Isolation, modeling and phytosome forms of antimicrobial and antiproliferative compounds from plectranthus spp.ectranthus spp

Laburpena

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,6βdihydroxyroyleanone, 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-6βhydroxyroyleanone. 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.