Combretum species are used in many cultures in folk medicine for treatment of microbial infections and several inflammatory conditions (abdominal pains, headache and toothache). There are two possible mechanisms to explain the use of plants extracts to treat microbial infections. A direct effect involves the action of active agents in the extracts on the microorganism tested and the indirect effect involves the stimulation of the host immune system to overcome the effects of microorganisms via the host immune system. Traditional healers use mainly aqueous extracts and in all Combretaceae we have studied, these extracts had hardly any in vitro antibacterial activity. A search of the literature confirmed our observations, aqueous plants extracts of many plant species usually have very little direct in vitro antimicrobial activity. One would expect that aqueous extracts would have higher anti-oxidant activities and may stimulate the immune system of patients thereby combating the infection indirectly. This possibility prompted the investigation of the antioxidant potential of more polar extracts of Combretum species. Methanol extracts of leaves of ten different Combretum species were evaluated for qualitative antioxidant activity by spraying TLC chromatograms of leaf extracts with 2, 2-diphenyl-1-picrylhydrazyl (DPPH). Compounds with anti-oxidant activity bleached the purple colour. Leaf extracts of Combretum apiculatum subspp apiculatumhad the most antioxidant compounds. This species was consequently selected for further examination. Anti-oxidant directed fractionation of the leaf extracts of C. apiculatum led to the isolation of four antioxidant compounds from ethyl acetate and butanol soluble fractions. The structures of the compounds were determined by spectral analyses (1H-NMR, 13C-NMR and MS) and identified as cardamomin (1), pinocembrin (2), quercetrin (3) and kaempferol (4). These compounds occur commonly in plant extracts, but the anti-oxidant activities of all these compounds were not known previously. In a quantitative antioxidant assay using DPPH with L-ascorbic acid as positive control, the more polar fractions (ethyl acetate and butanol) obtained by solvent-solvent fractionation had the highest activity among the extracts with EC50 values of 3.91 ± 0.02 and 2.44 ± 0.02 µg/mL respectively. Of the isolated compounds, quercetrin (3) and kaempferol (4) had strong antioxidant activity with EC50 values of 11.81 ± 85 and 47.36 ± 0.03 µM respectively. Cardamomin (1) and pinocembrin (2) did not have strong activity as these compounds could not scavenge 50% of the DPPH radical at the highest concentration (200 µM) tested. L-ascorbic acid was used standard antioxidant agent (EC50 = 13.37 ± 0.20 µM or 2.35 µg/mL). The antioxidant activity of the isolated compounds supported structure-activity relationships developed by other authors. The cytotoxicity of cardimonin and pinocembrim was evaluated using MTT assay with, berberine as positive control and DMSO as negative control. At higher concentrations than 50 µg/ml of cardimomin or pinocembrin the cells were not viable. Cardimomin was more toxic to the cells (LC50 of 1.97 µg/ml) than pinocembrin (LC50 of 29.47 µg/ml) and even the positive control, berberine (LC50 of 12.35 µg/ml). The presence of these antioxidants could provide a rationale for the ethnomedicinal use of this species for the treatment of inflammatory conditions in traditional medicine. It appears that the antimicrobial activity of aqueous plant extracts may be related to the antioxidant activity leading to a stimulated immune system rather than antimicrobial activity per se Because the crude polar extract had an antioxidant activity half that of L ascorbic acid, the rationale for using these plants by traditional healers becomes clear. The toxicity of cardomomin is a warning that the safety of these extracts should be evaluated in in vivo assays. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2007. / Paraclinical Sciences / unrestricted

Thesis (MSc (Biochemistry))--University of Limpopo (Medunsa Campus), 2010. / Background: Clausena anisata (C. anisata) (Willd) Hook [Family: Rutaceae] is one of the South African indigenous medicinal plant whose blood glucose lowering effect has been demonstrated in animal models of diabetes mellitus. However, the mechanism(s) by which extracts of these plants exert their blood glucose lowering effect have not been investigated Objectives of the study: The objectives of the current study were to investigate in vitro inhibitory effects of different C. anisata leaf extracts on the activities of human urinary α-amylase, Bacillus stearothermophilus α-glucosidase and rat hepatic glucose 6-phosphatase and to characterize the enzyme inhibitory effect of the extracts on these enzymes with respect to the mode of inhibition (type of inhibition) and IC50 values (measure of inhibition potency). Another objective of the study was to confirm the enzyme