Infectious diseases are a serious concern worldwide especially in immune-compromised patients. Some of these diseases are considered to be contagious and are spread via airborne transmission, while others are not contagious, i.e. non-communicable diseases. The problem is compounded by the emergence of pathogens resistant to currently used antimicrobial drugs. A wide range of microbes including bacteria, fungi, parasites, viruses and protozoans are implicated as causative agents of various diseases. Many patients without ready access to Western medical facilities rely on medicinal plants for the cure of various ailments including infectious diseases. Two plant species used in South African traditional medicine for treating infectious diseases, namely Calodendrum capense Thunb. (Rutaceae) and Lydenburgia cassinoides N. Robson (syn. Catha transvaalensis, Celastraceae) were screened for antimicrobial activities against a range of fungi, bacteria and mycobacteria. The test organisms included Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Mycobacterium smegmatis and M. bovis BCG. The L. cassinoides acetone extract generally had good activity, with MIC values ranging from 0.04 to 0.15 mg/ml while the C. capense extract MIC values ranged from 0.31 to 0.62 mg/ml against the fungi. The hexane extract of L. cassinoides had good activity (MIC = 0.04 mg/ml) against M. smegmatis and the methanol extract had MIC = 0.16 mg/ml against M. bovis BCG. The two plant species had reasonable antibacterial activity against S. aureus and P. aeruginosa, with MIC values ranging from 0.16 to 0.32 mg/ml. Antibacterial activity against Escherichia coli and Enterococcus faecalis resulted in MIC = 0.63 mg/ml for both plants. However in the bioautography assay, the hexane extracts of C. capense and L. cassinoides had good activity against S. aureus, showing active zones of bacterial growth inhibition. The aim of this study was to isolate and characterize the active compounds, with emphasis on antifungal activity, from Calodendrum capense and Lydenburgia cassinoides that may be useful in treating opportunistic infections in immunocompromised patients. Bioassay-guided evaluation of the antimicrobial active components of both hexane fractions using C. neoformans and C. albicans as test organisms led to the isolation and characterization of lupeol from C. capense and ß-amyrin from L. cassinoides. The MIC values of lupeol and ß-amyrin ranged from 1.5 to 6.2 µg/ml against all the tested organisms. Both compounds were also tested against a resistant strain of Candida albicans which resulted in MICs of 3.2 and 6.2 µg/ml respectively. Crude extracts and compounds were also tested for cytotoxicity against human liver (C3A) cells. The crude plant extracts had a low cytotoxicity with average LC50 values of 205.8 ± 8.38 µg/ml for L. cassinoides and 83.07 ± 44.66 µg/ml for C. capense. LC50 values for the isolated compounds were greater than 200 µg/ml, the highest concentration tested. Selectivity index (SI) values were calculated using the formula SI = LC50/MIC. The SI values of the crude extracts of the two plant species ranged between 0.18 and 0.91, showing that these extracts were relatively toxic compared to the antimicrobial activity as the SI values were less than 1. However, the compounds ß-amyrin and lupeol had good activity and low toxicity with SI values greater than 10. In conclusion both plant species showed broad-based antimicrobial activity against the standard ATCC strains of bacterial and laboratory isolates of fungal pathogens. Purified compounds with very good antifungal activity and negligible detectable cytotoxicity, namely lupeol from C. capense and ß-amyrin from L. cassinoides (both pentacyclic triterpenoids) were isolated. This is apparently the first report of these two compounds from these two plant species. In our research group, lupeol has been isolated from various other plant species and it is known to have antimicrobial and anti-inflammatory activities. The second compound, ß-amyrin, reportedly has anti-inflammatory, antitumor, antibacterial, gastroprotective and hepatoprotective effects. The findings from this study show that the two isolated compounds were highly active against fungal and bacterial pathogens, with the lowest MIC value of 0.015 mg/ml. Both compounds showed much better selectivity index values with regard to antifungal activity compared to those of the crude extracts. The compounds also had good activity against the two Mycobacterium strains tested, indicating potential application in antimycobacterial therapy. The results may validate to an extent the use of these two plants as anti-infectious agents in traditional medicine. The compounds have potential for development into therapeutic agents, but various factors will need to be investigated further, including in vivo efficacy and safety, as well as other aspects such as mode of administration. / Dissertation (MSc)--University of Pretoria, 2012. / Paraclinical Sciences / unrestricted

Submitted by Marilene Donadel (marilene.donadel@unioeste.br) on 2018-06-04T23:33:22Z No. of bitstreams: 1 Elissandro_Klein_2016.pdf: 4516260 bytes, checksum: 605fbf2e69fd746b1152b1431bf0dca5 (MD5) / Made available in DSpace on 2018-06-04T23:33:22Z (GMT). No. of bitstreams: 1 Elissandro_Klein_2016.pdf: 4516260 bytes, checksum: 605fbf2e69fd746b1152b1431bf0dca5 (MD5) Previous issue date: 2016-02-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Eugenia pyriformis Cambess. (Uvaia), typical of the Atlantic Forest belongs to the Myrtaceae family, the same family of cherry, jabuticaba and other plants, currently studied due to their antioxidant, antimicrobial and medicinal properties. The interest in bioactive compounds obtained from natural sources has led to an increase in researches aiming to find these compounds in plant extracts. The extractions using conventional methods generally have some drawbacks, and an interesting