Submitted by admin tede (tede@pucgoias.edu.br) on 2017-06-29T13:18:42Z No. of bitstreams: 1 MARIA JOSÉ BATISTA DE SOUSA.pdf: 2843064 bytes, checksum: f400a0df10a20e086f4f74a5b6cf9480 (MD5) / Made available in DSpace on 2017-06-29T13:18:42Z (GMT). No. of bitstreams: 1 MARIA JOSÉ BATISTA DE SOUSA.pdf: 2843064 bytes, checksum: f400a0df10a20e086f4f74a5b6cf9480 (MD5) Previous issue date: 2017-03-28 / The species Brosimum gaudichaudii (family Moraceae) and Caesalpinia ferrea (family Fabaceae) are widely distributed throughout Brazil and are considered medicinal plants. The extract of Brosimum gaudichaudii bark has been indicated for the treatment of skin blemishes and vitiligo. On the other hand, the extract of Caesalpinia ferrea fruit has been used due to its therapeutic properties as antibacterial, anti-inflammatory and analgesic action. Much of the medicinal plant extracts constituents are unknown and may be toxic to human and animal health, so it is necessary to study the qualitative phytochemical of secondary metabolites and to evaluate the cytotoxic, genotoxic and mutagenic potential of the extracts of these species. In this study, in order to evaluate the mutagenic and / or genotoxic effects, different concentrations of the extractive solutions of B. gaudichaudii and C. ferrea were evaluated in vivo in Astyanax sp and Allium cepa, and ex vivo, by the micronucleus test in T lymphocytes humans. Data were submitted to Kruskall-Wallis a non-parametric test and then to simple linear regression with a significance level of 5%. The Allium cepa test, micronucleus test for human T lymphocytes and erythrocytes of Astyanax sp did not indicate mutagenic and / or genotoxic potential of phytochemicals (p> 0.05) when compared to the non-exposed controls, except the concentration of 5 mg/L of B. gaudichaudii that showed cytotoxicity. On the other hand, the comet assay revealed genotoxic action for all concentrations evaluated for the tail length parameter of the comet. For the moment parameter of Olive's tail only the 20mg /L concentration of Caesalpinia ferrea extract was genotoxic. Therefore, apical meristematic cells from the roots of Allium cepa and human T lymphocytes did not present genotoxic and / or mutagenic changes induced by exposure to both plant extracts detectable by micronuclei tests or mitotic index reduction. Genotoxic effect was evidenced by the tail length and tail moment parameter of Olive in the Comet Assay only for C. ferrea extract in the erythrocytes of Astyanax sp. In order to understand the genotoxic and mutagenic activities of B. gaudichaudii and C. ferrea it is important to increase the number of studies to establish safer doses for human consumption. / As espécies Brosimum gaudichaudii (família Moraceae) e Caesalpinia ferrea da família Fabaceae são amplamente distribuídas pelo território brasileiro e são consideradas plantas medicinais. O extrato das cascas de Brosimum gaudichaudii tem sido indicado para tratamento de mancha de pele e vitiligo. Por outro lado, o extrato dos frutos de Caesalpinia ferrea tem sido usado devido suas propriedades terapêuticas como ação antibacteriana, antiinflamatória e analgésica. A maioria dos fitoquímicos presentes nos extratos de plantas medicinais ainda não foram completamente estudados e podem ser tóxicos para a saúde humana e animal. Nesse sentido, é necessário estudos fitoquímicos qualitativos de metabólitos secundários e avaliação do potencial citotóxico, genotóxico e mutagênico dos extratos destas espécies. Nesse estudo, visando avaliar os efeitos mutagênico e/ou genotóxico, diferentes concentrações das soluções extrativas de B. gaudichaudii e C. ferrea foram avaliadas in vivo em Astyanax sp e em Allium cepa, e em ex vivo, pelo teste de micronúcleos em linfócitos T humanos. Os resultados observados das análises foram submetidos ao teste não paramétrico Kruskall-Wallis e posteriormente a regressão linear simples com nível de significância 5%. O teste em Allium cepa, teste de micronúcleo em linfócitos T humanos e em eritrócitos de Astyanax sp não indicaram potencial mutagênico e/ou genotóxico dos fitoconstituintes (p>0,05) quando comparado aos controles não expostos, exceto a concentração de 5g/L de B. gaudichaudii que apresentou citotoxicidade (p=0,038). Por outro lado, o ensaio cometa, revelou ação genotóxica para todas as concentrações avaliadas no parâmetro comprimento da cauda do cometa, para o parâmetro momento da cauda de Olive, apenas a concentração de 20mg/L do extrato de Caesalpinia ferrea mostrou-se genotóxica. Nenhum dos parâmetros avaliados evidenciou danos genéticos resultantes da exposição aos extratos das cascas do caule de B. gaudichaudii. Portanto, as células meristemáticas apicais das raízes de Allium cepa e os linfócitos T humanos não apresentaram alterações genotóxicas e/ou mutagênicas induzidas pela exposição a ambos extratos vegetais que pudesse ser detectadas pelos testes do micronúcleos ou redução do índice mitóticos. Enquanto, nos eritrócitos de Astyanax sp foi evidenciado ação genotóxica pelo parâmetro comprimento da cauda do cometa e momento de da cauda de Olive somente para o extrato de C. ferrea. Diante do exposto, há necessidade de ampliar os estudos para melhor compreensão das atividades genotóxicas e/ou mutagênicas dos extratos de B. gaudichaudii e C. ferrea visando o estabelecimento de doses mais seguras para o consumo humano.

