Search results for: in vitro toxicity

Authors: Chiao-Cheng Huang, Pei-Te Chiueh, Ya-Hsuan Liou

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Chromium-contaminated wastewater from electroplating industrial activity has been a long-standing environmental issue, as it can degrade surface water quality and is harmful to soil ecosystems. The traditional method of treating chromium-contaminated wastewater has been to use chemical coagulation processes. However, this method consumes large amounts of chemicals such as sulfuric acid, sodium hydroxide, and sodium bicarbonate in order to remove chromium. However, a series of new methods for treating chromium-containing wastewater have been developed. This study aimed to compare the environmental impact of four different lab scale chromium removal processes: 1.) chemical coagulation process (the most common and traditional method), in which sodium metabisulfite was used as reductant, 2.) electrochemical process using two steel sheets as electrodes, 3.) reduction by iron-copper bimetallic powder, and 4.) photocatalysis process by TiO2. Each process was run in the lab, and was able to achieve 100% removal of chromium in solution. Then a Life Cycle Assessment (LCA) study was conducted based on the experimental data obtained from four different case studies to identify the environmentally preferable alternative to treat chromium wastewater. The model used for calculating the environmental impact was TRACi, and the system scope includes the production phase and use phase of chemicals and electricity consumed by the chromium removal processes, as well as the final disposal of chromium containing sludge. The functional unit chosen in this study was the removal of 1 mg of chromium. Solution volume of each case study was adjusted to 1 L in advance and the chemicals and energy consumed were proportionally adjusted. The emissions and resources consumed were identified and characterized into 15 categories of midpoint impacts. The impact assessment results show that the human ecotoxicity category accounts for 55 % of environmental impact in Case 1, which can be attributed to the sulfuric acid used for pH adjustment. In Case 2, production of steel sheet electrodes is an energy-intensive process, thus contributed to 20 % of environmental impact. In Case 3, sodium bicarbonate is used as an anti-corrosion additive, which results mainly in 1.02E-05 Comparative Toxicity Unit (CTU) in the human toxicity category and 0.54E-05 (CTU) in acidification of air. In Case 4, electricity consumption for power supply of UV lamp gives 5.25E-05 (CTU) in human toxicity category, 1.15E-05 (kg Neq) in eutrophication. In conclusion, Case 3 and Case 4 have higher environmental impacts than Case 1 and Case 2, which can be attributed mostly to higher energy and chemical consumption, leading to high impacts in the global warming and ecotoxicity categories.

Keywords: chromium, lab scale, life cycle assessment, wastewater

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Authors: A. Schander, T. Tessmann, H. Stemmann, S. Strokov, A. Kreiter, W. Lang

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For the chronic application of neural prostheses and other brain-computer interfaces, long-term stable microelectrodes for electrical stimulation are essential. In recent years many developments were done to investigate different appropriate materials for these electrodes. One of these materials is the electrical conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT), which has lower impedance and higher charge injection capacity compared to noble metals like gold and platinum. However the long-term stability of this polymer is still unclear. Thus this paper reports on the in-vitro evaluation of the long-term stability of PEDOT coated gold microelectrodes. For this purpose a highly flexible electrocorticography (ECoG) electrode array, based on the polymer polyimide, is used. This array consists of circular gold electrodes with a diameter of 560 µm (0.25 mm2). In total 25 electrodes of this array were coated simultaneously with the polymer PEDOT:PSS in a cleanroom environment using a galvanostatic electropolymerization process. After the coating the array is additionally sterilized using a steam sterilization process (121°C, 1 bar, 20.5 min) to simulate autoclaving prior to the implantation of such an electrode array. The long-term measurements were performed in phosphate-buffered saline solution (PBS, pH 7.4) at the constant body temperature of 37°C. For the in-vitro electrical stimulation a one channel bipolar current stimulator is used. The stimulation protocol consists of a bipolar current amplitude of 5 mA (cathodal phase first), a pulse duration of 100 µs per phase, a pulse pause of 50 µs and a frequency of 1 kHz. A PEDOT:PSS coated gold electrode with an area of 1 cm2 serves as the counter electrode. The electrical stimulation is performed continuously with a total amount of 86.4 million bipolar current pulses per day. The condition of the PEDOT coated electrodes is monitored in between with electrical impedance spectroscopy measurements. The results of this study demonstrate that the PEDOT coated electrodes are stable for more than 3.6 billion bipolar current pulses. Also the unstimulated electrodes show currently no degradation after the time period of 5 months. These results indicate an appropriate long-term stability of this electrode coating for chronic recording and electrical stimulation. The long-term measurements are still continuing to investigate the life limit of this electrode coating.

Keywords: chronic recording, electrical stimulation, long-term stability, microelectrodes, PEDOT

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Authors: Tella Toluwani, Adegbegi Ademuyiwa, Musei Chiedu, Adekunle Odola, Ayangbenro Ayansina, Adaramoye Oluwatosin

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Dithiocarbamates are very useful biological agents with antioxidant properties. Diclofenac (DIC) is a non-steroidal analgesic, anti-inflammatory, and antipyretic agent. The use of diclofenac has been linked with reproductive toxicity/damage. The purpose of this study is (i) To investigate the therapeutic potential of diisopropyldithiocarbamate sodium salt (Na(i-Pr₂dtc)) and vitamin E (VIT E) against diclofenac induced toxicity in the testes of male Wistar rats. (ii) To investigate the effect of (Na(i-Pr₂dtc)) and vitamin E on ameliorating damage done to the testes through histological analysis of the testes. Thirty-six (36) male Wistar rats were used for the experiment, they were divided into six (6) groups, the animals in group 1 served as control, animals in groups 2, 3, 4, 5 and 6 received DIC only, DIC and (Na(i-Pr₂dtc)), DIC and VIT E, (Na(i-Pr₂dtc) only and VIT E only respectively. A single dose of 100 mg/kg body weight of DIC was administered to male Wistar rats, while 30 mg/kg body weight of (Na(i-Pr₂dtc)) was used to treat both normal and DIC treated animals, control animals were treated with the vehicle, after 24 hrs of treatment the animals were euthanized and the testes were removed for analysis. The treatment of rats with Na(i-Pr₂dtc) significantly restored catalase (CAT) activity depressed by diclofenac. (Na(i-Pr₂dtc)) also restored glutathione levels reduced by DIC treatment and this was also accompanied by reduced lipid peroxidation (LPO) level. VIT E significantly restored superoxide dismutase (SOD) activity when compared with DIC only treated animals. Photomicrographs of testes from (Na(i-Pr₂dtc)) treated rats showed seminiferous epithelium with no lesions. We conclude that (Na(i-Pr₂dtc)) has an antioxidant effect, which might be related to the dose and duration of administration.

