Danio rerio) as a model organism, the objective of the present study was to characterize the effects of waterborne exposure to both SeMet and tert-butyl hydroperoxide (tBOOH) to early life stages of zebrafish pre-treated with the antioxidant tert-butyl hydroquinone (tBHQ) in an attempt to investigate the mechanism of Se toxicity as it relates to oxidative stress. During the initial concentration range finding experiment, recently fertilized embryos were exposed for five days to 5, 25, 125, and 625 µg Se/L (as SeMet). These exposures informed the second experiment in which embryos were exposed to two concentrations of SeMet (25 and 125 µg Se/L) and 75 mg/L tBOOH either with  or without  a 4 h 100 µg/L tBHQ pre-treatment. Survival, hatchability, time to hatch, the frequency and severity of deformities , and changes in the expression of seven antioxidant-associated genes were determined. Exposures to SeMet and tBOOH reduced hatchability, increased time to hatch, decreased survival, increased the incidence and severity of deformities, and increased glutathione-disulfide reductase (gsr) expression in the pre-treated tBOOH treatment group.Aqueous L-selenomethionine (SeMet) embryo exposures represent a rapid and simplified method for investigating the embryotoxic effects of SeMet. Using zebrafish ( Danio rerio) represents one of the most widely-used small fish models in toxicological research, due to beneficial traits such as a short life cycle, frequent egg production, ease of culture, and a transparent embryo chorion which allows for the determination of fertilization status and morphological analysis throughout development [The zebrafish , tert-Butyl hydroquinone , and tert-Butyl hydroperoxide  were purchased from Sigma-Aldrich .Newly fertilized zebrafish embryos were collected in house from an adult breeding colony maintained in an environmental chamber with controlled temperature (27 +/− 1 °C) and photoperiod (16:8 light:dark) at the Toxicology Centre, University of Saskatchewan . Embryos determined to be healthy/developmentally normal and within the 4-cell–high blastula stage  of embryonic development  were selEmbryos were randomly distributed into glass petri dishes containing 40 mL of test solution. Each petri dish contained 20 embryos, with 9–12 replicate petri dishes in each exposure group. Exposure proceeded for 5 days in facility water alone (control embryos) or facility water spiked with nominal SeMet concentrations of 5, 25, 125, and 625 µg Se/L. To maintain water quality, 75% solution changes were performed daily and any dead embryos were removed.The 100 µg/L concentration of tBHQ was chosen based on preliminary studies as a concentration that did not induce a rate of mortality or deformities greater than that of the control embryos (unpublished data), while 75 mg/L of tBOOH was chosen based on preliminary studies as a concentration that caused a significant increase in the rate of deformities (unpublished data) relative to control embryos. Based on the aqueous SeMet exposures, 25 and 125 µg Se/L were selected for use in the following exposures as concentrations that caused a significant elevation in both mortality and the frequency of deformities relative to control embryos.Zebrafish embryos were collected and sorted as described previously. Embryos receiving the antioxidant pre-treatment were randomly distributed into plastic petri dishes containing 40 mL of 100 µg/L tBHQ, while embryos not receiving the pre-treatment were randomly distributed into plastic petri dishes containing 40 mL of facility water. Following 4 h, all embryos were re-distributed into glass petri dishes containing 40 mL of facility water alone, 75 mg/L tBOOH, 25 µg Se/L, or 125 µg Se/L. This resulted in 8 treatment groups, including: facility water alone (control embryos), 100 µg/L tBHQ (tBHQ control), 75 mg/L of tBOOH with and without antioxidant pre-treatment , 25 µg Se/L with and without antioxidant pre-treatment , and 125 µg Se/L with and without antioxidant pre-treatment . Each individual petri dish contained 20 embryos, with 32–33 control replicates and 13–21 replicates in each exposure group. Exposure proceeded for 5 days, during which dead embryos were removed, and 75% solution changes were performed daily.Following the 5-day aqueous exposures, remaining live larvae destined for deformities analysis were euthanized with an overdose of buffered MS-222 , fixed in buffered 10% formalin for 18–24 h, and stored in 70% ethanol. The preserved larvae were then examined for malformations in a blind fashion using an Olympus model S261 dissecting microscope . Larvae destined for biochemical analysis were frozen at −80 °C until analysis.In the present study, both the frequency and severity of larval deformities were quantified. During frequency analysis, larvae in each replicate were individually assessed and categorized as either deformed or not deformed. An overall incidence was calculated by dividing the number of individuals classified as deformed by the total number of individuals in that replicate. This process was then repeated for specific types of deformities .The severity of abnormalities was assessed using the Graduated Severity Index (GSI) system ,19. SeveWater samples taken from the exposure stock solutions were filtered and acidified with 2% high purity nitric acid  for total Se analysis. Pooled larvae collected for total Se analysis were euthanized with MS-222, collected in microcentrifuge tubes and stored at −20 °C until they could be freeze dried. Dried samples were then digested using high purity nitric acid and 30% hydrogen peroxide. Inductively coupled plasma-mass spectrometry (ICP-MS)  was used to quantify total Se in both water and tissue samples as described previously . InstrumFour to five replicates of 11–20 zebrafish larvae exposed to the SeMet, pro- and antioxidant treatment combinations described previously were collected in microcentrifuge tubes and stored at −80 °C until analysis. Total RNA was extracted using the RNeasy Plus Universal Mini Kit  according to the manufacturer’s protocol, quantified using the QIAxpert® microfluidic UV/VIS Spectrophotometer , and stored at −80 °C until analyzed. First-strand cDNA synthesis was performed using the QuantiNova™ Reverse Transcription Kit (QIAGEN) according to the manufacturer’s protocol using 2.25 µg of total RNA. The cDNA samples were stored at −20 °C until analyzed. Quantitative Real-Time PCR was performed in optical, fast, clear 96-well plates using the PowerUp™ SYBR™ Green Master Mix qPCR Kit  and the QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems).nrf2a), glutathione peroxidase 1a (gpx1a), glutathione s-transferase piscine 1 (gst p1), glutathione-disulfide reductase (gsr), manganese superoxide dismutase (sod2), glutamate-cysteine ligase catalytic subunit (gclc), aryl hydrocarbon receptor 2 (ahr2), actin beta (actb), and elongation factor 1 alpha (ef1α) are shown in −∆∆CT method described by Applied Biosystems [actb and ef1α serving as reference genes. A slight deviation from the suggested calculations involved the use of the control group’s mean ΔCT to calculate a ΔΔCT value and fold change value for each replicate as opposed to each treatment group, allowing for the calculation of error terms.A 25 µL reaction mixture containing PowerUp™ SYBR™ Green Master Mix (Applied Biosystems), an optimized concentration of cDNA (100 ng) and gene-specific qPCR primers (0.6 µmol), as well as nuclease free water was used for each sample and primer combination. The PCR reaction mixture was incubated/denatured at 50 °C for 2 min, then 95 °C for 2 min before the first PCR cycle. The thermal cycle profile consisted of denaturing at 95 °C for 1 s and extension at 60 °C for 30 s for a total of 40 PCR cycles. This was immediately followed by a dissociation curve. Each reaction was conducted in duplicate. Target gene primer sequences, accession numbers, and efficiencies for nuclear factor erythroid 2-related factor 2 . All data were tested for normality and homogeneity of variance using the Shapiro-Wilk normality test and either the Browne-Forsythe test or Bartlett’s test as appropriate. When data distribution was normal and exhibited homogeneity of variance, one-way analysis of variance (ANOVA) followed by Holm-Sidak’s multiple comparisons test was used to test for significant differences between treatment groups. When data distribution was non-normal, or data could not be satisfactorily transformed, a non-parametric Kruskal–Wallis one-way ANOVA by ranks was used followed by Dunn’s multiple comparisons test. Alpha values were two-tailed and set at 0.05. A nonlinear regression, dose-response analysis without constraints was performed on the percent cumulative mortality and deformities data to determine an LCTotal Se quantified in pooled larvae following 5 days of aqueous exposure at concentrations of 5, 25, 125, and 625 µg Se/L had mean total Se tissue concentrations of 34.2, 144, 264, and 291 µg Se/g dry mass (d.m.), respectively, compared to 2.98 µg/g d.m. in control embryos, indicating bioaccumulation in larvae in a concentration-dependent manner. Test solutions were also analyzed for total Se using ICP-MS to verify exposure concentrations . Measurep ≤ 0.024), compared to 2.50% ± 1.2% in the controls. An LC50 value of 67.9 µg/L (95% CI = 43.4–108 µg/L) was calculated for SeMet-induced mortality  and 62.3% ± 14% (p ≤ 0.0185) compared to 15.2% ± 2.9% in the controls. An EC50 value of 43.8 µg/L (95% CI = 21.4–138 µg/L) was also calculated for SeMet-induced deformities (Representative images of the deformities observed in this study can be found in ormities .p ≤ 0.0098). The incidence of edema, craniofacial and fin malformations in the 25 and 125 µg/L treatments were 25.9% ± 7.2% and 39.4% ± 9.4% compared to 0.417% ± 0.42% in the controls; 28.3% ± 8.2% and 30.7% ± 7.2% compared to 3.38% ± 1.4% in the controls; and 31.9% ± 6.3% and 55.1% ± 16% compared to 7.15% ± 2.2% in the controls, respectively  were also calculated. While exposure to SeMet had no significant effect on the incidence of skeletal deformities in zebrafish larvae, the incidence of edema, fin, and craniofacial malformations increased in a concentration-dependent manner, with significant elevations observed at concentrations ≥25 µg/L (ectively .p ≤ 0.0036), compared to 0.237 ± 0.056 in the control larvae . The severity/GSI scores for edema, craniofacial and fin malformations in the 25 and 125 µg/L treatments were: 0.383 ± 0.11 and 0.460 ± 0.11 compared to 0.004 ± 0.004 in the controls; 0.332 ± 0.082 and 0.307 ± 0.072 compared to 0.0338 ± 0.014 in the controls; and 0.472 ± 0.10 and 0.743 ± 0.23 compared to 0.0882 ± 0.030 in the controls, respectively  compared to 97.5% ± 1.2% in the controls , with percentages of 46.5% ± 7.2%, 50.5% ± 6.6%, 54.5% ± 5.9%, and 73.1% ± 5.0% in the 25-t, 25, 125-t, and 125 