Дисертації з теми "Antifungal activiy"

La vaste diversité biologique et chimique dans l'environnement marin en fait une ressource précieuse pour la découverte de nouveaux antibiotiques, en réponse à l'émergence de la crise des antibiotiques à l'échelle mondiale. Cependant, le taux de découverte de nouveaux médicaments d'origine marine semble insuffisant par rapport à son potentiel. Dans un effort pour contribuer à la recherche de composés prometteurs pour le développement de nouveaux antibiotiques, nous avons étudié les métabolites secondaires et l'activité biologique de quatre souches de champignons : Fusarium equiseti, Anthracocystis flocculosa, Scedosporium dehoogii et Amesia nigricolor.En ce qui concerne les composants chimiques, 45 composés ont été isolés, appartenant principalement aux classes des chromones, des alcaloïdes, des polykétides cycliques, des glycolipides, des sesquiterpènes et des naphtalènes. 18 composés (représentant 40 %) ont été identifiés comme nouveaux composés. Les structures de ces composés ont été élucidées en utilisant une combinaison de HRMS, de RMN, de diffraction des rayons X, de méthode de Mosher modifiée et de calculs chimiques quantiques (de ECD et analyse de probabilité ML-J-DP4 et DP4+). Parmi eux, les déhoogiikétones A-B (C3.1-2) isolées du champignon S. dehoogii possédaient des squelettes de bergamotène réarrangés, décrits pour la première fois dans la nature.En ce qui concerne l'activité biologique, six dérivés de fusarochromanone (C2.1-6) isolés de F. equiseti (dont deux sont nouveaux : C2.2, C2.6) ont montré une cytotoxicité allant de forte à modérée sur trois lignées cellulaires testées (RPE1, HCT-116, U2OS). Seuls deux de ces composés ont présenté une activité d'inhibition contre trois (ABL1, JAK3, EphB1) des seize kinases protéiques testées. Seuls trois flocculosines A-C (C3.1-3) sur huit dérivés isolés d'A. flocculosa ont montré une activité antibactérienne contre S. aureus S25. Ces résultats ont révélé la relation structure-activité des dérivés de fusarochromanone et de flocculosine.La equisetin (C2.8) isolée de F. equiseti a présenté une forte activité antibactérienne contre S. aureus S25 mais n'a montré aucune cytotoxicité sur trois lignées cellulaires testées (RPE1, HCT-116, U2OS). Les chaetochromines A et B (C5.7-8) ont présenté une forte activité antibactérienne mais ont montré une toxicité cellulaire allant de faible à modérée sur la lignée cellulaire (THP-1) et les cellules primaires testées (RBC, PBMC), indiquant une possible fenêtre thérapeutique. Ces trois composés méritent d'être étudiés davantage dans les étapes de développement des antibiotiques
The enormous biological and chemical diversity in the marine environment is making it a valuable resource for the discovery of new antibiotics, in response to the emergence of antibiotic crisis worldwide. However, the rate of discovery of new marine-derived drugs seems insufficient compared to its potential. In an effort to contribute to the search for hit compounds for the development of new antibiotics, we investigated the secondảy metabolites and biological activity of four fungi strains: Fusarium equiseti, Anthracocystis flocculosa, Scedosporium dehoogii, and Amesia nigricolor.Regarding chemical components, 45 compounds were isolated, mainly belonging to chromones, alkaloids, cyclic polyketides, glycolipids, sesquiterpenes, and naphthalenes classes. 18 compounds (accounting for 40%) were identified as new compounds. The structures of these compounds were elucidated using a combination of HRMS, NMR, X-ray diffraction, modified Mosher's method, and quantum chemical calculations (ECD, ML-J-DP4, and DP4+ probability analysis). Among them, dehoogiiketones A-B (C3.1-2) isolated from the fungus S. dehoogii possessed rearranged bergamotene skeletons, described for the first time in nature.Regarding biological activity, six fusarochromanone derivatives (C2.1-6) isolated from F. equiseti (two of which are new: C2.2, C2.6) showed cytotoxicity ranging from strong to moderate on three tested cell lines (RPE1, HCT-116, U2OS). Only two of these compounds exhibited inhibition activity against three (ABL1, JAK3, EphB1) out of sixteen tested protein kinases. Only three flocculosins A-C (C3.1-3) out of eight derivatives isolated from A. flocculosa showed antibacterial activity against S. aureus S25. These findings revealed the structure-activity relationship of fusarochromanone and flocculosin derivatives.Equisetin (C2.8) isolated from F. equiseti exhibited strong antibacterial activity against S. aureus S25 but showed no cytotoxicity on three tested cell lines (RPE1, HCT-116, U2OS). Chaetochromins A and B (C5.7-8) exhibited strong antibacterial activity but showed cell toxicity ranging from weak to moderate on tested cell line (THP-1) and primary cells (RBC, PBMC) isolated from the blood of healthy donors, indicating a possible therapeutic window. These three compounds are worthy of further research stages in antibiotic development

The secondary metabolism of organisms, especially mycelial fungi, is attracting increasing attention since it may produce a reservoir of unique molecules from which therapeutic agents are formed or derived for clinical application The production of secondary metabolites is not well understood and involves a broad spectrum of metabolic processes that often have little in common. This study screened a number of New Zealand isolates, mostly of the Arthrinium genus, and assessed their antifungal activity. The primary screening used for this study consisted of agar diffusion tests with a range of filamentous fungi and yeast. Secondary screening was also started, with active cultures being chemically extracted. It was found that New Zealand isolates of Arthrinium phaeospermum display antifungal activity against yeasts and filamentous fungi, as well as bacteria. The teleomorphic state of an Arthrinium sp., Apiospora montagnei, was found to have activity against the yeast S. cerevisiae. In addition, activity against two filamentous fungi by Apiospora montagnei was demonstrated. In the course of this study, a contaminant fungus displaying antifungal activity was isolated and screened. It was found to have antifungal activity against C. albicans and filamentous fungi, as well as bacteria.

>Magister Scientiae - MSc
Nanotechnology is spreading rapidly across the world as an extremely powerful technology. Nanoscience and nanotechnology are innovative scientific advancements that have been introduced only in this century. Nanotechnology has developed as the scientific advancement to grow and transform the entire agri-food area, with the potential to elevate global food production, in addition to the nutritional value, quality, and safety of food and food products. It has gained recognition due to its variability in shape, size, and dimension and how it correlates to its possibilities. One of those functions is nanoparticles’ (NPs) ability to have antimicrobial activity, more specifically its antifungal activity. One particular pathway of synthesising NPs is through phytonanotechnology which is the use of biomaterial to synthesis the NPs.
2024

Les moisissures sont responsables de contaminations sur les produits de BVP et induisent des pertes économiques conséquentes. Dans ce contexte, les cultures bioprotectrices représentent un intérêt croissant comme alternative aux conservateurs chimiques. L’objectif de la première partie de cette étude a été d’évaluer in vitro et in situ l’activité antifongique de bactéries lactiques et propioniques contre cinq moisissures contaminants isolées de produits de BVP. Les bactéries les plus actives pendant les tests in vitro ont été testées in situ par pulvérisation de surface. Sur le milieu WFH, les isolats bactériens les plus actifs correspondent aux espèces Lactobacillus plantarum, reuteri et au groupe buchneri. Les souches plus actives après ces tests ont été testées par inclusion dans la recette du pain au lait et différentes souches ont montré un effet retard en particulier la souche Leuconostoccitreum qui semble retarder la croissance de Penicillium corylophilum après 10 jours. Dans la deuxième partie de cette étude, les surnageants de cultures actifs sont analysés pour identifier les composés antifongiques grâce à différentes traitements et différentes méthode telle que l’HPLC et la spectrométrie de masse. Les résultats suggèrent que les acides organiques jouent un rôle prépondérant dans l’activité antifongique et ont montré que les composés antifongiques retrouvés correspondaient aux acides lactique, acétique et propionique, à l’éthanol et au peroxyde d’hydrogène, ainsi que d’autres composés mais à plus faible échelle. Sur ces résultats, différentes combinaisons des composés identifiés ont été testées pour leur effet sur la germination et la croissance radiale de P. corylophilum et E. repens. Certaines de ces combinaisons ont montré les mêmes effets que le surnageant actif ce qui confirme l’implication des molécules identifiées dans l’activité. Les résultats suggèrent que l’acide acétique est responsable de la totalité de l’activité antifongique observée sur P. corylophilum et qu’il joue un rôle important dans l’inhibition de E. repens. La souche bactérienne sélectionnée pourrait représenter une possibilité de culture bioprotectrice pour les produits de BVP
Molds are responsible for the spoilage of bakery products and thus, cause substantial economic losses. In this context, bioprotective cultures represent a growing interest as an alternative to chemical preservatives. The aims of the first part of this study was to evaluate the in vitro and in situ antifungal activity of lactic acid bacteria (LAB) and propionibacteria against five moulds species isolated from bakery products. The most inhibitorybacteria found during the in vitro test were evaluated in situ after surface spraying. In WFH medium, the most active LAB isolates belonged to the Lactobacillus plantarum, reuteri and buchneri groups. The most active strains were added directly during “pains au lait” preparation and differents strains present delayed effect in particular a strain of Leuconostoc citreum which seems to delay the growth of Penicillium corylophilum after 10 days. In the second part, supernatants were analyzed to identified and quantified antifungal compounds by different treatments and different methods like HPLC, mass spectrometry. The results suggested that organic acids played the most important role in the antifungal activity and show that the main antifungal compounds corresponded to lactic, acetic and propionic acids, ethanol and hydrogen peroxide, as well as other compounds present at low levels. Based on these results, various combinations of the identified compounds were used to evaluate their effect on spore germination and fungal growth of P. corylophilum and E. repens. Some combinations presented the same activity than the bacterial culture supernatant thus confirming the involvement of the molecules in the antifungal activity. The results suggested that acetic acid was responsible of the entire antifungal activity against Penicillium corylophilum and played an important role in Eurotium repens inhibition. The selected bacteria provide a future prospect for use as bioprotective cultures on bakery products

Zemljište predstavlja bogat izvor različitih mikroorganizama čijiprodukti metabolizma mogu biti od izuzetnog značaja za čoveka.Dosadašnja ispitivanja mikrobnog diverziteta u zemljištu suotkrila bogati biosintetski potencijal za proizvodnju novihprirodnih proizvoda kod velikog broja mikroorganizama, naročitokada je u pitanju klasa Actinobacteria. Među zemljišnim izolatima,rod Streptomyces prednjači po broju identifikovanih bioaktivnihmolekula u odnosu na sve ostale bakterije. Stoga je jedan odciljeva u okviru ove doktorske disertacije izolacija streptomiceta izrizosfera medicinski značajnih biljaka sakupljenih na teritorijiRepublike Srbije (Papaver rhoeas, Matricaria chamomilla, i Urticadioica) i ispitivanje njihovog antifungalnog potencijala na različitevrste kandida. Morfološki različiti izolati (ukupno 103) su izolovaniiz uzoraka rizosfera i okarakterisani kao streptomicete. Dverazličite podloge i dve procedure za ekstrakciju su korišćene da bise pospešila detekcija antifungalnih jedinjenja. Ispitan je uticajukupno 412 ekstrakata na rast Candida albicans disk difuzionimesejem pri čemu je utvrđeno da 42% (43/103) izolata imajusposobnost proizvodnje antifungalnih jedinjenja pri ispitivanimuslovima. Pojedini ekstrakti su inhibirali rast važnih humanihpatogena poput Candida krusei, Candida parapsilosis, i Candidaglabrata. Na osnovu stepena i spektra antifungalne aktivnostidevet izolata je odabrano za dalja istraživanja. Ispitana jesposobnost njihovih ekstrakata da inhibiraju rast kandida u tečnojkulturi i u formi biofilma, a takođe je ispitan i njihov uticaj na većformirane biofilmove kandide u koncentracijama od 8 do 250pg/ml. Hromatografski profili ovih ekstrakata i uvid u njihovumetaboličku raznolikost dobijeni su korišćenjem tečnehromatografije visokih performansi. Tri ekstrakta sa specifičnomantifungalnom aktivnošću podvrgnuta su hemijskim analizama sciljem da se detektuju i strukturno okarakterišu molekuli koji sunosioci antifungalne aktivnosti. Na osnovu rezultata nuklearnomagnetno-rezonantne spektroskopije otkriveno je da su aktivnimolekuli genistein, daidzein i staurosporin. Genistein i daidzeinkoji su poznati fitoestrogeni poreklom iz sojinog brašna za koje jepoznato da inhibiraju ključne enzime u biosintetskom putusteroida. Njihovo prisustvo je u ovom istraživanju detektovanousled korišćenja sojinog brašna u hranljivoj podlozi. Kakostreptomicete u čijim ekstraktima su detektovani ovi molekulipokazuju sposobnost oslobađanja ovih važnih jedinjenja izkompleksne hranljive podloge, mogu se uzeti u razmatranje zabiotehnološku proizvodnju fitoestrogena. Staurosporin jedetektovan kao nosilac antifungalne aktivnosti kod ekstrakta sojaStreptomyces sp. BV410. Staurosporin je inhibitor protein kinaza injegovi derivati i analozi se koriste u kao antitumorski agensi.Biosinteza ovog molekula je optimizovana do prinosa od 36,94mg/l nakon 14 dana gajenja u hranljivoj podlozi koja sadržiglukozu, skrob, manitol i sojino brašno (JS). Dalja optimizacijahranljive podloge za biosintezu staurosporina ukazala je nasledeći sastav hranljive podloge: 20 g/l glukoze, 0,36 g/l skroba,21,46 g/l manitola, 17,32 g/l sojinog brašna. Primenomdefinisanih optimalnih vrednosti i korišćenjem odgovarajućihmatematičkih modela, predviđeno je da će se na ovaj načinpostići prinosi od 46,88 mg/l staurosporina i 12,05 mg/mlbiomase. Validnost predviđenih rezultata potvrđena jeizvođenjem bioprocesa u optimizovanoj hranljivoj podlozi (JSSta).Ispitana je kinetika biosinteze staurosporina i produkcije biomase,kao i potrošnje izvora ugljenika i razvijeni su odgovarajućiprocesni modeli. Dodatna optimizacija je podrazumevala dodataksuplemenata koji prema literature stimulišu sekundarnimetabolizam streptomiceta (joni cinka, gvožđa, fosfati, metiloleat, ulje semenki grožđa). Ovi eksperimenti su izvođeni na tri pHvrednosti (6,5, 7,5 i 8,5) a uspešnost bioprocesa je procenjivana 7.,10. i 14. dana gajenja. Dodatna optimizacija je dovela do podatkada dodatak soli gvožđa značajno pospešuje biosintezustaurosporina sa povećanjem prinosa od 25%. Dobijeni rezultatipotvrđuju da su rizosfere medicinski značajnih biljaka značajanizvor streptomiceta koje proizvode komponente saantifungalnom aktivnošću. Izolacija novog proizvođačastaurosporina i optimizacija procesa njegove biosintezeomogućiće dalja istraživanja ovog jedinjenja koje može bitiosnova za razvoj novih antifungalnih i jedinjenja koja inhibirajuangiogenezu. Rezultati dobijeni u okviru ovih istraživanjapredstavljaju početni korak ka potencijalnoj industrijalizacijiproizvodnje staurosporina.
Different soils are still a source of remarkable microbial diversitywhich also reflects in the unexplored chemical diversity. Recentadvances in assessment of microbial diversity from soil haverevealed the extraordinarily rich biosynthetic potential for theproduction of new natural products among different microbialstrains, especially within the group of Actinobacteria. Amongbacterial soil isolates, representatives of Streptomyces genus arethe most prolific producers of bioactive compounds. One of theobjectives of the present study was to isolate Streptomyces spp.from the rhizosphere soils of three ethno-medicinal plantscollected in Serbia (Papaver rhoeas, Matricaria chamomilla, andUrtica dioica) and to screen their antifungal activity againstCandida spp. Morphologically different sporulating isolates (103in total) were collected from rhizosphere soil samples anddetermined as Streptomyces spp. Two different media and twoextraction procedures were used to induce the production andfacilitate identification of antifungals. Overall, 412 crude cellextracts were tested against Candida albicans using diskdiffusion assays, with 42% (43/103) of the strains showing theability to produce antifungal agents. Also, extracts inhibitedgrowth of other important human pathogens: Candida krusei,Candida parapsilosis, and Candida glabrata. Based on theestablished degree and range of antifungal activity, nine isolateswere selected for further testing. Their ability to inhibit Candidagrowth in liquid culture, to inhibit biofilm formation, and todisperse pre-formed biofilms was assessed with activeconcentrations from 8 to 250 pg/ml. High-performance liquidchromatographic profiles of extracts derived from selectedstrains were recorded, revealing moderate metabolic diversity.The most potent extracts were subjected to comprehensiveidentification and structural characterization of antifungalcompounds. Applying a bioactivity-guided isolation approach,active compounds of three extracts were separated, and basedon NMR structure elucidation it was shown that activecompounds were genistein, daidzein and staurosporine.Genistein and daidzein, soy phytoestrogens, are known to inhibitkey enzymes in the steroid metabolism pathway and werecoming from the fermentation medium containing soy flower.Since isolated Streptomyces spp. showed good ability to extractthese molecules from complex medium, they can be furtherconsidered for biotechnological production of thesephytoestrogens. One of the isolates, Streptomyces sp. BV410,was characterized as an efficient staurosporine producer.Staurosporine is a potent inhibitor of protein kinases and isconsidered in anticancer therapy. The biotechnologicalproduction of staurosporine by strain BV410 was optimized toyield 36.94 mg/l after 14 days of incubation in soy flowerglucose-starch-mannitol based fermentation medium (JS).Further optimization of medium for biosynthesis ofstaurosporine indicated the following optimal values of theexamined factors: the content of glucose of 20 g/l, starch 0.36g/l, mannitol 21.46 g/l, soy flower 17.32 g/l. By applying thedefined optimal values and using the appropriate mathematicalmodels, the following responses were predicted: concentrationof staurosporine 46.88 mg/l and biomass yield 12.05 mg/ml. Thevalidity of the results was confirmed by performing thebiosynthesis of the staurosporine in the medium with optimalcomposition (JSSta). Kinetics of staurosporine and biomassproduction and carbon source consumption were examined andprocess models were developed. Additionally, optimization ofstaurosporine production was performed with differentsupplements which, according to literature data, had stimulativeeffect on secondary metabolism (Zn, Fe and P salts, methyloleate, grape seed oil). In order to improve the production ofstaurosporine, effects of pH (6.5, 7.5 and 8.5) and incubation time(7, 10 and 14 days) were also examined. It was found thataddition of FeS04 significantly improved the staurosporine yieldin comparison to the starting conditions (increase of 25%). Ourresults proved that rhizosphere soils of ethno-medicinal plantsare a prolific source of streptomycetes, producers of compoundswith good antifungal activity. Isolation of the new staurosporineproducing strain, allowed for its detailed bioactivity assessment.Staurosporine scaffold might serve as a lead structure for thedevelopment of new antifungal and antiangiogenic agents. Also,results obtained within this research represent the basis for thefurther scale-up and potential industrialization of the proposedproduction process.