Keywords: diisopropyldithiocarbamate sodium salt, diclofenac, vitamin E, testes

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Authors: Elena Maria Scalisi, Roberta Pecoraro, Martina Contino, Sara Ignoto, Carmelo Iaria, Santi Concetto Pavone, Gino Sorbello, Loreto Di Donato, Maria Violetta Brundo

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5G Technology is evolving to satisfy a variety of service requirements that may allow high data-rate connections (1Gbps) and lower latency times than current (<1ms). In order to support a high data transmission speed and a high traffic service for eMBB (enhanced mobile broadband) use cases, 5G systems have the characteristic of using different frequency bands of the radio wave spectrum (700 MHz, 3.6-3.8 GHz and 26.5-27.5 GHz), thus taking advantage of higher frequencies than previous mobile radio generations (1G-4G). However, waves at higher frequencies have a lower capacity to propagate in free space and therefore, in order to guarantee the capillary coverage of the territory for high reliability applications, it will be necessary to install a large number of repeaters. Following the introduction of this new technology, there has been growing concern over the past few months about possible harmful effects on human health. The aim of this preliminary study is to evaluate possible short term effects induced by 5G-millimeter waves on embryonic development and early life stages of Danio rerio by Z-FET. We exposed developing zebrafish at frequency of 27 GHz, with a standard pyramidal horn antenna placed at 15 cm far from the samples holder ensuring an incident power density of 10 mW/cm2. During the exposure cycle, from 6 h post fertilization (hpf) to 96 hpf, we measured a different morphological endpoints every 24 hours. Zebrafish embryo toxicity test (Z-FET) is a short term test, carried out on fertilized eggs of zebrafish and it represents an effective alternative to acute test with adult fish (OECD, 2013). We have observed that 5G did not reveal significant impacts on mortality nor on morphology because exposed larvae showed a normal detachment of the tail, presence of heartbeat, well-organized somites, therefore hatching rate was lower than untreated larvae even at 48 h of exposure. Moreover, the immunohistochemical analysis performed on larvae showed a negativity to the HSP-70 expression used as a biomarkers. This is a preliminary study on evaluation of potential toxicity induced by 5G and it seems appropriate to underline the importance that further studies would take, aimed at clarifying the probable real risk of exposure to electromagnetic fields.

Keywords: Biomarker of exposure, embryonic development, 5G waves, zebrafish embryo toxicity test

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Authors: Renata Rank Miranda, Arandi Ginane Bezerra, Ciro Alberto Oliveira Ribeiro, Marco AntôNio Ferreira Randi, Carmen Lúcia Voigt, Lilian Skytte, Kaare Lund Rasmussen, Francisco Filipak Neto, Frank Kjeldsen

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Synergetic and antagonistic effects of drugs are well-known concerns in pharmacological assessments of dose and toxicity. Similar approach should be used in assessing cellular uptake and cytotoxicity of nanoparticles. Since nanoparticles are released into the aquatic environment they may interact with existing xenobiotics. Here we used biochemical assays and quantitative proteomics to assess the cytotoxicity of silver nanoparticles (AgNP) when human hepatoma HepG2 cells were co-exposed to 2 nm AgNP together with either Cd2+ or Hg2+ ions. Time-course experiments (2h, 4h, and 24h) were conducted to assess the first response to the exposure studies. The general trend was that a synergetic toxicological response was observed in cells exposed to both AgNP and Cd2+ or Hg2+, with AgNP and Cd2+ being more toxic. This was observed by a significant increase in the ROS and superoxide level of >35% in the case of AgNP+Cd2+ compared to the sum of responses of AgNP and Cd2+, individually. Metabolic activity and viability also dropped more for AgNP+Cd2+ (>10%) than for AgNP and Cd2+ combined. We used inductively coupled plasma mass spectrometry to investigate if AgNP facilitates larger influx of toxic metal ions into HepG2 cells. Only Hg2+ ions was found to be more efficiently engulfed as the concentration of Hg2+ was found 2.8 times larger compared to exposure experiments with only Hg2+. This effect was not observed for Cd2+. We now continue with deep proteomics studies to obtain wider details on the mechanism of the toxicity related to AgNP, Cd2+, and AgNP+Cd2+, respectively.

Keywords: nanotoxicology, silver nanoparticles, proteomics, human cell line

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Authors: M. Youngsabanant-Areekijseree, C. Thepsithar, K. Sribuddhachart, J. Tananantayot

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Porcine oviductal fluid (pOF) from oviduct, an unused organ from the slaughterhouse, was effectively used for biotechnology studies. The fluid components consisted of micro- and macro-nutrients, amino acids, carbon source and proteins that played important roles in animal cell and embryo development. This was our knowledge on investigating pOF as growth promoting substance in culture medium of an orchid, Dendrobium mirbelianum. Two-leaf shoots were cultured in liquid Vacin and Went (VW) medium as a standard medium supplemented with 2 g/L peptone (Pe) or 100 g/ L boiled-potato water (Po) alone or in combinations, and added with 0, 1, 3 or 5 ml/L pOF. All explants were cultured in a stationary condition for 8 weeks. It was found that medium added with 100 g/L Po and 1 ml/L pOF provided the best results (1.02 g fresh weight, 4.2 shoots, 0.53 cm shoot height, 4.4 protocorms, 11.0 leaves and 5.7 roots with 100% survival) when compared to other medium, but not statistically significant difference from medium added with 100 g/L Po (0.86 g fresh weight, 4.3 shoots, 0.51 cm shoot height, 4.6 protocorms, 12.4 leaves and 6.6 roots with 100% survival). However, VW medium supplemented with 1 or 3 ml/L pOF alone showed the higher percentage of survival (100%) than VW medium (86.67%). It was shown the potential role of pOF as an organic supplement for promoting growth of plants. Acknowledgements—The project was funded by a grant from Silpakorn University Research & Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: Dendrobium mirbelianum, pig, oviductal fluid, in vitro growth

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Authors: Shagufta Gulraiz, Abu Saeed Hashmi, Muhammad Mohsin Javed

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Lysine required for poultry feed is imported in Pakistan to fulfil the desired dietary needs. Present study was designed to produce maximum lysine by utilizing cheap sources to save the foreign exchange. To achieve the goal of lysine production through fermentation, large scale production of lysine was carried out in 7.5 L stirred glass vessel fermenter with wild and mutant Brevibacterium flavum (B. flavum) using all pre-optimized conditions. The identification of produced lysine was carried out by TLC and amino acid analyzer. Toxicity evaluation of produced lysine was performed before feeding to broiler chicks. During biological trial concentrated fermented broth having 8% lysine was used in poultry rations as a source of Lysine for test birds. Fermenter scale studies showed that the maximum lysine (20.8 g/L) was produced at 250 rpm, 1.5 vvm aeration, 6.0% inoculum under controlled pH conditions after 56 h of fermentation with wild culture but mutant (BFENU2) gave maximum yield of lysine 36.3 g/L under optimized condition after 48 h. Amino acid profiling showed 1.826% Lysine in fermented broth by wild B. flavum and 2.644% by mutant strain (BFENU2). Toxicity evaluation report showed that the produced lysine is safe for consumption by broilers. Biological evaluation results showed that produced lysine was equally good as commercial lysine in terms of weight gain, feed intake and feed conversion ratio. A cheap and practical bioprocess of Lysine production was concluded, that can be exploited commercially in Pakistan to save foreign exchange.

Keywords: lysine, fermentation, broiler chicks, biological evaluation

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Authors: Venkat Garigapati

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Cell-based therapies are limited due to underlying host immune system activity. Microencapsulation of living cells to overcome this issue has some serious drawbacks, such as limitations of nutrient and oxygen diffusion, which pose a threat to the function and longevity of cells. The conformal coating could overcome the issues which are generally involved in traditional microencapsulation. Some of the theoretical advantages of conformal coating include superior nutrient and oxygen supply to cells, prolonged lifespan, improved drug-secreting cell functionality and an opportunity to load high cell doses in small volumes. Despite several advantages to the conformal coating, there are no suitable methods available to apply to living cells. The ultra-thin conformal coating was achieved utilizing click-reactive methacryloyloxyethyl phosphorylcholine (MPC) polymers, which are capable of specifically reacting one polymer to another at neutral pH in the aqueous isotonic system at the desired temperature suitable for living cells without the need of deleterious initiators. ARPE-19 (Adult Retinal Pigment Epithelial cell line-19) cell-spheroids and rat pancreatic islets were used in the formulation studies. The in vitro studies of coated ARPE-19 cell-spheroids and rat islets indicate that the coat was intact; cells were viable and functioning. The in vitro study results revealed that the conformal coating technology seems promising and in vivo studies are being planned.

