.82 ± 0.43 LogMAR (range 0.2–2.1 LogMAR), 0.82 ± 0.42 (range 0.2–2.1 LogMAR), and 0.83 ± 0.43 (range 0.2–2.1 LogMAR), respectively. The mean BCVA of the patients in the ranibizumab group at baseline, months 3, 6, 9, 12, and at the most recent followup was 0.97 ± 0.46 LogMAR (range 0.1–2.1 LogMAR), 0.83 ± 0.47 LogMAR (range 0.1–1.8 LogMAR), 0.81 ± 0.47 LogMAR (range 0.0–1.8 LogMAR), 0.81 ± 0.42 LogMAR (range 0.1–1.8 LogMAR), 0.82 ± 0.43 (range 0.0–1.8 LogMAR), and 0.82 ± 0.47 (range 0.0–2.1 LogMAR), respectively . The chaollowup) .P = 0.4). Sixty-eight of the 79 patients (86.1%) in the bevacizumab group and 64 of the 74 patients (86.5%) in the ranibizumab group had stable or improved vision  (P = 0.5). Eleven of the 79 patients (13.9%) in the bevacizumab group and 10 of the 74 patients (13.5%) in the ranibizumab group had VA loss ≥ 3 lines (P = 0.5). At the most recent followup, 34 of the 79 patients (43.0%) in the bevacizumab group and 30 of the 74 patients (40.5%) in the ranibizumab group gained VA ≥ 3 lines (μm (range 180–758 μm), 289 ± 118 μm (range 106–796 μm), 266 ± 79 μm (range 140–558 μm), 260 ± 93 μm (range 148–713 μm), 266 ± 100 (range 150–789 μm), and 262 ± 124 (range 103–979 μm), respectively. The mean CRT in the ranibizumab group at the baseline, and at months 3, 6, 9, and 12, and at the most recent followup was 315 ± 88 (range 139–681 μm), 250 ± 68 μm (range 133–487 μm), 243 ± 63 μm (range 135–410 μm), 254 ± 72 μm (range 135–540 μm), 247 ± 60 (range 140–415 μm), and 256 ± 73 (range 130–483 μm), respectively . However, the change in mean CRT from the baseline to months 3, 6, 9, and 12, and at the most recent follow-up was not statistically different between the two groups   .P = 0.8). The mean injection numbers were summarized in The mean number of injections at month 12 was 4.8 ± 1.2 (range 3–7) in the bevacizumab group and 4.7 ± 1.4 (range 3–8) in the ranibizumab group, and the difference was not statistically significant between the two groups (P = 0.7). The mean IOP at the most recent followup was 15.3 ± 2.4 mm Hg (range 10–22 mm Hg) and 15.6 ± 2.1 (range 11–22 mm Hg), in the bevacizumab and ranibizumab groups, respectively (P = 0.4). The mean baseline intraocular pressure (IOP) was 15.5 ± 2.3 mm Hg (range 9–19 mm Hg) and 15.4 ± 1.7 (range 12–20 mm Hg) in the bevacizumab and ranibizumab groups, respectively (No serious ocular or systemic complications were observed in any of the patients. Only mild complications like punctate keratitis, subconjunctival hemorrhage, and transient mild anterior uveitis were detected . Functional and anatomical outcomes of nAMD patients treated with bevacizumab and ranibizumab in a population of Turkish patients were reported in this study. In both of the groups, the mean BCVA and CRT were significantly better than that in baseline at every time point. The two groups were parallel to each other with respect to change in BCVA and CRT, injection numbers, and complications. In previous clinical studies, these two drugs, bevacizumab or ranibizumab, have been found to provide similar improvements in visual and anatomical outcomes to our study , 16–20. A short term efficacy and safety study by Landa et al.  reportedIn a retrospective study by Feng et al. , 371 nAMFong et al.  reportedIn a retrospective study concerning the efficacy of bevacizumab and ranibizumab in nAMD by Bellerive et al. , the meaThe CATT study was designed as a multicenter, noninferiority trial to compare the efficacy and safety of bevacizumab and ranibizumab. In the study, both monthly and as-needed treatment regimens were evaluated within and between the two drugs. At first year, it was reported that there was not a significant difference between the two drugs in regards of visual acuity and anatomical outcomes and the mean injection numbers. At month 12, as-needed bevacizumab was found to be equivalent to as-needed ranibizumab with a gain of 5.9 ETDRS letters and 6.8 ETDRS letters in visual acuity, respectively. At month 12, the eyes treated with as-needed bevacizumab and ranibizumab showed a significant decrease in CRT, and the difference was not statistically significant between the bevacizumab groups and ranibizumab groups. The mean injection numbers of as-needed bevacizumab group and as-needed ranibizumab group were 7.7 and 6.9 at month 12, respectively. The two-year results of the study were similar to the first-year results; however the mean gain in visual acuity was found to be greater in monthly groups than as-needed groups . The stuLimitation of the present study includes the relatively small numbers of patients. Strengths of the study include the fact that patients in the bevacizumab and ranibizumab groups had received no other forms of treatment and similar baseline characteristics of the two groups.In summary, in the present study we found that 86.1% of the patients in the bevacizumab group and 86.5% of the patients in the ranibizumab group had a stable or improved visual acuity after a mean follow-up time of approximately 18 months. The visual and anatomical outcomes and the number of injections were similar between the two drugs. No patients had suffered from a serious adverse event during the entire followup. The patients with nAMD treated with as-needed bevacizumab or as-needed ranibizumab showed significant improvements in visual acuity throughout at least 1.5 years compared to baseline visual acuity.


In vitro dissolution studies suggested a rapid disintegration, in which most of levocetirizine (93.54 ± 3.9%) dissolved within 90 seconds after insertion into the medium. Subsequently, Sprague–Dawley rats were used to compare the pharmacokinetic properties of the film preparation administered to the oral cavity, to those with oral administration of the pure drug solution. The pharmacokinetic parameters were similar between the two groups in which AUC0–t (ng h/ml), AUC0–∞ (ng h/ml) Cmax (ng/ml), Tmax (min), Kel (h−1), and t1/2 (h) of the reference were 452.033 ± 43.68, 465.78 ± 48.16, 237.16 ± 19.87, 30, 0.453 ± 0.051, and 1.536 ± 0.118, respectively, for the film formulation 447.233 ± 46.24, 458.22 ± 46.74, 233.32 ± 17.19, 30, 0.464 ± 0.060, and 1.496 ± 0.293, respectively. These results suggest that the present levocetirizine containing fast-dissolving film is likely to become one of the choices to treat different allergic conditions.A fast-dissolving film containing levocetirizine, a non-sedative antihistamine drug, was developed using pullulan, xanthan gum, propylene glycol, and tween 80 as the base materials. The drug content of the prepared films was within an acceptable limit as prescribed by the USP. The film exhibited excellent stability for four months when stored at 40 °C and 75% humidity.  