Search results for: cholinesterases enzymes inhibition

Authors: Alireza Jalalvand, Maryam Saleh, Somayeh Behjat Khatouni, Zahra Bahri Najafi, Foroozan Fatahinia, Narges Ismailzadeh, Behrokh Farahmand

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Object: In the last two decades, the recent outbreak of Coronavirus (SARS-CoV-2) imposed a global pandemic in the world. Despite the increasing prevalence of the disease, there are no effective drugs to treat it. A suitable and rapid way to afford an effective drug and treat the global pandemic is a computational drug study. This study used molecular docking methods to examine the potential inhibition of over 50 antiviral drugs against three fundamental proteins of SARS-CoV-2. METHODS: Through a literature review, three important proteins (a key protease, RNA-dependent RNA polymerase (RdRp), and spike) were selected as drug targets. Three-dimensional (3D) structures of protease, spike, and RdRP proteins were obtained from the Protein Data Bank. Protein had minimal energy. Over 50 antiviral drugs were considered candidates for protein inhibition and their 3D structures were obtained from drug banks. The Autodock 4.2 software was used to define the molecular docking settings and run the algorithm. RESULTS: Five drugs, including indinavir, lopinavir, saquinavir, nelfinavir, and remdesivir, exhibited the highest inhibitory potency against all three proteins based on the binding energies and drug binding positions deduced from docking and hydrogen-bonding analysis. Conclusions: According to the results, among the drugs mentioned, saquinavir and lopinavir showed the highest inhibitory potency against all three proteins compared to other drugs. It may enter laboratory phase studies as a dual-drug treatment to inhibit SARS-CoV-2.

Keywords: covid-19, drug repositioning, molecular docking, lopinavir, saquinavir

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Authors: Sakeena Naikwadi, K. Jagaluraiah Sannapapamma

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Vetiveria zizanioides is an aromatic grass and traditionally been used in aromatherapy and ayurvedic medicine. Vetiver root is multi-functional biopolymer and has highly aromatic, antimicrobial, UV blocking, antioxidant properties suitable for textile finishing. The vetiver root (Gulabi) powder of different concentration (2, 4, 6,8 percent) were extracted by aqueous and solvent methods subjected to bioassay for antimicrobial efficiency and GCMS spectral analysis. The organic cotton fabric was finished with vetiver root extract (8 percent) by exhaust and pad dry cure methods. The finished fabric was assessed for functional properties viz., UV protective factor, antimicrobial efficiency and aroma intensity. The results revealed that Ethanol extraction showed a greater zone of inhibition compared to aqueous extract in root powder. Among the concentrations, 8 percent root extract in ethanol showed a greater zone of inhibition against gram-positive organism S. aureus and gram-negative organism E. coli. The major compounds present in vetiver root extracts were diethyl pathalate with greater percentage (87.73 %) followed by 7- Isopropyl dimethyl carboxylic acid (4.05 %), 2-butanone 4-trimethyle cyclohexen (1.21 %), phenanthrene carboxylic acid (1.03 %), naphthalene pentanoic acid (0.99 %), 1-phenanthrene carboxylic acid and 1 cyclohexenone 2-methyl oxobuty (0.89 %). The sample finished by pad dry cure method exhibited better UV protection even after 10th wash as compared to exhaust method. Vetiver extract treated samples exhibited maximum zone of inhibition against S. aureus than the E. coli organism. The vetiver root extract treated organic cotton fabric through pad dry cure method possessed good antimicrobial activity against S. aureus and E. coli even after 20th washes compared to vetiver root extract treated by exhaust method. The olfactory analysis was carried out by 30 panels of members and opined that vetiver root extract treated fabric has very good and pleasant aroma with better tactile properties that provide cooling, soothing effect and enhances the mood of the wearer. Vetiver root extract finished organic cotton fabric possessed aroma, antimicrobial and UV properties which are aptly suitable for medical and healthcare textiles viz., wound dressing, bandage gauze, surgical cloths, baby diapers and sanitary napkins. It can be used as after finishing agent for variegated garments and made-ups and can be replaced with commercial after finishing agents.

Keywords: antimicrobial, olfactory analysis, UV protection factor, vetiver root extract

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Authors: Shivankar Agrawal, Sunil Kumar Deshmukh, Colin Barrow, Alok Adholeya

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A variety of genetic and environmental factors cause various cosmetics and dermatological problems. There are already claimed drugs available in market for treating these problems. However, the challenge remains in finding more potent, environmental friendly, causing minimal side effects and economical cosmeceuticals. This leads to an increased demand for natural cosmeceutical products in the last few decades. Plant derived ingredients are limited because plants either contain toxic metabolites, grow too slow or seasonal harvesting is a problem. The research work carried out in this project aims at isolation, characterization of marine fungal secondary metabolite and evaluating their potential use in future cosmetic skin care products. We have isolated and purified 35 morphologically different fungal isolates from various marine habitats of the India. These isolates have been functionally characterized for anti-tyrosinase, antioxidant and anti-acne activities. For molecular characterization, the Internal Transcribed spacer (ITS) region of 15 functionally active marine fungal isolates was amplified using universal primers, ITS1 and ITS4 and sequenced. Out of 15 marine fungal isolates crude extract of strains D4 (Aspergillus terreus) and P2 (Talaromyces stipitatus) showed 70% and 57% tyrosinase inhibition at 1mg/mL respectively. Strain D5 (Simplicillium lamellicola) has showed significant inhibition against Propionibacterium acnes and Staphylococcus epidermidis. In addition, all these strains also displayed DPPH- radical scavenging activity and may be utilized as skin cosmeceutical applications. Purification and characterization of crude extracts for identification of active lead molecule is under process.

Keywords: anti-acne, anti-tyrosinase, cosmeceutical, marine fungi

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Authors: F. Saleh, F. Lyall, A. Abdulsid, L. Marks

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Background: Labour in human is a complex biological process that involves interactions of neurological, hormonal and inflammatory pathways, with the placenta being a key regulator of these pathways. It is known that uterine contractions and labour pain cause physiological changes in gene expression in maternal and fetal blood, and in placenta during labour. Oxidative and inflammatory stress pathways are implicated in labour and they may cause alteration of placental gene expression. Additionally, in placental tissues, labour increases the expression of genes involved in placental oxidative stress, inflammatory cytokines, angiogenic regulators and apoptosis. Recently, Hydrogen Sulphide (H2S) has been considered as an endogenous gaseous mediator which promotes vasodilation and exhibits cytoprotective anti-inflammatory properties. The endogenous H2S is synthesised predominantly by two enzymes: cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). As the H2S pathway has anti-oxidative and anti-inflammatory characteristics thus, we hypothesised that the expression of CBS and CSE in placental tissues would alter during labour. Methods: CBS and CSE expressions were examined in placentas using western blotting and RT-PCR in inner, middle and outer placental zones in placentas obtained from healthy non labouring women who delivered by caesarian section. These were compared with the equivalent zone of placentas obtained from women who had uncomplicated labour and delivered vaginally. Results: No differences in CBS and CSE mRNA or protein levels were found between the different sites within placentas in either the labour or non-labour group. There were no significant differences in either CBS or CSE expression between the two groups at the inner site and middle site. However, at the outer site there was a highly significant decrease in CBS protein expression in the labour group when compared to the non-labour group (p = 0.002). Conclusion: To the best of author’s knowledge, this is the first report to suggest that, CBS is expressed in a spatial manner within the human placenta. Further work is needed to clarify the precise function and mechanism of this spatial regulation although it is likely that inflammatory pathways regulation is a complex process in which this plays a role.

