Search results for: in-vitro and in-vivo assay

Authors: Valdrina Ajeti, Icko Gjorgoski

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Stress can be a reason for some physiological and biological disorders in the body. The antioxidative defense system is necessary for the maintenance of redox homeostasis in organisms. Because of its antioxidant effect, alkaline water (AW) is the focus of scientific interest. Adding AW and co-treatment with sodium ascorbate (SA) is expected for the organism to act preventively to hyperthermic stress. To investigate the effect of AW and SA on glucose and cortisol levels during acute hyperthermic stress, white female Wistar laboratory rats, divided into three groups of 10 individuals, were exposed to heat for 80 min, for 21 days. Acute hyperthermic exposure at 41˚C was a cause for oxidative stress. The first group is the control group, the second group is treated with AW, and the third group with AW and SA. Plasma glucose levels were determined by colorimetric method and cortisol was measured using the enzyme-linked immunosorbent assay method. The comparison of the means was made using the Tukey test. Differences were considered significant at a level of p < 0.05. Our results show that levels of glucose and cortisol have been increased in the group treated with AW on the 21st day after treatment (p < 0.0001), but not on the 7th and 14th day as compared to the control group. Also, co-treatment of animals with AW and SA significantly increased the levels of glucose and cortisol on the 21st day after treatment showing a synergistic effect. The individual action of AW, as well as synergism with SA, caused a high protective effect on oxidative damage.

Keywords: alkaline water, sodium ascorbate, hyperthermic stress, glucose, cortisol

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Authors: Suphaket Saenthaweesuk, Nutiya Somparn, Atcharaporn Thewmore

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The present study aims to investigate the effects of Cymbopogon citratus, Stapf (CS) or lemon grass ethanol extract on antioxidant and vascular disorders parameters in rat. The CS ethanol extract was screened for its phytochemical contents and antioxidant activity in vitro. Moreover, the extract was studied in rats to evaluate its effects in vivo. Rats were orally administered with CS at 1,000 mg/kg/day for 30 days. Phytochemical screening of CS extract indicated the presence of tannins, flavonoids and phenolic compounds. The extract contained phenolic compounds 1,400.10 ± 0.47 mg of gallic acid equivalents per gram CS extract. The free radical scavenging activity assessed by DPPH assay gave IC50 of 168.77 ± 3.32µg/mL, which is relatively lower than that of BHT with IC50 of 12.34 ± 1.14 µg/mL. In the animals, the protein expression of antioxidant enzymes, γ-glutamylcysteine ligase (γ-GCL) in liver was significantly increased. This was consistent with elevation of serum catalase (CAT) and superoxide dismutase (SOD) activities. However, Protein expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule (ICAM-1) and endothelial nitric oxide synthase (eNOS) in heart and aorta were not differenced from normal control. Taken together, the present study provides evidence that CCS water extract exhibits direct antioxidant properties and can induce cytoprotective enzymes in vivo.

Keywords: antioxidant, Cymbopogon citratus Stapf, VCAM-1, γ-glutamylcysteine ligase

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Authors: Nabil Ghedadba, Abdessemed Samira, Leila Hambaba, Sidi Mohamed Ould Mokhtar, Nassima Fercha, Houas Bousselsela

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The overall objective of this study was to evaluate in vitro the hemostatic activity of secondary metabolites (polyphenols, flavonoids, and tannins) of Marrubium vulgare leaves, aromatic plant widely used in traditional medicine for the treatment of asthma, cough, diabetes (by its effect on the pancreas to secrete insulin), heart disease, fever has a high efficiency as against inflammation. Qualitative analysis of the aqueous extract (AQE) by thin layer chromatography revealed the presence of quercetin, kaempferol and rutin. Quantification of total phenols by Folin Ciocalteu method and flavonoids by AlCl3 method gave high values with AQE: 175±0.80 mg GAE per 100g of the dry matter, 23.86±0.36 mg QE per 100g of dry matter. Moreover, the assay of condensed tannins by the vanillin method showed that AQE contains the highest value: 16.55±0.03 mg e-catechin per 100 g of dry matter. Assessment of hemostatic activity by the plasma recalcification method (time of Howell) has allowed us to discover the surprising dose dependent anticoagulant effect of AQE lyophilized from leaves of M. vulgare. A positive linear correlation between the two parameters studied: the content of condensed tannins and hemostatic activity (r=0.96) were used to highlight a possible role of these compounds that are potent vasoconstrictor activity in hemostatic. From these results we can see that Marrubium vulgre could be used for the treatment of health.

Keywords: Marrubium vulgare L., aqueous extract, phenolic compounds dosing, hemostatic activity, condensed tannins

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Authors: Shalini Menon, K. Girish Kumar

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Norepinephrine (NE) is one of the naturally occurring catecholamines which act both as a neurotransmitter and a hormone. Catecholamine levels are used for the diagnosis and regulation of phaeochromocytoma, a neuroendocrine tumor of the adrenal medulla. The development of simple, rapid and cost-effective sensors for NE still remains a great challenge. Herein, a dual-channel sensor has been developed for the determination of NE. A mixture of AgNO₃, NaOH, NH₃.H₂O and cetrimonium bromide in appropriate concentrations was taken as the working solution. To the thoroughly vortexed mixture, an appropriate volume of NE solution was added. After a particular time, the fluorescence and absorbance were measured. Fluorescence measurements were made by exciting at a wavelength of 400 nm. A dual-channel optical sensor has been developed for the colorimetric as well as the fluorimetric determination of NE. Metal enhanced fluorescence property of nanoparticles forms the basis of the fluorimetric detection of this assay, whereas the appearance of brown color in the presence of NE leads to colorimetric detection. Wide linear ranges and sub-micromolar detection limits were obtained using both the techniques. Moreover, the colorimetric approach was applied for the determination of NE in synthetic blood serum and the results obtained were compared with the classic high-performance liquid chromatography (HPLC) method. Recoveries between 97% and 104% were obtained using the proposed method. Based on five replicate measurements, relative standard deviation (RSD) for NE determination in the examined synthetic blood serum was found to be 2.3%. This indicates the reliability of the proposed sensor for real sample analysis.