Some patients, particularly pediatric and geriatric patients, have difficulty in swallowing or chewing solid dosage forms. Many pediatric and geriatric patients are unwilling to take these solid preparations due to fear of choking . The fasAn allergy is a hypersensitive disorder of the immune system. Allergic reactions occur due to environmental substances known as allergens. An allergy is one of four forms of hypersensitivity and is called type I hypersensitivity. It is characterized by the excessive activation of certain white blood cells called mast cells and basophils by a type of antibody known as IgE, resulting in an extreme inflammatory response. Mild allergies like hay fever are highly prevalent in the human population and cause symptoms such as allergic conjunctivitis, itchiness, and runny nose. A variety of tests now exist to diagnose allergic conditions; these include testing the skin for responses to known allergens or analyzing the blood for the presence and levels of allergen-specific IgE. Treatments for allergies include allergen avoidance, use of antihistamines, steroids or other oral medications, immunotherapy to desensitize the response to the allergen, and targeted therapy –9.Levocetirizine (LCZ) is a third-generation non-sedative antihistamine, which works by blocking histamine receptors. It does not prevent the actual release of histamine from mast cells, but prevents it binding to its receptors. LCZ prevents the release of other allergic chemicals produced in response to allergens and the increased blood supply to the area, and provides relief from the typical symptoms of hay fever .Levocetirizine is bitter in taste. The bitterness of the drug was lessened by using hydroxyl propyl β-cyclodextrin complexes as reported by Mahesh in 2010 and Dinge in 2008 , 12.Aureobasidium pullulans. It is a white, tasteless, odourless, and water-soluble powder. Pullulan (PI-20 grade) is the deionised form of pullulan, having an average molecular weight of 200,000 daltons and possesses excellent film-forming properties [Pullulan is a natural polysaccharide produced from starch by cultivating the black yeast, operties , 14.Levocetirizine dihydrochloride (LCZ) was obtained as a gift sample from Cipla Ltd., Ahmedabad, Gujarat, India. Neotame, hydroxyl propyl β-cyclodextrin (HPβ-CD), and xanthan gum were provided by Alkem pharmaceutical Pvt Ltd. Mumbai, India. Pullulan gum was provided as a gift sample by Gengwal Chemicals, Mumbai, India. Propylene glycol (PG) and tween 80 were purchased from Sigma chemicals.2 and a 1.3 cm wall height. The dispersion was dried in a hot air oven at 45–50 °C. After drying, the films were removed with the help of a sharp blade and kept in a desiccator for 24 hrs before cutting it into small pieces, having an area of 6 cm2 for each film. Films with air bubbles, cuts, or imperfections were excluded from further study. Selected films were subjected to different evaluation parameters [The basic components of FDFs were LCZ : HPβ-CD complex , pullulan (2% w/v), xanthan gum (0.4% w/v), propylene glycol (0.2% w/w), and tween 80 (0.1% w/w) in 25 ml of water. All of these excipients were dissolved in water by using a magnetic stirrer to obtain a homogenous dispersion. Then it was allowed to be kept aside for 8 h to remove air bubbles, and then 25 ml of the dispersion was cast onto a glass petridish  having a surface area of 64 cmrameters , 12.The prepared films were evaluated for different parameters like: appearance, weight , tensilein vitro dissolution study was performed for selected films for three minutes in the USP paddle apparatus using a pH 6.4 buffer solution. The dissolution medium was kept at 37 ± 0.5 °C and rotated at 500 rpm. The samples (5 ml) were withdrawn after every 30 sec interval and replaced with a fresh buffer (pH 6.4) solution. One ml of the sample was then taken and diluted up to 10 ml in a volumetric flask. The samples were analyzed for the drug content using a UV spectrophotometer at 231 nm. Dissolution was performed three times for each formulation to calculate the drug-release profile [An  profile .Short-term stability studies were performed for optimized films, and were placed in plastic containers and exposed to 40 ± 0.5 C and 75± 5% RH (ICH guidelines) for a period of four months. Different film properties like physical appearance of the film, mechanical properties, and drug content were evaluated at intervals of one week .18  at ambient temperature. The mobile phase was a mixture of Acetonitrile : Buffer  70:30% v/v [In this study, Sprague–Dawley rats were used. Their mean weight was 264.66 ± 8.96 g in the range of 250–275 g. Animals were housed in a room maintained on a 12-h light/dark cycle at 23 ± 2 °C with free access to food and water. The experimental procedures were approved by the Committee for the Care and Use of Laboratory Animals at the IICP (Protocol number: IICP/PH/12-2010/02). For the administration of sample (film) preparation, 50 μl aliquot of distilled water was dropped into the rat oral cavity under light ether anesthesia, then two halves (1 cm×0.5 cm) of the film preparation were applied to the buccal cavity bilaterally. For oral administration, rats were given 1mg/kg of the LCZ dose  in solution containing 1 ml water, under light ether anesthesia . The HPL:30% v/v , 23.max) and the time to attain peak concentration (Tmax) were obtained from experimental points of the plot. The other pharmacokinetic parameters were determined by using Kinetica software . The data of the reference  and sample (film) were compared and statistically evaluated by Student’s t-test.Pharmacokinetic parameters were derived from the plasma concentration vs. time plot. The peak plasma concentration  as specified by the USP. Films were disintegrated in 16 ± 1.56 sec.Prepared films were opaque in appearance. The weight and tensile strength of the films were found to be 83 ± 6.9 mg and 6.18 ± 1.45 N/mmin vitro dissolution study was performed for the films for three minutes in a USP paddle apparatus using a pH 6.4 buffer solution. The prepared films released their 93.54 ± 3.9% of drug in 90 sec.An A stability study of selected films at 40 ± 2 °C/75% RH ± 5% RH was carried out for four months. The results are shown in The intra-assay and inter-assay precision were determined with a quality control sample spiked with three different concentrations of levocetirizine  as shown in As reported earlier, the HPLC determination of levocetirizine in human plasma was assayed by Macek in 1999 . In our Since the film and solution remain in the oral cavity for a few seconds (5–10 sec), there are fewer chances of pre-gastric absorption, so that this study was carried out only on blood profiles.0–t 452.033 ± 43.68 ng h/ml, AUC0–∞ 465.78 ± 48.16 ng h/ml, Cmax 237.16 ± 19.87 ng/ml, Tmax 30 minutes, Kel 0.453 ± 0.0519 h−1, and t1/2 1.536 ± 0.118 h. The pharmacokinetic parameters for the sample (film) were found as follows- AUC0–t 447.233 ± 46.24 ng h/ml, AUC0–∞ 458.22 ± 46.74 ng h/ml, Cmax 233.32 ± 17.198 ng/ml, Tmax 30 minutes, Kel 0.464 ± 0.0601 h−1, and t1/2 1.496 ± 0.293 h . The pharmacokinetic parameters for the reference  were found as follows: AUC 0.293 h .0–t, AUC0–∞, Cmax, and Tmax showed no statistically significant difference between the two formulations. This suggests that both of the formulations have similar blood profiles. The respective 95% CI of the ondansetron sample/reference percent ratios were 98.93% (91.14–118.36%) for AUC0–t, 97.39% (92.34–115.61) for AUC0–∞, and 98.38% (89.34–114.41%) for Cmax, while the acceptable range was 80–125% for AUC0–t and AUC0–∞, and 70–143% for C max as proposed by the FDA and the ANVISA  for data from AUCe ANVISA .The results of this study showed that there was no significant difference in the blood profile of the reference  and the sample (film) as shown in The prepared films of levocetirizine revealed excellent mechanical properties, drug content, and stability. The pattern of changes in plasma concentrations of the drug was similar between the two groups, although the concentrations were slightly, but not significantly, higher in the oral solution-treated group. No significant differences were observed in the pharmacokinetic parameters obtained from rats with the oral administration of levocetirizine solution of the pure drug and those with the film. Hence, the present fast-dissolving films containing levocetirizine are considered to be potentially useful for treating different allergic conditions.Based on the results, it is concluded that FDFs are convenient and reliable dosage forms that can get rid of the common problems associated with solid dosage forms.


