Atividade citotóxica in vitro dos metabólicos das folhas de Piper cernuum vell (Piperaceae) e derivados monoterpênicos naturais e semissintéticos
Data
2013-08-23
Autores
Capello, Tabata Molero 
Orientadores
Lago, João Henrique Ghilardi
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
No presente trabalho foi realizado o estudo químico biomonitorado pela atividade citotóxica (células tumorais B16F10-Nex2) do extrato metanólico das folhas de Piper cernuum vell. (Piperaceae). Para tanto, o extrato bruto foi submetido à cromatografia em coluna (SiO2) com diferentes fases móveis (hexano, acetato de etila e metanol). Após a separação em onze grupos (A – K), estes foram analisados frente ao potencial citotóxico, dos quais apenas três grupos (D, F e G) apresentaram atividade e foram novamente purificados através de cromatografia de camada delgada preparativa. Tal procedimento permitiu o isolamento dos seguintes compostos: ácido 3,4-dimetoxidiidrocinâmico (I), piplaróxido (II) e 4-hidroxi-3,5-dimetoxidiidrocinamato de metila (III), sendo que as estruturas foram definidas através de análise dos respectivos espectros de RMN de 1H e de 13C além de EM. Todos os compostos isolados foram analisados quanto atividade citotóxica frente a células tumorais B16F10-Nex2 (melanoma murino), A2058 (melanoma humano), HeLa (carcinoma cervical), HL-60 (leucemia), U-87 (glioblastoma humano) e HCT (carcinoma ileocecal-cólon), porém apenas o composto (II) mostrou uma concentração inibitória (CI50) de 58 µg/mL para linhagem B16F10-Nex2 (melanoma murino), enquanto que todos os outros compostos isolados não mostraram potencial citotóxico para nenhuma linhagem (CI50 ≥ 100 g/mL). Adicionalmente, o óleo volátil das folhas de P. cernuum foi extraído por arraste a vapor utilizando aparelho de Clevenger e analisado por CG-EM, o que possibilitou a identificação de dezenove compostos, dentre os quais β-elemeno, biciclogermacreno e (E)--cariofileno foram os compostos majoritários. Do mesmo modo que os compostos isolados do extrato bruto, o óleo volátil também foi analisado quanto à atividade citotóxica in vitro frente às mesmas linhagens tumorais citadas acima, apresentando potencial em todas as linhagens (CI50 variando de 15,5 ± 3,5 a 30 ± 2 µg/mL). Além disso, nesse trabalho foi avaliada a atividade citotóxica de onze monoterpenos naturais (α-pineno, β-pineno, canfeno, limoneno, carvacrol, timol, p-cimeno, mirceno, linalol, α-terpineol e mentol) frente às linhagens tumorais B16F10-Nex2 (melanoma murino), A2058 (melanoma humano), HeLa (carcinoma cervical) e HL-60 (leucemia), sendo os derivados α-pineno, β-pineno, canfeno, limoneno, carvacrol e timol os mais ativos. Foram preparados seus derivados semissintéticos hidrogenados (canfano e p-mentano) e acetilados (acetatos de carvacrol e de timol), cujas estruturas foram identificadas por análises dos espectros de RMN 13C e EM. Tais compostos semissintéticos foram também analisados quanto à atividade citotóxica frente às mesmas linhagens tumorais dos monoterpenos naturais, mostrando-se ativo apenas para o composto semissintético p-mentano (CI50 28 ± 6 µg/mL) o qual mostrou atividade similar ao monoterpeno natural limoneno (CI50 33 ± 3 µg/mL).
