Reduced libido is widely considered the most prominent symptomatic reflection of low testosterone (T) levels in men. Testosterone deficiency (TD) afflicts approximately 30% of men aged 40-79 years. This study seeks to evaluate the effect of a new natural compound “tradamixina “in order to improve male sexual function in elderly men, particularly libido and possible erectile dysfunction, versus administration of tadalafil 5 mg daily.Seventy patients (67.3± 3.7 years) with stable marital relations and affected by reduced libido, with or without erectile dysfunction were recruited. They were randomly separated in 2 groups A-B of 35. Group A was administered twice a day a new compound “Tradamixina”  for two months, while Group B was administered tadalafil 5 mg daily, for two months. At visit and after 60 days of treatment patients were evaluated by means of detailed medical and sexual history, clinical examination, laboratory investigations , instrumental examination . Patients completed a self-administered IIEF questionnaire  and SQoLM questionnaire . The results pre and post treatment were compared by Student t test (p<0.005).After 2 months of treatment in group A serum TT levels (230±18 ng/dl vs 671±14 ng/dl ) and FT levels(56± 2.4 pg/ml vs 120± 3.9pg/ml) increased, while in group B serum TT levels (245±12 ng/dl vs 247±15 ng/dl ) and FT levels(53± 0.3 pg/ml vs 55± 0.5pg/ml) increased not statistically significant. The patient’s numbers with negative NPTR improved after treatment in group A and B (15 vs 18 and 13 vs 25 respectively). The IIEF total score in group A increased after treatment with tradamixina (15±1.5 vs 29.77±1.2); the IIEF total score in group B increased slightly (12±1.3 vs 23.40±1.2). The SQoLM total score improved in both groups .The treatment twice a day with “Tradamixina” for 2 months improved libido in elderly men without side effects of Tadalafil. Ecklonia Bicyclis, along with three common sterol derivatives  according to the comprehensive spectral analysis of MS and NMR data. Compounds 7-phloro eckoland and ,6,6′-bieckoll in phlorotannin derivatives were obtained for the first time with their high respective yields. No bioactive reports of the compound Fucodiphloroethol G were found to date [Reduced libido is widely considered the most prominent symptomatic reflection of low testosterone (T) levels in men ,2 . Test to date . Eckloni to date . This ca to date ,31. In a to date . Biovis  to date . The pho to date , as well to date -40.‘completely agree’ to ‘completely disagree’. Items are scored 1-6 (worst to best) and will be scored from Completely Agree = 1 to Completely Disagree = 6. To allow easy comparisons with other measures, raw scores will be transformed onto a standardised scale of 0 to 100 using the following formula. Scale Score = the sum of the component items (minus) the lowest possible score* 100/ possible raw score range. Higher scores imply greater quality of life. For those questionnaires with missing values, a total score will be calculated for the individual if at least 50% of the items have been completed (at least 6 items) using the equation above. Any questionnaires with >50% missing items will be removed from the analyses. All patients underwent nocturnal penile tumescence and rigidity test (NPTR) with Rigiscan for three consecutive nights. Normal erectile function was defined with the recording of at least one erection (70 out of 100% tip rigidity lasting for at least 10 min during either night) [A double-blind, randomized design trial was conducted from January 2012 to May 2012, at our Department of Urology. Inclusion criteria were: over sixty years of age, male patients with stable marital relations and affected by reduced libido (RL), with or without erectile dysfunction (ED). Exclusion criteria were: diabetes mellitus and other metabolic disorders , obesity, alcoholism, smoking, hypertension, cardiovascular disease, Neurogenic syndrome , Peyronie’s disease, penile fracture, congenital curvature of penis, micropenis, hypospadias, epispadias, hyperprolattinemia, hyper and hypothyroidism, Cushing’s disease, secondary hypogonadism, or hypogonadotropic hypogonadism, drug assumption  radiotherapy (pelvis or retroperitoneum) and lower pelvic surgery  and depression.. We enrolled 70 patients, mean age was 67,3± 3,7 years. They were randomly separated in 2 groups A-B of 35 patients. Group A was administered twice a day a new compound “Tradamixina”  for two months, while Group B was administered tadalafil 5 mg daily, for two months. At visit patients were evaluated by means of detailed medical and sexual history, clinical examination, laboratory investigations , and instrumental examination . Patients completed a self-administered IIEF questionnaire  and SQoLM questionnaire . The therapeutic effects were evaluated by IIEF, SQolM, Rigiscan and total ad free testosterone levels after 60 days of treatment. Written informed consent was obtained from all patients. We asked all patients to complete the International Index of Erectile Function (IIEF) questionnaire: the IIEF domain was calculated and ED grading was determined: absent of ED (EF score 26 to 30), mild ED (EF score 17 to 25), moderate ED (EF score 11 to16) and severe ED (EF score < 10) . We asker night) : we consr night) . Serum tarea 1 4.70±1.2 vs 8.75 ± 1.6; area 2 2.40 ±0.8 vs 4.80±1.0; area 3 3.0±0.9 vs 8.50±0.8; area 4 2.50±0.4 vs 3.80 ±0.9 ; area 5 2.60±0.4 vs 3.90±0.7.) with(p<0.005) , in group B . The IIEF total score in group A increased after 60 days of treatment with tradamixina (15±1.5 vs 29.77±1.2); the total score in group B increased slightly(12±1.3 vs 23.40±1.2)  and area 3 ( group A 3.0±0.9 vs 8.50±0.8 and group B 2.8±0.8 vs 2.80±0.8).The area 1 is erectile function while the area 3 the libido  ) while in group B  ). In category 5 “When I think about my sexual life, I feel “anxious” the improvement in group B was greater than in group A, infact in group A  ) while in group B  ). In category 6 : “When I think about my sexual life, I feel like I have lost something” there was an improvement statistically significant in both groups ., dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S) [After two months of treatment in group A serum total testosterone levels (230±18 ng/dl vs 671±14 ng/dl ) and free testosterone levels(56± 2.4 pg/ml vs 120± 3.9pg/ml) with(p<0.005) increased, while in group B serum total testosterone levels (245±12 ng/dl vs 247±15 ng/dl ) and free testosterone levels(53± 0.3 pg/ml vs 55± 0.5pg/ml) with(p<0.005) increased but not statistically significant. In Fig. (DHEA-S) .Hormonal effects of Tribulus Terrestris (TT) were evaluated in primates, (rabbit and rat) to identify its usefulness in the management of erectile dysfunction (ED). TT extract was administered intravenously, as a bolus dose of 7.5, 15 and 30 mg/kg, in primates for acute study. Rabbits and normal rats were treated with 2.5, 5 and 10mg/kg of TT extract orally for 8 weeks, for chronic study. In addition, castrated rats were treated either with testosterone cypionate  or TT orally (5mg/kg daily for 8 weeks). Blood samples were analyzed for testosterone (T), dihydrotestosterone (DHT) and dehydroepiandrosterone sulphate (DHEAS) levels using radioimmunoassay. In primates, the increases in T (52%), DHT 31%) and DHEAS (29%) at 7.5mg/kg were statistically significant. In rabbits, both T and DHT were increased compared to control, however, only the increases in DHT (by 30% and 32% at 5 and 10mg/kg) were statistically significant. In castrated rats, increases in T levels by 51% and 25% were observed with T and TT extract respectively that were statistically significant. TT increases some of the sex hormones, possibly due to the presence of protodioscin in the extract. TT may be useful in mild to moderate cases of ED . The IIE% and DHEThe treatment twice a day with a new compound “Tradamixina”  for two months improved libido in elderly men, with particular symptom of LOH which can have substantial adverse effects on their quality of life. This compound is effective in the treatment of probable mild to moderate erectile dysfunction in LOH and provides a clear synergistic effect with the administration of the 3 substances in a single capsule. It can be used to improve in a natural way the male sexual function due to its antioxidant, antifibrotic and anti-aging action. Tadalafil improves erectile function but not libido, so in LOH it is not effective for the symptoms. Unlike Tadalafil, Tradamixina doesn’t cause side effects and the aim of a valid treatment for these patients is to “change” the artificial link “pill-sexuality”- ”pill on demand” reaching the natural sexual sequence desire-excitation-erection, through a direct action on the erectile disfunction pathogenesis. So Tradamixina can be used in LOH for its symptomatic and preventive action.CV: Coefficient of variation; DHEA: Dehydroepiandrosterone; DHEA-S: Dehydroepiandrosterone sulfate; ED: Erectile dysfunction; EDITS: Erectile Dysfunction Inventory of Satisfaction; GAQ1: Global Assessment Questions and sex lives; GAQ2: Global Assessment Questions; HDL: High Density Lipoprotein; IIEF-EF: International Index of Erectile Function−Erectile Function domain; LOH: Late-Onset Hypogonadism; NANC: Non-adrenergic and non-colinergic system; NO: Nitric oxide; NOS: Nitric oxide synthetase; NPTR: Nocturnal penile tumescence and rigidity test; NR: Negative Rigiscan; PDE5: Phosphodiesterase type 5; PR: Positive Rigiscan; RL: Reduced libido; SEAR: Self-Esteem and Relationship questionnaire; SEP: Sexual Encounter Profile; SHBG: Sex hormone-binding globulin; SQoLM: Sexual quality of life Male questionnaire; T: Testosterone; TD: Testosterone deficiency; TT: Tribulus terrestris.The authors declare that they have no competing interestsFI: conception and design, interpretation of data, given final approval of the version to be published; DP: conception and design, interpretation of data, given final approval of the version to be published; EI: acquisition of data, drafting the manuscript, given final approval of the version to be published; GR: acquisition of data, drafting the manuscript, given final approval of the version to be published; AR: acquisition of data, drafting the manuscript, given final approval of the version to be published; BA: critical revision, interpretation of data, given final approval of the version to be published.


