Το πρόβλημα με τις μυκοτοξίνες είναι σημαντικό και δικαιολογημένα προκαλεί ανησυχίες. Αναφέρεται ως παγκόσμιος κίνδυνος και θεωρείται ως μία από τις πλέον σοβαρές προκλήσεις για την ασφάλεια των τροφίμων, την υγεία των ανθρώπων, των ζώων, και για τη σύγχρονη τοξικολογία. Με τον «όρο» μυκοτοξίνες εννοούμε τοξίνες οι οποίες παράγονται από μύκητες. Συγκεκριμένα πρόκειται για προϊόντα δευτερογενούς μεταβολισμού των μυκήτων (Aspergillus spp Fusarium spp, Penicillium spp, κ.α). Υπάρχει μία πληθώρα από διάφορες μυκοτοξίνες οι οποίες απαντώνται σε πολλές τροφές, όπως στο γάλα, στα δημητριακά, στους ξηρούς καρπούς, στα αποξηραμένα φρούτα, στο αλεύρι κ.α. που καταναλώνουν καθημερινά οι άνθρωποι, αλλά και σε ζωικές τροφές. ωστόσο μόνο για κάποιες από αυτές υπάρχουν τεκμηριωμένες μελέτες ενώ ακόμα λιγότερες είναι αυτές, για τις οποίες έχουν προσδιοριστεί τα νόμιμα επιτρεπτά όρια συγκέντρωσης στις τροφές, που καταναλώνονται καθημερινά. Οι μυκοτοξίνες θεωρούνται γενικά επικίνδυνες ενώσεις που παράγονται από ορισμένα είδη μυκήτων, οι οποίοι αναπτύσσονται σε προϊόντα αγροτικών καλλιεργειών είτε πριν τη συγκομιδή, είτε κατά την αποθήκευσή τους σε γεωργικές εγκαταστάσεις και παραμένουν δραστικές για μεγάλο χρονικό διάστημα και μετά την καταστροφή των μυκήτων από τους οποίους προήλθαν. Η εμφάνισή τους στα τρόφιμα και στα ποτά έχει αναγνωριστεί ως απειλή για την υγεία τόσο του ανθρώπου όσο και των ζώων, είτε αυτή προέρχεται από την άμεση μόλυνση των φυτικών ιστών, είτε από προϊόντα που έχουν παραχθεί από αυτά, είτε από την μεταφορά των μυκοτοξινών και των μεταβολιτών τους στους ζωικούς ιστούς, και κατά συνέπεια στο γάλα, στα αυγά και αλλού. Μερικές από αυτές τις μυκοτοξίνες, όπως για παράδειγμα οι αφλατοξίνες, παρουσιάζουν εξαιρετικά υψηλή τοξικότητα, γεγονός που τις καθιστά ενώσεις που χρήζουν ιδιαίτερη προσοχή. ως εκ τούτου κρίνεται απαραίτητο όλα τα αγροτικά προϊόντα που προορίζονται για τον άνθρωπο ή για ζωοτροφές να υποβάλλονται σε συνεχή και σχολαστικό έλεγχο. Αντικείμενο μελέτης της παρούσας εργασίας είναι να μελετήσει τις παραμέτρους (θερμοκρασία και υγρασία) οι οποίοι επηρεάζουν την ανάπτυξη της μυκοτοξίνης ζεαραλενόνης χρησιμοποιώντας ως υπόστρωμα αλεύρι από αποθηκευμένο αραβόσιτο. Στόχος ήταν ο έλεγχος των συνθηκών καλλιέργειας, αποθήκευσης και συντήρησης των πρωτογενών γεωργικών προϊόντων έτσι ώστε να ελαττώνεται η ανάπτυξη μυκοτοξινών στα γεωργικά προϊόντα και στα τρόφιμα. Η δραστηριότητα βοήθησε στη διερεύνηση του μηχανισμού δράσης των συνθηκών που οδηγούν στην ανάπτυξη μυκοτοξινών τόσο κατά την αποθήκευση της πρώτης ύλης, όσο και κατά τα στάδια επεξεργασίας, παραγωγής και τυποποίησης του τελικού προϊόντος. / The problem with mycotoxins is a significant and legitimate concern. Referred to as global risk and is considered one of the most serious challenges to food security, human health, animals, and modern toxicology. With the "average" mean toxins mycotoxins produced by fungi. It is a secondary metabolic products of fungi (Aspergillus spp Fusarium spp, Penicillium spp, etc.). There is a plethora of different mycotoxins found in many foods such as milk, cereals, nuts, dried fruits, flour, etc. consumed daily by people, but also in animal feeds. But only some of them are documented studies and even fewer are those, which have been identified, legally permissible concentration limits in foods consumed daily. The mycotoxins are generally considered harmful compounds produced by certain species of fungi which grow on agricultural products or crops before harvest or during storage in agricultural systems and remain active for a long time after the destruction of fungi of which came. Their occurrence in foods and beverages has been recognized as a health threat to both human and animal, whether it comes from direct infection of plant tissues or products derived there from, or the transfer of mycotoxins and their metabolites in animal tissues, and hence in milk, eggs and elsewhere. Some of these mycotoxins such as aflatoxins, are extremely high toxicity, which makes compounds that deserve particular attention. Therefore it is essential that all agricultural products intended for human or animal feed can be subjected to constant and meticulous. The subject of this paper is to study the parameters (temperature and humidity) that influence the development of the mycotoxin zearalenone using as substrate flour stored maize. The aim was to check the growing conditions, storage and maintenance of primary agricultural products and thus reduce the development of mycotoxins in agricultural products and foodstuffs. The activity helped to investigate the mechanism of action of the conditions that lead to the development of both mycotoxins during storage of raw materials and in processing, production and packaging of finished product.

