Дисертації з теми "Antimicrobial activity/content relationship"

Antibiotic resistance is a growing health issue that necessitates development of alternative drugs with antimicrobial properties. Antimicrobial peptides are a promising group of compounds in this respect and are used by all varieties of living organisms to defend against invading or competing organisms. Hairpinin is an antimicrobial peptide isolated from Echinochloa crus-galli that has previously been found to have antifungal activity. In this study, truncated variants of hairpinin were synthesized and their antifungal activity tested against Candida albicans, Aspergillus fumigatus, and Saccharomyces cerevisiae to identify the minimum structural element of hairpinin required for maintained activity. Hairpinin was active against all three fungi with a minimum inhibitory concentration ranging between 0.6 μM - 5 μM depending on strain and growth media. Two truncated versions were synthesized in this study by solid-phase peptide synthesis, also resulting in a dimer of one of the derivatives, and their antifungal activity was assessed together with four other truncated peptides previously synthesized. The findings indicated that hairpinins C-terminal end together with an inflexible central part stabilized by at least one disulfide bond was vital for activity. The mechanism of action in which hairpinin inhibits fungi was examined by liposome leakage assay of Escherichia coli and Saccharomyces cerevisiae model membranes. It was concluded that the mechanism of action did not involve membrane disruption, a common mechanism among similar antimicrobial peptides. Although hairpinin displayed potent antifungal activity, it was found to be proteolytically unstable in serum. To improve hairpinins value in pharmaceutical context stability has to be improved while preserving the important structural elements.

Although the antomicrobial nature of essential oils and their major constituents, the terpenoids, has been widely investigated the mechanism of their antimicrobial action has not been subject to the same scrutiny. In this study the membrane disruptive nature of the terpenoids has been determined by experiments on the effects of terpenes on both microbial membrane and model lipid bilayer systems. These terpenes exhibited a range of membrane damaging effects. Experiments showed that the terpenoids were able to increase disorder in DPPC bilayers, and that antimicrobially active terpenoids cause increased membrane permeability in living cells. The effect of molecular structure on antimicrobial activity and activity differences between each organism were determined for 60 terpenoids. Terpenoids with a low water solubility were found to be inactive. Hydrogen bond donor capacity and hydrogen bond acceptor capacity were found to be the molecular parameters which most strongly discriminated between activities against individual organisms. Activity against Gram negative Escherichia coli and Pseudomonas aeruginosa was associated also with a molecular size parameter. The protective nature of the outer membrane of P. aeruginosa and E. coli was also investigated. The evidence presented in this thesis has enabled the postulation of a two stage process to explain the overall mode of action of these compounds.
Doctor of Philosophy (PhD)

Hypoxis (commonly known as African potato, Ilabatheka, Inkomfe, sterretjie or yellow starflowers and also as monna wa maledu or thitidi) is a genus of the family Hypoxidaceae. The rootstocks of Hypoxis species, particularly H. hemerocallidea, are well-known to be used in traditional medicine for the treatment of different ailments, such as urinary tract infection, epilepsy, prostate cancer and onset diabetes. Several visits have been made to the Faraday Medicinal Market and Abey Bailey Nature Reserve in Johannesburg to determine the availability of Hypoxis plant materials. From these visits, it was discovered that different Hypoxis species are harvested and sold as the same plant commonly referred to as the African potato, and the treatment with these plants might be questionable as the secondary metabolites might differ. This was proven when a number of rootstocks bought from the medicinal market grew into plants showing distinct morphological differences when planted at the Agricultural Research Council (Roodeplaat, Pretoria). It is possible that the plants sold are used as the substitutes for the commonly main plant, which is H. hemerocallidea and a reality is that many of the problems related to the quality of medicinal plants are based on the substitution of the declared plants and when the substitution occurs the quality of the plants becomes compromised leading to the risks of public health. It is however, not known whether the substitution is done deliberately or unintentionally since the outward morphology of Hypoxis species are not the same except their bright yellow flowers. The similarity within these species is on their underground rootstock. The dosage and toxicity of plant preparations is extremely important and, therefore adulteration is a concern where plant preparations are taken orally and the information about the plants used not being accurate. The aim of this study was to compare the secondary metabolite content of four Hypoxis species namely, H. acuminata, H. hemerocallidea, H. iridifolia and H. rigidula Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) were used to analyze the secondary metabolite content of the plant extracts. Differences were also noted as one green compound was observed only in H. acuminata and H. rigidula. The HPLC results showed major differences in retention time in fresh material. The antibacterial activity of extracts of all four Hypoxis species showed similar results, although the activity differed amongst the microorganisms. The species showed high level of antioxidant activity that increased with increasing concentration in all four Hypoxis species. The species also showed no toxicity when tested in vitro on Vero cells however, they seemed to be toxic to cancer cells (Hela cells) but with a higher concentration. Hypoxoside was isolated and identified as the purple colour band on the TLC fingerprint and was confirmed in all the species. It might be possible to replace or substitute different Hypoxis species for H. hemerocallidea for medicinal use as the chromatograms of the other three species correlate well with the chromatograms of H. hemerocallidea after storage.
Dissertation (MSc)--University of Pretoria, 2011.
Plant Science
unrestricted

Herein 47 2,4-disubstituted quinazolines were synthesized and tested against Leishmania donovani intracellular amastigotes. A structure-activity relationship was conducted and lead to the identification of quinazolines with EC50s in the single digit and high nanomolar range with favorable antileishmanial selectivity indexes. Quinazoline 2.6 and 2.31 underwent in vivo efficacy studies in murine models of visceral leishmaniasis, reducing liver parasitemia by 12 % and 24 %, respectively, when given by the intraperitoneal route at 15 mg/kg/day x 5 days. The antileishmanial efficacy and easy of synthesis make the 2,4-disubstituted quinazoline compound series a suitable platform for the future development of antileishmanial agents. A similar series of 50 N2,N4-disubstituted quinazoline-2,4-diamines has also been synthesized and tested against multi-drug resistant strains of Acinetobacter baumannii. Quinazolines with MICs in the single digit micromolar range were identified within the structure-activity relationship. The observed potencies of the top compounds and the easy of synthesis lend to the further investigation of in vivo efficacy studies and could be considered a suitable platform for the future development of anti-bacterial agents against A. baumannii.

Bacillus subtilis and related species have strong interest in agriculture because several strains are biocontrol agents of plant diseases, which have been recognized as biosafe. In this work, a selective enrichment procedure based on PCR targeting antimicrobial peptide (AMP) genes has been designed and evaluated to screen field samples and pure cultures of Bacillus spp. The procedure increased the yield of isolation of Bacillus strains compared to the standard procedure. The isolates were characterized according to the presence of six AMP genes, the capacity to produce AMPs, and their antimicrobial activity against fungal and bacterial plant pathogens. The relationships between AMP genes, products and antimicrobial activity have been established. Finally, a collection of 184 Bacillus has been obtained having multiple AMP genes simultaneously, and producing several cyclic lipopeptides, with a wide range of antimicrobial activity. It is expected that such strains would be suitable candidates to develop novel biopesticides for plant disease control
Bacillus subtilis i les espècies relacionades són de gran interès per l'agricultura, gràcies a la seva implicació en el biocontrol de malalties de plantes, i ser reconeguts com a biosegurs. En aquest treball s'ha dissenyat i avaluat un procés d'enriquiment selectiu basat en la detecció de gens relacionats amb la síntesis de pèptids antimicrobians, per tal d'analitzar mostres naturals i cultius purs de Bacillus. El procediment va permetre un increment d'aïllament de Bacillus en comparació al mètode estàndard d'aïllament. Els aïllats van ser caracteritzats segons la presencia de sis gens biosintètics, la capacitat de produir ciclolipopèptids i la seva activitat antimicrobiana contra fongs i bacteris patògens de plantes. Es van establir les relacions existents entre la presència del gens biosintètics, els productes i l'activitat antimicrobiana. Finalment, es va obtenir una col•lecció de 184 aïllats de Bacillus amb múltiple presència de gens biosintètics de pèptids antimicrobians simultàniament, productors de varis ciclolipopèptids, i amb un ampli espectre d'activitat antimicrobiana. És d'esperar que aquests aïllats resultin bons candidats per desenvolupar nous biopesticides per al control de malalties de plantes

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.