treatments, respectively, compared to 4.85% ± 0.74% in the controls. However, no differences were observed between embryos pre-treated with tBHQ and embryos exposed to SeMet alone , with percentages of 6.86% ± 2.2%, 32.9% ± 4.9%, 31.9% ± 4.3%, 39.1% ± 5.1%, 37.1% ± 5.6%, 36.2% ± 8.0%, and 48.1% ± 9.4% in the tBOOH-t, tBOOH, 25-t, 25, 125-t, and 125 treatments respectively, compared to 5.30% ± 1.5% in the controls. No significant differences were observed between embryos pre-treated with tBHQ and embryos exposed to either tBOOH or SeMet alone , with percentages of 7.36% ± 2.9%, 8.62% ± 3.3%, 12.8% ± 5.2%, and 25.6% ± 9.3% in the 25-t, 25, 125-t, and 125 treatments, respectively, compared to 0.479% ± 0.27% in the controls. Scoliosis only showed a significant increase in frequency in the pre-treated 25 µg/L group (p = 0.0072), with a percentage of 15.1% ± 3.5% compared to 3.71% ± 1.4% in the controls. The incidence of edema increased significantly in all treatment groups (p ≤ 0.025), with percentages of 15.5% ± 4.6%, 21.3% ± 4.4%, 26.3% ± 5.2%, 34.7% ± 5.6%, 24.0% ± 6.2%, and 30.6% ± 8.4% in the tBOOH-t, tBOOH, 25-t, 25, 125-t, and 125 treatments, respectively, compared to 0.470% ± 0.26% in the controls. The incidence of fin malformations increased significantly in the pre-treated tBOOH group and within all SeMet treatment groups compared to the controls (p ≤ 0.017), with percentages of 14.8% ± 3.1%, 17.9% ± 5.7%, 17.0% ± 3.6%, 14.4% ± 4.1%, and 27.0% ± 8.0% in the tBOOH-t, 25-t, 25, 125-t, and 125 treatments, respectively, compared to 1.58% ± 0.62% in the controls. Finally, craniofacial malformations increased significantly in frequency in both 25 µg/L SeMet treatment groups (p ≤ 0.022), with percentages of 11.2% ± 3.9% and 11.0% ± 3.2% in the 25-t and 25 treatments, respectively, compared to 0.614% ± 0.36% in the controls. No significant differences were observed between embryos pre-treated with tBHQ and embryos exposed to either tBOOH or SeMet alone , with severity/GSI scores of 0.470 ± 0.086, 0.417 ± 0.062, 0.709 ± 0.17, 0.875 ± 0.16, 0.710 ± 0.19, and 1.32 ± 0.41 in the tBOOH-t, tBOOH, 25-t, 25, 125-t, and 125 treatments, respectively, compared to 0.0766 ± 0.024 in the controls. However, no significant differences were observed between embryos pre-treated with tBHQ and embryos exposed to either tBOOH or SeMet alone , with severity/GSI scores of 0.0991 ± 0.035, 0.142 ± 0.053, and 0.340 ± 0.13 in the 25, 125-t, and 125 treatments, respectively, compared to 0.0048 ± 0.003 in the controls. The severity of scoliosis only increased significantly in the pre-treated 25 µg/L group (p ≤ 0.020), with a severity/GSI score of 0.162 ± 0.038 compared to 0.0422 ± 0.015 in the controls. Exposure to all treatment groups resulted in a significant increase in the severity of edema (p ≤ 0.030), with severity/GSI scores of 0.155 ± 0.046, 0.213 ± 0.044, 0.229 ± 0.044, 0.375 ± 0.061, 0.240 ± 0.062, and 0.337 ± 0.092 in the tBOOH-t, tBOOH, 25-t, 25, 125-t, and 125 treatments, respectively, compared to 0.0062 ± 0.004 in the controls. In contrast, the severity of craniofacial malformations increased significantly only in the 25 µg/L group (p ≤ 0.0080), with severity/GSI score of 0.110 ± 0.032 compared to 0.0077 ± 0.005 in the controls. Finally, the severity of fin malformations increased significantly within the pre-treated tBOOH group, 25 µg/L group, and both 125 µg/L treatment groups (p ≤ 0.014), with severity/GSI scores of 0.151 ± 0.031, 0.205 ± 0.052, 0.178 ± 0.063, and 0.351 ± 0.12 in the tBOOH-t, 25, 125-t, and 125 treatments, respectively, compared to 0.0158 ± 0.0062 in the controls. No significant differences were observed between embryos pre-treated with tBHQ and embryos exposed to either tBOOH or SeMet alone , with mean time to hatch values of 3.90 ± 0.13 days, 3.79 ± 0.11 days, 3.66 ± 0.11 days, and 3.58 ± 0.083 days in the tBOOH-t, tBOOH, 25-t, and 125 treatments, respectively, compared to 3.29 ± 0.045 days in the controls (p ≤ 0.078), with percentages of 40.7% ± 5.8%, 70.7% ± 5.5%, 51.0% ± 7.3%, 68.1% ± 5.2%, 47.0% ± 6.5%, and 38.6% ± 4.4% in the tBOOH-t, tBOOH, 25-t, 25, 125-t, and 125 treatments, respectively, compared to 95.2% ± 0.74% in the controls. No significant differences in time to hatch or hatchability were observed between embryos pre-treated with tBHQ and embryos exposed to either tBOOH or SeMet alone . Consistent with all previous endpoints, no significant differences were observed between embryos pre-treated with tBHQ and embryos exposed to either tBOOH or SeMet alone  [Pimephales promelas) [Previous aqueous SeMet embryo exposure work in zebrafish , Japaneslatipes) , and fatromelas)  reportedromelas) , it appeThe heightened sensitivity of zebrafish relative to other fish species used in aqueous embryo SeMet exposure experiments remained consistent during evaluations of larval deformities, as previous aqueous SeMet embryo exposure work in Japanese medaka  and fathConsistent with the mortality and deformities frequency data, comparisons between the present study and previous aqueous embryo exposure work suggest that zebrafish are more sensitive to aqueous Se exposures, as our previous work in fathead minnows  showed aThe lack of effect on time to hatch is consistent with previous work in fathead minnow  and JapaBased on the results of the first experiment, 25 and 125 µg Se/L were selected for use in the second experiment, along with two additional pharmacological agents, tert-butyl hydroperoxide (tBOOH), and tert-butyl hydroquinone (tBHQ).Consistent with the first experiment, exposure to the 25 and 125 µg/L SeMet treatment concentrations caused a significant increase in mortality compared to control embryos; however, no differences were observed between embryos pre-treated with tBHQ and embryos exposed to SeMet alone. This was consistent with previous aqueous SeMet embryo exposure work in zebrafish, where embryos were pre-treated with the antioxidant NAC for 24 h prior to SeMet exposure and no significant differences in mortality compared to embryos exposed to SeMet alone were observed [Consistent with the first experiment, exposure to the 25 and 125 µg/L SeMet treatment concentrations also resulted in a significant increase in both the incidence and severity of deformities compared to control embryos. However, no differences were observed between embryos pre-treated with tBHQ and embryos exposed to SeMet alone. This contrasted with the antioxidant rescue work conducted by Arnold et al. [While there were no clear patterns in terms of the occurrence or severity of certain types of deformities observed, we did note that significant elevations in the incidence and severity of skeletal deformities was only observed in the SeMet treatments, and not the tBHQ controls or tBOOH treatments. Furthermore, there seemed to be a general trend towards a higher proportion and severity of deformities observed in the SeMet treatments compared with the tBOOH treatments.In the present study, lordosis  was excluded from the deformity calculations due to an abnormally high incidence within both the control and tBHQ treatment groups , as it wIn the present study, no consistent patterns were evident during assessment of time to hatch. Furthermore, the biological relevance of these results was considered minimal, as the time to hatch in these treatments only differed from the controls by about a day at most. In terms of effects on hatchability, the reductions observed in the SeMet exposed embryos were consistent with the first experiment, as well as previous aqueous Se embryo work ,14,15. Wnrf2a), which is an oxidant-responsive transcription factor that regulates the transcription of antioxidant genes such as gst p1 [gpx1a), which is an enzyme that serves a protective role in cells by catalyzing the GSH-dependent reduction/degradation of various hydroperoxides [gst p1), which is one of the phase II detoxification enzymes that catalyze the conjugation of GSH to harmful/electrophilic compounds to aid with excretion [gsr), which is an enzyme that directly catalyzes the reduction of oxidized glutathione (GSSG) back to reduced glutathione (GSH) [sod2), which is an enzyme that catalyzes the reduction of the superoxide anion radical (O2∙-) into hydrogen peroxide and oxygen [gclc), which is a rate-limiting enzyme that catalyzes the first step in GSH synthesis from l-cysteine and glutamate; and finally aryl hydrocarbon receptor 2 (ahr2), which is a ligand-dependent transcription factor involved with the biotransformation of a range of contaminants [In an attempt to further reinforce the relationship between oxidative stress and Se induced toxicity, changes in the expression of 7 genes relevant to the antioxidant defense system were evaluated. These included: nuclear factor erythroid 2-related factor 2  ; manganed oxygen ; glutamaaminants ,28.gsr expression increased significantly out of the 7 genes evaluated, and only in the pre-treated tBOOH treatment group. Since neither tBOOH or tBHQ alone induced a response in gsr, it is difficult to conclude on which chemical was responsible for the up-regulation of this particular gene. Limited previous data exists for changes in gsr expression in fish embryos exposed to tBOOH or tBHQ; however, one study involving 4-dpf zebrafish eleutheroembryos  exposed to tBHQ for 6 hr reported a significant increase in gsr gene expression [As was mentioned previously, only pression .Exposure to the tBOOH treatment groups did not cause significant mortality but did result in significant increases in both the incidence and severity of deformities such as those observed in the SeMet treated embryos, potentially suggesting a similar pathway  is playing a role in the generation of deformities.tBHQ is not ideally suited for use as an antioxidant. As noted by Kobayashi et al. , tBHQ isThe concentration of tBHQ (100 µg/L) selected was low enough that no significant differences in mortality or deformities were observed relative to controls, but perhaps not high enough to induce the activation of the antioxidant defense system.The observed results were influenced by the duration and developmental stage of exposure. In the present study, embryos were pre-treated for 4h with 100 µg/L tBHQ immediately following collection. Previous work by Timme-Laragy et al.  treated No differences were observed between embryos pre-treated with tBHQ and embryos exposed to either SeMet or tBOOH alone for all endpoints evaluated in the present study. Here we offer a few potential reasons for this observation:To the best of our knowledge, there is no previous research that has looked at the production of deformities in fish embryos following exposure to these chemicals (tBHQ and tBOOH). Therefore, more research is required to better understand whether these pharmacological agents are helpful/suitable for investigating the mechanism of Se toxicity.


