Evaluation of bioactive components from natural products for prevention of toxicity and neurological disorders

Nature has created almost an inexhaustible array of molecular entities, which stands as an infinite resource for novel therapeutics and scaffolds for designing of efficacious drugs for a multitude of diseases. The synthetic products gained attention due to its cost and time effectiveness, easy quality control, stringent regulation and quick effects, but their safety and efficacy often remain questionable, resulting in the dependence on the natural products by more than 80% of the total population in the developing world. This study deals with the characterization of proteins, peptides and small organic molecules isolated from natural sources like a medicinal plant Aristolochia indica, Russell’s viper (Daboia russelli russelli) venom (RVV) and human placental extract towards possible development of drugs against various illness. The investigation was initiated on the aqueous extract of the root of A. indica, a wellknown medicinal plant used against snake envenomation in many parts of rural India. Our aim was to evaluate the root extract of this ethnomedicine from its biochemical characters. The aristolochic acid (AA) content of the extract is considerably low and the solution does not exert any detectable toxicity in animals. It contains a large number of proteins that appear to be clustered under native condition. It does not show nonspecific protease activity but strong gelatinolytic, collagenase, nuclease and peroxidase activities. It interacts with the components of RVV and partially inhibits proteolytic and L-amino acid oxidase (LAAO) activities of the venom. Collectively, the properties of the extract explain, at least in parts, neutralization of RVV-induced toxicity and also its application in wound healing and inflammatory diseases

Factor V Activator from Daboia russelli russelli Venom Destabilizes �-Amyloid Aggregate, the Hallmark of Alzheimer Disease*

Formation of plaque by fibrils of �-amyloid (A�) peptide in the brain is the characteristic feature of Alzheimer disease (AD). Inhibition of the process of aggregate formation from A�-monomer and destabilization of the aggregate could be useful for prevention and propagation of the disease respectively. Russell’s viper venom (RVV) contains protein(s) that destabilize A� aggregates as revealed from the thioflavin T assay. The active component was identified as factor V activator (RVV-V). Among the possible mechanisms of destabilization, RVV-V-mediated proteolysis was ruled out from mass spectrometric data and the thioflavinTassay. The alternate hypothesis that small peptides derived fromRVV-Vdestabilize the aggregate is better supported by experimental results. Six small peptides were synthesized using RVV-V as the template, and three unrelated peptides were synthesized to serve as controls. Destabilization ofA� aggregate by these peptides was studied using spectrofluorometric assays, atomic force microscopy, transmission electron microscopy, and confocal microscopy. Among the peptides, CTNIF and the mixture of the six peptides were most potent in converting the aggregates to the monomeric state and thus, preventing cytotoxicity in SH-SY5Y human neuroblastoma cells. The control peptides failed to show similar effects. Moreover, some of these peptides are stable in blood for 24 h. Therefore, these venomderived peptides offer an encouraging opportunity to prevent amyloidosis and may provide information to combat A

Characterization of the Aqueous Extractof the Root of Aristolochia Indica: Evaluation of its Traditional use as an Antidote for Snake Bites

Ethnopharmacological relevance: The aqueous extract of the roots of Aristolochia indica is used as a decoction for the ailment of a number of diseases including snake bite treatment. Though the alcoholic extract of the different parts of the plant are well studied,information on the aqueous extract is limited. We have estimated aristolochic acid,different enzymes,enzyme inhibitors and anti-snake venom potency of its root extract. potency of its root extract . Reverse phase–HPLC was used to quantify aristo lochic acid.Zymography,DQ- gelatin assay and atomic force microscopy were done to demonstrate gelatinase and collagenase activities of the extract

Hypothetical model of fibrin-Aβ42 co-aggregate formation and its destabilization by the plant enzymes (PE) or plasmin (PL).

<p>Fibrinogen and Aβ42 form aggregates independently that are degraded by PL. PE also degrades fibrin clot. When fibrinogen interacts with Aβ42, it forms abnormal co-aggregate. The efficiency of PE is superior to PL in destabilizing the co-aggregate. FDP stands for fibrin degradation products. Stable end products are marked bold.</p

Topographic AFM images of fibrin-plasma protein co-aggregates.

<p><b>(A-D)</b> co-aggregates of fibrin-HSA, fibrin-lysozyme, fibrin-transthyretin and fibrin-fibronectin respectively. <b>(E-H)</b> These co-aggregates were treated with plant enzyme for 24 hr and their corresponding morphological features were shown. <b>(I-L)</b> The morphology of the co-aggregates treated with plasmin for 24 hr are illustrated. Features have been described in the text. The instrumentation was same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141986#pone.0141986.g005" target="_blank">Fig 5</a>.</p

Effect of protease inhibitors.

