Analytical Grade Chemicals Research Articles

Determination of Phytochemicals and Some Elemental Compositions of Watermelon from Sokoto, Nigeria

Currently, many people are battling with infectious and chronic diseases in the country. More especially, the chronic diseases are associated with the level of ingredients in the food we take in, therefore, the easiest way to remedy our challenges is to monitor levels of chemicals in foods, especially the ones around us at affordable prices. Watermelon is a typical fruit that is conspicuous in the state, and can be a source of important natural chemicals for body health. The objective of this study was to perform a phytochemical and an elemental analysis of water melon grown in Sokoto state, Nigeria. The phytochemicals were determined with appropriate methods and chemicals of analytical grade, and elements are determined using atomic absorption spectroscopy. Regarding phytochemicals, flavonoids, alkaloids, saponins, and steroids were determined. Sodium levels in the seed, pulp, and peel reveals 0.40±0.02 (ppm), 3.0±0.01 (ppm), 2.00±0.01(ppm) respectively; potassium levels in the seed, pulp, and peel reveals, 5.000 ±0.04(ppm), 0.15±0.02 (ppm), 3.00±0.02 (ppm) respectively. Calcium determination shows values 20±1.0 (ppm), 20.0±1.00 (ppm), 20.30±0.3 (ppm) in seed, pulp, and peel respectively; and magnesium levels in seed, pulp, and peel are, 2.20±0.001 (ppm), 6.0±0.01 (ppm), and 0.40±0.03 (ppm) respectively. Iron levels of seed, pulp, and peel of watermelon reveals, 1.01±0.02 (ppm), 0.10±0.01(ppm), 15.00±0.2 (ppm) respectively; and Zinc 15±0.20 (ppm), 20.0±2.0 (ppm), and 10.00±1.05 (ppm) respectively. The watermelon contains significant amount of phytochemicals like flavonoids, alkaloids, saponins, and steroids useful in many purposes such as antimicrobial agents in neutralising the infectious diseases, a burden in the state. Also, the fruit contains useful elements in significant amount like, sodium, potassium, calcium, magnesium, iron, and zinc, that can be used in people that suffers specific nutritional problems.

Potential of cassava peel collected from Bauchi (Nigeria) as culture media for growing specific fungi

The objective of this paper is to assess the potential of using cassava peel for A. niger and penicillium growth using standard methods and chemicals of analytical grade. The result of the study indicates that, the peel is rich in proximate contents as the analyzed cassava peel is composed of 8.70% moisture, 4.89% crude protein, 6.9.00% carbohydrates, 4.00 % fat content, 8.93% ash, and 8.75% fibre. Considering the observed morphological and physical characteristics of the test fungi; therewith, A. niger, and penicillium were present. There is indication that, the two utilized media (control and formulated media) are in support of the two microbes growth and depicted positive results of degradation, as well as fermentation ability on the cassava peels. The enriched cassava agar supports higher growth of the two analyzed microbes (viz, A. niger, and penicillium). There was significant difference pertaining the colonies number made on the formulated media (cassava peel agar, and enriched cassava agar). Potato dextrose agar (PDA) shows lower colony count amounting to 3.4 0.1 CFU/ml for A. niger, and amounting to 3.3 0.2 CFU/ml for penicillium. Cassava peel agar (CPA) possessed 6.5 0.5 CFU/ml mean colony count (MCC) for A. niger, and 5.6 0.1 CFU/ml for penicillium. Enriched cassava agar media (ECA) had highest MCC as 7.8 0.1 CFU/ml with respect to A. niger, and 7.3 0.6 CFU/ml pertaining penicillium. Utilization of cassava peel as substrate due to its contents of nutrients that can meet the nutritional requirement of fungi growth is a better move. The peel may be considered as substitute and alternative substrate for producing culture media to cultivate fungi and consequently reducing pollution.