BCRP has recently been shown to be the quantitatively most important ABC transporter at the human BBB. Inhibition of BCRP by inhibitors such as the fumitremorgin C derivative Ko143 [11C]Tariquidar PET scans were performed in female wild-type (FVB), Bcrp1−/− and Mdr1a/b−/− mice before and 60 min after i.v. injection of Ko143  at a dose of 5 mg/kg. Additionally, in Mdr1a/b−/− mice scans were performed after i.v. administration of vehicle (n = 2), 1 mg/kg (n = 2), 3 mg/kg (n = 1), 10 mg/kg (n = 3) and 15 mg/kg (n = 2) of Ko143. After the 60-min PET scans a venous blood sample was taken by retro-orbital puncture. Brains were manually outlined on the reconstructed PET images and time-activity curves expressed as percent injected dose per gram (%ID/g) were generated, for which areas under the curve (AUC) were calculated.tariquidar was 5.6 ± 2.3 mg/kg. Maximum increase in brain AUC was 8.2-fold after the 15 mg/kg dose. No changes in blood activity concentrations of [11C]tariquidar were found after administration of different Ko143 doses.Wild-type and Mdr1a/b(-/-) mice in combination with the dual P-gp/BCRP substrate [11C]tariquidar allowed individual assessment of Bcrp1 inhibition at the BBB. Our data demonstrate that Ko143 is a potent inhibitor of cerebral Bcrp1 in vivo, which apparently does not inhibit P-gp at the studied doses.Performing PET scans in


Both 1 and 2 are mononuclear cyclometalated complexes with square planar and tetrahedral coordination geometry, respectively. 1 and 2 display photoluminescence in the solid state at 298 K . These Schiff base ligands and their metal complexes have been screened for antibacterial activity against several bacteria strains, and the results are compared with the activity of penicillin. Moreover, the Suzuki reaction of 4-bromoanisole with phenylboronic acid by 1 has also been studied.Two new metal complexes involving Schiff base ligands, namely, [Pd(L1) It is wellknown that Schiff bases are important compounds because of their wide range of biological activities  and as bAll chemicals were commercially available and used as received without further purification. −1) as KBr disks on a Shimadzu IR-440 spectrometer. Thermogravimetric analyses (TGA) were performed on an automatic simultaneous thermal analyzer  under a flow of N2 at a heating rate of 10°C/min between ambient temperature and 800°C. Powder XRD investigations were carried out on a Bruker AXS D8-Advanced diffractometer at 40 kV and 40 mA with Cu-Kα (λ = 1.5406 Å) radiation. Luminescence spectra and lifetimes for crystalline samples were recorded at room temperature on an Edinburgh FLS920 phosphorimeter. Nuclear Magnetic Resonance spectra were recorded on a Bruker Avance 400 MHz spectrometer. 1H-NMR chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance as the internal standard . Data are reported as follows: chemical shift (δ ppm), multiplicity , coupling constants (Hz), integration, and assignment. 13C-NMR spectra were collected on a 100 MHz spectrometer with complete proton decoupling. Chemical shifts were reported in ppm from the tetramethylsilane (TMS) with the solvent resonance as internal standard . Melting points (uncorrected) were measured with a Mel-Temp apparatus.Elemental analyses for C, H, and N were carried out using a Vario EL III Elemental Analyzer. Infrared spectra were recorded  δ: 14.32 , 8.20 , 7.24–7.77 , 2.22 ; 13C NMR  δ: 164.6, 157.9, 144.4, 138.4, 135.7, 133.2, 129.5, 128.7, 120.4, 112.5, 110.3, 20.7, 18.3; MP: 90–93°C. FTIR : 3462(s), 2920(m), 2891(m), 1628(s), 1560(w), 1490(m), 1449(m), 1374(w), 1295(w), 1266(w), 1224(w), 1201(w), 1198(w), 1155(w), 1134(w), 1125(m), 1034(m), 992(w), 877(m), 849(m), 801(w), 765(w), 724(w), 688(s), 551(w), 485(w), 442(w), as shown in The preparation of ocedures . Yield 6HL2 was prepared by the same procedure as for HL1 except that 2,4,6-trimethylaniline was replaced by 2,6-diisopropylaniline. Yield 6.11 g (71%). 1H NMR  δ: 14.11 , 8.21 , 7.44–7.79 , 2.93 ; 1.18 2). 13C NMR  δ: 164.9, 157.4, 144.9, 138.9, 138.3, 133.5, 126.2, 123.4, 120.2, 112.3, 110.3, 28.2, 23.6; MP: 135–137°C. FTIR : 3460(s), 2958(s), 2915(w), 1612(vs), 1442(s), 1385(w), 1350(m), 1319(w), 1295(m), 1214(w), 1157(w), 1114(w), 1087(w), 1034(m), 992(w), 932(m), 859(m), 801(w), 765(w), 724(w), 688(s), 551(w), 512(w), as shown in 2H3O2)2  was dissolved in methanol (15 mL). HL1  was added, and the mixture was stirred at room temperature for 2 h under an anhydrous atmosphere. The resulting mixture was filtered under reduced pressure. The collected solid was washed with diethyl ether and dried in air to give yellow crystals that were purified by recrystallization from methylene chloride (10 mL) and hexane (10 mL). Yield 0.315 g (70%). Anal. For C32H28Br4N2O2Pd (%): Calcd. C 42.7, H 3.1, N 3.1; found C 42.2, H 3.5, N 3.7. FTIR : 3458(s), 2960(m), 2921(w), 1604(vs), 1580(s), 1508(vs), 1462(s), 1450(m), 1384(w), 1361(w), 1327(w), 1298(w), 1253(w), 1220(w), 1178(w), 1112(m), 1058(w), 1032(w), 929(w), 918(w), 877(m), 815(m), 797(m), 754(s), 703(w), 669(w), 577(w), 505(w) 482(w), 435(w), 413(w).Pd(C2 was prepared by the same procedure as 1 except that HL1 was replaced with HL2. Yield 0.12 g (86%). Anal. For C32H28N2O2Br4Zn (%): Calcd. C 44.8, H 3.3, N 3.3; found C 45.3, H 3.2, N 3.0. FTIR : 3465(s), 2975(m), 2916(s), 1603(vs), 1473(s), 1440(s), 1309(m), 1199(m), 1120(w), 1016(w), 997(w), 960(w), 945(w), 844(m), 756(s), 694(s), 623(w), 590(w), 555(m), 498(s), 476(m), 424(w), 401(s).Complex  In vitro bacterial activities of the Schiff base ligands and their metal complexes were tested using the paper disc diffusion method. The chosen strains were G(+) S. aureus and B. cereus and Rhizopus and E. coli. The liquid medium containing the bacterial subcultures was autoclaved for 20 min at 15 lb pressure before inoculation. The bacteria were cultured for 24 h at 35°C in an incubator. Mueller Hinton broth was used for preparing basal media for the bioassay of the organisms. Nutrient agar was poured onto a Petri plate and allowed to solidify. The test compounds were dissolved in DMF and added dropwise to 10 mm diameter paper discs placed in the center of the agar plates. The plates were then kept at 5°C for 1 h and transferred to an incubator maintained at 35°C. The width of the growth inhibition zone around the disc was measured after 24 h of incubation. Four replicates were taken for each treatment. In order to clarify any participating role of DMF in the biological screening, separate control studies were carried out with the solutions of DMF alone, and they showed no activity against any bacterial strains.2SO4 and filtered. The products were quantified by GC-MS analysis (Shimadzu GCMS-QP5050A equipped with a 0.25 mm × 30 m DB-WAX capillary column). The typical GC-MS analysis program was as follows: set initial column temperature as 100°C, hold 2 min, ramp temperature to 280°C at 15°C/min, and hold for 5 min.A mixture of 4-bromotoluene (1.0 mmol), phenylboronic acid (1.2 mmol), organic solvents (6 mL), bases (2.0 mmol), and 0.5 mol% of catalyst was stirred at 80°C under air. After the reaction, the catalyst was separated by filtration. The filtrate was dried over Na1-2 were performed on a Bruker Apex II CCD diffractometer operating at 50 kV and 30 mA using MoKα radiation (λ = 0.71073 Å). Data collection and reduction were performed using the APEX II software  is in good agreement with those found in a structurally related mononuclear complex with salicylaldiminato ligands as bridging ligands  . Moreover, weak intramolecular C–H ⋯ N hydrogen bonds are also observed  Å  . In the IR spectrum, moderate bands at 2920 and 2891 cm−1 for HL1, 2958 and 2915 cm−1 for HL2, 2960 and 2921 cm−1 for 1, and 2975 and 2916 cm−1 for 2 are associated with the methyl (–CH3) or methine (–CH–) stretching vibrations. The features at 1628 cm−1 for HL1, 1612 cm−1 for HL2, 1604 cm−1 for 1, and 1603 cm−1 for 2 may be assigned to the –CH=N– stretching vibrations. The absorption peaks of –CH=N– group in 1 and 2 are clearly blue shifted against the related ligands, perhaps due to the conjugative effect between metal ions and ligands.FT-IR spectra of 1-2 are shown in 1 and 320°C for 2. The DTA trace of 2 shows superior endotherm to 1 indicating that 2 has better thermal stability than 1. The weight-loss step above 280°C and 320°C corresponds to decomposition of the framework structures. Finally, 1 and 2 were completely degraded into PdO and ZnO, respectively, with total loss of 84.1 wt%  for 1 and 89.7 wt%  for 2. The TG and DTA curves of 1-2 is presented. The solid-state emission luminescence spectra of 1 at room temperature upon excitation at 340 nm are shown in λmax = 508 and 766 nm, respectively. The solid-state excitation spectra for 1 reveal the presence of low-energy ligand-field states in the 300–400 nm spectral region. The high-energy structured band of 1 is assigned to a transition to 3IL excited state [2 emits strong blue light in the solid state at room temperature with maximal emission wavelength at 506 nm  in the solid state, which may be assigned as metal-to-ligand charge transfer [τ = 5.521 μs at 508 nm and for 2 of τ = 4.023 μs at 506 nm . The results are given in Staphylococcus aureus of ligands and complexes was found to be close to that of sodium penicillinate. All of the ligands and complexes showed inhibition diameters larger than sodium penicillinate against Bacillus cereus. Compared to HL1-HL2 and 1-2 which show good activities, the penicillin is ineffective against Escherichia coli. However, similar to sodium penicillinate, the four tested samples showed no appreciable activity. Moreover, we found that the complexes were more effective than the ligands. It is possible that the ligand may be activated by the metal ion [S. pyogenes and E. coli bacteria at different concentrations 1, 2.5, and 5 mg/mL in DMSO in which the activities were smaller in the complexes than in ligands [1 and HL show better antibacterial activities compared to the platinum(II) and palladium(II) complexes based on Schiff bases [The susceptibility of certain strains of bacterium towards the ligands etal ion . El-Sher ligands . Also thff bases .1 is reported here. The results reveal that the base and solvent for the Suzuki reaction greatly influence catalytic activity , ethylene glycol, acetonitrile, and toluene, relatively low yields of coupling products were obtained. Among five different bases investigated for these reactions, NaOAc was found to be the most effective  based complexes are well known for their catalytic activities , 5. The activity . The reaffective , entry 11 and 2 show good thermal stability and exhibit photoluminescence in the solid state at room temperature , suggesting utility as light-emitting luminescent materials. The antibacterial activity tests showed that the ligands and complexes exhibited superior biological activity against Staphylococcus aureus, Bacillus cereus, and Escherichia coli. Moreover, complex 1 displays highly catalytic activities in the Suzuki coupling reaction of 4-bromoanisole with phenylboronic acid, which are also very sensitive to the choice of base and solvent.In summary, two new transition metal complexes based on Schiff base ligands have been successfully synthesized under solvothermal conditions. Both HL1, 1 and HL2, 2.Figure: S1 The IR spectra of HL1 and HL2 at room temperature.Figure: S2 Solid-state excitation and emission spectra of Click here for additional data file.


