Antioxidant and anticholinesterase investigations of Rumex hastatus D. Don: potential effectiveness in oxidative stress and neurological disorders

Background

Rumex species are traditionally used for the treatment of neurological disorders including headache, migraine, depression, paralysis etc. Several species have been scientifically validated for antioxidant and anticholinestrase potentials. This study aims to investigate Rumex hastatus D. Don crude methanolic extract, subsequent fractions, saponins and flavonoids for acetylcholinestrase, butyrylcholinestrase inhibition and diverse antioxidant activities to validate its folkloric uses in neurological disorders. Rumex hastatus crude methanolic extract (Rh. Cr), subsequent fractions; n-hexane (Rh. Hex), chloroform (Rh. Chf), ethyl acetate (Rh. EtAc), aqueous fraction (Rh. Aq), crude saponins (Rh. Sp) and flavonoids (Rh. Fl) were investigated against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) at various concentrations (125, 250, 500, 1000 μg/mL) using Ellman’s spectrophotometric analysis. Antioxidant potentials of Rh. Sp and Rh. Fl were evaluated using DPPH, H₂O₂ and ABTS free radical scavenging assays at 62.5, 125, 250, 500, 1000 μg/mL.

Results

All the test samples showed concentration dependent cholinesterase inhibition and radicals scavenging activity. The AChE inhibition potential of Rh. Sp and Rh. Fl were most prominent i.e., 81.67 ± 0.88 and 91.62 ± 1.67 at highest concentration with IC₅₀ 135 and 20 μg/mL respectively. All the subsequent fractions exhibited moderate to high AChE inhibition i.e., Rh. Cr, Rh. Hex, Rh. Chf, Rh. EtAc and Rh. Aq showed IC₅₀ 218, 1420, 75, 115 and 1210 μg/mL respectively. Similarly, against BChE various plant extracts i.e., Rh. Sp, Rh. Fl, Rh. Cr, Rh. Hex, Rh. Chf, Rh. EtAc and Rh. Aq resulted IC₅₀ 165, 175, 265, 890, 92, 115 and 220 μg/mL respectively. In DPPH free radical scavenging assay, Rh. Sp and Rh. Fl showed comparable results with the positive control i.e., 63.34 ± 0.98 and 76.93 ± 1.13% scavenging at 1 mg/mL concentration (IC₅₀ 312 and 104 μg/mL) respectively. The percent ABTS radical scavenging potential exhibited by Rh. Sp and Rh. Fl (1000 μg/mL) were 82.58 ± 0.52 and 88.25 ± 0.67 with IC₅₀ 18 and 9 μg/mL respectively. Similarly in H₂O₂ scavenging assay, the Rh. Sp and Rh. Fl exhibited IC₅₀ 175 and 275 μg/mL respectively.

Conclusion

The strong anticholinesterase and antioxidant activities of Rh. Sp, Rh. Fl and various fractions of R. hastatus support the purported ethnomedicinal uses and recommend R. hastatus as a possible remedy for the treatment of AD and neurodegenerative disorders.     

Determination of carbohydrates in medicinal plants--comparison between TLC, mf-MELDI-MS and GC-MS

Introduction – Quality control in the pharmaceutical and phytopharmaceutical industries requires fast and reliable methods for the analysis of raw materials and final products. Objective – This study evaluates different analytical approaches in order to recognise the most suitable technique for the analysis of carbohydrates in herbal drug preparations. Methodology – The specific focus of the study is on thin‐layer chromatography (TLC), gas chromatography (GC), and a newly developed mass spectrometric method, i.e. matrix free material enhanced laser desorption/ionisation time of flight mass spectrometry (mf‐MELDI‐MS). Samples employed in the study were standards and microwave‐assisted water extracts from Quercus. Results – TLC analysis proved the presence of mono‐, di‐ and trisaccharides within the biological sample and hinted at the existence of an unknown carbohydrate of higher oligomerisation degree. After evaluation of different derivatisation techniques, GC‐MS confirmed data obtained via TLC for mono‐ to trisaccharides, delivering additionally quantified values under a considerable amount of time. A carbohydrate of higher oligomerisation degree could not be found. The application of mf‐MELDI‐MS further confirmed the presence of carbohydrates up to trisaccharides, also hinting at the presence of a form of tetrasaccharide. Besides this information, mf‐MELDI‐MS delivered further data about other substances present in the extract. Quantitative determination resulted in 1.750, 1.736 and 0.336 mg/mL for glucose, sucrose and raffinose respectively. Conclusion – Evaluation of all three techniques employed, clearly proved the heightened performance of mf‐MELDI‐MS for the qualitative analysis of complex mixtures, as targets do not need modification and analysis requires only a few minutes. In addition, GC‐MS is suitable for quantitative analysis. Copyright © 2011 John Wiley & Sons, Ltd.     

