Leishmania amazonensis and L. brasiliensis in vitro. The cell viability was determined, comparing the results with reference standards. The dichloromethane fractions of the roots, stems, and leaves of Allamanda schottii showed IC50 values between 14.0 and 2.0 μg/mL. Plumericin was the main active compound, with IC50 of 0.3 and 0.04 μg/mL against the two species of Leishmania analyzed. The hexane extract of Eugenia umbelliflora fruits showed IC50 of 14.3 and 5.7 μg/mL against L. amazonensis and L. brasiliensis, respectively. The methanolic extracts of the seeds of Garcinia achachairu and guttiferone A presented IC50 values of 35.9 and 10.4 μg/mL, against L. amazonensis, respectively. The ethanolic extracts of the stem barks of Rapanea ferruginea and the isolated compound, myrsinoic acid B, presented activity against L. brasiliensis with IC50 of 24.1 and 6.1 μg/mL. Chloroform fraction of Solanum sisymbriifolium exhibited IC50 of 33.8 and 20.5 μg/mL, and cilistol A was the main active principle, with IC50 of 6.6 and 3.1 μg/mL against L. amazonensis and L. brasiliensis, respectively. It is concluded that the analyzed plants are promising as new and effective antiparasitic agents.This study evaluated extracts, fractions, and isolated compounds from some selected Brazilian medicinal plants against strains of promastigotes of  Leishmania are protozoan parasites responsible for a spectrum of diseases known as leishmaniasis. There are two main forms of leishmaniasis: cutaneous, characterized by skin sores; and visceral, which affects the internal organs . This disease is classified as a neglected disease and is one of major public health problem, causing significant morbidity and mortality in various countries. Leishmaniasis is considered by the World Health Organization as one of six major infectious diseases, with a high detection rate and ability to produce deformities [ormities –3.Currently, the treatment of this disease is based on a limited number of chemotherapeutic agents, which represent high toxicity and cost. The search for new medicinal agents has become extremely important. The higher plants are a very rich source of new and selective substances, with therapeutic potential against these ailments. There are approximately 250.000 plant species worldwide, of which only a fraction has so far been studied. However, much research is needed to identify plants as sources of drugs or their phytoconstituents. The World Health Organization also advocates the use of traditional medicine for the treatment of these tropical diseases –6.Allamanda schottii, Rapanea ferruginea, Eugenia umbelliflora, Garcinia achachairu, and Solanum sisymbriifolium, have exhibited various biological properties, including anti-proliferative antinociceptive, anti-inflammatory, antimicrobial, and gastroprotective effects [Our research group participated in an Iberoamerican program (RIBIOFAR/CYTED/CNPq) to search for Brazilian plants with therapeutic potential to treat several diseases, including the so-called neglected diseases. Some species have been previously analyzed with respect to their chemical composition and biological properties. Now we are interested in exploring the potential of the crude plant extracts, semi-purified fractions, and chemically defined molecules, in terms of their activity against leishmaniasis.  effects –15.Allamanda schottii were collected in the city of Blumenau, in December of 2006. The fruits of Eugenia umbelliflora Berg were collected in June 2008, in the town of Itapema. Rapanea ferruginea, synonymously Myrsine coriacea (Ruiz and Pavon) Mez, was collected in Blumenau in July 2007. Garcinia achachairu was collected in Camboriú, in March 2007. Aerial parts of Solanum sisymbriifolium Lam. were collected in Itapema, in January 2006. All the species were identified or authenticated by Professor Oscar Benigno Iza , and vouchers were deposited at the Barbosa Rodrigues Herbarium , under numbers HBR 52525 (A. schottii), VC-Filho 50 (E. umbelliflora), HBR 52715 (R. ferruginea), HBR 52637 (M. coriacea), and VC-Filho 037 (S. sisymbriifolium).Different parts  of A. schottii was macerated with 95% ethanol at room temperature, for seven days. Solvent removal was carried out under reduced pressure at temperatures below 45°C, until the desired concentrations were achieved, in order to obtain the ethanolic extracts. Part of each extract was dissolved separately in methanol : water (90 : 10) and successively partitioned with hexane, dichloromethane, and ethyl acetate to obtain the respective fractions, after the removal of solvents. The samples were stored under refrigeration and protected from light until analysis.The dried vegetal material (100 g) of each part  of E. umbelliflora were dried under air circulation (37°C) for 2 days and powdered using a knife grinder. The dried fruits (570 g) were extracted twice by maceration with n-hexane for five days. Next, solvent was removed under vacuum to yield a dry residue of 2.62% hexane extract. The fruits were then reextracted using dichloromethane and methanol, following the methods described above. The separate extracts were submitted to concentration to obtain dichloromethane and methanolic extract 1 with yields of 1.16% and 4.47%, respectively. The methanolic extract 2 was obtained by extracting the dried fruits with methanol at room temperature for seven days.The fruits of R. ferruginea was macerated with 95% ethanol at room temperature, for seven days. Solvent removal was carried out under reduced pressure at temperatures below 45°C, until the desired concentrations were achieved, in order to obtain ethanolic extracts with yields of 16%, 15%, and 13% from leaves, stem, and fruits, respectively. The chloroform extract was obtained by the stem barks and is described in Hess et al. [The dried material 100 g of each part  of G. achachairu were separately extracted at room temperature with methanol for seven days. The macerated material was filtered and concentrated under reduced pressure, yielding 9.01 g (3.6%), 15.0 g (6%), and 12.0 g (4.8%) of crude methanolic extract, respectively. The air-dried and powdered seeds, leaves and branches (250 g each) of S. sisymbriifolium were extracted with MeOH for ten days. The concentrated methanolic extract was diluted with a water : methanol mixture  and extracted with hexane , chloroform , and ethyl acetate . The powdered leaves, and stem (680.0 g) of A. schottii. The ethanolic extract of A. schottii stems (15.1 g) was chromatographed in a silica-gel column with a hexane: ethylacetate: ethanol gradient. The fraction eluted with n-hexane : ethyl acetate 7 : 3 yielded plumericin (15.2 mg). Elution with a hexane : ethyl acetate ratio of 6.5 : 3.5 yielded ursolic acid (62.4 mg). The fraction eluted with ethyl acetate : ethanol 8 : 2 yielded plumieride (39.4 mg) as described previously by Schmidt et al. [t et al. .R. ferruginea. The CHCl3 extract of R. ferruginea (63.4 g) was subjected to column chromatography packed with silica gel 60–230 mesh and eluted with hexane gradually enriched in ethyl acetate. The fraction eluted with 40% ethyl acetate in hexane provides impure myrsinoic acid B. This fraction (8.5 g) was chromatographed in a silica-gel column with hexane : ethyl acetate to provide pure myrsinoic acid B (4.3 g) as described previously by Baccarin et al. [E. umbelliflora. The complete procedure used to obtain the compound isolated from E. umbelliflora fruits, the meroterpenoid Eugenial A, was described previously by Faqueti et al. [i et al. . G. achachairu. The methanolic extract of the seeds (5.0 g) was chromatographed on a silica-gel column  and eluted with a gradient of CHCl3–MeOH (100 -> 0) yielding guttiferone A as described previously by Dal Molin et al. [n et al. .S. sisymbriifolium. The chloroform fraction was purified by CC on silica gel, yielding Cilistol A (60.0 mg) and cilistadiol (25.0 mg) as described previously by Niero et al. [o et al. . The molLeishmania braziliensis (M2904 C192 RJA) obtained from in vitro cultures of IIFB (20 μL) were fixed with glutaraldehyde  and counted in a Neubauer chamber. The population was adapted to 3 × 106 parasites/mL with Schneider medium (pH = 6.8) and Fetal Bovine Serum (10%), distributed (100 μL/well) in 96-microwell plates. Solutions of the samples, at different concentrations, were added (100 μL). DMSO (1%) and Amphotericin B (0.5 μg/mL) were used as parasite growth control. Each test was performed in triplicate and the plates were incubated for 72 hours at 26°C.AML (MHOM/BR/76/LTB-012) and μL/well) a solution of XTT (1 mg/mL) in phosphate buffer  with PMS , which was incubated for 4 h at 26°C. The plates were read on a computer Stat Fax (Model 2100 Series-Plate Reader) at 450 nm. The IC50 values of 50% inhibitory concentration of parasites were calculated using Microsoft Excel 2000 [To each well of the plate was added . The data are mean ± stand error, and statistical differences were determined by the one-way ANOVA followed by Tukey-Kiover .The samples were analyzed in replicates with in vitro antiparasitic effect against promastigotes of L. amazonensis and L. brasiliensis. For the initial screening, extracts with IC50 less than 100 μg/mL were considered active. The results of the minimal inhibitory concentrations are shown in Tables In this work we tested extracts, fractions, and compounds of five Brazilian medicinal plants to determine their A. schottii  exhibited pronounced leishmanicidal activity  for L. amazonensis and 0.04 μg/mL (0.13 μM) for L. brasiliensis. Ursolic acid was active against the two species evaluated with IC50 of 66.1 μg/mL and 8.3 μg/mL for L. amazonensis and L. brasiliensis, respectively. Amphotericin B, used as positive control, exhibited activity with IC50 of 0.6 μg/mL and 0.7 μg/mL, respectively, for the two species evaluated  against Leishmania donovani with IC50 of 3.7 μg/mL [Allamanda genus are promising antileishmanial agents.On the other hand, ursolic acid was isolated as an active compound from extracts of .7 μg/mL . Our resL. amazonensis and L. brasiliensis with respect to E. umbelliflora fruits indicated that the hexane extract was the most active with IC50 of 14.3 ± 0.86 and 5.7 ± 0.92 μg/mL, respectively. When the fruits were directly submitted to methanol extraction, activity was observed with IC50 of 12.5 ± 0.5 and 7.8 ± 0.4 μg/mL against L. amazonensis and L. brasiliensis, respectively. Despite reports in the ethnobotanical literature on the medicinal uses of Eugenia species to treat some diseases, there have been few scientific studies that validate their antileishmanial activity. Previous studies with Eugenia genus have suggested that terpenic compounds found in the essential oil may have potential anti-leishmanial activity [The antiparasitic investigation against promastigotes of activity .G. achachairu for its leishmanicidal activity. Thus, the methanolic extract, some fractions, and isolated compounds were evaluated. As can be seen in  G. achachairu exhibited IC50 values of 35.9 and 100 μg/mL in promastigote forms of L. amazonensis and 28.2, 100, and 81.6 μg/mL against promastigote of L. brasiliensis, respectively. The most bioactive extract (from the seeds) was chromatographed on a silica-gel column yielding guttiferone A as the main constituent. This compound, after evaluation in promastigote forms of L. amazonensis and L. brasiliensis  need to be carried out, in order to understand the mechanisms of action and to evaluate the toxicity, searching for a clinical use for these bioactive compounds.Our results, together with those reported in the literature, strongly suggest that