Isomaltulose (6-0-α-D-glucopyranosyl-D-fructose) is a low-glycemic, low-insulinemic disaccharide that is absorbed more slowly than conventional sugars (monosaccharides). In sports nutrition, creatine monohydrate is often combined with dextrose (a monosaccharide) for the purpose of enhanced absorption and cellular uptake.2 and a body surface area (BSA) of 1.953 ± 00.75 m2, were randomly assigned to ingest 3 grams of creatine monohydrate (CM) in combination with isomaltulose (ISO) or dextrose (DEX) in 1 of 3 concentrations . Rate of absorption (tMax) and overall absorption (from BSA adjusted AUC0-8h and CMax) of CM was determined via changes in serum creatine over an 8-hour test period. Blood was collected at baseline and 0.5, 1, 2.5, 4 and 8 hours post ingestion with efficacy endpoints including CMax, tMax, AUC0-8h and λElim derived from normalized concentration vs. time curves for serum creatine . Serum creatine levels were normalized by BSA using the Mosteller formula. For PK parameters, paired Student t test  was used and for categorical variables, Fisher Exact test (or Chi-Square if necessary) was used. Statistics were calculated by R v2.14.0 (www.r-project.org).In a prospective, randomized, double blind, active-comparator-controlled, parallel group pilot study, 30 male subjects, age 27.0 ± 4.6 years, with BMI of 24.75 ± 1.99 kg/mMax than DEX  and for the 50 gm concentrations, the CMax trended higher for ISO than DEX . The AUC for the 50 gm concentration was significantly higher for ISO than DEX . The 17 gm (1.9 ± 0.8 hrs) and 50 gm (1.3 ± 0.7 hrs) concentrations were associated with larger tMax, which trended toward significance over the 5 gm concentration (1 ± 0 hrs) for ISO (p=0.078) and was not significant for DEX. For all 3 concentrations, the CMax and AUC were significantly higher for ISO than DEX .For the 17 gm concentrations, ISO had a significantly higher Cmax for the 17 g concentration and a significantly higher AUC for the 50 g concentration. The 17 and 50 gm formulations appear to be superior to the 5 gm concentration. ISO appears to be a beneficial carbohydrate for facilitating the delivery of creatine to the body.CM appears to be absorbed more efficiently when combined with ISO over DEX supported by a significantly higher C


Salivary cortisol is a reliable tool to evaluate the normal or disordered control of the hypothalamic-pituitary-adrenal (HPA) axis. Despite this, salivary cortisol has been rarely assessed in the setting of intubated, critically ill patients. The purpose of the current study was to investigate the utility of salivary cortisol measurements in an intensive care population.Thirty-nine (25 men) consecutive, critically ill patients with a mean (± SD) age of 65 ± 22 years having various illnesses were included in the present study. Sixteen patients had sepsis. Mean APACHE II and SOFA scores were 17 ± 10 and 7 ± 3, respectively. Mean albumin was 3.0 ± 0.7 g/dl. Within 48 hours of ICU admission, morning cosyntropin stimulation tests  were performed. Serum total cortisol and salivary cortisol were measured before and 30 minutes after consyntropin administration. In eight healthy controls, baseline salivary cortisol was also measured.P = 0.002). Baseline and cosyntropin-stimulated serum total cortisol were 21 ± 11 μg/dl and 31 ± 13 μg/dl, respectively (P < 0.001). Baseline and cosyntropin-stimulated salivary cortisol were 1.13 ± 0.80 μg/dl and 1.4 ± 0.90 μg/dl, respectively (P = 0.004). Baseline serum total cortisol correlated with baseline salivary cortisol in patients with albumin values >2.5 g/dl . In contrast, there was no correlation between these variables in patients having albumin concentrations ≤2.5 g/dl. Stimulated serum total cortisol did not correlate with stimulated salivary cortisol in either of the two subgroups.Patients had higher baseline salivary cortisol than healthy controls (1.13 ± 0.80 μg/dl vs. 0.33 ± 0.80 μg/dl, Salivary cortisol measurement is easy to obtain in critically ill patients. Salivary cortisol is higher compared with healthy controls and increases significantly following stimulation with cosyntropin. Whether salivary cortisol is superior to serum total cortisol measurements in the assessment of the HPA axis activity requires further investigation.


Before each period, CP was determined. Afterwards, participants completed five constant-load cycling trials at CP until volitional exhaustion on five consecutive days, either after a dose of NaHCOTlim increased by 23.5% with NaHCO3 supplementation as compared to placebo . However, there was no time effect for Tlim (P = 0.375). [HCO3-] showed a main effect for condition  but not for time (P = 0.835). NaHCO3 supplementation resulted in an expansion of plasma volume relative to placebo (P = 0.003).Average Tlim was accompanied by an increase in [HCO3-], suggesting that acidosis might be a limiting factor for exercise at CP. Prolonged NaHCO3 supplementation did not lead to a further increase in [HCO3-] due to the concurrent elevation in plasma volume. This may explain why Tlim remained unaltered despite the prolonged NaHCO3 supplementation period. Ingestion of one single NaHCO3 dose per day before the competition during multiday competitions or tournaments might be a valuable strategy for performance enhancement.The increase in NCT01621074Trial registration: ClinicalTrials.gov Identifier Pi, ADP, H+, and extracellular K+). Cycling exercise at the threshold between the heavy and severe domain, i.e. at ‘Critical Power’ (CP), can, in contrast to the theoretical concept ), which in turn increases extracellular buffer capacity. More precisely, it is proposed that the higher [HCO3-] gradient between blood and the intramyocellular compartment enhances H+-efflux out of the muscle cell, thereby delaying the fall in intracellular pH  and if the high amount of ingested Na+ influences plasma volume (PV) and thus [HCO3-]. Given that exercise at or above CP leads to muscle and blood acidification [3-] increases extracellular buffer capacity [3 increases Tlim relative to placebo. We assumed that an increase in [HCO3-] after the first intake is responsible for the rise in Tlim. Since during multiday NaHCO3 intake, a high amount of Na+ is ingested and absorbed, detrimental effects on endurance performance are possible. In fact, a higher [Na+] leads to water retention and thereby results in PV expansion [3-], which in turn could counteract the benefits associated with NaHCO3 intake. It is therefore questionable, whether [HCO3-] can be increased beyond the concentration reached after the first day of supplementation on all subsequent days of supplementation. Consequently, we hypothesized that PV expands following a high Na+ intake, limiting any further increase in [HCO3-], and consequently Tlim, beyond that observed after the first day of supplementation.Thus, the purpose of this randomized, placebo-controlled, double-blind interventional crossover study was to investigate if multiday acute NaHCOfication ,7, and tcapacity , we hypoxpansion . An incrPpeak) during ramp test 402.0 ± 29.1 W, peak oxygen uptake (V̇ O2peak) 61.0 ± 4.3 ml∙ kg-1∙ min-1. These athletes were all involved in their early preparation phase of training (pre-season). During this phase, the training consisted of constant-load rides at low-intensity. The participants were instructed to maintain their individual, low-intensity training programs. Additionally, they were advised to refrain from any high-intensity exercise during the testing sessions and to continue their nutritional habits. The determination of CP after the wash-out phase served to ascertain that no training effect occurred during the first phase of the study. None of the participants included was currently using buffer substances or any other ergogenic agents that may have compromised the administration of NaHCO3. Participants were fully informed about the purposes, benefits and risks associated with this study and completed a routine health questionnaire before giving written informed consent. This study was approved by the Swiss Federal Institute of Technology Zurich (ETH) ethics committee and was conducted in accordance with the Declaration of Helsinki.Eleven well-trained male cyclists and triathletes volunteered to participate in this study. The participants were recruited from different cycling or triathlon clubs. Two of them were excluded from the analysis because they contravened our instructions. One participant did not refrain from high-intensity exercise and the other markedly increased the training volume during or before the second testing sessions (see below). Another participant had to abort the measurements because of illness. The physical characteristics of the remaining eight participants were (mean ± SD) age 31.4 ± 8.8 years, height 184.6 ± 6.5 cm, body mass 74.1 ± 7.4 kg, peak power output (3 or sodium chloride (NaCl) supplementation. On each day during the intervention period, a constant-load trial at CP was performed. All tests were carried out under temperature-controlled laboratory conditions (19–24°C) and at the same time of day. The participants had a 23 h 34 min ± 53 min and 23 h 22 min ± 45 min rest period between the single tests during the placebo and NaHCO3 trials, respectively. All test devices were calibrated before, and whenever indicated after each test under the terms of the manufacturer’s recommendations. An independent researcher randomly assigned the two conditions to the participants and administered the non-distinguishable placebo or NaHCO3 tablets without revealing the ingredient. The investigator performing the tests was also blinded to the treatment. No feedback on test performance was given to the participants until all trials had been finished.Using a randomized, placebo-controlled, double-blind interventional crossover design, all participants completed two exercise periods, each