Keywords: anti-inflammatory, hydrogen sulphide, labour, oxidative stress

Procedia PDF Downloads 241

Authors: P. S. Percin, O. Inanli, S. Karakaya

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Bitter melon (Momordica charantia) is a medicinal vegetable, which is used traditionally to remedy diabetes. Bitter melon contains several classes of primary and secondary metabolites. In traditional Turkish medicine bitter melon is used for wound healing and treatment of peptic ulcers. Nowadays, bitter melon is used for the treatment of diabetes and ulcerative colitis in many countries. The main constituents of bitter melon, which are responsible for the anti-diabetic effects, are triterpene, protein, steroid, alkaloid and phenolic compounds. In this study total phenolics, total carotenoids and β-carotene contents of mature and immature bitter melons were determined. In addition, in vitro α-amylase and α-glucosidase activities of mature and immature bitter melons were studied. Total phenolic contents of immature and mature bitter melon were 74 and 123 mg CE/g bitter melon respectively. Although total phenolics of mature bitter melon was higher than that of immature bitter melon, this difference was not found statistically significant (p > 0.05). Carotenoids, a diverse group of more than 600 naturally occurring red, orange and yellow pigments, play important roles in many physiological processes both in plants and humans. The total carotenoid content of mature bitter melon was 4.36 fold higher than the total carotenoid content of immature bitter melon. The compounds that have hypoglycaemic effect of bitter melon are steroidal saponins known as charantin, insulin-like peptides and alkaloids. α-Amylase is one of the main enzymes in human that is responsible for the breakdown of starch to more simple sugars. Therefore, the inhibitors of this enzyme can delay the carbohydrate digestion and reduce the rate of glucose absorption. The immature bitter melon extract showed α-amylase and α-glucosidase inhibitory activities in vitro. α-Amylase inhibitory activity was higher than that of α-glucosidase inhibitory activity when IC50 values were compared. In conclusion, the present results provide evidence that aqueous extract of bitter melon may have an inhibitory effect on carbohydrate breakdown enzymes.

Keywords: bitter melon, in vitro antidiabetic activity, total carotenoids, total phenols

Procedia PDF Downloads 241

Authors: Ivana Cabarkapa, Zorica Tomicic, Olivera Duragic

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Antimicrobial resistance represents one of the major challenges facing humanity in the last decades. Increasing antibiotic-resistant pathogens indicates the need for the development of alternative antibacterial drugs and new treatment strategies. One of the innovative emerging treatments in overcoming multidrug-resistant pathogens certainly represents the inhibition anti-quorum sensing system. For most of the food-borne pathogens, the expression of the virulence depends on their capability communication with other members of the population by means of quorum sensing (QS). QS represents a specific way of bacterial intercellular communication, which enabled owing to their ability to detect and to respond to cell population density by gene regulation. QS mechanisms are responsible for controls the pathogenesis, virulence luminescence, motility, sporulation and biofilm formation of many organisms by regulating gene expression. Therefore, research in this field is being an attractive target for the development of new natural antibacterial agents. Anti-QS compounds are known to have the ability to prohibit bacterial pathogenicity. Considering the importance of quorum sensing during bacterial pathogenesis, this research has been focused on evaluation anti - QS properties of four essential oils (EOs) Origanum heracleoticum, Origanum vulgare, Thymus vulgare, and Thymus serpyllum, using biomonitor strain of Chromobacterium violaceum CV026. Tests conducted on Luria Bertani agar supplemented with N hexanol DL homoserine lacton (HHL) 10µl/50ml of agar. The anti-QS potential of the EOs was assayed in a range of concentrations of 200 – 0.39 µl/ml using the disc diffusion method. EOs of Th. vulgaris and T. serpyllum were exhibited anti-QS activity indicated by a non- pigmented ring with a dilution-dependent manner. The lowest dilution of EOs T. vulgaris and T. serpyllum in which they exhibited visually detectable inhibition of violacein synthesis was 6.25 µl/ml for both tested EOs. EOs of O. heracleoticum and O. vulgare were displayed different active principles, i.e., antimicrobial activity indicated by the inner clear ring and anti-QS activity indicated by the outer non-pigmented ring, in a concentration-dependent manner. The lowest dilution of EOs of O. heracleoticum and O. vulgare in which exhibited visually detectable inhibition of violacein synthesis was 1.56 and 3.25 µl/ml, respectively. Considering that, the main constituents of the tested EOs represented by monoterpenes (carvacrol, thymol, γ-terpinene, and p-cymene), anti - QS properties of tested EOs can be mainly attributed to their activity. In particular, from the scientific literature, carvacrol and thymol show a sub-inhibitory effect against foodborne pathogens. Previous studies indicated that sub-lethal concentrations of carvacrol reduced the mobility of bacteria due to the ability of interference using QS mechanism between the bacterial cells, and thereby reducing the ability of biofilm formation The precise mechanism by which carvacrol inhibits biofilm formation is still not fully understood. Our results indicated that EOs displayed different active principles, i.e., antimicrobial activity indicated by the inner clear ring and anti-QS activity indicated by an outer non- pigmented ring with visually detectable inhibition of violacein. Preliminary results suggest that EOs represent a promising alternative for effective control of the emergence and spread of resistant pathogens.

Keywords: anti-quorum sensing activity, Chromobacterium violaceum, essential oils, violacein

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Authors: Antonia Stergiou

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Background: Due to the developmental trajectory of executive function in preschool age, the majority of existing studies investigating the association between acute physical activity and cognitive control have focused on adolescents and adult population. Aim- The aim of this study was to investigate the possible effects of physical activity on the inhibitory control of pre-school children. Methods: This is a prospectively designed study that was conducted in a primary school in Bristol in June 2015. The total number of subjects was n=61 and 20 trials of a modified Eriksen Flanker Task were completed before and after a 30-minutes session of moderate exercise (including both 5 minutes of warm up and cool down). For each test a pre- and post-test assessment took place that included both congruent and incongruent trials. The congruent trials were considered as the control condition and the incongruent trials as those that measure inhibitory control (experimental condition). At the end of the assessment, the participants were instructed to choose the face that described their current feelings between three options (happy, neutral, sad). Results: There was a trend for increased accuracy following moderate exercise, but there was statistical significance (p > .05). However, there was statistically significant improvement in the reaction time following the same type of exercise (p = .005). Face board assessment revealed positive emotions after 30 minutes of moderate exercise. Conclusions: The current study supports findings from previous studies related to the benefits of physical activity on the children’s inhibitory control and provides evidence of those benefits in even younger ages. Further research should take place considering each child individually. Implementation of those findings could result in an improved curriculum in schools with additional time spent on physical education courses.

Keywords: cognitive control, inhibition, physical activity, pre-school children

Procedia PDF Downloads 256

Authors: Sangyeop Shin, D. C. M. Kulatunga, S. H. S. Dananjaya, Chamilani Nikapitiya, Jehee Lee, Mahanama De Zoysa

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Saprolegniasis is one of the most devastating fungal diseases in freshwater fish which is caused by species in the genus Saprolegnia including Saprolegnia parasitica. In this study, we isolated the strain of S. parasitica from diseased rainbow trout in Korea. Morphological and molecular based identification confirmed that isolated fungi belong to the member of S. parasitica, supported by its typical fungal features including cotton-like whitish mycelium, zoospores (primary and secondary) and phylogenetic analysis with internal transcribed spacer (ITS) region. Pathogenicity of isolated S. parasitica was developed in embryo, larvae, juvenile and adult zebrafish as a disease model. Up regulation of host genes encoding ZfTnf-α, Zfc-Rel, ZfIl-12, ZfLyz-c, Zfβ-def, and ZfHsp-70 was identified in zebrafish larvae after experimental challenge of S. parasitica showing the host immune responses against the S. parasitica. Survival of the juveniles upon fungal infection might be due to the increased immune protection in the host. Investigation of antifungal susceptibility of S. parasitica with natural lawsone (2-hydroxy-1,4-naphthoquinone) revealed the minimum inhibitory concentration (MIC) and percentage inhibition of radial growth (PIRG %) as 200 µg/mL and 31.8%, respectively. Lawsone was able to change the membrane permeability, and cause irreversible damage and disintegration to the cellular membranes of S. parasitica which might have effect on fungi growth inhibition. Moreover, the mycelium exposed to lawsone (MIC level) changed the transcriptional responses of S. parasitica genes. Overall results indicate that lawsone could be a potential and novel anti-S. parasitica agent for controlling S. parasitica infection.

