Search results for: In vitro

Authors: Sihem Benmimoune, Abdelbaki Lemgharbi, Ahmed Ait Yahia, Abdelkrim Kameli

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Our study is based on chemical and phytochemical characterization of Artemisia absinthium L and in vitro tests to demonstrate the biological activities of essential oil and natural extract. A qualitative and quantitative comparison of the essential oil extracted by two extraction procedures was performed by analysis of CG/SM and the yield calculation. The method of hydrodistillation has a chemical composition and provides oil content than the best training water vapor. These oils are composed mainly of thujone followed chamazulene and ρ-cymene. The antimicrobial activity of wormwood oil was tested in vitro by two methods (agar diffusion and microdilution) on four plant pathogenic fungi (Aspergillus sp, Botrytis cinerea, Fusarium culmorum and Helminthosporium sp). The study of the antifungal effect showed that this oil has an inhibitory effect counterpart the microorganisms tested in particular the strain Botrytis cinerea. Otherwise, this activity depends on the nature of the oil and the germ itself. The antioxidant activity in vitro was studied with the DPPH method. The activity test shows that the oil and extract of Artemisia absinthium have a very low antioxidant capacity compared to the antioxidants used as a reference. The extract has a potentially high antiradical power not from its oil. The quantitative determinations of phenolic compounds by the Folin-Ciocalteu revealed that absinthe is low in total polyphenols and tannins.

Keywords: artemisia absinthium, biological activities, essential oil, extraction processes

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Authors: Marek Halenár, Eva Tvrdá, Tomáš Slanina, Ľubomír Ondruška, Eduard Kolesár, Peter Massányi, Adriana Kolesárová

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The present in vitro study was designed to reveal whether amygdalin (AMG) is able to cause changes to the motility, viability and mitochondrial activity of rabbit spermatozoa. New Zealand White rabbits (n = 10) aged four months were used in the study. Semen samples were collected from each animal and used for the in vitro incubation. The samples were divided into five equal parts and diluted with saline supplemented with 0, 0.5, 1, 2.5 and 5 mg/mL AMG. At times 0h, 3h and 5h spermatozoa motion parameters were assessed using the SpermVision™ computer-aided sperm analysis (CASA) system, cell viability was examined with the metabolic activity (MTT) assay, and the eosin-nigrosin staining technique was used to evaluate the viability of rabbit spermatozoa. All AMG concentrations exhibited stimulating effects on the spermatozoa activity, as shown by a significant preservation of the motility (P<0.05 with respect to 0.5 mg/mL and 1 mg/mL AMG; Time 5 h) and mitochondrial activity (P< 0.05 in case of 0.5 mg/mL AMG; P< 0.01 in case of 1 mg/mL AMG; P < 0.001 with respect to 2.5 mg/mL and 5 mg/mL AMG; Time 5 h). None of the AMG doses supplemented had any significant impact of the spermatozoa viability. In conclusion, the data revealed that short-term co-incubation of spermatozoa with AMG may result in a higher preservation of the sperm structural integrity and functional activity.

Keywords: amygdalin, CASA, mitochondrial activity, motility, rabbits, spermatozoa, viability

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Authors: D. H. Tejavathi

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Medicinal plants are globally valuable sources of herbal products. Wild populations of many medicinal plants are facing threat of extinction because of their narrow distribution, endemicity, and degradation of specific habitats. Micropropagation is an established in vitro technique by which large number of clones can be obtained from a small bit of explants in a short span of time within a limited space. Mycorrhization can minimize the transient transplantation shock, experienced by the micropropagated plants when they are transferred from lab to land. AM fungal association improves the physiological status of the host plants through better uptake of water and nutrients, particularly phosphorus. Consequently, the growth performance and biosynthesis of active principles are significantly enhanced in AM fungal treated plants. Bacopa monnieri, Andrographis paniculata, Agave vera-curz, Drymaria cordata and Majorana hortensis, important medicinal plants used in various indigenous systems of medicines, are selected for the present study. They form the main constituents of many herbal formulations. Standard in vitro techniques were followed to obtain the micropropagated plants. Shoot tips and nodal segments were used as explants. Explants were cultured on Murashige and Skoog, and Phillips and Collins media supplemented with various combinations of growth regulators. Multiple shoots were obtained on a media containing both auxins and cytokinins at various concentrations and combinations. Multiple shoots were then transferred to rooting media containing auxins for root induction. Thus, obtained in vitro regenerated plants were subjected to brief acclimatization before transferring them to land. One-month-old in vitro plants were treated with AM fungi, and the symbiotic effect on the overall growth parameters was analyzed. It was found that micropropagation coupled with mycorrhization has significant effect on the enhancement of biomass and biosynthesis of active principles in these selected medicinal plants. In vitro techniques coupled with mycorrhization have opened a possibility of obtaining better clones in respect of enhancement of biomass and biosynthesis of active principles. Beneficial effects of AM fungal association with medicinal plants are discussed.

Keywords: cultivation, medicinal plants, micropropagation, mycorrhization

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Authors: Khaled S. Al Salhen

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Aldehyde oxidase is molybdo-flavoenzyme involved in the oxidation of hundreds of endogenous and exogenous and N-heterocyclic compounds and environmental pollutants. Uncharged N-heterocyclic aromatic compounds such phenanthridine are commonly distributed pollutants in soil, air, sediments, surface water and groundwater, and in animal and plant tissues. Phenanthridine as uncharged N-heterocyclic aromatic compound was incubated with partially purified aldehyde oxidase from rainbow trout fish liver. Reversed-phase HLPC method was used to separate the oxidation products from phenanthridine and the metabolite was identified. The 6(5H)-phenanthridinone was identified the major metabolite by partially purified aldehyde oxidase from fish liver. Kinetic constant for the oxidation reactions were determined spectrophotometrically and showed that this substrate has a good affinity (Km = 78 ± 7.6 µM) for hepatic aldehyde oxidase, coupled with a relatively high oxidation rate (0.77± 0.03 nmol/min/mg protein). In addition, the kinetic parameters of hepatic fish aldehyde oxidase towards the phenanthridine substrate indicate that in vitro biotransformation by hepatic fish aldehyde oxidase will be a significant pathway. This study confirms that partially purified aldehyde oxidase from fish liver is indeed the enzyme responsible for the in vitro production 6(5H)-phenanthridinone metabolite as it is a major metabolite by mammalian aldehyde oxidase.

