Dose-dependent cell necrosis induced by silica nanoparticles

In recent years, much attention has been given to nanoparticles (NPs) due to their many possible applications, and as research has progressed, these NPs have become valuable tools for medical purposes. Among many different types of NPs, silica nanoparticles (SiO₂NPs) have been specifically evaluated for medical purposes and have also been used in many different types of products. Although SiO₂NPs have already been applied and are believed to be nontoxic, there is still a concern regarding possible adverse effects that may be triggered after SiO₂NP exposure. Therefore, in the present study, we employed a recommended cell line (BALB/c 3T3) for the toxicity evaluation to investigate the cytotoxic effects of SiO₂NPs produced by chemical synthesis at a laboratory scale. First, we employed OECD guideline 129 in order to evaluate cytotoxicity effects and also estimate the starting doses for acute oral systemic toxicity tests. We evaluated the cytotoxic effects of two types of SiO₂NPs (nonfluorescent and fluorescent) and found that they were not significantly different (IC₅₀ = 1986.39 ± 237 μg/mL and IC₅₀ = 1861.13 ± 186.72 μg/mL, respectively). Then, we used the predicted LD₅₀ of both types of SiO₂NPs to suggest that they could be categorized as GHS category 4 substances. By ultrastructural evaluation, we found that SiO₂NPs are internalized by 3 T3 cells and are located in vacuole-like structures with no other significant changes in cell structure. We also found that SiO₂NPs lead to cell necrosis in a dose-dependent manner.     

In vitro toxicity screening of magnetite nanoparticles by applying mesenchymal stem cells derived from human umbilical cord lining

Despite the growing interest in nanoparticles (NPs), their toxicity has not yet been defined and the development of new strategies and predictive models are required. Human stem cells (SCs) offer a promising and innovative cell‐based model. Among SCs, mesenchymal SCs (MSCs) derived from cord lining membrane (CL) may represent a new species‐specific tool for establishing efficient platforms for primary screening and toxicity/safety testing of NPs. Superparamagnetic iron oxide NPs, including magnetite (Fe₃O₄NPs), have aroused great public health and scientific concerns despite their extensive uses. In this study, CL‐MSCs were characterized and applied for in vitro toxicity screening of Fe₃O₄NPs. Cytotoxicity, internalization/uptake, differentiation and proliferative capacity were evaluated after exposure to different Fe₃O₄NP concentrations. Data were compared with those obtained from bone marrow (BM)‐MSCs. We observed, at early passages (P3), that: (1) cytotoxicity occurred at 10 μg/mL in CL‐MSCs and 100 μg/mL in BM‐MSCs (no differences in toxicity, between CL‐ and BM‐MSCs, were observed at higher dosage, 100‐300 μg/mL); (2) cell density decrease and monolayer features loss were affected at ≥50 μg/mL in CL‐MSCs only; and (3) NP uptake was concentration‐dependent in both MSCs. After 100 μg/mL Fe₃O₄NP exposures, the capacity of proliferation was decreased (P5‐P9) in CL‐MSCs without morphology alteration. Moreover, a progressive decrease of intracellular Fe₃O₄NPs was observed over culture time. Antigen surface expression and multilineage differentiation were not influenced. These findings suggest that CL‐MSCs could be used as a reliable cell‐based model for Fe₃O₄NP toxicity screening evaluation and support the use of this approach for improving the confidence degree on the safety of NPs to predict health outcomes.     

Cytotoxic and proinflammatory responses induced by ZnO nanoparticles in in vitro intestinal barrier

