Trichophyton mentagrophytes-infected guinea pig onychomycosis model, decreasing the mean viable fungal cell count by 54.3% compared to the vehicle-treated group after once-daily treatment for 4 weeks. Thus, the accelerated skin and nail penetration effect of EFN-K is expected to achieve good patient compliance, and improve the complete cure rate of onychomycosis.Onychomycosis is a progressive fungal infection of the nails that involves the deeper nail layer and nail bed. It is important to maintain sufficient drug concentration in the diseased tissues after topical application. In this study, a stable topical delivery system for efinaconazole (EFN) was designed to enhance absorption potential through the skin and nail plate by incorporating ethanol, diethylene glycol monoethyl ether (Transcutol P) and isopropyl myristate, and cyclomethicone into the topical solution as a delivery vehicle, permeation enhancers, and a wetting agent, respectively. In addition, the stability of EFN in the formulation was significantly improved by adding butylated hydroxytoluene, diethylenetriamine pentaacetic acid, and citric acid as an antioxidant, chelating agent, and pH-adjusting agent, respectively, without discoloration. The optimum EFN formulation (EFN-K) showed 1.46-fold greater human skin permeation than that of the reference control . Furthermore, after a 24-hour incubation, the amount of infiltrated EFN from EFN-K in the human nail plate was 4.11-fold greater than that of the reference control, resulting in an 89.7% increase in nail flux at 7 days after treatment. EFN-K significantly accelerated structural recovery of the keratin layer in a  Finally, the in vivo antifungal efficacy of the selected EFN topical formulation was assessed in a T. mentagrophytes-infected guinea pig onychomycosis model.The main objective of the present study was to design a topical delivery system for EFN with highly penetrability, rapid absorption, and high stability during storage. To enhance the skin and nail permeability of EFN, Transcutol P and isopropyl myristate were used as penetration enhancers, along with cyclomethicone as a wetting agent. In addition, ethanol was used as a volatile solvent to enhance the solubility of EFN. Fast infiltration and permeation of EFN through human skin and nail plate were expected due to the enhanced solubility, effects of permeation enhancers, and lower binding of the drug to keratin. In addition, the stability of EFN topical solution during storage was improved by incorporation of an antioxidant, chelating agent, and pH-adjusting agent. After assessing the artificial membrane permeability of EFN using different topical solutions, the stability at 65 °C and 2.2.1.®  was obtained from Valeant Pharmaceuticals International Inc. . Fulcare®  was purchased from A. Menarini Industrie Farmaceutiche Riunite S.r.l. . Sabouraud’s dextrose agar (SDA) medium and yeast malt (YM) agar medium were obtained from BD Biosciences . Solvents for high-performance liquid chromatography (HPLC) were obtained from Merck Millipore  and Thermo Fisher Scientific Inc.EFN was obtained from Cipla Limited . Transcutol P was obtained from Gattefosse . Quercetin , isopropyl myristate, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), ethylenediaminetetraacetic acid (EDTA), bovine serum albumin (BSA), polyoxyethylene (20) oleyl ether (Brij O20), sodium azide, sorbic acid, citric acid, cycloheximide, and chloramphenicol were obtained from Sigma-Aldrich Inc. . Diethylenetriaminepentaacetic acid (DTPA) was purchased from Thermo Fisher Scientific . Jublia2.2.Hartley strain guinea pigs  were purchased from Saeronbio . Ethical approval for this study was obtained from the Institutional Animal Care and Use Committee (IACUC) of Chung-Ang University . All animal experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and the IACUC guidelines.2.3.The EFN-loaded topical formulations  developed in this study are listed in in vitro transdermal membrane permeability through a synthetic membrane  was evaluated using a Franz diffusion cell system . Each Strat-M® disc (25 mm in diameter) was mounted on the receiver compartment with a diffusion area of 0.785 cm2. After assembling the donor compartment, 5 mL of phosphate-buffered saline  containing 1.5% Brij O20 and 0.1% sodium azide was added to the receiver compartment, and the cells were allowed to equilibrate for at least 60 minutes to confirm membrane hydration. After loading, 127 µL/cm2 of each 10% EFN topical solution was applied to the donor compartment. The receiver compartment was stirred at 600 rpm in a heating system to maintain a membrane surface temperature of 32 °C throughout the experiment. Then, 300 μL of the receptor phase was withdrawn after 12 and 24 hours, and each aliquot was replaced with the same volume of fresh PBS (pH 7.4) containing 1.5% Brij O20 and 0.1% sodium azide. The collected samples were then filtered through a membrane filter  and the concentration of EFN that permeated through the artificial membrane was determined by HPLC with a C18 column  at 30 °C. EFN was measured using an ultraviolet (UV) detector at 205 nm by the isocratic elution method with acetonitrile:water  as the mobile phase at a flow rate of 1 mL/min.Next, to compare the skin permeability of the EFN topical solutions, 2.4.The results of the artificial membrane permeability tests informed the compositions of the permeation-enhancing agents used in the EFN topical solution; furthermore, the antioxidant and chelating agents included in the selected formulations (EFN-C and EFN-G) were modified to improve drug stability. After selecting the EFN topical formulations, EFN-J and EFN-K were stored at 65 °C ± 2 °C for an accelerated stability test. Physicochemical properties such as phase separation and precipitation, as well as physical appearance and drug contents, were analyzed weekly. In addition, discoloration of the EFN topical solutions was observed by measuring the absorbance values at 400, 500, and 600 nm; the acceptable absorbance values for drug stability were considered to be less than 0.4, 0.1, and 0.1 AU at 400, 500, and 600 nm, respectively.2.5.T. rubrum (ATCC® MYA-4438), obtained from the American Type Culture Collection , was cultured in SDA medium containing chloramphenicol (20 µg/mL) and cycloheximide (200 µg/mL) at 25 °C for 5 days. After incubation, the concentration of spores in the suspension was adjusted with 0.1 M PBS to 1 × 108 CFU/mL using a hemocytometer. Next, a 1-cm-dimaeter hole was punched in sterilized filter paper, which was then laid on the center of each SDA plate followed by uniform spreading of 100 µL of the prepared spore suspension on the plate. Each disk was then impregnated with 5 µL of sample solution. Control plates were prepared by incubating a disk soaked with sterilized water and spore suspension. After incubating at 25 °C for 14 days, the zone of inhibition (ZOI) was calculated by measuring the mean diameter of the area of growth inhibition around the disk.The antifungal activities of EFN-J and EFN-K were evaluated by disk diffusion assay and compared to reference control #1 . Briefly, 2.6.in vitro human skin permeability study was performed using the Franz diffusion cell system (Labfine). Excised full-thickness human skin  was mounted with the stratum corneum (SC) facing upward on the acceptor compartment of a Franz diffusion cell system, to which 5 mL of PBS (pH 7.4) containing 1.5% Brij O20 and 0.1% sodium azide was then added. Next, the donor compartment was clamped in place  and the cells were allowed to equilibrate for at least 60 minutes to confirm the integrity of the skin. After reaching equilibrium, 127 µL/cm2 of each 10% EFN topical solution was applied to the donor compartment. The acceptor compartment was stirred at 600 rpm and heated to maintain a skin surface temperature of 32 °C throughout the experiment. Then, aliquots of 300 µL of sample solution were collected at predetermined time intervals  from each diffusion cell and replaced with the same volume of fresh PBS (pH 7.4) containing 1.5% Brij O20 and 0.1% sodium azide. The collected samples were then filtered through a membrane filter , and the amount of EFN that permeated through the full-thickness human skin was determined by HPLC at 205 nm by the isocratic elution method, as described above.To compare the skin permeability of the selected EFN topical solution with the reference control solution, an 2.7.in vitro nail permeation and deposition were assessed using a Franz diffusion cell system. Human fingernails from different adult human cadavers were obtained from Science Care, Inc.  and kept in a closed container at −70 °C until use. One day before the experiment, nail plates were removed from a deep freeze (−70 °C) and kept at room temperature overnight. On the day of the experiment, nail plates were hydrated in normal saline for 3 hours at room temperature. The hydrated nails were firmly fixed between two Teflon gaskets of the nail holder in the donor compartment, which was then mounted and clamped onto the receptor compartment filled with PBS (pH 7.4) containing 4% (w/v) BSA and 0.01% (w/v) sodium azide . Next, the donor compartment was clamped in place; the cells were allowed to equilibrate for at least 60 minutes and were stirred at 600 rpm in an incubator at 32 °C. For the permeation study, 1.77 µL/mm2 of 10% EFN solution was applied to the human nail plate in the donor compartment once daily for 7 days, and the nail plate was washed with distilled water between doses. On day 1 after loading, 300 µL aliquots of the samples from each receptor compartment were withdrawn at different time points  and replaced with the same volume of fresh PBS (pH 7.4) containing 4% (w/v) BSA and 0.01% (w/v) sodium azide. From day 2, the receptor phase solution was withdrawn every 24 hours and replaced with the same volume of fresh PBS (pH 7.4) containing 4% (w/v) BSA and 0.01% (w/v) sodium azide. The withdrawn samples were stored at −70 °C.To evaluate the infiltration of EFN topical solution across human nail, the + = 349.3) and ([M + H]+ = 301.0) for EFN and quercetin, respectively.Furthermore, the nail-deposited EFN formulations were monitored for 24 hours after dosing. After 24-hour exposure to 50 µL of 10% EFN topical solution, the diffusion cells were disassembled and the nails were removed from the chamber, followed by washing twice with 5 mL of deionized water. Then, the nail area exposed to the formulation was cut and dissolved in 1 N sodium hydroxide:methanol  solution. The nail samples were then incubated at 60 °C for 4 hours, followed by the addition of 1 mL of methanol and 1 mL of ethanol with continuous vortex mixing. The mixture was centrifuged at 4,000 rpm for 10 minutes, and the clear supernatant was then withdrawn and evaporated using a centrifugal evaporator . Next, the dried residue was resuspended with 100 µL of a 1:1 (v/v) mixture of ethanol and mobile phase . The concentrations of EFN in the nail, as well as the receptor phase solution, were determined using an Agilent 6120 quadruple liquid chromatography/mass spectrometry (LC/MS) system with a Phenomenex Luna C18 column , and the samples were chromatographed using isocratic mobile phase at a flow rate of 1 mL/min. Aliquots of 20 µL were injected and EFN and quercetin were measured in positive and negative ionization modes, respectively, using an atmospheric pressure ionization-electron spray (API-ES) source. The following parameters were optimized for EFN analysis: capillary voltage, 3.0 kV; drying gas flow rate, 12.0 L/min; and drying gas temperature, 304 °C. The fragment ions at this capillary voltage were detected and quantified at ([M + H]2.8.T. mentagrophytes (ATCC 18748). After incubation of T. mentagrophytes at 25 °C for 7 days in YM agar media, a spore suspension was prepared at a concentration of 1 × 108 CFU/mL in YM broth. Then, the hind paw nails of guinea pigs were covered with sterilized gauze wetted with 100 μL of the T. mentagrophytes spore suspension, three times a week for 4 weeks. To increase the infection efficiency, the animals’ legs were sealed with latex socks for 4 weeks. The guinea pigs were randomly divided into five treatment groups (six