Assessment of Lipase Enzyme Production Potential of Bacteria Isolated from Oryza Sativa (Rice) Rhizosphere in Sokoto, Nigeria

The work analyzed a total five (5) different soil samples in the rhizosphere of rice from Sokoto metropolis. Therewith, the potentially of bacterial isolate was screened for the lipase production using standard methods and procedures, and chemicals of analytical grade. Bacillus spp had the highest percentage of occurrence 40%. The next was E. coli with percentage of 30%. Followed by, Flavobacterium with percentage of 20% which also show that flavobacterium appears in two locations. S.aureus had a percentage occurrence of 10% respectively. The results of this study outlined the potential of synthesizing lipase enzyme from bacteria (especially the Bacillus spp which had a highest lipase activity). In turn, microbes examined could be utilized as for industrial production and other benefits.

Physicochemical and sensory properties of cookies produced from malted sorghum and cassava grate composite flour blends

Malted sorghum - cassava flour was formulated into blends to produce cookies. Sorghum grains were sorted, washed, steeped (12 hours), malted (48 hours) and oven dried. Freshly harvested cassava roots were manually peeled, washed, grated and dried (using hybrid solar dryer at 50 oC). The dried malted sorghum grains and cassava were processed into flour and sieved. The blends were prepared by homogenously mixing malted sorghum flour and high quality cassava grate flour in varied proportion labelled as sample A (100 % Malted Sorghum Flour) Sample B (60 % Malted Sorghum Flour and 30 % Cassava Flour), sample C (70 % Malted Sorghum Flour and 30 % Cassava Flour) and Sample D (80 % Malted Sorghum Flour and 20 % Cassava Flour) separately prepared into cookies and subjected to proximate, mineral, anti-nutrients, physical and sensory analysis using standard analytical methods with chemicals of analytical grade. The result for moisture, Ash, Fat, Fibre, Protein, Carbohydrate and energy content for samples A, B, C and D respectively ranged from 3.11 % - 6.81 %; 1.41 % - 1.52 %; 21.66 % - 23.41 %; 2.20 % - 2.67 %; 4.41 % - 7.26 %; 60.86 % - 63.85 % 473.09 Kcal - 478.82 Kcal. The mineral result for sodium, potassium and calcium 15.06 mg/100 g, 12.70 mg/100 g, 14.40 mg/100 g and 17.00 mg/100 g; 24.70 mg/100 g, 28.17 mg/100 g, 21.60 mg/100 g and 25.17 mg/100 g; 11.30 mg/100 g, 13.10 mg/100 g, 10.09 mg/100 g and 10.09 mg/100 g respectively. The result recorded for colour ranged from 6.20 - 7.40; aroma was 6.30 - 7.30; taste ranged from 6.10 - 7.20; texture ranged from 5.60 - 5.90 and overall acceptability ranged from 5.70 - 6.50. It can be concluded that combination of Malted Sorghum and Cassava Flour in cookies production will help to enhance the utilization of both crops, nutritional intake, increase variety and reduce wheat importation.

Trace assay of insulin in a pharmacy drug with a paste electrode.

This paper describes the development of a voltammetric assay of insulin using a DNA immobilized onto a carbon nanotube paste electrode (CNPE), the peak potential of which was 0.2V, vs. Ag/AgCl on the CNPE. The cyclic voltammetry (CV) and square-wave (SW) stripping voltammetry parameters of the optimized conditions were determined. Low analytical working ranges of 10-80 ugL-1 CV and 0.01-0.1 ngL-1 SW were attained. The precision of the insulin concentration of 0.01 ugL-1 was 0.14 (n = 15) RSD using the optimum conditions, in which the detection limit was 0.004 ngL-1 (6.9 × 10-12M) (S/N = 3) using only an accumulation time of 400s. The developed method was applied to determine insulin in a pharmacy drug from analytical-grade chemicals (from Aldrich).