ZnO nanoparticles (NPs) are widely used nowadays, thus the gastrointestinal exposure to ZnO NPs is likely to be relevant and the effects on the intestinal barrier should be investigated. Polarized Caco‐2 cells were exposed from the apical (Ap) and basolateral (Bl) compartments to increasing concentrations (0, 10, 50 and 100 μg/mL) of sonochemical (sono) and commercial ZnO NPs. The transepithelial electrical resistance (TEER), cell viability, proinflammatory cytokine release and presence of protein oxidative damage were evaluated after exposure. TEER was not significantly affected by Ap exposure to either sono or commercial ZnO NPs at any tested concentrations. After Bl exposure to sono ZnO NPs (all the concentrations) and to 100 μg/mL of commercial ZnO NPs TEER was decreased (P < 0.05). Ap and Bl exposure to 100 μg/mL sono ZnO NPs and Ap exposure to 50 μg/mL commercial ZnO NPs induced a significant (P < 0.05) release of interleukin‐6. A significant (P < 0.05) release of interleukin‐8 was observed after Ap exposure to ZnO NPs at 100 μg/mL and after Bl exposure to sono ZnO NPs at 100 μg/mL. Ap or Bl exposure to sono or commercial ZnO NPs did not affect tumour necrosis factor‐alpha secretion or protein sulphydryl oxidation. In conclusion, the ZnO NP exposure from the Ap compartment appeared almost safe, while the exposure through the basal compartment appeared to be more hazardous and the different NP size and crystallinity seem to affect the mode of action, but further studies are necessary to elucidate better these toxicity mechanisms.     

In vitro cytotoxicity of hydrothermally synthesized ZnO nanoparticles on human periodontal ligament fibroblast and mouse dermal fibroblast cells

The use of metal oxide nanoparticles (NPs) in industrial applications has been expanding, as a consequence, risk of human exposure increases. In this study, the potential toxic effects of zinc oxide (ZnO) NPs on human periodontal ligament fibroblast cells (hPDLFs) and on mouse dermal fibroblast cells (mDFs) were evaluated in vitro. We synthesized ZnO NPs (particle size; 7–8 nm) by the hydrothermal method. Characterization assays were performed with atomic force microscopy, Braun–Emmet–Teller analysis, and dynamic light scattering. The hPDLFs and mDFs were incubated with the NPs with concentrations of 0.1, 1, 10, 50 and 100 μg/mL for 6, 24 and 48 h. Under the control and NP-exposed conditions, we have made different types of measurements for cell viability and morphology, membrane leakage and intracellular reactive oxygen species generation. Also, we monitored cell responses to ZnO NPs using an impedance measurement system in real-time. While the morphological changes were visualized using scanning electron microscopy, the subcellular localization of NPs was investigated by transmission electron microscopy. Results indicated that ZnO NPs have significant toxic effects on both of the primary fibroblastic cells at concentrations of ∼50–100 μg/mL. The cytotoxicity of ZnO NPs on fibroblasts depended on concentration and duration of exposure.     

Anti-inflammatory activity of edible brown alga Saccharina japonica and its constituents pheophorbide a and pheophytin a in LPS-stimulated RAW 264.7 macrophage cells

Anti-inflammatory activity of Saccharina japonica and its active components was evaluated via in vitro inhibitory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) expression in RAW 264.7 murine macrophage cells. Since the methanolic extract of S. japonica showed strong anti-inflammatory activity, it was fractionated with several solvents. Among the fractions, the ethyl acetate fraction demonstrated the highest inhibition of LPS-induced NO production (IC₅₀ = 25.32 μg/mL), followed by the CH₂Cl₂ fraction (IC₅₀ = 75.86 μg/mL). Considering the yield and anti-inflammatory potential together, the CH₂Cl₂ fraction was selected for chromatographic separation to yield two active porphyrin derivatives, pheophorbide a and pheophytin a, together with an inactive fucoxanthin. In contrast to fucoxanthin, pheophorbide a and pheophytin a showed dose-dependent inhibition against LPS-induced NO production at nontoxic concentrations in RAW 264.7 cells. Both compounds also suppressed the expression of iNOS proteins, while they did not inhibit the COX-2 expression in LPS-stimulated macrophages. These results indicate that pheophorbide a and pheophytin a are two important candidates of S. japonica as anti-inflammatory agents which can inhibit the production of NO via inhibition of iNOS protein expression. Thus, these compounds hold great promise for use in the treatment of various inflammatory diseases.     

