Phytochemical analysis of Moringa Oleifera leaves extracts by GC-MS and free radical scavenging potency for industrial applications

Natural extracts have been of very high interest since ancient time due to their enormous medicinal use and researcher’s attention have further gone up recently to explore their phytochemical compositions, properties, potential applications in the areas such as, cosmetics, foods etc. In this present study phytochemical analysis have been done on the aqueous and methanolic Moringa leaves extracts using Gas Chromatography-Mass spectrometry (GCMS) and their free radical scavenging potency (FRSP) studied using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical for further applications. GCMS analysis revealed an extraction of range of phytochemicals in aqueous and methanolic extracts. In aqueous, extract constituents found with high percent peak area are Carbonic acid, butyl 2-pentyl ester (20.64%), 2-Isopropoxyethyl propionate (16.87%), Butanedioic acid, 2-hydroxy-2-methyl-, (3.14%) (also known as Citramalic acid that has been rarely detected in plant extracts) and many other phytochemicals were detected. Similarly, fifty-four bio components detected in methanolic extract of Moringa leaves, which were relatively higher than the aqueous extract. Few major compounds found with high percent peak area are 1,3-Propanediol, 2-ethyl-2- (hydroxymethyl)- (21.19%), Propionic acid, 2-methyl-, octyl ester (15.02%), Ethanamine, N-ethyl-N-nitroso- (5.21%), and 9,12,15-Octadecatrienoic acid etc. FRSP for methanolic extract was also recorded much higher than aqueous extract. The half-maximal inhibitory concentration (IC₅₀) of Moringa aqueous extract observed is 4.65 µl/ml and for methanolic extract 1.83 µl/ml. These extracts can act as very powerful antioxidants, anti-inflammatory ingredient for various applications in diverse field of food, cosmetics, medicine etc.     

La3+-induced fusion of phosphatidylserine liposomes. Close approach, intermembrane intermediates, and the electrostatic surface potential.

The fusion of large unilamellar phosphatidylserine liposomes (PS LUV) induced by La3+ has been monitored using the 1-aminoapthalene-3,6,8-trisulfonic acid/p-xylenebis(pyridinium bromide) (ANTS/DPX) fluorescence assay for the mixing of aqueous contents. The fusion event is extensive and nonleaky, with up to 95% mixing of contents in the fused liposomes. However, addition of excess EDTA leads to disruption of the fusion products in a way that implies the existence of metastable intermembrane contact sites. The maximal fusion activity occurs between 10 and 100 microM La3+ and fusion can be terminated rapidly, without loss of contents, by the addition of excess La3+, e.g., 1 mM La3+ at pH 7.4. This observation is explained by the very large intrinsic binding constant (approximately 10(5) M-1) of La3+ to the PS headgroup, as measured by microelectrophoresis. Addition of 1 mM La3+ causes charge reversal of the membrane and a large positive surface potential. La3+ binding to PS causes the release of a proton. These data can be explained if La3+ can chelate to PS at two sites, with one of the sites being the primary amino group. This binding model successfully predicts that at pH 4.5 fusion occurs up to 2 mM La3+, due to reduced La3+ binding at low pH. We conclude that the general mechanism of membrane fusion includes three kinetic steps. In addition to (a) aggregation, there is (b) the close approach of the surfaces, or thinning of the hydration layer, and (c) the formation of intermembrane intermediates which determine the extent to which membrane destabilization leads to fusion (mixing of aqueous contents), as opposed to lysis. The lifetime of these intermembrane intermediates appears to depend upon La3+ binding to both PS sites.     