<p>^<b>a</b> The residual activity of the inhibitor treated sample was calculated with respect to the activity of enzyme in absence of any inhibitor which was considered as 100%. The concentrations of protease inhibitors were 1 mM for all the sets. The values represent mean ± S.D., where <i>p</i> < 0.05.</p><p>Effect of protease inhibitors.</p

Reduction of co-aggregate induced cytotoxicity by the plant enzymes.

<p><b>(A)</b> MTT assay: viability of cells was plotted against the concentration of plant enzyme (black bar) and plasmin (gray bar). The viability of the untreated cells was considered as 100%. <b>(B)</b> LDH assay. The % of LDH released from cells was plotted against the concentration of plant enzyme (black bar) and plasmin (gray bar). LDH released from the co-aggregate treated cells was considered as 100%. The values of the untreated cells were subtracted from the test samples. The bars represent mean ± S.D. of five independent experiments in each set. Probability values of <i>p</i> < 0.05 were considered to represent significant differences. The probability values <i>(p</i> > 0.05) of viability and % LDH release from cells treated with co-aggregate were compared with cells treated with co-aggregates preincubated with plasmin. Insignificant difference between these two groups was observed.</p

Inhibitory effect of plant enzyme on interaction of fibrin-Aβ42 co-aggregate with neuroblastoma cells

<p><b>(A)</b> Untreated cells; <b>(B)</b> deposition of preformed fibrin-Aβ42 co-aggregates on cell surface after incubation for 48 hr at 37°C; <b>(C)</b> removal of the co-aggregates from the cell surface after incubation with the plant enzyme; <b>(D)</b> residues of fibrin-Aβ42 co-aggregates on the cell surface after incubation with plasmin. The resolutions of images E-K were variable according to the area of selection to get a clear presentable image. <b>(E)</b> untreated cells with elongated morphology showing prominent blue nuclei (DAPI stained) and very faint expression of Aβ42 appearing as red as Alexa fluor 633 conjugated anti-rabbit secondary antibody was used against rabbit polyclonal antibody to Aβ1–42 peptide; <b>(F)</b> cells treated with fibrin-Aβ42 co-aggregate showing intense greenish-yellow fluorescence (as human fibrinogen conjugated with FITC showing green fluorescence and Aβ42 showing red fluorescence colocalized) indicating penetration of the co-aggregate inside the cells and deposition on the extracellular surface. The cells have markedly different morphology from untreated cells. <b>(G)</b> Cells treated with co-aggregate preincubated with plant enzyme showing morphology and absence of localization of the co-aggregate similar to that of the untreated cells as of <b>(E)</b>; <b>(H)</b> cells treated with co-aggregate preincubated with plasmin showing failure of prevention of its localization within the cells and deposition on the cell surface. The morphology also differs from the untreated cells. The magnification of all images was 60X and resolution was 10 nm.</p

Detection and purification of fibrinolytic component from the aqueous extract of the root of <i>Aristolochia indica</i>.

<p><b>(A)</b> fibrin zymography. Lane 1, plant extract (35.5 μg); lane 2, plasmin (5 μg). The lanes were selected from two different zymograms run under identical conditions. <b>(B)</b> 2-D fibrin zymography of the root extract (350 μg) showing the presence of multiple components; 5 spots in the acidic zone and one trailing lane in the alkaline zone. <b>(C)</b> Transverse 0–8 M urea gradient fibrin zymography showing presence of multiple enzymes which are stable up to variable denaturant concentrations. <b>(D)</b> dot blot analysis of 1, plasmin; 2, BSA and 3, Russell’s viper venom L-amino acid oxidase; 4, <i>A</i>. <i>indica</i> root extract developed against rabbit polyclonal human plasminogen antibody. <b>(E)</b> DEAE cellulose chromatography of the crude extract where a linear gradient of 0–1 M NaCl was applied. The bar represents the active fractions that were pooled; <b>(F)</b> substrate affinity chromatogram of the pooled fractions from DEAE-chromatography. The bound fractions were eluted by the application of 0-1M NaCl and the bar represents the active fractions. <b>(G)</b> SDS-PAGE of the plant extract (lane 1), unabsorbed fraction from DEAE cellulose column (lane 2), unabsorbed fraction from substrate affinity column (lane 3), absorbed fraction from substrate affinity column (lane 4) and fibrin zymography of the same sample (lane 5). The arrow indicates position of fibrinolysis. The position of the marker proteins is indicated at the left.</p