Thesis (MScFoodSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The phenolic composition of Cyclopia species is believed to be partially responsible for the numerous health promoting properties associated with their extracts. Current quality control measures do not accommodate variation in phenolic profiles of Cyclopia species. In this study, comprehensive high performance liquid chromatography (HPLC) methods were developed for the improved characterisation of the phenolic composition of aqueous extracts of two Cyclopia species (C. subternata and C. maculata). The methods were developed to be suitable for both routine quantitative analysis on conventional HPLC instrumentation, and the construction of chromatographic fingerprints for further data analysis. The latter entailed similarity analysis and prediction of total antioxidant capacity (TAC). Using a methodical approach, two separate HPLC methods, using diode array detection (DAD), were developed and validated for the analysis of aqueous extracts prepared from unfermented (green) and fermented plant material of C. subternata and C. maculata. Separation was achieved using the same method parameters (column, temperature, mobile phases), except for differing mobile phase gradients. Hyphenation of the developed HPLC methods with mass spectrometry (MS) and tandem MS allowed the confirmation of phenolic compounds previously identified in Cyclopia, and the tentative identification of several additional compounds in Cyclopia species, which are reported here for the first time. These included apigenin-6,8-di- C-glucoside, 3-hydroxyphloretin-30,50-di-C-hexoside, eriodictyol-di-C-glucoside, iriflophenone-di-O,C-hexoside, hydroxymangiferin and hydroxyisomangiferin. Subsequently, a large number of aqueous extracts of randomly selected green C. subternata (n = 64) and C. maculata (n = 50) plant material samples were analysed. Large quantitative variations were observed on intra- and inter-species levels. Cyclopia maculata extracts contained almost six times more mangiferin than extracts from C. subternata. HPLC-DAD analysis produced duplicate fingerprints for each extract which were consequently used for further analysis. The chromatographic fingerprint of a bioactive extract of each species was included in the respective data sets. Similarity analysis was conducted between the fingerprints from the randomly selected extracts and the corresponding active extract. For each species several extracts were determined to have similar “activity” as that of the active extract (n = 15 for C. subternata and n = 45 for C. maculata). Compounds potentially responsible for the activity were tentatively identified with the aid of principal component analysis (PCA) in combination with similarity analysis. PCA was more effective in identifying small differences between fingerprints than similarity analysis based on the correlation coefficients (r) alone. Furthermore, multivariate data analysis was used to construct partial least squares (PLS) regression models for the prediction of TAC from fingerprint data of each species, and available data from two microplate TAC assays. The construction of the models was successful with reasonable errors (< 10%), and permitted the determination of compounds of interest for future research. These included compounds of known identity that had large positive contributions toward the predictions of TAC, or unknown compounds that had small UV signals, but relatively large positive contributions to the models. / AFRIKAANSE OPSOMMING: Die talle gesondheidbevorderingseienskappe van ekstrakte van Cyclopia spesies word gedeeltelik geassosieer met hul fenoliese samestelling. Huidige kwaliteitskontrolemaatreëls is nie in staat om die variasie wat in die fenoliese profiele van die spesies voorkom, te akkommodeer nie. Omvattende hoë druk vloeistof chromatografiese (HPLC) metodes is vir twee Cyclopia spesies, naamlik C. subternata en C. maculata, in hierdie studie ontwikkel vir beter karakterisering van die fenoliese samestelling van waterekstrakte van dié spesies. Die metodes moes ook geskik wees vir roetine analise van C. subternata en C. maculata ekstrakte op konvensionele HPLC instrumentasie, en vir die opstel van chromatografiese vingerafdrukke (fenoliese samestellingsprofiele) vir verdere data analise, soos gelykvormigheidsanalise en die voorspelling van die totale antioksidantkapasiteit (TAC). Twee HPLC metodes, wat van ’n ultraviolet-diode detektor (DAD) gebruik maak, is ontwikkel deur ’n sistematiese benadering te volg. Die onderskeie metodes is vir die ontleding van waterekstrakte van groen (ongefermenteerde) en gefermenteerde plantmateriaal van C. subternata en C. maculata gevalideer. Ongeag die spesie is optimale skeiding met dieselfde kolom, mobiele fase en kolom-temperatuur bereik, maar met verskillende mobiele fase gradiënte. Analise met massaspektrometrie (MS) en tandem MS het die teenwoordigheid van fenoliese verbindings, wat voorheen in Cyclopia spesies geidentifiseer is, bevestig. Verder is ook ’n aantal verbindings vir die eerste keer in Cyclopia tentatief geidentifiseer. Dit sluit apigenien-6,8-di-C-glukosied, 3- hidroksiefloretien-30,50-di-C-heksosied, eriodiktiol-di-C-glukosied, iriflofenoon-di-O,C-heksosied, hidroksiemangiferien en hidroksie-isomangiferien in. Vervolgens is ’n groot aantal ewekansig gekose waterekstrakte van beide groen C. subternata (n = 64) en C. maculata (n = 50) plantmateriaal geanaliseer, en groot kwantitatiewe variasie op intra- en inter-spesievlak waargeneem. Cyclopia maculata ekstrakte het byvoorbeeld byna ses maal die mangiferieninhoud van C. subternata ekstrakte gehad. HPLC-DAD analise van die ekstrakte het duplikaat vingerafdrukke van elke ekstrak geproduseer, wat vir verdere data analise gebruik is. Die chromatografiese vingerafdruk van ’n bioaktiewe ekstrak van elke spesie was by die onderskeie datastelle ingesluit. Gelykvormigheidsanalise is tussen vingerafdrukke van die ewekansig gekose ekstrakte en die ooreenstemmende aktiewe ekstrak uitgevoer. Vir elke spesie is ’n aantal “aktiewe” ekstrakte aangewys (n = 15 vir C. subternata en n = 45 vir C. maculata). Die verbindings wat potensieel verantwoordelik kan wees vir die aktiwiteite is met behulp van hoofkomponentontleding (PCA) in kombinasie met gelykvormigheidsanalise, tentatief aangewys. PCA was egter meer effektief om klein verskille tussen vingerafdrukke aan te dui, in vergelyking met gelykvormigheidsanalise wat slegs op die korrelasie koëffisiënt (r) gebaseer is. Meerveranderlike data analiese is gebruik om “gedeeltelike kleinste kwadrate” (PLS) regressiemodelle, vir die voorspelling van die TAC van beide spesies te bou. Die voorspelling is gebaseer op hul vingerafdruk data en TAC data van twee TAC mikroplaat metodes. Die model-konstruksie was suksesvol met aanvaarbare voorspellingsfoute (< 10%). Verbindings van belang kon ook bepaal word. Dit sluit bekende verbindings in wat groot positiewe bydraes ten opsigte van die voorspelling van TAC getoon het, asook ongeidentifiseerde verbindings wat klein UV-seine getoon het, maar relatiewe groot bydraes tot die modelle gehad het.