Effect of Spent Tea Leave Dosages on the Removals of Chemical Oxygen Demand and Total Phosphorus from Synthetic Food Processing Wastewater

In the food processing industries, enormous amounts of water are utilized in the manufacturing process. Therefore, wastewater discharged from food industries must be characterized to develop viable treatment technologies. In this study, the adsorption method is used throughout the investigation and spent tea leave is used as a potential adsorbent to remove the concentrations of chemical oxygen demand (COD) and total phosphorus (TP). The synthetic wastewater sample containing COD, TP, suspended solids, and ammoniacal nitrogen was prepared to mimic the actual food processing wastewater as reported in the literature. The synthetic wastewater samples were formulated using analytical grade chemicals such as glucose anhydrous, sodium dihydrogen phosphate, kaolin, and ammonium chloride. The concentrations of the targeted parameters in the synthetic samples were first identified using the calibration curves. Then, this study was carried out in batch adsorption technique to determine the removal of COD and TP using varied adsorbent dosages between 100 mg/L to 500 mg/L. This study found that TP and COD were removed up to 75.04% and 15.5%, respectively, at the adsorbent dosage of 500 mg/L. Furthermore, the trend of TP and COD removals is increased as the adsorbent doses increased due to the availability of binding sites being higher at higher adsorbent dosage. Therefore, the results from this study suggest that spent tea leave has the potential to be used as an adsorbent to treat wastewater containing organic pollutants.

Light-Responsive Proton Conductor: Record High Gain of Proton Conductivity Achieved by Photoinduced Electron-Transfer Strategy

Light-Responsive Proton Conductor: Record High Gain of Proton Conductivity Achieved by Photoinduced Electron-Transfer Strategy

A Supersmall Single-Cell Nanosensor for Intracellular K + Detection

Intracellular potassium ions (K+) play pivotal roles in many physiological processes. Several K+ sensors have been developed for probing cellular K+ fluctuations. Nevertheless, the existing solutio...

Facile synthesis of CuxZn1−xFe2O4 nanoparticles and their thermo-physical properties evaluation

Since the advent of material science, nanomaterials have been the most attractive and alluring research domain of nanotechnology with a variety of applications. Considering the significance of nanomaterials specifically in industrial progressions, the present work demonstrates facile synthesis approach of copper–zinc ferrite nanoparticles and their thermo-physical characterization and evaluation. Analytical grade chemicals were used to synthesize the respective nanoparticles employing the co-precipitation method, with the base solution of NaOH to maintain pH of the solution within range of 12–14. A series of nanoparticles were synthesized varying the amount of copper and zinc precursors, and their thermal and physical properties were evaluated using various analytical tools including XRD (X-ray diffraction), SEM (Scanning Electron Microscope), FTIR (Fourier transform infrared spectroscopy) and thermal constant analyzer.

Ultratrace Metal Speciation Analysis by Coupling of Sector-Field ICP-MS to High-Resolution Size Exclusion and Reversed-Phase Liquid Chromatography.

Techniques for metal speciation analysis with subnanomolar (ppt) detection limits in complex matrices, with simultaneous quantification of matrix elements, have become a necessity for investigating targets of trace metal binding to macromolecules and pigments at environmentally relevant concentrations. In this work we optimized the analysis of such metal binding in a custom-built HPLC-ICP-MS system. Key elements of the optimization were the choice of components for the metal-free HPLC-DAD system and sector-field ICP-MS detection (ICF-sfMS) with desolvating injection and optimization of sample handling. Protein analysis was done using ammonium bicarbonate buffer and size exclusion chromatography (SEC-ICP-sfMS), with possible addition of anion exchange chromatography. Detection of metal exchange in pigments (chlorophylls and bacteriochlorophylls) was based on reversed-phase chromatography with a methanol-acetone gradient and coupling to the ICP-sfMS via a dedicated organic matrix interface (RPC-ICP-sfMS). The resulting HPLC-DAD-ICP-sfMS system has detection limits in the picomolar range in protein buffer, limited by the maximal achievable purity of buffers/solvents and not by system sensitivity. Tests for method optimization showed that sonication, meant to increase protein solubilization, leads to artifacts of metal loss from metalloproteins. Examples for Cd binding to soybean proteins and chlorophyll, Cr binding to Arabidopsis thaliana proteins, La binding to Desmodesmus quadricauda proteins, and Cu binding to Rhodospirillum rubrum proteins and pigments are shown. These application examples demonstrate that the system is sensitive enough to detect binding of metals to proteins and pigments at background concentration levels of typical nutrient solutions made from analytical grade chemicals, equivalent to ultratrace metal concentrations in nonpolluted environments.