guinea pigs per group). A daily 1 μL/mm2 topical treatment of vehicle control , reference control #1 , reference control #2 , EFN-J (10% EFN) or EFN-K (10% EFN) was applied for 4 weeks using a nail brush on all hind paw nails of each animal (six hind paw nails per guinea pig). To promote evaporation of the test solutions, the hind paws were held stable for 1 minute after application. Non-infected guinea pigs were treated as negative controls to allow comparison with normal paw nails. After infection followed by 4 weeks of treatment, to directly monitor and assess the antifungal effect, the nails from the guinea pigs were collected, homogenized in PBS (pH 7.4) containing 0.25% trypsin and 10 mmoL/L FeCl3, and digested at 37 °C for about 1 hour. After diluting with 0.1 M PBS, the nail samples were inoculated into YM agar medium containing 200 μg/mL cycloheximide and 20 μg/mL chloramphenicol, and cultured at 25 °C. After 14 days of incubation, viable fungal colonies were used as an index of the nail penetration and therapeutic efficacy of EFN-K for treatment of onychomycosis. Furthermore, the collected nails were stained with periodic acid-Schiff (PAS) for histological examination of fungal infection. Briefly, the nails were biopsied and fixed with 10% neutral buffered formalin for 1 day, and embedded in paraffin. Paraffin-embedded nail blocks were then sliced to a thickness of 3 μm, stained with PAS, and imaged under light microscopy. The remaining nails from each guinea pig were homogenized, diluted with 0.1 M PBS and inoculated into YM agar medium with 200 μg/mL of cycloheximide and 20 μg/mL of chloramphenicol. After 14 days of incubation at 25 °C, the living fungal colonies were counted to confirm the nail-penetrating and therapeutic effects of EFN-J and EFN-K for the treatment of onychomycosis.To evaluate the antifungal efficacy of EFN-J and EFN-K, onychomycosis was induced in guinea pigs by infecting their paw nails with 2.9.t-test for comparisons between two mean values, and one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test for comparisons between more than two mean values. All data are expressed as means ± standard deviation. In all analyses, p < .05 was taken to indicate statistical significance.Unpaired data were evaluated using Student’s 3.3.1.in vitro permeability of each formulation across an artificial transdermal membrane, to optimize each component.Based on the solubility of EFN and the known skin permeation-enhancing activity of ethanol, ethanol was selected as the drug delivery vehicle  and the reference control #1 through the artificial transdermal membrane were evaluated at 12 and 24 hours after loading the samples , or increased it by 3% (EFN-E) or 8% (EFN-F), with respect to EFN-A. Although the cumulative infiltrated concentration of each formulation (EFN-D − EFN-F) was higher than that of EFN-A, no significant differences in permeability were observed among them. In addition, due to the various advantages of Transcutol P, such as its solubilizing activity, permeation enhancement, colorless and nearly odorless nature, non-volatility, non-toxicity, biocompatibility with skin, and miscibility with polar and non-polar solvents, it has been used in topically applied FDA-approved products at proportions up to 49.9% .Based on the results of studies on artificial transdermal membrane permeability, the optimum EFN topical solutions were EFN-C and EFN-G; accordingly, these were selected for further study. However, EFN-C and EFN-G showed discoloration after storage for 1 month at 65 °C. Therefore, we replaced the antioxidant, chelating agent, and pH-adjusting agent in EFN-C with BHT, DTPA, and citric acid, respectively. As a result, the infiltration of EFN from EFN-J or EFN-K through an artificial transdermal membrane was significantly improved compared with reference control #1 . EFN was3.3.in vitro antifungal activities against T. rubrum of EFN-J and EFN-K were compared to those of the reference control #1 using the disk diffusion assay test method  are required to determine the clinical relevance of the skin permeability of EFN topical solution at a finite dose.The improvements in skin infiltration of EFN-J and EFN-K through human skin compared to the reference control #1 are shown in 3.5.2, which was 3.87-fold higher than that of reference control #1 (9.23 ± 5.68 ng/h/mm2)  . EFN-J eg/h/mm2) . EFN-J, After 7 days of application of EFN-J, EFN had significantly permeated through the nail plate, in an amount 1.22-fold greater than that of reference control #1; this resulted in a 1.53-fold increase in the nail flux of EFN-J, suggesting that it takes 24 hours to form a concentration gradient across channels within the nail plate filled with the second formulation . On day Based on the overall results, it appears that markedly enhanced skin and nail penetration of EFN-K are induced by low surface tension after topical application, as well as maintenance of sufficient solubility of EFN in the secondary formulation after evaporation of ethanol; this allowed EFN to penetrate through the nail plate and into the nail bed, by spreading under the nail plate and filling the air gap at the site of infection, and supplying a sufficient concentration of drug to the diseased tissues after topical application.3.6.T. mentagrophytes, and before drug treatment, the mean viable fungal cell counts in the vehicle control, reference control #1, reference control #2, EFN-J, and EFN-K groups were 4.74 ± 0.173, 4.53 ± 0.155, 4.70 ± 0.137, 4.79 ± 0.132, and 4.67 ± 0.129 log CFU/nail, respectively. After 4 weeks of treatment, the number of colonies in the infected nails treated with vehicle solution was not significantly reduced. In contrast, the number of viable fungal colonies of reference control #1 was decreased by approximately 50.8% compared to the initial value, which resulted in a 6.33% decrease in viable fungal colonies compared to the reference control #2 group. Furthermore, EFN-J and EFN-K also markedly inhibited the growth of T. mentagrophytes present in the keratin layer, resulting in decreases of 54.5% and 53.2% in the mean viable fungal cell counts compared to their initial values, respectively. At 4 weeks after treatment, the antifungal activities of EFN-J and EFN-K were still superior to the vehicle-treated and reference control #2 groups, and the reductions in number of living fungal colonies were 12.7% and 10.5% greater than reference control #2, respectively.Next, we investigated the curative effects of the antifungal activity of reference controls, EFN-J, and EFN-K on onychomycosis in guinea pigs. As shown in Regarding the histological evaluation of infected paw nails from guinea pigs treated with vehicle, reference controls, EFN-J, and EFN-K, PAS staining results showed similar trends . The negT. mentagrophytes-infected nails than reference control #2 (8% ciclopirox nail lacquer), which may have been due to the more potent antifungal activity of EFN, with a narrow range of minimum inhibitory concentration (MIC) values against T. rubrum (0.001 − 0.015 μg/mL) and T. mentagrophytes (0.001 − 0.03 μg/mL) compared to those of ciclopirox (0.03 − 0.5 μg/mL for T. rubrum and 0.03 − 0.5 for T. mentagrophytes), as well as rapid and enhanced drug absorption from the topical solutions compared to that from nail lacquers  increase in the penetration of EFN across full-thickness human skin compared to reference control #1  after a 24-hour incubation. Furthermore, after the 24-hour incubation EFN-K showed 1.46- and 1.33-fold greater human nail permeation and infiltration into the nail plate than the reference control #1, respectively, which in turn significantly increased the nail flux on day 7 (by 89.7%) compared to the reference control #1. As a result, EFN-K markedly decreased the mean viable fungal cell count (by 54.3%) compared to the vehicle-treated group after once-daily treatment for 4 weeks in the T. mentagrophytes-infected guinea pig onychomycosis model. These observations suggest that highly skin- and nail-permeable EFN-K can improve patient compliance and increase the cure rate of onychomycosis.In this study, 10% EFN topical solutions were prepared to improve the transungual permeability and antifungal efficacy of EFN through the introduction of Transcutol P and isopropyl myristate as permeation enhancers, and cyclomethicone as a wetting agent. In addition, the optimum EFN-K, which was stabilized by adding BHT, DTPA, and citric acid as antioxidant, chelating, and pH-adjusting agents, respectively, exhibited 1.18-fold higher


V = 160 kWh·m−3. The direct and technological effects, i.e., after UD and anaerobic digestion (AD) were investigated, respectively. Statistical analysis showed that characteristics and parameters of the WAS, which affects the magnitude of the direct effects create the following sequence: TS , VS (volatile solids), ΔT (temperature increase) > EPS (extracellular polymeric substances) > SCOD  > CST (capillary suction time) > NTOT , PTOT  > pH. Whereas, in the case of technological effects, the above sequence was as follows: TS, VS > CST > NTOT, PTOT > pH. Ultrasonic disintegration of WAS prior to AD increased total biogas production (from 13.0% to 19.7%) and reduced the content of TS (from 4.1% to 8.2%) and VS (5.8% to 9.5%) in comparison to the control sample. This confirms the usefulness of ultrasonic disintegration as an effective method of sludge digestion intensification. The obtained results showed that changes in the characteristics of WAS have a significant impact on the magnitude of the effects of ultrasonic disintegration, especially TS, VS, ΔT, EPS, SCOD and CST. Concluding, it can be inferred that the most promising conditions for ultrasonic pretreatment conducted under constant energy supply per sludge volume, are: low power, long sonication time, large surface area of the emitter, and high increase of sludge temperature while conducting the process.A comprehensive analysis of the effects obtained in the process of ultrasonic disintegration (UD) of waste activated sludge (WAS), was conducted. Sludge samples were collected periodically from Central Wastewater Treatment Plant (WWTP) in Gliwice (Poland) and disintegrated in the two ultrasonic devices of different construction and technical parameters, i.e., WK-2010 (A) and ultrasonic washer (B). The experiments were performed under a constant energy supply per sludge volume E TS, in which 950,000 MgTS would be in Poland alone [The development of new technologies and growing effectiveness of biological wastewater treatment observed in recent years, can be mainly accounted to the implementation of the European Council Directive  concernind alone . Moreovend alone ,7. AmongCOD) [V) or total solids content (ES), ultrasonic density (UD) and intensity (UI), sonication time (t) and process temperature (T) [There are different types of disintegration techniques, i.e., mechanical , thermal (hydrolysis—low and high temperature), chemical  and biological (with application of enzymes) ,9. CurreCOD) , etc. ThCOD) ,15, concCOD) ,16, biogCOD) ,18 and eCOD)  in sludgCOD) ,20 or miCOD) . In the COD) ,13. The ture (T) ,23.Therefore, taking into account the number of factors, which may have an impact on a magnitude of disintegration effects, a comprehensive analysis and selection of the most important sludge characteristics (parameters) and technical conditions of the process conducting are necessary. This is particularly important due to the fact that it is still unknown why in the similar conditions of sludge sonication, different effects are obtained. However, it is important to emphasize that there are no two identical sludges—their characteristics change over time. Therefore, it is important to carry out the ultrasonic disintegration in a cyclical manner, i.e., at an appropriate time intervals, which will allow observing changes in sludge characteristics and indicate the main parameters, that have the greatest impact on the obtained effects.The aims of this study were: (a) to evaluate direct and technological effects based on the values of selected indicators and optical microscopy examination; (b) to determine sludge characteristics (parameters) and technical conditions