Use of HPLC-DAD-MS/MS technique to detect secondary metabolites in Jatropha multifida L. (Euphorbiaceae)

The genus Jatropha is composed of about 175 tropical and subtropical species, belonging to the family Euphorbiaceae.1 The ornamental species J. multifida L., commonly known as coral flower and plant mertiolate, it’s a single-stem shrub with large leaves and red inflorescences, with small yellow flowers in the center.2 In Brazil, J. multifida L. has been used to treat injuries.3 Sabandar et al. (2013)4 showed that the genus Jatropha is responsible for exhibiting different pharmacological activities and for producing a great chemical diversity of secondary metabolites (diterpenes, triterpenes, lignans, coumarins, flavonoids, cyclopeptide alkaloids and cyanogenic glycosides). The objective of this work is to promote a comparative analysis of the chemical profile of extracts obtained from different organs of J. multifida by the replication method, focusing mainly on the class of macrocyclic diterpenes and biflavones. The species was identified by Pereira, O.J. and the exsiccata was deposited in the VIES Herbarium of the Federal University of Espírito Santo, under the registration number VIES 23404. The fresh organs of the species (roots, stems, leaves, bark and petioles) were weighed separately, dried in a circulating air oven at 40° C, reweighed, crushed and subjected to continuous extraction by static maceration with the solvent mixtures: CHCl3: MeOH 1:1 (maceration 1) and MeOH: H2O 8:2 (maceration 2). These extractive solutions were filtered, concentrated under reduced pressure on a rotary evaporator, lyophilized and weighed. All leaf extracts were analyzed by HPLC – DAD-MS/MS for the characterization of chemical profiles. Each extract was solubilized in about 400 mL MeOH:H2O (8:2) and some of these were already partitioned into hexane, dichloromethane, ethyl acetate and n-butanol, and the aqueous residue was finally obtained. The polar fractions are being analyzed by HPLC-DAD-MS/MS while the hexane and dichloromethane fractions are by CG-MS, and these data are compared to online libraries (NIST). Among the crude extracts analyzed, the leaves presented the highest yields, being 15.24% and 37.59% in extract 1 and 2, respectively. The extracts were analyzed by CCD to investigated the chemical profile of the different organs of J. multifida L., BAW (8:1:1) was used as eluent and as revelators anisaldehyde sulfuric acid and NP-PEG, being found the presence of terpenic skeletons and phenolic compounds, respectively. The chromatograms obtained by HPLC-DAD-MS/MS are being analyzed to propose possible substances present in the different organs. Until now, we identified isovitexin whose m/z [M-H]-is 433.00 in the retention time= 7.5 min. Furthermore, we detected the presence of alkaloids, flavonoids, and diterpenes. The liquid-liquid partition is being performed, the partition of Leaves 1 and Leaves 2, Petiole 2, Bark 2, where the fractions obtained were concentrated in a rotary evaporator and are being analyzed by HPLC-DAD-MS/MS and GC-EM according to the fraction profile. Currently, by analyzing the chromatograms obtained previously, a Countercurrent Chromatography was performed with the ethyl acetate fraction (supernatant) of Leaves 2. The results are still being analyzed, but the separation of some phenolic compounds and separate terpenic skeletons that are present in the fraction can be confirmed. Subsequently, these results will be analyzed again on HPLC-DAD-MS/MS and those of higher purity will be analyzed for later isolation and structural elucidation using the one- and two-dimensional NMR.