Assessment of the phytochemical constituents and antioxidant activity of a bloom forming microalgae Euglena tuba

Background

Unstable generation of free radicals in the body are responsible for many degenerative diseases. A bloom forming algae Euglena tuba growing abundantly in the aquatic habitats of Cachar district in the state of Assam in North-East India was analysed for its phytochemical contents, antioxidant activity as well as free radical scavenging potentials.

Results

Based on the ability of the extract in ABTS^•+ radical cation inhibition and Fe³⁺ reducing power, the obtained results revealed the prominent antioxidant activity of the algae, with high correlation coefficient of its TEAC values to the respective phenolic and flavonoid contents. The extract had shown its scavenging activity for different free radicals and 41.89 ± 0.41 μg/ml, 5.83 ± 0.07 μg/ml, 278.46 ± 15.02 μg/ml and 223.25 ± 4.19 μg/ml were determined as the IC₅₀ values for hydroxyl, superoxide, nitric oxide and hypochlorous acid respectively, which are lower than that of the corresponding reference standards. The phytochemical analysis also revealed that the phenolics, flavonoids, alkaloids, tannins and carbohydrates are present in adequate amount in the extract which was confirmed by HPLC analysis.

Conclusions

The results showed that 70% methanol extract of the algae possesses excellent antioxidant and free radical scavenging properties.     

A randomised dose-ranging study of tiotropium Respimat? in children with symptomatic asthma despite inhaled corticosteroids

Background

A considerable number of children with asthma remain symptomatic despite treatment with inhaled corticosteroids, resulting in significant morbidity, reduced quality of life, increased healthcare costs and lost school days. The aim of our study was to assess the efficacy, safety and tolerability of once-daily tiotropium Respimat® 5 μg, 2.5 μg and 1.25 μg add-on to medium-dose inhaled corticosteroids, with or without a leukotriene modifier, in children aged 6–11 years with symptomatic asthma.

Methods

In this Phase II, double-blind, placebo-controlled, incomplete-crossover, dose-ranging study, patients were randomised to receive three of the four treatments evaluated: once-daily tiotropium Respimat® 5 μg, 2.5 μg or 1.25 μg or placebo Respimat®, in the evening during the 12-week (three × 4-week) treatment period.

Results

In total, 76, 74, 75 and 76 patients aged 6–11 years received tiotropium Respimat® 5 μg, 2.5 μg, 1.25 μg and placebo Respimat®, respectively. For the primary end point (peak forced expiratory volume in 1 second measured within 3 hours post-dosing), the adjusted mean responses with tiotropium Respimat® 5 μg (272 mL), 2.5 μg (290 mL) and 1.25 μg (261 mL) were significantly greater than with placebo Respimat® (185 mL; p = 0.0002, p < 0.0001 and p = 0.0011, respectively). The safety and tolerability of all doses of tiotropium Respimat® were comparable with those of placebo Respimat®, with no serious adverse events and no events leading to discontinuation.

Conclusions

Tiotropium Respimat® add-on to medium-dose inhaled corticosteroids, with or without a leukotriene modifier, was efficacious in paediatric patients with symptomatic asthma and had comparable safety and tolerability with placebo Respimat®.

Trial registration

ClinicalTrials.gov identifier {"type":"clinical-trial","attrs":{"text":"NCT01383499","term_id":"NCT01383499"}}NCT01383499

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0175-9) contains supplementary material, which is available to authorized users.     

Randomised,double-blind,placebo-controlled crossover study to investigate different dosing regimens of olodaterol delivered via Respimat? in patients with moderate to severe persistent asthma

Background

A Phase II, multicentre, randomised, double-blind, placebo-controlled, crossover trial comparing the 24-h forced expiratory volume in 1 s (FEV₁) time profile after 3 weeks’ treatment with once-daily (QD) or twice-daily (BID) olodaterol (at the same total daily dose) versus placebo delivered via Respimat® in patients with moderate to severe asthma.

Methods

Patients were randomised to different sequences of olodaterol with 2-week washout, either as a total daily dose of 5 μg (5 μg QD [AM] or 2.5 μg BID) or placebo, or 10 μg (10 μg QD [AM] or 5 μg BID) or placebo. Primary end point was FEV₁ area under the curve from 0 to 24 h (AUC_0–24) response (defined as change from study baseline FEV₁) after 3 weeks. Key secondary end points were FEV₁ AUC_0–12 and AUC_12–24 responses.

Results

Two hundred and six patients received treatment. All olodaterol treatments demonstrated statistically significant improvements in FEV₁ AUC_0–24 response at 3 weeks versus placebo (p < 0.0001); adjusted mean treatment difference versus placebo was 0.191 L for olodaterol 2.5 μg BID (95 % confidence interval [CI] 0.152, 0.229), 0.150 L for 5 μg QD (95 % CI 0.111, 0.189), 0.228 L for 5 μg BID (95 % CI 0.190, 0.266) and 0.209 L for 10 μg QD (95 % CI 0.170, 0.247). These results were supported by the key secondary end points. Olodaterol 5 μg QD provided numerically lower mean values for 24-h bronchodilation than olodaterol 2.5 μg BID (p = 0.0465), with no statistically significant difference between treatment with olodaterol 10 μg QD and 5 μg BID. No relevant differences in morning and evening peak expiratory flow or Asthma Control Questionnaire scores at 3 weeks were observed between different doses and regimens. Adverse events were generally mild to moderate and comparable between groups.