of the process conducting, having the greatest impact on the direct and technological effects; (c) to conduct a comprehensive analysis of the disintegration effects, with including periodically changes of WAS characteristics and technical conditions of conducting the process, using selected statistical tests.The WAS samples were collected once a month over a 7-months period of time, starting from July 2013 up to January 2014. Sludge samples after mechanical thickening were collected from the advanced biological Central Wastewater Treatment Plant in Gliwice . The digested sludge (inoculum) was taken from a full scale anaerobic digester  in the same WWTP and used only in order to conduct anaerobic digestion. The collected sludge samples were stored in polypropylene tubes at 4 °C before further analysis. The operational parameters of the WWTP are reported in TOT , PTOT , proteins and carbohydrates concentrations, CST, flocs disruption , TS and VS content, as well as volume and composition of the evolved biogas. Prior and after UD or AD, sludge samples were centrifuged for 30 min at 20,000 rpm (18 °C) and subjected to vacuum filtration throughout membrane filters  [TOT and PTOT concentrations. In order to determine the concentration of extracellular polymeric substances the thermally extraction was conducted. At the beginning, WAS sample was centrifuged at 2000× g for 20 min. The residual sludge pellet was resuspended in the distilled water right to the original volume and placed into the water bath (at 80 °C) for 1 h. After incubation sludge was separated from extract by centrifugation at 2000× g and 4500× g over 20 min each time, respectively [4, CO2, H2S) were measured during sludge anaerobic digestion. The list of methods/devices used for sludge samples analysis, before and after UD and/or AD processes (including type of the obtained effects) are shown in In order to determine the changes of sludge samples characteristics—before and after UD/AD, selected parameters were considered, i.e., pH, SCOD, N, Korea) . Sludge ectively ,26. In tTwo different experimental ultrasonic devices were applied for WAS ultrasonic disintegration. The first device (A) consists of disintegrator of high power disintegrator WK-2010, equipped with “sandwich” head and “lens” emitter . This device is also equipped with a gauge allowing to read the frequency, as wells as with a steel chamber where the UD of sludge samples takes place. The second device (B) consists of ultrasonic washer which is a rectangular chamber, equipped with a single “flat” emitter of “sandwich” type placed in the bottom of the chamber . The technical characteristics and operating conditions of the experimental devices are shown in V = 160 kWh·m−3 . Sludge samples were disintegrated for: 267 s (WK-2010) and 1920 s (ultrasonic washer). The sample volume was constant and equal V = 0.3 L. The ultrasonic density (UD) for each of experimental device was as follows: 2.2 W·cm−3 (WK-2010) and 0.3 W·cm−3 (ultrasonic washer). Whereas, the ultrasonic intensity (UI) was: 2.6 W·cm−2 (WK-2010) and 1.9 W·cm−2, respectively. The above conditions were the most favorable ones and were determined in the course of previously carried out studies. The variable parameter during sludge UD was the amount of specific energy (ES), which depends on TS content. The values characterizing the ultrasound field and the amount of energy supplied to the process were calculated by the following Equations (1)–(4):V—energy supplied per sludge volume (kWh·m−3) [S—specific energy (kJ·kgTS−1) [D—ultrasonic density (W·cm−3) [I—ultrasonic intensity (W·cm−2) [3·cm3); t—sonication time (s); TS—total solids; (g·L−1); AE—surface area of the disintegration (cm2).The WAS samples pretreatment were performed under a constant energy supply (over time) per sludge volume, i.e., E·kgTS−1) ; UD—ultr(W·cm−3) ; UI—ultrThe anaerobic digestion was conducted in the installation consisting of the anaerobic glass digesters with a working volume of 0.5 L, water bath—in order to maintain a constant temperature, columns to collect and measure the volume of evolved biogas and MULTITEC 540 gauge —applied for composition analysis. AD was conducted under mesophilic conditions (37 °C ± 0.5 °C) over 20 days, each time. The examined sample was a mixture of: digested sludge (30%) collected from the fermentation chamber of the Central WWTP in Gliwice and ultrasonically pretreated one (70%) from the same WWTP. Control sample constituted mixture of untreated and digested sludge. During the AD process each digester was shaken manually three times per day to prevent the sludge from settling. The process was conducted once a month (for a period of 7 months). The research position was made by Wytwórnia Przyrządów Laboratoryjnych (WPL) Gliwice, according to DIN 38414-8:1985 [TOT, NTOT, proteins and carbohydrates, as well as CST measurement and sludge flocs optical microscopy analysis. Whereas, the technological effects observed after completion of sludge AD included the same parameters as for the direct once  and microscopic analysis). Moreover, process of AD was also controlled by the volume and composition of the evolved biogas, as well as by measuring the rate of TS and VS reduction.In order to determine the direct and technological effects of sludge UD process, selected indicators were used (defined in COD) was determined using indicator proposed by Müller [i, ITi, ITd) based on the ratio of the concentration or value of specific compound resulting from changes in the sludge characteristics, as a result of its pretreatment [COD—disintegration degree of (%), SCOD0 and SCODUD—supernatant COD of the original and disintegrated sample (mg·L−1), SCODNaOH—the maximum CODNaOH obtained by alkaline hydrolysis  (mg·L−1).NUD and CUD—concentration or value of a specific compound in the sludge/supernatant of non—disintegrated and disintegrated sludge, respectively (mg·L−1), (s).In order to evaluate the direct and technological effects of WAS ultrasonic disintegration, a comprehensive analysis of the obtained results was conducted. The disintegration degree . To determine whether any of the differences between the means are statistically significant, one-way analysis of variance (ANOVA) was used. Tests were carried out with a confidence level of 95%.In order to evaluate the obtained results, a comprehensive statistical analysis was conducted. The calculations were performed with Statistica 12.0 (StatSoft) and Excel 2013 (Microsoft Office Standard). To check the differences in the mean concentrations of specific compounds between related groups of variables (sludge characteristics before and after UD and AD processes), −1 (TS); 24.8–53.2 g·L−1 (VS); 46.6–82.7 mg·L−1 (SCOD); 8.2–12.4 mg·L−1 (NTOT), 16.2–37.7 mg·L−1 (PTOT); 624.6–1228.2 mg·L−1 (proteins); 241.7–821.7 mg·L−1 (carbohydrates) and 7–13 s (CST). Among all examined parameters, the pH was characterized by the lowest variability (CV = 2.0%), whilst the concentration of carbohydrates by the highest one (CV = 35.6%).The characteristics of WAS before and after ultrasonic pretreatment are shown in pH, which values were lower than 1.0  and ultrasonic washer (WAS_B), respectively. The highest variability was observed for sludge pretreated in the ultrasonic washer (CV = 2.7%). The decrease of pH was also confirmed by IDd−1 (WAS_A) and 1842.0–6935.0 mg·L−1 (WAS_B). Higher variability of examined parameter was observed for sludge undergone disintegration in the ultrasonic washer (CV = 39.2%). The biggest increase of SCOD concentration in the sludge supernatant was noted in July and September, when the TS content was in the range of 24.8–35.5 g·L−1. The above observations were confirmed by DDCOD and IDiSCOD, which maximum values amounted: 37.5 and 91.7% , higher increase of sludge temperature was obtained at lower TS content, i.e., from 27.2 to 38.2 °C at TS = 2.0% and 18.0 to 29.0 °C at TS = 4.2% [S = 15,880 kJ·kg−1 TS [The concentrations of SCOD in the supernatant after sludge pretreatment, were in the range of 741.0–1827.0 mg·Lnd 91.7% , as wellnd 91.7%  for WAS ed 57.9% ,16. HoweS = 4.2% . Moreove−1 (WAS_A) and 210.0 to 719.0 mg·L−1 (WAS_B), while for the total phosphorus from 91.5 to 187.0 mg·L−1 and 126.0 to 223.0 mg·L−1, respectively. The concentration of NTOT was characterized by higher variability during the experiment duration. The value of CV for NTOT and PTOT were in the range of 23.8–37.5% and 18.7–22.5%, respectively. Almost for all examined sludge samples, the highest values of IDiNTOT and IDIPTOT were obtained in October, i.e., 20.0 (WAS_A), 63.6 (WAS_B) and 8.7 (WAS_A); 11.4 (WAS_B) respectively (S = 9690 kJ·kg−1 TS) [This study revealed that ultrasonic pretreatment of WAS increased the concentration of biogenic substances in the sludge supernatant. The concentration of total nitrogen ranged from 99.5 to 196.0 mg·Lectively , while tkg−1 TS) ,18. The −1 (WAS_A) and 618.5 to 1310.9 mg·L−1 (WAS_B). The above results were confirmed by the values of IDiPROT. and IDdPROT., which ranged from: −1.0 to −1.4 (WAS_A) and −1.3 to 1.3 (WAS_B) (−1 (WAS_A) and 115.0–790.0 mg·L−1 (WAS_B). The values of IDdCARBS amounted: −1.1 to −2.2 (WAS_A) and −1.0 to −2.1 (WAS_B) (−1 TS) increased the concentration of above substances initially, but with increasing energy supplied to the process their content decreased [−1 TS) increased the concentration of EPS in the sludge supernatant [As a result of ultrasonic disintegration of WAS, for most of the analyzed sludge samples the decrease in proteins concentration were observed. The only exception was shown for the sludge samples pretreated in ultrasonic washer, where the increase of proteins content was observed . The proteins concentration in the supernatant fluctuated in the range from: 465.3 to 1213.5 mg·L (WAS_B) . Moreove (WAS_B) . The higecreased . Whereasernatant . Howeverernatant , which wernatant .−1 and 53.2 g·L−1, respectively. The values of IDiCST were in the range of 6.2–102.6 (WAS_A) and 78.2–199.4 (WAS_B)  and 1042 to 1396 (B). The highest values of CST were noted in September, while the lowest in January, when the TS content was 24.8 g·L (WAS_B) . Similar−1); second one (S2) by mean (35.5 g·L−1) and third one by a highest TS content (53.2 g·L−1), among of all samples analyzed during seven months of the experiment conducting. The collection of above samples was conducted in: September, July and January, respectively. The results were presented depending on the type of disintegrator used for sludge pretreatment, i.e., WK-2010 (A1–A3) and ultrasonic washer (B1–B3)  was characterized by a lowest TS (24.8 g·L (B1–B3) . The optby mean 3.5 g·L−1 The characteristics of inoculum, as well as samples containing untreated or disintegrated WAS (mixed with inoculum), before  and after the process of AD  are shown in TOT concentration and CST values. Similar relations were observed in with reference to mixtures after AD process. For both samples, before and after AD, the lowest variability was observed for pH value. It was indicated that mixtures containing WAS after ultrasonic pretreatment , were characterized by higher concentrations and values of examined parameters, compared to the control sample (S_0). The highest variability in the mixtures before AD was expressed by PTOT and PTOT concentration, as well as the reduction of TS and VS content and increase of biogas production. While, CST values were generally decrease. The above observations were confirmed by ITi and ITd (pH (<1.0), this parameter was not included in The conducted experiment indicated, that after AD process, following changes in the examined mixtures were observed: the increase of pH, N and ITd . MoreoveTOT and PTOT concentration, were observed. The values of ITiNTOT and ITiPTOT ranged: 3.2–5.5; 2.5–4.9; 1.6–4.0 and 3.1–4.9; 1.4–2.9; 1.2–2.5, for S_0*; S_A* and S_B*, respectively . The above observations were in a good agreement with the results obtained by other researchers. For example, it was stated that ultrasonic pretreatment of WAS at 20 kHz and P = 200 W, increased biogas production by 6.3% [−1 TS [The amount of total biogas production observed after the process of sludge anaerobic digestion is presented in  by 6.3% . Whereas% [−1 