Maximum Non-cytotoxic Concentrations Research Articles

Anti-microbial and anti-hepatitis-A activities of Asphodelus refractus boiss. extract and pure isolated metabolites

Four compounds, including N-trans-feruloyltyramine, were isolated from Asphodelus refractus extract and elucidated using UV, IR, MS, and NMR techniques. The trans-feruloyltyramine displayed advantageous structural characteristics, including a Log P of 2.58 and compliance with Lipinski's rules. With 7 rotatable bonds, 23 heavy atoms, 12 aromatic atoms, and a surface area of 78.79 Ų, it effectively binds to the target 3C-proteinase receptor. Trans-feruloyltyramine exhibited characteristic IR and 1H-NMR spectra, confirming the presence of a conjugated double bond, feruloyl and tyramine moieties, and an amide structure. The antimicrobial assay revealed mild antifungal effects for the plant extract and isolated compounds against Candida tropicalis with inhibition zone diameters (IZD) of 12–15 mm, compared to ketoconazole (IZD, 21 mm). The extract exhibited better inhibition for Gram-ve compared to the Gram+ve bacteria. The findings indicated that chloroform extract and trans-feruloyltyramine have marked anti-hepatitis-A activity, with inhibition percentages at 69.34 ± 3.92 and 56.91 ± 1.75, respectively, at their maximum non-cytotoxic concentrations (MNCC), compared to amantadine (83.24 %). They have also exhibited non-selective anti-HAV activity with EC50 values of 308.69 ± 8.43 and 317.94 ± 6.88 μg/mL, respectively. In comparison to other isolated compounds, docking predicted a higher binding affinity for trans-feruloyltyramine towards hepatitis A virus (HAV) 3C-proteinase, with binding energy at DG -8.3 Kcal/mol.

Baicalin Inhibits FIPV Infection In Vitro by Modulating the PI3K-AKT Pathway and Apoptosis Pathway.

Feline infectious peritonitis (FIP), a serious infectious disease in cats, has become a challenging problem for pet owners and the industry due to the lack of effective vaccinations and medications for prevention and treatment. Currently, most natural compounds have been proven to have good antiviral activity. Hence, it is essential to develop efficacious novel natural compounds that inhibit FIPV infection. Our study aimed to screen compounds with in vitro anti-FIPV effects from nine natural compounds that have been proven to have antiviral activity and preliminarily investigate their mechanisms of action. In this study, the CCK-8 method was used to determine the maximum noncytotoxic concentration (MNTC), 50% cytotoxic concentration (CC50), and 50% effective concentration (EC50) of natural compounds on CRFK cells and the maximum inhibition ratio (MIR) of the compounds inhibit FIPV. The effect of natural compounds on FIPV-induced apoptosis was detected via Annexin V-FITC/PI assay. Network pharmacology (NP), molecular docking (MD), and 4D label-free quantitative (4D-LFQ) proteomic techniques were used in the joint analysis the mechanism of action of the screened natural compounds against FIPV infection. Finally, Western blotting was used to validate the analysis results. Among the nine natural compounds, baicalin had good antiviral effects, with an MIR > 50% and an SI > 3. Baicalin inhibited FIPV-induced apoptosis. NP and MD analyses showed that AKT1 was the best target of baicalin for inhibiting FIPV infection. 4D-LFQ proteomics analysis showed that baicalin might inhibit FIPV infection by modulating the PI3K-AKT pathway and the apoptosis pathway. The WB results showed that baicalin promoted the expression of EGFR, PI3K, and Bcl-2 and inhibited the expression of cleaved caspase 9 and Bax. This study found that baicalin regulated the PI3K-AKT pathway and the apoptosis pathway in vitro and inhibited FIPV-induced apoptosis, thus exerting anti-FIPV effects.

