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Αποφρακτικός ίκτερος ως αιτία οξειδωτικού στρες στον εγκέφαλο και επίδραση αντιοξειδωτικών παραγόντωνΚαραγεώργος, Νικόλαος 03 August 2009 (has links)
Η ηπατική εγκεφαλοπάθεια είναι ένα πολύπλοκο νευροψυχιατρικό σύνδρομο που έχει συσχετισθεί με οξείες και χρόνιες ηπατοπάθειες. Τα τελευταία χρόνια συσσωρεύονται πληροφορίες που εμπλέκουν το οξειδωτικό στρες (φορτίο) ως παράγοντα-κλειδί στην παθογένεση της ηπατικής εγκεφαλοπάθειας σε μελέτες που χρησιμοποιούν ως μετρούμενους δείκτες την υπεροξείδωση λιπιδίων και το οξειδοαναγωγικό ζεύγος της γλουταθειόνης (GSH/GSSG).
Στην παρούσα μελέτη χρησιμοποιήθηκε ένα μοντέλο πειραματικού αποφρακτικού ίκτερου με απολίνωση του χοληδόχου πόρου. Αρσενικοί αρουραίοι χωρίστηκαν σε ομάδες ελέγχου, ψευδώς χειρουργημένων, και σε ομάδες απολίνωσης χοληδόχου πόρου που είτε θυσιάστηκαν την 5η ημέρα, είτε τη 10η ημέρα, ή τους χορηγήθηκαν αντιοξειδωτικοί παράγοντες (NAC, ALP, Vit-E). Στη συνέχεια, μελετήθηκε η θειολική οξειδοαναγωγική κατάσταση στον εγκέφαλο των αρουραίων, και μάλιστα ανά περιοχές (εγκεφαλικός φλοιός, στέλεχος, παρεγκεφαλίδα), καθώς και η επίδραση των επιλεγμένων αντιοξειδωτικών παραγόντων σε αυτήν.
Χρησιμοποιήθηκε για πρώτη φορά μια πιο αντιπροσωπευτική εκτίμηση του οξειδωτικού στρες, καθώς ποσοτικοποιήθηκαν συγκεκριμένοι δείκτες υψηλού (GSSG, NPSSR, NPSSC, PSSR, PSSC, PSSP, υπεροξείδια λιπιδίων) και χαμηλού (GSH, CSH, PSH) οξειδωτικού στρες. Τα αποτελέσματά μας δείχνουν αύξηση των πρώτων και μείωση των τελευταίων σε όλες τις εγκεφαλικές περιοχές καταδεικνύοντας έτσι την παρουσία αυξημένου οξειδωτικού φορτίου στον αποφρακτικό ίκτερο.
Το σημαντικότερο αποτέλεσμα αυτής της μελέτης είναι ότι κατέδειξε πρώιμα σημεία οξειδωτικού στρες στον εγκέφαλο ήδη από την 5η ημέρα μετά την απολίνωση του χοληδόχου πόρου. Οι μεταβολές των βιοχημικών δεικτών, και αυτό αφορά σε όλους τους δείκτες και σε όλες τις εγκεφαλικές περιοχές, αρχίζουν να φαίνονται από την 5η ημέρα και γίνονται στατιστικά σημαντικές τη 10η ημέρα από την απολίνωση του χοληδόχου πόρου.
Διαπιστώσαμε επιπλέον ότι η GSH είχε περίπου διπλάσιες τιμές στον εγκεφαλικό φλοιό από ό, τι στο στέλεχος και την παρεγκεφαλίδα, και ότι στο στέλεχος παρατηρήθηκε μια δραματική αύξηση των επιπέδων των NPSSR τη 10η ημέρα μετά την απολίνωση του χοληδόχου πόρου, τα οποία όμως παρέμειναν χαμηλά στις άλλες δύο περιοχές. Καθώς είναι γνωστό πως το οξειδωτικό στρες έχει ενοχοποιηθεί στην παθογένεση διαφόρων νευροεκφυλιστικών παθήσεων στον άνθρωπο, τα ευρήματα αυτά θα μπορούσαν να συσχετισθούν με διαφορές στη φυσιολογία και τη βιοχημεία των περιοχών αυτών και ενδεχομένως να σχετίζονται με τον τρόπο που το οξειδωτικό στρες εκφράζεται στην παθοφυσιολογία και άλλων νοσολογικών καταστάσεων όπως η πλάγια αμυοτροφική σκλήρυνση, η νόσος Parkinson, και η νόσος Alzheimer. Έχει ενδιαφέρον το γεγονός ότι τα βασικά γάγγλια και οι πυρήνες του στελέχους είναι θέσεις εκλεκτικής βλάβης στην εγκεφαλοπάθεια από χολερυθρίνη στο νεογνικό ίκτερο.