This thesis examined the antimicrobial activity of select neotropical plants from Costa Rica and traditional Q’eqchi Maya medicines from Belize. In particular the potential for interference with bacterial quorum sensing (QS) and biofilm formation as well as fungal growth were assessed. Of one hundred and twenty six extracts collected from Costa Rica, one third showed significant QS inhibition while 13 species displayed more biofilm inhibitory activities than the positive control allicin. The active species belonged to the Lepidobotryaceae, Melastomataceae, Meliaceae, Sapindaceae, and Simaroubaceae. Twelve Marcgraviaceae species were tested for the same biological activities; of these, three showed similar QS inhibition to that of the positive control Delisea pulchra (Greville) Montagne and five with at least 30% biofilm inhibition. Only one species inhibited fungal growth – Marcgravia nervosa Triana & Planch. Bioassay-guided isolation of this plant resulted in the identification of the active principle as a naphthoquinone, with a minimum inhibitory concentration (MIC) ranging from 85 to 100 μM against Saccharomyces cerevisiae. Similarly, sixty one Q’eqchi’ Maya medicinal plant species were evaluated for their antimicrobial activities. Of these, four species showed more QS inhibition than D. pulchra, seven with comparable biofilm inhibitory activities that of allicin, and two with similarly antifungal activity to berberine. Two spirostanol saponins were isolated from Cestrum schlechtendahlii G.Don, an active antifungal plant. The major saponin showed growth inhibition against Saccharomyces cerevisiae and Fusarium graminearum, with MICs of 16.5 μM and 132 μM, respectively. Further analyses of this compound using chemical genomics suggested that its antifungal mechanism of action is pleiotropic, affecting multiple targets. Taken together, these findings showed that neotropical plants and traditional Q’eqchi’ Maya medicines contain phytochemicals that interfere with bacterial biofilm formation and quorum sensing as well as fungal growth.

Cilj istraživanja doktorske disertacije bio je da se ispita pojedinačni i sinergistički uticajekstrakata kima (Carum carvi L.), bosiljka (Ocimum basilicum L.), origana (Origanumvulgare L.) i etarskih ulja crnog (Allium cepa L. kultivar Kupusinski jabučar) i belog luka(Allium sativum L. kultivar Bosut) na rast plesni izolovanih iz prehrambenih proizvoda, kao injihov uticaj na biosintezu mikotoksina.Ukupan broj plesni u uzorcima salata od povrća spremnih za konzumiranje kretao se od10,0 do 5,5´102 cfu/g, u uzorcima poslastičarskih proizvoda do 6,1´102 cfu/g i uproizvodima od mesa do 60,0 cfu/g. Najveći broj plesni izolovan je na DG18 podlozi(1,53´102 cfu/g), a najmanji na MY50G (42,0 cfu/g). U ukupnoj mikopopulaciji svih ispitivanihuzoraka dominirale su vrste rodova Penicillium (39,07%), Cladosporium (23,40%) iAspergillus (20,42%). Vrste iz rodova Alternaria, Fusarium i Eurotium su bile zastupljene sa5,85%, 4,97% i 2,76%. Dominantne vrste u ukupnoj mikopopulaciji bile su C.cladosporioides (21,63%), A. niger (16,0%) i P. aurantiogriseum (11,81%).Dominirali su potencijalni producenti ohratoksina A (31,89%), proizvoñači fumonizina(4,74%), moniliformina (1,43%) i sterigmatocistina (1,54%). Izolati A. versicolor subiosintetisali sterigmatocistin u koncentracijama od 56,3 i 109,2 ng/mL. Ostale potencijalnetoksin-produkujuće vrste nisu pokazale sposobnost produkcije mikotoksina.Mikotoksikološkim ispitivanjem hrane u dva uzorka salata spremnih za konzumiranje(kupus beli rezani i FIT salata) utvrñen je sadržaj sterigmatocistina u koncentracijama od 3,5i 5,5 mg/kg.Kao glavna komponenta u ekstraktu kima odreñen je karvon (43,98%), u ekstraktu bosiljkaestragol (metil kavikol) (86,72%), a u ekstraktu origana karvakrol (34,20%) i karvon (18,05%).Najveći deo etarskog ulja crnog luka činili su: dimetil-trisulfid, metil-propil-trisulfid, dimetiltetrasulfid,dietil-1,2,4-tritiolan, metil-(1-propenil)-trisulfid, metil-(1-propenil)-disulfid. Dialildisulfid,dialil-trisulfid, metil-alil-trisulfid i metil-alil-disulfid su glavne komponente koje suodreñene u etarskom ulju belog luka.Koncentracija od 0,35 mL/100 mL ekstrakta kima je bila fungicidna (MFC) prema C.cladosporioides, dok je 0,70 mL/100 mL potpuno inhibirala rast A. carbonarius, A. wentii, E.nidulans, Eurotium spp., C. cladosporioides, P. glabrum, P. brevicompactum, F.subglutinans i F. verticillioides. Na rast P. chrysogenum i P. aurantiogriseum istakoncentracija bila je inhibitorna (MIC). Najslabije delovanje ovaj ekstrakt ispoljio prema A.niger, A. versicolor, F. oxysporum i F. proliferatum.Primena ekstrakta bosiljka u koncentraciji od 0,70 mL/100 mL pokazala je fungicidnodelovanje na C. cladosporioides. Koncentracija od 1,50 mL/100 mL potpuno je inhibiralarast A. wentii, A. versicolor, E. nidulans, E. herbariorum, E. chevalieri, E. rubrum, P.chrysogenum i Fusarim spp. Ekstrakt bosiljka je najslabije delovao prema A. niger, A.carbonarius, P. aurantiogriseum, E. amstelodami, P. glabrum i P. brevicompactum.Ekstrakt origana je pokazao najslabije ihibitorno delovanje na rast ispitivanih plesni.Primena ekstrakta u koncentraciji od 1,50 mL/100 mL je bila fungicidna prema E. rubrum.Koncentracija od 2,50 mL/100 mL je pokazala fungicidno delovanje na E. rubrum, E.herbariorum, A. wentii, C. cladosporioides i P. aurantiogriseum, a inhibitorno prema E.nidulans, E. chevalieri, E. amstelodami, P. glabrum i P. brevicompactum. Ovaj ekstrakt jenajslabije delovao na A. niger, A. carbonarius, F. proliferatum, F. subglutinans i P.chrysogenum.Etarsko ulje crnog luka pokazalo je signifikantno jače antifungalno delovanje na ispitivaneplesni u odnosu na etarsko ulje belog luka. Koncentracija od 14,0 mL/100 mL ulja belog lukafungicidno je delovala prema E. rubrum, E. chevalieri i C. cladosporioides, dok je ulje crnogluka na ovoj koncentraciji pokazalo fungicidni efekat i na E. herbariorum i E. amstelodami.Za ostale plesni fungicidna koncentracija iznosila je 28,0 mL/100 mL, osim za A. niger i P.aurantiogriseum.Neke od ispitivanih smeša bosiljka i kima, bosiljka i origana, origana i kima i etarskih uljalukova pokazale su sinergističko delovanje na inhibiciju rasta A. wentii, E. herbariorum, F.verticilllioides i P. aurantiogriseum sa FICindex od 0,63 do 0,97.Začinski ekstrakti i etarska ulja lukova su pored ograničavanja rasta kolonija plesniuzrokovali i promene u makro i mikromorfologiji.Potpuna inhibicija biosinteze sterigmatocistina i rasta A. versicolor postignuta je prikoncentraciji od 0,20 mL/100 mL ekstrakta kima i origana u periodu od 21 dana. Na ovojkoncentraciji ekstrakt bosiljka je inhibirao biosintezu sterigmatocistina za 88,73% i rastplesni za 52,56%. Smeša koja je sadržavala 75% ekstrakta kima i 25% ekstrakta bosiljkapotpuno je inhibirala rast plesni i biosintezu sterigmatocistina u YES bujonu tokom 21 danainkubiranja.Pojedinačne koncentracije etarskih ulja crnog i belog luka od 5,0 i 10,0 mL/100 mL i u smešisa 1,50 mL/100 mL etarskog ulja crnog luka i 0,50 mL/100 mL etarskog ulja belog luka bile supotrebne za potpunu inhibiciju rasta A. versicolor i biosintezu sterigmatocistina.Dodatak smeše ekstrakata kima i bosiljka (0,35 mL/100 mL ekstrakta kima + 0,70 mL/100 mLekstrakta bosiljka) u svež kupus rezanac uticao je na smanjenje inicijalne kontaminacijeplesnima za 93,9%, uz pojavu intenzivnijeg, ali prihvatljivog mirisa i neznatne promene boje.Definisani matematički model za komparaciju uticaja ekstrakata i etarskih ulja na rast plesnimože se primenjivati u formiranju matrica inhibicije i optimizaciji vremena i koncentracijeantifungalnih agenasa.Dobijena saznanja o antifungalnom delovanju ekstrakata začina i etarskih ulja lukova mogubiti značajna u poboljšanju antifungalne zaštite namirnica, smanjenju biosintezemikotoksina i ukupnim smanjenju šteta izazvanih delovanjem plesni.
The aim of this PhD thesis was to study the individual and synergistic effects of extracts ofcaraway (Carum carvi L.), basil (Ocimum basilicum L.), oregano (Origanum vulgare L.) and theessential oils of onion (Allium cepa L. cultivar Kupusinski jabučar) and garlic (Allium sativumL. cultivar Bosut) on the growth of moulds isolated from food products. The study alsofocused on the impact of extracts and the essential oils on mycotoxins biosynthesis.The total number of moulds detected in samples of vegetable salads “ready for use” rangedfrom 10.0 to 5.5´102 cfu/g. In cake and pastries, as well as, meat samples, the numberreached 6.1´102 cfu/g and 60.0 cfu/g, respectively. The highest number of mould colonies wasisolated in DG18 medium (1.53 ´ 102cfu/g) and the lowest in MY50G medium (42.0 cfu/g). Thespecies of the genera Penicillium (39.07%), Cladosporium (23.40%) and Aspergillus (20.42%)prevailed in the entire mycopopulation of all tested samples. Species of the genera Alternaria,Fusarium and Eurotium were represented with 5.85%, 4.97% and 2.76%, respectively, while C.cladosporioides (21.63%), A. niger (16.0%) and P. aurantiogriseum (11.81%) were the mostdominant species in the entire mycopopulation.Potential producers of ochratoxin A (31.89%) accounted for the largest share of the isolatedmycopopulation. The share of producers of fumonisin, moniliformin and sterigmatocystinamounted to 4.74%, 1.43% and 1.54%, respectively. Sterigmatocystin was biosynthesised inthe concentration of 56.3 ng/mL and 109.2 ng/mL by both isolates of A. versicolor, while otherpotential toxin producers did not show the ability of mycotoxin production.Mycotoxicological investigation showed the sterigmatocystin content in two samples ofvegetable salads "ready for use" (shredded white cabbage and FIT salad - carrot, lettuce andred chicory) in concentrations of 3.5 mg/kg and 5.5 mg/kg, respectively.The major component in the extract of caraway was carvon with a share of 43.98%. The basilextract contained estragol (methyl cavicol) in the highest percentage (86.72%), whilecarvacrol (34.20%) and carvon (18.05%) were major components of the oregano extract. As forthe essential oil of onion, dimethyl trisulphide, methyl propyl trisulphide, dimethyl tetrasulfid,diethyl-1, 2, 4-tritiolan, methyl-(1-propenyl)-trisulphide, and methyl-(1-propenyl) – disulfideconstituted the largest share. The major components isolated in garlic essential oil werediallyl disulfide, diallyl-trisulphide, allyl methyl trisulphide and allyl methyl disulfide.The concentration of the caraway extract of 0.35 mL/100mL exhibited fungicidal effect (MFC)on C. cladosporioides, while the concentration of 0.70 mL/100mL completely inhibited thegrowth of A. carbonarius, A. wentii, E. nidulans, Eurotium spp., C. cladosporioides, P.glabrum, P. brevicopmactum, F. subglutinans and F. verticillioides. The same concentrationshowed the inhibitory effect (MIC) on the growth of P. chrysogenum and P. aurantiogriseum.The poorest effect of the caraway extract was expressed on the growth of A. niger, A.versicolor, F. oxysporum and F. proliferatum.The basil extract application in the concentration of 0.70 mL/100mL showed fungicidal effects(MFC) on the growth of C. cladosporioides. The concentration of 1.50 mL/100mL completelyinhibited (MFC) the growth of A. wentii, A. versicolor, E. nidulans, E. herbariorum, E.chevalierii, E. rubrum, P. chrysogenum and Fusarim spp. The poorest effect of the basilextract was exhibited on A. niger, A. carbonarius, P. aurantiogriseum, E. amstelodami, P.glabrum and P. brevicompactum.The oregano extract showed the weakest growth inhibition influence on all of the testedmoulds. The application of this extract in the concentration of 1.50 mL/100mL was fungicidal(MFC) to E. rubrum. The concentration of 2.50 mL/100mL showed fungicidal effects (MFC) onthe growth of E. rubrum, E. herbariorum, A. wentii, C. cladosporioides and P. aurantiogriseumand inhibitory effects (MIC) on E. nidulans, E. chevalieri, E. amstelodami, P. glabrum and P.brevicompactum. The weakest effect of this extract was expressed on the growth of A. niger,A. carbonarius, F. proliferatum, F. subglutinans and P. chrysogenum.Onion essential oil showed a significantly stronger antifungal effect on the tested moulds incomparison to garlic essential oil. While the concentration of 14.0 mL/100mL of garlic oil had afungicidal effect on E. rubrum, E. chevalieri and C. cladosporioides, the same concentrationof onion oil was also fungicidal to E. herbariorum and E. amstelodami. With an exception of A.niger and P. aurantiogriseum, the concentration that showed a fungicidal effect on theremaining moulds equalled 28.0 mL/100mL.Some of the tested mixtures of basil with caraway, basil with oregano, oregano with caraway,and essential oils of onion and garlic, showed a synergistic effect on the growth inhibition ofA. wentii, E. herbariorum, F. verticilllioides and P. aurantiogriseum with the FIC index rangingfrom 0.63 to 0.97.Apart from the inhibitory effect on the mould colony growth, the spices extracts and theessential oils of onion and garlic also caused changes in the macro- and micro- morphologyof the moulds.Complete inhibition of the growth of A. versicolor and sterigmatocystin biosynthesis wasachieved at a concentration of 0.20 mL/100mL of the extract of caraway and oregano in theperiod of 21 days. At this concentration the basil extract delayed the sterigmatocystinbiosynthesis by 88.73% while the mould growth was inhibited by 52.56%. Mixtures containing75% of the caraway extract and 25% of the basil extract completely inhibited the mouldgrowth and sterigmatocystin biosynthesis in YES broth during 21 days of incubation.The concentrations of 5.0 m L/100mL (onion essential oil) and 10.0 m L/100mL (garlic essentialoil) applied in a mixture containing 1.50 mL/100mL of onion and 0.50 mL/100mL of garlicessential oil were necessary for a complete inhibition of the growth of A. versicolor andsterigmatocystin biosynthesis.The addition of the mixture of caraway and basil extracts (0.35 mL/100mL of caraway + 0.70mL/100mL of basil) to fresh shredded cabbage influenced the reduction of initial mouldcontamination by 93.9%. This was accompanied by the occurrence of acceptable moreintense flavour and slight discoloration.The defined mathematical model for comparing the effects of extracts and essential oils onthe growth of moulds can be applied in establishing inhibition matrices and optimisation ofthe time and the concentration of antifungal agents.The obtained results on the antifungal effects of the spices extracts and onion and garlicessential oils can be beneficial for improving the antifungal protection of food and reducingthe mycotoxin biosynthesis as well as the overall damage caused by the action of moulds.

