Maximum Non-toxic Concentration Research Articles

Biomedical Promise of Aspergillus Flavus-Biosynthesized Selenium Nanoparticles: A Green Synthesis Approach to Antiviral, Anticancer, Anti-Biofilm, and Antibacterial Applications.

This study utilized Aspergillus flavus to produce selenium nanoparticles (Se-NPs) in an environmentally friendly and ecologically sustainable manner, targeting several medicinal applications. These biosynthesized Se-NPs were meticulously characterized using X-ray diffraction (XRD), Fourier-transform infrared (FT-IR) spectroscopy, transmission electron microscope (TEM), and UV-visible spectroscopy (UV), revealing their spherical shape and size ranging between 28 and 78 nm. We conducted further testing of Se-NPs to evaluate their potential for biological applications, including antiviral, anticancer, antibacterial, antioxidant, and antibiofilm activities. The results indicate that biosynthesized Se-NPs could be effective against various pathogens, including Salmonella typhimurium (ATCC 14028), Bacillus pumilus (ATCC 14884), Staphylococcus aureus (ATCC 6538), Clostridium sporogenes (ATCC 19404), Escherichia coli (ATCC 8739), and Bacillus subtilis (ATCC 6633). Additionally, the biosynthesized Se-NPs exhibited anticancer activity against three cell lines: pancreatic carcinoma (PANC1), cervical cancer (Hela), and colorectal adenocarcinoma (Caco-2), with IC50 values of 177, 208, and 216 μg/mL, respectively. The nanoparticles demonstrated antiviral activity against HSV-1 and HAV, achieving inhibition rates of 66.4% and 15.1%, respectively, at the maximum non-toxic concentration, while also displaying antibiofilm and antioxidant properties. In conclusion, the biosynthesized Se-NPs by A. flavus present a promising avenue for various biomedical applications with safe usage.

Radioprotective Effects of Thymus Vulgaris L. Essential Oil on Human Peripheral Blood Mononuclear Cells

Purpose: Generated free radicals by ionizing radiations, as powerful cytotoxic agents, can damage DNA and proteins. Thymus vulgaris L (thyme) plant is a rich source of antioxidant phenolic compounds, which makes it a preferable candidate for medical applications. Given this, we set out the present study to investigate the effectiveness of thyme essential oil on Human Peripheral Blood Mononuclear Cells (PBMCs) as a radioprotector agent against ionizing radiations.
 Materials and Methods: We extracted the thyme essential oil by the conventional Clevenger extraction method. Heparinized peripheral blood samples were also collected from five male volunteers, aged 22-25, without a history of smoking and irradiation. PBMCs were isolated and the maximum nontoxic concentrations (85µg/ml (of thyme essential oil were determined based on the result of the MTT method. In the next step, the PBMCs were cultured in the presence of thyme essential oil before and after X-irradiation with doses of 0.25 and 2.00 Gy.
 Results: The most radioprotective effect was observed in the dose of 2.00 Gy for thyme-treated cells 24 hours before the irradiation (p-value ≤ 0.001) by a survival enhancement factor of 1.67, compared to the control group.
 Conclusion: Our results showed that thyme essential oil can be used as an effective radioprotector agent for PBMCs against ionizing radiations. The most radioprotective effect was observed in the presence of thyme essential oil during irradiation.

Evaluation for Antiviral Potential of Ficus deltoidea Against Dengue Virus Type-2

Dengue is one of the most widespread arthropod-borne viral diseases that cause negative impact globally. Presently, no effective drug is available to safeguard people against dengue. Ficus deltoidea is Malaysia's famous traditional herb belonging to Moraceae family due to its pharmacological potential. F. deltoidea leaves (FDL) were extracted with n-hexane, ethyl acetate and methanol. Cell cytotoxicity study using MTT assay measuring the formazan absorbance was conducted to determine maximum non-toxic concentration on Vero cells. The antiviral activities of various concentrations of FDL extracts were assessed using virus reduction neutralisation tests against dengue virus type 2. The CC20 value of n-hexane, ethyl acetate and methanol FDL extracts were 2.99 ± 0.31, 22.15 ± 2.39, and 25.22 ± 0.42 µg/mL, respectively. Methanol FDL extract at maximum non-toxic concentration exerted strongest direct extracellular virucidal effect against DENV-2. In cell protection assay, ethyl acetate FDL extract achieved highest reduction in viral infectivity (98.17%), whereas n-hexane FDL extract showed strongest inhibition in DENV-2 viral replication in post-infection assay. Methanol FDL extract showed highest selectivity index value in direct virus inhibition, cell protection and post-infection assay. Conclusively, FDL extracts, especially methanol FDL showed potential antiviral activity against DENV-2, thus considered as promising anti-dengue agent.