Keywords: host-pathogen interactions, lawsone, rainbow trout, Saprolegnia parasitica, Saprolegniasis, zebrafish

Procedia PDF Downloads 248

Authors: Hajji Lobna, Chattaoui Mayssa, Regaieg Hajer, M'Hamdi-Boughalleb Naima, Rhouma Ali, Horrigue-Raouani Najet

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In this study, three local isolates of Trichoderma (Tr1: T. viride, Tr2: T. harzianum and Tr3: T. asperellum) were isolated and evaluated for their biocontrol effectiveness under in vitro conditions and in greenhouse. In vitro bioassay revealed a biopotential control against Fusarium oxysporum f. sp. radicis lycopersici and Meloidogyne javanica (RKN) separately. All species of Trichoderma exhibited biocontrol performance and (Tr1) Trichoderma viride was the most efficient. In fact, growth rate inhibition of Fusarium oxysporum f. sp. radicis lycopersici (FORL) was reached 75.5% with Tr1. Parasitism rate of root-knot nematode was 60% for juveniles and 75% for eggs with the same one. Pots experiment results showed that Tr1 and Tr2, compared to chemical treatment, enhanced the plant growth and exhibited better antagonism against root-knot nematode and root-rot fungi separated or combined. All Trichoderma isolates revealed a bioprotection potential against Fusarium oxysporum f. sp. radicis lycopersici. When pathogen fungi inoculated alone, Fusarium wilt index and browning vascular rate were reduced significantly with Tr1 (0.91, 2.38%) and Tr2 (1.5, 5.5%), respectively. In the case of combined infection with Fusarium and nematode, the same isolate of Trichoderma Tr1 and Tr2 decreased Fusarium wilt index at 1.1 and 0.83 and reduced the browning vascular rate at 6.5% and 6%, respectively. Similarly, the isolate Tr1 and Tr2 caused maximum inhibition of nematode multiplication. Multiplication rate was declined at 4% with both isolates either tomato infected by nematode separately or concomitantly with Fusarium. The chemical treatment was moderate in activity against Meloidogyne javanica and Fusarium oxysporum f. sp. radicis lycopersici alone and combined.

Keywords: trichoderma spp., meloidogyne javanica, Fusarium oxysporum f.sp.radicis lycopersici, biocontrol

Procedia PDF Downloads 278

Authors: Nijole Savickiene, Antonella Di Sotto, Gabriela Mazzanti, Rasa Starselskyte, Silvia Di Giacomo, Annabella Vitalone

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Plant lectins are non-enzymic and non-immune origin proteins that specifically recognize and bind to various sugar structures and possess the activity to agglutinate cells and/or precipitate polysaccharides and glycoconjugates. The emerging evidences showed that plant lectins contribute not only to tumour cell recognition but also to cell adhesion and localization, to signal transduction, to mitogenic cytotoxicity and apoptosis. Among chitin-binding lectins, the Urtica dioica agglutinin (UDA), which is a complex of different isoforms, has been poorly studied for its biological activity. In this context and according to the increasing interest for lectins as novel antitumor drugs, present paper aimed at evaluating the potential antimutagenic activity of a lectin-like glycoprotein-enriched fraction from aerial part of Urtica dioica L. Aim: to evaluate the potential chemopreventive properties of a protein - lectin fraction from the aerial part of Urtica dioica. Materials and methods: Protein – lectin fraction has been tested for the antimutagenic activity in bacteria (50–800 mg/plate; Ames test by the preincubation method) and for the cytotoxicity on human hepatoma HepG2 cells (0.06–2 mg/mL; 24 and 48 h incubation). Results: Protein – lectin fraction from stinging nettle was not cytotoxic on HepG2 cells up to 2 mg/mL; conversely, it exhibited a strong antimutagenic activity against the mutagen 2-aminoanthracene (2AA) in all strains tested (maximum inhibition of 56.78 and 61% in TA98, TA100, and WP2uvrA strains, respectively, at 800 mg/plate). Discussion and conclusions: Protein – lectin fraction from Urtica dioica L. possesses antimutagenic and radical scavenging properties. Being 2AA a pro-carcinogenic agent, we hypothesize that the antimutagenicity of it can be due to the inhibition of CYP450-isoenzymes, involved in the mutagen bioactivation.

Keywords: lectins, antimutagenicity, chemoprevention, Urtica dioica

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Authors: Sylwia K. Naliwajko, Justyna Moskwa, Patryk Nowakowski, Renata Markiewicz-Zukowska, Krystyna Gromkowska-Kepka, Anna Puscion-Jakubik, Maria H. Borawska

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Amygdalin is found in many fruit seeds, including apricot, peach, quince, apples, and almonds. Amygdalin (also named vitamin B17), as well as its sources, are commonly used as an alternative therapy or prevention of cancer. The potential activity of amygdalin is related to its enzymatic degradation to the hydrogen cyanide. Hydrogen cyanide is a toxic substance that causes liver and nerves damage, fever, coma or even death. Amygdalin is much better tolerated after intravenous than oral administration. The aim of this study was to examine the influence of amygdalin on glioblastoma multiforme cell lines. Three glioblastoma multiforme cell lines – U87MG, T98, LN18 were incubated (48 h) with amygdalin in concentrations 100, 250, 500, 1000 and 2000 µg/mL. The MTT (Thiazolyl Blue Tetrazolium Bromide) test and DNA binding test by [3H]-thymidine incorporation were used to determine the anti-proliferative activity of amygdalin. The secretion of metalloproteinases (MMP2 and MMP-9) from U87MG cells was estimated by gelatin zymography. The statistical analysis was performed using Statistica v. 13.0 software. The data was presented as a % of control. Amygdalin did not show significant inhibition of viability of all the glioblastoma cells in concentrations 100, 250, 500, 1000 µg/mL. In 2000 µg/mL there were significant differences compared to the control, but inhibition of viability was less than 20% (more than 80% of control). The average viability of U87MG cells was 92,0±4%, T98G: 85,8±3% and LN18: 94,7±2% of the control. There was no dose-response viability, and IC50 value was not recognized. DNA binding in U87MG cells was not inhibited (109,0±3 % of control). After treatment with amygdalin, we observed significantly increased secretion of MMP2 and MMP9 in U87MG cells (130,3±14% and 112,0±5% of control, respectively). Our results suggest that amygdalin has no anticancer activity in glioblastoma cell lines.

Keywords: amygdalin, anticancer, cell line, glioblastoma

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Authors: Pardis Abolghasemi, Benyamin Hatamsaz

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Background: Diabetic nephropathy is a serious health problem described by specific kidney structure and functional disturbance. Renoprotective effects of the stem cells secretase have been shown in many kidney diseases. The aim is to evaluate the capability of human Wharton’s jelly mesenchymal stem cells conditioned media (hWJMSCs-CM) to alleviate DN in streptozotocin (STZ)-induced diabetes. Methods: Diabetic nephropathy was induced by injection of STZ (60 mg/kg, IP) in twenty rats. Conditioned media was extracted from hWJMSCs at third passages. At week 8, diabetic rats were divided into two groups: treated (hWJMSCs-CM, 500 μl/rat for three weeks, IP) and not treated (DN). In the 11th week, three groups (control, DN and DN+hWJMSCs-CM) were kept in metabolic cages and urine was collected for 24h. Blood pressure (BP) and heart rate (HR) were continuously recorded. The serum samples were maintained for measuring BUN, Cr and angiotensin-converting enzyme (ACE) activity. The left kidney was kept at -80°C for ACE activity assessment. The right kidney and pancreas were used for histopathologic evaluation. Result: Diabetic nephropathy was detected by microalbuminuria and increased albumin/creatinine ratio, as well as the pancreas and renal structural disturbance. Glomerular filtration rate, BP and HR increased in the DN group. The ACE activity was elevated in the serum and kidneys of the DN group. Administration of hWJMSCs-CM modulated the renal functional and structural disturbance and decreased the ACE activity. Conclusion: Conditioned media was extracted from hWJMSCs may have a Renoprotective effect in diabetic nephropathy. This may happen through regulation of ACE activity and renin-angiotensin system inhibition.

