The Comprehensive Cancer Centre Eugène Marquis and the National Graduate School of Chemistry of Rennes (ENSCR) have jointly developed a stable and efficient labelling of Lipiodol with rhenium-188 (Eβmax = 2.1 MeV) for the treatment of HCC. The major “milestones” of this development, from the first syntheses to the recent first injection in man, are described.Hepatocellular carcinoma (HCC) is the 5th most common tumour worldwide and has a dark prognosis. For nonoperable cases, metabolic radiotherapy with Lipiodol labelled with  Indeed, rhenium-188 has ideal properties for molecular radiotherapy , t1/2 = 17 h) and has the added advantage of being available on a cost-effective day-to-day basis thanks to its generator mode of production  which structure is given in Previous studies at the ENSCR led to the preparation of a new class of complexes with perthiobenzoate and dithiobenzoate moieties [M (PhCSP = 3.33).The SSS complex was subsequently prepared with technetium-99 m, using a freeze-dried kit method , initially in a view of lymphocytes labelling . The com99mTc-SSS/Lipiodol is thus obtained with a 96 ± 2.8% yield and a final RCP of 92.5 ± 2.6% [It was, therefore, a good candidate to label Lipiodol. The method previously described by Jeong et al.  was used5 ± 2.6% . The pre188W/188Re generator, similar to the 99Mo/99mTc generator. The major difference lies in the necessity of postelution concentration to obtain high-volumic activity (up to 20 GBq/mL with a 37 GBq generator), due to the lower specific activity of 188 W compared to 99Mo, necessitating a bigger alumina column , 7.5 mg sodium gluconate, 30 mg ascorbic acid, and 40 mg potassium oxalate. This freeze-dried kit is reconstituted in 0.5 mL saline, and the perrhenate  is then added. After 15 min at room temperature, 20 mg of sodium dithiobenzoate is added, and the solution is heated for 30 min at 100°C, to provide the 188Re-SSS complex, as a precipitate. 2-3 mL of Lipiodol is added to the mixture, which is then centrifuged, as previously described with technetium-99 m. 188Re-SSS/Lipiodol was obtained with a 87 ± 9.1% yield and a final RCP of 93 ± 3.4%. Labelling was further optimised to reach 97.3 ± 2.1% yield and a final RCP of 94.1 ± 1.7% [Kit formulation: 0.8 mg SnCl1 ± 1.7% .A new efficient and stable labelling of Lipiodol was described. However, for activities above 1850 MBq (therapeutic activities), the labelling yield decreased dramatically. Consequently, the synthesis had to be further improved, in view of HCC treatment. Kit formulation was tuned, and reaction conditions were slightly modified.2·2H2O (dissolved in 0.1 mL HCl 1 M), 30 mg sodium gluconate, 30 mg ascorbic acid, 40 mg potassium oxalate) is reconstituted in 0.5 mL saline, and the perrhenate  is then added. After 15 min at room temperature, 40 mg of sodium dithiobenzoate is added, and the solution is heated for 15 min at 100°C, to provide the 188Re-SSS complex, as a precipitate. 2-3 mL of Lipiodol is added to the mixture, which is then stirred with a vortex. After 10 min of centrifugation (2200 g), both phases are separated, and the lower phase (radiolabelled Lipiodol) is carefully recovered. 188Re-SSS/Lipiodol is obtained with a 98.56 ± 1.2% yield and a final RCP of 92.52 ± 2.3% and is stable for at least 7 days [A freeze-dried kit  fixed at the finger tips, and, respectively, 80 and 58% decreases in the right-hand and left-hand doses were shown . Personain vivo, in healthy pigs, then in hepatoma-bearing rats. The radiotracer was injected through the hepatic artery, and biodistribution was checked by scintigraphy and ex vivo countings. Autoradiography was also done, to assess more precisely the fixation of the radiotracer.Good targeting and stability of radiolabelled Lipiodol were investigated 99mTc-SSS/Lipiodol could be useful for carrying pretherapeutic dosimetry studies, as is performed with 99mTc-MAA for 90Y-labelled microspheres [131I-labelled Lipiodol in human [ospheres . It was in human . It has 188Re-SSS/Lipiodol was also investigated in healthy pigs [ex vivo countings . The Re-SSS/Lipiodol was injected for less than 24 h after preparation.To assess the safety of the radiotracer, a toxicity study—acute and chronic—has been undertaken in dogs (Beagles), with the nonradioactive analogue The study comprised two phases. For the first phase (7 days), animals  received a single injection at D1, and for the second one, animals  received one dose at D1 and one at D30. Control group  received Lipiodol alone. Some results are summarised in Figures This study thus demonstrated lack of toxicity of Re-SSS/Lipiodol, opening the way for the injection in human.Comité de Protection des Personnes, CPP), and approval was eventually granted. A phase 1 escalation dose study was thus initiated . This study will comprise 4 dose stages, ranging from 1.85 to 7.4 GBq. Each stage will comprise 3 to 6 patients, depending on the toxicity (or lack thereof) of the compound. To date, 5 patients have been injected (3 with 1.85 GBq and 2 with 3.7 GBq). For illustration, SPECT/CT scans of the third patient 1 h after injection of 1.85 GBq of 188Re-SSS/Lipiodol are shown in Files were submitted to the relevant authorities, that is, French Agency for the Safety of Health Products (AFSSAPS), French Nuclear Safety Authority (ASN), and Ethical Committee (In conclusion, we have developed a potential HCC treatment by radioembolisation, with a phase 1 clinical trial currently in progress. This represents almost ten years of multidisciplinary research, from basic chemistry to clinic. Its clinical relevance has now to be demonstrated, and its efficiency and tolerance have to be compared to other existing therapeutic options.


Oryzias latipes (medaka) embryos to 0.1−1 mg/L of homogeneously dispersed AgNPs for 14 days. During this period, developmental endpoints, including lethality, heart rate, and hatching rate, were evaluated by microscopy for different stages of medaka embryonic development. To compare toxic sensitivity, acute adult toxicity was assessed. There was no difference in acute lethal toxicity between embryo and adult medaka. Interestingly, we found that the increase in stepwise toxicity was dependent on the developmental stage of the embryo. Lethal embryonic toxicity increased from exposure days 1 to 3 and exposure days 5 to 8, whereas there was no change from exposure days 3 to 5. In addition, 7 d exposure to 0.8 mg/L AgNPs resulted in significant heart beat retardation in medaka embryos. AgNPs also caused a dose-dependent decrease in the hatching rate and body length of larvae. These results indicate that AgNP exposure causes severe developmental toxicity to medaka embryos and that toxicity levels are enhanced at certain developmental stages, which should be taken into consideration in assessments of metallic NPs toxicity to embryos.The developmental toxicity of silver nanoparticles (AgNPs) was investigated following exposure of  Manufactured nanoparticles (NPs) are particles of 100 nm diameter or less. The applications of NPs have extended in recent years to areas such as medicine, pharmacology, electronic engineering, magnetic fields and semiconductors, biotechnology, materials and process development, energy, and environmental remediation . It is eSilver nanoparticles (AgNPs) have emerged as an important class of nanomaterials and are currently used in a wide range of industrial applications and healthcare products with their antimicrobial effects , 9. WithOryzias latipes). This fish species has been used as a model organism in a wide variety of research fields, particularly in embryonic development, because individuals are transparent and easily observed under a microscope.The extensive application of AgNPs might eventually lead to their release into the environment , causingAgNP colloid capped with citrate  was purchased from ABC NanoTech Co., Ltd. . To obtain the UV-Vis spectrum of AgNPs, the original colloid was diluted in deionized water (DW) or culture water at 10 mg/L, and scanned from 200 to 600 nm using a spectrophotometer . The zeta potentials of AgNP dispersions were measured using dynamic light scattering method  to check the pH-dependent variation in AgNP. The hydrodynamic sizes of AgNP were measured using a dynamic light scattering (DLS) method , and particle characteristics were measured by transmission electron microscopy .O. latipes were performed at 23 ± 1°C in 50-L aquariums containing 40 L of culture water prepared following the Organisation for Economic Co-operation and Development (OECD) guidelines [Artemia salina nauplii once a day. Eggs were pooled and washed in culture water and then screened under a stereo dissecting microscope . The embryonic developmental stage was determined as reported by a previous study [The medaka used in this study was cultured by the National Institute of Environmental Research . The culture and maintenance of idelines . Light cus study . FertiliAn acute adult lethality test was conducted as outlined by OECD Technical Guide 203 . Seven aA short-term test of AgNP toxicity on embryos and sac-fry stages (larvae) was conducted with fertilized eggs for 14 d, as outlined in OECD Technical Guide 212 . Thirty Exposure concentration was determined as a range of 0.1, 0.25, 0.5, 0.75, and 1.0 mg/L for both the adult 96 h acute toxicity tests and embryo toxicity tests.The median lethal concentrations (LC50) and associated 95% confidence intervals (CIs) were calculated by Probit analysis . The significant difference of LC50 value was considered to be present if the CI. One-way analyses of variance (ANOVA) followed by Dunnett's test as a post hoc test were performed for heart rate, hatching rate, and body length data. The results were reported as mean ± standard deviation.A representative TEM image of AgNPs is shown in http://dx.doi.org/10.1155/2013/494671) showed the acute toxicity of AgNP to adult and embryo medaka. LC50 values after 96 h exposure were calculated as 0.84 mg/L (CI: 0.67–1.00). In comparison, the acute toxicity level for adults was 0.80 mg/L (CI: 0.65–0.96). The present study demonstrates that the AgNPs used here were acutely lethal to both embryo and adult medaka. Many studies have reported variable acute toxicity levels of AgNPs on teleosts, with LC50s ranging from 0.0346 to 250 mg/L, depending on species and exposure duration  [Pimephales promelas were 0.0894 and 0.0461 mg/L, respectively, for 10 nm uncoated AgNP [P. promelas was 10.6 mg/L for ≤100 nm uncoated AgNPs [D. rerio fry, 96 h LC50s of uncoated AgNP, monodispersed AgNP, and PVP-AgNP were 0.210, 0.088, and 0.162 mg/L, respectively [D. rerio, the LC50 values for AgNPs of 3, 10, 50, and 100 nm were suggested 10.07, 13.55, 13.69, and 14.81 mg/L, respectively [Table S1  , 0.084 mdefined) . The 96 ted AgNP . For embectively . In anotectively . These rWhile the toxicity pattern of AgNPs to embryos dramatically increased during 96 h exposure, a rapid increase in toxicity occurred at 24 h exposure in adults. The LC50s of the embryo test after 24 to 96 h exposure decreased from 1.46 mg/L (CI: 0.81–2.10) to 0.84 mg/L (CI: 0.67–1.00). In comparison, the 24 h LC50 value in the acute adult toxicity test was not calculated (>1 mg/L), with no change in LC50s over the 48 h exposure period . These observations may be attributed to differences in medaka's developmental stage and the aggregation characteristics of AgNPs. It has been generally suggested that fish embryos are less affected by waterborne chemicals than larvae or adults, because of the protective barrier formed by the chorion –35. In aO. latipes, D. rerio, and P. promelas [LC50s values from the embryonic lethal test dramatically decreased from 1.46 mg/L (CI: 0.81–2.10) to 0.43 mg/L CI: 0.37–0.49) at 8 d after AgNP exposure when embryos started to hatch. Previous studies have reported the transport characteristics of AgNPs into the chorion layers of the embryos of various teleosts, including promelas , 19, 37.–0.49 at In addition to differences in the toxicity pattern between embryo and adult medaka, we observed an interesting stepwise increase in toxicity that was dependent on the developmental stage of the embryo . Lethal μg/L [Gottschalk et al.  modeled μg/L . The expOnly a few reports on AgNP toxicity have been published to date, despite the limited understanding about the unintended toxicity of AgNPs to aquatic organisms and its mechanism of contamination. In the present study, we provided experimental evidence regarding developmental stage-dependent sensitivity to AgNPs, as well as information about the different characteristics of toxicity between embryo and adult medaka. Oryzias latipes embryo on exposure to different AgNPs concentrations over a 14-d period were presented in Fig. S1 and Table S1, respectively. To compare sensitivity between embryo and adult, LC50s of adult medaka for 24h ~ 96h were also suggested (Table S1).The cumulative survival rate and LC50 values of Click here for additional data file.


