Preincubation Assay Research Articles

Optimizing the detection of N-nitrosamine mutagenicity in the Ames test

Accurately determining the mutagenicity of small-molecule N-nitrosamine drug impurities and nitrosamine drug substance-related impurities (NDSRIs) is critical to identifying mutagenic and cancer hazards. In the current study we have evaluated several approaches for enhancing assay sensitivity for evaluating the mutagenicity of N-nitrosamines in the bacterial reverse mutagenicity (Ames) test. Preincubation assays were conducted using five activation conditions: no exogenous metabolic activation and metabolic activation mixes employing both 10% and 30% liver S9 from hamsters and rats pretreated with inducers of enzymatic activity. In addition, preincubations were conducted for both 60 min and 30 min. These test variables were evaluated by testing 12 small-molecule N-nitrosamines and 17 NDSRIs for mutagenicity in Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537, and Escherichia coli strain WP2 uvrA (pKM101). Eighteen of the 29 N-nitrosamine test substances tested positive under one or more of the testing conditions and all 18 positives could be detected by using tester strains TA1535 and WP2 uvrA (pKM101), preincubations of 30 min, and S9 mixes containing 30% hamster liver S9. In general, the conditions under which NDSRIs were mutagenic were similar to those found for small-molecule N-nitrosamines.

Matrine and Oxymatrine: evaluating the gene mutation potential using in silico tools and the bacterial reverse mutation assay (Ames test).

The quinolizidine alkaloids matrine and its N-oxide oxymatrine occur in plants of the genus Sophora. Recently, matrine was sporadically detected in liquorice products. Morphological similarity of the liquorice plant Glycyrrhiza glabra with Sophora species and resulting confusion during harvesting may explain this contamination, but use of matrine as pesticide has also been reported. The detection of matrine in liquorice products raised concern as some studies suggested a genotoxic activity of matrine and oxymatrine. However, these studies are fraught with uncertainties, putting the reliability and robustness into question. Another issue was that Sophora root extracts were usually tested instead of pure matrine and oxymatrine. The aim of this work was therefore to determine whether matrine and oxymatrine have potential for causing gene mutations. In a first step and to support a weight-of-evidence analysis, in silico predictions were performed to improve the database using expert and statistical systems by VEGA, Leadscope (Instem®), and Nexus (Lhasa Limited). Unfortunately, the confidence levels of the predictions were insufficient to either identify or exclude a mutagenic potential. Thus, in order to obtain reliable results, the bacterial reverse mutation assay (Ames test) was carried out in accordance with OECD Test Guideline 471. The test set included the plate incorporation and the preincubation assay. It was performed with five different bacterial strains in the presence or absence of metabolic activation. Neither matrine nor oxymatrine induced a significant increase in the number of revertants under any of the selected experimental conditions. Overall, it can be concluded that matrine and oxymatrine are unlikely to have a gene mutation potential. Any positive findings with Sophora extracts in the Ames test may be related to other components. Notably, the results also indicated a need to extend the application domain of respective (Q)SAR tools to secondary plant metabolites.

Quercetin as a major component of Punica granatum L. effectively controls WSSV infection and transmission in the larval shrimp culture stage

White spot syndrome virus (WSSV) is a major pathogen in shrimp culture because of its high infectivity, fast transmission speed, and high fatality rate. White spot disease (WSD), caused by WSSV poses a substantial threat to shrimp production. Therefore, it is crucial to identify effective antiviral agents to mitigate the economic loss caused by WSD. In this study, we prepared 20 ethanol extracts of Chinese herbal medicines (CHMs) and evaluated their anti-WSSV effectiveness using Litopenaeus vannamei larvae as a model. Among the tested extracts, Punica granatum L. exhibited the highest inhibition of WSSV infection at 85%, followed by Gentiana macrophylla at 79% and Isatis tinctoria L. at 63%. Quercetin, an active ingredient found in P. granatum at a concentration of 6.3 μM, increased the survival rate of WSSV-infected larvae by 30% after 72 hpi, and significantly inhibited WSSV infection. Further data from pre-incubation assays indicated that quercetin exhibited a potentially destructive effect on WSSV virions, thereby reducing the virulence and blocking viral horizontal transmission. Moreover, the high stability of the aquacultural water facilitated the continuous exchange of quercetin treatment, which further prolonged the survival time of WSSV-infected shrimp such that the survival rate was still at 13% after 5 days. These results highlight the potential application of quercetin in eliminating WSSV during shrimp larval production.