Thesis (PhD (Biomedical Sciences))--Cape Peninsula University of Technology, 2017. / The use of medicinal plants in the management of various health problems date back to the ancient times. However, only in recent years, researchers are starting to focus on the use of natural plant products as alternative treatment in disease control. Basella alba (Ba), commonly called Ceylon or Indian spinach is one of such medicinal plants, wildly cultivated and consumed mostly as vegetable. Studies have established many beneficial effects of Ba, including androgenic effects as well as antidiabetic effects which have been described in rats following oral administration of the leave extract. However, the actual mechanisms underlying the antihyperglyceamic effect of Ba have not been reported in any study and little or no research details are yet available on the potential beneficial effects of Ba in reproductive dysfunction resulting from diabetes mellitus. This study was aimed at investigating the mechanisms underlying the antidiabetic effect of Ba and the possibility of a role for the plant in correcting diabetic-induced reproductive dysfunctions in male Wistar rats. The first part of the study involved comparing of three different solvent extracts of Ba leaves namely ethyl acetate, methanolic and aqueous extracts for their antioxidant potentials, after which the aqueous extract was selected for further use in the experiments. Animal experimentation involved male rats (n=40) aged 8-10 weeks, randomly divided into four equal groups as follows: Healthy Control, Diabetic Control, Healthy Treatment and Diabetic Treatment. Diabetes was induced via a single intraperitoneal injection of streptozotocin (55mg/kg) and all animals subsequently received treatment via gavage (Rats in Control groups received 0.5ml/100g normal saline daily and treatment groups received 200mg/kg plant extract daily) for a period of four weeks. Fasting blood sugar and body weights were recorded weekly throughout the study. Animals were sacrificed upon completion of the treatment and blood samples and tissues collected for further analysis which included computer aided sperm analysis, Luminex® technology and enzyme-linked immunosorbent hormonal assays, inflammatory cytokine assays, analysis of oxidative stress markers and Histopathological analysis. The single intraperitoneal injection of a high streptozotocin dose resulted in hyperglycaemia, weight loss, subnormal sperm parameters, negative balance of inflammatory cytokines and endogenous antioxidants and degenerative changes in the pancreas, testes and epididymis as observed in the diabetic control rats. Oral administration with the aqueous extract of Ba for four weeks in diabetic treatment rats led to a significant reduction in blood sugar and improvement of sperm parameters by modulating the production of gonadal hormones, in vivo antioxidants and inflammatory cytokines. There was also significant recovery of normal islet histology and reduction in testicular and epididymal degeneration in the diabetic treatment rats when compared to their diabetic control counterparts. It was concluded from the findings of this study that the antidiabetic and profertility effects of Ba are largely dependent on the modulation of in vivo production of antioxidants, gonadal hormones and inflammatory cytokines, probably stimulated by one or more phytochemical component(s) that can be isolated in the aqueous extract of the plant.

O objetivo deste trabalho é a análise do modo de vida camponês na comunidade Pau Rosa no projeto de assentamento Tarumã Mirim, Manaus-AM. Uma das características do etnoconhecimento camponês é o conhecimento da natureza. Neste sentido, são notórios os saberes ambientais adquiridos para a produção de remédios com base em plantas retiradas da mata, cultivadas e/ou domesticadas nos quintais. O vasto conhecimento sobre o ambiente é resultado das experiências acumuladas em virtude das trajetórias de vida diversificadas, pela transferência de conhecimentos de geração em geração e por constantes pesquisas necessárias para o tratamento de doenças. As informações sobre o etnoconhecimento foram colhidas em entrevistas aos camponeses nos sítios e nas feiras onde comercializam a produção, com registro fotográfico dos elementos constituintes dos sítios e das plantas medicinais. O recurso da história oral foi utilizado pelo fato de as entrevistas serem de extrema importância para o conhecimento da realidade local, tendo em vista, a transmissão dos saberes da cultura camponesa ser com base na oralidade. Para o entendimento do modo de vida dos camponeses residentes na comunidade Pau Rosa foi preciso resgatar o contexto maior da qual fazem parte. Dessa forma, é preciso ressaltar as transformações da questão agrária amazônica, principalmente no pós década de 1960, período no qual ocorreu o incentivo à entrada de capital estrangeiro pelo Estado brasileiro, como estratégia para a ocupação da região. Para tanto, houve a necessidade de atrair mão de obra para os chamados grandes projetos, constituindo assim o cenário favorável para o estímulo à migração. Com efeito, os migrantes são resultado da concentração fundiária no Brasil o qual fez emergir uma classe camponesa expropriada e em busca da terra de trabalho. O Estado, ao incentivar o campesinato sem terra a vir para a região diminuir-se-ia a pressão sobre a terra no país, no entanto, as comunidades locais foram menosprezadas, instaurando-se o conflito, resultado da ideia equivocada de vazio demográfico. A migração é, portanto, essencial para a compreensão da realidade local, na qual o camponês torna-se posseiro e passa a lutar pela terra negando a propriedade capitalista. O território camponês foi forjado no projeto de assentamento para reforma agrária a partir do etnoconhecimento. Dessa forma, os camponeses mantêm o etnoconhecimento dos seus modos de vida do ambiente de origem, as interações culturais possibilitaram um manejo da biodiversidade também pelos não amazônidas, assim, a identificação de espécies para fins medicinais e os remédios feitos a partir destas, retratam os saberes ambientais típicos das sociedades camponesas. / The aim of this study is the analysis of peasant life in the Pau Rosa community in Tarumã Mirim settlement project, Manaus-AM. One characteristic of peasant ethnoknowledge is knowledge of nature. In this sense, environmental wisdom acquired for the production of herbal medicines is notorious. The raw material for the preparation of medicines is taken from nature or grown in backyards. The vast