Isolation, modeling and phytosome forms of antimicrobial and antiproliferative compounds from plectranthus spp.
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2016
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Os produtos naturais têm sido uma valiosa fonte de novos produtos farmacêuticos. As plantas de género Plectranthus L'Her (Lamiaceae) possuem uma ampla diversidade de usos etnomedicinais, que correspondem a uma indicação da presença de potenciais fármacos na sua composição. Métodos inovadores para a veiculação dos produtos naturais, como os fitossomas, demostraram ser uma estratégia promissora para a melhora da veiculação e estabilidade destes produtos. Três plantas do género Plectranthus foram estudadas: P. madagascariensis, P. neochilus e P. porcatus. Extratos foram preparados pela conjugação de técnicas de extração como infusão, decocção, maceração, micro-ondas, ultrassons ou de extração por fluidos supercríticos, utilizando como solventes água, acetona, metanol ou dióxido de carbono supercrítico. Os extratos preparados foram perfilados por HPLC-DAD e identificados alguns dos principais componentes, por comparação com padrões, verificando-se na sua constituição polifenois, diterpenos e flavonoides. Os mesmos extratos foram avaliados em termos das suas atividades antimicrobiana (bactérias de Gram positivo, Gram negativo e leveduras), antioxidante (captura de radicais) e citotóxica (células MDA-MB-231). Extratos de P. madagascariensis (maceração e ultrassons em acetona) e P. neochilus (ultrassons em acetona) apresentaram efeitos antibacterianos contra Bacillus subtilis, Staphylococcus aureus, S. epidermidis e Klebsiella pneumoniae. O extrato de ultrassons de P. madagascariensis obtido com acetona apresentou potentes efeitos antibacterianos em espécies de Staphylococcus, incluindo uma estirpe resistente à meticilina, verificando-se uma concentração mínima inibitória no intervalo de 1,95 a 7,81 μg/mL. Esta atividade encontra-se de acordo com os usos tradicionais destas plantas como agente anti-infecioso. Os extratos metanolicos apresentaram potentes efeitos antioxidantes a uma concentração de extrato de 100 ng/mL (60,8-89,0%). O extrato de maceração em acetona de P. madagascariensis apresentou alguns efeitos citotóxicos na linha celular de cancro de mama MDA-MB-231 com um IC50 de 64,52 mg/mL. Uma vez que os extratos mais bioativos foram obtidos a partir de P. madagascariensis, estes foram caracterizados em maior detalhe com identificação e quantificação dos principais compostos nesses extratos. Quatro compostos foram identificados por comparação com padrões: ácido rosmarinico, 7α,6β-dihidroxiroyleanona, 7α-acetoxi-6β-hidroxiroileanona e coleona U. Um composto diterpenico foi isolado a partir do extrato de ultrassons com acetona de P. madagascariensis e caracterizado por RMN de 1H e 13C como 7α-formiloxi-6β-hidroxiroileanona. Este composto foi isolado pela primeira vez a partir desta planta. Os efeitos citotóxicos dos compostos purificados foram determinados numa bateria de linhas celulares de cancro e em conjugação com dados da literatura, foram estabelecidas algumas relações de estrutura-atividade na estrutura abietanica do tipo roileanona. Foi observada alguma seletividade para linhas celulares de cancro por parte da 7α,6β-dihidroxiroileanona e 7α-acetoxi-6β-hidroxiroileanona com com índice de seletividade de 4,3 e 3,2 respetivamente.O extrato com maior potência antibacteriana identificado na triagem inicial foi selecionado para sua incorporação numa formulação fitossomal com posterior encapsulação por quitosano. Os fitossomas obtidos apresentavam-se amorfos, uniformes na forma (SEM e AFM), com um tamanho médio de 1082 ± 363 nm e potencial zeta de 20,59 ± 12,02 mV. A eficiência de encapsulação determinada por HPLC (57,7 ± 0,06%). A existência de interações entre os componentes da formulação foi analisada por A ocorrência de encapsulação foi demonstrada por DSC e DRIFTS. Os fitossomas demonstraram uma libertação sustentada com redução da permeação trans-cutanea. Uma melhoria em até 4 vezes na atividade anti-estafilococo (concentração mínima inibitória de 0,49 a 31,25 mg/ml). A segurança desta formulação foi demonstrada por baixa citotoxicidade em queratinocitos humanos in vitro e por irritação negligenciável in vivo nos ensaios de irritação cutânea aguda e sub-crónica em ratinhos. Este estudo demonstrou o potencial do género Plectranthus como fonte de novos agentes antibacterianos justificando-se alguns dos usos etnomedicinais destas plantas. Os diterpenos do tipo abietano isolados a partir de P. madagascariensis possuem efeitos citotóxicos com alguma seletividade face a algumas linhas celulares de cancro. A formulação desenvolvida a partir do extrato de P. madagascariensis corresponde a um promissor candidato a antibacteriano tópico com amplo espectro de atividade e elevada potência.