Keywords: aldehyde oxidase, fish, phenanthridine, specificity

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Authors: Qiuyue Peng, Vladimir Zachar

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The application of adipose-derived stromal/stem cells (ASCs) in regenerative medicine is gaining more awareness due to their advanced translational potential and abundant source preparations. However, ASC-based translation has been confounded by high subpopulation heterogeneity, causing ambiguity about its precise therapeutic value. Some phenotypes defined by a unique combination of positive and negative surface markers have been found beneficial to the required roles. Therefore, the immunophenotypic repertoires of cultured ASCs and temporal changes of distinct subsets were investigated in this study. ASCs from three donors undergoing cosmetic liposuction were cultured in standard culturing methods, and the co-expression patterns based on the combination of selected markers at passages 1, 4, and 8 were analyzed by multi-chromatic flow cytometry. The results showed that the level of heterogeneity of subpopulations of ASCs became lower by in vitro expansion. After a few passages, most of the CD166⁺/CD274⁺/CD271⁺ based subpopulations converged to CD166 single positive cells. Meanwhile, these CD29⁺CD201⁺ double-positive cells, in combination with CD36/Stro-1 expression or without, feathered only the major epitopes and maintained prevailing throughout the whole process. This study suggested that, upon in vitro expansion, the phenotype repertoire of ASCs redistributed and stabilized in a way that cells co-expressing exclusively the strong markers remained dominant. These preliminary findings provide a general overview of the distribution of heterogeneous subsets residents within human ASCs during expansion in vitro. It is a critical step to fully characterize ASCs before clinical application, although the biological effects of heterogeneous subpopulations still need to be clarified.

Keywords: adipose-derived stromal/stem cells, heterogeneity, immunophenotype, subpopulations

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Authors: M. Areekijseree, W. Pongsawat, M. Pumipaiboon, C. Thepsithar, S. Sengsai, T. Chuen-Im

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Morphological interaction of porcine cumulus-oocyte complexes (pCOCs) was investigated on in vitro condition using electron microscope (SEM and TEM). The totals of 1,923 oocytes were round in shape, surrounded by zona pellucida with layer of cumulus cells ranging between 59.29-202.14 µm in size. They were classified into intact-, multi-, partial cumulus cell layer oocyte, and completely denuded oocyte, at the percentage composition of 22.80% 32.70%, 18.60%, and 25.90 % respectively. The pCOCs classified as intact- and multi cumulus cell layer oocytes were further culturing at 37°C with 5% CO2, 95% air atmosphere and high humidity for 44 h in M199 with Earle’s salts supplemented with 10% HTFCS, 2.2 mg/mL NaHCO3, 1 M Hepes, 0.25 mM pyruvate, 15 µg/mL porcine follicle-stimulating hormone, 1 µg/mL LH, 1µg/mL estradiol with ethanol, and 50 µg/mL gentamycin sulfate. On electron microscope study, cumulus cells were found to stick their processes to secrete substance from the sac-shape end into zona pellucida of the oocyte and also communicated with the neighboring cells through their microvilli on the beginning of incubation period. It is believed that the cumulus cells communicate with the oocyte by inserting the microvilli through this gap and embedded in the oocyte cytoplasm before secreting substance, through the sac-shape end of the microvilli, to inhibit primary oocyte development at the prophase I. Morphological changes of the complexes were observed after culturing for 24-44 h. One hundred percentages of the cumulus layers were expanded and cumulus cells were peeling off from the oocyte surface. In addition, the round-shape cumulus cells transformed themselves into either an elongate shape or a columnar shape, and no communication between cumulus neighboring cells. After 44 h of incubation time, diameter of oocytes surrounded by cumulus cells was larger than 0 h incubation. The effect of hormones in culture medium is exerted by their receptors present in porcine oocyte. It is likely that all morphological changes of the complexes after hormone treatment were to allow maturation of the oocyte. This study demonstrated that the association of hormones in M199 could promote porcine follicle activation in 44 h in vitro condition. This culture system should be useful for studying the regulation of early follicular growth and development, especially because these follicles represent a large source of oocytes that could be used in vitro for cell technology.

Keywords: cumulus cells, electron microscopy, in vitro, porcine oocyte

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Authors: Abhishek Kumar Sah, Preeti K. Suresh

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Effective drug delivery to the eye is a massive challenge, due to complicated physiological ocular barriers, rapid washout by tear and nasolachrymal drainage. Thus, most of the conventional ophthalmic formulations face the problem of low ocular bioavailability. Ophthalmic drug therapy can be improved by enhancing the precorneal drug retention along with improved drug penetration. The aim of the present investigation was to develop and evaluate a biodegradable polymer poly (D, L-lactide-co-glycolide) (PLGA) coated nanoparticulate carrier of loteprednol etabonate. PLGA nanoparticles were prepared by modified emulsification/solvent diffusion method using high-speed homogenizer followed by sonication. The nanoparticles were characterized for various parameters such as particle size, zeta potential, polydispersity index, X-ray powder diffraction (XRD), Transmission electron microscopy (TEM), in vitro drug release profile and stability. The prepared nanocarriers displayed mean particle size in the range of 271.7 to 424.4 nm, with zeta potential less than –10 mV. In vitro release in simulated tear fluid (STF) nanocarrier showed an extended release profile of loteprednol etabonate. TEM confirmed the spherical morphology and smooth surface of the particles. All the prepared formulations were found to be stable at varying temperatures.

