Monkey Kidney Epithelial Cell Line Research Articles

Anticancer and Neuroprotective Effects of the Triterpene Glycosides From Sea Cucumber Holothuria imitans

Introduction: Sea cucumbers has gained notoriety because possess a wide range of biological and pharmacological activities. In this sense, the aim of this work was to evaluate the anticancer and neuroprotective effects of the triterpene glycosides from sea cucumber Holothuria imitans. Methods: Triterpene glycosides were separated and purified by Reversed-phase high-performance liquid chromatography (RP-HPLC). Their structures were deduced by spectral analysis and chemical evidence. Cytotoxic activity was evaluated using normal African green monkey kidney epithelial cell line (VERO) and three cancer cell lines: cancer gastric (MKN-28), breast adenocarcinoma (MCF-7) and lung carcinoma (A-549). Besides, the neuroprotective effect was studied using the Cath.a-differentiated (CAD) cell line and human glial (Oligodendrocytic) hybrid cell line (MO3.13). Results: Two triterpene glycosides (Fuscocineroside C and Scabraside D) were isolated, which showed low cytotoxic activity against VERO cell line, and high cytotoxic activity against lines MKN-28, MCF-7 and A-549 cells, with IC50 between the ranges of 0.92 μmol/L to 2.61 μmol/L. The isolated triterpene glycosides showed the ability to regain mitochondrial viability in CAD and MO3.13 cells treated with neurotoxin (C2-ceramide) with statistically significant results (p<0.05). Conclusion: The triterpene glycosides Fuscocineroside C and Scabraside D isolated from sea cucumber Holothuria imitans show anticancer and neuroprotective potential and may be considered promising active principles for anticancer and neuroprotective drugs.

Antiviral effects and tissue exposure of tetrandrine against SARS-CoV-2 infection and COVID-19.

Tetrandrine (TET) has been used to treat silicosis in China for decades. The aim of this study was to facilitate rational repurposing of TET against SARS-CoV-2 infection. In this study, we confirmed that TET exhibited antiviral potency against SARS-CoV-2 in the African green monkey kidney (Vero E6), human hepatocarcinoma (Huh7), and human lung adenocarcinoma epithelial (Calu-3) cell lines. TET functioned during the early-entry stage of SARS-CoV-2 and impeded intracellular trafficking of the virus from early endosomes to endolysosomes. An in vivo study that used adenovirus (AdV) 5-human angiotensin-converting enzyme 2 (hACE2)-transduced mice showed that although TET did not reduce pulmonary viral load, it significantly alleviated pathological damage in SARS-CoV-2-infected murine lungs. The systemic preclinical pharmacokinetics were investigated based on in vivo and in vitro models, and the route-dependent biodistribution of TET was explored. TET had a large volume of distribution, which contributed to its high tissue accumulation. Inhaled administration helped TET target the lung and reduced its exposure to other tissues, which mitigated its off-target toxicity. Based on the available human pharmacokinetic data, it appeared feasible to achieve an unbound TET 90% maximal effective concentration (EC90) in human lungs. This study provides insights into the route-dependent pulmonary biodistribution of TET associated with its efficacy.

Isolation, Propagation and Genotyping of Human Rotaviruses Circulating among Children with Gastroenteritis in Two Egyptian University Hospitals.

Simple SummaryGroup A rotaviruses are the most common cause of acute gastroenteritis affecting Egyptian children under the age of five, with symptoms ranging from asymptomatic infection to severe dehydration or death. In the present work, diarrheal samples from Egyptian children admitted to gastrointestinal pediatric wards of two main governmental hospitals were collected and molecularly analyzed for Group A rotavirus. Our findings revealed that rotaviruses accounted for more than one-sixth of all cases under study, peaking in the winter. G1P[8] was the most prevalent rotavirus genotype in this study. The two cell lines used in our work coherently isolated and propagated rotavirus strains. Continuous rotavirus detection and genome sequencing of the successfully isolated strains will be recommended in the future in order to support the control of such viruses, and tackle the problem in Egypt.The most prevalent cause of infectious neonatal diarrhea is Group A rotavirus (RVA). Unfortunately, there is a dearth of data on the incidence of rotavirus-associated infections among Egyptian children. The present study aimed to isolate, propagate, and genotype human rotaviruses circulating among Egyptian children with acute gastroenteritis admitted to two main university pediatric hospitals, Abo El-Reesh and El-Demerdash, over two consecutive winters, 2018–2020. Diarrheal samples (n = 230) were screened for Group A rotavirus RNA using RT-PCR assay. In positive samples (n = 34), multiplex semi-nested PCR was utilized to determine G and P genotypes. Thirty-four (14.8%) of the collected samples tested positive. The genotype distribution revealed that G1P[8] was the predominant rotavirus genotype throughout the current study. All rotavirus-positive fecal samples were passaged twice on human colorectal adenocarcinoma cell line (Caco-2) and rhesus monkey kidney epithelial cell line (MA104). Both cell lines could successfully isolate 14.7% (n = 5 out of 34) of the identified strains; however, Caco-2 cell line was shown to be more efficient than MA104 in promoting the propagation of human rotaviruses identified in Egyptian children’s feces.

