Improving Rosmarinus officinalis L. extracts´ health potential with Bacillus G36 metabolites formulated in AgNP

Rosmarinus officinalis L. is a medicinal plant with a traditional use for healing several diseases ranging from inflammatory to neurological diseases to mention the most relevant. The healing potential relies on its antioxidant potential due to its bioactive components among which are monoterpenes, constituting the essential oil; diterpenes like carnosol and its derivatives; triterpenes like ursolic acid; and fenilpropanoids among which caffeic acid and rosmarinic acid and isoforms are the best known. All these components are secondary metabolites key for plant adaptation to environmental changes. As secondary metabolites, their synthesis is inducible so finding elements that trigger plant secondary metabolism is relevant to enhance healing potential of medicinal plants, as is the case in rosmarinus. In addition to plant’s genetic endowment to enhance adaptation, plants recruit soil bacterial strains to benefit growth. These strains are known as Plant growth promoting rhizobacteria (PGPR) and communicate with plants by specific chemical molecules termed elicitors. Some strains and specific elicitors are able to trigger secondary metabolism, increasing bioactive contents. Nanotechnology appears as an innovative approach to increase effectiveness of specific molecules when a nanoparticle is synthetized in order to profit from NP’s size, shape and characteristics to move through the system. So, we hypothetized that metabolites from Bacillus G36 would be able to reduce Ag+, biosynthetizing a functionalized AgNP able to trigger rosmarinus´ secondary metabolism, increasing bioactive contents on rosemary extracts, and therefore, its healing potential. AgNP biological synthesis. Metabolites from Bacillus G36, a consolidated beneficial strain, were incubated with AgNO3- solutions for 24h at 37 ºC, in different pH conditions, resulting in the biosynthesis of AgNP. AgNP characterization. Full characterization by UV spectrum, TEM, XRD and FTIR was carried out. Experimental plant set up. Rosmarinus branches were detached from the plants and immediately sprayed with treatment solution (control, strain suspension, metabolites and AgNP, n=3). Plants were allowed to dry and ethanolic extracts were prepared. Extracts were characterized by total phenolic contents, total flavonol contents, antioxidant potential; secondary metabolites (carnosol and rosmarinic acid) were analyzed by HPLC. Results. A brownish color after incubation indicative of reduced silver, confirmed AgNP synthesis with UV absorption at 410-420 nm, indicative of reduced silver. AgNP were spherical, with an average diameter of 7.5 nm showing a coating of organic matter from the bacterium. Hence, bacterial metabolites were able to reduce silver, creating a unique coating that confers special activity to the AgNP. NP under 10 nm were most suitable to penetrate to subcellular levels and therefore, cause changes in cell metabolism. When sprayed on rosmarinus, AgNP increased total phenols (40%), total flavonols (40%), antioxidant potential (60%), rosmarinc acid (30%), and carnosic acid (25%); bacterium and metabolites alone did not affect total phenols and total flavonols; interestingly, the bacterial metabolites enhanced antioxidant potential to similar levels than the NP. Finally, the unique characteristics of the AgNP coated with bacterial metabolites is evidenced