Dissertations / Theses on the topic 'Medicinal plants (In vitro study)'

The search for cheap, non toxic and readily available antidiabetic drugs has been a challenge for researchers and the pharmaceutical industries. Diabetes mellitus is a metabolic disease characterized by defects in the synthesis of insulin and/or insensitivity to the action of insulin at the target cells. The disease has been on the increase mostly in developing countries where large proportions of the population have little access to good medical care due to either accessibility or non availability of synthetic drugs. This has led to the use of medicinal plants to treat diabetes because it is safe, cheap and with few side effects. There is little scientific evidence on the dosages, active compounds, mechanisms of action and toxicity of these traditionally used plants. Two of the most frequently used plants; Albuca setosa and Albuca bracteata were investigated in this study. The qualitative analysis of different extractions of these plants revealed the presence of phenolics, alkaloids, tannins and saponins. The antioxidant properties of aqueous, acetone and methanollic extracts of Albuca setosa and Albuca bracteata were investigated using models such as Diphenyl-1-Picrylhydrazyl (DPPH), 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), Ferric ion reducing antioxidant potential (FRAP), Nitric Oxide and Hydrogen Peroxide (H2O2). Both plants revealed inhibitions against DPPH in a concentration - dependent manner with Albuca setosa (0.330 mg/ml) showing higher activity than Albuca bracteata (0.647 mg/ml) determined from the IC50. The aqueous extract of Albuca setosa showed a higher inhibition against DPPH radical compared to the Albuca bracteata aqueous extract at all concentrations investigated. The isolated saponins from Albuca bracteata had a higher DPPH scavenging activity than the crude methanolic extract of the plant in a concentration - dependent manner but are significantly different from each other at 0.4, 0.6 and 1.0 mg/ml only. The IC50 of the saponins was also observed to be higher than the crude extracts and standards.The Albuca setosa aqueous extract showed a higher percentage inhibition of ABTS radicals than Albuca bracteata at all the concentrations investigated. Overall, the Albuca setosa aqueous extract (0.0809 mg/ml) showed maximum activity against ABTS radicals. The iron reducing power was significantly higher (P < 0.05) in the methanolic extract of both plants compared to the aqueous counterpart. Overall, the Albuca bracteata aqueous extract (0.344 mg/ml) showed maximum activity as indicated by the IC50. The aqueous extracts of both plants also revealed percentage inhibitions in a concentration - dependent manner against NO2. The aqueous extract of Albuca bracteata bulb was more active against nitric oxide and hydrogen peroxide inhibition. In this study, the cytotoxicity of the extracts was evaluated at a high dose of 100 μg/ml on Chang liver cells and determined using MTT, crystal violet, glucose consumption, lactate production and lactate dehydrogenase release and FRAP. The aqueous extracts of both Albuca setosa and Albuca bracteata were non-toxic on Chang liver cells at the concentrations investigated. The MTT revealed that the aqueous extract of Albuca setosa bulb had the optimum cell viability of 108.09 percent while the acetonic extract of Albuca bracteata showed the least cell viability (37.72 percent) compared with the control. The crystal violet test also revealed the acetone extract of Albuca bracteata to have the least percentage of cell viability at 31.47 percent, while the aqueous extract of Albuca setosa showed the maximum cell viability at 112.5 percent. The aqueous extracts of both plants showed higher percentage cell density on the second day of incubation from the proliferation assay. All the tested samples were observed to consume more glucose than the blank except for the methanollic and acetone extracts of Albuca bracteata bulb. The aqueous and methanolic extracts of Albuca setosa bulbs produced the highest lactate with 120.2 μg/ml and 113.7 μg/ml respectively. The acetone extracts of both Albuca setosa and Albuca bracteata revealed toxicity with a higher lactate dehydrogenase release compared to the control.

As ervas e especiarias são fontes de antioxidantes. Essa capacidade antioxidante está relacionada aos compostos fenólicos que apresentam papel importante nos processos de inibição da peroxidação e podem atuar sobre o estresse oxidativo, relacionado com diversas patologias, inclusive o diabetes. O alecrim é bastante apreciado por seu aroma e sabor, tendo como constituintes os seguintes compostos fenólicos: ácido carnósico, carnosol, ácido rosmarínico, ácido caféico e éster do ácido hidroxicinâmico. Objetivou-se avaliar a capacidade antioxidante in vitro de extratos e frações de ácidos fenólicos obtidos das folhas de alecrim e seu efeito sobre ratos diabéticos induzidos por estreptozotocina. Constatou-se que tanto os extratos como as frações apresentaram altos teores de compostos fenólicos totais e expressiva atividade antioxidante in vitro nos três métodos utilizados: β-caroteno/ácido linoléico, varredura do radical DPPH e ORAC. No ensaio in vivo, foi utilizado modelo de diabetes tipo 1, apresentando as características principais: poliúria, polifagia, polidipsia e perda de peso. Observaram-se alterações na atividade das enzimas antioxidantes, nos parâmetros bioquímicos do sangue e aumento significativo (p<0,05) da glicemia, no percentual de hemoglobina glicosilada (Hb-G), nos níveis de triglicérides, de colesterol total, de creatinina, de AL T e de AST. O extrato aquoso de alecrim administrado por 30 dias na concentração de 50 mg/Kg aumentou a atividade das enzimas CAT e GPx, no fígado, e da SOD no cérebro de ratos diabéticos, diminuindo também o percentual de Hb-G. A mesma dose, quando administrada por 60 dias, reduziu o percentual de Hb-G, nos lipídios circulantes, na creatinina, na atividade das enzimas SOD e GPx dos tecidos avaliados e manteve os valores normais das enzimas de função hepática AL T e AST. Não foi observado efeito dose resposta nos parâmetros analisados, sugerindo que a maior dose (100mg/Kg) apresentou níveis de toxicidade que devem ser melhor caracterizados. Portanto, o extrato aquoso de alecrim apresentou capacidade antioxidante in vitro significativa e quando administrado na concentração de 50 mg/Kg pode ter papel importante sobre o estresse oxidativo presente no diabetes experimental.
Herbs and spices are potential sources of antioxidants. This antioxidant capacity is related to the presence of phenolic compounds that play an important role in the peroxidation inhibition processes, that can act on the oxidative stress, which is related to various diseases, including the diabetes. Rosemary is much appreciated due to its aroma and flavor properties, and has the following constituents as phenolic compounds: carnosic acid, carnosol, rosmannlc acid, cafeic acid, and hydroxycinnamic ester. This study aimed to evaluate the in vitro antioxidant capacity of the extracts and fractions of phenolic acids obtained from the leaves of rosemary and its effect on diabetic rats induced by streptozotocin. The results showed that both extract and fractions contains high leveis of total phenolic compounds, and a significant in vitro antioxidative activity evaluated by three methods: β-carotene / Linoleic acid system, DPPH radical scavenging and ORAC. For in vivo tests, it was used a type 1 diabetes model, which presented the main clinicai features: polyuria, polyfagia, polydipsia, and weight loss. It was also observed the following changes in biochemical markers: an altered antioxidant enzymatic activity; a significant increase (p<0.05) of blood glucose, percentage of glycosylated hemoglobin (Hb-G), triglycerides, total cholesterol, creatinine, and enzymes ALT and AST. The aqueous extract of rosemary administered daily for 30 days at dose of 50 mg/kg increased the activity of enzymes CAT and GPx in the liver and SOD in the brain of diabetic rats, also decreased the percentage of Hb-G. The same dose, when administered for 60 days, led to a reduction of percentage of Hb-G, circulating lipids, and creatinine, also reducing the activity of enzymes SOD and GPx in the analyzed tissues, and maintaining the normal function of liver enzymes AL T and AST. There was no dose¬response effect on the studied parameters. Some toxic effect was observed when higher doses were used (100 mg/kg) but this effect must be better characterized. Therefore, aqueous extracts of rosemary at dose of 50 mg/kg presented a significant in vitro antioxidant capacity that can be an important role on the oxidative stress in experimental diabetes.

Magister Artium (Development Studies) - MA(DVS)
Medicinal plants remain a very important natural resource used as traditional medicines for health needs in many developing countries. In the current deepening economic and political crises in Zimbabwe a significant number of the population has inevitably relied more on natural resources which has led to receding population and scarcity of many medicinal plant species in their natural habitat. It is against this background that this research, using Rural Eastern Zimbabwe that this study explored the extent to which use values of medicinal plants increased since the Zimbabwean crises and the different use values of these species among men and women. The rational choice theory, use value approach and concept of utility constituted a theoretical grounding of the research process. The study essentially used qualitative research methods with some quantitative data. A mix of interviews and focus group discussions were employed for this study. Interviews were conducted with community leaders, traditional healers, NGOs in the similar field and Government stakeholders eliciting their views on use values of medicinal plants and sustainable interventions that can be enacted in conserving these species. The findings of the study were shown through using tables, charts and the quantitative data was presented using STATA. The calculated total usevalues of the 11 medicinal plants showed that Kirkia ancuminata Oliv, Dicoma anomala Sond, Syzgium guineense DC, Zingiber offinale, Acacia Karoo Hayne were found to have “high total use-values” and Lannea edulis Engl, Aloe, Lippia javanica Spreng, Virtex payos merril, parinari curatelli and Coleochloa setiflora have “low total use-values”

Includes bibliographical references.
Tuberculosis is the leading cause of death due to a single organism; with a mortality of more than 3million people each year, worldwide. The emergence of multi-drug resistance and HIV/AIDS are the major causes of this problem. New therapeutic agents with a different mode of action, and thereby of resistance to Mycobacterium tuberculosis, the causative agent, are needed urgently. Amongst the methods used, the area of ethnopharmacology is explored in this study. Visits were performed to collect the plants used by traditional healers in 7 provinces of South Africa for the treatment of tuberculosis.

In South Africa part of the cultural and religious beliefs of the African people is the use of traditional remedies to treat diseases. These remedies are obtained from medicinal plants (Steenkamp, 2003). One of the most frequently traded plants in the Eastern Cape is Hypoxis, commonly known as Afrika patat, or African potato. South African traditional healers instruct patients to brew the fresh Hypoxis corm as a tea and then ingest it (Steenkamp, 2006a). This prompted an investigation into the digestive stability of a traditionally prepared Hypoxis extract. The H. sobolifera extracts were digested using a simulated gastric/small intestinal digestion and their biological activity determined. The hot water H. sobolifera extract before digestion only showed cytotoxic activity against cancer cell lines at very high concentrations which are not likely to be achieved under normal ingestion circumstances. In Chang liver cells on the other hand, chronic exposure to the hot water H. sobolifera extract increased glucose uptake in amounts similar to that of metformin. On the negative side, the glucose utilization stimulation was lost due to the simulated digestion process. The significant inhibition of AGEs by hot water H. sobolifera extract (IC50 of 6.3 Ig/ml) is a very encouraging result as treatment in the management of diabetes. This activity was only slightly reduced by the in vitro digestion process. Also observed was enzyme inhibition activity by traditionally prepared H. sobolifera, with ∝-amylase being inhibited (IC50 of approximately 250 Ig/ml) and therefore preventing or limiting starch breakdown. From the DPPH results it was clear that H. sobolifera, even when digested, is a potent anti-oxidant (IC50 of 134.4 Ig/ml when undigested compared to 162.9 when digested with β-glucosidase added to stomach digestive step). HPLC and TLC experiments revealed that rooperol which has previously been thought to be the compound responsible for the anti-oxidant activity in Hypoxis extracts, was absent from the traditional extract of H. sobolifera and therefore cannot be the sole compound exhibiting anti-oxidant activity; other compounds such as phenolics may be contributing. The phenolic and flavonoid content results revealed very highconcentrations of these compounds in the traditionally prepared H. sobolifera extract. These compounds may therefore play major roles in all of the biological activities observed from treatment with Hypoxis spp. The ROS results yielded interesting and promising results. Using standard or traditionally prepared H. sobolifera extracts, activation of differentiated U937 cells with PMA was greatly enhanced by cotreatment with the extracts, while extracts on their own did not cause significant activation. Future studies should investigate this property of the extracts as a promising immune boosterThe HPLC results showed that hypoxoside was undetectable in the hot water traditional extract and the TLC anti-oxidant experiment proved that rooperol is not present in the hot water traditional extract after treatment with β-glucosidase. This indicates that neither one of the Hypoxis compounds previously believed to be responsible for the biological activities observed are present in the extract when prepared the traditional way. Therefore, the biological activities observed in this study can be attributed to other phytochemical compounds.

Farmers generally collect fresh plant materials from the wild for ethnoveterinary uses. They are encouraged to harvest with caution and dry or cultivate important materials in order to protect the biodiversity. These recommendations are not validated scientifically. The microplate method for minimum inhibitory concentration (MIC) determination was used to compare wild with cultivated, and fresh with dry plant materials. The MIC values obtained ranged from 1.25 to 0.01 mg/ml. MIC values ≤0.3 mg/ml were considered as cut off point between effective and none effective inhibition. The multilevel linear models (hierarchical linear models), both unadjusted and adjusted models were employed. The plant (name) was considered as level-2 or higher level, while the actual observation was level-1 or lower level. The crude estimates of the odds ratio indicated that wild is significantly 0.57 times less likely than garden to yield MIC values of more than 0.3 mg/ml (p-value = 0.005). Also, fresh are about 4.195 times more likely than dry to yield MIC scores of more than 0.3 mg/ml (p-value < 0.001). Adjusting for conditions “dry and fresh”, microbe and solvent; wild is significantly 0.52 times less likely than garden to yield MIC values of more than 0.3 mg/ml (p-value = 0.003). On the other hand, when adjusting for “wild or garden”, type of solvents and type of microbes; fresh is significantly 4.202 times more likely than dry to yield MIC values of more than 0.3 mg/ml (p-value < 0.001). These results partially support farmers claiming that wild plant materials are more potent than the grown ones. On the contrary, the results are in favour of drying plant materials.