Wound healing activity of Pluchea indica leaf extract in oral mucosal cell line and oral spray formulation containing nanoparticles of the extract

Context: Pluchea indica (L.) Less (Asteraceae) is an herb used as a traditional medicine for wound healing. The chemical compounds found in Pluchea indica leaves are phenolic acids, flavonoids, anthocyanins and carotenoids.

Objective: This study investigates the effect of Pluchea indica leaf ethanol extract and its nanoparticles (NPs) on cytotoxicity, cell survival and migration of human oral squamous carcinoma cell line.

Materials and methods: Cell viability was measured using MTT assay to assess the effect of Pluchea indica leaf extract and NPs (1–500?μg/mL) on cytotoxicity and cell survival. The effect of Pluchea indica leaf extract and NPs on cell migration was determined by scratch assay. The % relative migration was calculated after 24, 48 and 72?h of treatment.

Results: The sizes of Pluchea indica leaf extract NPs were in a range of nanometers. NPs possessed negative charge with the polydispersity index (PDI) smaller than 0.3. After the treatment for 24, 48 and 72?h, Pluchea indica leaf extract had IC₅₀ value of 443.2, 350.9 and 580.5?μg/mL, respectively, whereas the IC₅₀ value of NPs after the treatment for 24, 48 and 72?h were 177.4, 149.2 and 185.1?μg/mL, respectively. The % relative migration of cells was significantly increased when the cells were treated with 62.5 and 125?μg/mL of the extract and 62.5?μg/mL of NPs.

Discussion and Conclusions: NPs increased cytotoxicity of the Pluchea indica leaf extract, increased the migration of cells at low concentration and increased colloidal stability of the extract in an oral spray formulation.     

Iron oxide nanoparticles mediated cytotoxicity via PI3K/AKT pathway: Role of quercetin

Recently Fe₂O₃ NPs (iron oxide nanoparticles) have been extensively used in medical imaging and in industry also. As a result, people are increasingly exposed day by day to those nanoparticles. The adverse effect of Fe₂O₃ NPs is not so significant at lower doses but at higher doses Fe₂O₃ NPs causes significant damage to cells. The present study investigates the cell signaling mechanism of Fe₂O₃ NPs induced oxidative stress and cytotoxicity in vitro using murine hepatocytes as the working model. In addition, the cytoprotective action of quercetin in this pathophysiology has also been investigated. Dose-dependent studies suggest that incubation of hepatocytes with 250 μg/ml Fe₂O₃ NPs for 4 h significantly decreased the cell viability and intra-cellular antioxidant ability. This study also showed that exposure to Fe₂O₃ NPs caused hepatocytes death via apoptotic pathway. Incubation of hepatocytes with quercetin (50 μmol/L) prior to 1 h of Fe₂O₃ NPs exposure protects the cells from the altering activities of antioxidant indices, cytotoxicity and apoptotic death. Results suggest that Fe₂O₃ NPs induced cellular damage and quercetin plays a protective role in Fe₂O₃ NPs induced cytotoxicity and apoptotic death.     

Genotoxic evaluation of titanium dioxide nanoparticles in vivo and in vitro

With the extensive application of titanium dioxide (TiO₂) nanoparticles (NPs) in food industry, there is a rising debate concerning the possible risk associated with exposure to TiO₂ NPs. The purpose of this study is to evaluate the genotoxicity of TiO₂ NPs using in vivo and in vitro test systems. In vivo study, the adult male Sprague-Dawley rats were exposed to anatase TiO₂ NPs (75 ± 15 nm) through intragastric administration at 0, 10, 50 and 200 mg/kg body weight every day for 30 days. The γ-H₂AX assay showed TiO₂ NPs could induce DNA double strand breaks in bone marrow cells after oral administration. However, the micronucleus test revealed that the oral-exposed TiO₂ NPs did not cause damage to chromosomes or mitotic apparatus observably in rat bone marrow cells. In vitro study, Chinese hamster lung fibroblasts (V79 cells) were exposed to TiO₂ NPs at the dose of 0, 5, 10, 20, 50 and 100 μg/mL. Significant decreases in cell viability were detected in all the treated groups after 24 h and 48 h exposure. Significant DNA damage was only observed at the concentration of 100 μg/mL after 24 h treatment using the comet assay. The obvious gene mutation was observed at the concentration of 20 and 100 μg/mL after 2 h treatment using hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutation assay. This study presented a comprehensive genotoxic evaluation of TiO₂ NPs, and TiO₂ NPs were shown to be genotoxic both in vivo and in vitro tests. The gene mutation and DNA strand breaks seem to be more sensitive genetic endpoints for the detection of TiO₂ NPs induced genotoxic effects.     

