[6]-Gingerol-induced cell cycle arrest, reactive oxygen species generation, and disruption of mitochondrial membrane potential are associated with apoptosis in human gastric cancer (AGS) cells.

Ginger (Zingiber officinale Roscoe), a monocotyledonous herb, is widely used as an herbal medicine owing to the phytoconstituents it possesses. In the current study, the quantity of [6]-gingerol, the major phenolic ketone, in the fresh ginger and dried ginger rhizome was found to be 6.11 µg/mg and 0.407 µg/mg. Furthermore, [6]-gingerol was assessed for its antiapoptotic effects in human gastric adenocarcinoma (AGS) cells evidenced by acridine orange/ethidium bromide staining technique and Annexin-V assay. An increase in reactive oxygen species (ROS) generation led to a decrease in mitochondrial membrane potential (MMP) and subsequent induction of apoptosis. Results disclose that perturbations in MMP are associated with deregulation of Bax/Bcl-2 ratio at protein level, which leads to upregulation of cytochrome-c triggering the caspase cascade. These enduringly suggest that [6]-gingerol can be effectively used for targeting the mitochondrial energy metabolism to manage gastric cancer cells.

Antiapoptotic effects of vitamins C and E against cypermethrin-induced oxidative stress and spermatogonial germ cell apoptosis.

Toxicological studies have demonstrated the relation between use of agrochemicals and fertility issues within males. Thus, the present study aimed to elucidate the propensity of cypermethrin (CYP) in bringing testicular germ cell apoptosis and effective attenuation by vitamins C and E in caprines. Reproductive toxicity of CYP was evaluated using histomorphological, cytological, and biochemical changes in the testicular germ cells in dose-dependent (1, 5, 10 µg/mL) and time-dependent (4, 6, 8 h) manner. Histological and ethidium bromide/acridine orange fluorescence staining exhibited that vitamins C and E (0.5 and 1.0 mM) successfully diminished the CYP-induced testicular germ cells apoptosis. CYP exposure along with vitamins C and E supplementation also resulted in significantly increased ferric reducing antioxidant power activity along with the antioxidant enzymes, namely catalase, superoxide dismutase, and glutathione-s-transferase, and decreased lipid peroxidation in testicular germ cells. Thus, vitamins C and E ameliorated CYP-induced testicular germ cell apoptosis, thereby preventing spermatogonial cells degeneration and male infertility.

Modulation of Hydrogen Peroxide-Induced Oxidative Stress in Human Neuronal Cells by Thymoquinone-Rich Fraction and Thymoquinone via Transcriptomic Regulation of Antioxidant and Apoptotic Signaling Genes.

Nigella sativa Linn. (N. sativa) and its bioactive constituent Thymoquinone (TQ) have demonstrated numerous pharmacological attributes. In the present study, the neuroprotective properties of Thymoquinone-rich fraction (TQRF) and TQ against hydrogen peroxide- (H2O2-) induced neurotoxicity in differentiated human SH-SY5Y cells were investigated. TQRF was extracted using supercritical fluid extraction while TQ was acquired commercially, and their effects on H2O2 were evaluated using cell viability assay, reactive oxygen species (ROS) assay, morphological observation, and multiplex gene expression. Both TQRF and TQ protected the cells against H2O2 by preserving the mitochondrial metabolic enzymes, reducing intracellular ROS levels, preserving morphological architecture, and modulating the expression of genes related to antioxidants (SOD1, SOD2, and catalase) and signaling genes (p53, AKT1, ERK1/2, p38 MAPK, JNK, and NF-κß). In conclusion, the enhanced efficacy of TQRF over TQ was likely due to the synergism of multiple constituents in TQRF. The efficacy of TQRF was better than that of TQ alone when equal concentrations of TQ in TQRF were compared. In addition, TQRF also showed comparable effects to TQ when the same concentrations were tested. These findings provide further support for the use of TQRF as an alternative to combat oxidative stress insults in neurodegenerative diseases.

Photo-induced biosynthesis of silver nanoparticles using aqueous extract of Erigeron bonariensis and its catalytic activity against Acridine Orange.