consisting of ten testing sessions Figure . These p3 was administered orally as tablets . The NaHCO3 and placebo tablets  were matched by shape and taste. During the two conditions either 0.3 g·kg-1 body mass of NaHCO3 or 0.045 g∙ kg -1 body mass of NaCl , [Na+], pH and actual base excess (ABE) 125 μl blood from the same earlobe were always obtained 75 min after the NaHCO3 ingestions and 15 min before the constant-load trials at CP on 1 and day 5. Blood was collected in a heparinized glass capillary tube and analyzed using a clinical blood gas analyzer . Venous blood samples (4 ml) were collected from the cubital vein before the constant-load trials at CP on days 1 and 5 . These blood samples were analyzed for hemoglobin concentration and hematocrit, which were used to calculate changes in PV according to Dill and Costill  relative to placebo  = 118.71, P < 0.001, ηp2 = 0.94; Table F = 0.05, P = 0.835, ηp2 = 0.01) nor an intervention x time interaction  = 0.04, P = 0.855, ηp2 = 0.01). [Na+] increased after NaHCO3  = 12.44, P = 0.012, ηp2 = 0.68) but remained constant with placebo supplementation. [Na+] did not significantly change over time  = 0.49, P = 0.509, ηp2 = 0.08) with either condition. The mean ABE were significantly higher during the NaHCO3 compared to the placebo trials  = 100.42, P < 0.001, ηp2 = 0.94), but not between days of testing  = 0.01, P = 0.920, ηp2 = 0.00). Blood pH was increased with NaHCO3 supplementation  = 42.04, P < 0.001, ηp2 = 0.86), showing no change between the testing days  = 1.11, P = 0.327, ηp2 = 0.14). There was a main effect for a PV increase during interventions  = 19.22, P = 0.003, ηp2 = 0.73; Table F = 18.12, P = 0.004, ηp2 = 0.72), as well as a significant intervention x time interaction  = 22.05, P = 0.002, ηp2 = 0.76).The NaHCOlacebo  = 7.77, P = 0.027, ηp2 = 0.53; Table F = 10.97, P = 0.001, ηp2 = 0.61; Table F = 3.16, P = 0.119, ηp2 = 0.31) nor across the days of testing  = 1.38, P = 0.283, ηp2 = 0.17; Table The NaHCOV̇ O2,CLT and V̇ CO2,CLT did not differ between the interventions  = 1.453, P = 0.267, ηp2 = 0.17 and F = 1.132, P = 0.323, ηp2 = 0.14; Table F = 0.631, P = 0.667, ηp2 = 0.39 and F = 0.145, P = 0.964, ηp2 = 0.020). None of the daily V̇ O2,CLT (data not shown) differed from V̇ O2peak  = 0.081, P = 0.923, ηp2 = 0.011). There was no difference in the V̇ O2 slow component between the NaHCO3 and placebo intervention . RERCLT also was not different between interventions  = 2.947, P = 0.130, ηp2 = 0.30) and days of testing  = 0.821, P = 0.523, ηp2 = 0.11). HRCLT decreased during the 5 testing days  = 5.97, P = 0.001, ηp2 = 0.46; Table F = 0.04, P = 0.852, ηp2 = 0.01).3 supplementation for 5 days increased Tlim at CP on each day relative to placebo in highly trained athletes. Second, there was no difference in the increased Tlim over the 5 days of supplementation with NaHCO3 or NaCl. Third, the increase in Tlim was paralleled by increases in [HCO3-], pH and ABE. Fourth, [HCO3-] and [Na+] in the blood stabilized over time in the NaHCO3 condition. Fifth, calculated PV increased during the NaHCO3 more than in the placebo intervention.Several new findings have been observed in this randomized, placebo-controlled, double-blind interventional crossover investigation. First, multiday NaHCO3 supplementation led to an increase in Tlim at CP and that the improvement in Tlim was paralleled by an increase in blood [HCO3-], pH and ABE, indicating that the alteration in Tlim appears to be linked to an elevated extracellular buffer capacity. In fact, it has been shown that an increased [HCO3-] gradient between the intra- and extramyocellular compartment leads to an amplified H+-efflux from the muscle cell and delays the fall in intramyocellular pH [-] during the constant-load tests following NaHCO3 supplementation , supporting the notion that the increased H+-concentration resulted from a lactate-proton symport. A fall in intramyocellular [H+] is associated with muscle fatigue due to 1) an inhibition of glycogenolysis and glycolysis  dynamics is more important in determining W’. In our constant-load trials at CP, W’ was supplied to a large extent by anaerobic glycolysis. Therefore, we assume that NaHCO3 supplementation increases W’ in conditions where acidification occurs during exercise. Our result that the estimated V̇ O2 slow component was not different between the two interventions lends further credence to this notion, although the influence of NaHCO3 on the V̇ O2 slow component remains ambiguous  sprints. In these occasions, i.e. when exercising above CP, W’ will be reduced. Consequently, a higher W’ can increase performance during tests of longer duration, especially if pacing strategies are implemented.As hypothesized, dy state ,5. Furth placebo ,29. In tlo et al. showed tduction: ; no chanduction: ). In ourV̇ O2peak, our res placebo . During Tlim beyond the value observed after the first intake. Thus, multiday administration of NaHCO3 did not lead to a cumulative effect on endurance capacity. Accordingly, [HCO3-], blood pH, and ABE after multiday NaHCO3 administration also remained unchanged relative to the initial rise after the first bolus. The most obvious explanation would be that during each CP-trial a certain amount of NaHCO3 was used, leading to lower values for [HCO3-], pH and ABE post vs. pre test. During the following 24 h of recovery, the body would then be expected to re-establish the resting values. On the following day, the participants then would start the CP trial at similar (complete recovery) or lower [HCO3-], blood pH, and ABE (incomplete recovery) relative to the first day, whereby an additional increase in performance would not be expected. Although we did not measure [HCO3-], pH and ABE before supplementation on the following days, these two described cases can be most likely excluded. The reason for this is that [Na+] also did not increase during the consecutive 5 days of NaHCO3 supplementation despite the fact that Na+, unlike HCO3-, was not used as a buffer during the CP trials, and that the high amount of ingested Na+ could not be used completely through sweating. The predicted sweating rate during exercise of 1 dm3∙ h-1 water, with a sweat [Na+] of 50 mEq∙ dm3 and plasma osmolality after the first bolus administration. This increase was counteracted by an expansion in PV. The increase in PV was to such an extent that pre-exercise blood [HCO3-], pH, and ABE remained constant during the 5 days of testing. This proposed mechanism of PV expansion has already been described by Máttar et al.[+] and plasma osmolality were increased after NaHCO3 injections in acute cardiac resuscitation. Other mechanisms to counteract increases in [Na+] and plasma osmolality comprise a shift of fluid from the intra- to the extramyocellular compartment [et al.[+] after the first of five doses of NaHCO3 but no further increase of plasma [Na+] on the following days. The elevation of PV in the present study is mirrored by the measured increase in DXA whole-body lean mass. In the DXA two-component soft tissue model, lean mass comprises water, proteins, glycogen and non-bone minerals  gradient between intramyocellular compartment and blood did not decrease over time, NaHCO3 can be taken daily in multiday competitions or tournaments lasting ≤ 5 d without the risk of reducing performance. Second, the apparent PV expansion in response to the high ion intake (see above) blunted any further increase in [HCO3-]. If the same mechanism would be true for the chronic supplementation protocol, the effectiveness of this protocol should be questioned, as it seems that [HCO3-] cannot be increased limitlessly, i.e. that it probably reaches a ceiling. The observed ceiling effect was probably based on a metabolic compensation mechanism preventing a disproportionate increase in [HCO3-]. A respiratory compensation mechanism is unlikely to have occurred in our study because there were no differences between the NaHCO3 and placebo intervention for V̇ CO2  and RER  during the resting measurements before the constant-load tests. Of further note is that the standard chronic protocol comprises a daily dose of 0.5 g NaHCO3 kg-1 body mass  gradient between the blood and the intramyocellular compartment, which stabilized over time in the NaHCO3 intervention. In contrast to the theoretical CP-model, where metabolites should reach a steady state during exercise at CP, and consequently, buffer substances should be ineffective in enhancing Tlim, we showed that in practice Tlim can be increased with NaHCO3 supplementation. Furthermore, the high amount of ingested Na+ caused a sustained elevation in PV, which inhibited a further increase in [HCO3-], and consequently limited the performance-enhancing effect. Therefore, this study indicates that NaHCO3 can be taken daily in multiday competitions or tournaments to maintain performance ability throughout the whole duration of the competition.In conclusion, multiple acute, consecutive day NaHCOV̇ O2 during the constant-load trials at CP; V̇ CO2,CLT: Carbon dioxide output during the constant-load trials at CP; W’: The finite work capacity above CPABE: Actual base excess; CP: Critical power; DXA: Dual-energy x-ray absorptiometry; [HCO3-]: Blood bicarbonate concentration; HRCLT: Heart rate during the constant-load trials at CP; NaCl: Sodium chloride; NaHCO3: Sodium bicarbonate; Ppeak: Peak power output; PV: Plasma volume; RERCLT: Respiratory exchange ratio during the constant-load trials at CP; Tlim: Time-to-exhaustion; V̇ O2peak: Peak oxygen uptake; V̇ O2,CLT: The authors declare that they have no competing interests.SMM, SMG, MT designed the study. SMG and SMM were involved in data collection. SMG, SMM, and MT were involved in statistical analysis and drafted the manuscript. SMM, SMG, SF, UB, CAW, and MT interpreted the data and reviewed the manuscript. All authors read and approved the final manuscript.


