Isolation and design of diterpenoids from Plectranthus species

Cancer is still a serious threat to public health and according to WHO in 2018, thre are 1 in 6 deaths due to cancer. Muilti-drug resistant remains a major challenge to cancer therapy and there for the need to develop new reversal MDR agents. Natural products from medicinal plants represent a major resource of novel therapeutic substances for combating serious diseases including cancer. Cytotoxicity screenings have identified Plectranthus plants as potential sources of cytotoxic compounds. Most importantly, abietane type diterpenoids known for their cytotoxicity. In this work, sixteen Plectranthus spp. were screened for their bioactivity. Their acetone extracts were prepared using the ultrasound-assisted extraction method (10 % (w/v) and screened for their antimicrobial, antioxidant, general toxicity. The most bioactive extracts were selected and tested against three different cancer cell lines. P. hadiensis and P. mutabilis were the most bioactive and their phytochemical studies was done to identify the compounds that may be responsible for their bioactivity. The HPLC analysis of P. hadiensis leaves showed that the known abietane diterpene, 7α-acetoxy-6β-hydroxyroyleanone (Roy) was the major compound in this extract. Roy was isolated and tested against the aggressive type of triple-negative breast cancer (MDA-MB-231S) cell lines. P. hadiensis extract and Roy reduced the viability of MDA-MB-231S cancer cell lines, showing an IC50 value of 25.6 μg/mL and 5.5 μM (2.15 μg/mL) respectively, suggesting that this lead molecule could be responsible for the bioactivity of this extract. Due to the poor aqueous solubility of royleanones, the need to decrease the site effects and enhance a target drug delivery; Roy, its hemisynthetic derivative 7α-acetoxy-6β-benzoyloxyroyleanone (12BzRoy) and 6,7-dehydroroyleanone (DHR) isolated from the essential oil of P. madagascariensis were used as lead molecules for the synthesis of self-assembled nanoparticles. Squalene (Sq), Oleic acid (OA) and 1-bromodecane were used was linkers. Roy-OA, DHR-sq, and 12BzRoy-sq conjugates were successfully synthesized and their nanoassemblies characterized based on size, Pdl, zeta potential, and morphology. The release profile of Roy was determined from Roy-OA NPs at physiological pH 7.4. The biological activity of DHR.sq and, Roy-OA NPs were evaluated, and both were found to have less bioactivity when compared with Roy and DHR respectively. These results suggested that these nanoassemblies act as prodrugs for the release of cytotoxic lead molecules. The second bioactive extract, P. mutabilis was subjected to a bio-guided fractionation of its acetone extract. From this extract, one new nor-abietane diterpene, (+)-(5S,10R)-10,11,12-trihydroxy-6,7-dioxo-20-nor-abieta-8,11,13-triene (1) alongside three known abietane-type diterpenoids Coleon-U-quinone (2), 8α,9α-epoxycoleon-U-quinone (3), and Coleon U (4) were isolated. The structure of the compounds was elucidated using different spectroscopic techniques (1D and 2D-NMR, LCMS, IR) and HRMS. From the ESI+ MS/MS fragmentation patterns analysis, an additional acetoxy derivative of an abietane diterpenoid, compound (5) was tentatively identified. Compounds 1-4 were quantified in the extract using HPLC-DAD. The isolated compounds were found to be unstable. Coleon U for instant was found to rapidly interchanges to its quinone derivative coleon U quinone. For this reason, a biosynthetic relation between the compounds was done. The results suggests that both the quinone (2) and the epoxyquinone (3) are formed directly from Coleon U (4). Cytotoxicity of the isolated compounds was done and their interaction with P-gp studied using the model systems human non-small cell lung carcinoma cells (NCI-H460), its MDR variant with P-gp expression (NCI-H460/R), and human embryonic pulmonary fibroblasts (MRC-5). In conclusion, this work provides important information about the biological activity of several Plectranthus spp. In a review, and published works, we identified the importance of using Artemia salina as a screening model in natural product chemistry. The phytochemical study of P. mutabilis was done for the first time in this study and we identified compounds with a unique way of interacting with P-gp. In addition, we proposed two paths for the possible formation of epoxide 3 from 4. We equally successively prepared self-assembly nanoparticles of royleanones using oleic and squalene linkers these by improving the the aqueous solubility of 7α-acetoxy-6β-hydroxyroyleanone, 7α-acetoxy-6β-benzoyloxyroyleanone and 6,7-dehydroroyleanone

Preliminary Biological Activity Screening of Plectranthus spp. Extracts for the Search of Anticancer Lead Molecules

Plectranthus species (Lamiaceae) have been employed in traditional medicine and this is now validated by the presence of bioactive abietane-type diterpenoids. Herein, sixteen Plectranthus acetonic extracts were prepared by ultrasound-assisted extraction and their biological activity was screened. The antimicrobial activity of each extract was screened against yeasts, and Gram-positive and Gram-negative bacteria. The P. hadiensis and P. mutabilis extracts possessed significant activity against Staphylococcus aureus and Candida albicans (microdilution method). Moreover, all extracts showed antioxidant activity using the DPPH method, with P. hadiensis and P. mutabilis extracts having the highest scavenging activities. Selected by the Artemia salina model, P. hadiensis and P.ciliatus possessed low micromolar anti-proliferative activities in human colon, breast, and lung cancer cell lines. Furthermore, the most bioactive extract of P. hadiensis leaves and the known abietane diterpene, 7α-acetoxy-6β-hydroxyroyleanone isolated from this plant, were tested against the aggressive type triple negative breast cancer (MDA-MB-231S). P. hadiensis extract reduced the viability of MDA-MB-231S cancer cell line cells, showing an IC50 value of 25.6 µg/mL. The IC50 value of 7α-acetoxy-6β-hydroxyroyleanone was 5.5 µM (2.15 µg/mL), suggesting that this lead molecule is a potential starting tool for the development of anti-cancer drugs.Fundação para a Ciência e a Tecnologia (FCT