Keywords: cells, hydrogel, conformal coating, microencapsulation, insulin

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Authors: A. Goma Amira, U. E. Mahrous

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Measuring the effect of perinatal lead exposure on learning ability of offspring is considered as a sensitive and selective index for providing an early marker for central nervous system damage produced by this toxic metal. A total of 35 Sprague-Dawley adult rats were used to investigate the effect of lead acetate toxicity on behavioral patterns of adult female rats and learning ability of offspring. Rats were allotted into 4 groups, group one received 1g/l lead acetate (n=10), group two received 1.5g/l lead acetate (n=10), group three received 2g/l lead acetate in drinking water (n=10), and control group did not receive lead acetate (n=5) from 8th day of pregnancy till weaning of pups. The obtained results revealed a dose-dependent increase in the feeding time, drinking frequency, licking frequency, scratching frequency, licking litters, nest building, and retrieving frequencies, while standing time increased significantly in rats treated with 1.5g/l lead acetate than other treated groups and control. On the contrary, lying time decreased gradually in a dose-dependent manner. Moreover, movement activities were higher in rats treated with 1g/l lead acetate than other treated groups and control. Furthermore, time spent in closed arms was significantly lower in rats given 2g/l lead acetate than other treated groups, while they spent significantly much time spent in open arms than other treated groups which could be attributed to occurrence of adaptation. Furthermore, number of entries in open arms was-dose dependent. However, the ratio between open/closed arms revealed a significant decrease in rats treated with 2g/l lead acetate than the control group.

Keywords: lead toxicity, rats, learning ability, behavior

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Authors: Navid Mosallaei, Mahmoud Reza Jaafari, Mohammad Yahya Hanafi-Bojd, Shiva Golmohammadzadeh, Bizhan Malaekeh-Nikouei

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Background: Docetaxel (DTX), a potent anticancer drug derived from the European yew tree, is effective against various human cancers by inhibiting microtubule depolymerization. Solid lipid nanoparticles (SLNs) have gained attention as drug carriers for enhancing drug effectiveness and safety. SLNs, submicron-sized lipid-based particles, can passively target tumors through the "enhanced permeability and retention" (EPR) effect, providing stability, drug protection, and controlled release while being biocompatible. Methods: The SLN formulation included biodegradable lipids (Compritol and Precirol), hydrogenated soy phosphatidylcholine (H-SPC) as a lipophilic co-surfactant, and Poloxamer 188 as a non-ionic polymeric stabilizer. Two SLN preparation techniques, probe sonication and microemulsion, were assessed. Characterization encompassed SLNs' morphology, particle size, zeta potential, matrix, and encapsulation efficacy. In-vitro cytotoxicity and cellular uptake studies were conducted using mouse colorectal (C-26) and human malignant melanoma (A-375) cell lines, comparing SLN-DTX with Taxotere®. In-vivo studies evaluated tumor inhibitory efficacy and survival in mice with colorectal (C-26) tumors, comparing SLNDTX withTaxotere®. Results: SLN-DTX demonstrated stability, with an average size of 180 nm and a low polydispersity index (PDI) of 0.2 and encapsulation efficacy of 98.0 ± 0.1%. Differential scanning calorimetry (DSC) suggested amorphous encapsulation of DTX within SLNs. In vitro studies revealed that SLN-DTX exhibited nearly equivalent cytotoxicity to Taxotere®, depending on concentration and exposure time. Cellular uptake studies demonstrated superior intracellular DTX accumulation with SLN-DTX. In a C-26 mouse model, SLN-DTX at 10 mg/kg outperformed Taxotere® at 10 and 20 mg/kg, with no significant differences in body weight changes and a remarkably high survival rate of 60%. Conclusion: This study concludes that SLN-DTX, prepared using the probe sonication, offers stability and enhanced therapeutic effects. It displayed almost same in vitro cytotoxicity to Taxotere® but showed superior cellular uptake. In a mouse model, SLN-DTX effectively inhibited tumor growth, with 10 mg/kg outperforming even 20 mg/kg of Taxotere®, without adverse body weight changes and with higher survival rates. This suggests that SLN-DTX has the potential to reduce adverse effects while maintaining or enhancing docetaxel's therapeutic profile, making it a promising drug delivery strategy suitable for industrialization.

Keywords: docetaxel, Taxotere®, solid lipid nanoparticles, enhanced permeability and retention effect, drug delivery, cancer chemotherapy, cytotoxicity, cellular uptake, tumor inhibition

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Authors: Mansi Patel, Priti Mehta

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Background: Ionizing radiations have detrimental effects on humans, and the growing technological encroachment has increased human exposure to it enormously. So, the safety issues have emphasized researchers to develop radioprotector from natural resources having minimal toxicity. A substance having anti-inflammatory, antioxidant, and immunomodulatory activity can be a potential candidate for radioprotection. One such plant with immense potential i.e. Bamboo was selected for the present study. Purpose: The study aims to evaluate the potential of Indian bamboo leaves for protection against the clastogenic effect of gamma radiation. Methods: The protective effect of bamboo leaf extract against gamma radiation-induced genetic damage in human peripheral blood lymphocytes (HPBLs) was evaluated in vitro using Cytokinesis blocked micronuclei assay (CBMN). The blood samples were pretreated with varying concentration of extract 30 min before the radiation exposure (4Gy & 6Gy). The reduction in the frequency of micronuclei was observed for the irradiated and control groups. The effect of various concentration of bamboo leaf extract (400,600,800 mg/kg) on the development of radiation induced sickness and altered mortality in mice exposed to 8 Gy of whole-body gamma radiation was studied. The developed symptoms were clinically scored by multiple endpoints for 30 days. Results: Treatment of HPBLs with varying concentration of extract before exposure to a different dose of γ- radiation resulted in significant (P < 0.0001) decline of radiation induced micronuclei. It showed dose dependent and concentration driven activity. The maximum protection ~ 70% was achieved at nine µg/ml concentration. Extract treated whole body irradiated mice showed 50%, 83.3% and 100% survival for 400, 600, and 800mg/kg with 1.05, 0.43 and 0 clinical score respectively when compared to Irradiated mice having 6.03 clinical score and 0% survival. Conclusion: Our findings indicate bamboo leaf extract reduced the radiation induced cytogenetic damage. It has also increased the survival ratio and reduced the radiation induced sickness and mortality when exposed to a lethal dose of gamma radiation.