In many developing countries, farmers and pastoralists still rely on their indigenous knowledge, practices and locally available plants to control nematode parasitic infections, both in livestock and humans. The overall aim of my thesis was to undertake bioassay-guided phyto-chemical study of extracts and their constituents from Ethiopian anti-parasitic plants used by healers to control gastrointestinal nematode parasites in livestock to validate their ethno-medicinal use and to characterise and identify their active ingredients. As a first experiment (Chapter Three), four types of crude extracts (water, 70% methyl-alcohol, absolute methanol and acetone) of four indigenous Ethiopian medicinal plants (Adenia species, Cissus ruspolii, Ipomoea eriocarpa and Euphorbia thymifolia) were screened against Teladorsagia circumcincta egg hatching in vitro, not only as a first step to validate the traditional healers claim but also to choose the most promising plant extract(s) for further phyto-chemical studies. The egg hatching inhibition (EHI) test results revealed that the anti-parasitic properties of these plants depended on plant species, dose, and solvent polarity. The water extracts of both C. ruspolii and Adenia sp. exhibited largest, up to 100% EHI but also larval migration inhibition activities, and were selected for further studies. The second experiment (Chapter Four) assessed the nature of active constituents in these extracts by physico-chemical methods. It was observed that the major constituents of both plant extracts responsible for the EHI activities are likely highly polar, water-soluble, small and moderately heat-labile molecules. The third and fourth experiments (Chapters Five and Six) consisted of separating Cissus ruspolii and Adenia sp. water extracts into discrete fractions by gel-permeation chromatography, EHI tests of Bio-Gel P-2 fractions followed by thin layer chromatography (TLC) profiling of these fractions to detect separated spots (in day light, under UV-light or after staining with various staining reagents) and also to see how elution patterns of separated spots affected by column parameters. The EHI tests on the fractions obtained revealed that the active constituents of C. ruspolii and Adenia sp. water crude extracts were eluted into few fractions based on their molecular sizes. The TLC profilings of these fractions identified spot patterns of active and inactive fractions, which allowed pooling of active constituents based on their EHI and TLC profiling into three pools for each plant. The fifth experiment (Chapter Seven) was to isolate and purify compounds from these pools using various preparative planar and column chromatographic methods. Sequential applications of column chromatography followed by preparative thin layer chromatography isolated and purified five active compounds from C. ruspolii and two active compounds from Adenia sp. The sixth experiment (Chapter Eight) was to characterize and propose/elucidate structures of compounds from the active fractions using chromatographic, analytical and spectroscopic methods. In this regard, the structures of two oleanane type triterpenoid saponins isolated from one of active fractions of Adenia sp. were proposed based on their mass spectrometry (MS) and nuclear magnetic resonance (NMR) data with support of compounds property, TLC and literature. Similar outcomes for C. ruspolii were not achieved due to lack of sufficient sample to run 13C-nuclear magnetic resonance spectroscopy and distortionless enhancement by polarization transfer (DEPT), contamination of some purified compounds with ill-characterised substance from the preparative TLC matrix and in some cases mass spectrometry (MS) and nuclear magnetic resonance (NMR) data did not support each other. The last experiment (Chapter Nine) was to assess anthelmintic efficacy and safety of C. ruspolii and Adenia sp. crude water extracts in Heligmosomoides bakeri infected mice. This in vivo test revealed that both plant extracts exhibited significant reduction in worm burdens and worm egg excretion, with moderate effects on haematology and organ weights at tolerated dosages. In conclusion, both in vitro and in vivo data revealed that Adenia sp. and C. ruspolii have anthelmintic properties, thus validating traditional healer claims and supporting ethno-medicinal use. The bioassay-guided phytochemical study resulted in the isolation of a number of active compounds from these plants, for some of which a structure has been proposed.

The African potato is used as an African traditional medicine for its nutritional and medicinal properties. Most research has been carried out on H. hemerocallidea, with very little or nothing on other Hypoxis spp. The main aim of this project was to provide scientific data on the anticancer, anti-inflammatory and antioxidant properties of H. hemerocallidea, H. stellipilis and H. sobolifera chloroform extracts and their active compounds. The hypoxoside and phytosterol contents of the three Hypoxis spp. were determined using TLC, HPLC and GC. H. hemerocallidea and H. sobolifera chloroform extracts contained the highest amounts of hypoxoside and β-sitosterol, respectively. For the anticancer properties, cytotoxicity of the Hypoxis extracts and its purified compounds were determined against the HeLa, HT-29 and MCF-7 cancer cell lines (using MTT), and PBMCs (using CellTiter-Blue®). H. sobolifera had the best cytotoxicity against the three cancer cell lines, whereas H. stellipilis stimulated HeLa and HT-29 cancer cell growth. IC50 values of hypoxoside and rooperol were determined. DNA cell cycle arrest (using PI staining) occurred in the late G1/early S (confirmed by increased p21Waf1/Cip1 expression) and G2/M phases after 15 and 48 hrs, respectively, when treated with Hypoxis extracts and rooperol. H. sobolifera and rooperol activated caspase-3 and -7 (using fluorescently labelled antibodies) in HeLa and HT-29 cancer cells, and caspase-7 in MCF-7 cancer cells after 48 hrs. Annexin V binding to phosphatidylserines in rooperol treated U937 cells confirmed early apoptosis after 15 hrs. The TUNEL assay showed DNA fragmentation in the three cancer cell lines when treated with H. sobolifera and rooperol for 48 hrs. A shift pass the G2/M phase has led to the investigation of endoreduplication, which was confirmed by cell/nucleus size, and anti-apoptotic proteins (Akt, phospho-Akt, phospho-Bcl-2 and p21Waf1/Cip1). U937 cell differentiation to monocyte-macrophages was optimized using PMA and 1,25(OH)2D3, which was confirmed by morphological and biochemical changes. For the anti-inflammatory properties, Hypoxis extracts and rooperol significantly increased NO production in monocyte-macrophages (pre-loaded with DAF-2 DA) and phagocytosis of pHrodoTM E. coli BioParticles®. The treatments had no effect on COX-2 expression in monocyte-macrophages. The phytosterols significantly increased IL-1β and IL-6 secretion xv (using the FlowCytomix Multiplex human Th1/Th2 10plex Kit I) in the PBMCs of one donor. For the antioxidant properties, Hypoxis extracts and rooperol significantly increased ROS production in undifferentiated and differentiated U937 cells, which were pre-loaded with DCFH-DA. Hypoxis extracts and purified compounds had ferric reducing activities, but only rooperol had ferric reducing activities significantly greater than ascorbic acid. β-sitosterol, campesterol and cholesterol significantly increased SOD activity in Chang liver cells, while H. stellipilis, H. sobolifera and rooperol decreased SOD activity. Anticancer, anti-inflammatory and antioxidant properties of the Hypoxis extracts may be attributed to the β-sitosterol content, because Hypoxis chloroform extracts contained very little or no hypoxoside. Unidentified compounds, and synergistic and additive effects of the compounds may have contributed to the biological effects. This study confirms previous reports that rooperol is the active compound. Results provide scientific data on the medicinal properties of one of the most frequently used medicinal plants in South Africa.

Artemisia afra is one of the oldest, most well known and widely used traditional medicinal plants in South Africa. It is used to treat many different medical conditions, particularly respiratory and inflammatory ailments. There is no reported evidence of its use for the treatment of cancer but due to its reported cytotoxicity, an investigation of the mode of cell death induced by an ethanol A. afra extract using two cancer cell lines was done. IC50 values of 18.21 and 31.88 μg/mL of ethanol extracts were determined against U937 and HeLa cancer cells, respectively. An IC50 value of the aqueous extract was greater than 250 μg/mL. The ethanol extract was not cytotoxic against confluent control cell lines, Chang Liver and Vero cells. The effect of the cytotoxic ethanol A. afra extract on U937 and HeLa cells and their progression through the cell cycle, apoptosis and mitochondrial membrane potential was investigated. After 12 hours of treatment with A. afra a delay in G2/M phase of the cell cycle was evident. Apoptosis was confirmed using the TUNEL assay for DNA fragmentation, as well as fluorescent staining with annexin V-FITC. Apoptosis was evident with the positive control and A. afra treatment at 24 and 48 hours. JC-1 staining showed a decrease in mitochondrial membrane potential at 24 hours. It was deduced that A. afra ethanol extract induces caspase-dependent apoptosis in a mitochondrial dependent manner. Plants harbour many compounds that are not only useful to the plants but also to mankind. Many metabolites have been isolated from A. afra and their biological activity characterised. Due to observed apoptosis induction, isolation of cytotoxic compounds was done and a new sesquiterpene lactone from A. afra was isolated. Structural elucidation of the compound was done by IR, 1D and 2D NMR, CD and mass spectrometry and it was identified as isoalantolactone. HeLa cancer cells were treated with isoalantolactone and cytotoxicity was exhibited in a dose-dependent manner. A low IC50 value of 8.15 ± 1.16 μM was achieved. This study showed that isoalantolactone is partly responsible for the observed A. afra cytotoxicity. Due to the evidence of G2/M arrest, the anti-mitotic potential and the possible onset of mitotic catastrophe by A. afra and isoalantolactone was investigated. It was evident from various flow cytometric analysis of cyclin B1 and phospho-H3 and confocal microscopy that A. afra does possess anti-mitotic activity by causing hyperpolymerisation of tubulin and cells progress into the mitotic phase where M arrest is experienced. The anti-inflammatory activity of sesquiterpene lactones is well documented; however, the anti-inflammatory activity of A. afra is not. Here, it is reported that the production of NO and COX-2 protein levels in RAW 264.7 cells decrease in the presence of A. afra and isoalantolactone after stimulation with LPS. The activated NF-κB subunit, p65 was also investigated. The results suggest that A. afra and isoalantolactone inhibit p65 activation as a decrease in the activated subunit was evident. Thus, the results indicate that exposure to A. afra and isoalantolactone induces an anti-inflammatory response. In conclusion, this study shows, for the first time, the mechanism of induced apoptosis, the anti-mitotic and anti-inflammatory activity of A. afra and its isolated compound, isoalantolactone. It also proves that although extensive research may have been done on a particular plant, as with A. afra, more can be discovered leading to the identification of new compounds and integration of signalling pathways that can be exploited for the treatment of various diseases and ailments.

Orientador: Eliana Aparecida Varanda
Banca: Francisca da Luz Dias
Banca: Denise Crispin Tavares
Banca: Iracilda Zeppone Carlos
Banca: Daisy Maria Fávero Salvador
Resumo: No presente trabalho, foram avaliadas dez espécies vegetais, nativas do cerrado brasileiro e utilizadas popularmente no tratamento de úlceras gástricas, quanto ao seu potencial mutagênico. Foram empregados os testes de Ames (in vitro) e do micronúcleo em células do sangue periférico de camundongos (in vivo). De cada espécie vegetal foram avaliados dois tipos de extratos brutos: um polar e um apolar. Nos ensaios in vitro foi verificada mutagenicidade especialmente para os extratos polares (metanólicos) das seguintes espécies vegetais: A. castaneifolia, A. glandulosa, A. triplinervia, M. pusa, Q. grandiflora, Q. multiflora e S. pseudoquina. Nesses mesmos ensaios, os únicos extratos apolares que apresentaram mutagenicidade foram os obtidos de Q. grandiflora e Q. multiflora. Foram avaliados in vivo apenas os extratos polares, sendo que apresentaram mutagenicidade os extratos metanólicos de A. castaneifolia, A. glandulosa, A. triplinervia, Q. multiflora e S. pseudoquina. De acordo com as caracterizações químicas realizadas com as espécies vegetais estudadas, ficou evidente a participação bastante efetiva dos compostos fenólicos (flavonóides e taninos) na mutagenicidade observada. Também foi possível verificar o papel que possíveis interações entre os diferentes compostos químicos presentes nos extratos podem ter em suas atividades biológicas. Tendo em vista as informações obtidas, ficou clara a necessidade que os estudos biológicos e fitoquímicos apresentam para se promover uma maior compreensão dos riscos que podem estar associados aos tratamentos medicinais baseados em plantas.
Abstract: In this work we evaluate the mutagenic potential of ten vegetable species native of the Brazilian savannah and used popularly in the treatment of gastric ulcers. The Ames test (in vitro) and the Micronucleus test (in vivo) were employed. We evaluate a polar extract and a non-polar extract to each vegetal species. The in vitro assays show the mutagenicity of the methanol extracts (polar) obtained from A. castaneifolia, A. glandulosa, A. triplinervia, M. pusa, Q. grandiflora, Q. multiflora e S. pseudoquina. The non-polar extracts from Q.grandiflora e Q. multiflora were mutagenic too. In vivo, only the polar extracts were studied and the methanol extracts from A. castaneifolia, A. glandulosa, A. triplinervia, Q. multiflora e S. pseudoquina were mutagenic. The phytochemical studies with the evaluated plants permit to infer the participation of the flavonoids and tannins in the mutagenic activities observed in vitro and in vivo. We also verified in this biological activity possible interactions between the different substances present in the raw extracts. In this way, we could to observe the importance of phytochemical and biological studies to promote a better comprehension of the risks that can be associated to the medicinal treatments based in the plants use.
Doutor

Cancer is a disease that imposes a heavy burden on public health and poses a challenge to science. The World Health Organization estimates that 80 percent of people in developing countries of the world rely on traditional medicine for their primary health needs, and about 85 percent of traditional medicine involves the use of plant extracts. This is particularly true in Africa where a large percentage of the population depends upon medicinal plants for health care. Therefore, detailed screening and evaluation of bioactive substances for chemotherapeutic purposes of African plants are urgently warranted. Furthermore, this will serve to validate the efficacy and safety of African traditional medicine. The current study investigated the in vitro cytotoxic effects of 17 ethanolic extracts of the following 16 plants used in traditional anticancer medicine in Nigeria: Sapium ellipticum leaves, Sapium ellipticum stembark, Combretum paniculatum, Celosia trigyna, Pupalia lappacea, Justica extensa, Hedranthera barteri leaves, Alternanthera sessilis, Ethulia conyzoides leaves, Lannea nigritana stembark, Combretum zenkeri root, Combretum molle leaves, Adenanthera parvoniana, Lannea acida, Cyathula achyranthoides, Drymaria cordata, Cyathula prostrata, against HeLa cancer cells. Five of the most promising extracts (Sapium ellipticum leaves, Combretum paniculatum, Celosia trigyna, Drymaria cordata, Cyathula prostrata) were selected for further screening against HT29 and MCF-7 cancer cells. Of the five, the first two were investigated further based on their activities in the screening phase. The S. ellipticum leaf extract yielded IC50 values of 88.60 ± 0.03 and 93.03 ± 0.03 μg/ml against HeLa and MCF-7, respectively. The toxicity was also evaluated on normal cells and an IC50 of 77.66 μg/ml was obtained for peripheral blood mononuclear cells (PBMCs). The IC50 values for proliferating and confluent Chang liver cells were both >125 μg/ml. These results suggest that the extract may be selective for specific cell types. Bio-assay guided fractionation of the S. ellipticum ethanolic extract yielded two active fractions; chloroform and ethyl acetate. Two compounds isolated from the chloroform extract were screened against the three cancer cell lines and found to be inactive. Three compounds were isolated from the ethyl acetate fraction and revealed IC50 values < 62.5 and < 31 μg/ml against MCF-7. Unfortunately these two compounds soon lost activity before any further work could be done on them and work was continued with the crude extract.