Στη δεύτερη πειραματική φάση, στους ικτερικούς αρουραίους χορηγήσαμε αντιοξειδωτικούς παράγοντες, που έχουν επανειλημμένα μελετηθεί τόσο in vitro όσο και σε ζωικά μοντέλα, σε μια προσπάθεια να αναστρέψουμε τις διαταραχές που είχαν παρατηρηθεί. Ένα πρώτο εύρημα ήταν η ευεργετική δράση στην υπεροξείδωση των λιπιδίων, που ποσοτικοποιήθηκε με τον υπολογισμό της MDA, στις ομάδες και των τριών αντιοξειδωτών αλλά κυρίως στις ALP και Vit-E. Και στις τρεις ομάδες που έλαβαν αντιοξειδωτικά, αντίθετα με την ομάδα απολίνωσης χοληδόχου πόρου, δεν παρατηρήθηκε εξάντληση του συνολικού κυτταρικού περιεχόμενου γλουταθειόνης. Επιπλέον, και στις τρεις ομάδες αντιοξειδωτικών δεν παρατηρήθηκε αύξηση των NPSSR στο εγκεφαλικό στέλεχος, γεγονός που υποδηλώνει ότι η συσσώρευση των μη-πρωτεϊνικών δισουλφιδίων στο στέλεχος εμποδίστηκε από τους αντιοξειδωτικούς παράγοντες. Η ανισορροπία των πρωτεϊνικών θειολών, όπως αυτή φάνηκε από τη μείωση της PSH και την αύξηση του PSSP, αναστράφηκε σημαντικά μόνο στην ομάδα NAC στην οποία η PSH αυξήθηκε στα φυσιολογικά επίπεδα.
Εν συντομία, η παρούσα μελέτη είναι η πρώτη που καταδεικνύει με σαφήνεια το οξειδωτικό στρες στον εγκέφαλο στο μοντέλο του αποφρακτικού ίκτερου και μάλιστα αρκετά πρώιμα. Χρησιμοποιεί μια συστοιχία βιοχημικών δεικτών που περιγράφουν την θειολική αναγωγική κατάσταση και την υπεροξείδωση των λιπιδίων και μελετά τις ευεργετικές επιδράσεις γνωστών αντιοξειδωτικών παραγόντων στον πειραματικό αποφρακτικό ίκτερο. Τα αποτελέσματά της θα μπορούσαν να προσφέρουν κάποια βοήθεια στην κατανόηση ορισμένων μηχανισμών της ηπατικής εγκεφαλοπάθειας στους ανθρώπους. Μελλοντικές έρευνες θα απαντήσουν ερωτήματα σχετικά με τα γενεσιουργά αίτια του οξειδωτικού στρες, την ίδια την παρουσία των ελευθέρων ριζών και την παθοφυσιολογία του φαινομένου. / Hepatic encephalopathy is a complex neuropsychiatric syndrome that has been associated with acute and chronic liver diseases. Accumulated evidence over the last several years has implicated oxidative stress a key factor in the pathogenesis of hepatic encephalopathy. These studies utilize measurements of lipid peroxidation products and glutathione (GSH) and its oxidized disulfide GSSG.
A model of experimental obstructive jaundice after ligation of the biliary duct has been used in the present study. Male Wistar rats were divided into control, sham operated and bile duct ligated groups that were sacrificed either on the 5th or the 10th day, or they have been treated with antioxidant agents (NAC, ALP, Vit-E). Subsequently, the thiol redox state of various areas (cortex, midbrain and cerebellum) of the rat brain and the effect of selected antioxidants were studied.
For the first time specific markers of high oxidative stress (GSSG, NPSSR, NPSSC, PSSR, PSSC, PSSP, lipid peroxides) and low oxidative stress (GSH, CSH, PSH) were quantified providing a more detailed assessment of the phenomenon. Our results show increase in the first and decrease in the latter group of markers in all studied brain areas, therefore demonstrating high oxidative stress in the obstructive jaundice.
The major impact of the present study is the demonstration of early signs of oxidative stress in the brain. Using this battery of biochemical markers, deviations from control and sham animals occurred as early as 5 days following bile duct ligation; by the 10th day the majority of these changes became statistically significant.
It was also observed that GSH values in cerebral cortex were twice as high as those in midbrain and cerebellum and a dramatic increase in the levels of NPSSR on the 10th day after bile duct ligation in midbrain that was not observed in the other brain areas. These findings could be attributed to specificities of metabolic or biochemical status of neurons and astrocytes and alterations of blood-brain barrier permeability in different brain areas and probably should be taken into account in further studies, since, as we know, oxidative stress has been implicated in the pathogenesis of many human diseases like Parkinson’s , Alzheimer’s and Amyotrophic Lateral Sclerosis (ALS). It is of interest that basal ganglia and brainstem nuclei are, as well, the sites of selective damage in bilirubin encephalopathy in jaundiced neonates.
Jaundiced rats were treated with agents that have frequently been used in vitro and in vivo for their antioxidant effects, in an effort to reverse the observed alterations in redox state. In the treated groups, there was no decrease in the total cell glutathione content unlike the bile duct ligated rats. There was also no significant difference in the levels of lipid peroxidation as compared with control and sham groups. The imbalance of protein thiols demonstrated by the decrease of PSH and the increase of PSSP was considerably reversed only in the NAC group. In all treated groups, no NPSSR increase was found suggesting that the antioxidant agents suppressed the accumulation of non-protein disulfides in the midbrain.