knowledge about the environment is the result of accumulated experience because of varied life trajectories, by the transfer of knowledge from generation to generation and constant research of plant remedies and treatments for diseases. The ethnoknowledge information was collected in peasant interviews in places and fairs where they sell the production, with photographic record of the constituents of medicinal plants and places. The use of oral history was used because the interviews were extremely important for the local knowledge; in order to transmit the knowledge of peasant culture which is based on oral tradition. For understanding the way of life of the peasants that are resident in the community Pau Rosa was necessary to rescue the larger context of which it belongs. Thus, we must emphasize the transformation of the Amazonian agrarian issue, especially in the post 1960 period, when there was the incentive for foreign capital by the Brazilian state, as a strategy for the occupation of the region. Therefore, it was necessary to attract labor to the \"big projects\" and thus constitute the favorable scenario for the stimulus to migration. Indeed, migrants are the result of land concentration in Brazil which did emerge an expropriated peasant class in search of land and work. This movement characterized the agrarian geography of Amazonia, which means, move the landless peasantry to the region would decrease the pressure on land in the country, however, local communities have been neglected, strengthening the mistaken idea of demographic void. Migration is therefore essential to understand the local reality, in which the peasant becomes \"posseiros\" and goes on to fight for the land. The formation of a peasant territory was constituted in a settlement project for agrarian reform and ethnoknowledge is a striking feature of the Amazon peasantry. Thus, the peasantry resident in the Pau Rosa Community keeps traces on their lifestyles environmental of origin and cultural interactions which enabled a biodiversity management also by non Amazonians, so the identification of species for medicinal purposes and medicines made from these, depict the typical environmental wisdom of peasant societies.

Na grande diversidade de plantas encontrada em todo o mundo, encontram-se as plantas medicinais com propriedades antivenenos. O estudo da relação dessas plantas com venenos de animais contribui muito para o desenvolvimento de novos medicamentos. A quantidade de dados a ser armazenada e a relação dessas informações é um processo que deve ser administrado por um sistema computacional. O desenvolvimento de sistemas de computadores tem se destacado nos últimos anos na Bioinformática e são muito úteis para organizar diferentes tipos de dados e, juntamente, com o uso de gerenciadores de conteúdo, eles contribuem, significantemente, no processo de desenvolvimento de softwares. O presente projeto teve como objetivo o desenvolvimento de um sistema computacional para Web, o qual relaciona dados de plantas medicinais com propriedades antivenenos e de animais venenosos, permitindo a integração dos mesmos, através de diferentes aplicativos de busca. O sistema foi denominado de Venom e está disponível no site http://gbi.fmrp.usp.br/venom/. Foram criadas categorias para a classificação dos dados de plantas e de animais. Essa categorização das informações é muito importante, pois possibilita o relacionamento das mesmas nas buscas por categorias. Os dados, tanto de plantas quanto de animais, foram extraídos de artigos científicos e de bases de dados públicos. Família, espécie, composto isolado e nome popular são algumas das informações referentes às plantas. Quanto aos animais venenosos, o sistema oferece informações tais como, espécie, seqüência de aminoácidos no formato FASTA, entre outras. Até o momento, encontram-se categorizados e disponíveis no sistema 97 dados de plantas medicinais com propriedades antiveneno, distribuídos em 42 famílias e 4.623 dados de animais venenosos, distribuídos em 392 espécies entre 10 diferentes organismos. Novas informações podem ser depositadas por colaboradores cadastrados no sistema. Tais depósitos entram em uma fila de espera e, se os campos requisitados estiverem preenchidos e os dados categorizados corretamente, o conteúdo é liberado de acordo com as regras de permissão estabelecidas pelo sistema de segurança. A interface do Venom é simples, contribuindo, assim, para um acesso rápido e funcional. / In the vast diversity of plants found around the world, there are medicinal plants with antivenom properties. The study that relates data from medicinal plants with poisons of animals contributes to the development of new medicines. These information and integration between them is a process that must be administered by a computer system, which helps significantly in the structure of storage. The development of computer systems has been highlighted in recent years in Bioinformatics and are very useful for organizing different types of data and, together with the use of content managers, they contribute, significantly, in the process of developing software. This project aimed to the development of a computer Web system, which related data of medicinal plants with anti-venom properties and venomous animals, allowing the integration of the data, through different search applications. The system was named Venom and is available on the web site http://gbi.fmrp.usp.br/venom/. Categories were created for the classification of the plants and animals data. This categorization is very important because it allows the use of the categories relationship in the searches. Data both of plants and animals were extracted from scientific articles and from public databases. Family, species, isolated composed and popular name are some of the information relating to the plants. About venomous animals, the system provides information such as species, amino acids sequence in FASTA format, among others. Until now, there are 97 categorized plants data available on the system, which are distributed in 42 families, and there are 4,623 data from venomous animals, distributed in 392 species of 10 different organisms. New information may be submitted by collaborators researches registered in the system. Such deposits come to a waiting queue, and whether all the requested fields are completed and corrected categorized, the content is released in accordance with the permission rules established by the system. The Venom\'s user interface is simple, contributing to a fast and functional access.