Keywords: diabetic nephropathy, mesenchymal stem cells, immunomodulation, anti-inflammation

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Authors: E. Heintz, H. J. van Lent, K. Glass, J. Lim

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The trend for sodium reduction in food products is clear. Following the World Health Organization (WHO) publication on sodium usage and intake, several countries have introduced initiatives to reduce food-related sodium intake. As salt is a common food preservative, this trend motivates the formulation of a suitable additive with comparable benefits of shelf life extension and microbial safety. Organic acid derivatives like acetates are known as generic microbial growth inhibitors and are commonly applied as additives to meet food safety demands. However, modern consumers have negative perceptions towards -synthetic-derived additives and increasingly prefer natural alternatives. Vinegar, for example, is a well-known natural fermentation product used in food preservation. However, the high acidity of vinegar often makes it impractical for direct use in meat products and a neutralized form would be desirable. This research demonstrates the efficacy of powdered vinegar (Provian DV) in inhibiting Listeria and spoilage organisms (LAB) to increase safety and shelf life of meat products. For this, the efficacy of Provian DV was compared to the efficacy of Provian K, a commonly used sodium free acetate-based preservative, which is known for its inhibition against Listeria. Materials & methods— Cured pork hams: Ingredients: Pork ham muscle, water, salt, dextrose, sodium tripolyphosphate, carrageenan, sodium nitrite, sodium erythorbate, and starch. Targets: 73-74% moisture, 1.75+0.1% salt, and pH 6.4+0.1. Treatments: Control (no antimicrobials), Provian®K 0.5% and 0.75%, Provian®DV 0.5%, 0.65%, 0.8% and 1.0%. Meat formulations in casings were cooked reaching an internal temperature of 73.9oC, cooled overnight and stored for 4 days at 4oC until inoculation. Inoculation: Sliced products were inoculated with approximately 3-log per gram of a cocktail of L. monocytogenes (including serotypes 4b, 1/2a and 1/2b) or LAB-cocktail (C. divergens and L. mesenteroides). Inoculated slices were vacuum packaged and stored at 4oC and 7°C. Samples were incubated 28 days (LAB) or 12 weeks (L. monocytogenes) Microbial analysis: Microbial populations were enumerated in rinsate obtained after adding 100ml of sterile Butterfield’s phosphate buffer to each package and massaging the contents externally by hand. L. monocytogenes populations were determined on triplicate samples by surface plating on Modified Oxford agar whereas LAB plate counts were determined on triplicate samples by surface plating on All Purpose Tween agar with 0.4% bromocresol purple. Proximate analysis: Triplicate non-inoculated ground samples were analyzed for the moisture content, pH, aw, salt, and residual nitrite. Results—The results confirmed the no growth of Listeria on cured ham with 0.5% Provian K stored at 4°C and 7°C for 12 weeks, whereas the no-antimicrobial control showed a 1-log increase within two weeks. 0.5% Provian DV demonstrated similar efficacy towards Listeria inhibition at 4°C while 0.65% Provian DV was required to match the Listeria control at 7°C. 0.75% Provian K and 1% Provian DV were needed to show inhibition of the LAB for 4 weeks at both temperatures. Conclusions—This research demonstrated that it is possible to increase safety and shelf life of cured ready-to-eat ham using preservatives that meet current food trends, like sodium reduction and natural origin.

Keywords: food safety, natural preservation, listeria control, shelf life extension

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Authors: Ajay Krishnamurthy, Mariyappan Mahesh Kumar, Sellamuthu Periyar Selvam

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Fruits are considered as functional foods due to the presence of various bioactive compounds available such as polyphenols, which are beneficial to health when consumed as part of our diet. The primary objective of this study was to analyze the various functional groups present in ethanolic extracts of Hylocereus undatus and Garcinia indica and also measure their antibacterial and antioxidant potential respectively thereby affirming its nutraceutical potential. To fulfill our objective, a Fourier - transform Infrared Spectroscopy (FTIR) was conducted for functional group analysis, Total Phenolic Content and DPPH free radical scavenging activity for measuring it anti-oxidant potential and agar-well diffusion assay for antibacterial potential. On careful observation and analysis of the spectrum it was found that both the fruit extracts contain similar compounds viz. Phenols, Alkanes, Alkenes, Aldehydes, Ketones, Carboxylic Acid and Amines. Total phenolic content of H.undatus and G.indica was estimated to be (26.85 ± 1.84 mg GAE/100g) and (32.84 ± 1.63 mg GAE/100g) respectively which corresponds to an inhibition of 84% and 81% respectively. H.undatus shows an inhibition of (3.4 ± 2.1mm) in gram-positive and (4.2 ± 2.24mm) in gram-negative organism on the other hand G.indica shows (2.1 ± 0.98mm) in gram-positive and (3.1 ± 1.44mm) in gram negative. The presence of such diverse compounds in the fruits helps us to understand the necessity for the inclusion of fruits in our daily diet and also helps the pharmaceutical industry in realizing the importance of exotic fruits as a potential nutraceutical.

Keywords: DPPH, fourier-transform infrared spectroscopy (FTIR), Hylocereus undatus, Garcinia indica

Procedia PDF Downloads 183

Authors: Bassma A. Abdel Haleem, Ehab K. Emam, George E. Yacoub, Ashraf M. Salem

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Background: Obesity is an increasingly common problem in childhood. Childhood obesity is considered the main risk factor for the development of metabolic syndrome (MetS) (diabetes type 2, dyslipidemia, and hypertension). Recent studies estimated that among children with obesity 30-60% will develop MetS. Visceral fat thickness is a valuable predictor of the development of MetS. Computed tomography and dual-energy X-ray absorptiometry are the main techniques to assess visceral fat. However, they carry the risk of radiation exposure and are expensive procedures. Consequently, they are seldom used in the assessment of visceral fat in children. Some studies explored the potential of ultrasound as a substitute to assess visceral fat in the elderly and found promising results. Given the vulnerability of children to radiation exposure, we sought to evaluate ultrasound as a safer and more cost-efficient alternative for measuring visceral fat in obese children. Additionally, we assessed the correlation between visceral fat and obesity indicators such as insulin resistance. Methods: A cross-sectional study was conducted on 46 children with obesity (aged 6–16 years). Their visceral fat was evaluated by ultrasound. Subcutaneous fat thickness (SFT), i.e., the measurement from the skin-fat interface to the linea alba, and visceral fat thickness (VFT), i.e., the thickness from the linea alba to the aorta, were measured and correlated with anthropometric measures, fasting lipid profile, homeostatic model assessment for insulin resistance (HOMA-IR) and liver enzymes (ALT). Results: VFT assessed via ultrasound was found to strongly correlate with the BMI, HOMA-IR with AUC for VFT as a predictor of insulin resistance of 0.858 and cut off point of >2.98. VFT also correlates positively with serum triglycerides and serum ALT. VFT correlates negatively with HDL. Conclusions: Ultrasound, a safe and cost-efficient technique, could be a useful tool for measuring the abdominal fat thickness in children with obesity. Ultrasound-measured VFT could be an appropriate prognostic factor for insulin resistance, hypertriglyceridemia, and elevated liver enzymes in obese children.