Conclusions

All doses and dose frequencies provided adequate 24-h bronchodilation superior to placebo. Based on the results of this study, it would be reasonable to include both posologies of 5 μg olodaterol daily (5 μg QD or 2.5 μg BID, both delivered in two puffs per dose from the Respimat® inhaler) in subsequent studies. Further studies are necessary to confirm the optimum dosing regimen in asthma. No safety concerns were identified.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01311661","term_id":"NCT01311661"}}NCT01311661

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0243-1) contains supplementary material, which is available to authorized users.     

Anti-inflammatory components of the Vietnamese starfish Protoreaster nodosus

Background

In the present study, we examined the inhibitory effects of a methanolic extract, dichloromethane fraction, water layer, and polyhydroxylated sterols (1–4) isolated from the Vietnamese starfish Protoreaster nodosus on pro-inflammatory cytokine (IL-12 p40, IL-6, and TNF-α) production in LPS-stimulated bone marrow-derived dendritic cells (BMDCs) using enzyme-linked immunosorbent assays (ELISA).

Results

The methanolic extract and dichloromethane fraction exerted potent inhibitory effects on the production of all three pro-inflammatory cytokines, with IC₅₀ values ranging from 0.60 ± 0.01 to 26.19 ± 0.64 μg/mL. Four highly pure steroid derivatives (1–4) were isolated from the dichloromethane fraction and water layer of P. nodosus. Potent inhibitory activities were also observed for (25S) 5α-cholestane-3β,4β,6α,7α,8β,15α,16β,26-octol (3) on the production of IL-12 p40 and IL-6 (IC_50s = 3.11 ± 0.08 and 1.35 ± 0.03 μM), and for (25S) 5α-cholestane-3β,6α,8β,15α,16β,26-hexol (1) and (25S) 5α-cholestane-3β,6α,7α,8β,15α,16β,26-heptol (2) on the production of IL-12 p40 (IC_50s = 0.01 ± 0.00 and 1.02 ± 0.01 μM). Moreover, nodososide (4) exhibited moderate inhibitory effects on IL-12 p40 and IL-6 production.

Conclusion

This is the first report of the anti-inflammatory activity from the starfish P. nodosus. The main finding of this study is the identification oxygenated steroid derivatives from P. nodosus with potent anti-inflammatory activities that may be developed as therapeutic agents for inflammatory diseases.     

In vitro antioxidant capacity and free radical scavenging evaluation of active metabolite constituents of Newbouldia laevis ethanolic leaf extract

Background

The aim of the present study was to evaluate the in vitro antioxidant and free radical scavenging capacity of bioactive metabolites present in Newbouldia laevis leaf extract.

Results

Chromatographic and spectrophotometric methods were used in the study and modified where necessary in the study. Bioactivity of the extract was determined at 10 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml and 400 μg/ml concentrations expressed in % inhibition. The yield of the ethanolic leaf extract of N.laevis was 30.3 g (9.93%). Evaluation of bioactive metabolic constituents gave high levels of ascorbic acid (515.53 ± 12 IU/100 g [25.7 mg/100 g]), vitamin E (26.46 ± 1.08 IU/100 g), saponins (6.2 ± 0.10), alkaloids (2.20 ± 0.03), cardiac glycosides(1.48 ± 0.22), amino acids and steroids (8.01 ± 0.04) measured in mg/100 g dry weight; moderate levels of vitamin A (188.28 ± 6.19 IU/100 g), tannins (0.09 ± 0.30), terpenoids (3.42 ± 0.67); low level of flavonoids (1.01 ± 0.34 mg/100 g) and absence of cyanogenic glycosides, carboxylic acids and aldehydes/ketones. The extracts percentage inhibition of DPPH, hydroxyl radical (OH^.), superoxide anion (O₂^.-), iron chelating, nitric oxide radical (NO), peroxynitrite (ONOO⁻), singlet oxygen (¹O₂), hypochlorous acid (HOCl), lipid peroxidation (LPO) and FRAP showed a concentration-dependent antioxidant activity with no significant difference with the controls. Though, IC₅₀ of the extract showed significant difference only in singlet oxygen (¹O₂) and iron chelating activity when compared with the controls.

Conclusions

The extract is a potential source of antioxidants/free radical scavengers having important metabolites which maybe linked to its ethno-medicinal use.     

Potential role of the lectin pathway of complement in the pathogenesis and disease manifestations of systemic sclerosis: a case-control and cohort study

Introduction

Repetitive episodes of ischemia and reperfusion (I/R) are a cardinal feature of the pathogenesis of systemic sclerosis (SSc), which precedes tissue fibrosis. The complement system is a key mediator of tissue damage after I/R, primarily by activation of the lectin pathway. This study investigated whether serum levels and polymorphisms of mannose-binding lectin (MBL) and ficolin-2 (FCN2), two pattern recognition receptors of the lectin pathway, are associated with the predisposition to and clinical features of SSc.

Methods

A case-control study was undertaken involving 90 patients with SSc from a single SSc outpatient clinic and 90 age- and sex-matched blood donors. MBL and FCN2 levels and polymorphisms were measured in both groups, and in cases correlated with clinical data.