Assessing the virucidal activity of essential oils against feline calicivirus, a non-enveloped virus used as surrogate of norovirus

Norovirus (NoV) causes serious gastrointestinal disease worldwide and is regarded as an important foodborne pathogen. Due the difficulties of in vitro cultivation for human NoV, alternative caliciviruses (i.e., feline calicivirus, FCV, or murine NoV) have long been used as surrogates for in vitro assessment of the efficacy of antivirals. Essential oils (EOs) are natural compounds that have displayed antimicrobial and antioxidant properties.We report in vitro the virucidal efficacy of four EOs, Melissa officinalis L. EO (MEO), Thymus vulgaris L. EO (TEO), Rosmarinus officinalis L. EO (REO), and Salvia officinalis L. EO (SEO) against FCV at different time contacts (10, 30 min, 1, 4 and 8 h). At the maximum non-cytotoxic concentration and at 10- and 100- fold concentrations over the cytotoxic threshold, the EOs did not decrease significantly FCV viral titers. However, MEO at 12,302.70 μg/mL exhibited a significant efficacy decreasing the viral titer by 0.75 log10 Tissue Culture Infectious Dose (TCID50)/50 μl after 10 min as compared to virus control.In this study, virucidal activity of four EOs against FCV, was investigated. A lack of virucidal efficacy of TEO, REO and SEO at different compound concentrations and time contacts against FCV was observed whilst MEO was able to significantly decrease FCV titer.

Matrine Targets BTF3 to Inhibit the Growth of Canine Mammary Tumor Cells.

The canine mammary tumor model is more suitable for studying human breast cancer, and the safety concentrations of matrine and the biotin-labeled matrine probe were determined in canine primary mammary epithelial cells, and then selected canine mammary tumor cell lines CHMm and CHMp were incubated with matrine, and cell viability was detected by CCK-8. The biotin-labeled matrine probe was used to pull-down the targets of matrine in canine mammary tumor cells, and the targets were screened in combination with activity-based protein profiling (ABPP) and Genecards database, and verified by qPCR and western blot. The results showed that the maximum non-cytotoxic concentrations of matrine and biotin-labeled matrine probe in canine primary mammary epithelial cells were 250 μg/mL and 500 μg/mL, respectively. Matrine and biotin-labeled matrine probe had a proliferation inhibitory effect time-dependently on CHMm and CHMp cells within a safe concentration range, and induced autophagy in cells. Then BTF3 targets were obtained by applying ABPP and Genecards screening. Cellular thermal shift assay (CETSA) findings indicated that matrine could increase the heat stability of BTF3 protein. Pull-down employing biotin-labeled matrine probe with CHMm and CHMp cell lysates revealed that BTF3 protein was detected in the biotin-labeled matrine probe group and that BTF3 protein was significantly decreased by the addition of matrine. The qPCR and western blot findings of CHMm and CHMp cells treated with matrine revealed that matrine decreased the expression of the BTF3 gene and protein with the extension of the action time, and the impact was more substantial at the protein level, respectively.

Salmonella mutant strains resistant to herbicides - Acetohydroxyacid synthase inhibitors and their use at the Ames test

