In vitro and in silico characterization of angiogenic inhibitors from <Emphasis Type="Italic">Sophora interrupta</Emphasis>

Sophora interrupta Bedd, (Fabaceae) is used in Indian folk medicine to treat cancer. Angiogenesis is one of the crucial characteristics of cancer metastasis and is regulated by vascular endothelial growth factor (VEGF). In this study, we examined the antiangiogenic properties of the root ethyl acetate extract of Sophora interrupta by various methods. In vitro antioxidant activity (100–600 μg/ml) of S. interrupta ethyl acetate (SEA) extract was evaluated by DPPH and ABTS, anti-inflammatory activity (50, 100 and 150 μg/ml) by estimating nitric oxide (NO) levels, anti-angiogenic activity (200 and 500 μg/ml) was validated by chorio allantoic membrane (CAM) assay and in silico molecular dynamic (MD) simulations analyses (25 ns) were performed to identify the anti-angiogenic compounds extracted from root extract. The antioxidative activity of SEA extract at IC₅₀ (200?±?0.6 μg/mL) is equal to that of ascorbic acid at IC₅₀ (50?±?0.6 μg/mL), and the anti-inflammatory activity of SEA extract at IC₅₀ (150?±?0.2 μg/mL) was inhibited significantly by nitric oxide (NO) production. The SEA extract significantly reduced the sprouting of new blood vessels at ID₅₀ 500?±?0.13 μg/mL in the CAM assay. Gas chromatography–mass spectrometry analysis of the SEA extract detected 34 secondary metabolites, of which 6a,12a-dihydro-6H-(1,3)dioxolo(5,6)benzofuro(3,2-c)chromen-3-ol (maackiain) and funiculosin formed strong hydrogen bond interactions with Lys 920, Thr 916 and Cys 919 (2H), as well as Glu 917 of VEGFR2, and these interactions were similar to those of the anti-angiogenic compound axitinib. Significant findings in all the assays performed indicate that SEA extract has potential anti-angiogenic compounds that may interfere with VEGF-induced cancer malignancy.     

Production and characterization of the agarase ofCytophaga flevensis

Cytophaga flevensis produced an inducible agarase which was extracellular under most conditions tested. The effect of cultural conditions on the production of enzyme was studied in batch and continuous culture. In batch culture, production was optimal whenCytophaga flevensis was incubated at 20C in a mineral medium with agar as the sole carbon source and ammonium nitrate as the nitrogen source at an initial pH of 6.6–7.0. The enzyme appeared to be subject to catabolite repression, since its synthesis was repressed when glucose was added to the medium in batch culture. Furthermore, in continuous culture, enzyme production decreased with increasing growth rate. Extracellular agarase was partially purified and the enzyme preparation obtained was very stable. The enzyme has a molecular weight of 26000 daltons. It is a β-agarase which is highly specific for polysaccharides containing neoagarobiose units. The final products of hydrolysis of agarose by the endo-acting enzyme were neoagarotetraose and neoagarobiose. Optimal conditions for its activity were pH 6.3 and 30C. When agarose was used as a substrate, an apparent temperature optimum of 35C was found, due to gelling of the substrate during the assay procedure.     

Heterologous expression of a newly screened β-agarase from Alteromonas sp. GNUM1 in Escherichia coli and its application for agarose degradation

The gene of agaG1 from Alteromonas sp. GNUM1 encoding a β-agarase (AgaG1) was heterologously expressed in E. coli BL21 (DE3). The recombinant strain was cultured at 37 °C and then AgaG1 was expressed at 25 °C and 0.5 mM IPTG. The optimum conditions for AgaG1 to hydrolyze agarose were pH 7.0 and 40 °C. The main products of agarose hydrolysis by AgaG1 were confirmed to be neoagarobiose and neoagarotetraose. A new agarose hydrolysis process using AgaG1 was developed, in which the reaction temperature was adjusted stepwise to avoid gelation problem with no chemical pretreatment step. The enzyme AgaG1 was found to be very effective and highly selective. When 10.0 g/L agarose was hydrolyzed, 98% of the agarose added was converted to 3.8 and 6.4 g/L of neoagarobiose and neoagarotetraose, respectively.     