Thesis (MScFoodSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: In this study quality control tools were developed for the rooibos industry, primarily to determine the quality of rooibos infusions. A considerable variation between samples of the same quality grade has been noted. As there are no guidelines or procedures in place to help minimise this inconsistency it was important to develop quality control tools, which could confront this problem. Both the sensory characteristics and phenolic composition of rooibos infusions were analysed in order to create and validate these quality control tools. Descriptive sensory analysis was used for the development of a targeted sensory wheel and sensory lexicon, to be used as quality control tools by the rooibos industry, and to validate the major rooibos sensory profiles. In order to ensure all possible variation was taken into account, 230 fermented rooibos samples were sourced from the Northern Cape and Western Cape areas within South Africa over a 3-year period (2011-2013). The aroma, flavour, taste and mouthfeel attributes found to associate with rooibos sensory quality were validated and assembled into a rooibos sensory wheel, which included the average intensity, as well as the percentage occurrence of each attribute. Two major characteristic sensory profiles prevalent within rooibos, namely the primary and secondary profiles, were identified. Both profiles had a sweet taste and an astringent mouthfeel, however, the primary sensory profile is predominantly made up of “rooibos-woody”, “fynbos-floral” and “honey” aroma notes, while “fruity-sweet”, “caramel” and “apricot” aroma notes are the predominant sensory attributes of the secondary profile. The predictive value of the phenolic compounds of the infusions towards the taste and mouthfeel attributes (“sweet”, “sour”, “bitter” and “astringent”) was examined using different regression analyses, namely, Pearson’s correlation, partial least squares regression (PLS) and step-wise regression. Correlations between individual phenolic compounds and the taste and mouthfeel attributes were found to be significant, but low. Although a large sample set (N = 260) spanning 5 years (2009-2013) and two production areas (Western Cape and Northern Cape, South Africa) was used, no individual phenolic compounds could be singled out as being responsible for a specific taste or mouthfeel attribute. Furthermore, no difference was found between the phenolic compositions of the infusions based on production area, a trend that was also seen for the sensory characterisation of rooibos infusions. Sorting, a rapid sensory profiling method was evaluated for its potential use as a quality control tool for the rooibos industry. Instructed sorting was shown to successfully determine rooibos sensory quality, especially based on the aroma quality of the infusions. However, determining the quality of the infusion based on flavour quality was more difficult, possibly due to the low sensory attribute intensities. Categorisation of rooibos samples based on the two major aroma profiles i.e. the primary and secondary characteristic profiles, was achieved with uninstructed sorting. The potential of using sorting as a rapid technique to determine both quality and characteristic aroma profiles, was therefore demonstrated, indicating its relevance as another quality control tool to the rooibos industry. / AFRIKAANSE OPSOMMING: Gehaltebeheer hulpmiddels is as deel van hierdie studie vir die rooibosbedryf ontwikkel, hoofsaaklik om die sensoriese kwaliteit van rooibostee te bepaal. Aansienlike verskille is tussen monsters van dieselfde gehaltegraad opgemerk, primêr omdat daar in die wyer rooibosbedryf beperkte riglyne of prosedures in plek is om kwaliteitsverskille effektief te bepaal. Dit is as belangrik geag om gehaltebeheer hulpmiddels te ontwikkel om laasgenoemde probleem aan te spreek. Spesifieke gehaltebeheer hulpmiddels is dus vir hierdie studie ontwikkel en gevalideer deur die sensoriese eienskappe en fenoliese samestelling van rooibostee te analiseer. Beskrywende sensoriese analise (BSA) is gebruik om ‘n sensoriese wiel en leksikon vir die rooibosbedryf te ontwikkel en te valideer. Om alle moontlike produkvariasie te ondervang, is 230 gefermenteerde rooibos monsters afkomstig van die Noord-Kaap en Wes-Kaap areas in Suid-Afrika oor ‘n tydperk van drie jaar (2011-2013) verkry. Die aroma, geur, smaak en mondgevoel eienskappe wat met rooibos se sensoriese kwaliteit assosieer, is bevestig en uiteindelik gebruik om die sensoriese wiel te ontwikkel. Die gemiddelde intensiteit en persentasie voorkoms van elke eienskap is in die wiel ingesluit. Twee belangrike “karakteristieke” sensoriese profiele wat met rooibos geassosieer word, is geïdentifiseer, nl. die primêre en sekondêre sensoriese profiele. Tipies van beide sensoriese profiele is ‘n kenmerkende soet smaak en vrank mondgevoel, daarenteen bestaan die primêre sensoriese profiel hoofsaaklik uit "rooibos-houtagtige", "fynbos-blomagtige" en "heuning" aromas, terwyl "vrugtige-soet", "karamel" en "appelkoos" aromas die oorheersende sensoriese eienskappe van die sekondêre profiel is. Die korrelasie tussen die fenoliese verbindings en die smaak en mondgevoel eienskappe van rooibos ("soet", "suur", "bitter" en "vrankheid") is ondersoek met behulp van verskillende tipe