Xanthones from the Roots of Moutabea guianensis Aubl.

The phytochemical investigation of Moutabea guianensis roots led to the isolation of five polyoxygenated xanthones, including two new ones named moutabeone B (1,8-dihydroxy-4,5,6,7-tetramethoxyxanthone) and moutabeone C (1-hydroxy-4,5,6,7,8-pentamethoxyxanthone), along with the three known xanthones, 1,8-dihydroxy-4,6-dimethoxyxanthone, 1,8-dihydroxy-4,5,6-trimethoxyxanthone and augustin A (1,8-dihydroxy-4,6,7-trimethoxyxanthone). Structural characterization of all compounds was established on the basis of spectroscopic methods, mainly 1D and 2D nuclear magnetic resonance (NMR) and comparison with literature data. The antioxidant activity of compounds was tested through a thin layer chromatography (TLC) bioautography assay using 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH·) as detection reagent. All tested compounds were more active (DL < 0.13–0.03 µg) than Trolox (DL < 0.15 µg), used as reference standard.


Introduction
Xanthones are secondary metabolites commonly occurring in a few higher plant families and their pharmacological activities have aroused great interest [1]. An important natural source of these compounds is the Polygalaceae family which includes about 22 genera and 1,300 species [2]. The genera Monnina, Bredemeyera, Securidaca and Polygala are the main sources of xanthones in the Polygalaceae [3][4][5]. On the other hand, there is only one report of the isolation of xanthones from the genus Moutabea, which described earlier chemical investigation of M. guianensis [6]. In continuation of the study of M. guianensis, the isolation and structural elucidation of two new xanthones and also other three known xanthones is now reported.
Thin-layer chromatography, combined with both biological and chemical detection methods, is an effective and inexpensive technique for the study of bioactive compounds [12]. In the antioxidant assay, the samples showed different values for the detection limit (DL), which were compared with a reference antioxidant Trolox (DL < 0.15 µg) (Figure 3). The increasing order of activity based on the observed detection limits was 1 (1.3 µg) < 5 (0.6 µg) < 3 and 4 (0.3 µg) < 2 (<0.15 µg). The detection limit of 2 was equivalent to Trolox (<1.5 µg).   Xanthones have been reported to inhibit lipid peroxidation, antioxidant activity, neuroprotective properties [13]. The xanthone nucleus is known as 9-xanthenone or dibenzo-γ-pyrone and its antioxidant activity depends of specific substituents and their positions to the basic structure of each molecule [14]. The xanthone 1 has only two substituents in the B ring and showed less activity. However, the highest activity was observed for xanthone 2, which has four substituents in the B ring (one hydroxyl and three methoxyl groups). The increase in antioxidant activity may be attributed to the number of hydroxyl and methoxyl groups, which are strong protons and electrons donors, respectively [15,16].

Plant Material
The roots of M. guianensis were collected in the experimental field of Embrapa Amazônia Oriental, located in Belé m, Pará State, on March 2012. A voucher specimen (195862) was kept in the Herbarium MG of the Museu Paraense Emí lio Goeldi (MPEG). Roots were dried under forced air circulation at 40 °C for five days and powdered in a knife mill.

Antioxidant Capacity by DPPH Assay
The antioxidant activity of xanthones was performed by TLC bioautography analysis [17]. The compounds were solubilized in chloroform at concentration of 2 mg/mL and diluted successively. For the assay, 10 µL of each solution were applied on the TLC plate corresponding to 20, 10, 5, 2.5, 1.3, 0.6, 0.3 and 0.15 µg/spot. The plate were sprayed with a DPPH solution 0.5 mM methanol solution for derivatization. Bands with the DPPH· scavenging activity were observed as white or yellow bands on a purple background. The antioxidant Trolox (Sigma Aldrich, St. Louis, MO, USA) was used as reference standard.

Conclusions
The chemical study of the roots of M. guianensis resulted in the isolation of five xanthones by chromatographic methods. The structures of the isolated compounds were proposed based on NMR methods. Compounds 1,8-dihydroxy-4,5,6,7-tetramethoxyxanthone (2), 1-hydroxy-4,5,6,7,8pentamethoxyxanthone (5) were identified as new natural products. Compound 1,8-dihydroxy-4,6,7trimethoxyxanthone (4) was isolated for the first time in the family Polygalaceae. This study shows that Moutabea genus is an important source of natural xanthones. All isolated compounds showed significant antioxidant activity in the thin layer chromatography (TLC) bioautography assay, equivalent to Trolox used as reference standard.