Keywords: metabolic syndrome, pediatric obesity, sonography, visceral fat

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Authors: José Maria, Vânia Laranjo, Luís Abrunhosa, António Inês

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The occurrence of mycotoxigenic moulds such as Aspergillus, Penicillium and Fusarium in food and feed has an important impact on public health, by the appearance of acute and chronic mycotoxicoses in humans and animals, which is more severe in the developing countries due to lack of food security, poverty and malnutrition. This mould contamination also constitutes a major economic problem due the lost of crop production. A great variety of filamentous fungi is able to produce highly toxic secondary metabolites known as mycotoxins. Most of the mycotoxins are carcinogenic, mutagenic, neurotoxic and immunosuppressive, being ochratoxin A (OTA) one of the most important. OTA is toxic to animals and humans, mainly due to its nephrotoxic properties. Several approaches have been developed for decontamination of mycotoxins in foods, such as, prevention of contamination, biodegradation of mycotoxins-containing food and feed with microorganisms or enzymes and inhibition or absorption of mycotoxin content of consumed food into the digestive tract. Some group of Gram-positive bacteria named lactic acid bacteria (LAB) are able to release some molecules that can influence the mould growth, improving the shelf life of many fermented products and reducing health risks due to exposure to mycotoxins. Some LAB are capable of mycotoxin detoxification. Recently our group was the first to describe the ability of LAB strains to biodegrade OTA, more specifically, Pediococcus parvulus strains isolated from Douro wines. The pathway of this biodegradation was identified previously in other microorganisms. OTA can be degraded through the hydrolysis of the amide bond that links the L-β-phenylalanine molecule to the ochratoxin alpha (OTα) a non toxic compound. It is known that some peptidases from different origins can mediate the hydrolysis reaction like, carboxypeptidase A an enzyme from the bovine pancreas, a commercial lipase and several commercial proteases. So, we wanted to have a better understanding of this OTA degradation process when LAB are involved and identify which molecules where present in this process. For achieving our aim we used some bioinformatics tools (BLAST, CLUSTALX2, CLC Sequence Viewer 7, Finch TV). We also designed specific primers and realized gene specific PCR. The template DNA used came from LAB strains samples of our previous work, and other DNA LAB strains isolated from elderberry fruit, silage, milk and sausages. Through the employment of bioinformatics tools it was possible to identify several proteins belonging to the carboxypeptidase family that participate in the process of OTA degradation, such as serine type D-Ala-D-Ala carboxypeptidase and membrane carboxypeptidase. In conclusions, this work has identified carboxypeptidase proteins being one of the molecules present in the OTA degradation process when LAB are involved.

Keywords: carboxypeptidase, lactic acid bacteria, mycotoxins, ochratoxin a.

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Authors: Sudeshna Mukherjee, Leena Fageria, R. Venkataramana Dilip, Rajdeep Chowdhury, Jitendra Panwar

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Metal nanoparticles in recent years have gained importance in cancer therapy due to their enhanced permeability retention effect. Among various nanomaterials, silver nanoparticles (AgNPs) have received considerable attention due to their unique properties like conductivity, chemical stability, relative lower toxicity and outstanding therapeutic potential, such as anti-inflammatory, antimicrobial and anti-cancerous activities. In this study, we took a greener approach to synthesize silver nanoparticle from fungus and analyze its effects on both epithelial and mesenchymal derived cancer cells. Much research has been done on nanoparticle-induced apoptosis, but little is known about its role in autophagy. In our study, the silver nanoparticles were seen to induce autophagy which was analyzed by studying the expression of several autophagy markers like, LC3B-II and ATG genes. Monodansylcadaverine (MDC) assay also revealed the induction of autophagy upon treatment with AgNPs. Inhibition of autophagy by chloroquine resulted in increased cell death suggesting autophagy as a survival strategy adopted by the cells. In parallel to autophagy induction, silver nanoparticles induced ROS accumulation. Interestingly, autophagy inhibition by chloroquine increased ROS level, resulting in enhanced cell death. We further analyzed MAPK signaling upon AgNP treatment. It was observed that along with autophagy, activation of JNK signaling served as pro-survival while ERK signaling served as a pro-death signal. Our results provide valuable insights into the role of autophagy upon AgNP exposure and provide cues to probabilistic strategies to effectively sensitize cancer cells.

Keywords: autophagy, JNK signalling, reactive oxygen species, silver nanoparticles

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Authors: Jung-Il Kang, Jeon Eon Park, Yu-Jin Moon, Young-Seok Ahn, Eun-Sook Yoo, Hee-Kyoung Kang

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This study was conducted to evaluate the effect of Undariopsis peterseniana, a seaweed native to Jeju Island, Korea, on the growth of hair. The dermal papilla cells (DPCs) have known to regulate hair growth cycle and length of hair follicle through interact with epithelial cells. When immortalized vibrissa DPCs were treated with the U. peterseniana extract, the U. peterseniana extract significantly increased the proliferation of DPCs. The effect of U. peterseniana extract on the growth of vibrissa follicles was also examined. U. peterseniana extract significantly increased the hair-fiber lengths of the vibrissa follicles. Hair loss is partly caused by dihydrotestosterone (DHT) binding to androgen receptor in hair follicles, and the inhibition of 5α-reductase activity can prevent hair loss through the decrease of DHT level. The U. peterseniana extract inhibited 5α-reductase activity. Minoxidil, a potent hair-growth agent, can induce proliferation in NIH3T3 fibroblasts by opening KATP channels. We thus examined the proliferative effects of U. peterseniana extract in NIH3T3 fibroblasts. U. peterseniana extract significantly increased the proliferation of NIH3T3 fibroblasts. Tetraethylammonium chloride (TEA), a K+ channel blocker, inhibited U. peterseniana-induced proliferation in NIH3T3 fibroblasts. These results suggest that U. peterseniana could have the potential to treat alopecia through the proliferation of DPCs, the inhibition of 5α-reductase activity and the opening of KATP channels. [Acknowledgement] This research was supported by The Leading Human Resource Training Program of Regional Neo industry through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT and future Planning (2016H1D5A1908786).

Keywords: hair growth, Undariopsis peterseniana, vibrissa follicles, dermal papilla cells, 5α-reductase, KATP channels

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Authors: Sebaa H., Cherifi F., Djabeur Abderrezak M.

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Argania spinosa, Sapotaceae sole representative in Algeria and Morocco; hence it is endemic in these regions. However, it is a recognised oil, forage, and timber tree highly adapted to aridity. The exploitation of the argan fruits produces considerable amounts of under or related products. These products, such as the endocarps of a fruit, recuperated after the use of kernels to extract oil. This research studies in detail the contents of total phenolic content was determined by Folin Ciocalteu reagent and Flavonoids by aluminum chloride colorimetric assay). Antioxidant activity of extracts was expressed as the percentage of DPPH radical inhibition and IC50 values (μg/mL). Antimicrobial activity evaluated using agar disk diffusion method against reference Pseudomonas aeruginosa ATTC 27453, Escherichia coli ATCC 23922. Immature endocarps showed a higher polyphenol content than mature endocarps. The total phenolic content in immature endocarps was found to vary from 983,75+ /- 0.45 to 980,1 +/- 0.43 mg gallic acid equivalents/g dry weight, whereas in mature endocarps, the polyphenol content ranged from 100,58 mg/g +/- 0.42 to 105 +/- 0.55% mg gallic acid equivalent / g dry weight. The flavonoid content was 16.5 mg equivalent catechin/g dry weight and 9.81mg equivalent catechin /g dry weight for immature and mature endocarp fruits, respectively. DPPH assay of the endocarps extract yielded a half-maximal effective concentration (IC50) value in the immature endocarps (549.33 μg/mL) than in mature endocarps (322 μg/mL). This result can be attributed to the higher phenolics and flavonoid compounds in the immature endocarps. Methanol extract of immature endocarps exhibited antibacterial activity against E.colie (inhibition zone, 11mm).