Cytotoxicity of some pesticides is a disadvantage for the Salmonella/microsome assay with regard to the equivalence assessment of pesticide technical grade active ingredients to the original products and detection of low-level impurities. The technical grade active ingredients (TGAIs) of pesticides from certain chemical classes were found to be toxic for Salmonella typhimurium strains. Among the highly cytotoxic compounds were sulfonylureas, which include 20 active ingredients. In addition, this class includes active pharmaceutical ingredients used for the manufacture of antidiabetics drugs.A traditional selection methodology was applied using the cultivation of S. typhimurium TA100 in the presence of high concentrations of thifensulfuronmethyl (TFSM) to obtain a resistant test strain insusceptible to sulfonylurea toxic effect. Two strains resistant not only to sulfonylureas (SFU) but also triazolepyrimidines were received. The first mutant strain (deposited as S. typhimurium VKPM B-14099 in the Russian National Collection of Industrial Microorganisms) demonstrated the TA100 phenotypic characteristics: hisG46, rfa, ΔuvrB-bio, pKM101. The second strain (deposited as S. typhimurium VKPM B-14359) showed the TA1535 phenotypic characteristics and probably lost the R-factor due to the selection using the poor Gm-media with TFSM. Positive controls caused pronounced mutagenic effects (±S9) in both strains, consequently the mutants did not lose the ability to respond to induction of the reverse gene mutations. The maximum non-cytotoxic concentrations of SFUs and triazole-pyrimidines for the Ames test strains did not exceed 0.05–0.125 mg/plate, while no evidence of cytotoxicity was observed for the mutants up to 5.0 mg/plate. Electron microscopy of the ultrathin sections of Salmonella cells grown with and without TFSM showed an obvious difference in the structure of the cell wall and cytoplasm in mutant and parental cultures. The concurrent resistance both to SFU and triazolepyrimidines was assumed to be mediated by the same mechanism of action of the pesticides from these classes – inhibition of acetohydroxyacid synthase. To confirm this hypothesis, the tests in the presence of branched-chain amino acids were carried out. The enrichment of agar with isoleucine prevented the toxic effects of SFU and triazolepyrimidines for all Ames test strains used in the study, while strong cytotoxicity was observed in the presence of valine and leucine.Considering the tolerance of strains both to SFU and triazolpyrimidines and the results with branched-chain amino acids, the modification of target acetohydroxyacid synthase was supposed the key to the acquired resistance. The new strains resistant to sulfonylureas and triazole-pyrimidines expands the possibilities to reveal mutagenic impurities that may occur in TGAIs in small amounts.

Target Discovery of Matrine against PRRSV in Marc-145 Cells via Activity-Based Protein Profiling.

Porcine reproductive and respiratory syndrome (PRRS) seriously endangers the sustainable development of the pig industry. Our previous studies have shown that matrine can resist porcine reproductive and respiratory syndrome virus (PRRSV) infection. This study aimed to explore the anti-PRRSV targets of matrine in Marc-145 cells. Biotin-labeled matrine 1 and 2 were used as probes. MTT assay was used to determine the maximum non-cytotoxic concentration (MNTC) of each probe in Marc-145 cells. The anti-PRRSV activity of each probe was evaluated via MTT, qPCR and Western blot, and its anti-inflammatory activity was evaluated via qPCR and Western blot. The targets of matrine in Marc-145 cells were searched using activity-based protein profiling (ABPP), and compared with the targets predicted via network pharmacology for screening the potential targets of matrine against PRRSV. The protein-protein interaction networks (PPI) of potential targets were constructed using a network database and GO/KEGG enrichment analysis was performed. ACAT1, ALB, HMOX1, HSPA8, HSP90AB1, PARP1 and STAT1 were identified as potential targets of matrine, and their functions were related to antiviral capacity and immunity. Matrine may play an anti-PRRSV role by directly acting on ACAT1, ALB, HMOX1, HSPA8, HSP90AB1, PARP1 and STAT1.

Exogenous nitric oxide generated from SNAP blocks porcine circovirus type 2 replication and regulates NF-κB activity in PK-15 cells

ObjectivesPCV2-associated disease (PCVAD), caused by porcine circovirus type 2 (PCV2) infection, is one of the major infectious diseases in the global swine industry. Nitric oxide (NO), as an important signalling molecule, has antiviral activities against a variety of viruses. To date, limited knowledge is available on the role of NO during PCV2 infection. MethodsThis study was conducted to investigate the effects of exogenous NO on PCV2 replication in vitro. To exclude the possibility that the detected antiviral effects were due to cell toxicity, maximum non-cytotoxic concentrations of the drugs were determined. Kinetics of NO production were assessed after drug treatment. The antiviral activities of NO at different concentrations and at different time points were carefully assessed by measuring the virus titers, viral DNA copies and percentage of PCV2-infected cells. Regulation of NF-κB activity by exogenous NO was also investigated. ResultsKinetics of NO production indicated that S-nitroso-acetylpenicillamine (SNAP) produced NO in a dose-dependent manner, while NO was scavenged by its scavenger haemoglobin (Hb). An in vitro antiviral assay demonstrated that exogenous NO strongly inhibited PCV2 replication in a time-dependent and dose-dependent manner, whereas the inhibitory effects could be reversed by Hb. Furthermore, inhibition of NF-κB activity induced by NO contributed to a notable decrease in PCV2 replication. ConclusionThese findings provide a new potential antiviral therapy against PCV2 infection, and the antiviral effects of exogenous NO may be partly achieved by regulating NF-κB activity.

