Poly(chitosan-ester-ether-urethane) Hydrogels as Highly Controlled Genistein Release Systems.

Polymeric hydrogels play an increasingly important role in medicine, pharmacy and cosmetology. They appear to be one of the most promising groups of biomaterials due to their favorable physicochemical properties and biocompatibility. The objective of the presented study was to synthesize new poly(chitosan-ester-ether-urethane) hydrogels and to study the kinetic release of genistein (GEN) from these biomaterials. In view of the above, six non-toxic hydrogels were synthesized via the Ring-Opening Polymerization (ROP) and polyaddition processes. The poly(ester-ether) components of the hydrogels have been produced in the presence of the enzyme as a biocatalyst. In some cases, the in vitro release rate of GEN from the obtained hydrogels was characterized by near-zero-order kinetics, without "burst release" and with non-Fickian transport. It is important to note that developed hydrogels have been shown to possess the desired safety profile due to lack of cytotoxicity to skin cells (keratinocytes and fibroblasts). Taking into account the non-toxicity of hydrogels and the relatively highly controlled release profile of GEN, these results may provide fresh insight into polymeric hydrogels as an effective dermatological and/or cosmetological tool.

A Selective Inhibitor of Ubiquitin-Specific Protease 4 Suppresses Colorectal Cancer Progression by Regulating ß-Catenin Signaling.

BACKGROUND/AIMS: Dysregulation of deubiquitinating enzymes (DUBs), which regulate the stability of key proteins, has been implicated in many human diseases, including cancers. Thus, DUBs can be considered as potential therapeutic targets for many diseases. Among them, USP4 has been proposed as a promising target for colon cancer drugs since USP4 controls the stability of ß-catenin, a key factor in the Wnt signaling involved in the tumorigenesis of colorectal cancer. However, developing potential DUB inhibitors has been hindered because many DUBs harbor similar active site structures and show broad substrate specificities. METHODS: By performing in vitro deubiquitinating activity assays using a chemical library, we identified several potential DUB inhibitors. Among them, only neutral red (NR) showed selective inhibitory activity on USP4 in a cell-based assay system. In colon cancer cells, NR affected the protein stability of ß-catenin, as shown by immunoblotting, and it affected the target gene expression of ß-catenin, as shown by quantitative real-time PCR. NR's potential as an anticancer drug was further estimated by colony formation and cell migration assays and by using a mouse xenograft model. RESULTS: We identified NR as an uncompetitive inhibitor of USP4 and validated its effects in colorectal cancer. NR-treated cells showed decreased ß-catenin stability and reduced expression of ß-catenin target genes. Additionally, treating colon cancer cells with NR significantly reduced colony formation and cell migration, and injecting NR into a mouse xenograft model reduced the tumor volume. CONCLUSION: The current results suggest that NR could be developed as an anticancer drug targeting USP4, and they support the possibility of developing specific DUB inhibitors as therapeutic agents.

Enhanced Bio-Electrochemical Reduction of Carbon Dioxide by Using Neutral Red as a Redox Mediator.

Microbial electrosynthetic cells containing Methylobacterium extorquens were studied for the reduction of CO2 to formate by direct electron injection and redox mediator-assisted approaches, with CO2 as the sole carbon source. The formation of a biofilm on a carbon felt (CF) electrode was achieved while applying a constant potential of -0.75 V versus Ag/AgCl under CO2 -saturated conditions. During the biofilm growth period, continuous H2 evolution was observed. The long-term performance for CO2 reduction of the biofilm with and without neutral red as a redox mediator was studied by an applied potential of -0.75 V versus Ag/AgCl. The neutral red was introduced into the systems in two different ways: homogeneous (dissolved in solution) and heterogeneous (electropolymerized onto the working electrode). The heterogeneous approach was investigated in the microbial system, for the first time, where the CF working electrode was coated with poly(neutral red) by the oxidative electropolymerization thereof. The formation of poly(neutral red) was characterized by spectroscopic techniques. During long-term electrolysis up to 17 weeks, the formation of formate was observed continuously with an average Faradaic efficiency of 4 %. With the contribution of neutral red, higher formate accumulation was observed. Moreover, the microbial electrosynthetic cell was characterized by means of electrochemical impedance spectroscopy to obtain more information on the CO2 reduction mechanism.