The fungus Botrytis cinerea is an opportunistic pathogen on a wide variety of crops, causing disease known as grey mould through infections via wounds or dead plant parts. Synthetic fungicides for controlling this disease are fast becoming ineffective due to the development of resistance. This, coupled with consumers world wide becomng increasingly conscious of potential environment and health problems associated with the build up of toxic chemicals, (particularly in food products), have resulted in pressure to reduce the use of chemical pesticide volumes as well as its residues. An emerging alternative to random synthesis is the study and exploitation of naturally occurring products with fungicidal properties. There have been reports on the uses of synthetic fungicides for the control of plant pathogenic fungi. When utilized in two-way mixtures, such fungicides may maintain or enhance the level of control of a pathogen at reduced rates for both components utilized in combinations, or alone at normal rates. For this study it was hypothesize that the addition of plant extracts may enhance the antifungal efficacy of the synthetic strobilurin fungicide, kresoxim-methyl against Botrytis cinerea.

Athrixia phylicoides (bush tea) is one of many plants from the Asteraceae family used as a traditional herbal medicine. With very few cultivated plants, natural growing plants currently serve as the main resource for plant material. The plant is not yet commercialised and its medicinal value is known and used only by a few people. With the long term aim at commercial scale propagation, this study consists of three parts. Firstly we developed a protocol for in vitro propagation of A. phylicoides. Secondly, the ultrastructure and morphology of leaves were studied microscopically and thirdly, comparisons were made between in vitro and ex vitro grown plants. Nodal segments of greenhouse plants were used to establish cultures. Better growth and less wilting was recorded on explants surface sterilised with NaOCl compared to Ca(OCl)2 after establishment. The addition of growth regulators IBA (indole-3-butyric acid) and BAP (6-benzylaminopurine) to the culture medium did not seem to affect the growth response of explants during the multiplication phase. Hyperhydricity was a problem throughout our trials. The development of hyperhydricity symptoms seems to be related to seasonal changes in the stock plant material used to initiate cultures, rather than the composition of growth medium or growth room temperatures. The occurrences of hyperhydricity symptoms were inconsistent and unpredictable. A rooting medium with added BAP and decreased sucrose levels resulted in a higher rooting percentage compared to the control medium, free from BAP and with a higher sucrose concentration, which yielded no rooting. However, in another experiment, in vitro rooting occurred spontaneously after subdividing and transfer of microshoots to fresh control medium. The addition of GA3 to the establishment medium (but not to the subsequently used multiplication and rooting media) yielded a slightly higher percentage of rooting. However, cultures initially established on GA3 medium yielded fewer roots per explant and roots were shorter than those of explants established on hormone free medium. The medicinal properties of plants are often linked to the production of essential oils. We hypothesised that the medicinal value of A. phylicoides can be linked to the production of the aromatic essential oils released by leaves. A microscopic study of leaves provided some preliminary insight of the mechanisms involved in the production of medicinally active products. Electron- and light microscopic examination of leaves were used to identify and study structures that are apparently involved in the production and secretion of essential oils. Two types of trichomes were identifyed – nonglandular and glandular trichomes. These glandular trichomes are multicellular with a subcuticular storage space and are present only on the adaxial surface of leaves. In the case of medicinal plants, it is essential that the medicinal properties of the plant are not altered by the method of propagation. This was our motivation for comparing the morphology and ultrastructure of leaves of plants that were grown in their natural environment to that of plants grown in vitro. Leaf surfaces of in vitro grown plants were smaller and the number of glandular trichomes per surface area was less on in vitro grown plants. There were no noticeable changes in the morphology of glandular trichomes.
Dissertation (MSc(Agric))--University of Pretoria, 2009.
Plant Production and Soil Science
unrestricted

This thesis presents the results of an ethnopharmacological survey conducted with Bedouin women in Egypt (South Eastern Desert, Siwa Oasis and North Sinai) to document their knowledge of using medicinal plants to treat women's health problems, such as dysmenorrhoea, perinatal problems, womb cleansing and urinary tract infections. Data collected revealed that the Bedouin women commonly use more than 45 different plant species. Four plant species (Haloxylon salicornicum, Achillea fragrantissima, Mentha longifolia and Acacia nilotica) were chosen for phytochemical and pharmacological investigations in this project.

Orientadores: João Ernesto de Carvalho, Mary Ann Foglio
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O câncer é um conjunto de mais de 100 doenças diferentes que afligem a população mundial e caracteriza-se pela perda de função celular e ausência de diferenciação, além da proliferação descontrolada. Somente no Brasil, estima-se que foram registrados 489.270 novos casos de câncer em 2010. O Brasil é o país com a maior diversidade genética vegetal do mundo, representando uma das mais ricas fontes de materiais farmacologicamente ativos. Os extratos de plantas medicinais e seus compostos derivados são conhecidos por serem efetivos agentes contra vários tipos de cânceres. Estima-se que 60% das drogas antitumorais sejam de origem vegetal. A partir da coleta aleatória realizada no projeto BIOPROSPECTA-FAPESP (04/15410-0), foram selecionadas 10 espécies que apresentaram efeitos antiproliferativos. Dessas espécies, 5 foram novamente coletadas, sendo elas: Miconia latecrenata (DC.)Naudin, Mollinedia schottiana (Spreng.)Perkins, Boehmeria caudata Sw., Boehmeria cylindrica Sw. e Acnistus arborescens. Foram preparados extratos diclorometânicos (EBD) e etanólicos (EBE) das folhas destas espécies os quais foram avaliados quanto à atividade antiproliferativa in vitro, em linhagens de células tumorais humanas. Todos os extratos apresentaram pelo menos efeito citostático no teste in vitro à exceção daqueles obtidos de Mollinedia schottiana. A partir desses resultados e da disponibilidade de material vegetal, foram selecionados para dar continuidade ao estudo Boehmeria caudata e a B. cylindrica. Assim, por existir dados referentes a alcalóides com efeitos citostáticos descritos na literatura, realizou-se extração ácido-base dos dois espécimes, onde se isolou alcalóides de polaridades diferentes. Os alcalóides isolados, e suas frações neutras, das duas plantas, EBAEN e EBCA, para Boehmeria cylindrica e FALCBC e FAENBC para Boehmeria caudata, foram encaminhados a testes in vitro, onde apenas os alcalóides da espécie B. caudata, retornou resultados positivos. O EBD de Boehmeria cylindrica e os extratos EBE e EBD de Boehmeria caudata, juntamente com a fração FALCBC foram submetidos a testes in vivo, utilizando o modelo do Tumor Sólido de Ehrlich. O EBD de Boehmeria cylindrica e de Boehmeria caudata apresentaram resultados significativos ( p<0,05) diminuindo a progressão tumoral.Os EBDs de B. cylindrica e de B. caudata apresentaram redução significativa (p<0,05) na progressão tumoral. Por outro lado, o EBE de B. caudata e a fração FALCBC não apresentaram atividade no teste in vivo, sugerindo que em função da maior polaridade dos compostos presentes nessas amostras, essas substâncias que apresentaram atividade antiproliferativa in vitro foram metabolizadas e excretadas antes de atingirem seus sítios de ação no modelo in vivo. Concluindo, as espécies estudadas apresentaram significativa atividade antitumoral, corroborando com os relatos existentes de estudos de atividade antitumoral para outras espécies do gênero Boehmeria, e indicando o potencial do gênero para o desenvolvimento de uma droga anticâncer
Abstract: Cancer is a group of 100 different diseases that afflict the world's population and is characterized by loss of cell function, due to absence of differentiation, in addition to uncontrolled proliferation. In Brazil alone, an estimated 489,270 new cancer cases were reported in 2010. Brazil is the country with the greatest plant genetic diversity of the world, representing one of the richest sources of pharmacologically active materials. Extracts of medicinal plants and their derived compounds are known to be effective agents against several types of cancers. It is estimated that 60% of anticancer drugs are of plant origin. Based on the random collection performed in the project BIOprospecTA-FAPESP (04/15410-0), 10 species that had significant anti-proliferative effects, were selected. From these, five were collected again, as follows: Miconia latecrenata (DC.)Naudin, Mollinedia schottiana (Spreng.)Perkins, Boehmeria caudata Sw., Boehmeria cylindrica Sw. and Acnistus arborescens. These species were submitted for extraction process, resulting in dichloromethane and ethanolic extracts, subsequently which was evaluated with in vitro tests of human tumor cell lines. All of them showed cytocidal or cytostatic effects with in vitro assays, except for Mollinedia schottiana that produced no positive results. The available specimens with positive results were selected to continue the study; they are Boehmeria caudata and Boehmeria cylindrica. Thus, since data of alkaloids with cytostatic effects have been described in the literature, an acid-base extraction was made of the two specimens, where alkaloids of different polarities were isolated. Isolated alkaloids, and the respective neutral fractions of the two plants, EBAEN and EBCA for Boehmeria cylindrica, FALCBC and FAENBC for Boehmeria caudata, were used in in vitro assays, where only the alkaloids from B. caudata, gave positive results. The DBE of Boehmeria cylindrica and the extracts, EBE and DBE of Boehmeria caudata, along with the FALCBC fraction were tested in vivo using the solid Ehrlich's tumor model. The DBE of Boehmeria cylindrica and of Boehmeria caudata showed significant results (p <0.05), decreasing tumor progression. The brute ethanol extract of Boehmeria caudata, along with its fraction, FALCBC, showed no anti-proliferative activity. The genus Boehmeria was subject to several studies for different species, indicating a potential of the genus for the discovery of a new anticancer drug. The species studied showed significant antitumor activity. The negative results of the alkaloidal fraction and the EBE of Boehmeria caudata in in vivo tests, do not invalidate its potential because these samples were possibly metabolized and excreted before reaching their action sites, probably due to the polarity of these substances
Mestrado
Biologia Celular
Mestre em Biologia Celular e Estrutural

Philosophiae Doctor - PhD
Over the years, microorganisms have become resistant to commonly used antimicrobial agents leading to multidrug resistance. This is believed to occur even with new classes of therapeutic agents thus creating a challenge on the global healthcare system. The study of medicinal plants allows for their possible use as alternative therapeutic agents. Galenia africana (G. africana) is a South African medicinal plant with numerous health benefits. The purpose of this study was to evaluate the potential antimicrobial, antifungal and anticancer properties of the ethanolic extract of G. africana. Prior to evaluating these properties, in vitro and in vivo acute toxicity studies were conducted to assess the toxicity profile of G. africana. The toxicity profile of the G. africana extract was evaluated using acute toxicity studies conducted in animal and reconstituted human epidermis skin models. The results of the acute oral and dermal toxicity studies revealed that the median lethal dosage (LD50) for G. africana extract in Sprague-Dawley rats was considered to exceed 2000 mg/kg. In the dermal sensitization study, the stimulation index (SI) values for the mice treated with the G. africana extract at concentrations of 25% (50 mg/ml), 50% (100 mg/ml), and 100% (200 mg/ml), when compared to the control group, were 1.3, 0.9 and 1.3, respectively which did not result in an SI value of ? 3 in any group. Hence, it did not elicit a hypersensitivity response. In the irritation test, the G. africana (concentrate) and G. africana (in-use dilution) extracts were non-irritant on the reconstituted human epidermis.

The use of medicinal plants in the treatment of infectious diseases is an acceptable and popular phenomenon in South Africa and worldwide. The potential of extracts from these plants as antimicrobial agents necessitates their scientific evaluation. Therefore, this study evaluated the antimicrobial activity of Carpobrotus edulis; Cotyledon orbiculata; Datura stramonium; Dodonaea angustifolia; and Zanthoxylum capense against Porphyromonas gingivalis; Tannerella forsythensis and Actinobacillus actinomycetemcomitan. Given that most currently used drugs are cytotoxic, the possible cytotoxic effect of these medicinal plants on human periodontal ligaments fibroblasts and human gingival fibroblasts was also determined. The modified broth micro dilution method incorporating resazurin as an indicator of cell growth in 96-well microtitre plates was used to determine the antibacterial activity of the test plants extracts. The extracts showed some significant antibacterialactivity against Porphyromonas gingivalis, Tannerella forsythensis and Actinobacillus actinomycetemcomitans. The activity varied with respect to individual test bacteria. Their minimum inhibitory concentration (MIC) values ranged from 10 to 0.01mg.mr-l. All bacteria tested were inhibited by the highest concentration of the selected plant extracts ( 1 Omg.ml-1). The MTT [3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] method was used to determine the cytotoxic effect of test extracts. All extracts tested with the exception of Carpobrotus edulis, inhibited the growth of both human periodontal ligament fibroblasts and human gingival fibroblasts at the tested dilutions, with the cytotoxicity levels being directly related to the concentration of the extracts. The extract of Carpobrotus edulis, inhibited the tested cells at 10-1 for human periodontal ligaments fibroblasts and ≥ 2: 10-2 for human gingival fibroblasts. All other tested concentrations of Carpobrotus edulis extracts enhanced the growth of both human periodontal ligaments and human gingival fibroblasts. The study provided a scientific evidence of the important role that medicinal plants play as antibacterial agents in the treatment of oral infections.
Dissertation (MSc)--University of Pretoria, 2009.
Community Dentistry
unrestricted

Bibliography: leaves 78-80.
Using the ethno-medicinal data approach, nine South African plants used traditionally in the treatment of malaria were collected and evaluated for cytotoxic and lymphocyte-proliferating effects. These included Acokanthera oppositofolia, Zanthoxylum capense, Glycyrrhiza glabra, Harpephyllum caffrum, Lippia javanica, Pentanisia prunneloides, Psidium guajava, Typha capensis and Cannabis sativa. The cytotoxic effect of the aqueous, methanol and dichloromethane extracts of these plants was evaluated in vitro in Rat-1 fibroblasts there was no observable difference in cytotoxic activity between cold-and hot-water extracts of all the plants investigated. Cell proliferation greater than 80% was observed for aqueous extracts of A. oppositofolia, H. caffrum, L. javanica, P. prunneloides, P. guajava and T. capensis which would suggest are considered safe for consumption. However, high cytotoxic effect was exhibited by dichloromethane extracts of Z. capense, L. javanica, C. sativa and P. guajava at 100 μg/ml. These findings suggest that dichloromethane extracted compounds that are not normally extractable by traditional methods.