TS .4, CO2 and H2S ranged from: 65.3 to 67.9% and 25.6 to 25.9%, as well as <1.0 ppm, respectively  and obtained effects, was expressed as Pearson’s correlation coefficient (r). Moreover, to indicate the differences between effects obtained in various technical conditions (disintegrators) of process conducting, the one-way analysis of variance (ANOVA) was carried out.In order to present the effects of ultrasonic disintegration as a function of WAS characteristics and technical conditions of the conducted process, a comprehensive analysis was carried out. In order to determine if there are any significant differences between examined variables, expressed as the changes in the characteristics of WAS before and after ultrasonic disintegration the T-Test confirmed existence of significant differences in the characteristics of WAS before and after ultrasonic pretreatment (p < 0.05). The only exceptions were: pH for samples disintegrated in WK-2010, as well as proteins for ultrasonic washer (p > 0.05) (The results of  > 0.05) .TOT, PTOT > pH. Moreover, data analysis also indicated that, the increase of sludge temperature during ultrasonic pretreatment of WAS exerted a significant impact on its final characteristics.The results of Pearson’s correlation are shown in T-Test results showed a significant differences in the characteristics of mixtures consisting of inoculum and pretreated WAS, prior and after anaerobic digestion (p < 0.05). The only exception was: CST for samples containing WAS after pretreatment in the ultrasonic washer (p > 0.05) (The  > 0.05) .TOT, PTOT > pH. The sequence of above parameters was determined based on the number of correlations between analyzed variables. Moreover, taking into account VS reduction and increase of biogas production, it can be said that increase of sludge temperature during ultrasonic disintegration most likely causes the difference in its biodegradation.Furthermore, the results of Pearson’s correlation revealed statistically significant differences between the characteristics of mixtures prior and after completion of anaerobic digestion . The abop < 0.05). Whereas, the results of statistical analysis in accordance to technological effects indicated existence of significant differences between analyzed variables, but only in the case of changes in the CST values  showed that in the case of direct effects, there is a basis for rejecting the null hypothesis (H0) on the about the absence of statistically significant differences between the variables in considered groups  . It meanT values . It meanIn conclusion, according to the results of statistical analysis and values of applied indicators, it could be inferred that the most promising conditions for ultrasonic pretreatment of WAS (conducting at constant energy input), are: low power, long sonication time, large surface area of the emitter.TOT, PTOT, proteins and carbohydrates concentrations, CST value, floc disruption, TS and VS content, before and after sludge ultrasonic disintegration or anaerobic digestion. To evaluation of direct and technological effects the commonly applied (DDCOD) and author’s  indicators were used.This article presents the results of a comprehensive analysis of direct and technological effects of disintegration as a function of periodical changes in the characteristics of WAS, as well as technical conditions of the conducted process. In this purpose selected parameters were considered, i.e., pH value, SCOD, NTOT and PTOT concentration, sludge dewaterability, as well as the reduction of TS and VS content and increases of biogas production were observed. Furthermore, the conducted experiment revealed that ultrasonic disintegration reduces the content of biogenic substances in the sludge supernatant after its anaerobic digestion. This information is positive, especially with respect to necessity of recycling the leachate, generated after dehydration of sludge undergone anaerobic digestion into the technological line.As a result of ultrasonic pretreatment, in the sludge or supernatant the increase of all examined parameters, except proteins and carbohydrates, were observed. Moreover, it was also showed that after completion of sludge anaerobic digestion, the increase of pH, NT-Test showed that there were significant differences between the characteristics of untreated and disintegrated WAS, regardless of the technological conditions of process conducting. The above observations were related both to direct and technological effects. Whereas, Pearson’s correlation confirmed, that changes in the characteristics of untreated WAS influencing effects of ultrasonic pretreatment. It was indicated that, the most important parameters, which affected the magnitude of the direct and technological effects, were: TS, VS, ΔT > EPS > SCOD > CST > NTOT, PTOT > pH and TS, VS > CST > NTOT, PTOT > pH, respectively. Furthermore, the analysis of variance showed, that the most significant differences between the effects obtained in various experimental devices were observed for: SCOD, NTOT, PTOT, CST, ΔT (direct effects) and CST . It was found, that the most favorable effects of sludge ultrasonic pretreatment can be obtained conducting the process in the device characterized with low power, long sonication time and large surface area of the emitter. The results obtained in this study also confirmed the significant impact of the increase of sludge temperature during ultrasonic disintegration on the obtained effects.The result of


Considering the lack of existing data on anaerobic power and neuromuscular fitness of female marathon runners, the findings reported in this study would be useful for strength and conditioning trainers to monitor the training of their athletes. Even if these parameters were not related to race time, they should be monitored regularly as they were either component of health-related physical fitness (muscle strength and flexibility) or could help runners (anaerobic power) under specific circumstances such as ascends during a race.Physical fitness components that relate with performance in marathon running, e.g., aerobic capacity and body composition, have been studied extensively. On the other hand, data on components of the health-related physical fitness, such as flexibility and muscle strength, were missing in this sport. Therefore, the aim of the present study was to profile force-velocity (F-v) characteristics, muscle strength and flexibility in female recreational marathon runners and to examine their relationship with age, race time and anthropometric characteristics . Thirty three female marathon runners , separated into three age groups  and three performance groups , performed sit-and-reach test (SAR), isometric muscle strength tests, squat jump, countermovement jump and F-v test on a cycle ergometer. The main findings of the present study were that (i) participants had moderate scores of body composition and physical fitness considering norms of the general population, (ii) the <35 age group had better jumping ability than 35–45 and >45 age group, and the older age group had lower F In addin ≤ 6) ; thus, in ≤ 6) .With regards to flexibility, an optimal level of this parameter is necessary for health; for example, the flexibility of the muscle, tendons and ligaments in the back might be associated with the range of motion and functional movement . A studyn = 33) were divided into three age groups  to study the effect of age on these performance parameters. Also, differences among three even performance groups, based on the best race time , were examined. The best race time was considered instead of the most recent race time as the latter could be influenced by non-performance conditions, e.g., injury. The relationship of physical fitness  with anthropometric characteristics  was examined using correlations.To examine the relationship of F-v characteristics, isometric muscle strength, jump ability and flexibility with age, performance and anthropometry, a cross-sectional study design was applied. Participants  mostly from the area of Athens volunteered to participate in this study, which had been advertised through popular websites for endurance runners. The participants reported sport experience 5.6 ± 4.6 years and were practicing endurance training for 4.1 ± 1.5 days weekly with each training session corresponding to 9.3 ± 2.8 km or 1.6 ± 0.5 h. During September and October 2017, the participants visited the laboratory where they were examined for anthropometric characteristics and performed a F-v test, isometric muscle strength, jump ability and flexibility. This study was approved by the local Institutional Review Board . The study was conducted in accordance with the Declaration of Helsinki. All participants gave written informed consent after having been provided detailed information about the risks and benefits of the research.Thirty-three female recreational marathon runners , and BF was estimated from the sum of 10 skinfolds, i.e., cheek, wattle, chest I, triceps, subscapular, abdominal, chest II, suprailiac, thigh, and calf (∗ BF/100).” CSA was calculated as “(4.68 ∗ midthigh circumference in cm) – (2.09 × anterior thigh skinfold in mm) – 80.99”  was employed for measurement of body mass (to the nearest 0.1 kg), a portable stadiometer  for height (0.001 m), and a caliper  for skinfolds (0.2 mm). BMI was calculated as the quotient of body mass (kg) to height squared  was used to assess low back and hamstring flexibility . It was To evaluate isometric muscle strength, the sum of four tests  in absolute and relative to body mass values was used. The handgrip test was performed with participants standing and having their elbow flexed at approximately 90°. They were instructed to squeeze the handle of the handgrip dynamometer  as hard as possible for 5 s. Two trials were performed for each hand, with a 1 min rest between trials. The best trial was recorded for each hand . ICC wasThe participants performed two trials for each jumping test  and the best result was recorded . There w−1 (rPmax), theoretical maximal velocity (v0) in revolutions per minute (rpm) and force (F0) in N, and v0/F0 was calculated in rpm.N−1. The participants performed four sprints, each one lasting 7 s, against braking force  on a leg cycle ergometer , interspersed by 5 min recovery periods. The seat height of the ergometer was adjusted to allow for a slight bend in the knee (approximately 175°) and in accordance with the participant’s satisfaction. Each sprint began with a flying start, i.e., as soon as velocity reached 50 rpm (revolutions per minute), the weight basket was released and the braking force was applied. For each participant an individual linear regression was determined between peak velocity and braking force for each of the four sprints. F0 and v0 corresponded to the intercepts with F and v axes in the F-v graph. Pmax was calculated as Pmax = 0.25⋅F0⋅v0  were used to examine the differences among age and performance groups, separately. To interpret effect size (ES) for statistical differences in the ANOVA, partial eta square classified as small (0.010 < ηp2≤ 0.059), medium (0.059 < ηp2≤ 0.138), and large (ηp2 > 0.138) was used (r). Magnitude of correlation coefficients was considered as trivial if r < 0.10, small if 0.10 ≤r < 0.30, moderate if 0.30 ≤r < 0.50, large if 0.50 ≤r < 0.70, very large if 0.70 ≤r < 0.90, nearly perfect if r ≥ 0.90, and perfect if r = 1.00  and GraphPad Prism v. 7.0 . Normality was examined using Kolmogorov-Smirnov test and visual inspection of normal Q-Q plots. Data were expressed as mean and standard deviation (SD). One-way repeated measures analysis of variance (ANOVA) and a subsequent Bonferroni was used . The relr = 1.00 . The lev0 of all participants was 167 ± 15 rpm (ranging from 132 to 195 rpm), F0 120 ± 20N (93–179 N), Pmax 507 ± 85W (340–704 W), rPmax 8.83 ± 1.17 W.kg−1 (6.0–11.1 W.kg−1) and v0.F0−1 1.43 ± 0.28 rpm.N−1 (0.87–1.93 rpm.N−1). With regards to neuromuscular fitness, SAR was 25.8 ± 8.3 cm (8.0–37.5 cm), SJ 17.7 ± 3.4 cm (10.3–25.2 cm) and CMJ 18.6 ± 3.7 cm (11.2–26.1 cm). In isometric muscle strength, right handgrip was 29.7 ± 4.5 kg (20.7–41.6 kg), left handgrip 29.7 ± 4.0 kg (23.4–39.7 