Aims. The objectives of this study were (i) to isolate and characterize by conventional column chromatography, structurally diverse triterpenes from G. lucidum to serve as chemical markers; (ii) to develop a high performance liquid chromatography (HPLC) method for the quality control and/or standardization of Lingzhi-containing products; (iii) to utilize and optimize operating conditions for the newer extraction technology: supercritical fluid extraction (SFE), in order to maximize yields of bioactive triterpenes, and to reduce time and costs. / Background. The dried fruiting body of Ganoderma lucidum, commonly known as Lingzhi, has been used extensively as a traditional Chinese medicine (TCM) for many centuries not only in China, but also in other countries such as Japan and Korea. In recent years, Lingzhi has also become a popular health supplement in many Western countries. The chemical composition of Lingzhi is complex, but it has been well documented that the lipophilic triterpenoid class of compounds possess a range of biological effects that include antitumor, immunomodulatory, cardiovascular, respiratory and antihepatotoxic activity. A major drawback in TCM research has been the lack of authentic chemical standards, and efficient methods for the extraction and analysis of bioactive fractions and/or single components. Conventional extraction methods for G. lucidum are time-consuming and laborious, and often result in low yields of useful chemical constituents. / Conclusion. This study enabled the development of a method for the simultaneous analysis of structurally diverse triterpenes with remarkably different chromatographic profiles. The isolated triterpenes, as chemical markers, and the HPLC method can readily be used for quality control and/or standardization purposes in evaluating Lingzhi-containing products. Optimization of operating conditions for SFE facilitated the rapid and selective extraction of acidic triterpenes from raw G. lucidum in significantly higher yields. / Methods. Raw material of G. lucidum was extracted with 80% ethanol; subjected to repeated column chromatography to purify triterpenes; and characterized by NMR (1H and 13C) and mass spectroscopy. Isolated lipophilic triterpenes were qualitatively and quantitatively analyzed by reversed-phase HPLC using an ODS column (150 x 4.6 mm) and PDA detection at 256 nm. The assay was validated over appropriate concentration ranges and benzophenone was used as an internal standard. Supercritical fluid extraction of G. lucidum was carried out using a commercial supercritical fluid extractor system Thar, SFE-1000M. Briefly, the raw powder of G. lucidum was soaked in ethanol containing 10% aqueous ammonia for 30 minutes prior to extraction. Extractions were performed at temperatures of 40, 50 and 60°C; and pressures of 200, 250, 300 and 350 bar; 5% ethanol was used as the co-solvent; and the flow rate of CO 2 was set at 20 g/min. / Results. Eight compounds were isolated and identified from G. lucidum: four triterpenes; namely, lucidenic acid N, ganoderic acid B, ganodermanontriol, and ganodermadiol; two steroids; ergosterol-7, 22-dien-3beta-ol and ergosterol peroxide; and two fatty acids, oleic acid and tetracosanoic acid. The four triterpenes were utilized as chemical markers, and the developed HPLC method was able to simultaneously analyze the structurally diverse components with good resolution. The total analysis run time was 110 min, and retention times (tR) were 14.88, 18.96, 63.88 and 90.73 min respectively, and the eluting system was a mixture of three solvents, methanol (A), acetonitrile (B) and 2% acetic acid solution (C): 0-22 min, 5% A, 25% B and 70% C; 22-85 min, gradient elution, the ratio changed gradually to 5% A, 80% B and 15% C; 85-110 min, gradient elution, the ratio changed gradually to 5% A, 85% B and 10% C. The validated HPLC method and the isolated chemical markers were effectively applied to determine the triterpenoid contents in a variety of commercial Lingzhi products. Supercritical fluid extraction conditions of: pressure 300 bar and temperature 50°C, gave the highest yields of triterpene-containing extracts. HPLC analysis of the SFE extracts showed predominantly acidic triterpenes such as lucidenic acid N and ganoderic acid B. / Hong Xin. / "December 2006." / Advisers: Ho Yee Ping; Albert H. L. Chow. / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5968. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 149-175). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Ha Wai-Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 166-180). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Abstract --- p.iii / Abbreviations --- p.vi / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- "Hstory, cultivation and trade" --- p.2 / Chapter 1.2 --- Botany --- p.4 / Chapter 1.3 --- Chemical Constituents and Pharmacological effects --- p.8 / Chapter 1.4 --- Authentication of Chinese herbal materials --- p.13 / Chapter 1.4.1 --- Morphological marker --- p.15 / Chapter 1.4.2 --- Histological marker --- p.18 / Chapter 1.4.3 --- Chemical marker --- p.20 / Chapter 1.4.4 --- Molecular markers --- p.24 / Chapter 1.4.4.1 --- Protein marker --- p.24 / Chapter 1.4.4.2 --- DNA-based markers --- p.26 / Chapter 1.4.4.2.1 --- PCR-based markers --- p.27 / Chapter 1.4.4.2.1.1 --- Random-primed PCR --- p.28 / Chapter 1.4.4.2.1.2 --- Simple Sequence Repeats (SSR) --- p.30 / Chapter 