Omega-3 fatty acids are important in treatment of severe primary hypertriglyceridemia (HTG). In 15 patients with severe primary HTG , we assessed efficacy-safety of sequential monthly treatment with Lovaza, 4 to 8 to 12 g/day.With TG >500 mg/dl despite Type V diet, hyperinsulinemia and diabetes control, and fibric acids, Lovaza (4 g/d) was added for 1 month, and if TG remained >500 mg/dl, increased to 8 g/d for 1 month, and then to 12 g/d for 1 month, and subsequently reduced to 4 g/day for 4 months.Primary HTG, median TG 884 mg/dl, 14 men, 1 woman, all white, age 50 ± 7 years, 12 non-diabetic, 3 with stable diabetes control. Weight and diet held stable throughout. In 5 patients, after 1, 2, and 3 months on 4 g/day, TG fell <500, mean 1390 to 234 , to 135 , and 158 mg/dl , with a negative TG slope, p=.0013. Non-HDLC fell from 320 to 177 , to 152 , and to 163 , with a negative slope, p=.01. In 10 patients, with Lovaza increased from 4 to 8 to 12 g, 3 failed to respond. In 7 of these 10 patients, TG fell 37% from 1075 to 672 on 4 g (p=.006), to 577 on 8 g , and to 428 mg/dl  on 12 g/day, with a negative TG slope, p=.0018. TG on 12 g/day was lower than on 8 g/day, p =.03. Non-HDLC fell from 245 to 217 mg/dl (−11%) on 4 g/day, to 203  on 8 g/day, and to 192  on 12 g/day, with a negative slope, p=.016. Compared to pre-Lovaza baseline, no abnormal measures developed in safety tests. The 4, 8, and 12 g/d Lovaza doses were well tolerated.Titration of Lovaza from 4 to 8 to 12 g/d safely offers an effective way to lower TG beyond conventional 4 g therapy. Omega-3 fatty acids of marine origin are safe, effective triglyceride (TG)-lowering agents -4, and aThere are many patients with primary and familial hypertrigyceridemia (HTG) where conventional treatment with diet, avoidance of alcohol, fibric acids, diabetes control, metformin (for hyperinsulinemia) , and 4 gOur specific aim in the current study was to assess effectiveness and safety of Lovaza 4, 8, and 12 g/day in 15 patients who were referred to us with primary HTG (TG>1000 mg/dl) and failed to lower TG below 500 mg/dl despite treatment with diet, avoidance of alcohol, fibric acids, treatment of type 2 diabetes mellitus, and, in the presence of hyperinsulinemia, metformin .th, 50th and 75th percentile TG of 791, 884, and 1640 mg/dl, Table There were 14 men, 1 woman, all white, mean age 50, baseline mean ± SD TG 1251 ± 671 mg/dl, with 25Of the 3 subjects with type 2 diabetes, hemoglobin A1C (HbA1C) was held stable below 7.2% throughout the study period in 2, while in the third subject, mean HbA1C was 8.2%, but stable throughout the study. All patients with type 2 diabetes were treated with metformin 2.5 g/day. Of the 12 non-diabetic patients, 5 had impaired fasting glucose , and another 5 patients had hyperinsulinemia (>24.9 uU/ml). All of these 10 patients were treated with branded Metformin 2.5 g/day.Two subjects had ApoE 2–3 heterozygosity, 1 ApoE 2–4, 6 were homozygous for the wild-type normal genotype (ApoE 3–3), and 6 were ApoE 3–4 heterozygotes, Table After 1 month on 4 g/day Lovaza, in 5 patients, TG fell well below 500 mg/dl, from a baseline mean of 1390 mg/dl on conventional TG-lowering therapy to 234 mg/dl , Figure Of the 10 patients , as Lovaza was increased from 4 to 8 to 12 g/day, 3 failed to consistently respond, with mean TG at baseline 1369 mg/dl, and after 3 months 1260 mg/dl while on Lovaza 12 g/day, Figure In 7 patients who responded with TG lowering as Lovaza was increased to 12 g/day, Figure Of the 12 subjects without type 2 diabetes, 5 of whom had impaired fasting glucose (3 of these 5 had hyperinsulinemia), and 5 had hyperinsulinemia without IFG. All 10 patients were treated with metformin 2.5 g/day. Changes in weight, insulin and glucose were not significant  from visit 0 to visit 1, and there was no correlation between the changes in insulin, weight, and TG from visit 0 to visit 1, p >0.6.In the 15 patients, there were 85 patient-months on Lovaza 4 g/day, 10 patient- months on 8 g/day, and 10 patient-months on 12 g/day. Compared to pre-Lovaza baseline, no abnormal measures developed in laboratory safety tests (p>0.1) on 4, 8, or 12 g/day Lovaza therapy. The 4, 8, and 12 g/d Lovaza doses were well tolerated, and adherence by pill count was >95% for all treatment groups.In the current study, after treating HTG with a conventional TG-lowering regimen , 5 patients were very sensitive to addition of 4 g Lovaza/day, with mean TG falling 90% from 1390 mg/dl to 135 mg/dl and mean non-HDLC falling 53% from 320 to 152 mg/dl in 2 months. Moreover, at 2 of 7 monthly visits on Lovaza, diastolic blood pressure was lower than at baseline, an additional beneficial outcome.In 10 patients with mean TG 1163 mg/dl despite conventional TG-lowering therapy, 3 patients (baseline mean TG 1369 mg/dl) failed to respond significantly to sequential increments of Lovaza from 4 to 8 to 12 g/day. In 7 of these 10 patients, however, TG fell 37% from 1075 mg/dl to 672 mg/dl on Lovaza 4 g/day, fell 46% to 577 mg/dl on 8 g/day, and fell 60% to 428 mg/dl on 12 g/day, with a negative TG slope. At the same time, mean non-HDLC fell 11% from 245 to 217 mg/dl, 17% to 203 mg/dl, and 22% to 192 mg/dl. Moreover, diastolic blood pressure was lower than baseline at last visits on Lovaza.Patients with the ApoE 3–4 genotype were less responsive to Lovaza therapy, congruent with the report by Christidis et al.  where trTitration of the Lovaza to 8 g/day (10 patient-months) and 12 g/day (10 patient- months) in 10 patients did not adversely affect any laboratory safety tests, did not produce petechiae or purpura, and was well tolerated. This is a limited experience, however, with regard to safety of 8 and 12 g/day of Lovaza, and is a limitation of the current study.Another limitation of the current study was the failure to measure plasma, red blood cell, and leukocyte fatty acid profiles and compare these with the patients’ responses to the stepwise increases in doses of Lovaza.Our findings in the current study are congruent with previous studies in hypertriglyceridemic patients given up to 15 g/day of omega-3 fatty acids -12. HarrA triglyceride concentration above 1,000 mg/dl is an indication for therapy to lower the risk of acute pancreatitis, while in subjects with TG levels below 1,000 mg/dl the indication for treatment is primarily to reduce the risk of clinical events caused by atherosclerosis ,8,16-18.Omega-3 fatty acids have successfully been used in treatment of severe primary HTG ,20,21. OBays et al.  carried Another approach to the stepped management of the type of severely hypertriglyceridemic patients described in this report would be to lower TG to < 1,000 mg/dL with Lovaza, and then add atorvastatin, 80 mg/day, or rosuvastatin, 40 mg/day .In patients with severe primary HTG despite conventional TG lowering therapy, addition of Lovaza 4 g per day, and where necessary, increasing to 8 or 12 g per day safely further lowers TG in a linear fashion.The study followed a protocol approved by the Jewish Hospital IRB, with signed informed consent, and after an entry visit, included 8 subsequent visits at monthly intervals. Patients were instructed in a eucaloric diet with total fat restricted to no more than 25% to 30% of calories [Inclusion criteria included primary HTG  with fas1. Patients with known allergy to fish.2. HTG secondary to alcoholism, exogenous corticosteroids or estrogens, nephrotic syndrome, hemochromatosis, glycogen storage disease, uncontrolled diabetes, Cushing’s syndrome, uremia.3. Bleeding gastric or duodenal ulcers, active inflammatory bowel disease.4. Pregnancy.5. Dementia.After an overnight fast, blood was obtained for entry measurement of lipid profile, complete blood count, fasting glucose, hemoglobin A1C (HbA1C), insulin, C-peptide, renal and liver function tests, T4 and TSH, and apo E genotype. At each of 8 subsequent visits, blood was drawn for lipid profile, complete blood count, glucose, HbA1C, renal and liver function, insulin, and C peptide. At entry, and at each subsequent visit, dermatologic examinations were carried out to record any petechiae or bruising. At entry, and at 8 subsequent visits, weight and blood pressure were measured, and a brief physical exam was carried out. At each visit, adherence to the targeted Lovaza dose was assessed by pill count, and adherence to diet by a 24-hour diet recall.Patients referred to our center with TG >1,000 mg/dl were instructed in a eucaloric diet with total fat restricted to no more than 25% to 30% of calories  to its uOne month after visit −1, if TG remained >500 mg/dl despite conventional TG lowering therapy, then patients were instructed to start Lovaza, 2 g with breakfast, 2 g with the evening meal, while continuing their antecedent TG lowering therapy unchanged, and dietary instruction was reinforced.One month after visit 0, if TG had fallen to < 500 mg/dl, Lovaza was held at 4 g/day. If TG remained >500 mg/dl, Lovaza was increased to 8 g/day, with other components of the TG lowering regimen held stable throughout.One month after visit 1, if TG had fallen to < 500 mg/dl on 8 g/day, the Lovaza dose was kept at 8 g/day, but if TG remained > 500 mg/dl, Lovaza was increased from 8 to 12 g per day, with other components of the TG lowering regimen held stable.One month after visit 2, Lovaza was diminished in all patients to 4 g/day.Four monthly visits after visit 3; Lovaza kept at 4 g/day.All statistical analysis was done using SAS 9.1.3.For each treatment group (according to Lovaza dosage sequences), mean ± SD and median TG, non-HDLC, body weight and blood pressure (BP) at each visit were calculated. Changes from baseline to follow-up were tested using MIXED model, with visit as fixed effect, subject as random effect, and comparison of least square means (SAS proc MIXED).For each treatment group, MIXED model for TG was constructed for the first 3 months on Lovaza, fixed effect was time (visit order), random effect was subject, with repeated measures had AR(1) variance structure. Using MIXED model, the TG slope was assessed.Paired Wilcoxon tests were used to assess the effect of changes of Lovaza dosage  on TG and non-HDLC.(HTG): Primary hypertriglyceridemia; (TG): triglyceride; (HDLC): high density lipoprotein cholesterol; (non-HDLC): non high density lipoprotein cholesterol.Beyond provision of support by way of drug and partial funding by GSK, the authors have no conflict of interest, including membership in speaker’s bureau or corporate advisory committee, stockholder, or travel support. No conflicts of interest with competing products.Drs NK, CJG, and PW participated in all aspects of the manuscript, study design, data collection, data editing, data analysis, production of manuscript. Drs ZK, MR, and JP participated in data collection, data editing, data analysis, production of manuscript. All authors read and approved the final manuscript.


