Submitted in fulfillment for the Degree of Doctor of Philosophy (Biotechnology), Durban University of Technology, Durban, South Africa, 2016. / Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB) has infected approximately one-third of the world population, with 9.6 million TB cases in 2014. The emergence of multi-drug resistant (MDR) and extensively-drug resistant (XDR) strains of MTB has further complicated the problem of TB control. It is now imperative that novel antimycobacterial compounds are discovered in order to treat infections and reduce the duration of current TB therapy courses. For centuries, medicinal plants have been used globally worldwide for the treatment and prevention of various ailments. This occurs particularly in developing countries where infectious diseases are endemic and modern health facilities and services are inadequate. In recent years, the use and search for plant drug derivatives have been fast-tracked. Ethnopharmacologists, botanists, microbiologists, and natural product chemists are trying to discover phytochemicals which could be developed for the treatment of infectious diseases, especially TB. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimycobacterial activity. In the search for new lead compounds, nine medicinal plant species, Buddleja saligna, Capparis tomentosa, Carpobrotus dimidiatus, Dichrostachys cinerea, Ekerbergia capensis, Ficus Sur, Gunnera perpensa, Leonotis leonurus and Tetradenia riparia were collected in Kwa-Zulu Natal (KZN) following report of their therapeutic use in traditional medicine to treat symptoms and infections related to TB. They were tested in vitro for their activity against Mycobacterium smegmatis, Mycobacterium tuberculosis H37Rv (ATCC 25177) and three well-characterized clinical isolates of MDR-TB and XDR-TB using the agar incorporation method. The minimum inhibitory concentration of the active plant extracts was determined using the broth microdilution method. Our findings show that five of the nine plants screened have antimycobacterial activity with concentrations ranging from 125 µg/ml to 1000 µg/ml. The aqueous extracts of G. perpensa and T. riparia; and the methanolic extracts of B. saligna, C. tomentosa, and C. dimidiatus possessed significant activity against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177) and the three well-characterized clinical isolates of MDR-TB and XDR-TB. The cytotoxic effect of the active plant extracts was evaluated against the mouse BALB/C monocyte-macrophage (J774.2) and peripheral blood mononuclear cells (PBMCs). The toxic effects of the active plant extracts were evaluated using the brine shrimp lethality assay. Except for a high concentration of G. perpensa none of the other plants which possessed antimycobacterial activity showed any toxic or cytotoxic activity. The active plant extracts were thereafter assessed to determine if they had any effect on the survival or death of mycobacterial species, M. smegmatis, bound within the macrophage (J774.2) cell line at a concentration of 100 µg/ml. B. saligna had inactivated most of the phagocytosed bacilli after 24 hours of treatment therefore, it has a bactericidal effect on the mycobacteria located within the mouse macrophage. A phytochemical investigation of the leaves of B. saligna led to the isolation of two isomeric pentacyclic triterpene compounds namely Oleanolic Acid (OA) and Ursolic Acid (UA) using thin layer chromatography followed by silica gel column chromatography. The structures of these compounds were fully characterized by detailed NMR investigations, which included 1H and 13C NMR. Ursolic acid was isolated from this plant for the first time. Two-dimensional (2D) and three-dimensional (3D) quantitative structure-activity relationship (QSAR) studies were carried out to provide insight on the interaction of the compounds with the enzyme. Molecular docking studies predicted the free binding energy of the triterpenes inside the steroid binding pocket of Mycobacterium tuberculosis fadA5 thiolase compared to a reported inhibitor. Thus, their ability to inhibit the growth of Mycobacterium tuberculosis was predicted and was confirmed to possess significant antimycobacterial activity when tested against M. smegmatis, M. tuberculosis H37Rv (ATCC 25177), clinical isolates of MDR-TB and XDR-TB using the Microplate Alamar Blue Plate (MABA) assay. The present study has scientifically validated the traditional use of medicinal plant B. saligna. / D

Orientador: Wagner Vilegas / Banca: Ian Castro-Gambôa / Banca: Fernando Batista da Costa / Banca: Mitsue Haraguchi / Banca: André Luiz Meleiro Porto / Resumo: Neste trabalho foram estudadas a composição química e atividades farmacológicas de folhas de Alchornea glandulosa e Alchornea triplinervia (Euphorbiaceae) e partes aéreas de Indigofera truxillensis e Indigofera suffruticosa (Fabaceae), usadas na medicina popular brasileira no tratamento de problemas gástricos. Nos extratos clorofórmicos das espécies de Alchornea foram identificados estigmasterol, campesterol, sitosterol, lupeol, friedelan-3-ol, friedelan-3-ona e pteroginidina. O estudo dos extratos metanólicos das espécies de Alchornea forneceu ácido gálico, galato de metila, ácido carboxílico da brevifolina, amentoflavona, isocorilagina, miricetina 3-O-α-L-raminopiranosídeo, quercetina