Potential of multi-strain probiotics extract as an anti-inflammatory agent through inhibition of macrophage migration inhibitory factor activity

Background: Probiotics are reported to have a role for improving health conditions and reduce the risk of diseases associated with inflammation. However, how they affect inflammation has not been well studied. The inflammation occurs during the progression of chronic diseases could damage the normal function of cells, tissues, and organs. Macrophage migration inhibitory factor (MIF) is recognized as a cytokine playing a key role in the inflammation process. Inhibition of its activity has been used by researchers as an approach for alleviating the inflammation. This study aims to evaluate the potential inhibitory effect of the extract of multi-strain probiotics consisting of Lactobacillus casei EMRO 002, L. casei EMRO 213, L. plantarum EMRO 009, L. fermentum EMRO 211, L. rhamnosus EMRO 014, L. bulgaricus EMRO 212,and Rhodopseudomonas palustris EMRO 201 on MIF tautomerase activity, the reversibility, and the mechanism of inhibition. Hence, the scope of this study is the evaluation of the functional characteristics of multi-strain probiotics extract on MIF activity.Methods:The multi-strain probiotics were centrifuged and the supernatant was separated from the pellet. The supernatant, i.e. the extract, was mixed with MIF and analyzed for its inhibition effect on MIF tautomerase activity. The reversibility of inhibition was evaluated by preincubation and dilution assays, and the mechanism of inhibition was determined by kinetic evaluation.Results:The multi-strain probiotics extract inhibited MIF tautomerase activity with an IC50 of 7.80 ± 1.96 mg/L. The preincubation and dilution assays showed that the inhibition is reversible, and the kinetic evaluation predicted that the extract components might bind to the enzyme active site and other site(s) of MIF.Conclusion:The findings show that the extract was able to inhibit MIF tautomerase activity reversibly and its components might bind to the enzyme active site and other site(s) of MIF suggest that the multi-strain probiotics extract has potential to be used as an anti-inflammatory agent for the treatment of chronic diseases associated with inflammation.Keywords: Multi-strain probiotics, anti-inflammatory, macrophage migration inhibitory factor, reversible inhibition, kinetic evaluation.

Assessment of mutagenicity of Boswellia serrata extract (Boswegex®) using Bacterial Reverse Mutation test method

Background: Extracted from the Indian branching tree Boswellia serrata, boswellic acids derived from the plant's resin hold promise in addressing inflammation, arthritis, and hyperlipidemia. Objective: In this study, the mutagenicity of B. serrata (Boswegex®) is evaluated. Materials and Methods: The potential genotoxic and mutagenic effects of Boswellia serrata's aqueous ethanolic extract, known as Boswegex®, were explored using a bacterial reverse mutation assay against Salmonella strains TA98, TA100, TA102, TA1535, and TA1537. Different concentrations ranging from 500 to 6000 μg/plate were tested using both Method 1 (Standard plate incorporation assay) and Method 2 (pre-incubation assay), with DMSO (Dimethyl sulfoxide) as the solvent control. The positive control, 2-aminoanthracene, was used alongside a metabolic activator (S-9 fraction). Given the prolonged use of supplements, an assessment for mutagenicity is vital. In this context, the mutagenicity of B. serrata (Boswegex®) was evaluated through the in vitro Bacterial Reverse Mutation Test, employing both the Standard Plate Incorporation Assay and the pre-incubation assay. Results: Notably, none of the mutagenic assays revealed a mutagenicity increase over background levels. Conclusion: These findings collectively indicate that Boswellia serrata (Boswegex®) does not exhibit mutagenic properties.

Elucidating the Kinetic Mechanism of Human METTL16.