In vitro genotoxicity assessment of nickel(II) oxide nanoparticles on lymphocytes of human peripheral blood

The current study was intended to elucidate the cytotoxicity, genotoxicity ability of nickel oxide (NiO) nanoparticles (NPs) and assessment of preliminary mechanism of the toxicity. Characterization studies showed that NiO‐NPs have a particle size of 17.94 (±3.48) nm. The particle size of the NPs obtained by dynamic light scattering method in Milli‐Q and RPMI 1640 media was 189.9 (±17.1) and 285.9 (±19.6) nm, respectively. The IC₅₀ concentration for NiO‐NPs after 24 hours of treatment was estimated as 23.58 μg/mL. Comet and cytokinesis‐block micronucleus assays revealed a significant dose‐ and time‐dependent genotoxic potential of NiO‐NPs. Morphological assessment of the lymphocytes upon exposure to NiO‐NPs showed that the mechanism of toxicity was apoptosis. Reactive oxygen species analysis and lipid peroxidation patterns were aligned with the cytotoxicity and genotoxicity endpoints. Thus, the preliminary mechanism of NiO‐NPs for cytotoxicity on lymphocytes was assumed to be oxidative stress‐mediated apoptosis and DNA damage. Furthermore, these NiO‐NPs are considered a potentially hazardous substance at environmentally significant levels. Further investigations are suggested to understand the immunotoxic effects of NiO‐NPs.     

Identification of sterols from the soft coral Dendronephthya gigantea and their anti-inflammatory potential

Exploration of anti-inflammatory phytochemicals has received tremendous attention worldwide owing to the rapid increase in inflammatory diseases. Current study reveals the identification of eight 3β-hydroxy-Δ⁵-steroidal congeners from a nonpolar column fraction of the ethanol solubles from the soft coral Dendronephthya gigantea collected from Jeju Island South Korea, using GC–MS/MS analysis. The sterol-rich fraction (DGEH21) showed a significant anti-inflammatory activity as exhibited by the inhibition of NO production (IC₅₀ 4.33 ± 0.50 μg/mL) and PGE₂ production in LPS-stimulated RAW 264.7 macrophages. It also suppressed the expression levels of proinflammatory cytokines, TNF-α, IL-1β, and IL-6 in a dose-dependent manner. Furthermore, DGEH21 effectively downregulated the expression levels of iNOS, and COX-2 and reduced NO and ROS production as well as cell death in LPS-stimulated in-vivo zebrafish embryo model. However, DGEH21 at relatively high concentrations indicated cytotoxicity in both RAW cells and zebrafish embryos with RAW cell viability being nearly 80% after treatment with 25 μg/mL DGEH21. This study highlights the synergistic anti-inflammatory activity of several steroids found in D. gigantea. Their actions may be useful in the development of anti-inflammatory cosmeceuticals, pharmaceutical agents, and other consumer products.     

Varying the morphology of silver nanoparticles results in differential toxicity against micro-organisms,HaCaT keratinocytes and affects skin deposition