Natural products have been used as a valuable source of new lead drugs. The Plectranthus L’Her (Lamiaceae) genus possess a wide diversity of ethnomedicinal uses which indicate the presence of bioactive molecules. Also, innovative methods for natural drug delivery, as phytosome, have showed to be a promising strategy for the improvement of delivery and stability. In this work, three plants from the Plectranthus genus were studied: P. madagascariensis, P. neochilus and P. porcatus. Several extracts were prepared by the combination of extraction methods (infusion, decoction, microwave, ultrasound, maceration and supercritical fluid extraction) with different polarity solvents (water, acetone, methanol and scCO2). Those extracts were profiled by HPLC-DAD and the main components were identified, including polyphenols (caffeic acid, chlorogenic acid and rosmarinic acid), diterpenes (7α-acetoxy,6β-hydroxyroyleanone and coleon U) and flavones (rutina y naringenina). The prepared extracts were screened for their antimicrobial (Gram positive and negative bacteria and yeasts), antioxidant (DPPH radical scavenging activity) and cytotoxic activities (MDA-MB-231 cell line). Extracts from P. madagascariensis (acetone maceration and acetone ultrasound) and P. neochilus (acetone ultrasound) showed antibacterial effects against Gram positive bacteria strains, namely, Bacillus subtilis, Staphylococcus aureus and S. epidermidis and a Gram negative bacteria strain, Klebsiella pneumonia (MIC values 1.95-250 μg/mL). The ultrasound extract of P. madagascariensis prepared with acetone showed potent antibacterial effect against Staphylococcus spp., including a methicillin-resistant strain (MRSA), with MIC values ranging from 1.95 to 7.81 μg/mL. These results validate the traditional uses of such plants as anti-infectious agents. All methanolic extracts showed potent antioxidant effects at 100 ng/mL (60.8-89.0%). The maceration acetone extract from P. madagascariensis showed moderate cytotoxic effects in the MDA-MB-231 breast cancer cell line with IC50 of 64.52 μg/mL. The organic solvent extracts from P. madagascariensis were the most bioactive and thus characterized (identification and quantification) using HPLC-DAD. Furthermore, the compounds were identified by authentic standard overlay: rosmarinic acid, 7α,6β-dihydroxyroyleanone, 7α-acetoxy-6β-hydroxyroyleanone and coleon U. A diterpenic compound was isolated from the ultrasound acetonic extract of P. madagascariensis and spectroscopically characterized (1H- and 13C-NMR) as 7α-formyloxy-6β-hydroxyroyleanone. This was the first time this compound was isolated from this plant. The cytotoxic effect of the identified compounds was evaluated in a battery of cancer cell lines (MDA-MB-231, MCF-7, HCT116, NCI-H460 and MCR-5). The diterpenic compounds showed moderate to potent cytotoxicity in the majority of tested cell lines. A high selectivity for cancer cell lines was observed for 7α,6β-dihydroxyroyleanone and 7α-acetoxy-6β-hydroxyroyleanone with selectivity index of 4.3 and 3.2, respectively. The combination of the observed results and literature data afforded the establishment of new structure-activity relationships of roylenanone abietanic compounds. The relevance of lipophilicity and of the presence of an electron donating group at 6 and/or 7 positions was observed. The antibacterial acetonic ultrasound extract from P. madagascariensis identified in the initial screening was selected for incorporation into a phytosomal formulation and subsequently coated by chitosan. Phytosomes were amorphous, uniform in shape as shown by AFM and SEM, and with an average size of 1082 ± 363 nm and zeta potential of +20.59 ± 12.02 mV. The encapsulation of the antibacterial extract was determined by HPLC (57.7±0.06%) and the chemical interactions between the formulation components was cofirmed by DSC and DRIFTS. Such phytosomes showed a sustained release of the extract 4 and lower skin-like permeation fluxes. An improvement up to a 4-fold factor in the anti-Staphylococci activity (MIC values 0.98-31.25 μg/mL) was observed. The safety of such formulation was verified by in vitro human keratinocytes cytotoxicity assays and by in vivo acute and sub-chronic dermal irritation tests in mice. This study showed the potential of the Plectranthus genus as source of lead antibacterial and antiproliferative agents and validate the ethnomedicinal uses of the studied plants. The isolated abietane diterpenes obtained from P. madagascariensis possess promising selective cytotoxic effects, namely, aginst the lung cancer lines tested. Also, the developed formulation of extract of P. madagascariensis corresponds to a potent topical antibacterial candidate with a broad spectrum of activity.