The stem bark of Peltophorum africanum and Bridelia micrantha are used in South Africa traditional medicine for treatment of intestinal parasites, relieve problems and human immunodeficiency virus/ acquired immune deficiency syndrome (HIV/AIDS). The growing problem of antibiotic resistance by Helicobacter pylori the major etiological agent in gastritis, gastric cancer, peptic and gastric ulcer demands the search for novel compounds from plant based sources. This study was aimed to determine the antimicrobial activity of five solvent (ethylacetate, acetone, ethanol, methanol and water) extracts of the stem bark of P. africanum and B. micrantha on clinical strains of H. pylori in a bid to identify potential sources of cheap starting materials for the synthesis of new drugs. H. pylori strains were isolated from patients presenting with gastric related morbidities at the Livingstone Hospital, Port Elizabeth for endoscopy and confirmed following standard microbiology procedures. The plant extracts including clarithromycin were tested against 31 clinical strains of H. pylori by the agar well diffusion method. The most potent extract was evaluated by the microdilution method to determine the Minimum Inhibitory Concentration (MIC50&90), followed by the rate of kill. Preliminary phytochemical analysis was carried out. The one way ANOVA test was used to statistically analyse the results. All the extracts demonstrated anti-H. pylori activity with zone diameters of inhibition that ranged from 0 to 23 mm for the extracts and 0 to 35 mm for clarithromycin. Marked susceptibility (100%) was recorded for the ethyl acetate extract of P. africanum (P. afr. EA) and the acetone extract of B. micrantha (B. mic. A), which were statistically significant (P < 0.05) compared to all other extracts and clarithromycin. For B. micrantha ethyl acetate extract, 93.5 percent susceptibility was observed while for the control iv antibiotic, clarithromycin it was 58.1 percent. The MIC50 ranged from 0.0048 to 0.313 mg/mL for P. afr. EA, and from 0.0048 to 0.156 mg/mL for B. mic. EA; MIC90 ranged from 0.156 mg/mL to 0.625 mg/mL and 0.0048 to 2.5 mg/mL for P. afr. EA and B. mic. EA respectively. There was a significant statistical difference observed in potency of both P. afr. EA and B. mic. A compared to the two antibiotics (P < 0.05). One hundred percent killing by P. afr EA was observed at 0.05 mg/mL (½ x MIC) and 0.2 mg/mL (2 x MIC) in 66 h for strain PE466C and PE252C respectively. For B. mic. EA, 100 percent killing effect of both strains (PE430C and PE369C) was observed at 0.1 mg/mL (2 x MIC) in 66 h. Qualitative phytochemical analysis confirmed the presence of alkaloids, flavonoids, steroids, tannins and saponins in the ethyl acetate extracts of both plants, which could be a potential template of lead molecule for the design of new anti- Helicobacter pylori therapies.

It is estimated by the World Health Organization (WHO) that by 2040 neurodegenerative disorders will collectively surpass cancer as the primary cause of death in industrialised countries (WHO,2006). Natural flora represents one of the most important therapeutic sources in modern drug discovery, however only a limited number of plant species have been screened for their neuroprotective value. The neuroprotective potential of eleven Algerian and two South African medicinal plant extracts were assessed in this study, aiming to identify promising candidates for future research. Neurodegenerative disorders such as Parkinson’s disease are characterised by distinct biochemical features, including protein misfolding/-aggregation, excessive oxidative stress and apoptotic cell death. As such, medicinal plant extracts were screened for biological properties directly relevant to neurodegeneration. The capacity to induce autophagy was also investigated as mounting evidence suggests that activation of this pathway may reduce abnormal protein aggregation and promote neuronal survival.

The n-Hexane, dichloromethane, methanol and aqueous crude extracts of Garcinia kola (Heckel) seeds were screened for their anti-Vibrio activities against 50 Vibrio bacteria isolated from wastewater final effluents. The 50 isolates consisted of different Vibrio species namely V. fluvialis (14), V. vulnificus (12), V. parahaemolyticus (12), V. metschnikovii (3) and 9 others unidentified to the specie level. The n-Hexane, dichloromethane and methanol extracts had activities against 16 (32 percent) of the Vibrio isolates, while the aqueous extracts had activities against 12 (24 percent) all at a screening concentration of 10 mg/ml. The minimum inhibitory concentrations (MICs) were 0.313-0.625 mg/ml, 0.313-0.625 mg/ml, 0.313-2.5 mg/ml and 10 mg/ml for n-Hexane, dichloromethane, methanol and aqueous extracts respectively. Rate of kill studies were carried out against three different Vibrio species namely V. vulnificus (AL042), V. parahaemolyticus (AL049) and V. fluvialis ( AL040) using the n-Hexane, dichloromethane and methanol extracts at 1× to 4 × MICs and 2 hour exposure. About 96.3 percent, 82.2 percent, and 78.1 percent (V. fluvialis AL040); 92.6 percent, 87.8 percent and 68.9 percent (V. parahaemolyticus AL049); and 91.6 percent, 64.4 percent, 60 percent (V. vulnificus AL042) of the bacteria were killed by the crude n-Hexane, dichloromethane and methanol extracts respectively after 2 hour exposure time at 4× MIC. The patterns of activity were bacteriostatic, with the n-Hexane extracts being most effective in activity. We conclude that the Garcinia kola seeds have promise in the treatment and management of infections caused by Vibrio species.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Universidade Estadual Paulista (UNESP)
No presente trabalho, foram avaliadas dez espécies vegetais, nativas do cerrado brasileiro e utilizadas popularmente no tratamento de úlceras gástricas, quanto ao seu potencial mutagênico. Foram empregados os testes de Ames (in vitro) e do micronúcleo em células do sangue periférico de camundongos (in vivo). De cada espécie vegetal foram avaliados dois tipos de extratos brutos: um polar e um apolar. Nos ensaios in vitro foi verificada mutagenicidade especialmente para os extratos polares (metanólicos) das seguintes espécies vegetais: A. castaneifolia, A. glandulosa, A. triplinervia, M. pusa, Q. grandiflora, Q. multiflora e S. pseudoquina. Nesses mesmos ensaios, os únicos extratos apolares que apresentaram mutagenicidade foram os obtidos de Q. grandiflora e Q. multiflora. Foram avaliados in vivo apenas os extratos polares, sendo que apresentaram mutagenicidade os extratos metanólicos de A. castaneifolia, A. glandulosa, A. triplinervia, Q. multiflora e S. pseudoquina. De acordo com as caracterizações químicas realizadas com as espécies vegetais estudadas, ficou evidente a participação bastante efetiva dos compostos fenólicos (flavonóides e taninos) na mutagenicidade observada. Também foi possível verificar o papel que possíveis interações entre os diferentes compostos químicos presentes nos extratos podem ter em suas atividades biológicas. Tendo em vista as informações obtidas, ficou clara a necessidade que os estudos biológicos e fitoquímicos apresentam para se promover uma maior compreensão dos riscos que podem estar associados aos tratamentos medicinais baseados em plantas.
In this work we evaluate the mutagenic potential of ten vegetable species native of the Brazilian savannah and used popularly in the treatment of gastric ulcers. The Ames test (in vitro) and the Micronucleus test (in vivo) were employed. We evaluate a polar extract and a non-polar extract to each vegetal species. The in vitro assays show the mutagenicity of the methanol extracts (polar) obtained from A. castaneifolia, A. glandulosa, A. triplinervia, M. pusa, Q. grandiflora, Q. multiflora e S. pseudoquina. The non-polar extracts from Q.grandiflora e Q. multiflora were mutagenic too. In vivo, only the polar extracts were studied and the methanol extracts from A. castaneifolia, A. glandulosa, A. triplinervia, Q. multiflora e S. pseudoquina were mutagenic. The phytochemical studies with the evaluated plants permit to infer the participation of the flavonoids and tannins in the mutagenic activities observed in vitro and in vivo. We also verified in this biological activity possible interactions between the different substances present in the raw extracts. In this way, we could to observe the importance of phytochemical and biological studies to promote a better comprehension of the risks that can be associated to the medicinal treatments based in the plants use.

Orientador: Jose Francisco Hofling
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: A cavidade oral abriga uma complexa comunidade de microrganismos que podem estar aderidos aos dentes, à mucosa epitelial ou formando biofilme. A maioria desses microrganismos são comensais, com uma minoria causando infecções oportunistas. Dentre esses microrganismos, encontramos as leveduras do gênero Candida, principalmente em indivíduos imunocomprometidos; o principal agente etiológico da cárie dental Streptococcus mutans e, apesar da presença controversa, o patógeno oportunista Staphylococcus aureus. Diante do desenvolvimento de resistência aos antimicrobianos, há uma grande preocupação relacionada a qual tipo de tratamento utilizar no combate às infecções provocadas por esses microrganismos. O emprego de extratos brutos de plantas tem merecido a atenção de pesquisadores de vários países, já que podem inibir o crescimento bacteriano e fúngico por diferentes mecanismos quando comparados aos antimicrobianos, justificando a pesquisa em questão. As plantas utilizadas nesta pesquisa foram a Mentha piperita, Rosmarinus officinalis, Tabebuia avellanedae, Arrabidaea chica, Syzygium cumini e Punica granatum, extraídas com os solventes diclorometano e metanol, os quais foram eliminados em rotaevaporador, para a obtenção dos extratos brutos, a fim de se avaliar sua concentração inibitória mínima (CIM) frente a essas cepas de microrganismos. Os resultados obtidos revelaram-se promissores com as espécies de Candida testadas, principalmente com o extrato metanólico, cuja inibição apresentou atividade com alta sensibilidade das leveduras, porém essa mesma atividade não ocorreu entre as bactérias. Esses dados indicam a necessidade da ampliação do conhecimento sobre as espécies de plantas com potencial antimicrobiano, além de se testar extratos mais purificados e com propriedades farmacológicas ativas contra organismos microbianos. De qualquer forma, estes resultados nos levam a considerar esses extratos vegetais como fontes valiosas para a descoberta de novas moléculas bioativas empregadas para o tratamento alternativo no combate aos agentes microbianos, principalmente àqueles resistentes às drogas antifúngicas e antibacterianas convencionais
Abstract: The oral cavity is a source of a complex microorganisms community that may adhere to teeth, epithelial mucosa or form biofilms. Most of these strains are thought to be commensal, and a small number cause opportunistic infections. Among these microorganisms Candida yeast is the most commom in immunocompromised patients; Sretptococcus mutans is regarded as the primary etiologic agent of dental caries and, although considered controversial the presence, Staphylococcus aureus is also mentioned as an opportunistic pathogen in the oral cavity. Considering the frequent use of antifungal and antibiotics as antimicrobial agents, the development of resistance to these drugs has become an important and worrying factor for the treatment of the infections caused by these microorganisms. The use of crude plants extracts has been a researcherâ?¿s concern from all over the world, since they may inhibit the bacteria and fungi growing by different mechanisms present in the conventional antimicrobials, becoming an important source of investigation. The plants used in this research were Mentha piperita, Rosmarinus officinalis, Tabebuia avellanedae, Arrabidaea chica, Syzygium cumini and Punica granatum, extracted with dichloromethane and methanol solvents, followed by a minimal inhibitory concentration (MIC) evaluation. The data obtained with Candida strains were promising, specially with the methanolic extract that showed activity in a higher sensibility, but the same activity did not occur among the bacterias. This information increases our knowledge of the crude plants extracts effect against microbial organisms. Nevertheless, the results of this study indicated that these extracts are a promising source for the alternative treatment against the microbial agents, specially for those resistant to the conventional antimicrobial agents
Mestrado
Microbiologia e Imunologia
Mestre em Biologia Buco-Dental

Thesis (MSc)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Sutherlandia frutescens (L.) R. Br., also known as Lessertia frutescens, is a leguminous shrub indigenous to southern Africa. Traditionally this plant has been used for the treatment of various ailments; current interest in this plant has escalated after it was announced that extracts could aid in the relief and treatment of HIV/AIDS. These extracts contain an array of metabolites, including sutherlandins, sutherlandiosides L-arginine, L-canavanine, asparagine, gamma-aminobutyric acid (GABA), and various other amino acids, which have been linked to medicinal uses. This study focused on the use of hormones to promote the growth and metabolite production of S. frutescens in vitro cultures. The growth promoting substances used in this study were synthetic analogues of strigolactones, GR24 and Nijmegen-1, and auxins, indole-3-butyric acid (IBA) and naphthalene acetic acid (NAA). The first part of this study focused on the effects strigolactones and auxins, alone and combined, had on the growth of S. frutescens in vitro nodal explants. The S. frutescens nodal explants had the most significant improvement in growth with treatments that contained 1 mg/L NAA. These treatments increased growth via fresh and dry mass and plant length. The metabolite content of these nodal explant cultures was evaluated using liquid chromatography/mass spectrometry (LC/MS) metabolite analysis. The treatments that contained 1 mg/L NAA differed in metabolite composition and showed an increase in metabolite quantity. The SU1 content of the treated plants was also quantified using LC/MS techniques and a combination of 1 mg/L NAA and Nijmegen-1 doubled the amount of SU1. The effect of strigolactones was also studied using hairy root cultures of S. frutescens. Strigolactones alone slightly inhibited the formation of lateral transgenic roots, but when these chemicals were used in combination with auxins, significant reduction in dry mass and lateral root outgrowth resulted. Of the treatments tested in this study, 0.1 mg/L IBA caused noticeable alterations to the metabolite pool, with amino acids such as GABA and arginine accumulating at higher levels than the control explants. The exploitation of hormones to up-regulate the growth and metabolism of the medicinally important plant, Sutherlandia frutescens, proved successful in this study. The use of in vitro nodal explants along with hairy root cultures has assisted in the establishment of a stable system for the up-regulation of metabolites.
AFRIKAANSE OPSOMMING: Sutherlandia frutescens (L.) R. Br., ook bekend as Lessertia frutescens, is 'n peulagtige struik inheems tot suider Afrika. Tradisioneel is die plant vir 'n groot verskeidenheid van kwale gebruik; huidige belangstelling in die plant het toegeneem nadat dit bekend gemaak was dat ekstraksies vanaf hierdie plant verligting kan bied vir MIV/VIGS. Hierdie ekstrakte bevat 'n verskeidenheid van metaboliete, insluitend sutherlandins, sutherlandiosiede L-arginien, L-kanavanien, asparagien, gamma-aminobottersuur (GABS), asook verskeie ander aminosure wat medisinale gebruike het. Die studie het gefokus op die gebruik van hormone om die groei en metaboliete van S. frutescens in vitro kulture te vermeerder. Die groei reguleerders wat in hierdie studie gebruik was, was die sintetiese analoë van strigolaktoon, GR24 en Nijmegen-1, asook die ouksiene, indool-3-bottersuur (IBS) en naftaleen asynsuur (NAS). Die eerste deel van die studie het gefokus op die effek van strigolaktoon en ouksien, alleen en in kombinasie, op die groei van S. frutescens in vitro nodale mikrostingels. Die S. frutescens nodale mikrostingels wat behandel was met 1 mg/L NAS het die aansienlikste toename in groei getoon. Hierdie behandeling het groei bevorder deur middel van vars en droë massa en plant lengte. Die metaboliet inhoud van die behandelde mikrostingels was met behulp van vloeistofchromatografie/massa spektrometrie (VC/MS) ondersoek. Al die behandelinge wat 1 mg/L NAS bevat het, het in metaboliet samestelling verskil en het ook 'n toename in metaboliet hoeveelheid getoon. Die SU1 inhoud van die behandelde plante was ook met behulp van VC/MS tegnieke gekwantifiseer en dit was gevind dat 'n kombinasie van 1 mg/L NAS en Nijmegen-1 die hoeveelheid SU1 verdubbel het. Die effek van strigolaktoon op harige wortel kulture van S. frutescens was ook ondersoek. Strigolaktoon alleen het die formasie van laterale transgeniese wortels effens inhibeer, maar wanneer hierdie chemikalieë saam met ouksiene gebruik was, was die aansienlike afname van die massa en inhibisie van die laterale wortel uitgroeisels meer prominent. Van al die behandelinge wat in hierdie studie getoets is, het 0.1 mg/L IBS die mees merkbare veranderinge in metaboliete meegebring en aminosure soos GABS en arginien het teen hoër vlakke versamel. Die uitbuiting van hormone om groei en metaboliet produksie te bevorder in die belangrike medisinale plant, Sutherlandia frutescens, was suksesvol in hierdie studie. Die gebruik van nodale mikrostingels asook harige wortel kulture het bygedra om 'n stabiele sisteem te vestig vir die vermeerdering van metaboliete.

