Great commercial value has been credited to the seeds of Dipteryx odorata WILLD. (AUBL.) (Leguminoseae) native from Amazon and also known as tonka beans or cumaru, because they are considered rich source of coumarins, used in cosmetics and perfumery.1) The presence of umbelliferone in seeds of tonka beans was described by Sullivan who also reported other natural compounds like isoflavonoids, triterpenoids, fatty acids and cassane diterpenoids on seeds of this species.2) Additionally some micromolecules specific of the heartwood of D. odorata as isoflavones retusin, retusin 8-methyl ether, 3Ј-hydroxyretusin 8-methyl ether, odoratin, dipteryxin, dipteryxine and odoratine; the triterpenes lupeol and betulin; and methyl esters of fatty acids have been isolated. 3,4) Isoflavonoids have gained considerable importance due to the diverse broad of biological activities correlated to them, including antioxidative, oestrogenic, inseticidal, piscicidal, antifungal, antimicrobial and contraceptive properties. 5,6) The pharmacological properties of isoflavonoids and the correlation between the consumption of isoflavonoids and reduction on the level of incidence of cardiovascular disease and cancer have increase the interest in the search for biologically active compounds from plant species of both, leguminous and non-leguminous sources.
7)In this work we report the establishment of D. odorata callus cultures focusing on a comparative study related to the production of tripanossomatidal GAPDH inhibitory isoflavones accumulated in roots of the intact plant and in callus cultures of this species.
ExperimentalPlant Material Dipteryx odorata plants were collected in the campus of Universidade do Amazonas in Manaus, Brazil. Voucher specimen is deposited under the number HUAM 7276 in the Herbarium of the Universidade do Amazonas.Seed Disinfestations Seeds of D. odorata were soaked in sterile distilled water and maintained under agitation overnight. Seeds were then immersed in 2% Benomyl solution for 4 h under agitation, washed in 70% hydroalcoholic solution, dipped in 1% CaClO for 40 min, washed for three times with sterile distilled water and inoculated into glass flasks (8.5 cmϫ2.5 cm i.d.) containing 5 ml MS medium 8) added with 30 g/l sucrose and 2.0 g/l Phytagel, pH was adjusted to 6.0 before autoclaving. Flasks were sealed with polypropylene closures (Bellco), autoclaved at 121°C and 105 kPa for 20 min and maintained at 25Ϯ2°C, 55-60% relative humidity under 16 h photoperiod with 40 mmol m Ϫ2 s
Ϫ1, 85 W coolwhite GE fluorescent lamps.Callus Induction Leaves, hypocotyl, and roots of D. odorata seedlings produced in vitro were used as explants for callus induction. Explants were inoculated onto semisolid MS medium supplemented with 2.0 mg/l 2,4-D, 0.5 mg/l 6-BAP and 340 mg/l KH 2 PO 4 and 2.0 g/l Phytagel, pH ajusted to 6.0. Callus were harvested and subcultured in 30-d intervals.General Analytical Procedures TLC was carried out on silica gel G pre-coated plates (Sigma-Aldrich) using CHCl 3 -MeOH (9 : 1) as solvent system. Spots...