Barbus barbus L.) were fed four purified casein-based diets for 6 weeks in outdoor cages. Besides control diet, these were supplemented with 0.3 mg kg−1 dw selenium (Se) from sodium selenite, or 0.3 and 1.0 mg kg−1 from Se-enriched microalgae biomass (Chlorella), a previously untested Se source for fish. Fish mortality, growth, Se accumulation in muscle and liver, and activity of selected enzymes in blood plasma, muscle, liver, and intestine were evaluated. There was no mortality, and no differences in fish growth, among groups. Se concentrations in muscle and liver, activity of alanine aminotransferase and creatine kinase in blood plasma, glutathione reductase (GR) in muscle, and GR and catalase in muscle and liver suggested that selenium from Se-enriched Chlorella is more readily accumulated and biologically active while being less toxic than sodium selenite.Yearling common barbel ( Oncorhynchus mykiss [Salmo salar [Ictalurus punctatus [Morone chrysops × M. saxatilis [Cyprinus carpio [Carassius auratus gibelio [γ-gluthamyl selenomethylselenocysteine and selenomethylselenocysteine, has been proposed as an alternative in producing Se-enriched meat of African catfish [Selenium (Se) is a trace element essential for all living organisms, where it is a constituent of selenoproteins , 2. In as mykiss , Atlantimo salar , 18, chaunctatus , hybrid axatilis , common s carpio , and cru gibelio . Althoug gibelio  confirme catfish , 25. HowBarbus barbus L.) with respect to effects on fish growth, Se accumulation, and the activity of selected enzymes in certain tissues.Feeding microalgae to fish has been used successfully to provide an important natural source of bioactive compounds, such as carotenoids, fatty acids, polysaccharides, amino acids, and vitamins . Progres−1 replaced with microalgae Chlorella spp. (cf. C. vulgaris Beijerinck) biomass (−1 dry weight (dw) from sodium selenite (Se 0.3) and 0.3 and 1.0 mg kg−1 from Se-enriched microalgae biomass, (Algadiet 0.3 and Algadiet 1.0)  . A contr species , while at status , 29.Components of artificial diets were sourced as follows: casein, gelatine, and dextrin were purchased from VWR International . Cellulose and carboxymethyl cellulose were obtained from Alfa Aesar GmbH & Co KG . Microalgae were cultivated at the Institute of Microbiology in Třeboň, Czech Republic. Pure sodium selenite was supplied by Sigma-Aldrich . Ingredients were mixed in a commercial food mixer. Oil was gradually added during mixing. Water was slowly blended into the mix to obtain dough of suitable texture for processing through a cold extruder. Obtained spaghetti-like strips were manually cut, dried in a household fruit dryer at 60°C for 24 h, and frozen at −20°C until feeding. The composition of the diets  was analBarbus barbus L.) yearlings weighing 48.4 ± 6.1 g (range 40.0–70.7 g) were stocked in outdoor experimental feeding cages (60 × 40 × 30 cm) supplied with microfiltrated (109 μm pore size) water from the Blanice River, South Bohemia. Initial standard body length and weight of stocked fish . Groups of 10 fish were acclimatized and fed the control diet for 10 days prior to the experimental period of 6 weeks. Fish were provided four equal feedings per day , initially at a rate of 1.5% of live weight to ensure that all feed was rapidly consumed. Feeding rate was adjusted according to batch weighing of fish every two weeks. Fish were not fed on the final day before sampling at the end of the experiment. Water temperature (19.2 ± 1.5°C) was registered hourly using Minikin loggers . Dissolved oxygen concentration (8.5 ± 0.8 mg L−1) and pH (7.5 ± 0.1) were measured daily by means of oximeter Oxi 330i and pH meter pH/Cond 340i , respectively. The trial was conducted in triplicate.Common barbel  and weighed to the nearest 1.0 g. Eight randomly selected fish from each replicate were sacrificed (stunned by a blow with a blunt object over the head) and sampled for biochemical analysis. The study was conducted according to the principles of the Ethical Committee for the Protection of Animals in Research of the University of South Bohemia, Faculty of Fisheries and Protection of Waters, Research Institute of Fish Culture and Hydrobiology, Vodňany, based on the EU harmonized animal welfare act of Czech Republic. The principles of laboratory animal care and the national laws 246/1992 and regulations on animal welfare were followed (Ref. number 22761/2009-17210).vena caudalis and samples stabilized with 40 IU sodium heparin per 1 mL. Blood was separated by centrifugation at 12 000 ×g for 10 min at 4°C, and plasma samples were held at −80°C until analysis. The following biochemical indices were evaluated: aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and creatine kinase (CK). For the biochemical analysis of plasma, the VETTEST 8008 analyzer  was used.Blood was drawn from the After blood sampling, the muscle, liver, and intestine were quickly dissected, immediately frozen, and stored at −80°C. For analysis, frozen tissue samples were weighed and homogenized  with an Ultra Turrax homogenizer  using 50 mM potassium phosphate buffer, pH 7.0, containing 0.5 mM EDTA. The homogenate was divided into two portions, one for measuring thiobarbituric acid reactive substances (TBARS) and a second was centrifuged at 12 000 ×g for 30 min at 4°C to obtain the postmitochondrial supernatant for other antioxidant parameter analyses.−1. The value was expressed as nanomoles of TBARS per mg of wet tissue.The TBARS method was used to evaluate lipid peroxidation . The con2O2 breakdown at 240 nm [−1 [−1 of substrate or generates 1 mol L−1 of product per min expressed in international units per mg of protein.Total superoxide dismutase  activity was determined by the method of S. Marklund and G. Marklund . This ast 240 nm . Glutath0 nm [−1 . Glutath0 nm [−1 . One uniProtein levels were estimated spectrophotometrically  using bovine serum albumin as a standard .n = 4 per replication). The detection limit of Se was 0.05 mg kg−1 dw in all matrices.Concentration of selenium was determined in the microalgae biomass, experimental diets, and fish tissues by means of ICP-MS analysis. Muscle of four randomly selected individuals per replication was analysed. Insufficient liver tissue for analysis required that we pair target tissue from two randomly chosen fish , Tukey's multiple comparison test was applied to identify which treatments were significantly different. If the conditions for ANOVA were not satisfied, the nonparametric Kruskal-Wallis test followed by multiple comparisons of mean ranks for all groups was used [Statistical analysis was carried out using Statistica software 9.0 for Windows . Data were first tested for normality and homoscedasticity with Kolmogorov-Smirnov and Levene's tests, respectively. If those conditions were satisfied, one-way analysis of variance (ANOVA) was employed to reveal significant differences in measured variables among control and experimental groups. When a difference was detected , and no clear trend of treatments was observed.No mortality was observed during the trial. Mean standard body length was 16.1 to 16.5 mm and weight was 52.1 to 54.6 g, at the end of the experiment . Values −1 dw. A concentration of 0.80 ± 0.10 and 0.88 ± 0.09 mg kg−1 dw was found in the muscle of fish fed Se 0.3 and Algadiet 0.3, respectively, with the latter being significantly higher than that of the control (P < 0.002). The Algadiet 1.0 group showed Se concentration of 1.18 ± 0.12 mg kg−1 dw, significantly higher than other groups . Similar trends with greater mean concentrations of Se were found in liver, 2.22, 2.92, 3.18, and 3.71 mg kg−1 dw in the control, Se 0.3, Algadiet 0.3, and 1.0 groups, respectively.The final Se concentration in muscle of fish from the control group was 0.71 ± 0.08 mg kgP < 0.01) greater in the Se 0.3 and Algadiet 1.0 groups as compared to the control group. The AST and LDH activity was similar in all groups  higher than the in the control in all Se-supplemented groups. In case of muscle, higher CAT activities were seen in the Se 0.3 and Algadiet 1.0 groups (P < 0.01) muscle GR activity was observed in all treatment groups compared to the control. GR activity observed in liver or intestine did not differ from the enzyme activity in the control group.The activity of CAT in liver was significantly  of the total Se in the Chlorella biomass according to Fan et al. [Chlorella [Chlorella in differing mineral-nutrient solutions. However, content of selenocysteine (Se-Cys) was higher, at 48 to 76%. Differing Se compounds in Se-enriched yeasts and Se-enriched microalgae might suggest using the former, as Se-Met is probably the most active compound. Se-enriched microalgae may, however, benefit from the presence of specific bioactive compounds such as antioxidants, pigments, fatty acids, polysaccharides, and immunoactive substances.Recently, Se-enriched yeasts containing 54–74% total Se in the form of selenomethionine (Se-Met)  have beed Se-Met . Reportsa et al.  assumed n et al. . Dimethyhlorella . Neumannhlorella  found 24−1 Se-enriched yeasts, suggesting that high Se levels in the diet exhibit a toxic effect related to liver and kidney dysfunction [Blood enzyme levels provide vital information to aid in fish health assessment , 52. Biofunction .Selenium is essential for the proper functioning of the antioxidant enzymes, which protect against oxidative stress , 57. MaiP < 0.01) lower muscle GR activity was observed in all Se-supplemented groups compared to the control. Decreased GR activity may lead to the depletion of glutathione if its loss cannot be compensated for by the synthesis of new glutathione molecules [Procambarus clarkii [Glutathione reductase activity maintains the cytosolic concentration of reduced glutathione , 64. Sigolecules . Lower molecules , largemoolecules , tench [olecules , and red clarkii , 67.Results of the present study support the hypothesis that alteration in activity of CAT and GR enzymes in muscle and liver can counteract the prooxidant effect induced by elevated dietary Se. Oxidative stress indicators in muscle and liver of inorganic Se and higher concentration of organic Se-treated common barbel suggest that these tissues may possess differing abilities to counteract Se-prooxidant effects, with muscle showing a higher level of oxidative stress. Se supplementation at levels equal to or higher than those employed in this study (Se 0.3 and Algadiet 1.0) may accelerate the antioxidant condition of barbell, and, therefore, the optimal Se dietary concentration may be lower in this species.Chlorella biomass is more effective than inorganic sodium selenite. Direct comparison of Se-enriched microalgae with organic forms  and Se-enriched yeasts is needed. Efficacy of Se-enriched microalgae when compared with Se-enriched yeasts may be lower in commonly investigated indices. Use of Se-enriched microalgae biomass in aquaculture feedstuffs could be supported by the presence of specific bioactive compounds like polyunsaturated fatty acids, pigments, and vitamins.Selenium concentrations and enzyme activity in tissues of common barbel suggest that supplementation with Se-enriched