In brief, this experimental study demonstrates the oxidative profile of the brain associated with obstructive jaundice at an early and later stage. A battery of biochemical markers that define the thiol redox state is utilized and the beneficial effects of known antioxidants are examined. The evidence presented supports the concept that oxidative stress is neither a uniform matter affecting brain in a general way nor that any antioxidant agent could prevent damage by enhancing equally well different defence system. It is also likely that oxidative stress is one of the important mechanisms of jaundice-induced encephalopathy. Further studies could provide with more evidence on the pathogenetic mechanisms and generative causes of the oxidative stress in obstructive jaundice.
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Effect of Dietary Antioxidants on Oxidative Stress, Inflammation and Metabolic Factors : Studies in Subjects with Overweight and with Type 2 DiabetesRytter, Elisabet January 2011 (has links)
Observational studies have indicated that fruit and vegetables, and dietary antioxidants may play an important role in reducing the risk of chronic diseases, potentially by affecting pathogenic mechanisms such as oxidative stress and inflammation. Clinical trials investigating the effects of supplementation with single or a few antioxidants in high doses have, however, shown inconsistent results and thus have not been able to support the observational findings. It was therefore hypothesised that a supplement, containing a combination of antioxidants mainly extracted from fruit and vegetables, and supplied at moderate doses, might act more beneficially than single antioxidants given at pharmacological doses. The effects of such a supplement were investigated in two interventional studies described in this thesis. The effects on antioxidant status, metabolic control, oxidative stress and inflammation were investigated in overweight men and in patients with type 2 diabetes, subjects that could be expected to have elevated levels of oxidative stress and inflammatory activity. The results of the studies did not support the hypothesis that supplementation with antioxidants from fruit and vegetables may have beneficial effects by counteracting oxidative stress and inflammation, despite markedly increased plasma antioxidant concentrations. However, interesting associations were observed in diabetes patients at baseline between intake of antioxidant rich food as well as levels of antioxidants in plasma, and markers of oxidative stress and inflammation. These associations are compatible with the hypothesis that a high intake of fruit and vegetables and dietary antioxidants decrease oxidative stress levels, have anti-inflammatory effects and a beneficial influence on glycaemic control. The results also indicated that glycaemic control may affect the level of oxidative stress. The absence of beneficial effects from antioxidants might to some extent be explained by the initial levels of oxidative stress and inflammation and by the antioxidative status in the subjects included in the studies. Since the levels generally were comparable with those observed in healthy subjects, this might have decreased the ability to observe any beneficial effects of supplementation with additional antioxidants. Continued investigations are needed to characterise the individuals who potentially might benefit from antioxidant supplementation. In view of apparent positive effects from a high intake of fruit and vegetables found in observational studies and until more knowledge is available from interventional trials about possible benefits and potential risks of antioxidant supplementation it still seems reasonable to recommend a diet rich in fruit and vegetables.
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Nutrition parentérale du nouveau-né : modulation du stress oxydant et conséquences hépatiquesMiloudi, Khalil 10 1900 (has links)
Introduction : Les enfants prématurés ont la particularité de naître alors que leur développement est souvent incomplet et nécessite la mise en œuvre de soins intensifs visant à poursuivre leur croissance en dehors de l’environnement utérin. Souvent cependant, le stade développemental de l’enfant ne lui permet pas d’assimiler une alimentation entérale du fait de l’immaturité de son système digestif. Le recours à une voie centrale délivrant les nutriments assurant le développement devient alors une nécessité. Ce type de nutrition, appelée nutrition parentérale (NP, ou total parenteral nutrition TPN), permet l’administration de molécules simples, directement dans le sang du prématuré. Il n’est toutefois pas exempt de risques puisqu’exposée à la lumière, la NP peut s’oxyder et générer des molécules oxydantes telles que des hydroperoxydes lipidiques susceptibles de se fragmenter par la suite en hydroxy-alkénals. Ceci devient problématique au vu de l’immaturité des systèmes de défenses antioxydants du nouveau-né prématuré. L’utilisation prolongée de la NP est d’ailleurs à l’origine de maladie hépatiques dans lesquelles le stress oxydant et la nécro-inflammation sont des composantes majeures. Nous avons émis l’hypothèse que l’infusion chez les enfants prématurés, d’aldéhydes d’origine lipidique est en relation avec le développement du stress oxydant et de l’inflammation hépatique. Objectif : Notre étude a consisté à évaluer la relation entre les quantités d’hydroxy-alkénals