Prenylated chromones and coumarins from the leaves of Billburttia capensoides Sales & Hedge (Apiaceae)

Billburttia Sales & Hedge is a new genus endemic to Madagascar, which belongs to the Apiaceae family and comprises of two species, namely Billburttia capensoides and B. vaginoides. The present work was undertaken in a view to explore secondary metabolites from the leaves of B. capensoides, contributing to have more knowledge on the chemical profile of this species. Liquid-liquid partition followed by chromatographic separation of its ethanolic extract led to the isolation of two new prenylated chromones, 2'R-hydroxy-7-O-methylallopeucenin and the known coumarins imperatorin and xanthotoxin. Their chemical structures were established on the basis of spectroscopic methods including nuclear magnetic resonance (NMR) and high resolution mass spectrometry, and by comparison with the reported data in the literature. The structure of the compound 3 was confirmed by single-crystal X-ray diffraction. This is the first report on the nonvolatile constituents of B. capensoides. The presence of imperatorin and xanthotoxin supported that the new endemic genus Billburttia belongs to the Apiaceae family. All these data may induce further research on this species in order to find new chemical entities with biological and chemotaxonomic interests.


Introduction
Billburttia (Apiaceae) is a new endemic genus of Madagascar and comprises only two species, namely B. capensoides Sales & Hedge and B. vaginoides S. & H [1]. These two species were initially placed in the genus Peucedanum (Apiaceae) before being transferred into Billburttia [2]. The present report is focusing on B. capensoides which is a shrub of 2m in height, encountered in the central part of Madagascar and locally known under the vernacular name Volotaratsitsina. The plant is traditionally used for the treatment of jaundice, convulsion and common cold [3]. The composition of the essential oils from the two Billburttia species has been previously reported with p-mentha-1,3,8-triene, terpinolene and dill apiole as the major components [4].
As a continuation of our study on medicinal plants endemic to Madagascar in a view to searching for new secondary metabolites and biologically active natural substances, we carried out a phytochemical investigation of the leaves of B. capensoides. Fractionation of the ethanol extract led to the isolation of three prenylated chromones (1-3) and the two coumarins (4 and 5). The isolation and structural identification of the two new chromone derivatives 1 and 2 as well as the X-ray analysis of 3 are described herein.

Plant material
The leaves of B. capensoides were collected in the region of Itasy, district of Arivonimamo, Commune of Manalalondo in the central part of Madagascar in September 2015. A voucher specimen (No. ST1494) is preserved in the Herbarium of the National Center of Applied Pharmaceutical Research, Antananarivo, Madagascar.

Extraction and Isolation
The dried leaves of B. capensoides (440 g) were ground into powder and extracted by maceration with EtOH (1.5 L) for 48 hours. After filtration and evaporation under reduced pressure of the solvent, the resulting ethanol extract (39.9 g) was taken in MeOH (250 mL) and defatted with n-hexane (3 x 200 mL). The methanol layer was evaporated to dryness and the residue was partitioned successively between chloroform and water, ethyl acetate and water, and n-butanol and water. Evaporation under reduced pressure of all the solvents used during this preliminary fractionation yielded 4.1 g of hexane-soluble, 10.1 g of chloroform-soluble, 2.6 g of ethyl acetate-soluble, 5.9 g of n-butanol-soluble (5.9 g) and 14 g of water-soluble fractions.

Results and discussion
The ethanol extract of the leaves from B. capensoides was liquid-liquid partitioned to give hexane-, chloroform-, butanoland water-soluble fractions. Fractionation of the chloroform-soluble part by a combination of chromatographic techniques, including normal phase silica gel and reversed phase silica gel (ODS C18) open columns, as well as reversed phase ODS-HPLC led to the isolation of two new compounds 1 and 2, 2'R-hydroxy-7-O-methylallopeucenin (3) and the two known coumarins imperatorin (4) and xanthotoxin (5) (Fig. 1). The structures of the known compounds 4 and 5 were identified by spectroscopic analyses including 1D and 2D NMR experiments and by comparison with data reported in the literature [5,6,7,8].
Prenylated chromones have been reported to exhibit anti-HIV, antiproliferative and anti-inflammatory activities [14,15]. Coumarins have been considered as one of the major biochemical markers of the Apiaceae family.

Conclusion
The present study suggests that B. capensoides is a new source of prenylated chromones, as exemplified by the two novel compounds 1 and 2. Moreover, the isolation of the prenylated chromone 3 was described herein for the first time although it was established to be the (2'R)-enantiomer of the reported 2'S-hydroxy-7-O-methylallopeucenin by X-ray crystallographic analysis. The presence of imperatorin (4) and xanthotoxin (5) in B. capensoides supported that the new endemic genus Billburttia belongs to the Apiaceae family. All the above data may induce further research on this species in order to find new chemical entities with biological and chemotaxonomic interests.