Aislamiento, Modelización y Fitossomas de Compuestos Antimicrobianos y Antiproliferativos de Plectranthus SPP

Abstract

The Plectranthus L’Her (Lamiaceae) genus possess a wide diversity of ethnomedicinal uses which indicate the presence of bioactive molecules. Also, natural drug delivery systems, as the phytosome, are promising strategies to improve bioactivity 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 with different polarity solvents. The obtained extracts were profiled by HPLC-DAD and the main components were identified, including polyphenols, diterpenes and flavones. Additionally, the 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). Acetonic extracts from P. madagascariensis (ultrasound and maceration) and P. neochilus (ultrasound) showed antibacterial effects against the majority of Gram positive bacteria strains tested (MIC values 1.95-250 μg/mL). The acetone ultrasound extract of P. madagascariensis showed potent antibacterial effect against Staphylococcus spp. tested, including a methicillin-resistant strain (MIC value 1.95-7.81 μg/mL). 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 (IC50 64.52 μg/mL). These results validate the traditional uses of such plants as anti-infectious agents. The bioactive organic solvent extracts from P. madagascariensis were characterized using HPLC-DAD (major compound identification and quantification). Rosmarinic acid, 7α,6β-dihydroxyroyleanone, 7α-acetoxy-6β-hydroxyroyleanone and coleon U were identified by authentic standard overlay. 7α-Formyloxy-6β-hydroxyroyleanone was isolated from the ultrasound acetonic extract of P. madagascariensis and spectroscopically characterized (1H- and 13C-NMR). The pure compounds were tested in a battery of cancer cell lines (MDA-MB-231, MCF-7, HCT116, NCI-H460 and MCR-5) and showed moderate to potent cytotoxicity in the majority of the tested cell lines. 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 showed the relevance of lipophilicity and of the presence of an electron donating group at 6 and/or 7 positions to obtain high cytotoxicity in roylenanone abietanic compounds. The antibacterial acetonic ultrasound extract from P. madagascariensis was selected for incorporation in a phytosomal formulation and subsequently coated by chitosan. Phytosomes were amorphous, uniform in shape (SEM and AFM) and with an average size of 1082 ± 363 nm and zeta potential of +20.59 ± 12.02 mV. The encapsulation efficiency of the extract was determined by HPLC (57.7±0.06%). Physico-chemical interactions between formulation components were studied by DSC and DRIFTS. The phytosomes showed a sustained release and lower skin-like permeation fluxes. An up to 4-fold improvement of the anti-Staphylococci activity was verified (MIC values 0.49-31.25 μg/mL). The safety of such formulation was verified by in vitro human keratinocytes cytotoxicity and by in vivo dermal irritation tests in mice. This study showed the potential of the Plectranthus genus as a source of lead antibacterial and antiproliferative agents and thus validate the ethnomedicinal uses of the studied plants. The isolated abietane diterpenes obtained from P. madagascariensis possess promising selective cytotoxic effects in the lung cancer lines tested. Additionally, the developed phytosomal formulation containing a P. madagascariensis extract corresponds to a potent topical antibacterial candidate with a broad spectrum of activity