The use of silver nanoparticles (Ag NPs) within the healthcare sector and consumer products is rapidly increasing. There are now a range of diverse-shaped Ag NPs that are commercially available and many of the products containing nanosilver are topically applied to human skin. Currently, there is limited data on the extent to which the antimicrobial efficacy and cytotoxicity of Ag NPs is related to their shape and how the shape of the Ag NPs affects their distribution in both intact and burn wounded human skin after topical application. In this study, we related the relative Ag NP cytotoxicity to potential skin pathogens and HaCaT keratinocytes in vitro with the shape of the Ag NPs. We employed multiphoton fluorescence lifetime imaging to map the distribution of the native and unlabeled Ag NPs after topical application to both intact and burn wounded human skin using the localized surface plasmon resonance signal of the Ag NPs. Truncated plate shaped Ag NPs led to the highest cytotoxicity against both bacteria (IC₅₀ ranges from 31.25 to 125?μg/mL depending on the bacterial species) and HaCaT keratinocytes (IC₅₀ 78.65?μg/mL [95%CI 63.88, 96.83]) thus both with similar orders of magnitude. All Ag NPs were less cytotoxic than solutions of silver nitrate (IC₅₀ of 7.85?μg/mL [95%CI 1.49, 14.69]). Plate-shaped Ag NPs displayed the highest substantivity within the superficial layers of the stratum corneum when topically applied to intact skin and the highest deposition into the wound bed when applied to burned ex vivo human skin relative to other Ag NP shapes.     

Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells

Primary cells are ideal for in vitro toxicity studies since they closely resemble tissue environment. Here, we report a detailed study on the in vitro interactions of 7-20 nm spherical silver nanoparticles (SNP) with primary fibroblasts and primary liver cells isolated from Swiss albino mice. The intended use of silver nanoparticles is in the form of a topical antimicrobial gel formulation for the treatment of burns and wounds.Upon exposure to SNP for 24 h, morphology of primary fibroblasts and primary liver cells remained unaltered up to 25 μg/mL and 100 μg/mL SNP, respectively, although with minor decrease in confluence. IC₅₀ values for primary fibroblasts and primary liver cells as revealed by XTT assay were 61 μg/mL and 449 μg/mL, respectively. Ultra-thin sections of primary cells exposed to 1/2 IC₅₀ SNP for 24 h, visualized under Transmission electron microscope showed the presence of dark, electron dense, spherical aggregates inside the mitochondria, and cytoplasm, probably representing the intracellular SNP. When the cells were challenged with ∼ 1/2 IC₅₀ concentration of SNP (i.e. 30 μg/mL and 225 μg/mL for primary fibroblasts and primary liver cells, respectively), enhancement of GSH (∼ 1.2 fold) and depletion of lipid peroxidation (∼ 1.4 fold) were seen in primary fibroblasts which probably protect the cells from functional damage. In case of primary liver cells; increased levels of SOD (∼ 1.4 fold) and GSH (∼ 1.1 fold) as compared to unexposed cells were observed. Caspase-3 activity assay indicated that the SNP concentrations required for the onset of apoptosis were found to be much lower (3.12 μg/mL in primary fibroblasts, 12.5 μg/mL in primary liver cells) than the necrotic concentration (100 μg/mL in primary fibroblasts, 500 μg/mL in primary liver cells). These observations were confirmed by CLSM studies by exposure of cells to 1/2 IC₅₀ SNP (resulting in apoptosis) and 2× IC₅₀) cells (resulting in necrosis).These results clearly suggest that although silver nanoparticles seem to enter the eukaryotic cells, cellular antioxidant mechanisms protect the cells from possible oxidative damage. This property, in conjunction with the finding that primary cells possess much higher SNP tolerance than the concentration in the gel (∼ 20 μg/g), indicates preliminary safety of the formulation and warrants further study for possible human application.     

In vitro cytogenetic evaluation of the particular combination of flurbiprofen and roxithromycin

Flurbiprofen (FLB) (anti-inflammatory and analgesic drug) and roxithromycin (RXM) (antibiotic) were widely used in world wide. This study deals with investigation of genotoxicity, cytotoxicity, and oxidative stress effects of a particular combination of these drugs in human cultured lymphocytes. Also, DNA damaging-protective effects of combination of these drugs were analyzed on plasmid DNA. Human lymphocytes were treated with different concentrations (FLB?+?RXM; 10?μg/mL?+?25?μg/mL, 15?μg/mL?+?50?μg/mL, and 20?μg/mL?+?100?μg/mL) of the drugs following by study of their genotoxic and cytotoxic effects by analysis of cytokinesis-block micronucleus test and nuclear division index, respectively. The effect of the combination in aspect of anti-oxidative and DNA damaging activity was evaluated on Pet-22b plasmid. According to our results, the combination of FLB and RXM did not show a notable genotoxic effect on cells. Although each of the substances had been shown as a cytotoxic agent by previous researchers, in this research, the combination of these drugs did not exhibit any adverse effect on cell division. FLB had DNA protection effect against H₂O₂ while in combination with RXM had not the same effect on the plasmid.     