dans la NP et les effets hépatiques engendrés sur les marqueurs de stress oxydant et les voies de signalisation responsables d’une induction de processus inflammatoire. Dans ce but, nous avons cherché à mesurer la peroxydation lipidique dans l’émulsion lipidique de la NP et la conséquence de l’infusion en continue d’hydroxy-alkénals sur les marqueurs de stress oxydant, sur la voie de signalisation médiée par le Nuclear Factor κB et sur le déclenchement du processus inflammatoire hépatique. A la suite de ce travail, nous avons également travaillé sur des alternatives à la photoprotection, qui est la seule méthode réellement optimale pour réduire la peroxydation des lipides de la NP, mais cliniquement difficilement praticable. Résultats : Nos résultats ont mis en évidence la génération de 4-hydroxynonenal in vitro dans la NP, ce phénomène est augmenté par une exposition lumineuse. Dans ce cadre, nous avons montré l’inefficacité de l’ajout de multivitamines dans l’émulsion lipidique comme alternative à la photoprotection. Dans la validation biologique qui a suivi sur un modèle animal, nos résultats ont permis de démontrer que l’augmentation des adduits glutathion-hydroxynonenal était imputable à l’augmentation de 4-hydroxynonenal (4-HNE) dans la NP, et non à une peroxydation endogène. Nos données indiquent que la probable augmentation hépatique des niveaux de 4-HNE a conduit à une activation du NFκB responsable de l’activation de la transcription des gènes pro-inflammatoires du Tumour Necrosis Factor-α (TNF-α) et de l’interleukine-1 (IL-1). Nous avons alors évalué la capacité d’une émulsion lipidique enrichie en acides gras polyinsaturés (AGPI) n-3 à baisser les concentrations de 4-HNE dans la NP, mais également à moduler le stress oxydant et les marqueurs pro-inflammatoires. Enfin, nous avons démontré, en collaboration avec l’équipe du Dr Friel, que certains peptides isolés du lait humain (par un processus mimant la digestion) permettent également une modulation du stress oxydant et du processus inflammatoire. Conclusion : Le stress oxydant exogène issu de la NP a conduit par activation de facteurs de transcription intra-hépatiques au déclenchement d’un processus inflammatoire potentiellement responsable du développement de maladies hépatiques reliées à la NP telle que la cholestase. Dans ce sens, les AGPI n-3 et les peptides antioxydants peuvent se poser en tant qu’alternatives crédibles à la photoprotection. / Introduction: Premature infants usually born before full term require intensive care to continue to grow up outside the uterine environment. Premature newborns are born with gastrointestinal systems that are too immature to absorb nutrients safely. Therefore they receive their initial nutrients through intravenous feeding, called total parenteral nutrition which delivers simple nutrients directly into bloodstream. However, light exposed-TPN can generate oxidant molecules such as lipid hydroperoxides, which can potently break up into hydroxy-alkenals. Prolonged use of TPN is also a cause of liver disease in which oxidative stress and necro-inflammation are major components. Thus, we hypothesize that lipid aldehydes contained in TPN are associated with oxidative stress and hepatic inflammation developments. Objectives: The aim of our study is to assess the relationship between quantities of hydroxyl-alkenals generated in TPN and effects on oxidative stress biomarkers and cell-signalling pathways molecules implicated in hepatic inflammation induction. To this end, we measure lipid peroxidation in the TPN lipid emulsion in and the consequence of continuous infusion of hydroxy-alkenals on markers of oxidative stress, on cell-signaling pathway mediated by the NFkB, and on liver inflammation induction. Following these data, we also worked on alternatives of photoprotection, which is the only optimal method for preventing lipid peroxidation, but unfortunately clinically impractical.
Results: In vitro studies have highlighted the generation of 4-HNE in the TPN, increased under light exposure. In this context, we have demonstrated that the addition of multivitamins in the lipid emulsion cannot be a valuable alternative to photoprotection. Concerning the biological validation in our guinea pig animal model, our results demonstrated that the increase of GS-HNE adducts was due to increased 4-HNE in the TPN, and does not provide from endogenous peroxidation. Our data also indicate that the increase of hepatic 4-HNE led to an activation of NFkB, responsible for the activation of the transcription of proinflammatory genes TNF-α, IL-1. In the next study, we have evaluated the ability of a lipid emulsion enriched with n-3 polyunsaturated fatty acids (PUFA) to reduce 4-HNE concentrations generated in TPN, and to modulate oxidative stress markers and pro-inflammatory process on the same animal model. We also have demonstrated, in collaboration with Dr Friel’s team, that two antioxidant peptides (derived from a process mimicking digestion process of human milk) allow also a modulation of oxidative stress and inflammatory process in the liver. Conclusion: This form of exogenous oxidative stress from the TPN led to an inflammatory process resulting from the activation of intrahepatic transcription, which is potentially responsible of liver disease development such as cholestasis. In this sense, the n-3 PUFA and antioxidant peptides may arise as a valuable alternative of photoprotection.