Rumex hastatus (Polygonaceae) are traditionally used for the treatment of various ailments including liver and lung diseases. In this study, various solvent extracts of R. hastatus roots, like methanolic, n-hexane, ethyl acetate, chloroform, butanol and aqueous fractions were assessed through their antioxidant properties in vitro and determination of phenolic contents.Roots of +, ˙OH, H2O2, superoxide free radical scavenging, iron chelating power, reducing power, β-carotene bleaching power, antioxidant capacity and total phenolics and flavonoids were evaluated. High Performance liquid Chromatography (HPLC) was also considered.Several parameters like DPPH˙, ABTS˙Though all the fractions exhibited dose dependant activity. The samples with the highest activity were the butanol and methanol fractions in all the assays except hydrogen peroxide radical scavenging assay where chloroform fraction showed the highest scavenging aptitude. On the other hand, aquous fraction showed significant beta carotene linoleic acid, while n-hexane and ethyl acetate fractions exhibited a lesser antioxidant activity in all the assays. HPLC revealed the presence of rutin, luteolin-7-glucoside, vitexin and luteolin.R. hastatus roots against different diseases, as an excellent basis of potential antioxidant due to the presence of sufficient amount of phenolics such as rutin and luteolin.These results have to some extent substantiated the use of  Rumex hastatus is suffrutescent richly branching shrub. It grows up to 90–120 cm tall and leaves with petioles of the same length as the blade; blade hastate, panicles terminal with erect-divergent, mostly simple branches, nut up to 2 mm long, brown, and long spindle-shaped roots. The plant is distributed in northern Pakistan, north east Afghanistan and south west of China, growing between 700-2500 m, sometimes grows as pure population [R. hastatus is used as medicine. It is laxative, alterative, tonic, used in rheumatism [Rumex hastatus is traditionally used in the treatment of sexually transmitted diseases including AIDS [R. hastatus were evaluated for phenolic compounds [R. hastatus, might have potent antioxidant properties against free radicals but no reports are available on the antioxidant activity of R. hastatus roots. Therefore, present study was conducted to explore the total phenolic content, HPLC characterizations and antioxidant activities of methanolic extracts and its various fractions through various in vitro models.eumatism . Plant ieumatism . Literating AIDS ,7. All ting AIDS . In caseing AIDS . Recentling AIDS . Phenoliing AIDS . Our preing AIDS -16. In oompounds , so it iR. hastatus was collected from Quaid-i-Azam University Islamabad, and Abbottabad of Northern Pakistan, respectively after identification by world renowed taxonomist Professor Dr. Mir Ajab Khan. Voucher specimens with Accession No. 27813 (R. hastatus) were deposited at the Herbarium of Pakistan Museum of Natural History, Islamabad for future correspondence. After complete cleaning of R. hastatus roots were cut into pieces and dried under shade. The dried samples were processed as mentioned [During September 2010 entioned ,18. The The total phenolics were assayed by the spectrophotometric method  using Foet al.[Total flavonoid content was determined following Yong et al. at 506 n50 mg of each fraction of both plants were extracted with 6 ml of 25% hydrochloric acid and 20 ml methanol for 1 hr. The obtained extract was filtered to a volumetric flask. The residue was heated twice with 20 ml of methanol for 20 min to obtain the extract. The combined extract was diluted with methanol to 100 ml. 5 ml portion of the solution was filtered and then was transferred to a volumetric flask and diluted with 10 ml of methanol. The sample (10 μL) was injected into the HPLC apparatus.The HPLC separation was performed using an Agilent HPLC system through column 20RBAX ECLIPSE, XDB-C18,  with UV–VIS Spectra-Focus detector, injector-auto sampler. The isocratic mobile phase, consisting of tetrahydro-furan/acetonitrile/ 0.05% phosphoric acid solution , was delivered at a flow-rate of1.0 mL/min with some amendments in time and wavelentgth . Prior tMethanol stock solution containing rutin, luteolin, vitexin and luteolin-7-glucoside was prepared and diluted to appropriate concentrations for the construction of calibration curves. At least six concentrations of the solution were analyzed in triplicate, and then the calibration curves were constructed by plotting the peak area versus the concentration of each analyte detected by HPLC.The stock solution containing rutin, luteolin, vitexin and luteolin-7-glucoside was diluted to a series of appropriate concentrations with methanol, and an aliquot of the diluted solutions was injected into HPLC for analysis. The LOD and LOQ under the present chromatographic conditions were determined at S/N of 3 and 10, respectively.Intra- and inter-day variations were chosen to determine the precision of the developed assay. The known concentrations of rutin, luteolin, and luteolin-7-glucoside were tested. For intra-day variability test, the solution was examined in duplicates for three consecutive days while for inter-day variability test, the mixed standards solution was analyzed for six times within one day, Variations were expressed as the RSD.To confirm the repeatability, BRR and MRR samples were extracted, respectively, and analyzed by HPLC as mentioned above. The RSD was used as the measurement of repeatability.Recovery test was used to evaluate the accuracy of the method. A known amount of standards was added to a certain amount of MRR and BRR, and then extracted and analyzed using the method described above. Three replicates were performed for the test. The recovery was calculated as follows:Identification of the different compounds was made by comparing their retention time and UV spectra with those of pure standards. Quantification was performed on the basis of external standard method. Peak Resolution value (R) was determined by using the following formula.1 and RT2 and W1and W2 are the times and widths, respectively, of the two immediately adjacent peaks.Where RTEach sample was dissolved in 95% methanol at a concentration1 mg/ml and then diluted to prepare the series concentrations for various antioxidant assays including DPPH , superox50 values were calculated using the Graph Pad Prism 5 software.All assays were carried out in triplicates and results are expressed as mean ± SD. Statistical comparisons were done with the ANOVA test. Differences were considered to be significant at p < 0.05. The ECR. hastatus and its different fractions is shown in Table The yield percent of methanol extract of roots of R. hastatus roots. Since the BRR and MRR exhibited the strongest antioxidant activity, they were quantified by assimilation of peak areas at 280 nm within runtime of 20 minutes  was linearly dependent on the concentration of the sample i.e. 5.0-250 μg/mL for rutin, 10–100 μg/mL for luteolin-7-glucoside, 2.0-200 μg/mL for vitexin and 2.0-250 μg/mL for luteolin. Calibration curves for standard analytes at different concentrations were found to be linear and summarized in Table To testify the precision and accuracy of the current method, RSD% values expressed as intra- and inter-day precisions and recoveries% of the standard analytes are determined in Table The results suggest that the activity of BRR and MRR is attributed to phenolic compounds and in particular to rutin, luteolin, luteolin-7–O-glucoside and vitexin however, unknown (peaks) compounds may also involve in the antioxidant activities of RH roots.R. hastatus roots extracts has DPPH free radical scavenging activity as BRR > MRR > AFC > CRR > ERR > HRR. The scavenging ability on DPPH radicals at 0.1 mg/ml of dried extract were 76.12 ± 2.11%, 61.13 ± 4.61%, 59.22 ± 2.34%, 50.23 ± 1.51%, 32.11 ± 2.13% and 30.51 ± 4.13% for BRR, MRR, ARR, CRR, ERR and HRR, respectively. The increase in activity at 0.25 mg/ml was 90.1 ± 2.13%, 86.2 ± 3.44%, 79.8 ± 1.19%, 77.7 ± 4.12%, 49.26 ± 1.34% and 43.21 ± 2.31% for the same order. The EC50 values of scavenging DPPH radicals for the BRR was 68.04 ± 1.7 μg/ml while for the HRR was > 246 μg/ml . The CRR and BRR fractions were equally potent in scavenging hydrogen peroxide, by 61.6 ± 1.21% and 57.1 ± 3.17% at a concentration of 100 μg/ml, respectively, while ERR and HRR were considerably less effective hydrogen peroxide scavengers (24.11 ± 1.22% and 27.03 ± 1.07%) at the same concentration. As