Icosandrin, a novel peltogynoid from the fruits of Phytolacca icosandra (Phytolaccaceae)

Besides the known compounds ( ± ) 3,3-bis-demethylpinoresinol (2), americanol A (3), spergulagenic acid (4), epi-acetylaleuritolic acid (5), 6′-palmityl-α-spinateryl-d-glucoside (6a) and 6′-palmityl-δ7-stigmastenyl-d-glucoside (6b), a novel peltogynoid (1) named icosandrin was obtained from the dried fruits of Phytolacca icosandra. This new compound was characterised by 1D-/2D-NMR, UV, IR and HR-MS techniques as 11ξ-methoxy-6,7-methylenedioxy-[2]benzopyrano-[4,3-b][1]-benzopyran-4-one. Toxicity of 1 was assessed through the Brine Shrimp Lethality Assay. Lignan 2 is reported for the first time in Phytolaccaceae family.


Introduction
Phytolacca icosandra (Phytolaccaceae) is a medium-high shrub, native of tropical regions of America (USA to Perú) and distributed also in Australia, New Zealand and South Africa regions (Steinmann 1997;Rzedowski & Calderón de Rzedowski 2000). Plants belonging to this genus have been used in folk medicine around the world, for the treatment of several affections such as oedema, rheumatism and dermatitis (Jolliffe 1982;Williams et al. 2002;Ravikiran et al. 2011) and also as molluscicidal in schistosomiasis prevention and control (Lemma 1970;Lambert et al. 1985). Several studies on P. icosandra for testing its antisecretory, anthelmintic, ovicidal and larvicidal activity have been reported (Santos-López et al. 2010;Hernández-Villegas et al. 2011, 2012. The chemistry of Phytolaccaceae is fairly wide and comprises a variety of secondary metabolites such as triterpenoidal saponins, flavonoids and lignans (Williams et al 2002). Previous phytochemical analysis of P. icosandra has lead to the isolation of serjanic and spergulagenic acid saponins (Treyvaud et al. 2000;Galarraga et al. 2014).
Peltogynoids (also known as cyclic homoflavonoids) are a type of natural products represented by a peltogynane skeleton, which is a rare type of modified flavonoid with specific cyclisation between 3-OH and C-2 0 of ring B; they can be formally related to anthocyanidins, flavan-3,4-diols, flavonols and dihydro-flavonols (Gottlieb & Rego de Souza 1972;Choudhary et al. 2001). The present article deals with the isolation and characterisation of a new natural peltogynane (1), and its chemotaxonomic implications. We also report on the in vitro toxicity of 1 against brine shrimps (Artemia salina).
2. Results and discussion 2.1. Structure elucidation of isolated compounds Compound 1 was isolated as a pale yellow solid m.p. , 3008C. The ESI mass spectra displayed a pseudo molecular-ion peak at m/z 325.07 [M þ H] þ suggesting the molecular formula C 18 H 12 O 6 , in agreement with thirteen degrees of unsaturation. The structure was first suggested to possess a flavone skeleton, based on the physicochemical properties and chromatography performance. IR absorptions at 1625 and 1495 (CvC), 1634 (CvO) and 1255, 1025 (CZOZC) cm 21 , along with UV maximal absorptions at 332 and 237 nm, confirmed that the compound was included in the flavone series. 13 C NMR and DEPT spectra of 1 displayed resonances for one carbonyl group (d C 171.0, C-4), fourteen sp 2 olefinic or aromatic carbons (d C 97.8-152.7), one acetalic carbon (d C 98.5, C-11), one dioxymethylene (d C 102.5) and one methoxyl group (d C 56.1). The 1 H NMR spectrum revealed the presence of two independent six carbon aromatic systems, the first characterising a tetra-substituted ring whose signals were located at d H 6.98 (1H, s, H-8) and 7.63 (1H, s, H-5), and the second identifying a 1,2-di-substituted ring with signals at d H 7.41 (1H, dd, J ¼ 1.2 and 7.2 Hz, H-3 0 ), 7.94 (1H, dd, J ¼ 1.2 and 7.2 Hz, H-6 0 ), 7.56 (1H, td, J ¼ 1.3 and 7.5 Hz, H-5 0 ) and 7.59 (1H, td, J ¼ 1.3 and 7.5 Hz, H-4 0 ), typical to the orto-coupled C-3 0 , C-4 0 , C-5 0 and C-6 0 protons. Methylenedioxy group was supported by the presence of a two-proton broad singlet at d H 6.12 and IR absorption at 930 cm 21 . This group was located at the tetrasubstituted aromatic system by the HMBC cross peaks between the ZOZCH 2 ZOZ protons and two sp 2 quaternary carbons at d C 146.0 (C-6) and 152.7 (C-7), which also showed interactions with the aromatic hydrogens H-5 and H-8, respectively.
HMBC interaction between one of the aromatic hydrogens of the 1,2-di-substituted ring at d H 7.41 (H-3 0 ) and the acetalic carbon at d C 98.5 (C-11), confirmed the pyrano-type structure representative of peltogynoids. Methoxyl group at d H 3.64 was also located at the C-11 position by a HMBC cross peak with the signal at d C 98.5. All the remaining assignments of 1 H and 13 C NMR data were achieved by HMQC and HMBC experiments. Stereochemistry at C-11 centre remained unknown due to the absence of vicinal protons with which H-11 may form a dihedral angle. From the above-described spectral evidence, compound 1 was identified as 11j-methoxy-6,7-methylenedioxy- Figure 1). The presence of the C-11 acetalic carbon, more commonly found in rotenoids at C-6 position (Crombie & Whiting 1998), has been seen so far only in the peltogynoid fasciculiferin, isolated from Acacia fasciculifera (van Heerden et al. 1979). Toxicity of 1 was assayed in the Brine Shrimp Lethality Assay (Meyer et al. 1982); it was found that icosandrin exhibited a toxic effect over A. salina (IC 50 ¼ 158^36 mg/mL).