The garlic extract ajoene is considered to have antimicrobial and antitumor effects against a variety of cell types, and it is suggested to have the potential to be used as an antifungal or antitumor drug clinically. The underlying mechanism of its inhibitory effects is still uncertain. In this project, the effects of ajoene on the growth of fungal and oomycete cells were studied on Candida albicans, Neurospora crassa and Achlya bisexualis. Endometrial cancer is the most common gynecologic cancer. A 3D spheroid model of endometrial cancer cells were for the first time used to investigate the antitumor effects of ajoene and selected antitumor agents. Ajoene was extracted from fresh garlic by chromatographic methods and the outcome of the extractions was verified with Mass spectrometry and NMR spectroscopy. Ajoene was then tested on the yeast form or germ tubes of C. albicans, and the cell division and germ tube formation was analyzed. N. crassa and A. bisexualis were treated with ajoene on plates or on glass slides to measure the hyphae radial extension or individual hyphal extension. 3D endometrial adenocarcinoma cell (Ishikawa) spheroids were treated with ajoene, paclitaxel, targeted drugs everolimus, sorafenib, gefitinib and canertinib alone or in combinations. The growth activity, metabolic activity, cell proliferation, apoptotic activity and the cytoskeletons were analyzed after the treatments. Cell division of C.albicans was inhibited by ajoene at 5µg/ml or higher concentrations. The length of C.albicans germ tubes was significantly shorter in ajoene treated groups than the untreated ones. Radial extension and individual hyphal extension of N. crassa and A. bisexualis were both inhibited by ajoene. Ajoene did not show any antitumor effects on the 3D cell model of Ishikawa cells. No synergistic effect was detected between ajoene and paclitaxel or ajoene and everolimus. The targeted drugs Canertinib and everolimus showed an inhibitory effect on growth activity of the spheroids, but no synergy with paclitaxel. In conclusion, ajoene was able to inhibit various forms of fungal and oomycete growth, but any antitumor activity of ajoene did not show on 3D culture of endometrial cancer cells.

Magister Chirurgiae Dentium - MChD (Oral Medicine and Periodontics)
Candida species are frequently isolated from oral mucosal surfaces of healthy individuals and is the most common genus responsible for up to 75% of all candidal infections. The most common problems associated management of oral candidiasis are antifungal drug resistance and side effects Natural medicine is an emerging field and is being explored to overcome drug resistance and to reduce side effects. Gymnemagenin (will be known as Gymnemic acid; GA) is a purified extract from Gymnema sylvestre, a slow growing, perennial, medicinal plant found in Central and Western India, Tropical Africa and Australia is regarded as one of the plants with potent antimicrobial and antifungal activity.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
Plants are constantly exposed to a variety of stresses conditions. However, they react to biotic stresses by triggering a set of defense mechanisms including the synthesis of defensive substances as the pathogenesis-related (PR) proteins. The PR-protein named osmotin can be induced under osmotic stress and water shortage conditions. Osmotin-like proteins have been purified from latex and some of them are related to antifungal activity. The aim of this study was to investigate the osmotin in the following species laticifers: C. grandiflora, P. rubra, T. peruviana, H. drasticus and C. papaya to isolate and evaluate its antifungal activities. Immunoaffinity column chromatography with anti-CpOsm antibodies were performed in order to purify these osmotin-like. They were detected in latex of C. grandiflora and P. rubra by immunoassays the ELISA, Dot Blot and Western Blot using anti-CpOsm antibody (the osmotin of C. procera latex). Osmotin of C. procera, C. grandiflora, P. rubra and H. drasticus were identified by mass spectrometry. However, the osmotin from C. procera was co-purified with cysteine proteases. The co-purified cysteine protease from C. procera was identified as Procerain B. The alignment and the 3-D structure analysis of Procerain B and CpOsm revealed the presence of a similar sequence in both proteins. This sequence might be an epitope which allows the anti-antibody recognition. The osmotin from C. grandiflora, and P. rubra did not show antifungal activity against Fusarium solani and Colletotrichum gloesporioides. Since no correlation between the antifungal activity and the presence of these osmotins were found, the proteolytic activities of these latex protein fractions were evaluated in order to correlate with the antifungal activity C. procera and C. grandiflora showed a strong proteolytic activity. In latex, the cysteine proteases are more often related to antifungal activity than osmotin, which might explain, at least in part, the antifungal activity performed by C. grandifora and not for its osmotin. Further studies on the role of osmotin in physiology laticifers plants are needed.
As plantas estÃo constantemente sujeitas a diversos tipos de estresse, tanto biÃticos como abiÃticos, resultando em respostas de defesa. Decorrente disto, os vegetais sintetizam certas proteÃnas denominadas de proteÃnas relacionadas à patogÃnese (PR proteÃna). As Pr-proteÃnas chamadas de osmotinas podem ser induzidas sob condiÃÃes de estresse osmÃtico, frio e escassez de Ãgua. Osmotinas tem sido purificadas de fluidos laticÃferos e algumas delas estÃo relacionadas com a atividade antifÃngica. O objetivo do presente trabalho foi prospectar osmotinas, bem como isolÃ- las e avaliar suas atividades antifÃngicas, nos fluidos laticÃferos das seguintes espÃcies: C. grandiflora, P. rubra, T. peruviana, H. drasticus e C. papaya. Nos lÃtex de C. grandiflora e P. rubra foram detectadas osmotinas atravÃs de imunoensaios em placa de ELISA, Dot Blot e Westen Blot, utilizando os anticorpos anti-CpOsm (osmotina do lÃtex de C. procera). Cromatografia de imunoafinidade em coluna com anticorpos anti-CpOsm foram realizadas com o intuito de purificar estas osmotinas. AnÃlises por meio de espectrometria de massas, revelaram a presenÃa de osmotina em C. procera, C. grandiflora, P. rubra e H. drasticus. No entanto, a osmotina de C. procera foram co-purificadas com proteases cisteÃnicas. A protease cisteÃnica co- purificada no lÃtex de C. procera foi identificada como Proceraina B. O alinhamento e a anÃlise da estrutura tridimensional da Proceraina B e CpOsm revelaram a presenÃa de uma sequÃncia semelhante em ambas as proteÃnas, que pode ser um epÃtopo disponÃvel ao reconhecimento do anticorpo anti-CpOsm. As osmotinas isoladas de C. grandiflora e P. rubra nÃo apresentaram atividade antifÃngica contra F. solani e C. gloesporioides. Desde que nÃo houve correlaÃÃo entre a atividade antifÃngica e à presenÃa destas osmotinas, as atividades proteolÃticas das fraÃÃes proteicas foram avaliadas a fim de correlaciona-las à atividade antifÃngica. Nos fluidos laticÃferos, as proteases cisteÃnicas estÃo mais frequentemente relacionadas à atividade antifÃngica do que as osmotinas. Estudos mais aprofundados sobre a funÃÃo das osmotinas na fisiologia de plantas laticÃferas sÃo necessÃrios.

Thesis (M.S.)--University of Tennessee Health Science Center, 2009.
Title from title page screen (viewed on August 11, 2009). Research advisor: Jegdish P. Babu, Ph.D. Document formatted into pages (ix, 32 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 28-31).

The continuous emergence of drug-resistance pathogens is a global concern. As a result, substantial effort is being expended to develop new therapeutics and mechanisms for controlling microbial growth to avoid entering a "post-antibiotic" era in which commonly used antibiotics are no longer effective in treating infections. In this work, we investigate the efficacy and application of ceragenins as non-peptide mimics of antimicrobial peptides (AMPs). First, this work examines the susceptibility of drug-resistant Gram-negative bacteria. The susceptibility of colistin-resistant clinical isolates of Klebsiella pneumoniae to ceragenins and AMPs suggests that there is little to no cross-resistance between colistin and ceragenins/AMPs. Furthermore, Lipid A modifications are found in bacteria with modest changes in susceptibility to ceragenins and with high levels of resistance to colistin. Next, we investigated the potential for cross resistance between chlorhexidine, colistin, AMPs and ceragenins as repeated exposure of bacteria to chlorhexidine might result in cross resistance with colistin, AMPs or ceragenins. Furthermore, a proteomics study on the chlorhexidine-resistant strains showed that chlorhexidine resistance is associated with upregulation of proteins involved in the assembly of LPS for outer membrane biogenesis and virulence factors in Pseudomonas aeruginosa.Second, this dissertation describes the antifungal activity of ceragenins against an emerging multidrug-resistant fungus, Candida auris. We found that lead ceragenins displayed activities comparable to known antifungal agents against C. auris isolates. We also found that fungal cell morphology was altered in response to ceragenin treatment, that ceragenins exhibited activity against sessile organisms in biofilms, and that gel and cream formulations including CSA-44 and CSA-131 resulted in a significant log reduction against established fungal infections in ex vivo mucosal tissues. Finally, a hydrogel film containing CSA-131 was generated on endotracheal tubes (ETTs). ETTs provide an abiotic surface on which bacteria and fungi form biofilms that cause serious infections. In this study, the eluting ceragenin prevented fungal and bacterial colonization of coated ETTs for extended periods while uncoated tubes were colonized by bacteria and fungi. Coated tubes were well tolerated in intubated pigs. The ability of ceragenins to eradicate established biofilms make them attractive potential therapeutics for persistent infections in the lung, including those associated with cystic fibrosis. In ex vivo studies, we initially found that this ceragenin, at concentrations necessary to eradicate established biofilms, also causes loss of cilia function. However, by formulating CSA-131 in poloxamer micelles, cilia damage was eliminated and antimicrobial activity was unaffected. These findings suggest that CSA-131, formulated in micelles, may act as a potential therapeutic for polymicrobial and biofilm-related infections in the lung and trachea.

The main aim of this study was to isolate and characterise antifungal and antibacterial compounds from leaf extracts of Combretum molle which belonging to the Combretaceae family. C. molle is one of the commonly used medicinal plants in southern Africa for numerous ailments. Three animal fungal pathogens, namely, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus and five plant fungal pathogens, namely, Aspergillus niger, Aspergillus parasiticus, Fusarium oxysporum, Penicillium janthinellum, Rhizoctonia solani and four nosocomial bacteria Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa were used as test microorganisms for bioactive compounds in leaf extracts of C.molle. Experiments for phytochemical analysis were done using different C. molle leaf extracts which were made using acetone, methanol, ethanol, ethyl acetate, chloroform, butanol and hexane as extractants. Thin Layer Chromatography (TLC) fingerprints of different leaf extracts were developed in three mobile phase systems, EMW, CEF and BEA and detected with vanillin-sulphuric acid spraying agent. The different extracts of C. molle showed the presence of many different compounds with distinct retardation factors (Rf), separated according to their polarities. Bioautography was carried out to determine the number of active compounds and their Rf values. The TLC plates were developed in three mobile systems, each sprayed with either fungal or bacterial strains. In BEA bioautograms of A. fumigatus, clear zones of inhibition were observed at Rf values of 0.12, 0.23, and 0.40. In EMW bioautogram of C. albicans, clear zones of inhibition were observed at Rf value of 0.73, 0.81, 0.87. C. neoformans had weak growth inhibition. Most of the fungal and bacterial strains tested in the bioautography displayed susceptibility to the active compounds, with P. janthinellum and P. aeruginosa showing exceptional sensitivity. The minimum inhibitory concentrations (MIC) values ranged from 0.02 to 2.5 mg/ml against the tested pathogens. The acetone and ethyl acetate extracts had the best inhibitory activity against P. janthinellum with an MIC value of 0.02 mg/ml. The acetone extract of C. molle gave the highest total activity (775 ml/g) against P. janthinellum. C. albicans was the most resistant pathogen with an average MIC value of 0.56 mg/ml compared with the other tested strains. Extracts were active against both Gram-positive and Gram-negative strains. P. aeruginosa extracts had the highest average MIC value (0.24 mg/ml) among the tested bacterial strains. In general, there was good overall inhibitory activity by different extracts of C. molle. Bioassay-guided fractionation of DCM extract of the leaves of C. molle yielded 32 fractions. Further fractionation led to the isolation of five compounds (C1, C2, C3, C4 and C5). Compound C1 was selected for structure elucidation due a larger quantity isolated and higher antimicrobial activity compared with the other isolated compounds. Nuclear magnetic resonance (NMR) spectroscopy and mass spectroscopy (MS) was used to show that compound C1 was taraxerol, belonging to the taraxerane group. Antimicrobial activity of the isolated compound against P. janthinellum had an MIC value of 0.08 ug/ml. Although the compound taraxerol have been discovered in other plant species, it is reported for the first time from C. molle in the study. The results illustrate that crude extracts and compound taraxerol from C. molle can be used as either an antibacterial or antifungal, and warrants further investigation.
Dissertation (MSc)--University of Pretoria, 2017.
Paraclinical Sciences
MSc
Unrestricted

Candida albicans is a yeast-like fungal pathogen that can cause infections ranging from superficial to life-threatening systemic candidiasis. Current treatments for systemic candidiasis are available but often ineffective and toxic. Consequently, it is necessary to develop new therapeutic approaches. The purpose of this study was to construct antibody-based fusion proteins that can bind to C. albicans cells and eliminate them. Two such fusion proteins were constructed. Each one is composed of M1 Fab as the antibody component that binds to C. albicans mannan and the antifungal peptide HPRP-A1. HPRP-A1 was attached via a 15-amino acid linker to either the C-terminus of the constant light chain of M1 Fab (M1 Fab-HPRP-CL) or the N-terminus of the variable light chain of M1 Fab (M1 Fab-HPRP-VL). Binding of the fusion proteins to purified C. albicans mannan was assessed with enzyme-linked immunosorbent assay and the half maximal effective concentration (EC₅₀) for each fusion protein was estimated. EC₅₀ for M1 Fab-HPRP-CL was 273.6 compared to 74.1 for the original M1 Fab (p < 0.05), whereas M1 Fab-HPRP-VL did not show any binding activity, indicating a negative impact on the antibody binding by the linked peptide. Similarly, M1 Fab-HPRP-CL also showed reduced binding for C. albicans cells when compared to M1 Fab as determined with immunofluorescence microscopy and flow cytometry. The effect of M1 Fab-HPRP-CL on the growth of C. albicans cells was analysed using microdilution and absorbance. At 16 µM, the growth of yeast cells treated with M1 Fab-HPRP-CL was 47.1 % of the growth control, compared to 43.5 % for M1 Fab (p > 0.05) and to 1.9 % for HPRP-A1 by itself (p < 0.001). Moreover, HPRP-A1 killed C. albicans at 32 µM and 64 µM, while M1 Fab and M1 Fab-HPRP-CL did not, indicating a loss of the antifungal activity of HPRP-A1 when attached to the antibody. These data together provide valuable insights into the development of novel antibody-based therapeutics as an alternative treatment for candidiasis.

