Isolation and design of diterpenoids from plectranthus species

Abstract

Despite the great development in human medicine, cancer is still a serious threat to public health. It is a well-known leading cause of morbidity and mortality worldwide, consequently, research on new anticancer agents ought to be continued. Natural products from medicinal plants represent a major resource of novel therapeutic substances for combating serious diseases including cancer. The Plectranthus genus (Family: Lamiaceae) represents a large and widespread group of species with a diversity of traditional uses for the treatment of several ailments. Cytotoxicity screenings have identified Plectranthus plants as potential sources of antitumor lead compounds. They are rich in diterpenoids which are reported to be responsible for various pharmacological activities such as cytotoxic activity. In this project, sixteen plants from the Plectranthus genus were studied. The acetonic extracts were prepared by the ultrasound-assisted extraction method (10 % (w/v)). The prepared extracts were screened for their antimicrobial, antioxidant, general toxicity, and cytotoxicity. The antimicrobial activity of each extract was screened against yeasts, Gram-positive and Gram-negative bacteria. P. hadiensis and P. mutabilis extracts were the most active using the well diffusion method. Their MIC and MBC (microdilution method) were determined, and they possessed significant activity against Staphylococcus aureus and Candida albicans with MIC values ranging from 3.91 µg/mL to 125 µg/mL and MBC from 62.5 to 250 µg/mL. The antioxidant activity of the extracts was quantitatively determined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging radical assay. P. hadiensis and P. mutabilis extracts having the highest scavenging activities of 46.14% and 36.24% respectively. The general toxicity of all the extracts was determined using the Brine Shrimp Lethality assay (BSLA) and the cytotoxicity of the most toxic extract was determined. P. hadiensis and P. mutabilis we found to be the most bioactive and the compounds responsible for their bioactivity identified. 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 using preparative TLC and tested against the aggressive type triple-negative breast cancer (MDA-MB-231S). P. hadiensis extract and 7α-acetoxy6β-hydroxyroyleanone reduced the viability of MDA-MB-231S cancer cell line cells, 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. The phytochemistry of the second bioactive extract (P. mutabilis) was done. Bio-guided fractionation of ultrasound-assisted acetonic extract of P. mutabilis leaves resulted in the isolation of a 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). From the ESI+ MS/MS fragmentation patterns analysis, compound (5) was tentatively identified as acetoxy derivative of an abietane diterpenoid. HPLC analysis revealed Coleon U (96 ± 0.048 µg/mg) to be the major compound of the P. mutabilis extract in the wavelengths analyzed. Computational data indicates a biosynthetic relation between 2, 3, and 4. These results suggest that both the quinone (2) and the epoxyquinone (3) are formed directly from Coleon U (4). Coleon U, coleon U quinone, and 8α,9α-Epoxycoleon U quinone were found to be selective towards the cancer cell lines and their anticancer effect was not compromised by P-gp activity in NCI-H460/R cells. Importantly, 2, 3, and 4 were able to inhibit P-gp activity in NCI-H460/R cells at longer exposure of 72 h and consequently revert doxorubicin (DOX) resistance in subsequent combined treatment. Compound 1 was inactive against all three cell lines in the range of concentrations (2 to 50 microM) tested. All compounds did not influence the ABCB1 expression in NCI-H460/R cells, while the extract significantly increased it. This work identified P. hadiensis leaves and P. mutabilis extracts as a potential source of bioactive compounds to fight MDR. Cytotoxic 7αacetoxy-6β-hydroxyroyleanone previously isolated from P. hadiensis, its hemisynthetic derivative 7α-acetoxy-6β-benzoyloxyroyleanone (12BzRoy) and 6,7-dehydroroyleanone (DHR) isolated from the essential oil of P. madagascariensis were employed in the present work as lead molecules for the synthesis of self-assembled nanoparticles. RoyOA, DHR-sq, and 12BzRoy-sq conjugates were successfully synthesized and their nanoassemblies characterized. Roy-OA NPs were most promising and was characterized based on size (509.33 nm), Pdl (0.249), zeta potential (-46.2mV), 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 DHR and Roy respectively. These results suggested that these nanoassemblies act as prodrugs for the release of cytotoxic lead molecules.