Biological Evaluation of 3-Azaspiro[Bicyclo[3.1.0]Hexane-2,5'-Pyrimidines] as Potential Antitumor Agents.

A series of heterocyclic compounds containing spirofused barbiturate and 3-azabicyclo[3.1.0]hexane frameworks have been studied as potential antitumor agents. Antiproliferative activity of products was screened in human erythroleukemia (K562), T lymphocyte (Jurkat), and cervical carcinoma (HeLa) as well as mouse colon carcinoma (CT26) and African green monkey kidney epithelial (Vero) cell lines. The most effective among the screened compounds show IC50 in the range from 4.2 to 24.1 μM for all tested cell lines. The screened compounds have demonstrated a significant effect of the distribution of HeLa and CT26 cells across the cell cycle stage, with accumulation of cells in SubG1 phase and induced apoptosis. It was found, using a confocal microscopy, that actin filaments disappeared and granular actin was distributed diffusely in the cytoplasm of up to 90% of HeLa cells and up to 64% of CT26 cells after treatment with tested 3-azaspiro[bicyclo [3.1.0]hexane-2,5′-pyrimidines]. We discovered that the number of HeLa cells with filopodium-like membrane protrusions was reduced significantly (from 91% in control cells to 35%) after treatment with the most active compounds. A decrease in cell motility was also noticed. Preliminary in vivo experiments on the impact of the studied compounds on the dynamics of CT26 tumor growth in Balb/C mice were also performed.

Hot Water Extract of Sasa borealis (Hack.) Makino & Shibata Abate Hydrogen Peroxide-Induced Oxidative Stress and Apoptosis in Kidney Epithelial Cells.

Sasa borealis (Hack.) Makino & Shibata or broad-leaf bamboo is famous for its richness of bioactive natural products and its uses in traditional medicine for its anti-inflammatory, diuretic, and antipyretic properties and preventive effects against hypertension, arteriosclerosis, cardiovascular disease, and cancer. The present study investigated the antioxidant activity of S. borealis hot water extract (SBH) and its effects in ameliorating hydrogen peroxide-induced oxidative stress, using an African green monkey kidney epithelial cell line (Vero). Known polyphenols in SBH were quantified by HPLC analysis. SBH indicated a dose-dependent increase for reducing power, ABTS+ (IC50 = 96.44 ± 0.61 µg/mL) and DPPH (IC50 = 125.78 ± 4.41 µg/mL) radical scavenging activities. SBH markedly reduced intracellular reactive oxygen species (ROS) generation in the Vero cells and increased the protective effects against H2O2-induced oxidative stress by reducing apoptosis. Other than the direct involvement in neutralizing ROS, metabolites in SBH were also found to induce NRF2-mediated production of antioxidant enzymes, HO-1, and NQO1. These findings imply that S. borealis hot water extract can be utilized to create nutraceutical and functional foods that can help to relieve the effects of oxidative stress in both acute and chronic kidney injury.

The Effect of Adding Spices to Green Walnut Tinctures on Their Polyphenolic Profile, Antioxidant Capacity and Action on Renal Cells

Green walnuts—unripe fruits of Juglans regia L. are known for their many biological activities and therapeutic potential. Tinctures based on unripe walnuts (samples 1 and 2) and tinctures with the addition of spices (ginger, cloves, bay leaves, juniper fruit: samples 3–6) were tested for polyphenol profile and antioxidant capacity. The effect on a normal monkey kidney epithelial cell line (VERO) was evaluated. For monitoring the changes in cell proliferation, real-time cell analysis (xCELLigence system) was employed and cell viability was measured by the MTS test. All tinctures showed the presence of polyphenols, mainly phenolic acids, flavonoids, and naphthoquinones, and in the sample with cloves, eugenol was found. Addition of spices increased the antioxidant capacity. Tinctures show a dose-dependent cytotoxic effect. The lowest concentrations (125 µg mL−1) of both tinctures without additives (1 and 2) and with bay leaves (4) did not adversely affect (p &gt; 0.05) and even supported cell proliferation (p &lt; 0.05) in comparison to the control cells without treatment. Viability was lower in all cells except for the cells treated with tincture with cloves addition (p &gt; 0.05). It indicates the beneficial effect of cloves enrichment and supports the assumption that the appropriate dosage of tinctures is necessary to be safe for the consumers.