Determination of Non-Cytotoxic Concentrations of Purine Analogues on Different Types of In Vitro-Incubated Embryonic Cells: A Pilot Study

Different concentrations of the methylxanthine/purine analogues aminophylline and 61-tartrat, were tested on in vitro-incubated embryonic avian cells from duck line DEC 99, as well as of mammalian embryonic cells from bovine line EBTr and mouse fibroblast line 3T3. In all cases, the C\(C_{50}\)/ml cell suspension, presenting the cytotoxic concentration, in which were observed 50\% death or changes, was determined. On the avian embryonic cells, the determined values of the methylxantine/purine analogues were 2.4x \(10^{-6}\) M/L about the aminophylline and 2.1x \(10^{-6}\) M/L about the 61-tartrat. The assessed C\(C_{50}\)/mL of the same compounds on the mammalian cells were 2.4x \(10^{-5}\) M/L about the aminophylline and 2.1x \(10^{-5}\) M/L about the 61-tartrat, respectively. The embryonic mammalian cells were more resistant to both substances than the embryonic avian cells. On the other hand, the assessed values of both C\(C_{50}\)/ml and maximal non-toxic concentration (MNC - in which no cellular mortality or other changes can be detected) of each one of the two methylxantine/purine analogues were very near about EBTr and 3T3 mammalian cell lines, and the mouse cells, which are proved as widely used experimental in vitro-model, showed some advantages in both cases. In this way, the current data suggest a possibility about application of methylxantine/purine derivatives about genetic and/or epigenetic reparations, on DNA- and mRNA-levels of low differentiated mammalian cells.

Production and characterization of polyhydroxybutyrate bioplastic precursor from Parageobacillus toebii using low-cost substrates and its potential antiviral activity

There is an increasing desire for bioplastics produced from renewable resources as an alternative to their petrochemical counterparts. These biopolymers have long-unnoticed antiviral properties. This study aimed to produce and characterize bioplastics by Parageobacillus toebii using low-cost substrates and determine their antiviral activity against coxsackievirus B4. Seven low-cost substrates (bagasse, water hyacinth, rice straw, rice water, sesame husks, molasses, and corn syrup) were compared with glucose for bioplastic precursor production. The highest bioplastic produced was from water hyacinth and glucose, followed by molasses, rice straw, rice water, sesame husks, and bagasse. Water hyacinth and glucose media were further optimized to increase the bioplastic precursor yield. The optimization of the media leads to increases in bioplastic precursor yields of 1.8-fold (3.456 g/L) and 1.496-fold (2.768 g/L), respectively. These bioplastics were further characterized by thermogravimetric analysis (TGA), Fourier-transformed infrared (FTIR) spectroscopy, proton nuclear magnetic resonance (1H NMR), and gas chromatography–mass spectrometry (GC–MS). They are thermostable, and their characterizations confirm the presence of polyhydroxybutyrate. The antiviral assay showed reasonable antiviral effects for bioplastics from water hyacinth (80.33 %) and glucose (55.47 %) media at 250 μg/mL maximum non-toxic concentrations (MNTC). The present investigation demonstrates a low-cost model for producing polyhydroxybutyrate bioplastic precursor for antiviral applications.

Fabrication of an effective peroxynitrite electrochemical sensing platform and studying the inhibitory effect of tea polyphenols on the oxidative damage of lead-treated cells

Peroxynitrite (ONOO−), as the main reactive nitrogen species, involves many physiological and pathological conditions. Particularly, aberrant expression levels of ONOO− can cause cancer and other diseases, so its analysis have attracted the attention and concern of many scientific researchers. Here, we present a new ONOO− sensing platform to monitor cells oxidative damage directly by utilizing the conjugated organic microporous polymer/chitosan (CMP/CS) modified glassy carbon electrode (GCE). The prepared CMP/CS/GCE shows excellent electrochemical performance for measuring ONOO− in the wide concentration range of 2.29 nM to 45.8 μM and the detection limit is as low as 0.763 nM (S/N = 3). Therefore, we used this sensor for the detection of ONOO− released from glioma cells (U87) during oxidative stress induced by lead stimulation. Significantly, the inhibitory effect of tea polyphenols (TP) on the oxidative damage of lead-treated cells was explored by electrochemical method for the first time, and the maximum non-toxic concentration range of TP was obtained. In the authors’ perception, the constructed sensing platform and the efficient analysis method may open a new path for their future use in living organisms.