Keywords: antioxidant activity, antimicrobial activity, total phenolic content, DPPH assay

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Authors: Driouiche Ali, Karmal Ilham

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The Agadir region is characterized by a dry climate, ranging from arid attenuated by oceanic influences to hyper-arid. The water mobilized in the agricultural sector of greater Agadir is 95% of underground origin and comes from the water table of Chtouka. The rest represents the surface waters of the Youssef Ben Tachfine dam. These waters are intended for the irrigation of 26880 hectares of modern agriculture. More than 120 boreholes and wells are currently exploited. Their depth varies between 10 m and 200 m and the unit flow rates of the boreholes are 5 to 50 l/s. A drop in the level of the water table of about 1.5 m/year, on average, has been observed during the last five years. Farmers are thus called upon to improve irrigation methods. Thus, localized or drip irrigation is adopted to allow rational use of water. The importance of this irrigation system is due to the fact that water is applied directly to the root zone and its compatibility with fertilization. However, this irrigation system faces a thorny problem which is the clogging of pipes and drippers. This leads to a lack of uniformity of irrigation over time. This so-called scaling phenomenon, the consequences of which are harmful (cleaning or replacement of pipes), leads to considerable unproductive expenditure. The objective set by this work is the search for green inhibitors likely to prevent this phenomenon of scaling. This study requires a better knowledge of these waters, their physico-chemical characteristics and their scaling power. Thus, using the "LCGE" controlled degassing technique, we initially evaluated, on pure calco-carbonic water at 30°F, the scaling-inhibiting power of some available plant extracts in our region of Souss-Massa. We then carried out a comparative study of the efficacy of these green inhibitors. The action of the most effective green inhibitor on real agricultural waters was then studied.

Keywords: green inhibitors, localized irrigation, plant extracts, scaling inhibition

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Authors: Sarah Almuhayya, Andrew Mcbain, Gavin Humphreys

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Background. The microbiome of the upper respiratory tract (URT) has received less research attention than other body sites. This study aims to investigate the microbial ecology of the human URT with a focus on the antagonism between the corynebacteria and staphylococci. Methods. Mucosal swabs were collected from the anterior nares and nasal turbinates of 20 healthy adult subjects. Genomic DNA amplification targeting the (V4) of the 16Sr RNA gene was conducted and analyzed using QIIME. Nasal swab isolates were cultured and identified using near full-length sequencing of the 16S rRNA gene. Isolates identified as corynebacteria or staphylococci were typed using (rep-PCR). Antagonism was determined using an agar-based inhibition assay. Results. Four major bacterial phyla (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria) were identified from all volunteers. The typing of cultured staphylococci and corynebacteria suggested that intra-individual strain diversity was limited. Analysis of generated nasal microbiota profiles suggested an inverse correlation in terms of relative abundance between staphylococci and corynebacteria. Despite the apparent antagonism between these genera, it was limited when investigated on agar. Of 1000 pairwise interactions, observable zones of inhibition were only reported between a single strain of C.pseudodiphtheriticum and S.aureus. Imaging under EM revealed this effect to be bactericidal with clear lytic effects on staphylococcal cell morphology. Conclusion. Nasal microbiota is complex, but culturable staphylococci and corynebacteria were limited in terms of clone type. Analysis of generated nasal microbiota profiles suggested an inverse correlation in terms of relative abundance between these genera suggesting an antagonism or competition between these taxonomic groups.

Keywords: nasal, microbiota, S.aureus, microbioal interaction

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Authors: Anil Kumar, Diwakar Aggarwal, M. Sudhakara Reddy

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The wood of Eucalyptus, Populus and bamboos are some important species used as raw material for the manufacture of pulp. However, higher levels of lignin pose a problem during Kraft pulping and yield of pulp is also lower. In order to increase the yield of pulp per unit wood and reduce the use of chemicals during kraft pulping it is important to reduce the lignin content and/or increase cellulose content in wood. Cellulose biosynthesis in wood takes place by the coordinated action of many enzymes. The two important enzymes are KORRIGAN and SUCROSE SYNTHASE. KORRIGAN (Endo-1,4--glucanase) is implicated in the process of editing growing cellulose chains and improvement of the crystallinity of produced cellulose, whereas SUCROSE SYNTHASE is involved in providing substrate (UDP-glucose) for growing cellulose chains. The present study was aimed at the cloning, characterization and overexpression of these genes in Eucalyptus and Populus. An efficient shoot organogenesis protocol from leaf explants taken from micro shoots of the species has been developed. Agrobacterium mediated genetic transformation using Agrobacterium tumefaciens strain EHA105 and LBA4404 harboring binary vector pBI121 was achieved. Both the genes were cloned from cDNA library of Populus deltoides. These were subsequently characterized using various bioinformatics tools. The cloned genes were then inserted into pBI121 under the CaMV35S promotors replacing GUS gene. The constructs were then mobilized into above strains of Agrobacterium and used for the transformation work. Subsequently, genetic transformation of these clones with target genes following already developed protocol is in progress. Four transgenic lines of Eucalyptus tereticornis overexpressing Korrigan gene under the strong constitutive promoters CaMV35S have been developed, which are being further evaluated. Work on development of more transgenic lines overexpressing these genes in Populus and Eucalyptus is also in progress. This presentation will focus on important developments in this direction.

Keywords: Eucalyptus tereticornis, genetic transformation, Kraft pulping Populus deltoides

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Authors: M. Marjaneh, M. Y. Narazah, H. Shahrul

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Array-based gene expression analysis is a powerful tool to profile expression of genes and to generate information on therapeutic effects of new anti-cancer compounds. Anti-apoptotic effect of thymoquinone was studied in MCF7 breast cancer cell line using gene expression profiling with cDNA micro array. The purity and yield of RNA samples were determined using RNeasyPlus Mini kit. The Agilent RNA 6000 Nano LabChip kit evaluated the quantity of the RNA samples. AffinityScript RT oligo-dT promoter primer was used to generate cDNA strands. T7 RNA polymerase was used to convert cDNA to cRNA. The cRNA samples and human universal reference RNA were labelled with Cy-3-CTP and Cy-5-CTP, respectively. Feature Extraction and GeneSpring software analysed the data. The single experiment analysis revealed involvement of 64 pathways with up-regulated genes and 78 pathways with down-regulated genes. The MAPK and p38-MAPK pathways were inhibited due to the up-regulation of PTPRR gene. The inhibition of p38-MAPK suggested up-regulation of TGF-ß pathway. Inhibition of p38 - MAPK caused up-regulation of TP53 and down-regulation of Bcl2 genes indicating involvement of intrinsic apoptotic pathway. Down-regulation of CARD16 gene as an adaptor molecule regulated CASP1 and suggested necrosis-like programmed cell death and involvement of caspase in apoptosis. Furthermore, down-regulation of GPCR, EGF-EGFR signalling pathways suggested reduction of ER. Involvement of AhR pathway which control cytochrome P450 and glucuronidation pathways showed metabolism of Thymoquinone. The findings showed differential expression of several genes in apoptosis pathways with thymoquinone treatment in estrogen receptor-positive breast cancer cells.

