Made available in DSpace on 2014-06-12T16:24:49Z (GMT). No. of bitstreams: 2 arquivo1084_1.pdf: 1041782 bytes, checksum: 575848b1ff08dd59252a4745034cc38f (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / Euclea natalensis A. DC. pertence a família Ebenaceae e é encontrada na África Austral desde o Quênia e República Democrática do Congo até a África do Sul. Esta planta destaca-se popularmente pela pronunciada ação antibacteriana ao impedir a formação da placa bacteriana e antiinflamatória de seus metabólitos secundários, principalmente as naftoquinonas. Em revisão de literatura, constatou-se a carência de informações detalhadas para a área odontológica. Neste sentido, este trabalho teve como objetivo avaliar a atividade antimicrobiana frente a microrganismos patogênicos para a cavidade oral e determinar a toxicidade aguda do extrato bruto etanólico da raiz de Euclea natalensis. Diante deste relato foi utilizado método de microdiluição em caldo Mueller Hinton para a determinação da concentração inibitória mínima (CIM) frente aos seguintes microrganismos Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Staphylococcus aureus, Porphyromonas gingivalis e algumas cepas de Candida albicans obtidas do Instituto Nacional de Controle de Qualidade em Saúde (INCQS) e Depto. de Antibióticos da UFPE e teste de toxicidade aguda. Na análise microbiológica, os resultados foram satisfatórios comparando com a clorexidina frente aos microrganismos ensaiados. Desta forma, foi observado que o extrato bruto das raízes de Euclea natalensis A.DC. possui atividade antimicrobiana possivelmente correlacionada a presença de naftoquinonas. Para o ensaio de toxicidade aguda por via oral em Ratos Wistar, realizado pelo método de Trevan (1927), foi administrada uma dose de 4mL de 6/6 horas, numa concentração de 5g/Kg, aos animais durante 24 horas e posteriormente foram observados em relação ao comportamento, consumo de água e alimento. Foi observado que os animais não apresentaram sinais clínicos de toxicidade, nem alteração no consumo de água e ração. Dessa forma conclui-se que o extrato bruto de Euclea natalensis A.DC. apresentou atividade antimicrobiana frente aos microorganismos testados e não possui toxicidade aguda

TB is still one of the world's biggest killers. Immunosuppresion induced by AIDS caused a rise in the incidence of TB during the past decade. The search for new drugs to effectively treat TB remains one of the big challenges facing the scientific community. Drugs from plants have been used for centuries to treat various human diseases with varying degrees of success. South Africa with its big resource of plants and ethnobotanical knowledge is an ideal place to screen for anti- TB compounds. The Zulu tribe of South Africa used the root bark of Euclea nata/ensis A.DC. to treat TB related symptoms. Naphthoquinones isolated from E. nata/ensis proved to have good activity against TB. Nine compounds were isolated from the chloroform extract of E. nata/ensis root material. Three of these compounds were newly isolated from this species (mamegakinone, neodiospyrin and 5-hydroxy-4-methoxy-2-napthaldehyde). The structures of the isolated compounds were confirmed using NMR methods and where possible the HPLC and TLC results were compared to authentic standards. Most of the compounds were tested for anti- TB activity with only mamegakinone, lupeol and betulin not showing any activity (5-hydroxy-4-methoxy-2-napthaldehyde still needs to be tested). The activity of the naphthoquinones, especially 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin, show promise that these compounds could develop into an affordable medicine to treat TB. The activity of the crude extract against the resistant DP48 110 1 TB strain showed that there are probably unknown active compounds remaining in the extract. The most active compound, 7 -methyljuglone, was synthesised and an improved synthetic pathway was developed. The synthesis of naphthoquinones remains important in order to produce the compounds on a larger scale. This will make further studies into the mode of action, biosynthesis, bioactivity etc. of these compounds possible. Attempts were made to synthesise diospyrin with 7 -methyljuglone as the starting material. These experiments failed up to now. By altering the reaction parameters such as pH and temperature it should be possible to synthesise diospyrin in future attempts. Neodiospyrin were synthesised from reduced 7 -methyljuglone. This synthesis will yield information on the naphthoquinone chemistry and on how to synthesise diospyrin and isodiospyrin. The enzymatic synthesis of naphthoquinones was also investigated with the use of a cell-free extract. These experiments indicated that it might be possible to enzymatically synthesise diospyrin and the other dimers. / Dissertation (MSc (Plant Physiology))--University of Pretoria, 2005. / Plant Science / unrestricted

