Yip Pui Ying. / Thesis submitted in: November 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 147-184). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgment --- p.v / Abbreviations --- p.vii / Table of contents --- p.viii / List of Figures --- p.xii / List of Tables --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- The importance of characterization of Chinese medicinal materials and the development of Chinese medicine in Hong Kong --- p.1 / Chapter 1.2. --- Methods for characterization of Chinese medicinal materials --- p.5 / Chapter 1.3. --- Molecular characterization of Chinese medicinal materials --- p.8 / Chapter 1.3.1. --- DNA sequencing --- p.9 / Chapter 1.3.2. --- DNA fingerprinting --- p.14 / Chapter 1.3.3. --- Nucleic acid hybridization --- p.19 / Chapter 1.4. --- Objectives --- p.20 / Chapter Chapter 2 --- Characterization of Plant and Fungal Materials by rDNA ITS Sequence Analysis --- p.22 / Chapter 2.1. --- Introduction --- p.22 / Chapter 2.2. --- Materials and Methods --- p.22 / Chapter 2.2.1. --- Chinese medicinal materials used in this study --- p.22 / Chapter 2.2.1.1. --- Plants and fungi for interspecific ITS study --- p.22 / Chapter 2.2.1.2. --- Plant for intraspecific ITS study and locality study --- p.33 / Chapter 2.2.2. --- Extraction of total DNA --- p.35 / Chapter 2.2.3. --- PCR amplification of ITS1 and ITS2 regions of rRNA gene --- p.35 / Chapter 2.2.4. --- Purification of PCR products --- p.38 / Chapter 2.2.5. --- Cloning using pCR-Script´ёØ Amp SK(+) Cloning Kit --- p.38 / Chapter 2.2.5.1. --- Polishing --- p.38 / Chapter 2.2.5.2. --- Ligation of inserts into pCR-Script´ёØ Amp SK(+) cloning vector --- p.38 / Chapter 2.2.5.3. --- Transformation --- p.40 / Chapter 2.2.5.4. --- PCR screening of white colonies --- p.40 / Chapter 2.2.5.5. --- Purification of PCR screening products --- p.41 / Chapter 2.2.6. --- Sequencing of ITS regions --- p.41 / Chapter 2.2.6.1. --- Cycle sequencing reaction --- p.41 / Chapter 2.2.6.2. --- Purification of sequencing extension products --- p.41 / Chapter 2.2.6.3. --- Electrophoresis by genetic analyzer --- p.42 / Chapter 2.2.7. --- Sequence analysis and alignment --- p.42 / Chapter 2.3. --- Results --- p.42 / Chapter 2.3.1. --- Extraction of total DNA --- p.42 / Chapter 2.3.2. --- PCR amplification of ITS1 and ITS2 regions of rRNA gene --- p.44 / Chapter 2.3.2.1. --- Interspecific ITS study --- p.44 / Chapter 2.3.2.2. --- Intraspecific ITS study --- p.46 / Chapter 2.3.3. --- Sequence analysis and alignment --- p.47 / Chapter 2.3.3.1. --- Interspecific ITS study --- p.47 / Chapter 2.3.3.2. --- Intraspecific ITS study --- p.56 / Chapter 2.4. --- Discussions --- p.60 / Chapter 2.4.1. --- rDNA regions used for studying Chinese medicinal materials --- p.60 / Chapter 2.4.2. --- The results agreed with previously published works --- p.60 / Chapter 2.4.3. --- Explanation of interspecific results within the Ganoderma genus --- p.60 / Chapter 2.4.4. --- Implications from interspecific comparisons --- p.60 / Chapter 2.4.5. --- Implications from intraspecific comparisons --- p.61 / Chapter Chapter 3 --- .Characterization of Astragalus membranaceus by DNA Fingerprinting / Chapter 3.1 --- Introduction --- p.62 / Chapter 3.2 --- Materials and Methods --- p.62 / Chapter 3.2.1 --- Extraction of total DNA --- p.62 / Chapter 3.2.2 --- Generation and detection of DNA fingerprints by AP-PCR --- p.63 / Chapter 3.2.3 --- Analysis of DNA fingerprints --- p.63 / Chapter 3.3 --- Results --- p.63 / Chapter 3.3.1 --- Generation of DNA fingerprints by AP-PCR --- p.63 / Chapter 3.3.2 --- Fingerprint analysis --- p.69 / Chapter 3.4 --- Discussion --- p.85 / Chapter 3.4.1 --- RP-PCR has been used on Chinese medicinal materials --- p.85 / Chapter 3.4.2 --- AP-PCR used instead of RAPD --- p.85 / Chapter 3.4.3 --- Reproducibility and amount of bands --- p.86 / Chapter 3.4.4 --- Alternatives of electrophoresis process --- p.88 / Chapter 3.4.5 --- Explanation of results --- p.88 / Chapter 3.4.6 --- Distinguishing Neimengu and Shanxi