inhibitory effects of C. anisata in vivo by measuring postprandial glucose levels in alloxan induced diabetic rats after oral administration of sucrose. Results: Aqueous and methanolic extracts of C. anisata leaves inhibited human urinary α-amylase with IC50 values of 1947 ± 50 and 2436 ± 62 µg/ml respectively. Inhibitions of α-amylase by these extracts were however, significantly less than that of the reference drug acarbose (84 ± 11 µg/ml) (P < 0.001). The mode of inhibition of these extracts on human urinary α-amylase appears to be a reversible non-competitive one. Acetone and hexane extracts of C. anisata inhibited Bacillus stearothermophilus α-glucosidase with IC50 values of 1020 ± 32 and 2068 ± 59 µg/ml respectively. Inhibition of Bacillus stearothermophilus α-glucosidase by these two extracts was also significantly less than that produced by acarbose (36 ± 11 µg/ml) (P < 0.001). The mode of inhibition of Bacillus stearothermophilus α-glucosidase by the acetone extract was found to be reversible competitive in this case. Aqueous and methanolic extracts of C. anisata leaves also inhibited hepatic glucose 6-phosphatase with IC50 values of 493.6 ± 12 and 1012 ± 58 µg/ml respectively. In this case the effect of the aqueous extract was significantly less than that of the reference inhibitor of glucose 6-phosphatase, sodium vanadate (1651 ± 46 µg/ml) (P < 0.05). The mode of inhibition of glucose 6-phosphatase by C. anisata aqueous leaf extract was found to be irreversible. Furthermore, oral administration of C. anisata acetone leaf extract to normal and alloxan induced diabetic rats, 30 minutes before sucrose loading failed to prevent the rise in postprandial glucose levels in these animals. Conclusions: Although both C. anisata aqueous and hexane leaf extract inhibited human urinary α-amylase and Bacillus stearothermophilus α-glucosidase in vitro, this does not appear to be the case in vivo. C. anisata aqueous leaf extract appeared to be a more potent inhibitor of rat hepatic glucose 6-phosphatase than the known inhibitor of the catalytic subunit of this multi-component enzyme system.

In preliminary work done in a tree leaf screening project in the Phytomedicine Programme (www.up.ac.za/phyto) Ochna pretoriensis acetone leaf extracts had good antibacterial activity against several important bacterial pathogens. The main aim of this study was to isolate and characterize antibacterial compounds present in the acetone leaf extract of Ochna species growing in South Africa. In a preliminary screening, the minimum inhibitory concentration of acetone leaf extracts of Ochna natalitia, Ochna pretoriensis, Ochna pulchra, Ochna gamostigmata, and Ochna. Serullata, against Staphylococcus aureus, Escherichia coli, Enterococcus faecalisand Pseudomonas aeruginosa were determined by using a serial microplate dilution assay. The number of antibacterial compounds in the extracts was also determined by bioautography against the same bacteria. The MIC values of the five species ranged from 0.039 mg/ml to 1.25 mg/ml. The lowest average MIC values observed were for O. Pretoriensis especially against E. Faecalis and E. Coli. The most sensitive organism to all the plants was E. coli. O. Pretoriensis had the lowest average MIC value and the highest total activity value of 1538 ml/g. Based on bioautography some of the Ochna species had antibacterial compounds with similar Rf values. The thin layer chromatography chemical profiles of the five plant extracts may be useful in the taxonomy of the genus. O. Pretoriensis was chosen for fractionation and isolation of antibacterial compound because it has the lowest average MIC values and highest total activity especially against E. Faecalis and E. coli. The acetone extract of O. Pretoriensis was fractionated into seven fractions (hexane, carbon tetrachloride, chloroform, ethyl acetate, 35% water in methanol, 70% water in methanol and butanol) by solvent-solvent fractionation. Only three of the seven fractions (carbon tetrachloride, chloroform, ethyl acetate fractions) had clearly defined antibacterial spots/lines on bioautograms. The three fractions were further fractionated using column chromatography from which three compounds were successfully isolated. The chemical structures of the isolated compounds were determined using NMR spectroscopy as β-Sitosterol (SS), ochnaflavone (OF) and ochnaflavone 7-O- methyl ether (OFME). Compounds that are related to sitosterol have activity against neurodegenerative disorders as well as estrogenic, analgesic, anti-inflammatory, anthelminthic and antimutagenic activity. OF and OFME are biflavonoids which belong to the group ochnaflavones previously characterized from Ochna obtusata. These compounds have anti-atherosclerotic, anti-inflammatory, and anti-tumor activity. They also inhibit lymphocytes proliferation, archidonic acid release and phospholipase activity. Moreover, OFME was reported to inhibit HIV-1 activity as well as