regressieontledings, nl. Pearson se korrelasie, gedeeltelike kleinstekwadrate regressie (PLS) en stapsgewyse regressie. Korrelasies tussen individuele fenoliese verbindings en die smaak en mondgevoel eienskappe was laag, maar steeds betekenisvol. Alhoewel die uitgebreide stel monsters (N = 260) verteenwoordigend was van vyf oesjare (2009-2013) en twee produksiegebiede (Wes-Kaap en Noord-Kaap, Suid-Afrika), kon geen individuele fenoliese verbindings uitgesonder word as betekenisvolle voorspellers van spesifieke smaak of mondgevoel eienskappe nie. Verder is daar ook geen verskil tussen die verskillende produksie-areas wat betref fenoliese samestelling gevind nie. Soortgelyke resultate is bevind vir die sensoriese karakterisering van rooibostee. Sortering, 'n vinnige sensoriese profileringsmetode, is geëvalueer vir sy potensiële gebruik as 'n gehaltebeheer hulpmiddel vir die rooibosbedryf. Gestrukteerde sortering was suksesvol om rooibos se sensoriese kwaliteit, veral die algemene aroma kwaliteit van rooibos, te bepaal. Hierdie profileringsmetode was egter nie so suksesvol om rooibos se algemene geur, smaak en mondgevoeleienskappe te bepaal nie. Hierdie tendens kan moontlik toegeskryf word aan die betekenisvolle laer intensiteite van laasgenoemde sensoriese eienskappe. Die kategorisering van die rooibos monsters op grond van hul karakteristieke primêre en sekondêre sensoriese profiele is suksesvol deur middel van ongestrukteerde sortering bepaal. In die geheel gesien is die potensiaal van die sorteringstegniek as ‘n vinnige metode om die algemene sensoriese kwaliteit, asook die karakteristieke aroma profiele van rooibos te bepaal, dus bewys. Hierdie vinnige sensoriese profileringstegniek hou dus besliste voordele in vir die rooibosbedryf as dit kom by sensoriese gehaltebeheer.

Há milênios as plantas são fontes naturais de substâncias utilizadas na indústria. Um exemplo dessas plantas é a erva mate, que por sua vez contém elevado conteúdo de compostos biologicamente ativos com potencial aplicação industrial. O objetivo do presente trabalho foi avaliar a atividade biológica do extrato de erva mate, quantificar seus compostos e aplicar em hambúrguer de peixe. O extrato seco de erva mate foi submetido a dois testes diferentes para avaliação da atividade antioxidante. No teste de DPPH (2,2-difenil-1-picrilhidrazila) foram observados resultados de IC50 de 7,91 μg mL-1 para o extrato de erva mate (EEM) e 1,04 μg mL-1 para o butil hidroxianisol (BHA). Já para o teste de FRAP (ferric reducing antioxidant power – poder antioxidante de redução do ferro) os resultados de poder de redução de Fe (II) por grama de amostra foram de 4922,67 para EEM e 15801,14 para BHA. A investigação do poder antioxidante da combinação de erva mate e BHA foi realizada por planejamento experimental pelo teste de DPPH, no qual os resultados obtidos nos permite prever a resposta de cada composto em concentrações pré-determinadas. A avaliação da atividade antimicrobiana foi avaliada isoladamente pelo teste de Concentração Inibitória Mínima (CIM) e em combinação pela técnica de checkerboard. Os resultados obtidos de CIM para o EEM frente a Escherichia coli, Staphylococcus aureus e Salmonella Typhi. foram de 10 mg mL-1, 5 mg mL-1 e 10 mg mL-1, respectivamente, e para o BHA foi de 0,313 mg mL-1 para todos os microrganismos testados. Quanto à análise de interação da combinação dos compostos observou-se atividade aditiva, de acordo com o valor de ICIF calculado que foi de 0,998 para S. aureus. A metodologia desenvolvida para a quantificação dos compostos da erva mate por cromatografia líquida de ultra eficiência (CLUE) se mostrou específica, linear, precisa e exata. Os resultados da quantificação dos compostos por CLUE foram de 49,6 mg g-1 de ácido clorogênico, 34,5 mg g-1 de cafeína e 23,1 mg g-1 de rutina. Por fim, foram preparados os hambúrgueres de peixe e realizadas as análises de composição centesimal, oxidação lipídica e microbiológica. Não foi observada oxidação lipídica no produto. Quanto à análise microbiológica de contagem de mesófilos no hambúrguer de peixe foi observado diferença estatística entre a fomulação controle e as formulações com BHA e extrato de erva mate a 1,0%. Contudo, estudos adicionais utilizando a erva mate devem ser realizados para comprovar seu efetivo poder conservante. / For thousand of years, plants have been natural sources of substances used in industry. An example of such plants is yerba mate, which in turn contains high content of biologically active compounds with potential industrial application. This paper aimed to assess the biological activity of yerba mate extract, to quantify its compounds and to apply in fish hamburger. The yerba mate dry extract was submitted to two different assays in order to evaluate the antioxidant activity. In the DPPH assay, IC50 results of 7.91 μg mL-1 for the yerba mate extract (YME) and 1.04 μg mL-1 for butyl hydroxyanisole (BHA) were observed. For the FRAP assay, the results of Fe (II) reduction per gram of sample were 4922.67 for YME and 15801.14 for BHA. The research of the antioxidant power of the combination of yerba mate and BHA was carried out by experimental design by the DPPH assay, in which the obtained results allow us to predict the response of each compound at predetermined concentrations. The assessment of the antimicrobial activity was carried out in isolation by