Keywords: drug delivery, ocular delivery, polymeric nanoparticles, loteprednol etabonate

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Authors: Dalia Jomehpour, Sara Sheikhlary, Esmaeil Heydari, Mohammad Hossien Majles Ara

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In this study, we report fulvic acid-coated iron oxide nanoparticles of 10.7 ± 2.7 nm size, which serve to inhibit amyloid fibrillation formation. Although the effect of fulvic acid on tau fibrils was investigated, to our best knowledge, its inhibitory impacts on amyloid aggregation formation have been assessed neither in-vitro nor in-vivo. On the other hand, iron oxide nanoparticles exhibit anti-amyloid activity on their own. This study investigates the inhibitory effect of fulvic acid coated iron oxide nanoparticles on amyloid aggregations formed from the commonly used in-vitro model, lysozyme from chicken egg white. FESEM, XRD, and FTIR characterization confirmed that fulvic acid was coated onto the surface of the nanoparticles. The inhibitory effects of the fulvic acid coated iron oxide nanoparticles were verified by Thioflavin T assay, circular dichroism (CD), and FESEM analysis. Furthermore, the toxicity of the nanoparticles on the neuroblastoma SH-SY5Y human cell line was assessed through an MTT assay. Our results indicate that fulvic acid coated iron oxide nanoparticles can efficiently inhibit the formation of amyloid aggregations while exhibiting negligible in-vitro toxicity; thus, they can be used as anti-amyloid agents in the development of the potential drug for neurodegenerative diseases.

Keywords: Alzheimer’s disease, fulvic acid coated iron oxide nanoparticles, fulvic acid, amyloid inhibitor, polyphenols

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Authors: Ira V. Stancheva, Ely G. Zayova, Maria P. Geneva, Marieta G. Hristozkova, Lyudmila I. Dimitrova, Maria I. Petrova

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The study describes a highly efficient and low-cost protocol for rapid and mass in vitro propagation of medicinal and aromatic plant species (Hyssopus officinalis L., Lamiaceae). Hyssop is an important aromatic herb used for its medicinal values because of its antioxidant, anti-inflammatory and antimicrobial properties. The protocol for large-scale multiplication of this aromatic plant was developed using young stem tips explants. The explants were sterilized with 0.04% mercuric chloride (HgCl₂) solution for 20 minutes and washing three times with sterile distilled water in 15 minutes. The cultural media was full and half strength Murashige and Skoog medium containing indole-3-butyric acid. Full and ½ Murashige and Skoog media without auxin were used as controls. For each variant 20 glass tubes with two plants were used. In each tube two tip and nodal explants were inoculated. Maximum shoot and root number were obtained on ½ Murashige and Skoog medium supplemented with 0.1 mg L-1 indole-3-butyric acid at the same time after four weeks of culture. The number of shoots per explant and shoot height were considered. The data on rooting percentage, the number of roots per plant and root length were collected after the same cultural period. The highest percentage of survival 85% for this medicinal plant was recorded in mixture of soil, sand and perlite (2:1:1 v/v/v). This mixture was most suitable for acclimatization of all propagated plants. Ex vitro acclimatization was carried out at 24±1 °C and 70% relative humidity under 16 h illuminations (50 μmol m⁻²s⁻¹). After adaptation period, the all plants were transferred to the field. The plants flowered within three months after transplantation. Phenotypic variations in the acclimatized plants were not observed. An average of 90% of the acclimatized plants survived after transferring into the field. All the in vitro propagated plants displayed normal development under the field conditions. Developed in vitro techniques could provide a promising alternative tool for large-scale propagation that increases the number of homologous plants for field cultivation. Acknowledgments: This study was conducted with financial support from National Science Fund at the Bulgarian Ministry of Education and Science, Project DN06/7 17.12.16.

Keywords: Hyssopus officinalis L., in vitro culture, micro propagation, acclimatization

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Authors: Mark Archei O. Javier

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The Fourth Industrial Revolution (FIR) is a major industrial era characterized by the fusion of technologies that is blurring the lines between the physical, digital, and biological spheres. Since this era teaches us how to thrive in the fast-paced developing world, it is important to be able to adapt. With this, there is a need to make learning and teaching in the bioscience laboratory more challenging and engaging. The goal of the research is to find out if using in silico and in vitro laboratory activities compared to the conventional conduct laboratory activities would have positive impacts on the academic performance of the learners. The potential contribution of the research is that it would improve the teachers’ methods in delivering the content to the students when it comes to topics that need laboratory activities. This study will develop a method by which teachers can provide learning materials to the students. A one-tailed t-Test for independent samples was used to determine the significant difference in the pre- and post-test scores of students. The tests of hypotheses were done at a 0.05 level of significance. Based on the results of the study, the gain scores of the experimental group are greater than the gain scores of the control group. This implies that using in silico and in vitro labs for the experimental group is more effective than the conventional method of doing laboratory activities.

Keywords: academic performance, general biology, in silico laboratory, in vivo laboratory, virtual laboratory

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Authors: Shayli Varasteh Moradi, Wayne A. Johnston, Dejan Gagoski, Kirill Alexandrov

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The demand for a rapid and more efficient technique to identify protein-protein interaction particularly in the areas of therapeutics and diagnostics development is growing. The method described here is a rapid in vitro protein-protein interaction analysis approach based on AlphaLISA technology combined with Leishmania tarentolae cell-free protein production (LTE) system. Cell-free protein synthesis allows the rapid production of recombinant proteins in a multiplexed format. Among available in vitro expression systems, LTE offers several advantages over other eukaryotic cell-free systems. It is based on a fast growing fermentable organism that is inexpensive in cultivation and lysate production. High integrity of proteins produced in this system and the ability to co-express multiple proteins makes it a desirable method for screening protein interactions. Following the translation of protein pairs in LTE system, the physical interaction between proteins of interests is analysed by AlphaLISA assay. The assay is performed using unpurified in vitro translation reaction and therefore can be readily multiplexed. This approach can be used in various research applications such as epitope mapping, antigen-antibody analysis and protein interaction network mapping. The intra-viral protein interaction network of Zika virus was studied using the developed technique. The viral proteins were co-expressed pair-wise in LTE and all possible interactions among viral proteins were tested using AlphaLISA. The assay resulted to the identification of 54 intra-viral protein-protein interactions from which 19 binary interactions were found to be novel. The presented technique provides a powerful tool for rapid analysis of protein-protein interaction with high sensitivity and throughput.