Keywords: cDNA microarray, thymoquinone, CARD16, PTPRR, CASP10

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Authors: Naveeda Akhtar Qureshi, Wajiha

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Coccidiosis is a protozoan parasitic disease of genus Eimeria. It is an avian infection causing a great economic loss of 3 billion USD per year globally. A number of anticoccidial drugs are in use however many of them have side effects and cost effective. With increase in poultry demand throughout the world there is a need of more drugs and vaccines against coccidiosis. The present study is based upon the use of F. racemosa a medicinal plant to be a potential source of anticoccidial agents. The methanolic leaves extract was fractionated by column and thin layer chromatography and got nineteen fractions. Each fraction different concentrations was evaluated for its anticoccidial properties in an invitro experiment against E. tenella, E. necatrix and E. mitis. The anticoccidial active fractions were further characterized by spectroscopy (UV-Vis, FTIR) and GC-MS analysis. The in silico molecular docking of active fractions identified compounds were carried out. Among all fractions significantly maximum sporulation inhibition efficacy was shown by F-19 (67.11±2.18) followed by F-15 (65.21±1.34) at concentration of 30mg/ml against E. tenella. The significantly highest sporozoites viability inhibition was shown by F-19 (69.23±2.11) followed by F-15 (67.14±1.52) against E. necatrix at concentration 30mg/ml. Anticoccidial active fractions 15 and 19 showed peak spectrum at 207 and 202nm respectively by UV analysis. Their FTIR analysis confirmed the presence of carboxylic acid, amines, phenols, etc. Anticoccidial active compounds like Cyclododecane methanol, oleic acid, Octadecanoic acid, etc were identified by GC-MS analysis. Identified compounds in silico molecular docking study showed that cyclododecane methanol of F-19 and oleic acid of F-15 showed highest binding affinity with target S-Adenosylmethionine synthase. Hence for further authentication in vivo anticoccidial studies are recommended.

Keywords: ficus racemosa, cluster fig, column chromatography, anticoccidial fractions, GC-MS, molecular docking., s-adenosylmethionine synthase

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Authors: Ibrahim M. Labouta, Gina N. Tageldin, Salwa M. Fahmy, Hayam M. Ashour, Mounir A. Khalil, Tamer M. Ibrahim, Nefertiti A. El-Nikhely

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Bruton’s tyrosine kinase (BTK) is a critical effector molecule in B cell antigen receptor (BCR) signaling transduction. It regulates B cell proliferation, development and survival. Since BTK is widely expressed in many B cell leukaemias and lymphomas, targeting BTK by small molecules inhibitors became an attractive idea as new treatment modalities for B cell mediated hematologic malignancies. Ibrutinib is the 1st generation BTK inhibitor, approved by FDA for treatment of relapsed mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). It binds irreversibly to the unique cysteine (Cys481) within the ATP-binding pocket of BTK. Besides ibrutinib, many irreversible covalent BTK inhibitors comprising pyrimidine nucleus such as spebrutinib (phase IIb) showed high selectivity and potency when compared to it. In this study, the designed compounds were based on 5-cyano-2-methylsulfanyl pyrimidine core and decorated with electrophilic warheads which are essential for the optimal activity for targeted covalent inhibition (TCI). However, modifications at pyrimidine C4 or C6 were made by introduction of substituted amines which are provided to behave differently. The synthesized derivatives were evaluated for their anticancer activity in leukemia cell lines (e.g. THP-1). Results showed that, some derivatives exhibited antiproliferative activity with IC50 ranged from 5-50 μM, The in vitro enzymatic inhibitory assay for these compounds against BTK is still under investigation. Nevertheless, we could conclude from the initial biological screening that, the synthesized 4 or 6-subsitituted aminopyrimidines represent promising and novel antileukemic agents. Meanwhile, further studies are still needed to attribute this activity through targeting BTK enzyme and inhibition of BCR signaling pathway.

Keywords: BTK inhibitors, hematologic malignancies, structure based drug design (SBDD), targeted covalent inhibitors (TCI)

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Authors: Muhammad Shahzad Tufail, Iram Liaqat, Umer Sohail Meer, Muhammad Ishtaiq, Muhammad Sattar

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Nanotechnology is a vibrant field with numerous applications in many different branches of science and technology. Several methods are used to synthesize nanoparticles (NPs), which have multiple range of applications. Comparatively, the biogenic synthesis of NPs is a more economical and environmentally favourable method than the traditional chemical method. The current study aims to synthesize biogenically silver nanoparticles (AgNPs) using bacterial isolates. Four bacterial strains Escherichia coli (MT448673), Pseudomonas aeruginosa (MN900691), Bacillus subtilis (MN900684) and Bacillus licheniformis (MN900686) were used for the synthesis of AgNPs from silver nitrate (AgNO3) solution. The biofilm time kinetics of four bacterial isolates (P. aeruginosa, E. coli, B. licheniformis and B. subtilis) was analysed by incubating bacterial cultures at 37◦C in test tubes over a period of different time intervals i.e., 2, 3, 5 and 7 days following crystal violet staining method. All the four strains had ability to form strong biofilms between 48 to 72 hours of incubation. Two strains (B. subtilis and B. licheniformis) formed significant (p < 0.05) biofilm after 3 days of incubation period. The other two strains (E. coli and P. aeruginosa) showed strong biofilm formation after 2 days of incubation. Next, the antibiofilm activity of biogenically synthesized AgNPs (10 - 100 µgmL-1) was analysed against biofilm forming human pathogenic bacteria. Findings of the work revealed that 60-90% inhibition was observed at 60 µgmL-1 of AgNPs, while maximum inhibition (i.e.,100%) was found at highest concentration (90 µgmL-1). It was evident that highly significant (p < 0.05) decrease in biofilm formation was observed with increasing concentration of AgNPs.

Keywords: antibiofilm, biofilm formation, nanotechnology, pathogenic bacteria, silver nanoparticles

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Authors: Cheng-Kuang Hsu, Chih-Hsiang Chang, Chi-Chih Wang

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Black soybeans contain high amount of antioxidants including polyphenols, anthocyanins and flavones. The protective function of black soybean against CCl4 (a strong oxidant) induced acute and chronic liver damage was investigated in vivo using SD rats or ICR mouse. The evaluation of CCl4 induced oxidative stress in the liver tissues included the measurements of the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the concentration of thiobarbituric acid reactive substances (TBARS), the activities of antioxidant enzymes (superoxide dismutase SOD, catalase, and glutathione peroxidase GPx), as well as the level of histological lesion in the liver tissues. For chronic experiment, a decoction at the concentration of 100 or 1000 mg/kg of body weight, produced by baking black soybean at 130°C for 5 min and followed by immerging in 100°C hot water for 20 min, showed the inhibitory effect against CCl4 induced liver damage in SD rats. Hot-water extract (80 °C for 30 min) from un-preheated black soybean at the concentration of 200 mg/kg of body weight could not reduce ALT and AST levels in CCl4 treated SD rats, but the hot-water extract from preheated black soybean did enhance antioxidant enzymes activities, decline ALT and AST levels. Specially, the hot-water extract from the seed cost of black soybean had the highest liver protective function since it can reduce vacuolization and necrosis in the liver tissues. For acute experiment, the hot-water extracts from black soybean and the seed coat, as well as pure cyanidin-3-glucoside (C3G) could reduce ALT and AST levels of CCl4 induced ICR mouse. The decoction and hot-water extract from the seed coat of black soybean had higher total polyphenols, anthocyanins and flavones contents than those extracts from whole black soybean. Such results agreed with high liver protective function in the decoction and hot-water from the seed coat of black soybean. Black soybean showed protective function only after preheating process (baking at 130°C for 5 to 10 min) because preheating treatment damaged the cell wall and made the extraction of the antioxidants more effectively.