e quatro derivados glicosilados. Análise por FIA-ESI-IT-MS indicou a presença de derivados do ácido galoilquínico, elagitaninos e proantodicianidinas em A. triplinervia. Nos extratos clorofórmicos das espécies de Indigofera foram identificados índigo (indigotina) e indirubina. O extrato metanólico de I. truxillensis forneceu índigo, indirubina, indicana, pinitol, sitosterol 3-O-β-D-glicopiranosídeo e cinco derivados glicosilados do kaempferol, entre eles a substância inédita kaempferol 3-O-[α-Larabinopiranosil-( 2→1)-α-L-apiofuranosil] -7-O-α-L-raminopiranosídeo. O extrato metanólico de I. suffruticosa forneceu as mesmas substâncias descritas para I. truxillensis, exceto derivados do kaempferol, além do ácido gálico, alantoína e quatro derivados glicosilados da quercetina. Deste extrato também foram isoladas as substâncias inéditas 6'-metoxi 2, 5, 6-triidroxiisoindirubina, indigotina-3-O-β-D- glicopiranosídeo, dioxindol-3-O-β-D-glicopiranosídeo e quercetina 3-O-β-Dglicopiranosil-[( 4→1)-α-L-raminopiranosil]- (6→1)-α-L-apiofuranosídeo. O uso de FIA-ESI-ITMS possibilitou a identificação de glicerolipídeos, enquanto o emprego de HPLC-ESI-IT-MS permitiu...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In this work we studied the chemical composition and pharmacological activities of the leaves of Alchornea glandulosa and A. triplinervia (Euphorbiaceae), as well as aerial parts of Indigofera truxillensis and I. suffruticosa (Fabaceae), used in Brazilian folk medicine against gastric problems. In the chloroform extracts of the Alchornea species we identified stigmasterol, campesterol, sitosterol, lupeol, friedelan-3-ol, friedelan-3-one and pterogynidine. The study of the methanolic extracts of the Alchornea species afforded gallic acid, methyl gallate, brevifolin carboxylic acid, amentoflavone, isocorilagin, miricetin 3-O-α-L-rhamnopyranoside, quercetin and four glycosilated derivatives. Analyses by FIA-ESI-IT-MS helped to detect the presence of galloylquinic acid derivatives, ellagitannins and proanthodicyanidins in A. triplinervia. The chloroform extract of both Indigofera species led to the identification of indigo (indigotin) and indirubin. The methanolic extract of I. truxillensis gave indigo, indirubin, indican, pinitol, sitosterol-3-O-β-D-glucopyranoside and five glycosilated kaempferol derivatives, among them the new substance kaempferol 3-O-[β-D-apiofuranosyl-(1→2)-α-L- arabinopyranoside]-7-O-α- L-rhamnopyranoside. The methanolic extract of I. suffruticosa contained the same substances described for I. truxillensis, except for kaempferol derivatives, besides gallic acid, allantoin and four quercetin derivatives, besides the new substances 6'-methoxy-2,5,6- trihydroxiisoindirubin, indigotin-3-O-β-D-glucopyranoside, dioxindol-3-O-β-D-glucopyranoside and quercetin-3-O-[α- L-rhamnopyranosyl-(1→4)-[β-D-apiofuranosyl-(1→6)]-β-D-glucoyranoside. The use of FIAESI- IT-MS led the identification of glicerolipids, whereas the use of HPLC-ESI-IT-MS allowed to establish the chemical fingerprint of these species. The Alchornea species presented ...(Complete abstract click electronic access below) / Doutor

Philosophiae Doctor - PhD / South Africa (SA) is home to one of the six floral kingdoms of the world, and hosts a very diverse flora comprising an astonishing ~30,000 species. Herbal medicines play an important role in many of the diverse cultures of this country. Yet, agricultural production systems for most of these species are missing, and medicinal plants are usually collected in the wild. The endemic medicinal plants of SA produce a wide range of rare medicinally active compounds, which could be developed into drugs. Knowledge on the genes involved in the biosynthesis of these compounds could not only promote establishment of plant production systems, but also their biotechnological exploitation. Transcriptomics has been revolutionized by Next Generation Sequencing technologies, which can cost-efficiently provide a lot of information on plant genes and biosynthetic pathways. This thesis focuses on the establishment of methodologies for high-throughput plant transcriptome research, including: 1) harvesting plant material suitable for high-quality RNA analysis from distant locations, 2) high-throughput, and inexpensive biochemical sample screening, 3) extraction of high-quality RNA from recalcitrant, polysaccharide- and polyphenol rich plant material, and 4) biocomputational analysis of Illumina sequencing data, including quality control and pre-processing of data, de novo assembly of reads, protein prediction and functional transcriptome annotation. Rooibos (Aspalathus linearis) was chosen as the pilot plant, because it is one of the few indigenous SA medicinal plants that has been successfully cultivated as a commercial crop. It produces a wide range of phenolic compounds with health promoting properties (e.g. aspalathin and a phenylpropenoic acid glucoside with scientifically verified antidiabetic and cardioprotective effects). In the course of this study, seven rooibos transcriptomes were produced, assembled and functionally annotated, providing a first extensive dataset for identification of genes associated with economically important traits such as medicinal compound production, rooibos growth type characteristics and stress resistance.