Listeria monocytogenes using peracetic acid, chlorhexidine, and organic acids as active agent, determining the respective D-, Z-, and F-values. From our knowledge, these important results from an industrial view point are not available in the current literature, mainly for organic acids, pointing out the main contribution of the present work. Lower D-values were obtained for peracetic acid and chlorhexidine, compared with the organic acids. For the reduction of 6 log10 of L. monocytogenes using peracetic acid, at 0.2, 0.1, and 0.05% are necessary 7.08, 31.08, and 130.44 min of contact, respectively. The mathematical models of F-values showed that at concentrations lower than 0.15% one can verify an exponential increase in F-values, for both de chlorhexidine and peracetic acid. The organic acids presented a linear behavior, showing slight variation in F-values, is even more effective in under dosage. The results obtained are of fundamental importance in terms of industrial strategy for sanitization procedure, permitting to choose the best relation product concentration/exposure time, aiming at reducing costs without compromising the disinfectant efficiency.The objective of this work was to evaluate the kinetic of inactivation of  The results obtained in the present work are of industrial importance and not available in the current literature, mainly for organic acids. The results obtained are of fundamental importance in terms of industrial strategy for sanitization procedure, permitting to choose the best relation product concentration/exposure time, aiming at reducing costs without compromising the disinfectant efficiency.Listeria monocytogenes has been considered the most important pathogenic microorganism transmitted by food due to the high death rate in risk group  or sterilization (10−6 CFUmL−1), considering the death kinetic curves as first order , kept in Luria Bertani medium  at 4°C was subcultured for inoculum preparation in counting standard medium  at 35°C for 24 h.The strain of 0 to 10−8) and in each replicate of dilution at different concentration of peracetic acid (20% of active principle), chlorhexidine (15% of principle active), or organic acids   was added and kept at 25°C. These dilutions were inoculated in counting agar standard  after different exposure times to disinfectant  and incubated at 35°C for 24 h. Dilutions without the addition of the disinfectant were also inoculated in counting agar standard and incubated at 35°C for 24 h to determine the initial number of CFU.From this inoculum, different dilutions were prepared in peptone distilled water at 0.1% :The mathematical model for determining the N is the number of CFU, t is the exposure time, and k the proportionality constant.where N (t = 0) = N0, where N0 is the initial number of CFU, we have:By the integration of equation Rearranging equation k = 1/D and D is defined as the constant of decimal reduction, which represents the time necessary to reduce a log10 cycle along the process.where D-value, a linear regression was performed among the different exposure time of the microorganism to the sanitizer and the CFU log10 of survivors. Following the procedure, the death resistance constant for L. monocytogenes in relation to the disinfectant  was calculated by equation For obtaining the D1 and D2 are the values of decimal reduction for concentrations C1 and C2, respectively. The Z constant represents the alteration in concentration necessary to occur a reduction in one log10 cycle (90% of reduction) on the death time caused by the disinfectant. For the Z-value determination, a linear regression among different chlorhexidine concentration (C) and the log10 of the respective D-value was carried out.where F-value was determined taking into account the D-value and the initial and final counting.It is worth to mention that equation 10 of CFU for L. monocytogenes and the exposure time to the peracetic acid, chlorhexidine, and organic acids at 0.2, 0.1, and 0.05% permitted to determine the D-values, presented in Table D-values were obtained for peracetic acid and chlorhexidine, compared with the organic acids .The linear regression for the logD-values using chlorhexidine for different bacteria. The vegetative strains that showed higher resistance to a solution of chlorhexidine 0.4% were Enterococcus cloacae (D = 8.3 min) and Staphylococcus aureus (D = 5.9 min) and the most sensible were Acinetobacter calcoaceticus (D = 4.1 min), Serratia marcescens (D = 4.0 min), and Escherichia coli (D = 3.0 min). Exposure times from 3 to 4 min were enough to reduce 90% the population of E. coli, S. marcescens, and A. calcoaceticus. Spores exposed to 2% of chlorhexidine showed D-values of 9.1 min for Bacillus stearothermophilus and 6.7 min for Bacillus subtilis. The same authors verified that the bacteria that presented more resistance to a solution of 1% Minncare (0.45% peracetic acid plus 2.2% of hydrogen peroxide) were B. stearothermophilus (D = 9.1 min), E.coli (D = 6.7 min), and B. subtilis (D = 5.9 min). The most sensitive strains with similar resistance were A. calcoaceticus (D = 3.4 min), E. cloacae (D = 3.5 min), and S. aureus (D = 3.6 min) .Considering the application of the results presented here to an industrial plant and a counting of 1 × 10n Stumbo  one can 10 of L. monocytogenes using peracetic acid, at 0.2, 0.1, and 0.05% are necessary 7.08, 31.08, and 130.44 min of contact , respectively. Under the same conditions, we obtained F-values of 8.28, 18.52, and 55.87 to chlorhexidine and 30.54, 32.58, and 33.6 to organic acids.Here, for the reduction of 6 logF-values from 9 to 12 min for reduction in 3 log10 for E. coli, S. marcescens, and A. calcoaceticus, using 0.4% of chlorhexidine.Mazzola et al.  obtainedSalmonella choleraesuis, S. aureus, E. coli, and L. monocytogenes), using concentration of 0.2% during 2 min. The chlorhexidine showed efficiency at 0.2% and 2 min for E. coli, 18 min for S. aureus, and L. monocytogenes, but a concentration of 0.5% and 18 min was necessary for S. choleraesuis. The organic acids were efficient after 15 min of exposure (0.2% for L. monocytogenes and 0.6% for E. coli and S. choleraesuis), did not showing disinfection under S. aureus at the concentration and exposure time evaluated.Beltrame et al. , evaluatF-values was obtained by the mathematical models for each different sanitizer . The reduction of peracetic acid and chlorhexidine concentration from 0.2% to 0.05% leads to an increase of 18.4 and 6.7 times on the exposure time necessary to reduce the L. monocytogenes concentration from 1.0 × 103 to 1.0 × 10−3 CFU/cm2. For the organic acids, the same reduction in the concentration results in an increase of only 1.6 times in the exposure time.The analysis of zer Fig. . we can Some works in the literature reports a nonlinear death rate for different microorganisms exposed to different disinfectants (Campbell and Dimmick A predictive model to evaluate the effect of a disinfectant in a nonspecific room is not available and the execution of tests by practical conditions necessary to determine the effect of each product can be difficult. The security of the process as a whole for a specific disinfectant is highly complex and a function of the kind of bacteria, metabolic phase, microorganisms biodiversity, influence of organic material, and processing conditions such as temperature and pH (Asselt and Giffel The results obtained here are of fundamental importance in terms of industrial strategy for sanitization procedure, permitting to choose the best relation product concentration/exposure time, aiming at reducing costs without compromising the disinfectant efficiency.None declared.