There was an error in affiliation number 1 for the first, second, fourth, fifth, sixth, seventh and last authors. The correct affiliation is: IIS-Fundación Jiménez Díaz, Autónoma University, Madrid.In addition, an affiliation for the last author was incorrectly omitted from the byline. In addition to institution number 1, Óscar Lorenzo is also affiliated with the following institution: Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM).


Studies focusing on Antarctic biodiversity must take into account soil geochemical and environmental factors that influence population and species heterogeneity.Terrestrial life in Antarctica has been described as some of the simplest on the planet, and mainly confined to soil microfaunal communities. Studies have suggested that the lack of diversity is due to extreme environmental conditions and thought to be driven by abiotic factors. In this study we investigated soil microfauna composition, abundance, and distribution in East Antarctica, and assessed correlations with soil geochemistry and environmental variables. We examined 109 soil samples from a wide range of ice-free habitats, spanning 2000 km from Framnes Mountains to Bailey Peninsula. Microfauna across all samples were patchily distributed, from complete absence of invertebrates to over 1600 specimens/gram of dry weight of soil (gdw), with highest microfauna abundance observed in samples with visible vegetation. Bdelloid rotifers were on average the most widespread found in 87% of sampled sites and the most abundant (44 specimens/gdw). Tardigrades occurred in 57% of the sampled sites with an abundance of 12 specimens/gdw. Nematodes occurred in 71% of samples with a total abundance of 3 specimens/gdw. Ciliates and mites were rarely found in soil samples, with an average abundance of 1.3 and 0.04 specimens/gdw, respectively. We found that microfaunal composition and abundance were mostly correlated with the soil geochemical parameters; phosphorus, NO Desert ecosystems are often regarded as some of the simplest on Earth, in terms of trophic levels and biodiversity, which is contrasted to temperate and tropical ecosystems Even within ice-free areas, the distribution of microfaunal populations remains irregular and taxonomically limited Studies of ice-free areas across Antarctica have shown variations in microfaunal composition according to location and habitat. Microfaunal abundance also shows seasonal variation with respect to abiotic factors. Higher moisture content in soil during summer, as a consequence of higher temperatures, has been associated to increase the growth of photosynthetic autotrophs, microbial and microfaunal species Plectus murrayi, P. frigophilusScottnema lindsayaeEudorylaimusPanagrolaimusHalomonhysteraHypodontolaimusDolichorhabditisEchiniscus and Pseudoechiniscus (Echiniscoidea) and the predatory species Milnesium tardigradum (Apochela). For rotifers, the Classes Bdelloidea and Monogononta have been reported for the Antarctic continent Adineta, Habrotrocha, Macrotrachela, Mniobia, Otostephanos and Philodina  and Framnes Mountains (67.78°S– 62.79°E) to Bailey Peninsula (66.28°S-110.54°E). To the best of our knowledge this is the first single study that correlates biotic and abiotic parameters for an area spanning more than 2,000 km from any region in Antarctica. Other studies have focused on diversity at a much smaller scale e.g.  includinAll field activities and sampling in Antarctica was undertaken with permits granted by the Australian Antarctic Division . Samples returned to Australia under required quarantine protocols with permits granted by Australian Quarantine Inspection Service . Under these permitted guidelines, sampling in EA was conducted during the 2009–2010 austral summer from Casey Station on 24 December 2009, and from all other locations from 14 January 2010 to 4 March 2010  at magnification 10× to 40×. Before isolation of specimens, presence of rotifers, nematodes, tardigrades, mites and ciliates were recorded and sorted coarsely within a gridded petri-dish. Individual specimens were then counted and abundance for each of the taxa assessed. In cases where samples were difficult to sort due to excessive amount of suspended material, further dilution was required. Samples with a high abundance of microfauna were sub-sampled and the total abundance was extrapolated for 100 g of soil.Taxa were divided into glass blocks using modified gel tips attached to micro-syringes. Representative morphotypes for each taxon were retained for subsequent morphological analyses. Specimens were carefully transferred with an Irwin loop into a water droplet on a slide and imaged under microscope  at 40× to 100× before placing in separate 2 ml Eppendorf tubes. The remaining microfauna not selected for imaging were stored at −20°C.Philodina (wheel-organ bearing bdelloid), Adineta (lacking wheel-organs) and a group including unidentified bdelloids (mostly comprising contracted specimens). Tardigrada were grouped according to their Order ; and Nematoda were categorised as Plectus, Eudorylaimus, Scottnema, Halomonhystera (genera) and cf. Panagrolaimidae (family). Plectus species were identified using de Man's ratios calculated from digital images , Ciliophora (ciliates) and Acari (mites) had their own separate categories, Rotifera were subdivided into monogononts (non-bdelloid rotifers) and bdelloids. Bdelloid identification was based on presence or absence of wheel-organs. The three bdelloid groups included es after  and veries after .3− and NH4+. Analyses for soil moisture (moist) and pH were performed at the University of Adelaide using the methods described by Rayment & Lyons Soil geochemical analyses were performed for each of the 109 samples collected across EA. These analyses were conducted in Australia by APAL  using standard chemical methods 3−, NH4+, moist and pH). In total, 16  abiotic variables were considered, with ten of these quantitative and six qualitative. The categories moss, algae-cyanobacteria , and soil samples from moss beds were qualitative dichotomous ; the categories region, geology and aspect were qualitative. Regions included: CS, VH, BP, SP, L-Isl, HI, MP, SI, FM and MS. Geology of terrain comprised three sub-categories as reported by tectonic studies Environmental variables were elevation, aspect (direction to which the slope faces), slope, vegetation content in soil , and proximity to moss beds when present. Other categories included region, geology of the terrain, amount of fine sediment in the sample and the soil geochemical parameters analysed  and then subjected to a Principal Component Analysis (PCA) based on Euclidean distances . CS samples were segregated to the right of PC1 ; BP and FM tend to segregate along the PC1 axis to the left of the cluster . No clear trend was observed for the other regions along PC1 and PC2 axes (4+) were positively correlated and presented the highest contribution for PC1 . For PC2 the highest contribution was observed for Cs_bed, moss and by EC . Cs_bed was positively correlated to moss samples but negatively correlated with pH. When examining other type of vegetation, we observed that algae-cyanobacteria  was better explained by PC2 (eigenvectors: 0.27). A positive correlation was also observed between al-cy and NO3−, but a negative correlation with elevation ((i) EC and NO3−; (ii) P, NH4+, C and soil moisture.The PCA is presented in PC2 axes . Overalllevation . To idenlevation  and geoclevation –6. In ge4+ (5–64 p.p.m.), and low NO3− (3.4 p.p.m.) and EC , NH4+ (4.8–345 p.p.m.), and P , C (0.01–0.22), EC (0.02–0.1 dS/m), and P  for 109 samples from the ten regions  were usePlectus morpho-types were compared with described populations for Plectus murrayi and P. frigophilus from continental Antarctica , Halomonhystera (three samples), and cf. Panagrolaimidae (one sample); while Eudorylaimus was always found in the presence of other nematode genera , EC (0.01–48 dS/m), NH4+ (4.2–372 p.p.m.), NO3− (3.4–19 p.p.m.), P (2–171 mg/kg), pH (4.3–8) and moisture (0.25–77%). Plectus frigophilus was less tolerant of extreme conditions as P. murrayi, occurring in only five sites with no visible moss filaments, limited EC range (0.04–0.88 dS/m), and diverse ranges of C (0.05–9.9%), NO3− (3.4–12 p.p.m.), NH4+ (5.1–372 p.p.m.), pH (4.7–7.6), P (7.3–99 mg/kg) and moisture (6.5–77%). The minimum soil moisture requirements for Plectus species were higher than for Scottnema and Eudorylaimus.All nematodes were identified using morphological measurements and de Man's ratios. For  samples . Plectuse genera .The genu regions . Althougnditions ; with P.Scottnema specimens were collected from VH to FM , NH4+ (4.5–18.6 p.p.m.), and C (0–0.55%), at low-moderate levels of moisture (0.1–15.4%), and various levels of P (3–44 mg/kg) and NO3− (18.4–3.4 p.p.m.). No visible cyanobacterial samples were associated with the presence of Scottnema. In samples where Scottnema was present, 58% of the time (14 samples) it occurred with Eudorylaimus  for Eudorylaimus than Scottnema (Eudorylaimus was found from VH to MS (4+ (4.2–63.6 p.p.m.), NO3− (3.4–11 p.p.m.), moisture (0.11–28.6%), and P (1.4–40 mg/kg).VH to FM  in sevenVH to FM . RecordsVH to FM . Scottnerylaimus  but nevecottnema . EudorylVH to MS  in soilsHalomonhystera was found in a total of five samples in VH, BP, and HI , moderate NO3− (3.4–7.5 p.p.m.), and various ranges of EC (0.04–3.02 dS/m), NH4+ (7–31 p.p.m.), moisture (5.8–24%), and P (4–67 mg/kg). Members of the family Panagrolaimidae have been recorded for EA Hypodontolaimus) was observed in a fine soil sample (HI-08) next to a saline lake (EC: 0.33 dS/m) without visible vegetation., and HI , and occ, and HI . It was rylaimus , but witAdineta and Philodina from some of the samples, but the remaining bdelloids were left as unidentified.. Bdelloids were present for all ten regions (3− (3.4–1163 p.p.m.), NH4+ (4.5–373 p.p.m.), moisture (0.11–77%) and pH  was only observed in two samples from the sides of lakes with similar NH4+ concentrations (7.5–7.8 p.p.m.) and close to neutral pH (6.7–7.6).We identified the Classes Bdelloidea and Monogononta from our Antarctic soils. It was only possible to morphologically discern live-mobile bdelloid specimens . Seven bdelloid species have been previously described in the literature for VH, Larsemann Hills, and CS  regions  in soil 4.3–9.2; . The rot4.3–9.2;  revealed3−, NH4+ and pH ranges as bdelloids, but in a narrower range of: EC (0.02–48 dS/m), C (0.01–9.9%), P (1.9–249 mg/kg), and moisture (0.28–77). Parachela was recorded from 56% of the 109 samples followed by Echiniscoidea 8% and Apochela 1.8% (Milnesium sp.) occurred in two samples from L-Isl and MS together with the other two tardigrade Orders (Plectus) and rotifers. Milnesium was found in fine soils containing visible moss filaments, high moisture (24–33%) and P (27–79 mg/kg), moderate organic C (2.4–3.6%) and