LPS-Induced Formation of Immunoproteasomes: TNF-α and Nitric Oxide Production are Regulated by Altered Composition of Proteasome-Active Sites

Stimulation of mouse macrophages with LPS leads to tumor necrosis factor (TNF-α) secretion and nitric oxide (NO) release at different times through independent signaling pathways. While the precise regulatory mechanisms responsible for these distinct phenotypic responses have not been fully delineated, results of our recent studies strongly implicate the cellular cytoplasmic ubiquitin-proteasome pathway as a key regulator of LPS-induced macrophage inflammatory responses. Our objective in this study was to define the relative contribution of specific proteasomal active-sites in induction of TNF-α and NO after LPS treatment of RAW 264.7 macrophages using selective inhibitors of these active sites. Our data provide evidence that LPS stimulation of mouse macrophages triggers a selective increase in the levels of gene and protein expression of the immunoproteasomes, resulting in a modulation of specific functional activities of the proteasome and a corresponding increase in NO production as compared to untreated controls. These findings suggest the LPS-dependent induction of immunoproteasome. In contrast, we also demonstrate that TNF-α expression is primarily dependent on both the chymotrypsin- and the trypsin-like activities of X, Y, Z subunits of the proteasome. Proteasome-associated post-acidic activity alone also contributes to LPS-induced expression of TNF-α. Taken together; our results indicate that LPS-induced TNF-α in macrophages is differentially regulated by each of the three proteasome activities. Since addition of proteasome inhibitors to mouse macrophages profoundly affects the degradation of proteins involved in signal transduction, we conclude that proteasome-specific degradation of several signaling proteins is likely involved in differential regulation of LPS-dependent secretion of proinflammatory mediators.     

Occult HCV or delayed viral clearance from lymphocytes of Chronic HCV genotype 3 patients after interferon therapy

Background

A recently discovered occult HCV entity reported by various investigators seems to be highly controversial. Especially, the clinical significance of these findings remains uncertain. For optimal outcome of antiviral therapy, investigation of occult HCV needs a broad-based probe in order to investigate the results of viral therapy and its host/viral interaction. The current study was aimed at determining the prevalence of occult HCV in peripheral blood lymphocytes of predominantly genotype 3 HCV-infected patients after completion of antiviral therapy and to investigate long term outcomes in the presence or absence of PBMC positivity.

Method

A total of 151 chronic, antiHCV and serum RNA-positive patients were enrolled in the study. Patients with a complete virological response at the end of treatment were screened for the presence of viral RNA in their PBMCs and were followed for up to one year for the presence of serum and PBMC viral genomic RNA.

Results

Out of 151 patients, 104 (70%) responded to the prescribed interferon treatment and showed viral-clearance from serum. These were screened for the presence of genomic RNA in their PBMCs. Sixteen samples were PBMC-positive for viral RNA at the end of treatment (EOT). All these patients had also cleared the virus from peripheral blood cells after the 6-12 month follow-up study.

Conclusion

True occult hepatitis C virus does not exist in our cohort. Residual viremia at the EOT stage merely reflects a difference in viral kinetics in various compartments that remains a target of immune response even after the end of antiviral therapy and is eventually cleared out at the sustained viral response (SVR).     

Continuous butanol fermentation and feed starch retrogradation: butanol fermentation sustainability using Clostridium beijerinckii BA101

Use of starch solution as feed for butanol bioconversion processes employing Clostridium beijerinckii BA101 may have added economic advantage over the use of glucose. Acetone butanol ethanol (ABE) was produced from 30 gL(-1) starch solution using a continuous process. The bioreactor was fed at a dilution rate of 0.02 h(-1) and starch solution/feed volume (3 L) was replaced every 72 h. The continuous reactor fed with cornstarch solution (feed temperature 19 degrees C) produced approximately 6.0 gL(-1) total ABE. Increasing the feed storage temperature to 37 degrees C improved ABE production to 7.2 gL(-1) suggesting that retrogradation was occurring more rapidly at 19 degrees C. In both these cases the fermentation drifted toward acid production after approximately 260 h, consistent with the retrogradation of starch overtime. The use of soluble starch, which is less prone to retrogradation, resulted in the production of 9.9 gL(-1) ABE at 37 degrees C feed storage temperature, as compared to 7.2 gL(-1) ABE when cornstarch was used. It should be noted that gelatinized starch retrogradation takes place after sterilization and prior to use of the feed medium, and does not occur during long-term storage of the raw corn material in the months leading up to processing. The degree of hydrolysis of gelatinized starch decreased from 68.8 to 56.2% in 3 days when stored at 37 degrees C. Soluble starch which does not retrograde demonstrated no change in the degree of hydrolysis.     