The structure of 1 was established on the basis of its extensive spectral data, and the absolute structures of 1 and 10 were determined by their CD data. The absolute structure of 10 was established for the first time. Among the obtained compounds, 5–8 inhibited the proliferation of K562 cells with inhibition rates of 26.6%, 65.7%, 40.4% and 45.6% at 100 µg/mL; 1 and 4–10 showed significant protective effects on anoxia-induced injury in cultured ECV304 or PC12 cells at 50 µg/mL; 8 and 9 showed antibacterial effects on Staphylococcus aureus ATCC6538 at the tested concentration of 150 µg/8 mm paper disc. Compounds 2 and 4–10 were isolated for the first time from this genus. The proliferation inhibiting activities of 7 and 8, the anti-hypoxia activities of 1 and 4–10, and the antibacterial effect of 8 and 9 on Staphylococcus aureus ATCC6538 are reported here for the first time.In the present study, a new flavanoid  Choerospondias axillaries, the only plant of the genus Choerospondias belonging to the family of Anacardiaceae, is mainly distributed in the Hubei, Guangdong, Guangxi, Yunnan, Fujian and Guizhou provinces of China. In China, the dried fruit of C. axillaries has been usually used for the treatment of cardiovascular diseases, especially in Mongolian traditional medicine +, and adduct peaks at 609.12290 [M+Na]+ and 625.09327 [M+K]+ corresponding to a formula of C28H26O14. Its UV spectrum showed a maximum absorption at 286 nm  arising from the hydroxylated phenyl groups. Its IR spectrum displayed characteristic absorptions for hydroxyl (3293 cm−1) and carbonyls (1685 and 1637 cm−1). The 1H-NMR spectrum showed aliphatic proton signals at δ 3.07  and at δ 2.66  from the axial and equatorial methylene protons at C-3 as well as a third aliphatic resonance at δ 5.31  from the methine proton, which bears an oxygen and phenyl function at C-2 of the flavanone moiety. The AA′BB′ system at δ 7.29  and at δ 7.06 , and an AX system at δ 5.90  and at δ 5.88  were in accordance with derivatives of 5,7,4′-trihydroxyflavonone, i.e., derivatives of naringenin. The anomeric proton signal at δ 4.91  revealed the presence of one sugar with a β configuration. The 13C-NMR signals at δ 101.5, 77.5, 75.1, 74.3, 71.5 and 64.4 indicated that the sugar should be glucosyl group  arising from a galloyl group, the 13C-NMR data of 1 were closely similar to those of naringenin-4′-O-β-d-glucopyranoside (3) (J = 7.6 Hz) of the glucopyranosyl moiety and C-4′ at δ 158.5  and 4.61 . Thus, compound 1 was identified as narigenin-4′-O-.Compound side (3) . The lin δ 158.5 . The loc δ 158.5 , which wS structure of narigenin was confirmed by the positive Cotton effect at 326.5 nm in the CD spectrum +, 609 [M+Na]+, 625 [M+K]+. Positive ion HR-ESI-MS m/z: 587.14907 [M+H]+, 609.12290 [M+Na]+, 625.09327 [M+K]+. UV (MeOH) λmax (log ε): 216 nm (4.41), 286 nm (4.03). IR (KBr) νmax: 3293, 2947, 2882, 1685, 1637, 1607, 1517, 1460, 1343, 1316, 1230, 1188, 1161, 1065, 1038 864, 744, 723 cm−1. CD λmax nm (mdeg) in MeOH at 1.0 mg/mL: 234.0 (0), 244 (6.4100), 255.5 (0), 272.5 (−25.0052), 301.5 (0), 326.5 (20.5810), 383.0 (0). 1H-NMR and 13C-NMR data see Naringenin-4′-O-β-D-glucopyranoside (3). White crystaline powder, m.p. 224–226 °C (MeOH), 2CO), showing a brown with ferric chloride reagent. Positive ion ESI-MS m/z: 435 [M+H]+, 457 [M+Na]+, 473 [M+K]+. 1H-NMR and 13C-NMR data see Dehydrodicatechin A (10). Yellow crystalline powder, m.p. 196–198 °C (MeOH), m/z: 577 [M+H]+, 599 [M+Na]+; Negative ion ESI-MS m/z: 575 [M−H]−. CD λmax nm (mdeg) in MeOH at 1.0 mg/mL: 190 (−0.3533), 232.5 (0), 237.5 (5.1513), 240 (0), 256.5 (−38.1595), 272.0 (0), 336.5 (53.8359), 358.5 (0), 403.0 (−60.7813), 456.0 (0). 1H-NMR and 13C-NMR data see 2 and 95% air.Human myeloid leukemia K562, human umbilical vein endothelial cell ECV304, and rat pheochromocytoma PC12 cell lines were used for bioassay. The K562, ECV304 and PC12 cells were routinely maintained in RPMI-1640 (for K562 and ECV 304 cells) or DMEM (for PC12 cells) medium containing 100 µg/mL penicillin and 100 µg/mL streptomycin supplemented with 10% FBS under a humidified atmosphere of 5% CO1–10 and DOC were dissolved in MeOH to prepare a 10.0 mg/mL solution, and serial dilutions were made for compounds 1–10. These solutions were subjected to the MTT assay. DOC was used as positive control and MeOH was used as blank control. The assay was run in triplicate on human cancer K562 cell lines by the method that we have previously reported [Compounds reported .1–10 were dissolved in the DMEM or RPMI 1640 medium to prepare a solution at 50.0 µg/mL. The anti-hypoxia activities of 2–5 were assayed using ECV304 cells, but the anti-hypoxia activities of 1 and 6–10 were assayed on PC12 cells.Compounds MTT assay on ECV 304 cells: Exponentially growing ECV 304 cells were suspended in fresh RPMI 1640 medium at the density of 1 × 105 cells/mL and then seeded into 96-well plates at 150 µL/well. The cells were incubated at 37 °C for 48 h, then discarded the medium and were assigned into normoxic control group, hypoxia control group and hypoxia administration group. Each well of the normoxic control group and hypoxia control group was added 150 µL fresh RPMI 1640 medium, and the hypoxia administration group was 150 µL sample solution. The hypoxia control group and hypoxia administration group were cultured in the atmosphere of 5% CO2 and 95% N2 for 24 h, while the normal control group was normally cultured for 24 h. Then, 15 µL MTT solution (5 mg/mL in PBS) was added to each well and incubated at 37 °C for 4 h. Then, the MTT solution were discarded, and 150 µL DMSO were added in each well; after the purple material were fully dissolved, the optical density (OD) of each well was determined on a Versa max plate reader at 490 nm. The cell viabilities were calculated using mean values from cell viabilities (%) = OD hypoxia control or OD sample/OD normal control × 100%.MTT assay on PC 12 cells: Exponentially growing PC12 cells were suspended in fresh DMEM medium at the density of 1 × 105 cells/mL and then seeded into 96-well plates at 150 µL/well. The cells were incubated at 37 °C for 24 h, then discarded the medium and were assigned into normoxic control group, hypoxia control group and hypoxia administration group. Each well of the normoxic control group and hypoxia control group was added 150 µL fresh DMEM medium, and the hypoxia administration group was 150 µL sample solution, then all were incubated at 37 °C for 1 h. Each well of hypoxia control group and hypoxia administration group were added 2 µL CoCl2 solution (306.14 µg/mL in water), all the three groups were incubated at 37 °C. After all were normally incubated for 24 h, 15 µL MTT solution (5 mg/mL in PBS) was added to each well and incubated at 37 °C for 4 h. Then, the MTT solution were discarded, and 150 µL DMSO were added in each well; after the purple material were fully dissolved, the optical density (OD) of each well was determined on a Versa max plate reader at 490 nm. The cell viabilities were calculated using mean values from cell viabilities (%) = OD hypoxia control or OD sample/OD normal control × 100%.Blastomyces albicans ATCC10231 and Staphylococcus aureus ATCC6538 were used to evaluate the antibacterial activities of compounds 1–10. Blastomyces albicans ATCC10231 was cultivated on Sabouraud’s agar medium, and Staphylococcus aureus ATCC6538 was cultivated on tryptose soya agar medium. All the compounds were dissolved in methanol to prepare 10 mg/mL test sample solutions. Samples (15 µL) were added to the paper discs (8 mm) and dried for 10 min. Then, the discs were put on the tested plate which contained either Staphylococcus aureus or Blastomyces albicans. Tested plates were cultured at 28 °C for 2 days and then the diameter of the inhibition zone was measured.C. axillaries. Among the obtained compounds, 1 is a new compound with a galloylglucosyl group on the B ring in the flavonoid skeleton, and compounds 2 and 4–10 were isolated from this genus for the first time. The absolute structures of 1 and 10 were established from their CD data, and the absolute configuration of 10 was determined exactly for the first time. The proliferation inhibiting activities of 7 and 8, the anti-hypoxia activities of 1 and 4–10, and the antibacterial effect of 8 and 9 on Staphylococcus aureus ATCC6538 are reported here for the first time.Ten flavonoids were isolated from the stem bark of


Despite the progress in colon cancer treatment, relapse is still a major obstacle. Hence, new drugs or drug combinations are required in the battle against colon cancer. α-Mangostin and betulinic acid (BA) are cytotoxic compounds that work by inducing the mitochondrial apoptosis pathway, and cisplatin is one of the most potent broad spectrum anti-tumor agents. This study aims to investigate the enhancement of BA cytotoxicity by α-mangostin, and the cytoprotection effect of α-mangostin and BA on cisplatin-induced cytotoxicity on HCT 116 human colorectal carcinoma cells. Cytotoxicity was investigated by the XTT cell proliferation test, and the apoptotic effects were investigated on early and late markers including caspases-3/7, mitochondrial membrane potential, cytoplasmic shrinkage, and chromatin condensation. The effect of α-mangostin on four signalling pathways was also investigated by the luciferase assay. α-Mangostin and BA were more cytotoxic to the colon cancer cells than to the normal colonic cells, and both compounds showed a cytoprotective effect against cisplatin-induced cytotoxicity. On the other hand, α-mangostin enhanced the cytotoxic and apoptotic effects of BA. Combination therapy hits multiple targets, which may improve the overall response to the treatment, and may reduce the likelihood of developing drug resistance by the tumor cells. Therefore, α-mangostin and BA may provide a novel combination for the treatment of colorectal carcinoma. The cytoprotective effect of the compounds against cisplatin-induced cytotoxicity may find applications as chemopreventive agents against carcinogens, irradiation and oxidative stress, or to neutralize cisplatin side effects. Colon cancer accounts for almost 10% of the total cases of cancer, and 8% of the total cancer deaths . DespiteGarcinia mangostana fruit rinds. The compound has been reported to possess multiple pharmacological effects, including antioxidant, anti-inflammatory, and anti-cancer properties [1,3,6-Trihydroxy-7-methoxy-2,8-bis(3-methyl-2-butenyl)-9H-xanthen-9-one -lupaene-28-oic acid BA, , is a pe-lupaene-Cis-diamminedichloroplatinum(II) . The purity of the compound was determined by HPLC and was found to be >97% . The retGarcinia mangostana. The compound’s purity was tested by HPLC, which was >97%  with the reference compound, which further confirms its identity. α-Mangostin was isolated at 60% yield (wt/wt) from the toluene extract of was >97% . The ide50s) were calculated from the dose response curves, and were found to be 4.0 ± 1.0 µg/mL, 8.9 ± 0.1 µg/mL, and 6.5 ± 0.1 µg/mL, respectively. α-Mangostin and BA showed a lower cytotoxic effect on the normal colonic cells (CCD-18Co) compared to their effect towards the cancer cells, and the IC50s were 11 ± 0.5 µg/mL and 47 ± 1.0 µg/mL, respectively.