Toxicity and differential protein analysis following destruxin A treatment of Spodoptera litura (Lepidoptera: Noctuidae) SL-1 cells

The cytotoxicity of a destruxin A (DA) treatment of Spodoptera litura SL-1 cells was investigated. An MTT assay showed that DA was highly toxic to SL-1 cells in a concentration- and time-dependent manner. The IC₅₀ values of DA, after 24 h and 48 h of treatment, were 17.86 μg/mL and 7.80 μg/mL, respectively. Under inverted phase contrast microscopy (IPCM), it was found that prolonged treatment with DA could induce cell rounding, cellular membrane shrinking, formation of apoptotic bodies, vacuole appearance and cytoplasm leak out. Apoptosis induced by DA was further confirmed by fluorescence microscopy (FM) and flow cytometry (FCM) studies. SL-1 cells entered early apoptosis following a treatment with 2.5 μg/mL DA and entered late apoptosis following a treatment with increasing concentrations of DA. Furthermore, two-dimensional gel electrophoresis (2-DE) analysis was used to identify 22 proteins which were differentially expressed (≥2-fold difference) between control cells and DA-treated cells, and the expression level of these proteins was significantly different between the treated and untreated cells. Our results suggest that these differentially expressed proteins may help explain the diverse biological effects caused by the destruxin A treatment of cells; additionally, some of the identified proteins may have roles in SL-1 cellular proliferation and apoptosis.     

Inhibitory effects of gypenosides on seven human cytochrome P450 enzymes in vitro

Among the various possible causes for drug interactions, pharmacokinetic factors such as inhibition of drug-metabolizing enzymes, especially cytochrome P450 (CYP) enzymes, are regarded as the most frequent and clinically important. Gypenosides is widely used as functional food and over-the-counter drug in East Asia. In this study, the in vitro inhibitory effects of gypenosides on the major human CYP enzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) activities in human liver microsomes were examined using liquid chromatography–tandem mass spectrometry. Gypenosides showed the strongest inhibition of CYP2D6, followed by CYP2C8, CYP3A4 and CYP2C9. The IC₅₀ values were 1.61 μg/mL, 20.06 μg/mL, 34.76 μg/mL (CYP3A4/midazolam), 46.73 μg/mL (CYP3A4/testosterone), and 54.52 μg/mL, respectively. Gypenosides exhibited competitive inhibition of CYP2D6 (Ki = 1.18). In conclusion, Gypenosides might cause herb–drug interactions via inhibition of CYP2D6. An in vivo study is needed to examine this further.     

Eudesmol Isomers Induce Caspase‐Mediated Apoptosis in Human Hepatocellular Carcinoma HepG2 Cells

Eudesmols are naturally occurring sesquiterpenoid alcohols that present cytotoxic effect to cancer cells. Herein, all eudesmol isomers displayed cytotoxicity to different tumour cell lines. α‐Eudesmol showed IC₅₀ values ranging from 5.38 ± 1.10 to 10.60 ± 1.33 μg/mL for B16‐F10 and K562 cell lines, β‐eudesmol showed IC₅₀ values ranging from 16.51 ± 1.21 to 24.57 ± 2.75 μg/mL for B16‐F10 and HepG2 cell lines, and γ‐eudesmol showed IC₅₀ values ranging from 8.86 ± 1.27 to 15.15 ± 1.06 μg/mL for B16‐F10 and K562 cell lines, respectively. In addition, in this work, we studied the mechanisms of cytotoxic action of eudesmol isomers (α‐, β‐ and γ‐eudesmol) in human hepatocellular carcinoma HepG2 cells. After 24‐hr incubation, HepG2 cells treated with eudesmol isomers presented typical hallmarks of apoptosis, as observed by morphological analysis in cells stained with haematoxylin–eosin and acridine orange/ethidium bromide. None of eudesmol isomers caused membrane disruption at any concentration tested. Moreover, eudesmol isomers induced loss of mitochondrial membrane potential and an increase in caspase‐3 activation in HepG2 cells, suggesting the induction of caspase‐mediated apoptotic cell death. In conclusion, the eudesmol isomers herein investigated are able to reduce cell proliferation and to induce tumour cell death by caspase‐mediated apoptosis pathways.     