Leishmania spp. are protozoa responsible for leishmaniasis, a neglected disease that kills up to 50,000 people every year. Current therapies mainly rely on antimonial drugs that are inadequate because of their poor efficacy and safety and increased drug resistance. An urgent need exists to find new and more affordable drugs. Our previous study demonstrated the antileishmanial activity of eupomatenoid-5, a neolignan obtained from leaves of Piper regnellii var. pallescens. The aim of the present study was to clarify the mode of action of eupomatenoid-5 against L. amazonensis. We used biochemical and morphological techniques and demonstrated that eupomatenoid-5 induced cell death in L. amazonensis promastigotes, sharing some phenotypic features observed in metazoan apoptosis, including increased reactive oxygen species production, hypopolarization of mitochondrial potential, phosphatidylserine exposure, decreased cell volume, and G0/G1 phase cell cycle arrest. Leishmaniasis remains a significant neglected tropical disease that puts 350 million people in 88 countries on four continents at risk for infection, with approximately 50,000 deaths per year . ApproxiCurrent chemotherapy for leishmaniasis is still based on the use of pentavalent antimonials as first-line drugs and pentamidine, amphotericin B , paromomycin, and miltefosine as second-line drugs. Although these drugs are usually effective, they have limitations, such as toxicity in the host and long-term treatment , 4. ThesLeishmania [Piper regnellii var. pallescens, representing a subclass with various biological activities, such as antifungal, antibacterial, insecticidal, and trypanocidal effects [L. amazonensis and elucidate the mechanism of action of eupomatenoid-5 involved in the cell death of this protozoan parasite.Natural compounds, known to be valuable sources of new medicinal agents, have been exhaustively evaluated against ishmania –7. One e effects –12. Our  effects . Conside2DCFDA), and 5-5′-dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich . Fetal bovine serum (FBS) was obtained from Invitrogen . Annexin-V FITC, 3,8-phenanthridine diamine-5-(6-triphenylphosphonium hexyl)-5,6-dihydro-6-phenyl (MitoSOX) and propidium iodide (PI) were obtained from Invitrogen . The protein assay kit was obtained from Bio-Rad . All of the other reagents were of analytical grade.Actinomycin D, antimycin A, bovine serum albumin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), digitonin, dimethylsulfoxide (DMSO), rhodamine 123 (Rh123), 2′,7′-dichlorofluorescin diacetate  in Parana, Brazil. A voucher specimen (no. HUM 8392) was deposited at the UEM Herbarium. Briefly, the dry plant material was extracted by exhaustive maceration at room temperature in the dark in an ethanol : water ratio of 90 : 10. Fractionation was performed from the ethyl acetate extract to obtain the hexane fraction, and a dihydrobenzofuran neolignan, eupomatenoid-5, was isolated from this fraction as described previously [1H nuclear magnetic resonance (NMR), 13C NMR, distortionless enhancement polarization transfer (DEPT), correlated spectroscopy (COSY), heteronuclear correlation (HETCOR), nuclear overhauser effect spectroscopy (NOESY), heteronuclear multiple bond correlation (HMBC), and gas chromatography/mass spectrometry (GC/MS) spectra. The data were compared with the literature [Eupomatenoid-5  was isolterature .μM, representing the IC50, IC90, and twofold IC90, respectively [Stock solutions of eupomatenoid-5 were prepared aseptically in DMSO and diluted in culture medium so that the DMSO concentration did not exceed 1% in the experiments. The concentrations of eupomatenoid-5 used in the assays were 30.0, 85.0, and 170.0 ectively .Leishmania amazonensis promastigotes (MHOM/BR/Josefa) were maintained at 25°C in Warren's medium  supplemented with 10% heat-inactivated FBS.7 cells/mL) were treated or untreated with 30.0, 85.0, and 170.0 μM eupomatenoid-5 for 24 h at 37°C, harvested, and washed with phosphate-buffered saline (PBS). The parasites were incubated with 1 mL (5 mg/mL in ethanol) of Rh123, a fluorescent probe that accumulates in mitochondria, for 15 min, resuspended in 0.5 mL PBS, and incubated for an additional 30 min. The assay was conducted according to the manufacturer's instructions. The parasites were analyzed using a BD FACSCalibur flow cytometer and CellQuest Pro software . A total of 10,000 events were acquired in the region that corresponded to the parasites. CCCP at 100 μM was used as a positive control [Promastigotes (1 × 107 cells/mL) were treated or untreated with 30.0, 85.0, and 170.0 μM for 3 and 24 h, centrifuged, washed, and resuspended in PBS (pH 7.4). Afterward, these parasites were loaded with 10 μM of a permeant probe, H2DCFDA, in the dark for 45 min [Promastigotes (1 × 10r 45 min . Reactiv7 cells/mL) were loaded with 5 μM of a fluorescent O2•−-sensitive, mitochondrial-targeted probe, MitoSOX, for 10 min at 25°C and then washed with Krebs-Henseleit (KH) buffer (pH 7.3) that contained 15 mM NaHCO3, 5 mM KCl, 120 mM NaCl, 0.7 mM Na2HPO4, and 1.5 mM NaH2PO4 [μM eupomatenoid-5, the fluorescence intensity was detected after 1, 2, 3, and 4 h of treatment using a fluorescence microplate reader , with an excitation wavelength of 510 nm and emission wavelength of 580 nm. Oxidized MitoSOX (oxMitoSOX) becomes highly fluorescent upon binding to nucleic acids. Cells were exposed to 10 μM antimycin A, a stimulus known to induce mitochondrial O2•− production.Promastigotes (2 × 10 NaH2PO4 . After t7 cells/mL) were treated or untreated with 30.0, 85.0, and 170.0 μM eupomatenoid-5 for 24, 48, and 72 h at 25°C. Afterward, the parasites were centrifuged, dissolved in 10 mM Tris-HCl buffer (pH 2.5), and sonicated. Acidic pH was used during sonication to prevent oxidation of the free thiol groups. Cellular debris was removed by centrifugation, and 100 μL of the supernatant and 100 μL of 500 mM phosphate buffer (pH 7.5) were taken in each microtiter well, followed by the addition of 20 μL of 1 mM DTNB to each well. Absorbance was measured at 412 nm [Thiol levels were determined using DTNB. Promastigotes (1 × 10t 412 nm .7 cells/mL) were treated or untreated with 30.0, 85.0, and 170.0 μM eupomatenoid-5 for 24 h at 25°C. Afterward, the parasites were washed and resuspended in 100 µL of binding buffer , followed by the addition of 5 μL of a calcium-dependent phospholipid binding protein, annexin-V FITC, for 15 min at room temperature. Binding buffer (400 μL) and 50 μL PI were then added. Antimycin A (125.0 μM) was used as a positive control. Data acquisition and analysis were performed using a BD FACSCalibur flow cytometer equipped with CellQuest software. A total of 10,000 events were acquired in the region that was previously established as the one that corresponded to the parasites. Cells that were stained with annexin-V (PI-positive or -negative) were considered apoptotic, and cells that were only PI-positive were considered necrotic [Promastigotes (1 × 10necrotic .7 cells/mL) were treated or untreated with 30.0, 85.0, and 170.0 μM eupomatenoid-5 for 24 h at 25°C, harvested, and washed with PBS. Subsequently, the parasites were analyzed using a BD FACSCalibur flow cytometer and CellQuest Pro software. Histograms were generated, and FSC-H represented the cell volume. A total of 10,000 events were acquired in the region that corresponded to the parasites. Actinomycin D (20.0 mM) was used as a positive control [Promastigotes (1 × 10 control .7 cells/mL) were treated or untreated with 30.0, 85.0, and 170.0 μM eupomatenoid-5 for 24 h at 32°C, harvested, and washed with PBS. The parasites were incubated with 50 mL of 2 mg/mL PI for 5 min according to the instructions provided by the manufacturer. Immediately thereafter, the parasites were analyzed using a BD FACSCalibur flow cytometer equipped with CellQuest software. A total of 10,000 events were acquired in the region that corresponded to the parasites. Digitonin (40.0 μM) was used as a positive control [Promastigotes (1 × 10 control .6 cells/mL) were treated or untreated with 30.0 and 85.0 μM eupomatenoid-5 for 48 h at 25°C and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1–3 h. Subsequently, the parasites were adhered on poly-L-lysine-coated coverslips and dehydrated in increasing concentrations of ethanol. The samples were critical point dried in CO2, coated with gold, and observed in a Shimadzu SS-550 (Japan) scanning electron microscope [Promastigotes (1 × 10croscope .7 cells/mL) were treated or untreated with 30.0, 85.0, and 170.0 μM eupomatenoid-5 for 24 h at 25°C. After incubation, the parasites were centrifuged and washed twice in PBS (pH 7.4). The resultant pellet was resuspended in 500 μL of a cold methanol/PBS (70% v/v) mixture and maintained at 4°C for 1 h. Afterward, the pellet was centrifuged, resuspended in PBS with 10 μg/mL PI and 20 μg/mL of DNase-free RNase (200 mg), and incubated for 45 min at 37°C. Data were acquired using a BD FACSCalibur flow cytometer and analyzed using CellQuest Pro software [Promastigotes .The data shown in the graphs are expressed as the means ± standard deviation (SD) of the mean of at least three independent experiments. The data were analyzed using one- and two-way analysis of variance (ANOVA). Significant differences among means were identified using the Tukey 90 (95.9%) than that at the IC50 (76.8%), and at the twofold IC90, the loss of ΔΨm remained the same as the IC90 (94.9%). The positive control, CCCP, induced a decrease of 62.5% in mitochondrial membrane potential.Our previous study used transmission electron microscopy and found that eupomatenoid-5 caused damage and significant changes in promastigote mitochondria . Based o2•− production in eupomatenoid-5-treated parasites. Mitochondrial-derived O2•− production was evaluated using MitoSOX reagent, which measures the mitochondrial accumulation of superoxide based on its hydrophobic nature and positively charged triphenylphosphonium moiety [2•− at most of the concentrations and times tested compared with the control group. After 1 and 2 h of treatment, eupomatenoid-5 increased O2•− production by approximately 40% only at higher concentrations. In contrast, after 3 and 4 h of treatment, eupomatenoid-5 increased O2•− production by more than 50% at all of the concentrations tested. The positive control, antimycin A, induced a two-fold increase in mitochondrial O2•− production.Based on our ΔΨm results, we evaluated Om moiety .  Figure2DCFDA. This probe primarily detects H2O2 and hydroxyl radicals and fluoresces after forming dichlorofluorescein [μM. After 24 h treatment, total ROS production increased by 16.3% even at the lower concentration.Based on the MitoSOX data, we evaluated the effects of total ROS production in eupomatenoid-5-treated parasites using a fluorescent probe, Horescein . Our resOur data suggest that eupomatenoid-5 induces oxidative imbalance, attributable to enhanced ROS production. However, oxidative imbalance conditions depend on both increased oxidant species and decreased antioxidant effectiveness . TherefoμM) compared with the control group, indicating phosphatidylserine exposure  and a 28% increase at the higher concentrations (85.0 and 170.0 μM) compared with the control group  . The incol group . In contP. regnellii leaves are responsible for the antileishmanial activity of this plant [Despite recent advances, the treatment of leishmaniasis continues to be unsatisfactory. Pentavalent antimonials remain the first-line treatment for this infection in most endemic areas, despite their limitations, such as high toxicity and increased drug resistance , 28. Thuis plant . Furtheris plant . Additiois plant . The preLeishmania is known to have a single mitochondrion, and so the maintenance of mitochondrial transmembrane potential is essential for parasite survival [We initially focused on investigating mitochondrial alterations and their consequences, especially with regard to parasite death. We found that parasites treated with eupomatenoid-5 exhibited a decrease in ΔΨm and increase in mitochondrial ROS production. survival . In factsurvival , 30. Eupsurvival , 32. In survival . Our datTrypanosoma cruzi after treatment with eupomatenoid-5 [Additionally, eupomatenoid-5 induced the externalization of phosphatidylserine and a reduction of parasite volume, indicated by flow cytometry and scanning electron microscopy. Altogether, our data showed that eupomatenoid-5-treated parasites exhibited apoptotic-like events. An increase in the generation of ROS in the cytosol, an established event in most apoptotic cells, might direct the cell toward this death pathway . Interestenoid-5 .Finally, we demonstrated that eupomatenoid-5 induced G0/G1 phase cell cycle arrest using flow cytometry and PI labeling, in which the amount of bound dye correlated with the DNA content, and thus DNA fragmentation in apoptotic cells is translated into fluorescence intensity that was lower than that of G0/G1 cells  , 37.Leishmania apoptosis-like death will provide insights into the future design of newer chemotherapeutic strategies.In parasites, apoptosis appears to be the predominant form of cell death , which h