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Impact of vanadium stress on physiological and biochemical characteristics in heavy metal susceptible and tolerant BrassicaceaeGokul, Arun January 2013 (has links)
There is an influx in heavy metals into soils and ground water due to activities
such as increased mineral mining, improper watering and the use of heavy metal
contaminated fertilizers. These heavy metals are able to increase the ROS species within plants which may result in plant metabolism deterioration and tissue damage. Heavy metals may also directly damage plants by rendering important
enzymes non-functional through binding in metal binding sites of enzymes. The
heavy metal focused on in this study was vanadium due to South Africa being
one of the primary produces of this metal. Two related Brassica napus L cultivars
namely Agamax and Garnet which are economically and environmentally
important to South Africa were exposed to vanadium. Physiological experiments
such as cell death, chlorophyll and biomass determination were conducted to
understand how these cultivars were affected by vanadium toxicity. A low cost,
sensitive and robust vanadium assay was developed to estimate the amount of
vanadium in samples such as water, soils and plant material. The oxidative state
as well as the antioxidant profile of the two cultivars were also observed under
vanadium stress. A chlorophyll assay which was conducted on the two cultivars
xiv exposed to vanadium showed a marked decrease in chlorophyll A in the
suspected sensitive cultivar which was Garnet. However, the suspected tolerant
cultivar Agamax fared better and the decrease in chlorophyll A was much less. A
similar trend was observed for the two cultivars when the cell death assay was
conducted. The vanadium assay showed that Garnet had higher concentrations
of vanadium within its leaves and lower concentrations in its roots when
compared to Agamax. This observation displayed that Agamax had inherent
mechanisms which it used to localize vanadium in its roots and which assisted in
its tolerance to the vanadium stress. The oxidative state was determined by doing assays for the specific reactive oxygen species namely hydrogen peroxide and superoxide. It was observed that vanadium treated Garnet leaves had higher reactive oxygen species (ROS) production when compared to the Agamax treated leaves. In-gel native PAGE activity gels were conducted to determine the antioxidant profile for the two cultivars which were exposed to vanadium. The antioxidant enzymes which were under investigation were ascorbate peroxide (APX), superoxide dismutase (SOD) and glutathione-dependent peroxidases (GPX-like) as these enzymes are known to be responsible for controlling the ROS produced in the plants. The GPX-like profile consisted of three isoforms. No isoforms were inhibited by vanadium treatments but one isoform had increased activity in both the Garnet and Agamax treated samples. The SOD profile for Garnet consisted of six isoforms xv and Agamax had seven isoforms. One isoform which was visualized in both Agamax as well as Garnet was inhibited by vanadium treatments. Agamax also had two isoforms which were up-regulated however the corresponding isoforms in Garnet showed no change. The Ascorbate peroxidase profile consisted of seven isoforms for both Garnet and Agamax. No isoforms were inhibited by vanadium treatment. Three isoforms were up-regulated in Garnet and Agamax under vanadium treatments. Here, it is illustrated that Garnet lacked certain mechanisms found in Agamax (and thus experienced more cell death, yield and chlorophyll loss) and performed worst under high vanadium concentrations. Although Garnet increased the activity of some of its antioxidant isoforms in response to increasing ROS levels it was not adequate to maintain a normal oxidative homeostasis. This disruption in oxidative homeostasis lead to plant damage. Agamax was observed to produce less ROS than Garnet and was able to control the ROS produced more effectively than Garnet and thus less damage was observed in Agamax. / Magister Scientiae - MSc
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Preparation, stability and in vitro evaluation of liposomes containing chloroquine / Stephnie NieuwoudtNieuwoudt, Stephnie January 2010 (has links)
Malaria is currently a huge treat worldwide, as far as infections are concerned, and is
responsible for thousands of deaths per annum. The dilemma associated with the development
of anti–malarial drug resistance over the past few decades should be addressed as a matter of
urgency. Novel drug delivery systems should be developed in order to employ new and existing
anti–malarial drugs in the treatment and management of malaria. The aim of these delivery
systems should include an improvement in the efficacy, specificity, acceptability and therapeutic
index of anti–malarial drugs.
Previous studies have suggested that liposomes have the ability to encapsulate, protect and to
promote the sustained release of anti–malarial drugs. Two liposome formulations, namely
liposomes and chloroquine entrapped in liposomes, were formulated during this thesis and
evaluated by conducting a stability study and an in vitro study with the main focus on cell
viability.
The stability study consisted of a series of stability tests regarding the stability of nine liposome
and nine chloroquine entrapped in liposome formulations over a period of twelve weeks. The in
vitro study included three assays such as a reactive oxygen species assay, a lipid peroxidation
assay and a hemolysis assay. The aims of these studies included the manufacturing of
liposomes, the incorporation of chloroquine into liposomes, the determination of the stability of
the formulations as well as the evaluation of the possible in vitro toxicity of liposomes.