Synthesis and cytotoxicity study of gold(III) porphyrin complexes and their derivative in breast cancer cells

Gold(III) [Au(III)] complexes exhibit potential anticancer activities. A series of Au(III) porphyrin complexes containing different meso-substituent groups (phenyl, methoxyphenyl, butyloxyphenyl, octyloxyphenyl, and decyloxyphenyl) was synthesized. The synthesized compounds were characterized using mass spectrometry, infrared spectroscopy, UV–visible and fluorescence spectroscopy, and electron paramagnetic resonance spectroscopy. Moreover, the in vitro cytotoxicity of these Au(III) porphyrin complexes was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide surrogate viability assay on an MCF7 human breast cancer cell line. The Au(III) complexes with 5,10,15,20-tetraphenylporphyrin (AuTPP) and 5,10,15,20-tetrakis(4-methyloxyphenyl)porphyrin (AuTOMPP) demonstrated high anticancer activity with IC50 values against MCF7 cells at 4.30 and 25.35 µM, respectively. The toxicity of the Au(III) porphyrin complexes against the LLC-MK2 rhesus monkey kidney epithelial cell line, a representative normal cell line, was investigated. All the tested Au(III) porphyrin complexes were non-cytotoxic in LLC-MK2 cells. For AuTPP, more than 80% LLC-MK2 cell viability was observed at concentrations lower than 5 μM.

Bromodomain and extraterminal (BET) protein inhibitor, apabetalone, reduces ACE2 expression and attenuates SARS-CoV-2 infection in vitro

Background/IntroductionSARS-CoV-2 causes life threatening COVID-19 complications including acute coronary syndrome, venous thromboembolism, hyperinflammation and damage in multiple tissues. The SARS-CoV-2 “spike protein” binds cell surface receptors including angiotensin-converting enzyme 2 (ACE2) for entry into host cells to initiate infection. Host cell dipeptidyl peptidase-4 (DPP4 / CD26) is implicated as a cofactor in uptake. Recent evidence indicates expression of factors involved in SARS-CoV-2 uptake into host cells is regulated by BET proteins, epigenetic readers modulating gene expression. Apabetalone, the most clinically advanced BET inhibitor (BETi), is in phase 3 trials for cardiovascular disease (CVD) (a,b). In cultured human cardiomyocytes, apabetalone suppressed infection with SARS-CoV-2 and prevented dysfunction of cardiac organoids induced by the cytokine-storm that arises in patients with severe symptoms (c). However, anti-viral properties of apabetalone in other cell types are not known.PurposeTo examine effects of apabetalone on SARS-CoV-2 infection in cell culture via downregulated expression of cell surface receptors involved in viral entry. Cell systems used mimic initial sites of infection in the lung as well as cell types contributing to complications in late stages of infection.MethodsGene expression was measured by real-time PCR, protein levels by immunoblot or flow cytometry, and binding of recombinant SARS-CoV-2 spike protein by flow cytometry. Infection with SARS-CoV-2 was determined in a BSL3 facility. Infectivity was quantified by determining levels of viral spike protein amongst total cells via imaging on an Operetta CLS.ResultsIn Calu-3, a human bronchial epithelial cell line, apabetalone dose-dependently downregulated ACE2 gene expression (up to 98%), reduced ACE2 protein levels (up to 84%) and diminished binding of SARS-CoV-2 spike protein (up to 77%, p<0.001 for all parameters). Further, apabetalone abolished infection of Calu-3 cells with live SARS-CoV-2, which was comparable to other antiviral agents. Apabetalone-driven ACE2 downregulation was also observed in extrapulmonary cell types including HepG2, Huh-7 or primary hepatocytes (up to 90%, p<0.001 for all cell types), and Vero E6, a monkey kidney epithelial cell line (up to 38%, p<0.05). DPP4/CD26, a potential cofactor for SARS-CoV-2 uptake, was also downregulated by apabetalone in Calu-3 cells (mRNA ∼65% and protein ∼40%, p<0.001), which may be synergistic with ACE2 reductions to impede SARS-CoV-2 infection.ConclusionsApabetalone, an investigational drug for CVD, reduced cell surface receptors (ACE2 and DPP4) involved in SARS-CoV-2 uptake into host cells and dramatically attenuated SARS-CoV-2 infection/propagation in vitro. Our results suggest apabetalone can mitigate SARS-CoV-2 replication in multiple organs, which together with an established safety profile supports clinical evaluation of apabetalone to treatFunding AcknowledgementType of funding sources: Public Institution(s). Main funding source(s): COVID-19 Rapid Response Grant from the College of Medicine at the University of Nebraska Medical Center (to DE and SPMR), and by University of Nebraska Medical Center start-up funds (to DE). Graphical