New possibilities of the Ames test for evaluation of mutagenicity of technical products of active ingredients of pesticides

Introduction. The Ames test is the one of the most popular methods for mutagenicity evaluation of environmental factors. In some cases, this method is suggested to be the only and sufficient assay for the first stage of the equivalence assessment of pesticide technical grade active ingredients (TGAI) to the original products. A limitation of the Ames test is related to the impossibility of an objective equivalence assessment of some cytotoxic TGAIs, in particular, sulfonylureas, and triazolpyrimidines. Based on the mode of action of the pesticides belongs to these chemical classes, we suggested a modification of the plate incorporation method protocol of the Ames test to the increase of maximal non-cytotoxic concentration up to the 5 mg/plate recommended by regulatory documents. Materials and methods. The five strains of Salmonella typhimurium TA98, TA100, TA1535, TA97, TA102 were used. The modification of the protocol included a supplementation of the top agar with isoleucine (1-5 mM). Results. The maximum non-cytotoxic concentrations of thifensulfuron-methyl and florasulam using the standard top agar did not exceed 0.05-0.125 mg/plate. The enrichment of the top agar with isoleucine allowed evaluating the mutagenicity of the substances up to the maximal recommended concentration of 5.0 mg/plate. The number of spontaneous revertants was within the historical limits of the laboratory control obtained under standard conditions. Positive controls showed pronounced mutagenic effects in case of all strains with and without metabolic activation (p≤0.05). Limitations. Mutagenicity was evaluated only for TGAIs, which are acetohydroxyacid synthase inhibitors. Conclusion. The application of the modified Ames test protocol for mutagenicity assessment of TGAIs from the classes of sulfonylureas and triazolpyrimidines under supplementation of the top agar with isoleucine is a more objective way to evaluate their mutagenicity. The proposed protocol expands the possibilities of revealing dangerous mutagenic impurities that may occur in TGAIs in the small quantities, and after entering the environment can cause the gain in the mutation level in living organisms.

Anti-Pollution Activity, Antioxidant and Anti-Inflammatory Effects of Fermented Extract from Smilax china Leaf in Macrophages and Keratinocytes

Air pollution has considerable effects on the human skin, showing that every single pollutant has a different toxicological impact on it. The oxidative stress that exceeds the skin’s antioxidant capacity can lead to oxidative damage and premature skin aging by repeated air pollutant contact. In this study, according to the generalized protocol available to objectively substantiate the ‘anti-pollution’ claim, we evaluated several biomarkers after pollutants exposure in Raw 264.7 macro-phages and HaCaT keratinocytes to investigate the possibility of anti-pollution cosmetic material of fermented extract from Smilax china leaves (FESCL). FESCL decreased pollutants-induced luciferase activity in a dose-dependent manner, and FESCL significantly inhibited XRE-luciferase activity at a concentration of 1%. The IC50 value of FESCL showed the same DPPH scavenging activity at 0.0625% as ascorbic acid, and the maximum DPPH scavenging activity (92.44%) at 1%. The maximum permissible non-cytotoxic concentrations of FESCL for a Raw 264.7 cell was determined to be 2%, where PGE2 production of FESCL was inhibited by 78.20%. These results show the anti-pollution activity of FESCL against the pollutant-stimulated human living skin explants. In conclusion, we confirmed the anti-pollution potential of FESCL as one of the functional materials in cosmetic formulation.