NH4+ (11–13 p.p.m.), and low pH (4.8–5.5). Echiniscoidea (represented by Echiniscus sp.) was present in nine samples  were identified in this study . In sampela 1.8% . Apochele Orders , nematod samples  with difParadileptus cf. elephantinus which was observed in a single soil sample collected at a bird moulting site in HI. Ciliates were observed in seven regions (3− (3.4–41.5 p.p.m.); and soils with a wide range in C (0.05–6.8%), P (1.9–310 mg/kg), NH4+ (4.8–222 p.p.m.), and pH . They were found in seven of the ten geographic regions (4+ (4.5–64 p.p.m.), moisture (1.8–27.7%); and low NO3− concentrations (3.4–8.2 p.p.m.), which was corroborated by a negative correlation for NO3− and mite presence , and the second highest P concentration (169.3 mg/kg) for all CS samples.The arthropod community in our EA soils was dominated by Prostigmata mites ; and the lowest for FM . The mosPlectus), a soil sample with visible moss filaments from Sansom Island , which accounted for 82% of the total microfaunal abundance. There were 12 samples containing only al-cy as the only visible type of vegetation and accounted for 38% of microfauna abundance. Soil samples including moss  were 26 and represented 29% of the abundance . Four samples included al-cy and moss (together) and contributed 14% of the abundance. Only two samples had lichen as the only form of vegetation and represented 2% of the abundance. Samples without visible vegetation (65 out of 109) included only 18% of the total microfauna abundance. Around 70% of the microfauna abundance was concentrated in six samples  followed by P (ρ = 0.108) and EC (ρ = 0.094) represented the abiotic variables with strongest correlations when considering each individually (4+ (ρ = 0.186) presented the highest correlation values when considered individually; while a combination of C and NO3− showed the highest two-variables correlation ; while abundance was better explained by NO3− (ρ = 0.206). A combination of P, NO3−, pH and al-cy had the strongest correlation values with nematode composition (ρ = 0.309); while the combined effect of P, NO3− and al-cy had the highest correlation with nematode abundance  we observed that the strongest correlations for Plectus were NO3− (ρ = 0.128) and al-cy (ρ = 0.149); for Eudorylaimus, P (ρ = 0.272), pH (ρ = 0.206) and C (ρ = 0.179); and for Scottnema, C (ρ = 0.283), NO3− (ρ = 0.213) and NH4+ . For our study it was observed that the highest correlation among abiotic categories and taxa composition was a combination of P, NOvidually . For mic3− was the best variable to explain composition (ρ = 0.075); and when combined, C, NO3−, elevation and region (ρ = 0.15) were the strongest variables. Rotifer abundance was better explained by P (ρ = 0.154) and by the combined effect of C, P and NO3−  and NO3− (ρ = 0.213); while for Philodina, corresponded to moist (ρ = 0.047) and slope ; and for ciliate abundance involved moist and slope , which was also the best value when correlated to mite abundance (ρ = 0.092); while the highest correlation value resulted for a combination of NO3− together with moist, slope and elevation  were considered separately, positive correlations were observed for vegetation and C, while moisture was positively correlated with tardigrade, rotifer and ciliate abundance but not with nematodes (Scottnema showing a negative correlation). NO3− was negatively correlated with mites, Plectus and Eudorylaimus presence, and with nematode abundance; while NH4+, P and C were all negatively correlated with Eudorylaimus and Scottnema presence. P and NH4+ were also found to have positive correlations with tardigrade and rotifer abundance (and ciliate abundance for NH4+); and EC showed correlations with Scottnema presence (negative) and ciliate presence and abundance (positive).Pearson correlation analysis (Table S3 in i) soil microfaunal composition and abundance; ii) soil geochemical data and vegetation content; and iii) correlations between soil geochemistry and other abiotic environmental variables with microfauna.The present study is the most comprehensive to date in Antarctica correlating microfauna and environmental data across more than 2,000 km from East Antarctica (EA). It provides et al.There have been few soil microfaunal surveys for EA with most focusing on extremely restricted populations. Current knowledge on microfauna composition and abundance in EA is still incomplete, and in need of appropriate sampling. Considering previous research in other Antarctic regions, further sampling and molecular work is likely to reveal new species, resolve taxonomic problems and extend the known ranges of species. Studies for other Antarctic regions (Victoria Land) have revealed that nematodes were the most extensively distributed and abundant metazoan in soils 4+ were inhabited predominantly by Plectus, while the opposite trend was observed for Eudorylaimus and Scottnema. Scottnema is reported to prefer dryer and saltier soils with lower organic matter than Eudorylaimus3−, P and EC have an important contribution explaining nematode composition and abundance (no significant contribution was seen for moss samples). Eudorylaimus and Scottnema (4+ as strong drivers determining their presence. We observed Scottnema in soils with the lowest average EC (0.1 dS/m) and Halomonhystera in the highest (0.92 dS/m). Our findings support studies by Andrássy Halomonhystera towards more saline environments. The distribution of Eudorylaimus from our study appears to correspond to their predatory habits on other nematode species Eudorylaimus presence was always linked to potential prey  in a variety of soils with low-moderate C levels and for only 7% in samples with visible algae. Wall et al.Eudorylaimus to be an algae-feeder and not an omnivore as previously recorded by others Eudorylaimus presence and al-cy in sample  indicating higher tolerance levels to environmental stresses. Plectus is a bacterial feeder, which potentially increases the range of habitats where it can be found. Nevertheless, denser populations were seen in presence of al-cy that could offer food as well as sheltered microhabitats. It is important to highlight that nematode presence was never as wide when considering NO3− levels. We observed that samples with high NO3− (23–1163 p.p.m.) only harbor tardigrades, ciliates and/or rotifers but no nematodes suggesting lower capabilities of the latter to adapt to NO3− rich environments.3−, NH4+ and pH) were observed for samples including bdelloids, suggesting that the effect of a single variable does not drive bdelloid composition and is more the result of a combination of abiotic factors  representing 15% of total samples and accounting for almost half of rotifer abundance. Bdelloid numbers also seem to be indirectly affected by high NH4+ concentration in soils (98–373 p.p.m.) with contrasting results for the top 11 samples; four of those samples contributed 51% of the bdelloid abundance; but for three of those 11 samples no rotifers were observed.The presence of bdelloid rotifers in 87% of soil samples  reflects factors , 5. Strobundance . Our resbundance  as in otbundance . P and NPseudechiniscus (Order Echiniscoidea), which has been reported as the most common tardigrade for the LH 3−  and bdelloid rotifers. Their presence is likely to be linked to presence of other microfaunal taxa, probably reflecting their feeding habits, but this is only based on two samples. Echiniscus sp. was observed in 8% of samples with various taxa, but never in samples where Scottnema occurred, suggesting that most suitable habitats comprise moderate to high soil moisture concentration (9–67%) that is likely to be outside the optimum requirements for Scottnema.The predatory and cosmopolitan species Due to their minute size (∼70–100 µm in length) and oval shape, Ciliophora were not easily discernible. We restricted our study to live-mobile ciliates with visible cilia, and abundance and presence of ciliates/protozoans in our samples is likely to have been underestimated (ciliates present in 15% of samples). Other studies, based on Victoria Land, also reveal low ciliate frequency 3− to be the strongest variable explaining mite presence and abundance  close to neutral pH, drier and inorganic soils (Scottnema), ii) low NO3, neutral pH, low-medium organic soils (Eudorylaimus), iii) saltier and less vegetated soils , iv) soils higher in phosphorous, NH4+, C and moist (Plectus), v) more acidic soils without vegetation (Echiniscus), vi) acid-neutral soils high in moisture content (Philodina), and vii) acidic soil (Adineta). Microfaunal abundance was significantly correlated with soil geochemistry (Question 3); we found that P, NO3−, EC and C are correlated with higher microfaunal densities of most common taxa ; whereas in habitats with low pH, low moisture, low C, and high EC, the ‘specialists’  Echiniscus, Scottnema, Eudorylaimus, and Halomonhystera seem to do better. Our data indicate that region, slope and aspect did not play a major role in determining abundance. Our ability to address whether the occurrence of taxa is correlated with the presence of other taxa (Question 4) was confounded by determining if there is a biotic correlation among taxa, or if taxa co-occurring together are the result of similar micro-habitat requirements. It is most likely that any correlation (at least for non-predatory species) is the result of a connection between suitable soil geochemical conditions with soil productivity levels and microbial activity. For predatory species , we could expect their distribution to reflect availability of prey, as they were always present with other taxa.We found that soil geochemical variables differed significantly among sites (Question 1) most likely as a result of variation in landscape formation/alteration, organic deposits from vegetation cover, ornithogenic inputs and shifting in nutrient accumulation due to melt-water runoff. Our study showed that abiotic variables are correlated with the composition of taxa (Question 2), with some taxa favouring 3−, P and salinity as the main drivers; and to a lesser extent by pH, C and soil moisture.To understand soil microfaunal abundance, taxa composition and distribution in Antarctica it is important to determine their correlation with soil geochemistry and other environmental parameters. Where a population exists is likely to be determined by a suite of soil geochemical factors, with NOFile S1Contains the following annexed supplementary tables.Table S1, Location, abiotic parameters and meiofauna abundance for 109 samples from East Antarctica. Table S2, Measurements and de Man's ratios for Plectus murrayi and Plectus murrayi and P. frigophilus females from East Antarctica compared to other regions from various studies. Table S3, Pearson correlation matrix for 109 sites and the most relevant environmental and biotic variables.(DOCX)Click here for additional data file.