Results

MBL levels and genotypes were equally distributed in cases and controls while there were some significant differences in FCN2 polymorphisms. Median MBL levels were higher in SSc cases with diffuse disease compared with controls (2.6 versus 1.0 μg/ml, P <0.001).In cases, higher MBL levels were associated with the presence of clinical findings associated with vascular dysfunction and local tissue damage (digital ulcers, calcinosis and pitting). Moreover, MBL levels were associated with fibrotic disease manifestations as evidenced by the presence of diffuse disease (median 2.6 versus 0.8 μg/ml, P = 0.002), the modified Rodnan skin score (r = 0.39, P <0.001), and interstitial lung disease as measured by forced vital capacity (r = −0.33, P = 0.001). Importantly, MBL levels also correlated with the Scleroderma Health Assessment Questionnaire scores (r = 0.33, P = 0.002). The results for FCN2 levels were less striking. Phenotypic MBL results were largely confirmed by analysis of MBL polymorphisms. MBL levels were not associated with the presence of autoantibodies or hypocomplementaemia.

Conclusions

Overall, predisposition to SSc was not influenced by the lectin pathway of complement in our matched case-control study. However, our preliminary data suggest that MBL, and to a lesser extent FCN2, may modulate disease manifestations of SSc, particularly in diffuse cutaneous disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0480-6) contains supplementary material, which is available to authorized users.     

Aclidinium bromide and formoterol fumarate as a fixed-dose combination in COPD: pooled analysis of symptoms and exacerbations from two six-month,multicentre, randomised studies (ACLIFORM and AUGMENT)

Background

The combination of aclidinium bromide, a long-acting anticholinergic, and formoterol fumarate, a long-acting beta₂-agonist (400/12 μg twice daily) achieves improvements in lung function greater than either monotherapy in patients with chronic obstructive pulmonary disease (COPD), and is approved in the European Union as a maintenance treatment. The effect of this combination on symptoms of COPD and exacerbations is less well established. We examined these outcomes in a pre-specified analysis of pooled data from two 24-week, double-blind, parallel-group, active- and placebo-controlled, multicentre, randomised Phase III studies (ACLIFORM and AUGMENT).

Methods

Patients ≥40 years with moderate to severe COPD (post-bronchodilator forced expiratory volume in 1 s [FEV₁]/forced vital capacity <70 % and FEV₁ ≥30 % but <80 % predicted normal) were randomised (ACLIFORM: 2:2:2:2:1; AUGMENT: 1:1:1:1:1) to twice-daily aclidinium/formoterol 400/12 μg or 400/6 μg, aclidinium 400 μg, formoterol 12 μg or placebo via Genuair™/Pressair®. Dyspnoea (Transition Dyspnoea Index; TDI), daily symptoms (EXAcerbations of Chronic pulmonary disease Tool [EXACT]-Respiratory Symptoms [E-RS] questionnaire), night-time and early-morning symptoms, exacerbations (Healthcare Resource Utilisation [HCRU] and EXACT definitions) and relief-medication use were assessed.

Results

The pooled intent-to-treat population included 3394 patients. Aclidinium/formoterol 400/12 μg significantly improved TDI focal score versus placebo and both monotherapies at Week 24 (all p < 0.05). Over 24 weeks, significant improvements in E-RS total score, overall night-time and early-morning symptom severity and limitation of early-morning activities were observed with aclidinium/formoterol 400/12 μg versus placebo and both monotherapies (all p < 0.05). The rate of moderate or severe HCRU exacerbations was significantly reduced with aclidinium/formoterol 400/12 μg compared with placebo (p < 0.05) but not monotherapies; the rate of EXACT-defined exacerbations was significantly reduced with aclidinium/formoterol 400/12 μg versus placebo (p < 0.01) and aclidinium (p < 0.05). Time to first HCRU or EXACT exacerbation was longer with aclidinium/formoterol 400/12 μg compared with placebo (all p < 0.05) but not the monotherapies. Relief-medication use was reduced with aclidinium/formoterol 400/12 μg versus placebo and aclidinium (p < 0.01).

Conclusions

Aclidinium/formoterol 400/12 μg significantly improves 24-hour symptom control compared with placebo, aclidinium and formoterol in patients with moderate to severe COPD. Furthermore, aclidinium/formoterol 400/12 μg reduces the frequency of exacerbations compared with placebo.

Trial registration

{"type":"clinical-trial","attrs":{"text":"NCT01462942","term_id":"NCT01462942"}}NCT01462942 and {"type":"clinical-trial","attrs":{"text":"NCT01437397","term_id":"NCT01437397"}}NCT01437397 (ClinicalTrials.gov)

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0250-2) contains supplementary material, which is available to authorized users.     

Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1β

Introduction

Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1β (IL-1β). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyclooxygenase-2 specific nonsteroidal pro-inflammatory drug carprofen.

Methods

Cartilage explant cultures were established by using metacarpophalangeal joints from horses euthanized for purposes other than research. Samples were treated as follows: no treatment (control), IL-1β (10 ng/ml), carprofen (100 μg/ml), and carprofen (100 μg/ml) + IL-1β (10 ng/ml). Explants were incubated (37°C, 5% CO₂) over twelve day time courses. High-throughput nano liquid chromatography/mass spectrometry/mass spectrometry uncovered candidate proteins for quantitative western blot analysis. Proteoglycan loss was assessed by using the dimethylmethylene blue (DMMB) assay, which measures the release of sulfated glycosaminoglycans (GAGs).

Results

Mass spectrometry identified MMP-1, -3, -13, and the ECM constituents thrombospondin-1 (TSP-1) and fibronectin-1 (FN1). IL-1β stimulation increased the release of all three MMPs. IL-1β also stimulated the fragmentation of FN1 and increased chondrocyte cell death (as assessed by β-actin release). Addition of carprofen significantly decreased MMP release and the appearance of a 60 kDa fragment of FN1 without causing any detectable cytotoxicity to chondrocytes. DMMB assays suggested that carprofen initially inhibited IL-1β-induced GAG release, but this effect was transient. Overall, during the two time courses, GAG release was 58.67% ± 10.91% (SD) for IL-1β versus 52.91% ± 9.35% (SD) with carprofen + IL-1β.