Inhibitory effects of cedar pine needle extract on H9N2 avian influenza virus in vitro and in vivo

H9N2 avian influenza virus causes significant economic losses to the poultry industry, due to its wide-spread prevalence and propensity to induce secondary and mixed infections. Antigenic drift limits vaccine efficacy. New anti-viral therapies are needed to complement existing control measures. At the maximum non-cytotoxic concentration (25 mg/mL), cedar pine needle extract inhibited H9N2 avian influenza virus proliferation in vitro and in vivo. Cedar pine needle extract reduced the haemagglutinin titre, inhibited H9N2 avian influenza virus nucleocapsid protein expression, and indirectly regulate type I and II interferon expression. Interleukin-6 expression increased during the pre-infection period but decreased during the mid-to-late stages of infection. Cedar pine needle extract may inhibit the proliferation of pathogens, regulate the immune response, and reduce host tissue damage and may serve as a potential target for drug development against H9N2 avian influenza virus.

A novel strategy for optimal component formula of anti-PRRSV from natural compounds using tandem mass tag labeled proteomic analyses

BackgroundPorcine Reproductive and Respiratory Syndrome (PRRS) is one of the most important porcine viral diseases which have been threatening the pig industry in China. At present, most commercial vaccines fail to provide complete protection because of highly genetic diversity of PRRSV strains. This study aimed to optimize a component formula from traditional Chinese medicine(TCM)compounds with defined chemical characteristics and clear mechanism of action against PRRSV.MethodsA total of 13 natural compounds were screened for the anti-PRRSV activity using porcine alveolar macrophages (PAMs). Three compounds with strong anti-PRRSV activity were selected to identify their potential protein targets by proteomic analysis. The optimal compound formula was determined by orthogonal design based on the results of proteomics. MTT assay was used to determine the maximum non-cytotoxic concentration (MNTC) of each compound using PAMs. QPCR and western blot were used to investigate the PRRSV N gene and protein expression, respectively. The Tandem Mass Tag (TMT) technique of relative quantitative proteomics was used to detect the differential protein expression of PAMs treated with PRRSV, matrine (MT), glycyrrhizic acid (GA) and tea saponin (TS), respectively. The three concentrations of these compounds with anti-PRRSV activity were used for orthogonal design. Four formulas with high safety were screened by MTT assay and their anti-PRRSV effects were evaluated.ResultsMT, GA and TS inhibited PRRSV replication in a dose-dependent manner. CCL8, IFIT3, IFIH1 and ISG15 were the top four proteins in expression level change in cells treated with MT, GA or TS. The relative expression of IFIT3, IFIH1, ISG15 and IFN-β mRNAs were consistent with the results of proteomics. The component formula (0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 μg/mL TS) showed synergistic anti-PRRSV effect.ConclusionsThe component formula possessed anti-PRRSV activity in vitro, in which the optimal dosage on PAMs was 0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 μg/mL TS. Compatibility of the formula was superposition of the same target with GA and TS, while different targets of MT. IFN-β may be one of the targets of the component formula possessed anti-PRRSV activity.

Structure-Based Virtual Screening and Identification of Potential Inhibitors of SARS-CoV-2 S-RBD and ACE2 Interaction.

The emergence and rapid spread of SARS-CoV-2 have caused a worldwide public health crisis. Designing small molecule inhibitors targeting SARS-CoV-2 S-RBD/ACE2 interaction is considered as a potential strategy for the prevention and treatment of SARS-CoV-2. But to date, only a few compounds have been reported as SARS-CoV-2 S-RBD/ACE2 interaction inhibitors. In this study, we described the virtual screening and experimental validation of two novel inhibitors (DC-RA016 and DC-RA052) against SARS-CoV-2 S-RBD/ACE2 interaction. The NanoBiT assays and surface plasmon resonance (SPR) assays demonstrated their capabilities of blocking SARS-CoV-2 S-RBD/ACE2 interaction and directly binding to both S-RBD and ACE2. Moreover, the pseudovirus assay revealed that these two compounds possessed significant antiviral activity (about 50% inhibition rate at maximum non-cytotoxic concentration). These results indicate that the compounds DC-RA016 and DC-RA052 are promising inhibitors against SARS-CoV-2 S-RBD/ACE2 interaction and deserve to be further developed.