Podophyllum hexandrum Royle (P. hexandrum) is a perennial herb and widely used in clinic. The present study was designed to separate and identify the biochemical composition and antioxidant capacity of extracts from P. hexandrum rhizome.P. hexandrum rhizome were analyzed by GC-MS (gas chromatography–mass spectrometry), and the antioxidant capacity of the extracts and the components was tested by using the DPPH  and FRAP (Ferric reducing/antioxidant power) assays.The ethyl acetate and ethanol extracts from 50 and FRAP values of 9.61 ± 0.81 and 2923.98 ± 21.89 μM, 9.98 ± 0.24 and 2847.27 ± 14.82 μM, and 13.37 ± 0.35 and 2404.32 ± 36.88 μM, respectively, than the positive control ASA (Ascorbic acid) with the values of 60.78 ± 1.22 and 1267.5 ± 30.24 μM (P < 0.01).The rhizome extracts had greater antioxidant capacity than the petiole extracts in DPPH and FRAP assays. About 16 kinds of main reactive oxygen components were identified in the extracts. Components of PADE , BADE , Polyneuridine, PODD , β-Sitosterol and POD (Podophyllotoxin) showed the antioxidant capacity in some degree. PODD, POD, and Polyneuridine showed stronger antioxidant capacity with the ICP. hexandrum rhizome. These results provide useful biochemical basis and information for the potential use of this plant.PODD, POD, and Polyneuridine are very critical for the antioxidant capacity in the extract of  Podophyllum hexandrum Royle (P. hexandrum), commonly named Himalayan Mayapple, is a perennial herb that grows in the Himalayan region and the southwest of China [P. hexandrum resin has been used topically for various skin lesions such as warts and condylomas [P.hexandrum rhizome. It has been used in the treatment of genital infection to sterilize noncervical human papilloma virus [et al. [P. hexandrum. POD is also used as starting compound for the chemical synthesis of etoposide (VP16-213) and teniposide (VM-26), and its congeners and derivatives have pronounced biological activity as anticancer, antineoplastic and anti-HIV drugs [P.hexandrum rhizome extracts. In our previous study, GC-MS showed that the ethyl ether extract of P. hexandrum seed contained some bioactive components, such as Hexanedioic acid, Oleic acid, and Octadecanoic acid [of China ,2. Sincendylomas . POD is ma virus . Penile ma virus . Goel et [et al.  reportedIV drugs -9. ReseaIV drugs . So far,oic acid .P.hexandrum by the DPPH and FRAP assays, separated and identified the biochemical compositions of the rhizome extracts by GC-MS, and then evaluated antioxidant capacity of some identified compositions with the parameters of IC50 and FRAP value.In the present study, we tested the antioxidant capacity of ethyl acetate and ethanol extracts in the rhizome and petiole of P. hexandrum was collected from a forest  in HuiChuan, WeiYuan of Gansu Province, China, after the plant fruit ripened in September 2011. The plant was dried in shade under room temperature. The species was identified by Professor Yanling Qi . A voucher specimen (No.0209069) was deposited in the herbarium of College of Agronomy, Gansu Agricultural University, Lanzhou, Gansu, P.R. China.3 · 6H2O, and HCl were purchased from Guangfu Chemical Research Institute . UV-1810  and Trace DSQ GC-MS  were used.DPPH, 2, 4, 6-tris (2-pyridyl)-s-triazine (TPTZ), PADE, BADE, Polyneuridine, PODD, β-Sitosterol, and POD were purchased from Sigma Chemical Company . Ethyl acetate, ethanol, methanol, ASA, FeClRhizome and petiole were washed, dried and grinded to powder. Then the powder was weighted (20.00 g) and soaked in ethyl acetate and ethanol (500 mg/mL) for 10 months at room temperature in dark, and was then filtered, evaporated and condensed to dryness under nitrogen to obtain extracts.Although there are numerous methods for determining the antioxidant capacity of soluble natural extracts and insoluble food components , no perfet al. [et al. [et al. [The free radical scavenging activity (FRSA) of DPPH was measured according to Ramadan et al.  and Nenc [et al. . which i [et al. ,19. Foti [et al.  suggeste [et al. ,19. This [et al. ,21. The −4 M DPPH methanol solution. Then the mixture was shaken and kept in dark for 30 min at room temperature. The decreased absorbance of DPPH solution was evaluated at 515 nm by a spectrophotometer UV-1018, and 500 μM 15% aqueous ethanol ASA was tested as a positive control. The test was carried out in triplicate, and the capability to scavenge the DPPH radicals was calculated as:The extracts were diluted with 15% aqueous ethanol with concentration of 10 mg/mL, and then 50 μL of the diluted extracts was added with 950 μL of 10A0 and A were the absorbance of DPPH without and with sample, respectively.where et al. [3+- TPTZ), in stoichiometric excess, to a blue ferrous form (Fe2+), with an increase in absorbance at 593 nm [et al. [The FRAP test was first introduced by Benzie et al.  for meast 593 nm . The spe [et al.  as folloex tempore by mixing 10 volumes of 300 mmol/L acetate buffer, pH 3.6, with 10 mmol/L TPTZ in 40 mmol/L HCl, and 20 mmol/L FeCl3 · 6H2O at 10:1:1 (v/v/v).The working FRAP reagent was prepared 4 · 7H2O, 500 μM) or test samples (10 mg/mL 15% aqueous ethanol) were added and mixed well. The reaction temperature was 37°C and the absorbance readings were taken at 593 nm immediately and 4 min later using a spectrophotometer UV-1018. And 500 μM 15% aqueous ethanol ASA was tested as a positive control. All tests were carried out in triplicate. The FRAP value of the test samples was calculated on the basis of 500 μM Fe2+ (FeSO4 · 7H2O) as follows:The 300μL FRAP reagent and the 10μL standard samples (FeSOÄA593nm was the absorbance of the sample minus the absorbance of the blank at the 4th minute.where The samples were the rhizome extracts of ethyl acetate and ethanol (500 mg/mL). Trace DSQ GC-MS from American Finnigan Company was used. The GC conditions were: 1 N NOWAX quartz capillary column: 30 m × 0.32 cm × 0.25 mm; column temperature: 50–190°C; procedure temperature: 5°C/min; carrier gas: He; vaporizer temperature: 280°C; the MS conditions were: Ion source: EI; temperature: 200°C; ionizing voltage: 70 eV; electric current of collection: 300 μA; electric current of emission: 1 mA; resolution: 600; mass: 10–600.50 (μM) and FRAP (μM) and the test methods were mentioned above (as the DPPH and FRAP assays).Reagents PADE, BADE, Polyneuridine, PODD, β-Sitosterol, POD, and including the positive control ASA were dissolved in 15% aqueous ethanol and tested at the same concentration of 500 μM. Antioxidant capacity was expressed ICThe results were presented as mean ± SD of triplicate determinations. Statistical analysis was performed by SPSS 11.5. One-way analysis of variance (ANOVA) was utilized to evaluate differences.P. hexandrum expressed as I % are showed in Figure p < 0.01).FRSAs from the rhizome and petiole extracts of P. hexandrum rhizome, the extracts of the ethyl acetate and ethanol were separated and identified by GC-MS. In total, About 16 kinds of reactive oxygen were identified. The compound retain time (RT), name, molecular formula, molecular weight, area and percentage of area (POA) were listed in Table In order to further study the biochemical compositions in The ethyl acetate and ethanol extracts contained five common components: PADE, BADE, PODD, β-Sitosterol, POD, and the POA of the five components in the ethyl acetate and ethanol extracts were 2.77 and 1.23, 2.25 and 7.05, 20.59 and 20.97, 1.61 and 1.22, 67.41 and 22.73%, respectively. The Polyneuridine also showed the high POA with 28.01% in the ethanol extract.50 and FRAP values of PADE, BADE, Polyneuridine, PODD, β-Sitosterol, and POD were 22.49 ± 0.60 and 1062.5 ± 23.39 μM, 16.32 ± 0.67 and 1025.00 ± 9.24 μM, 13.37 ± 0.35 and 2404.32 ± 36.88 μM, 9.61 ± 0.81 and 2923.98 ± 21.89 μM, 32.43 ± 0.81 and 891.37 ± 22.14 μM, 9.98 ± 0.24 and 2847.27 ± 14.82 μM, respectively. Both of the tested statistics of IC50 and FRAP values showed that PODD, POD and Polyneuridine had greater antioxidant capacity than the positive control ASA (60.78 ± 1.22 and 1267.5 ± 30.24 μM) (p < 0.01).Figure Podophyllum contained 4–20% of podophyllum resin, which is the source of POD [et al. [P.hexandrum that contains 6–12% of resin, of which the concentration of podophyllotoxin is around 40%. Chawla et al. [P.hexandrum rhizome, which revealed several bioactivities of direct relevance to radioprotection.e of POD ,25. Giri [et al.  reporteda et al.  reportedet al. [et al. [et al. [et al. [et al. [Eclipta prostrate by GC-MS. The six components PADE, BADE, Polyneuridine, PODD, β-Sitosterol, and POD presented high POA (pereentage of area) in the extracts, and the highest POAs of PODD, POD and Polyneuridine were 20.97, 67.41 and 28.01% in the rhizome, respectively, these results suggest that the components PODD, POD and Polyneuridine are very critical for the antioxidant capacity.In this work, about 16 kinds of reactive oxygen components were identified by GC-MS, some of which have been preliminarily studied for antioxidant capacity or identified from other plants. Zhang et al.  and Jin  [et al.  reported [et al.  revealed [et al. . Zhao et [et al.  identifi [et al.  isolatedAlthough the POAs of other components in the ethyl acetate and ethanol extracts are relatively small, antioxidant capacity was also reported in other plant extracts, such as D-allose , DibutylP.hexandrum, this was the first to report PADE and BADE from a plant product and further study the antioxidant capacity of the identified components PADE, BADE, Polyneuridine, PODD, β-Sitosterol, and POD. The results of tested data and statistical analysis proved that the extracts from P. hexandrum had strong antioxidant capacity, and the PODD, POD and Polyneuridine played an important role. Based on the above data it can be recommended as an alternative plant material of antioxidant and radical scavenging activity.The rhizome extracts had greater antioxidant capacity than the petiole extracts in DPPH and FRAP assays. About 16 kinds of reactive oxygen components were identified by GC-MS in the extracts of The authors declare that they have no competing interests.MFL designated the study, performed the statistical analysis and drafted the manuscript. LLZ and TTL participated in the extraction and the antioxidant assays. DLY did the isolation and structure elucidation part and helped in manuscript editing. WL has made substantial contribution to conception and design, 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/1472-6882/12/263/prepub


Stryphnodendron adstringens has a high tannin content and is used as an antiseptic and antimicrobial and in the treatment of leucorrhea, gonorrhea, wound healing, and gastritis. The present study evaluated the toxic effects of the heptamer prodelphinidin (F2) from the stem bark of S. adstringens in rodents. In the acute toxicity test, the mice that received oral doses exhibited reversible effects, with an LD50 of 3.015 mg · kg−1. In the chronic toxicity test at 90 days, Wistar rats were treated with different doses of F2 . In the biochemical, hematological, and histopathological examinations and open-field test, the different dose groups did not exhibit significant differences compared with controls. The present results indicate that F2 from the stem bark of S. adstringens caused no toxicity with acute and chronic oral treatment in rodents at the doses administered. Stryphnodendron sp belongs to the family Fabaceae (native savanna), approximately 48 species of which have been identified, including Stryphnodendron adstringens (Mart.) Coville (known as “barbatimão”). This species is found in the central savannah region of Brazil [The bioactivity of various compounds in medicinal plants has been assessed. These compounds are isolated and analyzed to determine biological activity, mechanisms of action, and toxicity , 2. The f Brazil –5. This f Brazil –9. Scienf Brazil , 10–18.S. adstringens is rich in proanthocyanidin polymers, including several flavan-3-ols, such as prodelphinidins and prorobinetinidins [The bark of tinidins –22. The tinidins .S. adstringens was the subject of a study by Rebecca et al. [S. adstringens had no genotoxic effects in Drosophila melanogaster or micronuclei (bone marrow) and Artemia salina tests in mice, respectively.The toxicity of a et al. , 24. Thea et al. , 26. Stua et al.  and Costa et al.  showed tS. adstringens, the present study sought to obtain more information about toxicity at therapeutic doses.Thus, considering the wide use of this plant and that few studies have been conducted to determine the toxicological profile of Stryphnodendron adstringens was collected in São Jerônimo da Serra, Paraná, Brazil , in March 2008. A voucher herbarium specimen was deposited at the Universidade Estadual de Maringá (no. HUM 14321).Stem bark from The bark was dried at room temperature and then pulverized. The crude extract was obtained by turboextraction of the bark at 1,000 ×g with 70% acetone in water for 15 min. The organic solvent was eliminated by rotavapor and lyophilized to yield a crude extract . F1 (50 g) was suspended in water (500 mL) and partitioned with ethyl acetate  to obtain a proanthocyanidin polymer-rich fraction .Adult Wistar rats (90 days old), weighing 230–240 g  and 355–365 g , and Swiss mice (60 days old), weighing 35–45 g  and 45–60 g , were used and housed in groups of five per cage, with food and water freely available. The animals were maintained on a 12 h/12 h light/dark cycle under controlled temperature (22 ± 1°C). The protocol was approved by the Ethical Committee of the State University of Maringá .The toxicity studies were performed according to the Brazilian National Health Surveillance Agency (ANVISA) –32.