H+- and Ca2+-induced fusion and destabilization of liposomes

A new liposome fusion assay has been developed that monitors the mixing of aqueous contents at neutral and low pH. With this assay we have investigated the ability of H+ to induce membrane destabilization and fusion. The assay involves the fluorophore 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and its quencher N,N'-p-xylylenebis(pyridinium bromide) (DPX). ANTS is encapsulated in one population of liposomes and DPX in another, and fusion results in the quenching of ANTS fluorescence. The results obtained with the ANTS/DPX assay at neutral pH give kinetics for the Ca2+-induced fusion of phosphatidylserine large unilamellar vesicles (PS LUV) that are very similar to those obtained with the Tb3+/dipicolinic acid (DPA) assay [Wilschut, J., & Papahadjopoulos, D. (1979) Nature (London) 281, 690-692]. ANTS fluorescence is relatively insensitive to pH between 7.5 and 4.0. Below pH 4.0 the assay can be used semiquantitatively by correcting for quenching of ANTS due to protonation. For PS LUV it was found that, at pH 2.0, H+ by itself causes mixing of aqueous contents, which makes H+ unique among the monovalent cations. We have shown previously that H+ causes a contact-induced leakage from liposomes composed of phosphatidylethanolamine and the charged cholesteryl ester cholesteryl hemisuccinate (CHEMS) at pH 5.0 or below, where CHEMS becomes protonated. Here we show that H+ causes lipid mixing in this pH range but not mixing of aqueous contents. This result affirms the necessity of using both aqueous space and lipid bilayer assays to comprehend the fusion event between two liposomes.     

Rheological and Physicochemical Studies on Emulsions Formulated with Chitosan Previously Dispersed in Aqueous Solutions of Lactic Acid

Chitosan, a natural, cationic polysaccharide, may be a hydrocolloid strategic to formulate acidic food products, as it can act as both bio-functional and technofunctional constituent. Typically, acetic acid is used to disperse chitosan in aqueous media, but the use of this acid is limited in food formulations due to its flavor. In this study, chitosan was firstly dispersed (0.1% m/V) in lactic acid aqueous solutions (pH 3.0, 3.5 or 4.0), and then evaluated regarding its thickener and emulsion stabilizer properties. O/W emulsions were prepared and characterized in terms of rheological properties, droplets average diameters and droplets ζ-potential. Emulsions containing chitosan were 3 times more viscous than controls without chitosan, and presented storage modulus (G’) higher than loss modulus (G”). Furthermore, they displayed two different populations of droplets (average diameters of 44 and 365 nm) and positive ζ-potential values (+50 mV). Droplets average diameters and ζ-potential did not present significant changes (p > 0.05) after storage at 25 °C during 7 days. This study showed that i) food organic acids other than acetic acetic acid can be used to disperse chitosan for technological purposes, and ii) chitosan dispersed at very low concentrations (0.1 m/V %) had relevant effects on rheological and physicochemical aspects of food-grade emulsions.     

Hydrogels obtained from an original catanionic system for efficient formulation of boron wood-preservatives

A new catanionic system associating amphiphilic carnosine (βAlaHisC8) and lauric acid forms supramolecular hydrogel at a very low concentration. This gel was investigated and we checked the validity of the concept of hydrogel utilization to reduce boron leachability and to develop new wood protection treatments. Impregnation with 5% aqueous borax solution (w/w) and 0.3% gelator agent (w/w) fosters improvement in the resistance of Scots pine sapwood subjected to water leaching toward the brown-rot fungus Poria placenta, while samples treated with 5% aqueous borax solution were partially degraded by the fungus. These results clearly indicate the effectiveness of hydrogel to retain boron in wood.     

High pressure liquid chromatographic identification of hyaluronic acid and chondroitin sulphate disaccharides.

In this report we describe a system capable of resolving all of the known unsaturated disaccharides derived from the chondroitin sulphates, dermatan sulphate and hyaluronic acid by chondroitinase digestion. This system is superior to others in that the non-sulphated and mono-, di- and tri-sulphated disaccharides can be separated with good resolution in approximately 40 min in an isocratic solvent. The system employs an amino-cyano silica gel column (Whatman Partisil 5 PAC, 25 cm) and is eluted with an isocratic solvent consisting of 48% (v/v) acetonitrile, 14% (v/v) methanol and 38% (v/v) aqueous buffer. This aqueous buffer contains 0.5 M Tris-HCl, 0.1 M boric acid, 23.4 mM sulphuric acid, pH 8.0. UV absorption is monitored at 229 nm and for most disaccharides as little as 150 ng can be reliably determined. The addition of boric acid to the eluent is essential for good resolution of all components and the addition of low concentrations of sulphuric acid is used to control the elution times of various components. The system was applied to the analysis of glycosaminoglycan standards and excellent agreement with previous compositional analyses was obtained.     