the Minimum Inhibitory Concentration (MIC) assay and in combination with the checkerboard technique. The results obtained from MIC for YME against E.coli, S. aureus and Salmonella sp. were 10 mg mL-1, 5 mg mL-1 and 10 mg mL-1, respectively, and for BHA was 0.313 mg mL-1 for all assessed microorganisms. In regard to the interaction analysis of the compound combination, additive activity was observed, according to the calculated FIC index value of 0.998 for S. aureus. The methodology developed for the quantification of yerba mate compounds by ultra performance liquid chromatography (UPLC) was specific, linear, precise and accurate. The results of the compounds quantification by UHPLC were 49.6 mg g-1 of chlorogenic acid, 34.5 mg g-1 of caffeine and 23.1 mg g-1 of rutin. Finally, the fish hamburgers were prepared and the analyzes of centesimal composition, lipid and microbiological oxidation were carried out. No lipid oxidation was observed in the product. Regarding the microbiological analysis of mesophilic counts, statistic difference was observed between the control formulation and the formulations with BHA and 1.0% yerba mate extract. However, additional studies using yerba mate should be performed to prove its effective preservative power.

A avaliação no perfil das impurezas de fármacos está sendo alvo de atenção das agências regulatórias nacionais e internacionais visto que podem gerar implicações na saúde da população. Frente a isso, as indústrias farmacêuticas devem adequarse às novas regulamentações para garantir a qualidade, segurança e eficácia dos medicamentos. O fostato de sitagliptina (STG) e vildagliptina (VLG), liberados para uso clínico no Brasil em 2006 e 2007, respectivamente, são utilizados para o tratamento do diabetes mellitus tipo 2. Na literatura pesquisada não foram encontrados relatos referentes à determinação quantitativa de impurezas de síntese da vildagliptina e sitagliptina. Desta forma, este trabalho teve por objetivo desenvolver e validar métodos analíticos para a determinação de impurezas dos fármacos sitagliptina e vildagliptina, além de avaliar a toxicidade dos mesmos. Foi desenvolvido e validado método analítico por cromatografia líquida de alta eficiência para determinação do fosfato de sitagliptina na presença de duas impurezas de síntese de acordo com os parâmetros de especificidade, linearidade, precisão, exatidão, robustez, limites de detecção e quantificação. Para tanto, foi utilizada coluna cromatográfica XBridgeTM Pheny (250 mm x 4,6 mm, 5 μm), vazão 1,0 mL/min e detecção em 207 nm. A fase móvel foi composta por acetonitrila: solução aquosa em 0,05% de ácido fórmico (40:60, v/v). A determinação quantitativa da vildagliptina e duas impurezas de síntese foi desenvolvida e validada utilizando a técnica por cromatografia líquida de ultra-eficiência. A coluna cromatográfica utilizada corresponde ACQUITI UPLC® BEH C8 (50 x 2,1 mm; 1,7 μm), vazão 0,3 mL/min, volume de injeção de 1 μL e temperatura de 35°C. A fase móvel foi composta por metanol em ácido fórmico 0,1%: solução aquosa em ácido fórmico 0,1%. A partir dos resultados obtidos, verificou-se que estes estão de acordo com os requisitos preconizados pelos códigos oficiais. Esta pesquisa também apresenta resultados referentes aos estudos de citotoxicidade e genotoxicidade tanto dos fármacos quanto das respectivas impurezas realizados a partir dos ensaios de MTT, vermelho neutro, óxido nítrico, espécies reativas de oxigênio e nitrogênio, potencial de membrana mitocondrial e teste cometa. / Impurities profiling have attention of national and international regulatory agencies as they may generate implications for the health of the population. Against this, pharmaceutical industry must be suitable for new regulations to ensure quality, safety and efficacy of medicines. The phosphate sitagliptin and vildagliptin, released for clinical use in Brazil in 2006 and 2007, respectively, and used for the treatment of diabetes mellitus type 2. In the literature, we not found any reports regarding the quantitative determination of impurities synthesis of vildagliptin and sitagliptin. Thus, this study aimed to develop and validate analytical methods for the determination of impurities of sitagliptin and vildagliptin in addition to performing toxicological tests. It was developed and validated an analytical method by high-performance liquid chromatography for determination of sitagliptin in presence of two impurities synthesis according to the specific parameters, linearity, precision, accuracy, robustness, limits of detection and quantification. The XBridgeTM Phenyl column (250 mm x 4.6 mm d.i., 5 μm), flow rate 1.0 mL/min and detection at 207 nm. The mobile phase was composed of acetonitrile: aqueous solution in 0.05% formic acid (40:60, v/v). The results obtained are in accordance with the requirements recommended by official guides. Quantitative determination of vildagliptin and its synthetic impurities was developed using liquid chromatography ultra efficiency. The chromatographic column ACQUITY UPLC® BEH C8 (50 x 2.1 mm, 1.7 μm) was used, flow rate 0.3 mL/min, injection volume 1 μL and temperature 35 °C. The mobile phase is composed of methanol in formic acid 0.1%: aqueous solution in formic acid 0.1%. This research presents results for the cytotoxicity and genotoxicity studies of both drugs and the impurities.

