Antimicrobial potential of metabolites from Peperomia obtusifolia and its endophytic fungus Phomopsis sp.

RUIZ MOSTACERO, N.; CASTELLI, M.V.; BATISTA JR., J.M.; SALAZAR, M.O.; FURLAN, R.L.E.; FURLAN, M.; FULGUEIRA, C.L.; LÓPEZ, S.N.

Araraquara

Congreso; Sao Pablo Avdanced School on Bioorganic Chemistry; 2013

School on Bioorganic Chemistry

Endophytes are microorganisms that live in inner tissues of plants, without causing apparent disease symptoms. Isolation and study of their metabolism constitute a promising source of substances with novel structures and interesting bioactivities1. Peperomia obtusifolia (Piperaceae) is an ornamental plant with a broad geographical distribution in America, has reports of antifungal, tripanocidal activity and a screening about its antibiotic potential2. Chemical studies indicated presence of secondary metabolites; amides, lignans, flavonoids, dihydrochalcones, benzoquinones and benzopyrans prenylated esters, among others3. We report here the isolation of fungal endophytes from P. obtusifolia, the antibacterial activity evaluation of organic extracts from the host plant and cultures of the endophyte identified as Phomopsis sp. Aerial parts of a healthy specimen of P. obtusifolia (Gattuso (901) 2032, UNR) were selected to isolate endophytes and to prepare organic extracts, which were performed sequentially with hexane, dichloromethane (DCM), ethyl acetate (EtOAc) and methanol. In addition, fresh collected leaves and stems were subjected to removal of microorganisms from surface and cut into small portions, subsequent incubation on water agar and potato dextrose agar plates. Cultures were kept until 45 days (28°C, darkness). 72 isolates were obtained, 9 bacteria and 63 filamentous fungi. Endophytic fungus PO045 was identified as Phomopsis sp. by molecular analysis of its rDNA using ITS1 and ITS4 primers. In order to evaluate its antimicrobial potential, the fungus was grown in Czapek broth (28 °C, darkness). Sucrose (Suc) and Glucose (Glu) concentrations were monitored by 1H NMR in static (SM) and stirring (AM, 150rpm) modes. After depletion of Carbon source (33 days at SM and 47 days at AM) cultures were filtered to separate biomass from broth and then extracted with DCM and EtOAc, respectively. Antibacterial activity was carried out with agar-overlay bioautography on thin-layer chromatography (Gel Silice 60 GF 254), showing inhibition only on Gram (+) bacteria (Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29312). Analysis of P. obtusifolia extracts, shown that inhibition of the most active extract (DCM) was almost due to 3,4-dihydro-5-hydroxy-2,7-dimethyl-8-(3??-methyl-2??-butenyl)-2-(4?-methyl-1?,3?-pentadienyl)-2H-1-benzopyran-6-carboxylic acid and Peperobtusin A, prenylated chromanes previously reported from this species. Regarding Phomopsis sp. extracts, the results indicated that SM extracts showed a higher inhibition than AM, especially the filtrate. The isolation, structural elucidation and quantification of antimicrobial compounds are carrying out. To our knowledge this is the first report about antibiotic activity of metabolites from P. obtusifolia and the study of its endophytic population.