−1). One group that received distilled water was included as a negative control. The volume administered by gavage in the mice was approximately 0.3 mL per animal. Water and food were freely available to the animals. The general behavior and number of survivors were observed at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, and 24 h and daily thereafter until day 14. Toxicological effects were assessed, including changes in locomotion, respiration, piloerection, diarrhea, drooling, altered muscle tone, hypnosis, convulsions, hyperexcitability, writhing , and mortality  [Swiss mice were divided into seven groups, with 10 animals per group . Six groups were orally treated by gavage with different doses of F2  . From thAt the end of this period, all of the animals were sacrificed. The kidneys, heart, lungs, spleen, and liver were removed, weighed, and evaluated for macroscopic abnormalities. When changes were observed in the autopsies, further histological examination of the organs was performed.S. adstringens  suspended in water and administered orally by gavage daily for 90 days. The volume administered by gavage in the rats was approximately 0.5 mL per animal. The choice of the doses was based on the estimated oral dose in popular use (10 mg·kg−1) [Wistar rats were divided into four groups . One group served as the control and received only water. The other groups received F2 of mg·kg−1)  and 10- mg·kg−1) , 30.ad libitum during the treatment. They were observed daily with regard to behavior and weighed weekly. At the end of the 90-day period, the animals were deprived of food for 15 h and then sacrificed. Their blood was collected for biochemical and hematological examination. The organs were carefully dissected and removed for weighing, macroscopic examination, and histopathological analysis.All of the animals received food and water Blood Analysis. Biochemical analyses were performed to determine glucose, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein, creatinine, uric acid, blood urea nitrogen (BUN), triglycerides, total cholesterol, γ-glutamyltransferase (γ-GT), bilirubin, sodium, and potassium. These were evaluated using the Dimension RXL Max system (Siemens).Hematological analyses were performed using the automatic counter Pentra 60 ABX (ABX Diagnostics) to evaluate the following parameters: erythrocyte count (red blood cells (RBCs)), hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count, and leucocyte count (white blood cells (WBCs)). Differential WBC counts  were made using a glass-slide method. Immediately after collection, blood smears were air-dried and stained with Leishman's stain. One hundred cells were randomly counted in each smear, and the percentage of each type was calculated. Any morphological change in the blood cells was noted.Organ Weights and Histopathological Analyses. The organs  of all of the animals were examined macroscopically. The positions, shapes, sizes, and colors of the internal organs were visually observed for signs of macroscopic abnormalities. The organs were weighed and fixed in Bouin's fixative and preserved in 70% ethanol. For the 200 mg·kg−1 dose of F2 and controls, tissue slides were prepared and stained with hematoxylin and eosin for microscopic examination.Open-Field Test (OFT). Locomotor behavior was assessed on day 86 of treatment in the open-field test. Each animal was placed in a round wooden arena (70 cm diameter) with 30 cm high walls. Luminosity at the center of the open field was 60 lux during a 5-min period. Rearing, self-cleaning, urination, the number of fecal pellets, and locomotion were recorded [recorded . For thepost hoc test. When the variance was not constant, the nonparametric Kruskal-Wallis test was performed to determine significant differences. The histopathological results were analyzed using Fisher's exact test. Differences were considered significant at P ≤ 0.05.The results are expressed as mean ± standard deviation (SD). The data were analyzed using Statistica 8.0 software. The statistical analyses were performed using one-way analysis of variance (ANOVA) followed by the Dunnett −1 showed no signs of toxicity. Only the 2,000 mg·kg−1 dose caused signs of toxicity, beginning with diarrhea and piloerection. At doses of 3,000–5,000 mg·kg−1, hypoactivity, hyperventilation, ptosis, hypothermia, motor impairment, sedation, and catatonia were observed, all of which were reversible after 48 h. Death occurred in the groups that received 2,000–5,000 mg·kg−1, resulting in an LD50 of 3,015 mg·kg−1. All of the groups had lower body weights in the first 24 h. After 7 days, however, body weights recovered , uric acid , and AST (186.0 ± 47.0 U·L−1 at 100 mg·kg−1). Other parameters were significantly decreased, including triglyceride (85.5 ± 28.3 mg·dL−1 at 200 mg·kg−1) and creatinine (0.4 ± 0.1 mg·dL−1 at 100 mg·kg−1). In female rats, only triglycerides  were significantly decreased compared with the control group.The effects of F2 of  changes . In maleS. adstringens on hematological parameters showed specific changes but only in male rats , decreased MCHC (35.1 ± 0.3% at 100 mg·kg−1), and increased platelet counts . No significant differences were observed in female rats compared with the control group.The effects of F2 of ale rats . SignifiNo macroscopic alterations, differences in relative body weight, or histopathological parameters were observed compared with controls.Locomotion , rearingS. adstringens without considering possible toxicity or efficacy can lead to serious health risks. The proanthocyanidin polymer-rich fraction (heptamer compound) of the stem bark of S. adstringens (F2) offers possible advantages, including antifungal activity against Candida spp., especially C. albicans [ The indiscriminate use of albicans . Vulvovaalbicans –38.S. adstringens is low  confirm its low acute toxicity that has been indicated by other parameters [50 of 2,700 mg·kg−1 of the crude extract with acute treatment, demonstrating that the aqueous fraction has less toxicity. The high LD50 values indicate that the extract can be administered with a high degree of safety. The high LDrameters . Moreoverameters  found an−1, significant differences in weight gain were observed.In the repeated-dose oral toxicity experiment, no significant changes in behavior or mortality were observed in rats. According to Raza et al. , changesAccording to Lewis et al. , “some fIn the present study, the biochemical results varied widely between doses and between sexes, with no linear profile. The altered values were within normal limits, and the results are considered normal for this animal species. Other values that changed did not show a dose response , 43.−1, triglycerides at 200 mg·kg−1, and uric acid at 100 and 200 mg·kg−1 significantly changed. In female rats, only triglycerides at 100 and 200 mg·kg−1 significantly changed compared with the control group. To evaluate kidney function, creatinine, urea, sodium, potassium, and uric acid were assessed [−1) [In male rats, potassium, AST, and creatinine at 100 mg·kgassessed . Urea ansed [−1) . The samγ-GT were assessed because these are considered markers of liver function, and liver changes have been reported after phytotherapeutic product use [To evaluate liver function, AST, ALT, ALP, total bilirubin, and duct use –48. In tduct use . HoweverThe levels of total protein, glucose, cholesterol, and triglycerides were measured to assess the general biochemical profile of the animals and determine the presence of metabolic changes. The values were not significantly different between the control and treatment groups in both female and male rats, with the exception of triglycerides, which appeared to be reduced in both females and males but remained within the normal range . High do−1), MCHC (100 mg·kg−1), and platelet counts (10 and 100 mg·kg−1) were observed in males. However, the changes in MCHC and platelets did not show a dose-response relationship, and the changes in MCH, despite being evident at the three doses tested, remained within the normal range (19.0 ± 1.09) [In the present study, hematological changes did not appear to be related to treatment with F2. No changes were observed in females, but changes in MCH  . These r ± 1.09) . This re ± 1.09) .The relative weights of the organs did not show significant changes in the macroscopic and histopathological examinations in the treatment groups compared with controls in either sex. In contrast to the study by Rebecca et al. , which fAnother interesting result was found in the open-field test, in which none of the parameters showed significant changes, indicating that F2 did not exert neurobehavioral alterations in the animals. S. adstringens, reflected by high LD50 values, suggests a wide safety margin at therapeutic doses. In the repeated-dose oral toxicity study, no serious signs or significant changes in hematological, biochemical, and histopathological parameters or other remarkable effects were observed in rats. These toxicity studies suggest that the fraction is safe at the doses administered. However, further studies are needed to evaluate other parameters, including carcinogenicity, teratogenicity, and neurotoxicity. Future clinical pharmacology studies should also be conducted to determine tolerance and substantiate its pharmacological use.The low toxicity of F2 from the stem bark ofS. adstringens in the acute and repeated-dose oral (chronic) toxicity studies suggests that F2 obtained from the stem bark of S. adstringens is safe at the concentrations tested.The low toxicity of F2 obtained from stem bark of


Changes to Treatment and Outcomes in Patients with Type 2 Diabetes Initiating Injectable Therapy (CHOICE) is a European prospective, observational cohort study assessing time to, and factors associated with, a significant change in therapy after type 2 diabetes patients initiate their first injectable glucose-lowering therapy, and these patients’ clinical outcomes over 24 months. The authors report baseline data and factors associated with the injectable treatment regimen.Demographic, clinical, and healthcare resource-use data were collected at initiation of injectable therapy and analyzed using univariate tests between cohorts and multivariate logistic regression analysis for treatment.2 higher: odds ratio (OR) 2.10, 95% confidence intervals (CI) 1.84–2.40], lower glycated hemoglobin , and lower age  were the variables most strongly associated with increased probability of receiving exenatide b.i.d. (P < 0.0001). Patients initiating exenatide b.i.d. had a mean BMI of 35.3 ± 6.5 kg/m2, HbA1c of 8.4 ± 1.4%, and age of 58 ± 10 years, compared with 29.7 ± 5.4 kg/m2, 9.2 ± 1.9%, and 64 ± 11 years, respectively, in patients initiating insulin (P < 0.0001). Other characteristics significantly associated with exenatide b.i.d. initiation were “disinhibited eating” , lower random blood glucose, less blood glucose self-monitoring, lower low-density lipoprotein cholesterol, and receipt of diet/exercise advice.Overall, 1,177 patients initiated exenatide twice daily (b.i.d.) and 1,315 initiated insulin. Most patients were recruited by secondary-care physicians. Univariate analyses revealed statistically significant differences between the characteristics of patients who initiated exenatide b.i.d. and patients who initiated insulin. On multivariate analysis, higher body mass index [BMI; 5 kg/m1c than those initiating insulin.Patients who initiated exenatide b.i.d. were on average