Cultura de calos e suspens??o celular de Duroia saccifera: estudo fitoqu??mico, cin??tica de crescimento e avalia????o das atividades biol??gicas

Autor: Sousa, Aline Bastos Brilhante de
Přispěvatelé: Nunez, Cec??lia Ver??nica, Albuquerque, Patricia Melchiona, Pohlit, Adrian Martin
Jazyk: portugalština
Rok vydání: 2018
Předmět:
Zdroj: Biblioteca Digital de Teses e Dissertações da UFAM
Universidade Federal do Amazonas (UFAM)
instacron:UFAM
Popis: Submitted by Aline Sousa (alinebbrilhante@gmail.com) on 2019-12-19T20:04:00Z No. of bitstreams: 3 Ata de defesa.pdf: 713680 bytes, checksum: c0846795bf4214f35a4c2ff3f3fbc28d (MD5) DISSERTA????O-ALINE-VERS??O FINAL 12.12.19.pdf: 3411580 bytes, checksum: 5dbb295aefdde54e571246305e2fc095 (MD5) IMG_5237.JPG: 2135269 bytes, checksum: 2bf27620a7ee0ef7fbd5e0cd376154b8 (MD5) Approved for entry into archive by PPGBIOTEC Biotecnologia (ppg_biotec.ufam@yahoo.com.br) on 2019-12-19T20:12:17Z (GMT) No. of bitstreams: 3 Ata de defesa.pdf: 713680 bytes, checksum: c0846795bf4214f35a4c2ff3f3fbc28d (MD5) DISSERTA????O-ALINE-VERS??O FINAL 12.12.19.pdf: 3411580 bytes, checksum: 5dbb295aefdde54e571246305e2fc095 (MD5) IMG_5237.JPG: 2135269 bytes, checksum: 2bf27620a7ee0ef7fbd5e0cd376154b8 (MD5) Approved for entry into archive by Divis??o de Documenta????o/BC Biblioteca Central (ddbc@ufam.edu.br) on 2019-12-26T18:22:06Z (GMT) No. of bitstreams: 3 Ata de defesa.pdf: 713680 bytes, checksum: c0846795bf4214f35a4c2ff3f3fbc28d (MD5) DISSERTA????O-ALINE-VERS??O FINAL 12.12.19.pdf: 3411580 bytes, checksum: 5dbb295aefdde54e571246305e2fc095 (MD5) IMG_5237.JPG: 2135269 bytes, checksum: 2bf27620a7ee0ef7fbd5e0cd376154b8 (MD5) Made available in DSpace on 2019-12-26T18:22:06Z (GMT). No. of bitstreams: 3 Ata de defesa.pdf: 713680 bytes, checksum: c0846795bf4214f35a4c2ff3f3fbc28d (MD5) DISSERTA????O-ALINE-VERS??O FINAL 12.12.19.pdf: 3411580 bytes, checksum: 5dbb295aefdde54e571246305e2fc095 (MD5) IMG_5237.JPG: 2135269 bytes, checksum: 2bf27620a7ee0ef7fbd5e0cd376154b8 (MD5) Previous issue date: 2018-07-27 CNPq - Conselho Nacional de Desenvolvimento Cient??fico e Tecnol??gico Tissue and plant cell culture is an efficient strategy for the safe and continuous production of secondary metabolites under controlled physical and chemical conditions. Duroia saccifera (Roem. & Schult.) K. Schum, as well as other species of the genus and the Rubiaceae family, is a potential target for phytochemical and pharmacological studies due to the occurrence of a wide variety of classes of biologically active molecules. Based on this, the objective of this work was to evaluate the growth potential, from the determination of the growth curves of the culture of calli and cell suspensions of the D. saccifera and to perform the phytochemical study of the extracts obtained from the callus and suspensions, and evaluate their chemical and biological activities. For this, calli previously established in vitro in Murashige and Skoog medium supplemented with 4 mg.L-1 of 2,4-dichlorophenoxyacetic acid (2,4D) and 2 mg.L-1 kinetin (KIN) were multiplied in successive subcultures every 30 days and kept in a growth room with controlled temperature of 26 ?? 2 ??C, under photoperiod of 16/8 hours (light/dark), provided by fluorescent lamps. The material was extracted with the organic solvents hexane, ethyl acetate (EtOAc) and methanol. The extracts were tested in antioxidant, antimicrobial and antimycobacterial assays and fractionated. Suspension cell culture was established, when 1 g of callus was inoculated into 100 mL of the liquid culture medium. The growth curves of both forms of cultivation were determined during 40 days, in order to perform a comparative study through the calculation of growth kinetic parameters. In the phytochemical study of the extracts it was possible to verify the presence of terpenoids, steroids, aromatic substances, fatty acids, among others secondary metabolites. In fraction 2 (6.3 mg) of the hexane extract the evidence of the presence of the lupen-3-one steroid was observed. Regarding the biological and chemical assays, the EtOAc extract presented high antimycobacterial activity against Mycobacterium tuberculosis, with minimal inhibitory concentration (MIC), 90% inhibitory concentration (IC90) and minimum bactericidal concentration (MBC) of 25, 19.5 and 200 ??g.L-1, respectively. The extracts were not active in the antimicrobial assays against the tested bacteria, as the extracts had no antioxidant potential against the DPPH free radical and the Fe3+/phenanthroline complex. The