Hepatocellular carcinoma (HCC), the major form of primary liver cancer, is a common cause of cancer-related death worldwide. Signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in the pathogenesis of HCC. Inhibition of STAT3 signaling has been proposed as a promising strategy for treating HCC. Due to the limitations of conventional therapeutics, increasing attention has been paid to complementary and alternative medicines (CAM) including traditional Chinese medicine (TCM) for the management of HCC. Antrodia camphorata (AC), a medicinal mushroom, is historically used for treating HCC. Pharmacological data showed that extracts and constituents of AC are able to inhibit STAT3 activation. Natural AC is scarce, cultured AC mycelia are becoming alternatives. AC mycelia have been demonstrated to possess anti-HCC properties. We hypothesize that inhibition of the STAT3 signaling pathway contributes to the anti-HCC mechanisms of AC mycelia. To test our hypothesis, we evaluated the safety and investigated the anti-HCC effects of the ethyl acetate fraction of an ethanolic extract of AC mycelia (EEAC); and we further explored the involvement of STAT3 signaling in EEAC's anti-HCC effects. Acute and repeated dose 28-day oral toxicity studies showed that EEAC had no toxicity in rats. The maximum tolerable dose for acute oral toxicity and the no-observed-adverse effects level for repeated dose 28-day oral toxicity of EEAC were higher than 5,000 mg/kg body weight and 1,000 mg/kg body weight, respectively, in rats. In cultured cells, EEAC is less toxic in normal liver-derived cells than in HCC cells. In HepG2 and SMMC-7721 cells, EEAC reduced viability, induced apoptosis, and retarded migration and invasion. In SMMC-7721 cell-bearing mice, EEAC significantly suppressed tumor growth. EEAC inhibited cell proliferation, induced apoptosis and suppressed angiogenesis in tumors. Mechanistic studies showed that EEAC downregulated protein levels of phosphorylated and total STAT3 and JAK2 (an upstream kinase of STAT3) in HCC cells and tumors. In cultured HCC cells, EEAC lowered the protein level of nuclear STAT3, decreased the transcriptional activity of STAT3, and downregulated protein levels of STAT3 targeted molecules. Over-activation of STAT3 in HCC cells diminished the cytotoxic effects of EEAC. STAT3 can be activated by receptor tyrosine kinases (RTKs). Phospho-RTK array assays showed that EEAC significantly inhibited the tyrosine phosphorylation of platelet-derived growth factor receptor-beta (PDGFR-β) in HepG2 cells. EEAC dose-dependently lowered mRNA levels of PDGF BB (a ligand of PDGFR-β) and protein levels of p-PDGFR-β and PDGFR-β in HCC cells. Activating PDGFR-β enhanced STAT3 activation, and inhibiting PDGFR-β blocked STAT3 activation in HCC cells. EEAC reversed PDGF BB induced STAT3 activation in HCC cells. Our data indicate that EEAC exerts anti-HCC effects, and inhibition of PDGFR-β/STAT3 signaling is, at least in part, responsible for these effects. In summary, we have demonstrated that EEAC exerts anti-HCC effects without significant toxicity in vitro and in vivo. We have also demonstrated that inhibition of PDGFR-β/STAT3 signaling contributes to the anti-HCC mechanisms of EEAC. Our findings provide a pharmacological basis for the development of EEAC as a modern anti-HCC agent and for the traditional use of AC in treating HCC. In addition, our data suggest that the PDGFR-β/STAT3 pathway plays a pathogenic role and presents a novel therapeutic target in HCC.