Phytochemical analysis of both HEA1 and the crude plant extract showed the presence of phenolics, tannins and saponins. Saponins were the predominant secondary metabolites and were mostly abundant in the plant extract and to a lesser extent in the active compound. Steroidal saponins, tannins and phenolics were also detected in the plant extract, but only the phenolics were detected in the active compound. The results of the phytochemical analysis showed that those compounds that were not present in the active compound could be removed from the crude extract during the TLC purification process. Investigation on the mechanism of action of the crude plant extract on the sterol production by A. flavus showed that the plant extract affected ergosterol biosynthesis by causing an accumulation of oxidosqualene in the ergosterol biosynthetic pathway resulting in a decline in ergosterol production. An oscillatory response in lanosterol production was observed in the presence of the plant extract, which may be an adaptation mechanism of A. flavus to unfavourable conditions and compensation for the loss of enzyme activity which may have occurred as a result of the accumulation of oxidosqualene. The antifungal activity of the plant extract on ergosterol production by A. flavus may also be due to saponins which target the cell membrane and ergosterol production in fungi. The effect of the plant extract on the fungal cell wall of A. flavus also showed that the plant extract caused a decline in β-(1, 3) glucan production by inhibiting β-glucan synthase. The plant extract also affected the chitin synthesis pathway of A. flavus, by causing a decline in chitin production, which was due to the inhibition of chitin synthase. Investigation of chitinase production using 4MU substrates showed that the plant extract caused an accumulation of chitobioses, by activating chitobiosidases and endochitinases. A decline in N-acetylglucosaminidase activity in the presence of the plant extract was observed and this prevented the formation of N-acetylglucosamine. The accumulation of chitobiosidase and endochitinase may be as a result of autolysis that may be triggered by A. flavus as a survival mechanism in the presence of the plant extract and as a compensatory mechanism for the loss of β-glucans and chitin. The antifungal effect of the plant extract on various components of the cell wall of A. flavus, makes T. violacea aqueous plant extract an ideal chemotherapeutic agent against both human and plant pathogens of Aspergillus. The broad spectrum of antifungal activity of T. violacea against A. flavus also eliminates any chances of the fungus developing resistance towards it and would make it a candidate for use as a potential antifungal agent. Further identification and possible chemical synthesis is needed to shed light on the safety and efficacy of the active compound for further development as a chemotherapeutic agent.

Food waste and food poisoning derived from food contamination are huge issues in a globalized world like ours, where the distances between producer and consumer are ever greater. Biofilm-forming fungi are among the microorganisms more common in this sector. In the present work, antimicrobial and antibiofilm efficacy of Ethyl Lauroyl Arginate (LAE) was evaluated on some strains of fungi, specifically, Cladosporium cladosporioides, Aspergillus ochraceus, Penicillium italicum, Botryotinia fuckeliana and Fusarium oxysporum. LAE is a surfactant derived from lauric acid and arginine, approved and generally recognized as safe (GRAS) for certain food applications, due to the rapidity with which it is hydrolyzed by the human body. The MIC of the LAE was identified and its activity as an antibiofilm was evaluated for the cited fungal strains. Subsequently, the antimicrobial was used as an active part of a varnish-based antibiofilm coating that could be used for surfaces and packaging. Finally, an edible pectin-based film with LAE was tested in vivo against grapes inoculated with B. fuckeliana.

The aim of this study was to investigate plant species to develop a product with the potential of protecting plants or plant products against plant fungal pathogens. Hexane, dichloromethane, acetone, and methanol leaf extracts of six plant species (Bucida buceras, Breonadia salicina, Harpephyllum caffrum, Olinia ventosa, Vangueria infausta and Xylotheca kraussiana) were evaluated for antifungal activity against seven plant fungal pathogens (Aspergillus niger, A. parasiticus, Colletotrichum gloeosporioides, Penicillium janthinellum, P. expansum, Trichoderma harzianum and Fusarium oxysporum). These plant species were selected from more than 400 plant species evaluated in the Phytomedicine Programme that had good activity against two animal fungal pathogens. All the leaf extracts were active against at least one or more of the phytopathogenic fungi in a serial microdilution assay. Of the six plant species, B. buceras had the best antifungal activity against four of the fungi, with MIC values as low as 0.02 mg/ml and 0.08 mg/ml against Penicillium expansum, P. janthinellum, Trichoderma harzianum and Fusarium oxysporum. The number of active compounds in the plant extracts was determined using bioautography with the above-mentioned plant pathogens. No active compounds were observed in some plant extracts against the fungal plant pathogens indicating possible synergism between metabolites responsible for the antifungal activity of the extract. B. salicina and O. ventosa were the most promising plant species, with at least three antifungal compounds. The antioxidant activities of plant extracts were determined using the qualitative method by spraying TLC chromatograms developed in three eluent systems BEA, CEF and EMW with 1, l-diphenyl -2 picrylhydrazyl (DPPH). The plant extracts of five of these species did not have a strong antioxidant activity. The methanol extract of X. kraussiana was the most active radical scavenger in the DPPH assay amongst the six medicinal plants screened. Based on good activity against Aspergillus niger and A. parasiticus, leaf extracts of the six plant species were also tested for antifungal activity against A. fumigatus, a very important animal fungal pathogen. The acetone extracts of B. buceras, B. salicina, V. infausta and X. kraussina had good antifungal activity against the animal pathogens, with MIC values ranging between 0.02 and 0.08 mg/ml. This indicates that crude extracts of these species may be more valuable in combating Aspergillus infections in animals than in humans. Based on the results discussed above, B. salicina was selected for in-depth study. Serial exhaustive extraction was used to extract plant material with solvents of increasing polarities namely, hexane, chloroform, acetone and MeOH. Amongst the four extractants, MeOH extracted the largest quantity of plant material 12.3% (61.5g), followed by acetone 5.6% (27.8 g), hexane 2.6% (12.8 g) and chloroform 2.1% (10.3 g). The chloroform fraction was selected for further work because it had the best antifungal activity against A. niger, C. gloeosporioides, P. janthinellum and T. harzianum and the bioautography assay showed the presence of several antifungal compounds in the chloroform fraction. Column chromatography was used in a bio-assay guided fractionation and led to isolation of four compounds. The antimicrobial activity was determined against seven plant pathogenic fungi and three bacteria, including the Gram-positive Staphylococcus aureus (ATCC 29213) and the Gram-negative Escherichia coli (ATCC 25922) and Pseudomonas aureus (ATCC 27853). The isolated compounds had good antifungal activity against A. parasiticus with an MIC of 10 μg/ml, while in other cases it ranged from 20 to 250 μg/ml. Amongst the four compounds tested, only three had a clear band, indicating that the growth of the pathogenic fungi was inhibited in the bioautography assay. Nuclear magnetic resonance spectroscopy (NMR) and mass spectroscopy (MS) were used for identification of isolated compounds. Only one compound was identified as the triterpenoid ursolic acid. Ursolic acid has been isolated from several plant species and has antifungal activity against Candida albicans (Shai et al. 2008). This is the first report on the isolation of antifungal compounds from leaves of Breonadia salicina. The other compounds isolated appeared to be mixtures of fatty acids based on mass spectroscopy and the structures were not elucidated. The cytotoxicity of acetone extracts and the four isolated compounds were determined against Vero cells using a tetrazolium-based colorimetric (MTT) assay. The acetone extract was selected based on good in vitro antifungal activity and was used in an in vivo fruit experiment. The acetone extract was less toxic toward the Vero cells with an LC50 of 82 μg/ml than ursolic acid and compound 4 which had LC50 values of 25 and 36 μg/ml respectively. Compounds 2 and 3 had low toxicity against the cells with LC50 values greater than 200 μg/ml. The potential use of the extract or isolated compound(s) against three plant fungal pathogens Penicillium expansum and P. janthinellum as well as P. digitatum (isolated from infected oranges) were tested after treating the oranges with the extract and ursolic acid. The model used gave good reproducible results. The concentration that inhibited growth correlated reasonably well with MIC values determined by serial microplate dilution. There were substantial differences in the susceptibility of the different isolates tested. The activity of ursolic acid was in the same order as that of the crude acetone leaf extract of B. salicina. The LC50 of the extract varied from 1 to 1.8 mg/ml. Penicillium digitatum was more resistant to amphotericin B in comparison to other Penicillium species. It has been reported that the fungus was resistant to the three fungicides: sodium ï-phenylphenate (ï-phenylphenol), imazalil, and thiabendazole used commercially in the fruit industry to reduce postharvest decay (Holmes and Eckert 1999). The toxicity of the extract to Vero cells was in the order of 10 times lower than the LC50 of the extracts to the fungal pathogens. Although much work still has to be done, there is good potential that a commercial product can be developed from an acetone leaf extract of B.salicina leaves, especially if the activity of this extract can be improved by removing inactive compounds. The results confirm the traditional use of B. salicina and demonstrate the potential value of developing biopesticides from plants.
Thesis (PhD)--University of Pretoria, 2009.
Paraclinical Sciences
unrestricted

The thesis entitled “Synthesis and biological activity evaluation of natural antifungals and their analogues” is divides into four chapters. Chapter 1 deals with important and challenging aspects of fungicide research and development of natural products as fungicides. Chapter 2 describes the first total synthesis of thiobutacin, a butanoic acid with antifungal activity, recently isolated from the culture broth of a soil actinomycete, Lechevalieria aerocolonigenes strain VK-A9. The five-step procedure involves readily available and cheap starting materials and can easily be transposed to the large scale. An approach towards the enantioselective synthesis of thiobutacin with the use of Amano lipase biotransformation strategy, optical pure menthol, and oxazolidinone for resolution is described. Chapter 2 deal also with biological activity evaluation of thiobutacin. The results show that fungal growth inhibition of thiobutacin is mediated by the pH of the growth medium. Maximum inhibitory activity was obtained between pH 6 and 7. Chapter 3 describes a short and efficient synthesis of cyrmenin B1, an antifungal metabolite of myxobacteria Cystobacter armeniaca and Archangium gephyra. To verify the feasibility of the synthetic strategy, the geometric isomer of cyrmenin B1 was first prepared. The crucial steps included the formation of the dehydroalanine moiety from the corresponding serine acetate and the formation of the β-methoxyacrylate system via trimethylsilyldiazomethane methylation of the corresponding β-hydroxy enamide. Chapter 4 describes synthesis, antifungal Activity, and Structure-Activity Relationship of Cyrmenin B1 analogues.

Microbe resistence is a serious issue, especially as they have become resistant to most well known drugs. Therefore this is considered as a global problem and is now dealt with at a poitical level. Since no new classes of antimicrobial agents have been discovered in the past three deacdes, the development of new drugs is extremely urgent. Therefore the aim of this project was to synthesise derivatives of benzimidazole, and then assesses their antimicrobial activities in vitro by using disc (well) diffusion and MICs tests. A total of 69 benzimidazole derivatives, with substituents at positions 1, 2, and 5, were synthesised, characterised and tested against selected bacteria and fungi. In addition, six bezimidazole silver complexes were prepared and evaluated for their antimicrobial behavior. The SAR showed that the antimicrobial activity of the compounds depended on the substituents attached to the bicyclic heterocycle. Some promising results were obtained. In particular, 5 compounds displayed antibacterial activity against two MRSA strains with MIC values corresponding to ciprofloxacin, which can be considered significant. The compounds have some common features; four possess 5-chloro or 5-bromo substituents; two are derivatives of (S)-2- ethanaminebenzimidazole and the others are derivative of one 2-(chloromethyl)-1Hbenzo[d]imidazole, (1H-benzo[d]imidazol-2-yl)methanethiol and 2-(methoxymethyl)-1-methyl-1H-benzo[d]imidazole. The results from the antifungal screening were very interesting as there were 26 compounds, including two silver complexes, which were potent fungicides against the selected fungal species. They showed equivalent or greater potentency in their MIC values than amphotericin B. In particular, the 5-fluoro, 5-chloro and 5-bromo benzimidazole showed broad spectrum activity.
Saudi Culture Bureau and King Saud University

Las infecciones fúngicas invasivas son una amenaza enorme, pero actualmente solo hay un limitado número de medicamentos antifúngicos disponibles. El aumento de la incidencia de infecciones fúngicas ha agravado la necesidad de nuevos enfoques para las terapias antifúngicas. Nuestro laboratorio descubrió en S. cerevisiae la proteína fosfatasa (PPasa) Ppz1, que participa en múltiples procesos celulares y es importante en la regulación de la homeostasis de los cationes monovalentes. En la levadura, Ppz1 está inhibida por dos proteínas reguladoras, Hal3 y Vhs3. Estas dos proteínas tienen propiedades moonlighting, ya que contribuyen a la formación de una PPC descarboxilasa heterotrimérica atípica, crucial para la biosíntesis de CoA. Ppz1 se encuentra solo en hongos, incluidos los patógenos y se ha identificado como un determinante de virulencia en Candida albicans y Aspergillus fumigatus. Además, cuando se sobreexpresa, parece ser la proteína más tóxica en las levaduras. Estas características definen a Ppz1 como un posible objetivo para el desarrollo de terapias antifúngicas. El primer objetivo de este proyecto es contribuir a dilucidar las bases moleculares de la toxicidad de Ppz1. Primero, mediante el uso de una cepa de sobreexpresión condicional de Ppz1 (ZCZ01, donde reemplazamos el promotor PPZ1 por el promotor GAL1-10), hemos confirmado que el efecto tóxico de Ppz1 se produce debido al aumento en su actividad fosfatasa y no al agotamiento de Hal3/Vhs3. Además, el defecto de crecimiento causado por la sobreexpresión de Ppz1 se caracterizó por crecimiento en líquido y en sólido, y por citometría de flujo. Dado que se sabe que Ppz1 inhibe la entrada de potasio al regular los transportadores de alta afinidad Trk1/Trk2, también hemos confirmado que un exceso de potasio no rescata la letalidad condicional causada por la sobreexpresión de Ppz1. Además, hemos definido los cambios transcriptómicos causados por la sobreexpresión de Ppz1 bajo el promotor GAL1-10, que han revelado la aparición de una respuesta a estrés oxidativo. Cryptococcus neoformans es un hongo patógeno que produce meningoencefalitis en pacientes inmunodeprimidos (principalmente HIV+) y es responsable de una alta tasa de mortalidad en infecciones invasivas. Aquí presentamos la caracterización funcional de CnPpz1 y dos posibles proteínas similares a Hal3: CnHal3a y CnHal3b. CnPpz1 es una PPasa funcional y parcialmente reemplaza la ScPpz1 endógena. Tanto CnHal3a como CnHal3b interactúan con ScPpz1 y CnPpz1 in vitro, pero no inhiben su actividad fosfatasa. Consistentemente, cuando se expresan en S. cerevisiae, reproducen pobremente las propiedades reguladoras de Ppz1 típicas de ScHal3. Por el contrario, ambas proteínas son PPCDC monogénicas funcionales. Ustilago maydis representa un importante organismo modelos eucariota de patógenos de plantas y se ha considerado como uno de los diez principales patógenos fúngicos de plantas. En este estudio mostramos la caracterización funcional de UmPpz1 y UmHal3. Hemos descubierto que UmPpz1 es una PPasa funcional y parcialmente imita a ScPpz1 in vivo. Tanto UmHal3 como su dominio PPCDC interactúan con ScPpz1 y UmPpz1 in vitro, mientras que no se observa la capacidad de inhibir la actividad de la fosfatasa. En S. cerevisiae, si bien UmHal3 no es capaz de inhibir la ScPpz1, se demuestra como una PPCDC funcional. En conjunto, los resultados obtenidos en este trabajo permitenestablecer las bases de la toxicidad de Ppz1 en S. cerevisiae y proporcionan los fundamentos para comprender la regulación y el papel funcional del sistema Ppz1-Hal3 en el hongo patógeno humano C. neoformans y el hongo patógeno de plantas U. maydis.
Invasive fungal infections are an enormous threat, while only a limited number of antifungal drugs are currently available. The increasing incidence of fungal infections has aggravated the need for novel approaches for antifungal therapies. Our laboratory has discovered in S. cerevisiae the protein phosphatase (PPase) Ppz1, which is involved in multiple cellular processes and is important in the regulation of monovalent cation homeostasis. In budding yeast, Ppz1 is inhibited by two regulatory proteins, Hal3 and Vhs3. These two proteins have moonlighting properties, as they contribute to the formation of an atypical heterotrimeric PPC decarboxylase enzyme, crucial for CoA biosynthesis. Ppz1 is found only in fungi, including pathogenic ones, and has been found to be a virulence determinant in Candida albicans and Aspergillus fumigatus. In addition, when overexpressed, it appears to be the most toxic protein in budding yeast. These characteristics define Ppz1 as a possible target for antifungal therapies. This project aims to elucidate the molecular basis of Ppz1 toxicity. First, by using a conditional Ppz1 overexpressing strain (ZCZ01, in which we replaced the PPZ1 promoter by the GAL1-10 promoter), we have confirmed that the toxic effect of Ppz1 occurs due to the increase in its phosphatase activity and not to depletion of Hal3/Vhs3. Besides, the growth defect caused by overexpression of Ppz1 was characterized by liquid growth assay, dot assay and flow cytometry. Since it is known that Ppz1 inhibits entry of potassium by regulating the Trk1/Trk2 high-affinity transporters, we also have confirmed that a potassium surplus does not rescue the conditional lethality caused by overexpression of Ppz1. In addition, we have defined the transcriptomic changes caused by overexpression of Ppz1 from the GAL1-10 promoter, which revealed the development of an oxidative stress response. Cryptococcus neoformans is a pathogenic fungus that produces meningoencephalitis in immunosuppressed patients (mainly HIV+) and is responsible for a high rate of mortality in invasive infections. Here we report the functional characterization of CnPpz1 and two possible Hal3-like proteins, CnHal3a and CnHal3b. CnPpz1 is a functional PPase and partially replaced endogenous ScPpz1. Both CnHal3a and CnHal3b interact with ScPpz1 and CnPpz1 in vitro but do not inhibit their phosphatase activity. Consistently, when expressed in S. cerevisiae, they poorly reproduced the Ppz1-regulatory properties of ScHal3. In contrast, both proteins were functional monogenic PPCDCs. Ustilago maydis represents an important eukaryotic plant pathogen model system and has been considered one of the top ten plant fungal pathogens. Here we describe the functional characterization of UmPpz1 and UmHal3. We show that UmPpz1 is a functional PPase that partially mimics ScPpz1 in vivo. Both UmHal3 and its PPCDC domain interact with ScPpz1 and UmPpz1 in vitro, but the capacity to inhibit the phosphatase activity is not observed. In S. cerevisiae UmHal3 is not able to inhibit the ScPpz1 whereas is demonstrated as a functional PPCDC. Overall, the results obtained in this work have established a basis of Ppz1 toxicity in S. cerevisiae and provide the foundations for understanding the regulation and functional role of the Ppz1-Hal3 system in the human pathogenic fungus C. neoformans and the plant pathogenic fungus U. maydis.