α-Mangostin, BA, and cisplatin inhibited the growth of HCT 116 cells in a dose dependent manner . The medThe cytotoxicity of α-mangostin/BA, α-mangostin/cisplatin, and BA/cisplatin drug pairs was investigated. Firstly, cells were treated simultaneously with the treatment compounds at inactive concentration of the first drug and various concentrations of the second. Secondly, cells were treated with cytotoxic concentration of the first drug with various concentrations of the other compound. The percentage inhibition was determined and the drug enhancement was determined by calculating the fold change in the cytotoxicity of the combination relative to their additive effect, the results are summarized in On the other hand, the α-mangostin/BA combinations did not enhance the BA cytotoxicity towards the normal colonic CCD-18Co cells. The fold change in BA cytotoxicity, relative to the additive effect of both compounds, in the following α-mangostin/BA combinations 2.5/2.5, 2.5/5.0, 2.5/7.5 and 2.5/10 µg/mL was 0.9 ± 0.1, 1.1 ± 0.1, 1.1 ± 0.1, and 1.0 ± 0.2, respectively. P = 0.99 and 0.96, respectively. Likewise, BA at 5 µg/mL did not induce significant changes in caspases 3/7 activity, P = 0.36. On the contrary, BA at 7.5 µg/mL significantly induced the caspases activity, P = 0.0 . The fold change in caspases 3/7 activity was 3.2 ± 0.7 and 1.3 ± 0.1 in the 2.5/5.0 and 2.5/7.5 α-mangostin/BA combinations, respectively, and the fold change in the 3.75/5.0 and 3.75/7.5 combinations was 8.0 ± 2.3 and 2.8 ± 0.2, respectively.The effect of α-mangostin/BA combinations was then investigated on caspases-3/7 of HCT 116 cells. α-Mangostin at 2.5 and 3.75 µg/mL did not show significant induction of the caspases 3/7 activity compared to the negative control,  P = 0.0 . The effP < 0.05 , or BA (5.0 and 7.5 µg/mL) alone caused minor changes on the nuclear morphology of HCT 116 cells. On the other hand, the combination of both compounds induced characteristic changes in the chromatin structure and nuclear morphology of the cells. Chromatin condensation with smaller nuclei, half moon or crescent-shaped nuclei, nuclear fragmentation, and formation of apoptotic bodies were all observed [P values < 0.05.In order to get deeper insights into the mechanism of action of α-mangostin, its effects on four cell signaling pathways was investigated by the luciferase assay. α-Mangostin at a concentration of 7.5 µg/mL caused significant enhancement of the p53/DNA damage (1.4 ± 0.2 folds), Myc/Max (1.6 ± 0.1 folds), and the MAPK/ERK (2.1 ± 0.4 folds) signaling pathways, and downregulation of the NFKB pathway (0.8 ± 0.1 folds), Garcinia mangostana Linn, and BA was isolated from a BA fraction from Syzygium campanulatum Korth. The identity of the compounds was confirmed by comparing their HPLC retention time and MS isotopic pattern with reference compounds. Both compounds have been reported to have anti-cancer activity, which is mediated via the mitochondrial apoptosis pathway [α-Mangostin and BA are cytotoxic compounds of natural origin. In this study α-mangostin was isolated at high purity from a xanthone extract from  pathway ,26. Prev pathway , consequThis study aimed to study the cytotoxic effect of α-mangostin/BA, α-mangostin/cisplatin, and BA/cisplatin combinations on colorectal carcinoma cells. HCT 116 human colorectal carcinoma cell line was selected because it was reported as an invasive and metastatic model of colorectal carcinoma , and CCDin vitro cytoprotective effect of α-mangostin and BA against cisplatin and other cytotoxic compounds on cancer and normal cells. Further investigation is needed in order to study their in vitro and in vivo cytoprotective effects on other cytotoxic compounds against other normal and cancer cell lines, and to study their mechanism of action. Previous reports showed that the DNA damaging agents such as cisplatin induce apoptosis through the accumulation of reactive oxygen species (ROS) . α-MangoThe α-mangostin/BA combination resulted in enhancement of their cytotoxic effect towards the colon cancer cells but not towards the normal colonic cells. This effect was further investigated on the early and late markers of apoptosis including cytoplasmic shrinkage and loss of attachment, enhancement of caspases-3/7 activity, loss of mitochondrial membrane potential, and chromatin condensation and nuclear morphology. Our data showed that the apoptotic effect α-mangostin/BA combination is significantly higher than their additive effect, which confirm the enhancement effect of the combination. It is noteworthy to mention that the effect on the mitochondrial membrane potential was stronger than that on chromatin condensation and nuclear morphology, which may indicate the direct effect of the combination on the mitochondria. We found that the apoptotic effects of α-mangostin are mediated via upregulation of p53, Myc/Max and MAPK/ERK signaling pathways and downregulation of the NFKB pathway. BA has been shown to work by inhibition of the hypoxia pathway , decreas® and DMEM cell culture media, heat inactivated fetal bovine serum (HI-FBS), and phosphate buffered saline (PBS) without calcium and magnesium were purchased from Bio-Diagnostics . The Cignal finder™ reporter-array system was purchased from SABiosciences . Caspases-3/7 reagent, and trans-fast liposome, and dual luciferase reporter system were purchased from Promega . Cisplatin, BA, Hoechst 33258, rhodamine 123, penicillin/streptomycin (PS), 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT), and phenazine methosulfate (PMS) were purchased from Sigma-Aldrich . α-Mangostin reference compound was purchased from ChromaDex . Human colorectal carcinoma cell line HCT 116, and human normal colonic cell line CCD-18Co were purchased from the American Type Culture Collection . RPMI 1640, Opti-MEMSyzygium campanulatum leaf extract as previously described [n-hexane-ethyl acetate at 8:2 ratio (v/v), and 10 mL fractions were collected. The purity of BA was determined by HPLC as previously described [3PO4 in water, the flow rate was set at 1 mL/min for 20 min, and the spectral data was collected at 210, 230 and 254 nm.BA was purified from a BA fraction that was prepared from escribed . The colescribed . BrieflyGarcinia mangostana fruit rinds. The column (25 × 1 inch) was packed with 60 g silica gel 60 . The xanthone extract (1 g) was dissolved in methanol and mixed with silica (5 g), and the dried mixture was loaded into the pre-packed column. Elution was then performed with 200 mL portions dichloromethane-n-hexane in 1:3, 1:2 and 2:1 ratios (v/v). Methanol was then added at 0.5% (v/v) with a two fold increment in each 200 mL cycle to reach 4% in the fourth one. Ten millilitre fractions were collected, and the purity of α-mangostin was tested using Dionex-Ultimate® 3000 Rapid Separation HPLC. The column was Nucleosil C18 , the column temperature was set at 30 °C, the mobile phase was 95% acetonitrile:5% 0.1% H3PO4 in water for 10 min at 1 mL/min, and data was collected at 244, 254, 316 and 320 nm (submitted manuscript). α-Mangostin was isolated by column chromatography from a toluene extract prepared from m/z, and the acquired mass spectra represent the average of five spectra.The identity of the isolated compounds was further confirmed by MS analysis. MS spectra of α-mangostin and BA were obtained using a LC-MSD Trap-VL Electrospray ion (ESI) mass spectrometer (Agilent Technologies) in the direct infusion mode. The samples were injected directly into the ESI source at 5 μL/min. The MS conditions were as follows: negative ion mode; gas (N2) temperature, 325 °C; flow rate, 5.0 L/min; nebulizer pressure, 15 psi; HV voltage, 4.0 kV; octopole RF amplitude, 150 Vpp; skim 1 voltage, −38.8 V; skim 2 voltage, −6.0 V; capillary exit, −113.8 V; cap exit offset, −75.0 V; scanning range, 350–550 2 humidified atmosphere. Cell viability was determined by the XTT test as described previously [50s were calculated from the dose response curves (n = 3).HCT 116 cells were propagated in RPMI 1640 medium supplemented with 10% HI-FBS and 1% PS. CCD-18Co cells were maintained in DMEM medium with 10% HI-FBS and 1% PS. Cells were cultured at 37 °C in a 5% COThe caspases 3/7 activity was measured by caspase Glo 3/7 as described previously . In brie® digital fluorescent inverted microscope . Cell morphology was evaluated by studying five randomly selected microscopic fields, and the apoptotic index was calculated. Rhodamine 123 and Hoechst 33258 were used as probes to study the effect on mitochondrial membrane potential and chromatin condensation ,40. BrieRenilla dual-luciferase assay according to manufacturer’s instructions. The Firefly/Renilla ratio was then calculated, and the fold change in the transcription factor activity was determined by dividing the results of the treated cells by that of untreated cells (0.5% DMSO).The effect of α-mangostin on four cell signaling pathways was investigated by the luciferase assay as described previously . In briet-test or One-way ANOVA, and were considered significant at P < 0.05. Data analysis was carried out using SSPS 16.0 software.The results are presented as mean ± SD. The additive effect of the individual treatments was calculated as described previously by Webb, the following equation was used :Additivein vivo investigation. The compounds may also provide a cytoprotective effect against oxidative stress, irradiation, and chemical carcinogens, which may benefit in prevention of several human diseases such as cancer, neurodegenerative disorders, aging, and cardiovascular diseases. The cytoprotective effect of BA and α-mangostin against cisplatin induced cytotoxicity may indicate a dual effect of these compounds in cancer chemotherapy; they may neutralize the nonselective cytotoxicity of cisplatin or other chemotherapeutics, or may reduce their overall anti-cancer effect, which needs further The enhancement of BA cytotoxic and apoptotic effects by α-mangostin may indicate this combination as a novel candidate for the treatment of colorectal carcinoma.