Assessment of reflectance confocal microscopy for non-invasive selection of optimal ovarian cortex fragments for autotransplantation.

RESEARCH QUESTION: Can reflectance confocal microscopy (RCM) be used to determine follicle density in human ovarian cortex fragments that are intended for fertility restoration? DESIGN: RCM was used on living cortex tissue fragments derived from five bovine ovaries and 13 human ovaries. All tissue fragments were cryopreserved and thawed before RCM analysis. Follicle numbers and distribution were determined by RCM and histology. Before and after RCM, general tissue viability and follicle integrity were assessed by a glucose uptake assay and neutral red staining, respectively. RESULTS: RCM can detect all stages of follicle development in living ovarian tissue to a maximum depth of 250 µm. In bovine tissue, all follicles were located within this 0-250 µm range. In human ovarian tissue, follicles were also present below the 250 µm RCM threshold, implying that only a percentage of the total number of follicles could be detected with RCM. The percentage of follicles detected by RCM appeared to be age dependent. The RCM procedure did not affect the glucose uptake by the tissue, whereas neutral red staining indicated a high level of follicle survival. CONCLUSION: In this proof of concept study, we have shown that RCM is a promising technique to determine the density of follicles ex vivo in living human ovarian cortex fragments, apparently without compromising the vitality of the tissue. Safety studies and further optimization of the RCM technique with a focus on increasing the penetration depth are required before clinical use of RCM.

Mechanistic Insight into the Photodynamic Effect Mediated by Neutral Red and a New Azine Compound in Staphylococcus aureus Cells.

The photodynamic activity of Neutral Red and the new monobrominated Neutral Red was studied in suspensions of Staphylococcus aureus. The effect of mannitol and sodium azide in the presence of 25 µm photosensitizer on lethal photosensitization were investigated. The results of the mechanistic evaluation of Neutral Red showed that both mannitol and sodium azide produced a completed protective effect after irradiation without significant differences between them. The evaluation of monobrominated Neutral Red also showed a protective effect of microorganisms with the addition of mannitol. Although sodium azide produced a protective effect of the photoinactivation, it was incomplete and less than that exhibited by mannitol. The results indicate that the starting reagent, Neutral Red, is a producer of radical species, acting through a type I mechanism, whereas the halogenated derivative of Neutral Red produced reactive oxygen species and a contribution of singlet molecular oxygen cannot be discarded in the photoinactivation of Staphylococcus aureus cells. These results, analyzed together with the previously evaluated properties of the dyes, allow us to explain the differences observed in the photoinactivation of Staphylococcus aureus mediated by both azine photosensitizers.

Chemical analysis and in vitro toxicological evaluation of aerosol from a novel tobacco vapor product: A comparison with cigarette smoke.

The recent rapid increase in the prevalence of emerging tobacco- and nicotine-containing products, such as e-cigarettes, is being driven in part by their reduced-risk potential compared to tobacco smoking. In this study, we examined emission levels for selected cigarette smoke constituents, so-called "Hoffmann analytes", and in vitro toxicity of aerosol from a novel tobacco vapor product (NTV). The NTV thermally vaporizes a nicotine-free carrier liquid to form an aerosol which then passes through tobacco, where it absorbs tobacco-derived flavors and nicotine. The NTV results were compared with those for 3R4F cigarette smoke. Chemical analysis of the NTV aerosol demonstrated that Hoffmann analyte levels were substantially lower than in 3R4F smoke and that the most were below quantifiable levels. Results from in vitro bacterial reverse mutation, micronucleus and neutral red uptake assays showed that, in contrast with 3R4F smoke, the NTV aerosol failed to demonstrate any measurable genotoxicity or cytotoxicity. The temperature of tobacco during NTV use was measured at approximately 30 °C, which may explain the lower Hoffmann analyte emission and in vitro toxicity levels. These results suggest that the aerosol from the NTV has a very different toxicological profile when compared with combustible cigarette smoke.