In Vitro and In Vivo Anti-Aging Effect of Coffee Berry Nanoliposomes.

Encapsulation of bioactive compounds in the liposome system provides several advantages, such as enhancing the stability and lowering the toxicity of active compounds. Coffee berry extract (CBE) has previously been established to have in vitro anti-aging properties and to retard the aging of human skin. The purposes of this study were to encapsulate CBE in nanoliposomes and to assess its stability and in vitro anti-aging potential in human dermal fibroblasts (HDF), as well as in healthy human skin. In the HDF model, anti-aging potential was determined by nitric oxide (NO) and collagenase inhibition assays and a superoxide dismutase (SOD) activity assay, whereas in healthy human skin (in vivo), the skin elasticity and brightness were examined. First, liposomal CBE (L-CBE) was created with a particle size of 117.33 ± 2.91 nm, a polydispersity index (PDI) of 0.36 ± 0.03, and a zeta potential of -56.13 ± 1.87 mV. The percentages of encapsulation efficacy (%EE) and loading efficacy (%LE) were 71.26 ± 3.12% and 2.18 ± 0.18%, respectively. After undergoing a 12-week stability test, the L-CBE retained more phenolic content than the free CBE when stored at 4 °C, room temperature, and 45 °C. Compared to free CBE, the L-CBE demonstrated a more consistent, elevated, and prolonged release of phenolics from the lipid system. In human dermal fibroblasts, L-CBE showed lower toxicity, and at its maximum nontoxic concentration (10 mg/mL), it exhibited slightly higher anti-aging effects than CBE, including NO inhibition, enhanced SOD activity, and anti-collagenase activities. In clinical trials (30 volunteer subjects), none of the participants' skin was irritated when the L-CBE, the CBE, or base creams were applied. After 2 weeks of application, the L-CBE and CBE creams both demonstrated an improvement in skin elasticity and a reduction in melanin levels, and after 4 weeks, L-CBE cream showed a significantly greater improvement in skin elasticity and lightening. The results demonstrate that the encapsulation of the CBE in liposomal systems could increase its stability and skin penetration, reduce its toxicity, and maintain its anti-aging effect, which is powerful enough to be exploited in anti-aging and whitening agents for application in cosmetics and cosmeceuticals.

Astragalus glycyphyllos L.: antiviral activity and tablet dosage formulation of a standardized dry extract

Astragalus glycyphyllos L. is widely used in Bulgarian folk medicine. Extracts from this plant have antiproliferative, antitumor and immune stimulating effects in vitro and in vivo. The aim of this study was to examine the potential antiviral activity of a standardized extract from the aerial parts of the plant and to formulate it in a suitable dosage form. A dry extract was obtained, and its phytochemical composition was determined by ultra-high-performance liquid chromatography–high resolution electrospray ionization mass spectrometry (UHPLC-HRESIMS). The extract contained flavonoids and predominantly saponins. 17(R),20(R)-3β,6α,16β-trihydroxycycloartanyl-23-carboxylic acid 16-lactone 3-O-β-D-glucopyranoside was used as a reference for quantitation. The extract had 3% saponins. The antiviral activity of the extract was tested against Simplexvirus humanalpha type 1 (acyclovir sensitive) and 2 (acyclovir resistant) in vitro. Between 60% and 70% of protection was reached against both virus strains when the extract (at the maximal non-toxic concentration of 0.6 mg/mL) was added simultaneously with and 1 h after Madine and Darby bovine kidney cell inoculation. The extract was also tested in combination with acyclovir only against Simplexvirus humanalpha type 1, in vitro. Further, the lyophilized extract was mixed with appropriate excipients aiming to prepare tablets via direct compaction. The optimized tablets fulfilled the requirements of the pharmacopoeia for disintegration and friability. Thus, the study demonstrated the possibility to formulate a standardized extract of A. glycyphyllos in tablets and their potential for adjuvant antiviral application in further trials.