1.4.4.2.1.3 --- Polymerase Chain Reaction Fragment Length Polymorphism (PCR-RFLP) --- p.31 / Chapter 1.4.4.2.2 --- Hybridization-based markers --- p.33 / Chapter 1.4.4.2.3 --- Sequencing-based markers --- p.35 / Chapter 1.5 --- Objectives and Strategies of the studies --- p.39 / Chapter Chapter 2 --- General Materials and Methods --- p.40 / Chapter 2.1 --- Reagents and Buffers --- p.41 / Chapter 2.1.1 --- Media for bacterial culture --- p.41 / Chapter 2.1.2 --- Reagents for preparation of competent cells --- p.42 / Chapter 2.1.3 --- Reagents for plasmid DNA preparation --- p.42 / Chapter 2.1.4 --- Reagents for agarose gel electrophoresis --- p.43 / Chapter 2.1.5 --- Reagents for polyacrylamide gel electrophoresis --- p.43 / Chapter 2.1.6 --- Reagents for Southern hybridization --- p.44 / Chapter 2.2 --- Agarose Gel electrophoresis of DNA --- p.46 / Chapter 2.3 --- Purification of PCR products --- p.46 / Chapter 2.3.1 --- From agarose gel using Geneclean® II kit --- p.46 / Chapter 2.3.2 --- Using Microspin´ёØ Column --- p.47 / Chapter 2.4 --- End modification of PCR amplified DNA --- p.47 / Chapter 2.5 --- Preparation of Escherichia coli Competent Cells --- p.48 / Chapter 2.6 --- "Ligation and Transformation of E, coli" --- p.49 / Chapter 2.7 --- Plasmid Preparation --- p.50 / Chapter 2.7.1 --- Minipreparation of plasmid DNA --- p.50 / Chapter 2.7.2 --- Preparation of plasmid DNA using Wizard® Plus SV Minipreps DNA Purification Kit (Promega) --- p.50 / Chapter 2.8 --- Screening for the Presence of insert in plasmid --- p.51 / Chapter 2.8.1 --- Rapid alkaline lysis --- p.51 / Chapter 2.8.2 --- PCR screening --- p.52 / Chapter 2.8.3 --- Restriction digestion of plasmid DNA --- p.53 / Chapter 2.9 --- DNA sequencing --- p.53 / Chapter 2.9.1 --- Plasmid sequencing using T7 Sequencing Kit --- p.53 / Chapter 2.9.2 --- Cycle Sequencing from PCR products or plasmid --- p.54 / Chapter 2.10 --- DNA Sequencing electrophoresis --- p.55 / Chapter 2.10.1 --- Preparation of 6 % polyacrylamide gel solution --- p.55 / Chapter 2.10.2 --- Gel casting --- p.55 / Chapter 2.10.3 --- Electrophoresis of Sequencing Gel --- p.56 / Chapter 2.10.4 --- Autoradiography --- p.57 / Chapter 2.11 --- DNA elution from dried sequencing gel --- p.57 / Chapter 2.12 --- Southern blot analysis --- p.58 / Chapter 2.12.1 --- Restriction digestion of genomic DNA --- p.58 / Chapter 2.12.2 --- Purification of digested DNA and agarose gel electrophoresis --- p.58 / Chapter 2.12.3 --- Capillary transfer of DNA to a Hybond´ёØ N+ nylon membrane --- p.59 / Chapter 2.12.4 --- DNA radiolabeling by nick translation --- p.60 / Chapter 2.12.5 --- Purificaiton of radiolabeled probe by NICK® Spin Column --- p.60 / Chapter 2.12.6 --- Hybridization of DNA --- p.61 / Chapter Chapter 3 --- Plant DNA extraction --- p.62 / Chapter 3.1 --- Introduction --- p.63 / Chapter 3.2 --- Reagents and buffer for total DNA extraction --- p.66 / Chapter 3.3 --- Extraction methods --- p.70 / Chapter 3.3.1 --- Sample preparation --- p.70 / Chapter 3.3.2 --- CTAB extraction method --- p.70 / Chapter 3.3.3 --- Potassium acetate/ SDS extraction method --- p.71 / Chapter 3.3.4 --- GIBRO Plant DNAzol® reagent for genomic DNA isolation --- p.72 / Chapter 3.4 --- Qualitative and quantitative analysis of DNA --- p.74 / Chapter 3.5 --- Results --- p.75 / Chapter 3.6 --- Discussion --- p.78 / Chapter Chapter 4 --- Amplified Fragment Length Polymorphism (AFLP) analysis of P. ginseng and P. quinquefolius --- p.81 / Chapter 4.1 --- Introduction --- p.82 / Chapter 4.2 --- Materials and methods --- p.88 / Chapter 4.2.1 --- Plant materials --- p.88 / Chapter 4.2.2 --- Choice of Primers and radiolabeling --- p.89 / Chapter 4.2.3 --- AFLP assay --- p.90 / Chapter 4.2.4 --- Electrophoresis of AFLP fingerprint --- p.91 / Chapter 4.2.5 --- Similarity Index (S.I.) analysis of AFLP profile --- p.91 / Chapter 4.2.6 --- Re-amplification of polymorphic DNA fragments isolated from dried sequencing gel --- p.92 / Chapter 4.2.7 --- Cloning and Sequencing of the AFLP fragments --- p.93 / Chapter 4.2.8 --- Conversion of AFLP marker into Directed Amplification of Minisatellite-region DNA polymorphism (DAMD) marker --- p.93 / Chapter 4.3 --- Results --- p.95 / Chapter 4.4 --- Discussion --- p.102 / Chapter Chapter 5 --- Direct Amplification of Length Polymorphisms (DALP) analysis of P. ginseng and P. quinquefolius --- p.107 / Chapter 5.1 --- Introduction --- p.108 / Chapter 5.2 --- Materials and methods --- p.112 / Chapter 5.2.1 --- Plant materials --- p.112 / Chapter 5.2.2 --- Choice of Primers --- p.113 / Chapter 5.2.3 --- Alternative labelled Amplification reaction --- p.114 / Chapter 5.2.4 --- Electrophoresis of the multi-locus amplification products --- p.114 / Chapter 5.2.5 --- Isolation and Re-amplification of polymorphic DALP fragments from dried sequencing gel --- p.115 / Chapter 5.2.6 --- Cloning and Sequencing --- p.115 / Chapter 5.2.7 --- Conversion of DALP marker to Sequence Tagged Site (STS) marker --- p.116 / Chapter 5.3 --- Results --- p.117 / Chapter 5.4 --- Discussion --- p.135 / Chapter Chapter 6 --- Sequence-characterized amplified region (SCAR): the sequel of random amplified polymorphic DNA (RAPD) --- p.137 / Chapter 6.1 --- Introduction --- p.138 / Chapter 6.2 --- Materials and methods --- p.140 / Chapter 6.2.1 --- Plant materials --- p.140 / Chapter 6.2.2 --- PCR reaction --- p.141 / Chapter 6.2.3 --- Cloning and sequencing --- p.143 / Chapter 6.3 --- Results --- p.144 / Chapter 6.4 --- Discussion --- p.157 / Chapter Chapter 7 --- Outlook --- p.159 / Chapter 7.1 --- Molecular authentication of Chinese medicinal materials --- p.160 / Chapter 7.2 --- Development of molecular markers for Ginseng --- p.161 / Appendix I --- p.164 / Appendix II --- p.165 / References --- p.166