Results obtained from these studies revealed that liposomes remained more stable over the
stability study period in comparison to chloroquine entrapped in liposomes. The entrapment of
chloroquine within liposomes was possible, although the initial entrapment efficiency (%) of
14.55 % was much too low. The production of reactive oxygen species occurred to a small
extent in the red blood cells and the infected red blood cells. Equal amounts of reactive oxygen
species (%) was observed within both the red blood cells and the infected red blood cells with a
maximum value of 23.27 % in the presence of the chloroquine entrapped in liposomes at
varying concentrations. Red blood cells experienced the highest degree of lipid peroxidation
(%) in the presence of chloroquine, at varying concentrations, entrapped in liposomes. The
maximum amount of lipid peroxidation (%) was 79.61 %. No significant degree of hemolysis
(%) was observed in the red blood cells neither in the presence of the liposomes nor in the
presence of the chloroquine entrapped in liposomes at varying concentrations.
It can be concluded that liposomes are a more stable formulation and have less toxic effects on
red blood cells and infected red blood cells in comparison to the chloroquine entrapped in liposome formulations. Future studies should investigate the possibility of a more stable and
less toxic chloroquine entrapped in liposome formulation. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.
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Preparation, stability and in vitro evaluation of liposomes containing amodiaquine / Jacques C. ScholtzScholtz, Jacques Coenraad January 2010 (has links)
Malaria is a curable disease that claims nearly one million lives each year. Problems with the treatment of malaria arise as resistance spreads and new treatment options are becoming less effective. The need for new treatments are of the utmost importance. Liposomes combined with antimalarials are a new avenue for research as liposomes can increase the efficacy of drugs against pathogens, as well as decreasing toxicity. Amodiaquine is a drug with known toxicity issues, but has proven to be effective and is, therefore, a prime candidate to be incorporated into the liposomal drug delivery system.
The aim of this study was to prepare, characterize and evaluate the toxicity of the liposomes with incorporated amodiaquine. The solubility of amodiaquine was determined and liposomes formulated with, and without, amodiaquine entrapped. Accelerated stability studies (at 5 'C, 25 'C with relative humidity of 60% and 40 'C with a relative humidity of 40%) were conducted during which the size, pH, morphology and the entrapment efficacy was determined. The toxicity was determined in vitro by analysing the levels of reactive oxidative species and lipid peroxidation caused by the formulations to erythrocytes infected with P. falciparum as well as uninfected erythrocytes with flow cytometry.
The solubility study of amodiaquine in different pH buffers showed that amodiaquine was more soluble at lower pH values. Solubility in solution with pH 4.5 was 36.3359 ± 0.7904mg/ml when compared to the solubility at pH 6.8, which was 15.6052 ± 1.1126 mg/ml. A buffer with a pH of 6 was used to ensure adequate solubility and acceptable compatibility with cells. Liposomes with incorporated amodiaquine were formulated with entrapment efficacies starting at 29.038 ± 2.599% and increasing to 51.914 ± 1.683%. The accelerated stability studies showed the median sizes and span values remained constant for both liposome and amodiaquine incorporated liposomes at 5 'C. The higher temperatures, i.e. 25 'C and 40 'C, displayed increases in the median size, and decreases in the span for both formulations. The conclusion can, therefore, be made that both liposome and amodiaquine incorporated liposomes are stable at lower temperatures. The entrapment efficacy increased from initial values to nearly 100% during the course of the stability study. This was attributed to amodiaquine precipitating from the solution. The pH values of the liposomes and amodiaquine incorporated liposomes remained constant for each formulation; though the amodiaquine incorporated liposomes had a lower starting pH, the formulations are both thought to be stable in terms of the pH.
Toxicity studies revealed low levels of reactive oxygen species as well as low levels of lipid peroxidation for both liposome and amodiaquine incorporated liposomes, on both erythrocyte and Plasmodium infected erythrocytes. From the toxicity studies it can be concluded that liposomes and amodiaquine incorporated liposomes are not toxic to erythrocytes and infected erythrocytes.
It was concluded that liposomes incorporating amodiaquine could possibly be used as a treatment option for malaria. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.
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Effects of Antioxidants and Pro-oxidants on Oxidative Stress and DNA Damage using the Comet Assay : Studies on Blood Cells from Type 2 Diabetes Subjects and Mouse Lymphoma CellsÅsgård, Rikard January 2014 (has links)
Diet and oral supplements comprise two distinct sources of antioxidants known to prevent oxidative stress. Beneficial effects from antioxidants have been seen for patients at risk for type 2 diabetes. The aim of this thesis was to evaluate the positive effects of antioxidants against oxidative stress and DNA damage in type 2 diabetes subjects. We also used antioxidants as tools to determine the mechanisms behind genotoxicity induced by mutagenic pro-oxidative agents in mouse lymphoma cells. Several techniques were used to measure oxidative stress and DNA damage, but the main technique used was alkaline comet assay. The results showed that the fruit and vegetable intake was inversely related to oxidative stress in type 2 diabetes subjects. However, oral supplementary intake of 20 antioxidants did not decrease oxidative stress biomarkers. In studies on mouse lymphoma cells, using the alkaline comet assay, DNA damage was induced by catechol and o-phenylenediamine (OPD), while 4-nitro-o-phenylenediamine (4-NOPD) induced only oxidative damage, showing different mechanisms of action behind the mutagenicity of the compounds. Also, oxidative stress was induced by catechol and 4-NOPD, whereas imbalances in the nucleotide pool were seen after exposure to OPD or 4-NOPD. Addition of antioxidants together with these pro-oxidants showed that β-carotene was able to reduce DNA damage at low concentrations of catechol, but increased DNA damage at high concentration. In comparison, addition of α-tocopherol slightly decreased catechol-induced DNA damage at all concentrations of catechol. However, no effect of α-tocopherol was seen on OPD-or 4-NOPD-induced DNA damage. In conclusion, antioxidants from fruits and vegetables, but not from oral supplements, reduced oxidative stress in type 2 diabetes patients, suggesting fruits and vegetables being a healthier source for antioxidant-intake, as compared to oral supplements. Different mechanisms of action for mutagenic pro-oxidants were shown in mouse lymphoma cells, introducing the nucleotide pool as an interesting target for oxidative stress. Reduction of catechol-induced DNA damage by β-carotene or α-tocopherol was shown, with a pro-oxidative action of β-carotene at high concentration of catechol, Interestingly, α-tocopherol was not able to decrease OPD- or 4-NOPD-induced DNA damage, supporting different mechanisms of action behind the genotoxicity from the three pro-oxidants.