Conclusions

Carprofen exhibits beneficial anti-inflammatory and anti-catabolic effects in vitro without causing any detectable cytotoxicity. Combining proteomics with this explant model provides a sensitive screening system for anti-inflammatory compounds.     

Remifentanil added to sufentanil-sevoflurane anesthesia suppresses hemodynamic and metabolic stress responses to intense surgical stimuli more effectively than high-dose sufentanil-sevoflurane alone

Background

Even extremely high-doses of the potent opioid, sufentanil, cannot reliably suppress stress responses to intense surgical stimuli such as sternotomy. The chemically related opioid remifentanil with its different pharmacokinetics and binding affinities for delta- and kappa-opioid receptors might be more effective in attenuating these responses.

Methods

ASA I-III patients scheduled for a surgical procedure with sternotomy under balanced anesthesia (sevoflurane and sufentanil 3 μg.kg^-1 bolus, 0.017 μg.kg^-1.min^-1 infusion) were randomized into two groups. Patients in the study group were supplemented with remifentanil (2 μg.kg^-1 bolus, 2–7 μg.kg^-1.min^-1 infusion) starting ten minutes before sternotomy. Heart rate, arterial blood pressures, cardiac index, ejection fraction, systemic vascular resistance index (SVRI), total body oxygen uptake (VO₂) and electric dermal response were measured and compared between the groups.

Results

62 patients were studied (study group 32, control group 30). Systolic and mean arterial blood pressures, SVRI, VO₂ and skin conductance increased during sternotomy and sternal spread in the control group but not in the study group. Systolic blood pressure increase: 7.5 ± 19 mmHg vs. -3.4 ± 8.9 (p = 0.005); VO₂ increase: 31 ± 46% vs. -0.4 ± 32%; incidence of systolic blood pressure increase greater than 15 percent: 20% vs. 3% (p = 0.035) (control vs. study group).

Conclusion

High-dose remifentanil added to sevoflurane-sufentanil anesthesia suppresses the sympathoadrenergic response to sternotomy and sternal spread better than high-dose sufentanil alone.

Trial registration

Clinical Trial number: DRKS00004327, August 31, 2012

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2253-15-3) contains supplementary material, which is available to authorized users.     

The effects of salbutamol on epithelial ion channels depend on the etiology of acute respiratory distress syndrome but not the route of administration

Introduction

We investigated the effects of intravenous and intratracheal administration of salbutamol on lung morphology and function, expression of ion channels, aquaporin, and markers of inflammation, apoptosis, and alveolar epithelial/endothelial cell damage in experimental pulmonary (p) and extrapulmonary (exp) mild acute respiratory distress syndrome (ARDS).

Methods

In this prospective randomized controlled experimental study, 56 male Wistar rats were randomly assigned to mild ARDS induced by either intratracheal (n = 28, ARDSp) or intraperitoneal (n = 28, ARDSexp) administration of E. coli lipopolysaccharide. Four animals with no lung injury served as controls (NI). After 24 hours, animals were anesthetized, mechanically ventilated in pressure-controlled mode with low tidal volume (6 mL/kg), and randomly assigned to receive salbutamol (SALB) or saline 0.9% (CTRL), intravenously (i.v., 10 μg/kg/h) or intratracheally (bolus, 25 μg). Salbutamol doses were targeted at an increase of ≈ 20% in heart rate. Hemodynamics, lung mechanics, and arterial blood gases were measured before and after (at 30 and 60 min) salbutamol administration. At the end of the experiment, lungs were extracted for analysis of lung histology and molecular biology analysis. Values are expressed as mean ± standard deviation, and fold changes relative to NI, CTRL vs. SALB.

Results

The gene expression of ion channels and aquaporin was increased in mild ARDSp, but not ARDSexp. In ARDSp, intravenous salbutamol resulted in higher gene expression of alveolar epithelial sodium channel (0.20 ± 0.07 vs. 0.68 ± 0.24, p < 0.001), aquaporin-1 (0.44 ± 0.09 vs. 0.96 ± 0.12, p < 0.001) aquaporin-3 (0.31 ± 0.12 vs. 0.93 ± 0.20, p < 0.001), and Na-K-ATPase-α (0.39 ± 0.08 vs. 0.92 ± 0.12, p < 0.001), whereas intratracheal salbutamol increased the gene expression of aquaporin-1 (0.46 ± 0.11 vs. 0.92 ± 0.06, p < 0.001) and Na-K-ATPase-α (0.32 ± 0.07 vs. 0.58 ± 0.15, p < 0.001). In ARDSexp, the gene expression of ion channels and aquaporin was not influenced by salbutamol. Morphological and functional variables and edema formation were not affected by salbutamol in any of the ARDS groups, regardless of the route of administration.

Conclusion

Salbutamol administration increased the expression of alveolar epithelial ion channels and aquaporin in mild ARDSp, but not ARDSexp, with no effects on lung morphology and function or edema formation. These results may contribute to explain the negative effects of β2-agonists on clinical outcome in ARDS.     

Small airway dysfunction is associated to excessive bronchoconstriction in asthmatic patients

Background

We investigated whether a relationship between small airways dysfunction and bronchial hyperresponsiveness (BHR), expressed both in terms of ease of airway narrowing and of excessive bronchoconstriction, could be demonstrated in asthma.