Application of a Biosurfactant Produced by Bacillus cereus UCP 1615 from Waste Frying Oil as an Emulsifier in a Cookie Formulation

Biosurfactants have attracted increasing interest from the food industry due to their emulsifying, foaming and solubilizing properties. However, the industrial use of microbial biosurfactants has been hampered by the high production costs related mainly to the use of expensive substrates. The search for low-cost alternative substrates is one of the strategies adopted to overcome this problem. In the present study, a biosurfactant produced by Bacillus cereus UCP1615 by fermentation in a medium supplemented with waste frying soybean oil as a low-cost substrate was evaluated as a bioemulsifier for the production of cookies. The biosurfactant was evaluated for its emulsifying capacity against different vegetable oils, antioxidant activity and toxicity, demonstrating favorable results for use in food. In particular, it showed satisfactory antioxidant activity at the tested concentrations and no cytotoxicity to the L929 (mouse fibroblast) and Vero (monkey kidney epithelial) cell lines using the MTT assay. The biosurfactant was then added at different concentrations (0.25%, 0.5% and 1%) to a standard cookie dough formulation to evaluate the physicochemical characteristics of the product. Cookies formulated with the biosurfactant exhibited similar energy and physical characteristics to those obtained with the standard formulation but with a lower moisture content. The biosurfactant also ensured a good preservation of the cookie texture after 45 days of storage. These results suggest that the biosurfactant has a potential application as a green emulsifier in accordance with the demands of the current market for biocompatible products.

In vitro maintenance of Mansonella perstans microfilariae and its relevance for drug screening

BackgroundMansonellosis arises from infections with threadlike filarial nematodes in millions of individuals, especially in sub-Saharan Africa. Since infections present no overt clinical symptoms but attenuate immune responses that might lead to increased susceptibility and worsened disease course of concomitant infections, it is truly a neglected tropical disease. Nevertheless, only few studies focus on identifying suitable safe drugs for its control and little is known about the requirements for in vitro maintenance of the Mansonella perstans transmission stage. This study, therefore, evaluated the survival of M. perstans microfilariae (mf) using in vitro conditions that have been shown to promote survival of Loa loa, a closely related filarial nematode. Furthermore, the in vitro microfilaricidal effect of 15 agents was assessed on this helminth. MethodsThe ability of two basic culture media; Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute (RPMI-1640) supplemented with 10% fetal bovine serum (FBS) and a monkey kidney epithelial cell line (LLC-MK2) to support the survival of M. perstans microfilariae was investigated. Subsequently, 6 anti-helminthics, 5 anti-malarials, 1 anti-microbacterial, 2 trypanocidals and 1 anti-cancer agent were tested in vitro against mf. The suitability of the culture media as well as the effect of the anti-infective agents on mf survival was assessed by scoring their motility. ResultsFBS supplement and additional LLC-MK2 cells significantly improved the survival of mf in DMEM and RPMI-1640 culture. In detail, RPMI-1640 supplemented with 10% FBS and LLC-MK2 cells sustained the maintenance of mf for at least 20 days (100.00 ± 0.00% survival). In co-cultures with LLC-MK2 cells without serum, M. perstans mf were maintained in DMEM and RPMI-1640 medium with a motility above 99% by day 5. Mefloquine displayed the highest microfilaricidal effect in vitro followed by artesunate. ConclusionBoth RPMI and DMEM in the presence of LLC-MK2 cells are suitable for the maintenance of M. perstans mf in vitro. In absence of the feeder cells, the addition of 10% FBS to RPMI-1640 medium improved the parasite survival rate and motility. The microfilaricidal activity of mefloquine and artesunate on M. perstans mf was documented for the first time in this study and can therefore be considered as reference for further screening of agents against this parasite stage.