The in vitro genotoxicity of benzoic acid in human peripheral blood lymphocytes

The chromosomal aberration (CA), sister chromatid exchange (SCE) and micronucleus test (MN) were employed to investigate the in vitro effect of antimicrobial food additive benzoic acid on human chromosomes. Lymphocytes were incubated with various concentrations (50, 100, 200 and 500 μg/mL) of benzoic acid. The results of used assays showed that benzoic acid significantly increased the chromosomal aberration, sister chromatid exchange and micronucleus frequency (200 and 500 μg/mL) without changing the pH of the medium in a dose-dependent manner. Also this additive significantly decreased the mitotic index (MI) at the highest concentration for 24 h and 100, 200 and 500 μg/mL for 48 h. This decrease was dose-dependent as well. However, it did not effect the replication (RI) and nuclear division (NDI) indices.     

Particulate Matter Facilitates C6 Glioma Cells Activation and the Release of Inflammatory Factors Through MAPK and JAK2/STAT3 Pathways

It has been widely accepted that astrocytes, play a role in regulating almost every physiological system. In the present study, we investigated the role of particulate matter (PM) in regulating activation of astrocytes. The glial cell strain C6 was cloned from a rat glioma which was induced by N-nitrosomethylurea. The C6 cells were plated at a density of 5 × 10⁶ cells/10 cm diameter dish and incubated with different concentrations (0, 12, 25, 50, 100, 200, and 400 μg/mL) of PM for 24 h and different time (0, 1, 3, 6, 8,12, and 24 h) with 100 μg/mL at 37 °C. The study revealed that PM stimulated the expression of inducible nitric oxide synthase (iNOS) as well as the production of IL-1β in a dose- and time-dependent manner. Furthermore, activation of JAK2/STAT3 and p38/JNK/ERK MAPKs was found in astrocytes following PM treatment. Blockage of JAK and p38/JNK/ERK MAPKs with their specific inhibitors, AG490, SB202190, SP600125 and U0126 significantly reduced PM-induced iNOS expression and IL-1β production. In addition, it was demonstrated that inhibition of p38, JNK and JAK prevented STAT3 tyrosine phosphorylation induced by PM, while blocking ERK did not. MAPKs (p38 and JNK) could regulate tyrosine STAT3 phosphorylation, which suggested that the JAK2/STAT3 pathway might be the downstream of p38/JNK MAPK pathways.     

Antioxidant Activity of Purified Protein Hydrolysates from Northern Whiting Fish (Sillago sihama) Muscle

In the present study, northern whiting fish (Sillago sihama) muscle was hydrolyzed with gastrointestinal enzymes (pepsin, trypsin and α-chymotrypsin) separately and the resulted protein hydrolysates were tested for antioxidant activities using DPPH radical scavenging activity and reducing power assays. The protein hydrolysate obtained from trypsin exhibited highest antioxidant activity. Further, it was fractionated by consecutive chromatography using anion exchange and gel filtration chromatography; the separated fractions were collected and evaluated for antioxidant activity. The results showed that fraction 2 exhibited high chelating activity (73.15 % at 0.5 mg/mL) and best radical scavenging activity for DPPH radical (55.16 % at 0.5 mg/mL), ABTS radical (57.98 % at 50 μg/mL), superoxide radical (39.55 % at 200 μg/mL) and hydroxyl radical (51.33 % at 100 μg/mL). In addition, the active fraction showed strong antioxidant activity in the inhibition of linoleic acid autooxidation (60 % at 0.5 mg/mL) and also it exhibited significant protective effect on DNA damage caused by hydroxyl radicals. The size of the active fraction was found to be <360.2 Da using mass spectroscopy. These results demonstrate that muscle protein hydrolysate from northern whiting fish could be a best alternative to produce natural antioxidant peptides.     