Methods

63 (36 F; mean age 42 yr ± 14) stable, mild-to-moderate asthmatic patients (FEV₁ 92% pred ±14; FEV₁/FVC 75% ± 8) underwent the methacholine challenge test (MCT). The degree of BHR was expressed as PD₂₀ (in μg) and as ∆FVC%. Peripheral airway resistance was measured pre- and post-MCT by impulse oscillometry system (IOS) and expressed as R5-R20 (in kPa sL⁻¹).

Results

All patients showed BHR to methacholine (PD₂₀ < 1600 μg) with a PD₂₀ geometric (95% CI) mean value of 181(132–249) μg and a ∆FVC% mean value of 13.6% ± 5.1, ranging 2.5 to 29.5%. 30 out of 63 patients had R5-R20 > 0.03 kPa sL⁻¹ (>upper normal limit) and showed ∆FVC%, but not PD₂₀ values significantly different from the 33 patients who had R5-R20 ≤ 0.03 kPa sL⁻¹ (15.8% ± 4.6 vs 11.5% ± 4.8, p < 0.01 and 156(96–254) μg vs 207 (134–322) μg, p = 0.382). In addition, ∆FVC% values were significantly related to the corresponding pre- (r = 0.451, p < 0.001) and post-MCT (r = 0.376, p < 0.01) R5-R20 values.

Conclusions

Our results show that in asthmatic patients, small airway dysfunction, as assessed by IOS, is strictly associated to BHR, expressed as excessive bronchoconstriction, but not as ease of airway narrowing.     

The Tiotropium Safety and Performance in Respimat? (TIOSPIR?) Trial: Spirometry Outcomes

Background

Tiotropium Safety and Performance in Respimat® (TIOSPIR®) compared the safety and efficacy of tiotropium Respimat® and tiotropium HandiHaler® in patients with chronic obstructive pulmonary disease (COPD). A prespecified spirometry substudy compared the lung function efficacy between treatment groups.

Methods

TIOSPIR® was a large-scale, long-term (2.3-year), event-driven, randomized, double-blind, parallel-group trial of 17,135 patients with COPD. In the spirometry substudy, trough forced expiratory volume in 1 second (FEV₁) and forced vital capacity (FVC) were measured at baseline and every 24 weeks for the duration of the trial.

Results

The substudy included 1370 patients who received once-daily tiotropium Respimat® 5 μg (n = 461), 2.5 μg (n = 464), or tiotropium HandiHaler® 18 μg (n = 445). Adjusted mean trough FEV₁ (average 24–120 weeks) was 1.285, 1.258, and 1.295 L in the Respimat® 5 μg, 2.5 μg, and HandiHaler® 18 μg groups (difference versus HandiHaler® [95 % CI]: −10 [−38, 18] mL for Respimat® 5 μg and, −37 [−65, −9] mL for Respimat® 2.5 μg); achieving noninferiority to tiotropium HandiHaler® 18 μg for tiotropium Respimat® 5 but not for 2.5 μg (prespecified analysis). Adjusted mean trough FVC was 2.590, 2.544, and 2.593 L in the Respimat® 5 μg, 2.5 μg, and HandiHaler® 18 μg groups. The rates of FEV₁ decline over 24 to 120 weeks were similar for the three treatment arms (26, 40, and 34 mL/year for the tiotropium Respimat® 5-μg, 2.5-μg, and HandiHaler® 18-μg groups). The rate of FEV₁ decline in GOLD I + II patients was greater than in GOLD III + IV patients (46 vs. 23 mL/year); as well as in current versus ex-smokers, in patients receiving combination therapies at baseline versus not, and in those experiencing an exacerbation during the study versus not.

Conclusions

The TIOSPIR® spirometry substudy showed that tiotropium Respimat® 5 μg was noninferior to tiotropium HandiHaler® 18 μg for trough FEV₁, but Respimat® 2.5 μg was not. Tiotropium Respimat® 5 μg provides similar bronchodilator efficacy to tiotropium HandiHaler® 18 μg with comparable rates of FEV₁ decline. The rate of FEV₁ decline varied based on disease severity, with a steeper rate of decline observed in patients with moderate airway obstruction.

Trial registration

{"type":"clinical-trial","attrs":{"text":"NCT01126437","term_id":"NCT01126437"}}NCT01126437.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0269-4) contains supplementary material, which is available to authorized users.     

Protective role of frizzled-related protein B on matrix metalloproteinase induction in mouse chondrocytes

Introduction

Our objective was to investigate whether a lack of frizzled-related protein B (FrzB), an extracellular antagonist of the Wnt signaling pathways, could enhance cartilage degradation by facilitating the expression, release and activation of matrix metalloproteinases (MMPs) by chondrocytes in response to tissue-damaging stimuli.

Methods

Cartilage explants from FrzB^−/− and wild-type mice were challenged by excessive dynamic compression (0.5 Hz and 1 MPa for 6 hours). Load-induced glycosaminoglycan (GAG) release and MMP enzymatic activity were assessed. Interleukin-1β (IL-1β) (10, 100 and 1000 pg/mL for 24 hours) was used to stimulate primary cultures of articular chondrocytes from FrzB^−/− and wild-type mice. The expression and release of MMP-3 and −13 were determined by RT-PCR, western blot and ELISA. The accumulation of β-catenin was assessed by RT-PCR and western blot.