compared with the EC50 values, the hydrogen peroxide- scavenging activities of CRR (75.11 ± 1.7 μg/ml) and BRR (77.02 ± 1.5 μg/ml) were comparable, and more effective (p < 0.05) than that of ERR (> > 250 μg/ml) and HRR (>250 μg/ml)  was significantly different (p < 0.05) from the EC50 values obtained for the ERR (>250 μg/ml) and HRR (> > 250 μg/ml), which were comparable  flower [R. hastatus root to hamper oxidation of linoleic acid emulsion is an indication of complex composition of fractions to interact with emulsion components. This data suggested that BRR and ARR have a notable propensity to scavenge free radicals that result in more stable non-reactive substances and to terminate radical chain reactions. Iron chelating data shows that various fraction possibly have talent to act against oxidative damage by chelating iron ions that may otherwise participate in decomposition of metal- catalyzed hydro peroxide and Fenton-Type reactions [Hydrogen peroxide itself is not very reactive, but it may produce hydroxyl free radicals that are very toxic to cells . Inspiteet al. phenolic) flower . The effeactions . The iro+3/ferric cyanide complex to ferrous form that necessitate antioxidant. The total ferrous complex can be scrutinized by computing the development of Perl’s Prussian blue at 700 nm. Data suggested that BRR and MRR have a noteworthy power to react with free radicals by converting them into more stable substances and to stop radical chain reactions. Activity may be endorsed with the collective antioxidant effects of phytochemicals especially phenolics and flavonoids. The results indicate that phenolics and flavonoids are responsible for the antioxidant activities of fractions of R. hastatus roots, and decorated the importance of phenolic compounds in the antioxidant measures of plant fractions. A noticeable correlation among different tests confirmed the viability and consistency of selected antioxidant assay systems. Our results are in agreement with Ao et al.[In the reducing power assay, donation of an electron is required to convert FeAo et al. who repoR. hastatus roots.In conclusion, the proposed HPLC method showed a good linearity, precision, repeatability, accuracy and recovery for the determination of four active compounds  and could be used for the quantitative analysis of RH roots. In this study, determination of phenolics and flavonoids, HPLC quantification and 9 antioxidant activity method with 6 extraction systems of different polarities, i.e., n-hexane, ethyl acetate, chloroform, butanol, methanol and water were compared. To our best knowledge this is the first record on the antioxidant potential of R. hastatus roots extracts were capable of inhibiting and directly quenching free radicals to terminate the radical chain reaction which might be attributed to the presence of flavonoid contents such as luteolin, rutin, vitexin and luteolin-7-glucoside.We have demonstrated that The authors declare that they have no conflict of interests.SS made a significant contribution to acquisition of data, analysis, drafting of the manuscript. MRK and RAK (ORCID ID: 0000-0003-0453-2090) have made a substantial contribution to conception and design, interpretation of data, drafting and revising the manuscript for intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/47/prepub


Background. While heat and moisture exchangers (HMEs) have been increasingly used for humidification during mechanical ventilation, the efficacy of pediatric HMEs has not yet been fully evaluated. Methods. We tested ten pediatric HMEs when mechanically ventilating a model lung at respiratory rates of 20 and 30 breaths/min and pressure control of 10, 15, and 20 cmH2O. The expiratory gas passed through a heated humidifier. We created two rates of leakage: 3.2 L/min  and 5.1 L/min (large) when pressure was 10 cmH2O. We measured absolute humidity (AH) at the Y-piece. Results. Without leakage, eight of ten HMEs maintained AH at more than 30 mg/L. With the small leak, AH decreased below 30 mg/L (26.6 to 29.5 mg/L), decreasing further (19.7 to 27.3 mg/L) with the large leak. Respiratory rate and pressure control level did not affect AH values. Conclusions. Pediatric HMEs provide adequate humidification performance when leakage is absent. In spontaneous breathing subjects, inspiratory gases are heated and humidified in the nasal cavity and pharynx; by the time, they reach the second bronchial bifurcation, temperature reaches 37°C, and absolute humidity reaches (AH) 44 mg/L . In mechHeat and moisture exchangers (HMEs) and heated humidifiers (active humidification) are commonly employed to warm and humidify medical gases. While HMEs are widely used during the mechanical ventilation of adults , their pUsing a pediatric model lung, we carried out a bench study to evaluate the effects of ventilator settings and leakage volume on the humidification performance of pediatric HMEs.2O. By inserting connectors with calibrated holes, we created two levels of leakage: 3.2 L/min  and 5.1 L/min (large) under airway pressure of 10 cmH2O. Ventilatory settings were assist-control mode, respiratory rates of 20 and 30 breaths/min, pressure control of 10, 15, and 20 cmH2O, inspiratory time of 0.8 s, positive end-expiratory pressure of 3 cmH2O, and FIO2 of 0.21. At the airway opening of the simulated respiratory system, we measured AH, RH, and temperature using a rapid response capacitance-type moisture sensor  described previously [Ten different models of HME were investigated . All HMEeviously , 9. Ambi2O, Nihon-Koden, Tokyo, Japan) placed between a flow generator and the HMEs. Thirty liters of flow was created by the flow generator and measured with a pneumotachometer  connected to a differential pressure transducer . Flow and pressure signals were amplified, digitized, and recorded at 100 Hz per signal using data acquisition software .The resistance of all HMEs was measured with a pressure sensor . Results are expressed as mean ± SD. Analysis of variance was performed using repeated measures ANOVA, and Scheffé's test was performed as a post hoc test for multiple comparison. For each pediatric HME we tested, we examined the relationship between dead space and obtained AH values. The relationship between variables was assessed using Spearman's rho (rs). We also calculated the AH delivered/dead space (volume of HME) as an index of humidification efficiency. Comparison between our measured data and manufacturer data for resistance was obtained by Wilcoxon signed-rank test. All statistical tests were two sided, and a 2O/L, RH was 100%, and temperature was 33.4 ± 0.4°C. Without leakage, recorded AH varied (28.4 to 32.4 mg/L) among the tested HMEs, the average being 30.9 ± 1.5 mg/L. Eight of the ten HMEs maintained AH at more than 30 mg/L. The levels of pressure control  did not statistically significantly affect AH (data are not shown). As the respiratory rate increased, AH increased in three HMEs, but the percentage difference between respiratory settings of 20 and 30 breaths/min always remained less than 3%; changes in AH values with the other seven HMEs were negligible (P = 0.018) (AH of expired gas at the end of the limb was about 36.5 ± 0.6 mgHgligible . With thgligible . When th= 0.018) .2O/L/s, with a maximum of 13.6 cmH2O/L/s and a minimum of 0.6 cmH2O/L/s. The mean difference between our values and those of the manufacturers' reports was −1.0 ± 2.6 cmH2O/L/s (P = 0.27). Values were summarized in The average resistance of 10 HMEs was 4.3 ± 4.0 cmHWe investigated the effects of leakage volume and ventilatory settings on humidification performance in ten HMEs for pediatric use. Without leakage, eight of the ten pediatric HMEs performed within the AARC guideline recommendation . When there was leakage, whether large or small, AH decreased below 30 mg/L in all pediatric HMEs. After investigating adult HMEs and antibacterial filters, Lellouche et al. found that 18 of 48 (38%) units were able to maintain AH at 30 mg/L or more, and the highest level of AH attained 31.9 mg/L . There hThe HMEs we tested were able to maintain AH at a level in the range from 28.4 to 32.4 mg/L. AH ranging from 30 to 25 mg/L is usually considered reluctantly acceptable but not desirable, while AH of less than 25 mg/L exceeds threshold and should be avoided during mechanical ventilation . With a 2O, the pressure control setting was increased to 20 cmH2O, AH tended to fall by approximately 3% although this decrease was not statistically significant. HMEs with little dead space may struggle to humidify gases flowing in large tidal volumes. Lucato et al. have reported that HMEs were more efficient when used with low tidal volume ventilation [Comparing manufacturer data with actual performance, Lellouche et al. have reported that different HMEs have different performance, some performing so poorly that they should not be used . While ttilation .For mechanically ventilated adults with humidification provided with an HME, when air leakage increases, inspiratory gas humidification decreases , 15. ConThis study has several limitations. Naturally, because it is based on a model-lung simulation, the findings do not directly correspond to clinical situations. Moreover, our observation period was relatively short; the performance and safety of HMEs in prolonged mechanical ventilation remain to be proven. We simulated real-life ventilatory conditions using HH and AH of expired gas which were slightly higher than those of previous study . This miIn conclusion, without respiratory circuit leakage, the majority of the HMEs for pediatric use provided sufficient humidification. With leakage, however, humidification decreased and was not maintained at guideline values. If there is leakage around the tracheal tube, extra caution is advised when using an HME for humidification for pediatric patients.