For centuries Aloe vera has been exploited for several verified and unverified medicinal uses such as wound healing, treatment of gastrointestinal ulcers and for its many biological effects including anti-microbial, laxative, anti-inflammatory and immunostimulatory activities. Studies have generally focused on its effects in vitro and in adults. When nursing mothers use Aloe vera extracts, their suckling infants are at risk of indirect exposure to Aloe vera via breast feeding or directly as dietary/health supplements. The gastrointestinal tract (GIT) of the neonate is sensitive to dietary manipulations during the suckling period with long lasting effects that can be irreversible. Thus babies may be at risk if administered Aloe vera extracts directly as dietary supplements or indirectly via breast milk. The main objectives of this study were to evaluate the effects of orally administered aqueous and alcohol extracts of Aloe vera on growth performance, the morphometry and morphology of the gastrointestinal tract and accessory organs, and liver function of suckling rats. Suckling Sprague-Dawley rats (77), males (n=38) and females (n=39) of 6 days old were randomly assigned to one of five treatment groups and given once daily by oral gavage a suspension of lyophilized crude alcohol or aqueous extracts of Aloe vera suspended in distilled water. Group I (control) was gavaged with distilled water (vehicle). Group II received a low dose of the aqueous extract (AqL) at 50mg. kg-1; Group III received a high dose of the aqueous extract (AqH) at 500mg. kg-1; Group IV received a low dose of the alcohol extract (AlcL) at 50mg. kg-1 whilst Group V received a high dose of the alcohol extract (AlcH) at 500mg. kg-1. The extracts and distilled water were 2 administered at a volume of 10ml.kg-1. The pups remained with their dams for the duration of the study and after 8 days on the treatments, the pups were humanely killed to harvest their tissues for measurements and physiological analysis. All data were expressed as mean ± SD and analyzed by one way ANOVA, the values were considered statistically significant when p < 0.05 and then a Bonferroni Post hoc test was applied. The suckling rats fed respectively with high doses of AlcH and AqH had a significantly higher body mass gain than the other groups (p < 0.05, one way ANOVA). Linear growth as measured by tibial length was significantly increased in the AqH group compared to the other groups. There was no significant difference in the mass and relative density of the tibia bones of the rats from the different treatment groups. The differences in growth could not be attributed to circulating concentrations of the somatotrophic hormone, Insulin-like growth factor-1 (IGF-1) which was not significantly different between the groups. The treatments did not result in any significant differences in lengths, and mass of the small and large intestine, however the caecum was significantly enlarged (hypertrophy of muscularis, submucosa and mucosa) in the rats that received the Aloe vera extracts. Although, there was no significant difference in the mass of the rats’ livers, the lipid and glycogen content were significantly higher (p < 0.001) for the AqH group compared to the other groups. Histologically, the hepatocytes showed enlarged nuclei, granular cytoplasm and dilated sinusoids for AqH and AlcH as compared to the control group. An indirect assessment of liver function by measurement of blood concentrations of alkaline phosphatase (ALP) and alanine amino transaminase (ALT) did not reveal a significant difference between the groups. The non fasting concentration of metabolic substrates (glucose and triglycerides) was also not significantly different between the groups. The pups given high doses of the extracts had a significantly greater (p < 0.05) thymus mass (hyperplastic) than the other groups. The short term administration of Aloe vera extracts has shown a growth promoting effect, enhanced hepatic storage of metabolic substrates and hypertrophy of the caecum and thymus of neonatal rats. These effects need to be explored further to enhance animal production and health.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg, 2014. / Heavy metal pollution is an increasing phenomenon and contamination of these heavy metals has detrimental effects on the environment and humans. The concentrations of metals in the soil, leaves, stem bark and flowers of Moringa ovalifolia sampled from Okahandja, Okaukuejo, Halali and Tsumeb in Namibia were investigated. Acid digestion of all samples was performed utilising the microwave technique. Determination of elements from the extracts were analysed by inductively coupled plasma - optical emission spectroscopy (ICP-OES). No lethal amounts of heavy metals were found in the soil, leaves, stem bark and flowers of Moringa ovalifolia. The results from Moringa ovalifolia was compared to spinach from another similar study. On average, Moringa leaves contained