Thirty-three 1,4-dihydropyridine diastereomeric pairs were synthesized and the structure-activity relationship studied in a Plasmodium falciparum in vitro model. Twenty-nine of these derivatives contained a 6-position oxygen, with 2.31, 2.32, 2.52 and 2.53 having single and double digit nanomolar activities. This SAR study revealed some insightful information about the 1,4-dihydropyridine substitution pattern. Substitution at the 7-position other than 3,4-dimethoxy severely reduced the activity. 4-phenyl substitution with 2- or 4- halo or methyl formed active compounds while substitution at the 3-position or with methoxy or conjugated aryl systems resulted in inactive compounds. The 2-position was found to majorly affect the activity, with groups larger than methyl being the most active. The other four derivatives contained a 6-position methylene, with 2.1, 2.59 and 2.60 having single nanomolar activities. Lastly, stereochemistry was revealed to play an important role in the activity of 2.1. One stereoisomer, (+)-trans-2.1, had subnanomolar activity in two assays. Another stereoisomer, (4S,7S)-2.1, had nanomolar activity. The other two stereoisomers were inactive. A series of N2,N4-disubstituted quinazoline-2,4-diamines has been synthesized and tested against Leishmania donovani and Leishmania amazonensis intracellular amastigotes. A structure-activity and structure-property relationship study was conducted in part using the Topliss operational scheme to identify new lead compounds. This study led to the identification of quinazolines with EC50s in the single digit micromolar or high nanomolar range in addition to favorable physicochemical properties. Quinazoline 3.23 also displayed efficacy in a murine model of visceral leishmaniasis, reducing liver parasitemia by 37% when given by the intraperitoneal route at 15 mg/kg/day for five consecutive days. Their antileishmanial efficacy, ease of synthesis, and favorable physicochemical properties make the N2,N4-disubstituted quinazoline-2,4-diamine compound series a suitable platform for future development of antileishmanial agents. A similar series of N2,N4-disubstituted quinazoline-2,4-diamines has been synthesized and tested against methicilin-resistant Staphylococcus aureus (MRSA) and multi-drug resistant strains of Acinetobacter baumannii. Quinazolines with MICs in the single digit micromolar or high nanomolar range were identified via SAR. In a murine model of MRSA infection, 1x the MIC for quinazoline 4.47 allowed for the survival of all tested mice at the end of a one week study. An in vivo model of A. baumannii was also undertaken using a Galleria mellonella model of infection. Quinazolines 4.74-4.76 afforded an increased protection of 87.5% when compared to the control experiments, with 70% of the wax worms surviving until day three. The observed potencies of frontrunner compounds in in vivo assays and their ease of synthesis make N2,N4-disubstituted quinazoline-2,4-diamines a suitable platform for the future development of anti-bacterial agents.

5-nitrofuraldehido darinių antimikrobinio aktyvumo tyrimas, toksiškumo, farmakokinetinių savybių ir struktūros aktyvumo ryšio įvertinimas. K. Gaivelytės magistro baigiamasis darbas. Moksliniai vadovai: dr. V. Petrikaitė, dr. J. Šarlauskas, prof. habil. dr. A. Pavilonis; Kauno medicinos universiteto, Farmacijos fakulteto, Vaistų chemijos katedra. Kaunas, 2010. Darbo tikslas – įvertinti 5-nitrofuraldehido darinių struktūros įtaką jų antimikrobiniam aktyvumui ir parinkti perspektyviausius antimikrobinius junginius tolimesniems tyrimams. Tyrimo metodai. Junginių antimikrobinio aktyvumo prognozė atlikta panaudojant PASS programą. Antimikrobinis aktyvumas ištirtas in vitro serijinio skiedimo standžioje terpėje metodu. Junginių farmakokinetinių savybių ir toksiškumo prognozė atlikta, panaudojant ADME/Tox Boxes programą. Tyrimo rezultatai. PASS programa antibakterinį ir priešgrybelinį aktyvumą prognozavo visiems tiriamiems junginiams. Atlikus tyrimus in vitro, nustatyta, kad junginių aktyvumas prieš įvairius mikroorganizmus skyrėsi, nitrofurano fragmento neturintys junginiai buvo visai neaktyvūs. Nitrofurano bisdariniai yra gana aktyvūs prieš visas bakterijas (MSK = 0,5 100 μg/ml), išskyrus P. aeruginosa, K. pneumoniae ir P. mirabilis. Bisjunginys BIC-34, turintis butilo fragmentą, buvo aktyviausias prieš S. aureus, E. faecalis ir B. subtilis, o piridino liekaną turintis bisjunginys BIC 67 – prieš K. pneumoniae, P. aeruginosa, P. mirabilis (MSK = 50 μg/ml). Gali būti, kad šio... [toliau žr. visą tekstą]
Analysis of 5-nitrofuraldehyde derivatives antimicrobial activity, evaluation of toxicity, pharmacokinetic properties and structure – activity relationship. K. Gaivelytė Master Thesis. Scientific supervisors: Dr. V. Petrikaitė, Dr. J. Šarlauskas, Prof. Habil. Dr. A. Pavilonis; Kaunas University of Medicine, Faculty of Pharmacy, Department of Medicinal Chemistry. Kaunas, 2010. The Aim of the Research – to evaluate the influence of the structure of 5-nitrofuraldehyde derivatives and identify the most promising compounds for the further research. Methods. Prognosis of antimicrobial activity of all compounds was carried out by using PASS software. Antimicrobial activity was tested in vitro by using a serial dilution in agar technique. Pharmacokinetic properties and toxicity were predicted by using ADME/Tox Boxes program. Results. PASS program predicted antibacterial and antifungal activity for all tested compounds. The results of experiments in vitro showed that activity against various microorganisms was different; compounds without nitrofuran fragment were not active. Biscompouds were active enough against all bacteria (MIC = 0,5-100 μg/ml), except P. aeruginosa, K. pneumoniae and P. mirabilis. Biscompoud possessing butyl fragment in its structure was the most active against S. aureus, E. faecalis and B. subtilis and biscompound BIC-67 with the moiety of pyridine was the most active against K. pneumoniae, P. aeruginosa, P. mirabilis (MIC = 50 μg/ml). It could be that the... [to full text]