Application of sodium ferrate produced from industrial wastes for TOC removal of surface water.

This study aimed to investigate the effect of sodium ferrate synthesized from industrial effluents (SF-W) and that of synthetized from analytical grade chemicals (SF-O) on total organic carbon (TOC) removal from surface water. Response surface methodology (RSM) was used to optimize the operating variables such as pH, dosing rate, rapid mixing time, and gentle mixing speed on TOC removal. A TOC removal of 89.805% and 79.79% was observed for SF-O and SF-W, respectively. Ferrate as SF-O and SF-W demonstrated 26.67% and 8.51% more TOC removal at a lower dosage compared to conventional chemicals such as chlorine, ozone, poly aluminum chloride (PAC) and polyelectrolyte. The optimum conditions of the independent variables including sodium ferrate (SF-O and SF-W), pH, rapid mixing time and gentle mixing speed were found to be 1.54 mg/L and 2.68 mg/L, 8.5, 30 s at 120 rpm for coagulation followed by 20 min of gentle mixing. Economic analysis showed that the application of SF instead of conventional chemicals provides a significant reduction in operational costs by about 68%, mainly because of the reduction of chemicals and energy consumption.

Can Mesoporous TiO2-Al2O3-Supported NiMoS OR CoMoS Effectively Perform in Ultra-Deep Desulfurization of Gas Oil?

Waste aluminum foil was used for preparation of mesoporous TiO2-Al2O3 using starch as a textural modifier. The catalytic species, Mo and Ni or Co were loaded onto the mesoporous support, following incipient wetness sequential impregnation. To gain an insight into the pore dimensions effect, Ni and Mo species with the same mass ratio were loaded onto the TiO2-Al2O3, prepared from analytical grade chemicals without templating. TPR spectra, TEM images and BET analysis showed how the promoter (Ni or Co), TiO2 and the template (starch) affect the ease of reduction of Mo species, the morphology of the active MoS2 phase and the pore dimensions of the catalysts. The catalysts were employed in hydro-desulfurization process of gas oil using a fixed bed down flow microreactor at varying operating conditions, viz., temperature (320–400°C), Liquid hourly space velocity (0.5–4 h–1), H2/oil ratio of 450 v/v, and 6 MPa operating pressure. The results showed that the promotion effect prevails over the textural effect, where Ni promoted catalyst (with lower surface parameters) exhibits higher activity than Co promoted one. The dual layer catalytic bed system achieved the sulfur level less than 10 ppm.

Effect of nickel sulphate hexahydrate addition on crystal growth, structural, optical and biological applications of glycine single crystals

ABSTRACTIn the present study, single crystals of α- and γ-glycine have been successfully grown in the presence of nickel sulphate hexahydrate as an additive for the first time by using slow solvent evaporation method. The analytical grade chemicals of glycine and nickel sulphate hexahydrate were taken in five different molar ratios: 1:0.2, 1:0.4, 1:0.6, 1:0.8 and 1:1 respectively to find out the α, and γ-polymorph of glycine. The lower molar proportion of nickel sulphate hexahydrate yield only α-polymorph whereas the higher molar proportion of nickel sulphate hexahydrate yields only γ-polymorph of glycine which was confirmed by powder XRD studies. UV–Visible–NIR transmittance spectra were recorded for the samples to analyze the transparency in visible and near infrared region (NIR). The optical band gap Eg was estimated for grown nickel sulphate hexahydrate (NSH) added glycine crystals using UV–Visible–Transmission study. Functional groups present in the sample were identified by FTIR spectroscopic analysis. The in vitro antimicrobial activities of the synthesized nickel sulphate added glycine single crystals were investigated against gram positive, gram negative bacterial strains using the agar disk diffusion method.