Methyltransferase-like protein 16 (METTL16) is one of four catalytically active, S-adenosylmethionine (SAM)-dependent m6A RNA methyltransferases in humans. Well-known methylation targets of METTL16 are U6 small nuclear RNA (U6 snRNA) and the MAT2A mRNA hairpins; however, METTL16 binds to other RNAs, including the 3' triple helix of the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). Herein, we investigated the kinetic mechanism and biochemical properties of METTL16. METTL16 is a monomer in complex with either the MALAT1 triple helix or U6 snRNA and binds to these RNAs with respective dissociation constants of 31 nM and 18 nM, whereas binding to the methylated U6 snRNA product is 1.1 μM. The MALAT1 triple helix, on the other hand, is not methylated by METTL16 under in vitro conditions. Using the U6 snRNA to study methylation steps, preincubation and isotope partitioning assays indicated an ordered-sequential mechanism, whereby METTL16 binds U6 snRNA before SAM. The apparent dissociation constant for the METTL16·U6 snRNA·SAM ternary complex is 126 μM. Steady-state kinetic assays established a kcat of 0.07 min-1, and single-turnover assays established a kchem of 0.56 min-1. Furthermore, the methyltransferase domain of METTL16 methylated U6 snRNA with an apparent dissociation constant of 736 μM and a kchem of 0.42 min-1, suggesting that the missing vertebrate conserved regions weaken the ternary complex but do not induce any rate-limiting conformational rearrangements of the U6 snRNA. This study helps us to better understand the catalytic activity of METTL16 in the context of its biological functions.

Inhibitory Effects of Varespladib, CP471474, and Their Potential Synergistic Activity on Bothrops asper and Crotalus durissus cumanensis Venoms.

Snakebite is a neglected tropical disease that causes extensive mortality and morbidity in rural communities. Antivenim sera are the currently approved therapy for snake bites; however, they have some therapeutic limitations that have been extensively documented. Recently, small molecule toxin inhibitors have received significant attention as potential alternatives or co-adjuvant to immunoglobulin-based snakebite therapies. Thus, in this study, we evaluated the inhibitory effects of the phospholipase A2 inhibitor varespladib and the metalloproteinase inhibitor CP471474 and their synergistic effects on the lethal, edema-forming, hemorrhagic, and myotoxic activities of Bothrops asper and Crotalus durissus cumanensis venoms from Colombia. Except for the preincubation assay of the lethal activity with B. asper venom, the mixture showed the best inhibitory activity. Nevertheless, the mix did not display statistically significant differences to varespladib and CP471474 used separately in all assays. In preincubation assays, varespladib showed the best inhibitory activity against the lethal effect induced by B. asper venom. However, in independent injection assays, the mix of the compounds partially inhibited the lethal activity of both venoms (50%). In addition, in the assays to test the inhibition of edema-forming activity, the mixture exhibited the best inhibitory activity, followed by Varespladib, but without statistically significant differences (p > 0.05). The combination also decreased the myotoxic activity of evaluated venoms. In these assays, the mix showed statistical differences regarding CP471474 (p < 0.05). The mixture also abolished the hemorrhagic activity of B. asper venom in preincubation assays, with no statistical differences to CP471474. Finally, the mixture showed inhibition in studies with independent administration in a time-dependent manner. To propose a mode of action of varespladib and CP471474, molecular docking was performed. PLA2s and SVMPs from tested venoms were used as targets. In all cases, our molecular modeling results suggested that inhibitors may occupy the substrate-binding cleft of the enzymes, which was supported by specific interaction with amino acids from the active site, such as His48 for PLA2s and Glu143 for the metalloproteinase. In addition, varespladib and CP471474 also showed interaction with residues from the hydrophobic channel in PLA2s and substrate binding subsites in the SVMP. Our results suggest a synergistic action of the mixed inhibitors and show the potential of varespladib, CP471474, and their mixture to generate new treatments for snakebite envenoming with application in the field or as antivenom co-adjuvants.

In vitro evaluation of antiviral activity of Shouchella clausii probiotic strain and bacterial supernatant against herpes simplex virus type 1.

Herpes simplex virus-1 (HSV-1) is an important human neurotropic virus infecting 70% of the world population. Due to the emergence of viral resistance via mutations in HSV-1 genes and some of the adverse effects of antiviral compounds, there is a growing need for safe, novel, and effective therapeutic and preventive strategies. The aim of the present study was to investigate for the first time the potential antiviral activity of Shouchella clausii probiotic strain and bacterial supernatant against HSV-1. The MTT assay was used to determine the possible cytotoxicity of the S. clausii and bacterial supernatant. Vero cells were treated by S. clausii, bacterial supernatant, and HSV-1 under pre-treatment (incubation of Vero cells with S. clausii then HSV-1 inoculation), pre-incubation (mixture of co-incubated HSV-1/S. clausii added to Vero cell), competition (adding HSV-1 and S. clausii into Vero cells simultaneously) and post-treatment (Vero cells inoculated with HSV-1 then incubated with S. clausii) assays. Viral titer reduction (TCID50) and viral DNA relative quantification by real-time PCR were measured in each experimental condition. The results indicated that S. clausii and its supernatant had the greatest inhibitory activity toward HSV-1 in pre-treatment assay. The HSV-1 titer treated with S. clausii, and bacterial supernatant was 3.6 and 2.2 Log10TCID50/mL lower compared to the control (7.66 Log10TCID50/mL). Results showed an antiviral effect of S. clausii and its supernatant. S. clausii could be considered as a novel inhibitor for HSV-1 infection.