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-09-20T20:31:49Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) gabriele_azevedo_cardoso.pdf: 4324631 bytes, checksum: 3efd70a64b9fe1de1234371d61299990 (MD5)
Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-10-09T13:35:54Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) gabriele_azevedo_cardoso.pdf: 4324631 bytes, checksum: 3efd70a64b9fe1de1234371d61299990 (MD5)
Made available in DSpace on 2017-10-09T13:35:54Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) gabriele_azevedo_cardoso.pdf: 4324631 bytes, checksum: 3efd70a64b9fe1de1234371d61299990 (MD5) Previous issue date: 2017
Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES)
Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq)
Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG)
A necessidade de desenvolvimento de novos agentes antimicrobianos cresce ? medida que se torna maior a resist?ncia de microrganismos aos antibi?ticos usualmente empregados. Nesse sentido, os pept?deos antimicrobianos (PAMs) surgem como uma excelente alternativa para o desenvolvimento de novos antibi?ticos. O presente trabalho prop?s a s?ntese do pept?deo antimicrobiano LyeTx-I para estudos de mecanismo de a??o em membranas bacterianas, empregando diferentes t?cnicas biof?sicas e estruturais. O pept?deo LyeTx-I, composto por 24 res?duos de amino?cidos, foi isolado pela primeira vez do veneno de aracn?deos da esp?cie Lycosa erythrognata. Utilizando como t?cnicas principais a ITC e a RMN para obten??o de par?metros cin?ticos, termodin?micos e da intera??o pept?deo-membrana, foi poss?vel avaliar a rela??o estrutura e atividade do pept?deo LyeTx-I. Foram utilizadas ainda, t?cnicas complementares de CD, extravasamento de CF, fluoresc?ncia de Trp, DLS e, potencial zeta para obter informa??es adicionais acerca do modo de intera??o do pept?deo. Observou-se a predomin?ncia de conforma??o helicoidal do pept?deo LyeTx-I, tanto em meios biomim?ticos zwitteri?nicos, quanto em meios ani?nicos. Em meios ani?nicos, observou-se maior conte?do de ?-h?lice, bem como maior constante de intera??o, enquanto que em presen?a de ambientes zwitteri?nicas foram observadas menor helicidade e constante de intera??o. Os dados termodin?micos, obtidos para ambos os meios, mostraram que o processo de intera??o pept?deo-membrana ? dirigido principalmente pela componente entr?pica, uma vez que a componente ent?lpica ? menor. Os dados estruturais e termodin?micos foram coerentes com os demais estudos biof?sicos. Foi observada a partir da an?lise de extravasamento de CF maior capacidade de forma??o de poros no meio ani?nico. Os dados de fluoresc?ncia intr?nseca de Trp e de supress?o de fluoresc?ncia por acrilamida mostraram maior mudan?a de ambiente qu?mico para apolar, do res?duo de Trp-2, quando em presen?a de meio biomim?tico ani?nico. Dessa forma, o pept?deo apresenta maior capacidade de permeabilizar a membrana ani?nica. Al?m disso, o estudo comparativo entre os meios zwitteri?nicos e ani?nicos, permitiu verificar que, embora a intera??o eletrost?tica seja importante para a intera??o pept?deo-membrana, a permeabiliza??o do LyeTx-I na membrana fosfolip?dica ? fundamental para a lise celular. Dessa forma, este estudo mostra que o pept?deo LyeTx-I apresenta elevada prefer?ncia por intera??o com bicamadas fosfolip?dicas ani?nicas, o que faz dele um potencial agente bactericida.
Disserta??o (Mestrado) ? Programa de P?s-Gradua??o em Qu?mica, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017.
The demand for the development of new antimicrobial agents increases in line with the resistance of microorganisms to the antibiotics usually employed. In this sense, antimicrobial peptides (AMPs) appear as an alternative to the classical antibiotics. The present work proposed the synthesis of the antimicrobial peptide LyeTx-I for studies of mechanism of action in bacterial membranes using a set of biophysical and structural techniques. LyeTx-I peptide is composed of 24 amino acid residues and was isolated for the first time from the venom of the Lycosa erythrognata arachnid species. In order to evaluate the structure-activity relationship of the LyeTx-I, we have employed ITC and NMR as main techniques to obtain the kinetic, thermodynamic and structural parameters of the peptide-membrane interaction. Complementary measurements of CD, CF extravasation, Trp fluorescence, DLS and zeta potential were also used as additional information about the mode of action of the peptide. The ?-helical conformation of the LyeTx-I peptide was observed either in presence of zwitterionic and anionic biomimetic media. Nevertheless, a higher ?-helix content and interaction constant was observed for LyeTx-I in all anionic media when compared to the zwitterionic environments. The thermodynamic data gathered in both media, showed that the peptide-membrane interaction is driven mainly by the entropic contributions, since the enthalpic component is smaller. The structural and thermodynamic data were consistent with the complementary biophysical experiments. It was observed from the CF extravasation a greater capacity of pore formation in the anionic medium. Intrinsic Trp fluorescence showed also a greater change of the residue of Trp-2 to the apolar chemical environment in the presence of anionic biomimetic medium. In this way, the peptide presents a higher capacity to permeabilize the anionic membrane. In addition, the comparative study between the zwitterionic and anionic media, reveals that, although the electrostatic interaction is important to the peptide-membrane interaction, the permeabilization of the LyeTx-I peptide in the phospholipid membrane is fundamental for the cellular lysis. Finally, the study clearly shows the high preference of LyeTx-I for interacting anionic phospholipid bilayers, which makes it a potential bactericidal agent.

A hemoglobina (Hb) é uma fonte reconhecida de peptídeos com funções biológicas diversas. O fragmento 33-61 da cadeia α da Hb, isolado do trato gastrointestinal do carrapato Boophilus microplus, foi o primeiro a ser descrito com ação antimicrobiana. O seu análogo sintético amidado, Hb33-61a, mostrou-se ativo contra bactérias Gram-positivas e fungos [Fogaça et al. (1999) J. Biol. Chem. 274, 25330-4]. O estudo de análogos do Hb33-61 nas formas amidada e com carboxila livre revelou que a amidação provoca aumento significativo da atividade frente a Candida albicans. Por apresentar propriedades biológicas e estruturais idênticas às do Hb33-61a, o Hb40-61a pareceu ser a sua porção mÌnima ativa [Sforça et al. (2005) Biochemistry 44, 6440- 51; Machado et al. (2007) Biopolymers 88, 413-26]. Para comprovar tal sugestão, no presente trabalho, sintetizamos, purificamos e caracterizamos novos an·logos do Hb33-61a, bem como os avaliamos quanto às suas atividades frente a C. albicans e Micrococcus luteus. Os resultados confirmaram a sugestão apenas para a ação antifúngica. O análogo Hb40-61a também se mostrou ativo frente a C. albicans resistente a fluconazol. A sua atividade antifúngica se mostrou fortemente dependente da força iônica do meio. A sua baixa atividade hemolítica foi confirmada mesmo em meio de baixa força iônica. O peptídeo Hb40-61a não apresentou sinergismo com o fluconazol frente a C. albicans. A cinética de morte celular mostrou que ele mata a levedura de forma rápida. Portanto, esta hemocidina sintética pode apresentar valor comercial se a via de administração for tópica ou se o seu uso envolver meios de baixa força iônica. Além disso, ela é um modelo valioso para o estudo do mecanismo de ação de peptídeos antimicrobianos com características estruturais similares e pode servir de base para o desenho de novos agentes antibiôticos.
It is well known that hemoglobin (Hb) is a source of biologically active peptides. The fragment 33-61 of bovine hemoglobin α-chain, isolated from the gut contents of the tick Boophilus microplus, was the first identified with antimicrobial activity . Its amidated analogue, Hb33-61a, showed to be active against Gram-positive bacteria and fungi strains [FogaÁa et al. (1999) J. Biol. Chem. 274, 25330-4]. The study of a series of carboxyl-free and amidated synthetic analogues of Hb33-61 revealed that C-terminus amidation enhances the activity against Candida albicans. Since Hb33-61a and Hb40-61a presented identical biological and structural properties, it seemed that Hb40-61a was Hb33-61a minimal active motif [SforÁa et al. (2005) Biochemistry 44, 6440- To test this suggestion, in the present study 51; Machado et al. (2007) Biopolymers 88, 413-26]. we synthesized, purified and characterized Hb40-61a analogues and assayed them against C. albicans and Micrococcus luteus. The results confirmed the suggestion only for the antifungal activity. When tested against fluconazole-resistant C. albicans, Hb40-61a was also active. Its antifungal activity showed to be dependent on the ionic strength of the medium. Its low hemolytic activity was confirmed even under low ionic strength conditions. Hb40-61a had no synergic effect with fluconazole on C. albicans. In vitro time-kill assays demonstrated that Hb40-61a kills the yeast rapidly. Therefore, this synthetic hemocidin may be of commercial interest for topical application or other uses involving low ionic strength medium. Moreover, it can serve as a template for the study of the mechanism of action of structurally related antimicrobial peptides or for the design of novel antibiotic drugs.