In the present work has been carried out the study of the bioguided fractionation by cytotoxic activity (tumour cell type B16F10-Nex2) of the methanolic extract from the leaves of Piper cernuum vell. (Piperaceae). Therefore, the crude extract was subjected to column chromatography (SiO2) with different moving stages (hexane, ethyl acetate and methanol). After its separation into eleven groups (A - K), these groups were evaluated against the cytotoxic potential in which only three groups (D, F and G) displayed activity and were further purified by preparative thin layer chromatography. This procedure allowed the isolation of the following substances: 3,4-dimethoxydihydrocinnamic acid (I), piplaroxide (II) and 4-hydroxy-3,5-dimethoxydihydrocinnamate of methyl (III), and the structures were established by analysis of the their 1H and 13C NMR spectra as well as MS. All of the isolated compounds were analyzed for cytotoxic activity against the tumour cells B16F10-Nex2 (murine melanoma), A2058 (human melanoma), HeLa (cervical carcinoma), HL-60 (leukemia), U-87 (human glioblastoma) and HCT (ileocecal carcinoma-colon), but only the compound (II) showed an inhibitory concentration (IC50) of 58 g/mL to the line B16F10-Nex2 (murine melanoma), while all other isolated compounds showed no cytotoxic to any line (IC50 ≥ 100 g/mL). Additionally, the volatile oil from leaves of P. cernuum was extracted by steam distillation using Clevenger apparatus and was analyzed by GC-MS, allowing the identification of nineteen compounds, among which β-elemene, bicyclogermacrene and (E)- β-caryophyllene were the major compounds. Similarly to the compounds isolated from the crude extract, the volatile oil has also been assayed for cytotoxic activity in vitro against the same tumour cell lines mentioned above, showing potential in all lineages (IC50 ranging from 15.5 ± 3.5 to 30 ± 2 g/mL). Moreover, in this work was evaluated the cytotoxic activity of eleven natural monoterpenes (α-pinene, β-pinene, camphene, limonene, carvacrol, thymol, p-cymene, myrcene, linalool, α-terpineol, and menthol) against tumour cell lines B16F10-Nex2 (murine melanoma), A2058 (human melanoma), HeLa (cervical carcinoma) and HL-60 (leukemia), being α-pinene, β-pinene, camphene, limonene, carvacrol, and thymol the most active compounds. Their semi-synthetic derivatives were prepared, including hydrogenated (camphane and p-menthane) and acetylated (carvacrol and thymol acetates), the structures of which were identified by analysis of 13C NMR spectra and MS. Such semi-synthetic compounds were also analyzed for cytotoxic activity against the same tumour cell lines, presenting themselves active only for the semi-synthetic compound p-menthane (IC50 28 ± 6 g/mL), which showed similar activity to the natural monoterpene limonene (IC50 33 ± 3 g/mL)
In the present work has been carried out the study of the bioguided fractionation by cytotoxic activity (tumour cell type B16F10-Nex2) of the methanolic extract from the leaves of Piper cernuum vell. (Piperaceae). Therefore, the crude extract was subjected to column chromatography (SiO2) with different moving stages (hexane, ethyl acetate and methanol). After its separation into eleven groups (A - K), these groups were evaluated against the cytotoxic potential in which only three groups (D, F and G) displayed activity and were further purified by preparative thin layer chromatography. This procedure allowed the isolation of the following substances: 3,4-dimethoxydihydrocinnamic acid (I), piplaroxide (II) and 4-hydroxy-3,5-dimethoxydihydrocinnamate of methyl (III), and the structures were established by analysis of the their 1H and 13C NMR spectra as well as MS. All of the isolated compounds were analyzed for cytotoxic activity against the tumour cells B16F10-Nex2 (murine melanoma), A2058 (human melanoma), HeLa (cervical carcinoma), HL-60 (leukemia), U-87 (human glioblastoma) and HCT (ileocecal carcinoma-colon), but only the compound (II) showed an inhibitory concentration (IC50) of 58 g/mL to the line B16F10-Nex2 (murine melanoma), while all other isolated compounds showed no cytotoxic to any line (IC50 ≥ 100 g/mL). Additionally, the volatile oil from leaves of P. cernuum was extracted by steam distillation using Clevenger apparatus and was analyzed by GC-MS, allowing the identification of nineteen compounds, among which β-elemene, bicyclogermacrene and (E)- β-caryophyllene were the major compounds. Similarly to the compounds isolated from the crude extract, the volatile oil has also been assayed for cytotoxic activity in vitro against the same tumour cell lines mentioned above, showing potential in all lineages (IC50 ranging from 15.5 ± 3.5 to 30 ± 2 g/mL). Moreover, in this work was evaluated the cytotoxic activity of eleven natural monoterpenes (α-pinene, β-pinene, camphene, limonene, carvacrol, thymol, p-cymene, myrcene, linalool, α-terpineol, and menthol) against tumour cell lines B16F10-Nex2 (murine melanoma), A2058 (human melanoma), HeLa (cervical carcinoma) and HL-60 (leukemia), being α-pinene, β-pinene, camphene, limonene, carvacrol, and thymol the most active compounds. Their semi-synthetic derivatives were prepared, including hydrogenated (camphane and p-menthane) and acetylated (carvacrol and thymol acetates), the structures of which were identified by analysis of 13C NMR spectra and MS. Such semi-synthetic compounds were also analyzed for cytotoxic activity against the same tumour cell lines, presenting themselves active only for the semi-synthetic compound p-menthane (IC50 28 ± 6 g/mL), which showed similar activity to the natural monoterpene limonene (IC50 33 ± 3 g/mL)
Descrição
Citação
CAPELLO, Tabata Molero. Atividade citotóxica in vitro dos metabólicos das folhas de piper cernuum vell (piperaceae) e derivados monoterpênicos naturais e semissintéticos. 2013. 116 f. Dissertação (Mestrado) - Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo (UNIFESP), Diadema, 2013.