Synthesis and characterization of hydrotalcite from South African Coal fly ash

The synthesis of adsorbents such as hydrotalcites (HT) from mineral waste (e.g. blast furnace slag) is receiving increasing attention. This study reports a novel procedure to prepare HT from a South African coal-combustion fly ash, an ubiquitous waste generated by coal-fired power stations. The second point of novelty is the optimization process, which highlights the boundary conditions for crystallization of this mineral phase. The parameters investigated for the optimization of HT synthesis from fly ash included HCl concentration, aging time and temperature, pH during the aging step, and crystallization time and temperature. The most suitable synthesis conditions were: HCl concentration of 3M, aging time of 30min, aging temperature of 65°C, pH of 11.5 during aging, crystallization time of 12h, and crystallization temperature of 70°C. HT had a large external surface area and very low microporosity. Morphologically, synthesized HT was mostly made up of sub-micron plate-like particles. The structural characteristics of HT synthesized from fly ash were similar to those of HT produced from analytical grade chemicals, except for the presence of calcite. A novel use of the waste South African Class F fly ash was a suitable feedstock for the synthesis of high quality HT under the optimized process conditions that minimized the formation of secondary undesired mineral phases such as calcite or hydrogarnet.

Structural and Dielectric Properties of Lanthanum Doped CaCu<sub>3</sub>Ti<sub>4</sub>O<sub>12</sub> for Capacitor Application

CaCu3Ti4O12 (CCTO) is a novel material with high relative dielectric constant and low loss tangent, CCTO belongs to a family of oxides of the type ACu3Ti4O12 (A=Ca, Cd, Sr, Na, Th). CaCu3Ti4O12 was first reported by Subramanian et al., to have an unusually high dielectric constant (~10,000) at 1 kHz. It is well known that dielectric properties of CCTO are strongly dependent upon the processing conditions as well as on doping. Semi-wet route was used to synthesise samples of CaCu3Ti4O12 and Ca(1-3x/2)LaxCu3Ti4O12 (x=0.01). Analytical grade chemicals, Ca(NO3)2.4H2O, La(NO3)3.6H2O, Cu(NO3)2.3H2O, titanium dioxide and citric acid having purity better than 99.95% were used as starting materials. The formation of single-phase solid solutions was confirmed by the absence characteristic lines of constituent’s oxides in the XRD patterns. Dielectric measurement of undoped and La doped CCTO has been performed using four probe novocontrol set up (ZG4) in a wide range of temperature starting from the room temperature. With La doping in CCTO there is an increase in the value of dielectric constant in comparison to undoped CCTO.

Diphenidine and its metabolites in blood and urine analyzed by MALDI-Q-TOF mass spectrometry