Elucidating the Kinetic Mechanism of Human METTL16

Over 140 RNA modifications have been discovered, yet only recently have they been studied in depth due to recent technological advancements. N6‐methyladenosine (m6A) is an abundant RNA modification in messenger RNA (mRNA) and long non‐coding RNA that affects various cellular functions such as mRNA stability, phase‐separation of RNAs, and others. Methyltransferase‐like protein 16 (METTL16) is one of four catalytically active m6A RNA methyltransferases in humans. Two well‐known methylation targets of METTL16 are U6 spliceosomal RNA and hairpins in the 3′ untranslated region of MAT2A mRNA. However, METTL16 binds to many other RNAs, including the 3′ triple helix of MALAT1. Using in vitro assays, we have started to investigate the kinetic mechanism and other fundamental properties of METTL16. Thus far, we have determined that METTL16 is a monomer in complex with either U6 snRNA or the MALAT1 triple helix. The METTL16•RNA complex has a dissociation constant (KD) of 18 nM with the U6 snRNA and 31 nM with the MALAT1 triple helix. The apparent dissociation constant for S‐adenosylmethionine (SAM), the methyl donor, with the METTL16•U6 snRNA binary complex is 112 µM. Under in vitro conditions, the cancer‐associated MALAT1 triple helix is not a substrate of METTL16 at position A8290 and other adenosine residues. Preincubation assays suggest that there is an ordered mechanism by which U6 snRNA binds to METTL16 before SAM. Steady‐state assays established a kcat of 0.074 min‐1 and single‐turnover assays established a kchem of 0.56 min‐1. This difference in the rates suggests conformational rearrangements and/or product release may be rate limiting. Ongoing work includes the characterization of METTL16 mutants. Mutations in the METTL16 K‐loop led to a 7‐fold increase of SAM binding to METTL16•U6 snRNA. Future studies will focus on more METTL16 mutants, including critical residues in other structures and those identified in cancer patients, to ascertain how these residues affect the kinetic mechanism of METTL16. This study will enable future research on METTL16 as a therapeutic target.

SpgC1qR interacts with WSSV VP28 exhibiting antiviral activity

Although human gC1qR is a multi-ligand binding protein with diverse biological functions, the functions of invertebrate gC1qR homologues remain largely unknown. In the present study, we characterized a novel gC1qR homologue, namely SpgC1qR, from mud crab Scylla paramamosain. SpgC1qR shared high identity and similar three-dimensional structure with human gC1qR. After challenge with White spot syndrome virus (WSSV), the transcripts of SpgC1qR were significantly increased, suggesting that SpgC1qR may be involved in antiviral immune response. To reveal the likely antiviral activity of SpgC1qR, the proliferation profile of WSSV in SpgC1qR-silenced crabs was examined. The result showed that knockdown of SpgC1qR by RNAi facilitated viral proliferation in vivo. This result was further confirmed by a SpgC1qR pre-incubation assay, in which pre-incubating WSSV particles with rSpgC1qR dramatically suppressed viral replication. Moreover, a GST pull-down assay revealed that SpgC1qR specifically bound to the viral envelope protein VP28. These findings clearly demonstrated that SpgC1qR specifically interacted with viral envelope protein VP28 and restricted WSSV replication, suggesting that it played a crucial role in anti-WSSV immune response of mud crab. This study provided new insights into the antiviral mechanism mediated by SpgC1qR and the biological functions of invertebrate gC1qR homologues.