Objectives. To compare the analgesic efficacy of intrathecal clonidine and fentanyl added to bupivacaine after cesarean section. Methods. Ninety patients scheduled for cesarean section under spinal anesthesia were randomly allocated to one of the three following groups to receive bupivacaine 10 mg combined with 75 µg clonidine (group C), bupivacaine 10 mg combined with 0.5 mL fentanyl (group F), and bupivacaine 10 mg combined with 0.5 mL distilled water (group P), intrathecally. The time to first analgesic request, analgesic requirement in the first 24 hours after surgery, sensory and motor blockade onset time, duration of sensory and motor blockade, the incidence of hypotension, ephedrine requirements, bradycardia, and hypoxemia were recorded. Results. The duration of anesthesia in clonidine group (275.10 ± 96.09) was longer compared to the placebo (211.73 ± 74.80) and fentanyl (192.33 ± 30.36) groups. This difference between group C versus F (P = 0.006) and P groups (P < 0.001) was significant. Similarly, the mean time to first analgesic request was also longer in group C (519.44 ± 86.25) than in groups F (277.88 ± 94.25) and P (235.43 ± 22.35 min). This difference between group C versus F (P < 0.001) and P groups (P < 0.001) was significant. Conclusion. Intrathecal clonidine 75 µg with bupivacaine prolonged the time to first analgesic request compared to fentanyl; however, the total analgesic consumption within the first 24 h postoperative was similar in fentanyl and clonidine groups following cesarean section. This trial is registered with ACTRN12611000909921 and ClinicalTrials.gov NCT01425658. In addition, inadequate analgesia leads to elevated plasma catecholamine concentrations, resulting in adverse effect on all organ systems . Neuraxioperties –9. Clonioperties . The safoperties , 11–16. operties , 16.We hypothesized that clonidine may provide a better pain relief after cesarean section compared to fentanyl. In addition, unlike spinal opioids, clonidine does not produce pruritus, hyperalgesia, or respiratory depression. To test our hypothesis, we designed this randomized-double-blind, placebo-controlled study to compare the postoperative analgesic effect of intrathecal clonidine and fentanyl added to bupivacaine in patients undergoing cesarean section.μg of preservative free clonidine; the fentanyl group received bupivacaine 10 mg combined with 25 μg fentanyl; and the placebo group received bupivacaine 10 mg combined with 0.5 mL distilled water, intrathecally. All patients received an intravenous preload of 5–7 mL/kg lactated Ringer's solution before a subarachnoid block. Later, using an aseptic technique, a 25-gauge Quincke needle was inserted intrathecally via a midline approach into the L4-5 interspaces by the anesthetist who was unaware of patient assignment while the patient was in sitting position. After a successful dural puncture, the anesthetic solution was injected. The primary outcomes of this randomized, double-blind and placebo-controlled clinical trial are to evaluate the time to first requirement of analgesic supplement and total analgesic consumption in the first 24 h postoperative. The secondary outcomes included the assessment of sensory block onset time, onset of motor block, duration of blockade, hemodynamic variables, the incidence of hypotension, ephedrine requirements, bradycardia, hypoxemia  < 90), and adverse events such as sedation, dizziness, pruritus, and postoperative nausea and vomiting.After approval of the Institutional Ethical Committee and written informed consent, Ninety-six patients 18–45 years old ASA physical status I or II, scheduled for cesarean section under spinal anesthesia, were enrolled in a prospective, double-blind, randomized parallel study. The recommendations by the Consolidated Standards of Reporting Trials (CONSORT) for reporting a randomized, controlled clinical trial  were folIn this study, the postoperative analgesia was defined as the time to first requirement of analgesic supplement from the time of injection. No additional analgesic was administered unless requested by the patient. Sensory block was assessed by a pinprick test. The onset of sensory block was defined as the time between the end of injection of the intrathecal anesthetic and the absence of pain at the T10 dermatome; the duration of sensory block was defined as the time for regression of the sensory from the maximum block height to the T10 dermatome as evaluated by pinprick. The maximum level of sensory block was evaluated by pinprick after 20 min following completion of injection. Motor block was assessed by the modified Bromage score ; the onset of motor block was defined as the time from intrathecal injection to Bromage block 1, whereas the duration of motor block was assumed when the modified Bromage score was zero. The duration of spinal anesthesia was defined as the period from spinal injection to the first occasion when the patient complained of pain in the postoperative period. Patients were preoperatively instructed to use the verbal rating scale (VRS) from 0 to 10  for pain assessment. If the VRS exceeded four and the patient requested a supplement analgesic, diclofenac Na sup. 100 mg every 8 hours was given to relieve the postoperative pain as needed (q 8 h PRN). If the time course following the administration of diclofenac Na decreased to less than 8 h and the patient made another request for supplement analgesic, pethidine 25 mg IV was given.2) were recorded by an anesthetist blinded to the patient group 5 min before the intrathecal injection and also 2, 4, 6, 8, 10, 15, and 20 min after injection. If systolic blood pressure (SBP) was 20% below the baseline  or less than 90 mmHg, ephedrine 5 mg was administered intravenously. Also, if HR was less than 50 beats/min, 0.5 mg of atropine sulfate was administered intravenously. A follow-up telephone call was made 24 h after surgery and again 1 and 6 months later, during which the patients were asked about the side effects and dysesthesia of the lower limbs or buttocks. To calculate the sample size, data from previous similar studies were taken into consideration [U test, with a power of 0.9 and an α equal to 0.05. We included 30 patients in each group to allow for dropouts and protocol violations. Data were analyzed using SPSS . Continuous variables were tested for normal distribution by the Kolmogorov-Smirnov test. Parametric data were expressed as mean and standard deviation (SD) and analyzed by independent t-test. Nonparametric data were expressed as median and interquartile range (IQR) and analyzed using the Mann-Whitney U test. The effect of time on hemodynamic parameters was analyzed using repeated measurement analysis of variance. The χ2 test was used to analyze the incidence of side effects. Pain scores, motor scores, and sensory level were evaluated within the groups using the Wilcoxon's signed rank test. A P value <0.05 was considered as significant, statistically.The mean arterial pressure (MAP), heart rate (HR), and peripheral oxygen saturation SpO were recderation , 12, 14.A total of 96 patients initially enrolled in this study, 6 patients had to be excluded because of logistical reasons or other violations of the study protocol. Ninety patients were included and randomly assigned to their treatment groups .There were no significant differences in age, height, and weight among the three groups. The duration of surgery was also similar .P = 0.523) and P (0.075) was insignificant. Similarly, this difference in groups F and P was also insignificant (P = 0.055). The mean duration of sensory block in group C (169.66 ± 25.69 min) was longer than group F (122.23 ± 32.78 min) and group P (133.53 ± 32.68 min). The difference between group C versus group F (P < 0.001) and P (P < 0.001) was significant, but the difference between groups F and P (P = 0.186) was found to be insignificant. The mean onset of motor block was 81.33 ± 26.71 in group C, 80.00 ± 30.62 in group F, and 81.83 ± 27.21 sec in group P. The difference between group C versus group F (P = 0.858) and P (P = 0.943) was insignificant. Similarly, the difference in groups F and P was insignificant (P = 0.807). The median value found for the maximum height of block was T6 for all three groups. The mean duration of motor blockade time was significantly longer in group C (182.66 ± 33.12 min) than F (136.76 ± 28.85 min) and P groups (143.16 ± 33.94). The difference in mean duration of motor blockade time between group C versus F (P < 0.001) and P groups (P < 0.001) was significant whereas no significant difference in duration of motor block between F and P groups was found (P = 0.435). The duration of anesthesia in clonidine group (275.10 ± 96.09) was longer compared to the placebo (211.73 ± 74.80) and fentanyl (192.33 ± 30.36) groups. As shown in P < 0.001) and F groups (P = 0.006). As to the duration of anesthesia, the mean time to first analgesic request was also significantly longer in group C (519.44 ± 86.25) than in groups F (277.88 ± 94.25) and P (235.43 ± 22.35 min). This difference between group C versus F (P < 0.001) and P groups (P < 0.001) was significant. Likewise, the difference between groups F and P was also significant (P = 0.022). The total number of analgesic request by patients during 24 hours after surgery in clonidine group was significantly smaller than in control group (P = 0.002). Total analgesic consumption during 24 hours after surgery failed to demonstrate a significant difference between F and C groups (P = 0.318).The mean onset of sensory block was 90 ± 23 sec in group C 95.33 ± 39.17 sec in group F and 78.5 ± 26.00 sec in group P. The difference between group C versus group F (P < 0.001) was significant whereas no significant difference between F versus C (P = 0.962) and P (P = 0.251) groups was found. The overall difference in ephedrine requirement between the three groups was significant, statistically (P < 0.001). The mean variation of HR was 34.33 ± 9.7 in group C, 33.43 ± 10.73 in group P, and 32.86 ± 10.17 in group F. The difference between group C versus P (P = 0.761) and F (P = 0.571) groups was also insignificant as it was for the difference between groups F and P (P = 0.851). As shown in As shown in μg with bupivacaine prolonged intraoperative anesthesia and the time to first analgesic request after cesarean delivery compared to fentanyl and control groups. These findings are consistent with previous studies [α2 receptors located in the dorsal horn. Presynaptic stimulation of α2 receptors inhibits neurotransmitter release and postsynaptic stimulation prevents neuronal transmission through hyperpolarisation [Based on the data found in our study, it was concluded that administration of intrathecal clonidine 75  studies , 19. Anarisation .