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Pertencente à família Meliaceae, a espécie Trichilia catigua A. Juss é conhecida popularmente como catigua, cataguá, argelim-rosa e mangalto-catingam. Sua casca apresenta propriedades adstringente, inseticida, purgativa, tônica, bactericida, antiinflamatória e antidepressiva. Com o objetivo de propagar a espécie T. catigua foram desenvolvidos experimentos testando o enraizamento de estacas e a micropropagação com explantes de matrizes e sementes. Os experimentos de enraizamento de estacas foram realizados na primavera 2004, verão 2004/2005, outono, inverno e primavera 2005 e primavera 2006. Em todos os experimentos, estacas com aproximadamente 15 cm de comprimento foram coletadas de árvores adultas e preparadas da parte apical e mediana dos ramos. A seguir, foram submetidas aos reguladores vegetais IBA (ácido indolbutírico), NAA (ácido naftalenoacético) e IAA (ácido 3-indolacético), variando as dosagens. Para a avaliação dos experimentos determinou-se a percentagem de estacas enraizadas, não enraizadas e mortas e quando enraizadas, seu comprimento e diâmetro. No experimento primavera de 2004 foram testadas as concentrações de 1000 e 2000 mg L-1 dos reguladores vegetais IBA, NAA e IAA. As avaliações aos 90 dias após sua instalação revelaram maiores percentagens de enraizamento e iguais a 33,33, 25,00, 22,91 e 23,43 % para estacas submetidas a IBA 1000, 2000 mg L-1 e NAA 1000 e 2000 mg L-1, respectivamente. No verão 2004/2005, outono, inverno e primavera 2005 os experimentos foram conduzidos com as concentrações dos reguladores IBA, NAA e IAA iguais a 1000, 2000 e 3000 mg L-1 e as avaliações foram realizadas após 120 dias. Não houve enraizamento no outono e inverno. A análise conjunta dos resultados obtidos na primavera e no verão mostrou percentagem de enraizamento superior na primavera. A maior percentagem de enraizamento, igual a 19,17%...
The Trichilia catigua A. Juss from the Meliaceae family is popularly known as catigua, cataguá, argelim-rose and mangalto-catingam. Its bark has astringent, insecticide, purgativa, tonic, bactericide, anti-inflammatory and antidepressant properties. With the aim of propagate T. catigua, experiments of rooting with stem cuttings and of micropropagation with explants of trees and seeds were carried out. In all the rooting experiments the stem cuttings with approximately 15 cm of length were collected from adult trees and prepared from the apical and intermediate parts. The cuttings were immersed in the vegetable regulators IBA (Indole-3- butyric acid), ANA (Naphthalene acetic acid) and IAA (Indole-3 acetic acid). The rooted stem cutting and not rooted stem cutting percentage and, when rooted, the length and diameter of roots, were evaluated. In the experiment spring 2004 the concentrations of 1000 and 2000 mg L-1 of IBA, ANA and IAA were tested, with evaluations 90 days after installation. The highest rooting percentage were 33,33, 25,00, 22,91 and 23,43% for IBA 1000, 2000 mg L-1 and ANA 1000 and 2000 mg L-1, respectively. In the summer of 2004/2005, autumn, winter and spring of 2005 IBA, ANA and AIA, with concentration of 1000, 2000 and 3000 mg L-1, were tested. The evaluation was carried out at 120 days. No rooting was observed in autumn and winter. The analysis of data from summer and spring showed higher rooting percentage in spring. The highest rooting percentage was obtained with IBA 3000 mg L-1 (19,17%). In the spring 2006 IBA (1000, 2000, 3000, 4000 and 5000 mg L-1) and ANA (1000, 2000, 3000 mg L-1) were tested. The highest rooting percentage (41,67%) was obtained with IBA 5000 mg L-1. In the in vitro cultivation, explantes obtained from trees were submitted to asepsis treatments with HgCl2, CaOCl2 and NaOCl and inoculated in Murashige & Skoog culture medium (MS) with 25%... (Complete abstract click electronic access below)

Orientador: Hérida Regina Nunes Salgado
Banca: Hérida Regina Nunes Salgado
Banca: Eloir Paulo Schenkel
Banca: Taís Maria Bauab
Resumo: As espécies Vismia guianensis (Aubl.) (Clusiaceae) e Senna reticulata (Willd.) Irwin & Barneby (Caesalpiniaceae), conhecidas popularmente por lacre e mata-pasto, respectivamente, são encontradas na região amazônica, onde são utilizadas na medicina popular para o tratamento de diversas moléstias, principalmente infecções e dermatomicoses. Diante do exposto, o presente trabalho objetivou avaliar a atividade antimicrobiana dos extratos e frações das referidas espécies vegetais, através da bioautografia e da determinação da concentração inibitória mínima (CIM) pelo método de microdiluição. Foi avaliada também a atividade imunológica dos extratos hidroetanólicos das cascas do caule e das folhas de Senna reticulata e de Vismia guianensis através da produção e inibição de óxido nítrico por células peritoneais de camundongos. Todos os extratos apresentaram atividade inibitória frente às cepas padrão (ATCC) e clínicas dos fungos Trichophyton rubrum, Trichophyton mentagrophytes e Microsporum canis. O extrato hidroalcoólico das cascas de V. guianensis apresentou melhor atividade antimicrobiana, com CIM de 62,5 μg/mL, de 125 μg/mL e de 500 μg/mL frente às cepas padrão de T. mentagrophytes, T. rubrum e M. canis, respectivamente; CIM de 125 μg/mL para as cepas clínicas de T. rubrum e T. mentagrophytes e, de 15,63 μg/mL frente à cepa clínica de M. canis. O extrato hidroetanólico das cascas de V. guianensis também apresentou atividade antimicrobiana contra todas as bactérias testadas, sendo as cepas de S. aureus, S. epidermidis e B. subtilis foram as mais sensíveis. As cepas de C. albicans foram resistentes aos extratos de ambas espécies vegetais. Através dos ensaios de atividade imunológica in vitro observou-se que na concentração de 50 μg/mL os extratos não apresentaram citotoxicidade e que apresentaram potencial atividade antiinflamatória
Abstract: The Vismia guianensis (Aubl.) (Clusiaceae) and Senna reticulata (Willd.) Irwin & Barneby (Caesalpiniaceae), popularly known as seal and obtusifolia, respectively, are found in the Amazon region, where they are used in folk medicine for the treatment of various diseases, especially infections and dermatomycoses. In this light, this study aimed to evaluate the antimicrobial activity of extracts and fractions of these plant species by bioautography and determination of minimum inhibitory concentration (MIC) by microdilution method. We also evaluate the immunological activity of the hydroethanolic from the stem bark and leaves of Senna reticulata and Vismia guianensis and production through the inhibition of nitric oxide by peritoneal cells of mice. All the extracts showed activity against the standard strains (ATCC) and clinical fungi Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. The hydroalcoholic extract of the bark of V. guianensis showed better antimicrobial activity, with MIC of 62.5 μg/mL, 125 μg/mL and 500 μg/mL against the standard strains of T. mentagrophytes, T. rubrum and M. canis, respectively, MIC of 125 μg/mL for clinical strains of T. rubrum and T. mentagrophytes and of 15.63 μg/mL against the clinical strain of M. canis. The hydroalcoholic extract of the bark of V. guianensis also showed antimicrobial activity against all bacteria tested, with strains of S. aureus, S. epidermidis and B. subtilis were the most sensitive. The strains of C. albicans were resistant to the extracts of both species. Through the in vitro testing of immune activity was observed that the concentration of 50 μg/mL extracts showed no cytotoxicity and showed potential anti-inflammatory activity
Mestre

Currently, the approved anti-HIV drugs on the market only target the three HIV enzymes: reverse transcriptase, protease and more recently, integrase. Due to the limited nature of the current therapy, it is possible that a multi-drug resistant virus can emerge. The main concerns in developing countries however, are the expense and availability of the drugs and because of this, it is essential to investigate all alternatives. Traditional medicine offers many advantages as compared to allopathic treatment in so far as being relatively cheaper, accessible and it is broadly accepted in the population groups of the developing countries. Little is known though, of the exact efficacy and toxicity of these remedies so it is vital that these possible leads be investigated thoroughly. For the purpose of this study, two plants, Sutherlandia frutescens and Lobostemon trigonum were studied to ascertain their potential anti-HIV activity. Sutherlandia has received international attention as a possible cheap herbal remedy to improve the health of AIDS sufferers. Anecdotal evidence from health workers claim that HIV- infected patients on Sutherlandia treatment have shown improved CD4 counts, decreased viral loads and a general improvement in well-being. Extracts were prepared from dried leaves and flowers in methanol, ethanol, acetone, methylene dichloride or distilled water. Sulphated polysaccharides have been described extensively in literature with regards to their anti-HIV activity, so as a form of dereplication; an ethanol precipitation was performed on the aqueous extracts to remove sulphated polysaccharides. A toxicity study was performed on all crude extracts using uninfected peripheral mononuclear blood cells (PBMCs) isolated from whole blood. To measure anti-HIV activity, HIV-infected PBMCs were cultured with each of the crude extracts and cell viability measured using the tetrazolium salt, XTT. HIV-infected CEM-NKR-CCR5 cells were also used and supernatant from the viral studies was tested for the HIV antigen p24. xii Results varied greatly between assays but with the inclusion of a point-scale system to evaluate the extracts it was clear that overall the organic extracts of the Sutherlandia flowers, especially the acetone extract (SFA), showed great anti-HIV potential. SFA in every case decreased p24 levels and in the toxicity study did not decrease cell proliferation. With the HIV-infected PBMCs SFA actually helped improve cell proliferation despite the infection. To determine the specific anti- HIV activity, all crude extracts were tested for inhibition of HIV-I reverse transcriptase, the glycohydrolase enzymes: a-glucosidase, ß-glucosidase, ßglucuronidase, HIV-I integrase and HIV-II protease. No significant inhibition was seen with these experiments except for the HIV-I RT assay. The aqueous extract of the Lobostemon leaves produced an inhibitor of HIV-RT with a very low IC50 value of 0.049mg/ml. Some inhibitory effect was lost with the removal of the sulphated polysaccharides and the addition of BSA to the assay, but still 64% inhibition of the HIVRT remained, which confirmed that the inhibitor could be something novel, and not of the polysaccharide or tannin compounds.

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The present study was developed from 2013 to 2016 in the Laboratory of Zoology of the Universidade Federal Rural do Rio de Janeiro and the Department of Animal Parasitology of the Universidade Estadual do Norte Fluminense Darcy Ribeiro, State of Rio de Janeiro. The objective was to test in vitro and in vivo the efficiency of the medicinal plants Baccharis trimera (Less.) DC., Eucalyptus globulus Labill., Mentha piperita Linnaeus, and Spigelia anthelmia Linnaeus, in phytotherapeutic and homeopathic forms, as an alternative way to control endoparasites of the Gallus gallus Linnaeus, 1758 (red junglefowl). Endoparasites are a serious problem that affects domestic bird rearing and development. They cause growth retardation, decrease in the food conversion index, increase in the susceptibility to infectious diseases, and culminate in death. The methodologies used were recommended by Coles et al. (1992), supported by the World Association for the Advancement of Veterinary Parasitology (WAAVP), reference to anthelmintic tests, and by Hubert and Kerboeuf (1992). The plants B. trimera and M. piperita showed moderate efficiency rate in in vitro tests, with maximum values of 80.00%, and low efficiency rate in in vivo test, with maximum values of 9.31%. The plants E. globulus and S. anthelmia, showed high efficiency rate in in vitro and in vivo tests, with values above 80.00%. The present study recorded the presence of the genera Ascaridia, Capillaria, and Heterakis. The plants sometimes showed indices above the traditional product used (Febendazol).
A pesquisa foi desenvolvida no Laborat?rio de Zoologia da Universidade Federal Rural do Rio de Janeiro e Setor de Parasitologia Animal da Universidade Estadual do Norte Fluminense Darcy Ribeiro, estado do Rio de Janeiro, no per?odo de 2013 a 2016. O objetivo foi testar in vitro e in vivo a efic?cia das plantas medicinais Baccharis trimera (Less.) DC., Eucalyptus globulus Labill., Mentha piperita Linnaeus e Spigelia anthelmia Linnaeus, nas formas fitoter?pica e homeop?tica, como meios alternativos para o controle de endoparasitos de Gallus gallus Linnaeus, 1758 (Galinha Caipira), um s?rio problema que afeta a cria??o e desempenho de aves dom?sticas, ocasionando morte quando muito intenso, retardo de crescimento, redu??o de ?ndice de convers?o alimentar e aumento na suscetibilidade ?s doen?as infecciosas. As metodologias utilizadas foram preconizadas por Coles et al. (1992), creditada pela World Association for the Advancement of Veterinary Parasitology (WAAVP), organiza??o refer?ncia para testes anti-helm?nticos, e Hubert e Kerboeuf (1992). Para as plantas B. trimera e M. piperita, os ensaios in vitro demonstraram moderada taxa de efic?cia com valores m?ximos de 80,00%, e o ensaio in vivo, baixa taxa de efic?cia, com valores m?ximos de 9,31%. Para E. globulus e S. anthelmia, os ensaios in vitro e in vivo demonstraram alta taxa de efic?cia, com valores acima de 80,00%. A pesquisa evidenciou a presen?a dos g?neros Ascaridia, Capillaria e Heterakis. As plantas demonstraram em certos momentos ?ndices superiores ao produto tradicional utilizado (Febendazol).