Effects of a Standardized Phenolic-Enriched Maple Syrup Extract on β-Amyloid Aggregation,Neuroinflammation in Microglial and Neuronal Cells,and β-Amyloid Induced Neurotoxicity in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Published data supports the neuroprotective effects of several phenolic-containing natural products, including certain fruit, berries, spices, nuts, green tea, and olive oil. However, limited data are available for phenolic-containing plant-derived natural sweeteners including maple syrup. Herein, we investigated the neuroprotective effects of a chemically standardized phenolic-enriched maple syrup extract (MSX) using a combination of biophysical, in vitro, and in vivo studies. Based on biophysical data (Thioflavin T assay, transmission electron microscopy, circular dichroism, dynamic light scattering, and zeta potential), MSX reduced amyloid β_1?42 peptide (Aβ_1?42) fibrillation in a concentration-dependent manner (50–500 μg/mL) with similar effects as the neuroprotective polyphenol, resveratrol, at its highest test concentration (63.5?% at 500 μg/mL vs. 77.3?% at 50 μg/mL, respectively). MSX (100 μg/mL) decreased H₂O₂-induced oxidative stress (16.1?% decrease in ROS levels compared to control), and down-regulated the production of lipopolysaccharide (LPS)-stimulated inflammatory markers (22.1, 19.9, 74.8, and 87.6?% decrease in NOS, IL-6, PGE₂, and TNFα levels, respectively, compared to control) in murine BV-2 microglial cells. Moreover, in a non-contact co-culture cell model, differentiated human SH-SY5Y neuronal cells were exposed to conditioned media from BV-2 cells treated with MSX (100 μg/mL) and LPS or LPS alone. MSX-BV-2 media increased SH-SY5Y cell viability by 13.8?% compared to media collected from LPS-BV-2 treated cells. Also, MSX (10 μg/mL) showed protective effects against Aβ_1?42 induced neurotoxicity and paralysis in Caenorhabditis elegans in vivo. These data support the potential neuroprotective effects of MSX warranting further studies on this natural product.     

The anti-tumor activity of Mikania micrantha aqueous extract in vitro and in vivo

Aqueous extract obtained from Mikania micrantha (MMAE) is commonly used as traditional medicine in some countries. We hypothesized that MMAE may inhibit tumor cell growth, both in an in vitro and in vivo setting. In in vitro experiments, two kinds of human cancer cell lines, K562 and Hela were used to test the anti-tumor activity. Inhibitory concentrations (IC₅₀) were obtained from the inhibition curves fitted by regression analysis, inhibitory rates (%) were calculated by MTT assay, morphological changes were observed by transmission electron microscope (TEM), cell cycles were analyzed by flow cytometry (FCM), and DNA ladders were determined by agarose gel electrophoresis. The in vivo anti-tumor activity was evaluated by calculating the tumor inhibitory rates, thymus index and spleen index of S180-bearing mice. Paraffin-embedded sections were used to test the pathologic changes. The result displayed that the growth of K562 and Hela were enhanced when treated with MMAE at 20 μg/mL after 48 h. Other concentrations of MMAE (50, 100, 200, 400 μg/mL) inhibited the proliferation of both kinds of cells. The IC₅₀ values of K562 and Hela at 48 h were 167.16 and 196.27 μg/mL and at 72 h 98.07 and 131.56 μg/mL, respectively. The effects showed time-dose dependence. MMAE led to damages of organelles and induced apoptosis. These results were confirmed by ladder DNA fragmentation profile. MMAE also increased the percentage of cells in G2/M phase and decreased the percentage of cells undergoing G0/G1 and S phase in in vivo tests using S180 cells. MMAE showed antitummor activity in vivo, with its tumor inhibitory rate ranging from 12.1 to 46.9 %. MMAE also induced necrosis, as shown by pathological examination of Hematoxilin-Eosin stained tumor sections. Meanwhile, compared with the control group, the changes of thymus index and spleen index in MMAE treated group were not obvious. This study suggests that MMAE may be an effective agent for cancer therapy with low toxicity.     

Evaluation of an enzyme immunoassay for the detection of the mycotoxin tenuazonic acid in sorghum grains and sorghum-based infant food

An enzyme-linked immunosorbent assay (ELISA) for the Alternaria mycotoxin tenuazonic acid (TeA) was evaluated by comparative analysis of naturally contaminated sorghum grains and sorghum-based infant food, using a stable isotope dilution LC-MS assay (SIDA; limit of detection (LOD) 1.0 μg/kg) as the reference method. LODs of the ELISA were 30 μg/kg in sorghum grains and 220 μg/kg in sorghum-based infant cereals. With SIDA, 100% of the samples (n = 28) had been positive for TeA in a concentration range of 6–584 μg/kg (mean 113 μg/kg). The ELISA consistently detected TeA in all naturally contaminated samples at cut-off levels of 30–60 μg/kg (sorghum) and 200–300 μg/kg (infant cereals), as based on corresponding to SIDA values. Although the ELISA was much less sensitive than the SIDA method, it may be useful as a screening method for sorghum and sorghum-based infant foods and can be employed to identify samples containing elevated concentrations of TeA in food, well below the proposed level of concern (500 μg/kg).     