Wheat (Triticum aestivum) kernel texture (hardness) is the most important determinant of milling and end-product quality. Recent data indicate that the only difference between soft and hard textured wheat is a single amino acid mutation in one protein, puroindoline-b (PIN-b). A rare tryptophan-rich domain in this protein consists of five tryptophan residues among a stretch of seven amino acid residues. To understand how this single mutation makes hard wheat possible, thus enabling bread making, it is crucial to have a high-resolution three-dimensional structure of this protein. The prerequisite for structural elucidation of any protein is the high-quality sample preparation. In this thesis PIN-b from a diploid wheat (Triticum monococcum ) was chosen as the model system because its grain is soft and it has the simplest genome of all wheats. The coding sequence of PIN-b was amplified from the diploid wheat using PIN-b specific primers. It was cloned into a protein expression vector. PIN-b was expressed as a protein behind the thioredoxin (TRX-a) tag. The TRX-a-PIN-b fusion protein was purified using nickel chelating chromatography. The immunological identity of the fusion protein was confirmed by Western blot. The PIN-b was released from the fusion protein by enterokinase proteolysis and purified using ion exchange chromatography. After glutathione treatment to facilitate full formation of the potential five disulfide bonds, PIN-b demonstrated higher fungicidal activity when compared to the non-treated PIN-b.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico
In recent years there has been a significant increase in the incidence of fungal infections caused by Candida species. Although C. albicans be considered the principal representing of the genus, other species have been gaining prominence. C. tropicalis, for example, has been associated with serious invasive cadidiases, being the first or second type of non-Candida albicans Candida most commonly isolated in humans with candidemia and candiduria and is frequently isolated from healthy animals and animals with candidiasis. To establish infection, C. tropicalis expresses many virulence factors such as the secretion of enzymes phospholipases and proteases, biofilm production, among others. This study aimed to evaluate the in vitro antifungal susceptibility profile and production of virulence factors in strains of C. tropicalis (n=100) isolated from several animal species. The strains were subjected to in vitro susceptibility testing by broth microdilution test, M27-A3 protocol, standardized by the Clinical and Laboratory Standards Institute against amphotericin B, itraconazole and fluconazole. We also evaluated the virulence attributes, such as proteases and phospholipases production and biofilm formation. Regarding the susceptibility of C. tropicalis strains, 38% were resistant to itraconazole, 40% were resistant to fluconazole and 34% were resistant to both azoles. None of the strains were resistant to amphotericin B. Regarding the production of proteases, 84% of the strains secreted these enzymes in the medium with pH 5.0, whereas only 40% of the strains were active at pH 3.5. Only 8% of the strains produced phospholipases. The strains showed different pattern in biofilm production, which 63,2% were strong producers, 17,6% were moderate producers, and 13,3% were weak producers. In sumary, the C. tropicalis strains isolated from animals showed high rate of resistance to azoles and expressed important virulence factors, indicating a potential threat to human and animal health.
Nos Ãltimos anos houve um aumento significativo na incidÃncia de infecÃÃes fÃngicas causadas por leveduras do gÃnero Candida. Apesar de C. albicans ser considerada a principal representante do gÃnero, outras espÃcies vÃm ganhando destaque. C. tropicalis, por exemplo, tem sido associada à cadidÃases invasivas graves, sendo a primeira ou segunda espÃcie de Candida nÃo-albicans mais comumente isolada em candidemia e candidÃria em humanos, alÃm de ser frequentemente isolada da microbiota de animais saudÃveis e com candidÃase. Para estabelecer a infecÃÃo, C. tropicalis expressa diversos fatores de virulÃncia, como a secreÃÃo de enzimas protease e fosfolipase, a produÃÃo de biofilme, dentre outros. O presente trabalho buscou avaliar o perfil de sensibilidade antifÃngica in vitro e produÃÃo de fatores de virulÃncia de cepas de C. tropicalis (n=100) isoladas de diferentes espÃcies animais. As cepas foram submetidas a teste de sensibilidade in vitro por meio do mÃtodo de microdiluiÃÃo em caldo, protocolo M27-A3, padronizado pelo Clinical and Laboratory Standards Institute, frente anfotericina B, itraconazol e fluconazol. Foram avaliados ainda os atributos de virulÃncia: produÃÃo de enzimas proteases e fosfolipases e produÃÃo de biofilme. Quanto ao perfil de sensibilidade das cepas de C. tropicalis, 38% foram resistentes a itraconazol, 40% resistentes a fluconazol e 34% foram resistentes a ambos os derivados azÃlicos. Nenhuma cepa apresentou resistÃncia a anfotericina B. Quanto a produÃÃo de proteases, 84% das cepas secretaram estas enzimas em meio com pH 5,0, enquanto somente 40% das cepas foram ativas em pH 3,5. Somente 8% das cepas produziram fosfolipases. As cepas apresentaram padrÃo diferenciado na produÃÃo de biofilme, em que 63,2% foram consideradas fortes produtoras, 17,6% foram consideradas moderadas produtoras e 13,3% foram consideradas fracas produtoras. Em suma, os isolados de C. tropicalis provenientes de animais apresentaram resistÃncia a derivados azÃlicos e expressaram fatores de virulÃncia importantes, indicando potencial risco à saÃde humana e animal.

2012 - 2013
This PhD project is focused on the development of new polymeric materials with antibacterial and antifungal activity. The first part of the work was dedicated to the synthesis and the insertion of a modified amino acid into an antimicrobial peptide (AMP), with the aim to retain the action mechanism on bacterial membranes. Understanding the mechanism of action of AMPs is important for the rational design of new drugs. For this reason, I have collected structural properties, antimicrobial activity values and biological origin of antimicrobial peptides from published data. I have calculated the most relevant chemical physical properties like charge, hydrophobic moment, helicity, flexibility, isoelectric point, Boman and instability index and penetration capabilities. This data collection work permitted us to create YADAMP (www.yadamp.unisa.it), a web database with detailed informations on AMPs. YADAMP database contains the highest number of active sequences with proven antimicrobial activity. YADAMP peremitted me to do a work of data mining that end up with the choice of a peptide, a defensine, to be used as template for developing a new photoresponsive peptide. The peptide, hereafter indicated as ALY, is a short α-helix, membrane active AMP with a tyrosine in the sequence. I have developed the modified analogue replacing the tyrosine by a modified tyrosine with azobenzene group in the side chain. The modified amino acid, named Fmoc-azoTyr, was synthesized according to the classic scheme of diazocopulation reactions. I have chosen the azobenzene group because it permits a reversible trans to cis photochemical isomerization... [edited by author]
XII n.s.

The n-butanol extract of aerial parts of Cordyline australis demonstrated antifungal activity. n-Butanol and chloroform extracts of dried or fresh leaves of C. australis afforded a steroidal glycoside, which was identified as 5α-spirost-25(27)-en-3β-ol 3-O{O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside}, saponin 1. This spirostanol glycoside showed strong antifungal activity towards Trichophyton mentagrophytes and some aspecific activity and cytotoxicity against MRC5 cell. The chloroform extract of fresh leaves of C. australis yielded a second new spirostanol glycoside which was identified as 5α-spirost-25(27)-ene-1β,3β-diol 1-{O-α-L-rhamnopyranosyl-(1→2)-β-D-fucopyranoside}, saponin 2. The n-butanol extracts of senescent leaves of C. australis afforded a third new spirostanol glycoside that was identified as 5α-spirost-25(27)-ene-1β,3β-diol 1-{O-β-D- fucopyranoside, saponin 3. A mixture of two isomeric flavonoid glycosides was isolated from dried leaves of C. australis and shown to be a ca 1:1 mixture of isorhamnetin-3-O-{O-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside}, 4 and isorhamnetin-3-O-{O-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside}, 5. Three other known steroidal glycosides, β-sitosterol glucoside, 6, prosapogenin A of dioscin, 7, and trillin, 8 were also isolated from the leaves of C. australis. The n-butanol extract of dried stems of C. australis afforded (25S)-5α-spirostane-1β,3α-diol 1-{O-β-D-glucopyranoside}, 9. This spirostanol glycoside showed moderate cytotoxicity against Herpes simplex type I virus (ATCC VR733) and Polio Virus Type I (Pfiser vaccine strain).

Dans le cadre d’une convention de thèse CIFRE en collaboration de recherche entre la société Pollenergie et le Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), le potentiel antifongique de la propolis a été étudié. A cet effet, six échantillons de propolis provenant de régions géographiques différentes (France et Brésil) et d’origines botaniques variées (genre Populus, Dalbergia ecastophyllum et Baccharis dracunculifolia) ont été analysés.Les Extraits Ethanoliques de Propolis (EEP) mis au point, riches en composéspolyphénoliques (de 12,8 ± 0,4 à 16,2 ± 0,3 g EAG.l-1) présentent une grande diversité de classes de molécules bioactives selon leur origine botanique. Les polyphénols identifiés dans les extraits de propolis de Populus (France) sont principalement des flavonoïdes et leurs dérivés (galangine, pinobanksine, chrysine) et des acides-phénols (acides p-coumarique et caféique) et dans les extraits de propolis verte de Baccharis dracunculifolia et rouge de Dalbergia ecastophyllum (Brésil) ce sont respectivement des acides-phénols et leur dérivés prénylés (artépilline C, acide p-coumarique) et des composés isoflavonoïdiques (vestitol, médicarpine).Tous les EEP ont démontré un potentiel antifongique in vitro, variable selon les espèces botaniques, à deux stades de développement d’une souche phytopathogène (Colletotrichum musae), isolée de la pourriture de couronne de la banane. Deux extraits de Populus (France) en particulier ont montré la plus forte efficacité contre C. musae.Un extrait aqueux de propolis rouge de Dalbergia ecastophyllum a démontré la plus grande efficacité in vitro. Cet extrait, testé ensuite in vivo sur trois maladies de conservation de la banane a prouvé tout son potentiel antifongique comme traitement alternatif et son efficacité en augmentant la durée de conservation des bananes à l’export
In the framework of a CIFRE thesis agreement in collaboration between Pollenergie and the Center for International Cooperation in Agronomic Research for Development (CIRAD), the antifungal potential of propolis has been studied. For this purpose, six samples of propolis from different geographical regions (France and Brazil) and various botanical origins (genus Populus, Dalbergiaecastophyllum and Baccharis dracunculifolia) were analyzed.The Ethanolic Extracts of Propolis (EEP) finalized, rich in polyphenolic compounds (from 12.8 ± 0.4 to 16.2 ± 0.3 g EAG.L-1), exhibit a great diversity of classes of bioactive molecules according to their botanical origin. The polyphenols identified in propolis extracts of Populus (France) are mainlyflavonoids and their derivatives (galangin, pinobanksin, chrysin) and acids-phenols (p-coumaric and caffeic acids). In contrast, the extracts of green propolis of Baccharis dracunculifolia and red from Dalbergia ecastophyllum (Brazil) they are respectively phenol acids and their prenylated derivatives(artepillin C, p-coumaric acid) and isoflavonoid compounds (vestitol, medicarpin).All EEPs have demonstrated an in vitro antifungal potential, which varies according to botanical species, at two stages of development of a phytopathogenic strain (Colletotrichum musae), isolated from the crown rot of banana. Two extracts of Populus (France) in particular showed the greatest efficacy against C. musae.An aqueous extract of red propolis from Dalbergia ecastophyllum demonstrated the greatest efficiency in vitro. This extract, which was then tested in vivo on three banana conservation diseases, proved its antifungal potential as an alternative treatment and its effectiveness in increasing the shelf life of bananas for export