Chronic non-communicable diseses are a global public health challenge that continuously threatens the development and health of humans. Risk factors such as unbalanced diet-the high consumption of processed food or food from animal origin are responsible for NCDs. NCDs result in weakened immune system, making the host susceptible to opportunistic infections. Thus, the NCDs burden is most times chronic and multiple with the illness and suffering of the affected person numerous. The lack of cure for NCDs, the high cost of drugs, their high side-effects, and the emergence of multiple drug resistance has given rise to the investigation of other sources for therapeutic cure such as medicinal plants. The ethanol, n-hexane and ethyl acetate extracts of Olea europaea were analysed for their antioxidant and antimicrobial activities. The essential oil was also analysed for their chemical constituents. The n-hexane extracts of O. europaea exhibited no inhibition against all of the microorganisms tested, while the ethyl acetate and ethanol extracts exhibited inhibition, with minimum inhibitory concentration values between 0.625 mg/ml to 1.25 mg/ml. The ethanol leaf and ethyl acetate stem extracts exhibited significant activity in the inhibition of 2, 2-azinobis-(3-ethylbenzothiazolin - 6-sulfonic acid diammonium salt (ABTS) free radical, the n-hexane leaf extract had the overall significant lipid peroxidation inhibition activity, while in the inhibition of 2, 2- diphenyl-1-picrylhydrazyl radical (DPPH), the ethanol and ethyl acetate leaf extracts had strong activity. Nonanal, phytol, α-Pinene, α-Phellandrene, spatulenol and farnesol were some of chemical components identified after the GC-MS analysis of O. europaea oil. In the final part of the dissertation, Euryops brevipapposus essential oil was assessed for the antioxidant activities using free radical scavenging assays. In addition to this, the antimicrobial activities were assessed and the chemical composition was analysed using GC-MS. The essential oil demonstrated significant antioxidant activity against 2, 2-diphenyl-2-picryl-hydrazyl free radical (DPPH), 2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and lipid peroxides with IC50 value of 0.0000000671 mg/ml, 1.05 mg/ml, and 1.170 mg/ml respectively. The essential oil also showed significant activity against all microorganisms tested with minimum inhibitory concentration (MIC) values between 0.055 mg/ml to 0.5 mg/ml. α-pinene, α- Phellandrene, germacrene D, β-pinene, trans- β.-Ocimene, bicyclogermacrene and β -Phellandrene were some of the chemical compounds identified in E. brevipapposus oil. The study has shown that E. brevipapposus and O. europaea are abundant in phytochemical compounds which were thought to be the root cause for the activities demonstrated. Therefore, these therapeutic properties observed validate and elucidate the traditional usage of the both plants in the treatment /management of diseases.

Previous studies in Native American ethnobotany on the shared use of medicinal and cultural plants between communities fail to clearly reveal if these shared uses are part of changing culture or remain a stabilizing connection between old and new tribes. During the late 1700's to early 1800's, various factions of the Creek tribes of Georgia migrated into Florida, forming a new tribe called the Seminoles. This event provides the unique opportunity to study the changing cultural and medicinal uses of plants by a new tribe in a new geographic location, revealing if cultural purposes were passed from one group to another. A list of plants used for medicinal purposes by the Creek and Seminole tribes was produced from previous studies. Utilizing these lists, comparisons were drawn to determine if cultural practices were carried on between tribes as they changed locations and lifestyles. This study examines the use of 465 plants in 125 plant families. Of these, 39 plants were found to be used by both tribes for different treatment purposes. In contrast, only 15 plants where used by both tribes for similar treatments. The small number of shared use of plants indicates the newly formed Seminole tribe developed new cultural and medicinal practices. These findings indicate that the plants used for medicinal purposes by the Native American tribes of the southeast were a part of a changing culture, not a stabilizing connection between old and new tribes as previously thought.

Despite the significant advances achieved in lessening the burden of malaria and other tropical diseases in recent years, protozoal infections remain a major cause of mortality in many developing countries, with malaria accounting for a large proportion of the recorded mortality. The main aim of this study was to bioprospect indigenous plant species for novel antiplasmodial plant products by means of NMRbased metabolomics. In this study, an ethnobotanical criterion was followed in collecting twenty indigenous plant species used to treat malaria or its symptoms by Vha-Venda people living in Mutale Municipality of Limpopo Province, South Africa. Plant samples were extracted in dichloromethane:50% methanol (1:1), separated into polar and nonpolar fractions, and tested on Trypanosoma brucei rhodesiense, T. cruzi, Leishmania donovani and Plasmodium falciparum. Plant extracts were correspondingly subjected to an antiproliferative bioassay against mammalian skeletal myoblast cells. The current study is the first scientific account on the significant antileishmanial efficacy (IC50 ? 5 ?g/ml) of Bridelia mollis (Phyllanthaceae), Vangueria infausta subsp. infausta (Rubiaceae), Syzygium cordatum (Myrtaceae) and Xylopia parviflora (Annonaceae), as well as high antitrypanosomal activity (IC50 = 3.45 ?g/ml) of Albizia versicolor (Fabaceae). Ten plant extracts exhibited significant in vitro antiplasmodial activity (IC50 ? 5 ?g/ml), with Tabernaemontana elegans (IC50 = 0.331 ?g/m and IC50 = 0.834 ?g/m) and V. infausta subsp. infausta (IC50 = 1.84 ?g/ml) being the best samples. This is the first scientific report to document significant antiplasmodial activity of extracts from T. elegans. The findings of this study substantiate the rationale for adopting an ethnopharmacological approach when bioprospecting medicinal plants for antiplasmodial compounds. Dichloromethane extracts were subjected to 1H NMR-based metabolomic analysis, where each crude extract was reconstituted in CDCl3, analysed on a Varian 600 MHz spectrometer and the acquired 1H NMR spectra were then analysed collectively using multivariate data analysis (MDA). Principal Component Analysis (PCA) could not separate the analysed profiles according to the detected antiplasmodial bioactivity. Application of supervised Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) on the 1H NMR profiles resulted in a discrimination pattern that could be correlated to the observed antimalarial bioactivity. A contribution plot generated from the OPLS-DA scoring plot illustrated the classes of compounds responsible for the observed grouping. Further phytochemical analyses were conducted on lipophilic extracts of T. elegans and V. infausta subsp. infausta. These best candidates were fractionated, purified and identified based on conventional chromatographic and spectroscopic techniques. Two known indole alkaloids isolated from T. elegans, were identified as dregamine and tabernaemontanine. The antiplasmodial activity of these acyl indole alkaloids has previously been established and ranges from moderate to good. Three compounds were isolated from V. infausta subsp. infausta of which two were identified as friedelin (IC50 = 3.01 ?g/ml) and morindolide (IC50 = 18.5 ?g/ml). While these compounds have been previously identified, this is the first account of their occurrence in the genus Vangueria and their antiplasmodial activity. An unidentified compound with significant antiplasmodial activity (IC50 = 0.143 ?g/ml) was also isolated. The study demonstrated the potential of discovering novel antiplasmodial scaffolds from medicinal plants.
Thesis (PhD)--University of Pretoria, 2016.
Plant Science
PhD
Unrestricted

É grande o interesse em substâncias que apresentem ação inibitória sobre leveduras do gênero Candida. Várias plantas utilizadas na medicina popular têm sido estudadas, com esta finalidade. O presente trabalho teve por finalidade avaliar a atividade inibitória in vitro de extratos de Arrabidaea chica (Verlot.), conhecida popularmente por crajirú em Rondônia (Brasil), sobre Candida albicans. Foi avaliada a concentração inibitória mínima (CIM) de extratos aquoso e hidroalcoólico, autoclavados e filtrados, utilizando a técnica de diluição em ágar, sobre vinte e cinco cepas de C. albicans. Os extratos brutos foram submetidos à Cromatografia de Camada Delgada (CCD). O extrato hidroalcoólico apresentou sete componentes e o aquoso autoclavado, dois. Tanto o extrato hidroalcoólico autoclavado quanto o filtrado inibiram todas as cepas de C. albicans na concentração de 20%. O extrato aquoso filtrado não apresentou efeito inibitório até a concentração de 50% e o extrato autoclavado, na concentração de 30%, inibiu 84% das cepas testadas. Os extratos, hidroalcoólico e aquoso, apresentaram atividade sobre C. albicans. O extrato hidroalcoólico apresentou desempenho superior, em menor concentração e maior número de componentes.
There is a great interest in substances that have inhibitory action on yeasts of the genus Candida. Several plants, used in popular medicine, have been studied for this purpose. This aim of this work was to evaluate in vitro the inhibitory activity of Arrabidaea chica (Verlot.) extracts, known in Rondônia (Brazil) as crajirú, against Candida albicans. The minimum inhibitory concentration (MIC) of aqueous and hydroalcoholic extracts, autoclaved and filtered was assessed, using the technique of agar dilution. Twenty-five strains of C. albicans were studied. The crude extracts were submitted to a Fine Chromatography Analysis (FCA). The hydroalcoholic extract presented seven compounds and the aqueous autoclaved extract two. Hydroalcoholic extracts inhibited all strains of C. albicans in the concentration of 20%. The aqueous filtered extract not showed inhibitory effect until 50%. The aqueous autoclaved extract, in the concentration of 30%, inhibited 84% of the strains. Hydroalcoholic and aqueous extracts showed activity against C. albicans. The hydroalcoholic extract presented superior performance in a lower concentration and had a greater number of compounds.

Tuberculosis (TB) still remains a health problem globally with over a million new infections and a mortality rate of 1.5 million individuals annually (Hawn et al., 2014). The emerging multi-drug resistant (MDR) strains that accompany human immune deficiency virus (HIV) infection in high-incidence populations contribute significantly to the health burden of TB (Areeshi et al., 2014). The standard treatment that is advocated by the World Health Organization (WHO) for active tuberculosis includes long-term therapy that incorporates the use of isoniazid, rifampicin, pyrazinimide and ethambutol as front line drugs (WHO, 2013). Drug resistance against established treatment options for TB makes research into new forms of therapy an imperative in health care (Ntulela et al., 2009). South Africa is currently witnessing a high number of cases of drug-resistant TB. In some parts of the country, one in ten cases of TB is resistant to treatment. It is therefore essential to have new anti-tuberculosis agents, which can be readily and simply produced from some local source (Warner et al., 2014). A logical starting point for this research of new agents would be the herbal medicines which have been used for centuries in rural areas by local healers. Western developed countries have harvested ethno botanical knowledge and have produced drug therapies for conventional medicines for other ailments. The activity of extracts of the active plants and their properties still require study in animal models in order to assess their future as new anti-tuberculosis agents (Lall and Meyer, 1999). This study focuses on qualitative and quantitative experimental findings after the administration of a medicinal plant extract to animals. This will include daily observation of animals, recording of feed consumption, recording of animal weights, macroscopic examination of animals at necropsy, tissue harvesting, histological procedures and microscopy.

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As espécies Vismia guianensis (Aubl.) (Clusiaceae) e Senna reticulata (Willd.) Irwin & Barneby (Caesalpiniaceae), conhecidas popularmente por lacre e mata-pasto, respectivamente, são encontradas na região amazônica, onde são utilizadas na medicina popular para o tratamento de diversas moléstias, principalmente infecções e dermatomicoses. Diante do exposto, o presente trabalho objetivou avaliar a atividade antimicrobiana dos extratos e frações das referidas espécies vegetais, através da bioautografia e da determinação da concentração inibitória mínima (CIM) pelo método de microdiluição. Foi avaliada também a atividade imunológica dos extratos hidroetanólicos das cascas do caule e das folhas de Senna reticulata e de Vismia guianensis através da produção e inibição de óxido nítrico por células peritoneais de camundongos. Todos os extratos apresentaram atividade inibitória frente às cepas padrão (ATCC) e clínicas dos fungos Trichophyton rubrum, Trichophyton mentagrophytes e Microsporum canis. O extrato hidroalcoólico das cascas de V. guianensis apresentou melhor atividade antimicrobiana, com CIM de 62,5 μg/mL, de 125 μg/mL e de 500 μg/mL frente às cepas padrão de T. mentagrophytes, T. rubrum e M. canis, respectivamente; CIM de 125 μg/mL para as cepas clínicas de T. rubrum e T. mentagrophytes e, de 15,63 μg/mL frente à cepa clínica de M. canis. O extrato hidroetanólico das cascas de V. guianensis também apresentou atividade antimicrobiana contra todas as bactérias testadas, sendo as cepas de S. aureus, S. epidermidis e B. subtilis foram as mais sensíveis. As cepas de C. albicans foram resistentes aos extratos de ambas espécies vegetais. Através dos ensaios de atividade imunológica in vitro observou-se que na concentração de 50 μg/mL os extratos não apresentaram citotoxicidade e que apresentaram potencial atividade antiinflamatória
The Vismia guianensis (Aubl.) (Clusiaceae) and Senna reticulata (Willd.) Irwin & Barneby (Caesalpiniaceae), popularly known as seal and obtusifolia, respectively, are found in the Amazon region, where they are used in folk medicine for the treatment of various diseases, especially infections and dermatomycoses. In this light, this study aimed to evaluate the antimicrobial activity of extracts and fractions of these plant species by bioautography and determination of minimum inhibitory concentration (MIC) by microdilution method. We also evaluate the immunological activity of the hydroethanolic from the stem bark and leaves of Senna reticulata and Vismia guianensis and production through the inhibition of nitric oxide by peritoneal cells of mice. All the extracts showed activity against the standard strains (ATCC) and clinical fungi Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. The hydroalcoholic extract of the bark of V. guianensis showed better antimicrobial activity, with MIC of 62.5 μg/mL, 125 μg/mL and 500 μg/mL against the standard strains of T. mentagrophytes, T. rubrum and M. canis, respectively, MIC of 125 μg/mL for clinical strains of T. rubrum and T. mentagrophytes and of 15.63 μg/mL against the clinical strain of M. canis. The hydroalcoholic extract of the bark of V. guianensis also showed antimicrobial activity against all bacteria tested, with strains of S. aureus, S. epidermidis and B. subtilis were the most sensitive. The strains of C. albicans were resistant to the extracts of both species. Through the in vitro testing of immune activity was observed that the concentration of 50 μg/mL extracts showed no cytotoxicity and showed potential anti-inflammatory activity