Electrochemical DNA Sensor Based on Carbon Black-Poly(Neutral Red) Composite for Detection of Oxidative DNA Damage.

Voltammetric DNA sensor has been proposed on the platform of glassy carbon electrode covered with carbon black with adsorbed pillar[5]arene molecules. Electropolymerization of Neutral Red performed in the presence of native or oxidatively damaged DNA resulted in formation of hybrid material which activity depended on the DNA conditions. The assembling of the surface layer was confirmed by scanning electron microscopy and electrochemical impedance spectroscopy. The influence of DNA and pillar[5]arene on redox activity of polymeric dye was investigated and a significant increase of the peak currents was found for DNA damaged by reactive oxygen species generated by Cu2+/H2O2 mixture. Pillar[5]arene improves the electron exchange conditions and increases the response and its reproducibility. The applicability of the DNA sensor developed was shown on the example of ascorbic acid as antioxidant. It decreases the current in the concentration range from 1.0 µM to 1.0 mM. The possibility to detect antioxidant activity was qualitatively confirmed by testing tera infusion. The DNA sensor developed can find application in testing of carcinogenic species and searching for new antitumor drugs.

Structural and Biophysical Characterization of the Mycobacterium tuberculosis Protein Rv0577, a Protein Associated with Neutral Red Staining of Virulent Tuberculosis Strains and Homologue of the Streptomyces coelicolor Protein KbpA.

Mycobacterium tuberculosis protein Rv0577 is a prominent antigen in tuberculosis patients, the component responsible for neutral red staining of virulent strains of M. tuberculosis, a putative component in a methylglyoxal detoxification pathway, and an agonist of toll-like receptor 2. It also has an amino acid sequence that is 36% identical to that of Streptomyces coelicolor AfsK-binding protein A (KbpA), a component in the complex secondary metabolite pathways in the Streptomyces genus. To gain insight into the biological function of Rv0577 and the family of KpbA kinase regulators, the crystal structure for Rv0577 was determined to a resolution of 1.75 Å, binding properties with neutral red and deoxyadenosine were surveyed, backbone dynamics were measured, and thermal stability was assayed by circular dichroism spectroscopy. The protein is composed of four approximate repeats with a ßαßßß topology arranged radially in consecutive pairs to form two continuous eight-strand ß-sheets capped on both ends with an α-helix. The two ß-sheets intersect in the center at roughly a right angle and form two asymmetric deep "saddles" that may serve to bind ligands. Nuclear magnetic resonance chemical shift perturbation experiments show that neutral red and deoxyadenosine bind to Rv0577. Binding to deoxyadenosine is weaker with an estimated dissociation constants of 4.1 ± 0.3 mM for saddle 1. Heteronuclear steady-state {1H}-15N nuclear Overhauser effect, T1, and T2 values were generally uniform throughout the sequence with only a few modest pockets of differences. Circular dichroism spectroscopy characterization of the thermal stability of Rv0577 indicated irreversible unfolding upon heating with an estimated melting temperature of 56 °C.

Neutral Red versus MTT assay of cell viability in the presence of copper compounds.

Copper is essential for numerous physiological functions, and copper compounds may display therapeutic as well as cytotoxic effects. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay is a standard test largely used in cytotoxicity studies. This report shows that low micromolar levels of copper compounds such as Cu(II)Urea2, Cu(II)Ser2 and CuCl2 can interfere with the MTT assay making improper the detection of formazan product of MTT reduction. Comparatively, the Neutral Red assay appears to be sensitive and showing no interference with these compounds. The lactate dehydrogenase alternative assay cannot be used because of inhibitory effect of these copper compounds on the enzyme activity.

New approach to biosensing of co-enzyme nicotinamide adenine dinucleotide (NADH) by incorporation of neutral red in aluminum doped nanostructured ZnO thin films.