A introdução de um grupo substituinte na molécula de um fármaco promove alterações químico-estruturais que, por sua vez, modificam suas propriedades físicoquímicas. O arranjo espacial de átomos ou grupos de átomos, em especial grupos funcionais, na molécula de um fármaco, expressos por meio de suas propriedades físico-químicas, influenciam direta ou indiretamente na interação fármaco-receptor. Esta, por sua vez, determina aspectos farmacológicos e farmacocinéticos que influem na eficácia terapêutica do medicamento. Assim, uma série de compostos 2-[5-nitro-tiofen-2-il]-3-acetil-5-[4-fenil-substituído]-2,3-diidro-1,3,4-oxadiazolínicos foi planejada e os análogos foram sintetizados, identificados estruturalmente e avaliados in vitro quanto a atividade antimicrobiana frente a Staphylococcus aureus (cepa ATCC 25923), expressa pela determinação da concentração inibitória mínima. Cepas desta bactéria são comuns em infecções hospitalares e frequentemente apresentam caráter de multi-resistência, portanto, alternativas aos fármacos comumente empregados na terapia antibacteriana, especialmente em infecções multi-resitentes, são alvo de estudos e desenvolvimento. Relações entre mudanças estruturais de 2-[5-nitro-tiofen-2-il]-3-acetil-5-[4- fenil-substituído]-2,3-diidro-1,3,4-oxadiazolinas e suas respectivas concentrações inibitórias mínimas podem fornecer informações sobre a influência de propriedades físico-químicas na ação antibacteriana destes compostos, informações estas que podem contribuir para o entendimento das relações entre a estrutura química e a atividade biológica desta classe de compostos, visando a identificação qualitativa e quantitativa das propriedades físico-químicas que influenciam no perfil farmacológico desta classe de compostos. Os grupos substituintes introduzidos nos derivados de 2,3-diidro-1,3,4-oxadizolinas-2,3,5-substituídas contribuem para alterações em suas propriedades físico-químicas, sendo representadas por parâmetros físico-químicos que descrevem a natureza e intensidade da alteração observada. O presente trabalho objetivou identificar, partindo das propriedades físico-químicas, fatores da estrutura química que favoreçam a atividade antimicrobiana dos compostos estudados. A atividade antimicrobiana, expressa pela concentração inibitória mínima, foi determinada pelo procedimento de microdiluição sucessiva, no qual diferentes concentrações de composto a ser analisado foram incubadas em presença de inóculo de Staphylococcus aureus, em meio de cultura líquido. As microdiluições originam concentrações decrescentes a fim de se determinar a menor concentração do composto testado onde o crescimento microbiano foi inibido. Foram realizadas comparações entre as concentrações inibitórias mínimas dos compostos analisados e destas com parâmetros estruturais que representam as propriedades físico-químicas dos compostos. Baseado nas análises comparativas, identificou-se tendências que evidenciam a preponderância de propriedades físicoquímicas sobre a atividade antimicrobiana desempenhada. Constatou-se que a hidrofobicidade influi significativamente na atividade antimicrobiana. Observou-se também que o efeito eletrônico por indução e o volume do grupo substituinte em posição para-fenila também influenciam a ação antimicrobiana, mas estas ainda não foram conclusivas, carecendo de estudos mais aprofundados que levem à compreensão dos fatores estruturais que mais influenciam a ação antimicrobiana de 2-[5-nitro-tiofen-2-il]-3-acetil-5-[4-fenilsubstituído]- 2,3-diidro-1,3,4-oxadiazolinas, de forma a fundamentar o planejamento de futuros candidatos a fármacos antimicrobianos a partir desta classe de compostos.
The introduction of a substitute group within a drug molecule promotes chemical-structure shifts, which by consequence, alters its physical-chemical properties. The atomic special design, particularly the one related to functional groups, assumed in a drug molecule, expressed by the physical-chemical properties, interferers directly or indirectly on drug-receptor interaction. Theses effects collaborate to pharmacologic and pharmacokinetics aspects of drugs, interfering with its therapeutic efficient. Therefore, a set of 2-[5-nitro-thiophen-2-yl]-3-acetyl-5-[4- substitued-phenyl]-3,4-dihydro-1,3,4-oxadiazolines compounds was designed and its analogs synthesized, structurally identified, and in vitro assayed due to its antimicrobial activity against ATCC 25923 Staphylococcus aureus strains, through the determination of minimum inhibitory concentration. Strains of such bacterial species are commonly present within hospital infections, and frequently appear as multi-resistant variant. Consequently, alternatives to the agents usually applied in antimicrobial chemotherapy, particularly to multi-resistant infections, are target for drug research and development. Relations among structural shifts on 2-[5-nitro-thiophen-2-yl]-3-acetyl-5-[4-substitued-phenyl]-3,4-dihydro-1,3,4-oxadiazolines compounds and its respective minimum inhibitory concentration may lead to information about the influence of physical-chemical properties in antimicrobial activity. Continually, such information might contribute to the elucidation of chemical structure-biological activity relationship of these compounds. The substitutes groups inserted on 2,3,5-substituted-3,4-dihydro-1,3,4-oxadiazolines derivates contribute to differs the molecule physical-chemical properties, which is represented by its physical-chemical parameters, which describe the nature and intensity of the observed modification. Therefore, the following study aimed to identify, throughout physical-chemical properties, chemical structure factors that favor the antimicrobial activity of the synthesized compounds. The antibacterial activity, expressed as the minimum inhibitory concentration, was quantified by the microdilution method, where gradual concentrations of the tested conpound were incubated within ATCC 25923 Staphylococcus aureus cells suspended in broth medium. The microdilutions originated descending concentrations, in order to spot the minimum concentration whose microbial growth was inhibited. The minimum inhibitory concentration regarded to each compound was correlated to its physical-chemical parameters. Based on observed relations, it could be identified evidences assuming the physical-chemical properties relevance on antimicrobial assay. It was verified that hydrophobic effects interferes with antimicrobial activity, as well as inductive electronic effects and molecular volume of substitute group in para-phenyl position. These factors contribute to physical-chemical properties shifts and therefore play a role on the antimicrobial action. However, these latest two influences were enable to conclude, suggesting the need of more extensive studies. So that, a more profound comprehension about structure factors that interfere with 2,3,5-substitued-3,4-dihydro-1,3,4-oxadiazolines derivates might lead to the rational design of potential antimicrobial agents among this class of compounds.