μg with bupivacaine prolonged intraoperative anesthesia and the time to first analgesic request compared to fentanyl yet the total analgesic consumption in the first 24 h postoperative was similar in fentanyl and clonidine groups after elective cesarean delivery. The possible explanation for this finding is that the analgesic effect of clonidine follows a dose-dependent manner. Eisenach et al. reported that a dose of 150 μg clonidine is required to observe antihyperalgesic effect, while a lower dose (50 μg) is ineffective [μg/kg was not associated with hemodynamic disturbance [μg. However, most studies have reported that although clonidine at a lower intrathecal dose less than 0.5 μg/kg body weight was devoid of its diverse side effects, at the same time the antinociceptive effect of this drug was also reduced significantly [μg and higher doses could only increase the duration but not the intensity of analgesia [The second observation which should be emphasized is that although intrathecal clonidine 75 ffective , 18, 20.turbance . Also, Bturbance  found thficantly –14, 16. The third finding which should be considered is that intrathecal clonidine clearly increases the duration of both sensory block and motor block as well as postoperative pain relief. This finding is also consistent with the previous studies , 23, 24.μg failed to cause a significant decrease in blood pressure when added to a high dose of bupivacaine (18 mg) [μg with bupivacaine prolonged intraoperative anesthesia and the time to first analgesic request compared to fentanyl, however, the total analgesic consumption in the first 24 h postoperative was similar in fentanyl and clonidine groups following elective cesarean delivery. Further studies are needed to evaluate the analgesic efficacy of clonidine with other neuraxial drug combinations such as epinephrine, ketamine, and magnesium to provide better analgesia and reduce the incidence and severity of side effects.The fourth finding which should be taken into account is that transient hypotension episodes and vasopressor requirement in clonidine group were significantly greater than F and P groups, a finding in agreement with previous studies , 28. Exc (18 mg) , 29. How (18 mg) , 31, a f (18 mg) . The pos


Escherichia coli (E. coli) among the as-synthesized AgNPs. Handwash solution has been prepared using Na lauryl sulfate as surfactant, hydroxyethyl cellulose as binder, and 15 mg/L of AgNPs/alginate as antimicrobial agent. The obtained results on the antibacterial test of handwash for the dilution to 3 mg AgNPs/L showed that the antibacterial efficiency against E. coli was of 74.6%, 89.8%, and 99.0% for the contacted time of 1, 3, and 5 min, respectively. Thus, due to the biocompatibility of alginate extracted from seaweed and highly antimicrobial activity of AgNPs synthesized by gamma Co-60 irradiation, AgNPs/alginate is promising to use as an antimicrobial agent in biomedicine, cosmetic, and in other fields.Colloidal solutions of silver nanoparticles (AgNPs) were synthesized by gamma Co-60 irradiation using different stabilizers, namely polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), alginate, and sericin. The particle size measured from TEM images was 4.3, 6.1, 7.6, and 10.2 nm for AgNPs/PVP, AgNPs/PVA, AgNPs/alginate, and AgNPs/sericin, respectively. The influence of different stabilizers on the antibacterial activity of AgNPs was investigated. Results showed that AgNPs/alginate exhibited the highest antibacterial activity against  The common stabilizers used in bottom-up approach of metallic nanoparticle synthesis  are polymers and surfactants such as sodium dodecyl sulfate (SDS) and Tween 80 . Furtherbacillus species. The zeta potential of AgNPs/citrate was −38 mV, whereas the zeta potential of AgNPs/PVP and AgNPs/BPEI were −10 and +40 mV, respectively [The study results of Kvítek et al. showed that the antibacterial activity of silver nanoparticles (AgNPs) was significantly enhanced by using the suitable stabilizers such as the following surfactants: SDS, Tween 80, and PVP K90 . Moreoveectively . Therefoectively ,20,21.w/v). Subsequently, the antibacterial activity of these colloidal AgNP solutions was investigated. To further demonstrate the effect of AgNPs on antibacterial activity and apply the development in practice, the AgNPs were added into a handwash solution, and the antibacterial activity was also tested.In this study, we prepared four colloidal AgNP solutions at a concentration of 1-mM Ag in different stabilizers, namely PVP, PVA, alginate, and sericin with the same concentration of 0.5%  medium purchased form Himedia, Mumbai, India contains 10 g triptone, 5 g yeast extract, 10 g sodium chloride, and 1 L distilled water.Pure-grade AgNOw/v) stabilizers of PVP, PVA, alginate, and sericin were prepared by gamma Co-60 irradiation method as described in our previous papers [3 was then dissolved in the above prepared solution to obtain a final concentration of 1-mM Ag+. The mixture was poured into glass bottles with plastic caps. The irradiation of these solutions at dose of 6 kGy for the synthesis of AgNPs was carried out on a Co-60 irradiator with a dose rate of approximately 1.2 kGy/h at VINAGAMMA Center, Ho Chi Minh City. Absorption spectra of the irradiated AgNP solutions with dilution by water to 0.1-mM AgNPs were taken on an UV-vis spectrophotometer, Jasco V-630 . The AgNP sizes were measured using a transmission electron microscope .Four colloidal solution samples of 1-mM AgNPs stabilized in 0.5%  was added to each flask. The cultures were shaken at 150 rpm, and the bacterial growth curves were determined by measuring optical density (OD) at 600 nm on a UV-vis Jasco V-630 with 30-min interval [A 99-mL sterilized LB medium and AgNPs in different stabilizers as prepared for the final concentration of 1-mg/L AgNPs were added into conical flasks (250 mL), and the control sample just contained a 99-mL LB medium. A 1-ml interval ,22,23.E. coli was carried out by culture medium toxicity method [E. coli suspension of 107 CFU/mL was injected to each sample. The samples were shaken at 150 rpm at room temperature for 1, 3, and 5 min. After that, the number of bacteria in each mixture was quantified by spread plate technique on LB agar plates.A handwash solution was prepared using Na lauryl sulfate (Na-LS) as surfactant, hydroxyethyl cellulose (HEC) as binder, and 15 mg/L of AgNPs/alginate as antimicrobial agent. The bactericidal activity assay of the handwash against y method ,13 as foλmax) of 413 nm, TEM image with quasi-spherical particles, and narrow size distribution of AgNPs stabilized by alginate were typically described in Figure λmax at 410 to 420 nm . Therefore, antibacterial activity of the resulted AgNP solutions, namely AgNPs/PVA, AgNPs/PVP, AgNPs/sericin, and AgNPs/alginate was tested.The gamma Co-60 irradiation method is fairly suitable to create the smaller AgNPs compared to chemical reduction method . It is g7E. coli cells/100 mL and 1-mg/L AgNPs in different stabilizers. ODo and ODt  > AgNPs/PVA (6.1 nm) > AgNPs/PVP (4.3 nm) > AgNPs/sericin (10.2 nm). The obtained results also proved that the antibacterial effect of AgNPs depends not only on the size but also on the stabilizer used.Figure  Figure  are the In addition, Sondi and Salopek-Sondi  and TiwaE. coli; the η value reached approximately 6.2% only. The bactericidal efficiency with only 3-mg/L AgNPs diluted from the handwash solution against E. coli with a bioburden of approximately 107 CFU/100 ml  was 74.6%, 89.8%, and 99.0% for 1, 3, and 5 min of contacting time, respectively (Table Photographs of handwash solutions and bactericidal activity were showed in Figure E. coli, particularly the η value of 10-mg/L AgNPs which was approximately 99.9% for 2 min of contacting time [2S that has extremely low solubility and exhibits a much lower toxicity than other forms of silver [2S that is considered to have no significant impact to the environment [Wei et al. also reported the high bactericidal effect of AgNPs with sizes of 6 to 8 nm against ing time . Our obting time . Concernf silver ,28. Therironment .The colloidal AgNP solutions stabilized by PVA, PVP, sericin, and alginate were successfully synthesized by gamma Co-60 irradiation method. Results on antibacterial activity test demonstrated that AgNPs/alginate with an average size of 7.6 nm exhibited the highest antibacterial activity among the as-synthesized AgNP solutions. The as-prepared handwash with 15-mg/L AgNPs/alginate showed a high antibacterial efficiency with rather short contacting time. Thus, handwash/AgNPs can be potentially used as a daily sanitary handwash to prevent transmission of infectious diseases.The authors declare that they have no competing interests.NQH came up with the idea. DVP and LAQ designed and set up the experimental procedure. NND, LQL, and BDD planed the experiments and agreed to the publication of the paper. NTKL conducted the size measurement of the as-prepared silver nanoparticles by TEM. NND, LQL, and LAQ performed the UV-vis measurement of the AgNP solutions stabilized by different polymers and evaluated the antibacterial efficiency of AgNP solutions and handwash solution containing AgNPs. NQH, LQL, and DVP analyzed the data, drafted the manuscript, revised the manuscript critically, and made a few changes. All authors read and approved the final manuscript.