O cloridrato de tizanidina é um relaxante muscular esquelético de ação central, utilizado no tratamento de espasticidade. Esse fármaco é comercializado apenas sob a forma de comprimidos, o que evidencia a necessidade do desenvolvimento de formulações líquidas orais. No ambiente hospitalar, este aspecto é contornado com a preparação de suspensões derivadas, de modo a permitir a administração em crianças e em adultos com deglutição prejudicada, mas não há dados sobre sua estabilidade. O objetivo deste trabalho foi avaliar a estabilidade físico-química e microbiológica de formas farmacêuticas líquidas preparadas em ambiente hospitalar a partir da derivação de comprimidos de cloridrato de tizanidina, empregando como metodologia a cromatografia líquida de alta eficiência (CLAE) e análise microbiológica. Um método simples e indicativo de estabilidade foi desenvolvido e validado por CLAE quanto a especificidade, linearidade, limite de detecção e quantificação, precisão, exatidão e robustez. As formulações líquidas foram preparadas em triplicata e acondicionadas em frasco de polietileno tereftalato (PET) âmbar e de vidro âmbar, que foram armazenados sob três diferentes condições: em temperatura ambiente, sob refrigeração e à 40 °C. Foram coletadas amostras a cada 7 dias por um período de 56 dias para a estabilidade físico-química. As formulações líquidas foram analisadas e demonstraram ser quimicamente estáveis durante 56 dias, permitindo um período de utilização prolongado. Entretanto, a determinação da estabilidade microbiológica mostrou que estas formulações estão propensas à contaminação microbiana, o que reduziu drasticamente a sua estabilidade para 7 dias, em ambos os frascos e em todas as temperaturas avaliadas. A qualidade microbiológica foi uma questão crítica, uma vez que as formulações foram produzidas apenas em água para injetáveis e não havia conservantes presentes na formulação. Os resultados deste estudo podem servir de referência para preparar derivações de cloridrato de tizanidina em uso hospitalar, uma vez que se demonstrou a confiabilidade do intervalo de tempo de armazenamento e as condições adequadas para o uso. De acordo com os resultados deste estudo de estabilidade, este fármaco pode ser incorporado na rotina de preparo das derivações dos hospitais, atendendo a uma demanda já há tempos sinalizada. / Tizanidine hydrochloride is a centrally acting skeletal muscle relaxant, used in the manegement of spasticity. This drug is sold only as tablets, which highlights the need to develop oral liquid formulations. In the hospital environment, this aspect is circumvented by the preparation of derived suspensions, to allow administration to children and adults with impaired swallowing, but there is no data regarding their stability. The objective of this work was to evaluate the physicochemical and microbiological stability of liquid dosage forms prepared in hospital environment from tizanidine hydrochloride tablets, applying high performance liquid chromatography (HPLC) and microbiological analysis. A simple and stability-indicating HPLC method was developed and validated for specificity, linearity, limits of detection and quantification, precision, accuracy and robustness. The liquid formulations were prepared in triplicate and placed in amber PET and glass bottles, which were stored under three different conditions: at room temperature, under refrigeration and at 40 °C. Samples were collected every 7 days along 56 days for physicochemical stability. The liquid formulations were analyzed and demonstrated chemical stability for 56 days, allowing the use for long period. However, the determination of microbiological stability showed that these formulations are prone to microbial contamination, which has dramatically reduced its stability to 7 days, in both bottles and all evaluated temperatures. Microbiological quality was a critical issue, since formulations were produced only in water for injectables and there was no preservatives present in the formulation. Results of this study could be applied as a reference for tizanidine hydrochloride derived preparation in hospital, once it was demonstrated the reliability of storage time interval and proper conditions for use. According to the results of this stability study, this drug could be incorporated into the routine of hospital derivations preparation, meeting a demand already indicated.