Différents extraits ont été préparés à partir de racines de H. johannis et différents tests biologiques ont été appliqués en vue de rechercher différentes activités. L'extrait aqueux s'est montré particulièrement actif sur Enterococcus fecalis, Staphylococcus aureus et Bacillus. Les extraits aqueux dépourvus de tanins et les tanins isolés ne présentent pas d'activité antibactérienne. L'effet synergétique des composés serait donc responsable de l'activité antibactérienne de la plante. Une activité antifongique sur Microsporum canis, une propriété antiradicalaire et une activité antiglycation ont été constatées avec les deux extraits. Une étude toxicologique de la poudre de plante et de l'extrait éthanolique sur des rats révèle une toxicité au niveau du foie et de la rate. Cinq composés ont été isolés puis identifiés. Il s'agit de 3',4',5-Trihydroxy-6,7-diméthoxyflavone ; 3,5-Dihydroxy-4,7-diméthoxy dihydroflavonol, Catéchine, Vanilline et l'acide Protocatechuic. Du stigmastérol, de l'acide oléique, de l'acide myristique et de l'acide palmitique ont été également identifiés. Le travail sur C. lanatus var. citroides a montré que l'extrait méthanolique (70%) des pulpes de fruits possède une activité contre B. subtilis, S. aureus et E. coli. Les extraits butanolique et à l'acétate d'éthyle ne sont pas toxiques contre les larves de crevettes. L'extrait butanolique possède une propriété significative antiradicalaire. Deux composés ont été isolés et identifiés. Ce sont la Cucurbitacine E 2-O-[bêta]-glucopyranoside et la Cucurbitacine L 2-O-[bêta]-glucopyranoside. Ces composés montrent une activité antibacterienne contre E. coli. La Cucurbitacine L 2-O-[bêta]-glucopyranoside possède une activité antibactérienne contre P. aeruginosa et une propriété modérée anti-radicaux libres
Different extracts were prepared from the roots of H. johannis and different biological tests were performed. Water extract exhibited significant activity against Enterococcus fecalis, Staphylococcus aureus and Bacillus. Water extract devoid from tannin or the tannin fraction did not show any antibacterial activity reflecting the synergistic property of active compounds. Both extracts showed antifungal, antiradical capacity as well as antiglycation activity. Toxicological study of the powder and ethanol extract on rats showed toxicity to the liver and kidney tissues. Five compounds were isolated namely; 3,4,5- Trihydroxy- 6,7-dimethoxy flavone ; 3,5-Dihydroxy- 4,7- dimethoxy dihydroflavonol, Catechin, Vanillin and Protocatechuic acid. Stigmasterol, Oleic acid, Myristic acid and Palmitic acid were also identified. A study on the fruit pulps of C. lanatus var. citroides revealed that the methanolic extract displayed an antibacterial activity against B. subtilis, S. aureus and E. coli. The butanolic extract showed antiradical capacity and was not toxic to brine shrimps larvae. Two compounds were isolated namely; Cucurbitacine E 2-O-[bêta]-glucopyranoside and Cucurbitacine L 2-O- [bêta] -glucopyranoside. Both compounds showed antibacterial activity against E.coli whereas, Cucurbitacine L 2-O-[bêta]-glucopyranoside showed antibacterial activity against P. aeruginosa as well as antiradical activity

Microbe resistence is a serious issue, especially as they have become resistant to most well known drugs. Therefore this is considered as a global problem and is now dealt with at a poitical level. Since no new classes of antimicrobial agents have been discovered in the past three deacdes, the development of new drugs is extremely urgent. Therefore the aim of this project was to synthesise derivatives of benzimidazole, and then assesses their antimicrobial activities in vitro by using disc (well) diffusion and MICs tests. A total of 69 benzimidazole derivatives, with substituents at positions 1, 2, and 5, were synthesised, characterised and tested against selected bacteria and fungi. In addition, six bezimidazole silver complexes were prepared and evaluated for their antimicrobial behavior. The SAR showed that the antimicrobial activity of the compounds depended on the substituents attached to the bicyclic heterocycle. Some promising results were obtained. In particular, 5 compounds displayed antibacterial activity against two MRSA strains with MIC values corresponding to ciprofloxacin, which can be considered significant. The compounds have some common features; four possess 5-chloro or 5-bromo substituents; two are derivatives of (S)-2- ethanaminebenzimidazole and the others are derivative of one 2-(chloromethyl)-1Hbenzo[d]imidazole, (1H-benzo[d]imidazol-2-yl)methanethiol and 2-(methoxymethyl)-1-methyl-1H-benzo[d]imidazole. The results from the antifungal screening were very interesting as there were 26 compounds, including two silver complexes, which were potent fungicides against the selected fungal species. They showed equivalent or greater potentency in their MIC values than amphotericin B. In particular, the 5-fluoro, 5-chloro and 5-bromo benzimidazole showed broad spectrum activity.

Validamycin A (Val-A) is an aminoglycoside's antibiotic with anti-fungal activity. Val-A synthesized by Streptomyces hygroscopicus strain depending on the growth and development of this actinomyces. In this study, the effects of Mn and Zn on the antifungal activity and biomass of the Streptomyces hygroscopicus were conducted. The results showed that micronutrients Mn, Zn had significant effects on biomass as well as antifungal activity of strain Streptomyces hygroscopicus- DA15. With the addition of Mn at a concentration of 1μg/l of the nutrient medium, biomass of Streptomyces hygroscopicus was 2.85±0.02g/ml, the anti-fungal Rhizoctoniasolani (R. solani) round diameter reached 3.5±0.2cm. With the addition of Zn=3μg/l of the nutrient medium, biomass of Streptomyces hygroscopicus DA15 was 4.5±0.02g/ml, the anti-fungal R. solani round diameter reached 3.4±0.2cm.
Validamycin A (val-A) là một loại kháng sinh có khả năng kháng nấm, được tổng hợp bởi xạ khuẩn Streptomyces hygroscopicus và phụ thuộc vào quá trình sinh trưởng, phát triển của xạ khuẩn. Bài báo này đánh giá ảnh hưởng của nguyên tố vi lượng Mn, Zn đến hoạt tính kháng nấm Rhizoctonia solani (R. solani) và sinh khối của chủng Streptomyces hygroscopicus DA15. Khi bổ sung Mn vào môi trường nuôi cấy với nồng độ 1μg/l, sinh khối của Streptomyces hygroscopicus- DA15 đạt 2,85±0,02g/ml, đường kính vòng kháng nấm đạt 3,5±0,2cm. Bổ sung Zn vào môi trường nuôi cấy với hàm lượng Zn=3μg/l, sinh khối của Streptomyces hygroscopicus DA15 đạt 4,5±0,02g/ml và đường kính vòng kháng nấm đạt 3,4±0,2cm.

In iron-rich conditions, Pseudomonas aureofaciens PA147-2 produces an antibiotic-like compound that inhibits the growth of a number of plant fungal pathogens. To contribute to the potential use of PA147-2 as a biocontrol organism, I report the identification of a genetic locus important for antibiotic biosynthesis. Mutants defective for fungal inhibition (Af-), were generated by Tn5 mutagenesis. Southern hybridisation of genomic DNA from the Af- mutants indicated that in each case loss of fungal inhibition was due to a single Tn5 insertion. Restriction mapping of cloned DNA showed that in two mutants, PA1 and PA109, the Tn5 insertions were in the same 16kb EeoRI fragment, separated by 2.1kb. Allele replacement, by homologous recombination with cosmids from a genomic library of PA147-2, restored one mutant (PA109) to antifungal activity. The 16kb EeoRI wildtype fragment complemented PA109 and PA1 in trans to antifungal activity. Saturation mutagenesis using a miniTn10 transposon identified a region of at least 13kb that is required for fungal inhibition in culture. Under growth room conditions, PA147-2 protected asparagus seedlings from Phytophthora megasperma root rot while the Af- mutant, PA109, did not suppress the pathogen. An inhibitory compound isolated from PA147-2 and analysed by HPLC, was absent in three Af- mutants. The region flanking Tn5 insertion in PA109 was partially sequenced. Thia region in mutant PA109 has significant homology to several two-component regulators and appears to be necessary for wildtype antifungal activity on PBPDA and in planta. The homologous wildtype region was sequenced and a single open reading frame encoding a putative regulatory protein was identified as a "hybrid kinase", a member of the family of two-component signal proteins in bacteria. The regulatory gene is located within the putative cluster that is involved in antibiotic-mediated biological control of fungal pathogens.

Thesis (MTech Biomedical Technology)) ---Cape Peninsula University of Technology, 2008
Resistance to antibiotics has been acknowledged as a major global public health problem. The use of peptides to provide alternatives to combat multi drug resistant organisms is of current relevance to overcome antibiotic resistance. The high deversity of amphibian skin peptides render these animals a potential source for the discovery of novel drugs.

[EN] Chitosan films and coatings have been obtained, by incorporating different essential oils (EO) and using different homogenization conditions of the film forming emulsions, in order to obtain antifungal materials for fruit preservation. The effect of oleic acid (OA) on the stability of the initial emulsions and on the film properties was analysed. Coatings were applied to control fungal decay in strawberries. The blending of chitosan with methylcellulose (MC) was also used in coating applications to tomato plants and fruits to prevent fungal infections. The films' functional properties as a function of their composition were analysed, as well as their antimicrobial activity through in vitro and in vivo tests. OA incorporation in the chitosan films (1:1 wt. ratio) reduced water vapour permeability (WVP) values to about 50 % of those of net chitosan films, with a small positive effect of the microfluidization process. EO (cinnamon, thyme and basil) did not notably reduced the WVP values of the chitosan films but a significant improvement in water barrier capacity was induced when OA was also added at 1:1 or 1:0.5 CH:OA ratios. In contrast, lipids slightly promoted oxygen permeability of the films. Lipid addition decreased the film stretchability and stiffness, with a lesser impact on the resistance to break, slightly depending on the droplet sizes. Essential oils also modulated the mechanical behaviour of the films, depending on their composition. Thyme and basil EO greatly promoted film stiffness and resistance to break, whereas cinnamon oil slightly reduced these mechanical attributes. Optical properties of the chitosan films were also affected by lipid incorporation. OA reduced the film transparency and gloss depending on the concentration, but provoked small changes in the colour parameters and whiteness index. EO affected transparency to a lesser extent, but had greater impact on the colour coordinates and whiteness index of the chitosan films due to the selective light absorption of their compounds. EO blend with oleic acid mitigated the colour changes in the films. Likewise, blending of OA with EO significantly reduced the losses of volatiles during the film formation due to the promotion of the stability of the film forming emulsions. Films containing cinnamon EO were effective in reducing the growth of Aspergillus niger, Botrytis cinerea and Rhizopus stolonifer, although thyme and basil EO encapsulated in the films did not exhibit antifungal action against these three fungi. When chitosan-cinnamon EO coatings were applied to strawberries inoculated with R. stolonifer, they reduced the fungal decay of the fruits during 14 days, at 10 °C, at the same time that total coliform counts were maintained at the initial levels. Chitosan coatings with lemon essential oil were also active at controlling fungal decay in strawberries. These did not significantly affect the physicochemical parameters of strawberries throughout cold storage, while they slowed down the respiration rate of the fruits and enhanced the chitosan antifungal activity against B. cinerea. The coatings, with and without lemon EO, affected the strawberry volatile profile, although it was only sensory appreciated for samples coated with chitosan-lemon oil. Blend films of CH and MC (1:1) containing oregano EO caused phytotoxic problems at "3 Leaves" stage of tomato plants, although the total biomass and crop yield was not affected. In the "Fruit" stage, the treatments had no negative effects. Coatings reduced the respiration rate of tomatoes, diminished weight loss during postharvest storage and were effective to decrease the fungal decay of tomatoes inoculated with R. stolonifer. Migration of thymol and carvacrol from CH-MC films in food simulants could overcome the stablished specific migration limit (60 mg/kg) for food contact packaging materials in aqueous and low pH systems if films contain a 1:1 polymer essential oil weight ratio.
[ES] En la presente tesis doctoral se han desarrollado diferentes materiales antifúngicos para su uso en conservación de frutas. Para ello, se han incorporado diferentes aceites esenciales (AE) en recubrimientos y películas de quitosano (Q). Se ha analizado el efecto de la adición de ácido oleico (AO) y las condiciones de homogeneización sobre la estabilidad de las emulsiones y sobre las propiedades de las películas. Se ha estudiado el efecto de los recubrimientos de Q sobre el deterioro fúngico en fresas y el efecto preventivo frente a infecciones fúngicas de las mezclas de metilcelulosa (MC) con Q en plantas de tomate. Se ha estudiado el efecto de la composición de las películas sobre las propiedades funcionales de las mismas, así como su actividad antimicrobiana in vitro e in vivo. La incorporación de AO en las películas de Q (proporción 1:1) redujo la permeabilidad al agua (PVA) en un 50% comparado con la de Q puro. La microfluidización indujo un efecto positivo sobre dicha reducción. La adición de AE (hoja de canela, tomillo o albahaca) no supuso una disminución notable de los valores de permeabilidad obtenidos para las películas de Q. Cuando se añadió AO a las formulaciones de Q y AE (proporciones 1:1 o 1:2), se promovió una mejora significativa en la PVA de las películas. En cambio, la adición de lípidos aumentó ligeramente la permeabilidad al oxígeno, disminuyó la elasticidad y la rigidez, y produjo un menor impacto sobre la resistencia a la rotura. A su vez, la adición de AE modificó el comportamiento mecánico de las películas. Los AE de tomillo y albahaca aumentaron considerablemente la rigidez y la resistencia a la rotura, mientras que el AE de hoja de canela redujo estos parámetros ligeramente. La adición de lípidos a las películas de Q afectó las propiedades ópticas de las mismas. El AO redujo la transparencia y el brillo, en función de la concentración añadida. La adición de AE tuvo un mayor impacto sobre los parámetros de color y el índice de blancura. Las mezclas de AE y AO mitigaron estos cambios de color. Además, la incorporación de las mezclas AE-OA redujo las pérdidas de volátiles del AE durante la formación de las películas. Las películas formuladas con el AE de hoja de canela fueron efectivas contra el crecimientos de A. niger, B. cinerea y R. stolonifer, aunque los AE de tomillo y albahaca encapsulados en las películas no mostraron ninguna actividad antifúngica. La aplicación de los diferentes recubrimientos de Q AE de C en fresas inoculadas con R. stolonifer dio lugar a una reducción en el deterioro fúngico de los frutos almacenados durante 14 días a 10°C. Los recubrimientos de Q-AE de limón también fueron efectivos en el control del deterioro fúngico en fresas. Estos recubrimientos no afectaron significativamente los parámetros físico-químicos de las fresas durante el almacenamiento en refrigeración, disminuyeron la tasa de respiración de los frutos y acentuaron la actividad antifúngica del Q frente a B. cinerea. Tanto los recubrimientos con AE como los de Q puro modificaron el perfil de volátiles de las fresas, aunque estos cambios solo fueron apreciados sensorialmente en el caso de los frutos recubiertos con AE. Las mezclas de Q y MC que contenían AE de orégano causaron efectos fitotóxicos en plantas de tomate en el estadio "3 hojas", aunque no afectaron a la biomasa total. En el estadio "frutos" los tratamientos no tuvieron ningún efecto negativo. Los recubrimientos redujeron la tasa de respiración de los tomates, disminuyeron la pérdida de peso durante el almacenamiento post-cosecha y fueron efectivos contra el deterioro fúngico de tomates inoculados con R. stolonifer. La migración de los compuestos fenólicos timol y carvacrol, contenidos en las películas de Q-MC, podría superar el límite de migración específica establecido (60 mg/Kg) para materiales de envase en contacto con alimentos en los casos de sistemas acuosos y d
[CAT] En la present tesi doctoral s'han desenvolupat diferents materials antifúngics per al seu ús en conservació de fruites. Per a açò, s'han incorporat diferents olis essencials (OE) en recobriments i pel·lícules de quitosano (Q). S'ha analitzat l'efecte de l'addició d'àcid oleic (AO) i les condicions d'homogeneïtzació sobre l'estabilitat de les emulsions i sobre les propietats de les pel·lícules obtingudes. S'ha estudiat l'efecte dels recobriments de Q sobre la deterioració fúngica en maduixes i l'efecte preventiu enfront d'infeccions fúngiques de les mescles de metilcelulosa (MC) amb Q en plantes de tomaca. S'ha estudiat l'efecte de la composició de les pel·lícules sobre les propietats funcionals de les mateixes, així com la seua activitat antimicrobiana in vitro i in vivo. La incorporació de AO en les pel·lícules de Q (1:1) va reduir la permeabilitat al vapor d'aigua (PVA) en un 50% comparat amb la de Q pur. La microfluidització va induir un petit efecte positiu sobre aquesta reducció. L'addició de AE (fulla de canyella, C, timó, T, i alfàbrega, A) no va suposar una disminució notable dels valors de permeabilitat obtinguts per a les pel·lícules de Q. Quan es va afegir AO a les formulacions de Q i AE, es va promoure una millora significativa en la PVA de les pel·lícules. Per contra, l'addició de lípids va augmentar lleugerament la permeabilitat a l'oxigen, va disminuir l'elasticitat i la rigidesa, i va produir un menor impacte sobre la resistència al trencament. Al seu torn, l'addició de OE va modificar el comportament mecànic de les pel·lícules. Els OE de T i d'A van augmentar considerablement la rigidesa i la resistència al trencament, mentre que l'OE de C va reduir aquests paràmetres lleugerament. L'addició de lípids a les pel·lícules de Q també va afectar les propietats òptiques de les mateixes. L'AO va reduir la transparència i la lluentor, en funció de la concentració afegida. L'addició d'OE va tenir un major impacte sobre el paràmetres de color i l'índex de blancor. Les mescles d'OE i AO van mitigar aquests canvis de color. A més, la incorporació de les mescles OE-AO va reduir les pèrdues de volàtils de l'OE durant la formació de les pel·lícules. Les pel·lícules formulades amb l'OE de C van ser efectives contra el creixements d'A. niger, B. cinerea i R. stolonifer, encara que els OE de T i A encapsulats en les pel·lícules no van mostrar cap activitat antifúngica. L'aplicació dels diferents recobriments de Q OE de fulla de canyella en maduixes inoculades amb R. stolonifer va donar lloc a una reducció en la deterioració fúngica dels fruits emmagatzemats durant 14 dies a 10°C. Els recobriments de Q-OE de llimó també van ser efectius en el control de la deterioració fúngica en maduixes. Aquests recobriments no van afectar significativament els paràmetres fisicoquímics de les maduixes durant l'emmagatzematge en refrigeració, van disminuir la taxa de respiració dels fruits i van accentuar l'activitat antifúngica del Q enfront de B. cinerea. Tant els recobriments amb OE com els de Q pur van modificar el perfil de volàtils de les maduixes, encara que aquests canvis sol van ser apreciats sensorialment en el cas dels fruits recoberts amb OE. Les mescles de Q:MC que contenien OE d'orenga van causar efectes fitotòxics en plantes de tomaca en l'estadi "3 fulles", encara que no van afectar a la biomassa total. En l'estadi "fruits" els tractaments no van tenir cap efecte negatiu. Els recobriments van reduir la taxa de respiració de les tomaques, van disminuir la pèrdua de pes durant l'emmagatzematge post collita i van ser efectius contra la deterioració fúngica de tomaques inoculades amb R. stolonifer. La migració dels compostos fenòlics timol i carvacrol, continguts en les pel·lícules de Q-MC, podria superar el límit de migració específica establit (60 mg/Kg) per a materials d'envàs en contacte amb aliments en els casos de sistemes aquosos i d
Perdones Montero, Á. (2015). ANTIFUNGAL CHITOSAN-BASED FILMS AND COATINGS CONTAINING ESSENTIAL OILS FOR FRUIT APPLICATIONS [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/59413
TESIS