kg), back 82.5 ± 16.2 kg (40.0–114.0 kg), back-and-leg 94.9 ± 19.0 kg (48.5–150.0 kg), sum 236.9 ± 40.1 kg (134.2–339.3 kg) and 4.12 ± 0.56 kg.kg−1 (2.90–5.23 kg.kg−1).The anthropometric characteristics of participants, in total and by age group, can be seen in Table p = 0.002, ηp2 = 0.361) and CMJ  was observed with higher values in the <35 age group than in the 35–45 and >45 age group, whereas no difference was shown in SAR  , F0  with higher score in 35–45 than <35 and >45 age group, Pmax  with higher score in 35–45 than >45 age group, rPmax  with higher score in <35 and 35–45 than >45 age group and v0/F0  with higher score in <35 than 35–45 age group  with higher score (+8.3 cm) in the slowest than in the average performance group.Anthropometric characteristics and physical fitness by performance level can be seen in Table SJ and the sum of isometric strength tests  correlated inversely moderately with BF Table . All the0, Pmax and rPmax than their younger counterparts, (iii) the slowest performance group scored the highest in SAR, and (iv) isometric strength, F0 and Pmax correlated largely with body mass and FFM.The main findings of the present study were that (i) participants had average scores of body composition and physical fitness compared to the general population, (ii) the <35 age group had better jumping ability than 35–45 and >45 age group, and the older age group had lower FCMJ was in similar levels as sex- and age-matched general population . Partici0, Pmax and rPmax than their younger counterparts, suggesting a decline of these physical fitness components with aging and indicating that probably the chronic endurance exercise does not prevent from loss in muscle strength and power. It should be highlighted that the participants had sport experience in endurance running for 5.6 years. These findings confirmed previous studies in male participants which showed a decline of anaerobic power with aging   .Among all anthropometric characteristics and physical fitness components examined in the present study, flexibility was the only one observed to differ among performance groups with the slowest one presenting the better score in SAR than the average group. This observation might be due to that running economy (which is related with performance) is inversely correlated with sit-and-reach test score . An incrMost indices of muscle strength and power correlated with both body mass, FFM and CSA, which might explain why female marathon runners are not characterized by high levels of muscle strength and power as their body dimensions are relatively small compared to other sports. An excess of FFM, even if this is “active mass,” is a load that marathon runners need to carry with them; thus, their profile consists of small anthropometric characteristics and corresponding moderate levels of muscle strength and power. Although an increased Pmax might improved the cost of running , its assA limitation of the present study was the specificity of protocols that assessed the various physical fitness components as caution would be needed to compare their findings with studies using other protocols. For instance, F-v test and WAnT reflect different aspects of anaerobic power and capacity and their findings should not be used interchangeably . StrengtProfiling physical fitness characteristics of marathon runners is of great practical importance for strength and conditioning coaches working in this sport. The assessment of physical fitness assists to evaluate the effectiveness of training. So far, a lack of reference data on female marathon runners’ physical fitness, especially with regards to anaerobic power, muscle strength and flexibility, has been observed. Therefore, the data reported in this study would be useful for strength and conditioning trainers to monitor the training of female marathon runners. Strength and conditioning coaches may work with female marathon runners differing for age, performance level and anthropometric characteristics; thus, knowledge about the effect of age, performance and anthropometry on physical fitness would assist to accurately evaluate and prescribe training program. The findings highlighted the lower leg strength in the older age group; thus, strength and conditioning coaches should focus on the development of age-tailored training programs to enhance the jump ability of older female runners. On the other hand, flexibility should be monitored regularly targeting and be within a physiological range, whereas a high flexibility should be alarming as it associates with reduced performance in marathon. Considering the increased number of female finishers in marathon races during the last years, the findings have practical applications to a large number of recreational marathon runners. It should be also highlighted that the age and performance level of participants in the present study  was close to the average of finishers in large marathon races such as the “New York City marathon” .PN performed the laboratory tests, analyzed the data and drafted the manuscript, TR and BK helped in drafting the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite the development of new therapies for leishmaniasis, among the 200 countries or territories reporting to the WHO, 87 were identified as endemic for Tegumentary Leishmaniasis and 75 as endemic for Visceral Leishmaniasis. The identification of antileishmanial drug candidates is essential to fill the drug discovery pipeline for leishmaniasis. In the hit molecule LQB-118 selected, the first generation of pterocarpanquinones was effective and safe against experimental visceral and cutaneous leishmaniasis via oral delivery. In this paper, we report the synthesis and antileishmanial activity of the second generation of pterocarpanoquinones.L. amazonensis, L. braziliensis, and L. infantum. Toxicity was assessed in peritoneal macrophages and selective index calculated by CC50/IC50. Oxidative stress was measured by intracellular ROS levels and mitochondrial membrane potential in treated cells.The second generation of pterocarpanquinones 2a-f was prepared through a palladium-catalyzed oxyarylation of dihydronaphtalen and chromens with iodolawsone, easily prepared by iodination of lawsone. The spectrum of antileishmanial activity was evaluated in promastigotes and intracellular amastigotes of L. amazonensis and promastigotes of L. infantum. As to the oxygen in ring B of the new generation, we observed that the oxygenated compound 2b is approximately twice as active against L. braziliensis promastigotes than its deoxy derivative 2a. Another modification that improved the activity was the addition of the methylenedioxy group. A variation in the susceptibility among species was evident in the clinically relevant form of the parasite, the intracellular amastigote. L. amazonensis was the species most susceptible to novel derivatives, whilst L. infantum was resistant to most of them. The pterocarpanoquinones (2b and 2c) that possess the oxygen atom in ring B showed induction of increased ROS production.In this work, we answered two pertinent questions about the structure of the first-generation pterocarpanquinones: the configuration and positions of rings B (pyran) and C (furan) and the presence of oxygen in the B ring. When rings B and C are exchanged, we noted an improvement of the activity against promastigotes and amastigotes of The data presented indicate that the pterocarpanoquinones are promising compounds for the development of new leishmanicidal agents. Leishmania, Trypanosoma, and Plasmodium. Pterocarpan derivatives, such as maackiain and others, have frequently shown antiprotozoal activity  + calcd for C21H16O4Na: 355.0940; found: 355.0944.After column chromatography using n-hexane/ethyl acetate (95:5) as eluant, this compound was obtained as a yellow solid in 46% yield in PEG-400 under heating at 90 °C. 1H NMR : δ = 8.09 , 7.74 , 7.68 , 7.12 , 7.09 , 6.87 , 5.87 , 3.90 , 3.84 , 2.71 , 2.65–2.56 , 2.25–2.14 , 2.02–1.91 . 13C NMR : δ = 182.37, 177.99, 160.00, 158.34, 134.17, 133.14, 132.96, 131.73, 131.45, 131.16, 129.48, 126.85, 126.31, 125.97, 115.78, 114.51, 84.96, 55.46, 39.83, 26.49, 25.71. HRMS: m/z [M + Na] + calcd for C21H16O4Na: 355.0940; found: 355.0935.The studies were performed in accordance with protocols approved by the Ethics Committee for Animal Use of the Oswaldo Cruz Foundation (LW07/2010) under the protocol 044/2009, and approved by the Ethics Committee on Animal Use (CEUA) of the Instituto de Biologia Roberto Alcantara Gomes of the Universidade do Estado do Rio de Janeiro-UERJ.Leishmania amazonensis (MHOM/BR/77/LTB0016) was maintained as promastigotes at 26 °C in Schneider’s insect medium (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (HIFCS), streptomycin at 100 μg/mL and penicillin at 100 U/mL. New cultures of promastigotes were obtained from infected Balb/C mice.L. braziliensis (MCAN/BR/98/R619) was routinely isolated from hamster lesions and maintained as promastigotes in Schneider’s insect medium containing 20% HIFCS and 100 μg/ml gentamicin (Schering-Plough). New cultures of promastigotes were obtained from an infected hamster.Leishmania infantum (MHOM/MA67ITMAP263) parasites were isolated from female BALB/c mice infected and cultured at 26 °C in Schneider’s insect medium supplemented with 20% HIFCS, streptomycin at 100 μg/ml and penicillin at 100 U/ml. New cultures of promastigotes were obtained from infected Balb/C mice.Leishmania promastigotes of three species were incubated in 96-well plates  under the conditions described above in either the absence or presence of different concentrations of pterocarpanquinones 2a-f (0-100 μM). A stock solution of the compounds was prepared at 50 mM in dimethylsulfoxide (Sigma Aldrich). The maximum solvent concentration used in the assays was 0.4% in a final volume per well of 200 μL. The cultures were initiated with 1.0 × 106 cells/ml and maintained at 26 °C for 72 h. Inhibition of parasitic growth was determined by cell viability indicator, resazurin [L. braziliensis). The 50% inhibitory concentration (IC50) was determined by nonlinear regression analysis in the software GraphPad Prism 5.esazurin , with exL. amazonensis and L. infantum, resident peritoneal macrophages (swiss mouse) were plated in RPMI (Sigma-Aldrich) at 2 × 106/mL (0.4 mL/well) in Lab-Tek eight-chamber slides  and incubated at 37 °C in 5% CO2 for 1 h. Stationary-phase L. amazonensis and L. infantum promastigotes were added at respective parasite/macrophage ratios of 3:1 and 5:1 for 4 h. After washing three times, pterocarpanquinones 2a-f at various concentrations were added for 72 h. Next, the slides were stained using an Instant Prov hematological dye system . The number of amastigotes was determined by counting at least 100 macrophages per sample.For the test against intracellular amastigotes of L. braziliensis due to the natural resistance of mice. Resident macrophages were obtained from the peritoneal cells of golden hamsters after a peritoneal injection of 10 mL of Dulbecco’s Modified Eagle’s medium (DMEM) (Sigma-Aldrich). The peritoneal macrophages were plated onto glass coverslips placed within the wells of a 24-well culture plate and incubated at 37 °C in 5% CO2 for 1 h. After removing the nonadherent cells, the monolayers were infected with stationary-phase L. braziliensis promastigotes at a 5:1 parasite/macrophage ratio for 4 h. The infected macrophages were washed and incubated with several pterocarpanquinone concentrations (0-25 μM) for 72 h. The monolayers were then stained with Giemsa, and at least 100 infected macrophages per sample were counted under optical microscopy.Macrophages of golden hamsters were used in assays against intracellular amastigotes of 50) was determined by nonlinear regression analysis in the software GraphPad Prism 5.The results were expressed as an infection index . The 50% inhibitory concentration (IC6/mL) were incubated with pterocarpanquinones (1.25–100 μM) for 72 h at 37 °C/5% CO2. Viability was evaluated with resazurin assay as described for the antipromastigote test. The 50% cytotoxicity concentration (CC50) was determined by nonlinear regression analysis in the software GraphPad Prism 5.To