Natural products have been used as a valuable source of new lead drugs. The Plectranthus L’Her (Lamiaceae) genus possess a wide diversity of ethnomedicinal uses which indicate the presence of bioactive molecules. Also, innovative methods for natural drug delivery, as phytosome, have showed to be a promising strategy for the improvement of delivery and stability. In this work, three plants from the Plectranthus genus were studied: P. madagascariensis, P. neochilus and P. porcatus. Several extracts were prepared by the combination of extraction methods (infusion, decoction, microwave, ultrasound, maceration and supercritical fluid extraction) with different polarity solvents (water, acetone, methanol and scCO2). Those extracts were profiled by HPLC-DAD and the main components were identified, including polyphenols (caffeic acid, chlorogenic acid and rosmarinic acid), diterpenes (7α-acetoxy,6β-hydroxyroyleanone and coleon U) and flavones (rutina y naringenina). The prepared extracts were screened for their antimicrobial (Gram positive and negative bacteria and yeasts), antioxidant (DPPH radical scavenging activity) and cytotoxic activities (MDA-MB-231 cell line). Extracts from P. madagascariensis (acetone maceration and acetone ultrasound) and P. neochilus (acetone ultrasound) showed antibacterial effects against Gram positive bacteria strains, namely, Bacillus subtilis, Staphylococcus aureus and S. epidermidis and a Gram negative bacteria strain, Klebsiella pneumonia (MIC values 1.95-250 μg/mL). The ultrasound extract of P. madagascariensis prepared with acetone showed potent antibacterial effect against Staphylococcus spp., including a methicillin-resistant strain (MRSA), with MIC values ranging from 1.95 to 7.81 μg/mL. These results validate the traditional uses of such plants as anti-infectious agents. All methanolic extracts showed potent antioxidant effects at 100 ng/mL (60.8-89.0%). The maceration acetone extract from P. madagascariensis showed moderate cytotoxic effects in the MDA-MB-231 breast cancer cell line with IC50 of 64.52 μg/mL. The organic solvent extracts from P. madagascariensis were the most bioactive and thus characterized (identification and quantification) using HPLC-DAD. Furthermore, the compounds were identified by authentic standard overlay: rosmarinic acid, 7α,6β-dihydroxyroyleanone, 7α-acetoxy-6β-hydroxyroyleanone and coleon U. A diterpenic compound was isolated from the ultrasound acetonic extract of P. madagascariensis and spectroscopically characterized (1H- and 13C-NMR) as 7α-formyloxy-6β-hydroxyroyleanone. This was the first time this compound was isolated from this plant. The cytotoxic effect of the identified compounds was evaluated in a battery of cancer cell lines (MDA-MB-231, MCF-7, HCT116, NCI-H460 and MCR-5). The diterpenic compounds showed moderate to potent cytotoxicity in the majority of tested cell lines. A high selectivity for cancer cell lines was observed for 7α,6β-dihydroxyroyleanone and 7α-acetoxy-6β-hydroxyroyleanone with selectivity index of 4.3 and 3.2, respectively. The combination of the observed results and literature data afforded the establishment of new structure-activity relationships of roylenanone abietanic compounds. The relevance of lipophilicity and of the presence of an electron donating group at 6 and/or 7 positions was observed. The antibacterial acetonic ultrasound extract from P. madagascariensis identified in the initial screening was selected for incorporation into a phytosomal formulation and subsequently coated by chitosan. Phytosomes were amorphous, uniform in shape as shown by AFM and SEM, and with an average size of 1082 ± 363 nm and zeta potential of +20.59 ± 12.02 mV. The encapsulation of the antibacterial extract was determined by HPLC (57.7±0.06%) and the chemical interactions between the formulation components was cofirmed by DSC and DRIFTS. Such phytosomes showed a sustained release of the extract 4 and lower skin-like permeation fluxes. An improvement up to a 4-fold factor in the anti-Staphylococci activity (MIC values 0.98-31.25 μg/mL) was observed. The safety of such formulation was verified by in vitro human keratinocytes cytotoxicity assays and by in vivo acute and sub-chronic dermal irritation tests in mice. This study showed the potential of the Plectranthus genus as source of lead antibacterial and antiproliferative agents and validate the ethnomedicinal uses of the studied plants. The isolated abietane diterpenes obtained from P. madagascariensis possess promising selective cytotoxic effects, namely, aginst the lung cancer lines tested. Also, the developed formulation of extract of P. madagascariensis corresponds to a potent topical antibacterial candidate with a broad spectrum of activity.
Descrição
Diretores de Tese : Dra. Patrícia Rijo ; Dra. Catarina Reis ; Dra. Ana Mª Díaz-Lanza
Palavras-chave
ANTIMICROBIANOS, DITERPENOS, FITOQUÍMICA, FARMACOLOGIA, DOUTORAMENTO EM CIÊNCIAS DA SAÚDE, CIÊNCIAS DA SAÚDE, HEALTH SCIENCES, ANTIMICROBIAL, PLANT CHEMISTRY, PHARMACOLOGY
Citação
Matias , D H C 2016 , Isolation, modeling and phytosome forms of antimicrobial and antiproliferative compounds from plectranthus spp. .