Optimization of acetonitrile co-solvent and copper stoichiometry for pseudo-ligandless click chemistry with nucleic acids

The copper(I) catalyzed azide-alkyne cycloaddition 'click' reaction yields a specific product under mild conditions and in some of the most chemically complex environments. This reaction has been used extensively to tag DNA, proteins, glycans and only recently RNA. Click reactions in aqueous buffer typically include a ligand for Cu(I), however we find that acetonitrile as a minor co-solvent can serve this role. Here we investigate the click labeling of RNA and DNA in aqueous buffer to determine the relationship between the stoichoimetry of Cu(I) and the acetonitrile co-solvent that affects nucleic acid stability. We find that very low concentrations of acetonitrile perform equally well and obviate the need for any additional Cu(I) stabilizing ligand. These pseudo-ligandless reaction conditions are optimal for nucleic acids click conjugations.     

Sequencing Batch Reactor Biodegradation of Hydrolyzed Sarin as Sole Carbon Source

The chemical nerve agent sarin (o-isopropyl methylphosphonofluoridate) was hydrolyzed at 7.18 weight percent in aqueous sodium hydroxide yielding primarily o-isopropyl methylphosphonic acid (IMPA). This hydrolysate was diluted and fed as sole carbon source to activated sludge in an aerobic sequencing batch reactor. Feed chemical oxygen demand (COD) concentrations ranged from approximately 2500 mg/L (initial) to 5000 mg/L (final). The reactor was operated essentially on a 15-day hydraulic residence time. Overall COD removal efficiency was 86.2% and the IMPA in the feed was converted to methylphosphonic acid. MICROTOX® and Daphnia magna aquatic toxicity testing showed the effluent to be of very low toxicity to aquatic test organisms. The final MPA product was effectively absorbed by Phoslock™, which is a lanthanide modified clay.     

Reduction of dehydroascorbic acid at low pH

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.     

Structure of strand-separted DNA in different environments studied by linear dichroism

We have studied the denaturation of DNA by linear dichroism (LD) techniques in the following systems: (1) in aqueous solution at low pH, (2) in 90% (w/w) ethylene glycol and in 90% glycerol solutions, and (3) in a matrix of polyvinyl alcohol (PVA). We report that denatured DNA at low ionic strength can be oriented by shear forces in all these systems and that it exhibits positive LD with markedly varying LD/A over the absorption band centered at 260 nm (A being the ordinary absorbance) as compared to the negative LD with approximately constant LD/A of ordinary native DNA. These results are interpreted in terms of a large tilt of the planes of the DNA bases. DNA adopts denatured forms in (1) aqueous solutions at pH < 3.5, (2) ethylene glycol and glycerol at low Na⁺ concentrations, and (3) PVA after heating. If the denaturation is done in dry PVA, a complete re-formation of the double helix is obtained after humidifying (60% w/w) the matrix. This shows that the matrix can keep the two strands in a position allowing a perfect fitting on reassociation. Previous attempts to determine any intrinsic LD of denatured DNA have failed, probably due to the fact that, unless a very low ionic strength is used, the flexibility of the single strands prevents the orientation required to give a detectable LD signal even with recently developed high-sensitivity measurement techniques.     

Reduction of dehydroascorbic acid at low pH

Ascorbic acid and dehydroascorbic acid are unstable in aqueous solution in the presence of copper and iron ions, causing problems in the routine analysis of vitamin C. Their stability can be improved by lowering the pH below 2, preferably with metaphosphoric acid. Dehydroascorbic acid, an oxidised form of vitamin C, gives a relatively low response on the majority of chromatographic detectors, and is therefore routinely determined as the increase of ascorbic acid formed after reduction. The reduction step is routinely performed at a pH that is suboptimal for the stability of both forms. In this paper, the reduction of dehydroascorbic acid with tris-[2-carboxyethyl] phosphine (TCEP) at pH below 2 is evaluated. Dehydroascorbic acid is fully reduced with TCEP in metaphosphoric acid in less than 20 min, and yields of ascorbic acid are the same as at higher pH. TCEP and ascorbic acid formed by reduction, are more stable in metaphosphoric acid than in acetate or citrate buffers at pH 5, in the presence of redox active copper ions. The simple experimental procedure and low probability of artefacts are major benefits of this method, over those currently applied in a routine assay of vitamin C, performed on large number of samples.     