Inactivation of Herpes Simplex Virus by Photosensitizing Anthraquinones Isolated from Heterophyllaea pustulata.

Heterophyllaea pustulata is a phototoxic plant from Argentina. Aerial parts extracts, high in photosensitizing anthraquinones, have shown in vitro antiviral activity. The purpose of this study was to study the antiherpetic activity of the main purified anthraquinones, even evaluating their competence as photodynamic sensitizers to photo-stimulate the antiviral effect. In vitro antiviral activity against Herpes Simplex virus type I and the photo-inactivation of viral particle were studied by the Neutral Red uptake test and observation of the cytopathic effect. Rubiadin 1-methyl ether and 5,5'-bisoranjidiol produced a significant effect (≥ 80% inhibition) with minimal damage to host cells (subtoxic concentration). Anthraquinones with poor antiherpetic activity at its maximum noncytotoxic concentration showed an important photo-stimulated effect, such is the case of soranjidiol and 5,5'-bisoranjidiol (28.0 ± 6.3 vs. 81.8 ± 2.1% and 15.5 ± 0.3 vs. 89.8 ± 1.7%, respectively). The study also proved the decrease of viral particles, necessary to reduce infection. Therefore, photosensitizing anthraquinones from natural resources could be proposed to develop new treatments for localized viral lesions with antimicrobial photodynamic therapy.

English

The jackfruit tree (Artocarpus heterophyllus Lam.) is considered one of the most versatile trees and is used from wood production to traditional medicine. In traditional medicine, leaves are used to treat asthma, wounds and abscesses, and prevent ringworm infection. Several biological activities have been documented for jackfruit extracts confirming its popular use; however, its in vitro cytotoxicity and anti-herpesvirus activity are rarely discussed. The objective of this study was to explore the in vitro anti-herpesviral potential of A. heterophyllus leaf extracts at non-cytotoxic levels. Leaves were obtained from specimens located in the Atlantic forest biome. Dried and fresh ethanolic, methanolic and hexanic leaf extracts were obtained from the partition of a crude ethanolic extract. The cytotoxicity was performed on erythrocyte and kidney cell lines. The antiviral test was against equine, suid, and bovine herpesviruses. The contents of phenolic compounds were established from methanolic leaf extracts. Fresh leaf extracts did not show toxicity to erythrocytes in any concentration tested; however, dry leaf extracts showed >10% of hemolysis at ≥200 µg/ml. The maximum non-cytotoxic concentrations for Vero and MDBK cells ranged from 7.8 to 125 µg/ml. The content of phenolic compounds was 22.02% higher in fresh leaf than in dry leaf methanolic extracts. Methanolic extracts showed antiviral activity against BoHV-1 (PI of 99.20%) and SuHV-1 (PI of 94.38%). A. heterophyllus fresh and dry leaf extracts are remarkable as anti-herpesviruses and candidates to proceed for in vivo tests. Key words: Artocarpus heterophyllus Lam., Jackfruit tree, antiviral activity, kidney cell-line, erythrocyte, medicinal plant.

Cytotoxicity and antiviral activity evaluation of Cymbopogon spp hydroethanolic extracts

Cymbopogon citratus and C. nardus are noteworthy among the several existing plant species displaying medicinal properties, due to the potential pharmacological activity of these species, including antiviral, antibacterial, antifungal and anti-trypanosomal activities. The objective of this study was to carry out in vitro toxicity tests of plant extracts from both species and analyze potential antiviral activity against Human mastadenovirus serotype 5 (HAdV-5). Two cell lines (A549 and VERO) were used and mitochondrial and lysosomal viability were determined by the MTT and neutral red assay, respectively, after two exposure times (24 hours and six days). The aim of these assays was to counteract the behavior of the extracts against the different cell lines and determine their non-toxic concentration range, in order to evaluate possible antiviral activity against HAdV-5. Plaque reduction and inhibition index of viral titer assays were performed using the maximum non-cytotoxic concentrations (MNCC) of each extract. The results indicate MNCC at 625 μg/mL for all extracts, except for Cymbopogon nardus obtained with 80% ethanol (CN80), which showed toxicity at concentrations higher than 312.5 μg/mL. CN80 was the only extract that displayed potential activity against HAdV-5, at a concentration of 75 μg/mL, becoming a candidate for extract fraction purification and/or the isolation of substances related to the observed antiviral activity.