In vitro and in vivo studies on anti-malarial activity of Commiphora africana and Dichrostachys cinerea used by the Maasai in Arusha region, Tanzania

BackgroundTraditional medicinal plants are one of the potential sources of anti-malarial drugs and there is an increasing interest in the use and development of traditional herbal remedies for the treatment of malaria and other ailments. This study was carried out with the aim to investigate the phytochemical screening, cytotoxic effect and antiplasmodial activities of Dichrostachys cinerea and Commiphora africana. Both plants are used by the Maasai in Tanzania in suspected malaria and other diseases. No previous work appears to have investigated the potential anti-malarial activity of the two plants.MethodsThis study aimed to investigate the in vitro anti-malarial activity of methanol and dichloromethane extracts of the two plants against chloroquine sensitive (D6) and chloroquine resistant (Dd2) strains of Plasmodium falciparum. The anti-malarial property was assessed by the lactate dehydrogenase method (pLDH). The in vivo anti-malarial study was carried out using the Peters’ 4-day suppressive test in Plasmodium berghei in Balb/c mice. Cytotoxic tests were carried out using monkey kidney epithelial cell line in [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Qualitative phytochemical screening was carried out using standard methods of analysis.ResultsThe phytochemical screening of plant extracts revealed the presence of alkaloids, flavonoids, tannins, steroids, triterpenoids, glycosides and saponins. However, alkaloids were absent in most plant extracts. The dichloromethane extracts of C. africana (stem bark); D. cinerea (stem bark) and methanol extracts of D. cinerea (whole stem) all showed promising in vitro anti-malarial activities. All other extracts did not show any significant anti-malarial activity. The two most promising extracts based on in vitro studies, DCM extracts of C. africana (stem bark) and D. cinerea (stems bark), equally exhibited very significant anti-malarial activities in the mouse model. They exhibited parasite suppression rates of 64.24 and 53.12%, respectively, and considerable improvement in weight and survival rate. Most plant extracts were not cytotoxic except for DCM extract of D. cinerea (whole stem) CC50 (29.44 µg/mL).ConclusionThe findings of this study provide scientific evidence supporting the traditional use of the plants in the treatment of malaria by the Maasai in Arusha region, Tanzania. Consequently, further work including bioassay-guided fractionation and advanced toxicity testing may yield new anti-malarial drug candidates from the two plants.

Evaluation of in vitro culture systems for the maintenance of microfilariae and infective larvae of Loa loa

BackgroundSuitable and scalable in vitro culture conditions for parasite maintenance are needed to foster drug research for loiasis, one of the neglected tropical diseases which has attracted only limited attention over recent years, despite having important public health impacts. The present work aims to develop adequate in vitro culture systems for drug screening against both microfilariae (mf) and infective third-stage larvae (L3) of Loa loa.MethodsIn vitro culture conditions were evaluated by varying three basic culture media: Roswell Park Memorial Institute (RPMI-1640), Dulbecco’s modified Eagle’s medium (DMEM) and Iscove’s modified Dulbecco’s medium (IMDM); four sera/proteins: newborn calf serum (NCS), foetal bovine serum (FBS), bovine serum albumin (BSA) and the lipid-enriched BSA (AlbuMax® II, ALB); and co-culture with the Monkey Kidney Epithelial Cell line (LLC-MK2) as a feeder layer. The various culture systems were tested on both mf and L3, using survival (% motile), motility (T90 = mean duration (days) at which at least 90% of parasites were fully active) and moulting rates of L3 as the major criteria. The general linear model regression analysis was performed to assess the contribution of each variable on the viability of Loa loa L3 and microfilarie. All statistical tests were performed at 95% confidence interval.ResultsOf the three different media tested, DMEM and IMDM were the most suitable sustaining the maintenance of both L. loa L3 and mf. IMDM alone could sustain L3 for more than 5 days (T90 = 6.5 ± 1.1 day). Serum supplements and LLC-MK2 co-cultures significantly improved the survival of parasites in DMEM and IMDM. In co-cultures with LLC-MK2 cells, L. loa mf were maintained in each of the three basic media (T90 of 16.4–19.5 days) without any serum supplement. The most effective culture systems promoting significant moulting rate of L3 into L4 (at least 25%) with substantial maintenance time were: DMEM + BSA, DMEM + NCS, DMEM-AlbuMax®II, DMEM + FBS all in co-culture with LLC-MK2, and IMDM + BSA (1.5%), DMEM + FBS (10%) and DMEM + NCS (5%) without feeder cells. DMEM + 1% BSA in co-culture scored the highest moulting rate of 57 of 81 (70.37%). The factors that promoted L. loa mf viability included feeder cells (β = 0.490), both IMDM (β = 0.256) and DMEM (β = 0.198) media and the protein supplements NCS (β = 0.052) and FBS (β = 0.022); while for L. loa L3, in addition to feeder cells (β = 0.259) and both IMDM (β = 0.401) and DMEM (β = 0.385) media, the protein supplements BSA (β = 0.029) were found important in maintaining the worm motility.ConclusionsThe findings from this work display a range of culture requirements for the maintenance of Loa loa stages, which are suitable for developing an effective platform for drug screening.