Cetoconazol é um agente antifúngico que possui ação tópica e sistêmica, podendo ser incorporado em diferentes formas farmacêuticas. Como exemplo, pode-se citar o xampu de cetoconazol, que é efetivo no tratamento da dermatite seborréica e pitiríase versicolor. É reconhecido que o cetoconazol, nas formas farmacêuticas xampu e solução, rapidamente altera a coloração se tornando avermelhado. A exposição de fármacos à radiação pode influenciar a estabilidade da formulação, levando a modificações nas propriedades físico-químicas do produto. O objetivo deste trabalho foi avaliar a fotoestabilidade do cetoconazol em xampu e solução. As formulações foram expostas à radiação UV-A (352 nm), UV-C (254 nm) e luz diurna. Métodos foram desenvolvidos e validados para a determinação quantitativa do cetoconazol: cromatografia líquida de alta eficiência e ensaio microbiológico utilizando o método de cilindros em placa. Estudos comparativos entre xampu de cetoconazol e xampu de cetoconazol contendo produtos de degradação foram feitos para avaliar a atividade antifúngica e a segurança biológica in vivo (teste de Draize) e in vitro (teste de citotoxicidade). Os resultados demonstraram diminuição na atividade antifúngica, mas não demonstraram alteração na segurança biológica. Os métodos utilizados para análise do cetoconazol (ensaio microbiológico e CLAE) demonstraram ser lineares, precisos e exatos, sendo úteis no controle de qualidade do fármaco. Os resultados indicam alteração do cetoconazol em presença da luz. Dois produtos de degradação foram isolados e identificados: 1-acetil-4-{4-[2-(2-clorofenil)-2-(1H-imidazolil-1- metil)-1,3-dioxolanil-4-metoxi]fenil}piperazina e 1-acetil-4-{4-[2-(4-clorofenil)-2- (1H-imidazolil-1-metil)-1,3-dioxolanil-4-metoxi]fenil}piperazina. Conseqüentemente, adequada proteção da luz deve ser adotada durante armazenamento das duas formas farmacêuticas contendo o fármaco. / Ketoconazole is an antifungal agent with topic and systemic action and can be incorporated into several dosage forms. As an example, it could be mentioned ketoconazole shampoo, which is effective against seborrhoeic dermatitis and pityriasis versicolor. It is remarkable that ketoconazole is rapidly altered in shampoo and solution dosage forms, since its colour turns into red. Exposure of a drug to radiation can influence the stability of the formulation, leading to changes in the physicochemical properties of the product. The aim of this work was to assess the photostability of ketoconazole in shampoo and solution. The formulations were exposed to UV-A (352 nm) and UV-C (254 nm) radiations and daylight. Methods were developed and validated for the quantitative determination of ketoconazole: high performance liquid chromatography and microbiological assay using the cylinder-plate method. Comparative studies between ketoconazole shampoo and ketoconazole shampoo containing degradation products were conducted to evaluated the antifungal activity and biological reactivity in vivo (Draize test) and in vitro (cytotoxity test). The results showed a decrease in antifungal activity but not an alteration on the biological reactivity. The methods used for ketoconazole analysis (microbiological assay and HPLC) were linear, precise and accurate, providing valuable methods for the quality control of this drug. The results indicate alteration in ketoconazole in presence of light. Two photodegradation products were isolated and identified: 1-acetyl-4-{4-[2-(2-chlorophenyl)-2-(1H-imidazol- 1-ylmethyl)-1,3-dioxolan-4-ylmethoxy]phenyl}piperazine and 1-acetyl-4-{4-[2- (4-chlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-ylmethoxy]phenyl} piperazine. Consequently, adequate light protection should be adopted for storage of these two dosage forms containing the drug.

O cloridrato de tizanidina é um relaxante muscular esquelético de ação central, utilizado no tratamento de espasticidade. Esse fármaco é comercializado apenas sob a forma de comprimidos, o que evidencia a necessidade do desenvolvimento de formulações líquidas orais. No ambiente hospitalar, este aspecto é contornado com a preparação de suspensões derivadas, de modo a permitir a administração em crianças e em adultos com deglutição prejudicada, mas não há dados sobre sua estabilidade. O objetivo deste trabalho foi avaliar a estabilidade físico-química e microbiológica de formas farmacêuticas líquidas preparadas em ambiente hospitalar a partir da derivação de comprimidos de cloridrato de tizanidina, empregando como metodologia a cromatografia líquida de alta eficiência (CLAE) e análise microbiológica. Um método simples e indicativo de estabilidade foi desenvolvido e validado por CLAE quanto a especificidade, linearidade, limite de detecção e quantificação, precisão, exatidão e robustez. As formulações líquidas foram preparadas em triplicata e acondicionadas em frasco de polietileno tereftalato (PET) âmbar e de vidro âmbar, que foram armazenados sob três diferentes condições: em temperatura ambiente, sob refrigeração e à 40 °C. Foram coletadas amostras a cada 7 dias por um período de 56 dias para a estabilidade físico-química. As formulações líquidas foram analisadas e demonstraram ser quimicamente estáveis durante 56 dias, permitindo um período de utilização prolongado. Entretanto, a determinação da estabilidade microbiológica mostrou que estas formulações estão propensas à contaminação microbiana, o que reduziu drasticamente a sua estabilidade para 7 dias, em ambos os frascos e em todas as temperaturas avaliadas. A qualidade microbiológica foi uma questão crítica, uma vez que as formulações foram produzidas apenas em água para injetáveis e não havia conservantes presentes na formulação. Os resultados deste estudo podem servir de referência para preparar derivações de cloridrato de tizanidina em uso hospitalar, uma vez que se demonstrou a confiabilidade do intervalo de tempo de armazenamento e as condições adequadas para o uso. De acordo com os resultados deste estudo de estabilidade, este fármaco pode ser incorporado na rotina de preparo das derivações dos hospitais, atendendo a uma demanda já há tempos sinalizada. / Tizanidine hydrochloride is a centrally acting skeletal muscle relaxant, used in the manegement of spasticity. This drug is sold only as tablets, which highlights the need to develop oral liquid formulations. In the hospital environment, this aspect is circumvented by the preparation of derived suspensions, to allow administration to children and adults with impaired swallowing, but there is no data regarding their stability. The objective of this work was to evaluate the physicochemical and microbiological stability of liquid dosage forms prepared in hospital environment from tizanidine hydrochloride tablets, applying high performance liquid chromatography (HPLC) and microbiological analysis. A simple and stability-indicating HPLC method was developed and validated for specificity, linearity, limits of detection and quantification, precision, accuracy and robustness. The liquid formulations were prepared in triplicate and placed in amber PET and glass bottles, which were stored under three different conditions: at room temperature, under refrigeration and at 40 °C. Samples were collected every 7 days along 56 days for physicochemical stability. The liquid formulations were analyzed and demonstrated chemical stability for 56 days, allowing the use for long period. However, the determination of microbiological stability showed that these formulations are prone to microbial contamination, which has dramatically reduced its stability to 7 days, in both bottles and all evaluated temperatures. Microbiological quality was a critical issue, since formulations were produced only in water for injectables and there was no preservatives present in the formulation. Results of this study could be applied as a reference for tizanidine hydrochloride derived preparation in hospital, once it was demonstrated the reliability of storage time interval and proper conditions for use. According to the results of this stability study, this drug could be incorporated into the routine of hospital derivations preparation, meeting a demand already indicated.