Keywords: black soybean, liver protective function, antioxidant, antioxidative stress

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Authors: Ruolan Zhang, Ryo Imai, Naoko Senda, Tomoyuki Sakai

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Enzymes have attracted increasing attentions in industrial manufacturing for their applicability in catalyzing complex chemical reactions under mild conditions. Directed evolution has become a powerful approach to optimize enzymes and exploit their full potentials under the circumstance of insufficient structure-function knowledge. With the incorporation of cell-free synthetic biotechnology, rapid enzyme synthesis can be realized because no cloning procedure such as transfection is needed. Its open environment also enables direct enzyme measurement. These properties of cell-free biotechnology lead to excellent throughput of enzymes generation. However, the capabilities of current screening methods have limitations. Fluorescence-based assay needs applicable fluorescent label, and the reliability of acquired enzymatic activity is influenced by fluorescent label’s binding affinity and photostability. To acquire the natural activity of an enzyme, another method is to combine pre-screening step and high-performance liquid chromatography (HPLC) measurement. But its throughput is limited by necessary time investment. Hundreds of variants are selected from libraries, and their enzymatic activities are then identified one by one by HPLC. The turn-around-time is 30 minutes for one sample by HPLC, which limits the acquirable enzyme improvement within reasonable time. To achieve the real high-throughput enzyme screening, i.e., obtain reliable enzyme improvement within reasonable time, a widely applicable high-throughput measurement of enzymatic reactions is highly demanded. Here, a high-throughput screening method using broadband coherent anti-Stokes Raman spectroscopy (CARS) was proposed. CARS is one of coherent Raman spectroscopy, which can identify label-free chemical components specifically from their inherent molecular vibration. These characteristic vibrational signals are generated from different vibrational modes of chemical bonds. With the broadband CARS, chemicals in one sample can be identified from their signals in one broadband CARS spectrum. Moreover, it can magnify the signal levels to several orders of magnitude greater than spontaneous Raman systems, and therefore has the potential to evaluate chemical's concentration rapidly. As a demonstration of screening with CARS, alcohol dehydrogenase, which converts ethanol and nicotinamide adenine dinucleotide oxidized form (NAD+) to acetaldehyde and nicotinamide adenine dinucleotide reduced form (NADH), was used. The signal of NADH at 1660 cm⁻¹, which is generated from nicotinamide in NADH, was utilized to measure the concentration of it. The evaluation time for CARS signal of NADH was determined to be as short as 0.33 seconds while having a system sensitivity of 2.5 mM. The time course of alcohol dehydrogenase reaction was successfully measured from increasing signal intensity of NADH. This measurement result of CARS was consistent with the result of a conventional method, UV-Vis. CARS is expected to have application in high-throughput enzyme screening and realize more reliable enzyme improvement within reasonable time.

Keywords: Coherent Anti-Stokes Raman Spectroscopy, CARS, directed evolution, enzyme screening, Raman spectroscopy

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Authors: Dmitry Nosik, Nickolay Nosik, Elli Kaplina, Olga Lobach, Marina Chataeva, Lev Rasnetsov

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Background and aims: Human Immunodeficiency Virus (HIV) infection is very often associated with Herpes Simplex Virus (HSV) infection but HIV patients are treated with a cocktail of antiretroviral drugs which are toxic. The use of an antiviral drug which will be active against both viruses like ferrovir found in our previous studies is rather actual. Earlier we had shown that Fullerene poly-amino capronic acid (FPACA) was active in case of monoinfection of HIV-1 or HSV-1. The aim of the study was to analyze the efficiency of FPACA against mixed infection of HIV and HSV. Methods: The peripheral blood lymphocytes, CEM, MT-4 cells were simultaneously infected with HIV-1 and HSV-1. FPACA was added 1 hour before infection. Cells viability was detected by MTT assay, virus antigens detected by ELISA, syncytium formation detected by microscopy. The different multiplicity of HIV-1/HSV-1 ratio was used. Results: The double viral HIV-1/HSV-1 infection was more cytopathic comparing with monoinfections. In mixed infection by the HIV-1/HSV-1 concentration of HIV-1 antigens and syncytium formations increased by 1,7 to 2,3 times in different cells in comparison with the culture infected with HIV-1 alone. The concentration of HSV-1 increased by 1,5-1,7 times, respectively. Administration of FPACA (1 microg/ml) protected cells: HIV-1/HSV-1 (1:1) – 80,1%; HIV-1/HSV-1 (1:4) – 57,2%; HIV-1/HSV-1 (1:8) – 46,3 %; HIV-1/HSV-1 (1:16) – 17,0%. Virus’s antigen levels were also reduced. Syncytium formation was totally inhibited in all cases of mixed infection. Conclusion: FPACA showed antiviral activity in case of mixed viral infection induced by Human Immunodeficiency Virus and Herpes Simplex Virus. The effect of viral inhibition increased with the multiplicity of HIV-1 in the inoculum. The mechanism of FPACA action is connected with the blocking of the virus particles adsorption to the cells and it could be suggested that it can have an antiviral activity against some other viruses too. Now FPACA could be considered as a potential drug for treatment of HIV disease complicated with opportunistic herpes viral infection.

Keywords: antiviral drug, human immunodeficiency virus (hiv), herpes simplex virus (hsv), mixed viral infection

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Authors: Pauline Nyssen, Ange Mouithys-Mickalad, Maryse Hoebeke

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Inflammation is a complex physiological phenomenon involving chemical and enzymatic mechanisms. Polymorphonuclear neutrophil leukocytes (PMNs) play an important role by producing reactive oxygen species (ROS) and releasing myeloperoxidase (MPO), a pro-oxidant enzyme. Released both in the phagolysosome and the extracellular medium, MPO produces during its peroxidase and halogenation cycles oxidant species, including hypochlorous acid, involved in the destruction of pathogen agents, like bacteria or viruses. Inflammatory pathologies, like rheumatoid arthritis, atherosclerosis induce an excessive stimulation of the PMNs and, therefore, an uncontrolled release of ROS and MPO in the extracellular medium, causing severe damages to the surrounding tissues and biomolecules such as proteins, lipids, and DNA. The treatment of chronic inflammatory pathologies remains a challenge. For many years, MPO has been used as a target for the development of effective treatments. Numerous studies have been focused on the design of new drugs presenting more efficient MPO inhibitory properties. However, some designed inhibitors can be toxic. An alternative consists of assessing the potential inhibitory action of clinically-known molecules, having antioxidant activity. Propofol, 2,6-diisopropyl phenol, which is used as an intravenous anesthetic agent, meets these requirements. Besides its anesthetic action employed to induce a sedative state during surgery or in intensive care units, propofol and its injectable form Diprivan indeed present antioxidant properties and act as ROS and free radical scavengers. A study has also evidenced the ability of propofol to inhibit the formation of the neutrophil extracellular traps fibers, which are important to trap pathogen microorganisms during the inflammation process. The aim of this study was to investigate the potential inhibitory action mechanism of propofol and Diprivan on MPO activity. To go into the anti-inflammatory action of propofol in-depth, two of its oxidative derivatives, 2,6-diisopropyl-1,4-p-benzoquinone (PPFQ) and 3,5,3’,5’-tetra isopropyl-(4,4’)-diphenoquinone (PPFDQ), were studied regarding their inhibitory action. Specific immunological extraction followed by enzyme detection (SIEFED) and molecular modeling have evidenced the low anti-catalytic action of propofol. Stopped-flow absorption spectroscopy and direct MPO activity analysis have proved that propofol acts as a reversible MPO inhibitor by interacting as a reductive substrate in the peroxidase cycle and promoting the accumulation of redox compound II. Overall, Diprivan exhibited a weaker inhibitory action than the active molecule propofol. In contrast, PPFQ seemed to bind and obstruct the enzyme active site, preventing the trigger of the MPO oxidant cycles. PPFQ induced a better chlorination cycle inhibition at basic and neutral pH in comparison to propofol. PPFDQ did not show any MPO inhibition activity. The three interest molecules have also demonstrated their inhibition ability on an important step of the inflammation pathway, the PMNs superoxide anions production, thanks to EPR spectroscopy and chemiluminescence. In conclusion, propofol presents an interesting immunomodulatory activity by acting as a reductive substrate in the peroxidase cycle of MPO, slowing down its activity, whereas PPFQ acts more as an anti-catalytic substrate. Although PPFDQ has no impact on MPO, it can act on the inflammation process by inhibiting the superoxide anions production by PMNs.

Keywords: Diprivan, inhibitor, myeloperoxidase, propofol, spectroscopy

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