CHEMICAL COMPOSITIONS AND BIOLOGICAL PROPERTIES OF ESSENTIAL OILS FROM ZANTHOXYLUM RHETSA (ROXB.) DC AND ZANTHOXYLUM LIMONELLA ALSTON

Background: Zanthoxylum rhetsa (Roxb.) DC and Zanthoxylum limonella Alston are spices for flavouring in indigenous Thai food. They are traditionally used as an aromatic, astringent, antimicrobial, antiseptic and antidiabetic agent. The purpose of this study is to examine their chemical compositions and evaluate antibacterial, antioxidant and anticancer properties of the essential oils.&#x0D; Materials and Methods: The essential oils of Z. rhetsa and Z. limonella were analysed for phytochemical constituents by Gas chromatography–mass spectrometry (GC-MS). The antibacterial activity was determined against several bacteria using the microdilution method. Antioxidant capacity was determined by free radical scavenger 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2-azinobis-3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) methods. The anticancer activity was determined with two breast cancer cell lines (MCF-7 and MDA-MB-231) and the normal African green monkey kidney epithelial (Vero) cell line and using MTT assay.&#x0D; Results: Sabinene (22.51%) and terpinene-4-ol (32.33%) were found to be major components of Z. rhetsa essential oil while limonene (57.94%) and alpha-phelladrene (15.54%) were the major components of Z. limonella essential oil. Essential oil from Z. limonella exhibited broad spectrum antibacterial activity. Z. rhetsa and Z. limonella essential oils exhibited moderate antioxidant activity. The essential oil from Z. rhetsa possessed the ability to inhibit breast cancer cell (MCF-7 and MDA-MB-231) proliferation and cell viability.&#x0D; Conclusion: This study suggest that the essential oils from Z. limonella and Z. rhetsa could be applied as safe antibacterial and antioxidant agents for food and have the potential for further development of new anticancer agents.

The Effect of Combining Natural Terpenes and Antituberculous Agents against Reference and Clinical Mycobacterium tuberculosis Strains.

Background: On account of emergence of multi- and extensively drug-resistant Mycobacterium tuberculosis (Mtb) strains, combinations of drugs with natural compounds were tested to search for antibiotic activity enhancers. In this work we studied terpenes (α-pinene, bisabolol, β-elemene, (R)-limonene, (S)-limonene, myrcene, sabinene), which are the main constituents of essential oil obtained from Mutellina purpurea L., a plant with described antitubercular activity, to investigate their interactions with antibiotics against reference Mtb strains and multidrug-resistant clinical isolates. Methods: The serial dilution method was used to evaluate the minimal inhibitory concentration (MIC) of tested compounds, while the fractional inhibitory concentration index (FICI) was calculated for characterization of interactions. Moreover, IC50 values of tested compounds were determined using monkey kidney epithelial cell line (GMK). Results: The combinations of all studied terpenes with ethambutol or rifampicin resulted in a synergistic interaction. Bisabolol and (R)-limonene decreased the MIC for rifampicin at least two-fold for all tested strains, however no synergistic action was observed against virulent strains. The tested terpenes showed slight (bisabolol) or no cytotoxic effect against normal eukaryotic cells in vitro. Conclusions: The obtained enhanced activity (FICI < 0.5) of ethambutol and rifampicin against H37Ra strain under the influence of the studied terpenes may be correlated to the capability of essential oil constituents to modify bacterial resistance mechanisms in general. The observed differences in avirulent and virulent bacteria susceptibility to terpenes tested separately and in combinations with antibiotics can be correlated with the differences in the cell wall structure between H37Ra mutant and all virulent strains.

Antimicrobial Properties of Tris(homoleptic) Ruthenium(II) 2-Pyridyl-1,2,3-triazole "Click" Complexes against Pathogenic Bacteria, Including Methicillin-Resistant Staphylococcus aureus (MRSA).