Azadirachta indica A. Juss (neem) extracts have been used in pharmaceutical applications as antitumor agents, due to their terpenes and phenolic compounds. To obtain extracts from neem leaves with potential antiproliferative effect, a sequential process of pressurized liquid extraction was carried out in a fixed bed extractor at 25 °C and 100 bar, using hexane (SH), ethyl acetate (SEA), and ethanol (SE) as solvents. Extractions using only ethanol (EE) was also conducted to compare the characteristics of the fractionated extracts. The results obtained by liquid chromatography-electrospray ionization mass spectrometry suggested a higher concentration of terpenes in the SEA extract in comparison to SH, SE, and EE extracts. Therefore, antiproliferative activity showed that SEA extracts were the most efficient inhibitor to human tumor cells MCF-7, NCI-H460, HeLa, and HepG2. Hepatocellular cells were more resistant to SH, SEA, SE, and EE compared to breast, lung, hepatocellular, and cervical malignant cells. Neem fractioned extracts obtained in the present study seem to be more selective for malignant cells compared to the non-tumor cells. Azadirachta indica A. Juss) is a tree of the Meliaceae family found worldwide in semi-tropical and tropical climates + peak at m/z 467.211 +, has been identified in neem leaves +. Other fragments can result from the rupture of ester bonds from the [M + H]+ species [The mass of nimbolide is 466.199 with a molecular formula Cm leaves . The nee species , thereby+ at m/z 611 [Among the 10 compounds identified in  m/z 611 . However m/z 611 ,33. AccoNeem extracts have demonstrated activity against tumor cells ,34. Due 50. SEA extracts show the highest potential to inhibit the growth of tumor cells, presenting GI50 value smaller than those found for SH, SE, and EE. The result suggests that the clean-up process performed by the sequential PLE extraction was able to produce fractions with high antitumor effects. NCI-H460, HeLa, and HepG2 cells were more sensitive to SEA than the other studied cells. Some investigations have demonstrated that plant-derived fractions obtained by high pressure show an antiproliferative potential against cancer cells [As presented in er cells ,37,38,39er cells .50 values were of 350 µg/mL on MCF-7 cells and 175 µg/mL on HeLa cells. Nevertheless, these GI50 values were higher than GI50 values found in the present study for SEA, that were 82.3 ± 4.3 µg/mL and 48.8 ± 4.3 µg/mL on MCF-7 and Hela, respectively. Moreover, SEA extract concentrations were also more cytotoxic to the MCF-7 and HeLa cells than 50 and 100 µg/mL of neem ethanolic extract combined with 5 µM cisplatin (antitumor agent). According to Sharma et al., these combinations have a synergistic effect on cancer cell growth inhibition in 52.2 (MCF-7) and 65% (HeLa) [Sharma et al., tested a vast range of concentrations (10–500 µg/mL) of ethanolic neem leaf extract against MCF-7 and Hela, and their GI50 values with 83 ± 9 (MCF-7), 262 ± 4 (NCI-H460), 160 ± 13 (HeLa) and 100 ± 10 (HepG2) µg/mL of Thymus vulgaris leaves and 154 ± 7 (MCF-7), 229 ± 16 (NCI-H460), 224 ± 12 (HeLa) and 111 ± 12 (HepG2) µg/mL of Mentha x piperita leaves [SEA  also exha leaves . Accordia leaves .Non-tumor liver PLP2 cells have been used to evaluate the tumor selectivity effect ,41. As cAzadirachta indica A. Juss) were collected during the spring from Germplasm Bank (GBN) of Embrapa Coastal Tablelands . All leaves were mixed and dried at 45 °C for 36 h in an oven with hot-air circulation. After that, the leaves were milled and classified according to its size in the range of 8 to 16 mesh using the Tyler sieves series. The material was stored under refrigeration and protected from light until the extractions. Young and old leaves of neem  during 10 min to remove residual n-hexane. After that, the system was depressurized and the second PLE step was performed at 100 bar and 25 °C, using ethyl acetate as an extraction solvent for 60 min at 1 mL/min. The system was then flushed again with carbon dioxide during 10 min to remove residual ethyl acetate. The third PLE step was conducted using water/ethanol (20:80 v/v) mixtures as solvent at 100 bar, 25 °C, 1 mL/min for 60 min of extraction. One-step pressurized liquid extraction using water/ethanol (20:80 v/v) mixtures as a solvent was also carried out for analyzing the effects of the sequential extraction sequential. After removing the organic solvents of the obtained solution in a rota-evaporator, the neem extracts were named SH, SEA, and SE for the sequential process using n-hexane, ethyl acetate, and water/ethanol as solvent, respectively. The one-step PLE produced an extract named EE. All extractions were performed in triplicate.® RP-18 column   was used. The mobile phase was acetonitrile/water (60:40 v/v) at a flow rate of 0.50 mL min−1, and the runtime was 40 min with a sample volume injection of 25 µL. The mass spectrometry analysis was performed under positive electrospray ionization (ESI+). The mass spectra were obtained in the scan range of 250–1200 m/z [Liquid chromatography-mass spectrometry has been used to characterize metabolic in the pharmaceutical analysis . In this1200 m/z , control3 cells/well for MCF-7 and NCI-H460, and 1.0 × 104 cells/well for HeLa, HepG2, and PLP2. The cells were allowed to attach for 24 h. After this period, distinct neem extract concentrations (1.56–250 µg/mL) or ellipticine (positive control) were added to the cells and incubated for 48 h. After that, prechilled trichloroacetic acid  was added and incubated for 60 min at 4 °C to improve the adherence of the cells. The plates were washed with deionized water, dried and after the addition of a sulforhodamine B solution , the mixture was incubated for 30 min at room temperature. Subsequently, the plates were washed with acetic acid (1%) to remove the unbound SRB and dried. The bounded SRB was solubilized with Tris  and the absorbance measured at 540 nm using an ELX800 microplate reader  [The cell lines used were: MCF-7 (breast adenocarcinoma), NCI-H460 , HeLa , HepG2 (hepatocellular carcinoma) and PLP2 (non-tumor liver primary culture). Each cell line was grown in a 96-well microplate, at a density of 7.5 × 10VT, USA) .p < 0.05.Statistical analysis of the results was conducted by using one-way ANOVA, followed by a post hoc Tukey’s test using Prism version 5.0 software . Statistical significance was concluded with 50 dose of 52.3 ± 4.8, 48.8 ± 4.3, 60.6 ± 4.3, and 82.3 ± 4.3 µg/mL to HepG2, HeLa, NCI-H460, and MCF-7, respectively. Nevertheless, results indicated that PLP2 non-tumor cells were more resistant to all extracts obtained in the present study with a GI50 dose higher than 200 µg/mL. The present study provides a process to obtain extracts of neem leaves with potential application as antiproliferative of malignant cells.This study demonstrated that sequential-PLE is an efficient methodology for extraction of bioactive compounds from neem leaves. The use of three different solvents for the extraction process provides extracts with different antiproliferative potential. SEA extract was the most efficient growth inhibitor for the tumor cells, with a GI


As an acute febrile and inflammatory disease, Kawasaki disease (KD) could develop Kawasaki disease shock syndrome (KDSS) sometimes. However its pathogenesis was still not well known. This study was to learn more about the clinical features and evaluate the role of cytokines in the pathogenesis of KDSS.n = 27)who were hospitalized at our hospital from Jan 2014 to Oct 2017. For patient with KDSS, we randomly identified 43 patients with KD as control subjects . Clinical features, laboratory evaluations were collected. Cytokines IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ in serum were assayed using flow cytometric bead array.We collected clinical and laboratory data retrospectively for all patients with KDSS, longer duration of fever , higher WBC count, neutrophils, CRP, ESR, PCT and D-dimer, and lower hemoglobin and albumin, more severe hyponatremia and hypokalemia, more refractory to IVIG therapy, more coronary artery abnormalities (CAAs), aseptic meningitis, and longer duration of hospitalization than patients with KD . The levels of serum IL-6 [184.1 (27.7–2577.3) vs 54.1 (4–425) pg/ml], IL-10 [42.6 (5–236.7) vs 9.4 (3–94) pg/ml], TNF-α [2.6 (1.0–23.4) vs 2.1 (1–6) pg/ml] and IFN-γ [18.3 (4.5–94.4) vs 6.7 (2–56) pg/ml] in KDSS patients were significant higher than KD patients . ROC curves showed that 66.7 pg/ml of IL-6, 20.85 pg/ml of IL-10 and 8.35 pg/ml of IFN-γ had sensitivity and specificity for identifying KDSS as 85.2 and 62.8%; 66.7 and 83.7%; 74.1 and 74.4% respectively. No fatality was recorded in this series.The patients