Diversos estudos vêm demonstrando o potencial de plantas medicinais no controle de fitopatógenos, tanto por sua ação fungistática direta, quanto pela capacidade de induzir a defesa das plantas, indicando a presença de moléculas com características elicitoras. Nesse sentido foram desenvolvidos três experimentos no ano de 2009 e 2010, na Universidade Tecnológica Federal do Paraná – Campus de Dois Vizinhos, com objetivos de avaliar o potencial de preparados a base de calêndula (Calendula officinalis L.) na indução de fitoalexinas em cotilédones de soja, na indução de resistência em frutos de morango e o efeito fungistático sobre Botrytis cinerea in vitro. O delineamento experimental utilizado para os experimentos foi inteiramente casualizado com 15 tratamentos resultantes da combinação de três formas de extração (extrato alcoólico, infusão e maceração) e cinco concentrações (zero; 1,25; 2,5; 5 e 10%), sendo a concentração zero água destilada, em arranjo fatorial (3 x 5) com 4 repetições. O primeiro experimento avaliou o potencial fungistático dos preparados sobre B. cinerea in vitro. Em placas de Petri® foram adicionados no meio de cultura BDA (Batata Dextrose Ágar) os preparados nas diferentes concentrações. Após a solidificação, um orifício de 8 mm foi realizado no centro da placa e introduzindo-se 2μL da suspensão de conídios de B. cinerea. As placas foram mantidas em câmara de crescimento durante 7 dias a 25ºC, e no sétimo dia mediu-se o diâmetro do halo de crescimento do fungo. O segundo experimento avaliou a indução de fitoalexinas em cotilédones de soja em resposta aos preparados à base de calêndula. Sementes de soja foram semeadas em areia autoclavada e mantidas em temperatura ambiente por 10 dias. Em seguida os cotilédones das plântulas foram removidos e na face abaxial destes foram aplicados os tratamentos. Após seguir os procedimentos metodológicos da técnica de extração, obteve-se via espectrofotometria a quantificação da fitoalexina gliceolina. O terceiro experimento avaliou o efeito dos preparados a base de C. officinalis sobre os parâmetros físico-químicos e bioquímicos de frutos de morango relacionados com a indução de resistência. Os frutos foram acondicionados em bandejas plásticas e pulverizados com os diferentes tratamentos. Após 6 horas da pulverização dos preparados foi pulverizada solução contendo cerca 104 conídios do fungo B. cinerea. As avaliações foram realizadas após 3 dias da implantação do experimento. Os parâmetros avaliados foram: perda de massa, incidência de podridões, acidez titulável, firmeza de polpa e sólidos solúveis totais, e as bioquímicas foram: açúcares totais, antocianinas, flavonóides e atividade da enzima fenilalanina amônia-liase (FAL). Os resultados demonstraram que a maceração em todas as suas concentrações inibiu o crescimento do fungo B. cinerea in vitro, sendo que a partir de 2,5% observou-se inibição total. O tratamento com infusão na sua maior concentração (10%) também apresentou resposta positiva na inibição do crescimento de B. cinerea. Os preparados de C. officinalis apresentaram capacidade de indução das fitoalexinas gliceolinas em cotilédones de soja. Na aplicação dos preparados em pós-colheita de morangos, não ocorreu influência dos tratamentos sobre perda de massa, incidência de podridões, firmeza de polpa, acidez titulável, sólidos solúveis totais, açúcares totais e antocianina. Para flavonóides o extrato alcoólico em todas as concentrações e a infusão a partir de 5% estimularam sua produção. A atividade da enzima FAL foi estimulada pela aplicação dos extratos.
Several studies have demonstrated the potential of medicinal plants in the pathogens control, through its direct fungistatic action or its ability to induce plant defense, it suggesting the presence of molecules with characteristics elicitors. Three experiments were carried out on 2009 and 2010, in the Universidade Tecnológica Federal do Paraná, Dois Vizinhos city (PR – Brazil). The aim of this work was to evaluate prepared caledula (Calendula officinalis L.) potential for phytoalexins induction in the soybean cotyledons, resistance induction in the strawberry fruits and fungistatic effect of Botrytis cinerea in vitro. The experimental design was completely randomized with 15 treatments resulting from the combination of three forms of extraction (alcohol extract, infusion and maceration) and five concentrations (zero, 1.25, 2.5, 5 and 10%), with zero concentration distilled water in a factorial (3 x 5) with four replications the distilled water was used as control. The first experiment evaluated the potential fungistatic preparations in the B. cinerea control. The preparations with the different concentrations were put in Petri dishes with BDA culture media. After the media solidification, a hole of 8 mm was punched in the dishes Petri center and in this hole was introduced 2μL of spore B. cinerea suspension. The Petri dishes were kept in growth chamber for 7 days at 25°C. In the seventh day, the halo diameter fungus growth was evaluated. The second experiment evaluated the phytoalexins induction in the soybean cotyledons soybean as result of C. officinalis derivatives. Soybean seeds were sown in sterilized sand and it were kept at room temperature during 10 days. Then, the seedlings cotyledons were removed and on the abaxial part it was applied the treatments. After methodological procedures of extraction technique, it was obtained the phytoalexins glyceolin quantification by for spectrophotometry. The third study evaluated the C. officinalis solutions effect on the strawberry fruits physic-chemical and biochemical characteristics. The fruits were packed in plastic trays and it sprayed with different treatments. After six hours of the treatments spraying, it was sprayed a solution containing about 10-4 spore of B. cinerea fungus. After 3 days, it was realized the evaluations. The mass loss, rots incidence, titratable acidity, flesh firmness, total soluble solids, totals sugars, anthocyanins, flavonoids and activity of phenylalanine-amonialiase (PAL) were evaluated. The results showed that the maceration with all over the concentrations used, it inhibited of the B. cinerea fungus in vitro growth. Concentrations higher that 2.5%, presented total inhibition ability. Treatment with infusion using the highest concentration (10%) also showed positive response for B. cinerea growth inhibiting. The C. officinalis solutions presented induction capacity of the phytoalexins glyceolin in soybean cotyledons. It was observed that the solutions applied during the strawberry postharvest didn’t influence in the physico-chemical characteristics (mass loss, rots incidence, titratable acidity, flesh firmness and total soluble solids). In the biochemical analyzes for totals sugars and anthocyanins wasn’t observed statistical difference among the treatments. The alcoholic extract all over the concentrations and the infusion with concentrations higher than 5% stimulated the flavonoids productions. The PAL enzyme activity was stimulated by the application of extracts.

Traditional Chinese medicine (TCM) plays an important role in the Chinese healthcare system for over five thousand years. It includes the use of herbal medicine, acupuncture, Tui Na (推拿), and diet therapy. TCM helps to maintain a balance of Yin-Yang (阴阳), Five Phases (五行), Meridians (经络) and Qi (气) inside the body. In practise, pregnant women take tocolytic drugs to tonify the blood and qi to provide a continuous supply of nutrients for baby. Traditional Chinese herbal medicines usually prescribed as a complex formula to produce synergistic or agonistic effect to maintain a well balance of the above components in human bodies. Moreover, TCM usually cannot produce immediate effect on patients, therefore, the efficacy of individual component remains largely unknown. This study aims to investigate whether Chinese tocolytic drug components could modulate fertility by affecting the in vitro spheroid (blastocyte surrogate) attachment process by using trophoblastic (JEG-3) and endometrial epithelial (Ishikawa) cells to mimic the embryo-endometrial implantation process. Nine Chinese herbal medicinal compounds (Atractylenolide I(白术内酯), Atractylenolide II(白术内酯II), Atractylenolide III(白术内酯III), Paeoniflorin(芍药苷), Albiflorin(芍药内酯苷), Nuzhenide(女贞子甙), Ecliptasaponin A(旱莲甙A), Wedelolactone(蟛蜞菊内酯) and Columbianadin(二氢欧山芹醇当归酸酯)) which are commonly found in traditional Chinese tocolytic drug formula were selected to study (1) the toxicity of the drugs on trophoblastic (JEG-3) and endometrial epithelial (Ishikawa) cells growth, (2) the effect of three tocolytic drugs (Atractylenolide I, Atractylenolide II and Atractylenolide III) on spheroid attachment, and (3) their effect of the expression of Wingless (Wnt) signaling molecules (Active-β-Catenin, Axin-2, β-catenin, E-cadherin, GSK-3β, and Mucin-1). It was found that the nine compounds, Atractylenolide I, Atractylenolide II, Atractylenolide III, Paeoniflorin, Albiflorin, Nuzhenide, Ecliptasaponin A, Wedelolactone and Columbianadin did not affect cell viability at 25μM, 25μM, 5μM, 0.2μM, 125μM, 125μM, 125μM, 5μM and 25μM, respectively, by cell proliferation assay. However, at these concentrations, the spheroid attachment was not significantly increased by Atractylenolide I, Atractylenolide II and Atractylenolide III. Interestingly, the protein expression of GSK-3β and Active-β-catenin were up-regulated by the three compounds in both cells and JEG-3 cells respectively. The expressions of Axin-2 and E-cadherin were up-regulated by Atractylenolide III in Ishikawa cells and Atractylenolide II in JEG-3 cells. Atractylenolide I and Atractylenolide III increase the Ishikawa cells expression of Active-β-catenin and β-catenin respectively and together suppress the JEG-3 cells Mucin-1 and β-catenin expression. In conclusion, the nine tocolytic compounds have different effect on cell proliferation. Atractylenolide I, Atractylenolide II and Atractylenolide III did not enhance the attachment rate of JEG-3 spheroid onto Ishikawa monolayer. However, they affected Wnt-signaling molecules expression, suggesting that they may modulate endometrial receptivity. Further experiments are needed to study their combined effect on co-culture and expression of Wnt-signaling molecules.
published_or_final_version
Obstetrics and Gynaecology
Master
Master of Medical Sciences

Orientador: Carmen Silvia Fernandes Boaro
Banca: João Domingos Rodrigues
Banca: Giuseppina Pace P. Lima
Banca: Marcos Roberto Furlan
Banca: Antonio Natal Gonçalves
Resumo: Pertencente à família Meliaceae, a espécie Trichilia catigua A. Juss é conhecida popularmente como catigua, cataguá, argelim-rosa e mangalto-catingam. Sua casca apresenta propriedades adstringente, inseticida, purgativa, tônica, bactericida, antiinflamatória e antidepressiva. Com o objetivo de propagar a espécie T. catigua foram desenvolvidos experimentos testando o enraizamento de estacas e a micropropagação com explantes de matrizes e sementes. Os experimentos de enraizamento de estacas foram realizados na primavera 2004, verão 2004/2005, outono, inverno e primavera 2005 e primavera 2006. Em todos os experimentos, estacas com aproximadamente 15 cm de comprimento foram coletadas de árvores adultas e preparadas da parte apical e mediana dos ramos. A seguir, foram submetidas aos reguladores vegetais IBA (ácido indolbutírico), NAA (ácido naftalenoacético) e IAA (ácido 3-indolacético), variando as dosagens. Para a avaliação dos experimentos determinou-se a percentagem de estacas enraizadas, não enraizadas e mortas e quando enraizadas, seu comprimento e diâmetro. No experimento primavera de 2004 foram testadas as concentrações de 1000 e 2000 mg L-1 dos reguladores vegetais IBA, NAA e IAA. As avaliações aos 90 dias após sua instalação revelaram maiores percentagens de enraizamento e iguais a 33,33, 25,00, 22,91 e 23,43 % para estacas submetidas a IBA 1000, 2000 mg L-1 e NAA 1000 e 2000 mg L-1, respectivamente. No verão 2004/2005, outono, inverno e primavera 2005 os experimentos foram conduzidos com as concentrações dos reguladores IBA, NAA e IAA iguais a 1000, 2000 e 3000 mg L-1 e as avaliações foram realizadas após 120 dias. Não houve enraizamento no outono e inverno. A análise conjunta dos resultados obtidos na primavera e no verão mostrou percentagem de enraizamento superior na primavera. A maior percentagem de enraizamento, igual a 19,17%... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The Trichilia catigua A. Juss from the Meliaceae family is popularly known as catigua, cataguá, argelim-rose and mangalto-catingam. Its bark has astringent, insecticide, purgativa, tonic, bactericide, anti-inflammatory and antidepressant properties. With the aim of propagate T. catigua, experiments of rooting with stem cuttings and of micropropagation with explants of trees and seeds were carried out. In all the rooting experiments the stem cuttings with approximately 15 cm of length were collected from adult trees and prepared from the apical and intermediate parts. The cuttings were immersed in the vegetable regulators IBA (Indole-3- butyric acid), ANA (Naphthalene acetic acid) and IAA (Indole-3 acetic acid). The rooted stem cutting and not rooted stem cutting percentage and, when rooted, the length and diameter of roots, were evaluated. In the experiment spring 2004 the concentrations of 1000 and 2000 mg L-1 of IBA, ANA and IAA were tested, with evaluations 90 days after installation. The highest rooting percentage were 33,33, 25,00, 22,91 and 23,43% for IBA 1000, 2000 mg L-1 and ANA 1000 and 2000 mg L-1, respectively. In the summer of 2004/2005, autumn, winter and spring of 2005 IBA, ANA and AIA, with concentration of 1000, 2000 and 3000 mg L-1, were tested. The evaluation was carried out at 120 days. No rooting was observed in autumn and winter. The analysis of data from summer and spring showed higher rooting percentage in spring. The highest rooting percentage was obtained with IBA 3000 mg L-1 (19,17%). In the spring 2006 IBA (1000, 2000, 3000, 4000 and 5000 mg L-1) and ANA (1000, 2000, 3000 mg L-1) were tested. The highest rooting percentage (41,67%) was obtained with IBA 5000 mg L-1. In the in vitro cultivation, explantes obtained from trees were submitted to asepsis treatments with HgCl2, CaOCl2 and NaOCl and inoculated in Murashige & Skoog culture medium (MS) with 25%... (Complete abstract click electronic access below)
Doutor