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É notável a crescente demanda comercial pelos extratos das folhas de “Chá de Bugre” (Cordia ecalyculata Vell.) principalmente para a sua aplicação como supressor de apetite e agente cicatrizante. Trata-se de uma planta de origem sul americana sendo no Brasil muito comum nos estados de Minas Gerais, Bahia, Goiás, Paraná e Santa Catarina. É uma planta da família boraginaceae utilizada na fitoterapia brasileira como diurético, febrífugo, estimulante circulatório e também como inibidora do apetite, embora poucos relatos científicos comprovem tais atividades. Uma ampla revisão bibliográfica foi realizada e até o momento verificou- se que são poucos os estudos referentes aos constituíntes químicos presentes na espécie, destacando-se alcalóides como a cafeína, a alantoína e ácido alantóico; glicosídeos como a consolidina; taninos e pigmentos, além de compostos inorgânicos como o potássio. Órgãos regulatórios nacionais e internacionais responsáveis pela avaliação dos parâmetros de qualidade dos extratos comercializados tem sido cada vez mais incisivos quanto à padronização focando em marcadores específicos, metodologiasanalíticas validadas para a determinação de tais marcadores, estudos de estabilidade e também comprovações científicas que atestem as atividades biológicas esperadas. De acordo com os objetivos propostos, realizou-se inicialmente uma ampla revisão bibliográfica focando aspectos botânicos, químicos e farmacológicos da espécie. Posteriormente, realizou-se o desenvolvimento e também a validação de uma metodologia analítica cromatográfica capaz de determinar a alantoína nas folhas e também nos extratos obtidos de chá de bugre. Por fim, foi realizada a avaliação farmacológica da espécie, focando em ensaios do tipo antihemorrágico antifúngico, antioxidante e citotóxico para células normais e tumorais...
The increasing demand for commercial extracts of Chá de Bugre” (Cordia ecalyculata Vell.) is remarkable, especially for its use as appetite suppressant and wound healing agent. The specie is a plant originated from South America and widely spread in Brazil, in the states of Minas Gerais, Bahia, Goiás, Parana and Santa Catarina. It is a plant of the family boraginaceae, used in the Brazilian phytotherapy as diuretic, antipyretic, circulatory stimulant and appetite suppressant, but few scientific reports corroborate such activities. An extensive literature review has been carried out and so far it appears that there are few studies regarding the chemical constituents present in this species like alkaloids such as caffeine, allantoin and allantoic acid, glycosides as consolidine, in addition to tannins, pigments, and inorganic compounds such as potassium. National and international regulatory departments responsible for assessment of parameters related to the quality of the extracts have been increasingly incisive focusing on the standardization of specific markers, validation of analytical methods for the determination of such markers, stability studies and also scientific evidence that demonstrates the expected biological activities. According to the proposed objectives, a deep literature review has been initially carried out focusing on botanical, chemical and pharmacological aspects of C. ecalyculata. Subsequently, a chromatographic analytical methodology capable of determining allantoin in the leaves and also in extracts of “chá de bugre” has been developed and validated. Finally, we performed a pharmacological evaluation of the species, focusing on the antihemorrhagic, antifungal, antioxidant and cytotoxic activities to both normal and tumor cells. Biological assays evidenced that the ethyl acetate phase of aqueous ethanol extract of leaves showed hemorrhage inhibition ... (Complete abstract click electronic access below)

Urease of Helicobacter pylori is an important virulence factor implicated in the pathogenesis of many clinical conditions, such as chronic gastritis, peptic ulceration, and gastric cancer. Many urease inhibitors have been discovered, like phosphorodiamidates, hydroxamic acid derivatives, and imidazoles. Despite good activities at the enzyme level and excellent kinetic properties most of them have not been used as therapeutic agents in vivo because of their side effects, toxicity and instability. This has led to much attention to focus on exploring the novel urease inhibitory activities of natural products because of their low toxicity and good bioavailability. Honey, a natural product has been used in folk medicine due to its antitumor, antioxidant, antimicrobial and anti-inflammatory properties. The aims of this study were to isolate, characterise, purify urease produced by H. pylori and investigate the inhibitory effects of solvent honey extracts on its enzymatic activity. Urease was found to be both surface-associated and cytoplasmic. Maximum cytoplasmic urease activity was found to occur after 72 hr whereas maximum extracellular urease activities were found to occur after 96 hr. Characterization of the crude cytoplasmic urease revealed optimal activity at a pH of 7.5 and temperature of 40°C. The kinetic parameters Vmax and Km were 45.32 U ml-1 and 61.11 mM respectively.The honey extracts inhibited the activity of the crude urease in a concentration dependent manner. The Lineweaver-Burk plots indicated a non-competitive type of inhibition against H. pylori urease. The two honey extracts gave promising inhibitory activities against urease of H. pylori. Thus the results of this study delineates that inhibition of urease can ease development in therapeutic and preventative approaches based on the enzymatic activity of this Helicobacter protein.

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Resumo: A epidemiologia das infecções causadas por Candida sofreu mudanças nos últimos anos. Embora Candida albicans ainda seja o principal patógeno causador de candidíase, relatos recentes reportam o aumento da incidência de infecções causadas por espécies de Candida não-albicans. A toxicidade dos fármacos antifúngicos utilizados juntamente com o desenvolvimento da resistência tem se tornado um sério problema clínico. Com isso, atualmente existe a urgência na descoberta de novos compostos com atividades antifúngicas. Objetivos: avaliar a atividade antifúngica do fármaco Cloridrato de Verapamil, determinar a Concentração Inibitória Mínima (CIM) e a Concentração Fungicida Mínima (CFM), frente à Candida albicans ATCC 18804, Candida krusei ATCC 6258, Candida parapsilosis ATCC 90018 e Candida glabrata ATCC 9030. Além de avaliar a citotoxicidade do fármaco em queratinócitos humanos (HaCaT). Metodologia: a determinação da CIM foi realizada pela técnica de microdiluição de acordo com The European Committee on Antimicrobial Susceptibility Testing (EUCAST). A CFM foi determinada por plaqueamento em ágar Sabouraud de alíquotas provenientes das diluições do ensaio de microdiluição. A avaliação da citotoxicidade foi realizada pela técnica de redução da resazurina, sendo possível avaliar a atividade mitocondrial das células HaCaT. Resultados: o fármaco Cloridrato de Verapamil demonstrou atividade antifúngica contra as quatro espécies patogênicas de Candida, com o valor de CIM de 1250 µM; valor de CFM maior que 1250 µM, sendo assim este fármaco foi considerado fungistático. Além disso, o Cloridrato de Verapamil não apresentou citotoxicidade nas concentrações avaliadas no estudo, pois, a redução de células viáveis nas concentrações mais elevadas não ultrapassa 30%. Com relação à redução de biomassa em biofilme de Candida, após tratamento com Cloridrato de Verapamil, houve redução (de 10 a 20%) para as quatro espécies de Candida em estudo, quando utilizadas concentrações de fármaco correspondentes a CIM; quando utilizadas concentrações de fármaco correspondentes a cinco vezes o valor de CIM, houve um aumento significativo na redução de biomassa (de 25% a 60%) em biofilme formado pelas quatro espécies de Candida. Conclusão: o fármaco Cloridrato de Verapamil apresentou atividade antifúngica para Candida albicans e Candida não-albicans, sendo considerado um fármaco fungistático, além de não apresentar citotoxicidade em queratinócitos humanos, e demonstrar atividade na redução de biomassa em biofilme formado pelas quatro espécies de Candida. Demais estudos são necessários para verificar a ação desse fármaco em diferentes mecanismos de virulência frente a células de Candida spp.
Abstract: The epidemiology of Candida infections has changed in recent years. Although Candida albicans is still the main pathogen causing candidiasis, recent reports have reported an increase in the incidence of infections caused by nonalbicans Candida species. The toxicity of the antifungal drugs used together with the development of resistance has become a serious clinical problem. With this, there is now an urgency in the discovery of new compounds with antifungal activities. Objectives: To evaluate the antifungal activity of the drug Verapamil Hydrochloride, to determine the Minimum Inhibitory Concentration (MIC) and the Minimum Fungicidal Concentration (CFM) against Candida albicans ATCC 18804, Candida krusei ATCC 6258, Candida parapsilosis ATCC 90018 and Candida glabrata ATCC 9030 In addition to evaluating the cytotoxicity of the drug in human keratinocytes (HaCaT). Methodology: MIC determination was performed by the microdilution technique according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST). The CFM was determined by plating on Sabouraud agar from aliquots from the dilutions of the microdilution assay. The evaluation of cytotoxicity was performed using the resazurin reduction technique, and it was possible to evaluate the mitochondrial activity of HaCaT cells. Results: The drug Verapamil Hydrochloride demonstrated antifungal activity against the four pathogenic species of Candida, with MIC value of 1250 μM; CFM value greater than 1250 μM, so this drug was considered fungistatic. In addition, Verapamil Hydrochloride did not present cytotoxicity in the units evaluated in the study, because the reduction of viable cells in the most cells is not exceeded by 30%. Regarding the biomass reduction in Candida biofilm, after treatment with Verapamil Hydrochloride, there was a reduction (from 10 to 20%) for the four Candida species in study, when the use of drug corresponding to MIC; when the use of drug corresponding to 5 times of MIC, there was a significant increase in the biomass reduction (from 25% to 60%) in the biofilm molded by the four Candida species. Conclusion: Thus, the drug Verapamil Hydrochloride led to the antifungal activity of Candida albicans and non-albicans Candida species, being considered a fungicidal drug, besides not presenting cytotoxicity in human serotonizers, and were submitted to biomass reduction in biofilm molded by four Candida species. More studies are needed to verify the action of the drug on different mechanisms of virulence against Candida spp cells.

The need to develop new fungicides remains a major driving force as fungal plant pathogens continue to develop resistance against existing fungicides at great speed, and also because new disease situations continually arise. Natural products have been and still are large reservoirs of new biologically active substances for crop protection development, by serving as lead structures for the discovery of active molecules, often featuring novel and unique modes of action. Many pesticides have been developed from naturally occurring lead compounds. An outstanding example is the class of fungicides that has been generated from strobilurins, antifungal metabolites of myxobacteria. Aim of the PhD work was the study of new naturally derived antifungal compounds and the development of synthetic sequences which might, in principle, have value in the preparation of the natural products themselves as well as in synthesizing various analogues. Among a number of possible candidates we have selected a natural products, Leopolic acid A as synthetic target, as it has been reported to be endowed with antifungal activity. During the PhD period the synthesis of this natural product and structurally related analogues has been carried out. Antifungal and antimicrobial activities of the compounds have been evaluated, and preliminary SAR have emerged.

A incidÃncia de infecÃÃes oportunistas causadas por fungos, com destaque para as leveduras do gÃnero Candida vem aumentando substancialmente. Estudos relatam que tem sido observado um notÃvel crescimento de infecÃÃes por espÃcies nÃo albicans (Candida tropicalis, Candida glabrata, Candida parapsilosis e Candida krusei). Isolados clÃnicos resistentes aos azÃlicos, em especial ao fluconazol, sÃo cada vez mais relatados. As naftoquinonas sÃo uma importante classe de molÃculas biologicamente ativas, apresentando atividades antibacteriana, antifÃngica, antiviral, anti-inflamatÃria, antipirÃtica, anticancerÃgena e tripanocida e tem sido amplamente testadas em vÃrios estudos farmacolÃgicos. Nos Ãltimos anos intensificou-se o interesse nestas substÃncias, nÃo sà devido à sua importÃncia em processos bioquÃmicos vitais, como tambÃm ao destaque cada vez maior que apresentam em variados estudos farmacolÃgicos. O presente trabalho buscou avaliar e comparar o efeito antifÃngico de naftoquinonas frente a cepas de Candida tropicalis resistentes e sensÃveis ao fluconazol, utilizando diferentes tÃcnicas tais como mÃtodos de microdiluiÃÃo em caldo, procedimentos de citometria de fluxo e ensaio do Cometa. Utilizamos sete cepas de Candida tropicalis resistentes ao fluconazol para esse estudo, as quais foram isoladas de sangue e faziam parte da colecÃÃo de leveduras do LaboratÃrio de BioprospecÃÃo Experimental em leveduras (LABEL), afiliado com a Faculdade de FarmÃcia da Universidade Federal do Cearà . Foi utilizado trÃs compostos de naftoquinonas frente à 07 cepas de Candida tropicalis e foram submetidas aos testes de Sensibilidade in vitro , onde apresentaram uma potente atividade antifÃngica. AtravÃs da Citometria de Fluxo, foi possÃvel avaliar alteraÃÃes morfolÃgicas e de integridade da membrana significativas desses compostos frentes Ãs cepas, alÃm de disfunÃÃo mitocondrial e produÃÃo de espÃcies reativas de oxigÃnio. AtravÃs do ensaio do Cometa foi possÃvel encontrar danos significativos ao DNA. Em sÃntese, os resultados sugerem que estes compostos podem ser usados como agentes antifÃngicos para o tratamento de candidemias
The incidence of opportunistic infections caused by fungi, with emphasis on Candida species has increased substantially. Studies report that there has been a notable increase of infections by non-albicans species (Candida tropicalis, Candida glabrata, Candida parapsilosis and Candida krusei). Clinical isolates resistant to azoles, particularly fluconazole, are increasingly reported. The naphthoquinones are an important class of active molecules biologically presenting antibacterial, antifungal, antiviral, antiinflammatory, antipyretic, anti-cancer and trypanocidal and has been tested extensively in several pharmacological studies. In recent years intensified the interest in in this substances, not only due to your importance in vital biochemical processes as well as the increasing emphasis that they show in various pharmacological studies. This study aimed to evaluate and compare the effect of antifungal naphthoquinones against strains of Candida tropicalis resistant and susceptible to fluconazole, using different techniques such as broth microdilution methods, procedures Flow Cytometry procedures and Comet assay. We use seven strains of Candida tropicalis resistant to fluconazole for this study, which were isolated from blood and were part of the collection of yeasts Experimental Laboratory Bioprospecting in yeast (LABEL), affiliated with the Faculty of Pharmacy, Federal University of CearÃ. We used three compounds of naphthoquinones against the seven strains of Candida tropicalis and were subjected to in vitro sensitivity tests, which showed an antifungal activity potent. Through Flow Cytometry, it was possible to assess morphological changes and membrane integrity of these compounds fronts to significant strains in addition to mitochondrial dysfunction and production of reactive oxygen species. Through the Comet assay was possible to find significant damage to DNA. In summary, the results suggest that these compounds may be used as antifungal agents for the treatment of candidemia