Keywords: norepinephrine, colorimetry, fluorescence, silver nanoparticles

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Authors: Eva Tvrdá, Barbora Babečková, Michal Ďuračka, Róbert Kirchner, Július Árvay

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This study was designed to assess the in vitro effects of Origanum vulgare L. (oregano) extract on the testicular steroidogenesis. We focused on identifying major biomolecules present in the oregano extract, as well as to investigate its in vitro impact on the secretion of cholesterol, testosterone, dehydroepiandrosterone and androstenedione by murine testicular fragments. The extract was subjected to high performance liquid chromatography (HPLC) which identified cyranosid, daidzein, thymol, rosmarinic and trans-caffeic acid among the predominant biochemical components of oregano. For the in vitro experiments, testicular fragments from 20 sexually mature Institute of Cancer Research (ICR) mice were incubated in the absence (control group) or presence of the oregano extract at selected concentrations (10, 100 and 1000 μg/mL) for 24 h. Cholesterol levels were quantified using photometry and the hormones were assessed by ELISA (Enzyme-Linked Immunosorbent Assay). Our data revealed that the release of cholesterol and androstenedione (but not dehydroepiandrosterone and testosterone) by the testicular fragments was significantly impacted by the oregano extract in a dose-dependent fashion. Supplementation of the extract resulted in a significant decline of cholesterol (P < 0.05 in case of 100 μg/mL; P < 0.01 with respect 100 μg/mL extract), as well as androstenedione (P < 0.01 with respect to 100 and 1000 μg/mL extract). Our results suggest that the biomolecules present in Origanum vulgare L. could exhibit a dose-dependent impact on the secretion of male steroids, playing a role in the regulation of testicular steroidogenesis.

Keywords: mice, Origanum vulgare L., steroidogenesis, testes

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Authors: Raju Ranjha, Surbhi Aggarwal

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Background: Inflammation plays an important role in infectious and non-infectious diseases. MiRNA is also reported to play role in inflammation and associated cancers. Chemokine CXCL12 is also known to play role in inflammation and various cancers. CXCL12/CXCR4 chemokine axis was involved in pathogenesis of IBD specially UC. Supplementation of CXCL12 induces homing of dendritic cells to spleen and enhances control of plasmodium parasite in BALB/c mice. We looked at the regulation of CXCL12β by miRNA in UC colitis. Prolonged inflammation of colon in UC patient increases the risk of developing colorectal cancer. We looked at the expression differences of CXCl12β and its targeting miRNA in cancer susceptible area of colon of UC patients. Aim: Aim of this study was to find out the expression regulation of CXCL12β by miRNA in inflammation. Materials and Methods: Biopsy samples and blood samples were collected from UC patients and non-IBD controls. mRNA expression was analyzed using microarray and real-time PCR. CXCL12β targeting miRNA were looked by using online target prediction tools. Expression of CXCL12β in blood samples and cell line supernatant was analyzed using ELISA. miRNA target was validated using dual luciferase assay. Results and conclusion: We found miR-200a regulate the expression of CXCL12β in UC. Expression of CXCL12β was increased in cancer susceptible part of colon and expression of its targeting miRNA was decreased in the same part of colon. miR-200a regulate CXCL12β expression in inflammation and may be an important therapeutic target in inflammation associated cancer.

Keywords: inflammation, miRNA, regulation, CXCL12

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Authors: Hajar Hosseini Khorami

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Porphyrins are organic, aromatic compounds found in heme, cytochrome, cobalamin, chlorophyll , and many other natural products with essential roles in biological processes that their cationic forms have been used as groups of favorable non-viral vectors recently. Cationic porphyrins are self-chromogenic reagents with a high capacity for modifications, great interaction with DNA and protection of DNA from nuclease during delivery of it into a cell with low toxicity. In order to have high efficient gene transfection into the cell while causing low toxicity, genetically manipulations of the non-viral vector, cationic porphyrin, would be useful. In this study newly modified cationic porphyrin derivative, 5, 10-dihexyl-15, 20bis (N-methyl-4-pyridyl) porphyrin was applied. Cytotoxicity of synthesized cationic porphyrin on Chinese Hamster Ovarian (CHO) cells was evaluated by using MTT assay. This cationic derivative is dose-dependent, with low cytotoxicity at the ranges from 100 μM to 0.01μM. It was uptake by cells at high concentration. Using direct non-viral gene transfection method and different concentration of cationic porphyrin were tested on transfection of CHO cells by applying derived transfection reagent with X-tremeGENE HP DNA as a positive control. However, no transfection observed by porphyrin derivative and the parameters tested except for positive control. Results of this study suggested that applying different protocol, and also trying other concentration of cationic porphyrins and DNA for forming a strong complex would increase the possibility of efficient gene transfection by using cationic porphyrins.

Keywords: cationic porphyrins, gene delivery, non-viral vectors, transfection reagents

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Authors: Tais Silva Lopes, Danilo Caneppele, Elizabeth Romagosa

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Surubim-do-Paraíba, Steindachneridion parahybae is a species of South American fish in critical conditions of extinction. Researches have been developed with the objective of conserving the biological material of this species. We evaluated the cooling of mature oocytes in the cryoprotective solutions containing the following alcohols: methanol, Propylene glycol and DMSO, each at concentrations of 1M, 2M and 4M, totaling nine treatments. After being submitted to treatments, the oocytes were maintained for 120 minutes in cooling to -5.52±2.58⁰C. A sample of oocytes was submitted to negative control (NC), kept in 90% L-15 solution, and positive control (PC), fertilized and taken directly to the incubator. Fertilization and hatching rates were evaluated. In order to compare the sensitivity of oocytes to embryos of the same species, the embryos maintained as CP in the previous assay were used in the free-flow stage (about 22 hours post fertilization) and submitted to the same treatments (prepared in distilled water) and also cooled for 120 min. The evaluation was done by the hatch rate. There was no fertilization rate of the oocytes submitted to the cooling with propylene glycol; the other cryoprotectants presented values of at most 3.7% of fertilization (Methanol 1M), and no treatment completed development until hatching. The cooled embryos had a significant percentage of normal larvae in all treatments, but inversely proportional to the increase in the concentration of the alcohols. DMSO 1M was the most promising treatment for embryo cooling, with 41.7% ± 20.2 of normal larvae, while mature oocytes were highly sensitive to cold.