HIV-1 reverse transcriptase activity. The antibacterial activity, and potential cytotoxic, genotoxic and antigenotoxic effects of the isolated compounds were determined. The MIC values ranged from 31.3 to 250 μg/ml. SS was more active against P. Aeruginosa with an MIC of 62.5 μg/ml, OF against P. Aeruginosa and E. Faecalis with MICs of 0.03 mg/ml and OFME against P. Aeruginosa with an MIC of 31.3 μg/ml. The isolated compounds were much less active than the positive control gentamycin. The compounds had low cytotoxic activity, with LC50 values of 193.8 μg/ml for β-Sitosterol, 125.9 μg/ml for OF and 125.9 μg/ml for OFME against Vero cells. The therapeutic indexes of the crude extract and the isolated compounds varied between 0.77 and 3.27, which is an indication of non-specific antibacterial activity i.e. general toxicity, thus the crude plant extract and compounds isolated from O. Pretoriensis can only be recommended for external applications. e.g. topical treatments. None of the compounds tested had potential genotoxic and/or antigenotoxic effects. The number of revertants in the mutagenicity experiments was less than twice the number of revertants in the negative control. The percentage inhibition of 4NQO in the antimutagenicity experiments were less than 45%. The results obtained in this case may be principally associated with the general toxicity of the test samples to the bacteria used in this study. Comparison of the total activity of the crude extract and the fractions gave a clear indication of synergic interaction of compounds in the crude extract to successfully inhibit the growth of the test pathogens. Approximately 76% of activity was lost in the 34% of dry mass lost during fractionation. Twelve percent of activity was present in the chloroform fraction and 6% in the carbon tetrachloride fraction. Despite the evidence for synergistic activity, the crude extract was also relatively toxic to the Vero cells with a therapeutic index of 0.8. As far as could be established, the antibacterial activity of members of the Ochna genus and the cytotoxicity of ochnaflavones were determined for the first time in the current study. The two most active antibacterial compounds (ochnaflavone and ochnaflavone 7-O- methyl ether) are being reported from this species for the first time. The relative safety of the crude extract and the compounds isolated from this plant was relatively low. Preparations of O. Pretoriensis may be safe in a topical application but internal use cannot be recommended for treating antibacterial infections before animal toxicity studies have been carried out. Caution is also required in using the isolated compounds or crude extracts for other applications. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Paraclinical Sciences / unrestricted

Medicinal plants have played an important role in drug discovery, with many pharmaceutical products originating from plants. Isolation and characterization of antibacterial compounds is still relevant today because of continuing development of resistance of bacteria to antibiotics. The aim of the study was to evaluate the antibacterial activity of leaf extracts of nine tree species (Acalypha sonderiana, Androstachys johnsonii, Dracaena mannii, Drypetes natalensis, Funtumia africana, Necepsia casteneifolia, Oncinotus tenuiloba, Turraea floribunda, and Xylia torreana) selected from the Phytomedicine Programme Database based on good antimicrobial activities. The next step was to select the most active plant species and to isolate and characterize the antibacterial compounds. A serial microplate dilution method was used to determine the minimal inhibitory concentration and bioautography was used to determine the number of antibacterial compounds in the extract and their Rf values. Four nosocomial infection pathogens (Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus) were used as test organisms. Extracts of all the plant species were active with average MIC values ranging from 0.13 to 2.0 mg/ml against the four bacteria. MIC values as low as 0.08 mg/ml was obtained with F. africana and O. tenuiloba extracts against S. aureus. In bioautography seven of the nine leaf extracts had activity with clear zones of inhibition on bioautograms against the red background. F. africana was active against all four bacteria while O. tenuiloba had selective activity against P. aeruginosa with clear bands on the bioautogram. F. africana was chosen for further investigation because (a) it had good antibacterial activity against the four tested bacteria with MIC value as low as 0.08 mg/ml, (b) there were several active compounds against all the tested bacteria based on bioautography, (c) it is common in nature, and (d) as far as our literature survey could ascertain there was no published information on the antimicrobial activity of this plant species. The bulk powdered leaves of F. africana were extracted with acetone. The acetone extract was fractionated