Characterization of Carnosine Effect on Human Microglial Cells under Basal Conditions

The activity of microglia is fundamental for the regulation of numerous physiological processes including brain development, synaptic plasticity, and neurogenesis, and its deviation from homeostasis can lead to pathological conditions, including numerous neurodegenerative disorders. Carnosine is a naturally occurring molecule with well-characterized antioxidant and anti-inflammatory activities, able to modulate the response and polarization of immune cells and ameliorate their cellular energy metabolism. The better understanding of microglia characteristics under basal physiological conditions, as well as the possible modulation of the mechanisms related to its response to environmental challenges and/or pro-inflammatory/pro-oxidant stimuli, are of utmost importance for the development of therapeutic strategies. In the present study, we assessed the activity of carnosine on human HMC3 microglial cells, first investigating the effects of increasing concentrations of carnosine on cell viability. When used at a concentration of 20 mM, carnosine led to a decrease of cell viability, paralleled by gene expression increase and decrease, respectively, of interleukin 6 and heme oxygenase 1. When using the maximal non-toxic concentration (10 mM), carnosine decreased nitric oxide bioavailability, with no changes in the intracellular levels of superoxide ion. The characterization of energy metabolism of HMC3 microglial cells under basal conditions, never reported before, demonstrated that it is mainly based on mitochondrial oxidative metabolism, paralleled by a high rate of biosynthetic reactions. The exposure of HMC3 cells to carnosine seems to ameliorate microglia energy state, as indicated by the increase in the adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio and energy charge potential. The improvement of cell energy metabolism mediated by 10 mM carnosine could represent a useful protective weapon in the case of human microglia undergoing stressing conditions.

Investigation of the Inhibition Activity of Aluminium Oxide Nanoparticles for Herpes Simplex Type 1.

Several studies have shown that Herpes simplex type 1 )HSV-1 (is one of the viruses resistant to medications, so potential antiherpetic agents need to be evaluated. This study aimed to evaluate the impact of Aluminum Oxide Nanoparticles (Al2O3-NPs) on HSV-1 infection. Characterization of Al2O3-NPs was performed using field emission scanning electron microscopy (FESEM), X-ray diffraction (XRD), dynamic light scattering (DLS), and high-resolution transmission electron microscopy (HRTEM). The MTT test was used to investigate the toxicity action of Al2O3-NPs on viable cells. Quantitative Real-Time PCR (qRT-PCR)and TCID50 assays were used to achieve the antiherpetic performance Al2O3-NPs.Indirect immunofluorescence assay (IFA) was performed to determine the inhibitory impact of Al2O3-NPs on viral antigen expression, and acyclovir was utilized as a standard agent in all tests. HSV-1 subjected to Al2O3-NPs at the maximum non-toxic concentration (100 μg / mL) leads to a decrease of 0.1, 0.7, 1.8, and 2.5 log10 TCID50 in the infectious titer relative to virus control (P<0.0001). This concentration of Al2O3-NPs was correlated with 16.9%, 47.1 %, 61.2 %, 72.5 % and 74.6 % inhibition rates, calculated based on HSV-1 viral load compared to virus control. Our results have shown that Al2O3-NPs have a robust antiviral activity against HSV-1. This function demonstrates excellent potential for using Al2O3-NP in topical formulations for treating orolabial or genital herpetic lesions.

Ammi-visnaga extract; a novel phyto-antiviral agent against bovine rotavirus.

The spread of bovine rotavirus has a great impact on animal productivity, milk products, and human public health. Thus, this study aimed to develop a novel, effective and accessible Phyto-antiviral treatment made from methanolic Ammi-visnaga seed extract against rotavirus infection. Rotaviruses were isolated from raw milk and cottage cheese samples randomly collected from Cairo and Qalubia governorates. They were all identified serologically, however, only three of them were both biologically and molecularly confirmed. The methanolic extract derived from Khella seeds (MKSE) was chemically analyzed with mass chromatography. The cellular toxicity of MKSE was tested on Caco-2 cells and its antiviral activity against one of the isolated bovine rotaviruses (BRVM1) was tested by both the cytopathic inhibition assay and the plaque reduction assay. Our results showed that 17.3% of the total collected 150 dairy samples were bovine rotavirus antigen positive. Three representatives of them were phylogenetically identified to be included in group A based on a 379bp coat protein gene. Visnagin, Benzopyran, Khellin, and Benzenepropanoic acid were the major active components found in the MKSE. The maximum non-toxic concentration of MKSE was 5µg/mL and the CC50 value was 417µg/mL. The MKSE exhibited in-vitro antiviral activity against BRVM1 indicated by inhibition of the viral cytopathic effect (SI = 204.5, IP = 98%), causing a 1.5 log decrease in BVRM1 TCID50 and reducing the viral plaques count by the percentage of 93.14% at MNTC (5 ug/ml). In conclusion, our study showed that bovine rotavirus represents a severe health problem that needs attention in Egypt, and it supports using MKSE as a potential natural anti-rotavirus agent.