Cross-reactivity of some Micrurus venoms against experimental and therapeutic anti-Micrurus antivenoms

We developed experimental equine polyvalent and monovalent antivenoms against the venoms of Micrurus (M.) fulvius, M. nigrocinctus and M. surinamensis and studied their immunochemical reactivity on the venoms used as immunogens and on M. pyrrhocryptus, M altirostris and M. balyocoriphus venoms. Assessment of the neutralizing capacity of the polyvalent experimental antivenom was based on inhibition of lethality (preincubation and rescue assay experiments in mice) and indirect hemolytic and phospholipase activities. The immunochemical reactivity and neutralizing capacity were compared with those of two therapeutic antivenoms used for the treatment of coral snake envenomation in North America and in Argentina. In general, the experimental antivenom conferred a comparable level of neutralization against the venoms used as immunogens when compared to the therapeutic antivenoms and a certain level of cross-neutralization against the other venoms. The results suggest the need for additional venoms in the immunogenic mixture used, in order to obtain a broad spectrum anti-Micrurus antivenom with a good neutralizing potency. Paraspecific neutralization of South American coral snake venoms, although present at a higher level than the neutralization conferred by available nonspecific Micrurus therapeutic antivenoms, was rather low in relation to the specific neutralizing capacity.

14-3-3 is a VP3-binding protein involved in Macrobrachium rosenbergii Taihu virus infection in shrimp

Macrobrachium rosenbergii Taihu virus (MrTV) is a fierce pathogen that causes high mortality in M. rosenbergii larvae. Little is known about the pathogenesis of MrTV and host-virus interactions. In this study, a virus overlay protein binding assay (VOPBA), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, was carried out to search for novel host molecules that bind with VP3, one of the main capsid proteins of MrTV. Macrobrachium rosenbergii 14-3-3 protein (Mr14-3-3) was identified as the binding protein of VP3, which was further confirmed by co-immunoprecipitation (Co-IP) and co-localization assay. A preincubation assay was developed, which indicated that preincubation with recombinant Mr14-3-3 (rMr14-3-3) could significantly decrease the expression level of VP3 in MrTV-infected M. rosenbergii larvae, suggesting that preincubation with rMr14-3-3 could partially block MrTV infection. This study revealed that Mr14-3-3 acts as a binding protein for MrTV–VP3 and plays an important role in MrTV infection, offering a potential target for the development of anti-MrTV therapies.

Evaluation of antiviral activity of Andrographis paniculata and Tinospora cordifolia using in silico and in vitro assay against DENV-2

Background: Dengue is one of the most widespread arthropod-borne viral infections without any effective treatment. The anti-DENV-2 mechanism of plants Andrographis paniculata (whole plant), Tinospora cordifolia (stem & leaves), their bioactive synthetic compounds depend on acute febrile treatment, is poorly understood for new anti-dengue therapy development. Objectives: The current study was undertaken to evaluate in silico and in vitro study on crude extracts, bioactive fractions, bioactive synthetic compounds of A. paniculata, T. cordifolia against anti-DENV-2. Methods: In silico study was evaluated by Lipinski’s rule of five, drug-likeness score and molecular docking against DENV-2 NS2B-NS3. After in silico study, the antiviral activity was performed under in vitro conditions with cytotoxicity, pre-incubation, post-incubation, and protective assay. Findings: It was observed that in in silico studies, the best docked compounds andrographolide (-11.58 kcal/mol), magnoflorine (-9.22 kcal/mol) and their combination (50:50); ethanolic extract of A. paniculata, aqueous-ethanolic (50:50) extract of T. cordifolia and their combination (50:50) extract, their bioactive fractions with possible phenolic glycosides, pyridinecarboxylic acid, flavone, phenols, phenylpropanoids, flavonoids, phenolic acid, alkaloids, isopalmitic acid, diterpenoids, quinic acid, isopalmitic acid and sesquiterpenoids compound class category, showed 50% minimum effective and inhibitory concentration. Conclusions: The crude extracts, bioactive fractions and bioactive synthetic compounds of A. paniculata and T. cordifolia and combination (50:50) could be the potential anti-DENV-2 therapy in in silico and in vitro infection model.

Novel Snakebite Therapeutics Must Be Tested in Appropriate Rescue Models to Robustly Assess Their Preclinical Efficacy.