Results

Cartilage degradation, as revealed by a significant increase in GAG release (2.8-fold, P = 0.014) and MMP activity (4.5-fold, P = 0.014) by explants, was induced by an excessive load. Load-induced MMP activity appeared to be enhanced in FrzB^−/− cartilage explants compared to wild-type (P = 0.17). IL-1β dose-dependently induced Mmp-13 and −3 gene expression and protein release by cultured chondrocytes. IL-1β-mediated increase in MMP-13 and −3 was slightly enhanced in FrzB^−/− chondrocytes compared to wild-type (P = 0.05 and P = 0.10 at gene level, P = 0.17 and P = 0.10 at protein level, respectively). Analysis of Ctnn1b and Lef1 gene expression and β-catenin accumulation at protein level suggests that the enhanced catabolic response of FrzB^−/− chondrocytes to IL-1β and load may be associated with an over-stimulation of the canonical Wnt/β-catenin pathway.

Conclusions

Our results suggest that FrzB may have a protective role on cartilage degradation and MMP induction in mouse chondrocytes by attenuating deleterious effects of the activation of the canonical Wnt/β-catenin pathway.     

Roflumilast improves corticosteroid resistance COPD bronchial epithelial cells stimulated with toll like receptor 3 agonist

Background

Chronic obstructive pulmonary disease (COPD) is characterised by chronic pulmonary inflammation punctuated by periods of viral exacerbations. Recent evidence suggests that the combination of roflumilast with corticosteroids may improve the compromised anti-inflammatory properties of corticosteroids in COPD. We analyzed differential and combination anti-inflammatory effects of dexamethasone and roflumilast N-oxide in human bronchial epithelial cells (HBECs) stimulated with viral toll like receptor (TLR) agonists.

Methods

Lung tissue and HBECs were isolated from healthy (n = 15), smokers (n = 12) and smokers with COPD (15). TLR3 expression was measured in lung tissue and in HBECs. IL-8 secretion was measured in cell cultures after TLR3 stimulation with poly I:C 10 μg/mL.

Results

We found that TLR3 expression was increased by 1.95 fold (protein) and 2.5 fold (mRNA) in lung tissues from smokers with COPD and inversely correlated with lung function. The TLR3 agonist poly I:C 10 μg/mL increased the IL-8 release in HBECs that was poorly inhibited by dexamethasone in smokers (24.5%) and smokers with COPD (21.6%). In contrast, roflumilast showed similar inhibitory effects on IL-8 release in healthy (58.8%), smokers (56.6%) and smokers with COPD (50.5%). The combination of roflumilast N-oxide and dexamethasone showed additive inhibitory effects. Mechanistically, roflumilast N-oxide when combined with dexamethasone increased the expression of MKP1, and enhanced the inhibitory effects on phospho-p38, AP1 and NFκB activities which may explain the additive anti-inflammatory effects.

Conclusions

Altogether, our data provide in vitro evidence for a possible clinical utility to add roflumilast on top of inhaled corticosteroid in COPD.     

Dose-finding evaluation of once-daily treatment with olodaterol,a novel long-acting β2-agonist,in patients with asthma: results of a parallel-group study and a crossover study

Background

Olodaterol is a novel, inhaled long-acting β₂-agonist (LABA) with >24-hour duration of action investigated in asthma and chronic obstructive pulmonary disease.

Methods

Two multicentre studies examined the efficacy and safety of 4 weeks’ once-daily (QD) olodaterol (2, 5, 10 and 20 μg, with background inhaled corticosteroids) in patients with asthma. One randomised, double-blind, parallel-group study (1222.6; 296 patients) administered treatment in the morning. Pulmonary function tests (PFTs) were performed pre-dose (trough) and ≤3 hours post-dose (weeks 1 and 2), and ≤6 hours post-dose after 4 weeks; primary end point was trough forced expiratory volume in 1 second (FEV₁) response (change from baseline mean FEV₁) after 4 weeks. A second randomised, double-blind, placebo- and active-controlled (formoterol 12 μg twice-daily) incomplete-block crossover study (1222.27; 198 patients) administered QD treatments in the evening. PFTs were performed over a 24-hour dosing interval after 4 weeks; primary end point was FEV₁ area under the curve from 0–24 hours (AUC_0–24) response (change from study baseline [mean FEV₁] after 4 weeks).

Results

Study 1222.6 showed a statistically significant increase in trough FEV₁ response with olodaterol 20 μg (0.147 L; 95 % confidence interval [CI]: 0.059, 0.234; p = 0.001) versus placebo, with more limited efficacy and no evidence of dose response compared to placebo across the other olodaterol doses (2, 5 and 10 μg). Study 1222.27 demonstrated increases in FEV₁ AUC_0–24 responses at 4 weeks with all active treatments (p < 0.0001); adjusted mean (95 % CI) differences from placebo were 0.140 (0.097, 0.182), 0.182 (0.140, 0.224), 0.205 (0.163, 0.248) and 0.229 (0.186, 0.272) L for olodaterol 2, 5, 10 and 20 μg, respectively, and 0.169 (0.126, 0.211) for formoterol, providing evidence of increased efficacy with higher olodaterol dose. Olodaterol was generally well tolerated, with a few events associated with known sympathomimetic effects, mainly with 20 μg.