L’usage abusif et excessif des antibiotiques a exacerbé le phénomène d’antibiorésistance, à travers la planète. Aujourd’hui, la lutte contre l’antibiorésistance est devenue une priorité mondiale en matière de santé publique. En effet, chaque année, elle est responsable de plus de 700,000 décès dans le monde et d'ici 2050, elle causera plus de 10 millions de décès par an, si des actions concrètes ne sont pas mises en place afin de freiner le développement de ce phénomène. En plus des pertes humaines, le coût financier des soins afférents à l’antibiorésistance pourra atteindre 100,000 milliards de dollars dans le monde. Pour faire face à cette crise annoncée, plusieurs stratégies innovantes, parmi lesquelles l’usage des peptides antimicrobiens (PAMs), ont été proposées. Dans cette perspective, les bactériocines, qui sont des PAMs synthétisés par voie ribosomique pourraient contribuer à la solution thérapeutique. Récemment, la souche Lacticaseibacillus paracasei CNCM I-5369 s’est particulièrement distinguée pour son activité contre des germes pathogènes à Gram-négatif. Cette activité est due à 5 nouvelles bactériocines codées par des gènes chromosomiques orf010, orf012, orf023, orf030 et orf038. Toutefois, l’activité est pH-dépendante, c’est-à-dire qu’elle est exercée, uniquement à une valeur de pH ≤ 5. Dans le cadre de cette thèse, nous avons dans un premier temps, vérifiée l’expression de ces gènes lors de la croissance de la bactérie, en utilisant la technique de qPCR. Ainsi, nous avons observé que ces 5 gènes sont exprimés après 24 h de croissance, de façon concomitante à l’apparition de l’activité antimicrobienne indiquant ainsi un lien entre l’expression génique et la production des cinq bactériocines. Ces 5 bactériocines ont été exprimées en système hétérologue chez Escherichia coli Rosetta. Seule la bactériocine codée par orf030, appelée lacticaséicine 30, a pu être produite en grande quantité, contrairement aux autres bactériocines qui ont été produites mais demeurent piégées dans la fraction insoluble. Dans un deuxième temps, nous avons étudié la relation entre la structure de la lacticaséicine 30 et son activité contre les bactéries à Gram-négatif. Les prédictions de structure ont suggéré une organisation en 5 hélices  de la lacticaséicine 30. La proportion d’hélice-α était plus importante à pH 5 qu’à pH 7. Pour identifier les régions impliquées dans l’activité contre les bactéries à Gram-négatif, nous avons produit des peptides dérivés de la lacticaséicine 30 par une approche de biologie moléculaire. Ces peptides dérivés sont obtenus en réduisant leurs tailles, ou en insérant des mutations ciblées dans différentes régions. Ainsi, nous avons généré des formes plus courtes de la lacticaséicine 30, contenant soit sa région N-terminale (acides aminés 1 à 39), soit les régions centrale et C-terminale (acides aminés 40 à 111). De la même manière, un peptide dérivé contenant uniquement la première hélice de la région N-terminale a été également produite. Les mutations introduisant des substitutions des acides aminés ont été introduites au sein des hélices-α. Au regard de leurs activités, ces peptides dérivés ont permis de localiser l’activité antibactérienne essentiellement dans la région N-terminale, et nécessite à minima deux hélices-α. Par ailleurs, l’activité de ces variants peptidiques E32G, T33P, T52P et D57G reste sensiblement identique, contrairement à celles des variants E6G, T7P, D57G T52P, A74P, Y78S, Y93S et A97P qui ont été significativement altérées. Dans la dernière partie, nous avons testé l’activité de la lacticaséicine 30 contre un panel de souches cliniques à Gram-négatif et portant des résistances à la colistine. Les résultats obtenus ont mis en lumière une synergie entre la lacticaséicine 30 et la colistine et la réduction significative de l’expression des gènes mcr-1 et mcr-9, responsables de la résistance à la colistine
The excessive use of antibiotics has exacerbated the phenomenon of antibiotic resistance throughout the world. Today, the fight against antibiotic resistance has become a global public health priority. Indeed, every year, it is responsible for more than 700,000 deaths in the world and by 2050, it will cause more than 10 million deaths per year, if concrete actions are not implemented to curb the development of this phenomenon. In addition to the human losses, the financial cost of antibiotic resistance-related care could reach 100,000 billion dollars worldwide. To face this crisis, several innovative strategies, including the use of antimicrobial peptides (AMPs), have been proposed. In this perspective, bacteriocins, which are ribosomally APMs, could contribute to the therapeutic solution. Recently, the Lacticaseibacillus paracasei CNCM I-5369 strain has been particularly distinguished for its activity against Gram-negative pathogens. This activity is due to 5 new bacteriocins encoded by chromosomal genes orf010, orf012, orf023, orf030 and orf038. However, the activity is pH-dependent, i.e., it is exerted, only at a pH value ≤ 5. In the framework of this thesis, we first verified the expression of these genes during the growth of the bacteria, using the qPCR technique. Thus, we observed that these 5 genes were expressed after 24 h of growth, concomitantly with the appearance of antimicrobial activity indicating a possible link between gene expression and the production of the five bacteriocins. These 5 bacteriocins were expressed in a heterologous system in Escherichia coli Rosetta. It should be noted that only the bacteriocin encoded by orf030, called lacticaseicin 30, could be produced in large quantities, in contrast to the other bacteriocins which were produced but remain trapped in the insoluble fraction. In a second step, we investigated the relationship between the structure of lacticaseicin 30 and its activity against Gram-negative bacteria. Structural predictions suggested a 5-helix organization of lacticaseicin 30. The proportion of helix-α was greater at pH 5 than at pH 7. To identify the regions involved in the activity against Gram-negative bacteria, we produced lacticaseicin 30-derived peptides by a molecular biology approach. These derived peptides are obtained by reducing their sizes, or by inserting targeted mutations in different regions. Thus, we generated shorter forms of lacticaseicin 30, containing either its N-terminal region (amino acids 1 to 39), or the central and C-terminal regions (amino acids 40 to 111). Similarly, a derivative peptide containing only the first helix of the N-terminal region was also produced. Mutations introducing amino acid substitutions were introduced within the α-helices. With regard to their activities, these derived peptides localized antibacterial activity mainly in the N-terminal region, and requires at least two helix-αs. Furthermore, the activity of these peptide variants E32G, T33P, T52P, and D57G remained essentially the same, unlike to those of the variants E6G, T7P, D57G T52P, A74P, Y78S, Y93S, and A97P, which were significantly impaired. In the last part, we tested the activity of lacticaseicin 30 against a panel of Gram-negative clinical strains with colistin resistance. The results obtained highlighted a synergy between lacticase 30 and colistin and a significant reduction of the expression of the mcr-1 and mcr-9 genes, responsible for colistin resistance