Zanthoxylum budrunga Wall enjoy a variety of uses in ethnobotanical practice in Bangladesh. In the present study, a number of phytochemical and pharmacological investigations were done on the ethanol extract of Z. budrunga seeds (ZBSE) to evaluate its antinociceptive and antioxidant potential. ZBSE was also subjected to HPLC analysis to detect the presence of some common antioxidants. In acetic acid induced writhing test in mice, ZBSE showed 65.28 and 74.30% inhibition of writhing at the doses of 250 and 500 mg/kg and the results were statistically significant (P < 0.001). In hot-plate test, ZBSE raised the pain threshold significantly (P < 0.001) throughout the entire observation period. In DPPH scavenging assay, the IC50 of ZBSE was observed at 82.60 μg/mL. The phenolic content was found to be 338.77 mg GAE/100 g of dried plant material. In reducing power assay, ZBSE showed a concentration dependent reducing ability. HPLC analysis indicated the presence of caffeic acid with a concentration of 75.45 mg/100 g ZBSE. Present investigation supported the use of Zanthoxylum budrunga seed in traditional medicine for pain management. Constituents including caffeic acid and other phenolics might have some role in the observed activity.Different parts of the medicinal plant  Zanthoxylum budrunga Wall (Syn: Zanthoxylum rhetsa Roxb.) (Rutaceae) is a deciduous medium-sized tree native to subtropical areas of the world and widely distributed in Bangladesh, India, Sri Lanka, Myanmar, China, Thailand, Malaysia, Indonesia, Philippines, and Papua New Guinea. It grows up to 35 m with sparsely armed branches having straight or ascending prickles. In Bangladesh, it is distributed in Sylhet, Chittagong Hill Tracts, Cox's Bazar, Gazipur, and Tangail and enjoys a number of uses in ethnobotanical practice. In traditional medicine, the fruit of the plant is used in the treatment of rheumatism, cholera, dysentery, asthma, infections, heart disease, bronchitis, and piles. The essential oil extracted from leaves is used in cholera. Juice of the bark is considered beneficial in vomiting, cough, dysentery, and headache [α-terpineol (8.2%), sabinene (8.1%), β-phellandrene (7.4%), and 2-undecanone (7.1%) [α-sanshool, β-phellandrene, β-pinene and pipertone, mullilam diol) and alkaloids  were isolated from the fruits [p-menthane, was reported from the roots, [Z. budrunga seeds.headache . The seeheadache . In a prheadache . Severale 7.1%) . Volatil.1% . Vole fruits . A monote roots,  while the roots, . The preZ. budrunga was collected from Tangail, Bangladesh, in September 2011 and identified by experts at Bangladesh National Herbarium, Dhaka, Bangladesh. A voucher specimen (DACB 37522) has been submitted there for future reference. The seeds were removed from the fruit rind and separated from other parts and extraneous materials.The seeds of Z. budrunga seed extract (ZBSE).The dried seeds were grinded into coarse powder (210 g) and macerated in ethanol  for three days with occasional shaking. It was filtered through a clear cotton plug to remove marc. Filtrate was evaporated to get gummy reddish crude extract. The seeds yielded 1.45% extract of dried plant material and designated as ad libitum. The investigations were carried out following the animal ethics guidelines provided by ICDDR,B.Randomly screened young Swiss-Albino mice aged 4-5 weeks, weighing 20–25 g, were selected for the pharmacological investigation. The mice were purchased from the Animal Resources Branch of International Centre for Diarrhoeal Disease and Research, Bangladesh , and fed with ICCDR,B formulated commercial rodent food and water p-coumaric acid, rutin hydrate, ellagic acid, quercetin, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich, USA. Folin-Ciocalteu's reagent, ascorbic acid, trichloroacetic acid, potassium ferricyanide, sodium carbonate, ferric chloride, acetic acid, and dimethyl sulfoxide (DMSO) were purchased from Merck, Germany. Tween-80 was purchased from Loba Chemie, India. Diclofenac sodium, morphine, and vincristine sulphate were obtained from Beximco Pharmaceuticals Ltd Bangladesh, Popular Pharmaceuticals Ltd Bangladesh, and Cipla Pharmaceuticals India, respectively.Gallic acid, (+)-catechin hydrate, vanillic acid, caffeic acid, (−)-epicatechin, ZBSE was subjected to a number of standard tests to detect the presence of major phytochemical groups , 13.In vitro antioxidant activity was determined based on the ability of the seed extract to scavenge the stable free radical DPPH [μg/mL, respectively. From each concentration, 1 mL was mixed with 2 mL DPPH solution (0.004% in ethanol). The mixture was kept for 30 minutes to complete the reaction. The absorbance was measured at 517 nm in a UV-visible spectrophotometer. Ascorbic acid was used as standard antioxidant. The percent inhibition was calculated according to the formula (1 − A1/A0) × 100, where A0 is the absorbance of control and A1 is the absorbance of ZBSE or standard. IC50 value was determined from the graph obtained by plotting % inhibition versus concentration.cal DPPH . Stock s2CO3 was added and adjusted with distilled water to make the final volume of 25 mL. It was kept for 30 min to complete any reaction that occurs. The absorbance was measured at 750 nm against blank. Blank was prepared by following the same steps mentioned above without adding ZBSE or gallic acid. Standard curve of gallic acid was prepared by plotting absorbance versus concentration. The total phenolic content of ZBSE was determined using the standard calibration curve of gallic acid and the value was expressed as gallic acid equivalent (GAE)/100 g of dried plant material.Grinded plant material (0.5 g) was mixed with 50 mL of 80% aqueous methanol and sonicated for 20 min. An aliquot of 2 mL was taken from it and centrifuged for 15 min at 14,000 rpm. Total phenolic content was determined by Folin-Ciocalteu's reagent [μg/mL were mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide with vigorous shaking. The mixtures were incubated for 20 min at 50°C. After cooling at room temperature, 2.5 mL of 10% trichloroacetic acid was added and centrifuged at 3000 rpm for 10 min. An aliquot (2.5 mL) of the supernatant was mixed with 2.5 mL of distilled water and 0.5 mL of ferric chloride (0.1%). After 5 min, the absorbance was measured at 700 nm. Ascorbic acid was used as standard.Reducing power of the extract was determined according to the method described by Oyaizu . ZBSE of18 (5 μm) Dionex column (4.6 × 250 mm) at 30°C with a flow rate of 1 mL/min and an injection volume of 20 μL. The mobile phase consisted of acetonitrile (solvent A), acetic acid solution pH 3.0 (solvent B), and methanol (solvent C) with the gradient elution program of 5%A/95%B (0–9 min), 10%A/80%B/10%C (10–19 min), and 20%A/60%B/20%C (20–30 min) with postrun equilibration of the system with 100%A (5 min). The UV detector was set to 280 nm for 18.0 min, changed to 320 nm for 6 min and finally to 380 nm, and held for the rest of the analysis period while the diode array detector was set at an acquisition range from 200 nm to 700 nm. For the preparation of calibration curve, a standard stock solution was prepared in methanol containing gallic acid, vanillic acid, (+)-catechin, (−)-epicatechin, p-coumaric acid, rutin, ellagic acid (20 μg/mL each), caffeic acid (8 μg/mL), and quercetin (6 μg/mL). A solution of ZBSE was prepared in methanol having a concentration of 5 mg/mL. Prior to HPLC analysis, all solutions  were filtered through 0.2 μm nylon syringe filter  and degassed in an ultrasonic bath  for 15 min. Data acquisition, peak integration, and calibrations were performed with Dionex Chromeleon software (Version 6.80 RS 10).Detection of selected phenolic compounds in ZBSE was determined by HPLC analysis as described by Chuanphongpanich and Phanichphant with some modifications . It was Antinociceptive activity was evaluated by acetic acid induced writhing model in mice . ControlIn hot-plate test, experimental mice were subjected to pain stimulus when placed on hot-plate maintained at the temperature of 55 ± 0.5°C . Mice, ht-test. Experimental results were considered statistically significant when P < 0.05.All the