with KDSS were older age (43.41 ± 31.42 vs 28.81 ± 21.51 months, KDSS were characteristic as more cytokine production and prone to developing IVIG non-responsiveness and CAAs. KD patients with IL-6 above 66.7 pg/ml, IL-10 above 20.85 pg/ml, and IFN-γ above 8.35 pg/ml suggested higher risk for KDSS. Kawasaki disease (KD) is an acute, febrile vasculitis affecting children of younger than 5 years of age . This diSporadic cases of KD with a shock syndrome have been described in the literature as early as in the mid 1990s –8. DominClinical characteristics of KDSS include poor perfusion or a shock-like state and typical signs of KD may not be obvious in the early phase of KDSS. So this syndrome is often difficult to diagnose . DelayedKD is a self-limiting disease, even in KD patients with severe CAAs or KDSS. During the self-limiting clinical course of KD, the intensity of systemic inflammation in the acute febrile stage of KD gradually increases and reaches a peak, after which the inflammation gradually decreases and the disease progresses to the convalescent stage. It has previously been postulated that the host immune reactions preceding this peak of inflammation process mediate tissue cell injury, whereas the immune reactions following the peak mediate tissue cell repair. The protein-homeostasis- system of the host is important for recovery from KD . PathogeCytokines may work mainly at cell level, and increased level of them may mean the numbers of immune cells involved in various pathologic lesions of KD. In a self-limiting disease like KD, pro-inflammatory reaction and counter-reaction of immune system act against insults from the disease and cytokines in plasma may be easily affected by stage of KD and severity of inflammation in KD. Therefore, study for cytokines’ role in KD is need in serial examinations during natural processes of the disease .To document the distinguishing clinical features of this syndrome and the role of cytokines in its pathogenesis, we compared the clinical characteristics, treatment outcomes and levels of serum cytokines in children with KDSS or the children with KD but without the complicating shock syndrome.From January 2014 to October 2017, 2203 children were diagnosed with KD in our hospital according to the standard diagnostic criteria . KDSS isOf the 2203 patients with KD, 27 were identified as satisfying clinical criteria of KDSS . For patThe following clinical data were collected: gender, age, race, clinical features and outcomes. Laboratory data were collected prior to treatment with IVIG. These included white blood cell count (WBC), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), procalcitonin (PCT), liver and kidney and coagulation function and other parameters. Echocardiography and electrocardiogram were collected as appropriate.Serum cytokine determination was routinely done as part of a comprehensive laboratory evaluation in our hospital laboratory. All patients were tested for the six cytokines including IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ at admission and at follow-up time points. Two milliliter(ml) of blood were collected, transferred to a serum separating tube and centrifuged at 1000 g at 20 °C for 20 min after clotting. The serum was carefully harvested, and was used to determine the cytokine level immediately, or stored at 2 °C to 8 °C until later for analysis . Concentrations of the six cytokines were quantitatively determined using the CBA Human Th1/Th2 Cytokine Kit II  according to the manufacturer’s instructions. The minimum and maximum limits of detection for all six cytokines were 1 and 5000 pg/ml, respectively. The cytokine concentrations at the time of KD or KDSS diagnosis were used for analysis in this study.The area under the ROC curve, referred to as the AUC, is an appropriate measure for describing the overall accuracy of a diagnostic test, with the higher AUC value signifying better diagnostic value. AUCs of IL-2, IL-4, IL-6, IL-10, TNF-α and IFN-γ were calculated respectively.P value < 0.05 was considered statistically significant.Two-sample Student’s t-test was used to test for significant differences in the means of continuous variables. Mann-Whitney U test was used for comparing medians in skewed continuous variables. Proportions were compared using Fisher’s exact test appropriately. Receiver operating characteristic (ROC) curve analysis was used to select the optimal cutoff values of parameters. All statistical analyses were performed using SPSS version 20.0 . A two-sided P < 0.05), longer duration of fever than those patients with KD. There was no significant difference in sex-distribution, clinical KD findings such as conjunctivitis, oral mucous membrane changes, cervical lymphadenopathy, changes in the peripheral extremity, polymorphous rash between 2 groups  . All patients were of Han descent in China. The patients with KDSS was older age , PMNs (79.44 ± 17.56 vs 60.76 ± 14.34%), CRP (137.15 ± 54.80 vs 76.11 ± 50.66 mg/dl), ESR[101 (24–140) vs 67 (6–135) mm/h], PCT [2.33 (0.46–19) vs 0.56 (0.001–21.88) ng/ml] and lower hemoglobin (99.37 ± 12.69 vs 110.44 ± 10.17 g/L) were observed in patients with KDSS compared to patients with KD . Hypoalbuminemia (30.12 ± 6.54 vs 36.28 ± 3.73 mmol/L), hyponatremia (132.85 ± 3.73 vs 135.62 ± 3.68 mmol/L), hypokalemia (3.38 ± 0.50 vs 3.81 ± 0.46 mmol/L), higher level of ALT [53 (12–415) vs 18 (10–477) U/L], CK-MB (34.12 ± 16.05 vs 20.01 ± 8.46 mmol/L), creatinine [47.5 (34–456) vs 35 (25–58) μmol/L], BUN [5 (2–16) vs 2.62 (1.38–4.84) mmol/L], triglyceride (1.78 ± 0.60 vs 1.30 ± 0.51 mmol/L), and D-dimer [3.45 (1.13–7.88) vs 1.16 (0.36–1.43) mg/L] were found more frequently in patients with KDSS compared to patients with KD . Platelet count was lower but there was no statistical difference between groups (P > 0.05). Also, no statistical differences between groups were identified for lactic acid, amylase and fibrinogen . There were no significant differences in IL-2 and IL-4 between two groups  (Table Significantly higher levels of serum IL-6 [184.1 (27.7–2577.3) vs 54.1 (4–425) pg/ml], IL-10 [42.6 (5–236.7) vs 9.4 (3–94) pg/ml], TNF-α [2.6 (1.0–23.4) vs 2.1 (1–6) pg/ml] and IFN-γ [18.3 (4.5–94.4) vs 6.7 (2–56) pg/ml] were found in patients with KDSS . More common of IVIG-resistance (70.37% vs 2.30%), CAAs (55.56% vs 9.30%), and aseptic meningitis (48.15% vs 4.65%) were found in KDSS than KD . The male/female ratio for CAAs was 2:1 in KDSS. There were more other organ damage in KDSS group  and renal damage  The diagnosis of incomplete KD was more frequent in KDSS compared to KD group . No fatality was seen in our study (Table All 27 KDSS patients (100%) received systemic steroids,fluid resuscitation and conventional therapy with aspirin and IVIG. Hypotension was recovered quickly after treatment in 14 patients. Other 13 (48.15%) of KDSS patients were admitted to the ICU due to hypotension or shock. Eight of cases (29.63%) required vasopressor including dobutamine, dopamine, and epinephrine because of persistent hypotension or shock. The other 5 patients can maintained normal blood pressure by intravenous normal saline. The hospital length of stay in patients with KDSS was significantly longer than KD patients . CRP and the mean ESR in the KDSS group were higher. Our study differed from the other as we had found that the KDSS patients had higher number of WBC and PMNs, which was not described in other studies [Like the other studies, we did find that the platelet count in KDSS group tended to be lower  . SCLS isThere are also shortcomings in this study. The number of cases in the control group is not enough, which may lead to sample bias. Since complete KD, incomplete KD, and KDSS are diagnosed by clinical manifestations, there could be a possibility of patient-selection bias. Although the control KD subsets could not match to KDSS group for age and fever duration, previous studies from the hematological department study group of our hospital showed that cytokine levels were more related to severe status of disease, but not to age and fever duration , 62.KDSS is an uncommon complication of KD but can lead to significant sequelae and poor outcome. The incidence of KDSS in our study was lower than the Western countries. KDSS were characteristic as hyperinflammation in serum and more prone to developing IVIG non-responsiveness and CAAs and aseptic meningitis. Increased production of IL-6, IL-10, TNF-α and IFN-γ cytokines may pay a key role in the pathogenesis of KDSS. IL-6 above 66.7 pg/ml, IL-10 above 20.85 pg/ml, and IFN-γ above 8.35 pg/ml suggested higher risk for KDSS. The paired cytokine studies may be helpful to define the cytokines’ role in KD and KDSS, but we did not analysis.