Euclea natalensis A.DC. belongs to the Ebenaceae family, and is extensively distributed along the eastern coast of southern Africa. Many Euclea species are widely gathered by indigenous people because of their medicinal properties. Roots of these plant species are frequently used to treat respiratory complications such as chest pains, bronchitis, pleurisy and asthma. Ground root powder is topically applied in cases of leprosy and is used by some ethnic groups to treat toothache and headache. The bioactivity encountered is attributable to naphthoquinones, which are common phenolic compounds in the Ebenaceae family. Naphthoquinones isolated from E. natalensis (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) have exhibited a broad spectrum of antimicrobial activity. The demand for these products will escalate due the amount of plant material required to further research. We need to explore techniques that can maximize their productivity. The present study was conducted on E. natalensis, in an attempt to establish if there exists any correlation between the accumulation of naphthoquinones and stages of seedling growth, seasonal fluctuations and application of fertilizers. A possible correlation between seedling growth stages and the accumulation of naphthoquinones (shinanolone, 7-methyljuglone and diospyrin) was investigated in seeds and seedlings of Euclea natalensis. In this study, the seeds represented the first stage, whereas the second seedling stage was defined as the stage when the radicles were about 6 cm long. The lengths of the seedlings at the third, fourth and fifth seedling stages were 9 cm, 12 cm and 16 cm respectively. Plant materials collected from the five seedling stages were separately extracted using chloroform and the naphthoquinones were then quantified by means of High Performance Liquid Chromatography (HPLC). Mobile phase of MeCN: H2O: AcOH (62.5: 32.5: 5) was used as an eluent in an isocratic mode and at a flow rate of 0.8 ml/min. Standard curves of each of the four compounds were obtained by making a series of dilutions in the concentration range of 22.5 µg/ml to 2.25 µg/ml. Ten microlitres of each dilution was injected three times into the HPLC, and the run time for each injection was 20 minutes. Calibration curves were then generated and used for the quantification of each compound. Shinanolone, which was the only naphthoquinone detectible in seeds, accumulated at variable rates (P<0.01) and no trend could be established between its synthesis and seedling growth. The content of shinanolone ranged from 87.5 mg/kg dry weight (dw) in seeds to a high mean value of 1047 mg/kg (dw) during the fourth seedling stage. A significant correlation (P<0.01) was found between the mean concentrations of 7-methyljuglone and seedling growth. 7-Methyljuglone was quantified at a high mean level of 5003 mg/kg during the third seedling stage and was not detected in the seed samples. A positive correlation (P<0.01) was established between the concentration of diospyrin and seedling stages. Diospyrin was detected at an elevated mean concentration of 6182 mg/kg during the fifth seedling stage, which was higher than the other quantified naphthoquinones. Seasonal variation of naphthoquinones (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) was investigated from eleven plants of E. natalensis subsp. natalensis growing in natural populations, over a period of four seasons. The roots were harvested, dried, extracted and analysed as in the previous study. The mean levels of shinanolone and 7-methyljuglone were found to be uniform in all the seasons and no statistically significant variation could be found between seasonal changes and their mean concentrations. Accumulation of isodiospyrin and neodiospyrin varied significantly with seasonal changes (P<0.05). These two bioactive naphthoquinones were detected only in summer and autumn respectively, and not in winter. A statistically significant variation (P<0.05) was established between the levels of diospyrin and seasonal fluctuations. Diospyrin was detected at a mean concentration of 3190 mg/kg (dw) during spring, which was higher than the other naphthoquinones