Conclusions

The LABA olodaterol has >24-hour duration of action. In patients with asthma, evidence of bronchodilator efficacy was demonstrated with statistically and clinically significant improvements in the primary end point of trough FEV₁ response measured in clinics over placebo for the highest administered dose of 20 μg in Study 1222.6, and statistically and clinically significant improvements versus placebo in FEV₁ AUC_0–24 responses at 4 weeks for all doses tested in Study 1222.27, which also exhibited a dose response. Bronchodilator efficacy was seen over placebo for all olodaterol doses for morning and evening peak expiratory flow in both studies. All doses were well tolerated.

Trial registrations

{"type":"clinical-trial","attrs":{"text":"NCT00467740","term_id":"NCT00467740"}}NCT00467740 (1222.6) and {"type":"clinical-trial","attrs":{"text":"NCT01013753","term_id":"NCT01013753"}}NCT01013753 (1222.27).

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0249-8) contains supplementary material, which is available to authorized users.     

Methanol extract from Vietnamese Caesalpinia sappan induces apoptosis in HeLa cells

Background

This study evaluated the cytotoxic activity of extracts from Caesalpinia sappan heartwood against multiple cancer cell lines using an MTT cell viability assay. The cell death though induction of apoptosis was as indicated by DNA fragmentation and caspase-3 enzyme activation.

Results

A methanol extract from C. sappan (MECS) showed cytotoxic activity against several of the cancer cell lines. The most potent activity exhibited by the MECS was against HeLa cells with an IC₅₀ value of 26.5 ± 3.2 μg/mL. Treatment of HeLa cells with various MECS concentrations resulted in growth inhibition and induction of apoptosis, as indicated by DNA fragmentation and caspase-3 enzyme activation.

Conclusion

This study is the first report of the anticancer properties of the heartwood of C. sappan native to Vietnam. Our findings demonstrate that C. sappan heartwood may have beneficial applications in the field of anticancer drug discovery.     

Anticholinesterse and antioxidant investigations of crude extracts,subsequent fractions,saponins and flavonoids of atriplex laciniata L.: potential effectiveness in Alzheimer’s and other neurological disorders

Background

Atriplex laciniata L. was investigated for phenolic, flavonoid contents, antioxidant, anticholinesterase activities, in an attempt to explore its effectiveness in Alzheimer’s and other neurological disorders. Plant crude methanolic extract (Al.MeF), subsequent fractions; n-hexane (Al.HxF), chloroform (Al.CfF), ethyl acetate (Al.EaF), aqueous (Al.WtF), Saponins (Al.SPF) and Flavonoids (Al.FLVF) were investigated for DPPH, ABTS and H₂O₂ free radical scavenging activities. Further these extracts were subjected to acetylcholinesterase (AChE) & butyrylcholinesterase (BChE) inhibitory activities using Ellman’s assay. Phenolic and Flavonoid contents were determined and expressed in mg Gallic acid GAE/g and Rutin RTE/g of samples respectively.

Results

In DPPH free radicals scavenging assay, Al.FLVF, Al.SPF and Al.MeF showed highest activity causing 89.41 ± 0.55, 83.37 ± 0.34 and 83.37 ± 0.34% inhibition of free radicals respectively at 1 mg/mL concentration. IC₅₀ for these fractions were 33, 83 and 82 μg/mL respectively. Similarly, plant extracts showed high ABTS scavenging potential, i.e. Al.FLVF (90.34 ± 0.55), Al.CfF (83.42 ± 0.57), Al.MeF (81.49 ± 0.60) with IC₅₀ of 30, 190 and 70 μg/ml respectively. further, H₂O₂ percent scavenging was highly appraised in Al.FLVF (91.29 ± 0.53, IC₅₀ 75), Al.SPF (85.35 ± 0.61, IC₅₀ 70) and Al.EaF (83.48 ± 0.67, IC₅₀ 270 μg/mL). All fractions exhibited concentration dependent AChE inhibitory activity as; Al.FLVF, 88.31 ± 0.57 (IC₅₀ 70 μg/mL), Al.SPF, 84.36 ± 0.64 (IC₅₀ 90 μg/mL), Al.MeF, 78.65 ± 0.70 (IC₅₀ 280 μg/mL), Al.EaF, 77.45 ± 0.46 (IC₅₀ 270 μg/mL) and Al.WtF 72.44 ± 0.58 (IC₅₀ 263 μg/mL) at 1 mg/mL. Likewise the percent BChE inhibitory activity was most obvious in Al.FLVF 85.46 ± 0.62 (IC₅₀ 100 μg/mL), Al.CfF 83.49 ± 0.46 (IC₅₀ 160 μg/mL), Al.MeF 82.68 ± 0.60 (IC₅₀ 220 μg/mL) and Al.SPF 80.37 ± 0.54 (IC₅₀ 120 μg/mL).

Conclusions

These results stipulate that A. laciniata is enriched with phenolic and flavonoid contents that possess significant antioxidant and anticholinestrase effects. This provide pharmacological basis for the presence of compounds that may be effective in Alzheimer’s and other neurological disorders.     

Synergistic activity and mechanism of action of Stephania suberosa Forman extract and ampicillin combination against ampicillin-resistant Staphylococcus aureus

Background

Ampicillin-resistant S. aureus (ARSA) now poses a serious problem for hospitalized patients, and their care providers. Plant-derived antibacterial that can reverse the resistance to well-tried agents which have lost their original effectiveness are the research objectives of far reaching importance. To this aim, the present study investigated antibacterial and synergistic activities of Stephania suberosa extracts (SSE) against ARSA when used singly and in combination with ampicillin.