values were expressed as mean ± SEM. Statistical analysis was performed by Student's Phytochemical screening of ZBSE revealed the presence of reducing sugars, alkaloids, glycosides, flavonoids, and gums while saponins, tannins, and steroids were absent .50 value for the extract was 82.60 μg/mL while the value was 12.58 μg/mL for ascorbic acid, used as standard in this assay -catechin, (−)-epicatechin, caffeic acid,  Figures  and 3.In acetic acid induced writhing, ZBSE showed 65.28 and 74.30% inhibition of writhing at the doses of 250 and 500 mg/kg body weight, respectively, whereas diclofenac sodium (25 mg/kg) showed 81.95% inhibition .In hot-plate test, ZBSE increased the latency time to pain stimulus. The maximum latency time for the extract at the doses of 250 and 500 mg/kg body weight was 5.90 ± 0.09 and 6.61 ± 0.07 sec, respectively, whereas morphine (3 mg/kg) showed a maximum latency time of 9.41 ± 0.16 sec .Z. budrunga seed extract possesses moderate antioxidant activity. Phenolic compounds are one of the major groups of natural antioxidants that neutralize free radicals by redox reactions [Phytochemicals belonging to the class of alkaloids, glycosides, and flavonoids are often associated with various bioactivities including antioxidant and antinociceptive activities . Resultseactions . Phenolieactions . The exteactions . The exteactions .2) by the enzyme phospholipase A2. Synthesis of prostaglandins including PGI2 through cyclooxygenase pathway is one of the major causes of pain sensation [Acetic acid induced writhing is an easy but reliable method to evaluate peripherally acting antinociceptive activity . Acetic ensation . Common ensation . In addiensation . Caffeicensation . Howeverensation . Antioxiensation . Thus, tThe extract showed pain inhibitory activity in hot-plate test which represents centrally acting algesia by inducing thermal pain stimulus through changes in spinal cord level . NaturalZ. budrunga seeds in the management of pain and antioxidants present in the seeds might have some role in the observed activity.Present investigation supported the traditional use of


New thionaphthoquinones and their hydroxyl derivatives, bearing alkyl side chains that match the phospholipids POPC and POPE, were synthesized in order to investigate their interactions with lipids. It was observed that, in general, these additives destabilize the lipid bilayer and induce less organized structures with higher curvature, in particular the induction of an hexagonal phase on aqueous POPC mixtures. Moreover, cubic phases, not normally observed in the pure lipids when fully hydrated, were detected. Coexistence of lamellar phases was interpreted as a consequence of microsegregation of the components in the mixtures. These results are in line with previous observations on the effect of structurally similar (hydro)quinones in phase behavior of these lipids. Staphylococcus aureus [in vitro antibacterial agents than gentamycin against Staphylococcus aureus and markedly in vitro antifungal against Cryptococcus neoformans, Sporothrix schenckii, and Trichophyton mentagrophytes, when compared with fluconazole [Mycobacterium tuberculosis [ Quinones are structures present in many naturally occurring compounds . 1,4-Naps aureus , better conazole . They arrculosis . Furtherrculosis  and alsorculosis  also learculosis .  Here, we describe some newly prepared thionaphthoquinones, to be used as additives for interacting with lipids normally present in cell membranes, and their influence on the structural behavior of the lipid matrix.16H33 chains, similar to those present in the lipids used. The lipids were selected for having different polarities  in water solution. Attached to the quinonoid ring of the newly prepared thionaphthoquinones, there is one  and POPE  were acquired from Avanti Polar Lipids .Generally, all chemicals, commercially available, were of pure grade and used without further purification. Lipids POPC 3 (5 mmol in 5 mL of water) was added. The solid was filtered and recrystallized from hexane/EtOH (20 : 1) yielding 76% of yellow crystals (4.8 mmol); m.p. 103–105°C; 1H-NMR : δ = 0.88 , 1.18–1.81 , 2.83 , 6.61 , 7.29 , and 8.10 ; I.R. (KBr): ν = 1650, 1669 cm−1; elemental analysis (%) calculated for C26H38O2S: C 75.3; H 9.3; found C 75.2; H 9.6.To a stirred solution of 1,4-naphthoquinone (6.3 mmol) in EtOH (50 mL), hexadecanethiol (12.0 mmol) was added. After stirring for 1 h at r.t., a solution of Fe(NO3N (3.0 mmol). The mixture turned red and, after stirring for 3 h at r.t., the solvent was removed under vacuum. A red solid was obtained in 28% yield (0.34 mmol), after crystallization from hexane; m.p. 83–85°C; 1H-NMR : δ = 1.15–1.72 , 3.29 , 7.69 , and 8.05 ; I.R. (KBr): ν = 1657 cm−1; elemental analysis (%) calculated for C42H70O2S2: C 75.1; H 10.5; found C 74.9; H 10.7. To a stirred solution of ANQ (1.2 mmol) in EtOH (2 mL), hexadecanethiol (1.5 mmol) was added, followed by Et2Cl2. The extract was dried (MgSO4) and concentrated under vacuum, yielding a white solid  that becomes yellow when exposed to air. 1H-NMR : δ = 0.84–1.76 , 2.73 , 6.86 , 7.54 , 8.09 , and 8.20 . ANQ (0.30 mmol) was dissolved in AcOH  and treated with Zn (dust) until colour fading. Water (10 mL) was added and the mixture was extracted with CH2Cl2 , and, under vigorous stirring, a saturated aqueous solution of Na2S2O4 was slowly added until color fading. CH2Cl2 (10 mL) was added and the organic layer was separated and dried (MgSO4). After concentration under vacuum, a white solid , that becomes red when exposed to air, was obtained. 1H-NMR : δ = 0.75–2.10 , 2.82 , 7.57 , and 8.21 . BANQ (0.12 mmol) was dissolved in a mixture of acetone/CH2Cl2) of additive and lipid and producing an homogeneous mixture, prior to removing the solvent under vacuum. After hydration, samples were left at least 5 minutes at each set temperature prior to exposure to the synchrotron radiation. X-rays scattering (SAXS) measurements were conducted at beamline SAXS of LNLS, Campinas, Brazil, equipped with a MAR 165 CCD detector, and using a thermostated sample holder. The original 2D small-angle diffraction data were linearized using Fit2D software [d, of each structure by the relationship Q = 2πs = 2π/d, where Q is associated with the scattering angle. The results were grouped according to lipid/additive interaction at variable conditions: molar ratio (mr), pH, and temperature . Samples were prepared by mixing solutions , assigned as lamellar, Lα, and induced gel phases, respectively, together with the onset peak of the hexagonal phase at Q = 1.01 nm−1  (d = 6.22 nm). Heating (45 < T ≤ 70) leads to a well-defined hexagonal phase (d ~ 6.3 nm) accompanied by several low-intensity broad peaks, at Q < 0.9 nm−1 values. For these broad peaks, a cubic phase can be proposed. For mr = 100, a less well-defined lamellar (d = 5.94 nm) and hexagonal (d = 6.91 nm) structures, that vanish upon heating, are observed  together with weak peaks related to a hexagonal phase (d = 6.99 nm) is observed. The system evolves to more complex structures upon heating that causes a decrease in both lattices and promotes the formation of a cubic phase with onset at T = 60, compatible with Pn3m (Q224). For T > 70, the hexagonal phase is no longer seen and only a minor peak of the lamellar phase remains. For mr = 50(T = 30), only a lamellar phase can be seen with Q = 1.18 nm−1  (d = 5.33 nm). Slow melting of this phase is completed at T = 65, while a hexagonal phase, with onset for 40 < T < 50, shows well-defined and intense peaks up to T = 70. The lattice parameter of the hexagonal structure decreases upon heating, going from Q = 0.98 nm−1  (d = 6.41 nm) at T = 50 to Q = 1.02 nm−1  (d = 6.16 nm) at T = 70. Interestingly, together with the hexagonal phase, one also sees several weak and broad peaks at Q ≤ 1 that are compatible with a cubic phase, however, of the Pm3n group (Q223) [T = 30), probably due to the higher hydration of the quinone carbonyl groups. Moreover, the head-group interactions can be partially screened, hindering the formation of the hexagonal phase.In these cases, complex diffraction patterns highlight an out-of-equilibrium system. These