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Preparation, stability and in vitro evaluation of liposomes containing chloroquine / Stephnie NieuwoudtNieuwoudt, Stephnie January 2010 (has links)
Malaria is currently a huge treat worldwide, as far as infections are concerned, and is
responsible for thousands of deaths per annum. The dilemma associated with the development
of anti–malarial drug resistance over the past few decades should be addressed as a matter of
urgency. Novel drug delivery systems should be developed in order to employ new and existing
anti–malarial drugs in the treatment and management of malaria. The aim of these delivery
systems should include an improvement in the efficacy, specificity, acceptability and therapeutic
index of anti–malarial drugs.
Previous studies have suggested that liposomes have the ability to encapsulate, protect and to
promote the sustained release of anti–malarial drugs. Two liposome formulations, namely
liposomes and chloroquine entrapped in liposomes, were formulated during this thesis and
evaluated by conducting a stability study and an in vitro study with the main focus on cell
viability.
The stability study consisted of a series of stability tests regarding the stability of nine liposome
and nine chloroquine entrapped in liposome formulations over a period of twelve weeks. The in
vitro study included three assays such as a reactive oxygen species assay, a lipid peroxidation
assay and a hemolysis assay. The aims of these studies included the manufacturing of
liposomes, the incorporation of chloroquine into liposomes, the determination of the stability of
the formulations as well as the evaluation of the possible in vitro toxicity of liposomes.
Results obtained from these studies revealed that liposomes remained more stable over the
stability study period in comparison to chloroquine entrapped in liposomes. The entrapment of
chloroquine within liposomes was possible, although the initial entrapment efficiency (%) of
14.55 % was much too low. The production of reactive oxygen species occurred to a small
extent in the red blood cells and the infected red blood cells. Equal amounts of reactive oxygen
species (%) was observed within both the red blood cells and the infected red blood cells with a
maximum value of 23.27 % in the presence of the chloroquine entrapped in liposomes at
varying concentrations. Red blood cells experienced the highest degree of lipid peroxidation
(%) in the presence of chloroquine, at varying concentrations, entrapped in liposomes. The
maximum amount of lipid peroxidation (%) was 79.61 %. No significant degree of hemolysis
(%) was observed in the red blood cells neither in the presence of the liposomes nor in the
presence of the chloroquine entrapped in liposomes at varying concentrations.
It can be concluded that liposomes are a more stable formulation and have less toxic effects on
red blood cells and infected red blood cells in comparison to the chloroquine entrapped in liposome formulations. Future studies should investigate the possibility of a more stable and
less toxic chloroquine entrapped in liposome formulation. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.
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Preparation, stability and in vitro evaluation of liposomes containing amodiaquine / Jacques C. ScholtzScholtz, Jacques Coenraad January 2010 (has links)
Malaria is a curable disease that claims nearly one million lives each year. Problems with the treatment of malaria arise as resistance spreads and new treatment options are becoming less effective. The need for new treatments are of the utmost importance. Liposomes combined with antimalarials are a new avenue for research as liposomes can increase the efficacy of drugs against pathogens, as well as decreasing toxicity. Amodiaquine is a drug with known toxicity issues, but has proven to be effective and is, therefore, a prime candidate to be incorporated into the liposomal drug delivery system.
The aim of this study was to prepare, characterize and evaluate the toxicity of the liposomes with incorporated amodiaquine. The solubility of amodiaquine was determined and liposomes formulated with, and without, amodiaquine entrapped. Accelerated stability studies (at 5 'C, 25 'C with relative humidity of 60% and 40 'C with a relative humidity of 40%) were conducted during which the size, pH, morphology and the entrapment efficacy was determined. The toxicity was determined in vitro by analysing the levels of reactive oxidative species and lipid peroxidation caused by the formulations to erythrocytes infected with P. falciparum as well as uninfected erythrocytes with flow cytometry.