Pricralima nitida and the Sonchus oleraceus whole plant respectively.Diabetes mellitus (DM) is a metabolic disease characterized by chronic hyperglycaemia generally associated with oxidative stress. The present study aims at evaluating the antioxidant and antidiabetic potential of methanol and hydroethanol extracts of the stem bark and leaves of in vitro antioxidant activity was assessed using 1,1-Diphenyl-2-picrilhydrazyl (DPPH) for free radical-scavenging properties of the extracts, and the Folin-Ciocalteu method in determining their phenol contents. The antidiabetic activity was tested in mice following streptozotocin diabetes induction, and selected oxidative stress markers  were measured in order to evaluate the level of oxidative stress in treated animals.The in vitro antioxidant activity using DPPH showed IC50 ranging from 0.19 ± 0.08 to 1.00 ± 0.06 mg/mL. The highest activity was obtained with the hydroethanol extracts of S. oleraceus (0.19 mg/mL and P. nitida (0.24 mg/mL). Polyphenol contents ranged from 182.25 ± 16.76 to 684.62 ± 46.66 μg Eq Cat/g. The methanol extract of P. nitida showed the highest activity, followed by the hydroethanol extract of S. oleraceus (616.89 ± 19.20 μEq Cat/g). The hydroethanol extract of whole plants (150 mg/Kg) and methanol leave extract of P. nitida (300 mg/Kg) exhibited significant antidiabetic activities with 39.40% and 38.48% glycaemia reduction, respectively. The measurement of stress markers in plasma, liver and kidney after administration of both extracts showed significant reduction in MDA and hydrogen peroxide levels, coupled with a substantial increase in catalase activity.The S. oleraceus whole plant and P. nitida leaves possess both antidiabetic and antioxidant properties, and therefore could be used as starting point for the development of herbal medicines and/or source of new drug molecules against diabetes.These findings suggest that  Sonchus oleraceus and Picralima nitida, two multipurpose medicinal plants of Cameroonian pharmacopeia, are widely used to treat DM across the African continent  × 100 where Ao was the absorbance of the control and A1 was the absorbance in the presence of the sample of extract or standard.% scavenged [Free radicals] = [. The antidiabetic profiles of the extracts with significant antiradical and antioxidant activities was assessed using two complementary approaches: the glucose intolerance test (hypoglycaemic activity), and the evaluation of activity in rats with induced diabetes.Wistar rats  with body weight of 285-310 g, were divided into 9 groups with comparable average body weight. The different treatments were administered orally as indicated in Table ad libitum. Fasting blood sugar (FBS) was first measured for all the animals before administration of the different treatments. The different extracts and Glibenclamide (positive control) were then administered orally 30 min after administration of glucose. FBS was then taken 0.5, 1.5, 2.5, 3.5 and 4.5 hours post hyperglycaemia induction. Each time, FBS was measured from a blood drop collected from the rat’s tail, using a glucometer . The hypoglycaemic potential of the extracts was evaluated as their ability to correct the hyperglycaemia 4.5 hours after induction.A total of 45 three month-old The acute and sub-acute activities of the different extracts were determined in rats with streptozotocin-induced diabetes, following the method of Al-Shamaony et al. . Male raSeven groups of 5 rats each were based on their body weight, and treated as shown by Table A total of 35 rats were divided into 7 groups of 5 each and treated similarly as for the acute activity study. But in this case, the different drugs were administered daily, for 14 consecutive days, and FBS measured on days 0 (before drug administration), 4, 8 and 14 (end of the assay). At the end of the assay, the rats were sacrificed, blood collected in heparin to prepare plasma. The liver and kidneys were equally extracted and ground to form homogenates. The plasma and organ homogenates prepared were stored at -20°C until required for measurement of the markers of oxidative stress .Serum MDA levels were estimated by the method of Yagi  using th1 was the absorbance of the test sample; Ao, absorbance of the negative control; ℇ = 1.53.105 M-1/Cm; l the diameter of the cuvette (l = 1 cm); and V was the total volume of the supernatant collected.Where AThe hydrogen peroxide content was determined by a methods previously described by Jiang et al. . Briefly1 was the absorbance of the test sample; Ao, the absorbance of the blank; ℇ = 1.53.105 M-1/Cm; l the diameter of the cuvette (l = 1 cm), and V was the total volume of the mixture.Where A2O2, and the reaction stopped after 0, 30 and 60 seconds for tube 1, 2, and 3 respectively. The reaction was stopped by adding 1 mL of dichromate-acetic acid mixture (100 mL of potassium dichromate dissolved in 300 mL of acetic acid). The tubes were then boiled at 100°C for 10 min, allowed to get cold and the absorbance recorded at 620 nm, for each incubation time. The OD values obtained were plotted against time in Microsoft Excel 2010 and the catalase activity calculated.The catalase activity in blood and organ homogenate was measured by the method of Sinha . For eact test. Correlations between the different activities were assessed. All the statistical analysis was conducted in SPSS at 95% and 99% confidence intervals.Each test was conducted at least in triplicate and all the replicate values pooled together into Mean ± Standard variation. The different test groups were compared to each other using ANOVA, whereas variations within the same group were evaluated using pair-P. nitida scored the highest extraction yield (28.17%), followed by the hydroethanol and methanol extracts of S. oleraceus (19.11% and 13.02% respectively), whereas methanol extraction of P. nitida stem bark had the lowest yield (5.39%). The phytochemical analysis of the different extracts revealed the presence of a diversity of chemical families, including flavonoids, terpenes, sterols, saponins, alkaloids and polyphenols. However, anthocyanines and lipids were absent in all the extracts.The methanol extract of the leaves of P. nitida leaves and the hydroethanol extract of S. oleraceus whole plant (p < 0.01); unlike the P. nitida stem bark and the methanol extract of S. oleraceus which did not show any significant change in activity with the extract concentration. Based on their IC50s, the hydroethanol extract of S. oleraceus exhibited the highest activity with a value of 0.19 ± 0.08 mg/mL which is very close to the positive control catechine (IC50 = 0.13 ± 0.00 mg/mL). The methanol extract of P. nitida leaves and the hydroethanol extract of S. oleraceus showed moderate activities with IC50s of 0.24 ± 0.03 and 0.28 ± 0.04 mg/mL, respectively, whereas the stem bark of P. nitida had the lowest activity (IC50 = 1 mg/mL).Figure P. nitida leaves showed the highest concentration (684.62 ± 46.66 μg Eq cat/g of dry matter), followed by the hydroethanol extract of S. oleraceus (616.89 ± 19.20 μg Eq cat/g) whereas the methanol extract of the stem bark of P. nitida and whole plant of S. oleraceus had the lowest polyphenol concentrations .Regarding polyphenol content, methanol extract of the P. nitida leaves and the hydroethanol extract of S. oleraceus were considered for in vivo antidiabetic studies.Based on their high polyphenol contents, combined with the significant free radical-scavenging activities, and their phytochemical profiles, the methanol extract of P. nitida extract. As from 1 hr post-induction, a significant drop was observed with all the extracts, with a higher activity with S. oleraceus extract at 150 and 300 mg/kg and Glibenclamide (p < 0.01). The dose of 75 mg/Kg therefore did not appear to have any significant effect on the glycaemia in postprandial rats. The doses of 150 and 300 mg/Kg were then retained for subsequent work. With regards to the P. nitida extract, there was a continuous decrease in glycaemia in the animals receiving 150 and 300 mg/Kg, but no significant effect observed with 75 mg/Kg. This lower dose was therefore left out in subsequent studies, like in the case of S. oleraceus.The variations of glycaemia following oral glucose administration, in the presence of the different extract and in controls are summarized in Figure P. nitida extract and the positive control (p < 0.05). The 300 mg/Kg dose showed 34.75% reduction, compared with 22.40% obtained with 150 mg/Kg, and 39.78% for Glibenclamide. The glycaemia remained statistically constant all through the experiment, in both the untreated rats and those receiving distilled water. A significant reduction in glycaemia was observed in all treated groups, but not dose-dependent: 35.89% for 150 mg/Kg against 16.96% for 300 mg/Kg.A dose-dependent and time-dependent drop in glycaemia was observed with the S. oleraceus at 150 mg/kg (39.40%), P. nitida at 300 mg/kg (38.48%) 150 mg/kg (29.62%), while S. oleraceus at 300 mg/kg instead showed a very weak activity, with 5.91% reduction only.Like in acute activity, glycaemia remained unchanged in untreated rats as well as in those under distilled water during entire period of the experiment Figure . GlibencS. oleraceus at 150 mg/Kg where the extract was ineffective in reducing the MDA production in the liver, though active in the kidney and plasma. In almost all the groups and considering all the three organs, MDA values were slight higher than in non-diabetic animals. Concerning hydrogen peroxide  than in the positive control Glibenclamide (0.47 ± 0.10 μmol H2O2/min/mg). In the kidney, P. nitida at 300 mg/kg had the highest catalase activity (1.80 ± 0.70 μmol H2O2/min/mg) followed by S. oleraceus at 150 mg/kg (1.70 ± 0.29 μmol H2O2/min/mg), still being more active than the reference drug (1.40 ± 0.40 μmol H2O2/min/mg). The activity was dose-dependent for P. nitida, but not for S. oleraceus.Figure e Figure C, generay Figure A signifiP. nitida and S. oleraceus, was achieved in the present study, through determination of 1) the phytochemical profiles and polyphenol contents of the extracts, 2) their free radical-scavenging activity, 3) the hypoglycaemic activity in postprandial rats, their acute and sub-acute antidiabetic activity in streptozotocin-induced rats, and 4) the ability of the extracts to stimulate fight against oxidative stress in diabetic rats. The phytochemical screening revealed the presence of flavonoids, terpenes, sterols, saponins, alkaloids and polyphenols which are potential bioactive molecules of these plants [et al. [P. nitida leaves and the hydroethanol extract of S. oleraceus whole plant showed higher polyphenol contents, free radical-scavenging activity, significant blood sugar reduction capacity, and reduced the levels of oxidative stress markers  pointedly. There was a significant correlation between the polyphenol content and both oxidative and hypoglycaemic activity, suggesting polyphenols as the main determinant of antioxidant and hypoglycaemic effects. Phenolic compounds are thought to be produced by plants to protect them from abiotic and biotic stresses, but they are also beneficial to humans under disease induced oxidative stress [P. nitida leaves and the hydroethanol extract of S. oleraceus were considered for in vivo antidiabetic studies. The