more or less similar amounts of Zn (29.4 mg kg-1) and Cr (13.2 mg kg-1) but higher higher amounts of Fe (263.8 mg kg-1) concentrations than spinach, with values of Zn (30.0 mg kg-1), Cr (20.0 mg kg-1) and Fe (190 mg kg-1) from a previous study. In this study, selected statistical methods such as correlation analysis and principal component analysis were used to identify the origin of these metals in the soil, leaves and stem bark collected from the Moringa farm, national parks and the road side in Namibia. From the correlation analysis, it was found that Co, Mg, Cr and Mg pairs were highly significant to each other. The principal component analysis revealed that heavy metals gave 53.41% of the total variance in factor 1. Sources of Mg and Ni could be from vehicle emissions. Factor 2 revealed As, Co, Al, and Si contributing to a total variance of 25.15% Co suggesting that the metals present could be as a result of geologic sources. This iii study proves that statistical analysis may provide a scientific foundation for the observation of heavy metal accumulation in samples. Pressurised hot water extraction (PHWE) was used for the extraction of bioactive compounds from the leaves and stem bark of Moringa ovalifolia and Moringa oleifera samples. The aim of the study was to assess the effect of temperature and extraction process of the Moringa extracts on Staphylococcus aureus (S. areus), Enterococcus faecalis (E. faecelis) and Pseudomonas aeruginosa (P. aeruginosa). The antibacterial activities of the extracts at varying temperatures were evaluated using the disk diffusion method where the zones of inhibition were recorded. ANOVA, which was calculated at a significance level of 0.05, was used to analyse the data. Antimicrobial activity of the stem bark extracts and leaf extracts from different sites extracted at 25˚C and 80˚C showed significant difference. As temperature increased, a decrease in inhibition was observed for stem bark extracts from different sites tested again S. areus, E. faecelis and P. aeruginosa. When the PHWE stem bark extracts collected in Tsumeb, were compared with ethanol and cold water extractions, the PHWE had more activity than other two methods. This proves that PHWE may be used as a novel extraction to get essential compounds from indigenous plant materials.

As raízes de Pfaffia paniculata (Ginseng brasileiro) são comercialmente encontradas como cápsulas contendo as raízes pulverizadas, misturadas ou não ao extrato etanólico destas raízes. Estas raízes são popularmente recomendadas para vários propósitos, e têm sido utilizadas na terapia contra o câncer pela medicina popular. Os principais componentes encontrados nestas raízes já isolados incluem o ácido pfáffico e os pfaffosideos A, C, D, E e F; estes componentes inibiram o crescimento de células do melanoma B-16, demonstrado em um outro estudo. O objetivo deste estudo foi investigar os efeitos dos extratos etanólico, butanólico ou aquoso das raízes de Pfaffia paniculata sobre o crescimento das linhagens tumorais de células mamárias humanas MCF-7 e SKBR-3, utilizando método colorimétrico (cristal violeta) e quantificação das células que incorporaram bromodeoxiuridina (BrdU). A coloração por laranja de acridina/brometo de etídeo foi utilizada para avaliar morte celular, e as alterações subcelulares foram avaliadas por microscopia eletrônica. O extrato butanolico apresentou efeitos citotóxicos nas linhagens MCF-7 e SKBR-3. Morte celular foi observada pelo tratamento com o extrato butanólico por 1 h; alterações morfológicas foram observadas com 500&micro;g/mL deste extrato, após 24 h de tratamento. Após o tratamento por 48 h com o extrato butanólico nesta mesma concentração, foi observado degeneração de componentes citoplasmáticos e alterações morfológicas sugestivas de morte celular. O tratamento com 1000&micro;g/mL deste extrato levou a profundas alterações citoplasmáticas e nucleares, incompatíveis com a viabilidade celular. O tratamento com o extrato etanólico não causou efeitos significantes no crescimento das células MCF-7 e SKBR-3; o extrato aquoso, por outro lado, estimulou o crescimento das células MCF-7, após o tratamento por 1 h. Estes resultados indicam efeitos citotóxicos exercidos pelo extrato butanólico das raízes de Pfaffia paniculata em linhagens celulares de tumor de mama humano / The roots of Pfaffia paniculata (Brazilian ginseng) are commercially available as capsules containing the powdered roots, mixed or not with the ethanolic extract of the roots. These roots have been populary recommended for many purposes, and have also been used on cancer therapy by folk medicine. The main components found in these roots that have already been isolated include pfaffic acid and pfaffosides A, C, D, E and F; these components inhibited the growth of melanoma B-16 cells, as shown in another study. The aim of this study was to investigate the effects of ethanolic, butanolic or aqueous extract of the roots of Pfaffia paniculata