New psychoactive substances that are different from synthetic cannabinoids or cathinone derivatives have appeared very recently on illicit drug markets. In 2014, three reports were published on diphenidine distribution in Japan. The first study characterized diphenidine and 1-benzylpiperidine in a powdered product called ‘‘fragrance powder’’ [1], while the second determined diphenidine and 5-fluoro-AB-PINACA in a dubious herbal product [2]. The third study dealt with a fatal case in which a victim smoked a herbal product containing diphenidine together with a herbal product containing AB-CHMINACA and 5-fluoroAMB [3]. The postmortem distributions of these substances determined by gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS-MS) indicated that the main cause of death was diphenidine poisoning [3]. To date, the toxicity and metabolism of diphenidine have not been studied in any scientific context because it is a research chemical [4] with very little history of use in humans [5]. To tentatively identify unreported molecules, such as metabolites, their exact masses with sensitivities better than 0.001 Da are necessary [6]. Matrix-assisted laser desorption ionization (MALDI) can achieve a softer ionization than electrospray ionization under some conditions, and hence is suitable for the detection of intact molecules. Therefore, in the present work we examined diphenidine and its metabolites by MALDI quadrupole time-of-flight mass spectrometry (Q-TOF-MS) with a resolution better than 0.001 Da. Diphenidine [1-(1,2-diphenyl-ethyl)piperidine]-HCl, acetonitrile (ACN), and ethanol that were suitable for LC– MS, and other chemicals of analytical grade were obtained from Wako Pure Chemicals (Osaka, Japan). a-Cyano-4hydroxycinnamic acid (CHCA) was obtained from SigmaAldrich (St. Louis, MO, USA), and amitriptyline, to be used as internal standard (IS) for diphenidine quantitation, was obtained from Astellas Pharma (Tokyo, Japan). Pure water with a specific resistance of 18 MX cm was used (Millipore, Bedford, MA, USA). Blood samples from healthy volunteers under permission were used as blank samples, and those spiked with several amounts of diphenidine were used as positive samples. The postmortem blood and urine samples of a victim intoxicated mainly with diphenidine were obtained at autopsy performed in our laboratory [3]. Individual stock solutions of diphenidine and amitriptyline were prepared separately by dissolving the appropriate amount of each compound in ethanol at 1 mg/ ml. The solutions were stored at -20 C. Working calibration solutions and quality control solutions were prepared daily by diluting the stock solution of diphenidine with blank blood at 1–100 ng/ml. Amitriptyline at 50 ng/ ml in blood was used as IS for the determination. Diphenidine and its metabolites in a sample were extracted and detected together. One microliter of IS and 59 ll of water were added to 20 ll of blood or urine placed in a tube (Eppendorf AG, Hamburg, Germany) using a MICROMAN M10 pipette (Gilson S.A.S., Villiers le Bel, France), and the mixture was centrifuged at 10,000 g for 3 min. The supernatant (72 ll) was placed in a new tube, & Kayoko Minakata kminakat@hama-med.ac.jp

Antiepileptic and antipsychotic activities of standardized Śilājatu (Shilajit) in experimental animals.

Background:Śilājatu (Shilajit; SJ) is claimed in traditional Indian medical practice to be useful in the treatment of nervous disorders, epilepsy and as antistress.Aim:To investigate whether SJ possesses antiepileptic and antipsychotic activities in rodents.Materials and Methods:Isonicotinyl hydrazine (INH), pentylenetetrazole (PTZ), apomorphine, phenytoin, diazepam, haloperidol and other chemicals of analytical grade were procured from standard companies. The antiepileptic activity of SJ was assessed using maximal electro shock (MES)-induced seizures in rats, INH and PTZ-induced seizures in mice. The antipsychotic effect of SJ was evaluated using apomorphine-induced climbing and stereotyped behaviours respectively, in mice and rats.Settings and Designs:SJ (25 and 50 mg/kg, p.o.) was given orally once daily for 15 days in all the rodent models. On the test day, SJ was administered 1 h prior to electric shock or chemical inducers (INH/PTZ/apomorphine) in experimental animals; the animals were then observed for different phases of seizures and psychotic behaviours. In addition, gamma-aminobutyric acid (GABA) content in the brain of rats and mice was estimated in seizure models.Statistical Analysis:The data were expressed as mean ± standard error of mean. Statistical comparisons were performed by one-way ANOVA followed by Tukey's post-test using Graph Pad Prism version 5.0, USA. A P < 0.05 was considered significant.Results and Conclusions:SJ pretreatment significantly inhibited the seizures induced by MES, INH and PTZ in a dose dependent manner. Further, SJ augmented brain GABA levels to normal, decreased by INH and PTZ in mice brain. SJ pretreatment also significantly inhibited the climbing and stereotyped behaviours induced by apomorphine. The present data seems to confirm the antiepileptic activity of SJ which may be because of enhancing the GABAergic system. The antipsychotic activity observed may be due to anti-dopaminergic and/or GABA-mimetic actions.