[EN] Persimmon (Diospyros kaki L.) 'Rojo Brillante' is an astringent variety characterised by good growing conditions, excellent colour, size, sensory characteristics and good nutritional properties. In the last decade, its production has grown substantially in Spain given the application of high levels of CO2 to remove astringency while firmness is preserved. This technology has also increased its potential as a fresh-cut commodity. However, physical damage during processing result in degradation of the colour and firmness of the product and a higher susceptibility to microbial spoilage that significantly reduces the fruit's shelf life. The objective of the present thesis was to develop optimum procedures for processing and marketing 'Rojo Brillante' persimmon into a fresh-cut product with the maximum shelf life and best physicochemical, nutritional, sensory and microbiological quality. Firstly, the objective was to evaluate the effect of the maturity stage (MS) at harvest, storage time at 15 ºC before processing, and the application of different antioxidant treatments on enzymatic browning, sensory and nutritional quality of fresh-cut 'Rojo Brillante' persimmon during storage at 5 ºC. Concentrations of 10 g L-1 ascorbic acid (AA) or 10 g L-1 citric acid (CA) controlled tissue browning and maintained the visual quality of fresh-cut persimmon above the limit of marketability for 6-8 storage days at 5 ºC, depending on the MS. However, these acidic solutions reduced fruit firmness as compared to control samples. Further studies showed that the combination of these antioxidants with 10 g L-1 CaCl2 maintained firmness of the persimmon slices within the same range as the control samples. In another work, the application of 1-methylcyclopropene (1-MCP) allowed to process fruits after 45 days of storage at 1 ºC with commercial firmness and the antioxidant solution (10 g L-1 CA + 10 g L-1 CaCl2) extended the limit of marketability up to 9 days of storage at 5 ºC. Different controlled atmosphere conditions in combination with AA or CA dips were also evaluated as a first step to select optimum O2 and CO2 concentrations for modified atmosphere packaging (MAP) of fresh-cut 'Rojo Brillante' persimmons. Overall, the combination of antioxidant dips and a controlled atmosphere composed of 5 kPa O2 (balance N2) was proved to be the most effective combination to control enzymatic browning. This atmosphere maintained the visual quality of persimmon slices within the limit of marketability during 7- 9 days at 5 ºC. On the contrary, high CO2 concentrations (10 or 20 kPa) induced darkening in some tissue areas, associated with a flesh disorder known as 'internal flesh browning'. Later studies confirmed the beneficial effect of an active MAP in 5 kPa O2 compared to passive MAP to improve the visual quality of fresh-cut 'Rojo Brillante' persimmon, showing a synergic effect with the antioxidant dip (10 g L-1 CA + 10 g L-1 CaCl2). Antioxidant edible coatings were prepared from whey protein isolate (WPI), soy protein isolate (SPI), hydroxylpropyl methylcellulose (HPMC) and apple pectin as the polymeric matrix. All edible coatings were amended with the antioxidant combination selected (10 g L-1 CA + 10 g L-1 CaCl2). All the edible coatings tested proved effective to control enzymatic browning of persimmon slices. However, the samples treated with the HPMC- and pectin- based coatings were scored with a better visual quality that the rest of the treatments. In general, free radical scavenging activity and total carotenoid content increased in late-season persimmons; whereas, processing (cutting and storage at 5 ºC), antioxidant dips, controlled atmosphere storage or edible coatings had no clear effect on nutritional quality (vitamin C, free radical scavenging activity, total phenolic content, and carotenoids) of fresh-cut persimmons.
[ES] El caqui persimmon (Diospyros kaki L.) 'Rojo Brillante' es un cultivar astringente que presenta unas propiedades organolépticas y nutricionales excelentes. En la última década, su cultivo en el área mediterránea de España se ha incrementado de manera exponencial con el desarrollo de la tecnología que permite eliminar la astringencia, manteniendo la firmeza del mismo. Esta nueva forma de presentación, aporta numerosas ventajas, entre la que se incluye la posibilidad de ser comercializado como fruta fresca cortada. Sin embargo, el éxito comercial del producto está limitado por el pardeamiento enzimático, la pérdida de firmeza y al crecimiento microbiano. En este contexto, el objetivo de la Tesis ha sido el desarrollo de caqui 'Rojo Brillante' fresco cortado mediante un enfoque que integra el estudio de las características del producto en el momento del procesado y de distintas tecnologías que mantengan la calidad físico-química, sensorial, nutricional y microbiológica del producto durante un periodo que permita su comercialización. En primer lugar, se evaluó el efecto del estado de madurez (MS) en el momento de recolección, el tiempo de almacenamiento a 15 ºC antes del procesado y la aplicación de diferentes antioxidantes en el pardeamiento enzimático y la calidad sensorial y nutricional del caqui 'Rojo Brillante' cortado y almacenado a 5 ºC. La aplicación de 10 g L-1 de ácido ascórbico (AA) ó 10 g L-1 ácido cítrico (CA) controló el pardeamiento enzimático y mantuvo la calidad visual del caqui por encima del límite de comercialización entre 6 y 8 días de almacenamiento a 5 ºC, dependiendo del MS. Sin embrago, la aplicación de estos antioxidantes redujo de manera significativa la firmeza del fruto respecto al control. La combinación de estos antioxidantes con 10 g L-1 de CaCl2 permitió mantener la firmeza en el mismo rango que las muestras control. En un trabajo posterior, la aplicación de 1-metilciclopropeno (1-MCP) permitió procesar caqui almacenado 45 días a 1 ºC con una buena firmeza comercial y el tratamiento antioxidante (10 g L-1 CA + 10 g L-1 CaCl2) consiguió alcanzar un límite de comercialización del producto de 9 días a 5 ºC. La evaluación de distintas atmósferas controladas en combinación con tratamientos antioxidantes (AA o CA), como paso previo al envasado en atmósfera modificada (MAP) del caqui, mostró como más efectiva en el control del pardeamiento enzimático la atmósfera compuesta por 5 kPa O2 (balance N2). Esta atmósfera mantuvo la calidad visual del caqui cortado dentro del límite de comercialización durante 7-9 días a 5 ºC. Por el contrario, la aplicación de altas concentraciones de CO2 (10 ó 20 kPa) dio lugar a un pardeamiento en ciertas zonas de la pulpa que se conoce como 'internal flesh browning'. Estudios posteriores confirmaron el efecto beneficioso del envasado de caqui cortado y tratado con solución antioxidante (CA-CaCl2) en una MAP activa de 5 kPa O2 en la calidad visual del fruto frente a la aplicación de una MAP pasiva. El desarrollo de recubrimientos comestibles con capacidad antioxidante se realizó mediante la incorporación de antioxidantes (10 g L-1 CA + 10 g L-1 CaCl2) a formulaciones a base de proteína de suero lácteo (WPI), proteína de soja (SPI), hidroxipropilmetilcelulosa (HPMC) y pectina. Todos los recubrimientos fueron efectivos controlando el pardeamiento enzimático del caqui cortado, siendo las muestras recubiertas con HPMC y pectina las mejor evaluadas visualmente. En general, el procesado, la aplicación de antioxidantes, el envasado en atmósferas controladas y los distintos recubrimientos comestibles estudiados, si bien no mostraron un efecto claro en los parámetros de calidad nutricional evaluados, no tuvieron un efecto negativo en los mismos. Por otra parte, los frutos cosechados a final de campaña tuvieron mayor actividad antioxidante y contenido en carotenoides.
[CAT] El caqui persimmon (Diospyros kaki L.) 'Rojo Brillante' és un cultiu astringent que presenta unes propietats organolèptiques i nutricionals excel¿lents. En la última dècada, el seu cultiu en l'àrea mediterrània d'Espanya s'ha incrementat de manera exponencial amb el desenvolupament de la tecnologia que permet eliminar l'astringència, mantenint la fermesa del mateix. Esta nova forma de presentació, aporta un gran nombre d'avantatges, entre els quals s'inclou la possibilitat de comercialitzar-lo com fruita fresca processada. No obstant, l'èxit comercial del producte està limitat per pardetjament enzimàtic, la pèrdua de fermesa i el creixement microbià. L'objectiu de la Tesis ha estat en el desenvolupament de caqui 'Rojo Brillante' tallat en fresc mitjançant un enfocament que integra l'estudi de les característiques del producte en el moment del processat i de diferents tecnologies en el manteniment de la qualitat físico-química, sensorial, nutricional i microbiològica del producte durant un període que permeta la seua comercialització. En primer lloc, es va avaluar l'efecte de l'estat de maduresa (MS) en el moment de recol¿lecció, el temps d'emmagatzemament a 15ºC abans del processat i l'aplicació de diferents tractaments antioxidants en el pardetjament enzimàtic i la qualitat sensorial i nutricional del caqui 'Rojo Brillante' tallat i emmagatzemat a 5 ºC. L'aplicació de 10 g L-1 d'àcid ascòrbic (AA) o 10 g L-1 d'àcid cítric (CA) va controlar el pardetjament enzimàtic i va mantenir la qualitat visual del caqui per damunt del límit de comercialització entre 6-8 dies d'emmagatzemament a 5 ºC, depenent del MS. No obstant, l'aplicació d'antioxidants va reduir de manera significativa la fermesa del fruit comparat amb el control. La combinació d'aquestos antioxidants amb 10 g L-1 de CaCl2 va permetre mantenir la fermesa en el mateix rang que les mostres control. En un treball posterior, l'aplicació de 1-metilciclopropeno (1-MCP) va permetre processar caqui emmagatzemat 45 dies a 1 ºC amb una bona fermesa comercial i a més, el tractament antioxidant (10 g L-1 CA + 10 g L-1 CaCl2) va aconseguir un límit de comercialització del producte tallat de 9 dies a 5 ºC. L'avaluació de diferents atmosferes controlades en combinació amb tractaments antioxidants (AA o CA), com a pas previ a l'envasament en atmosfera modificada (MAP) del caqui 'Rojo Brillante, va mostrar com a més efectiva en el control del pardetjament enzimàtic l'atmosfera composta per 5 kPa O2 (balanç N2). Aquesta atmosfera va mantenir la qualitat visual del caqui tallat dins del límit de comercialització durant 7-9 dies a 5 ºC. Per contra, l'aplicació d'altes concentracions de CO2 (10 ó 20 kPa) va donar lloc a un pardetjament en certes zones de la polpa, el qual és conegut com 'internal flesh browning'. Estudis posteriors van confirmar l'efecte beneficiós de l'envasament de caqui tallat i tractat amb solució antioxidant (CA-CaCl2) en una MAP activa de 5 kPa O2 millorant la qualitat visual de la fruita front a l'aplicació de una MAP passiva. El desenvolupament de recobriments comestibles amb capacitat antioxidant es va realitzar mitjançant la incorporació d'antioxidants (CA-CaCl2) en formulacions a base de proteïna de sèrum làctic (WPI), proteïna de soia (SPI), hidroxipropilmetilcel-lulosa (HPMC) i pectina. Tots els recobriments van ser efectius controlant el pardetjament enzimàtic del caqui tallat. No obstant, les mostres recobertes amb HPMC i pectina van ser millor avaluades visualment que la resta de tractaments. En general, el processat, l'aplicació d'antioxidants, l'envasament en atmosferes controlades i els distints recobriments comestibles estudiats, si bé no van mostrar un efecte clar en els paràmetres de la qualitat nutricional avaluats, no van tindre un efecte negatiu en els mateixos. Per altra banda, els fruits recol¿lectats a final de temporada van tenir major activitat antioxidant i contingut en
Sanchís Soler, E. (2016). Effect of processing on the physicochemical, sensory, nutritional and microbiological quality of fresh-cut 'Rojo Brillante' persimmon [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/62588
TESIS