A avaliação no perfil das impurezas de fármacos está sendo alvo de atenção das agências regulatórias nacionais e internacionais visto que podem gerar implicações na saúde da população. Frente a isso, as indústrias farmacêuticas devem adequarse às novas regulamentações para garantir a qualidade, segurança e eficácia dos medicamentos. O fostato de sitagliptina (STG) e vildagliptina (VLG), liberados para uso clínico no Brasil em 2006 e 2007, respectivamente, são utilizados para o tratamento do diabetes mellitus tipo 2. Na literatura pesquisada não foram encontrados relatos referentes à determinação quantitativa de impurezas de síntese da vildagliptina e sitagliptina. Desta forma, este trabalho teve por objetivo desenvolver e validar métodos analíticos para a determinação de impurezas dos fármacos sitagliptina e vildagliptina, além de avaliar a toxicidade dos mesmos. Foi desenvolvido e validado método analítico por cromatografia líquida de alta eficiência para determinação do fosfato de sitagliptina na presença de duas impurezas de síntese de acordo com os parâmetros de especificidade, linearidade, precisão, exatidão, robustez, limites de detecção e quantificação. Para tanto, foi utilizada coluna cromatográfica XBridgeTM Pheny (250 mm x 4,6 mm, 5 μm), vazão 1,0 mL/min e detecção em 207 nm. A fase móvel foi composta por acetonitrila: solução aquosa em 0,05% de ácido fórmico (40:60, v/v). A determinação quantitativa da vildagliptina e duas impurezas de síntese foi desenvolvida e validada utilizando a técnica por cromatografia líquida de ultra-eficiência. A coluna cromatográfica utilizada corresponde ACQUITI UPLC® BEH C8 (50 x 2,1 mm; 1,7 μm), vazão 0,3 mL/min, volume de injeção de 1 μL e temperatura de 35°C. A fase móvel foi composta por metanol em ácido fórmico 0,1%: solução aquosa em ácido fórmico 0,1%. A partir dos resultados obtidos, verificou-se que estes estão de acordo com os requisitos preconizados pelos códigos oficiais. Esta pesquisa também apresenta resultados referentes aos estudos de citotoxicidade e genotoxicidade tanto dos fármacos quanto das respectivas impurezas realizados a partir dos ensaios de MTT, vermelho neutro, óxido nítrico, espécies reativas de oxigênio e nitrogênio, potencial de membrana mitocondrial e teste cometa. / Impurities profiling have attention of national and international regulatory agencies as they may generate implications for the health of the population. Against this, pharmaceutical industry must be suitable for new regulations to ensure quality, safety and efficacy of medicines. The phosphate sitagliptin and vildagliptin, released for clinical use in Brazil in 2006 and 2007, respectively, and used for the treatment of diabetes mellitus type 2. In the literature, we not found any reports regarding the quantitative determination of impurities synthesis of vildagliptin and sitagliptin. Thus, this study aimed to develop and validate analytical methods for the determination of impurities of sitagliptin and vildagliptin in addition to performing toxicological tests. It was developed and validated an analytical method by high-performance liquid chromatography for determination of sitagliptin in presence of two impurities synthesis according to the specific parameters, linearity, precision, accuracy, robustness, limits of detection and quantification. The XBridgeTM Phenyl column (250 mm x 4.6 mm d.i., 5 μm), flow rate 1.0 mL/min and detection at 207 nm. The mobile phase was composed of acetonitrile: aqueous solution in 0.05% formic acid (40:60, v/v). The results obtained are in accordance with the requirements recommended by official guides. Quantitative determination of vildagliptin and its synthetic impurities was developed using liquid chromatography ultra efficiency. The chromatographic column ACQUITY UPLC® BEH C8 (50 x 2.1 mm, 1.7 μm) was used, flow rate 0.3 mL/min, injection volume 1 μL and temperature 35 °C. The mobile phase is composed of methanol in formic acid 0.1%: aqueous solution in formic acid 0.1%. This research presents results for the cytotoxicity and genotoxicity studies of both drugs and the impurities.

The aim of this thesis was to study the effect of the acetylsalicylic acid on the stimulation of plant immunity and thus the influence on the content of the active constituents in Silybum marianum L. plants. The main active constituents of Silybum marianum L. seeds are silybine, silydianine, isosilybine, silycristine, usually expressed as silymarine content, and taxifoline. These constituents have antihepatotoxic effect and many different protective effects on numerous organs and cells. Knowledge about stress and elicitation, origin, botanical characterization, growth, development and cultivation of Silybum marianum L. were summarized before the research.The small-plot experiment was set up in Hluboká nad Vltavou in 2013. Plants of Silybum marianum were divided into four groups and then three goups were treated three times during the vegetation with the acetylsalicylic acid of three different concentrations - 10-3 mol.l-1 (high), 10-4 mol.l-1 (medium) a 10-5 mol.l-1 (low). Every single one group was treated with only one concentration of the acetylsalicylic acid. The last group was treated only with water and served as a control group. The preparation of the extracts was being held with using mixture of acetone, methanol and water. The extracts were determined by High Performance Liquid Chromatography.The effects of each chosen concetration of acetylsalicylic acid (high, medium, low) on the active constituents in seeds were not statistically proven compared to seeds without aplication of the elicitor (control group). The ineffectiveness of the elicitor should have been also caused by nonoptimal condition of plants due to various abiotic and biotic stressors.