Endotoxin-induced uveitis (EIU) is an animal model for acute ocular inflammation. Several substances play major roles in the development of inflammatory changes in EIU, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. These inflammatory cytokines trigger the degradation of IκB by activating IκB kinases (IKKs). Released nuclear factor kappaB (NFκB) subsequently translocates to the nucleus, where NFκB expresses its proinflammatory function. IMD-0354, N--5-chloro-2-hydroxybenzamide, selectively inhibits IKKβ, particularly when induced by proinflammatory cytokines, such as TNF-α and IL-1β. In the present study, we examined whether IKKβ inhibition has therapeutic effects on EIU by using IMD-0354 and its prodrug IMD-1041.Escherichia coli that had been diluted in 0.1 ml of phosphate-buffered saline. IMD-0354 was administered intraperitoneally at 30, 10, 3, or 0 mg/kg, suspended in 1.0 ml of 0.5% carboxymethyl cellulose sodium. The prodrug IMD-1041 (100 mg/kg) was also administered orally. The rats were euthanized 24 h after LPS injection, and EIU severity was evaluated histologically. The number of infiltrating cells and the protein, TNF-α, and monocyte chemoattractant protein-1 (MCP-1) concentrations in the aqueous humor were determined. TNF-α and MCP-1 concentrations were quantified with enzyme-linked immunosorbent assay. Eye sections were also stained with anti-NFκB and phosphorylated I-κBα antibodies.Six-week-old male Lewis rats were used. EIU was induced with subcutaneous injections of 200 μg of lipopolysaccharide (LPS) from 5, 72.5±17.0×105, 127.25±32.0×105, and 132.0±25.0×105 cells/ml in rats treated with 30, 10, 3, or 0 mg/kg of IMD-0354, respectively. The total protein concentrations of aqueous humor were 92.6±3.1 mg/ml, 101.5±6.8 mg/ml, 112.6±1.9 mg/ml, and 117.33±1.8 mg/ml in rats treated with 30, 10, 3, and 0 mg/kg of IMD-0354, respectively. Infiltrating cells and protein concentrations were significantly decreased by treatment with IMD-0354 (p<0.01). IMD-0354 treatment significantly reduced the concentration of TNF-α (p<0.05) and MCP-1 (p<0.01) in aqueous humor. The number of NFκB positive nuclei was reduced when treated with IMD-0354. Furthermore, IMD-0354-treated EIU rats showed only background levels of phosphorylated I-κBα; however, it was strongly expressed in the iris-ciliary body cell cytoplasm of the IMD-0354 untreated EIU rats. Oral administration of IMD-1041 also decreased the cell number (p<0.01) and protein concentration (p<0.05) of aqueous humor in EIU.The number of infiltrating cells in aqueous humor was 53.6±9.8×10Acute uveitis was ameliorated by inhibition of IKKβ in rats. IMD-0354 and its prodrug IMD-1041 seem to be promising candidates for treating intraocular inflammation/uveitis. Phosphorylated IκBα is then ubiquitinated, which targets it for degradation by the 26S proteasome [IκB complex and allowing them to translocate to the nucleus. NFκB then binds to κB-enhancer elements of target genes, inducing transcription of proinflammatory genes. Proinflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), are regulated by NFκB activation and are known to be the stimuli that activate IκB kinase. Since NFκB is the main factor in the positive feedback loop of inflammation, inhibiting its activation may be an effective therapy for intraocular inflammation.Endotoxin-induced uveitis (EIU) is an animal model of acute anterior segment intraocular inflammation induced by injection of endotoxin, the lipopolysaccharide (LPS) component of the Gram-negative bacterial cell wall . Cellulaoteasome , thus reIMD-0354, IUPAC name N--5-chloro-2-hydroxybenzamide, was originally designed to competitively interrupt the access of ATP to its docking site on IKKβ, resulting in suppressing the activity of the IKK complex . IMD-035IMD-1041 is a prodrug of IMD-0354, and oral administration of IMD-1041 has prevented kidney injury in rats . These fN--5-chloro-2-hydroxy-benzamide, and its prodrug IMD-1041 were kindly given by the Institute of Medical Molecular Design . They were dissolved in 0.5% carboxymethylcellulose (CMC) vehicle  immediately before use. Drug-free vehicle (0.5% CMC solution) was used as a control.IMD-0354, Escherichia coli  that had been diluted in 200 μl phosphate-buffered saline 12H20 2.9 g, KCl 0.2 g, KH2PO4 0.2 g, in H2O 1 l; pH 7.4). At the same time, the rats were injected intraperitoneally with 30, 10, or 3 mg/kg of IMD-0354, diluted in 500 μl of 0.5% CMC. Control EIU rats were intraperitoneally administered 500 μl of CMC alone (no IMD-0354). Naïve rats were used as controls. All experiments were performed in triplicate with five animals in each group. Unless stated otherwise, all section slide-imaging and evaluation were performed with a BZ-9000 fluorescence microscope  and software bundled with the apparatus.Eight-week-old male Lewis rats (180–220 g) were used. All procedures involving animals were performed in accordance with the ARVO resolution on the use of animals in research. EIU was induced with subcutaneous injection with 200 μg LPS from Rats were euthanized 24 h after LPS administration by single intravenous injection of sodium pentobarbital . The eyes were enucleated immediately and stored in 4% paraformaldehyde in 0.1 M PBS for 24 h, and were then embedded in paraffin. Sagittal sections (5 μm thick) were cut through the optic nerve head and stained with hematoxylin and eosin. The number of infiltrating cells was counted in the two iris-ciliary body (ICB) areas of each slide as well as near the retinas in the posterior area of the eyes, and the results were averaged.At 24 h after LPS injection, rats were euthanized, and the aqueous humor was collected immediately from both eyes with an anterior chamber puncture (15–20 μl/rat) using a 30-gauge needle , under Olympus SZ61 microscope  under a surgical microscope. For cell counting, the aqueous humor sample was suspended in an equal amount of Türk stain solution , and the cells were counted with a hemocytometer under Olympus CKX31 microscope (Olympus). The number of cells was manually counted in each field, and the number of cells per microliter was obtained by averaging the results of four fields from each sample. The total protein concentration in the aqueous humor samples was measured with a bicinchoninic acid protein assay kit . The aqueous humor samples were stored in ice water until testing, and cell counts and total protein concentrations were measured on the day of sample collection.The levels of TNF-α and MCP-1 in the aqueous humor were assessed with commercially available ELISA (ELISA) kits: Rat TNF-α ELISA and Rat MCP-1 ELISA according to the manufacturer’s instructions . The detection limits of the ELISA kits were 15 pg/µl and 5 pg/ml for TNF-α and MCP-1, respectively. The ELISA assay was performed in triplicate, with five or more wells involved in each experiment.At 3 h after LPS injection, the rats were euthanized by intravenous injection of sodium pentobarbital , and the eyes were fixed with an intracardiac perfusion of 4% paraformaldehyde in 0.1 M PBS. The eyes were then enucleated, immersed in the same fixative for 12 h, and then embedded in paraffin. Next, 5-μm sagittal sections were cut near the optic nerve head. Sections were dewaxed with xylene and rehydrated with ethanol. Antigen retrieval was performed by heating sections in a microwave oven in citrate buffer. To evaluate the NFκB-positive cells in the ICB cells, sections were applied with a 1:50 dilution of NFκB P65 antibody  for 12 h, washed with PBS, and then applied with 1:1000 dilution of secondary goat–anti-rabbit antibody dye conjugate , which gives red fluorescence. Since NFκB is widely present in intercellular tissue, to detect its expression in cell nuclei, we stained the section slides with 1:1000 dilution of YO-PRO-1 (Invitrogen), which produces a green signal from the cell nuclei. YO-PRO-1 is a sensitive detector of membrane permeabilization, and a normally cell-impermeant, monomeric, cyanine dye with a strong binding affinity to nucleic acids. NFκB positive (yellow) nuclei were quantified in merged images of the ICB area of the eyes. The results for the two areas were averaged for each sample and in each group. Slide-imaging and evaluation were performed with a FluoView 1000 confocal microscope  and software bundled with the apparatus.Rat eye fixation and harvesting as well as histological section preparation were identical to the NFκB immunohistochemical staining described above. To evaluate phosphorylated I-κBα expression in the ICB cell cytoplasm, sections were applied with a 1:50 dilution of phosphorylated I-κBα antibody  for 12 h, washed with PBS, and then applied with 1:1000 dilution of secondary goat–anti-mouse antibody dye conjugate (Invitrogen), which gives green fluorescence. To detect cell nuclei, we stained the section slides with 1:1000 dilution of 4',6-diamidino-2-phenylindole , which produces a blue signal. Sections from EIU rats without phosphorylated I-κBα antibody applied were used as a negative control. The staining protocol was based on recent reports on immunohistochemical detection and evaluation of phosphorylated I-κBα .E. coli (Sigma-Aldrich). Control EIU rats were orally administered 500 μl of CMC alone. All experiments were performed in triplicate with four animals in each group. The protein concentration and the number of infiltration cells of the aqueous humor samples were then evaluated.IMD-0354 is available only in injection form; however, the prodrug, IMD-1041 can be administered orally. We performed an additional experiment to examine the effect of oral prodrug IMD-1041 administration on EIU in rats. Eight-week-old male Lewis rats (180–220 g) were orally treated with 100 mg/kg of IMD-1041 dissolved in 1 ml of 0.5% CMC vehicle (Sigma) immediately before use. Drug-free vehicle (0.5% CMC solution) was used as a control. Disposable feeding needles for oral administration were purchased from Fuchigami Inc. . EIU was induced with subcutaneous injection with 200 μg LPS from t test. P values less than 0.05 are considered significant.All values are expressed as mean±standard error of mean (SEM) from the respective groups of experimental or control data. Statistical significance was evaluated with the unpaired Student Only a few inflammatory cells infiltrated in the eyes of rats treated with 30 mg/kg  and 10 mThe infiltrating cells in the ICB area of the sections were counted and averaged. The mean numbers of the infiltrating cells were 6.0±1.0, 18.3±5.2, 23.3±2.8, and 51.7±5.6 in eyes treated with 30, 10, 3, and 0 mg/ml of IMD-0354, respectively. The numbers of infiltrating cells in the ICB area were significantly reduced in eyes treated with 30 mg/ml (p<0.01), 10 mg/ml (p<0.05), and 3 mg/ml (p<0.05) of IMD-0354 compared with those of vehicle alone in the EIU rats .The infiltrating cells in the posterior area of the histological sections were also counted and averaged. The mean number of infiltrating cells was 64.0±2.1 in vehicle-given EIU eyes, and 9.0±1.5, 18.3±3.4, and 57.0±5.3 in eyes treated with 30, 10, and 3 mg/ml of IMD-0354, respectively. The number of infiltrating cells was significantly reduced in eyes treated with 30 or 10 mg/kg of IMD-0354 mg/ml compared to those of the positive controls p<0.01, . The num5 cells/ml. The number of aqueous infiltrating cells was 53.6±9.8×105, 72.5±17.0×105, and 127.25±32.0×105 cells/ml, when treated with 30, 10, and 3 mg/kg of IMD-0354, respectively. The mean number of aqueous inflammatory cells was significantly reduced by treatment with 30 mg/kg (p<0.01) and 10 mg/kg (p<0.05) of IMD-0354, whereas 3 mg/kg of IMD-0354 did not result in a significant reduction compared with the LPS group  and 10 mg/kg  of IMD-0354 were significantly lower than that in the LPS group, whereas 3 mg/kg of IMD-0354 did not result in a significant reduction in protein concentration (112.6±1.9 mg/ml) compared with the LPS group . The decConsiderable production of TNF-α and MCP-1 in the aqueous humor was seen in the untreated EIU rats. Treatment with 30 mg/kg of IMD-0354 significantly reduced TNF-α (p<0.05) and MCP-1 (p<0.01) concentrations in aqueous humor . The TNFThree hours after LPS was injected, activated NFκB p65 immunoreactivity was examined. NFκB p65 was strongly expressed in the ICB of untreated EIU rats . In contThree hours after LPS was injected, phosphorylated I-κBα immunoreactivity was examined. No phosphorylated I-κBα signal was detected in the negative controls . Only ba5 and 31.33±5.2×105 cells/ml in rats untreated and treated with 100 mg/kg of IMD-1041, which significantly reduced the number of inflammatory cells , an animal model for human endogenous uveitis ,26. HoweIn summary, inhibiting IκB phosphorylation to prevent nuclear translocation of NFκB attenuates inflammatory response in the eyes of rats. NFκB plays central and multiple roles in the immune response, as a mediator of many proinflammatory signal transductions, and therefore, an inhibitor of IKKβ may function as a multifaceted suppressor of inflammatory disorders in the eye. IMD-0354 and IMD-1041 seem to be promising candidate agents for ocular inflammatory disorders since they showed significant effects with no systemic side effects in this study; in addition, oral agents are less stressful for patients.