Submitted by Natalie Mendes (nataliermendes@gmail.com) on 2015-07-25T15:23:13Z No. of bitstreams: 1 Disserta??o Melina PPGRGV.pdf: 1355642 bytes, checksum: 38055ea3e94c1f1eaec922c55c2d09bf (MD5) / Made available in DSpace on 2015-07-25T15:23:13Z (GMT). No. of bitstreams: 1 Disserta??o Melina PPGRGV.pdf: 1355642 bytes, checksum: 38055ea3e94c1f1eaec922c55c2d09bf (MD5) Previous issue date: 2014-03-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The species Psychotria ipecacuanha (Brot.) Stokes, popularly known as ipeca or poaia, has attracted interest from the pharmaceutical industry for many decades due to the presence of cephalin and emetine alkaloids in their roots that have expectorant properties , emetic and amebicides. The extinction risk and difficulty of conventional crop production, front of the large market demand, encourage studies of cultivation methods that enable the economic exploitation and, consequently , the creation of new productive chains in rainforest areas . This study was performed at the Environmental Preservation Area Pratigi - APA Pratigi - located on the territory of Southern Bahia and aimed to quantify the growth and production of emetine in ipeca plants under agroforestry crop type cabruca , evaluating the effect of different lighting conditions , age and nutrient sources. Quantification of emetine was made by high performance liquid chromatography (HPLC) and the experimental results were subjected to analysis of variance , comparing treatment means by the Tukey test and Skot Knot . The experimental results showed that the lighting conditions affect plant growth of ipeca , being higher in environments with shading above 90 % . Ipecc had an average yield of 207 kg ha - 1 of fresh root and 80 kg ha - 1 dry at 16 months of growing root ; and 240 kg ha - 1 of fresh root and 93 kg ha - 1 at 28 months. It was possible to obtain levels of emetine , the conditions of agroforestry cultivation cabruca kind in Southern Bahia territory , equivalent to the levels obtained from plants collected extractive in other regions of the country . Age affects the content of emetine in agroforestry cultivation conditions, with higher concentrations in younger plants, after 16 months of cultivation. Considering the sources of fertilizer, with and without liming, gave higher plant height and higher No leaves / plant in treatments without liming , however, yielded better results for No / plant roots in limed areas . For the accumulation of root dry weight, the commercial part of the plant , treatments with nitrogen, with and without liming , promoted the highest averages. The levels of emetine were higher in treatments without lime and subjected to potassium fertilization. / A esp?cie Psychotria ipecacuanha (Brot.) Stokes, conhecida popularmente como ipeca ou poaia, tem despertado interesse da ind?stria farmac?utica a muitas d?cadas, devido ? presen?a dos alcaloides emetina e cefalina em suas ra?zes, que possuem propriedades expectorantes, em?ticas e amebicidas. O risco de extin??o e a dificuldade do cultivo convencional da esp?cie, frente ? grande demanda de mercado, fomentam estudos de m?todos de cultivo que possibilitem a explora??o econ?mica e, consequentemente, a cria??o de novas cadeias produtivas em ?reas de floresta tropical. O presente trabalho foi realizado na ?rea de Preserva??o Ambiental do Pratigi - APA do Pratigi ? localizada no territ?rio do Baixo Sul da Bahia e teve como objetivos quantificar o crescimento e a produ??o de emetina em plantas de ipeca sob cultivo agroflorestal tipo cabruca, avaliando o efeito de diferentes condi??es de luminosidade, idade e fontes de aduba??o. A quantifica??o da emetina foi feita por meio de cromatografia l?quida de alta efici?ncia (CLAE) e os resultados experimentais foram submetidos ? an?lise de vari?ncia, comparando-se as m?dias dos tratamentos por meio dos testes Tukey e Skot Knot. Os resultados experimentais demonstraram que as condi??es de luminosidade afetaram o crescimento das plantas de ipeca, sendo maior em ambientes com sombreamento acima de 90%. A ipeca apresentou rendimento m?dio de 207 kg Ha-1 de raiz fresca e 80 kg Ha-1 de raiz seca aos 16 meses de cultivo; e de 240 kg Ha-1 de raiz fresca e 93 kg Ha-1 aos 28 meses. Foi poss?vel obter teores de emetina, nas condi??es de cultivo agroflorestal tipo cabruca no territ?rio Baixo Sul da Bahia, equivalente aos teores obtidos de plantas coletadas extrativistamente nas demais regi?es do pa?s. A idade afeta o teor de emetina nas condi??es de cultivo agroflorestal, com maiores teores em plantas mais jovens, aos 16 meses de cultivo. Considerando as fontes de aduba??o, com e sem calagem, obteve-se maior altura de plantas e maior n? folhas/planta nos tratamentos que n?o receberam calagem, entretanto, obteve-se melhores resultados para n? ra?zes/planta nas ?reas com calagem. Para o ac?mulo de massa seca de raiz, parte comercial da planta, os tratamentos com nitrog?nio, com e sem calagem, promoveram as maiores m?dias. Os teores de emetina foram maiores nos tratamentos sem calagem e submetidas ? aduba??o com pot?ssio.