Simultaneous improvement of saccharification and ethanol production from crystalline cellulose by alleviation of irreversible adsorption of cellulase with a cell surface-engineered yeast strain

Enzymatic hydrolysis of cellulosic material is an essential step in the bioethanol production process. However, complete cellulose hydrolysis by cellulase is difficult due to the irreversible adsorption of cellulase onto cellulose. Thus, part of the cellulose remains in crystalline form after hydrolysis. In this study, after 96-h hydrolysis of Avicel crystalline cellulose, 47.1 % of the cellulase was adsorbed on the cellulose surface with 10.8 % crystalline cellulose remaining. In simultaneous saccharification and fermentation of 100 g/L Avicel with 1.0 filter paper unit/mL cellulase, a wild-type yeast strain produced 44.7 g/L ethanol after 96 h. The yield of ethanol was 79.7 % of the theoretical yield. On the other hand, a recombinant yeast strain displaying various cellulases, such as β-glucosidase, cellobiohydrolase, and endoglucanase, produced 48.9 g/L ethanol, which corresponds to 87.3 % of the theoretical yield. Higher ethanol production appears to be attributable to higher efficiency of cellulase displayed on the cell surface. These results suggest that cellulases displayed on the yeast cell surface improve hydrolysis of Avicel crystalline cellulose. Indeed, after the 96-h simultaneous saccharification and fermentation using the cellulase-displaying yeast, the amount of residual cellulose was 1.5 g/L, one quarter of the cellulose remaining using the wild-type strain, a result of the alleviation of irreversible adsorption of cellulases on the crystalline cellulose.     

DNA damage in human germ cell exposed to the some food additives in vitro

The use of food additives has increased enormously in modern food technology but they have adverse effects in human healthy. The aim of this study was to investigate the DNA damage of some food additives such as citric acid (CA), benzoic acid (BA), brilliant blue (BB) and sunset yellow (SY) which were investigated in human male germ cells using comet assay. The sperm cells were incubated with different concentrations of these food additives (50, 100, 200 and 500 μg/mL) for 1 h at 32 °C. The results showed for CA, BA, BB and SY a dose dependent increase in tail DNA%, tail length and tail moment in human sperm when compared to control group. When control values were compared in the studied parameters in the treatment concentrations, SY was found to exhibit the highest level of DNA damage followed by BB > BA > CA. However, none of the food additives affected the tail DNA%, tail length and tail moment at 50 and 100 μg/mL. At 200 μg/mL of SY, the tail DNA% and tail length of sperm were 95.80 ± 0.28 and 42.56 ± 4.66, for BB the values were 95.06 ± 2.30 and 39.56 ± 3.78, whereas for BA the values were 89.05 ± 2.78 and 31.50 ± 0.71, for CA the values were 88.59 ± 6.45 and 13.59 ± 2.74, respectively. However, only the highest concentration of the used food additives significantly affected the studied parameters of sperm DNA. The present results indicate that SY and BB are more harmful than BA and CA to human sperm in vitro.     

Antioxidant and Neuroprotective Effects of Scrophularia striata Extract Against Oxidative Stress-Induced Neurotoxicity

In this study, the neuroprotective effect of Scrophularia striata Boiss (Scrophulariaceae) extract, a plant growing in northeastern of Iran, against oxidative stress-induced neurocytotoxicity in PC12 was evaluated. The PC12 cell line pretreated with different concentrations (10, 50, 100, and 200 μg/ml) of the extract and then treated with H₂O₂ to induce oxidative stress and neurotoxicity. Survival of the cells, reactive oxygen species (ROS) generation, and apoptosis were measured using MTT assay, fluorescent probe 2′,7′-dichlorofluorescein diacetate, and annexin V/propidium iodide, respectively. Moreover, the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) was used to evaluate the antioxidant capacity of the plant extract. Phytochemical assay by thin layer chromatography showed that the main components, including phenolic compounds, phenyl propanoids and flavonoids, were presented in the S. striata extract. The extract in concentrations of 50–200 μg/ml protected PC12 cells from H₂O₂-induced toxicity. The survival of the cells at concentration of 200 μg/ml was 64 % compared to that of H₂O₂ alone-treated cells (48 %) (p < 0.001). The extract also dose-dependently reduced intracellular ROS production (p < 0.001). Moreover, the extract showed antioxidative effects and decreased apoptotic cells. Collectively, these findings indicated the ability of S. striata to decrease ROS generation and cell apoptosis and also suggest the presence of the neuroprotective agents in this plant.     