The solubility study of amodiaquine in different pH buffers showed that amodiaquine was more soluble at lower pH values. Solubility in solution with pH 4.5 was 36.3359 ± 0.7904mg/ml when compared to the solubility at pH 6.8, which was 15.6052 ± 1.1126 mg/ml. A buffer with a pH of 6 was used to ensure adequate solubility and acceptable compatibility with cells. Liposomes with incorporated amodiaquine were formulated with entrapment efficacies starting at 29.038 ± 2.599% and increasing to 51.914 ± 1.683%. The accelerated stability studies showed the median sizes and span values remained constant for both liposome and amodiaquine incorporated liposomes at 5 'C. The higher temperatures, i.e. 25 'C and 40 'C, displayed increases in the median size, and decreases in the span for both formulations. The conclusion can, therefore, be made that both liposome and amodiaquine incorporated liposomes are stable at lower temperatures. The entrapment efficacy increased from initial values to nearly 100% during the course of the stability study. This was attributed to amodiaquine precipitating from the solution. The pH values of the liposomes and amodiaquine incorporated liposomes remained constant for each formulation; though the amodiaquine incorporated liposomes had a lower starting pH, the formulations are both thought to be stable in terms of the pH.
Toxicity studies revealed low levels of reactive oxygen species as well as low levels of lipid peroxidation for both liposome and amodiaquine incorporated liposomes, on both erythrocyte and Plasmodium infected erythrocytes. From the toxicity studies it can be concluded that liposomes and amodiaquine incorporated liposomes are not toxic to erythrocytes and infected erythrocytes.
It was concluded that liposomes incorporating amodiaquine could possibly be used as a treatment option for malaria. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.
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L'acide carnosique et le carnosol, deux super-antioxydants du romarin (Rosmarinus officinalis) : rôles, mécanismes, physiologie et applications / Carnosic acid and carnosol, two supra-antioxidant of rosemary (Rosmarinus officinalis) : roles, mecanisms, physiology and applicationsLoussouarn-Yvon, Margot 07 November 2017 (has links)
L’acide carnosique (CA) et le carnosol (CARN), deux diterpènes spécifiques des Lamiacées, sont en abondance dans le romarin. Le CA extrait de cette plante est largement utilisé dans l’industrie pour ses propriétés antioxydantes portées par le groupe catéchol. Malgré beaucoup d’applications, les rôles et les modes d'action de ces composés in planta n’ont reçu que peu d’attention. Des analyses par HPLC-UV et imagerie d’autoluminescence révèlent que le CA et le CARN protègent les lipides contre des oxydations in vitro par les ERO. Lors de la préservation des lipides, des analyses de MS indiquent que le CA est oxydé en une variété de dérivés alors que le CARN résiste. L’utilisation d’une sonde de spin et de la spectroscopie RPE montre que le CA est un piégeur chimique des ERO. L’action inhibitrice du CARN sur des oxydations de lipides induites in vitro ou in vivo indique que le CARN interfère avec le processus de peroxydation lipidique. Des études in vivo de deux variétés de romarin contrastées en CA exposées à un stress photooxydant montrent que le CA protège les lipides in planta. Une étude des variations de CA et de CARN en fonctions des facteurs abiotiques met en avant qu’une forte intensité lumineuse et des fluctuations de températures favorisent la réponse antioxydante du CA qui s’oxyde en CARN. Des analyses de RT-qPCR montrent que les facteurs abiotiques ne stimulent pas la voie de biosynthèse du CA. En condition de stress oxydant, le CA du romarin préserve les membranes par piégeage des ERO produisant des dérivés d’oxydation, dont le CARN, qui protègent aussi les membranes en bloquant la réaction en chaîne de la peroxydation lipidique. / Carnosic acid (CA) and carnosol (CARN), two diterpenes specific of the Lamiaceae, are highly abundant in rosemary species. CA extracted from rosemary is used by industries for its antioxidative features, endowed by it catechol group. Despite numerous applications, the role and the mode of action of CA in planta has received little attention. Analyses, using HPLC-UV and luminescence imaging revealed that CA and CARN protect lipids from in vitro oxidation by ROS. Upon ROS oxidation of lipids, MS analyses indicated that CA was oxidized into various derivatives while CARN resisted. Using spin probes and EPR detection, we confirmed that CA, rather than CARN, is a ROS quencher. The inhibitory effect of CARN on lipid peroxidation induced in vitro or in vivo indicated that CARN interferes with lipid peroxidation. In vivo studies of two rosemary varieties contrasted in their CA content exposed to photooxidative stress showed that CA protects lipids in planta. A study of CA and CARN variations in response to abiotic factors showed that high light and temperature fluctuations lead to CA oxidation into CARN. RT-qPCR analyses revealed that abiotic factors do not stimulate CA biosynthesis genes. Under oxidative stress condition, rosemary CA preserves biological membranes by ROS scavenging, hence producing a set of oxidative derivatives, including CARN, which protect biological membranes by blocking the lipid peroxidation chain reaction.
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