samples --- p.89 / Chapter 3.4.7 --- Further studies --- p.90 / Chapter Chapter 4 --- Characterization of Plant and Fungal Materials by DNA-DNA Hybridization on Microarrays --- p.91 / Chapter 4.1 --- Introduction --- p.91 / Chapter 4.2 --- Materials and Methods --- p.92 / Chapter 4.2.1 --- Samples for microarray study --- p.92 / Chapter 4.2.2 --- Extraction of total DNA --- p.95 / Chapter 4.2.3 --- Amplification and sequencing of ITS 1 region of rRNA gene --- p.95 / Chapter 4.2.4 --- Preparation of labeled probe --- p.95 / Chapter 4.2.5 --- Amplification of ITS1 fragments --- p.97 / Chapter 4.2.6 --- Preparation of slides --- p.103 / Chapter 4.2.7 --- Hybridization and washing --- p.104 / Chapter 4.2.8 --- Scanning and data analysis --- p.105 / Chapter 4.3 --- Results --- p.105 / Chapter 4.3.1 --- DNA extraction --- p.105 / Chapter 4.3.2 --- Amplification and sequencing of ITS1 region of rRNA gene --- p.107 / Chapter 4.3.3 --- Preparation of labeled probe and amplification of ITS1 fragments… --- p.112 / Chapter 4.3.4 --- Preparation of slides --- p.112 / Chapter 4.3.5 --- Scanning and data analysis --- p.116 / Chapter 4.4 --- Discussion --- p.134 / Chapter 4.4.1 --- Implications --- p.134 / Chapter 4.4.2 --- Applying the findings --- p.134 / Chapter 4.4.3 --- Ways to maximize specificity --- p.137 / Chapter 4.4.4 --- Optimisation --- p.138 / Chapter 4.4.5 --- Microarray may be more advantageous over sequencing --- p.138 / Chapter Chapter Five --- General Discussion and Summary --- p.140 / Chapter 5.1. --- Objectives of this study --- p.140 / Chapter 5.2. --- rDNA ITS sequencing --- p.140 / Chapter 5.2.1. --- Description of the approach and summary of the results --- p.140 / Chapter 5.2.2. --- Implications from the results --- p.140 / Chapter 5.2.3. --- Advantages and limitations of DNA sequencing --- p.141 / Chapter 5.3. --- AP-PCR fingerprinting --- p.141 / Chapter 5.3.1. --- Description of the approach and summary of the results --- p.141 / Chapter 5.3.2. --- Advantages and limitations of DNA fingerprinting --- p.142 / Chapter 5.4. --- DNA-DNA hybridization on microarrays --- p.143 / Chapter 5.4.1. --- Description of the approach and summary of the results --- p.143 / Chapter 5.4.2. --- Implications from the results --- p.143 / Chapter 5.4.3. --- Advantages and limitations of DNA hybridization on microarrays. --- p.144 / Chapter 5.5. --- Overall summary --- p.144 / Chapter 5.6. --- Future studies --- p.146 / References --- p.147 / Appendix --- p.185

Objective To evaluate the cytotoxic and antibacterial effect of Bixa orellana L. (B. orellana) (achiote) methanol extract against Streptococcus mutans (ATCC 25175) (S. mutans) and Streptococcus sanguinis (ATCC 10556) (S. sanguinis). Methods Two methanol extracts of B. orellana were prepared in vitro, from the seeds and leaves. The antibacterial activity of extracts against S. mutans and S. sanguinis was evaluated using the cup-plate agar diffusion method. The minimum inhibitory concentration (MIC) was determined using the microdilution method and the cytotoxic activity was determinated by using the cell line MDCK. Results A stronger antibacterial effect was observed with the leaves methanolic extract with an inhibition zone of (19.97 ± 1.31) mm against S. mutans and (19.97 ± 1.26) mm against S. sanguinis. The methanolic extract of the seeds had an activity of (15.11 ± 1.03) mm and (16.15 ± 2.15) mm against S. mutans and S. sanguinis, respectively. The MIC of the leaf and the seed extracts against S. sanguinis was 62.5 and 125 μg/mL, respectively, and the MIC of the leaf extract against S. mutans was 62.5 μg/mL, and for the seed extract it was 31.25 μg/mL. The 50% cytotoxic concentration was 366.45 and 325.05 μg/mL for the leaves and seeds extracts, respectively. Conclusions The experimental findings demonstrated the antibacterial effect of the methanolic extract of B. orellana (achiote) on S. mutans and S. sanguinis. The extract of this plant is cytotoxic at high concentrations. / Peer review