Effect of zinc acetate addition on crystal growth, structural, optical, thermal properties of glycine single crystals

In the present study, γ-glycine has been crystallized by using zinc acetate dihydrate as an additive for the first time by slow solvent evaporation method. The second harmonic conversion efficiency of γ-glycine crystal was determined using Kurtz and Perry powder technique and was found to be 3.66times greater than that of standard inorganic material potassium dihydrogen phosphate (KDP). The analytical grade chemicals of glycine and zinc acetate dihydrate were taken in six different molar ratios: 1:0.2, 1:0.4, 1:0.6, 1:0.7, 1:0.8, and 1:0.9 respectively to find out the γ-polymorph of glycine. The lower molar concentration of zinc acetate yield only α-polymorph where as the higher molar concentration of zinc acetate inhibits the γ-polymorph of glycine which was confirmed by single crystal XRD and powder XRD studies. Inductively coupled plasma optical emission spectrometry (ICP-OES) was carried out to quantify the concentration of zinc element in the grown glycine single crystals. The concentration of zinc element in the presence of grown γ-glycine single crystal is found to be 0.73ppm. UV–Visible–NIR transmittance spectra were recorded for the samples to analyse the transparency in visible and near infrared region (NIR). The optical band gap Eg was estimated for γ-glycine single crystal using UV–Visible–NIR study. Functional groups present in the samples were identified by FTIR spectroscopic analysis. Differential scanning calorimetry technique was employed to determine the phase transition, thermal stability and melting point of the grown crystal.

Preliminary Phytochemical Screening and Antispasmodic Activity of Artemisia macrocephala Jacquem

The current work describes the antispasmodic action of Artemisia macrocephala, which is achieved via blocking of the calcium channels. This explains its traditional use as an antispasmodic.The crude methanolic extract of A. macrocephala was studied for possible relaxant effect(s) on spontaneous rabbits’ jejunum preparations. Analytical-grade chemicals were used in the experimental protocols. A. macrocephala gave positive tests for flavonoids, saponins, glycosides, alkaloids, and terpenes. A. macrocephala caused relaxation of spontaneous rabbits’ jejunum preparations (n=6) at a dose of 10.0 mg/mL (EC50 = 6.95 ± 0.20 mg/mL; 95% CI: 6.2 to 7.5). Contractions induced by 80 mM potassium chloride (KCl) were also relaxed by the A. macrocephala at dose of 10.0 mg/mL. Attempting to find an explanation for the possible mode of action, we found that, A. macrocephala at concentration of 1.0 mg/mL produced rightward shift in the calcium chloride curves, with EC50 value of –1.65 ± 0.02 log [Ca++] M vs control with EC50 value of –2.44 ± 0.043 for calcium chloride curves. At a concentration of 1.0 mg/mL it could produce 52.4% of the control response at log [Ca++] M = –1.6. Similarly, verapamil at a concentration of 0.1 μM produced a rightward shift, with EC50 value of –1.74 ± 0.026 log [Ca++] M (95% CI: –1.66 to –1.82; n=6) vs control with EC50 value of –2.45 ± 0.05 log [Ca++] M (95% CI: – 2.23 to –2.91; n=6). The right shift of the EC50 values is justification for the folkloric use of A. macrocephala as an antispasmodic, suggesting that the possible mode of action is through calcium channel blockade.

Two Atypical l-Cysteine-regulated NADPH-dependent Oxidoreductases Involved in Redox Maintenance, l-Cystine and Iron Reduction, and Metronidazole Activation in the Enteric Protozoan Entamoeba histolytica

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular L-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and L-cystine to L-cysteine and also function as ferric and ferredoxin-NADP(+) reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower K(m) and higher k(cat)/K(m) values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred L-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, L-cysteine/L-cystine homeostasis, iron reduction, and the activation of metronidazole.