The aim of the thesis entitled “Synthesis and biological activity evaluation of natural products and their analogues” is to synthesize some natural products and their analogues in order to study SAR and evaluate their biological activity as fungicides or herbicides. The thesis reports the first enantioselective synthesis of crassinervic acid in 12 steps and 11.8% overall yield. Crassinervic acid, recently isolated from leaves of Piper crassinervium by Kato and co-workers, shows high antifungal activity mainly against Cladosporium cladosporioides and Cladosporium sphaerospermum (minimum amount required for the inhibition of fungal growth on TLC: 0.5 microg). Crucial steps for our synthetic strategy included an enantioselective Sharpless epoxidation of easily available geraniol, followed by a regioselective reduction of the corresponding epoxyalcohol, and a condensation of the monoterpene fragment with lithiated 4-hydroxymethylphenol. The successful coupling of the two moieties required an accurate choice of the protecting groups. On the basis of the developed strategy, the absolute configuration of the compound was assigned as (S). A short and efficient synthesis of racemic Crassinervic acid was also carried on and applied to the preparation of analogues. All the synthesized compounds were successively tested for antifungal activity and SAR studies were developed. Synthetic efforts towards Ascaulitoxin were also carried on. Ascaulitoxin, a new unusual phytotoxic bis-aminoacid N-glucoside, was isolated from the culture filtrate of Ascochyta caulina (P. Karst.) v.d. Aa and v. kest, the causal agent of leaf and stem necrosis of Chenopodium album (also known as lambsquarter or fat hen, a common world-wide weed of many arable crops such as sugar beet and maize), a promising microherbicide for the biological control of this common noxious weed. The proposed route is concise and modular, making it convenient for large scale preparation. Finally, an approach towards the synthesis of Harzianic acid was developed. This compound is a new antimicrobial antibiotic from a Trichoderma harzianum strain which was isolated for the first time in 1994 by Sawa et al.. Very recently Vinale et al., reported its isolation, absolute configuration and antifungal as well as plant growth promoting activity. Harzianic acid showed antifungal activity against Pythium irregulare, Sclerotinia sclerotiorum, and Rhizoctonia solani. The synthesis is now at a final stage.

Klinikinėje praktikoje infekcinėms ligoms gydyti vartojama daugiau kaip 200 priešmikrobinių medžiagų, tačiau infekcinės ligos yra viena dažniausių mirties priežasčių ir naujų priešmikrobinių vaistų poreikis nemažėja. Modifikuojant 4-tiazolidinono žiedą susintetinami įvairaus biologinio aktyvumo junginiai. 4-tiazolidinono dariniai su sulfanilamido farmakoforu yra aktyvesni už sulfanilamidus prieš bakterijas ir pasižymi priešgrybeliniu poveikiu; su nitrofurano farmakoforu yra vieni aktyviausių priešmikrobinių junginių. Alilaminą įjungus į molekulę kartu su jau anksčiau žinomais farmakoforais, tikėtąsi geresnio naujų junginių priešmikrobinio aktyvumo. Pritaikius pasirinktas metodikas naujų 4-tiazolidinono darinių su pasirinktų farmakoforų (sulfanilamido, nitrofurano, alilamino) struktūriniais fragmentais sintezei, susintetinti 39 4-tiazolidinono ciklą turintys junginiai. Įvertinus fiziko-chemines savybes ir toksiškumo riziką in silico, nustatyta junginių, kaip biologiškai aktyvių medžiagų, vertė. In silico ir in vitro patvirtinta, kad nauji 4-tiazolidinono dariniai su sulfanilamido, nitrofurano ir alilamino farmakoforais pasižymi prieš¬mikrobinėmis savybėmis prieš skirtingas bakterijų (S. aureus, E. faecalis, E. coli, P. aeruginosa, K. pneumonia, B. subtilis, P. mirabilis) ir grybelio C.albicans kultūras. Apibendrinus mikrobiologinių tyrimų rezultatus, nustatyti aktyviausi antibakteriniai ir priešgrybeliniai junginiai.
More than 200 antimicrobial agents are used in clinical practice for the treatment of infectious diseases, however, infectious diseases are still one of the most common causes of death, and the need for new antimicrobials isn’t decreasing. Compounds with different biological activity are synthesized by modifying 4-thiazolidinone. 4-thiazolidinones with sulfanilamide pharmacophore are more active against bacteria than sulfanilamides, and are characterized as antifungals. 4-thiazolidinones with nitrofuran pharmacophore are one of the most active antimicrobials. Higher antimicrobial activity of new compounds was expected after attaching allylamine to the molecule together with the previously known pharmacophores. By applying the selected methodologies for the synthesis of new 4-thiazolidinones with the fragments of selected pharmacophores (sulfanilamide, nitrofurane, allylamine), 39 derivatives of 4-thiazolidinone were synthesized. The assessment of physico-chemical properties and toxicity risk in silico helped to determine the value of compounds as biologically active substances. In silico and in vitro studies confirmed that new 4-thiazolidinone’s with sulfanilamide, nitrofurane and allylamine pharmacophores showed antimicrobial activity against various bacteria (S. aureus, E. faecalis, E. coli, P. aeruginosa, K. pneumonia, B. subtilis, P. mirabilis) and fungal cultures of C.albicans. Summarising microbiological tests, the most active antibacterial and antifungal compounds... [to full text]

Synthesizing bioactive small molecules by structural modification of 1,4-naphthoquinone was the primary goal of this research. Several bioactive compounds with anticancer, antifungal, and antibacterial activities were synthesized. All the synthetic protocols were optimized in such ways that do not require cumbersome purification. First, a new protocol for the synthesis of NQM111 was developed. NQM111 is a highly potent anticancer agent developed in our laboratory, but the old protocol does not provide enough quantity for in vivo study. Therefore, a new safe and improved method was developed which provides enough quantity for in vivo study. The second project involves the synthesis of 1,4-naphthoquinone conjugated with an aromatic group. These compounds are a highly potent anticancer agent with ~8-fold selectivity towards cancer cell lines than the non-cancer cell line. A mode of action study of this compound was identified, and it was observed that these compounds generate reactive oxygen species,which triggers apoptosis. The final project involves the synthesis of 1,4-naphthoquinone based antifungal, and antibacterial compounds. These compounds are multi-cationic in nature with a hydrophobic tail. Six different analogs with varying hydrophobic tails were synthesized and tested for their antibacterial and antifungal activity. These compounds showed excellent activity against wide range of fungi including resistant strains.

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Os micro-organismos têm demonstrado serem valiosas fontes de princípios ativos de uso clínico, no qual a penicilina é o exemplo mais conhecido de metabólito secundário, de importância medicinal, produzido por fungos. A espécie vegetal Eugenia jambolana Lam., popularmente conhecida como Jambolão, é utilizada na medicina popular brasileira, principalmente no tratamento de diabetes. O fungo Botryosphaeria parva, isolado das folhas da E. jambolana (L.) e codificado como Ej_F01, foi cultivado em escala ampliada em três meios de cultivo: Czapek®, PDB e Milho. Após o período de fermentação do endófito no meio de milho, foi feita uma extração direta com MeOH, seguida de filtração e evaporação do solvente. O extrato obtido foi solubilizado em AcOEt e fez-se três partições líquido-líquido com H2O, após a evaporação do solvente orgânico, o extrato foi dissolvido em CH3CN e foi feita uma nova partição líquido-líquido com Hexano, após a evaporação da CH3CN, foi obtido o extrato de interesse. Após o período de fermentação do endófito nos meios líquidos (PDB e Czapek®), a suspensão micelar foi filtrada a pressão reduzida, separando os micélios dos meios fermentados. Em seguida, os filtrados aquosos foram submetidos a três partições líquido-líquido com AcOEt, e após a evaporação do solvente orgânico, foram obtidos os extratos brutos de interesse. Todos os extratos foram fracionados utilizando técnicas cromatográficas, como Cromatografia em Coluna e/ou Cromatografia Líquida de Alta Eficiência preparativa. Após o fracionamento dos extratos brutos, foi possível obter sete substâncias oriundas do extrato do milho, seis substâncias do extrato do Czapek®, e uma do extrato de PDB, que foram submetidas a análises espectrométricas (EM, RMN de 1H, 13C, 1D e 2D) para a determinação/identificação estrutural. As substâncias 05, 08, 09 e 11 foram identificadas como 5-hidroximeleina, rel. (3S, 4S)-4-hidroximeleina, meleina e rel. (3S, 4R)-4-hidroximeleina, respectivamente, pertencentes à classe das isocumarinas; as substâncias 01, 03, 06 e 07 foram identificadas como botryosphaerin A, 13,14,15,16-tetranorlabd-7-eno-19,6β:12,17-diolídeo, CJ-14445 e oidiolactona E, respectivamente, pertencentes à classe dos diterpenos tetranorlabdanos; a substância 12 foi identificada como botryosphaerona D, pertencente à classe das naftalenonas. A substância 02, inédita, foi nomeada como rel. (4S, 5R, 6R, 10S)-18-hidroxi-13,14,15,16-tetranorlabd-7,9-dieno-19,6β:12,17-diolídeo, codificada como botryosphaerin I. Os estudos relacionados ao B. parva têm como finalidade verificar sua produção metabólica em diferentes meios de cultivo, bem como a avaliação do potencial biológico dos extratos brutos, frente aos ensaios antioxidante, antifúngico e anticolinesterásico.
Microorganisms have proven to be valuable sources of active principles for clinical use, wherein the penicillin is the best known example of a secondary metabolite of medicinal importance, produced by fungi. The plant species Eugenia jambolana Lam., commonly called Black Plum, is used in traditional Brazilian medicine, especially in the treatment of diabetes. The Botryosphaeria parva fungus, isolated from leaves of E. jambolana (L.) encoded as Ej_F01, was grown on an enlarged scale in three different culture media: Czapek®, PDB and corn. After the fermentation period in the corn media, a direct extraction was done with MeOH followed by filtration and evaporation of the solvent. The extract was disolved in AcOEt and made three liquid-liquid partition with H2O, after evaporation of the organic solvent, the extract was dissolved in CH3CN and was made a new liquid-liquid partition with Hexane, after evaporation of the CH3CN, it was obtained extract of interest. After the fermentation period in liquid media (PDB and Czapek®), the micellar suspension was filtered under reduced pressure, separating the mycelium from the fermented broth. Then, the aqueous filtrates were subjected to three liquid-liquid partition with AcOEt, and after evaporation of the organic solvent, there were obtained the crude extracts of interest. All extracts were fractionated using chromatographic techniques such as column chromatography and/or preparative High Performance Liquid Chromatography efficiency. After the fractioning of crude extracts, it was possible to obtain seven substances derived from corn extract, six substances obtained from Czapek extract, and a substance obtained from the PDB extract, which were submitted to spectrometric analysis (MS-ESI, NMR 1H, 13C, 1D and 2D) for determining and structural identification. The substances 05, 08, 09 and 11 were identified as 5-hydroxymelein, rel. (3S, 4S)-4-hydroxymelein, Melein and rel. (3S, 4R)-4-hydroxymelein, respectively, belonging to the class of isocoumarins; the substances 01, 03, 06 and 07 were identified as botryosphaerin A, 13,14,15,16-tetranorlabd-7-ene-19,6β:12,17-diolide, CJ-14445 and oidiolactone E, respectively, belonging to the class of tetranorlabdanes diterpenoids, and substance 12 was identified as botryosphaerone D, belonging to the class of naftalenones. A new tetranorlabdane diterpenoid was obtained, substance 02, named rel. (4S, 5R, 6R,10S)-18-hydroxy-13,14,15,16-tetranorlabd-7,9-dien-19,6β:12,17-diolide and encoded botryosphaerin I. These studies related to the B. parva are intended to verify your metabolic production in different culture media as well as the evaluation of the biological potential of extracts compared to antioxidant, antifungal and anticholinesterase trials.

Ce travail de doctorat porte sur la synthèse et utilisation de bicyclolactones glycidiques, de façon à accéder des dérivés de sucres contenant un système carbonylé α,β-insaturé. Trois types de bicyclolactones ont été étudiés: butenolides liés à des cycles furanose, butenolides fusionnés à des cycles pyranose, comprenant S- et NH-analogues et carboxyméthyle glycosides lactones (CMGLs). La méthodologie de synthèse de butenolides sur motif sucre est basée sur l’oléfination de Wittig de 3 ou 5-cétosucres et lactonisation intramoléculaire spontanée de gamma-hydroxyesters α,β-insaturés intermédiaires. Pour la synthèse des systèmes fusionnés, des furano-3-uloses protégés ont été convertis en 3-C-(éthoxycarbonyl)méthylène furanoses. Une hydrolyse acide finale permet la transestérification intramoléculaire et aussi l’isomérization du cycle en forme pyranose. Des précurseurs 5-S et 5-aminofuranosidiques ont conduit à des analogues thiosucres ou à des dérivés glycidiques ayant une fonction amide et un système carbonylé α,β-insaturé, respectivement. Les CMGLs ont été converties en 3-enopyranosid-2-uloses par l’ouverture de la lactone avec une amine et oxydation/élimination du 2-hydroxy pyranoside tri-O-acétylé obtenu. L’oléfination de Wittig subséquente a conduit aux diènes conjugués pyranosidiques ramifiés en C-2. Les glycosides contenant un groupement propargyle ont permis de préparer des 1,2,3-triazoles par ‘click’ chemistry. Quelques molécules ont été soumises à évaluation antimicrobienne et les énulosides de (N-dodécylcarbamoyl)méthyle ont montré les meilleures activités. Le composé le plus actif est l’énuloside-α qui a montré un très fort effet contre des espèces de Bacillus et une forte activité contre Enterococcus faecalis et Penicillium aurantiogriseum. Les diènepyranosides ont révélé une activité forte et sélective contre E. faecalis. Les dérivés triazolés n'ont montré aucune activité. Parmi les composés bioactifs, trois sont avérés peu toxiques chez les cellules eucaryotes
This PhD work was focused on the synthesis and the uses of carbohydrate bicyclic lactones for the access to sugar derivatives comprising an alpha, beta-unsaturated carbonyl function. Three types of bicyclic lactones were investigated: furanose C-C-Iinked butenolides, pyranose-fused butenolides, including S-or NH-analogues and carboxymethyl glycoside lactones (CMGLs). The synthetic methodology for butenolide containing-sugars was based on the Wittig olefination of 3- or 5-keto sugars and spontaneous intramolecular lactonization of the intermediate gamma-hydroxy axy alpha,beta-unsaturated esters. In the case of the fused systems, protected furanos-3-uloses were converted into 3-C-(ethoxycarbonyl)methylene furanoses. Further acid hydrolysis elicited both intramolecular transesterification and isomerization to the pyranose ring. Introduction of a sulfur or a nitrogen function at C-5 of the furanose precursors led to thiosugar analogues or to carbohydrate derivatives comprising both an amide function and an alpha,beta-unsaturated system, respectively. CMGLs were converted into 3-enopyranosid-2-uloses by a sequence involving opening of the lactone moiety by amines and oxidation/elimination of the resulting tri-0-acetylated 2-hydroxy pyranosides. Further Wittig olefination afforded 2-C-branched-chain conjugated dienepyranosides. Glycosides bearing a propargyl moiety were engaged in "click" chemistry reactions leading to 1,2,3-triazoles. Some of the new molecules were submitted to antimicrobial evaluation and (N-dodecylcarbamoyl)methyl enulosides proved to display the best efficacy. The most active one was the a-enuloside which showed very strong effect towards Bacillus species and strong activity against Enterococcus faecalis and the fungal pathogen Penicillium aurantiogriseum. Dienepyranosides exhibited a strong activity selectively towards E. faecalis. Triazole derivatives were virtually ineffective. Three of the bioactive compounds showed low acute toxicity in eukaryotic cells