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Abietane Cationic Amphiphiles (ACA) - loaded polymeric microspheres to tackle resistant bacteria
| Resumo: | This dissertation focused on the development of novel abietane cationic amphiphiles (ACAs) by chemical modification of dehydroabietic acid (DHA), leading to cationic amide derivatives by condensation of the DHA carboxyl function with the amine group of biogenic polyamines (spermine) and basic proteinogenic amino acids (arginine). After ACAs several synthesis procedures were performed, and subsequently TLC and NMR analyses, it was concluded that no molecular form was identified in the form of an amide, but rather the intermediary product and products of degradation. The presented dissertation performed a screening of antimicrobial activities of the compounds DHA and ACA hemi-synthesis intermediary (ACA-Int) against a collection of Gram-positive, Gram-negative bacteria and yeasts; toxicological studies to assess if the compounds had antimicrobial and not citotoxic properties; and microencapsulation and stability studies to evaluate the chemical behavior and stability of the tested compounds. The antimicrobial activity screening of DHA and ACA-Int showed that both compounds had inhibited the planktonic and biofilm growth of a collection of Gram-positive and Gram-negative bacteria and a yeast. In addition, the MIC, MIC with organic interfering substances , MBC, MBIC, MBIC with organic interfering substances and time-kill kinetic study against standard and clinical isolates of sensitive and resistant bacterial strains was determined for DHA and ACA-Int. In MICs studies DHA demonstrated to have higher efficacy than the ACA-Int in Gram-positive bacteria S. epidermidis ATCC 12228 and M. smegmatis ATCC 607 both with the lowest MIC value (7.81 μg/mL); and in Gram-negative bacteria, all clinical isolates of K. pneumoniae and clinical isolate strain E. coli 3023 with a MIC value of 125.00 μg/mL. The studies of MIC with organic interfering substances have showed that organic challenges have influence in MIC values thus increasing them. Regarding to MBC values, the results showed low MBC/MIC ratios within each strain, which is typical for bactericidal agents. In biofilm formation assay, most of the tested microorganisms were biofilm producer except for clinical isolate Ent. Cloacae 1264. Concerning the biofilm inhibition properties of DHA and ACA-Int, where the lowest MBICs value registered of DHA was achieved against Gram-positive S aureus ATCC 43866 (0.49 μg/mL; BI = 75.13±8.82%) which is a strong biofilm producer. In Gram-negative bacteria, the lowest MBIC was showed against clinical isolates K. pneumoniae 701 (0.98 μg/mL; BI = 92.75±5.69%) and K. pneumoniae 703 (0.98 μg/mL; BI = 94.13±2.37%). DHA and ACA-Int were also tested against C. albicans ATCC 10231 and they achieved MBIC values of 62.50 μg/mL; BI = 81.83±7.05% and 125.00 μg/mL; BI = 57.85±24.64%, respectively. With MBIC organic interfering substances, in Gram-positive bacteria only HBS has more influence in MBIC and BI, yet in Gram-negative bacteria all challenges had influence in MBIC and BI values. Time-kill kinetics profiles of DHA against the test organisms (S. aureus ATCC 25923) at concentrations of 31.25, 62.50 and 93.75 μg/mL showed that this compound has bacteriostatic properties. Nonetheless, as a future perspective regarding antimicrobial assays, it would be important to better understand the mechanisms of action of these diterpenes. Regarding to the microencapsulation of the compounds, it was achieved a EE% of 99.49±0.05%, and the stability studies demonstrated that DHA remains stable within alginate beads (PP% = 60.00±0.05%; PE% = 88.12±0.05%). In the Artemia salina toxicological study, both DHA and ACA-Int demonstrate not to have toxicity, but then again in SRB assay they demonstrate to have IC50 values of 19.69±7.40 μg/mL and 23.67±3.75 μg/mL, which demonstrates the need for further investigation in this area using other methodologies as well. According to the references of this dissertation, this is the first report on these type of hemi-synthesis biological activities with these compounds, with preliminary scientific validations upon their known ethnopharmacological uses. Perspective is to improve the studies of DHA and its derivatives, to scientifically validate their uses, understand their safety, and unravel new bioactive compounds with therapeutic potential and specific targets. |
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| Autores principais: | Neto, Íris Raquel Branco |