Objective: To evaluate the susceptibility of Pseudomonas aeruginosa (P. aeruginosa) in vitro to the ethanolic extracts obtained from five different Peruvian medicinal plants. Methods: The plants were chopped and soaked in absolute ethanol (1:2, w/v). The antibacterial activity of compounds against P. aeruginosa was evaluated using the cupplate agar diffusion method. Results: The extracts from Maytenus macrocarpa (“Chuchuhuasi”), Dracontium loretense Krause (“Jergon Sacha”), Tabebuia impetiginosa (“Tahuari”), Eucalyptus camaldulensis Dehn (eucalyptus), Uncaria tomentosa (“Uña de gato”) exhibited favorable antibacterial activity against P. aeruginosa. The inhibitory effect of the extracts on the strains of P. aeruginosa tested demonstrated that Tabebuia impetiginosa and Maytenus macrocarpa possess higher antibacterial activity. Conclusions: The results of the present study scientifically validate the inhibitory capacity of the five medicinal plants attributed by their common use in folk medicine and contribute towards the development of new treatment options based on natural products. / This work has been partially supported by Universidad Peruana de Ciencias Aplicadas and Nutrition Research Institute, Lima, Peru. / Peer review

O presente trabalho teve como objetivo realizar o levantamento da entomofauna associada a plantas medicinais, aromáticas e condimentares desidratadas no comércio atacadista na cidade de São Paulo, empregar diferentes doses de radiação gama visando a desinfestação dos materiais e determina a dose letal de radiação gama de indivíduos adultos de Sphaericus gibboides. Foram coletados em 10 estabelecimentos no período de abril a maio de 2011 os materiais: Melissa officinalis L. (ervacidreira), Mentha piperita L. (hortelã), Ocimum basilicum L. (manjericão), Origanum vulgare L. (orégano), Rosmarinus officinalis L. (alecrim), Thymus vulgaris L. (tomilho), Senna alexandrina Mill (sene), Coriandrum sativum L. (coentro), Petroselinum crispum (Mill.) Fuss(salsa) e Pimpinella anisum L. (ervadoce), Baccharis trimera (Less.) DC. (carqueja), Matricaria chamomilla L. (= M. recutita L.) (camomila), Laurus nobilis L. (louro) (Lauraceae); Capsicum annuum L. (pápricadoce), Bixa orellana L. (colorífico) (Bixaceae); e Peumus boldus Molina (boldo). Na primeira triagem constatou-se que todos os materiais analisados não apresentaram presença de insetos adultos. Após o período de 45 dias foram encontrados 1838 insetos adultos, oriundos de ovos e larvas. Dos substratos analisados Matricaria recutita apresentou o maior índice de infestação, com 70,6 %. Pelmus boldus, Laurus nobilis, Matricaria recutita e Capsicum annuum, tiveram a maior diversidade de espécies. Baccharis strimera, Bixa orellana, Melissa officinalis, Origanum vulgares e Coriandrum sativum não apresentaram infestação. A espécie Lasioderma serricorne foi o inseto com maior número de indivíduos encontrados (936), maior porcentagem de infestação nos diferentes materiais (62,5 %) e maior ocorrência (68,75 %) nos materiais (M. piperita, S. alexandrina, P. anisum, M. chamomilla, P. crispum, L. nobilis, C. sativum, C. annuum, O. basilicum, P. boldus e T. vulgaris). Os seguintes materiais foram selecionados para os ensaios de desinfestação pelo processo de irradiação: Bixa orellana, Capsicum annuum, Cassia angustifolia, Coriandrum sativum, Mentha piperita, Petrosolium sativum, Matricaria recutita. Os materiais foram infestados com adultos de L. serricorne e Sphaericus gibboides, divididos em 5 parcelas de 30g com 20 indivíduos cada. Utilizando o Irradiador Multiprpósito cobalto-60 com taxa de dose no período dos ensaios de 6 kGy/h, as parcelas foram submetidas a doses crescentes de radiação gama de 0 (grupo controle); 0,25; 0,50; 0,75; 1,00; 1,25; 1,50; 1,75 e 2,00 kGy. A determinação da dose letal para adultos de Sphaericus gibboides, procedeu-se irradiando 4 repetições por tratamento contendo 25 indivíduos cada. As doses utilizadas foram: 2,4; 2,2; 2,0; 1,8; 1,6; 1,4; 1,2; 1,0; 0,8; 0,6 kGy. A menor dose para a não emergência de adultos de L. serricorne e S. gibboides em todos os materiais analisados foi a de 0,25 kGy. Não foi observada a presença de adultos nas demais doses crescentes. Foi observado que os materiais adquiridos no comércio apresentaram diferentes porcentagens de infestação justificando assim a aplicação de métodos de controle. A utilização do Irradiador Multipropósito Cobalto-60 com taxa de dose de 6 kGy/h foi eficiente no controle de L. serricorne e S. gibboides na dose de 0,25 kGy. A dose de 2,2 kGy atingiu 100% letalidade em indivíduos adultos de S. gibboides. / The present study aimed to survey the insect fauna associated with medicinal plants, aromatic dehydrated and seasoning trade in São Paulo city, using different doses of gamma radiation with the aim of disinfestation of the material and determine the lethal dose of gamma radiation on Sphaericus gibboides. From April to May 2011 were collected in 10 establishments the following sample materials: Melissa officinalis L. (Lemongrass), Mentha piperita L. (Mint), Ocimum basilicum L. (Basil), Origanum vulgare L. (Oregano), Rosmarinus officinalis