capacity of the extract to stimulate glucose storage in case of sudden hyperglycaemia was assessed in rats submitted to induced-hyperglycaemia, through ingestion of considerable amount of sugar. Efficient doses in both extracts were 150 mg/Kg and 300 mg/Kg. The drop in blood sugar observed may be due to either a stimulation of insulin secretion and activity, or other factors. Again, polyphenols and other metabolites present in these extracts  were previously shown to have hypoglycaemic activity [The assessment of the antioxidant and antidiabetic potential of e plants . Stanley [et al.  earlier e stress . Afolabie stress  also conactivity ,23,24.S. oleraceus at 300 mg/Kg. In general, the hydroethanol extract of S. oleraceus exhibited the highest activity, reaching 39.40% reduction in blood sugar at 150 mg/Kg, compared to 38.48% for the methanol leave extract of P. nitida at 300 mg/Kg. However, a much surprising finding noticed was that this activity of S. oleraceus was not dose-dependent, the reduction being more pronounced at the lower dose (150 mg/Kg). Furthermore, no sign of toxicity was observed both in the animal behaviour and at organ level. Further investigation may be needed to clarify this situation, by testing the extract using a different animal model for example.In acute and sub-acute studies, significant effects were observed as from 3 h. On day 14, all the extracts showed good activity, except for The oxidative stress has been associated with diabetic states in humans and streptozotocin (STZ)-induced diabetic rats -27. DuriP. nitida and S. oleraceus were previously studied using different solvent systems. Working on the seeds, Okonta and Aguwa [P. nitida seeds. However, in the present work, there was no glycoside in the leaves or stem bark extracts used. This observation highlights the significant difference between the phytochemical composition of the leaves and seeds of P. nitida. Polyphenol, alkaloids and sterols were noticed as the main metabolites of the leaves, whereas glycosides were shown to be predominant in the seeds [P. nitida seeds was also confirmed by Nwakile and Okore [The antioxidant and antidiabetic potential of other plant parts of nd Aguwa  observedhe seeds . The hypnd Okore .S. oleraceus, a treatment of diabetic rats with the whole plant infusions was shown to produce a marked amelioration of the impaired glucose tolerance at all examined periods after oral glucose loading and the lowered insulin and C-peptide levels, improvement in serum lipid profile including decrease in serum total lipid, total cholesterol, triglyceride, LDL-cholesterol and vLDL-cholesterol levels and increase in HDL-cholesterol level [Concerning previous work on ol level . The antIn summary, the antioxidant and anti-diabetic activities observed with these two medicinal plants could be explained by: 1) the presence of free radical scavenging ingredients fighting oxidative stress, and the inhibitory effects of phenolic compounds exerted against glucose generating pathways  guidelines.Animals were handled according to the ethical guidelines of the Cameroon National Veterinary Laboratory  and the ARRIVE (Animal Research: Reporting The authors declare that they have no competing interests.CMT conceived the project and supervised the work all through, PCM collected the plants parts and took part in biological tests, DZ participated in work design and drafted the manuscript, BTT participated in biological tests, FFB took part of work design and biological tests. All the authors proofread and approved the manuscript before submission.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/175/prepub1. Plant extracts; 2. Polyphenols and other secondary metabolites; 3. Free radical scavenging molecules; 4. Free radicals.Click here for file


Coagulation tests and factors measurements have been widely applied in clinical practice. Pre-analytical conditions are very important in laboratory assessment.Here,we aim to determine the effects of storage time and temperature on activated partial thromboplastin time (APTT), fibrinogen (Fbg), prothrombin time (PT), the international normalized ratio (INR), thrombin time (TT), factor VIII activity (FVIII:C), and factor IX activity (FIX:C) in fresh plasma. Seventy-two blood samples were tested after storage for 0 (baseline), 2, 4, 6, 8, 12, and 24 h at 25°C (room temperature) and 4°C (refrigeration) in two centers. The mean percentage change of greater than 10% and the numbers of samples with greater than 10% percentage changes more than 25% were used to determine clinically relevant difference. We demonstrated that samples for Fbg, PT/INR, and TT could be safely stored for ≤24 h; FVIII:C for ≤2 h; FIX:C for ≤4 h both at 4°C and 25°C; and APTT for ≤12 h at 4°C and ≤8 h at 25°C. Pre-analytical conditions are very important in laboratory assessment of hemostatic and coagulation systems114567Many studies have suggested acceptable storage temperatures and times for routine coagulation tests810Between- and within-batch imprecision of coagulation tests and factors were all consistent with the manufacturer's product information. Within- and between-batch imprecision were <3% and 10%, respectively.The mean percentage changes in the values of Fbg, PT/INR, and TT were all less than 10%, and the mean percentage changes in the values of APTT following sample storage for 2, 4, 6, 8, and 12 h at 4°C and 2, 4, 6, and 8 h at 25°C were all less than 10%, compared to the baseline values. However, the mean percentage changes of FVIII:C and FIX:C all exceeded 10%, except FVIII:C storage for 2 h at 25°C and 4°C; FIX:C storage for 2, 4, 6, and 8 h at 4°C; and FIX:C storage for 2, 4, and 6 h at 25°C. Coagulation tests and factors measurements have been widely applied in clinical practice; therefore, it is necessary to evaluate the effects of temperature and time from collection on the outcome of these results. Our multicenter study investigated the effects of split tube storage for 2, 4, 6, 8, 12, and 24 h at 25°C and 4°C. Of note, this study was the first to investigate the stability of FVIII:C and FIX:C at 25°C and 4°C in fresh plasma. Although many domestic and foreign scholars have studied the effects of pre-analytical variables on coagulation test analysis, no studies have described the stability of FVIII:C and FIX:C at 25°C and 4°C. A summary of these studies is listed in Table 32810121315161213In our study, although significant differences shown in  were obs1316Although many studies have analyzed the stability of coagulation factor activity in FFP61920146225Our multicenter study has some limitations. First, the study was based on asymptomatic individuals. Second, we used only one type of reagent, instrument, and collection container. Our conclusions can perhaps be corroborated by further study based on different study populations, reagents, instruments, and collection containers.In conclusion, our results demonstrated that plasma samples tested for Fbg, PT/INR, and TT determination could be safely stored for up to 24 h both at 4°C and 25°C; those tested for APTT measurement could be safely stored for 12 h at 4°C and 8 h at 25°C; those tested for FIX:C measurement could be safely stored for 4 h at 4°C and 25°C; and those tested for FVIII:C could be safely stored for only 2 h at 4°C and 25°C, suggesting that FVIII:C should be immediately measured within 2 h after collection in our laboratory. Thus, clinical samples should only be stored for these time frames prior to testing.The study was carried out in two laboratories, including the First Affiliated Hospital of Zhejiang University (Center 1) and The First People's Hospital of Hangzhou (Center 2). Both Centers, 1 and 2, used the same blood collection system , Sysmex CA7000 system , Siemens reagents , and International Sensitivity Index .Sample size estimates were calculated using a post hoc power of analysis with two size effects (0.8/0.9) and alpha levels (0.05/0.1). A sample size of 72 patients (36 per group) was appropriate.The study involved 36 asymptomatic individuals who visited the First Affiliated Hospital of Zhejiang University (Center 1) and 36 asymptomatic individuals who visited the First People's Hospital of Hangzhou (Center 2) for physical examination in September 2013. The patients of Center 1 included 18 men and 18 women, with a median age of 44 years old ; the patients of Center 2 included 18 men and 18 women, with a median age of 45 years old .This study was approved by the Ethics Committees of the First Affiliated Hospital of Zhejiang University and The First People's Hospital of Hangzhou, China. Patients provided written informed consent for their samples to be used in the study.g) to obtain fresh plasma without platelets and cells, and each sample was divided into 13 Eppendorf tubes and capped. The Eppendorf tubes used for the aliquots were composed of a nonactivating plastic. One split tube was tested immediately, which served as the baseline (0 h) results, while the remaining 12 split tubes were tested, respectively, after storage for 2, 4, 6, 8, 12, and 24 h at 25°C and 4°C.Venipunctures were performed in the morning following a 12-h fast. From each patient, a 5.4-ml venous whole blood sample was collected into a tube containing 0.109 M sodium citrate as an anticoagulant  at a blood to anticoagulant ratio of 9:1. The 72 samples were centrifuged , g/l (Fbg level), and % (FVIII:C and FIX:C).In Centers 1 and 2, the plasma samples were tested by a coagulation method for APTT, PT/INR, TT, Fbg, FVIII:C, and FIX:C using a Sysmex CA7000 system  and Siemens reagents : Dade Actin activated cephaloplastin reagent (lot 547713), Dade Thrombin reagent (lot 538065), Thromborel S (lot 545515), Test Thrombin reagent (lot 42780), Coagulation factor VIII-deficient plasma (lot 546576), and Coagulation factor IX-deficient plasma (lot 500870B), respectively. In addition, INR was transformed by PT according to the formula: INR = Two mixed pools having normal and abnormal target results were prepared from freshly collected plasma and were used to measure APTT, Fbg, PT, TT, FVIII:C, and FIX:C 20 times in 2 h to assess within-batch imprecision. Commercial quality control products (SIEMENS) with target results in normal/abnormal ranges were used to measure APTT, Fbg, PT, TT, FVIII:C, and FIX:C on 20 separate days to calculate between-batch imprecision. The manufacturer's product information for the assays claims that between- and within-batch imprecision should be <15% and <5%, respectively, for PT, APTT, Fbg, and TT; between-batch imprecision should be <10% for FVIII:C and FIX:C; and within-batch imprecision should be <10%  and <3%  for FVIII:C and FIX:C, respectively.The coagulation test and factor activity results were reported as mean ± standard deviation. The results following storage for 2, 4, 6, 8, 12, and 24 h at 25°C and 4°C were compared with the baseline results by using paired t-tests. To assess the stability of the coagulation tests and factor activities, the percentage changes compared to the baseline results were calculated [(result at storage time X – result at baseline)/result at baseline] and averaged for each time point21213Z.X.S. designed the experiments. L.M.F., Y.Z. and H.C.Z. performed the experiments. L.M.F. and Y.Z. wrote the main manuscript text. All authors reviewed the manuscript.