Biosensing of NADH on bare electrodes has drawbacks such as high over-potential and poisoning during the oxidation reaction. To overcome this challenge a different approach has been undertaken by incorporating neutral red (NR) in Al doped ZnO (AZO) thin films using one-pot chemical bath deposition (CBD). The surface morphology of the films was hexagonal nanorods along the c-axis, perpendicular to the substrate. The thickness of the thin films were ranging from 400 to 3000nm varying dependent on time of deposition (30 to 150min). The average diameter of the nanorods was larger in the presence of neutral red (NR-AZO) with ~300nm in contrast to its absence (AZO) with ~200nm. The density of the packing of nanorods was dependent on the citrate concentration used during deposition. Control over the dopant concentration in the films was achieved by varying the area of Al foil used in the deposition solution. The selected area diffraction (SAED) and X-ray diffraction (XRD) indicated 002 plane of orientation in the nanorods. FTIR and FT-Raman analysis revealed conserved structure of NR and AZO. Chronoamperometric (CA) analysis showed a sensitivity of 0.45µAcm-2mM-1 and LoD of 22µM within the range 0.075-4mM of NADH. The biological sensing of NADH was validated by physical adsorption of NAD+ dependent-lactate dehydrogenase (LDH) on NR-AZO. CA showed sensitivity of 0.56µAcm-2mM-1 and LoD for lactate was 27µM in the range of 0.1-1mM of lactate. Further validation with real-time serum sample shows that LDH/NR-AZO correlates with the clinical values. The distinction in this study is that the organic mediator like neutral red has been incorporated into the grain structure of the ZnO thin film whereas other study with the mediators have only attempted surface functionalization. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.

Metal ion-induced supramolecular pKa tuning and fluorescence regeneration of a p-sulfonatocalixarene encapsulated neutral red dye.

The host-guest interactions and the consequent modulation in the prototropic equilibrium of a phenazine dye, neutral red, with p-sulfonatocalix[4]arene (SCX4) and p-sulfonatocalix[6]arene (SCX6) macrocyclic hosts have been investigated. Both the neutral (NR) and cationic (NRH+) forms of dyes formed inclusion complexes with SCX6, with a larger binding constant for the latter (K = 8.6 × 105 M-1versus 4.8 × 103 M-1) due to the cation receptor behavior of the calixarenes. The distinct differences in the binding constant of NR and NRH+ provided a finite tuning of pKa between 6.5 and 8.8, through a competitive binding with metal ions. Importantly, the fluorescence quenching observed in the SCX-neutral red interactions stands in contrast to the fluorescence enhancement observed with other macrocyclic hosts, such as ß-cyclodextrin and cucurbit[7]uril. This is due to the unique portal stacking interaction of NRH+ with the SCXs, compared to the axial inclusion geometry documented for the other macrocycles. The electron transfer from the SCX to the neutral red dye is adjudged to be the effective excited-state relaxation pathway leading to fluorescence quenching. In combination with the metal-ion induced fluorescence regeneration and tuning the pKa value, the SCX-neutral red system finds potential applications in drug delivery, photodynamic therapy, catalysis, and sensor applications.

Sublethal effects induced by morphine to the freshwater biological model Dreissena polymorpha.

Opioids are considered as emerging contaminants in aquatic ecosystems, mainly due to their large illicit consume worldwide. Morphine (MOR) is the main opiate and it was commonly found at measurable concentrations in freshwaters. Even though its occurrence is well documented, just limited information is available regarding its hazard to nontarget organisms. The aim of this study was of the evaluation of sublethal effects induced by MOR to the freshwater bivalve Dreissena polymorpha. We exposed mussels to two MOR concentrations (0.05 µg/L and 0.5 µg/L) for 14 days and we investigated the sublethal effects by a suite of biomarkers. The Neutral Red Retention Assay (NRRA) was used as a test of cytotoxicity, while the oxidative stress was evaluated by the activity of antioxidant and detoxifying enzymes, namely catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), and by measuring the levels of lipid peroxidation (LPO) and protein carbonylation (PCC). The genetic damage was assessed by the Single Cell Gel Electrophoresis (SCGE) assay, the DNA diffusion assay and the micronucleus test (MN test). Finally, the filtration rate of D. polymorpha was evaluated in order to investigate possible physiological effects. Both tested concentrations reduced the lysosome membrane stability of bivalves, but only the highest MOR concentration induced significant changes in the activity of antioxidant enzymes (SOD, CAT, and GPx) and increase in lipid peroxidation levels. Slight increase in primary DNA fragmentation was noticed, while no fixed genetic damage and alterations of the filtering rate were found.