O uso de plantas medicinais no tratamento de doenças tem aumentado de forma significativa nos últimos anos, bem como as pesquisas sobre a caracterização química destas plantas. Neste estudo, foram determinados os constituintes inorgânicos presentes nas folhas e nos extratos de três espécies da planta do gênero Casearia (C. sylvestris, C. decandra e C. obliqua) coletadas em regiões distintas da Mata Atlântica, SP. As composições elementares dos solos em que estas plantas foram cultivadas também foram determinadas. Tradicionalmente, estas plantas são utilizadas devido às suas propriedades antiinflamatórias, antiácidas, anti-sépticas e cicatrizantes. Além disso, as atividades antiulcerogênicas e antitumorais do gênero Casearia, e sua capacidade de neutralizar venenos de serpentes e abelhas, também tem sido comprovadas cientificamente. A metodologia analítica empregada foi a análise por ativação com nêutrons. Irradiações longas e curtas das amostras e dos padrões foram realizadas utilizando fluxos de nêutrons do reator de pesquisas IEA-R1 do IPEN/CNEN-SP. Nas folhas, o elemento K foi encontrado em níveis de porcentagens, os elementos Ca, Cl, Mg e Na na ordem de mg g-1, Br, Fe, Mn, Rb e Zn em μg g-1 e os elementos As, Co, Cr, Cs, La, Sb, Sc e Se em teores mais baixos, da ordem de ng g-1. Os resultados obtidos nas análises dos extratos indicaram que os mesmos elementos determinados nas folhas de Casearia foram também encontrados em seus respectivos extratos. A comparação das composições elementares das folhas de Casearia sylvestris coletadas em três regiões diferentes da Mata Atlântica mostrou que as concentrações dos elementos encontrados nas plantas dependem da composição dos solos em que elas foram cultivadas. Por outro lado, espécies diferentes do gênero Casearia cultivadas em uma mesma região apresentaram composições elementares similares. Estes resultados mostram a importância da realização de estudos que avaliem as atividades farmacológicas de Casearias cultivadas em diferentes tipos de solos. A qualidade analítica dos resultados foi avaliada pela análise dos materiais de referência certificados NIST 1573a Tomato Leaves, NIST 1515 Apple Leaves, INCT-TL-1 Tea Leaves, IAEA-Soil-7 e USGS W1. Os desvios padrão relativos e os erros relativos obtidos nestas análises indicaram boa precisão e exatidão dos resultados. / The use of medicinal plants in the treatment of diseases has increased significantly in the last years, as has research concerning chemical characterization of these plants. In this study, inorganic constituents were determined in leaves and in extracts from three medicinal plant species of the Casearia genus (C. sylvestris, C. decandra and C. obliqua) collected in distinct regions of the Atlantic Forest, SP. The elemental compositions of the soils in which these plants were grown were also determined. Traditionally, these plants are used due to their antiinflamatory, antiacid, antiseptic and cicatrizing properties. The antiulcer and the antitumor activities of the Casearia genus and its capacity to neutralize snake and bee venoms, have also been scientifically confirmed. The analytical methodology used was neutron activation analysis. Long and short irradiation periods of the samples and the standards were carried out at IPEN’s IEA-R1 nuclear research reactor. In the leaf K was found at the percentage levels, Ca, Cl, Mg and Na at mg g-1 levels and the elements Br, Fe, Mn, Rb and Zn at the μg g-1 levels. As, Co, Cr, Cs, La, Sb, Sc and Se at the ng g-1 levels. Results obtained in the extracts indicated that the same elements present in the leaves are also found in their extracts. The comparison between the inorganic composition of Casearia sylvestris leaves collected from three different regions of the Atlantic Forest showed that the elemental concentrations in the plants leaves varied depending on the place where they were grown. Different Casearia species cultivated in a same region presented similar elemental compositions. Based on these findings it can be concluded that the studies about the pharmacological effect of Casearia genus plants grown in different types of soil are of great importance. The quality of the obtained results was assured by the analyses of the certified reference materials NIST 1573a Tomato Leaves, NIST 1515 Apple Leaves, INCT-TL-1 Tea Leaves, IAEA-Soil-7 and USGS W1. The relative standard deviations and the relative errors obtained in these analyses indicated good precision and accuracy of the results.