APPLICATION OF BIOSURFACTANTS IN WASTEWATER BIOFILTRATION TECHNOLOGY USING DAPHNIA MAGNA

When solving water purification problems, including for the needs of industrial fish farming, biofiltration methods involving planktonic organisms, in particular branchiopod crustaceans, deserve special attention. A specific filtration apparatus feeding daphnia can provide highly efficient wastewater treatment from suspended fine particles that have a low settling velocity and are not able to be retained on mechanical filters. An important advantage of this method of water treatment is the ability to use the resulting zooplankton biomass as live feed for fish farming. The filtration properties of zooplankton can be improved by growing it in the presence of surfactants of biological origin, which, in addition, improve the absorption of nutrients by cladocerans and intensify their growth. In this study, the possibility of using a preparation of trehalosolipid biosurfactants obtained from the culture fluid of Rhodococcus erythropolis AU-1 for intensive growth of Daphnia magna culture for the purpose of biological treatment of water bodies was evaluated. It was established that the toxicity of trehalosolipids is dose-dependent, in particular, their minimum concentration at which the death of daphnia was observed within 24 hours of exposure was 300 mg/l, and the LC50 at the same duration of exposure was 479 mg/l. The maximum non-toxic concentration of the trehalosolipid biosurfactant is 200 mg/l. Concentrations of trehalosolipids in the range of 1-3 mg/l provide an increase in the density of daphnia culture by 1.3-1.7 times compared to the control. To evaluate the efficiency of biofiltration of Daphnia magna in the model experiment, water from the mechanical filter of the experimental recirculation system of the Yuriy Fedkovych Chernivtsi National University was used. The value of the optical density of the discharge water decreases by more than half after one day of daphnia presence in it, and after 4 days - by 7 times. The addition of biosurfactants leads to a more efficient filtration process, with the best results obtained when using the preparation with a concentration of 1 mg/l. The use of biosurfactants in concentrations of 1, 2 and 3 mg/l leads to a decrease in electrical conductivity and total mineralization compared to the control. At the same time, the pH and soluble oxygen content did not change compared to the control.

Ayapana triplinervis Vahl: Potential toxicity and therapeutic effects assessed in a zebrafish model

Background: In Reunion, 27 medicinal plants have recently been registered in the French Pharmacopoeia. Among them, Ayapana triplinervis Vahl (A. triplinervis) is traditionally used to combat inflammation and oxidative stress, as well as to promote healing and tissue regeneration. Purpose: This work aims to investigate the toxicity of A. triplinervis Aqueous Extract (AAE) using the zebrafish model, and to determine its Maximum Non-Toxic Concentration (MNTC). Then, using several pathological models established in zebrafish, the beneficial effects of AAE at its MNTC on inflammation, oxidative stress, tissue regeneration, and on lipid accumulation were investigated. Methods: AAE toxicity was evaluated according to the OECD (Organization for Economic Cooperation and Development) guidelines in the zebrafish eleutheroembryos (0 to 96 hpf) and by gene/transgene expression analyses of toxicity biomarkers. The impact of AAE on inflammation, oxidative stress and regeneration was studied by immunohistochemistry and reactive oxygen species staining methods in a caudal fin amputation model. Finally, the beneficial effects of AAE on lipid accumulation were investigated in high-fat diet (HFD)treated larvae. Results: AAE increased immune cell recruitment as well as tail regeneration after caudal fin amputation. It also reduced oxidative stress. In the HFD larva model, AAE showed interesting lipid reduction effects. Conclusion: Using rapid and robust zebrafish models, we demonstrated the beneficial properties of AAE, highlighting its pro-regenerative, antioxidant and lipid-lowering effects.