on the growth of human breast tumor cell lines MCF-7 and SKBR-3, using a colorimetric method (crystal violet) and quantification of bromodeoxiuridine (BrdU) positive cells. Acridine orange/ethidium bromide staining was utilized to evaluate cell death, and subcellular alteration was evaluated by electron microscopy. The butanolic extract presented cytotoxic effects in MCF-7 and SKBR-3 cell lines. Cell death was observed following treatment with the butanolic extract for 1 h; morphologic alterations were observed with 500&micro;g/mL of this extract, after 24 and 48 h of treatment. Following treatment for 48 h with the butanolic extract with this same concentration, degeneration of cytoplasmic components and morphological alterations suggestive of cell death were observed. Treatment with 1000&micro;g/mL of this extract lead to profound cytoplasmic and nuclear alterations, incompatible with cell viability. The treatment with ethanolic extract didn´t lead to significant effects on the growth of MCF-7 and SKBR-3 cells; the aqueous extract, on the other hand, stimulated the growth of MCF-7 cells, following treatment for 1 hour. These results point to cytotoxic effects exerted by the butanolic extract from the roots of Pfaffia paniculata on human breast tumor cell lines

Considerando que diversas atividades biológicas dos ácidos clorogênicos já foram descritas na literatura, uma melhor compreensão da biossíntese e do acúmulo desses polifenois nas plantas está diretamente vinculada aos parâmetros de qualidade de fitoterápicos. Este trabalho teve o objetivo de monitorar a variação na concentração de metabólitos secundários do grupo dos ácidos clorogênicos no tecido foliar de diversas espécies diante de diferentes tratamentos pós-coleta. Este monitoramento foi realizado através da quantificação das substâncias por cromatografia líquida de ultra eficiência acoplada à espectrometria de massas em sequência (CLUE-EM/EM). Na primeira fase do estudo, foram coletadas folhas de cada espécie selecionada para o estudo (Caju - Anacardium occidentale L.; Graviola - Annona muricata L.; Pata-de-Vaca - Bauhinia variegata L.; Limão - Citrus limon (L.) Burm. f.; Café - Coffea arabica L.; Pitanga - Eugenia uniflora L.; Alecrim - Rosmarinus officinalis L., e Jambolão - Syzygium cumini (L.) Skeels). As folhas foram submetidas a um processo de extração e os extratos obtidos foram analisados por CLAE-EM/EM. Foram encontradas nove substâncias monossubstituídas nas posições 3, 4 e 5 pertencentes aos subgrupos dos ácidos p-cumaroilquínicos, cafeoilquínicos e feruloilquínicos dos ácidos clorogênicos, além de ácidos dicafeoilquínicos. A identificação dos sinais cromatográficos foi realizada por meio dos padrões de fragmentação de cada composto. Na segunda fase do estudo, foram coletadas quatro folhas dos indivíduos em estudo. As folhas foram congeladas em nitrogênio líquido (para interrupção do metabolismo) em tempos diferentes: imediatamente após a coleta (T0), 30 min após a coleta (T0,5), 1 h após a coleta (T1) e 2 h após a coleta (T2). Este procedimento foi realizado uma vez por mês durante seis meses. Em outro momento foram retiradas seis folhas de três indivíduos de espécies selecionadas (Alecrim, Pitanga, Pata-de-Vaca, Limão e Café) e cada folha foi congelada nos tempos T0 (imediatamente após a coleta), T6 (6 h após a coleta), T12 (12 h após a coleta), T24 (24 h após a coleta), T48 (48 h após a coleta) e Ts (após secagem das folhas - aproximadamente 30 dias após a coleta). O material coletado foi extraído e analisado por CLUE-EM/EM, no modo MRM. O método analítico quantitativo foi validado, considerando os parâmetros de linearidade, exatidão e precisão. As concentrações dos ácidos clorogênicos em estudo não apresentaram um padrão de variação que se relacione com os tempos de tratamento ou um aumento linear nas respostas dentro do intervalo de 2 h após o início do catabolismo do tecido. As amostras com tempos de tratamento mais longo, por sua vez, apresentaram aumento significativo de diversos compostos nas folhas que não tiveram seu metabolismo interrompido 30 dias após a coleta. Este acúmulo de ácidos clorogênicos corrobora a hipótese de que o aumento da concentração destes compostos pode estar relacionado ao catabolismo de polímeros fenólicos de maior massa. / Considering that diverse chlorogenic acids\' biological activities have been described, a better understanding of their biosynthesis and accumulation is closely related to phytotherapics\' quality parameters. This research aimed to monitor the variation on the concentration of secondary metabolites from the chlorogenic acids\' group on the leaf\'s tissue of diverse species, towards different post-harvest treatments. The monitoring was performed through the quantification of the compounds by UPLCMS/ MS. On the first phase of the project, leaves from each selected species (Cashew - Anacardium occidentale L.; Soursop - Annona muricata L.; Pata-de-Vaca - Bauhinia variegata L.; Lemon - Citrus limon (L.) Burm. f.; Coffee - Coffea arabica L.; Cherry - Eugenia uniflora L.; Rosemary - Rosmarinus officinalis L., and Jambolan - Syzygium cumini (L.) Skeels) were harvested. This material was submitted to an extraction process, and the obtained extracts were analyzed by HPLC-MS/MS. Nine substances were found, monossubstituted on positions 3-, 4- and 5-, belonging to the subgroups of p-coummaroylquinic, caffeoylquinic and feruloylquinic acids, in addition to dicaffeoylquinic acids. The chromatographic signals\' identification was preceded observing the fragmentation pattern of each compound. On the second phase of the study, four leaves were collected from each individual. The leaves were frozen with liquid nitrogen (to interrupt the metabolism) on different moments - immediately after the collection (T0), 30 min after de collection (T0,5), 1 h after the collection (T1) and 2 h after the collection (T2). This procedure happened once a month during six months. On August, 2016, a different collection was performed for a broader observation: six leaves were harvested from three individuals from selected species (Rosemary, Cherry, Pata-de-Vaca, Limon and Coffee), and each leave was frozen on T0 (immediately after the collection), T6 (6 h after the collection), T12 (12 h after the collection), T24 (24 h after the collection), T48 (48 h after the collection) and Ts (after leaf\'s drying - approximately 30 days after collection). The harvested material was extracted and analyzed by UPLC-MS/MS, on MRM mode. The analytical method was validated for linearity, accuracy and precision. Chlorogenic acids\' concentrations did not show a pattern of variation that related to the different treatments or a linear increase on response until 2 hours after the beginning of the tissue catabolism. The samples submitted to a broader treatment interval, however, showed a significant rise on the concentration of different compounds, on leaves that did not have their metabolism interrupted until 30 days after the collection. This accumulation of chlorogenic acids agrees with the hypothesis that the raise on the concentration of these substances can be related to the catabolism of polymeric phenols of higher mass.

Cordia ecalyculata Vell. (Boraginaceae), espécie medicinal vulgarmente conhecida como porangaba e chá-de-bugre, é utilizada popularmente como emagrecedor, diurético, para curar tosses catarrais e reumatismos além de ser usada no tratamento de úlceras externas em forma de banhos. No Brasil existe grande confusão com relação aos nomes populares das espécies medicinais, fato que motivou a realização do presente trabalho. A espécie estudada é confundida com outras espécies vegetais, particularmente com Casearia sylvestris Swartz (Flacourtiaceae), também conhecida pelos nomes de guaçatonga e erva-de-bugre. O estudo farmacobotânico de C. ecalyculata revelou estruturas papilosas na epiderme e células esclereificadas no parênquima cortical, que não haviam sido descritas anteriormente. Foram listadas as principais diferenças morfoanatômicas entre as folhas de C. sylvestris e C. ecalyculata. auxiliares na diagnose da droga vegetal comercializada. As amostras adquiridas no comércio como porangaba não apresentaram características de C. ecalyculata, mas sim, de C. sylvestris, como: duas a três camadas de parênquima paliçádico; cavidades secretoras; drusas e cristais prismáticos em abundância; estômatos paracíticos; células epidérmicas poligonais; ausência de litocistos e areia cristalina. Fotomicrografias ilustram o trabalho. A triagem fitoquímica do extrato hidroetanólico liofilizado (EHEL) de C. ecalyculata apresentou resultado positivo para taninos, flavonóides e saponinas. A análise cromatográfica permitiu verificar a presença de substância coincidente com a alantoína no extrato de C. ecalyculata, além de possibilitar o desenvolvimento do perfil cromatográfico, auxiliar na diferenciação de C. sylvestris. Os ensaios de quantificação de alantoína no EHEL foram realizados por meio de dois métodos espectrofotométricos, cujos valores encontrados foram 0,68% e 0,70% de alantoína. O EHEL não apresentou róxicidade no ensaio de toxicidade aguda, na dose de 5 g/kg de massa corpórea do animal, por via oral. O experimento de atividade antiúlcera do EHEL de C. ecalyculata não apresentou diferenças significativas entre os animais controle e tratados. / Cordia ecalyculata Vell. (Boraginaceae) is a medicinal species most known as porangaba and chá-de-bugre. It is commonly used as diuretic, weight controller, for phleem cough healing, rheumatism healing and also in baths for externai ulcers treatment. The present work aims at clarifying the popular names of medicinal species often misnamed in Brazil. For example, the species cited above has always been mistaken as Casearia sylvestris Swartz. (Flacourtiaceae), also known as guaçatonga and erva-de-bugre. The pharmacological-botanical study of C. ecalyculata revealed papillary structures on