Enzymatic hydrolysis of agar: purification and characterization of neoagarobiose hydrolase and p-nitrophenyl alpha-galactoside hydrolase.

The mixture of polysaccharides in the gelling component of agar (agarose) is hydrolyzed to D-galactose and 3,6-anhydro-L-galactose by a series of hydrolytic enzymes obtained from Pseudomonas atlantica. The final degradative step in the pathway of agarose decomposition is the hydrolysis of the alpha-linkage in the dissaccharide neoagarobiose yielding D-galactose and 3,6-anhydro-L-galactose. Pseudomonas atlantica when grown on agar produces two specific enzymes, p-nitrophenyl alpha-galactose hydrolase and neoagarobiose hydrolase. The purification and partial characterization of both enzymes are presented.     

Gastrodin Ameliorates Depressive-Like Behaviors and Up-Regulates the Expression of BDNF in the Hippocampus and Hippocampal-Derived Astrocyte of Rats

Gastrodin (GAS), a main constituent of a Chinese herbal medicine Tian ma, has been shown to be effective in treating various mood disorders. The purpose of the present study was to assess the effects of GAS on alleviating depressive-like behaviors in a rat model of chronic unpredictable stress (CUS) and regulating the expression of BDNF in the hippocampus and hippocampal-derived astrocyte from Sprague–Dawley (SD) rats. Following CUS, rats were intraperitoneally administered gastrodin (50, 100, or 200 mg/kg daily) or vehicle for 2 weeks. Rats were then experienced sucrose preference test and forced swim test. The expressions of GFAP and BDNF in the hippocampus were evaluated. In addition, hippocampal astrocytes were isolated from neonatal SD rats and exposed to different concentrations of GAS (sham, 5, 10, 20, 50 and 100 μg/mL) for 48 and 72 h before the cell viability and the levels of pERK1/2 and BDNF were analyzed. Furthermore, the cell viability was also tested after exposure to serum-free condition that contain different concentrations of GAS for 48 and 72 h. GAS administration (100 and 200 mg/kg daily) reversed depressive-like behaviors in rats exposed to CUS paradigm and restored the expression of GFAP and BDNF in the hippocampus. Moreover, in vitro experiments revealed that GAS did not increase the cell viability of astrocytes but protected it from 72 h’s serum-free damage at the dosage 20 μg/mL. Increased levels of ERK1/2 phosphorylation and BDNF protein were also observed after GAS (20 μg/mL) treatment for 72 h. These results indicate that gastrodin possesses antidepressant effect. The changes of the astrocyte activation and the level of BDNF may play a critical role in the pharmacological action of GAS.     

Variation in protein levels obtained from human blood cells and biofluids for platelet, peripheral blood mononuclear cell, plasma, urine and saliva proteomics

Blood cells and biofluid proteomics are emerging as a valuable tool to assess effects of interventions on health and disease. This study is aimed to assess the amount and variability of proteins from platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva from ten healthy volunteers for proteomics analysis, and whether protein yield is affected by prolonged fasting. Volunteers provided blood, saliva and morning urine samples once a week for 4 weeks after an overnight fast. Volunteers were fasted for a further 24 h after the fourth sampling before providing their final samples. Each 10 mL whole blood provided 400–1,500 μg protein from platelets, and 100–600 μg from PBMC. 30 μL plasma depleted of albumin and IgG provided 350–650 μg protein. A sample of morning urine provided 0.9–8.6 mg protein/dL, and a sample of saliva provided 70–950 μg protein/mL. None of these yields were influenced by the degree of fasting (overnight or 36 h). In conclusion, in contrast to the yields from plasma, platelets and PBMC, the protein yields of urine and saliva samples were highly variable within and between subjects. Certain disease conditions may cause higher or lower PBMC counts and thus protein yields, or increased urinary protein levels.     