younger and more obese with lower HbA During the enrollment period for the CHanges to Treatment and Outcomes in Patients with Type 2 Diabetes Initiating Injectable Therapy (CHOICE) study, exenatide twice daily (b.i.d.), the first approved glucagon-like peptide-1 (GLP-1) receptor agonist, was available in Europe for use in combination with metformin and/or a sulfonylurea in patients with type 2 diabetes mellitus (T2DM) with insufficient glycemic control on maximal doses of these medications. Indications for exenatide b.i.d. in Europe have subsequently been expanded to include use in combination with thiazolidinediones (in 2010) and as adjuvant therapy with basal insulin, with or without metformin and/or pioglitazone, in patients who had not achieved adequate glycemic control with these agents (in 2012). In head-to-head phase 3 clinical trials, exenatide b.i.d. and insulin (glargine and biphasic insulin aspart) provided similar glycemic control in patients whose diabetes was not controlled with oral antidiabetic medications (OADs). Exenatide b.i.d. treatment was associated with weight loss, while patients randomized to insulin typically gained weight .While randomized, controlled trials are the reference standard for assessing the efficacy and safety of therapy, large, observational studies are necessary to determine how glucose-lowering medications are used in clinical practice and to evaluate their effectiveness and safety in this setting , 6. The CHOICE is the first European observational study conducted specifically to address this lack of evidence. CHOICE is an ongoing, prospective cohort study designed to assess the time to a significant subsequent change in therapy among patients who initiate their first injectable glucose-lowering therapy with either exenatide b.i.d. or insulin (the only injectable treatments available when this study commenced) in Europe. The study also aimed to describe the characteristics of patients with T2DM initiating injectable therapy, factors associated with treatment changes, and clinical outcomes and common adverse events observed over 24 months. CHOICE is being conducted in six European countries and will be completed in 2012. This paper reports the baseline characteristics of enrolled patients and measured variables associated with injectable treatment regimen.CHOICE is a prospective, multinational, non-interventional observational study being conducted in Belgium (31 sites), Denmark (eight sites), France (71 sites), Germany (130 sites), Greece (49 sites), and Sweden (33 sites). The primary endpoint is time spent on initial injectable regimen (exenatide b.i.d. or insulin) before significant treatment change. Significant treatment change is defined as at least one of the following (and does not include switching between brands of the same class/type of insulin): addition of a new medication (any route of administration) for treatment of T2DM; a change in the number of times insulin is administered per day; discontinuation of any exenatide b.i.d./insulin initiated at baseline; substitution of a human insulin for an analog insulin or vice versa.Secondary objectives include assessment of characteristics of patients initiating each treatment, factors associated with injectable treatment regimen, and clinical and patient-reported outcomes.Eligible for inclusion were adults aged ≥18 years initiating their first injectable glucose-lowering therapy with exenatide b.i.d. or insulin for the treatment of T2DM in routine clinical practice. At study entry patients could be taking any OADs. Patients were initiated on either exenatide b.i.d. or insulin according to clinical decision making, and were then informed about CHOICE and invited to participate. Patients gave written informed consent for the use of their data. Appropriate ethical review board approval was obtained for this study.Patients have been assessed at study visits at the time of initiation of injectable therapy  and approximately 3, 6, 12, 18, and 24 months thereafter, as per routine care. Patients referred from the study site to another healthcare provider during the study have been followed up by contacting, and obtaining the consent of, the new provider and by postal patient questionnaires. Interim analyses are planned at baseline, 6, and 12 months, with the final analysis after the 24-month visit.1c; at initiation and over previous 2 years), weight, blood pressure, lipid levels, and diabetes complications; retrospectively recalled incidence of self-reported hypoglycemic episodes (over the preceding 3 months) and gastrointestinal symptoms (over the preceding 4 weeks); previous and ongoing diabetes therapy and care; concomitant medications; and patient-reported outcome measures of health status and functioning, including the Diabetes Health Profile (DHP)-18 instrument .2 and HbA1c of 8.9 ± 1.7%. Mean duration of diagnosed diabetes was 9 ± 7 years. Univariate analyses revealed statistically significant differences between patients whom clinicians initiated on exenatide b.i.d. and starter insulin regimens . This trend was consistent across all countries except Denmark, wherein the cohorts did not differ significantly in age . Patients initiating exenatide b.i.d. had significantly higher mean body weight  and mean BMI  than those initiating insulin . This was consistent across all countries except Belgium (wherein the durations were similar) and was statistically significant in France  and Greece . Injectable therapy was initiated following an increase in HbA1c during the previous 9–12 months, a trend consistent across countries. This increase in HbA1c prior to initiation of injectable therapy was more pronounced in patients initiating insulin rather than exenatide b.i.d. At the point of initiation, patients who initiated exenatide b.i.d. had a significantly lower mean HbA1c than those who initiated insulin . This was consistent across countries  and 13.6% (Denmark) for exenatide b.i.d.  and between 0% (Denmark) and 6.4% (France) for insulin . Patients who initiated exenatide b.i.d. also had significantly lower random blood glucose values than those who initiated insulin  or microvascular complication , compared with patients initiating insulin. Differences in the prevalence of macrovascular complications at the time of initiation of injectable therapy were particularly notable in Germany , Greece , and Sweden . Differences in the frequency of microvascular complications were especially notable in France  and Greece .Significantly fewer patients initiating exenatide b.i.d. had reported one or more macrovascular complication , i.e., a diagnosis of hypertension  and hyperlipidemia . Overall, 85.5% of patients were using at least one concomitant (nondiabetes) medication  at initiation of injectable therapy and this was consistent across countries. There were no significant differences between cohorts in the use of cardiovascular, lipid-lowering, or antiplatelet agents (Table P < 0.0001).Patients initiating exenatide b.i.d. had a statistically higher incidence of comorbidities than patients initiating insulin P = 0.004, i.e., Overall, 79.4% of patients self-monitored blood glucose, using a mean of 9.6 ± 8.3 test strips/week. There were no statistically significant differences between the cohorts in the number of contacts with healthcare professionals in the 6 months prior to baseline Table .1c as the variables most strongly associated with a propensity for treatment assignment, with higher BMI, lower age, and lower HbA1c indicating an increased probability of receiving exenatide b.i.d. (Table 2 higher) 2.10, 95% CI 1.84–2.40; HbA1c (1% higher): OR 0.77, 95% CI 0.69–0.86; age (5 years older) OR 0.82, 95% CI 0.76–0.88; all P < 0.0001). Other statistically significant factors associated with the treatment regimen were the “Disinhibited Eating Subscale” of the DHP-18  identified BMI, age, and HbAd. Table ; [odds rd. Table  higher 2A propensity score analysis based on baseline demographic and clinical variables underlined the differences between the treatment cohorts. The mean propensity score value for the probability of receiving exenatide b.i.d. was 0.63 ± 0.23, and of receiving insulin was 0.33 ± 0.23  studies are essential to enhancing the evidence upon which the management of T2DM is based . The CHO1c), and duration of diabetes. The differences between the two cohorts in key variables such as age, obesity measures, and HbA1c were consistent across the participating countries, although the statistical significance of some inter-cohort differences was limited by the sample size in individual countries. Overall BMI, HbA1c, and age were factors significantly associated with differentiation between exenatide b.i.d. and insulin (P < 0.0001).Overall, patients initiating exenatide b.i.d. were characterized by: younger age; higher body weight, BMI, and waist circumference; higher diastolic blood pressure, lower total and LDL cholesterol levels; shorter time since diabetes diagnosis; and better glycemic control. A lower proportion of patients initiating exenatide b.i.d. had microvascular and macrovascular complications (compared with patients initiating insulin), a finding that might reflect their younger mean age, glycemic control /European Association for the Study of Diabetes (EASD) consensus statement for T2DM management specifies exenatide b.i.d. as an option when hypoglycemia is a particularly important consideration [The risk of treatment-induced hypoglycemia is an important consideration during treatment selection –3, especderation . ConsistThe finding that exenatide b.i.d. was favored in patients with higher body weight is not surprising. Patients treated with exenatide b.i.d. show significant weight loss compared with those treated with insulin –3. The A1c level in both cohorts in CHOICE exceeded the recommended target level of <7% [1c levels rose gradually during the 18–24 months prior to baseline. A steeper rise occurred in the 6 months prior to baseline and this appeared more marked in the insulin cohort (reaching 9.2%) than in the exenatide b.i.d. cohort (reaching 8.4%). Although this finding may reflect different disease progression in the two cohorts prior to the initiation of either exenatide b.i.d. or insulin, missing pre-baseline HbA1c records in some patients make interpretation of this phenomenon difficult. These findings are consistent with previous observational evidence that insulin initiation is very often delayed for years despite poor glycemic control on oral glucose-lowering medications [The mean HbAreaching .2% than ications –15.1c levels than those initiating insulin is consistent with US observational data [1c including high values (>9%) and not only where HbA1c is close to target levels [1c of <7%.The finding that patients initiating exenatide b.i.d. in CHOICE had lower HbAnal data  and withnal data . Howevert levels –19. The 1c and weight. Patients who initiated insulin had significantly higher creatinine levels than those who initiated exenatide b.i.d. This could reflect the higher mean age in the insulin cohort. It is also possible that renal complications, as well as other complications, might have contributed to the initiation of insulin. The lack of data on glomerular filtration rate hampers interpretation of the creatinine data.The observation  of a higher proportion of diagnosed dyslipidemia or hypertension inpatients initiating exenatide b.i.d. is of unclear significance but may be related to body weight. Similar findings were observed in the aforementioned retrospective database analysis in the United States . In commUnivariate analyses showed that patients receiving exenatide b.i.d. were more likely than insulin recipients to be employed. Primary occupational status was correlated with age, and only age showed a significant association with treatment selection in the multivariate analysis. The higher educational status in the exenatide b.i.d. cohort compared with the insulin cohort on univariate analysis may reflect the possibility that more educated patients are more likely to ask for newer treatments, but this is speculation. Educational status was not included in the multivariate analysis owing to a relatively high proportion of missing data.1c at the initiation of insulin (9.6% vs. 9.2% in CHOICE). The small sample size in some countries reduces the statistical power of the inter-cohort analysis. The variation in sample sizes between the six countries also limits the robustness of inter-country comparisons, although the inter-cohort comparisons nevertheless showed a considerable degree of international