Dendrobium tosaense (DT) extract. Ground DT was extracted using deionized water (W) or 50% ethanol (50E) at room temperature (RT) or 50 °C (50T) for 20 min. The 50T + 50E extract exhibited the highest total phenol content 47.0 ± 4.0 mg gallic acid equivalent/g DT extract, the highest level of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) free-radical scavenging 66.0 ± 3.0 mg Trolox equivalent/g DT extract, and the highest reducing power 12.00 ± 0.50 mg vitamin C equivalent/g DT extract. The RT + W extract had the highest total flavonoid content 110.0 ± 3.0 mg quercetin equivalent/g DT extract. The RT + 50E extract had the lowest half maximal inhibitory concentration 1.30 ± 0.00 mg/mL for 2,2-diphenyl-1-picrylhydrazyl free-radical scavenging, and the lowest half maximal inhibitory concentration 6.40 ± 0.30 mg/mL for mushroom tyrosinase inhibition activity. DT extracts, especially RT + W and 50T + W, exhibited potent inhibitory effects on melanogenesis of B16/F10 cells. These results demonstrated the application potential of DT extract for skincare.This study investigated the polyphenol content, antioxidant activity, and inhibition ability of mushroom tyrosinase and melanogenesis of  Dendrobium, known as “Shihu” in China, is the second largest genus in Orchidaceae [Dendrobium species are epiphytic and grown in tropical and subtropical regions [Dendrobium serves numerous functions in traditional Chinese medicine, including as an antipyretic, as an anti-inflammatory agent, for benefit of the eyes and digestive system, and for lowering blood sugar [Dendrobium has also attracted attention for its potential inhibition and prevention of cancer, its immune-system stimulation, and its antioxidant activity [Dendrobium tosaense (DT), Dendrobium linawamum (DL), and Dendrobium moniliforme (DM) are well-known medicinal plants, but DT and DM are more commonly used than DL [The origins of orchids (Orchidaceae) can be traced back 120 million years [od sugar . Dendrobactivity . Dendrob than DL . DT is n than DL . Dendrobium species include alkaloids, bibenzyls, fluorenones, phenanthrenes, sesquiterpenoids, trace elements, polysaccharides, and amino acids [Dendrobium is also rich in active polysaccharides, which exhibit antitumor and antioxidation properties and contribute to enhanced immunity [The chemical compounds in the no acids ,6,7,8,9.no acids ,11. Gigano acids  that funno acids . Dendrobimmunity . Tyrosinase acts as the critical enzyme in the melanin biosynthesis pathway ,16,17. TSolvent extraction is a simple and efficient technique for obtaining extracts from plants. However, few reports have evaluated DT extracts that have been prepared through various methods. Therefore, the aim of the present study was to determine the polyphenol contents, antioxidant activity, mushroom tyrosinase inhibition ability and melanogenesis inhibition of DT samples obtained using various solvent extraction methods.− molecular ion at m/z 273 was detected for all extracts except the room temperature plus water (RT + W) extract. The order of samples according to amounts of gigantol from high to low level was RT + 50E, 50T + 50E, and 50T + W.Gigantol in four extracts was identified through mass spectrometry as illustrated in Phenolics have significant antioxidation and anticancer effects and contribute to prevention of cardiovascular diseases. The experimental results indicate that the 50T + 50E extraction had the highest total phenol content, at 47.0 ± 4.0 mg gallic acid equivalent/g DT extract . PhenoliFlavonoids have more than 3000 varieties and are potent antioxidants with free-radical-scavenging capabilities . As depi50), 1.30 ± 0.00 mg/mL for DPPH  and then oxidizes l-DOPA to form dopachrome, which can be converted into melanin. Therefore, inhibition of tyrosinase is generally evaluated in the process of screening potential inhibitors of melanogenesis [50 6.40 ± 0.30 mg/mL  assays , all DT Stems of DT were powdered using a pulverizer. A total of 5 g of DT powder was extracted using 50 mL of deionized water (this method is labeled W) or 50% ethanol (50E) at room temperature (RT) or 50 °C (50T) for 20 min. After centrifugation with 3000 rpms for 10 min at room temperature and filtration, a liquid extract was obtained. The lyophilization process was further applied to obtain the dry extract to determine the concentration of DT extract. Finally, the concentrations of DT extract in original working solutions of RT + W, 50T + W, RT + 50E, and 50T + 50E were 6.23, 6.60, 4.38, and 4.70 mg/mL, respectively.2) was used for analysis. The mobile phase consisted of a mixture of 0.1% formic acid solution and acetonitrile containing 0.1% formic acid. The proportion of 0.1% formic acid in the mobile phase was 95% at 0 min, 90% at 0–2 min, 82% at 2–3 min, 62% at 3–4 min, 0% at 4–6 min, and 95% at 6–10 min. The flow rate was 0.35 mL/min. The MS/MS settings were a negative electrospray ion source, 3 kV capillary voltage, 3 V cone voltage, 350 °C desolvation temperature, and 0.15 mL/min collision gas flow.A liquid chromatography–tandem mass spectrometry system (UPLC-Xevo-TQMS) with a reverse-phase column , 100 μL of 1% potassium ferricyanide, and 200 μL of DT extract was obtained and subjected to a water bath at 50 °C. After 20 min of reaction, the mixture was rapidly cooled for 3 min. After the addition of 100 μL of 10% trichloroacetic acid and the application of centrifugation with 7000 rpms for 10 minutes at room temperature, 400 μL of supernatant was withdrawn and added to 400 μL of deionized water and 100 μL of 0.1% iron chloride. After 10 min in the dark, Prussian blue formed in the mixture. The absorbance of the resulting mixture was measured at 700 nm. The ferric-reducing power was evaluated from a vitamin C standard curve.A 96-well plate was filled with 40 μL of 3 mM L-Dopa dissolved in 67 mM phosphate buffer at pH 6.8, 20 μL of DT extract, and 40 μL of mushroom tyrosinase solution (200 U). In this experiment, DT extract obtained using 50E was freeze-dried and then redissolved into dimethyl sulfoxide (DMSO). The mixture was homogenized and incubated at 37 °C for 30 min. The absorbance of the resulting mixture was measured at 475 nm for monitoring dopachrome which appeared during the reaction catalyzed by mushroom tyrosinase. Kojic acid was used as a standard, and pure water solution was used as the control.2. The viability of cells treated with 20 μL of DT extract with a quintuple dilution was determined using a methylthiazol tetrazolium (MTT) assay. In brief, 3 × 104 B16/F10 cells were seeded on 96-well plates. After 24 h, DMEM was removed, and 200 μL of fresh DMEM and DT sample were added. The cells were then incubated for 24 h. After incubation, the medium was removed, and 100 μL of MTT in a PBS solution (0.5 mg/mL) was added to each well. The cells were then incubated at 37 °C for 40 min. Subsequently, the MTT solution was removed, 100 μL of DMSO was added to each well, and the plate was gently shaken to dissolve the formazan crystals. The absorbance of each well was measured at 570 nm.B16/F10 cells were cultured in Dulbecco’s minimum essential medium (DMEM) supplemented with 100 units/mL of penicillin G, 10% fetal bovine serum, 0.25 μg/mL of amphotericin, and 100 μg/mL of streptomycin, and then incubated at 37 °C with 5% CO5 B16/F10 cells were seeded in 24-well plates and cultured at 37 °C with 5% CO2 for 48 h. Subsequently, cells were treated with 200 nM α-MSH for 24 h, and then incubated with 20 mg/mL DT extract or 1 mg/mL kojic acid for 24 h. The cells were washed twice with PBS, and the cell pellets were then dissolved in 200 μL of 1 N NaOH in 10% DMSO for 1 h at 80 °C. The relative melanin content was determined by measuring the absorbance at 405 nm. The melanin content was calculated using the following equation, (absorbance of sample/absorbance of α-MSH) × 100%.The cellular melanin content was determined as previously described . In briep ≤ 0.05. Treatment means with the same letter are not significantly different at the 5% level.The experiments were laid out in Completely Randomized Design (CRD) with three replications. The evaluated data are presented as the mean ± standard error. The data collected were analyzed by analysis of variance (ANOVA) using SAS version 9.4 . If significant, means were separated by least significant difference (LSD) at 50 for DPPH free-radical scavenging and mushroom tyrosinase inhibition ability. DT extracts showed no cytotoxicity to the B16F10 cells, and the RT + W and 50T + W exhibited better effects on anti-melanogenesis than the RT + 50E and 50T + 50E. The results in this study indicated that DT extracts may be a potential material in skincare.In summary, this study confirmed that DT is rich in polyphenols and displays antioxidant and inhibition on mushroom tyrosinase and melanogenesis properties. The DT extract obtained using 50% ethanol at 50 °C exhibited the highest level of total phenol content and ABTS free-radical scavenging, and had the highest ferric-reducing power. The DT extract by water at RT exhibited the highest total flavonoid content. The DT extract by 50% ethanol at RT had the lowest IC


Leishmania, Trypanosoma, and Toxoplasma. Their anti-histone deacetylase (HDAC) potency was determined. Moderate to no antileishmanial or antitrypanosomal activity was found (IC50 > 10 μM) that contrast with the highly efficient anti-Toxoplasma activity (IC50 < 1.0 μM) of these compounds. The antiparasitic activity of the synthetized compounds correlates well with their HDAC inhibitory activity. The best-performing compound (named 363) express a high anti-HDAC6 inhibitory activity (IC50 of 0.045 ± 0.015 μM) a moderate cytotoxicity and a high anti-Toxoplasma activity in the range of known anti-Toxoplasma compounds (IC50 of 0.35–2.25 μM). The calculated selectivity index (10–300 using different human cell lines) of the compound 363 makes it a lead compound for the future development of anti-Toxoplasma molecules.A series of aminophenylhydroxamates and aminobenzylhydroxamates were synthesized and screened for their antiparasitic activity against   •A series of aminophenylhydroxamates and aminobenzylhydroxamates were synthetized.•Toxoplasma activity.They show moderate anti-Trypanososmatids and high anti-•T. gondii activity correlates with their HDAC inhibitory.The •The best-performing compound had high anti-HDAC6 (class II) activity.•Toxoplasma.This compound represents a “lead” for the development of new anti- Seventy million people risk to contract HAT  play key roles in diverse intracellular processes and epigenetic regulation through the modification of histone and nonhistone proteins to repress transcription. In human cells, 18 HDACs have been identified  and clasactivity . Accumulactivity  and reviin vitro antileishmanial activity associated with a moderate cytoxicity  that have demonstrated an toxicity .22.13 and brine, dried over Na2SO4, filtrated and concentrated to afford the amide or sulfonamide derivatives. The products were used such as in the next step. Amide or sulfonamide derivatives (1 eq.) were dissolved in THF (3 ml). A solution of LiOH (3 eq.) in 3 ml of water was added and the reaction mixture was heated at 40 °C overnight. The reaction mixture was concentrated. The residue was taken up with 30 ml of water and the aqueous phase was washed with 20 ml of EtOAc, then the aqueous phase was acidified to pH 2 with a solution of 1 M HCl. The aqueous phase was extracted three times with 20 ml of EtOAc. The organic phases were combined and dried over Na2SO4, filtrated and concentrated to afford the acid derivatives. The products were used such as in the next step. Acids derivatives (1 eq.) were dissolved in dry DMF (5 ml). Ethyl chloroformate (1.2 eq.) and N-methyl-morpholine (1.3 eq.) were added successively at 0 °C. After 10 mn, a solution of hydroxylamine (2 eq.) in MeOH (10 ml) was added and the reaction mixture was warm up to room temperature and let overnight. The reaction mixture was concentrated. The residue was taken up with EtOAc and the organic phase was washed with a saturated solution of NaHCO3 and brine, dried over Na2SO4, filtrated and concentrated. The crude products were purified by flash chromatography to afford the hydroxamate. The characterization data of compound ST3 was reported in a previous work  . Synthesous work , and the2.1.11H NMR (DMSO-d6) δ 11.17 , 9.19 , 9.00 , 7.70 , 7.56 , 7.39 , 4.51 . HPLC purity 99%. MS ESI+H+ calc. 289.28 exp. 289.10.2.1.21H NMR (DMSO-d6) δ 10.40 , 7.75 , 7.58 , 7.45 .