View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by FADA - Birzeit University PHYTOCHEMISTRY Phytochemistry 68 (2007) 652–662 www.elsevier.com/locate/phytochem Covalent anthocyanin–flavone dimer from leaves of Oxalis triangularis Torgils Fossen a,*, Saleh Rayyan a, Maya H. Holmberg a, Manfred Nimtz b, Øyvind M. Andersen a a Department of Chemistry, University of Bergen, Alle´gt. 41, N-5007 Bergen, Norway b Helmholz Centre for Infection Research, Department of Structural Biology, Inhoffenstrasse 7, D-38124 Braunschweig, Germany Received 20 July 2006; received in revised form 30 October 2006 Available online 19 December 2006 Abstract The anthocyanin–flavone C-glycoside, (malvidin 3-O-(6II-O-a-rhamnopyranosylAIV-b-glucopyranosideAII)-5-O-b-glucopyrano- sideAIII) (apigenin 6-C-(2II-O-b-glucopyranosylFIII-b-glucopyranosideFII)) malonateAV (AIV-4 ! AV-1, FIII-6 ! AV-3) (1), has been iso- 1 lated from leaves of Oxalis triangularis A. St.-Hil. In the 1D H NMR spectrum of 1 dissolved in CD3OD–CF3CO2D (95:5), MTFA, recorded 45 min after sample preparation, this covalently linked dimer occurred mainly as flavylium cation (38%) and two equilibrium forms assigned to be quinonoidal bases (54%), whereas only minor amounts of the hemiacetal forms were present. After five days storage at 300 K, the hemiacetals (39%) and flavylium cation (38%) constituted the main forms of 1. More simple anthocyanins are normally considered to be on the flavylium cation form in acidified deuterated methanol. The cross-peaks observed in NOESY NMR spectra of 1 indicated the presence of vertical ’p–p’ stacking between the B-ring of the flavone unit and the A-ring of each of the two forms assigned to be quinonoidal bases. It was not possible to discriminate between inter- or intramolecular association mechanisms. The equi- libria between the various forms of 1 were studied by two-dimensional NOESY and ROESY NMR spectroscopy. 2D HSQC-TOCSY NMR spectroscopy was among the methods used for characterization of the various forms. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Oxalis triangularis; Leaves; Anthocyanin–flavone C-glycoside dimer; Malvidin 3-rutinoside-5-glucoside (malonate) apigenin 6-C-sophoroside; Colour; Equilibrium forms; HSQC-TOCSY; 13CNMR 1. Introduction from the blue-violet flowers of Eichhornia crassipes (Ponte- deriaceae) (Toki et al., 1994, 2004). These structures were III II More than 34% of the reported anthocyanins have been established as (6 -O-(delphinidin 3-O-(6 -O-(b-D-gluco- II found to be acylated with aliphatic acyl moieties (Andersen pyranosyl)-b-D-glucopyranosyl))) (6 -O-(apigenin 7-O-(b- and Jordheim, 2006). Dicarboxylic acids, among which D-glucopyranosyl))) malonate and its corresponding luteolin malonic acid is the most common type (25%), make possi- derivative. The existence of other anthocyanin-flavonol ble a covalent linkage between the anthocyanin and complexes has been indicated in orchids (Strack et al., another flavonoid unit through the dicarboxylic acid. 1989), lupins (Takeda et al., 1993)andSalvia patens (Takeda Covalently linked anthocyanin–flavonol pigments have et al., 1994). been isolated from blue flowers of Agapanthus (Bloor and Anthocyanidins are reckoned to be involved in a series Falshaw, 2000) and from pale-purple flowers of chive (Fos- of equilibria giving rise to different forms, which exhibit sen et al., 2000), while disubstituted dicarboxylic acids their own properties including colour (Brouillard and Dan- involving an anthocyanin and a flavone have been isolated gles, 1994). One- and two-dimensional 1H NMR have to some extent been used to characterize the various forms * Corresponding author. Tel.: +47 55 58 82 44; fax: +47 55 58 94 90. of the 3-glucoside and 3,5-diglucoside of malvidin present E-mail address: [email protected] (T. Fossen). in acidic aqueous solutions, and to determine their molar 0031-9422/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2006.10.030 T. Fossen et al. / Phytochemistry 68 (2007) 652–662 653 fractions as a function of pH (Hoshino et al., 1982; Chemi- When dissolved in acidified methanol, 1 occurred in vari- nat and Brouillard, 1986; Hoshino, 1991; Mistry et al., ous equilibrium forms depending on storage time, and 1991; Santos et al., 1993). Vitisin A has been assumed to one aim of this paper was to characterize these forms by be formed from malvidin 3-glucoside by condensation both 1Hand13C NMR spectroscopy. Some considerations reaction with pyruvic acid in red wine during storage (Bak- regarding intra- or intermolecular association of 1 are ker et al., 1997; Fulcrand et al., 1998). Several forms, reported. including hemiacetals, quinonoidal bases, chalcones and the flavylium