mixtures proved to be the most sensitive to variations in mr, pH, and p (Q223) . Also toT < 12 upon cooling from the gel phase, with a lattice size of 6.08 nm. Prior to the splitting, their WAXS patterns showed a peak corresponding to 0.417 nm, characteristic for a hexagonal chain arrangement. Although the phase transition is of similar shape, the dimensions observed in the SAXS are at least one order of magnitude larger than on WAXS. Therefore, the Y shape must have different origins for each system. In our mixture, at T = 30, above the Lβ > Lα phase transition of pure POPE, two sets of peaks are observed. They are compatible with microsegregation into lamellar phases, one poor and one rich in ANQ. The poor one (d = 5.92 nm) resembles the Lα phase of pure hydrated POPE (T = 25) but with enhanced thermal sensitivity, considering that ANQ has a significant hydration shell but not with tightly bound water molecules. Moreover, the separation between ANQ molecules should be significant and, therefore, the interaction weak, making the overall structure prone to non-linear expansion upon T increase. This indicates a liquid-like behavior in the core of each bilayer that forms the lamellar structure. The richer one in ANQ (d = 5.76 nm), that is more insensitive to thermal variations, can be compared to the gel structure of pure hydrated POPE at T < 25, with tight interactions between lipid and naphthoquinone molecules, and contracts linearly upon heating , decreasing to d = 5.24 nm, at T = 50. For mr = 50  is identified. At pH = 9, the mixture shows very similar patterns but with an expanded lamellar structure (d = 7.55 mm). It becomes evident that the second-SC16H33 chain in BANQ alters the interactions between the POPC layers, leading to the formation of structures with limited long-range order in their lattices. In order to evaluate the influence of increasing hydrophobicity of the additive, we performed measurements with BANQ . For POP mr = 50 , low-defT = 30(d = 5.55 nm), and a hexagonal phase at T > 60(d = 6.44 nm) can be seen. For T > 60, weak and broad peaks at smaller Q values are associated with a cubic structure, probably P4332 (Q212), which has been observed in a previous study using additives of similar structure [T = 25 (d = 5.24 nm) is seen together with traces of a hexagonal phase (d = 7.00 nm). Heating causes the lamellar phase to melt, and the hexagonal phase prevails for >40(d = 5.96 nm). Very interesting is the observation that the cubic phase is suppressed and only the extinction of the lamellar structure for T > 40 is observed. At pH = 9, the Y shape sequence of patterns mentioned before  is again observed. Heating (25 < T < 60) causes the initially single broad diffraction peak, observed at d = 5.91 nm, to split into two; one of them moves slightly towards larger Q values while the second one decreases its position linearly . For T < 35, second-order peaks are also observed, allowing for unequivocal assignment to lamellar phases. The smaller structure is almost identical to the one observed using ANQ as additive to the lipid matrix , a clear lamellar phase at y P4332 Q2, which  [T = 25 , a well-inlet in , thus su For the equivalent reduced forms of naphthoquinones ANQ and BANQ, mixtures of ANHQ and BANHQ, with the same lipids, lead to results following a common trend, that is, destabilizing the lipid bilayer, mostly by inducing curvature. These additives have not necessarily the same chemical stability of the naphthoquinones equivalents, and we cannot exclude the possibility of a small degree of oxidation, due to the combination of factors such as pH, variation of temperature, and exposure to strong X-rays radiation. The overall characteristic of this group of samples is the low definition of the scattering patterns, showing very broad and weak signals that cannot be assigned to a unique structure.Q = 1.58 nm−1, d = 3.98 nm) related to a cluster of insoluble ANHQ. A mixture of mr = 50, at pH = 4, provides the temperature scan seen in  Increasing mr to 100, at pH = 4, leads to similar scattering patterns, with even lower resolution, precluding structural identification, with no indication of insoluble ANHQ in the sample.d = 7.48 nm at T = 25. The signal associated with this phase shows also a broad profile extending to lower Q values. For T > 40, the intensity of the peaks decreases significantly leading to profiles of no conclusive structure, indicating a “melting” of the structures, reflecting a weaker interaction between the components of the mixture. A very weak signal associated with insoluble ANHQ is observed at T < 40. Changing to pH = 9, the mixture of mr = 50 shows a thermal scattering profile similar to the ones observed at pH = 4, but with lower resolution. A large lamellar structure can be identified at low temperature T = 25, two phases consisting of a well-defined lamellar (d = 5.32 nm) containing traces of hexagonal . The lamellae in this system have a smaller lattice than the POPE/ANQ mixture at the same conditions (d = 5.93 nm), while in the hexagonal phase  are equivalent. No signals below Q = 0.9 nm−1 are observed. The mixture POPE/ANHQ at pH = 4 and mr = 50 shows, at exagonal . Above TQ = 1.15 nm−1, d = 5.46 nm at T = 25) and above T = 50 a hexagonal structure of similar dimensions as for mr = 50 . Heating causes shifting of the peaks from the lamellar structure towards larger Q values, indicating contraction of this phase while the peak, attributed to insoluble ANHQ , remains at a constant position until it vanishes above T = 60, coinciding with the onset of the hexagonal phase  shows patterns containing only two small and broad signals that shift to larger the extra aromatic ring present on the quinone ring of ANQ, ANHQ, BANQ, and BANHQ;meta for BATH and ortho in BANQ;the relative position of the second hydrophobic chain; using a classical nomenclature: the higher oxidation degree of quinones ANQ and BANQ employed in this work as compared to hydroquinones 2,5-ATH and 2,6-BATH.Previous results obtained with similar mixtures of the same lipids with both 2-hexadecylthio-3,6-dimethylbenzene-1,4,diol    and  2,6-bis   show smaT = 71 [Lα to hexagonal phase in POPE-based mixtures, containing any of these additives, takes place at significantly lower temperatures. Finally, it is noteworthy to mention that the peaks on SAXS patterns show low resolution, indicating that the additives studied here generally promote structures with much shorter long-range order. The overall observation about the influence of all these additives over the lipid matrices is that they do modify them in a similar way, that is, promoting curvature and therefore destabilizing the lipid bilayer. These effects can be inferred from the observation that our mixtures form structures that contain a high degree of curvature in their mesogenic units, for example, hexagonal and cubic phases. It should be noted that fully hydrated POPC does not show such phases over the temperature range studied. Moreover, fully hydrated POPE shows a hexagonal phase above T = 71 . The pha Sulfurated naphthoquinones ANQ or BANQ alter the phospholipid bilayer significantly, due to strong interactions between both entities depending, more or less on the effective polarity of the lipid head group. In acidic conditions (pH = 4), POPC forms, at low temperature, a structure compatible with a gel phase that, upon heating, induces curvature as seen by the formation of hexagonal and cubic phases. POPE-based mixtures form different cubic structures. In basic medium (pH = 9) and lower mr, microsegregation and tendency towards lamellae supersede the promotion of curvature in POPE. In this case, the interactions at the region of the head-groups give place to others in the hydrophobic core that become strong enough to determine the structural behavior. The scattering patterns showing complex mixtures of phases, cubic in most cases, present limited resolution disabling us to assign a set of diffraction peaks to a unique structure. Therefore, we conclude that the overall balance of interactions between the components of these mixtures is not strong, leading to structures containing only limited long-range order.