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

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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 500µg/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 1000µg/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 500µg/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 1000µg/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

O uso de plantas medicinais no tratamento de doenças tem aumentado de forma significativa nos últimos anos, bem como as pesquisas sobre a caracterização química destas plantas. Neste estudo, foram determinados os constituintes inorgânicos presentes nas folhas e nos extratos de três espécies da planta do gênero Casearia (C. sylvestris, C. decandra e C. obliqua) coletadas em regiões distintas da Mata Atlântica, SP. As composições elementares dos solos em que estas plantas foram cultivadas também foram determinadas. Tradicionalmente, estas plantas são utilizadas devido às suas propriedades antiinflamatórias, antiácidas, anti-sépticas e cicatrizantes. Além disso, as atividades antiulcerogênicas e antitumorais do gênero Casearia, e sua capacidade de neutralizar venenos de serpentes e abelhas, também tem sido comprovadas cientificamente. A metodologia analítica empregada foi a análise por ativação com nêutrons. Irradiações longas e curtas das amostras e dos padrões foram realizadas utilizando fluxos de nêutrons do reator de pesquisas IEA-R1 do IPEN/CNEN-SP. Nas folhas, o elemento K foi encontrado em níveis de porcentagens, os elementos Ca, Cl, Mg e Na na ordem de mg g-1, Br, Fe, Mn, Rb e Zn em μg g-1 e os elementos As, Co, Cr, Cs, La, Sb, Sc e Se em teores mais baixos, da ordem de ng g-1. Os resultados obtidos nas análises dos extratos indicaram que os mesmos elementos determinados nas folhas de Casearia foram também encontrados em seus respectivos extratos. A comparação das composições elementares das folhas de Casearia sylvestris coletadas em três regiões diferentes da Mata Atlântica mostrou que as concentrações dos elementos encontrados nas plantas dependem da composição dos solos em que elas foram cultivadas. Por outro lado, espécies diferentes do gênero Casearia cultivadas em uma mesma região apresentaram composições elementares similares. Estes resultados mostram a importância da realização de estudos que avaliem as atividades farmacológicas de Casearias cultivadas em diferentes tipos de solos. A qualidade analítica dos resultados foi avaliada pela análise dos materiais de referência certificados NIST 1573a Tomato Leaves, NIST 1515 Apple Leaves, INCT-TL-1 Tea Leaves, IAEA-Soil-7 e USGS W1. Os desvios padrão relativos e os erros relativos obtidos nestas análises indicaram boa precisão e exatidão dos resultados. / The use of medicinal plants in the treatment of diseases has increased significantly in the last years, as has research concerning chemical characterization of these plants. In this study, inorganic constituents were determined in leaves and in extracts from three medicinal plant species of the Casearia genus (C. sylvestris, C. decandra and C. obliqua) collected in distinct regions of the Atlantic Forest, SP. The elemental compositions of the soils in which these plants were grown were also determined. Traditionally, these plants are used due to their antiinflamatory, antiacid, antiseptic and cicatrizing properties. The antiulcer and the antitumor activities of the Casearia genus and its capacity to neutralize snake and bee venoms, have also been scientifically confirmed. The analytical methodology used was neutron activation