Inhibition of Herpes Simplex Virus-1 by the Modified Green Tea Polyphenol EGCG-Stearate

Epigallocatechin gallate (EGCG), a green tea polyphenol possesses antioxidant, antibacterial, anticancer and antiviral properties. EGCG-Stearate (EGCG-S) is of interest for this study because of its stability and lipophilic properties. The chemical modification of EGCG-S increased its lipid solubility. Herpes simplex virus-1 (HSV-1), a member of the family Herpesviridae, and Alphaherpesvirinae subfamily is a leading cause of human viral diseases in the United States. In this study, 25 μM, 50 μM, 75 μM, and 100 μM of EGCG and EGCG-S were used to carry out cytotoxicity, cell viability and cell proliferation assays to determine the maximum non-cytotoxic concentrations on cultured A549 cells. The results suggested that 75 μM of EGCG and EGCG-S is the appropriate concentration to further study the effect on the infection of HSV-1 in A549 cells. Infectivity, antiviral, and inverted microscopy assays were performed to study the effects of EGCG and EGCG-S on HSV-1 infection. An antiviral assay was performed using luminescence and it indicated that EGCG-S treated HSV-1 showed up to 90% inhibition. Confocal microscopy images further supported the inhibitory effects of 75 μM EGCG-S on HSV-1 infection in A549 cells. The long-term goal of this research is to use EGCG-S as a possible novel topical therapeutic treatment to limit the spread of HSV-1 infections.

English

This study aimed to determine the in vitro toxicity of Genipa americana and its antiviral and anti-microbial activities. Due to lack of knowledge on G. americana validation as medicinal plant, biological studies were performed. For the cytotoxicity experiments, decreasing concentrations of ethanolic extracts of G. americana branches, fruits, and leaves were added to a confluent monolayer of Vero cells. After 72 h, it was determined, the first concentration of each extract in which any cellular change was observed (maximum non-cytotoxic concentration- MNCC). The MNCC of fruit and branches extracts was of 0.5 and 0.25 mg/mL, respectively. The leaves extract did not show cytotoxicity in the tested dilutions. The antiviral activities of G. americana extracts were tested on the suid (SuHV-1) and equine (EHV-1) alpha herpes viruses through reduction of their viral titers. The fruit extract presented a viral inhibition on EHV-1 of 86%, but it was not effective for SuHV-1. Instead, the leaves and branch extracts showed antiviral activity against SuHV-1, with a viral inhibition of 73 and 79%, respectively, but no inhibition on EHV-1. The antimicrobial action on Escherichia coli and Staphylococcus aureus was performed by the agar diffusion and the microdilution methods; and by agar diffusion method against Candida albicans, Candida parapsilosis, Candida glabrata and Candida krusey. The fruits extract showed inhibition of bacterial growth for E. coli and S. aureus with a bacteriostatic effect on E. coli and a bactericidal effect on S. aureus. There was no effect on any Candida species. Ethanolic extract of G. americana fruits seemed to be a promising source of phytotherapic drugs with a spectrum of action on diverse microorganisms in non-toxic concentrations. Key words: Alphaherpes virus, medicinal plant, antimicrobial, antiviral, cell line, broad spectrum.

