Snezana Agatonovic-Kustrin1,2, David Morton1,2
1Department of Pharmaceutical and Toxicological Chemistry named after Arzamastsev of the Institute of Pharmacy, I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow 119991, Russia
2Department of Rural Clinical Sciences, La Trobe University, Edwards Rd, Bendigo 3550, Australia
This email address is being protected from spambots. You need JavaScript enabled to view it.
Olea europaea L. or European olive, is one of the oldest known agricultural fruit trees cultivated to produce olives and olive oil. Cultivation of olive trees for extraction of olive oil generates large quantities of olive leaves as a by-product. Due to their high polyphenolic content, olive leaves present an important raw material. It has been reported by many researchers that olive leaf extract has antimicrobial activity due to its high phenolic content. The aim of this study was to analyse the effect of spontaneous microbial maceration on the release and extraction of the flavonoids and phenolics from olive leaves, and to identify compounds responsible for the antimicrobial activity of olive leaf extracts via thin layer chromatography - direct bioautography (TLC-DB). Results show that fermentation significantly enhances extraction of phenolic compounds and flavonoids. Polyphenolic content was increased from 6.7 µg GAE (gallic acid equivalents) to 25.5 µg GAE, antioxidants from 10.3 µg GAE to 25.3 µg GAE, and flavonoid content from 42 µg RE (rutin equivalents) to 238 µg RE per 20 µL of extract. Increased antioxidant activity of fermented ethyl acetate extracts was attributed to the increased extraction of flavonoids and phenolic terpenoids, while increased antioxidant activity in fermented ethanol extract was due to increased extraction of flavonoids. This increase in extraction may be due, in part, to in situ generated lactic acid based natural deep eutectic solvents (NADES) from the plant sample. Lactic acid that is released during fermentation and glycine present in the olive leaves form a Natural Deep Eutectic Solvent (NADES) with significantly increased solubility for flavonoids. TLC screening and effect-directed analysis of olive leaf extracts revealed two zones that inhibit the growth of different Enterococcus and Staphylococcus bacterial strains. The active compounds were further identified as maslinic and oleanolic acid by comparison of the RF values of the bands with the standard reference materials and confirmed with ATR-FTIR and NMR spectra after isolation with flash chromatography.