Keywords: AlphaLISA technology, cell-free protein expression, epitope mapping, Leishmania tarentolae, protein-protein interaction

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Authors: Vishnu Varthan Vaithiyalingamjagannathan, M. N. Sathishkumar, K. S. Lakhsmi, D. Satheeshkumar, Srividyaammayappanrajam

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Background:Naringenin, aglycone flavonoid possess certain activities like anti-oxidant, anti-estrogenic, anti-diabetic, cardioprotective, anti-obesity,anti-inflammatory, hepatoprotective and also have anti-cancer characteristics like carcinogenic inactivation, cell cycle arrest, anti-proliferation, apoptosis, anti-angiogenesis and enhances anti-oxidant activity. Methodology:The inhibitory effect of Naringenin in lung tumor progression estimated with adenocarcinoma (A549) cell lines (in vitro) and C57BL/6 mice injected with 5 X 106A549 cell lines (in vivo) in a tri-dose manner (Naringenin 100mg/kg,150mg/kg, and 200mg/kg) compared with standard chemotherapy drug cisplatin (7mg/kg). Results:The results of the present study revealed a dose-dependent activity in Naringenin and combination with cisplatin at a higher dose which showed decreased tumor progression in mice. In vitro studies carried out for estimation of cell survival and Nitric Oxide (NO) level, shows dose dependent action of Naringenin with IC50 value of 42µg/ml. In vivo studies were carried out in C57BL/6 mice. Naringenin satisfied the condition of an anti-cancer molecule with its characteristics in fragmentation assay, Zymography assay, anti-oxidant, and myeloperoxidase studies, than cisplatin which failed in anti-oxidant and myeloperoxidase effect. Both in vitro and in vivo establishes dose dependent decrease in NO levels. But whereas, Naringenin showed adverse results in Matrix Metalloproteinase (MMP) enzymatic levels with increase in dose levels. Conclusion:From the present study, Naringenin could suppress the lung tumor progression when given individually and also in combinatorial with standard chemotherapy drug.

Keywords: naringenin, in vitro, cell line, anticancer

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Authors: A. M. Plante, F. O’Halloran, A. L. McCarthy

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By 2050 it is projected that 2 billion of the global population will be more than 60 years old. Older adults have unique dietary requirements and aging is associated with physiological changes that affect appetite, sensory perception, metabolism, and digestion. Therefore, it is essential that foods recommended and designed for older adults promote healthy aging. To assess cheese as a functional food for the elderly, a range of commercial cheese products were selected and compared for their antioxidant properties. Cheese from various milk sources (bovine, goats, sheep) with different textures and fat content, including cheddar, feta, goats, brie, roquefort, halloumi, wensleydale and gouda, were initially digested with two different simulated in vitro gastrointestinal digestion (SGID) models. One SGID model represented a validated in vitro adult digestion system and the second model, an elderly SGID, was designed to consider the physiological changes associated with aging. The antioxidant potential of all cheese digestates was investigated using in vitro chemical-based antioxidant assays, (2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP) and total phenolic content (TPC)). All adult model digestates had high antioxidant activity across both DPPH ( > 70%) and FRAP ( > 700 µM Fe²⁺/kg.fw) assays. Following in vitro digestion using the elderly SGID model, full-fat red cheddar, low-fat white cheddar, roquefort, halloumi, wensleydale, and gouda digestates had significantly lower (p ≤ 0.05) DPPH radical scavenging properties compared to the adult model digestates. Full-fat white cheddar had higher DPPH radical scavenging activity following elderly SGID digestion compared to the adult model digestate, but the difference was not significant. All other cheese digestates from the elderly model were comparable to the digestates from the adult model in terms of radical scavenging activity. The FRAP of all elderly digestates were significantly lower (p ≤ 0.05) compared to the adult digestates. Goats cheese was significantly higher (p ≤ 0.05) in FRAP (718 µM Fe²/kg.fw) compared to all other digestates in the elderly model. TPC levels in the soft cheeses (feta, goats) and low-fat cheeses (red cheddar, white cheddar) were significantly lower (p ≤ 0.05) in the elderly digestates compared to the adult digestates. There was no significant difference in TPC levels, between the elderly and adult model for full-fat cheddar (red, white), roquefort, wensleydale, gouda, and brie digestates. Halloumi cheese was the only cheese that was significantly higher in TPC levels following elderly digestion compared to adult digestates. Low fat red cheddar had significantly higher (p ≤ 0.05) TPC levels compared to all other digestates for both adult and elderly digestive systems. Findings from this study demonstrate that aging has an impact on the bioactivity of cheese, as antioxidant activity and TPC levels were lower, following in vitro elderly digestion compared to the adult model. For older adults, soft cheese, particularly goats cheese, was associated with high radical scavenging and reducing power, while roquefort cheese had low antioxidant activity. Also, elderly digestates of halloumi and low-fat red cheddar were associated with high TPC levels. Cheese has potential as a functional food for the elderly, however, bioactivity can vary depending on the cheese matrix. Funding for this research was provided by the RISAM Scholarship Scheme, Cork Institute of Technology, Ireland.