Keywords: bamboo leaf extract, Cytokinesis blocked micronuclei (CBMN) assay, ionizing radiation, radio protector

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Authors: Šárka Hradilová, Aleš Panáček, Radek Zbořil

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Nanotechnologies are considered the most promising fields with high added value, brings new possibilities in various sectors from industry to medicine. With the growing of interest in nanomaterials and their applications, increasing nanoparticle production leads to increased exposure of people and environment with ‘human made’ nanoparticles. Nanoparticles (NPs) are clusters of atoms in the size range of 1–100 nm. Metal nanoparticles represent one of the most important and frequently used types of NPs due to their unique physical, chemical and biological properties, which significantly differ from those of bulk material. Biological properties including toxicity of metal nanoparticles are generally determined by their size, size distribution, shape, surface area, surface charge, surface chemistry, stability in the environment and ability to release metal ions. Therefore, the biological behavior of NPs and their possible adverse effect cannot be derived from the bulk form of material because nanoparticles show unique properties and interactions with biological systems just due to their nanodimensions. Silver and gold NPs are intensively studied and used. Both can be used for instance in surface enhanced Raman spectroscopy, a considerable number of applications of silver NPs is associated with antibacterial effects, while gold NPs are associated with cancer treatment and bio imaging. Antibacterial effects of silver ions are known for centuries. Silver ions and silver-based compounds are highly toxic to microorganisms. Toxic properties of silver NPs are intensively studied, but the mechanism of cytoxicity is not fully understood. While silver NPs are considered toxic, gold NPs are referred to as toxic but also innocuous for eukaryotic cells. Therefore, gold NPs are used in various biological applications without a risk of cell damaging, even when we want to suppress the growth of cancer cells. Thus, gold NPs are toxic or harmless. Because most studies comparing particles of various sizes prepared in various ways, and testing is performed on different cell lines, it is very difficult to generalize. The novelty and significance of our research is focused to the complex biological effects of silver and gold NPs prepared by the same method, have the same parameters and the same stabilizer. That is why we can compare the biological effects of pure nanometals themselves based on their chemical nature without the influence of other variable. Aim of our study therefore is to compare the cytotoxic effect of two types of noble metal NPs focusing on the mechanisms that contribute to cytotoxicity. The study was conducted on murine fibroblasts by selected common used tests. Each of these tests monitors the selected area related to toxicity and together provides a comprehensive view on the issue of interactions of nanoparticles and living cells.

Keywords: cytotoxicity, gold nanoparticles, mechanism of cytotoxicity, silver nanoparticles

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Authors: Mohamed M. Ibrahima, Abdul Aziz A.AL Sahli, Ibrahim A. Alaraidh, Ali A. Al-Homaidan, E.M. Mostafa, G.A. EL-Gaaly

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Traditionally, in Middle Eastern countries, many cultures use chewing sticks of arak for medicinal purposes especially, for oral cleanliness care. It was used by Muslims for the treatment of teeth and highly recommended to be used by muslims during the whole day. Therefore, the present work aimed to determine the total phenolic content and total flavonoids in two Miswak extracts obtained from arak roots collected from two different localities in Saudi Arabia. They were extracted with aqueous ethanol (80%) and used to estimate in vitro their antioxidative abilities. The new findings showed that the two tested extracts contained significantly different amounts of both total phenolic content and total flavonoids. According to the increase of total phenolic contents and total flavonoids obtained from the two extracts, Miswak collected from the southern region was found to contain more contents than those collected from the middle region. The results of antioxidant activities of Miswak root extract obtained by using different in vitro methods were varied depending on the technique used. According to the malondialdehyde (MDA) method, hydrogen peroxide (H2O2) scavenging ability and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods, the two Miswak extracts exhibited to have high to very high antioxidant activities. Mostly, the values of antioxidant activities of Southern region have been shown to be always the highest.

Keywords: Arak, antioxidant, medicinal plants, Saudi Arabia

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Authors: Aberdam Edith, Sangari Linda, Petit Isabelle, Aberdam Daniel

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Background and Rationale: Transparent avascularised cornea is essential for normal vision and depends on limbal stem cells (LSC) that reside between the cornea and the conjunctiva. Ocular burns or injuries may destroy the limbus, causing limbal stem cell deficiency (LSCD). The cornea becomes vascularised by invaded conjunctival cells, the stroma is scarring, resulting in corneal opacity and loss of vision. Grafted autologous limbus or cultivated autologous LCS can restore the vision, unless the two eyes are affected. Alternative cellular sources have been tested in the last decades, including oral mucosa or hair follicle epithelial cells. However, only partial success has been achieved by the use of these cells since they were not able to uniformly commit into corneal epithelial cells. Human pluripotent stem cells (iPSC) display both unlimited growth capacity and ability to differentiate into any cell type. Our goal was to design a standardized and reproducible protocol to produce transplantable autologous LSC from patients through cell reprogramming technology. Methodology: First, keratinocyte primary culture was established from a small number of plucked hair follicles of healthy donors. The resulting epithelial cells were reprogrammed into induced pluripotent stem cells (iPSCs) and further differentiate into corneal epithelial cells (CEC), according to a robust protocol that recapitulates the main step of corneal embryonic development. qRT-PCR analysis and immunofluorescent staining during the course of differentiation confirm the expression of stage specific markers of corneal embryonic lineage. First appear ectodermal progenitor-specific cytokeratins K8/K18, followed at day 7 by limbal-specific PAX6, TP63 and cytokeratins K5/K14. At day 15, K3/K12+-corneal cells are present. To amplify the iPSC-derived LSC (named COiPSC), intact small epithelial colonies were detached and cultivated in limbal cell-specific medium. In that culture conditions, the COiPSC can be frozen and thaw at any passage, while retaining their corneal characteristics for at least eight passages. To evaluate the potential of COiPSC as an alternative ocular toxicity model, COiPSC were treated at passage P0 to P4 with increasing amounts of SDS and Benzalkonium. Cell proliferation and apoptosis of treated cells was compared to LSC and the SV40-immortalized human corneal epithelial cell line (HCE) routinely used by cosmetological industrials. Of note, HCE are more resistant to toxicity than LSC. At P0, COiPSC were systematically more resistant to chemical toxicity than LSC and even to HCE. Remarkably, this behavior changed with passage since COiPSC at P2 became identical to LSC and thus closer to physiology than HCE. Comparative transcriptome analysis confirmed that COiPSC from P2 are similar to a mixture of LSC and CEC. Finally, by organotypic reconstitution assay, we demonstrated the ability of COiPSC to produce a 3D corneal epithelium on a stromal equivalent made of keratocytes. Conclusion: COiPSC could become valuable for two main applications: (1) an alternative robust tool to perform, in a reproducible and physiological manner, toxicity assays for cosmetic products and pharmacological tests of drugs. (2). COiPSC could become an alternative autologous source for cornea transplantation for LSCD.

Keywords: Limbal stem cell deficiency, iPSC, cornea, limbal stem cells

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Authors: Befekadu Urga Wakayo