Chemotaxonomic significance
Despite Phytolacca genus is characterised for biosynthesise mainly triterpenoidal saponins, some related peltogynane-type compounds, such as flavonoids, have been obtained from Phytolacca americana (Bylka & Matlawska 2001), Phytolacca thyrsiflora (Haraguchi et al. 1988) and Phytolacca dioica (Soliman & Sobieh 1999). On the other hand, peltogynanes are metabolites with some restricted distribution in nature and to the best of our knowledge, they have been isolated mainly from genus belonging to the Leguminosae family such as Acacia  (Ferreira et al. 1974). The other sources outside of Leguminosae family are Cassine (Celastraceae) (Drewes & Mashimbye 1993), Iris (Iridaceae) (Choudhary et al. 2001), Macaranga and Croton (Euphorbiaceae) (Li et al. 2009;Zou et al. 2010) and Ophioglossum (Ophioglossaceae) (Lin et al. 2005). As observed, peltogynoids are limited to a few families, making the presence of 1 in Phytolaccaceae a very uncommon feature that could be used in chemotaxonomic studies for P. icosandra.
The presence of lignans in Phytolacca is also limited, with reports only for P. americana (Sick Woo et al. 1980) and P. thyrsiflora (Haraguchi et al. 1988). Compound 2 has been previously isolated from Joannesia princeps (Euphorbiaceae) (Waibel et al. 2003), and this is the first report for Phytolaccaceae. Compounds 3, 6a and 6b were found in P. americana (Woo 1974;Sick Woo et al. 1980), whilst 5 in P. acinosa (Razdan et al. 1982). Compound 4 has only been characterised as the aglycone of several saponins from P. icosandra (Treyvaud et al. 2000).

General procedures
Melting points were determined with a Fisher-Johns apparatus and were not corrected. Optical rotation was measured in Rudolph Research Autopole III equipment. UV spectra were obtained in chloroform using a Perkin-Elmer, Lambda-3B spectrophotometer. IR spectra were recorded on a Perkin-Elmer FT-1725X spectrophotometer using KBr pellets. 1D and 2D NMR spectra were acquired with a Bruker-Avance DRX-400 instrument, using CDCl 3 as solvent. ESI-MS was run on a TSQ QUANTUM, Ultra AM, Thermo Scientific Spectrophotometer. The HR-MS analysis was conducted in a JEOL JMS-AX505WA spectrometer with direct inlet and dual approach mass analyser, using electron impact (EI) method. TLC was carried out on 0.25 mm layers of silica gel PF 254 (Merck, Kenilworth, NJ, USA).

Identification of known compounds
Known compounds were identified by comparison of their physical constants and NMR spectroscopic data with those reported in the literature (Chakrabarty et al. 1968;Woo 1974;Fukuyama et al. 1992;Razdan et al. 1982;Waibel et al. 2003).

Brine shrimp lethality assay
The assay was performed as described previously by Meyer et al. (1982) with some minor modifications. Brine shrimp eggs (Gulf Breeze w ) were hatched in artificial seawater prepared with commercial salt mixture (Instant Ocean w ), illuminated and oxygenated with an aquarium pump. After 48 h incubation at 278C, 10 shrimps were transferred with a Pasteur pipette to three sample vials for each of three doses (100, 50 and 10 mg/mL) for a total of nine vials. The sample was prepared by dissolving the compound (3 mg) in CHCl 3 (5 mL) and transferring the solution to each vial (833, 417 or 83 mL solution for 100, 50 or 10 ppm doses) followed by high vacuum for 1 h. After the solvent was evaporated, the compound was redissolved in 20 mL of Tween 80 w and 5 mL of artificial sea water were added to achieve the correct concentration. Survivors were counted and the percent deaths at each dose and control were determined. Tween 80 w at this concentration did not affect this bioassay. The IC 50 and 95% confidence intervals were calculated from 24 h counts, using the Probit analysis method (Finney 1971).

Supplementary material
Supplementary material relating to this article is available online