In the field of antivenom research, development, and manufacture, it is often advised to follow the World Health Organization’s (WHO) guidelines for the production, control, and regulation of snake antivenom immunoglobulins, which recommend the use of preincubation assays to assess the efficacy of snakebite therapeutics. In these assays, venom and antivenom are mixed and incubated prior to in vivo administration to rodents, which allows for a standardizable comparison of antivenoms with similar characteristics. However, these assays are not necessarily sufficient for therapeutics with significantly different pharmacological properties than antibody-based antivenoms, such as small molecule inhibitors, nanoparticles, and other modalities. To ensure that the in vivo therapeutic utility of completely novel toxin-neutralizing molecules with no history of use in envenoming therapy and variable pharmacokinetics is properly evaluated, such molecules must also be tested in preclinical rescue assays, where rodents are first challenged with appropriate doses of venoms or toxins, followed by the administration of neutralizing modalities after an appropriate time delay to better mimic the real-life scenarios faced by human snakebite victims. Such an approach takes the venom (or toxin) toxicokinetics, the drug pharmacokinetics, and the drug pharmacodynamics into consideration. If new modalities are only assessed in preincubation assays and not subjected to evaluation in rescue assays, the publication of neutralization data may unintentionally misrepresent the actual therapeutic efficacy and suitability of the modality being tested, and thus potentially misguide strategic decision making in the research and development of novel therapies for snakebite envenoming.

Antifungal and Antivirulence Activity of Vaginal Lactobacillus Spp. Products against Candida Vaginal Isolates.

Candida yeasts are generally found in the vaginal microbiota; however, disruption of the balance maintained by host factors and microorganisms results in vulvovaginal candidiasis (VVC). This study evaluated the antagonistic activity of vaginal Lactobacillus spp. on Candida albicans to verify whether active compounds of Lactobacillus spp. had antifungal and antivirulence activity. The antagonism assay showed that 15 out of 20 Lactobacillus strains had an inhibitory effect on C. albicans. Biosurfactants displayed surface-tension-reducing activity, with the best value obtained for Lactobacillus gasseri 1. Lactobacillus rhamnosus ATCC 9595, Lactobacillus acidophilus ATCC 4356, and Lactobacillus paracasei 11 produced biosurfactants that decreased C. albicans adhesion and disrupted biofilm formation. The best results were obtained in the pre-incubation assay for L. gasseri 1 and L. paracasei 11. Overall, Lactobacillus strains showed significant anti-Candida activity, and their biosurfactants exhibited considerable anti-adhesion and antibiofilm activity against C. albicans. To be considered safe for use in vivo, the safety of biosurfactant (BS) should be investigated using cytotoxicity assays.

Evaluation of mutagenic and antimutagenic potential of stem bark aqueous extracts of eight trees by the bacterial reverse mutation assay

Stem bark aqueous extracts of eight woody plants Brachychiton populneus, Ceiba pentandra, Bombax malabaricum, Chorisia speciosa, Albizia lebbeck, Bauhinia variegata, Kigelia africana and Pinus halepensis were tested for their mutagenic and antimutagenic potential in the Ames test with Salmonella typhimurium strains TA98 and TA100. The aqueous extracts were neither toxic nor mutagenic in S. typhimurium tester strains. All of the tested extracts showed detectable antimutagenic effect towards the direct acting mutagens 2-nitrofluorene (2-NF) in TA98 as well as sodium azide in TA100. The extract from Kigelia africana was the most effective in reducing the mutagenicity caused by the direct mutagen 2-NF in the TA98 with 85.42% inhibition rate. A. lebbeck stem bark extract demonstrated the highest antimutagenic activity reducing the base substitution mutations rate for strain TA100 by 94.66% in pre-incubation assay. The results obtained showed that the stem bark aqueous extracts tested can protect cells against induced gene mutations.

P-331 - Cytoprotective effect of Hyptis spp. extracts against oxidative insults

Oxidative stress is known to be one of the major causes of the development of several diseases. Since the genus Hyptis has been reported to possesses a diverse range of biological activities, this study aimed the evaluation of the antioxidative potential of extracts of Hyptis marrubioides (Hm) and Hyptis pectinata (Hp) in HepG2 cell line using t-BHP (1 mM) and paraquat (2 mM) as oxidative insults (4 h). Hyptis extracts were firstly evaluated regarding their cytoxicity on HepG2 cells showing no significant toxicity at 100 g/ml. Then, they were evaluated for HepG2 cytoprotection against oxidative insults in co-incubation or pre-incubation (addition of the extract 2 h before the insult) assays. Cellular viability (MTT assay) was assessed immediately or 16 hours later, after t-BHP or paraquat insults, respectively. In both co- and pre-incubation assays, Hp and Hm showed significant cytoprotection against t-BHP and paraquat toxicity respectively (e.g. 1.8 and 0.4-fold-increase, respectively at 100 g/ml in co-incubation assays). Phytochemical analysis of Hyptis extracts showed several phenolic compounds e.g caffeyoilquinic acids and flavonoids that can be responsible for the cytoprotective effects.