Keywords: cryoconservation, cooling, embryos, freezing, oocytes, south American fish

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Authors: Eva Tvrdá, Marek Halenár, Hana Greifová, Alica Mackovich, Faridullah Hashim, Norbert Lukáč

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Oxidative stress associated with semen cryopreservation may result in lipid peroxidation (LPO), DNA damage and apoptosis, leading to decreased sperm motility and fertilization ability. Curcumin (CUR), a natural phenol isolated from Curcuma longa Linn. has been presented as a possible supplement for a more effective semen cryopreservation because of its antioxidant properties. This study focused to evaluate the effects of CUR on selected oxidative stress parameters in cryopreserved bovine semen. 20 bovine ejaculates were split into two aliquots and diluted with a commercial semen extender containing CUR (50 μmol/L) or no supplement (control), cooled to 4 °C, frozen and kept in liquid nitrogen. Frozen straws were thawed in a water bath for subsequent experiments. Computer assisted semen analysis was used to evaluate spermatozoa motility, and reactive oxygen species (ROS) generation was quantified by using luminometry. Superoxide generation was evaluated with the NBT test, and LPO was assessed via the TBARS assay. CUR supplementation significantly (P<0.001) increased the spermatozoa motility and provided a significantly higher protection against ROS (P<0.001) or superoxide (P<0.01) overgeneration caused by semen freezing and thawing. Furthermore, CUR administration resulted in a significantly (P<0.01) lower LPO of the experimental semen samples. In conclusion, CUR exhibits significant ROS-scavenging activities which may prevent oxidative insults to cryopreserved spermatozoa and thus may enhance the post-thaw functional activity of male gametes.

Keywords: bulls, cryopreservation, curcumin, lipid peroxidation, reactive oxygen species, spermatozoa

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Authors: Assia Belfar, Mohamed Hadjadj, Messaouda Dakmouche, Zineb Ghiaba

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Introduction: This study aims to phytochemical screening; Extracting the active compounds and estimate the effectiveness of antioxidant in Medicinal plants desert Limoniastrum guyonianum (Zeïta) from South Algeria. Methods: Total phenolic content and total flavonoid content using Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. The total antioxidant capacity was estimated by the following methods: DPPH (1.1-diphenyl-2-picrylhydrazyl radical) and reducing power assay. Results: Phytochemical screening of the plant part reveals the presence of phenols, saponins, flavonoids and tannins. While alkaloids and Terpenoids were absent. The acetonic extract of L. guyonianum was extracted successively with ethyl acetate and butanol. Extraction of yield varied widely in the L. guyonianum ranging from (0.9425 %to 11.131%). The total phenolic content ranged from 53.33 mg GAE/g DW to 672.79 mg GAE/g DW. The total flavonoid concentrations varied from 5.45 to 21.71 mg/100g. IC50 values ranged from 0.02 ± 0.0004 to 0.13 ± 0.002 mg/ml. All extracts showed very good activity of ferric reducing power, the higher power was in butanol fraction (23.91 mM) more effective than BHA, BHT and VC. Conclusions: Demonstrated this study that the acetonic extract of L. guyonianum contain a considerable quantity of phenolic compounds and possess a good antioxidant activity. Can be used as an easily accessible source of Natural Antioxidants and as a possible food supplement and in the pharmaceutical industry.

Keywords: limoniastrum guyonianum, phenolics compounds, flavonoid compound, antioxidant activity

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Authors: Marwa T. Badawy, Alaa F. Bakr, Nesrine Hegazi, Mohamed A. Farag, Ahmed Abdellatif

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Diabetes Mellitus (DM) is a chronic disease affecting a large population worldwide. Africa is rich in native medicinal plants with myriad health benefits, though less explored towards the development of specific drug therapy as in diabetes. This study aims to determine the in vivo antidiabetic potential of the well-reported and traditionally used fruits of Baobab (Adansonia digitata L.) using STZ induced diabetic model. The in-vitro cytotoxic and antioxidant properties were examined using MTT assay on L-929 fibroblast cells and DPPH antioxidant assays, respectively. The extract showed minimal cytotoxicity with an IC50 value of 105.7 µg/mL. Histopathological and immunohistochemical investigations showed the hepatoprotective and the renoprotective effects of A. digitata fruits’ extract, implying its protective effects against diabetes complications. These findings were further supported by biochemical assays, which showed that i.p., injection of a low dose (150 mg/kg) of A. digitata twice a week lowered the fasting blood glucose levels, lipid profile, hepatic and renal markers. For a comprehensive overview of extract metabolites composition, ultrahigh performance (UHPLC) analysis coupled to high-resolution tandem mass spectrometry (HRMS/MS) in synchronization with molecular networks led to the annotation of 77 metabolites, among which 50% are reported for the first time in A. digitata fruits.