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Introduction: Bacterial infections represent major human and animal health problems in Ethiopia. In the face of poor antibiotic regulatory mechanisms, development of antimicrobial resistance (AMR) to commonly used drugs has become a growing health and livelihood threat in the country. Monitoring and control of AMR demand close coloration between human and veterinary services as well as other relevant stakeholders. However, risk of AMR transfer from animal to human population’s remains poorly explored in Ethiopia. This systematic research literature review attempted to give an overview on AMR challenges of bovine mastitis bacteria in Ethiopia. Methodology: A web based research literature search and analysis strategy was used. Databases are considered including; PubMed, Google Scholar, Ethiopian Veterinary Association (EVA) and Ethiopian Society of Animal Production (ESAP). The key search terms and phrases were; Ethiopia, dairy, cattle, mastitis, bacteria isolation, antibiotic sensitivity and antimicrobial resistance. Ultimately, 15 research reports were used for the current analysis. Data extraction was performed using a structured Microsoft Excel format. Frequency AMR prevalence (%) was registered directly or calculated from reported values. Statistical analysis was performed on SPSS – 16. Variables were summarized by giving frequencies (n or %), Mean ± SE and demonstrative box plots. One way ANOVA and independent t test were used to evaluate variations in AMR prevalence estimates (Ln transformed). Statistical significance was determined at p < 0.050). Results: AMR in bovine mastitis bacteria was investigated in a total of 592 in vitro antibiotic sensitivity trials involving 12 different mastitis bacteria (including 1126 Gram positive and 77 Gram negative isolates) and 14 antibiotics. Bovine mastitis bacteria exhibited AMR to most of the antibiotics tested. Gentamycin had the lowest average AMR in both Gram positive (2%) and negative (1.8%) bacteria. Gram negative mastitis bacteria showed higher mean in vitro resistance levels to; Erythromycin (72.6%), Tetracycline (56.65%), Amoxicillin (49.6%), Ampicillin (47.6%), Clindamycin (47.2%) and Penicillin (40.6%). Among Gram positive mastitis bacteria higher mean in vitro resistance was observed in; Ampicillin (32.8%), Amoxicillin (32.6%), Penicillin (24.9%), Streptomycin (20.2%), Penicillinase Resistant Penicillin’s (15.4%) and Tetracycline (14.9%). More specifically, S. aurues exhibited high mean AMR against Penicillin (76.3%) and Ampicillin (70.3%) followed by Amoxicillin (45%), Streptomycin (40.6%), Tetracycline (24.5%) and Clindamycin (23.5%). E. coli showed high mean AMR to Erythromycin (78.7%), Tetracycline (51.5%), Ampicillin (49.25%), Amoxicillin (43.3%), Clindamycin (38.4%) and Penicillin (33.8%). Streptococcus spp. demonstrated higher (p =0.005) mean AMR against Kanamycin (> 20%) and full sensitivity (100%) to Clindamycin. Overall, mean Tetracycline (p = 0.013), Gentamycin (p = 0.001), Polymixin (p = 0.034), Erythromycin (p = 0.011) and Ampicillin (p = 0.009) resistance increased from the 2010’s than the 2000’s. Conclusion; the review indicated a rising AMR challenge among bovine mastitis bacteria in Ethiopia. Corresponding, public health implications demand a deeper, integrated investigation.

Keywords: antimicrobial resistance, dairy cattle, Ethiopia, Mastitis bacteria

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Authors: J. B. Minari, O. B. Oloyede

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Polymorphonuclear neutrophils (PMNs) play a crucial role in a variety of infections caused by bacteria, fungi, and parasites. Indeed, the involvement of PMNs in host defence against Plasmodium falciparum is well documented both in vitro and in vivo. Many of the antimalarial drugs such as chloroquine used in the treatment of human malaria significantly reduce the immune response of the host in vitro and in vivo. Myeloperoxidase is the most abundant enzyme found in the polymorphonuclear neutrophil which plays a crucial role in its function. This study was carried out to investigate the effect of chloroquine on the enzyme. In investigating the effects of the drug on myeloperoxidase, the influence of concentration, pH, partition ratio estimation and kinetics of inhibition were studied. This study showed that chloroquine is concentration-dependent inhibitor of myeloperoxidase with an IC50 of 0.03 mM. Partition ratio estimation showed that 40 enzymatic turnover cycles are required for complete inhibition of myeloperoxidase in the presence of chloroquine. The influence of pH on the effect of chloroquine on the enzyme showed significant inhibition of myeloperoxidase at physiological pH. The kinetic inhibition studies showed that chloroquine caused a non-competitive inhibition with an inhibition constant Ki of 0.27mM. The results obtained from this study shows that chloroquine is a potent inhibitor of myeloperoxidase and it is capable of inactivating the enzyme. It is therefore considered that the inhibition of myeloperoxidase in the presence of chloroquine as revealed in this study may partly explain the impairment of polymorphonuclear neutrophil and consequent immunosuppression of the host defence system against secondary infections.

Keywords: myeloperoxidase, chloroquine, inhibition, neutrophil, immune

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Authors: Zahid Manzoor, Shaukat Hussain Munawar, Zahid Iqbal, Imran Ahmad Khan, Abdul Aziz, Hafiz Muhammad Qasim

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The present study was aimed to investigate the pharmacokinetics behavior and optimal dosage regimen of danofloxacin in 8 adult healthy buffaloes of local breed (Nili Ravi) following single intravenous administration at the dose of 2.5 mg/kg body weight. Plasma drug concentrations at various time intervals were measured by HPLC method. In vitro plasma protein binding was determined employing the ultrafiltration technique. The distribution and elimination of danofloxacin was rapid, as indicated by the values (Mean±SD) of distribution half-life (t1/2α = 0.25±0.09 hours) and elimination half life (t1/2β = 3.26±0.43 hours), respectively. Volume of distribution at steady state (Vss) was 1.14±0.12 L/kg, displaying its extensive distribution into various body fluids and tissues. The high value of AUC (9.80±2.14 µg/ml.hr) reflected the vast area of the body covered by drug concentration. The mean residence time was noted to be 4.78±0.52 hours. On the basis of pharmacokinetic parameters, a suitable intravenous regimen for danofloxacin in adult buffaloes would be 6.5 mg/kg to be repeated after 12 hours intervals. The present study is the foremost pharmacokinetic study of danofloxacin in the local species which would provide the valueable contribution in the local manufacturing of danofloxacin in Pakistan in future.

Keywords: danofloxacin, pharmacokinetics, plasma protein binding, buffaloes, dosage regimen

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Authors: Joan O. Ogundele, Aladesanmi A. Oshodi, Adekunle I. Amoo

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Abstract In-vitro multi-enzyme protein digestibility (IVMPD) and some anti-nutritional factors (ANF) of five melon (egusi) seed flours (MSF) and their protein isolates (PI) were carried out. Their PI have potentials comparable to that of soya beans. It is important to know the IVMPD and ANF of these protein sources as to ensure their safety when adapted for use as alternate protein sources to substitute for cow milk, which is relatively expensive in Nigeria. Standard methods were used to produce PI of Citrullus colocynthis, Citrullus vulgaris, African Wine Kettle gourd (Lageneria siceraria I), Basket Ball gourd (Lagenaria siceraria II) and Bushel Giant Gourd (Lageneria siceraria III) seeds and to determine the ANF and IVMPD of the MSF and PI unheated and at 37oC. Multi-enzymes used were trypsin, chymotrypsin and peptidase. IVMPD of MSF ranged from (70.67±0.70) % (C. vulgaris) to (72.07± 1.79) % (L.siceraria I) while for their PI ranged from 74.33% (C.vulgaris) to 77.55% (L.siceraria III). IVMPD of the PI were higher than those of MSF. Heating increased IVMPD of MSF with average value of 79.40% and those of PI with average of 84.14%. ANF average in MSF are tannin (0.11mg/g), phytate (0.23%). Differences in IVMPD of MSF and their PI at different temperatures may arise from processing conditions that alter the release of amino acids from proteins by enzymatic processes. ANF in MSF were relatively low, but were found to be lower in the PI, therefor making the PI safer for human consumption as an alternate source of protein.

Keywords: Anti-nutrients, Enzymatic protein digestibility, Melon (egusi)., Protein Isolates.

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Authors: Alok Raghav, Jamal Ahmad

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Background: Serum albumin glycation and advanced glycation end products (AGE) formation correlates in diabetes and its associated complications. Extensive modified human serum albumin is used to study the biochemical, electrochemical and functional properties in hyperglycemic environment with relevance to diabetes. We evaluate Spectroscopic, side chain modifications, amino acid analysis, biochemical and functional group properties in four glucose modified samples. Methods: A series four human serum albumin samples modified with glucose was characterized in terms of amino acid analysis, spectroscopic properties and side chain modifications. The diagnostic technique employed incorporates UV Spectroscopy, Fluorescence Spectroscopy, biochemical assays for side chain modifications, amino acid estimations, electrochemical and optical characterstic of glycated albumin. Conclusion: Glucose modified human serum albumin confers AGEs formation alters biochemical, electrochemical, optical, and functional property that depend on the reactivity of glucose and its concentration used for in-vitro glycation. A biochemical, electrochemical, optical, and functional characterization of modified albumin in-vitro produced AGE product that will be useful to interpret the complications and pathophysiological significance in diabetes.