Plasmodium-infected red blood cells. This water-soluble fluorescent probe is photostable, excitable in the visible region and cell membrane-permeable, and its photophysical properties are not notably pH-sensitive. Fluorescence imaging microscopy of erythrocytes infected with Plasmodium falciparum, a causative agent of malaria in humans, showed that only the parasite was stained. Z-stacking analysis suggested that the probe accumulates proximal to the nucleus of the parasite. Indicaxanthin, one of the natural fluorescent betalains found in the petals of certain flowers, did not stain the parasite or the red blood cell.A model betalainic dye was semisynthesized from betanin, the magenta pigment of the red beet, and was effective for live-cell imaging of  BvDODA1 gene HBt) and amines or amino acids. Betalains are water-soluble pigments responsible for the visible fluorescence of flowering succulent plants. Plasmodium species are the causative agents of malaria. These obligate, intracellular organisms have a complex life cycle and switch between a mosquito vector and a vertebrate host. Plasmodium spp. merozoites infect erythrocytes and undergo a differentiation process that starts with the ring stage, followed by the trophozoite stage and finally the schizont stage. Differentiation inside the erythrocyte occurs within the parasitophorous vacuole (PV), a vacuolar membrane that surrounds the intracellular parasite. The sequential pathway for the entry of exogenous nutrients required for parasite survival and multiplication includes crossing the red blood cell membrane (RBCM), the parasitophorous vacuole membrane and the parasite plasma membrane. BtC via aldimine coupling of the fluorescent hydrophobic chromophore 7-amino-4-methylcoumarin (C120) to HBt. This dye was used for the identification of erythrocytes infected by Plasmodium spp. using fluorescence imaging.Inspired by the biological role of betalains, we sought to create a new small, tunable and water-soluble molecular framework for dyes suitable for live-cell fluorescence imaging. Due to the presence of at least two carboxyl groups in the betalain framework, such dyes are negatively charged at near-neutral pH and favor cell permeation through anion channels, such as the NPP. The functional group attached to the betalainic moiety modulates the amphiphilic character of the dye and influences its electronic properties. Therefore, we prepared the artificial coumarinic betalain International Guiding Principles for Biomedical Research Involving Animals, CIOMS, 1985). The protocol was approved by the Committee on the Ethics in Research of the Federal University of São Paulo (Permit Number: 1364/09). All surgery was performed under anesthesia, and all efforts were made to minimize animal suffering.This study was carried out in strict accordance with the recommendations of the Council for International Organizations of Medical Sciences  ppm, TFE δH = 3.88 (m) and 5.02 (s) ppm. High-resolution mass spectra were obtained using a Bruker MicroTOF-QII mass spectrometer. Flash column chromatography was performed using 70–230 mesh silica gel. Absorption spectra were recorded in the visible region of the electromagnetic spectra (380–780 nm) at 25±1°C using a Varian Cary 50 Bio spectrophotometer equipped with a Peltier thermostated cell holder. Fluorescence spectroscopy studies were performed using a Varian Cary Eclipse spectrometer connected to a thermostatic bath. The excitation and emission spectra were recorded at 25°C.All chemicals were purchased from Sigma-Aldrich and used without further purification, except where noted. The NMR spectra were recorded using a Bruker Avance  or Bruker Avance III  spectrometer. Chemical shifts are reported as δ values (ppm) referenced to the solvent residual signals: DBeta vulgaris subsp. vulgaris var. vulgaris, 0.5 kg) were peeled, sliced and homogenized in a centrifugal juice extractor  at the maximum speed. The juice was centrifuged  and filtered , and the supernatant was stored at –20°C and used within 5 days. The betanin/isobetanin mixture was purified from beetroot juice by reversed-phase column chromatography . The concentration of betanin was determined by assuming a molar absorption coefficient (ε) of 6.5×104L mol–1cm–1 at 536 nm. Beetroots , ε430 nm = 2.65×104 L mol–1 cm–1 ; Fl: λFlmax 500 nm .HBt absorption band at 430 nm and the concomitant appearance of the BtP band at 485 nm. After completion (ca. 30 min), the solution was cooled (0°C), and HOAc (conc.) was slowly added until the solution reached pH 5. The resulting solution was then stirred at RT for 1 h. The product was purified by reversed-phase column chromatography , and the fractions containing BtP were combined and lyophilized. The resulting orange powder was stored at −20°C, and the purity of the product was assessed by RP-HPLC analysis prior to use. In a typical run, 0.5 mg (2.5 µmol) of betalamic acid yielded 0.7 mg  of BtP.L-Proline (100 equiv.) was added to an aqueous solution of betalamic acid , and the resulting solution was stirred at room temperature (RT). The reaction was spectrophotometrically monitored by the depletion of the 1H-NMR : δ 8.34 , 8.24 , 6.20 , 6.16 , 6.08 , 5.80 , 4.47 , 3.76–3.71 , 3.33 , 3.11 , 2.20–2.27 , 2.27–2.37 , 2.43–2.54 , 2.04–2.20 , 2.00–2.15 .HRMS (m/z): [M+H]+ calculated for C14H17N2O6+: 309.1087; observed: 309.1082 (dif.: 1.62 ppm).LC-ESI(+)-MS:tR = 6.2 min, (m/z) [M+H]+ observed: 309.1.UV/Vis: λmax 485 nm (water), ε485 nm = 4.8×104 L mol–1cm–1 (water) Fluorescence: λmaxfl 520 nm , ΦFl = 4.6×10–3  vs. fluorescein.I.4 BtC. 7-Amino-3-methylcoumarin (100 equiv.) was added to an aqueous solution of betalamic acid (0.7 mg (3.2 µmol) in 1.0 mL, pH 10), and the solution was submitted to ultrasonic irradiation for 5 min and stirred at RT for an additional 30 min. The reaction was spectrophotometrically monitored for the appearance of the BtC band at 520 nm. After completion, the solution was cooled (0°C), and HCl (conc.) was slowly added until the solution reached pH 3. The resulting solution was then stirred at RT in the dark for 2 hours. The product was purified via gel-permeation chromatography using lipophilic Sephadex LH-20 with water as the eluent. The fractions containing BtC were combined and lyophilized. The resulting purple powder was stored at −20°C, and the purity of the product was verified by RP-HPLC analysis prior to use. In a typical run, 0.7 mg (3.2 µmol) of betalamic acid yielded 0.4 mg  of BtC.1H-NMR , 7.73 , 7.17 , 7.12 , 6.27 , 6.23 , 6.07 , 4.40 , 3.30 , 3.10 , 2.43 .1H-NMR , 8.02 , 7.55 , 7.44 , 6.76 , 6.52 , 6.50 , 4.50 , 3.63 , 3.34 , 2.72 .13C-NMR -MS:tR = 5.6 min, (m/z) [M+H]+ observed, 369.1.HRMS (m/z): [M+H]+ calculated for C19H17N2O6+, 369.1078; observed, 369.1082 (dif.: –1.08 ppm).UV/Vis: λmax 520 nm (water), ε520 nm = 6.6×104 L mol–1cm–1 (water).Color: CIE Lab D65/10°: L* = 90.4, a* = 25.9, b* = –3.9, C* = 26.2 and h° = 351.4.Fluorescence: λmaxfl 570 nm , ΦFl = 4.3×10–3 (water) vs. fluorescein.molar absorption coefficient (ε) of BtC was determined using an end-point method by performing a set of betalain degradation experiments, as previously described. BtC  was spectrophotometrically monitored for 30 min at 1-min intervals. The initial pigment concentration and ε of BtC were determined by comparing the absorption of the resulting betalamic acid solution with that of betanin solutions of known concentrations. The betalamic acid solutions were stable under the experimental conditions, and no appreciable change in the spectral properties could be detected after 30 min.The Color analysis was performed using the CIE L*a*b D-65/10° color space employing the Color software . L* indicates the lightness, and its value ranges from 0  to 100 . In the CIE L*a*b* system, a* and b* are the chromaticity coordinates. A positive a* value indicates the red direction, and a negative a* value is the green direction; a positive b* value is the yellow direction, and a negative b* value is the blue direction. Two derived color parameters, the hue angle ×180/π) and the Chroma value (C* = (a*2+ b*2)½), were calculated.FL)fluorescence quantum yield (Φ was measured relative to fluorescein (NaOH(aq) 0.1 mol L–1, ΦFL = 0.79, EX 470–500 nm, EM 500–600 nm) with excitation at 475 nm. Fluorescence spectra of the standard and BtC were obtained under identical spectrometer conditions in triplicate and averaged  and 5.0 nm (EM), photomultiplier power 600 V). The optical density at the peak was maintained below 0.1 to avoid inner-filter effects, and the integrated intensities of the emission spectra, corrected for differences in the indexes of refraction and concentration, were used to calculate the ΦFL using Eq. 1,A is the absorbance at 475 nm, n is the refractive index of the solvent, S is the integrated area under the fluorescence spectrum and ΦFL are the absolute quantum yields; the superscripts X and P refer to the sample and standard, respectively.The LC analysis conditions were as follows: Solvent A, water with 0.1% v/v TFA; Solvent B, 60% v/v MeCN/water with 0.1% v/v TFA, linear gradient from 5% to 95% B in 20 min at 25°C, flow rate: 1 mL min–1, injection volume: 10 µL, spectrophotometric detection set at 254/536 nm for Bn, 484 nm for BtP and 520 nm for BtC. The analysis of BtC was performed in the absence of acid.Analytical RP-HPLC separations and analyses were performed using a Waters 2695 Alliance system instrument with a UV-Vis detector  and a Supelcosil LC-18  C18 column. The HPLC-DAD-ESI(+)-MS/MS analysis was performed using a Bruker Daltonics Esquire HCT ion trap mass spectrometer equipped with an electrospray source and coupled to a Shimadzu Prominence liquid chromatograph. The chromatograph was equipped with a Luna C18 column  maintained at 30°C and a PDA SPD-M20A detector. Nitrogen was used as the nebulizing (45 psi) and drying gas , and helium was used as the buffer gas (4×10–6 mbar). The capillary high voltage was set to 3,500 V. To avoid space-charge effects, the smart ion charge control (ICC) was set to the arbitrary value of 50,000.High-resolution mass spectrometric (HRMS) analysis was performed using a Bruker MicroTOF-QII spectrometer with an electrospray ionization source with a capillary potential of 4.5 kV. Nitrogen was used as the nebulizing (5.8 psi) and drying gas . An internal calibration was performed using a sodium formate solution (1.0 mg mL–1) before and after each acquisition. The samples were introduced into the spectrometer by direct infusion at a constant flux of 3.0 µL min–1. The data were acquired and analyzed using