| Assunto: | Abietanos Catiónicos Anfifílicos Ácido desidroabiético Resistência antimicrobiana Microencapsulação Alginato Teses de mestrado - 2017 |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | This dissertation focused on the development of novel abietane cationic amphiphiles (ACAs) by chemical modification of dehydroabietic acid (DHA), leading to cationic amide derivatives by condensation of the DHA carboxyl function with the amine group of biogenic polyamines (spermine) and basic proteinogenic amino acids (arginine). After ACAs several synthesis procedures were performed, and subsequently TLC and NMR analyses, it was concluded that no molecular form was identified in the form of an amide, but rather the intermediary product and products of degradation. The presented dissertation performed a screening of antimicrobial activities of the compounds DHA and ACA hemi-synthesis intermediary (ACA-Int) against a collection of Gram-positive, Gram-negative bacteria and yeasts; toxicological studies to assess if the compounds had antimicrobial and not citotoxic properties; and microencapsulation and stability studies to evaluate the chemical behavior and stability of the tested compounds. The antimicrobial activity screening of DHA and ACA-Int showed that both compounds had inhibited the planktonic and biofilm growth of a collection of Gram-positive and Gram-negative bacteria and a yeast. In addition, the MIC, MIC with organic interfering substances , MBC, MBIC, MBIC with organic interfering substances and time-kill kinetic study against standard and clinical isolates of sensitive and resistant bacterial strains was determined for DHA and ACA-Int. In MICs studies DHA demonstrated to have higher efficacy than the ACA-Int in Gram-positive bacteria S. epidermidis ATCC 12228 and M. smegmatis ATCC 607 both with the lowest MIC value (7.81 μg/mL); and in Gram-negative bacteria, all clinical isolates of K. pneumoniae and clinical isolate strain E. coli 3023 with a MIC value of 125.00 μg/mL. The studies of MIC with organic interfering substances have showed that organic challenges have influence in MIC values thus increasing them. Regarding to MBC values, the results showed low MBC/MIC ratios within each strain, which is typical for bactericidal agents. In biofilm formation assay, most of the tested microorganisms were biofilm producer except for clinical isolate Ent. Cloacae 1264. Concerning the biofilm inhibition properties of DHA and ACA-Int, where the lowest MBICs value registered of DHA was achieved against Gram-positive S aureus ATCC 43866 (0.49 μg/mL; BI = 75.13±8.82%) which is a strong biofilm producer. In Gram-negative bacteria, the lowest MBIC was showed against clinical isolates K. pneumoniae 701 (0.98 μg/mL; BI = 92.75±5.69%) and K. pneumoniae 703 (0.98 μg/mL; BI = 94.13±2.37%). DHA and ACA-Int were also tested against C. albicans ATCC 10231 and they achieved MBIC values of 62.50 μg/mL; BI = 81.83±7.05% and 125.00 μg/mL; BI = 57.85±24.64%, respectively. With MBIC organic interfering substances, in Gram-positive bacteria only HBS has more influence in MBIC and BI, yet in Gram-negative bacteria all challenges had influence in MBIC and BI values. Time-kill kinetics profiles of DHA against the test organisms (S. aureus ATCC 25923) at concentrations of 31.25, 62.50 and 93.75 μg/mL showed that this compound has bacteriostatic properties. Nonetheless, as a future perspective regarding antimicrobial assays, it would be important to better understand the mechanisms of action of these diterpenes. Regarding to the microencapsulation of the compounds, it was achieved a EE% of 99.49±0.05%, and the stability studies demonstrated that DHA remains stable within alginate beads (PP% = 60.00±0.05%; PE% = 88.12±0.05%). In the Artemia salina toxicological study, both DHA and ACA-Int demonstrate not to have toxicity, but then again in SRB assay they demonstrate to have IC50 values of 19.69±7.40 μg/mL and 23.67±3.75 μg/mL, which demonstrates the need for further investigation in this area using other methodologies as well. According to the references of this dissertation, this is the first report on these type of hemi-synthesis biological activities with these compounds, with preliminary scientific validations upon their known ethnopharmacological uses. Perspective is to improve the studies of DHA and its derivatives, to scientifically validate their uses, understand their safety, and unravel new bioactive compounds with therapeutic potential and specific targets. |
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