A series of tris(homoleptic) ruthenium(II) complexes of 2-(1-R-1H-1,2,3-triazol-4-yl)pyridine "click" ligands (R-pytri) with various aliphatic (R = butyl, hexyl, octyl, dodecyl, and hexdecyl) and aromatic (R = phenyl and benzyl) substituents was synthesized in good yields (52%-66%). The [Ru(R-pytri)3]2+(X-)2 complexes (where X- = PF6- or Cl-) were characterized by elemental analysis, high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), 1H and 13C nuclear magnetic resonance (NMR) and infrared (IR) spectroscopies, and the molecular structures of six of the compounds confirmed by X-ray crystallography. 1H NMR analysis showed that the as-synthesized materials were a statistical mixture of the mer- and fac-[Ru(R-pytri)3]2+ complexes. These diastereomers were separated using column chromatography. The electronic structures of the mer- and fac-[Ru(R-pytri)3]2+ complexes were examined using ultraviolet-visible (UV-Vis) spectroscopy and cyclic and differential pulse voltammetry. The family of R-pytri ligands and the corresponding mer- and fac-[Ru(R-pytri)3]2+ complexes were tested for antimicrobial activity in vitro against both Staphylococcus aureus and Escherichia coli bacteria. Agar-based disk diffusion assays indicated that two of the [Ru(R-pytri)3](X)2 complexes (where X = PF6- and R = hexyl or octyl) displayed good antimicrobial activity against Gram-positive S. aureus and no activity against Gram-negative E. coli at the concentrations tested. The most active [Ru(R-pytri)3]2+ complexes ([Ru(hexpytri)3]2+ and Ru(octpytri)3]2+) were converted to the water-soluble chloride salts and screened for their activity against a wider range of pathogenic bacteria. As with the preliminary screen, the complexes showed good activity against a variety of Gram-positive strains (minimum inhibitory concentration (MIC) = 1-8 μg/mL) but were less effective against Gram-negative bacteria (MIC = 16-128 μg/mL). Most interestingly, in some cases, the ruthenium(II) "click" complexes proved more active (MIC = 4-8 μg/mL) than the gentamicin control (MIC = 16 μg/mL) against two strains of methicillin-resistant S. aureus (MRSA) (MR 4393 and MR 4549). Transmission electron microscopy (TEM) experiments and propidium iodide assays suggested that the main mode of action for the ruthenium(II) R-pytri complexes was cell wall/cytoplasmic membrane disruption. Cytotoxicity experiments on human dermal keratinocyte and Vero (African green monkey kidney epithelial) cell lines suggested that the complexes were only modestly cytotoxic at concentrations well above the MIC values.

&lt;i&gt;In vitro&lt;/i&gt; anticancer activity and cytotoxicity of some papaver alkaloids on cancer and normal cell lines

Background: The purpose of this study is to determine the effect of in vitro anticancer activity and cytotoxicity of 13 Papaver alkaloids (amurine, armepavine, berberine, isocorydine, isothebaine, macranthine, mecambrine, mecambridine, narkotine, orientalidine, oripavine, salutaridine and thebaine) against the human cervical cancer cell line (HeLa) compared to the normal African green monkey kidney epithelial cell line (Vero) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Materials and Methods: The Vero and HeLa cell lines were treated with various concentrations (1-300 μg/mL) of alkaloids for 48 h. Values for cytotoxicity measured by MTT assay were expressed as the concentration that causes a 50% decrease in cell viability (IC50) (μg/mL). Results: Berberine and macranthine were the most active alkaloids. Salutaridine exhibited no cytotoxic activity against two types of cell lines. Dose-dependent studies presented IC50 of 12.08 μg/mL and IC50 of 71.14 μg/mL for berberine and IC50 of 24.16 μg/mL and IC50 of >300 μg/mL for macranthine on the HeLa cells and the Vero cells respectively. Conclusion: The degree of selectivity of the compounds can be expressed by its Selectivity Index (SI) value. High SI value (>2) of a compound gives a selective toxicity towards cancer cells (SI = IC50 for normal cells/IC50 for cancer cells). Two alkaloids showed significant SI values, which are 12.42 for macranthine and 5.89 for berberine. Hence, macranthine and berberine display potential to be further exploited in the discovery and development of new anticancer agents.