evaluate the toxicity of pterocarpanquinones 2a-f, peritoneal macrophages of Swiss mice . The fluorescence was monitored at 1 h intervals for 4 h using 485 and 530 nm as excitation and emission wavelengths, respectively, using the spectrofluorometer Spectra Max Gemini XPS . Antimycin A 10 μM was used as positive control.Intracellular ROS levels were measured in treated and untreated cells as described previously . BrieflyL. infantum (5 × 106 cells/mL) were incubated in the presence of 0–5 μM of derivatives at 26 °C. After 4 h, the parasites were incubated for 15 min with 10 μg/mL rhodamine 123 (Rh123) (Sigma-Aldrich). Data acquisition of 10,000 events and analysis were performed using the flow cytometer FACSCalibur . Alteration in ΔΨm was quantified using an index of variation (IV) obtained by the eq. IV = (MT − MC)/MC [p-Trifluoromethoxyphenylhydrazone) 20 μM was used as positive control [To determine the effect of pterocarpanquinones 2a-f on the ΔΨm, promastigotes of − MC)/MC , where M control .p < 0.05. Dose-response significance was evaluated by one-way ANOVA test.Antipromastigote and macrophage toxicity assays were repeated three times in triplicate. Antiamastigote assays were repeated three times in duplicate. The results reported for these assays are presented by the Mean ± SEM. Measurements of ROS and ΔΨm were performed in triplicate three times. The ROS and index of variation data of the Ψm are represented as Mean ± SD. Significant differences between pairs of groups were assessed using Student’s t-test with the significance level set at 2CO3 was used, did not improve 2a yields. Therefore, we decided to run the others experiments using 1.5 times the previous salt amount. The yield additionally increased when pinacolone (bp 110 °C) was used as the solvent instead of acetone. Under conventional heating, a 57% yield was obtained from 2a after 18 h of reaction (entry 5) while under microwave irradiation 2a was isolated at 50% yield after 40 min of reaction. When PEG-400 was utilized as solvent at 140 °C, 3a reacts with 4 and 2a could be isolated at a 65% yield after 10 min. of reaction  and chromens 3b-d with 3-iodolawsone (4), easily prepared by iodination of lawsone under three reaction conditions . Herein, we used the oxyarylation to obtain type 2 compounds. We first studied the reaction between 3a and 4 that leads to pterocarpanquinone 2a Scheme , Table 1In fact, pterocarpanquinone 2a could also be obtained by this unprecedented oxyarylation of 3a with lawsone 5  showed the lowest IC50  for the species causing tegumentary leishmaniasis, which is in relation to second generation derivatives and LQB-118. In addition to improved potency, derivative 2d showed less toxicity to peritoneal macrophages, with a 4.2-fold lower cytotoxic concentration than LQB-118 (CC50 = 77.7 μM and CC50 = 18.46 μM). This decrease in cytotoxicity aligned with increased potency of pterocarpquinone 2d resulted in a selectivity index of 172.6 for L. amazonensis. Despite the potency in intracellular amastigotes of L. amazonensis, compound 2d showed no significant activity in L. infantum amastigote. Only derivative 2b displayed activity against the three species despite the low selectivity index for L. braziliensis and L. infantum, as presented by the reference drug pentamidine, which showed low index selectivity for L. amazonensis and L. braziliensis, and 10-fold greater selectivity  was evaluated with the probe H2DCFDA for 4 h. The production of ROS after 4 h of treatment was significant for compounds 2b, 2c, and 2e  production in promastigotes of inones 1.5–5.0 μM 2e and 2f induced greater depolarization in L. infantum promastigotes.Promastigotes treated with second-generation pterocarpanquinones for 4 h had their mitochondrial membrane potential (ΔΨm) evaluated with rhodamine 123 by flow cytometry. The displacement of populations of treated cells, to the left in the histogram, represents the depolarization of the cells, as verified with the positive control used (FCCP), a classical uncoupler of ΔΨm  and a species that causes visceral leishmaniasis (L. infantum). In this work, we answered two pertinent questions about the structure of the first generation pterocarpanquinones: (i) the configuration of the position of rings B (pyran) and C (furan) of the pterocarpan core and (ii) the presence of oxygen in the B ring. When rings B and C are inverted (2b in comparison to 1), we noted an improvement of about 1.6 times in the activity against promastigotes and amastigotes of L. amazonensis and 4 times against promastigotes of L. infantum. Notwithstanding, the opposite was observed in promastigotes of L. braziliensis, which presented a decrease of 3.2 times in the antileishmanial activity with the second generation compound. As to the oxygen in the ring B of the new generation, we observed that the oxygenated compound 2b is approximately two times more active against promastigotes of L. braziliensis than its deoxy derivative 2a. Furthermore, on average, the oxygenated compounds 2b, 2c and 2d were more potent against amastigotes of L. amazonensis than the deoxy derivatives 2a, 2e, and 2 f. Another modification that improved the activity was the addition of the methylenedioxy group (2d). This group has demonstrated the ability to potentiate the activity of triazole compounds [Rollinia emarginata [L. amazonensis and L. braziliensis; however, this effect was not seen in promastigotes of L.infantum. The chlorine atom as the substituent in R2 in 2c increased cytotoxicity when compared to 2b by 2.6 times; this increased cytotoxicity profile was also observed with the insertion of one or two chlorine atoms in the 4-Phenyl-1,3-thiazol-4-amines series [Leishmania species show a significant variation in their sensitivity to established and experimental drugs [L. amazonensis was the species most susceptible to novel derivatives, whilst L. infantum was resistant to most of them. In fact, the compound 2b was the only one active against L. infantum amastigotes. The mechanism of induction by pterocarpanquinone LQB-118 of L. amazonensis parasite death involved oxidative stress with hallmarks of apoptosis, similar to cancer-induced death [2b and 2c) that possess the oxygen atom in ring B, showed induction of increased ROS production. However, the pterocarpanquinone (2d) with methylenedioxy substituent altered this ROS-increasing ability, as also presented by the pterocarpanquinones with an absence of oxygen in the B . Natural and synthetic quinones may undergo redox cycling and induce oxidative stress with ROS production [Limited therapeutic options in leishmaniasis make its treatment very challenging. This small arsenal has resulted in long-term treatment and severe adverse effects. It is still important to emphasize that there are no vaccines or chemoprophylaxis currently available for humans , 29. Givhe world , there ilubility . Recent lubility ; thus, tompounds , analogsompounds , thiosemompounds  and secoarginata . We veris series . Croft aal drugs . As expeed death . The secoduction , 38.The data presented indicate that the second-generation pterocarpanoquinones are promising scaffolds for the development of new leishmanicidal agents.


H. annuus plants is traditionally used as medicinal remedies for several diseases, including malaria. Antimalarial activity of the leaf and the seed has already been observed; however, there is no report about antimalarial activity of the other parts of H. annuus plants. In this study, we assess in vitro and in vivo antimalarial activity of each part of the plants and its mechanism as antimalarial agent against inhibition of heme detoxification.  Each part of H. annuus.  To investigate the antimalarial activity of various parts of H. annuus plant were tested for in vitro antimalarial activity against Plasmodium falciparum 3D7 strain (chloroquine-sensitive), in vivo antimalarial activity against P. berghei using Peters' 4-day suppressive test in BALB/c mice, curative and prophylaxis assay, and inhibition of heme detoxification by evaluating β-hematin level.  Various parts of the 50 values of 2.3 ± 1.4 and 4.3 ± 2.2 μg/mL, respectively and the percentage inhibition of P. berghei of 63.6 ± 8.0 and 59.3 ± 13.2 at a dose of 100 mg/kg, respectively. Ethanol extract of roots produced an ED50 value of 10.6 ± 0.2 mg/kg in the curative test and showed an inhibition of 79.2% at a dose of 400 mg/kg in the prophylactic assay. In inhibition of heme detoxification assay, root and leaf ethanol extracts yielded a lower IC50 value than positive (chloroquine) control with a value of 0.4 ± 0.0 and 0.5 ± 0.0 mg/mL, respectively.  Ethanol extract of the roots showed the highest antimalarial activity, followed by ethanol extract of leaves, with ICH. annuus as a new source for the development of a new plant-based antimalarial agent. There were promising results of the ethanol extracts of root of  Helianthus annuus (Asteraceae), known as sunflower or “bunga matahari” in local language. Traditionally, each part of H. annuus plants is widely used in several healthcare applications, including the treatment of malaria. Sunflower tea is reported to be useful in the treatment of malarial and lung disease [Most developing countries, including Indonesia, still depend on using plants in traditional medicine, including antimalarial agents. One of these plants is  disease , and the disease .H. annuus leaf shows good antimalarial activity with an ED50 value of 4.64 mg/kg. Moreover, the combination of 80% ethanol extract of H. annuus leaf and Acalypha indica Linn. leads to synergetic effect in in vivo antimalarial assay with a CI value of 0.46 (CI < 1) and an ED50 value of 1.23 mg/kg [H. annuus seeds showed good inhibition activity against P. falciparum strain KI with an EC50 of 0.19 and 0.34 μg/mL, respectively.Muti'ah et al.  report tH. annuus plants are potential source of antimalarial drugs. However, antimalarial activity of the other parts of H. annuus plants has not been reported to date. Thus, in this study, we assess in vitro and in vivo antimalarial activity of each part of the plant and its mechanism as antimalarial agent against the inhibition of heme detoxification. The inhibition of heme detoxification leads to growth disorder and malaria parasites' mortality as a result of membrane lysis and activity disorder of some enzymes [H. annuus plant parts and its ability to inhibit heme detoxification are expected to be obtained through this study as it can be a new source of antimalarial drugs.Based on the data above, it is recognized that  enzymes –9. Hence enzymes , 11. TheH. annuus plants were obtained from Oro-oro Ombo, Malang, East Java, Indonesia, in November 2016, and determined by Materia Medica Batu, Malang, East Java. Each part of the plant, including roots, stems, seeds, flowers, and leaves, were air-dried separately. Samples were then ground to powder, once they are completely dried.Adult male BALB/c mice with 20–30 g of bodyweight were obtained from Faculty of Veterinary Medicine, Universitas Airlangga. The mice were fed with mice pellet diet and given free access to drink clean water. The animals were allowed to acclimatize for two weeks before being treated. The permission and approval for animal studies were obtained from Faculty of Veterinary Medicine, Universitas Airlangga.H. annuus plants (50 gram) were macerated by 96% ethanol and then evaporated on a rotary evaporator.Powders from each part of Plasmodium falciparum strain 3D7 (chloroquine-sensitive) were used and cultivated using the Trager and Jensen method [+ red blood cells with 5% hematocrit in RPMI 1640  added with 22.3 mM HEPES (Sigma), hypoxanthine, sodium bicarbonate, and 10% human O+ plasma. Chloroquine diphosphate was used as the positive control.In this research, cultures of n method . Culture50 value was calculated using Probit Analysis.Antimalarial assay was done using a 24-well microplate with 1% initial and experimental parasitaemia (1 mL/well of suspension), followed by incubation in a multigas incubator at 37°C for 48 hours. The incubated materials were then collected and used to make a thin smear in a glass slide, fixated in methanol, and stained with Giemsa. The number of parasites was then