Amazonian plants from ethnomedicine to biotechnology through pharmaceutical biology approaches: a PhD experience in connecting forest with laboratory

The South american Natives, Shuar and Achuar people and their ethnomedical culture constitute the background subject of the Phd research, performed both in Ecuador (Salesian Politechnic University, Quito), and in Italy (Pharmaceutical biology labs, University of Ferrara). Based on ethnomedical responses, Piper aduncum, Maytenus macrocarpa, Schinus molle, Tecoma stans and Eugenia hallii were chosen as amazonian plant species subject of the research. AIMS The research has been focused on: − checking the presence of endophytic fungi in plants; − isolating and subculturing pure endophytic strains; − checking the biotransformation capacity of the isolated endophytes on pure compounds; the most performing endophytes were also tested on phytocomplexes and pure chemicals obtained by the plant from which the fungi were isolated; − phytochemical characterization and bioactivity assays of plant extracts: P. aduncum. − METHODS Biotransformations. Fresh aerial plant parts were properly washed in sanitizing solutions and in vitro cultured using adequate solid media to isolate endophytes. (+/-)-cis-bicyclo[3.2.0]hept-2-en-6-one, acetophenone, 1-indanone, 2-furyl methyl ketone, 2-methylcyclopentanone, 2-methylcyclohexanone, 2- methoxycyclohexanone were chosen as substrate model for biotransformations. The cultures were sampled after 1, 3, 7, 10 days of culturing, and ethyl acetate extracted to verify by GC-MS the presence of possible biotransformation products. Biotransformations were also checked on P. aduncum whole essential oil and on dillapiol, cis-ocimene, piperitone, (-)-terpinen-4-ol as most abundant chemicals. Chemical fingerprinting of P. aduncum essential oil. Steam distillation was adopted to obtain the essential oil, then characterized by GC-MS, NMR analyses. In vitro bioassays of P. aduncum essential oil. Antimicrobial activities were checked in vitro using proper agarized media to reach MIC. Antioxidant capacities were checked through DPPH test, ABTS and photochemiluminescence assays. Born's turbidimetric method and Writhing test were respectively adopted to check platelet-aggregation and anti-nociceptive properties. Mutagenic, antimutagenic properties and toxicity were assayed using classical and modified Ames test. MAIN RESULTS 364 fungal strains were in vitro isolated. Among all, 5 strains performed biotransformations on acetophenone to (S)-1-phenylethanol, with important yields (78-97%) and enantiomeric excess (78- 100%). Three strains gave also phenols probably by enzymatic reactions (Baeyer-Villiger oxidations). 15 fungal strains gave the lactones (-)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (-)-(1R,5S)-3- oxabicyclo[3.3.0]oct-6-en-2-one from (+/-)-cis-bicyclo[3.2.0]hept-2-en-6-one, probably as result of monooxygenase activation. Phytochemical characterization of P. aduncum essential oil has evidenced dillapiol as the most abundant terpene, followed by cis-ocimene, piperitone and terpinen-4-ol. Only cisocimene and piperitone gave several biotransformation products through dehydrogenation and hydroxylation reactions. The essential oil has evidenced non-mutagenic properties and interesting antifungal and antioxidant activities. CONCLUSIONS Several endophytic fungal strains from Amazonian plants were isolated and checked for biotransformations on pure chemicals and on P. aduncum essential oil. Data obtained will be useful for possible following patents about micro-organisms able to transform pharmaceutically interesting chemicals. Taxonomical characterization of the most performing fungal strains is still in progress. P. aduncum essential oil can be considered genotoxically safe and provides interesting antifungal and antioxidant properties, supporting its ethnomedical use as cicatrising and disinfectant crude drug and suggesting an extension of its employ as preservative ingredient.