Keywords: adansonia digital, diabetes mellitus, metabolomics, streptozotocin, Sprague, dawley rats

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Authors: N. Boussoualim, H. Trabsa, I. Krache, S. Aouachria, S. Boumerfeg, L. Arrar, A. Baghiani

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The aim of this study consisted in evaluating the antioxidant in vivo properties, anti-hemolytic and XOR inhibitory effect of Globularia alypum L. (GA) extracts. GA was submitted to extraction and fractionation to give crude (CrE), chloroformique (ChE), ethyle acetate (EAE) and aqueos (AqE) extracts. Total polyphenols contents of GA extracts were determined; EAE is the most rich in polyphenols (157,74±5,27 mg GAE/mg of extract). GA Extracts inhibited XO in a concentration-dependent manner, the EAE showed the highest inhibitory properties on the XOR activity (IC50=0,083±0,001 mg/ml), followed by CrE and ChE. The antioxidant activities of the CrE, EAE, and AqE were tested by an in vivo assay in mice, the plasma ability to inhibit DPPH radical was measured, The CrE was found to exhibit the greatest scavenger activity with 48.41±2.763%, followed by AqE and EAE (40.54±7.51% and 41.79±1.654%, respectively). Total antioxidant capacity of red blood cells was measured, from the kinetics of hemolysis obtained. The calculated HT50 reveal an extension of time for half hemolysis in all treated groups compared with the control group. CrE increase significantly HT50 (112,8±2,427). The hemolysis is lagged, indicating that endogenous antioxidants in the erythrocytes can trap radicals to protect them against free-radical-induced hemolysis. Antimicrobial activities of the extracts were determined by the disc diffusion method. Test microorganisms were; 4 Gram positive, 7 gram negative bacteria, most active extracts were EAE and CrE. We deduce a great relationship between the effect on the extracts antibacterial effect and their contents in flavonoid.

Keywords: Globularia alypum, Xanthine oxidoreductase, in vivo-antioxidant activity, hemolysis, polyphenol

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Authors: Funmilola O. Omoya, Abdul O. Momoh

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Malaria constitutes one of the major health problems in Nigeria. One of the reasons attributed for the upsurge was the development of resistance of Plasmodium falciparum and the emergence of multi-resistant strains of the parasite to anti-malaria drugs. A continued search for other effective, safe and cheap plant-based anti-malaria agents thus becomes imperative in the face of these difficulties. The objective of this study is therefore to evaluate the in vivo anti-malarial efficacy of ethanolic extracts of Chromolaena odorata and Androgaphis paniculata leaves. The two plants were evaluated for their anti-malaria efficacy in vivo in a 4-day curative test assay against Plasmodium berghei strain in mice. The group treated with 500mg/ml dose of ethanolic extract of A. paniculata plant showed parasite suppression with increase in Packed Cell Volume (PCV) value except day 3 which showed a slight decrease in PCV value. During the 4-day curative test, an increase in the PCV values, weight measurement and zero count of Plasmodium berghei parasite values was recorded after day 3 of drug administration. These results obtained in group treated with A. paniculata extract showed anti-malarial efficacy with higher mortality rate in parasitaemia count when compared with Chromolaena odorata group. These results justify the use of ethanolic extracts of A. paniculata plant as medicinal herb used in folklore medicine in the treatment of malaria.

Keywords: anti-malaria, curative, plant-based anti-malaria agents, biology

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Authors: B. Nageshwar Rao

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Urinary tract infection (UTI) represents the most commonly acquired bacterial infection worldwide, with substantial morbidity, mortality, and economic burden. The objective of the study is to characterize the microbial profiles of uropathogenic in the obstetric population by RTPCR. Study design: An observational cross-sectional study was performed at a single tertiary health care hospital among 50 pregnant women with UTIs, including asymptomatic and symptomatic patients attending the outpatient department and inpatient department of Obstetrics and Gynaecology.Methods: Serotyping and genes detection of various uropathogens were studied using RTPCR. Pulse filed gel electrophoresis methods were used to determine the various genetic profiles. Results: The present study shows that CsgD protein, involved in biofilm formation in Escherichia coli, VIM1, IMP1 genes for Klebsiella were identified by using the RTPCR method. Our results showed that the prevalence of VIM1 and IMP1 genes and CsgD protein in E.coli showed a significant relationship between strong biofilm formation, and this may be due to the prevalence of specific genes. Finally, the genetic identification of RTPCR results for both bacteria was correlated with each other and concluded that the above uropathogens were common isolates in producing Biofilm in the pregnant woman suffering from urinary tract infection in our hospital observational study.

Keywords: biofilms, Klebsiella, E.coli, urinary tract infection

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Authors: Sehrish Iftikhar, Kiran Nawaz, Ahmad A. Shahid, Waheed Anwar, Muhammad S. Haider

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Early blight caused by Alternaria solani Sorauer is one of the most serious foliage diseases of the potato (Solanum tuberosum L.). This disease causes huge crop losses and has major economic importance worldwide. The antifungal activity for three medicinal plants (Foeniculum vulgare, Syzygium aromaticum, and Eucalyptus citriodora) against Alternaria solani has been evaluated. The inhibitory potential of selected essential oils on the radial mycelial growth and germination of spore was measured in vitro at various concentrations (5%, 2.5%. 1.25%, 0.625%, and 0.312%) using agar well diffusion assay. Essential oil of E. citriodora was most effective causing 85% inhibition of mycelial growth and 88% inhibition of spore germination at 0.625% and 1.25% concentrations. Essential oil of Foeniculum vulgare also caused 80% and 82% inhibition of the above mentioned parameters but at double the concentrations 1.25% and 2.5%. While essential oil of Syzygium aromaticum was least effective in controlling the mycelial growth and spore germination with 76% and 77% inhibition at 1.25% and 2.5%. All the selected essential oils, especially E. citriodora, showed marked antimicrobial activity significant at higher concentration. These results suggest that the use of essential oils for the control of A. solani can reduce environmental risks related with commercial fungicides, lower cost for control, and the chances for resistance development. Additional studies are essential to evaluate the potential of essential oils as natural treatments for this disease.