analysis. Long and short irradiation periods of the samples and the standards were carried out at IPEN’s IEA-R1 nuclear research reactor. In the leaf K was found at the percentage levels, Ca, Cl, Mg and Na at mg g-1 levels and the elements Br, Fe, Mn, Rb and Zn at the μg g-1 levels. As, Co, Cr, Cs, La, Sb, Sc and Se at the ng g-1 levels. Results obtained in the extracts indicated that the same elements present in the leaves are also found in their extracts. The comparison between the inorganic composition of Casearia sylvestris leaves collected from three different regions of the Atlantic Forest showed that the elemental concentrations in the plants leaves varied depending on the place where they were grown. Different Casearia species cultivated in a same region presented similar elemental compositions. Based on these findings it can be concluded that the studies about the pharmacological effect of Casearia genus plants grown in different types of soil are of great importance. The quality of the obtained results was assured by the analyses of the certified reference materials NIST 1573a Tomato Leaves, NIST 1515 Apple Leaves, INCT-TL-1 Tea Leaves, IAEA-Soil-7 and USGS W1. The relative standard deviations and the relative errors obtained in these analyses indicated good precision and accuracy of the results.

Mestrado em Controlo de Qualidade / MSc in Quality Control / Piper betle L., conhecida como bétele, é uma espécie que se desenvolve largamente nos países do Sudeste Asiático, onde as suas folhas são económica e medicinalmente importantes. Para determinar o maior número possível de compostos voláteis e semivoláteis, as folhas foram sujeitas as diferentes processos de extracção, nomeadamente headspace - microextracção em fase sólida (HS-SPME), hidrodestilação e extracção por Soxhlet, e posteriormente analisadas por GC/MS, o que permitiu identificar 50 compostos, distribuídos por várias classes químicas, 23 dos quais foram descritos pela primeira vez. As diferentes técnicas permitiram a extracção de compostos distintos, sendo a HS-SPME aquela com que se obteve o perfil mais completo e com que se determinou maior teor de compostos. Dentro deste processo os melhores resultados foram obtidos utilizando a fibra revestida com divinilbenzeno/polidimetilsiloxano (DVB/PDMS). Considerando que a espécie é vulgarmente mascada, o seu extracto aquoso também foi analisado, tendo sido caracterizados apenas sete compostos, sendo o eugenol o composto principal. A composição em ácidos orgânicos deste extracto foi determinada por HPLC/UV e os ácidos oxálico, aconítico, cítrico, pirúvico, málico, chiquímico, acético e fumárico foram descritos pela primeira vez. O extracto aquoso também revelou capacidade para inibir a acetilcolinesterase de modo dependente da concentração. / Piper betle L., popularly known as “Paan”, is a species widely growing in South East Asia, where its leaves are economically and medicinally important. In order to screen the highest possible number of volatile and semi-volatile components, the leaves were subjected to headspace solid-phase microextraction (HS-SPME), hydrodistillation and Soxhlet extraction, prior to their analysis by GC/MS, which allowed the identification of 50 compounds, distributed by several chemical classes, 23 of them described for the first time. The different techniques lead to distinct compounds' extraction, with HS-SPME extracting highest amounts and providing the most complete profile. Within this procedure, best results were obtaine using Divinylbenzene/Polydimethylsiloxane (DVB/PDMS) fibre. Considering the use of the species as masticator, an aqueous extract was also analysed, in which only seven compounds were characterize, being eugenol the main one. The organic acids composition of this extract was determine by HPLC/UV and oxalic, aconitic, citric, pyruvic, malic, shikimic, acetic and fumaric acids are reported for the first time in this species. The aqueous extract also displayed AChE inhibitory capacity, in a concentration-dependent way.