quantified in all four seasons. The effect of NPK fertilizers on growth performance and accumulation of naphthoquinones (shinanolone, 7-methyljuglone, diospyrin, isodiospyrin and neodiospyrin) in seedlings of E. natalensis grown in shade and under field conditions was investigated. Each group was subdivided into four subgroups, which were then subjected to four respective treatments of water-soluble foliar feed (2:1:2 (44) NPK) at three different concentrations. Treatments tested were as follows: Treatment 1 at 40 g/l, Treatment 2 and Treatment 3 at 20 g/l and 10 g/l respectively. The control group received only supplemental water. The first harvest was conducted after 6 months of application of fertilizers and the second one was done after 12 months of treatment. Roots and shoots were harvested and analysed separately. The naphthoquinones were quantified as previously described. The bioactivity of root extracts from seedlings was tested against Mycobacterium smegmatis and extracts with lower MIC were further tested on M. tuberculosis. Growth parameters differed between the two groups, with seedlings from the shadehouse showing more plant vigour than the field grown plants. No significant interaction could be established between the measured growth factors and treatment. A significant interaction (P<0.001) was found between Treatment 2 and shadehouse seedlings. Treatment 2 enhanced vegetative performance with the mean values of fresh weight of shoots and roots being twice as much as their respective control mean values. A significantly positive correlation was established between the concentration of shinanolone (P<0.01), isodiospyrin (P<0.05) and neodiospyrin (P<0.05) with fertilization from field-grown seedlings. Application of NPK fertilizers significantly (P<0.05) increased the accumulation of neodiospyrin in seedlings subjected to shadehouse conditions. The most potent naphthoquinone, 7-methyljuglone, was found to be abundant in all the extracts and was quantified at a high mean concentration of 10200 mg/kg from shadehouse seedlings. Root extracts of E. natalensis seedlings grown under field conditions were generally more active against the bacterial strain of M. smegmatis as compared to extracts acquired from roots of seedlings maintained under a shadehouse setting. A lowest minimum inhibitory concentration (MIC) of 0.78mg/ml against M. smegmatis was observed from the second harvest of field-cultivated seedlings of the control and Treatment 1 subgroups. The MIC values for shadehouse seedlings ranged from 1.6 to 6.3 mg/ml. Minimum bactericidal concentration (MBC) values from all the extracts tested were relatively higher than their respective MIC’s. Root extracts of E. natalensis were more active against M. tuberculosis and their MIC values were lower than the tested concentrations. Extracts acquired from field-grown seedlings were more active against M. smegmatis with a lowest MIC value of 0.78 mg/ml. Extracts from the control group and Treatment 1, which had less application of fertilizers were more active against strains of M. tuberculosis with MIC value of 10 µg/ml. This shows the selectivity of E. natalensis against the mycobacterial strain of M. tuberculosis. Based on the findings, synthesis and accumulation of naphthoquinones in E. natalensis is highly variable within individuals of the species investigated. Naphthoquinones accumulate in relatively higher amounts in roots of E. natalensis than in the aboveground structures, which validate their harvest by indigenous people. The concentration of shinanolone varied slightly and its production increased with seedling growth. The synthesis of 7-methyljuglone is independent of fertilisation as its accumulation was enhanced in seedlings subjected to control treatment. Neodiospyrin and isodiospyrin were always present in every sample obtained from the seedlings but they were not detectible in every profile of samples from mature plants. Diospyrin is the only naphthoquinone that was detected in every sample analysed and also quantified in high concentrations from mature plants harvested in spring. The study showed that depending on the requirement of a particular naphthoquinone for research, one could target the seasons and seedling stages recommended from this study. This study also showed that field-cultivated seedlings produced more potent naphthoquinones than the ones subjected to controlled environments. / Dissertation (MSc (Plant Science))--University of Pretoria, 2008. / Plant Production and Soil Science / unrestricted