Keywords: antioxidants, cheese, in-vitro digestion, older adults

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Authors: Sulaiman B. Ali Alharbi, Bassam H. Mashat, Naif Abdullah Al-Harbi, Milton Wainwright, Abeer S. Aloufi, Sulamain Alnaimat

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Bismuth salicylate was found to inhibit the growth of a range of bacteria and yeast, Candida albican. In general the growth of bacteria did not result in the increase in bismuth solubilisation, in contrast, bismuth solubilisation increased following the growth of C. albicans. A significant increase in the biomass (dry weight) of Aspergillus niger and Aspergillus oryzae occurred in vitro when these fungi were grown in the presence of bismuth salicylate. Biomass increase occurred over a range of bismuth compound additions, which in the case of A. oryzae was associated with the increase in the solubilisation of the insoluble bismuth compounds.

Keywords: bacterial inhibition, fungal growth stimulation, medical uses of bismuth, yeast inhibition

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Authors: Gulay Oncu Ince, Sibel Karakaya

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Food is a worldwide concern. Among the factors that have influences on human health, food, nutrition and life style have been regarded as the most important factors since they can be intervened. While some parts of the world has been faced with food shortages and hence, chronic metabolic diseases, the other part of the world have been emerged from over consumption of food. Both situations can result in shorter life expectancy and represent a major global health problem. Hunger, satiety and appetite sensation form a balance ensures the operation of feeding behavior between food intake and energy consumption. Satiety is one of the approaches that is effective in ensuring weight control and avoid eating more in the postprandial period. By manipulating the microstructure of food macro and micronutrient bioavailability may be increased or reduced. For the food industry appearance, texture, taste structural properties as well as the gastrointestinal tract behavior of the food after the consumption is becoming increasingly important. Also, this behavior has been the subject of several researches in recent years by the scientific community. Numerous studies have been published about changing the food matrix in order to increase expected impacts. In this study, yogurts were enriched with caseinomacropeptide (CMP), whey protein (WP), CMP and sodium alginate (SA), and WP + SA in order to produce goat yogurts having different food matrices. SDS Page profiles of the samples after in vitro digestion and viscosities of the stomach digesta at different share rates were determined. Energy values were 62.11kcal/100 g, 70.27 kcal/100 g, 70.61 kcal/100 g, 71.20 kcal/100 g and 71.67 kcal/100 g for control, CMP added WP added, WP + SA added, and CMP + SA added yogurts respectively. The results of viscosity analysis showed that control yogurt had the lowest viscosity value and this was followed by CMP added, WP added, CMP + SA added and WP + SA added yogurts, respectively. Protein contents of the stomach and duedonal digests of the samples after subjected to two different in vitro digestion methods were changed between 5.34-5.91 mg protein / g sample and 16.93-19.75 mg protein /g of sample, respectively. Viscosity measurements of the stomach digests showed that CMP + SA added yogurt displayed the highest viscosity value in both in vitro digestion methods. There were differences between the protein profiles of the stomach and duedonal digests obtained by two different in vitro digestion methods (p<0.05).

Keywords: caseinomacropeptide, protein profile, whey protein, yogurt

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Authors: Benjamin Bonsu Bruce, Marian Dorcas Quain David Appiah-Kubi, Gertrude Osei-Diko, Harrison Kwame Dapaah

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Taro [Colocasia esculenta (L.) Schott] is a major staple food and remains a significant crop to many cultural and agricultural customs worldwide. In Ghana, taro is mostly propagated using vegetative material, which is conserved in field collection and recycled from their farms to establish new fields. However, this practice promotes the accumulation of systemic pathogens. Prior exposure to pests and subsequent expression of disease symptoms can also be a huge constraint to sustainable conservation and utilization of taro genetic resources. In vitro, slow growth is one of the most promising techniques to be utilized for conservation. The objective of this study was to find a medium-term in vitro conservation protocol for local and exotic taro genotypes. The medium-term conservation study was conducted using actively growing shoots obtained from in vitro cultures. Explants were cultured to full strength in complete Murashige and Skoog medium supplemented with Mannitol at different concentrations (0g/l, 20g/l, 25g/l, and 30g/l). Another medium that was tested as an additional treatment is the White’s medium. The highest number of shoots (6.33) and leaves (22.67) occurred on medium containing 20 and 25g/l mannitol in genotype SAO 006 as compared to other genotypes, whereas 30g/l mannitol was the best to restrict growth for the entire 6 months period in terms of shoot height (22.50cm). The study reveals that mannitol supplemented culture media could reduce the growth of Colocasia plantlets, especially in stem height. Culture growth following 6 months of conservation, showed that healthy shoot cultures of Taro were obtained after 6 months of storage in a medium containing 20gl⁻¹ and 25gl⁻¹ mannitol.

Keywords: complete murashige, skoog medium, culture conditions, mannitol, slow growth conservation

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Authors: Akwu N. A., Naidoo Y., Salau V. F., Olofinsan K. A.

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Grewia lasiocarpa E. Mey. ex Harv. (Malvaceae) is a Southern African medicinal plant indigenously used with other plants for birthing problems. The anti-diabetic properties of the hexane, chloroform, and methanol extracts of Grewia lasiocarpa stem bark were assessed using in vitro α-glucosidase enzyme inhibition assay. The predictive in silico drug-likeness and toxicity properties of the phytocompounds were conducted using the pKCSM, ADMElab, and SwissADME computer-aided online tools. The highest α-glucosidase percentage inhibition was observed in the hexane extract (86.76%, IC50= 0.24 mg/mL), followed by chloroform (63.08%, IC50= 4.87 mg/mL) and methanol (53.22%, IC50= 9.41 mg/mL); while acarbose, the standard anti-diabetic drug was (84.54%, IC50= 1.96 mg/mL). The α-glucosidase assay revealed that the hexane extract exhibited the strongest carbohydrate inhibiting capacity and is a better inhibitor than the standard reference drug-acarbose. The computational studies also affirm the results observed in the in vitroα-glucosidaseassay. Thus, the extracts of G. lasiocarpa may be considered a potential plant-sourced compound for treating type 2 diabetes mellitus. This is the first study on the anti-diabetic properties of Grewia lasiocarpa hexane, chloroform, and methanol extracts using in vitro and in silico models.