Economic comparison of multiple techniques for recovering leaf protein in biomass processing

Leaf protein concentrates (LPC) can be used as a valuable co-product to cellulosic biofuel production and can also mitigate the food versus fuel controversy. Two major approaches have been considered for LPC production: a well-characterized mechanical pressing method and a less studied method involving aqueous extraction with recovery using ultrafiltration. Experimental results with switchgrass extracts show low protein recovery after filtration, particularly if protein is recovered after cellulose hydrolysis. Economic modeling suggests that aqueous extraction costs less than mechanical pressing, but due to lower protein yields and lower quality, overall profit is higher for mechanical pressing versus aqueous extraction ($26/Mg feedstock vs. $14/Mg). If modest improvements can be made in extraction yields, filtration recovery, and protein quality, then the profitability of the aqueous extraction approach can be increased to $37/Mg feedstock. This study suggests that aqueous extraction is a viable alternative for LPC co-production in a biorefinery if key improvements can be made in the process.     

Simple procedures for the rapid cleavage of ester lipids and for the large-scale isolation from brain of cerebroside sulfate

The ester groups of glycerophospholipids in tissue extracts can be cleaved in less than 10 min at room temperature if the lipids are extracted with hexane-isopropanol and the filtrate is treated with methanolic NaOH. The resulting mixture can be treated with aqueous Na-sulfate containing sulfuric acid and partitioned to remove the inorganic reagents and hydrophilic ester degradation products. When the procedure is applied to brain lipid extracts, the addition of alkali produces a second, lower phase that contains much of the hydroxycerebroside, virtually all of the sulfatide in the extract, and small amounts of other lipids. The sulfatide can be isolated from the lower phase by neutralizing it with HCl in aqueous methanol, and partitioning with chloroform to remove nonlipid components. The lower phase is evaporated to dryness and treated with periodic acid to convert the cerebroside to a less polar product. The lipids recovered from the reaction mixture are then fractionated with a Florisil column, which yields highly purified sulfatide. Starting with 300 g of pig brain one can obtain about 1.1 g of sulfatide in 4 working days, using conventional, compact equipment. Since the precipitation step is almost complete, and the procedure can be scaled down to very low levels, the method has promise for quantitation methods and isotopic studies of sulfatide metabolism.     

Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent K_m for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.     

Use of thionitrobenzoic acid to characterize the stability of nitric oxide in aqueous solutions and in porcine aortic endothelial cell suspensions

Nitric oxide is an important vasodilator which can be biologically produced from leukocytes and endothelial cells. However, it is highly unstable, which is an obstacle to detection and quantitation. We have exploited the reactivity of nitric oxide with thiols to establish an assay based on oxidation of thionitrobenzoic acid (TNB). The oxidation of thionitrobenzoic acid and the reaction with oxygen, which was measured by employing an oxygen electrode, were examined after the addition of nitric oxide solutions. The inhibition of aggregation of human platelets after challenge with 2.5 microM adenosine diphosphate was also investigated. These studies show the following properties of nitric oxide in aqueous solutions. (i) Nitric oxide is highly reactive to oxygen. (ii) Thiols react with a labile, highly reactive nitric oxide-oxygen product. (iii) Medium with very low oxygen content increases the life span of nitric oxide in aqueous solution. We also used the nitric oxide quantitation using TNB to study the metabolism of nitric oxide by porcine aortic endothelial cells and the results show that nitric oxide added to these cells in low oxygen content solution is stable. From these studies, we conclude that deoxygenated solutions stabilize nitric oxide. An important consequence of low oxygen content at localized tissue sites may be to augment biological effects mediated by nitric oxide.     

Silicone/graphite coating for on-target desalting and improved peptide mapping performance of matrix-assisted laser desorption/ionization-mass spectrometry targets in proteomic experiments

