, 2006). Additionally, the isoorientin content in the extracts was determined by HPLC–UV/DAD. P. edulis Sims f. flavicarpa Degener (Passifloraceae)

fruits were picked on São Luiz farm in the municipality of Bauru, São Paulo, Brazil, in January 2008. P. alata Dryander fruits were picked in April 2008 on Morada da Paz farm in the municipality of Arealva, São Paulo, Brazil. The species were identified by Dr. Luís Carlos Bernacci (Herbarium IAC, INCB024360 ic50 Campinas-SP, Brazil) and voucher specimens of P. edulis and P. alata were deposited in the herbarium of the Campinas Agronomic Institute, São Paulo, Brazil (IAC 49929, IAC 50283, respectively). The plants were evaluated individually for incidence of the PWV virus in the fruit, based on typical symptoms, such as wrinkles, deformations and blisters on the leaf and rind surfaces. The fruits pulp and seeds were separated by sieving, after which the pulp was stored in jars and immediately frozen at −20 °C prior to preparation. The rinds (epicarp and mesocarp) were first washed in distilled water, then sliced and dried at 40 °C

until they reached a constant PD98059 supplier weight. The dried rinds were then ground in a food processor and sieved through a 16-mesh sieve to separate the material with a particle size of 1.0 mm. The ground rinds were stored in plastic containers protected from moisture and heat. Analytical-grade ethanol (Merck, VWRI, Leuven, Belgium) and methanol (J.T. Baker, Phillipsburg, NJ) were used in the extraction procedure. Dimethylsulfoxide (DMSO), NaCl, KCl, CaCl2, hydrogen peroxide (30% v/v) and Tween 20 were supplied by Merck. p-Nitrophenyl phosphate, sodium nitrite, bovine serum albumin (BSA), EDTA disodium salt, bis-N-methylacridinium nitrate (lucigenin), and phorbol 12-myristate 13-acetate (PMA) were purchased from

Sigma (Bornem, Belgium). Amplex red was purchased from Molecular Probes (Invitrogen, Merelbeke, Belgium). Isoorientin standard (purity ⩾99%) was purchased from Carl Roth (Karlsruhe, Germany). All the solutions were prepared with water purified in a Milli-Q system (Millipore, Bedford, Amino acid MA). The flavonoids of P. edulis and P. alata pulp were extracted under pre-optimised conditions ( Zeraik & Yariwake, 2010): passion fruit pulp (10.0 mL) was sonicated for 1.5 min with 30.0 mL of 60% ethanol at room temperature. The extracts were centrifuged at 5000g, 25 °C, for 20 min and the supernatant was concentrated to 2.0 mL using a rotary evaporator. The resulting aqueous solution was purified by solid-phase extraction, using Sep-Pak C18 cartridges (400.0 mg, Waters Associates, Milford, MA), which were preconditioned with 5.0 mL of methanol and 5.0 mL of water. The flavonoid fractions were eluted with 60% methanol to a precise volume of 2 mL. The extracts were evaporated to dryness using a rotary evaporator, and solubilised in DMSO to obtain stock solutions of 1.0, 10.0 and 100.0 mg L−1.