Keywords: grewia lasiocarpa, α-glucosidase inhibition, anti-diabetes, ADMET

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Authors: Abobkar Saad, Qasmia Abdalla, Fatma Emhemed

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Eighty-four of Calligonum comosum were cultured on Murashige and Skoog medium on every combination supplemented with different concentrations of IAA, BA, Zeatin, and GA3. When 84 seeds were inoculated on MS free hormones, different types of cells contain dense cytoplasm were observed ater 23 days and long thick wall cells arranged in layers. In case of using MS +BA(0.5mg/L), different types and shapes of parenchyma cells contain dense cytoplasm were detected after four weeks. In the case of using MS + BA(1mg/L) + GA3 (3mg/L), thick wall parenchyma cells contain dense cytoplasm after 19 days, but many layers of parenchyma cells contain dense cytoplasm after 28 days. When MS +kin(0.5mg/L) a thick cells wall as Sclereids were observed after 29 days. No any response were observed on Zeatin (0.5, 1 mg/L).

Keywords: anatomy, Calligonum comosum, in vitro, aeeds

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Authors: Mengjie Qu, Yi Wang, Jiawei Ding, Siyu Chen, Yanan Di

Abstract:

Marine ecosystem is facing intensified multiple stresses caused by environmental contaminants from human activities. Xenobiotics, such as benzo(α)pyrene (BaP) have been discharged into marine environment and cause hazardous impacts on both marine organisms and human beings. As a filter-feeder, marine mussels, Mytilus spp., has been extensively used to monitor the marine environment. However, their genomic alterations induced by such xenobiotics are still kept unknown. In the present study, gills, as the first defense barrier in mussels, were selected to evaluate the genetic instability alterations induced by the exposure to BaP both in vivo and in vitro. Both random amplified polymorphic DNA (RAPD) assay and comet assay were applied as the rapid tools to assess the environmental stresses due to their low money- and time-consumption. All mussels were identified to be the single species of Mytilus coruscus before used in BaP exposure at the concentration of 56 μg/l for 1 & 3 days (in vivo exposure) or 1 & 3 hours (in vitro). Both RAPD and comet assay results were showed significantly increased genomic instability with time-specific altering pattern. After the recovery period in 'in vivo' exposure, the genomic status was as same as control condition. However, the relative higher genomic instabilities were still observed in gill cells after the recovery from in vitro exposure condition. Different repair mechanisms or signaling pathway might be involved in the isolated gill cells in the comparison with intact tissues. The study provides the robust and rapid techniques to exam the genomic stability in marine organisms in response to marine environmental changes and provide basic information for further mechanism research in stress responses in marine organisms.

Keywords: genotoxic impacts, in vivo/vitro exposure, marine mussels, RAPD and comet assay

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Authors: Vanessa Funk, Steven Blum, Stephanie Cole, Jorge Maciel, Matthew Lux

Abstract:

Paper-based synthetic gene circuits offer a new paradigm for programmable, fieldable biodetection. We demonstrate that by freeze-drying gene circuits with in vitro expression machinery, we can use complimentary RNA sequences to trigger colorimetric changes upon rehydration. We have successfully utilized both green fluorescent protein and luciferase-based reporters for easy visualization purposes in solution. Through several efforts, we are aiming to use this new platform technology to address a variety of needs in portable detection by demonstrating several more expression and reporter systems for detection functions on paper. In addition to RNA-based biodetection, we are exploring the use of various mechanisms that cells use to respond to environmental conditions to move towards all-hazards detection. Examples include explosives, heavy metals for water quality, and toxic chemicals.

Keywords: cell-free lysates, detection, gene circuits, in vitro

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Authors: Uftah Ali M. Shushni, Viola Stuppec, Ulrike Lindequist

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Because of the evolving resistance of microorganisms to existing antibiotics, there is an increasing need for new antibiotics not only in human but also in veterinary medicine. As part of our ongoing work on the secondary metabolites produced by marine fungi, the organic extract of the culture filtrate of an Ascomycetous fungus, which was found on driftwood collected from the coast of the Greifswalder Bodden, Baltic Sea, Germany displayed antimicrobial activity against some fish and human pathogenic bacteria. Bioactivity-guided column chromatographic separation led to the isolation of 6-Deoxybostrycoidin. The structure was determined from the interpretation of spectroscopic data (UV, MS, and NMR). 6-Deoxybostrycoidin exhibited in vitro activity against Bacillus subtilis, Staphylococcus aureus and Flexibacter maritimus with minimal inhibitory concentrations of 25, 12.5 and 12.5 μg/ml respectively.

Keywords: marine fungi, fish pathogenic bacteria, microorganism, medicine

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Authors: Zishan Ahmad, Anwar Shahzad

Abstract:

The present studies describe a protocol for high frequency in vitro propagation through nodal segments and shoot tips in D. arayalpathra, a critically endangered medicinal liana of the Western Ghats, India. Nodal segments were more responsive than shoot tips in terms of shoot multiplication. Murashige and Skoog’s (MS) basal medium supplemented with 2.5 µM 6-benzyladenine (BA) was optimum for shoot induction through both the explants. Among different combinations of plant growth regulator (PGRs) and growth additive screened, MS medium supplemented with BA (2.5 µM) + indole-3-acetic acid (IAA) (0.25 µM) + adenine sulphate (ADS) (10.0 µM) induced a maximum of 9.0 shoots per nodal segment and 3.9 shoots per shoot tip with mean shoot length of 8.5 and 3.9 cm respectively. Half-strength MS medium supplemented with Naphthaleneacetic acid (NAA) (2.5 µM) was the best for in vitro root induction. After successful acclimatization in SoilriteTM, 92 % plantlets were survived in field conditions. Acclimatized plantlets were studied for chlorophyll and carotenoid content, net photosynthetic rate (PN) and related attributes such as stomatal conductance (Gs) and transpiration rate during subsequent days of acclimatization. The rise and fall of different biochemical enzymes (SOD, CAT, APX and GR) were also studies during successful days of acclimatization. Moreover, the effect of acclimatization on the synthesis of 2-hydroxy-4-methoxy benzaldehyde (2H4MB) was also studied in relation to the biomass production. Maximum fresh weight (2.8 gm/plant), dry weight (0.35 gm/plant) of roots and 2H4MB content (8.5 µg/ ml of root extract) were recorded after 8 weeks of acclimatization. The screening of in vitro raised plantlet root was also carried out by using GC-MS analysis which witnessed more than 25 compounds. The regenerated plantlets were also screened for homogeneity by using RAPD and ISSR. The proposed protocol surely can be used for the conservation and commercial production of the plant.

Keywords: 6-benzyladenine, PGRs, RAPD, 2H4MB

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Authors: Rajesh A. Rane, Shital S. Naphade, Rajshekhar Karpoormath

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Thiozolidin-4-one, a mimic of thiazolobenzimidazole (TBZ) has drawn many attentions due to its potent and selective inhibition against the HIV-1 and low toxicity by binding to the allosteric site of the reverse transcriptase (RT) as a non-nucleoside RT inhibitor (NNRTI). Similarly, marine bromopyrrole alkaloids are well known for their diverse array of anti-infective properties. Hence, we have reported synthesis and in vitro HIV-1 RT inhibitory activity of a series of 4-thiazolidinone-bromopyrrole alkaloid hybrids tethered with amide linker. The results of in vitro HIV-1 RT kit assay showed that some of the compounds, such as 4c, 4d, and 4i could effectively inhibit RT activity. Among them, compounds 4c having 4-chlorophenyl substituted 4-thiazolidione ring was the best one with the IC50 value of 0.26 µM. The sturdy emerges with key structure-activity relationship that pyrrole-NH-free core benefited inhibition against HIV-1 RT inhibition. This study identified conjugate 4c with potent activity and selectivity as promising compound for further drug development to HIV.

Keywords: antiviral drugs, bromopyrrole alkaloids, HIV-1 RT inhibition, 4-thiazolidinone

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Authors: Alessandra N. Santos, Max P. Ferreira, Alexandra R. P. Silva, Agda A. R. de Oliveira, Marivalda M. Pereira

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The literature describes experiments, in which ceria nanoparticles in the bioactive glass significantly improve differentiation of stem cells into osteoblasts and increase production of collagen. It is not known whether this effect observed due to the presence of nanoceria can be also observed in the presence of cerium in the bioactive glass network. The effect of cerium into bioactive glasses using the sol–gel route is the focus of this work, with the goal to develop a material for tissue engineering with the potential to enhance osteogenesis. A bioactive glass composition based on 70% SiO2–30% CaO is produced with the addition of cerium. The analyses XRD, FTIR, SEM/EDS, BET/BJH, in vitro bioactivity test and the Cell viability assay were performed. The results show that cerium remains in the bioactive glass structure. The obtained material present in vitro bioactivity and promote the cell viability.

Keywords: bioactive glass, bioactivity, cerium salt, material characterization, sol-gel method

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Authors: P. S. Rajinikanth, Shobana Mariappan, Jestin Chellian

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The objective of the study was to prepare and characterize a water-in-oil nano emulsion of 5-Fluorouracil (5FU) to enhance the skin penetration. The present study describes a nano emulsion of 5FU using Capyrol PGMC, Transcutol HP and PEG 400 as oil, surfactant and co-surfactant, respectively. The optimized formulations were further evaluated for heating cooling cycle, centrifugation studies, freeze thaw cycling, particle size distribution and zeta potential in order to confirm the stability of the optimized nano emulsions. The in-vitro characterization results showed that the droplets of prepared formulation were ~100 nm with ± 15 zeta potential. In vitro skin permeation studies was conducted in albino mice skin. Significant increase in permeability parameters was also observed in nano emulsion formulations (P<0.05). The steady-state flux (Jss), enhancement ration and permeability coefficient (Kp) for optimized nano emulsion formulation (FU2, FU1, 1:1 S mix were found to be 24.21 ±2.45 μg/cm2/h, 3.28±0.87 & 19.52±1.87 cm/h, respectively), which were significant compared with conventional gel. The in vitro and in vivo skin deposition studies in rat indicated that the amount of drug deposited from the nano emulsion (292.45 µg/cm2) in skin was significant (P<0.05) an increased as compared to a conventional 5FU gel (121.42 µg/cm2). The skin irritation study using rat skin showed that the mean irritation index of the nano emulsion reduced significantly (P<0.05) as compared with conventional gel contain 1% 5FU. The results from this study suggest that a water-in-oil nano emulsion could be safely used to promote skin penetration of 5FU following topical application.

Keywords: nano emulsion, controlled release, 5 fluorouracil, skin penetration, skin irritation

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Authors: Rudra Vaghela, P. K. Kulkarni

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The aim of the present research was to develop oral Amphotericin B (AmB) nanocrystals (Nc) grafted with suitable ligand in order to enhance drug transport across the intestinal epithelial barrier and subsequently, active uptake by macrophages. AmB Nc were prepared by liquid anti-solvent precipitation technique (LAS). Poloxamer 188 was used to stabilize the prepared AmB Nc and grafted with mannose for actively targeting M cells in Peyer’s patches. To prevent shedding of the stabilizer and ligand, N,N’-Dicyclohexylcarbodiimide (DCC) was used as a cross-linker. The prepared AmB Nc were characterized for particle size, PDI, zeta potential, X-ray diffraction (XRD) and surface morphology using scanning electron microscope (SEM) and evaluated for drug content, in vitro drug release and cell uptake studies using caco-2 cells. The particle size of stabilized AmB Nc grafted with WGA was in the range of 287-417 nm with negative zeta potential between -18 to -25 mV. XRD studies revealed crystalline nature of AmB Nc. SEM studies revealed that ungrafted AmB Nc were irregular in shape with rough surface whereas, grafted AmB Nc were found to be rod-shaped with smooth surface. In vitro drug release of AmB Nc was found to be 86% at the end of one hour. Cellular studies revealed higher invasion and uptake of AmB Nc towards caco-2 cell membrane when compared to ungrafted AmB Nc. Our findings emphasize scope on developing oral delivery system for passively targeting M cells in Peyer’s patches.