HPTLC analysis,antioxidant, anti-inflammatory and antiproliferative activities of Arisaema tortuosum tuber extract

Context: Oxidative stress and inflammation are related to several chronic diseases including cancer and atherosclerosis. Arisaema tortuosum (Wall.) Schott (Araceae) is an Indian folk medicinal herb traditionally used for treatment of various diseases related to inflammation and stress.

Objective: This study was carried out for HPTLC analysis and evaluation of antioxidant, anti-inflammatory and antiproliferative activities of a methanol extract of A. tortuosum tuber.

Materials and methods: The antioxidant activities of methanol extract of A. tortuosum tuber (1?mg/mL) were evaluated by DPPH, ABTS and FRAP assays and anti-inflammatory effects by diene-conjugate and β-glucuronidase assays, with in vitro tumor growth inhibition on HeLa cancer cells. The results for antioxidant and anti-inflammatory effects were compared using Trolox and salicylic acid as reference compounds, respectively.

Results: The TLC and HPTLC analysis showed the presence of quercetin, rutin, luteolin and lectin (R_f values 0.97, 0.53, 0.59 and 1.58, respectively). The methanol fraction of tuber exhibit higher activity in each antioxidant system with a special attention for DPPH (IC₅₀?=?852?μg/mL), ABTS (IC₅₀?=?532?μg/mL), and FRAP (IC₅₀?=?458?μg/mL), as compared with Trolox as standard, with a remarkable amount of phenolics (86.2?mg/100?g) and flavonoids (175.5?mg/100?g), along with potent anti-inflammatory activity indicated by diene-conjugate (86.20%) and β-glucuronidase (92.92%) inhibition, as compared with salicylic acid as reference compound. The antiproliferative activity at 100?mg/mL was 88% inhibition with HeLa cells. The inhibition of HeLa cell proliferation was greatest (p?A. tortuosum tuber extract treatments and least with the 25?mg/mL dose.

Discussion and conclusion: Our results suggested that A. tortuosum tuber might be used as a promising and potent antioxidant, anti-inflammatory, and antiproliferative agent and might be used for standardization of potential drug after successful isolation and characterization of bioactive compounds.     

Antiproliferative Activity of King Cobra (Ophiophagus hannah) Venom l‐Amino Acid Oxidase

King cobra (Ophiophagus hannah) venom l ‐amino acid oxidase (LAAO), a heat‐stable enzyme, is an extremely potent antiproliferative agent against cancer cells when compared with LAAO isolated from other snake venoms. King cobra venom LAAO was shown to exhibit very strong antiproliferative activities against MCF‐7 (human breast adenocarcinoma) and A549 (human lung adenocarcinoma) cells, with an IC₅₀ value of 0.04 ± 0.00 and 0.05 ± 0.00 μg/mL, respectively, after 72‐hr treatment. In comparison, its cytotoxicity was about 3–4 times lower when tested against human non‐tumourigenic breast (184B5) and lung (NL 20) cells, suggesting selective antitumour activity. Furthermore, its potency in MCF‐7 and A549 cell lines was greater than the effects of doxorubicin, a clinically established cancer chemotherapeutic agent, which showed an IC₅₀ value of 0.18 ± 0.03 and 0.63 ± 0.21 μg/mL, respectively, against the two cell lines. The selective cytotoxic action of the LAAO was confirmed by phycoerythrin (PE) annexin V/7‐amino‐actinomycin (AAD) apoptotic assay, in which a significant increase in apoptotic cells was observed in LAAO‐treated tumour cells than in their non‐tumourigenic counterparts. The ability of LAAO to induce apoptosis in tumour cells was further demonstrated using caspase‐3/7 and DNA fragmentation assays. We also determined that this enzyme may target oxidative stress in its killing of tumour cells, as its cytotoxicity was significantly reduced in the presence of catalase (a H₂O₂ scavenger). In view of its heat stability and selective and potent cytotoxic action on cancer cells, king cobra venom LAAO can be potentially developed for treating solid tumours.     