Comparison of three different cell viability assays for evaluation of vanadyl sulphate cytotoxicity in a Chinese hamster ovary K1 cell line.

Previously, evaluation of sodium metavanadate (NaVO3) cytotoxicity after 24 h exposure of Chinese hamster ovary K1 (CHO-K1) cells revealed different sensitivity of the in vitro assays used starting from the neutral red (NR, 3-amino-7-dimethylamino-2-methylphenazine hydrochloride) test (detecting lysosomal and possibly the Golgi apparatus damage) as the most sensitive followed by the 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt (XTT) and resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide) tests (mitochondrial disruption). The trypan blue (TB) staining (plasma membrane permeability) showed cytotoxicity of NaVO3 at a much higher NaVO3 concentration than the above-mentioned assays. In the current study, using the same experimental approach, we have assessed the toxicity of vanadyl sulphate (VOSO4) and compared the obtained results with NaVO3 action. Unlike metavanadate, VOSO4 treatment at 24 h resulted in similar sensitivity of the NR and resazurin tests. Nevertheless, following the 48-h incubation with VOSO4, the NR test showed markedly higher sensitivity than the resazurin test when comparing the half maximal inhibitory concentration values (61 and 110 µM for the NR and resazurin test, respectively, p < 0.05). The TB staining method was the least susceptible for detecting vanadyl cytotoxicity at each exposure time point. In summary, both the NR and resazurin tests can be advocated as similarly sensitive in detection of VOSO4-induced cytotoxicity in the CHO-K1 cell line at 24 h. However, the longer incubation time with VOSO4 showed that the NR test is more sensitive than the resazurin assay. The differences in the results between the cytotoxicity tests employed probably arise from dissimilar susceptibility of the endpoints (targets) measured with these tests to the damage by vanadium. Considering this, the current and the previous studies highlight the role of lysosomes (and possibly the Golgi apparatus) apart from mitochondria in the toxicity mechanism induced by inorganic vanadium in mammalian cells.

Fabrication and Optimization of Stable, Optically Transparent, and Reusable pH-Responsive Silk Membranes.

The fabrication of silk-based membranes that are stable, optically transparent and reusable is yet to be achieved. To address this bottleneck we have developed a method to produce transparent chromogenic silk patches that are optically responsive to pH. The patches were produced by blending regenerated silk fibroin (RSF), Laponite RD (nano clay) and the organic dyes neutral red and Thionine acetate. The Laponite RD played a central role in the patch mechanical integrity and prevention of dye leaching. The process was optimized using a factorial design to maximize the patch response to pH by UV absorbance and fluorescence emission. New patches of the optimized protocol, made from solutions containing 125 µM neutral red or 250 µM of Thionine and 15 mg/mL silk, were further tested for operational stability over several cycles of pH altering. Stability, performance, and reusability were achieved over the tested cycles. The approach could be extended to other reporting molecules or enzymes able to bind to Laponite.

In vitro evaluation of inorganic and methyl mercury mediated cytotoxic effect on neural cells derived from different animal species.