+ calc. 275.25 exp. 275.10.HPLC purity 98%. MS ESI+H2.1.31H NMR (DMSO-d6) δ 11.17 , 9.02 , 8.37 , 7.60 , 7.50 , 7.29 , 4.07 . HPLC purity 97%. MS ESI+H+ calc. 325.30 exp. 325.10.2.1.41H NMR (DMSO-d6) δ 11.07 , 10.75 , 8.95 , 7.55 , 7.48 , 7.15 . HPLC purity 99%. MS ESI+H+ calc. 311.31 exp. 311.10.2.1.51H NMR (DMSO-d6) δ 11.18 , 9.13 , 9.01 , 8.09 , 7.98 , 7.95 , 7.70 , 7.38 , 4.52 , 2.73 . HPLC purity 96%. MS ESI+H+ calc. 368.43 exp. 368.10.2.1.61H NMR (DMSO-d6) δ 11.13 , 10.46 , 8.95 , 8.14 , 8.10 , 8.03 , 7.87 , 7.76 , 2.75 .+ calc. 354.40 exp. 354.10.HPLC purity 99%. MS ESI+H2.1.71H NMR (DMSO-d6) δ 11.17 , 8.99 , 8.58 , 7.67 , 7.29 , 7.19 , 6.80 , 4.30 , 3.73 , 3.45 . HPLC purity 99%. MS ESI+H+ calc. 315.34 exp. 315.10.2.1.81H NMR (DMSO-d6) δ 11.09 , 10.35 , 8.93 , 7.70 , 7.64 , 7.22 , 6.89 , 6.82 , 3.74 , 3.63 . HPLC purity 99%. MS ESI+H+ calc. 301.32 exp. 301.10.2.2Leishmania parasites were cultured at 26 °C in SDM-79 medium supplemented with 10% fetal calf serum (FCS), and the antileishmanial activity was determined according to a previously published protocol for LUC-expressing and wild-type Leishmania , 100 U/ml penicillin, 100 μg/ml streptomycin and 20 mg/ml bovine hemin. The epimastigote form of Trypanosoma cruzi   were grown at 26 °C in liver infusion tryptose (LIT) medium, supplemented with 10% FCS, 2 mM GlutaMAX™-I, 100 U/ml penicillin, 100 μg/ml streptomycin and bovine hemin (20 mg/l) (Gibco). The antitrypanosomatids activity was determined using a protocol that was initialy set up for wild-type Leishmania  monolayers were infected with YFP-type I Rh kindly provided by B. Striepen, see /PBS for 30 min at room temperature. Nuclei were stained with Hoechst 33258  for 20 min and then washed three times with water for 10 min each time. The number of infected cells, i.e., cells harboring a parasitophorous vacuole, and the number of parasites per vacuole were determined using an Olympus ScanR microscope ( × 20 objective) and ScanR software. The parasitic index in % compared to the control was calculated as follows: (PI=((number of parasite/100 cells in treated wells) x (% of infected macrophage in treated well)/(number of parasite/100 cells in untreated wells) x (% of infected macrophage in untreated wells))*100). Then, the 50% inhibitory concentration (IC50) was calculated using Prism software .The antitrypanosomatid activity of the compounds was ascertained as follows. ishmania . Trypanoishmania . To assepen, see  or Tomat2 incubator at 37 °C. Cytotoxicity was evaluated via the MTT assay, and the IC50 values were calculated using Prism software .The human monocytic cell line THP-1 was cultivated in RPMI medium supplemented with 10% FCS, 2 mM GlutaMAX, 100 U/ml penicillin, and 100 μg/ml streptomycin. HFF cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) with Earle's salts containing 10% FCS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM GlutaMAX, 100 U/ml penicillin, and 100 μg/ml streptomycin. The THP-1 and HFF cells were maintained in a 5% CO2.3The HDACi effect against HeLa nuclear extracts and recombinant proteins were measured using a fluorometric HDAC assay kit  according to the manufacturer's instructions. Briefly, 30 μL of HeLa nuclear extract  or various HDAC recombinant proteins  was mixed with 5 μL of a 10 × compound and 10 μL of assay buffer. A negative control containing trichostatin A at a final concentration of 2 μM was analyzed for each independent experiment. A fluorogenic substrate (10 μL) was added, and the reaction was allowed to proceed for 30 min at room temperature and then stopped by the addition of a developer containing trichostatin A. The fluorescence was monitored after 30 min at excitation and emission wavelengths of 360 and 460 nm, respectively.T. gondii. Confluent cell cultures (T-175 cm2) were infected with 5 × 107 tachyzoïtes in DMEM supplemented with 1% (v/v) FCS. Tachyzoïtes were harvested after 72 h, resuspended in RIPA buffer  and incubated for 30 min at 4 °C. After centrifugation at 13000 rpm for 30 min at 4 °C, the supernatant was collected, adjusted to a total protein concentration of 312 μg/ml and then used for determining the HDAC activity as described above.HDAC activity was determined from total protein extracted from 2.4T. gondii (http://toxodb.org/) and from UNIPROT for the leishmanial and human enzymes. Unfortunately the leishmanial enzymes provided poor sequence-structure alignment precluding further analysis. Meanwhile, the sequences from both strains, GT1 and ME49 revealed similar trends, so the docking study was performed on three GT1 sequences only. The best results were obtained for three sequences from T. gondii  but not for two other sequences (S7UW66 and S7UZC5). The latter showed only partial alignment and significant sequence divergence with known crystal structures of HDACs. For those two sequences, the truncated structural alignment did not allow complete modeling of the active site, which prevented further ligand docking studies. Otherwise, for the three other sequences, active site boundaries in each structural model was deduced as the vicinity of the co-crystallized ligand (or hydroxamate compounds transferred from related HDACs by protein-protein superposition) using @TOME-2 comparative option. The same chemical entities served, in addition, to define a shape restraint to guide the docking, in absence of the zinc atom, in the automatically computed models.Protein sequences were recovered from the appropriate databases for two toxoplasmal strains GT1 and Me49 as representatives of type I and type II T. gondii (GT1 and Me49 strains) as well as for Leishmania infantum and human HDACs, can be found on the following web pages (the links therein):http://atome3.cbs.cnrs.fr/AT23/DB/HDAC_TOXGG.htmlhttp://atome3.cbs.cnrs.fr/AT23/DB/HDAC_TOXGM.htmlhttp://atome3.cbs.cnrs.fr/AT23/DB/HDAC_LEIIN.htmlhttp://atome3.cbs.cnrs.fr/AT23/DB/HDAC_HUMAN.htmlAll the results for the HDACs from 3The synthetic routes of aminophenylhydroxamate and aminobenzylhydroxamate derivatives are summarized in L. infantum and L. braziliensis in the range of 20 μM but is less potent against L. tropica (T. brucei but do not display anti-T. cruzi activity (IC50 > 100 μM). It is moderately cytotoxic for THP-1 cell line or for HFF, with a determined cytotoxicity above 250 μM (Leishmania it discloses a low antiparasitic activity (IC50 of 140 μM and 111 μM against L. braziliensis and L. infantum respectively). As the calculated index of selectivity for ST3 is of about 5.4, this small compound (MW: 138) was considered as an interesting lead from which a series of derivative targeting trypanosomatid parasites was developed. Unexpectedly, almost all the aminophenylhydroxamates and aminobenzylhydroxamates synthetized exert low or no antitrypanosomatid activity (T. brucei or Leishmania spp.). The sole exception is the compound named 345, which exhibited moderate antileishmanial activity in the range of 15–50 μM, L. braziliensis being the most susceptible Leishmania species. Nevertheless, this compound is highly cytotoxic to THP-1 and HFF cells.The compound ST3, a benzohydroxamate, exert antileishmanial activity against promastigote forms of  tropica . In addie 250 μM . Neverthactivity , irrespeToxoplasma gondii, an intracellular parasite belonging to Phyllum Apicomplexa. For T. gondii, the IC50 of ST3 is above 50 μM. The 8 compounds synthesized express IC50 values ranging from more than 60 μM to less than 1 μM   to 0.085 μM for the more active compound (363). This latter inhibitory concentration is lower than the IC50 that we determined for SAHA and more than 20-fold lower than those of well-known HDACis. To further characterize the HDACi effect of our series of compounds, we first ascertained the inhibitory effect of all the compounds at a single concentration of 1.0 μM against HeLa nuclear extract and recombinant HDAC1, 3, 6, and 8. As shown in T. gondii HDAC activity. As shown in T. gondii. Compound 363 inhibited 44% of the T. gondii deacetylase activity, whereas SAHA inhibited only 22% of this activity . We obtained an IC μM) see , which a μM) see . For allactivity B. The coactivity  and it wToxoplasma activity, i.e IC50 < 50 μM , are among the best HDACis of the series. A high correlation between HDACi and T. gondii activity is recorded (R2 = 0.9950). In addition, their cytotoxic activity against HFF and THP-1 correlates also well with their HDACi (R2 = 0.9000 and R2 = 0.9933) respectively  were included in the analysis, R2 = 0.3758, 0.4437 and 0.6667 for T. gondii, HFF and THP-1 respectively (see Supplementary data S2). Therefore, improved anti-T. gondii potency of the series is largely dependent on potent HDACi activity. However, since three compounds exhibit improved HDACi activity that does not lead to improved anti-T. gondii activity it is likely that other factors are also involved in the modulation of their cytotoxicity and anti T. gondii potency. Among them, the capacity of each compound to reach their intracellular targets and/or to readily inhibit HDAC activity might play a role. The THP-1 cell line, which was derived from the peripheral blood of a 1-year-old human male with acute monocytic leukemia, is known to be highly susceptible towards hydroxamate derivatives , 3 are class I HDACs, one is a class IV HDAC, and 1 is a class II HDAC. Fold recognition using the server @TOME-2 (Toxoplasma gondii TgHDACs with Homo sapiens HsHDAC2 (with 56% and 64% of sequence identity over ∼360 residues). Two other TgHDACs showed weaker similarities with bacterial HDACs (40% identity), being far more distant to the Homo sapiens HsHDADC6 (∼20% identity). The last one appeared to encode only a partial HDAC fold and might correspond to a pseudogene. Taking advantage of the comparative docking implemented in @TOME-2, active site delimitations and pharmacophoric shape restraints could be defined to allow focused screening performed using PLANTS (Danio rerio (PDB5G0J) (The currently identified HDACi-based anti- @TOME-2  was usedg PLANTS . The docPDB5G0J) . SatisfaPDB5G0J) . A neighT. gondii compound. The molecular target now needs to be further characterized in T. gondii. Unexpectedly, among the 8 derivatives synthesized there was minimal efficacy against Trypanasomes and only one compound with demonstrated antileishmanial activity (345), but this compound also exhibited high in vitro cytotoxicity. In order to address whether these HDAC inhibitors can be developed to target intracellular parasites more broadly, we will synthesize new compounds from the ST3 and or the 345 molecules in order to improve their efficacy against Leishmania parasites and decrease their cytotoxicity.For the first time, we have demonstrated that a potent inhibitor of the mammalian class IIb of HDACs (HDAC6) is effective as an anti-T. gondii exists in two life stages, the rapidly proliferating tachyzoïte form and the latent encysted bradyzoïte form, the latter of which remains in the body for the lifetime of the host, maintaining the risk of recurrence. There are currently no effective treatments against the bradyzoïte form   . We prop