Investigation of Biological Activities of Dichloromethane and Ethyl Acetate Fractions of Platonia insignis Mart. Seed

Platonia insignis Mart., a native species of the Brazilian Amazon more commonly known as bacuri, is a member of the Clusiaceae family. In this study, we evaluated the chemical composition and the antioxidant and toxicity activities of the dichloromethane and ethyl acetate fractions from P. insignis seed ethanolic extract using different experimental models. Our results demonstrate in vitro antioxidant effects, by 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid) diammonium salt and 1,1‐diphenyl‐2‐picryl‐hydrazyl assays, as well as in vivo effects in antioxidant‐defective Saccharomyces cerevisiae strains to both fractions. Toxicity was evaluated against the micro‐crustaceous Artemia salina Leach. and promastigote Leishmania amazonensis. The dichloromethane fraction was the most active fraction evaluated on A. salina and promastigote L. amazonensis (IC₅₀ = 24.89 μg/mL and 2.84 μg/mL, respectively). In addition, a slight cytotoxicity was observed in mammalian V79 cells using ethyl acetate and dichloromethane fractions with MTT assays. Both fractions displayed genotoxicity up to 25 μg/mL (dichloromethane) and 10 μg/mL (ethyl acetate) in V79 cells, as evaluated by the alkaline comet assay. Thus, in this study, we demonstrate for the first time that ethyl acetate and dichloromethane fractions from P. insignis seeds display antioxidant effects, a toxic effect against A. salina and L. amazonensis and induce genotoxicity in V79 mammalian cells. The observed activities can be attributed to the phenolic compounds present in these fractions and to the presence of xanthones (alpha‐ and gamma‐mangostin).     

Comparative study of cyto‐ and genotoxic potential with mechanistic insights of tungsten oxide nano‐ and microparticles in lung carcinoma cells

The exigency of semiconductor and super capacitor tungsten oxide nanoparticles (WO₃ NPs) is increasing in various sectors. However, limited information on their toxicity and biological interactions are available. Hence, we explored the underlying mechanisms of toxicity induced by WO₃ NPs and their microparticles (MPs) using different concentrations (0–300 μg ml^–1) in human lung carcinoma (A549) cells. The mean size of WO₃ NPs and MPs by transmission electron microscopy was 53.84 nm and 3.88 μm, respectively. WO₃ NPs induced reduction in cell viability, membrane damage and the degree of induction was size‐ and dose‐dependent. There was a significant increase in the percentage tail DNA and micronuclei formation at 200 and 300 μg ml^–1 after 24 hours of exposure. The DNA damage induced by WO₃ NPs could be attributed to increased oxidative stress and inflammation through reactive oxygen species generation, which correlated with the depletion of reduced glutathione content, catalase and an increase in malondialdehyde levels. Cellular uptake studies unveiled that both the particles were attached/surrounded to the cell membrane according to their size. In addition, NP inhibited the progression of the cell cycle in the G₂/M phase. Other studies such as caspase‐9 and ‐3 and Annexin‐V‐fluorescein isothiocyanate revealed that NPs induced intrinsic apoptotic cell death at 200 and 300 μg ml^–1 concentrations. However, in comparison to NPs, WO₃ MPs did not incite any toxic effects at the tested concentrations. Under these experimental conditions, the no‐observed‐significant‐effect level of WO₃ NPs was determined to be ≤200 μg ml^–1 in A549 cells.