The green synthesis of silver nanoparticles (AgNPs) has reduced the pollution load in the environment to a greater extent by avoiding the use of hazardous chemicals. In the present work we have developed an ecofriendly and zero cost approach for the green synthesis of more stable and spherical AgNPs using aqueous extract of Erigeron bonariensis (AEE) which act as both reducing and stabilizing agent. The reaction of AEE and AgNO3 was carried out in direct sunlight for the instant biosynthesis of AgNPs within minutes. The biosynthesis was monitored by UV-vis spectroscopy which exhibited a sharp SPR band at 442 nm and 435 nm after 5 and 35 min of sunlight exposure. The optimum conditions for biosynthesis of AgNPs were found to be 2.5mM AgNO3 concentration, 1.5% (v/v) of AEE inoculum dose and 35 min of sunlight exposure. Presence of spherical AgNPs with average size 13 nm was confirmed by SEM and TEM analysis. The XRD and SAED analysis confirmed the crystalline nature of the AgNPs where the Bragg's diffraction pattern at (111), (200), (220) and (311) corresponded to face centered cubic crystal lattice of metallic silver. The average roughness of the synthesized AgNPs was 3.21 nm which was confirmed by AFM analysis. FTIR analysis was recorded between 4000 and 400 cm(-1) which confirmed the involvement of various functional groups in the synthesis of AgNPs. The AgNPs thus obtained showed catalytic activity towards degradation of Acridine Orange (AO) without involvement of any hazardous reducing agent. The concentration dependent catalytic activity of the synthesized AgNPs was also monitored using 1, 2 and 3 mL of silver colloids and was found that the degradation of AO followed pseudo first-order kinetics.

Monitoring stepwise proteolytic degradation of peptides by supramolecular domino tandem assays and mass spectrometry for trypsin and leucine aminopeptidase.

A label-free optical detection method has been designed that allows direct monitoring of enzymatic peptide digestion in vitro. The method is based on the addition of a reporter pair, composed of the macrocyclic host cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO), to detect the proteolytic degradation of peptides. The enzymatic activity of trypsin and leucine aminopeptidase (LAP) was investigated using H-LSRFSWGA-OH as a substrate. The substrate as well as the intermediary and final products (i.e., H-FSWGA-OH and phenylalanine) formed during its enzymatic hydrolysis differ in their binding affinity to the receptor CB7, which results in varying degrees of dye displacement and, therefore, different fluorescence intensities. CB7 showed a relatively weak binding constant of K approximately 10(4) M(-1) with the substrate, a relatively strong binding constant of K > or = 10(6) M(-1) with H-FSWGA-OH (which is a final product formed by trypsin digestion and the intermediary product formed during the enzymatic activity of LAP), and a moderate binding constant of K < or = 10(5) M(-1) with phenylalanine. Owing to this differential binding affinity of CB7 with the substrate and the corresponding products, the digestion of a peptide by trypsin was followed as a decrease in fluorescence signal, while the complete degradation of the peptide by LAP was monitored as a decrease and a subsequent increase in fluorescence signal. The k(cat)/K(M) value for trypsin (2.0 x 10(7) min(-1) M(-1)) was derived from the change in fluorescence signal with time. Additionally, the complete degradation of the peptide by LAP was also followed by mass spectrometry. The use of a supramolecular sensing ensemble (macrocyclic host and dye) as a fluorescent reporter pair gives this method the flexibility to adapt for monitoring the stepwise degradation of different biologically relevant peptides by other proteases.

Screening and mechanism study of components targeting DNA from the Chinese herb Lonicera japonica by liquid chromatography/mass spectrometry and fluorescence spectroscopy.