Keywords: clove, essential oils, fennel, potato

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Authors: Houda Haddad, Radhia Mejri, , Alyne Rodrigues de Araujo, Amira Zairi

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Nosocomial infections represent one of the biggest health problems nowadays. Acinetobacter baumannii is known as an opportunistic pathogen in humans, affecting people with compromised immune systems, and is becoming increasingly important as a hospital-derived infection. It is known that in recent years, more and more bacteria have become multidrug-resistant (MDR), and for this reason, the development of new drugs is a priority. However, these products must not affect the human body, and therefore, cytotoxicity studies are mandatory. In this context, antimicrobial peptides with potential antibacterial proprieties could be an alternative. In this research, we describe the synthesis and the bioactivity of dermaseptins and their derivatives against Acinetobacter baumannii. The cytotoxicity of these dermaseptins was investigated on the HEp-2 cell line by the MTT cell viability assay. Thereafter, we studied morphological alterations caused by the action of one of the active peptides on the bacterial membrane using atomic force microscopy (AFM). The cytotoxicity of dermaseptins was concentration-dependent at microgram concentrations. It was observed that all tested analogs exhibit antibacterial activity with Minimum Inhibitory Concentrations (MICs) ranging from 3.125 to 12.5 μg/mL and Minimum Bactericidal Concentrations (MBCs) ranging from 6.25 to 25 μg/mL. Microscopic images obtained by AFM revealed morphological changes on the surface of treated bacteria caused by K4S4(1-16), as well as significant surface alterations. Overall, these findings demonstrate that dermaseptins might constitute new lead structures for the development of potent antibacterial agents against Acinetobacter baumannii infections.

Keywords: dermaseptin B2, dermaseptin S4, analogs, Acinetobacter baumannii, healthcare-associated infections, antibacterial activity

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Authors: Recep Keşli, Onur Türkyılmaz, Hayriye Tokay, Kasım Demir

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Objective: Coeliac disease (CD) is a primary small intestine disorder caused by high sensitivity to gluten which is present in the crops, characterized by inflammation in the small intestine mucosa. The goal of this study was to determine and to compare the sensitivity and specificity values of anti-endomysial IgA (EMA IgA) (IFA) and anti-tissue transglutaminase IgA (anti-tTG IgA) (ELISA) antibodies in the diagnosis of patients suspected with the CD. Methods: One thousand two hundred seventy three patients, who have applied to gastroenterology and pediatric disease polyclinics of Afyon Kocatepe University ANS Research and Practice Hospital were included into the study between 23.09.2010 and 30.05.2016. Sera samples were investigated by immunofluorescence method for EMA positiveness (Euroimmun, Luebeck, Germany). In order to determine quantitative value of Anti-tTG IgA (EIA) (Orgentec Mainz, Germany) fully automated ELISA device (Alisei, Seac, Firenze, Italy) were used. Results: Out of 1273 patients, 160 were diagnosed with coeliac disease according to ESPGHAN 2012 diagnosis criteria. Out of 160 CD patients, 120 were female, 40 were male. The EMA specificity and sensitivity were calculated as 98% and 80% respectively. Specificity and sensitivity of Anti-tTG IgA were determined as 99% and 96% respectively. Conclusion: The specificity of EMA for CD was excellent because all EMA-positive patients (n = 144) were diagnosed with CD. The presence of human anti-tTG IgA was found as a reliable marker for diagnosis and follow-up the CD. Diagnosis of CD should be established on both the clinical and serologic profiles together.

Keywords: anti-endomysial antibody, anti-tTG IgA, coeliac disease, immunofluorescence assay (IFA)

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Authors: Deepika Saini, Sandeep Jain, Ajay Kumar

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The quinoline scaffold is one of the most widely studied in the discovery of derivatives with various heterocyclic moieties due to its potential antimalarial activities. In the present study, a chalcone series of quinoline derivatives clubbed with pyrazole were synthesized to evaluate their antimalarial property by in vitro schizont maturation inhibition assay against both chloroquine sensitive, 3D7 and chloroquine resistant, RKL9 strain of Plasmodium falciparum. Further, top five compounds were studied for in vivo preclinical study for antimalarial potential against P. berghei in Swiss albino mice. To understand the mechanism of synthesized analogues, they were screened computationally by molecular docking techniques. Compounds were docked into the active site of a protein receptor, Plasmodium falciparum Cysteine Protease Falcipain-2. The compounds were successfully synthesized, and structural confirmation was performed by FTIR, 1H-NMR, mass spectrometry and elemental analysis. In vitro study suggested that the compounds 5b, 5g, 5l, 5s and 5u possessed best antimalarial activity and further tested for in vivo screening. Compound 5u (CH₃ on both rings) with EC₅₀ 0.313 & 0.801 µg/ml against CQ-S & CQ-R strains of P. falciparum respectively and 78.01% suppression of parasitemia. The molecular docking studies of the compounds helped in understanding the mechanism of action against falcipain-2. The present study reveals the binding signatures of the synthesized ligands within the active site of the protein, and it explains the results from in vitro study in their EC₅₀ values and percentage parasitemia.

Keywords: antimalarial activity, chalcone, docking, quinoline

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Authors: Bhavana V. Mohite, Satish V. Patil

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Bacterial cellulose (BC) is an alternative for plant cellulose (PC) that prevents global warming leads to preservation of nature. Although PC and BC have the same chemical structure, BC is superior with its properties like its size, purity, porosity, degree of polymerization, crystallinity and water holding capacity, thermal stability etc. On this background the present study focus production and applications of BC as antimicrobial wound dressing material. BC was produced by Gluconoacetobacter hansenii (strain NCIM 2529) under shaking condition and statistically enhanced upto 7.2 g/l from 3.0 g/l. BC was analyzed for its physico mechanical, structural and thermal characteristics. BC produced at shaking condition exhibits more suitable properties in support to its high performance applications. The potential of nano silver impregnated BC was determined for sustained release modern antimicrobial wound dressing material by swelling ratio, mechanical properties and antimicrobial activity against Staphylococcus aureus. BC in nanocomposite form with other synthetic polymer like PVA shows improvement in its properties such as swelling ratio (757% to 979%) and sustainable release of antibacterial agent. The high drug loading and release potential of BC was evidenced in support to its nature as antimicrobial wound dressing material. The nontoxic biocompatible nature of BC was confirmed by MTT assay on human epidermal cells with 90% cell viability that allows its application as a regenerative biomaterial. Thus, BC as a promising new generation antimicrobial wound dressing material was projected.