Results

The majority chemical compounds of SSE were alkaloid (526.27 ± 47.27 mg/1 g of dried extract). The Minimum inhibitory concentration (MICs) for ampicillin and SSE against all ARSA strains were >512 μg/ml and 4 mg/ml, respectively. Checkerboard assay revealed synergistic activity in the combination of ampicillin (0.15 μg/ml) and SSE (2 mg/ml) at fractional inhibitory concentration index (FICI) <0.5. The killing curve assay had confirmed that the viability of ARSA was dramatically reduced from 5x10⁵ cfu/ml to 10³ cfu/ml within 6 h after exposure to SSE (2 mg/ml) plus ampicillin (0.15 μg/ml) combination. Electron microscopic study clearly revealed that these ARSA cells treated with this combination caused marked morphological damage, peptidoglycan and cytoplasmic membrane damage, and average cell areas significant smaller than control. Obviously, Immunofluorescence staining and confocal microscopic images confirmed that the peptidoglycan of these cells were undoubtedly disrupted by this combination. Furthermore, the CM permeability of ARSA was also increased by this combination. Enzyme assay demonstrated that SSE had an inhibitory activity against β-lactamase in concentrations manner.

Conclusions

So, these findings provide evidence that SSE has the high potential to reverse bacterial resistance to originate traditional drug susceptibility of it and may relate to three modes of actions of SSE: (1) inhibits peptidoglycan synthesis, resulting in morphological damage, (2) inhibits β-lactamases activity, and (3) increases CM permeability. It is widely recognized that many types of drugs are derived from alkaloids. So, this SSE offers the prominent potential to develop a novel adjunct phytopharmaceutical to ampicillin for the treatment of ARSA. Further active ingredients study, toxicity of it, and the synergistic effect on blood and tissue should be performed and confirmed in an animal test or in humans.     

Extraction of saponins and toxicological profile of Teucrium stocksianum boiss extracts collected from District Swat,Pakistan

Background

The current era is facing challenges in the management of neoplasia and weeds control. The currently available anti-cancer and herbicidal drugs are associated with some serious side effects. Therefore numerous researchers are trying to discover and develop plant based alternative particularly for the rational management of cancer and weed control. Teucrium stocksianum possess antioxidant and analgesic activities. The current study was designed to evaluate crude saponins (CS), methanolic extract and sub-fractions of T. stocksianum for cytotoxic and phytotoxic potentials. CS, methanolic extract and sub-fractions were extracted from powdered plant material using different solvents. Cytotoxic potential of the extracts at a dose of 10, 100 and 1000 μg/ml were evaluated against Brine shrimp’s nauplii. Phytotoxic assay also performed at the same concentration against Lemna minor. Etoposide and Paraquat were used as positive controls in cytotoxic and phytotoxic assays respectively.

Results

The percent yield of crude saponins was (5%). CS demonstrated tremendous brine shrimp lethality showing < 10 μg/ml LC₅₀. The n-hexane (HF) and chloroform fractions (CF) demonstrated excellent cytotoxicity with 80 and 55 μg/ml LC₅₀ respectively. Whereas the methanolic extract (TSME), ethyl acetate (EAF) and aqueous fractions (AF) revealed moderate cytotoxicity showing 620, 860 and 1000 μg/ml LC₅₀ values respectively. In phytotoxic assay profound inhibition was displayed by HF (96.67%) and TSME (95.56%, 30 μg/ml LC₅₀) against the growth of Lemna minor at 1000 μg/ml respectively. Both CF and EAF demonstrated profound phytoxicity (93.33%) respectively at highest concentration (1000 μg/ml), while AF and CS demonstrated weak phytotoxicity with 1350 and 710 μg/ml LC₅₀ values respectively.

Conclusion

Cytotoxicity and phytotoxicity assays indicated that the crude saponins, n-hexane and chloroform fractions of T. stocksianum could play a vital role in the treatment of neoplasia and as potential natural herbicides. Therefore these sub-fractions are recommended for further investigation with the aim to isolate novel anti-cancer and herbicidal compounds.     

Plasma levels of alpha-1-antichymotrypsin are elevated in patients with chronic heart failure,but are of limited prognostic value

Background

There is increasing interest in utilising novel markers of cardiovascular disease risk in patients with chronic heart failure (HF). Recently, it was shown that alpha-1-antichymotrypsin (ACT), an acute-phase protein and major inhibitor of cathpesin G, plays a role in the pathophysiology of HF and may serve as a marker for myocardial distress.

Objective

To assess whether ACT is independently associated with long-term mortality in chronic HF patients.

Methods

ACT plasma levels were categorised into quartiles. Survival times were analysed using Kaplan-Meier curves and Cox proportional hazards regression, without and with correction for clinically relevant risk factors, including sex, age, duration of HF, kidney function (MDRD), ischaemic HF aetiology and NT-proBNP.

Results

Twenty healthy individuals and 224 patients (mean age 71 years, 72 % male, median HF duration 1.6 years) with chronic HF were included. In total, 159 (71 %) patients died. The median survival time was 5.3 (95 % CI 4.5–6.1) years. ACT was significantly elevated in patients (median 433 μg/ml, IQR 279–680) in comparison with controls (median 214 μg/ml, IQR 166–271; p < 0.001). Cox regression analysis demonstrated that ACT was not independently related to long-term mortality in chronic HF patients (crude HR = 1.03, 95 % CI 0.75–1.41, p = 0.871; adjusted HR = 1.12, 95 % CI 0.78–1.60, p = 0.552), which was confirmed by Kaplan-Meier curves.

Conclusion

ACT levels are elevated in chronic HF patients, but no independent association with long-term mortality can be established.