To extend the current understanding of the mercury-mediated cytotoxic effect, five neural cell lines established from different animal species were comparatively analyzed using three different endpoint bioassays: thiazolyl blue tetrazolium bromide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (MTT), neutral red uptake assay (NRU), and Coomassie blue assay (CB). Following a 24-hr exposure to selected concentrations of mercury chloride (HgCl2) and methylmercury (II) chloride (MeHgCl), the cytotoxic effect on test cells was characterized by comparing their 50% inhibition concentration (IC50) values. Experimental results indicated that both these forms of mercury were toxic to all the neural cells, but at very different degrees. The IC50 values of MeHgCl among these cell lines ranged from 1.15±0.22 to 10.31±0.70µmol/L while the IC50 values for HgCl2 were much higher, ranging from 6.44±0.36 to 160.97±19.63µmol/L, indicating the more toxic nature of MeHgCl. The IC50 ratio between HgCl2 and MeHgCl ranged from 1.75 to 96.0, which confirms that organic mercury is much more toxic to these neural cells than inorganic mercury. Among these cell lines, HGST-BR and TriG44 derived from marine sea turtles showed a significantly high tolerance to HgCl2 as compared to the three mammalian neural cells. Among these neural cells, SK-N-SH represented the most sensitive cells to both chemical forms of mercury.

Lysosomal membrane stability of the mussel, Mytilus galloprovincialis (L.), as a biomarker of tributyltin exposure.

The effect of tributyltin (TBT) on the stability of hemocytic lysosome membranes of the mussel, Mytilus galloprovincialis, and the use thereof as a biomarker of TBT-induced stress, was investigated. Mussels were exposed to 0.1 and 1.0 µg/L tributyltin respectively for 4 weeks. Lysosomal membrane stability of hemocytes was tested weekly by means of the neutral red retention time (NRRT) assay, after which the mussel samples were analyzed for TBT content. The two exposed groups exhibited significantly increased (p < 0.05) whole body TBT concentrations with concomitant significant decreases (p < 0.05) in NRRT (R(2) values of 0.85 and 0.971 for lower and higher exposure groups, respectively). The higher exposure group showed a typical dose-response curve. For the control, no TBT was detected and NRRT remained stable. It was concluded that the NRRT assay could be considered as a useful technique, and lysosomal membrane destabilization a useful early warning and cellular biomarker of stress due to TBT exposure in M. galloprovincialis.

A highly sensitive NADH sensor based on a mycelium-like nanocomposite using graphene oxide and multi-walled carbon nanotubes to co-immobilize poly(luminol) and poly(neutral red) hybrid films.

Hybridization of poly(luminol) (PLM) and poly(neutral red) (PNR) has been successfully performed and further enhanced by a conductive and steric hybrid nanotemplate using graphene oxide (GO) and multi-walled carbon nanotubes (MWCNTs). The morphology of the PLM-PNR-MWCNT-GO mycelium-like nanocomposite is studied by SEM and AFM and it is found to be electroactive, pH-dependent, and stable in the electrochemical system. It shows electrocatalytic activity towards NADH with a high current response and low overpotential. Using amperometry, it has been shown to have a high sensitivity of 288.9 µA mM(-1) cm(-2) to NADH (Eapp. = +0.1 V). Linearity is estimated in a concentration range of 1.33 × 10(-8) to 1.95 × 10(-4) M with a detection limit of 1.33 × 10(-8) M (S/N = 3). Particularly, it also shows another linear range of 2.08 × 10(-4) to 5.81 × 10(-4) M with a sensitivity of 151.3 µA mM(-1) cm(-2). The hybridization and activity of PLM and PNR can be effectively enhanced by MWCNTs and GO, resulting in an active hybrid nanocomposite for determination of NADH.

Biological safety studies of gemifloxacin mesylate and related substances.

PURPOSE: The aim of this study was to evaluate the cytotoxic, phototoxic, genotoxic and photogenotoxic potential of gemifloxacin mesylate (GFM), its main synthetic impurity (SI) and one isolated and structurally elucidated degradation product (DP). METHODS: The neutral red uptake (NRU) and reduction of 2,5-diphenyl-3,-(4,5-dimethyl-2-thiazolyl)tetrazolium bromide (MTT) assays were performed as in vitro endpoints to evaluate cytotoxicity and phototoxicity in a 3T3 cell line, and predict toxicity and/or phototoxicity after systemic administration of the drug. The in vitro alkaline single-cell electrophoresis (comet) assay was used to evaluate the genotoxic and photogenotoxic potential of the substances using the same cell line. RESULTS: The results showed that the SI and the DP are more cytotoxic and phototoxic than the drug GFM using the 3T3 cell line. In the comet assay, the drug GFM was found to be more genotoxic and photogenotoxic than its related substances. CONCLUSIONS: Our findings highlight the relevance of the biological safety studies to increase the knowledge regarding the toxic potential of the related substances, which can be associated with the drug side effects and toxicity.