Keywords: agitated culture, biopolymer, gluconoacetobacter hansenii, nanocomposite

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Authors: D. J. Kalita

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Cancer is one of the prime causes of early death worldwide. Mutation of the gene involve in DNA repair and damage, like BRCA2 (Breast cancer gene two) genes, can be detected efficiently by PCR-RFLP to early breast cancer diagnosis and adopt the suitable method of treatment. Host Defense Peptide can be used as blueprint for the design and synthesis of novel anticancer drugs to avoid the side effect of conventional chemotherapy and chemo resistance. The change at nucleotide position 392 of a -› c in the cancer sample of dog mammary tumour at BRCA2 (exon 7) gene lead the creation of a new restriction site for SsiI restriction enzyme. This SNP may be a marker for detection of canine mammary tumour. Support vector machine (SVM) algorithm was used to design and predict the anticancer peptide from the mature functional peptide. MTT assay of MCF-7 cell line after 48 hours of post treatment showed an increase in the number of rounded cells when compared with untreated control cells. The ability of the synthesized peptide to induce apoptosis in MCF-7 cells was further investigated by staining the cells with the fluorescent dye Hoechst stain solution, which allows the evaluation of the nuclear morphology. Numerous cells with dense, pyknotic nuclei (the brighter fluorescence) were observed in treated but not in control MCF-7 cells when viewed using an inverted phase-contrast microscope. Thus, PCR-RFLP is one of the attractive approach for early diagnosis, and synthetic cationic peptide can be used for the treatment of canine mammary tumour.

Keywords: cancer, cationic peptide, host defense peptides, Breast cancer genes

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Authors: Abdollah Jamshidi, Tayebeh Zeinali, Mehrnaz Rad, Jamshid Razmyar

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Listeria infections occur worldwide in variety of animals and man. Listeriae are widely distributed in nature. The organism has been isolated from the feces of humans and several animals, different soils, plants, aquatic environments and food of animal and vegetable origin. Listeria monocytogenes is recognized as important food-borne pathogens due to its high mortality rate. This organism is able to growth at refrigeration temperature, and high osmotic pressure. Poultry can become contaminated environmentally or through healthy carrier birds. In recent decades, prophylactic use of antimicrobial agents may be lead to emergence of antibiotic resistant organisms, which can be transmitted to human through consumption of contaminated foods. In this study, from 200 fresh chicken carcasses samples which were collected randomly from different supermarkets and butcheries, 80 samples were detected as contaminate with Listeria spp. and 19% of the isolates identified as Listeria monocytogene using multiplex PCR assay. Conventional methods were used to differentiate other species of the listeria genus. The results showed the most prevalent isolates as L. monocytogenes (48.75%). Other isolates were detected as Listeria innocua (28.75%), Listeria murrayi (20%), Listeria grayi (3.75%) and Listeria welshimeri (2.5%).The Majority of the isolates had multidrug resistance to commonly used antibiotics. Most of them were resistant to erythromycin (50%), followed by Tetracycline (44.44%), Clindamycin (41.66%), and Trimethoprim (25%). Some of them showed resistance to chloramphenicol (17.65%). The results indicate the resistance of the isolates to antimicrobials commonly used to treat human listeriosis, which could be a potential health hazard for consumers.

Keywords: listeria species, L. monocytogenes, antibiotic resistance, chicken carcass

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Authors: Seon Yeong Byeon, Hwa Sung Shin

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Nanofibrous sheets are of interest in the beauty industries due to the properties of moisturizing, adhesion to skin and delivery of nutrient materials. The benefit and function of the cosmetic products should not be considered without safety thus a non-toxic manufacturing process is ideal when fabricating the products. In this study, we have developed cosmetic patches consisting of alginate and Spirulina extract, a marine resource which has antibacterial and antioxidant effects, without addition of harmful cross-linkers. The patches obtained their structural stabilities by layer-upon-layer electrospinning of an alginate layer on a formerly spread polycaprolactone (PCL) layer instead of crosslinking method. The morphological characteristics, release of Spirulina extract, water absorption, skin adhesiveness and cytotoxicity of the double-layered patches were assessed. The image of scanning electron microscopy (SEM) showed that the addition of Spirulina extract has made the fiber diameter of alginate layers thinner. Impregnation of Spirulina extract increased their hydrophilicity, moisture absorption ability and skin adhesive ability. In addition, wetting the pre-dried patches resulted in releasing the Spirulina extract within 30 min. The patches were detected to have no cytotoxicity in the human keratinocyte cell-based MTT assay, but rather showed increased cell viability. All the results indicate the bioactive and hydro-adhesive double-layered patches have an excellent applicability to bioproducts for personal skin care in the trend of ‘A mask pack a day’.

Keywords: alginate, cosmetic patch, electrospun nanofiber, polycaprolactone, Spirulina extract

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Authors: Gabriel S. De Oliveira, Patricia P. Adriani, Christophe Moriseau, Bruce D. Hammock, Felipe S. Chambergo

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Epoxide hydrolases (EHs) are enzymes that are present in all living organisms and catalyze the hydrolysis of epoxides to the corresponding vicinal diols. EHs have high biotechnological interest for the drug design and chemistry transformation for industries. In this study, we describe the identification of substrates and inhibitors of epoxide hydrolase enzyme from the filamentous fungus Trichoderma reesei (TrEH), and these inhibitors showed the fungal growth inhibitory activity. We have used the cloned enzyme and expressed in E. coli to develop the screening in the library of fluorescent substrates with the objective of finding the best substrate to be used in the identification of good inhibitors for the enzyme TrEH. The substrate (3-phenyloxiranyl)-acetic acid cyano-(6-methoxy-naphthalen-2-yl)-methyl ester showed the highest specific activity and was chosen for the next steps of the study. The inhibitors screening was performed in the library with more than three thousand molecules and we could identify the 6 best inhibitors. The IC50 of these molecules were determined in nM and all the best inhibitors have urea or amide in their structure, because It has been recognized that these groups fit well in the hydrolase catalytic pocket of the epoxide hydrolases. Then the growth of T. reesei in PDA medium containing these TrEH inhibitors was tested, and fungal growth inhibition activity was demonstrated with more than 60% of inhibition of fungus growth in the assay with the TrEH inhibitor with the lowest IC50. Understanding how this EH enzyme from T. reesei responds to inhibitors may contribute for the study of fungal metabolism and drug design against pathogenic fungi.