observed under a microscope and compared with the negative control to determine the percentage of parasite growth inhibition. ICPlasmodium berghei strain ANKA was obtained from Eijkman Institute of Molecular Biology, Jakarta, Indonesia. About 20% of parasitaemia-infected mice were dissected and then the intracardial blood was taken and diluted with the PBS. Each mouse used in this research was inoculated priorly intraperitoneally with 0.2 mL of solution containing 1 × 107P. berghei-infected erythrocytes [hrocytes .In vivo antimalarial assay of the plant extract was done using Peter's method [s method , which h7P. berghei-infected erythrocytes. Negative control (group 1) was treated with 0.5% Na CMC suspension. Groups 2, 3, 4, and 5 were treated with 96% ethanol extract suspension of H. annuus root with 1, 10, 100, and 250 mg/kg dose per mouse. Suspension was given orally once a day for 4 days. Percentage of parasitaemia was calculated by making a thin blood smear on the fourth day, which is then fixated in methanol and stained with Giemsa. The calculation was then compared with the control group. ED50 value was obtained by using Probit Analysis.Thirty adult male BALB/c mice (8 weeks old) were randomly divided into five groups (six mice per group). Each mouse was infected intraperitoneally with 0.2 mL of solution containing 1 × 10H. annuus root with 100, 200, 400, and 800 mg/kg dose per mouse. Group 5 as the negative control group was treated with 0.5% Na CMC suspension, while the positive control (group 6) was given 13 mg/kg of doxycycline suspension [7P. berghei-infected erythrocytes by intraperitoneal injection. Percentage of parasitaemia was determined on the third day by observing the thin blood smear and compared with the control group. Mean survival time of mice in each group was determined during 14 days (D0–D13). Mean survival time was calculated using the following equation:Thirty adult male BALB/c mice (8 weeks old) were randomly grouped into six groups (five mice per group). Groups 1, 2, 3, and 4 were treated with 96% ethanol extract suspension of spension . Each suβ-hematin was calculated and compared with the negative control [Inhibition of heme detoxification was assessed using the Basilico method  with mod control , 18.50 values were calculated using Probit Analysis. Statistical significance was determined by Kruskal–Wallis nonparametric independent t-test and ANOVA (one-way).Data are expressed as mean ± standard deviation (SD). ICH. annuus has been planted widely in Indonesia and long been used traditionally as anti-inflammatory, antimalarial, antiasthma, antioxidant, antitumor, and antimicrobial agent. It has been reported that several chemical compounds were already isolated and identified from various parts of this plant, such as heliangolides (sesquiterpene lactones), α-pinene, sabinene (monoterpene), helikauranoside (diterpene), and alkaloid and phenolic group in the leaf part; helianthoside (triterpene) and saponin group in the flower part; fatty acid, tocopherol, tannin, and polyphenol group in the seed part; alkaloid and phenolic group in the stem and root parts [H. annuus plants macerated with 96% ethanol is shown in ot parts . ExtractIn vitro screening to observe P. falciparum inhibition activity of the plant extract with IC50 value less than 10 μg/mL is an important primary step in the development of new antimalarial drugs. Plant extracts showing IC50 values ≤10 μg/mL can be considered to be active, IC50 values in the range 10 < IC50 ≤ 25 μg/mL can be considered to be moderately active, and those exhibiting IC50 values >25 μg/mL can be considered to be inactive [50 values of 96% ethanol extract of various H. annuus plant parts are shown in in vitro antimalarial activity, followed by leaf and flower extracts with an IC50 value of 2.3 ± 1.4, 4.3 ± 2.2, and 4.8 ± 0.0 μg/mL, respectively.inactive . IC50 vaIn vivo antimalarial activity was also determined for each extract from various parts of H. annuus plants, and the results are shown in P. berghei, followed by leaf extract with the percentage inhibition of 63.6 ± 8.0 and 59.3 ± 13.2, respectively.P. berghei-infected mice given orally 50–250 mg/kg/day of extract exhibiting inhibition percentage >60% are considered to be active or very active, and those exhibiting inhibition percentage >30% are considered to be moderately active [H. annuus extract is used as an initial experiment and observation of parasite growth up to day 4 using mice model.Method and criteria are varied among the treatment groups examining antimalarial potency of plants by using the rodent animal model. y active –24. In tin vivo assay, it is shown that H. annuus root extract has the highest activity. Thus, the experiment is continued to assess curative and prophylaxis antimalarial activity of the ethanol root extract of H. annuus.Based on the first screening of in vivo curative assay, P. berghei-infected mice are treated orally with 96% ethanol root extract at a dose of 1, 10, 100, and 250 mg/kg. The results of the 4-day suppressive test show that the ethanol extract of H. annuus root has an ED50 value of 10.6 ± 0.2 mg/kg  tends to have toxicity.The 400 mg/kg dose group shows the highest percentage of prophylactic inhibition (79.2%) at day 3, and it declines to 62.5% at a dose of 800 mg/kg. Calabrese and Baldwin  report tt-test and one-way ANOVA analysis show that there are significant differences  in inhibition percentage of the positive control group compared with all the sample groups and the negative control group. There is no significant difference between 100, 200, 400, and 800 mg/kg doses.The results of Kruskal–Wallis nonparametric independent H. annuus root in vivo, it can be concluded that the 96% ethanol extract of H. annuus root has more potential activities as curative antimalarial than prophylactic.H. annuus root (IC50 = 0.4 ± 0.0 mg/mL) and leaf (IC50 = 0.5 ± 0.0 mg/mL) has higher activity than chloroquine diphosphate, a standard antimalarial drug (IC50 = 0.6 ± 0.0 mg/mL).One of the potential biochemical targets for the development of new antimalarial drugs is heme detoxification. The results of heme detoxification inhibition assay see  show thaIt was reported that free heme is toxic to parasites as it causes lysis of the parasite vacuolar membrane . Thus, mH. annuus root shows the highest antimalarial activity among the other parts of plants, either in in vitro, in vivo, or in heme detoxification inhibition assay. Therefore, the ethanol extract of H. annuus root is a potential source of new antimalarial drugs from natural resources.Based on the entire assay undertaken in this research, it is established that the ethanol extract of H. annuus root has the highest antimalarial activity compared to stems, seeds, flowers, and leaves of H. annuus. 96% ethanol extract of H. annuus root has more potential activity as a curative antimalarial drug than a prophylactic one. Similarly, for the antimalarial mechanism in inhibiting heme detoxification, 96% ethanol extract of H. annuus root has the greatest inhibitory activity compared to the other parts of H. annuus; even the inhibition is greater than chloroquine, a standard drug. So, it can be concluded that the 96% ethanol extract of H. annuus root contains a potential antimalarial substance which is a new source for the development of a new plant-based antimalarial agent.Based on the results of the study, they show that 96% ethanol extract of


Venenum Bufonis, exerts antitumor effects but has low bioavailability and adverse effects when administered as a single agent. The purpose of this study was to evaluate the physical and chemical properties, antitumor efficacy, general pharmacology, acute toxicity, and tissue distribution profile of bufalin-loaded PEGylated liposomes (BF/PEG-LP), which were prepared in a previous study. To evaluate the safety of the preparation, a red blood cell hemolysis test was performed, which indicated that the hemolysis rate of BF/PEG-LP was significantly lower than that of bufalin alone. Cell viability assay revealed that the blank liposomes were nontoxic. In an in vitro experiment, BF/PEG-LP dose-dependently induced the apoptosis of HepG2, HCT116, A549, and U251 cancer cells, with half-maximal inhibitory concentration (IC50) values of 21.40 ± 2.39, 21.00 ± 3.34, 43.39 ± 6.43, and 31.14 ± 2.58 ng/mL, respectively, at 24 h. Tumor xenograft experiments in nude mice showed that BF/PEG-LP significantly inhibited the growth of U251 cells. Pharmacological evaluation revealed that BF/PEG-LP impacted the general behavior, independent activities, and coordination of mice after a week of administration compared with those of mice in the control group. In an acute toxicity test, the median lethal concentration (LD50) of BF and BF/PEG-LP in mice was 0.156 and 3.03 mg/kg, respectively. Tissue distribution profiles showed that the BF concentration in brain tissue was 20% higher, whereas that in heart tissue was 30% lower when BF/PEG-LP was administered to mice compared with BF. Thus, BF/PEG-LP exhibited lower hemolysis and cytotoxicity and improved pharmacokinetic and antitumor properties compared with bufalin alone, indicating its potential for future pharmacological application, particularly for glioma treatment.Bufalin, derived from The online version of this article (10.1186/s11671-019-3057-0) contains supplementary material, which is available to authorized users. Venenum Bufonis, has anti-inflammatory, cardiotonic, antiviral, acesodyne, and antitumor activities [Bufalin -bufadienolide, 5β,20(22)-bufadienolide-3β, 14-diol), extracted from tivities ..) injection. In the BF-treated group, almost no binuclear kernels and mitosis were observed; in addition, nucleoli were smaller, and cell morphology was more regular than in the control group. Although BF has many potential pharmacological effects and the results obtained thus far are promising, BF as a single agent is still far from clinical application. Furthermore, BF treatment is associated with undesirable adverse and side effects such as immunoreaction, injury of normal tissues, and high toxicity [Lan et al.  previoustoxicity .Several drug delivery systems, such as lipid emulsions, nanoparticles, and liposomes, have been employed to promote the solubility, pharmacological activity, and bioavailability of BF , 11. MorIn previous research, we prepared PEGylated liposomes loaded with bufalin (BF/PEG-LP) and characterized the same, including its cytotoxicity and pharmacokinetic profiles, in an attempt to overcome the abovementioned challenges, increase the efficacy of drug delivery, and decrease toxicity. To extend that research, we further evaluated the physical and chemical properties, antitumor efficacy, acute toxicity, and tissue distribution profiles of BF/PEG-LP.BF and cinobufagin (CBG) were purchased from BaoJi Chenguang Technology Development Co., Ltd.  and dissolved in DMSO for storage at − 20 °C as stock solutions. CBG was used as the internal standard (IS). Doxorubicin (DOX) was purchased from Beijing Solarbio Science & Technology Co., Ltd.  and dissolved in DMSO for storage at − 20 °C as stock solutions. BF/PEG-LP were prepared in our laboratory  [Male Sprague-Dawley rats, weighing approximately 230–250 g; Kunming mice, weighing 18–22 g; and 6-week-old nude Balb/c mice were supplied by the Experimental Animal Research Center, Fourth Military Medical University . The animals were kept separately in an individually ventilated cage (IVC) system and fed standard laboratory food and water ad libitum for 5 days. All animals were fasted (except for water) overnight before the experiments. Experimental procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University .Calibration curves were prepared by spiking 100 μL of tissue homogenate with 20 μL each of the working solutions to produce samples containing 20, 50, 200, 500, and 2000 ng/mL of BF. The spiked standard homogenate samples were prepared in advance and evaluated with each analytical batch of unknown samples.g for 5 min to remove the RBCs and fragments thereof. The supernatant was precipitated in 2 mL ethanol/hydrochloric acid solution :37% hydrochloric acid (w/v)). The supernatant was centrifuged at 750g for 5 