In two-dimensional gel electrophoresis-based proteomic experiments matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting is often the technique of choice in identifying proteins. Here, we present a novel surface coating technique for MALDI-MS targets that improves manual and automatic sample analysis. A mixture of silicone and graphite is spread in the form of a thin layer over the target. Due to the hydrophobicity of the coating, aqueous solutions can be applied to relatively small spots very precisely using a robotic system. At least four times more liquid can be concentrated on the same area compared to uncoated steel targets. alpha-cyano-4-hydrocinnamic acid crystallizes in form of very small crystals evenly distributed over the surface. The search for "hot spots" during the analysis is not necessary, which supports the automatic acquisition of data. The homogeneous crystal layer can be very effectively washed on-target without encountering major sample losses. This efficient washing and the focused application of aqueous samples replace expensive and time-consuming reversed phase micro column based sample clean-ups. When analyzing peptide mixtures, the signal intensities are up to five times higher than with preparations of the same un-desalted samples on steel targets, since four times more sample can be loaded. The mass resolution remains unaffected by the surface coating. After usage the coating can be removed, followed by a new coating avoiding any carry-over of sample to the next analysis. All these properties make the precoating of MALDI-MS targets with a silicone/graphite layer an ideal technique for routine analysis in large-scale proteomic experiments.     

Simple and green approach for photoluminescent carbon dots prepared from faba bean seeds as a luminescent probe for determination of Hg+ ions and cell imaging

In this research, for the first time, a dedicated sensor was designed to detect Hg⁺ ions using photoluminescent carbon dots (CDs). Due to the preferred green synthesis of CDs from bio-resources, carbohydrate-rich faba bean seeds as a potential carbon precursor were applied to the synthesis of CDs. The CDs were prepared from the faba bean seeds using the hydrothermal method in an aqueous solution in the absence of substances such as an acid or base and any other additives. The synthesized CDs exhibited maximum emission intensity at 387 nm when excited at 310 nm and their luminescence quantum yield was calculated to be ~5.94%. Then, the fluorescence emission of CDs was examined in the presence of different metal ions. Results revealed that the CDs had good selectivity towards the Hg⁺ ions, so the fluorescence emission was significantly changed in the presence of these ions with a limit of detection (LOD) as low as 0.35 μM. Furthermore, because of their very low cytotoxicity, these CDs can be applied for cell imaging.     

A rapid column chromatographic method for the isolation of catechol-type siderophores.

The ester groups of glycerophospholipids in tissue extracts can be cleaved in less than 10 min at room temperature if the lipids are extracted with hexane-isopropanol and the filtrate is treated with methanolic NaOH. The resulting mixture can be treated with aqueous Na-sulfate containing sulfuric acid and partitioned to remove the inorganic reagents and hydrophilic ester degradation products. When the procedure is applied to brain lipid extracts, the addition of alkali produces a second, lower phase that contains much of the hydroxycerebroside, virtually all of the sulfatide in the extract, and small amounts of other lipids. The sulfatide can be isolated from the lower phase by neutralizing it with HCl in aqueous methanol, and partitioning with chloroform to remove nonlipid components. The lower phase is evaporated to dryness and treated with periodic acid to convert the cerebroside to a less polar product. The lipids recovered from the reaction mixture are then fractionated with a Florisil column, which yields highly purified sulfatide. Starting with 300 g of pig brain one can obtain about 1.1 g of sulfatide in 4 working days, using conventional, compact equipment. Since the precipitation step is almost complete, and the procedure can be scaled down to very low levels, the method has promise for quantitation methods and isotopic studies of sulfatide metabolism.     

Large-scale synthesis of a persistent trityl radical for use in biomedical EPR applications and imaging

Tetrathiatriarylmethyl radicals are ideal spin probes for biological electron paramagnetic resonance (EPR) spectroscopy and imaging. The wide application of trityl radicals as biosensors of oxygen or other biological radicals was hampered by the lack of affordable large-scale syntheses. We report the large-scale synthesis of the Finland trityl radical using an improved addition protocol of the aryl lithium monomer to methylchloroformate. A new reaction for the formal one-electron reduction of trityl alcohols to trityl radicals using neat trifluoroacetic acid is reported as well. Initial applications show that the compound is very sensitive to molecular oxygen. It has already provided high-resolution EPR images on large aqueous samples and should be suitable for a broad range of in vivo applications.     

Sonication and Other Improvements on Jeffrey's Technique for Macerating Wood

Large, up to pencil-size pieces of wood, rather than slivered wood, are macerated in Jeffrey's fluid, which consists of ca. 1:1 aqueous nitric acid and aqueous chromic acid (each 10% or weaker). Staining is done in ca. 1% solution of safranin in absolute alcohol. The intact pieces of wood can be very briefly sonicated in xylene to release isolated elements and connected cell groups. Various alternative steps are noted. Discussion focuses on the practical and theoretical advantages derived from the aforementioned innovations.