Antifungal Activity of Chitosan-Coated Poly(lactic-co-glycolic) Acid Nanoparticles Containing Amphotericin B

Amphotericin B (AmB) is one of the most used drugs for the treatment of systemic fungal infections; however, the treatment causes several toxic manifestations, including nephrotoxicity and hemolytic anemia. Chitosan-coated poly(lactide-co-glycolide) (PLGA) nanoparticles containing AmB were developed with the aim to decrease AmB toxicity and propose the oral route for AmB delivery. In this work, the antifungal efficacy of chitosan-coated PLGA nanoparticles containing AmB was evaluated in 20 strains of fungus isolates from patients with vulvovaginal candidiasis (01 Candida glabrata and 03 Candida albicans), bloodstream infections (04 C. albicans and 01 C. tropicalis) and patients with urinary tract infection (04 Candida albicans, 02 Trichosporon asahii, 01 C. guilhermondii, 03 C. glabrata) and 01 Candida albicans ATCC 90028. Moreover, the cytotoxicity over erythrocytes was evaluated. The single-emulsion solvent evaporation method was suitable for obtaining chitosan-coated PGLA nanoparticles containing AmB. Nanoparticles were spherical in shape, presented mean particle size about 460 nm, positive zeta potential and encapsulation efficiency of 42%. Moreover, nanoparticles prolonged the AmB release. All the strains were susceptible to plain AmB and nanostructured AmB, according to EUCAST breakpoint version 8.1 (resistant > 1 μg/mL), using broth microdilution method. In C. albicans (urine, blood, and vulvovaginal secretion isolates, and 1 ATCC), the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.5 μg/mL. In urine and vulvovaginal secretion isolates of C. glabrata, the MIC value of AmB-loaded nanoparticles varied from 0.25 to 0.5 μg/mL and EUCAST varied from 0.03 to 0.015 μg/mL. In urine isolates of C. guilhermondii, the MIC value of AmB-loaded nanoparticles was 0.12 μg/mL and EUCAST was 0.06 μg/mL. In blood isolates of C. tropicalis, the MIC value of AmB-loaded nanoparticles was 0.5 μg/mL and EUCAST was 0.25 μg/mL. Finally, in urine isolates of T asahii, the MIC value of AmB-loaded nanoparticles was 1 μg/mL and EUCAST varied from 0.5 to 1 μg/mL. In the cytotoxicity assay, plain AmB was highly hemolytic (100% in 24 h) while AmB-loaded chitosan/PLGA nanoparticles presented negligible hemolysis.     

Optimization of Dilute Acid Pretreatment and Enzymatic Hydrolysis of <Emphasis Type="Italic">Phalaris aquatica</Emphasis> L. Lignocellulosic Biomass in Batch and Fed-Batch Processes

Phalaris aquatica L., a rich in holocellulose (69.80 %) and deficient in lignin (6.70 %) herbaceous, perennial grass species, was utilized in a two-step (biomass pretreatment-enzymatic hydrolysis) saccharification process for sugars recovery. The Taguchi methodology was employed to determine the dilute acid pretreatment and enzymatic hydrolysis conditions that optimized hemicellulose conversion (75.04 %), minimized the production of inhibitory compounds (1.41 g/L), and maximized the cellulose to glucose yield (69.69 %) of mixed particulate biomass (particles <1000 μm) under batch conditions. The effect of biomass particle size on saccharification process efficiency was also investigated. It was found that small-size biomass particles (53–106 μm) resulted in maximum hemicellulose conversion (81.12 %) and cellulose to glucose yield (93.24 %). The determined optimal conditions were then applied to a combined batch pretreatment process followed by a fed-batch enzymatic hydrolysis process that maximized glucose concentration (62.24 g/L) and yield (92.48 %). The overall efficiency of the saccharification process was 88.13 %.     

