Selective and cost‑effective protocol to separate bioactive triterpene acids from plant matrices using alkalinized ethanol: Application to leaves of Myrtaceae species

Background: Triterpenes as betulinic (BA), oleanolic (OA) and ursolic acids (UA) have increasingly gained therapeutic relevance due to their wide scope of pharmacological activities. To fit large‑scale demands, exploitable sources of these compounds have to be found and simple, cost‑effective methods to extract them developed. Leaf material represents the best plant sustainable raw material. To obtain triterpene acid‑rich extracts from leaves of Eugenia, Psidium and Syzygium species (Myrtaceae) by directly treating the dry plant material with alkalinized hydrated ethanol. This procedure was adapted from earlier methods to effect depolymerization of the leaf cutin. Materials and Methods: Extracts were prepared by shaking the milled dry leaves in freshly prepared 2% NaOH in 95% EtOH solution (1:4 w/v) at room temperature for 6 h. Working up the product in acidic aqueous medium led to clear precipitates in which BA, OA and UA were quantified by gas chromatography. Results: Pigment‑free and low‑polyphenol content extracts (1.2–2.8%) containing 6–50% of total triterpene acids were obtained for the six species assayed. UA (7–20%) predominated in most extracts, but BA preponderated in Eugenia florida (39%). Carried out in parallel, n‑hexane defatted leaves led to up to 9% enhancement of total acids in the extracts. The hydroalcoholate treatment of Myrtaceae species dry leaves proved to be a cost‑effective and environmentally friendly method to obtain triterpene acids, providing them be resistant to alkaline medium. These combined techniques might be applicable to other plant species and tissues.