the Bruker Compass software.Plasmodium chabaudi (clone AS) was maintained in BALB/C mice by weekly transfers from previously infected mice. Animals infected at the trophozoite stage (parasitaemia approximately 60%) were sacrificed, the blood was collected and leukocytes and platelets were removed from the whole blood by filtration through a powdered cellulose column (Whatman CF11). The trophozoite-infected erythrocytes were then washed three times by centrifugation  in PBS. The parasites (107 cells mL–1) were isolated by the lysis of the erythrocyte membranes with saponin in PBS (10 mg mL–1). After pelleting to remove the red blood cell membranes, the parasites were washed twice in PBS by centrifugation . Plasmodium falciparum (3D7 strain) was cultured as previously described 2, 5% CO2 and 90% N2.BtC for 2 min, washed once with PBS and imaged within 10 min at 20 °C using a Zeiss Axio Observer Z1 epifluorescence microscope  with a 63×/1.25 oil-immersion objective, image acquisition time was 10,000 ms. Differential interference contrast (DIC) images were obtained using transmitted light and recorded using the Axio Vision software.The parasitized erythrocytes were mounted wet on a glass-bottom petri dish (MatTek Corporation) treated with poly-L-lysine, incubated with BtC (35 µmol L–1) and washed three times with PBS. The cells were subsequently stained with Hoechst 33258 dye (3.6 µmol L–1) for 2 min. The coverslips were mounted on slides using 4 µL of ProLong Gold (Invitrogen) and observed in a Leica SP5 TS confocal microscope with a 100×/1.44 oil-immersion objective; the Z series was acquired according to the sampling criteria built into the software for sequential imaging of the Hoechst 33258 dye, which stains the nucleus  and the BtC . All images  were acquired with the same exposure time and sensibility. The raw images were processed with the ImageJ software.Cells were treated as described for the epifluorescence imaging and incubated for 2 min in the dark at 4°C with BtC in seven different protonation states are provided. The SMD parameterization of the IEFPCM was employed to model the effect of the solvent (water). Molecular descriptors of the betalains were estimated as the means of the Marvin Sketch 5.10.3 prediction program . The LogD was predicted according to the method of Viswanadhan and collaborators. Ka values of the carboxylic portions of BtC were obtained based on the partial charges, Gaussian09 was used for all of the quantum-chemical calculations. Bn, purified from red beet juice BtC. The dye was purified by gel-permeation chromatography on lipophilic Sephadex LH-20 and subsequently characterized (File S1). The absorption and fluorescence spectra of BtC , εW = 66,000 L mol–1 cm–1) are red-shifted relative to that of C120  BtC. Furthermore, the Stokes shift of the betalain is smaller than that of C120  vs. ΔνBtC = 1687 cm–1 (50 nm), BtC is water-soluble and excitable with visible light, two relevant improvements over the majority of coumarins used as fluorescent probes. However, BtC is several orders of magnitude less fluorescent in solution than is C120 (fluorescence quantum yield (ΦFL) of BtC in water = 4.3×10–3vs. ΦFLC120, MeOH = 0.51). The lack of a fluorescence signal from the uninfected erythrocytes is likely a result of the low quantum yield of BtC. However, a weak fluorescence signal is convenient for the quantification of the probe uptake and accumulation inside the parasite. BtC is reasonably stable in aqueous media  = 50 µmol L–1; EM 570 nm.(TIFF)Click here for additional data file.Figure S5Fluorescence profiles of BtC in aqueous solution (control) and in suspension containing P. chabaudi–1, 5 min incubation and washing (5×), EX 520 nm).-infected erythrocytes ([BtC] = 50 µmol L(TIFF)Click here for additional data file.File S1NMR and HRMS spectra of BtP and BtC and cartesian coordinates of optimized structures.(DOCX)Click here for additional data file.Scheme S1A) Chemical structures of betanin (Bn) and indicaxanthin (BtP); (B) semisynthesis of BtC.((TIFF)Click here for additional data file.Scheme S2Acid–base equilibria of BtC.(TIFF)Click here for additional data file.Scheme S3Attribution of NMR signals of BtC; s: singlet, d: doublet, t: triplet, bt: broad triplet, bs: broad singlet, dd: double doublet, m: multiplet.(TIFF)Click here for additional data file.Video S13D Rotation of an erythrocyte infected with two parasites. Staining: BtC (pseudo-colored green) and Hoechst dye (pseudo-colored blue).(AVI)Click here for additional data file.


Artemisia species, A. armeniaca Lam. and A. aucheri Boiss, were screened for their antimalarial properties by in vitro  β-hematin formation assay. Dichloromethane (DCM) extracts of both plants showed significant antimalarial activities with IC50 values of 1.36 ± 0.01 and 1.83 ± 0.03 mg/mL and IC90 values of 2.12 ± 0.04 and 2.62 ± 0.09 mg/mL for A. armeniaca and A. aucheri, respectively. Bioactivity-guided fractionation of DCM extracts of both plants by vacuum liquid chromatography (VLC) over silica gel with solvent mixtures of increasing polarities afforded seven fractions. Two fractions from DCM extract of A. armeniaca and four fractions from DCM extract of A. aucheri showed potent antimalarial activity with reducing IC50 and IC90 values compared to extracts. The most potent fraction belonged to DCM extract of A. armeniaca with IC50 and IC90 values of 0.47 ± 0.006 and 0.71 ± 0.006 mg/mL, respectively.Ten extracts with different polarity from two Iranian  Plasmodium and kills more than one million individuals in the tropical and subtropical zones annually  × 100, where AN: absorbance of negative control and AS is absorbance of test samples.The potential antimalarial activity of plant extracts was evaluated by the method described by Afshar et al.  with som50 and IC90 values were calculated from nonlinear regression analysis.All experiments were conducted in triplicate measurements and presented as the Mean ± SD. Data were analyzed by Excel 2010 Microsoft. The ICin vitro  β-hematin formation assay of five different extracts from A. armeniaca and A. aucheri and seven fractions of their respective DCM extracts as well as the extraction and fractionation yields are listed in β-hematin formation expressed as percentage (I%) and standard deviations (n = 3) are given for each extract/fraction. IC50 and IC90 values were measured graphically by plotting concentrations versus percentage of inhibition. Three extracts  of both plants had no anti-malarial activity at all while the DCM extracts of A. armeniaca and A. aucheri compared to the standard anti-malarial compound, chloroquine , showed the most potent anti-malarial activity with IC50 values of 1.36 ± 0.01 and 1.83 ± 0.03 mg/mL and IC90 values of 2.12 ± 0.04, 2.62 ± 0.09 mg/mL, respectively. PE extracts possessed a weak activity with IC50 values of 3.51 ± 0.11 and 4.79 ± 0.92 mg/mL and IC90 values of 7.96 ± 0.17, 10.25 ± 2.91 mg/mL for A. armeniaca and A. aucheri, respectively. Among the seven different polarity fractions obtained from the DCM extract of A. armeniaca, 80% EtOAC/PE and 100% EtOAC fractions showed considerable anti-malarial activity with IC50 values of 0.47 ± 0.006 and 0.94 ± 0.006 mg/mL and IC90 values of 0.71 ± 0.006, 1.26 ± 0.02 mg/mL, respectively. In the case of fractions separated from DCM extract of A. aucheri four polar fractions  showed remarkable anti-malarial effects with close IC50 and IC90 values  have been described and used for screening of novel synthetical [A. armeniaca and A. aucheri were evaluated for their antimalarial activity by an in vitro  β-hematin formation assay developed by Afshar et al. [A. armeniaca, DCM extract showed the most potent activity (IC50 = 1.36 ± 0.01 and IC90 = 2.12 ± 0.04 mg/mL) and indicated that compounds with strongest antimalarial activity have medium polarity. Subsequent bioactivity-guided fractionation of DCM extract by VLC over silica gel with solvent mixtures of increasing polarities afforded seven fractions. The activity of 80% EtOAC/PE and 100% EtOAC fractions was observed to be significantly higher than that of DCM extract and 80% EtOAC/PE fraction was determined the most active fraction. The inhibitory activity of these two fractions was comparable with that of standard drug chloroquine (IC50 = 0.04 ± 0.002 and IC90 = 0.35 ± 0.006 mg/mL) while the remaining fractions were considered inactive. TLC analysis of the DCM extract and its fractions indicated the presence of terpenes, fatty acids, methylated coumarins, and methoxylated flavonoids as major constituents. Previous investigations showed that methoxylated flavonoids [A. armeniaca DCM extract and its active fractions might be related to the presence of these compounds. As observed in A. aucheri extracts and fractions. DCM extract was found to be more potent  than the corresponding PE extract  while the other three polar extracts were inactive. The DCM extract was selected for further investigation because of its potent activity and subjected to fractionation by the above-mentioned procedure. Among the seven different polarity fractions, 10% EtOAC/PE, 20% EtOAC/PE, and 40% EtOAC/PE fractions revealed no activity in this assay system, while the last four polar fractions showed the potent activity with close IC50 and IC90 values (A. aucheri [A. aucheri [Artemisia species act as inhibitors of heme crystallization pathway and A. armeniaca illustrated more potent activity than A. aucheri. A comparison of DCM fractions of two tested plants based on IC50 and IC90 values (A. armeniaca were about 3-fold and 4-fold more potent than 80% EtOAC/PE fraction of A. aucheri, respectively. This difference might have been derived from the high concentration of antimalarial components in this fraction and removing as much the lipid-like compounds from it. As shown in Figures 50 and IC90 values could be reduced by completely removing the lipids and purifying the active antimalarial principles.The malaria parasite, feration , 27. Freferation . To protferation . Thus, tferation . Many inthetical , 32 and thetical  antimalar et al. . As showavonoids , terpensavonoids  exhibite0 values . Methoxy aucheri  that str aucheri . Therefo0 values  revealedA. armeniaca was the most active extract in β-hematin formation assay followed by the DCM extract of A. aucheri. This preliminary study and its data persuade us to focus on purifying the active components of these extracts and investigating further on animal models for in vivo evaluation.From the selection of 10 extracts with different polarity, results showed that the DCM extract of