min, after which the absorbance was measured at 398 nm by a UV spectrophotometer. A positive control consisting of distilled water and a negative control consisting of 0.9% saline were prepared and measured in the same manner.Eight milliliters of blood was collected from rats and subsequently heparinized and diluted with 10 mL 0.9% normal saline. The whole blood samples were then mixed with 100 μL of BF solution or 1 mL of BF/PEG-LP suspension. After 40 min of immersion in a water bath maintained at 37 °C, the samples were centrifuged at 750The hemolysis rate (HR%) of the nanoparticle suspension was calculated as follows:Asample is the absorbance of the sample, Anegative is the absorbance of the negative control, and Apositive is the absorbance of the positive control.where 4 cells/well) were inoculated into a 96-well plate and cultured by RPMI-1640 culture medium until the logarithmic phase. Different concentrations of blank liposomes were added to the culture medium. Six replicate wells were prepared for each concentration. After incubation for 24 h, the medium was discarded, and 100 μL of CCK-8 working solution  was added. After 2 h of incubation, the absorbance was detected and quantified at 450 nm by a microplate reader .CCK-8 was used to detect the cytotoxicity of blank liposomes in human hepatocellular carcinoma HepG2 cells and human colon cancer HCT116 cells. Suspended HepG2 cells and HCT116 cells  at pH 7.4 and pH 5.0, respectively. The liposomes were stored at 4 °C under dark conditions. The absorbance of samples at 296 nm was measured at 0, 5, 15, 30, 60, and 120 min.4 cells/well in 96-well plates at 37 °C with 5% (v/v) CO2. After 24 h, different concentrations of BF/PEG-LP were added to the wells. After another 24 h of incubation, the cell medium was discarded, and 100 μL of CCK-8 working solution was added. After 2 h of incubation, the absorbance was detected and quantified at 450 nm by a microplate reader (Bio-Rad 680). The percentage of viable cells and IC50 values were estimated from a dose-response curve. Values of each sample and control were determined by the average of six replicate wells.Cell proliferation was evaluated by CCK-8 proliferation assay. HepG2, HCT116, A549, and U251 cells were individually seeded at a density of 1 × 107) in 0.15 mL PBS were inoculated subcutaneously in nude mice and allowed to grow for 7 days to reach a tumor size of more than 50 mm3. Then, mice were randomly divided into two groups with six mice each. In the treatment group, 0.5, 1.0, or 2.0 mg/kg/day of BF or BF/PEG-LP was i.p. injected for 21 consecutive days. PBS containing 0.1% DMSO was injected in the vehicle control group, while 2 mg/kg of cisplatin (DPP) was injected in the positive control group.U251 cells . The growth and necrosis of tumor cells and infiltration of surrounding tissues were observed under light microscopy.n = 10 per group) that were i.p. administered the following at a single dose: saline at the volume administered to the control group, 20 mg/kg pentobarbital sodium as the positive control group, and 1.0, 1.5, and 2.0 mg/kg as the low-, medium-, and high-dose BF/PEG-LP groups. The general behavior, posture, gait, salivation, myofibrillation, nystagmus, and secretions of mice were then observed. After 1 h of administration, the mice were placed in an open-field box. The number of mice moving in the box was counted for 10 min after the mice were allowed to adapt for 1 min.Fifty Kunming mice were randomly divided into five groups  that were i.p. administered the following: saline at the volume administered to the blank control group, 2.5 mg/kg chlorpromazine hydrochloride as the positive control group; and 1.0, 1.5, and 2.0 mg/kg BF/PEG-LP as the low-, medium-, and high-dose BF/PEG-LP groups, respectively.The mice were randomly divided into five groups  was placed perpendicularly to the ground and fixed at the bottom to keep upright. After 1 week of treatment, the climbing test was performed for 30, 60, and 120 min, respectively. The mice were placed on the top of the metal rod and allowed to crawl down naturally. The coordination of mice as they climbed down the metal rod was observed and scored. The scoring criteria were as follows: 0 points, step-by-step descent; 1 point, sliding descent; 2 points, inability to descend; and 3 points, no reflex observed.50) of BF and BF/PEG-LP was calculated to evaluate acute toxicity. A total of 110 Kunming mice  were randomly divided into 11 groups (n = 10). Five groups received a single i.v. dose of BF at 1.0, 0.37, 0.14, 0.05, or 0.02 mg/kg, and five groups received a single i.v. dose of BF/PEG-LP at 4.0, 2.83, 2.0, 1.41, or 1.0 mg/kg. The injection volume was 0.2 mL. BF was first dissolved in normal saline with 1% DMSO; BF and BF/PEG-LP were then diluted with normal saline to obtain the desired concentration. The control group was administered normal saline containing 1% DMSO. After treatment, the general behavior of the mice was observed for 14 days, and the death rate was recorded. The pathological changes in the mice after i.v. administration were observed by optical microscopy .The median lethal concentration (LDn = 6 per group). These two sections were i.p. administered 0.5 mg/kg BF and BF/PEG-LP, respectively. Samples of major tissues, including the heart, liver, spleen, lung, kidney, and brain, were collected at 5, 15, 45, and 90 min after administration. Tissue samples were quickly rinsed with saline solution (0.9%), removing the blood and contents as appropriate. After cleaning, filter paper was used to blot the samples. The samples were weighed and then homogenized in 0.9% saline solution . The obtained homogenates were stored at – 80 °C until analysis.Sprague-Dawley rats were randomly assigned to two sections  though vortex-mixing for 5 min and centrifugation at 12000 g for 10 min. Then, a 10-μL aliquot of the supernatant was injected into the HPLC system . The optimized chromatograph conditions for detection of BF and IS were presented previously [The target compounds were extracted, and the interference of endogenous protein was eliminated by a liquid-liquid preparation method . We founn = 3), and differences between formulations were compared by one-way analysis of variance (ANOVA), using GraphPad Prism 5 software. P < 0.05 denotes significance in all cases.All results were expressed as mean ± SD . When the concentration of BF/PEG-LP was increased to 200 μg/mL, the hemolysis rate was obviously higher than that of blank liposomes  revealed that BF/PEG-LP had a uniform spherical shape, as shown in Additional file P < 0.01).The effect of environmental pH on the stability of BF/PEG-LP was evaluated by UV spectrophotometry. Figure 50 values of BF/PEG-LP for all tested cells are presented in Table 50 values, the sensitivity of cells to BF/PEG-LP was ranked as follows: HCT116 > HepG2 > U251 > A549.Apoptosis in HepG2, HCT116, A549, and U251 cells induced by BF/PEG-LP was determined by CCK-8 assay. The ICP < 0.05). As shown in Fig. P < 0.01), while chlorpromazine had a significant promoting effect on the positive control group (P < 0.05), as shown in Fig. After administration of BF/PEG-LP, the general behavior of mice was different from that of the blank group. In particular, the mice in the high-dose BF/PEG-LP group exhibited tremors and salivation. After administration, the mice remained in the open-field box for 10 min, and the number of active grids was recorded. In the positive control  group, the number of active grids was only 342 ± 35, which was significantly lower than that in the control group , as shown in Fig. P < 0.01), as shown in Fig. With prolonged administration, mice in the positive control, BF, and BF/PEG-LP groups showed mental retardation, reduced food intake, slow reaction, and dull fur. Mouse death occurred in both the BF and BF/PEG-LP groups. The antitumor rate of the positive control group was 36.5%; that of the high-dose BF group was 27.6%; and that of the low-, medium-, and high-dose BF/PEG-LP groups was 11.4%, 28.9%, and 38.7%, respectively. Except for the low-dose BF group, there was a significant difference between the antitumor rates of the drug-treated groups and the negative control group ; this effect was attributed to the liposomal formulation, which controlled BF release into the blood.The acute toxicity of a single i.v. administration were observed by optical microscopy , was used to assay the selectivity of the method, as shown in Additional file . injection of BF/PEG-LP, the tissue distribution thereof is influenced by several variables, such as composition, particle size, and zeta potential [. administration, the concentration of BF in both groups was very low in some tissues, such as the heart, liver, spleen, lung, and kidney, indicating that the distribution and elimination of BF occurred quickly. The concentration of BF in the brain, though not other tissues, could still be detected at 90 min after i.v. administration in both groups, indicating that the distribution and elimination of BF were relatively slower in the brain than other tissues.The raw data of the tissue distribution experiments are shown in Additional file otential . The conP < 0.01). A similar phenomenon, which is mainly caused by a dramatic increase in BF in the blood before metabolism and excretion, was observed in previous studies [Meanwhile, 1.20-fold higher BF (333 ± 92.5 ng/g at 5 min) was detected in the brain tissues of the BF/PEG-LP group compared with that in the BF group. In addition, a higher concentration of BF was found in the liver, spleen, and brain tissues in the BF/PEG-LP group. Significant accumulation of BF was detected in the liver, with concentrations of 187 ± 31.0 ng/g in the BF/PEG-LP group and 163 ± 35.0 ng/g in the BF group .According to the results of the tissue distribution experiments, BF itself could traverse the BBB, possibly owing to its high lipid solubility, low molecular weight 386.52 g/mol), and chemical structure, which is similar to that of cholesterol, with which it shares the same mother nuclear structure. However, the clinical application of BF is limited because of its high lipid solubility and poor water solubility. The bioavailability of BF is low if not administered in an alternative form. In a previous study [.52 g/molvs. 134 ± 17.8 ng/g at 5 min, P < 0.01; 15.32 ± 5.56 vs. 63.79 ± 28.21 ng/g at 15 min, P < 0.01). Whether cardiac toxicity can be attenuated with a lower dose of BF requires further verification. Nevertheless, the antitumor effect of BF was sufficiently strong in this study . For in vivo application, it is critical to decrease the toxicity  and increase the water solubility of BF, which was our focus in this study.In a previous study, BF was demonstrated to exert a digitalis-like effect on the heart, increasing the heart rate and myocardial contractive power in rat . TherefoIn general, PEGylated liposomes are considered to have no or low immunogenicity. However, according to recent studies, when PEGylated liposomes were repeatedly injected into the same animal, an immune response was triggered . In factIn the present study, we evaluated hemolysis induced by BF/PEG-LP and the cytotoxicity of blank liposomes to verify the safety thereof. Moreover, the effect of environmental pH on the release of BF from liposomes was investigated to assess the stability of BF/PEG-LP. BF encapsulation in a liposomal suspension might affect its antitumor activity in vitro and in vivo and general behavior, acute toxicity, and tissue distribution in vivo. Nonetheless, we believe that the development of the liposomal formulation described in this study has laid a foundation for the future clinical application of BF. Intensive investigation of the relevance of the ABC phenomenon to BF/PEG-LP is warranted in the future.Additional file 1:Figure S1. Characterization of PEGylated liposomes containing bufalin. Figure S2. Representative chromatograms of BF and CBG (IS) in rat tissue homogenate samples. Table S1. The precision, accuracy and recovery experiments of bufalin in biological samples (n = 5). Table S2. The stability experiments of bufalin in biological samples (n = 5). Table S3. The tissue distribution of BF in rat organs. (DOCX 822 kb)