alternative has been the use of non-conventional extraction technologies, such as the supercritical fluid and the ultrasound-assisted extraction. This work aims to study unconventional methods of obtaining uvaia extracts in order to obtain extracts rich in bioactive compounds. Factors such as the influence of type of extraction (supercritical fluid and ultrasound) and the used conditions on the yield and composition were evaluated. The results were compared to those obtained using the extraction by maceration, a conventional extraction method. After drying and milling the uvaia leaves, the plant material was subjected to extraction with the supercritical CO2 solvent under different conditions of temperature (40, 50 and 60 °C) and pressure (100, 150 and 200 bar), with a fixed flow of 2.0 kg min-1. In the ultrasound-assisted extraction, the effects of temperature (40, 50 and 60 °C), power (150, 250 and 350 W) and ratio between the mass of leaves and the volume of solvent (1:10, 1:15 and 1:20) were evaluated. The extracts obtained were chemically characterized by GC-MS giving β-amyrin (53.72 % for SFE and 71.25 % for UAE) and α-amyrin (24.63 % for SFE and 22.69 % for UAE) as main compounds. In the supercritical extraction, the pressure and the interaction between pressure and temperature showed statistically significant effects on the yield, with the best result being achieved at 200 bar and 60 °C, reaching 1.69%. The data obtained from the supercritical fluid extraction were used to adjust the extraction curves simulated by using two empirical models available in the literature. The tested models adjusted well to the experimental data and the analysis of the estimated parameters allowed to define which model best describes the kinetics of each experimental condition. For the ultrasound-assisted extraction, the best yield result found in the determination of the kinetics was 1.81% in 40 minutes, although in the experimental design a 3 minute time extraction was used due to the better characteristics showed by the extracts at this time. The best result obtained in experimental design with ultrasound was 1.79% provided 30% power, 60 °C and mas/solvent ratio 1:20. Statistical analysis of the experimental design of the ultrasound-assisted extraction indicated significant effects from the variables temperature, mass/solvent ratio and the interaction between power and mass/solvent ratio. The best results for amyrin content by extract weight was found for supercritical extraction, with 97.43%. The ultrasound-assisted extraction showed the best amyrin amount per mass of leaves, 12.13 g of the mixture of isomers per kg of dry leaf. / Eugenia pyriformis Cambess. (Uvaia), típica da Mata Atlântica pertence à família Myrtaceae, mesma família da pitanga, jabuticaba e outras plantas, atualmente estudadas devido às suas propriedades antioxidantes, antimicrobianas e medicinais. O interesse por compostos bioativos obtidos de fontes naturais tem levado ao aumento no número de pesquisas que visam encontrar esses compostos em extratos de plantas. As extrações que utilizam os métodos convencionais geralmente apresentam inconvenientes, e uma alternativa interessante tem sido a utilização de tecnologias não convencionais de extração, como a extração utilizando fluidos supercríticos e a extração assistida por ultrassom. O presente trabalho tem como objetivo estudar métodos não convencionais de obtenção de extratos de uvaia visando obter-se extratos ricos em compostos bioativos. Foram avaliados fatores como influência do tipo de extração (fluido supercrítico e ultrassom) e das condições utilizadas no rendimento e na composição. Os resultados foram comparados com os obtidos utilizando a extração por maceração, um método convencional de extração. Após secagem e moagem das folhas de uvaia, o material vegetal foi submetido à extração com o solvente CO2 supercrítico sob diferentes condições de temperatura (40, 50 e 60 oC) e de pressão (100,150 e 200 bar), com vazão fixa de 2,0x10-3 kg min-1. Na extração assistida por ultrassom, foram avaliados os efeitos da temperatura (40, 50 e 60 oC), da potência (150, 250 e 350 W) e razão entre massa de folhas e volume de solvente (1:10, 1:15 e 1:20). Os extratos obtidos foram caracterizados quimicamente por CG-EM apresentando com principais compostos β-amirina (53,72 % para EFS e 71,25 % para EAU) e α-amirina (24,63 % para EFS e 22,69 % para EAU). Na extração supercrítica, a pressão e a interação entre pressão e temperatura apresentaram efeitos estatisticamente significativos em relação ao rendimento, sendo que o melhor resultado foi encontrado com 200 bar e 60 oC, obtendo-se 1,69 %. Os dados obtidos na extração com fluido supercrítico foram utilizados para ajustar as curvas de extração simuladas utilizando dois modelos empíricos disponíveis na literatura. Os modelos testados ajustaram-se de forma satisfatória aos dados experimentais e a análise dos parâmetros estimados permitiu definir qual dos modelos melhor se ajustou à cinética de cada condição experimental. Para a extração assistida por ultrassom o melhor resultado de rendimento encontrado na determinação da cinética foi 1,81 % com 40 minutos, porém no planejamento experimental foi utilizado um tempo de extração de 3 minutos pois com esse tempo os extratos apresentavam melhores características, sendo portanto o melhor resultado obtido igual a 1,79 % na condição de 30 % de potência, 60 oC e razão massa/solvente de 1:20. A análise estatística do planejamento experimental da extração assistida por ultrassom indicou efeitos significativos das variáveis temperatura, razão massa/solvente e a interação entre potência e razão massa/solvente. Os melhores resultados de teor de amirina por massa de extrato foi encontrada para a extração supercrítica, com 97,43 %. A extração assistida por ultrassom apresentou a melhor quantidade de amirina por massa de folha, 12,13 g da mistura de isômeros por kg de folha seca.