Keywords: human serum albumin, glycated albumin, adavanced glycation end products, associated pathologies

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Authors: Amlan Kumar Das, Avinash Marwal, Vikram Pareek

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Liposome plays an important role in medical and pharmaceutical science as e.g. nano scale drug carriers. Liposomes are vesicles of varying size consisting of a spherical lipid bilayer and an aqueous inner compartment. Magnet-driven liposome used for the targeted delivery of drugs to organs and tissues1. These liposome preparations contain encapsulated drug components and finely dispersed magnetic particles. Liposomes are vesicles of varying size consisting of a spherical lipid bilayer and an aqueous inner compartment that are generated in vitro. These are useful in terms of biocompatibility, biodegradability, and low toxicity, and can control biodistribution by changing the size, lipid composition, and physical characteristics2. Furthermore, liposomes can entrap both hydrophobic and hydrophilic drugs and are able to continuously release the entrapped substrate, thus being useful drug carriers. Magnetic liposomes (MLs) are phospholipid vesicles that encapsulate magneticor paramagnetic nanoparticles. They are applied as contrast agents for magnetic resonance imaging (MRI)3. The biological synthesis of nanoparticles using plant extracts plays an important role in the field of nanotechnology4. Green-synthesized magnetite nanoparticles-protein hybrid has been produced by treating Iron (III)/Iron(II) chloride with the leaf extract of Dhatura Inoxia. The phytochemicals present in the leaf extracts act as a reducing as well stabilizing agents preventing agglomeration, which include flavonoids, phenolic compounds, cardiac glycosides, proteins and sugars. The magnetite nanoparticles-protein hybrid has been trapped inside the aqueous core of the liposome prepared by reversed phase evaporation (REV) method using oleic and linoleic acid which has been shown to be driven under magnetic field confirming the formation magnetic liposome (ML). Chemical characterization of stealth magnetic liposome has been performed by breaking the liposome and release of magnetic nanoparticles. The presence iron has been confirmed by colour complex formation with KSCN and UV-Vis study using spectrophotometer Cary 60, Agilent. This magnet driven liposome using nanoparticles-protein hybrid can be a smart vesicles for the targeted drug delivery.

Keywords: nanoparticles-protein hybrid, magnetic liposome, medical, pharmaceutical science

Procedia PDF Downloads 250

Authors: A. Kazbekova, A. Kudaibergenov, G. Atazhanova, S. Adekenov

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In recent years, it achieved some progress such a direction as to study the possibility of correlation between different types of biological activity. In particular, in our work, we consider questions such as: the impact of the qualitative composition of total substances in the example of plant extracts on antioxidant and antiradical activity, the presents of correlation between these types of activity, etc. It is known that there is a relationship between the values of optical density of working solutions of extracts and corresponding bioactivity in vitro, in particular, the antioxidant and hepatoprotective effects. In this study, we have identified that among some studied species of wormwood (Artemisia viridis Wild, Artemisia jacutica Drob, Artemisia annua L, Artemisia siversiana Wild, Artemisia adamsii Bess, Artemisia tianschanica, Artemisia obtusiloba Ledeb., Artemisia heptopotamica), as well as extracts of Inula caspica, Аjania tenuifolia, Abies sibirica, Galatella songorica, Mentha asiatica and Thymus mugodzharicus it was identified that the highest content of polyphenol compounds is in Thymus mugodzharicus. At the same time, we determined the antioxidant and antiradical activity, which was the highest for the Thymus mugodzharicus. Butylhydroxyanisole and ascorbic acid were used as comparison substances. Also, it was established that antioxidant and anti-radical activities depend on the concentration of the of all investigated samples. Based on obtained data, we believe that the extract of Thymus mugodzharicus can be recommended for further study on the antioxidant and antiradical activity in vivo, as well as the opportunity of this sample to demonstrate hepatoprotective effect. The study was sponsored by SANTO academic program.

Keywords: in vitro, in vivo, antioxidant, hepatoprotective effect

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Authors: Narine Ghazaryan, Joana Catarina Macedo, Sara Vaz, Naira Ayvazyan, Elsa Logarinho

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Macrovipera lebetina obtusa (MLO) is a poisonous snake in Armenia. Obtustatin represents the shortest known monomeric disintegrin, isolated from the snake venom of MLO, and is known to specifically inhibit α1β1 integrin. Its oncostatic effect is due to the inhibition of angiogenesis, which likely arises from α1β1 integrin inhibition in the endothelial cells. To explore the therapeutic potential of the MLO snake venom and obtustatin, we studied activity of obtustatin and MLO venom in vitro, by testing their efficacy in human dermal microvascular endothelial cells (HMVEC-D) and in vivo, using chick embryo chorioallantoic membrane assay (CAM assay). Our in vitro results showed that obtustatin in comparison with MLO venom did not exhibit cytotoxic activity in HMVEC-D cells in comparison to MLO venom. But in vivo results have shown that 4µg /embryo (90 µM) of obtustatin inhibited angiogenesis induced by FGF2 by 17% while MLO snake venom induced 22% reduction of the angiogenic index. The concentration of obtustatin in the crude MLO venom was 0.3 nM, which is 300.000 times less than the concentration of the obtustatin itself. Given this enormous difference in concentration, it is likely that some components of the crude venom contribute to the observed anti-angiogenic effect. Hypotheses will be ascertained to justify this action: components in the MLO venom may increase obtustatin efficacy or have independent but synergic anti-angiogenic activities.

Keywords: angiogenesis, alpa1 beta 1 integrin, Macrovipera lebetina obtusa, obtustatin

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Authors: Shwu-Ling Peng, Chiung-Man Tsai, Chia-Jui Weng

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Chronic micro-inflammation is a hallmark of many aging-related neurodegenerative and metabolic syndrome-driven diseases. In high glucose (HG) environment, reactive oxygen species (ROS) is generated and the ROS induced inflammation, cytokines secretion, DNA damage, and cell cycle arrest to lead to cellular senescence. Water chestnut shell (WCS) is a plant hull which containing polyphenolic compounds and showed antioxidant and anticancer activities. Orchid, which containing a natural polysaccharide compound, possesses many physiological activities including anti-inflammatory and neuroprotective effects. These agricultural plants might be able to reduce oxidative stress and inflammation. This study was used HG-induced human normal dermal fibroblasts (HG-HNDFs) as an in vitro model to disclose the effects of water extract of Phalaenopsis orchid flower (WEPF) and ethanol extract of water chestnut shell (EEWCS) on the anti-aging and their underlying molecular mechanisms. The toxicity of extracts on human normal dermal fibroblasts (HNDFs) was determined by MTT method. The senescence of cells was assayed by β-galactosidase (SA-β-gal) kit. ROS and nitrate production was analyzed by Intracellular ROS contents and ELISA, respectively. Western blotting was used to detect the proteins in cells. The results showed that the exposure of HNDFs to HG (30 mM) for 72 h were caused cellular senescence and arrested cells at G0/G1 phase. Indeed, the treatment of HG-HNDFs with WEPF (200 μg/ml) and EEWCS (10 μg/ml) significantly released cell cycle arrest and promoted cell proliferation. The G1/S phase transition regulatory proteins such as protein retinoblastoma (pRb), p53, and p16ᴵᴺᴷ⁴ᵃ depressed by WEPF and EEWCS were also observed. Additionally, the treatment of WEPF and EEWCS increased the activity of HO-1 through upregulating Nrf2 as well as decreased the ROS and NO of HG-HNDFs. Therefore, the senescence marker protein-30 (SMP30) in cells was diminished. In conclusion, the WEPF and EEWCS might inhibit HG-induced aging of HNDFs by reducing oxidative stress and free radicals.