Mutagenicity and Antimutagenic Activities of Lactic Acid Bacteria (LAB) Isolated from Fermented Durian (Tempoyak)

Mutagenic and antimutagenic activities of lactic acid bacteria (LAB) Lactobacillus plantarum isolated from the local fermented durian (tempoyak) was determined by Ames test (Salmonella/microsome mutagenicity assay). Our study also involved pre-incubation assay against Salmonella typhimurium TA 98 and TA 100 bacterial strain in the presence and absence of metabolic activator S9 system. It was found that the L. plantarum showed no mutagenic activity on both S. typhimurium strain TA 98 and TA 100 in the presence and absence of metabolic activator. Significant antimutagenic activity (p < 0.05) was observed in both cell-free supernatant and bacterial cell suspension of L. plantarum as compared to the mutagenicity induced by 2-Aminoanthracene in the presence of metabolic activator. Meanwhile, in the absence of metabolic activator, only the bacterial cells of L. plantarum showed antimutagenicity acitivity against Sodium Azide and 2-Nitrofluorene. In conclusion, L. plantarum could play a vital role as chemopreventive agent by binding to mutagens and suppressing mutagenesis. Thus, L. plantarum could be consider as a good candidate for functional food development as a supplement product to prevent development of colon cancer. DOI : http://dx.doi.org./10.17576/JSKM-2018-04

Antiviral effects of Lactobacillus crispatus against HSV-2 in mammalian cell lines

BackgroundHerpes simplex virus type 2 (HSV-2) infectious disease is one of the most common viral sexually transmitted diseases. As regards, vaginal lactobacilli play an important role in protecting host against the urogenital pathogens; here we assessed the potential antiviral activity of Lactobacillus crispatus against HSV-2 infection in vitro. MethodsBoth Vero and HeLa cell lines were treated by L. crispatus before, during and after HSV-2 infection. The pre-incubation assay was also performed for the evaluating of virus adsorption by L. crispatus. Virus titer reduction in each stage was determined by a plaque reduction assay. ResultsL. crispatus significantly decreased the infectivity of the HSV-2 in initial steps on both cell lines; however, no significant inhibition was ascertained during adsorption and multiplication process. The lactobacilli adhere on Vero cells two-fold stronger than HeLa and subsequently protect the Vero cells nearly 2.5 fold higher than HeLa cell against the virion. Co-incubation of HSV-2 with bacterial cells prior to virus inoculation significantly decreased the virus titer. ConclusionL. crispatus appears to inhibit the entry of the virus into cells by trapping HSV-2 particles. In addition, formation of L. crispatus microcolonies in the cell surface could block HSV-2 receptors and prevent viral entry to cells in initial infection steps.

Citrus aurantium (bitter orange) extract: Safety assessment by acute and 14-day oral toxicity studies in rats and the Ames Test for mutagenicity

The primary active constituent in bitter orange extract (BOE) is p-synephrine. This study assessed the safety of a BOE standardized to 50% p-synephrine following short-term exposure to rats and by the Ames Test. Following 5000 mg/kg of the extract orally to female rats all animals survived. Administration at 2000 mg/kg to female rats for four days yielded no signs of toxicity. Five male and five female rats were administered the BOE at 0, 250, 500, 1000 and 2000 mg/kg/day for 14 days. No significant effects were observed at any dose with respect to body weights, food intake, absolute and relative organ weights, hematology, clinical chemistry, and pathology. Two male rats died after 2000 mg/kg with gastrointestinal impaction at necropsy. During week two of 1000 mg/kg and 2000 mg/kg/day, rats exhibited transient signs of repetitive burrowing of heads in the bedding material (hypoactivity) for about 15 and 45 min, respectively. The no-observed-effect-level (NOEL) was 500 mg/kg/day. The mutagenic potential was assessed at and up to the limit dose of 5000 μg/plate in a Salmonella typhimurium reverse mutation (Ames) test, performed in duplicate as a pre-incubation assay in the presence and absence of metabolic activation (S9). The BOE did not induce an increase in the frequency of revertant colonies at any dose in the five tester strains, and was therefore non-mutagenic.