In-vitro antiviral activity of Carica papaya formulations against dengue virus type 2 and chikungunya virus

Dengue and chikungunya are diseases of global health significance and currently, no antivirals are available to treat these arboviral diseases. Carica papaya leaves extract is traditionally used to treat thrombocytopenia in patients infected with the dengue virus. The current study was undertaken to study the antiviral activity of commercially available Carica papaya leaves extract (CPLE) based products and CPLE prepared in four formulations against dengue virus type 2 (DENV-2) and chikungunya virus (CHIKV). Maximum nontoxic concentrations of the commercially available CPLE based products and CPLE based formulations (Carica papaya leaves in powder form, Carica papaya leaves in lyophilized form, Carica papaya leaves based silver nanoparticles and supercritical fluid extract of Carica papaya leaves) were used for screening the antiviral activity. The antiviral activity against DENV-2 and CHIKV were assessed post infection using focus forming unit assay. Effective formulations were tested under different conditions i.e. pretreatment, cotreatment and posttreatment. The virus output after treatment was assessed by real-time RT-PCR, immunofluorescence assay and focus forming unit assay. The results revealed Carica papaya leaves based silver nanoparticles and supercritical fluid extract of Carica papaya leaves formulations showed significant inhibition in case of DENV while papaya leaves in powder form showed significant reduction in case of CHIKV. This study demonstrates the antiviral activity of CPLE formulations against DENV-2 and CHIKV infection in in-vitro system and needs further validation in in-vivo models.

Production, characterization, and application of phage-derived PK34 recombinant anti-microbial peptide.

PK34 is a D29 mycobacteriophage-derived anti-microbial peptide (AMP) with anti-Mycobacterium tuberculosis activity. It is expected to become an auxiliary drug for the treatment of M. tuberculosis infection, or as a template for the development of anti-M. tuberculosis drugs. The focus of this paper is to obtain recombinant PK34 by a novel method of prokaryotic expression and purification by affinity chromatography. The minimum inhibitory concentration (MIC) of recombinant PK34 was better than that of synthetic PK34 as measured by the microplate-based Alamar Blue assay (MABA). In order to further compare the different anti-bacterial effects of PK34 obtained by the two methods on M. tuberculosis, the bacterial changes after drug incubation were observed at the microscopic level by transmission electron microscopy (TEM). In order to apply PK34 to clinical treatment earlier in the future, this paper tested the maximum non-toxic concentration of recombinant PK34 to the two most studied immune cells, RAW264.7 and THP-1, through cytotoxicity experiments. The maximum non-toxic concentration was the same as the MIC of recombinant PK34 to M. tuberculosis H37Rv, and both were 12.5μg/mL. The monoclonal antibodies against PK34 and their hybridoma cell lines were prepared using recombinant PK34 as the antigen. Next, we obtained the gene sequence of the monoclonal antibody, which was prepared for the basic research of PK34 in M. tuberculosis treatment. In addition, the possible molecular docking mode between PK34 and trehalose-6,6-dimycolate (TDM) was predicted by AI simulation. To sum up, this paper provides a new idea for the birth of more new AMPs of the same type as PK34 in the future. KEY POINTS: • Design and prepare a novel recombinant PK34 anti-microbial peptide. • Recombinant PK34 has higher purity and anti-bacterial activity than synthetic PK34. • The monoclonal antibody against recombinant PK34 was prepared and sequenced.

Antiviral Potentialities of Chemical Characterized Essential Oils of Acacia nilotica Bark and Fruits against Hepatitis A and Herpes Simplex Viruses: In Vitro, In Silico, and Molecular Dynamics Studies.