consistency. There was also variation between countries in the ratios of exenatide b.i.d. and insulin patients.The CHOICE study has several limitations. While the data allow a multivariate analysis to help improve the understanding of the measured variables most strongly associated with the choice of injectable therapy, other variables that were not captured, such as clinical guidelines and patient preference, may be at least as clinically relevant. As significant differences between the cohorts are present, outcome results observed in these groups are not directly comparable. The matched population refers to those patients in whom outcomes can be statistically compared. However, this largely represents the upper tail of the exenatide b.i.d. propensity score distribution and the lower tail of the insulin distribution. Therefore, caution is advised when interpreting comparative outcomes. Although CHOICE was designed to recruit a representative sample of patients, the degree to which the data can be generalized is unclear for several reasons. Firstly, patients were mostly recruited in secondary-care centers and hence the data may not be generalizable to the primary-care setting. Secondly, the similarity in the size of the exenatide b.i.d. and insulin cohorts does not reflect the ratio in routine clinical practice, wherein many more patients initiate insulin than exenatide b.i.d. The CHOICE population appears similar to that of the observational “Insulin titration; gaining an understanding of type 2 diabetes in Europe” (INSTIGATE) study , which l1c than those who initiated insulin. These differences appear to reflect the recommendations for the use of these two therapies, with exenatide b.i.d. identified as an option when weight gain is a particular concern and when HbA1c is modestly raised [In conclusion, this analysis has improved understanding of which patients are initiated on exenatide b.i.d. or insulin in routine clinical practice. The cohort of patients who initiated exenatide b.i.d. in CHOICE was younger, more obese, and had a lower HbAy raised , 12. The


Montelukast is a leukotriene receptor antagonist for treatment of asthma, gliclazide is an oral hypoglycemic antidiabetic agent, and nifedipine is a calcium channel blocker used for treatment of angina pectoris and hypertension. These drugs may be prescribed to patients suffering from these chronic diseases. A survey of the literature reveals that there is no reported method for the simultaneous determination of montelukast, gliclazide, and nifedipine in pharmaceutical preparations or biological fluids.18 (50 × 4.6 mm) 1.8 μm particle size column. The mobile phase consisted of acetonitrile: 0.1% formic acid (84:16). The three drugs were simultaneously extracted from plasma by protein precipitation with acetonitrile using zaferolukast as an internal standard. The method was validated according to FDA guidelines with good reproducibility and linearity of 0.999 and the limits of quantification were 0.11, 0.04, and 0.07 ng/mL for montelukast, gliclazide, and nifedipine, respectively. The accuracies of the three QCs for the three drugs were 99.48% (montelukast), 106.53% (gliclazide), and 108.03% (nifedipine) in human plasma. The validated method was applied to a pharmacokinetic study in human volunteers after oral administration of the three drugs. The applied LC/MS/MS method was shown to be sufficiently sensitive and suitable for pharmacokinetic studies.A simple, sensitive, and rapid method for the simultaneous quantification of montelukast, gliclazide, and nifedipine in human plasma was developed and validated. Montelukast, gliclazide, and nifedipine were resolved using rapid resolution LC/MS/MS Agilent system and SB-CThe LC/MS/MS method was validated and successfully applied for the determination of montelukast, gliclazide, and nifedipine concentrations in human plasma. Montelukast, gliclazide, and nifedipine are drugs used for the management of chronic asthma and as prophylactic agents for exercise-induced asthma, treatment of non-insulin-dependent diabetes mellitus, and treatment of angina pectoris and hypertension, respectively.This study aimed to determine serum concentrations of these three drugs, which are often jointly prescribed to patients with chronic diseases such as asthma, hyperglycemia, and hypertension.N-(4-methylbenzenesulfonyl)-N- (3-azabicyclo- [3.3.0] oct-3-) urea , 1-[({(R)-m-[(E)-2-(7-chloro-2-quinolyl)-vinyl]-α-[o-(1-hydroxy-1-methylethyl) phenethyl]-benzyl}thio)methyl]cyclopropane acetate  and zaferolukast (ZA) (I.S.) (99.3% potency) were kindly supplied by Merck. GL and NI standard material  was kindly supplied by Sigma-Aldrich Co. (UK). Singulair 10 mg tablets (MO) (Merk&Co. Inc-USA), Diamicron 80 mg tablets (GL) (Servier), and Epilat retard 20 mg tablets (NI) by EPICO, Egypt.Formic acid was purchased from Romil chemicals, England) and acetonitrile and methanol (HPLC grade) were purchased from, Alpha Chemicals, Egypt). Deionized water was obtained from a Milli-Q water purification system 3 , and human plasma was supplied by VACSERA, Egypt). The mobile phase was filtered through a 0.45-μm Whatman membrane filter.An Agilent triple quadrupole mass spectrometer with an API source coupled with an Agilent pump controlled by an Agilent 1200 controller and equipped with an Agilent 1200 autosampler injector was used for analysis. Separation was performed using an analytical Agilent SB-C18 column (50 × 4.6 mm) with particle size of 1.8 μm. The mobile phase consisting of acetonitrile: 0.1% formic acid (84:16), was delivered at a flow rate of 0.6 mL/min.2) least square calibration curves in which the peak area ratios of the calibration standards were plotted versus their concentrations.Mass spectra were obtained using an electrospray ionization source operated in the multiple reaction monitoring (MRM) mode. Sample introduction and ionization were both performed in the positive ion mode  and negative ion mode (ZA). The cone voltage was set at 135 V for both MO and GL and 80 V and 150 V for NI and ZA, respectively. The capillary voltage was optimized at 4000 V. Argon was used as the collision gas. The collision energy was set at 20, 15, 5, and 25 MeV for MO, GL, NI, and ZA, respectively. Optimal gas flow during tuning was 8 L/min and nebulizer pressure was 30 psi. The source temperature was 325°C. The selected mass transitions ion pairs were 586.2/568.3, 324.4/127.2, 347.3/315.2 at (positive ion) and 574.6/462.2 at (negative ion) for MO, GL, NI, and ZA (I.S.), respectively. Agilent Mass Hunter software was used for data acquisition. For quantification, the peak area ratios of the target ions of the drugs to those of the internal standard were compared with weighted  samples were prepared in methanol at a concentration of 100.0 μg/mL and stored at 4°C. Handling and analysis of all samples were performed under diffused light conditions . Evaluations of the assay were performed by seven point calibration curves made by serial dilutions of the stock solution of each drug at the nominal concentration ranges of 10.0–800.0 ng/mL, 10.0–5000.0 ng/mL, and 10.0–600.0 ng/mL for MO, GL, and NI, respectively, in human plasma. The slopes and intercepts of the calibration lines were determined.Sample preparation was performed by liquid–liquid extraction of 100 μL of plasma by protein precipitation using 300 μL of acetonitrile. A 5.0-μL aliquot of the internal standard solution was added. The mixture was then shaked by vortex for 30 s and centrifuged for 10 min at 4000 rpm. A 20 μL aliquot of the supernatant was injected into the LC/MS/MS instrument.This method was applied for the analysis of plasma samples after the administration of a single dose of 10 mg MO (Singulair tablets), 80 mg GL (Diamicron tablets), and 20 mg NI (Epilat retard tablets) to six healthy male volunteers. The study protocol was approved by the Ethics Committee of Bioavailability Studies (NODCAR). The age of the volunteers ranged from 29 to 39 years and the body weights of the subjects ranged from 58 to 71 kg. All subjects provided written informed consent. The study was conducted in accordance with the provisions of the Declaration of Helsinki. After an overnight fast for 10 h, all volunteers received a single dose of the three drugs with 200 mL of water. Blood samples (3 mL) from a suitable antecubital vein were collected into heparin-coated tubes at 0.0 (before dose), 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3, 4, 6.0, 8.0, 10.0, 12.0, 24.0, and 48.0 h post dosing. The blood samples were centrifuged at 3000 rpm for 5 min at room temperature and the plasma was removed and stored at −80°C until assayed for MO, GL, and NI content. All samples from a single volunteer were analyzed in the same run in order to avoid inter-assay variations.el) was estimated by linear regression from the points describing the elimination phase on a log-linear plot. The maximum observed plasma concentration (Cmax) and the time taken to achieve this maximum level (tmax) were directly obtained from the curves.Pharmacokinetic parameters from the human plasma samples were calculated by a noncompartmental model using the WinNonlin 5.3 software. Blood samples were taken for a period of 48 h. and it was considered as the area under the concentration–time curve (AUC) ratios were higher than 80% as per FDA guidelines ,56. The 0–48) were calculated using the trapezoidal method. Extrapolation of this area to infinity (AUC0–∞) was performed by adding the value C48/Kel to the calculated AUC0–48 where C48 is the MO, GL, and NI plasma concentration at 48 h. Kel is the first-order terminal elimination rate constant.The areas under the time-concentration curve for MO, GL, and NI plasma concentration versus time for 48 h  was evaluated to obtain a better response from the analytes. The best signal was achieved using an ESI-positive ion mode for MO, GL, and NI, and negative ion mode for ZA. The product ion spectrum for MO, GL, NI, and ZA yielded a high abundance of fragment ions of m/z 568.3, 127.2, 315.2, and 462.2, respectively  and limit of quantitation (LOQ) were calculated according to the ICH guidelines for validation of analytical procedures based on the standard deviation of the response and the slope of the calibration curve  using thThe accuracy and precision of the proposed method were determined by intra-day and inter-day replicate analyses of plasma spiked with different concentrations of each drug covering the working linear ranges. The inter-day assays were carried out on three different days with the same concentration levels for spiked plasma samples. The accuracy values from the intra-day analysis were 94.59% –105.58%, 89.75% −114.86%, and 83.46%–110.22%, whereas the values for the inter-day analysis were 93.11%, −109.32%, 99.93%–106.16%, and 97.90%–114.82% for MO (Table The method described here was successfully applied to a pharmacokinetic study of MO, GL, and NI in plasma samples from healthy human volunteers. None of the volunteers exhibited any general adverse reactions. Following oral drug administration, plasma concentration–time profiles of the three drugs best fit a non-compartment in all subjects. A representative MRM chromatogram of a plasma sample extracted from a healthy volunteer at 6 h is shown in Figure The results of this study showed that the validated LC/MS/MS method proved to be a simple, rapid, reliable, selective, and sensitive method sufficient for simultaneous monitoring of pharmacokinetic parameters of montelukast, gliclazide, and nifedipine. A small plasma sample volume and LOQ were sufficiently sensitive to detect terminal phase concentrations of the drugs.MO: Montelukast sodium; GL: Gliclazide; NI: Nifedipine; LC/MS/MS: Liquid chromatography/Mass/Mass spectrometry; LOD: Limit of detection; LOQ: Limit of quantification; I.S: Internal standard.The authors declare that they have no competing interests.EE proposed the subject, designed the study, conducted pharmacokinetic and statistical analysis, participated in the results discussion and revised manuscript. NFA participated in study design, assay design, literature review, conducted the validation of the assay, analysis of the samples, participated in the results discussion and participated in preparing the manuscript. MHT participated in study design, assay design, literature review, conducted the validation of the assay, analysis of the samples, participated in the results discussion and participated in preparing the manuscript. AAA participated in the writing the manuscript. All authors read and approved the final manuscript