into five fractions (hexane, chloroform, butanol, H2O and 30% H2O in methanol) using solvent-solvent fractionation, to group the phytochemicals based on their polarity. Hexane and chloroform fractions were the most active with MIC values as low as 0.02 mg/ml for the chloroform fraction. One of the traditional uses of F. africana is to treat burns. As a result, the crude extract and its five fractions were also tested for anti-inflammatory activity using both the COX-1 and COX-2 assays. The crude extract and the hexane and chloroform fraction had moderate activity against both cyclooxygenase 1 and 2. The chloroform fraction was more active than the crude extract (59.7 ± 1.4%)with an inhibition of 68.2 ± 6.6%. Because there was no activity in the aqueous extracts and traditional healers usually use water as extractant, the pain relief experiences traditionally must be due to another anti-inflammatory mechanism. One antibacterial compound was isolated from the hexane fraction using column chromatography with silica gel as the stationary phase and a hexane ethyl acetate gradient as the mobile phase from low to high polarity. The isolated compound was identified as methyl ursolate using nuclear magnetic resonance (NMR) and mass spectrometry. Methyl ursolate has been isolated from a number of plant species. However, this is the first report on the isolation from Funtumia genus and the first report of its antimicrobial activity. Previous phytochemical investigation from the stem bark of F. africana led to the isolation of steroidal alkaloids of the conanine group. Methyl ursolate had a low activity with MIC values of >250 μg/ml against the four tested bacteria, but had better activity against five fungal (Candida albicans, Cryptococcus neomeforms, Fusarium oxysporum, Penicillium janthinellium, and Rhizoctonia solani) species with an MIC value of 63 μg/ml against F. oxysporum. The chloroform fraction had excellent activity with an MIC of 20 μg/ml and may be developed to become a useful complex drug. The more than one hundred fold lower activity of the isolated methyl ursolate compared to the activity of the chloroform fraction from which it was isolated, provides strong evidence of synergism. This may be good model system for studying synergism in antimicrobial preparations. / Dissertation (MSc)--University of Pretoria, 2010. / Paraclinical Sciences / unrestricted

Made available in DSpace on 2016-06-02T19:30:13Z (GMT). No. of bitstreams: 1 6774.pdf: 4696015 bytes, checksum: 084c02944eff3cb2f25b1db2ce63280b (MD5) Previous issue date: 2015-03-31 / Financiadora de Estudos e Projetos / Plants species from Brazilian savanna often have defense mechanisms in response to the low nutrient content of the soil, the light intensity, the competition for resources and drought, pronounced by deep water deficit, to which they are subjected. This area is a potential source of bioactive compounds that still little explored and potential phytochemical studies of these species are important to this preservation. Biological invasions are one of the main factors threatening the Brazilian savanna biodiversity. Many plants are initially introduced as ornamental species and subsequently become invasive. Invasive plants are generally more competitive than the other species and tolerate unfavorable conditions. This advantage can be explained according to the "Novel weapons hypothesis". It proposes that invasive plants have secondary metabolites that act as allelochemicals and present new challenges to native species. Thus, secondary metabolites of invasive species in the Brazilian savanna are also an important area of phytochemistry being investigated. Therefore, we hypothesized that native species from Brazilian savanna, Myrsine umbellata and Myrsine coriacea, and the alien specie, Tithonia diversifolia, produces secondary metabolites that have different biological activities, including insecticidal, antifungal and phytotoxic acitivities, favoring their establishment in this environment and have economic importance for agriculture and pharmaceutic industry. In the Chapter 1, the biological activity of leaf extracts of M. umbellata and M. coriacea, was evaluated. The in vitro antifungal effect of leaf extracts of these two species was evaluated against Pythium aphanidermatum, Fusarium oxysporum and Colletotrichum acutatum, three important crop pathogens. We also evaluated the insecticide potential of these extracts against Spodoptera frugiperda, known as fall armyworn, an important pest that causes damages to many crops. Finally, the total polyphenols and antioxidant activity of the leaf extracts of these two species was quantified by the DPPH method. In the Charpter 2 we evaluated the phytotoxic potential of leaf, stem and root extracts from T. diversifolia. Bioassays of wheat elongation coleoptile seed germination and seedling growth are performed. The Ethyl Acetate leaf extract was the most active extract and was