Antiviral activity of Thiosemicarbazones derived from α‐amino acids against Dengue virus

The endemicity and seasonal outbreaks of Dengue disease in most tropical and subtropical countries underscores an urgent need to develop effective prevention and control measures. Development of a Dengue vaccine, which is complicated by the Antibody Dependent Enhancement effect (ADE), a viral inhibitor, seems prudent as it would inhibit the spread of the virus. In vitro methods such as MTT assay and plaque formation unit reduction assays were employed for screening the viral inhibitory property of α-amino acid based Thiosemicarbazides. The results elicits that at concentrations not exceeding the maximum non cytotoxic concentration (MNCC), these compounds completely prevented Dengue virus infection in vero cells as indicated by the absence of cytopathic effects in a dose-dependent manner. The high potency of Bz-Trp-TSC against all four types of Dengue virus infection elevates Thiosemicarbazide as a lead antiviral agent for Dengue disease. Screening small molecules for antiviral activity against the most rapidly spreading mosquito-borne viral disease is being explored by several research groups. Our findings would help to augment the efforts to identify the lead compounds for antiviral therapy to combat the Dengue disease. J. Med. Virol. 89:546-552, 2017. © 2016 Wiley Periodicals, Inc.

Definition of transcriptome-based indices for quantitative characterization of chemically disturbed stem cell development: introduction of the STOP-Toxukn and STOP-Toxukk tests

Stem cell-based in vitro test systems can recapitulate specific phases of human development. In the UKK test system, human pluripotent stem cells (hPSCs) randomly differentiate into cells of the three germ layers and their derivatives. In the UKN1 test system, hPSCs differentiate into early neural precursor cells. During the normal differentiation period (14 days) of the UKK system, 570 genes [849 probe sets (PSs)] were regulated >fivefold; in the UKN1 system (6 days), 879 genes (1238 PSs) were regulated. We refer to these genes as ‘developmental genes’. In the present study, we used genome-wide expression data of 12 test substances in the UKK and UKN1 test systems to understand the basic principles of how chemicals interfere with the spontaneous transcriptional development in both test systems. The set of test compounds included six histone deacetylase inhibitors (HDACis), six mercury-containing compounds (‘mercurials’) and thalidomide. All compounds were tested at the maximum non-cytotoxic concentration, while valproic acid and thalidomide were additionally tested over a wide range of concentrations. In total, 242 genes (252 PSs) in the UKK test system and 793 genes (1092 PSs) in the UKN1 test system were deregulated by the 12 test compounds. We identified sets of ‘diagnostic genes’ appropriate for the identification of the influence of HDACis or mercurials. Test compounds that interfered with the expression of developmental genes usually antagonized their spontaneous development, meaning that up-regulated developmental genes were suppressed and developmental genes whose expression normally decreases were induced. The fraction of compromised developmental genes varied widely between the test compounds, and it reached up to 60 %. To quantitatively describe disturbed development on a genome-wide basis, we recommend a concept of two indices, ‘developmental potency’ (Dp) and ‘developmental index’ (Di), whereby Dp is the fraction of all developmental genes that are up- or down-regulated by a test compound, and Di is the ratio of overrepresentation of developmental genes among all genes deregulated by a test compound. The use of Di makes hazard identification more sensitive because some compounds compromise the expression of only a relatively small number of genes but have a high propensity to deregulate developmental genes specifically, resulting in a low Dp but a high Di. In conclusion, the concept based on the indices Dp and Di offers the possibility to quantitatively express the propensity of test compounds to interfere with normal development.

Inhibition of Curcumin-Treated Herpes Simplex Virus 1 and 2 in Vero Cells

The purpose of this study was to investigate the effect of curcumin-treated Herpes simplex virus-1 (HSV-1) and Herpes simplex virus-2 (HSV-2) virions in cultured Vero cells. Previous studies have indicated that curcumin, a polyphenol extracted from the plant Curcuma longa, has demonstrated antiviral properties against a variety of viruses. After establishing the maximum non-cytotoxic concentrations of curcumin on Vero cells, HSV-1 and HSV-2 virions were treated with varying concentrations of curcumin. The effect on infectivity was determined by antiviral assays, using WST-1, plaque assays, adsorption and penetration assays. Treating HSV-1 and HSV-2 viruses with curcumin, at a concentration of 30 μM, reduces the production of infectious HSV-1 and HSV-2 virions in cultured Vero cells by interfering with the adsorption process. These results support the potential of curcumin to be used as a therapeutic agent to reduce the transmission of HSV-1 and HSV-2.