Keywords: epoxide hydrolases, fungal growth inhibition, inhibitor, Trichoderma reesei

Procedia PDF Downloads 181

Authors: Hassan Ali Abood Nassrullah, Jabbar Fadeel Mahdi, Mohammed Salih Mahdi Alkurdi, Ali Al Mousawi, Saad Ibrahim Al-Ghabban, Abdul Amir H. Kadhum, Ahmed Al-Amiery

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Background: Q fever is a zoonotic disease characterized by its clinical polymorphism and can present acutely as fever, pneumonia, hepatitis, and chronically as infective endocarditis, arthritis, osteomyelitis, or hepatitis. Objective: The aim of this study is To estimate the prevalence of cases of Q fever in hospitalized febrile patients in Imam Al Hussain Teaching Medical City in Karbala. Methods: One hundred patients with pyrexia were admitted to the medical ward from 1st August to 31st December 2019. Serological procedures fortified by Enzyme-linked Immunosorbent Assay test. Patients were considered to have acute Q fever when the specific antibodies (IgM and IgG) of phase II of Coxiella burnetii were positive. Results: The mean age of the patients was 35.05±12.93 years; females constituted 60% of them. Eighteen patients (18%) showed positive results for IgM, a lower proportion (13% n=13) had positive IgG levels, and 9% showed equivocal results. Statistical analysis revealed a significant association between positive IgM levels of the female gender and in patients consuming unpasteurized milk. One patient (female aged 60 years) died in the hospital, while all other patients were discharged well. Two female patients were pregnant, and one of them had an abortion. Conclusions: Q fever is more common in febrile patients. The study indicates that this disease should not be overlooked in the differential diagnosis of acute fever. Serological testing should be performed in all patients with acute febrile illness with an unsettling diagnosis.

Keywords: antibodies, frequency, immunoglobulin IgM, Q fever

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Authors: Carol Yue-En Ong, Angelina Hui-Min Tan, Quan Liu, Paul Chi-Lui Ho

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A public general hospital in Singapore has recently implemented an automated unit-dose machine in their inpatient dispensary, Swisslog, with the objective of reducing human error and improving patient safety. However, a concern in stability arises as tablets are removed from their original packaging (bottled loose tablets/capsules) and are repackaged into individual, clear plastic wrappers as unit doses in the system. Drugs that are light-sensitive and hygroscopic would be more susceptible to degradation as the wrapper does not offer full protection. Hence, this study was carried out to study the stability of haloperidol tablets and phenytoin capsules that are light-sensitive and hygroscopic respectively. Validated HPLC-UV assays were first established for quantification of these two compounds. The medications involved were put in the Swisslog and sampled every week for one month. The collected data was analysed and showed no degradation over time. This study also explored an alternative approach for drug stability determination-Raman spectroscopy. The advantage of Raman spectroscopy is its high time efficiency and non-destructive nature. The results suggest that drug degradation can indeed be detected using Raman microscopy, but further research is needed to establish this approach for quantification or qualification of compounds. NanoRam®, a portable Raman spectrocope was also used alongside Raman microscopy but was unsuccessful in detecting degradation in this study.

Keywords: drug stability, haloperidol, HPLC, phenytoin, raman spectroscopy, Swisslog

Procedia PDF Downloads 319

Authors: Samuel B. Dulay, Sandra Julich, Herbert Tomaso, Ciara K. O'Sullivan

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An early, rapid and definite detection for the presence of biowarfare agents, pathogens, viruses and toxins is required in different situations which include civil rescue and security units, homeland security, military operations, public transportation securities such as airports, metro and railway stations due to its harmful effect on the human population. In this work, an electrochemical genosensor array that allows simultaneous detection of different biowarfare agents within an integrated microsystem that provides an easy handling of the technology which combines a microfluidics setup with a multiplexing genosensor array has been developed and optimised for the following targets: Bacillus anthracis, Brucella abortis and melitensis, Bacteriophage lambda, Francisella tularensis, Burkholderia mallei and pseudomallei, Coxiella burnetii, Yersinia pestis, and Bacillus thuringiensis. The electrode array was modified via co-immobilisation of a 1:100 (mol/mol) mixture of a thiolated probe and an oligoethyleneglycol-terminated monopodal thiol. PCR products from these relevant biowarfare agents were detected reproducibly through a sandwich assay format with the target hybridised between a surface immobilised probe into the electrode and a horseradish peroxidase-labelled secondary reporter probe, which provided an enzyme based electrochemical signal. The potential of the designed microsystem for multiplexed genosensor detection and cross-reactivity studies over potential interfering DNA sequences has demonstrated high selectivity using the developed platform producing high-throughput.