碩士
國立宜蘭大學
食品科學系碩士班
106
Pecan is the most valuable nut native to North America. The United States is the world leader in pecan nut production, accounting for about 50% of total production. In past decades, pecan has been introduced to many other countries, including South Africa, Brazil, Australia, Argentina, and China. The pecan nut shell is rich in phenolic compounds, such as phenolic acids, flavonoid acids and proanthocyanidins, which have been extensively studied due to their antioxidant properties. Moreover, phenolic compounds such as caffeic acid, gallic acid, p-coumaric acid, catechin and ferulic acid have been reported to function as antibacterial agents against microorganisms. Therefore, this study discussed the total phenolics, total flavonoids, six phenolic acids and antimicrobial activities of pecan. The results showed that the total phenolics of pecan kernel and shell were 6.39 ± 0.40 mg GAE / g d.w. and 10.17 ± 0.15 mg GAE / g d.w. The contents of gallic acid, vanillic acid, caffeic acid, syringic acid, p-coumaric acid and ferulic acid were 151.07 ± 2.10 mg / 100 g d.w., 146.81 ± 14.22 mg / 100 g d.w., 64.81 ± 4.16 mg / 100 g d.w., 38.83 ± 2.5 mg / 100 g d.w., 2.46 ± 1.48 mg / 100 g d.w. and 183.94 ± 4.41 mg / 100 g d.w.. Pecan shell hot water and ultrasound extraction can effectively inhibit the growth of S. aureus (BCRC 10451) and P. aeruginosa (BCRC 11633). Also had the MIC at 0.625 mg mL-1 on S. aureus (BCRC 10451). However, the corresponding concentrations to E. coli (BCRC 11634) and S. mutan (BCRC 10793), were all located at 2.5 mg mL-1. Similarly, among the experimental extracts, two extract samples also got activity to the bacterium inhibition in the mouth environment.

碩士
義守大學
生物技術與化學工程研究所
100
Flavonoids are a class of natural polyphenol compounds existing in many natural plants，among which chalcone is one with high bioactivity. Up to now, it has been found to have many pharmacological activities, including anti-inflammation, anti-oxidation, antimicrobial action，inhibition of tumor growth etc. In this research, a series of chalcone derivatives were synthesized from acetophenones and aromatic aldehydes with different substituents by the method of Claisen-Schmidt Condensation. And then, we conducted the research on their bacteriostatic activity and anti-oxidative ability，and discussed the structure-activity relationship with the expectation of finding active ingredients for either new antibiotics or new antioxidants. Analysis of bacteriostatic activity was done through the disc agar diffusion method：Disk Inhibition Zone（DIZ） and Minimum inhibitory concentration(MIC） test. For the analysis of anti-oxidative activity, three assay methods were used: DPPH radical scavenging ability, reductive ability, and ABTS radical scavenging ability. According to the experimental results, bioactivity of chalcone derivatives have a close relationship with the position and type of radicals. Experiment results indicate that hydroxyl group on the A cycle plays an important role in both bacteriostatic activity and anti-oxidative activity. Compounds with hydroxyl group on the A cycle （DIZ ＝12 mm〜20 mm）have higher bacteriostatic activity than those without any（DIZ ＝0 mm）. Furthermore，for the derivatives’ anti-oxidative activity, there is no significant action on DPPH radical scavenging. Vanillin（IC50＝5.51 ppm） lost its original anti-oxidative activity after the reaction. In the reductive ability test, we conjectured that this may be the result of structure change after the reaction, which can lead to the increase in steric hindrance. Therefore, in terms of bacteriostasis, it’s much harder for the compound to combine with bacteria. From the anti-oxidative perspective, the scavenging process is also blocked as a result of difficulties in combining with radicals.

碩士
東海大學
化學系
104
The extensive use of antibiotics in medicine results in the multidrug-resistance of bacteria, making the development of new antimicrobial agents an urgent need. Antimicrobial peptides (AMPs) are considered as a new class of antibiotics with characteristics including an ability to kill target cells rapidly and an unusually broad spectrum of activity. In our previous study, MAP-04-03 was discovered as a potent antimicrobial peptide (MIC= 5 μM), but showed high hemolytic activity (70.7%). In the current study, to increase its antimicrobial activity and reduce the hemolytic side effect, three peptides analogs of MAP-04-03 (JJ-01, JJ-02 and JJ-03) were designed by substitution of certain amino acids in its sequence. These peptide analogs were synthesized by solid phase method, purified by RP-HPLC and characterized by MALDI-TOF MS. Structure - activity relationships of these analogs were studied by using circular dichroism and biological assays. The content of helical structure in JJ-01, JJ-02 and JJ-03 was lower than that of MAP-04-03 and the antimicrobial activity of JJ-01 was the same as that of MAP-04-03 (MIC = 5 μM), JJ-02 exhibited the highest activity (MIC= 2.5 μM), while JJ-03 showed the same activity as MAP-0403 (MIC= 5 μM). The hemolytic activity of JJ-01 and JJ-03 decreased slightly (46.2% and 55.6%, respectively), while JJ-02 showed the lowest hemolytic activity (3.4%). The potent peptide JJ-02 with little hemolytic activity is promising and becomes the lead for further development of antimicrobial agents.