L. (Rosemary), Thymus vulgaris L. (Thyme), Senna alexandrina Mill (senna), Coriandrum sativum L. (Coriander), Petroselinum crispum (Mill.) Fuss (salsa) and Pimpinella anisum L. (Fennel), Baccharis trimera (Less.) DC. (Gorse), Chamomilla recutita L. (= M. recutita L.) (chamomile), Laurus nobilis L. (Blonde) (Lauraceae); Capsicum annuum L. (Sweet paprika), Bixa orellana L. (Spice) (Bixaceae) and Peumus boldus Molina (Boldo). The first screening showed that all the tested materials did not show the presence of adult insects. After 45 days 940 adult insects were found and larvae from eggs. The substrates analyzed Chamomilla recutita showed the highest rate of infestation, with 70,6%. Pelmus boldus, Laurus nobilis, Chamomilla recutita and Capsicum annuum, had the highest species diversity. Baccharis trimera, Bixa orellana, Melissa officinalis, Origanum vulgare and Coriandrum sativum showed no infestation. The species was Lasioderma serricorne the insect with the largest number of individuals found (936), higher percentage of infestation in different materials (62.5%) and lots, and highest occurrence (68,75%) materials (M. piperita, S. alexandrian, P. anisum, Chamomilla recutita, P. crispum, L. nobilis, C. sativum, C. annuum, O. basilicum, P. boldus and T. vulgaris). The following materials were selected for testing disinfestation by irradiation process: Bixa orellana, Capsicum annuum, Cassia angustifolia, Coriandrum sativum, Mentha piperita, Petrosolium sativum, Chamomilla recutita. The materials were infested with adults of L. serricorne and S. gibboides, divided into 5 portions of 30g with 20 individuals each. Using Radiator Multiprpósito Cobalt-60 dose rate during the tests of 6 kGy/h, the plots were subjected to increasing doses of gamma radiation of 0 (control), 0,25; 0,50; 0,75; 1,00; 1,25; 1,50; 1,75 and 2,00 kGy. The determination of the lethal dose for adults S. gibboides, proceeded radiating 4 replicates per treatment each containing 25 individuals. The doses used were: 3,0; 2,8; 2,6; 2,4; 2,2; 2,0; 1,8; 1,6; 1,4; 1,2; 1,0; 0,8; 0,6 kGy. The lowest dose at no emergence of adult L. serricorne and S. gibboides in all materials tested was to 0,25 kGy. We did not observe the presence of other adults in increasing doses. It was observed that the materials acquired in trade showed different percentages of infestation thus justifying the application of control methods. Using Radiator Multiprpósito Cobalt-60 with dose rate of 6 kGy was effective in controlling L. serricorne and S. gibboides at a dose of 0,25 kGy. The dose of 2,2 kGy reached 100% mortality for adults of S. gibboides.

A number of recent research studies have been done on different species of the plants of the genus Phyllanthus. The plants are widely distributed in most tropical and subtropical countries and have long been used for the treatment of liver diseases in China and India. / Hepatitis B virus (HBV) is a major pathogen of human viral hepatitis. It has been estimated that 350 million people are chronic carriers of HBV throughout the world. Increasing evidence indicates that persistent viral infection of the liver is associated with cirrhosis and hepatocellular carcinoma. Hepatitis B virus belongs to a family of DNA viruses called hepadnaviruses. The current treatments of HBV infection with interferon or lamivudine have several disadvantages, and there appears to be much room for improvement in terms of medical treatment. / My project research focuses on two poorly characterized Indian Phyllanthus species called Phyllanthus nanus ("PN") and Phyllanthus niruri ("PI"). In my studies, random amplified polymorphic DNA ("RAPD") technique and high performance liquid chromatography ("HPLC") fingerprinting were used to authenticate different species of Phyllanthus. Both aqueous and ethanolic extracts of PN and PI were prepared to study their cytotoxicity in hepatoma cell lines. The effect of these extracts on hepatitis B virus was also examined in the HBV-genome integrated cell lines - PLC/PRF/5 (Alexander) and HepG2 2.2.15. Microparticle enzyme immunoassay ("MEIA") and enzyme-linked immunosorbent assay were used to measure the amount of hepatitis B surface antigen ("HBsAg") and hepatitis B e antigen ("HBeAg") secretion from the cell lines. RT-PCR was used to detect the change in HBsAg mRNA's expression level in the drug-treated cell lines. Real-time PCR was also employed to examine the effect of drug treatment on the level of HBV DNA replication and the amount of virions secreted into the medium. The experimental results showed that both the aqueous and ethanolic extracts of PN and PI exerted suppressive effect on HBsAg secretion and HBsAg mRNA level. The PN and PI ethanolic extracts also showed mild suppression of viral replication in vitro. The ethanolic extract of PN seemed to be more potent in suppressing HBV than the other extracts tested. (Abstract shortened by UMI.) / Lam Wai Yip. / "June 2005." / Advisers: Mary Waye; Vincent Ooi. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3594. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 217-234). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.