A method which involves combination of centrifugal ultrafiltration sampling with high-performance liquid chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD-MS) analysis was established for screening bioactive compounds binding to calf thymus deoxyribonucleic acid (ct-DNA) from the extracts of Lonicera japonica. Four compounds were screened out and identified as rutin, quercetin-3-O-glucoside, luteolin-7-O-glucoside and lonicerin, based on the comparison of retention time, UV spectra and MS data with those of standards. The DNA-binding capabilities of the latter three flavonoids were found for the first time. The binding mechanisms of rutin, quercetin-3-O-glucoside and luteolin-7-O-glucoside with ct-DNA at the molecular level were explored using acridine orange (AO) as a fluorescence probe. Groove binding is the most appropriate binding mode of these three flavonoids to DNA, according to ultraviolet absorption and fluorescence spectra, as well as melting temperature (T(m)) curves and viscosity measurements. The binding constants of rutin, quercetin-3-O-glucoside and luteolin-7-O-glucoside with DNA-AO complex were 3.81 x 10(3), 3.37 x 10(3) and 5.50 x 10(3) L/mol, respectively.

Kinetics and thermodynamics of basic dye sorption on phosphoric acid esterifying soybean hull with solid phase preparation technique.

In this paper, the solid phase preparation method of a cationic sorbent, which bears hydroxyl groups of phosphoric acid derived from esterified soybean hull (ESH), was reported. The sorption kinetics and thermodynamics of two basic dyes, acridine orange (AO) and malachite green (MG), from aqueous solution onto ESH were investigated with a batch system. The isothermal data of dye sorptions followed the Langmuir model better than the Freundlich model. The maximum sorption capacity (Q(m)) of ESH for AO and MG was 238.1 mg/g and 178.57 mg/g, respectively. The dye sorption processes could be described by the pseudo-second-order kinetic model. The thermodynamic study indicated that the dye sorptions were spontaneous and exothermic. Lower temperatures were favorable for the sorption processes.

[Fluorescence resonance energy transfer quenching method for determination of arsenic with acridine orange-rhodamine B].

A new method for the determination of trace arsenic was developed by using fluorescence resonance energy transfer from acridine orange (AO) to rhodamine B (RB). It was found that under the condition of lambda(ex)/lambda(em) = 470/580 nm, effective energy transfer could occur between AO and RB in the dodecyl benzene sodium sulfonate solution. The fluorescence intensity of RB was diminished by molybdoarsenide which was formed by the reaction of arsenic (V) with molybdate in sulfuric acid medium. The detection limit of this method was 2. 56 microg x L(-1). This method was used for the determination of trace arsenic in tea. The range of determination for arsenic was 0.01-0.25 mg x L(-1). The relative standard deviation for the determination of arsenic was 0.48%-0.64%. The recoveries for the addition of 0.01-0.03 mg x L(-1) arsenic were 98%-103%. The method has been applied to the determination of arsenic with satisfactory results.

Identification of inhibitors of ribozyme self-cleavage in mammalian cells via high-throughput screening of chemical libraries.

We have recently described an RNA-only gene regulation system for mammalian cells in which inhibition of self-cleavage of an mRNA carrying ribozyme sequences provides the basis for control of gene expression. An important proof of principle for that system was provided by demonstrating the ability of one specific small molecule inhibitor of RNA self-cleavage, toyocamycin, to control gene expression in vitro and vivo. Here, we describe the development of the high-throughput screening (HTS) assay that led to the identification of toyocamycin and other molecules capable of inhibiting RNA self-cleavage in mammalian cells. To identify small molecules that can serve as inhibitors of ribozyme self-cleavage, we established a cell-based assay in which expression of a luciferase (luc) reporter is controlled by ribozyme sequences, and screened 58,076 compounds for their ability to induce luciferase expression. Fifteen compounds able to inhibit ribozyme self-cleavage in cells were identified through this screen. The most potent of the inhibitors identified were toyocamycin and 5-fluorouridine (FUR), nucleoside analogs carrying modifications of the 7-position and 5-position of the purine or pyrimidine bases. Individually, these two compounds were able to induce gene expression of the ribozyme-controlled reporter approximately 365-fold and 110-fold, respectively. Studies of the mechanism of action of the ribozyme inhibitors indicate that the compounds must be incorporated into RNA in order to inhibit RNA self-cleavage.