Acacia nilotica (synonym: Vachellia nilotica (L.) P.J.H.Hurter and Mabb.) is considered an important plant of the family Fabaceae that is used in traditional medicine in many countries all over the world. In this work, the antiviral potentialities of the chemically characterized essential oils (EOs) obtained from the bark and fruits of A. nilotica were assessed in vitro against HAV, HSV1, and HSV2. Additionally, the in silico evaluation of the main compounds in both EOs was carried out against the two proteins, 3C protease of HAV and thymidine kinase (TK) of HSV. The chemical profiling of the bark EOs revealed the identification of 32 compounds with an abundance of di- (54.60%) and sesquiterpenes (39.81%). Stachene (48.34%), caryophyllene oxide (19.11%), and spathulenol (4.74%) represented the main identified constituents of bark EO. However, 26 components from fruit EO were assigned, with the majority of mono- (63.32%) and sesquiterpenes (34.91%), where trans-caryophyllene (36.95%), Z-anethole (22.87%), and γ-terpinene (7.35%) represented the majors. The maximum non-toxic concentration (MNTC) of the bark and fruits EOs was found at 500 and 1000 µg/mL, respectively. Using the MTT assay, the bark EO exhibited moderate antiviral activity with effects of 47.26% and 35.98% and a selectivity index (SI) of 2.3 and 1.6 against HAV and HSV1, respectively. However, weak activity was observed via the fruits EO with respective SI values of 3.8, 5.7, and 1.6 against HAV, HSV1, and HSV2. The in silico results exhibited that caryophyllene oxide and spathulenol (the main bark EO constituents) showed the best affinities (ΔG = -5.62, -5.33, -6.90, and -6.76 kcal/mol) for 3C protease and TK, respectively. While caryophyllene (the major fruit EO component) revealed promising binding capabilities against both proteins (ΔG = -5.31, -6.58 kcal/mol, respectively). The molecular dynamics simulation results revealed that caryophyllene oxide has the most positive van der Waals energy interaction with 3C protease and TK with significant binding free energies. Although these findings supported the antiviral potentialities of the EOs, especially bark EO, the in vivo assessment should be tested in the intraoral examination for these EOs and/or their main constituents.

Tunable Toxicity of Bufadienolides is Regulated through a Configuration Inversion Catalyzed by a Short-Chain Dehydrogenase/Reductase.

Bufadienolides are toxic components widely found in amphibious toads that exhibit a wide range of biological activities. Guided by UPLC-QTOF-MS analysis, several 3-epi-bufadienolides with unique structures were isolated from the bile of the Asiatic toad, Bufo gargarizans. However, the enzymatic machinery of this epimerization in toads and its significance in chemical ecology remains poorly understood. Herein, we firstly compared the toxicities of two typical bufadienolides, bufalin (featuring a 14β-hydroxyl) and resibufogenin (containing a 14, 15-epoxy group), with their corresponding 3-epi isomers in a zebrafish model. The results of the toxicology assays showed that the ratio of maximum non-toxic concentrations of these two pairs of compounds are 256 and 96 times, respectively, thereby indicating that 3-hydroxyl epimerization leads to a significant decrease in toxicity. Aiming to investigate the biotransformation of 3-epi bufadienolides in toads, we applied liver lysate to transform bufalin and found that it could stereoselectively catalyze the conversion of bufalin into its 3α-hydroxyl epimer. Following this, we cloned and characterized a short-chain dehydrogenase/reductase, HSE-1, from the toad liver cDNA library and verified its 3(β→α)-hydroxysteroid epimerization activity. To the best of our knowledge, this is the first hydroxyl epimerase identified from amphibians that regulates the toxicity of animal-derived natural products.

Vathasura Kudineer, an Andrographis based polyherbal formulation exhibits immunomodulation and inhibits chikungunya virus (CHIKV) under invitro conditions