Antioxidant potential, cytotoxic activity and total phenolic content of Alpinia pahangensis rhizomes.

BACKGROUND: Alpinia pahangensis, a wild ginger distributed in the lowlands of Pahang, Malaysia, is used by the locals to treat flatulence. In this study, the antioxidant and cytotoxic activities of the crude aqueous methanol and fractionated extracts of Alpinia pahangensis against five different cancer and one normal cell lines were investigated. The total phenolic content of each extract and its fractions were also quantified. This is the first report on the antioxidant and cytotoxic activities of Alpinia pahangensis extract. METHODS: In the current study, the crude methanol and fractionated extract of the rhizomes of Alpinia pahangensis were investigated for their antioxidant activity using four different assays namely, the DPPH scavenging activity, superoxide anion scavenging, ß-carotene bleaching and reducing power assays whilst their phenolic contents were measured by the Folin-Ciocalteu's method.In vitro neutral red cytotoxicity assay was employed to evaluate the cytotoxic activity against five different cancer cell lines, colon cancer (HCT 116 and HT-29), cervical cancer (Ca Ski), breast cancer (MCF7) and lung cancer (A549) cell lines, and one normal cell line (MRC-5). The extract that showed high cytotoxic activity was further investigated for its chemical constituents by GC-MS (gas chromatography-mass spectrometry) analysis. RESULTS: The ethyl acetate fraction showed the strongest DPPH radical scavenging (0.35 ± 0.094 mg/ml) and SOD activities (51.77 ± 4.9%) whilst the methanol extract showed the highest reducing power and also the strongest antioxidant activity in the ß-carotene bleaching assays in comparison to other fractions. The highest phenolic content was found in the ethyl acetate fraction, followed by the crude methanol extract, hexane and water fractions. The results showed a positive correlation between total phenolic content with DPPH radical scavenging capacities and SOD activities. The hexane fraction showed potent cytotoxic effect against KB, Ca Ski and HCT 116 cell lines with IC50 of 5.8 ± 0.1 and 9.1 ± 2.0 ug/ml, respectively. The major components of hexane fraction analysed by GC-MS analysis were mostly methyl esters. CONCLUSIONS: The current study suggests that the methanol extract and ethyl acetate fraction of A. pahangensis is a potential source of natural antioxidant for protective as well as prevention of life-threatening diseases. The hexane fraction of A. pahangensis may have the potential to be developed into therapeutic option for treating cancer.

Adsorption of neutral red and malachite green onto grapefruit peel in single and binary systems.

This study characterized the properties of NaOH-modified grapefruit peel (MGP) and investigated its adsorption properties, specifically the adsorption of the synthetic dyes neutral red (NR) and malachite green (MG) onto MGP, in single and binary systems by means of batch techniques. The adsorption equilibrium data of NR onto MGP fit well with both the Langmuir and Koble-Corrigan models, while the Koble-Corrigan and Dubinin-Radushkevich models seemed to agree better with MG adsorption. The maximum equilibrium quantities of NR and MG from the Langmuir model were 640.3 and 314.9 mg/g at 298 K, respectively. The Elovich model was a better fit with the kinetic process, which suggested that ion exchange was one of the main mechanisms at work. The thermodynamic parameters of adsorption systems indicated spontaneous and endothermic processes. In the binary system experiments, NR and MG exhibited competitive adsorption. The quantity of MG adsorbed was more strongly influenced by NR, due to the higher affinity of MGP for the latter.