Search and destroy: ER quality control and ER-associated protein degradation

Proteins synthesized in the endoplasmic reticulum (ER) encounter quality control checkpoints that verify their fitness to proceed in the secretory pathway. Molecules undergoing folding and assembly are kept out of the exocytic pathway until maturation is complete. Misfolded side products that inevitably form are removed from the mixture of conformers and returned to the cytosol for degradation. How unfolded proteins are recognized and how irreversibly misfolded proteins are sorted to ER-associated degradation pathways was poorly understood. Recent developments from a combination of genetic and biochemical analyses has revealed new insights into these mechanisms.The emerging view shows distinct pathways working in collaboration to filter the diverse range of unfolded proteins from the transport flow and to divert misfolded molecules for destruction.     

An inter-platform repeatability study investigating real-time amplification of plasmid DNA

Background  

The wide variety of real-time amplification platforms currently available has determined that standardisation of DNA measurements is a fundamental aspect involved in the comparability of results.     

Modulation of proinflammatory responses to Pneumocystis carinii f. sp. muris in neonatal mice by granulocyte-macrophage colony-stimulating factor and IL-4: role of APCs

Clearance of Pneumocystis carinii f. sp. muris (PC) organisms from the lungs of neonatal mice is delayed due to failure of initiation of inflammation over the first 3 wk after infection. The ability of neonatal lung CD11c(+) dendritic cells (DCs) to induce Ag-specific T cell proliferative responses was significantly reduced compared with adult lung DCs. However, neonatal bone marrow-derived DCs were as competent at presenting PC Ag as were adult bone marrow-derived DCs. Because GM-CSF mRNA expression and activity were significantly reduced in neonatal lungs compared with adults, we treated neonates with exogenous GM-CSF and IL-4 and found enhanced clearance of PC compared with untreated neonates. This was associated with increased lung TNF-alpha, IL-12p35, and IL-18 mRNA expression, indicating enhanced innate immune responses. Cytokine-treated mice had marked expansion of CD11c(+) DCs with up-regulated MHC-II in the lungs. Moreover, increased numbers of activated CD4(+)CD44(high)CD62L(low) cells in the lungs and draining lymph nodes suggested improved Ag presentation by the APCs. Together these data indicate that neonatal lungs lack maturation factors for efficient cellular functioning, including APC maturation.     

Effects of selenium on altered mechanical and electrical cardiac activities of diabetic rat

Since selenium compounds can restore some metabolic parameters and structural alterations of diabetic rat heart, we were tempted to investigate whether these beneficial effects extend to the diabetic rat cardiac dysfunctions. Diabetes was induced by streptozotocin (50mg/kg body weight) and rats were then treated with sodium selenite (5 micromol/kg body weight/day) for four weeks. Electrically stimulated isometric contraction and intracellular action potential in isolated papillary muscle strips and transient (I(to)) and steady state (I(ss)) outward K(+) currents in isolated cardiomyocytes were recorded. Sodium selenite treatment could reverse the prolongation in both action potential duration and twitch duration of the diabetic rats, and also cause significant increases in the diminished amplitudes of the two K(+) currents. Treatment of rats with sodium selenite also markedly increased the depressed acid-soluble sulfhydryl levels of the hearts. Our data suggest that the beneficial effects of sodium selenite treatment on the mechanical and electrical activities of the diabetic rat heart appear to be due to the restoration of the diminished K(+) currents, partially, related to the restoration of the cell glutathione redox cycle.     

Butanol production from corn fiber xylan using Clostridium acetobutylicum

Acetone, butanol, and ethanol (ABE) were produced from corn fiber arabinoxylan (CFAX) and CFAX sugars (glucose, xylose, galactose, and arabinose) using Clostridium acetobutylicum P260. In mixed sugar (glucose, xylose, galactose, and arabinose) fermentation, the culture preferred glucose and arabinose over galactose and xylose. Under the experimental conditions, CFAX (60 g/L) was not fermented until either 5 g/L xylose or glucose plus xylanase enzyme were added to support initial growth and fermentation. In this system, C. acetobutylicum produced 9.60 g/L ABE from CFAX and xylose. This experiment resulted in a yield and productivity of 0.41 and 0.20 g/L x h, respectively. In the integrated hydrolysis, fermentation, and recovery process, 60 g/L CFAX and 5 g/L xylose produced 24.67 g/L ABE and resulted in a higher yield (0.44) and a higher productivity (0.47 g/L x h). CFAX was hydrolyzed by xylan-hydrolyzing enzymes, and ABE were recovered by gas stripping. This investigation demonstrated that integration of hydrolysis of CFAX, fermentation to ABE, and recovery of ABE in a single system is an economically attractive process. It is suggested that the culture be further developed to hydrolyze CFAX and utilize all xylan sugars simultaneously. This would further increase productivity of the reactor.