results showed that the callus followed a slow growth pattern, reaching a higher fresh (FM) and dry mass (DM) on the 40th day, respectively, 72.6 and 2.808 g.L-1. The accumulation of biomass and the growth of suspension cell culture was significantly higher, reaching higher MF and MS, on the 36th day of cultivation, respectively of 413.7 and 12.995 g.L-1. It was not possible to determine the stationary stages in the growth, however, it was inferred that the maximum production of secondary metabolites occurred on the 40th day of the culture of callus and from the 24th to the 36th day in the culture of cell suspension. The calculated kinetic parameters demonstrate that the cell suspension has a high potential for the supply of plant biomass of the species, becoming a promising source for the in vitro secondary metabolites production. A cultura de tecidos e c??lulas vegetais ?? uma estrat??gia eficiente para a produ????o segura e cont??nua de metab??litos secund??rios em condi????es f??sicas e qu??micas controladas. A Duroia saccifera (Roem. & Schult.) K. Schum, assim como outras esp??cies do g??nero e da fam??lia Rubiaceae, ?? alvo potencial para estudos fitoqu??micos e farmacol??gicos devido ?? ocorr??ncia de uma grande variedade de classes de mol??culas biologicamente ativas. Com base nisso, esse trabalho teve como objetivo avaliar o potencial de crescimento, a partir da determina????o das curvas de crescimento, de culturas de calos e de suspens??es celulares de D. saccifera, e realizar o estudo da composi????o qu??mica dos extratos obtidos dos calos e suspens??es, bem como avaliar as atividades qu??micas e biol??gicas. Para isso, calos previamente estabelecidos in vitro em meio Murashige e Skoog suplementado com 4 mg.L-1 de ??cido 2,4-diclorofenoxiac??tico (2,4D) e 2 mg.L-1 de cinetina (KIN) foram multiplicados em sucessivos subcultivos a cada 30 dias e mantidos em sala de crescimento com temperatura controlada de 26 ?? 2 ??C, sob fotoper??odo de 16/8 horas (claro/escuro), providos por l??mpadas fluorescentes. O material foi extra??do com os solventes org??nicos hexano, acetato de etila (AcOEt) e metanol. Os extratos foram testados nos ensaios antioxidante, antimicrobiano e antimicobacteriano, e fracionados. As culturas em suspens??o celular foram estabelecidas a partir de 1 g de calos inoculados em 100 mL de meio de cultura l??quido, em agita????o cont??nua. Foram determinadas as curvas de crescimento de ambas as formas de cultivo, ao longo de 40 dias, com intuito de realizar um estudo comparativo atrav??s do c??lculo dos par??metros da cin??tica de crescimento. No estudo fitoqu??mico dos extratos foi poss??vel verificar a presen??a de terpenoides, esteroides, subst??ncias arom??ticas, ??cidos graxos, entre outros metab??litos secund??rios. Na fra????o 2 (6,3 mg) do extrato hex??nico foi observado o ind??cio da presen??a do esteroide lupen-3-ona. Com rela????o aos ensaios biol??gicos e qu??micos, o extrato AcOEt apresentou alta atividade antimicobacteriana frente a Mycobacterium tuberculosis, com uma concentra????o inibit??ria m??nima (CIM) de 25 ??g.mL-1, uma concentra????o inibit??ria a 90% (IC90) de 19,5 ??g.mL-1 e uma concentra????o bactericida m??nima (CBM) de 200 ??g.mL-1. Os extratos n??o foram ativos nos ensaios antimicrobianos frente ??s bact??rias testadas, assim como os extratos n??o apresentaram potencial antioxidante frente ao radical livre DPPH e o complexo Fe3+/fenantrolina. Os resultados obtidos demonstraram que o calo seguiu um padr??o de crescimento lento, atingindo maior massa fresca (MF) e seca (MS) no 40?? dia de cultivo com concentra????o de 72,6 e 2,808 g.L-1, respectivamente. J?? para a suspens??o celular, o ac??mulo de biomassa e o crescimento da cultura de c??lulas foi expressivamente maior e mais r??pido, atingindo maior MF e MS no 36?? dia de cultivo, sendo de 413,7 e 12,995 g.L-1, respectivamente. N??o foi poss??vel determinar com exatid??o as fases estacion??rias dos cultivos celulares, no entanto, foi inferido que a produ????o m??xima de metab??litos secund??rios ocorreu no 40?? dia para as culturas de calos e entre o 24?? ao 36?? dia para as culturas em suspens??o. Os par??metros cin??ticos calculados demonstram que a suspens??o celular possui um elevado potencial para o fornecimento de biomassa vegetal da esp??cie, tornando-se uma fonte promissora para a produ????o de metab??litos secund??rios obtidos in vitro. Achei o sistema claro, autoexplicativo e f??cil de usar.
Databáze: OpenAIRE
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