In vitro anticancer properties of selected <Emphasis Type="Italic">Eucalyptus</Emphasis> species

In spite of the recent advancements in oncology, the overall survival rate for pancreatic cancer has not improved over the last five decades. Eucalypts have been linked with cytotoxic and anticancer properties in various studies; however, there is very little scientific evidence that supports the direct role of eucalypts in the treatment of pancreatic cancer. This study assessed the anticancer properties of aqueous and ethanolic extracts of four Eucalyptus species using an MTT assay. The most promising extracts were further evaluated using a CCK-8 assay. Apoptotic studies were performed using a caspase 3/7 assay in MIA PaCa-2 cells. The aqueous extract of Eucalyptus microcorys leaf and the ethanolic extract of Eucalyptus microcorys fruit inhibited the growth of glioblastoma, neuroblastoma, lung and pancreatic cancer cells by more than 80% at 100 μg/mL. The E. microcorys and Eucalyptus saligna extracts showed lower GI₅₀ values than the ethanolic Eucalyptus robusta extract in MIA PaCa-2 cells. Aqueous E. microcorys leaf and fruit extracts at 100 μg/mL exerted significantly higher cell growth inhibition in MIA PaCa-2 cells than other extracts (p < 0.05). Statistically similar IC₅₀ values (p > 0.05) were observed in aqueous E. microcorys leaf (86.05 ± 4.75 μg/mL) and fruit (64.66 ± 15.97 μg/mL) and ethanolic E. microcorys leaf (79.30 ± 29.45 μg/mL) extracts in MIA PaCa-2 cells using the CCK-8 assay. Caspase 3/7-mediated apoptosis and morphological changes of cells were also witnessed in MIA PaCa-2 cells after 24 h of treatment with the extracts. This study highlighted the significance of E. microcorys as an important source of phytochemicals with efficacy against pancreatic cancer cells. Further studies are warranted to purify and structurally identify individual compounds and elucidate their mechanisms of action for the development of more potent and specific chemotherapeutic agents for pancreatic cancer.     

The Effect of Antimicrobial Peptide Temporin-Ra on Cell Viability and Gene Expression of Pro-inflammatory Factors in A549 Cell Line

Antimicrobial peptide Temporin-Ra was isolated from the skin secretions of marsh frog Rana ridibunda. Temporin-Ra was chemically synthesized and purified using RP-HPLC technique. The cytotoxicity of peptide on lung airway epithelial cell line (A549) and peripheral blood mononuclear cells (PBMC) was studied by MTT assay. Furthermore, the effect of Temporin-Ra on the expression of pro-inflammatory factors such as IL-1β and IL-8 in A549 cell line was evaluated at peptide concentrations of 15, 30 and 50 μg/mL for 6, 12 and 24 h using semi-quantitative RT-PCR and real-time PCR methods. The result of our experiments revealed that Temporin-Ra decreased cell viability about 3–13 % in A549 cells and 4–6 % in PBMC cells. Moreover, Temporin-Ra induced the mRNA expression of IL-1β and IL-8 genes in a dose- and time-dependent manner. According to our results, maximum IL-8 mRNA expression was observed after a 24-h treatment of cancer cells with 50 μg/mL peptide with approximately 18-fold increase in expression. The least expression level of IL-1β was observed after 6-h of incubation in the presence of 15 μg/mL peptide with 2.5-fold increase in expression whereas the most expression level was obtained following 24 h-treatment with 50 μg/mL peptide with 26-fold increase in mRNA expression. Eventually, the present study highlights the role of Temporin-Ra as a potent antimicrobial peptide in the activation and maintenance of inflammatory processes.     

Selenious-β-lactoglobulin induces the apoptosis of human lung cancer A549 cells via an intrinsic mitochondrial pathway

In this study, the cytotoxic activity of selenious-β-lactoglobulin (Se-β-Lg) and the anticancer mechanism were investigated in human lung cancer A549 cells in vitro. MTT assay showed that Se-β-Lg at 200 μg/mL exhibited a significant suppression effect on A549 cells and the maximum inhibition rate reached 90% after 72 h treatment. Flow cytometry analysis revealed that 200 μg/mL of Se-β-Lg induced cell cycle arrest at G₀/G₁ phase. Cell apoptosis was induced via the generation of reactive oxygen species (ROS) and the decrease of mitochondrial membrane potential (ΔΨm) in a time-dependent manner. Furthermore, Se-β-Lg suppressed the expression of Bcl-2 and improved the level of Bax, leading to the release of cytochrome c and a higher expression of caspase-3 in A549 cells. In summary, Se-β-Lg could induce apoptosis in A549 cells via an intrinsic mitochondrial pathway and it might serve as a potential therapeutic agent for human lung cancer.