Keywords: leishmaniasis, amphotericin b nanocrystals, macrophage targeting, LAS technique

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Authors: Michael Graz, Natasha Hurril, Andrew Shearer

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Research on feed supplementation with natural compounds is currently being intensively pursued with a view to improving energy utilisation in ruminants and mitigating the production of methane by these animals. Towards this end, a novel combination of extracts from garlic and bitter orange was therefore selected for trials on the basis of their previously published in vitro anti-methanogenic potential. Three separate in vitro experiments were conducted to determine energy utilisation and greenhouse gas production. These included use of rumen fluid from fistulated cows and sheep in batch culture, the Hohenheim gas test, and the Rusitec technique. Experimental and control arms were utilised, with 5g extracts per kilogram of total dietary dry matter (0.05g/kg active compounds) being used to supplement or not supplement the in vitro systems. Respiratory measurements were conducted on experimental day 1 for the batch culture and Hohenheim gas test and on day 14-21 for the Rusitec Technique (in a 21-day trial). Measurements included methane (CH4) production, total volatile fatty acid (VFA) concentration, molar proportions of acetate, propionate and butyrate and degradation of organic matter (Rusitec). CH4 production was reduced by 82% (±16%), 68% (±11%) and 37% (±4%) in the batch culture, Hohenheim gas test and Rusitec, respectively. Total VFA production was reduced by 13% (±2%) and 2% (±0.1%) in the batch culture and Hohenheim gas test whilst it was increased by 8% (±2%) in the Rusitec. Total VFA production was reduced in all tests between 2 and 10%, whilst acetate production was reduced between 10% and 29%. Propionate production which is an indicator of weight gain was increased in all cases between 16% and 30%. Butyrate production which is considered an indicator of potential milk yield was increased by between 6 and 11%. Degradation of organic matter in the Rusitec experiments was improved by 10% (±0.1%). In conclusion, the study demonstrated the potential of the combination of garlic and citrus extracts to improve digestion, enhance body energy retention and limit CH4 formation in relation to feed intake.

Keywords: citrus, garlic, methane, ruminants

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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: Ilzira A. Minigalieva, Tatiana V. Bushueva, Eleonore Frohlich, Vladimir Panov, Ekaterina Shishkina, Boris A. Katsnelson

Abstract:

Background: The overwhelming majority of the experimental studies in the field of metal nanotoxicology have been performed on cultures of established cell lines, with very few researchers focusing on animal experiments, while a juxtaposition of conclusions inferred from these two types of research is blatantly lacking. The least studied aspect of this problem relates to characterizing and predicting the combined toxicity of metallic nanoparticles. Methods: Comparative and combined toxic effects of purposefully prepared spherical NiO and Mn₃O₄ nanoparticles (mean diameters 16.7 ± 8.2 nm and 18.4 ± 5.4 nm respectively) were estimated on cultures of human cell lines: MRC-5 fibroblasts, THP-1 monocytes, SY-SY5Y neuroblastoma cells, as well as on the latter two lines differentiated to macrophages and neurons, respectively. The combined cytotoxicity was mathematically modeled using the response surface methodology. Results: The comparative assessment of the studied NPs unspecific toxicity previously obtained in vivo was satisfactorily reproduced by the present in vitro tests. However, with respect to manganese-specific brain damage which had been demonstrated by us in animal experiment with the same NPs, the testing on neuronall cell culture showed only a certain enhancing effect of Mn₃O₄-NPs on the toxic action of NiO-NPs, while the role of the latter prevailed. Conclusion: From the point of view of the preventive toxicology, the experimental modeling of metallic NPs combined toxicity on cell cultures can give non-reliable predictions of the in vivo action’s effects.

Keywords: manganese oxide, nickel oxide, nanoparticles, in vitro toxicity

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Authors: Mala Nath, Nagamani Kompelli, Partha Roy, Snehasish Das

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Two new metal-based anticancer chemotherapeutic agents, [(Ph2Sn)2(HGuO)2(phen)Cl2] 1 and [(Ph3Sn)(HGuO)(phen)]- Cl.CH3OH.H2O 2, were designed, prepared and characterized by analytical and spectral (IR, ESI-Mass, 1H, 13C and 119Sn NMR) techniques. The proposed geometry of Sn(IV) in 1 and 2 is distorted octahedral and distorted trigonal-bipyramidal, respectively. Both 1 and 2 exhibit potential cytotoxicity in vitro against MCF-7, HepG-2 and DU-145 cell lines. The intrinsic binding constant (Kb) values of 1 (2.33 × 105 M-1) and 2 (2.46 × 105 M-1) evaluated from UV-Visible absorption studies suggest non-classical electrostatic mode of interaction via phosphate backbone of DNA double helix. The Stern-Volmer quenching constant (Ksv) of 1 (9.74 × 105 M-1) and 2 (2.9 × 106 M-1) determined by fluorescence studies suggests the groove binding and intercalation mode for 1 and 2, respectively. Effective cleavage of pBR322 DNA is induced by 1. Their interaction with DNA of cancer cells may account for potency.

Keywords: anticancer agents, DNA binding studies, NMR spectroscopy, organotin

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