the epidermis and sclerenchymatous cells on the cortical parenchyma which had not yet been described in the literature. The main morphological-anatomical differences between the C. sylvestris and the C. ecalyculata leaves were listed. These differences have been of great help concerning the diagnosis of commercialized vegetal drug. The commercialized samples do not show the features of the C. ecalyculata, but the features of the C. sylvestris, such as: two or three palisade parenchyma layers; secretive cavities; plenty of druses and prismatic crystals; paracytic stomata; polygonal epidermical cells; absence of lythocysts and crystal-sand, as it can be seen in the illustrative photomicrographies. The phytochemical screening of the lyophilized hydroethanolical extract (EHEL) of the C. ecalyculata showed a positive outcome for tannins, flavonoids and saponins. The chromatographic analysis not only allowed the evaluation of the coincident substance with the allantoin in the C. ecalyculata extract, but also enable the development of its chromatographic aspect, what eventually helped to differenciate it from C. sylvestris. The quantification assays of allantoin in the EHEL extract of the C. ecalyculata were performed through two spectrophotometric methods and the values found were 0,68% and 0,70% of allantoin. The EHEL extract displayed no toxicity in the acute toxicity trial in the 5 g/kg oral dosage per animal body weight. The anti-ulcer assay of this extract showed no meaningful differences among the subject and the treated animais.

Au cours des dernières décennies, un certain nombre de brevets déposés sur des médicaments à base de plantes se sont vus dénoncés comme des cas de « biopiratage ». Ils sont en effet accusés de constituer une appropriation indue des connaissances de peuples autochtones sur les vertus thérapeutiques de plantes. Il est vrai qu'en droit positif, les produits phytopharmaceutiques peuvent répondre aux conditions légales de la brevetabilité, quand les savoirs médicinaux ne font l'objet d'aucun mécanisme de réservation. En réaction à ces pratiques prolifèrent donc des initiatives visant à définir les instruments juridiques qui permettraient de faire bénéficier les peuples autochtones des fruits de l'exploitation de leurs connaissances. Le droit international s'est ainsi récemment enrichi d'accords visant à la protection des savoirs traditionnels, parmi lesquels la Convention sur la Diversité Biologique de 1992, prolongée par le Protocole de Nagoya en octobre 2010. Les principes posés doivent cependant être traduits en des règles concrètes pour être mis en œuvre. C'est pourquoi, à différents niveaux, les acteurs proposent voire développent leurs propres instruments pour tenter d'apporter une réponse au problème du biopiratage, à la fois dans et en dehors du système de protection de la propriété intellectuelle. Ce sont les modalités d'introduction de ces différents mécanismes en droit positif qu'il s'agit ici d'étudier. Pour ce faire, l'analyse économique du droit est convoquée pour évaluer les tenants et les aboutissants de réformes qui, pour se trouver à la frontière entre les droits de la propriété intellectuelle, de l'environnement, du développement et du commerce international, soulèvent des questions juridiques imprégnées de considérations éthiques, politiques et économiques. / Over the past decades, several patents granted for plant-based medicine have been denounced as “biopiracy” based on the assertion that they misappropriated indigenous people's knowledge over plants' therapeutic properties. Indeed, in the current state of law, while the pharmaceuticals may be the subject of patent law, medicinal knowledge is not the subject of any proprietary regime. Therefore, many initiatives have been suggested that aim at defining the appropriate legal instruments to ensure that a sharing of the benefits is imposed on pharmaceutical companies when utilising medicinal knowledge. The principle that the traditional knowledge associated with medicinal plants requires legal protection has already found expression in a number of political outputs, including such major international agreements as the “Convention on Biological Diversity” signed in 1992. This has been further developed with the Nagoya Protocol adopted in October 2010. However, the principles in these conventions still need to be translated into a concrete legal framework to be fully enforceable. This is why it is equally important to study legal practice to see how various actors can explore ways of dealing with the biopiracy issue, both within and outside the existing intellectual property system. This study assesses the legal accuracy of these posited options at the intersection of intellectual property, environmental, development and international trade laws. It uses an economic analysis of law to evaluate the background and the outcomes of reforms that raise not only legal, but also economic, political, and ethical burning issues.