Keywords: agricultural plant extract, anti-aging, high glucose, Phalaenopsis orchid flower, water chestnut shell

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Authors: Guadalupe Pérez, Jorge Islas, Mirna Guevara, Raúl Suárez

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Jatropha curcas is a perennial oleaginous plant that is currently considered an energy crop with high potential as an environmentally sustainable biofuel. During the last decades, research in biofuels has grown in tropical and subtropical regions in Latin America. However, as far we know, there are no reports on the growth and yield patterns of Jatropha curcas under the specific agro climatic scenarios of the State of Morelos, Mexico. This study presents the results of 52 months monitoring of 10 toxic and non-toxic ecotypes of Jatropha curcas (E1M, E2M, E3M, E4M, E5M, E6O, E7O, E8O, E9C, E10C) in an experimental plantation with minimum watering and fertilization resources. The main objective is to identify the ecotypes with the highest potential as biodiesel raw material in the select region, by developing experimental information. Specifically, we monitored biophysical and growth parameters, including plant survival and seed production (at the end of month 52), to study the performance of each ecotype and to establish differences among the variables of morphological growth, net seed oil content, and toxicity. To analyze the morphological growth, a statistical approach to the biophysical parameters was used; the net seed oil content -80 to 192 kg/ha- was estimated with the first harvest; and the toxicity was evaluated by examining the phorbol ester concentration (µg/L) in the oil extracted from the seeds. The comparison and selection of ecotypes was performed through a methodology developed based on the normalization of results. We identified four outstanding ecotypes (E1M, E2M, E3M, and E4M) that can be used to establish Jatropha curcas as energy crops in the state of Morelos for feasible agro-industrial production of biodiesel and other products related to the use of biomass.

Keywords: biodiesel production, Jatropha curcas, seed oil content, toxic and non-toxic ecotypes

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Authors: Shabnam Alizadeh, Hatice Dumanoglu

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Plant tissue culture is a substantial plant propagation technique for mass clonal production throughout the year, regardless of time in fruit species. However, the rooting achievement must be enhanced in the difficult-to-root genotypes. Classical auxin applications in clonal propagation of these genotypes are inadequate to solve the rooting problem. Nanoparticles having different physical and chemical properties from bulk material could enhance the rooting success of controlled release of these substances when loaded with auxin due to their ability to reach the active substance up to the target cells as a carrier system.The purpose of this study is to investigate the effects of zinc oxide nanoparticles loaded with indole-3-acetic acid (IAA-nZnO) and indole-3-butyric acid (IBA-nZnO) on in vitro rooting of microcuttings in a difficult-to-root apple genotype (Malus domestica Borkh.). Rooting treatments consisted of IBA or IAA at concentrations of 0.5, 1.0, 2.0, 3.0 mg/L; nZnO, IAA-nZnO and IBA-nZnO at doses of 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 mg/L were used. All components were added to the Murashige and Skoog (MS) basal medium at strength ½ with 2% sucrose and 0.7% agar before autoclaving. In the study, no rooting occurred in control and nZnO applications. Especially, 1.0 mg/L and 2.0 mg/L IBA-nZnO nanoparticle applications (containing 0.5 mg/L and 0.9 mg/L IBA), respectively with rooting rates of 40.3% and 70.4%, rooting levels of 2.0±0.4 and 2.3±0.4, 2.6±0.7 and 2.5±0.6 average root numbers and 20.4±1.6 mm and 20.2±3.4 mm average root lengths put forward as effective applications.

Keywords: Auxin, Malus, nanotechnology, zinc oxide nanoparticles

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Authors: Mohamed A. Morsy, Azza A. El-Sheikh, Abdulla Y. Al-Taher

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Resveratrol (RES) is a well-known polyphenol antioxidant. We have previously shown that testicular protective effect of RES against the anticancer drug methotrexate (MTX)-induced toxicity involves transporter-mediated mechanisms. Here, we investigated the effect of RES on MTX-induced nephrotoxicity. Rats were administered RES (10 mg/kg/day) for 8 days, with or without a single MTX dose (20 mg/kg i.p.) at day 4 of the experiment. MTX induced nephrotoxicity evident by significantly increase in serum blood urea nitrogen and creatinine compared to control, as well as distortion of kidney microscopic structure. MTX also significantly increased renal nitric oxide level, with induction of inducible nitric oxide synthase expression. MTX also significantly up-regulated fas ligand and caspase 3. Administering RES prior to MTX significantly improved kidney function and microscopic picture, as well as significantly decreased nitrosative and apoptotic markers compared to MTX alone. RES, but not MTX, caused significant increase in expression of breast cancer resistance protein (BCRP), an apical efflux renal transporter that participates in urinary elimination of both MTX and RES. Interestingly, concomitant MTX and RES caused further up-regulation of renal Bcrp compared to RES alone. Using Human BCRP ATPase assay, both RES and MTX exhibited dose-dependent increase in ATPase activity, with Km values of 0.52 ± 0.03 and 30.9 ± 4.2 µM, respectively. Furthermore, combined RES and MTX caused ATPase activity which was significantly less than maximum ATPase activity attained by the positive control; sulfasalazine (12.5 µM). In conclusion, RES exerted nephro-protection against MTX-induced toxicity through anti-nitrosative and anti-apoptotic effects, as well as via up-regulation of renal Bcrp.

Keywords: methotrexate, resveratrol, nephrotoxicity, breast cancer resistance protein

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Authors: Alija Uzunovic, Sasa Pilipovic, Aida Sapcanin, Zahida Ademovic, Berina Pilipović

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Capsaicin is a naturally occurring alkaloid extracted from capsicum fruit extracts of different of Capsicum species. It has been employed topically to treat many diseases such as rheumatoid arthritis, osteoarthritis, cancer pain and nerve pain in diabetes. The high degree of pre-systemic metabolism of intragastrical capsaicin and the short half-life of capsaicin by intravenous administration made topical application of capsaicin advantageous. In this study, we have evaluated differences in the dissolution characteristics of capsaicin patch 11 mg (purchased from market) at different dissolution rotation speed. The proposed patch area is 308 cm2 (22 cm x 14 cm; it contains 36 µg of capsaicin per square centimeter of adhesive). USP Apparatus 5 (Paddle Over Disc) is used for transdermal patch testing. The dissolution study was conducted using USP apparatus 5 (n=6), ERWEKA DT800 dissolution tester (paddle-type) with addition of a disc. The fabricated patch of 308 cm2 is to be cut into 9 cm2 was placed against a disc (delivery side up) retained with the stainless-steel screen and exposed to 500 mL of phosphate buffer solution pH 7.4. All dissolution studies were carried out at 32 ± 0.5 °C and different rotation speed (50± 5; 100± 5 and 150± 5 rpm). 5 ml aliquots of samples were withdrawn at various time intervals (1, 4, 8 and 12 hours) and replaced with 5 ml of dissolution medium. Withdrawn were appropriately diluted and analyzed by reversed-phase liquid chromatography (RP-LC). A Reversed Phase Liquid Chromatography (RP-LC) method has been developed, optimized and validated for the separation and quantitation of capsaicin in a transdermal patch. The method uses a ProntoSIL 120-3-C18 AQ 125 x 4,0 mm (3 μm) column maintained at 600C. The mobile phase consisted of acetonitrile: water (50:50 v/v), the flow rate of 0.9 mL/min, the injection volume 10 μL and the detection wavelength 222 nm. The used RP-LC method is simple, sensitive and accurate and can be applied for fast (total chromatographic run time was 4.0 minutes) and simultaneous analysis of capsaicin and dihydrocapsaicin in a transdermal patch. According to the results obtained in this study, we can conclude that the relative difference of dissolution rate of capsaicin after 12 hours was elevated by increase of dissolution rotation speed (100 rpm vs 50 rpm: 84.9± 11.3% and 150 rpm vs 100 rpm: 39.8± 8.3%). Although several apparatus and procedures (USP apparatus 5, 6, 7 and a paddle over extraction cell method) have been used to study in vitro release characteristics of transdermal patches, USP Apparatus 5 (Paddle Over Disc) could be considered as a discriminatory test. would be able to point out the differences in the dissolution rate of capsaicin at different rotation speed.