Quantification was subsequently performed by liquid chromatography–tandem mass spectrometry. Using this methodology, residual levels of isocyanates and amines in commercial PUR products were quantified. Although the recoveries of certain isocyanates and amines were low, the main compounds used as monomers in the production of PUR products, and their decomposition species, were clearly identified at quantifiable levels. 2,4-and 2,6-toluenediisocyanate were detected in most PUR foam samples and a pastry bag in the range of 0.02–0.92 mg/kg, with their decomposition compounds, 2,4-and 2,6-toluenediamine, detected in all PUR foam samples in the range of 9.5–59 mg/kg. PUR-coated gloves are manufactured using 4,4′-methylenebisphenyl diisocyanate as the main raw material, and a large amount of this compound, in addition to 4,4′-methylenedianiline and dicyclohexylmethane-4,4′-diamine were found in these samples.An analytical method for the identification and quantification of 10 different isocyanates and 11 different amines in polyurethane (PUR) foam and PUR-coated products was developed and optimized. Isocyanates were extracted and derivatized with di- Polyurethane (PUR) is obtained from the chemical reaction between diisocyanates and polyols. The reaction of isocyanates with water results in the formation of amines and carbon dioxide, which then acts as a blowing agent. Flexible PUR foam is used as a cushioning for fruits, absorbent pad for fish and meats, and in kitchen sponges. In addition, PUR-coated products such as gloves and pastry bags are used in applications involving contact with food.PUR is commonly made from 2,4-or 2,6-toluene diisocyanate  and sometimes from 4,4′-methylenebisphenyl diisocyanate   detection can allow for highly sensitive and selective identification. In this study, we have optimized this analytical method for 10 isocyanates and 11 amines in PUR products. We have then quantitatively evaluated the residual levels of isocyanates and amines in commercial flexible PUR foams and PUR-coated products. The relationship between the levels of the different components is also discussed.Nine PUR foam products and three PUR-coated products were purchased from Japanese retail outlets. The PUR foam products consisted of three cushionings for fruits and six kitchen sponges. The PUR-coated products consisted of two types of nylon fiber glove that were coated with PUR on the palm side, and one polyester pastry bag (otherwise known as a piping bag or forcing bag) that was coated with PUR on one side. The kitchen sponges consisted of two or three parts, which increased the sample number to 12.μg/mL) were prepared in acetone , and those of each isocyanate (1000 μg/mL) were prepared in toluene (Sigma–Aldrich).The isocyanate and amine standards used in this study are listed in Table n-butylamine (DBA) was purchased from Tokyo Kasei Kogyo . Water was purified using the Milli-Q Gradient A10 system .Analytical grade methanol and acetonitrile were purchased from Merck . Analytical grade acetone, acetic acid, formic acid, and dichloromethane were purchased from Sigma–Aldrich. Di-2 of coated area) were placed in a 50 mL glass tube (24 mm i.d. × 220 mm) equipped with a screw cap, and 20 mL of DBA solution (0.1 mg/mL in dichloromethane) was added. After derivatization and extraction overnight at 60°C, the samples were removed and washed with 5 mL of dichloromethane. The solutions were combined and the dichloromethane was evaporated in a vacuum centrifuge . The residues (∼200 μL of DBA) were then diluted to 1 mL with acetonitrile. The test solutions were subsequently evaluated using LC/MS/MS .Samples were cut into small pieces. PUR foam samples (50 mg) or PUR-coated samples (10 cmμm particle diameter); column temperature: 40°C; mobile phase: A, 0.1% formic acid; B, 0.1% formic acid/acetonitrile, A:B (40:60)—linear gradient (8 min)—A:B (5:95) (5 min); flow rate: 0.4 mL/min; injection volume: 1 μL; method of ionization: electrospray ionization (+); capillary voltage: 3 kV; ion source temperature: 150°C; cone gas and flow rate: 50 L/h (nitrogen); desolvation temperature: 400°C; collision gas: 0.1 mL/min (argon); measurement mode: multiple-reaction monitoring (MRM). Cone voltages and collision energies for the MRM of each compound are shown in Table The LC/MS/MS analysis was performed with an Acquity Series LC–MS, Waters Corporation. The conditions were as follows: column: Acquity BEH C18  were placed in a 50 mL glass tube equipped with a screw cap, and 20 mL of methanol was added. After extraction overnight at 60°C, the samples were removed and washed with 5 mL of methanol. The solutions were combined, and 1 mL of 4% acetic acid was added. The solution was evaporated to a volume of ∼1 mL, and then diluted to 10 mL with 4% acetic acid. The test solutions were subsequently evaluated by LC/MS/MS.The samples were cut into small pieces. PUR foam samples (50 mg) or PUR-coated samples (10 cmμm particle diameter); mobile phase: A, 0.1% formic acid; B, 0.1% formic acid/methanol, A:B (99:1)—linear gradient (5 min)—A:B (5:95) (2 min); flow rate: 0.3 mL/min. Cone voltages and collision energies for the MRM of each compound are shown in Table The LC/MS/MS conditions were as follows: column: Acquity HSS T3  No 10/2011. Of these, diphenylmethane-2,4′-diisocyanate  and 2,4-TDI dimer cannot be used as standard reagents. In addition, 4,4′-oxybis(diphenyldiisocyanate), 1,5-diisocyanatonaphthalene, and 4,4′-diisocyanato-3,3′-dimethylbiphenyl standards are not soluble in toluene or dichloromethane; thus, they were not analyzed. Phenyl isocyanate (PI), a base compound of aromatic isocyanates, was added, producing 10 different isocyanates for quantification.Many isocyanate analysis methods have been previously reported. In these methods, the use of DBA derivatization is advantageous in its high reactivity, and the fact that it allows for extremely sensitive and selective LC/MS/MS detection. The conditions for the derivatization were optimized for quantification of the residual isocyanate in PUR products according to the literature . Performing MS/MS product ion scan of the [M+H]+ of the isocyanate–DBA derivatives, the [DBA+H]+ (m/z 130) ions were all present as the most abundant ion. Therefore, [DBA+H]+ ions from [M+H]+ were used as quantitative ion. The [M+H-DBA]+ (m/z = M + 1–129) ions were used as qualifying ions for identification (Table When performing electrospray ionization (ESI)–MS of the isocyanate–DBA derivatives, the most abundant ions were [M+H]+H]+ m/z 30 ions wLC separation was performed with a gradient mobile phase using water and acetonitrile with 0.1% formic acid. Most isocyanate–DBA derivatives were separated. The chromatograms of the standard solutions are shown in Figure Dichloromethane was used for extraction of isocyanate in consideration of the solubility and stability of the compound. Preliminary analysis of kitchen sponge 1 showed the presence of 2,4-and 2,6-TDI; therefore, the derivatization and extraction conditions were optimized using this sample. Fifty milligrams of sample was immersed in 0.001–0.2 mg/mL of DBA solution, and left for 6, 12, 18, or 24 h at 60°C. Test solutions were then prepared from each extract, and the peak areas of the 2,4-TDI and 2,6-TDI derivatives were compared. The peak areas remained constant between 12 and 24 h of extraction at DBA concentrations of 0.05–0.2 mg/mL. However, when the 0.2 mg/mL DBA solution was used, unreacted DBA interfered with the separation of PI and cyclohexyl isocyanate (CHI). Therefore, the extraction and derivatization of isocyanates were performed for 18 h with a 0.1 mg/mL DBA solution.Measurements were attempted for the 13 amines obtained from the isocyanates listed in Regulation (EU) No 10/2011, in addition to PI. However, trimethylhexamethylene-1,6-diamine could not be obtained as a standard reagent, and octadecylamine was not soluble in 4% acetic acid. Therefore, 11 different amines were quantified.+, and the main product ions from [M+H]+ were used for identification, as shown in Table LC/MS/MS detection without derivatization has been previously reported for amine analysis  were spiked into 20 mL of methanol for the test procedure. The corresponding amines were not detected in significant quantities (<1% of spiked amount). H12MDA was detected at a level of 14% in spiked H12MDI; therefore, the H12MDA was presumed to include some hydrolyzed H12MDI when carrying out the quantification.Methanol was used for extraction of amines in consideration of the solubility of the compound. Moreover, methanol would react with the free isocyanates and prevent them from hydrolyzing to amines. The eight different isocyanates, hexamethylene diisocyanate (HDI), 2,4-and 2,6-TDI, IPDI, PI, CHI, 4,4′-MDI, and H12MDI  was spiked directly into the sample (50 mg of kitchen sponge 1 or 10 cm2 of glove 1), their recoveries were poor. Although the multiple extraction steps were tried, there was no improvement effect of residual levels and recoveries. It was speculated that the isocyanates reacted with functional groups present on the sponge/glove polymer, or that they decomposed to amines. Therefore, in this study, the isocyanates were spiked immediately after immersing the sample in the extraction solution.When 100 2) based on the residual levels in samples. Recoveries of 2,4-and 2,6-TDI, and 4,4′-MDI, which were used as the main monomers, were found to be in the range of 90–124% (Table Isocyanates were spiked at 10 ng (0.2 mg/kg or 1 ng/cm2), as shown in Table 2) based on the residual levels in the samples. Recoveries of 2,4-and 2,6-TDA, and 4,4′-MDA, which were the main residual amines, were 66–93%, with standard deviations of 1–5%. The recoveries of 2,4-and 2,6-TDA from the glove sample were quite low. Recoveries of HDA were not sufficient for quantification to be carried out. Other amines were found at levels of 83–100%, with standard deviations of 0–8%. In the case of 50 ng spiking, recoveries of HDA, 2,4-and 2,6-TDA, and IPDA from glove 1 were <50%.Evaluation of the recovery of the amines was carried out by directly spiking into kitchen sponge 1 (50 mg) and glove 1 10 cm, as show2. From these results, the LOQ of the isocyanates determined to 0.02–0.1 mg/kg or 0.1–0.5 ng/cm2, those of the amines (excluding HDA) determined to 0.2–1 mg/kg or 1–50 ng/cm2.The limits of quantification (LOQ) defined as signal to noise ratio of 10. However, the recoveries of 2,6-TDA, IPDA, and 2,4-TDA for glove 1 were not enough (less than 30%) by the recovery test spiked at 5 ng/cmThe residual levels of isocyanates and amines in three cushionings for fruits and 12 samples of kitchen sponge are quantified. Although kitchen sponges are not food contact material, since their materials were similar to cushionings for fruits. Therefore, the residual levels in kitchen sponges were compared with the regulation value as reference of cushionings. The results are shown in Tables Levels of 2,4-TDI and 2,6-TDI were found to be 0.04–0.92 and 0.02–0.26 mg/kg, respectively, in most samples. In addition, 4,4′-MDI, which is used as an adhesive to bond different parts of the sponges, was detected in three kitchen sponges at levels of 0.03–0.30 mg/kg. PI and CHI were detected in cushioning 1 at levels below the LOQ, and were deduced to be present as impurities or decomposition products of 2,4-and 2,6-TDI. No other types of isocyanate were detected. The total amount of isocyanates was found to be 0.03–1.15 mg/kg (0.01–0.55 mg/kg as NCO). No samples exceeded 1.0 mg/kg (as NCO), which is the maximum level specified in Regulation (EU) No 10/2011 for food contact materials. It should be noted that kitchen sponges are not classified as food contact materials.Levels of 2,4-TDA and 2,6-TDA were quantified to be 10–59 and 7.0–26 mg/kg, respectively, in all samples. In addition, small amounts of aniline (ANL) or cyclohexylamine (CHA) were detected in cushionings 1 and 2. No other types of amines were detected. The total amount of amines was 14–89 mg/kg.R2 = 0.976). Flexible PUR foam is usually manufactured from a mixture of 2,4-and 2,6-TDI , and one pastry bag (119 mg/cm2). The levels of the isocyanates and amines detected in the samples are shown in Table The residual levels of isocyanates and amines were quantified in the PUR-coated section of two types of gloves . The residual levels in the product were 0.4, 18.7, and 7.4 ANL, CHA, 4,4′-MDA, and H12MDA were all identified in both glove types, with total amine quantities of 30 and 5.6 mg/kg, respectively. The major amine contribution in glove 1 was 4,4′-MDA, while that in glove 2 was found to be H12MDA. It was speculated that H12MDI was used for the surface treatment when producing glove 2. In contrast, no amines were detected in the pastry bag. When the residual levels of amines and isocyanates in the two gloves were compared, no correlation was observed, the same as in the case of the PUR foam samples.An analytical method for identifying 10 different isocyanates and 11 different amines in PUR foam and PUR-coated products using LC/MS/MS was developed and optimized. Since isocyanates decompose to amine easily, isocyanates do not exist in the product surface. The residual isocyanates existed in the inside of the PUR material. In this study, we quantify the extractable isocyanate with dichloromethane. The recoveries of certain isocyanates and amines were low. Although we tried the improvement of these recoveries, they have not improved. Therefore, the residual levels of these compounds were quantified as reference value. Furthermore, the identification of suitable internal standards for the isocyanates recovered at unsatisfactory levels would greatly improve the accuracy of the methodology. But, the main compounds used as monomers in the production of PUR products, and their decomposition species, were clearly identified at quantifiable levels.The residual levels of isocyanates and amines in commercial PUR products were quantified using the LC/MS/MS technique. Commercial PUR foam is manufactured using 2,4-and 2,6-TDI as raw materials; however, these were only detected at low levels. On the other hand, 2,4-and 2,6-TDA, which are hydrolysates of 2,4-and 2,6-TDI, were detected at levels 18–633 times that of the corresponding isocyanates. PUR-coated gloves are manufactured using 4,4′-MDI as the main raw material, and a large amount of this compound, in addition to 4,4′-MDA and H12MDA, remained in these samples. 2,4-and 2,6-TDI, and 4,4′-MDI were detected in the pastry bag; however, no amines were detected.In this study, the residual levels in kitchen sponges were quantified as reference of cushionings. As a result, there were no samples that exceeded the level of 1.0 mg/kg (as NCO) as specified in Regulation (EU) No 10/2011. Since PUR foam and PUR-coated gloves are mainly used for dry foods, it is thought that there is almost no release of amines. However, as absorbent pads for fish and meats contain similar levels of amine to kitchen sponges, caution should be applied when using these in case of migration of the aromatic compounds 2,4-and 2,6-TDA, and 4,4′-MDA, in particular.