selected to continue the isolation studies and the identification of compounds. The chromatographic separation of this extract allowed us the isolation of 12 sesquiterpene lactones (STSs), 1 secosesquiterpeno, and 1 diterpene. Two STSs are reported for the first time in this study, 8&#946;-O-(2-methylbutyroyl) and tirotundin 8&#946;-O-(isovaleroyl)tirotundin. The high phytotoxic activity of the extracts and compounds, especially the majority, 1&#946;-methoxydiversifolin, tagitinin A and tagitinin C, indicates that allelopathy may be responsible for their establishment in this habitat. In the Chapter 3, the T. diversifolia Ethyl Acetate fractions were tested for insecticidal activity against S. frugiperda and it was observed a significant reduction in the pupal weight of the caterpillar. This may be an indicative of the presence of toxic substances to this insect in these fractions, however, this reduction in the pupal weight is not associated with the majority compounds tested. Thus, we conclude that the studied plant species have secondary metabolites with promising biological activities and economic importance for agriculture and pharmaceutical industry. / Espécies vegetais provenientes do Cerrado costumam apresentar mecanismos de defesa em resposta ao baixo teor de nutrientes do solo à que estão submetidas, a intensa luminosidade, a maior competição por recursos e ao longo período de estiagem, marcada por profundo déficit hídrico. Este domínio é uma fonte potencial de compostos bioativos ainda pouco explorada e estudos do potencial fitoquímico destas espécies são importantes para a preservação da sua biodiversidade. Entre os principais fatores que ameaçam a biodiversidade do Cerrado, está a invasão biológica. As plantas invasoras são geralmente mais competitivas que as demais espécies e suportam condições adversas. Essa vantagem pode ser explicada segundo a hipótese das novas armas , que afirma que as plantas invasoras possuem metabólitos secundários que agem como aleloquímicos e apresentam novos desafios para as espécies nativas. Dessa forma, metabólitos secundários de espécies invasoras do Cerrado também são uma importante área da fitoquímica a ser investigada. Sendo assim, foi proposta a hipótese de que espécies nativas, Myrsine umbellata e Myrsine coriacea e uma invasora do cerrado, Tithonia diversifolia, produzem metabólitos secundários em resposta ao estresse à que estão submetidas e que estes apresentam diferentes funções biológicas, incluindo, atividade antifúngica, antioxidante, inseticida e fitotóxica, que favoreçam seu estabelecimento nesse ambiente e apresentam importância econômica para uso na agricultura e na indústria farmacêutica. Para isso, no primeiro capítulo, foi avaliada a atividade biológica de extratos foliares de M. umbellata e M. coriacea. Foi avaliado o efeito antifúngico in vitro de extratos foliares destas espécies sobre Pythium aphanidermatum, Fusarium oxysporum e Colletotrichum acutatum, importantes patógenos de culturas agrícolas. Também foi avaliado o potencial inseticida destes extratos sobre Spodoptera frugiperda, conhecida como lagarta-do-cartucho do milho, importante praga causadora de danos a esta cultura. E por último, foi determinado a concentração de fenóis totais e atividade antioxidante dos extratos foliares destas duas espécies pelo método de redução do radical DPPH (2,2-difenil-1-picrilidrazil). No segundo capítulo foi avaliado o potencial fitotóxico de extratos de folha, caule e raiz de T. diversifolia. Foram realizados bioensaios com coleóptilos de trigo, germinação de sementes e crescimento de plântulas. O extrato foliar preparado com Acetato de Etila foi selecionado para continuar os estudos de isolamento e identificação de compostos por apresentar melhor atividade. A separação cromatográfica deste extrato nos permitiu isolar 12 lactonas sesquiterpênicas (STS), 1 secosesquiterpeno, e um diterpeno. Duas STSs estão sendo relatadas pela primeira vez neste trabalho, 8&#946;-O-(2-metilbutiroil)tirotundina e 8&#946;-O- (isovaleroil)tirotundina. A alta atividade fitotóxica desta espécie, bem como dos compostos avaliados e, principalmente, os compostos majoritários 1&#946;-metoxidiversifolina, tagitinina A e tagitinina C, indica que a alelopatia pode ser uma das formas de seu estabelecimento nesse ambiente. No capítulo 3 foram testados a atividade inseticida de frações do extrato Acetato de Etila de T. diversifolia e seus compostos majoritários contra S. frugiperda e foi observado uma redução significativa no peso pupal da lagarta. A redução da massa pupal pode ser o indicativo da presença de substâncias tóxicas ao inseto nessas frações, no entanto, essa redução não está associada à presença dos compostos majoritários testados. Dessa forma, concluímos que as espécies vegetais estudadas, possuem metabólitos secundários com atividades biológicas promissoras e importância econômica para serem utilizados na agricultura e na indústria farmacêutica.