Keywords: biowarfare agents, genosensors, multipled detection, microsystem

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Authors: Atefeh Esmailnejad, Mohammad Abbaszadeh Hasiri

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Coxiella burnetii is a gram-negative obligatory intracellular bacterium that causes Q fever, a significant zoonotic disease. Sheep, cattle, and goats are the most commonly reported reservoirs for the bacteria, but infected cats and dogs have also been implicated in the transmission of the disease to human. The aim of present study was to investigate the presence of antibodies against Coxiella burnetii among companion dogs in Fars province, South of Iran. A total of 181 blood samples were collected from asymptomatic dogs, mostly referred to Veterinary Hospital of Shiraz University for regular vaccination. The IgG antibody detection against Coxiella burnetii was made by indirect Enzyme-linked Immunosorbent Assay (ELISA), employing phase I and II Coxiella burnetii antigens. A logistic regression model was developed to analyze multiple risk factors associated with seropositivity. An overall seropositivity of 7.7% (n=14) was observed. Prevalence was significantly higher in adult dogs above five years (18.18 %) compared with dogs between 1 and five years (7.86 %) and less than one year (6.17%) (P=0.043). Prevalence was also higher in male dogs (11.21 %) than in female (2.7 %) (P=0.035). There were no significant differences in the prevalence of positive cases and breed, type of housing, type of food and exposure to other farm animals (P>0.05). The results of this study showed the presence of Coxiella burnetii infection among the companion dogs population in Fars province. To our knowledge, this is the first study regarding Q fever in dogs carried out in Iran. In areas like Iran, where human cases of Q fever are not common or remain unreported, the public health implications of Q fever seroprevalence in dogs are quite significant.

Keywords: Coxiella burnetii, dog, Iran, Q fever

Procedia PDF Downloads 285

Authors: Vinod Nair, C. Sadasivan

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Beta-lactam antibiotics are the most frequently prescribed medications in modern medicine. The antibiotic resistance by the production of enzyme beta-lactamase is an important mechanism seen in microorganisms. Resistance to beta-lactams mediated by beta-lactamases can be overcome successfully with the use of beta-lactamase inhibitors. New generations of the antibiotics contain mostly synthetic compounds, and many side effects have been reported for them. Combinations of beta-lactam and beta-lactamase inhibitors have become one of the most successful antimicrobial strategies in the current scenario of bacterial infections. Plant-based drugs are very cheap and having lesser adverse effect than synthetic compounds. The synergistic effect of eugenol acetate with beta-lactams restores the activity of beta-lactams, allowing their continued clinical use. It is reported here the enhanced inhibitory effect of phytochemical, eugenol acetate, isolated from the plant Syzygium aromaticum with beta-lactams on beta-lactamase. The compound was found to have synergistic effect with the antibiotic amoxicillin against antibiotic-resistant strain of S.aureus. The enzyme was purified from the organism and incubated with the compound. The assay showed that the compound could inhibit the enzymatic activity of beta-lactamase. Modeling and molecular docking studies indicated that the compound can fit into the active site of beta-lactamase and can mask the important residue for hydrolysis of beta-lactams. The synergistic effects of eugenol acetate with beta-lactam antibiotics may justify, the use of these plant compounds for the preparation of β-lactamase inhibitors against β-lactam resistant S.aureus.

Keywords: betalactamase, eugenol acetate, synergistic effect, molecular modeling

Procedia PDF Downloads 229

Authors: David Guillermo Piedrahita Marquez, Hector Suarez Mahecha, Jairo Humberto Lopez

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There is a need to preserve aquaculture matrices due to their high nutritional value, and its broad consumption, one of those species is the white cachama (Piaractus brachypomus), this fish is located in the rivers of eastern Colombia, and the previously mentioned species needs more study. Therefore, in a paper the effects of an alternative method of preservation of shell life were investigated, the method used is the application of an edible coating made from chitosan and ethanolic extract of propolis (EEP) encapsulated in maltodextrin. The coating was applied by immersion, and after that, we investigated the post mortem quality changes of the fish performing physicochemical and microbiological analysis. pH, volatile bases, test thiobarbituric acid and peroxide value were tested; finally, we studied the effect of the coating on mesophilic strains, coliforms and other microorganisms such as Staphylococcus, and Salmonella. Finally, we concluded that the coating prolongs the shelf life because it acts as a barrier to oxygen and moisture, the bioactive compounds trap free radicals and the coatings changes the metabolism and cause the cell lysis of the microorganisms. It was determined that the concentration of malonaldehyde, the volatile basic nitrogen content and pH are the variables that distinguish more clearly between the samples with the treatment and the control samples.

Keywords: antimicrobial activity, lipid oxidation, texture profile analysis (TPA), sensorial analysis, peroxide value, thiobarbituric acid assay (TBA), total volatile basic nitrogen (TVB-N)

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Authors: Mustapha Abdulsalam, R. N. Ahmed

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Nauclea latifolia is one of the medicinal plants used in traditional Nigerian medicine in the treatment of various diseases such as fever, toothaches, malaria, diarrhea among several other conditions. Nauclea latifolia leaf extract acts as a capping and reducing agent in the formation of silver nanoparticles. Silver nanoparticles (AgNPs) were synthesized using a combination of aqueous extract of Nauclea latifolia and 1mM of silver nitrate (AgNO₃) solution to obtain concentrations of 100mg/ml-400mg/ml. Characterization of the particles was done by UV-Vis spectroscopy and Fourier transform infrared (FTIR). In this study, aqueous as well as ethanolic extract of leaf of Nauclea latifolia were investigated for antibacterial activity using the standard agar well diffusion technique against three clinical isolates (Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa). The Minimum Inhibitory Concentration (MIC) was achieved by microbroth dilution method and Minimum Bactericidal Concentration (MBC) was also determined by plate assay. Characterization by UV-visible spectrometry revealed peak absorbance of 0.463 at 450.0nm, while FTIR showed the presence of two functional groups. At 400mg/ml, the highest inhibitory activities were observed with S.aureus and E.coli with zones of inhibition measuring 20mm and 18mm respectively. The MIC was obtained at 400mg/ml while MBC was at a higher concentration. The data from this study indicate the potential of silver nanoparticle of Nauclea latifolia as a suitable alternative antibacterial agent for incorporation into orthodox medicine in health care delivery in Nigeria.

Keywords: agar well diffusion, antimicrobial activity, Nauclea latifolia, silver nanoparticles

Procedia PDF Downloads 188