Biocompatibility of composite membranes composed of anodic aluminium oxide (AAO) and Poly (2-hydroxyethylmethacrylate) for use as a cell culture substrate

In this study we investigate the biomedical potential of composite membranes composed of anodic aluminium oxide (AAO) and Poly (2-hydroxyethylmethacrylate) (pHEMA). The nano-porous AAO membranes were produced using a temperature controlled two-step electrochemical anodization technique. The AAO/pHEMA composite membranes were formed using the solution template wetting technique. The Cercopithecus aethiops (African green monkey) Kidney (Vero) epithelial cell line was used to demonstrate the applicability of the synthesised membranes and composites. Investigating cell adhesion, morphology and proliferation over a 72h period assessed cellular interactions and responses of this cell line to the various membranes types. The novelty of the results stems from the use of pHEMA as a constituent, a material which normally does not promote cell adhesion, hence its use in contact lens and catheters.

Cratoxylum formosum (Jack) Dyer ssp. pruniflorum (Kurz) Gogel. (Hóng yá mù) extract induces apoptosis in human hepatocellular carcinoma HepG2 cells through caspase-dependent pathways

BackgroundCratoxylum formosum (Jack) Dyer ssp. pruniflorum (Kurz) Gogel. (Hóng yá mù) (CF) has been used for treatment of fever, cough, and peptic ulcer. Previously, a 50% ethanol-water extract from twigs of CF was shown highly selective in cytotoxicity against cancer cells. This study aims to investigate the molecular mechanisms underlying the apoptosis-inducing effect of CF.MethodsThe cytotoxicity of CF was evaluated in the human hepatocellular carcinoma (HCC) HepG2 cell line in comparison with a non-cancerous African green monkey kidney epithelial cell line (Vero) by a neutral red assay. The apoptosis induction mechanisms were investigated through nuclear morphological changes, DNA fragmentation, mitochondrial membrane potential alterations, and caspase enzyme activities.ResultsCF selectively induced HepG2 cell death compared with non-cancerous Vero cells. A 1.5-fold higher apoptotic effect compared with melphalan was induced by 120 μg/mL of the 50% ethanol-water extract of CF. The apoptotic cell death in HepG2 cells occurred via extrinsic and intrinsic caspase-dependent pathways in dose- and time-dependent manners by significantly increasing the activities of caspase 3/7, 8, and 9, decreasing the mitochondrial membrane potential, and causing apoptotic body formation and DNA fragmentation.ConclusionsCF extract induced a caspase-dependent apoptosis in HepG2 cells.

Can Anodic Aluminium Oxide Nanomembranes Treated with Nanometre Scale Hydroxyapatite be Used as a Cell Culture Substrate

In this study we investigate the biomedical potential of composite membranes composed of anodic aluminium oxide (AAO) and nanometre scale hydroxyapatite (HAP). The nano-porous AAO membranes were produced using a temperature controlled two-step anodization technique. The AAO/HAP composite membranes were formed using the solution template wetting technique. The Cercopithecus aethiops (African green monkey) Kidney (Vero) epithelial cell line was used to demonstrate the biocompatibility of the synthesised membranes and composites. Investigating cell adhesion, morphology and proliferation over a 72 h period assessed cellular interactions and responses of the cell line to the various membranes types.

Chemical Synthesis, Characterisation, and Biocompatibility of Nanometre Scale Porous Anodic Aluminium Oxide Membranes for Use as a Cell Culture Substrate for the Vero Cell Line: A Preliminary Study

In this preliminary study we investigate for the first time the biomedical potential of using porous anodic aluminium oxide (AAO) membranes as a cell substrate for culturing the Cercopithecus aethiops (African green monkey) Kidney (Vero) epithelial cell line. One advantage of using the inorganic AAO membrane is the presence of nanometre scale pore channels that allow the exchange of molecules and nutrients across the membrane. The size of the pore channels can be preselected by adjusting the controlling parameters of a temperature controlled two-step anodization process. The cellular interaction and response of the Vero cell line with an in-house synthesised AAO membrane, a commercially available membrane, and a glass control were assessed by investigating cell adhesion, morphology, and proliferation over a 72 h period. The number of viable cells proliferating over the respective membrane surfaces revealed that the locally produced in-house AAO membrane had cells numbers similar to the glass control. The study revealed evidence of focal adhesion sites over the surface of the nanoporous membranes and the penetration of cellular extensions into the pore structure as well. The outcome of the study has revealed that nanometre scale porous AAO membranes have the potential to become practical cell culture scaffold substrates with the capability to enhance adhesion and proliferation of Vero cells.