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Doctoral Thesis
DOI
https://doi.org/10.11606/T.46.2004.tde-07102014-115557
Document
Author
Full name
Ana Paula Santos da Conceicao
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2004
Supervisor
Committee
Moreno, Paulo Roberto Hrihorowitsch (President)
Carvalho, Lilian Rothschild Franco de
Fischer, Dominique Corinne Hermine
Morgante, Patrícia Gleydes
Rezende, Daisy de Brito
Title in Portuguese
Estabelecimento de culturas in vitro de Pilocarpus pennatifolius Lemmaire e estudos iniciais sobre a biossíntese do alcalóide pilocarpina
Keywords in Portuguese
Alcalóides (Biossíntese)
Biossíntese
Cultura in vitro
Óleos essenciais
Pilocarpina
Pilocarpus
Pilocarpus pennatifolius
Produtos naturais
Rutaceae
Abstract in Portuguese
Pilocarpus pennatifolius Lemmaire (Rutaceae) é uma espécie nativa que ocorre nas regiões Sul, Sudeste, Centro-Oeste e Nordeste do Brasil, e é popularmente conhecida por jaborandi. Essa e outras espécies de Pilocarpus encontram-se em risco de extinção devido ao extrativismo (uso como planta medicinal) e ao desmatamento. O cultivo in vitro de P. pennatifolius foi realizado como uma alternativa para a produção de pilocarpina (princípio ativo do jaborandi). A cultura de células foi obtida em meio MS semi-sólido, suplementado com sacarose, como fonte de carbono, e tendo como reguladores de crescimento BAP, IAA e NAA. O tempo de duplicação da cultura de calos foi de 22 dias. A concentração do alcalóide, determinada por técnicas cromatográficas, foi de aproximadamente 0,1 µg/ mg de biomassa. Os ensaios enzimáticos referentes à biossíntese da pilocarpina tiveram como objetivo elucidar a etapa inicial de formação do anel imidazólico, além da localização desta reação na planta. Os experimentos realizados indicaram a presença da enzima histidina amino-transferase (HAT, EC 2.6.1.38) cuja atividade catalítica foi de 46,09 nKat/ mg de proteína, apenas no extrato protéico das raízes de P. pennatifolius. A determinação da composição do óleo essencial por CG e CG/EM indicou como constituintes majoritários os hidrocarbonetos tridecano (56,8 %) e pentadecano (25,5 %). Os sesquiterpenos não oxigenados constituíram cerca de 15 % do óleo obtido.
Title in English
Establishment of cultures in vitro of Pilocarpus pennatifolius Lemmaire and initial studies about biosynthesis of alkaloid pilocarpine
Keywords in English
Alkaloids (Biosynthesis)
Biosynthesis
Essential oils
In vitro culture
Natural products
Pilocarpine
Pilocarpus
Pilocarpus pennatifolius
Rutaceae
Abstract in English
Pilocarpus pennatifolius Lemmaire (Rutaceae) is a native Brazilian species which is commonly known as jaborandi. This species is endangered due to its exploitation as medicinal plant and the increasingly deforestation. A callus culture of P. pennatifolius was established as an alternative source for pilocarpine production (the active compound of jaborandi). The cell culture was obtained in a semi-solid media, supplemented with sucrose as carbon source and as growth regulators, BAP, IAA e NAA. The doubling time for the callus culture was determined as 22 days. The concentration of the alkaloid, obtained by chromatographic techniques was ca. 0.1 µg/ mg dry weight. The enzymatic assays related to pilocarpine biosynthesis were carried out with the aim to elucidate the imidazole ring formation beyond identify the site were this reaction takes place. The results indicated the presence of the enzyme histidine ammonia transferase (HAT, EC 2.6.1.38) only in the protein extract obtained from the roots of P. pennatifolius. The catalytic activity for this enzyme was 46.09 nKat/ mg protein. Volatile constituents from the leaves were analyzed by GC and GC/MS and the major compounds were determined as the hydrocarbons tridecane (56.8 %) and pentadecane (25.5 %). Almost 15 % of the total composition was constituted of non-oxygenated sesquiterpenes.
 
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Publishing Date
2014-10-07
 
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