Our results marginally support the hypothesis that genetic susceptibility to brain tumors may be associated with mEPHX gene polymorphisms.mEH is a critical biotransformation enzyme that catalyzes the conversion of xenobiotic epoxide substrates into more polar diol metabolites: it is also capable of inactivating a large number of structurally different molecules. Two polymorphisms affecting enzyme activity have been described in the exon 3 and 4 of the mEH gene. The hypothesis of this study is that inherent genetic susceptibility to a primary brain tumor is associated with mEH gene polymorphisms. The polymorphisms of the mEH gene were determined with PCR-RFLP techniques and 255 Turkish individuals. Our results indicate that the frequency of the mEH exon 4 polymorphism (in controls) is significantly higher than that of primary brain tumor patients . This report, however, failed to demonstrate a significant association between mEH exon 3 polymorphism and primary brain tumor susceptibility in this population. Analysis of patients by both histological types of primary brain tumor and gene variants showed no association, although analysis of family history of cancer between cases and controls showed a statistically significant association ( In recent years, genetic polymorphism in a variety of xenobiotic-metabolizing enzymes (phase I and II enzymes) has been studied extensively. In general, these polymorphisms do not always lead to changes in protein expression and catalytic activities. However, the gene mutations cause alterations in expression and function of the enzymes. For this reason, several polymorphic genes, encoded for enzymes involved in phase I and II reactions, partially explain individual susceptibility to cancer. One of the enzymes is mEH, which catalyzes xenobiotic biotransformation in mammalian tissues and supports detoxification capability of the organism .trans-dihydrodiols and is responsible for the detoxification of PAH [The mEH EPHX1 EC 3.3.2.3) is a smooth endoplasmic reticulum enzyme that catalyzes the hydrolysis of epoxides into .3.2.3 isn of PAH . These pAn epoxide is a three-membered cyclic ether, while its ring system is highly strained, and its oxygen-carbon bond is polarized. Some reactive epoxides are responsible for electrophilic reactions, with critical biological targets such as DNA and protein, leading to mutagenic, toxic, and carcinogenic effects . The mEHThe development of disease is an outcome of interactions between human genes and the environment. Therefore, genetic variability may be responsible for individual susceptibility to carcinogenic factors in primary brain tumors. Brain tumor etiology indicates that certain occupations involving exposure to polycyclic aromatic hydrocarbons (or other aromatic hydrocarbons) may be associated with increased risk . Viral aSOD3 IVS1+186C>T polymorphism is associated with a significantly increased risk of meningioma, while a possible increased risk of glioma was observed by Rajaraman et al. [A number of investigations have been conducted to examine the potential association of the mEH genotype with altered susceptibility to disease incidence. These investigations yield highly confusing results. However, negative or positive results have been observed in similar studies for genotypes that involve different genes. From this perspective, Badr El-Din et al. found benign brain tumors that showed a significantly higher frequency of IL-10-1082 A/A and IL-6-174 C/C homozygous genotypes, compared to controls . The SODn et al. . On the n et al. . In somen et al. . Some stn et al. .The hypothesis of this study is that inherent genetic susceptibility to primary brain tumor is associated with mEH gene polymorphisms. To investigate this, we analyzed epidemiologic and molecular data from a case-controlled study in the Turkish population. We believe that there is a great need for large and well-designed epidemiological studies of potential genetic and environmental risk factors.n = 105) who attended the Neurosurgery Department at Cumhuriyet University Hospital in Sivas. Only those patients with newly diagnosed primary brain tumors (without any previous radiotherapy and chemotherapy) were included in the study. There was no sex, age, or tumor type restriction. The diagnosis of brain tumor was histologically confirmed, and tumor types were classified according to WHO guidelines. Age- and sex-matched controls were recruited mainly from patients  at the same hospital (n = 150). All study subjects agreed to participate and filled out a short questionnaire about occupation, tobacco use, alcohol consumption, and a family history of cancer. The local university ethics committee on human research approved the study.In this study, 255 Turkish people were assessed. Cases included brain tumor patients  were detected with the PCR and RFLP assay . The tarμL with 50 ng of DNA, 50 pmol of exon 3 or 4 primer, 5 μL dNTPs, 4 μL MgCI2, 5 μL PCR buffer, and 0.5 μL Taq DNA polymerase. The amplification of exon 3 was an initial denaturation at 95°C for 7 min followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 53°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 10 min. The amplification of exon 4 was an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 45 s, and final extension at 72°C for 5 min. Amplified products  were resolved by using 1.5% wt/vol agarose gels with ethidium bromide.PCR was carried out in a volume of 50 μL PCR product (163 bp) with 10 U PsyI, these products were resolved with 2.5% agarose gel containing ethidium bromide. Following digestion, the homozygous wild-type (Try 113) was identified by two DNA bands (140 and 23 bp), whereas the homozygous mutant allele (His 113) produced one band (163 bp), and the heterozygotes displayed a combination of all three  DNA fragments.Following overnight digestion of the 14 μL PCR product (357 bp) with 10 U RsaI, the digested products resolved with 2.5% agarose gel containing ethidium bromide. After digestion, homozygous wild-type (His 139) was identified by two DNA bands (295 and 62 bp), whereas the homozygous mutant allele (Arg 139) produced three bands ; the heterozygotes displayed a combination of all four bands  DNA fragments.Again, after overnight digestion of the 14 χ2 test or Fisher's exact test (when the expected number in any cell was less than five). In addition, the association between histopathologic type of brain tumor and mEPHX genotypes was evaluated using the χ2 test. The strength of the association was assessed for each polymorphism individually with OR and 95% CI from logistic regression models. All analyses were conducted using the Statistical Package for Social Sciences Program .The distribution of the mEPHX genotypes, tobacco use, and family history of cancer between cases and controls was evaluated using the In this study, samples were collected from 150 controls  and 105 cases  of primary brain tumor patients. χ2 = 3.5, P = 0.1)  .χ2 = 7.0, P = 0.01)  .χ2 = 0.05, P = 0.8, Crude OR = 1.0, 95% CI = 0.6−1.7) (The mEPHX exon 3 genotype distributions in controls and cases can be seen in 0.6−1.7) . We evalχ2 = 4.3, P = 0.03, Crude OR = 1.8, 95% CI = 1.0−3.4) (The mEPHX exon 4 genotype distributions in controls and cases are given in 1.0−3.4) .The polymorphisms in the mEH exon 4 and 3 were in HWE .We subdivided patients according to histopathologic type of brain tumor, so that we could evaluate the association between the polymorphisms of mEPHX and specific types of primer brain tumors. Histopathologic data were available for 102 cases: 29% were meningioma, 38% were astrocytoma, 9% were hypophysis adenoma, and 24% were other types  of exon 3. Histopathologic data were available for 105 cases: 30% were meningioma, 37% were astrocytoma, 9% were hypophysis adenoma, and 24% were other types in exon 4. There was no statistically significant association between mEPHX gene variants and tumor types .Cancer is the most common cause of death after diseases of the cardiovascular system. Brain tumor is a predominant problem worldwide, in terms of both incidence and prevalence, and is a leading cause of tumor death. Brain tumors affected more than 18,300 American males in 2003, and the mortality rate was 72% . The most common brain tumors are gliomas, which originate in the supportive glia tissue: astrocytomas arise from astrocytes . Brain tχ2 = 0.05, P = 0.8, OR = 1.0, 95% CI = 0.6−1.7)  and proto-oncogenes  activation [χ2 = 4.3, P = 0.03, OR = 1.8%, 95 CI = 1.0−3.4)  . Some sttivation , 29. We 1.0−3.4) . Thus, eχ2 = 3.5, P = 0.1)  . Our resχ2 = 7.0, P = 0.01)  . This reIn our case-control study, an increase in risk was not observed for any histological type of brain tumor associated with mEPHX variant genotypes. This finding is inconsistent with other studies and may be due to the small number of brain tumor cases in our study.These results add to the literature about the contribution of variation in metabolic genes to the incidence of adult brain tumors. Overall, these data do not provide strong evidence for the importance of genes involved in biotransformation of PAHs and their metabolites for brain carcinogenesis; however, specific genes may play a role in the context of both PAHs and other exposures. We believe that the association between mEPHX enzyme polymorphisms and brain tumor incidence must be investigated by studies with a larger number of cases and controls.In conclusion, our results somewhat support the hypothesis that genetic susceptibility to brain tumors may be associated with mEPHX gene polymorphisms. Additional research is still necessary to determine an individual's susceptibility to brain tumor development.