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: Sutherlandia frutescens (L.) R. Br., also regarded as Lessertia frutescens, is a leguminous, perennial shrub indigenous to South Africa. Extracts prepared from the leaves have traditionally been used for the treatment of various diseases. Reports have also indicated that S. frutescens provides certain health benefits to cancer and HIV/AIDS patients. Analysis of extracts indicated the presence of several compounds (bitter triterpenoid glycosides, several flavonoids, amino acids, small amounts of saponins (no alkaloids though), asparagine, Larginine, canavanine, gamma-aminobutyric acid (GABA) and pinitol) which contribute to the medicinal properties of this plant. The first part of this study involved testing the effect of six treatments (light, dark, soaking of seeds, physical scarification, chemical scarification and flaming of seeds) on the in vitro germination of Sutherlandia seeds to elucidate the factors which control seed germination. Those treatments which removed the seed coat were most successful for germination with physical scarification being the most efficient method, resulting in 98.6% of the seeds germinating after 21 days. Although the organogenesis of Sutherlandia explants (cotyledons and hypocotyls) in vitro were investigated (results not included in this thesis), omitting plant growth regulators (PGR) in the cultivation medium was best for shoot multiplication. However, this PGR-free system successfully provided a continuous supply of plant material for further studies. It would be possible to successfully adopt it for commercial production of plants to assist with cultivation of Sutherlandia as a field crop. Another advantage of this system is spontaneous rooting with 85% of the in vitro microshoots rooting in PGR-free medium. These rooted plants were acclimated in the glasshouse using vented lids to harden off the shoots and this method resulted in 100% survival of plants. The second part of this study investigated the induction of hairy root cultures of S. frutescens using Agrobacterium-mediated transformation. The efficiency of three Agrobacterium strains (A4T, LBA9402 and C58C1) to transform different S. frutescens explants (cotyledons and hypocotyls) was analyzed. All three strains were equally efficient at inducing hairy roots in both hypocotyls and cotyledons. However, transformation of S. frutescens was dependent on the type of explant used with the hypocotyls being more efficiently transformed than the cotyledons. Overall the transformation of both the hypocotyl (93%) and cotyledon (47%) was highest when the strain A4T was used. Four hairy root clones were selected and their cultivation in a liquid system was optimized by investigating their growth in four different types of media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934; White, 1954), MS (Murashige and Skoog, 1962) and half strength MS medium). All the growth of hairy root clones was best in the B5 and MS medium, with White’s medium being the least effective cultivation medium. Molecular analysis of hairy roots was used to prove the transgenic status of these four putative transgenic clones. This was achieved using polymerase chain reaction (PCR) amplification of rol A (320 bp), B (780 bp) and C (600 bp) genes to determine the presence of the TL-DNA in the plant genome. During Southern hybridization a radioactively labeled rol A probe was used to determine the copy number of the rol A gene. The three rol genes were present in all four hairy root clones. The third part of this study focused on the effect of three abiotic stress factors (nitrogen availability, salinity and drought) on the synthesis of four metabolites (gamma-aminobutyric acid (GABA), asparagine, arginine and canavanine). The effect of nitrogen availability on metabolite synthesis and the morphology was determined using in vitro shoot cultures as well as the hairy root clone C58C1-g. Nitrogen availability studies were conducted by cultivating the microshoots or root tips on modified MS medium. The MS medium contained either the normal amount of nitrogen (1.9 g L-1 KNO3 and 1.65 g L-1 NH4NO3) in the MS medium (1x nitrogen), half the normal nitrogen concentration in MS medium (0.5x nitrogen) or twice the normal nitrogen concentration in MS medium (2x nitrogen). The arginine and asparagine levels in the roots and shoots and the canavanine level in the shoots were directly correlated with the amount of nitrogen in the medium (as the nitrogen level increased, the metabolite levels increased). The GABA level in the shoots was inversely correlated with the amount of nitrogen in the medium. Several reasons may explain these metabolic changes including the assimilation of extra nitrogen into asparagine, canavanine and arginine in the shoots. The reduced GABA levels may indicate the preferential flux of the free GABA into other nitrogen assimilatory pathways such as protein synthesis as well as its rapid utilization to replenish the tricarboxilic acid cycle intermediates. The effect of water (induced by including 3% (w/v) PEG in the medium) and salt stress (induced by including either 50 or 100 mM NaCl in the medium) was only investigated in the shoot cultures as the root cultures lacked the synthesis of canavanine. Water stress did not significantly alter the metabolite levels, but resulted in a significant decrease in the growth (fresh weight and total shoot length) and the rooting response of these microshoots. Salt stress only resulted in a significant increase in arginine levels with increasing salinity and also caused a reduction in the rooting and growth response. Lowered plant vigour may be the first visual sign of water stress. Addition of NaCl may lead to ion toxicity and requires osmotic adjustment resulting in changes at the metabolic level concomitant to physiological growth changes. Finally, the anti-bacterial activity and the phytochemistry of transgenic root cultures and untransformed in vitro and ex vitro plant material was examined. Only the extracts prepared from the wild harvested leaf material exhibited moderate anti-bacterial activity (1.25 mg ml-1) against all the bacteria (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis and Staphylococcus aureus) tested. Changes to the secondary metabolism of hairy roots were investigated using TLC and LC-MS analysis. Several of the compounds in the hairy root extracts were present in higher levels than in the control root extracts. Transformation also increased the complexity of the phytochemical pattern of the hairy roots, either due the synthesis of novel compounds or upregulated synthesis of existing metabolic pathways. The production of hairy roots and the establishment in a liquid system during this study was an important step towards upscaling these cultures to a bioreactor. In future these roots can assist in developing cultures which produce a high yield of the desired metabolites.
AFRIKAANSE OPSOMMING: Sutherlandia frutescens (L.) R. Br., ook bekend as Lessertia frutescens is ‘n peulagtige meerjarige struik, inheems tot Suid Afrika. Ekstrakte wat van die blare voorberei word, is tradisioneel gebruik vir die behandeling van verskeie siektes. Berigte het ook daarop gedui, dat S. frutescens sekere gesondheidsvoordele vir kanker en HIV/VIGS pasiënte inhou. ‘n Ontleding van die ekstrakte, dui op die teenwoordigheid van verskeie verbindings (bitter triterpenoïed glikosiede, verskeie flavonoïede, aminosure, klein hoeveelhede saponiene (alhoewel geen alkaloïede), asparagien, L-arginien, canavanien, gamma-aminobottersuur (GABS) en pinitol) wat tot die medisinale eienskappe van hierdie plant bydrae. Die eerste deel van die studie het die effek van ses behandelings (lig, donker, week van sade, fisiese skarifikasie, chemiese skarifikasie en die vlam van sade) op die in vitro ontkieming van Sutherlandia sade getoets met die doel om die faktore wat saadontkieming beheer, te identifiseer. Die beste behandeling vir saadontkieming was dié behandelings wat die saadhuid verwyder het. Die mees effektiewe metode van saadhuidverwydering was die fisiese skarifikasie van sade, wat gelei het tot ‘n 98.6% ontkieming van sade na 21 dae. Alhoewel in vitro organogenese van Sutherlandia eksplante (kotiel en hipokotiel) ondersoek was (resultate nie ingesluit in die tesis nie), was plant groei reguleerders (PGR) uitgesluit in die groeimedium om stingelvermeerdering te bevorder. Nie te min was die PGR-vrye sisteem suksesvol om ‘n voortdurende bron van plant material vir verder studies te verskaf. Dit sou egter moontlik wees om die PGR-vrye sisteem suksesvol te kon aanpas vir die kommersiële produksie van plante met die doel om Sutherlandia as ‘n landbougewas te bevorder. ‘n Verdere voordeel van dié sisteem, is die spontane wortelvorming, met 85% van die in vitro mikrostingels wat wortels in die PGR-vrye medium produseer het. Hierdie bewortelde plante was in die glashuis geakklimatiseer met behulp van geventileerde deksels (vir stingel afharding) en het tot ‘n 100% oorlewing gelei. Die tweede deel van die studie het die induksie van S. frutescens harige wortelkulture met behulp van Agrobacterium-bemiddelde transformasie ondersoek. Die effektiwiteit van drie Agrobacterium stamme (A4T, C58C1 en LBA9402) om verskillende S. frutescens eksplante (kotiel en hipokotiele) te transformeer, was geanaliseer. Al drie stamme was ewe effektief om harige wortels op beide hipokotiel en kotiele te induseer. S. frutescens transformasie blyk egter tog van die tipe eksplant afhanklik te wees, aangesien die hipokotiele meer effektief as die kotiele getransformeer kon word. Met inagneming van beide die hipokotiel (93%) en kotiel vii (47%), was transformasie optimaal met die gebruik van die A4T stam. Vier harige wortelklone was geselekteer en hulle produksie in ‘n vloeibare sisteem was geoptimiseer deur hulle groei in vier verskillende tipe media (Gamborg B5 (Gamborg et al., 1968), White’s (White, 1934; White, 1954), MS (Murashige and Skoog, 1962) en half-sterkte MS medium) te ondersoek. B5 en MS medium was beskou as die beste vir alle die harige wortelklone se groei, terwyl White’s medium die minste doeltreffende groeimedium was. Molekulêre analise van die harige wortels was gebruik ten einde die transgeniese status van die vier vermoedelike transgeniese klone te bewys. Dit was behaal deur polimerase kettingreaksie amplifisering (PKR) van die rol A, B en C gene ten einde die teenwoordigheid van die TL-DNS in die plant genoom aan te toon. Tydens Southern hibridisasie was ‘n radioaktief gemerkte peiler gebruik om die aantal rol A geen kopieë te bepaal. Die drie rol gene was teenwoordig in al vier harige wortelklone. Die derde deel van die studie het gefokus op die effek van drie abiotiese stress faktore (stikstof beskikbaarheid, sout- en droogte stres) op die produksie van vier metaboliete (GABS, asparagien, canavanien en arginien). Die effek van stikstof beskikbaarheid op die metaboliet produksie asook die morfologie was bestudeer deur gebruik te maak van in vitro mikrostingels asook die harige wortel kloon C58C1-g. Stikstof beskikbaarheidstudies was uitgevoer deur die mikrostingels of wortelpunte in ‘n gewysigde MS medium te groei. Die MS medium was aangepas om die normale hoeveelheid stikstof (1.9 g L-1 KNO3 en 1.65 g L-1 NH4NO3) in MS medium (1x stikstof), of die helfte van die normale stikstof konsentrasie (0.5x stikstof) of twee keer die normale stikstof konsentrasie in MS medium (2x stikstof) te bevat. Die arginien en asparagien vlakke in die wortels en stingels, asook die canavanien vlak in die stingels was positief gekorreleerd aan die stikstof konsentrasie in die medium. Die GABS vlak in die stingels was egter omgekeerd eweredig aan die stikstof konsentrasie in die medium. Verskeie redes kan aangevoer word om die metaboliet veranderinge te verduidelik, insluitende die assimilasie van addisionele stikstof in asparagien, canavanien en arginien in die stingels. Die verlaagde GABS vlakke kan dui op die voorkeur van vrye GABS vloei na ander stikstofassimilerende metaboliese paaie soos proteïen sintese, asook die snelle benutting van GABS ten einde die Trikarboksielsuursiklus intermediêre produkte aan te vul. Die effek van droogte (geïnduseer deur die byvoeging van 3% (m/v) PEG tot die medium) en sout stres (geïnduseer deur 50 of 100 mM NaCl byvoeging tot die medium) was slegs in die stingel kulture ondersoek weens die afwesigheid van canavanien produksie in die wortel kulture. Water stres het nie ‘n betekenisvolle verandering in die metaboliet vlakke meegebring nie, maar dit het wel tot ‘n beduidende afname in groei (vars massa en totale stingel lengte) en bewortelingsreaksie in die mikrostingels gelei. Sout stres het slegs tot ‘n betekenisvolle viii toename in arginien vlakke asook ‘n afname in die wortelvorming en groeireaksie tydens die toenemende sout vlakke gelei. ‘n Verlaging in plant groeikragtigheid mag ‘n eerste visuele teken van water stres wees. Die toevoeging van NaCl tot die medium kan tot ioontoksisiteit lei en plante reageer deur middel van osmotiese aanpassing wat tot veranderinge in die metaboliet vlakke asook veranderinge in fisiologiese groei, lei. Die finale deel van die studie het die anti-bakteriële aktiwiteit en die fitochemie van die transgeniese wortel kulture asook die ongetransformeerde in vitro en ex vitro plant materiaal ondersoek. Slegs die ekstrakte verkry vanaf blaar materiaal geoes uit die natuur, het matige anti-bakteriële aktiwiteit (1.25 mg ml-1) teen al die bakterië (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis en Staphylococcus aureus) wat ondersoek is, getoon. Aanpassings in die sekondêre metabolisme van die harige wortels is deur middel van dunlaag chromatografie (DLC) en vloeibare chromatografie-massa spektroskopiese (VC-MS) analise ondersoek. Verskeie verbindings was in hoër vlakke in die harige wortels teenwoordig, as in die kontrole wortel ekstrakte. Transformasie het ook die kompleksiteit van die harige wortels se fitochemiese patroon verhoog, moontlik weens die produksie van nuwe verbindings of weens die opregulasie van bestaande metaboliese paaie. Die produksie van harige wortels en die vestiging daarvan in ‘n vloeibare sisteem tydens hierdie studie word beskou as ‘n belangrike stap na die opskalering van die kulture na bioreaktore. Hierdie wortels kan toekomstig tot die ontwikkeling van kulture met ‘n hoë produksie van gewenste metaboliete lei.

UA Open Access Publishing Fund
Background: Ethnobotanical knowledge continues to be important for treating illness in many rural communities, despite access to health care clinics and pharmaceuticals. However, access to health care clinics and other modern services can have an impact on the distribution of medical ethnobotanical knowledge. Many factors have been shown to be associated with distributions in this type of knowledge. The goal of the sub-analyses reported in this paper was to better understand the relationship between herbal remedy knowledge, and two such factors, age and social network position, among the Yucatec Maya in Tabi, Yucatan. Methods: The sample consisted of 116 Yucatec Maya adults. Cultural consensus analysis was used to measure variation in herbal remedy knowledge using competence scores, which is a measure of participant agreement within a domain. Social network analysis was used to measure individual position within a network using in-degree scores, based on the number of people who asked an individual about herbal remedies. Surveys were used to capture relevant personal attributes, including age. Results: Analysis revealed a significant positive correlation between age and the herbal medicine competence score for individuals 45 and under, and no relationship for individuals over 45. There was an insignificant relationship between in-degree and competence scores for individuals 50 and under and a significant positive correlation for those over 50. Conclusions: There are two possible mechanisms that could account for the differences between cohorts: 1) knowledge accumulation over time; and/or 2) the stunting of knowledge acquisition through delayed acquisition, competing treatment options, and changes in values. Primary ethnographic evidence suggests that both mechanisms may be at play in Tabi. Future studies using longitudinal or cross-site comparisons are necessary to determine the whether and how the second mechanism is influencing the different cohorts.