Keywords: capsaicin, in vitro, patch, RP-LC, transdermal

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Authors: Joulié Aurélien, Jourdain Elsa, Bailly Xavier, Gasqui Patrick, Yang Elise, Leblond Agnès, Rousset Elodie, Sidi-Boumedine Karim

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Q fever is a worldwide zoonosis caused by the gram-negative obligate intracellular bacterium Coxiella burnetii. Following the recent outbreaks in the Netherlands, a hyper virulent clone was found to be the cause of severe human cases of Q fever. In livestock, Q fever clinical manifestations are mainly abortions. Although the abortion rates differ between ruminant species, C. burnetii’s virulence remains understudied, especially in enzootic areas. In this study, the infectious potential of three C. burnetii isolates collected from French farms of small ruminants were compared to the reference strain Nine Mile (in phase II and in an intermediate phase) using an in vivo (CD1 mice) model. Mice were challenged with 105 live bacteria discriminated by propidium monoazide-qPCR targeting the icd-gene. After footpad inoculation, spleen and popliteal lymph node were harvested at 10 days post-inoculation (p.i). The strain invasiveness in spleen and popliteal nodes was assessed by qPCR assays targeting the icd-gene. Preliminary results showed that the avirulent strains (in phase 2) failed to pass the popliteal barrier and then to colonize the spleen. This model allowed a significant differentiation between strain’s invasiveness on biological host and therefore identifying distinct virulence profiles. In view of these results, we plan to go further by testing fifteen additional C. burnetii isolates from French farms of sheep, goat and cattle by using the above-mentioned in vivo model. All 15 strains display distant MLVA (multiple-locus variable-number of tandem repeat analysis) genotypic profiles. Five of the fifteen isolates will bee also tested in vitro on ovine and bovine macrophage cells. Cells and supernatants will be harvested at day1, day2, day3 and day6 p.i to assess in vitro multiplication kinetics of strains. In conclusion, our findings might help the implementation of surveillance of virulent strains and ultimately allow adapting prophylaxis measures in livestock farms.

Keywords: Q fever, invasiveness, ruminant, virulence

Procedia PDF Downloads 362

Authors: Loren S. Bernal., Catalina Castillo, Carel E. Carvajal, José F. Ibla

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Plastic pollution, specifically microplastics, has become a significant issue in aquatic ecosystems worldwide. The large amount of plastic waste carried by water tributaries has resulted in the accumulation of microplastics in water bodies. The polymer aging process caused by environmental influences such as photodegradation and chemical degradation of additives leads to polymer embrittlement and properties change that require degradation or reduction procedures in rivers. However, there is a lack of such procedures for freshwater entities that develop over extended periods. The aim of this study is evaluate the potential of Pleurotus ostreatus a fungus, in reducing lowdensity polyethylene microplastics present in freshwater samples collected from the middle basin of the Magdalena River in Colombia. The study aims to evaluate this process both in vitro and in silico by identifying the growth capacity of Pleurotus ostreatus in the presence of microplastics and identifying the most likely interactions of Pleurotus ostreatus enzymes and their affinity energies. The study follows an engineering development methodology applied on an experimental basis. The in vitro evaluation protocol applied in this study focused on the growth capacity of Pleurotus ostreatus on microplastics using enzymatic inducers. In terms of in silico evaluation, molecular simulations were conducted using the Autodock 1.5.7 program to calculate interaction energies. The molecular dynamics were evaluated by using the myPresto Portal and GROMACS program to calculate radius of gyration and Energies.The results of the study showed that Pleurotus ostreatus has the potential to degrade low-density polyethylene microplastics. The in vitro evaluation revealed the adherence of Pleurotus ostreatus to LDPE using scanning electron microscopy. The best results were obtained with enzymatic inducers as a MnSO4 generating the activation of laccase or manganese peroxidase enzymes in the degradation process. The in silico modelling demonstrated that Pleurotus ostreatus was able to interact with the microplastics present in LDPE, showing affinity energies in molecular docking and molecular dynamics shown a minimum energy and the representative radius of gyration between each enzyme and its substract. The study contributes to the development of bioremediation processes for the removal of microplastics from freshwater sources using the fungus Pleurotus ostreatus. The in silico study provides insights into the affinity energies of Pleurotus ostreatus microplastic degrading enzymes and their interaction with low-density polyethylene. The study demonstrated that Pleurotus ostreatus can interact with LDPE microplastics, making it a good agent for the development of bioremediation processes that aid in the recovery of freshwater sources. The results of the study suggested that bioremediation could be a promising approach to reduce microplastics in freshwater systems.

Keywords: bioremediation, in silico modelling, microplastics, Pleurotus ostreatus

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Authors: N. S. Aggangan, B. Dell, P. Jeffries

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Two moderately nickel-tolerant isolates of Pisolithus were compared with a non-Ni tolerant isolate for the ability to increase the growth of Eucalyptus urophylla seedlings in the presence of nickel (Ni) in pots in a glasshouse. Seedlings, either inoculated with mycorrhizal fungi or uninoculated, were transplanted into pots containing 3 kg steam-pasteurized yellow sand amended with five concentrations of nickel (0, 6, 12, 24 and 48 mg Ni kg-1 soil). Within a day after transplanting, all seedlings subjected to Ni rates greater than 12 mg Ni kg-1 showed symptoms of wilting and all died within two weeks. At lower nickel concentrations, inoculation with all 3 Pisolithus strains increased rates of seedling survival after 12 weeks. Inoculation with all 3 isolates Pisolithus significantly increased the growth of plants in Ni-free soils between 2 to 4 fold dependent on isolate. However, seedlings growing in soils containing 12 mg Ni kg-1 grew poorly, mycorrhizal development was inhibited and no beneficial effects of inoculation were noted. In contrast, in soils containing 6mg Ni kg-1, inoculated seedlings did not show the reduced root growth and severe toxicity symptoms (chlorosis on young leaves and shoot tips) of uninoculated seedlings. Only the Ni-tolerant Pisolithus strains conferred a significant growth benefit compared to non-inoculated controls, and plants inoculated with one of these strains grew twice the size as those inoculated with the other Ni-tolerant strain. Inorganic plant analysis revealed that inoculation increased plant growth through improved P uptake but did not prevent Ni uptake. However, toxicity may have been minimized by dilution due to an increase in plant biomass. The results suggest that only one of the Ni-tolerant strains of Pisolithus has the potential to improve the growth and survival of E. urophylla seedlings in serpentine soils in the Philippines.

Keywords: ectomycorrhizas, Eucalyptus urophylla, nickel tolerance, pisolithus

Procedia PDF Downloads 304