碩士
國立臺灣大學
農業化學研究所
97
Abstract This study applied ultrafine-milling technology on bamboo leaf. The post-milled particle size of bamboo leaf was examined. The chemical content, bioactive material of bamboo leaf extract, and its ability to suppress bacterial growth were investigated. The antimicrobial material in bamboo leaf was also preliminarily determined. The extracting solvent used in this study included water and 60% ethanol. After a 1 hour ultrafine-milling treatment, using either water or 60% ethanol, the mean particle size and d90 of bamboo leaf decreased obviously. With even longer ultrafine-milling treatment, the mean particle size continued to decrease but changed more slowly. After a 4 hour ultrafine-milling treatment, the mean particle size and d90 were 12 μm and 25 μm, reaching the goal of destructed cell wall. After a 2 hour ultrafine-milling treatment with water, the total soluble carbohydrates and total flavonoids of bamboo leaf extract elevated obviously, but the total phenolics showed no apparent differences. Under the same milling condition except using 60% ethanol, the total soluble carbohydrates of bamboo leaf extract decreased, but the total flavonoids increased to 1.76 mg catechin equivalent /g ground bamboo leaf powder(GP), suggesting that 60% ethanol is a good extracting solvent of flavonoids. The DPPH scavenging capacity of ethanol extract was the highest than that of water extract under all the conditions in this study. The result of total cyanide content analysis showed that dried ground bamboo leaf powder contained 2.5 μg HCN. After ultrafine-milling treatment, the total cyanide content of residue of bamboo leaf increased to 3-4 μg HCN/g GP. But the total cyanide content of bamboo leaf extract decreased obviously after rotary evaporation and cetrifuge treatment. Bamboo leaf extract composition analysis performed by silica gel column, 70% methanol elution and High-performance liquid chromatography (HPLC) demonstrated that there were three major peaks on the HPLC chromatograms of bamboo leaf extract. According to the UV scanning spectra of bamboo leaf extract from 200 to 400 nm wavelength, the components were very likely to be flavonoids, while the Mass spectrometry (MS) was determined as isoorientin (or orientin) and isovitexin (or vitexin). Antimicrobial assay was performed by conducting agar diffusion assay, serial dilution method, and hole-plate diffusion method. The results showed that bamboo leaf ethanol extract can apparently suppress the growth of Bacillus cereus and Staphylococcus aureus at the dilution of 10, but it was ineffective for Escherichia coli and Salmonella typhimuriun. Time-kill method showed that bamboo leaf ethanol extract at the dilution of 10 could persist antimicrobial effects within 12 hours. The inhibitory concentration of bamboo leaf ethanol extract for S. aureus was compared by using colony size method. On the contrary, bamboo leaf water extract, even at the original concentration, had no antimicrobial effect for all the 4 kinds of bacteria in this study. Bamboo leaf treated with ultrafine-milling using 60% ethanol as extracting solvent could achieve higher flavonoids content and antimicrobial activity for Bacillus cereus and Staphylococcus aureus. According to this study and considering the fact that there are abundant bamboo resources in Taiwan, bamboo leaf extract has great potential to be excellent food additives as antimicrobial agent.

碩士
大葉大學
食品工程研究所
88
Shrimp shell derived chitosan was chemically modified to obtain sulfonated chitosan ( SC ) and sulfobenzoyl chitosan ( SBC ) in pyridine and methanol solvent system respectively. Antimicrobial activity and aqueous solubility of these two chitosan derivatives as functions of sulfur content ( ranged from 1 to 5% ) were studied. Test microorganism included Escherichia coli（CCRC 10674）, Salmonella typhimurium（CCRC 10746） and Staphylococcus aureus（CCRC 12652）. Generally speaking, solubility increased with increasing sulfur content. However, increased sulfur content did not result in higher antimicrobial activity. It was also found that there existed optimal sulfur content in terms of antimicrobial activity and the sensitivities toward sulfur content were strain dependent. Degree of deacetylation also affected these two properties of SC,SBC and underivatised chitosan﹒ Similar conclusion that the influence was strain dependent was obtained. Relatively speaking, SC and SBC demonstrated superior antimicrobial activity and aqueous solubility than underivatised chitosan．

Tese de doutoramento em Chemistry (especialização em Organic and Biological Chemistry)
A investigação de compostos bioactivos que possam levar ao desenvolvimento de novas alternativas terapêuticas para algumas das patologias mais nefastas a nível mundial continua, nos dias de hoje, a ser uma necessidade no ramo da Química Medicinal. Nesta pesquisa, algumas moléculas presentes na natureza são usadas como ponto de partida para o desenvolvimento de novos compostos promissores. Neste âmbito, os cromenos e os imidazóis são amplamente reconhecidos como estruturas interessantes. Estão descritos na literatura vários derivados de cromenos e imidazóis que apresentam actividade biológica no tratamento de uma vasta gama de patologias (p. ex. anticancerígeno, antibiótico, antimicrobiano, antiviral, etc). Neste trabalho, foi explorada a reactividade de 2H- e 4H-cromenos, que levou ao desenvolvimento de novos derivados, como por exemplo cromeno[2,3-d]pirimidin-4(5H)-onas, cromeno[2,3- d]pirimidin-4(3H)-onas e 5-imino-N,2-diaril-5H-cromeno[3,4-c]piridin-4-aminas. Foram também preparados outros compostos promissores como por exemplo 5-(2-hidroxiaril)-7-arilpirido[2,3- d]pirimidin-4(1H)-ones e 1-aril-2-(4-(arilamino)-5H-cromeno[2,3-d]pirimidin-5-il)etanonas, e novos derivados de 5-ciano-1-(2-hidroxibenzil)-1H-imidazol-4-carboxamidas. Foi estudado o mecanismo que levou à formação de 5-imino-N,2-diaril-5H-cromeno[3,4-c]piridin-4-aminas, 5-(2-hidroxiaril)-7- arilpirido[2,3-d]pirimidin-4(1H)-onas e 5-ciano-1-(2-hidroxibenzil)-1H-imidazol-4-carboxamidas e o mecanismo deste último foi também estudado com recurso a metodologias computacionais. Foi avaliada uma selecção de compostos relativamente ao seu potencial no tratamento do cancro da mama triplo negativo (TNBC), tuberculose (M.tuberculosis) e malária (plasmodium falciparum), e algumas moléculas apresentaram resultados promissores. Foi ainda realizado um conjunto de estudos de docking molecular e de dinâmica molecular, tendo como ponto de partida os resultados de actividade biológica obtidos para as cromeno[2,3-d]pirimidin-4(5H)-onas contra o cancro da mama triplo negativo, o que permitiu o desenvolvimento de um modelo preliminar de relação estrutura-actividade para este grupo de compostos, nesta patologia.
The search for bioactive compounds that can lead to novel therapeutic alternatives to some of the most devastating diseases is still an important research topic in Medicinal Chemistry. In this search, some naturally occurring scaffolds are used as inspiration for the development of new drug candidates and among those, chromenes and imidazoles are widely studied and recognized as interesting scaffolds. Several chromene and imidazole derivatives have been described in the literature, where their biological activity against a wide range of diseases (e.g. anticancer, antibiotic, antimicrobial, antiviral, etc) is identified. In this work, the reactivity of 2H- and 4H-chromenes was explored, which led to the development of novel derivatives, such as chromene[2,3-d]pyrimidin-4(5H)-ones, chromene[2,3-d]pyrimidin- 4(3H)-ones and 5-imino-N,2-diaryl-5H-chromeno[3,4-c]pyridin-4-amines. Other promising compounds based on other scaffolds, such as 5-(2-hydroxyaryl)-7-arylpyrido[2,3-d]pyrimidin-4(1H)- ones and 1-aryl-2-(4-(arylamino)-5H-chromeno[2,3-d]pyrimidin-5-yl)ethanones, as well as 2- substituted 5-cyano-1-(2-hydroxybenzyl)-1H-imidazole-4-carboxamides were also prepared. The mechanism that leads to the formation of 5-imino-N,2-diaryl-5H-chromeno[3,4-c]pyridin-4-amines, 5-(2-hydroxyaryl)-7-arylpyrido[2,3-d]pyrimidin-4(1H)-ones and 2-substituted 5-cyano-1-(2- hydroxybenzyl)-1H-imidazole-4-carboxamides was studied, and for the imidazole derivatives the mechanistic proposal was supported by computational studies. A selection of the prepared compounds was evaluated for their potential as anticancer agents using triple-negative breast cancer cell lines (TNBC), as antituberculosis agents (M.tuberculosis) and as antimalarial agents (Plasmodium falciparum), some of them with promising results. Studies on molecular docking and molecular dynamics were also performed, based on the results obtained for the anticancer activity of chromene[2,3-d]pyrimidin-4(5H)-ones, allowing the development of a preliminary structure-activity relationship (SAR) model for this group of compounds and for this disease.
I would also like to acknowledge the financial support from Programa NORTE 2020 - CCDR-N (ref. NORTE-08-5369-FSE-000033; scholarship UMINHO/BD/42/2016) and thank for the opportunity to develop my work and my PhD.