Ethnopharmacological relevanceChikungunya disease (CHIKD) is caused by the alphavirus, chikungunya virus (CHIKV) and is characterized by acute fever and joint inflammation; the inflammation continues even after clearance of the virus from the system, persisting for several months to years. Currently, there are no modern medicines/vaccines available for its treatment and use of over-the-counter anti-inflammatory generic medicines to relieve symptoms is generally practiced. In India, Indian traditional medicines hold a lot of promise to treat this infection and are routinely used during outbreaks. Aim of the studyIn the present study, we characterized the phytochemical and physicochemical properties of aqueous and ethanol extracts of the Vathasura Kudineer (VSK), a Andrographis based Siddha polyherbal formulation. Additionally, we evaluated its immunomodulatory and antiviral potential using an in vitro system. Materials and methodsAqueous and ethanolic extracts of VSK were prepared and their physico and phytochemical properties were obtained by biochemical and biophysical assays, HPTLC and FTIR. The aqueous extracts of VSK and several of its ingredients were evaluated for their cytotoxicity in Vero cells and using the maximum non-toxic concentration (MNTC), were processed further for evaluating their ability to inhibit CHIKV infection in Vero cells. We performed the co-treatment assay with ethanol extract of VSK and several of its ingredients to assess the antiviral activity against chikungunya virus on Vero cells and through pre-treatment assay (anti-adhesive effect), co-incubation assay (virucidal effect) and post-treatment assay (post-entry effect) were evaluated. Further, we tested the aqueous extract of VSK along with some of its ingredients for their immunomodulatory properties. We performed antioxidant and anti-inflammatory assays using LPS-simulated RAW 264.7 cells. For antioxidant capacity of extracts, we performed extra-cellular ABTS radical scavenging activity and intra-cellular effects on ROS generation and SOD activity. We assessed the effect on most important inflammatory mediators like Nitric oxide (NO) and Prostaglandin E2 (PGE2) and pro-inflammatory cytokines like interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNFα). ResultsWe provided the fingerprint of the phytochemicals of both ethanol and aqueous extracts of VSK that can be used for identification. We observed that ethanol extract was able to inhibit CHIKV infection at MNTC with 48 h of treatment on Vero cells. Its ingredient VSKI-As (Anethum sowa) found to be most effective to show virucidal effect while VSKI-Cs (Clerodendrum serratum) and VSKI-Pn (Pipper nigrum) found to be effective in post-entry effect. VSK was able to show ABTS radical scavenging activity, reduce ROS generation, inhibit the inflammatory mediators (NO and PGE2) and pro-inflammatory cytokines (IL-1β and TNFα) production in LPS-stimulated RAW 264.7 cells. ConclusionsWe provided the evidence that VSK has both immunomodulatory as well as antiviral potential. It shows virucidal as well as post-entry effects on chikungunya virus. VSK can inhibit pro-inflammatory cytokines, IL-1β and TNFα production by suppressing the inflammatory mediators, NO and PGE2.

Anti-herpes simplex virus type Ⅰ of tectorigenin derivative and effect on Toll-like receptors in vitro

The study investigated the inhibitory effect and mechanism of tectorigenin derivative(SGY) against herpes simplex virus type Ⅰ(HSV-1) by in vitro experiments. The cytotoxicity of SGY and positive drug acyclovir(ACV) on African green monkey kidney(Vero) cells and mouse microglia(BV-2) cells was detected by cell counting kit-8(CCK-8) method, and the maximum non-toxic concentration and median toxic concentration(TC_(50)) of the drugs were calculated. After Vero cells were infected with HSV-1, the virulence was determined by cytopathologic effects(CPE) to calculate viral titers. The inhibitory effect of the tested drugs on HSV-1-induced cytopathy in Vero cells was measured, and their modes of action were initially explored by virus adsorption, replication and inactivation. The effects of the drugs on viral load of BV-2 cells 24 h after HSV-1 infection and the Toll-like receptor(TLR) mRNA expression were detected by real-time fluorescence quantitative PCR(RT-qPCR). The maximum non-toxic concentrations of SGY against Vero and BV-2 cells were 382.804 μg·mL~(-1) and 251.78 μg·mL~(-1), respectively, and TC_(50) was 1 749.98 μg·mL~(-1) and 2 977.50 μg·mL~(-1), respectively. In Vero cell model, the half maximal inhibitory concentration(IC_(50)) of SGY against HSV-1 was 54.49 μg·mL~(-1), and the selection index(SI) was 32.12, with the mode of action of significantly inhibiting replication and directly inactivating HSV-1. RT-qPCR results showed that SGY markedly reduced the viral load in cells. The virus model group had significantly increased relative expression of TLR2, TLR3 and tumor necrosis factor receptor-associated factor 3(TRAF3) and reduced relative expression of TLR9 as compared with normal group, and after SGY intervention, the expression of TLR2, TLR3 and TRAF3 was decreased to different degrees and that of TLR9 was enhanced. The expression of inflammatory factors inducible nitric oxide synthase(iNOS), tumor necrosis factor-α(TNF-α), and interleukin-1β(IL-1β) was remarkably increased in virus model group as compared with that in normal group, and the levels of these inflammatory factors dropped after SGY intervention. In conclusion, SGY significantly inhibited and directly inactivated HSV-1 in vitro. In addition, it modulated the expression of TLR2, TLR3 and TLR9 related pathways, and suppressed the increase of inflammatory factor levels.

Autonomous Chemistry Enabling Environment-Adaptive Electrochemical Energy Storage Devices

Autonomous Chemistry Enabling Environment-Adaptive Electrochemical Energy Storage Devices