cation form were tentatively identified when vitisin A was dissolved in DMSO-d6–TFA-d (9:1; v/v) 2. Results and discussion (Bakker et al., 1997). Increasing proportions of the chal- cone forms and decreasing proportions of the flavylium The aqueous concentrate of the acidified methanolic form, respectively, were observed during storage, suggest- extract of leaves of O. triangularis was purified by partition ing that the chalcone forms were favourable under these against ethyl acetate followed by Amberlite XAD-7 column solvent conditions. The flavylium cation and chalcone chromatography. Pigment 1 co-eluted (HPLC) with malvi- forms of vitisin A were partly characterised by 1H and din 3-O-(6-O-(4-O-malonyl-a-rhamnopyranosyl-b-gluco- 13C NMR spectroscopy (Bakker et al., 1997). In acidified pyranoside)-5-O-b-glucopyranoside (Table 1), which is methanolic solutions and in aqueous solutions at pH lower one of the main anthocyanins of O. triangularis, thus pre- than 5-6, pyranoanthocyanins like vitisin A have been venting the detection of 1 in the crude extract. However, reported to occur mainly as flavylium cations (Bakker 1 was separated from the monomeric anthocyanins by and Timberlake, 1997; Fulcrand et al., 1998; Fossen and Sephadex LH-20 chromatography, and thereafter purified Andersen, 2003). The only previously reported complete by preparative HPLC. The UV–Vis spectrum of 1 recorded assignment of 1H and 13C NMR chemical shifts of another on-line during HPLC analysis (Fig. 2) showed visible max- anthocyanin equilibrium form than the flavylium cation imum at 558 nm, the A440/Avis-max ratio of 10%, and local form, described one 2-OH hemiacetal form of malvidin 3- UV maxima at 346 nm, 310 nm and 276 nm, respectively, II O-(6 -O-(E)-p-coumaroyl-b-D-glucopyranosyl)-5-O-(2-O- indicating both a 3,5-disubstituted anthocyanin and acetyl-b-D-xyloopyranoside), isolated from Tibouchina another flavonoid unit. Analysis of the NMR spectra of urvilleana flowers, using DMSO-d6–TFA-d (9:1; v/v) as 1 recorded in CF3COOD–CD3OD (5:95; v/v) revealed that solvent (Terahara and Suzuki, 1993). 1 existed in several equilibrium forms (denoted as 1b–1e)in Anthocyanins in solutions can be stabilized by: (i) copig- addition to the flavylium cation form, 1a (Figs. 3 and 4). mentation with other aromatics like flavones and cinnamic The equilibria between the different forms of 1, and poten- acids, (ii) intramolecular sandwich ‘p–p’ stacking of the tial inter- or intramolecular associations of 1, were exam- anthocyanidin and aromatic acyl residues of anthocyanins ined by 2D nuclear Overhauser and exchange di- or poly-acylated with aromatic acyl groups and (iii) spectroscopy (NOESY) and rotational frame nuclear Over- intermolecular self-association by vertical hydrophobic hauser effect (ROESY) spectroscopy performed at 271 K ‘p–p’ stacking of the aromatic anthocyanidin nuclei (Hosh- and 300 K. The relative proportions of the various equilib- ino et al., 1982; Goto et al., 1987; Goto and Kondo, 1991; rium forms of 1 in the NMR solvent varied with storage Hoshino, 1991, 1992; Nerdal and Andersen, 1991, 1992; time. Kondo et al., 1998; Gakh et al., 1998). Based on circular dichroism (CD) spectroscopy, 1D 1H NMR spectra 2.1. The flavylium cation form, 1a recorded at different temperatures and with various antho- cyanin concentrations (Hoshino et al., 1982; Hoshino, The downfield regions of the 1D 1H NMR, 1H–13C 1991, 1992), and NOESY NMR cross-peaks obtained at HMBC and 1H–13C HSQC spectra of 1 showed signals in different temperatures (Nerdal and Andersen, 1991, agreement with both malvidin and C-substituted apigenin 1992), intermolecular self-association has been reported (Table 2). The diagnostic 13C chemical shift value of C- for the anhydrobase equilibrium forms occurring in weakly 8F (d 93.1) revealed 6-C substitution (Rayyan et al., acidic–neutral aqueous solvents and for the flavylium cat- 2005) of the apigenin moiety. A molecular ion (M+)at ion in acidified methanol, respectively. Based on distance m/z 1463.36 in the positive ion ESI mass spectrum was in geometry calculations self-association structures were pro- accordance with covalently linked malvidin, apigenin, mal- posed (Nerdal and Andersen, 1991, 1992). The impact of onyl, rhamnosyl and four hexoses. inter- or intra-molecular copigmentation in covalent antho- The anomeric coupling constants (7.7 Hz, 7.7 Hz, cyanin–flavone and anthocyanin–flavonol pigments on 7.7 Hz, 10.3 Hz and 1.5 Hz), the 13C resonances in the flower colour, has to some extent been discussed previously sugar region of the 2D 1H–13C HSQC spectrum, and the (Figueiredo et al., 1996; Bloor and Falshaw, 2000; Fossen cross-peaks in the 1H–1H DQF-COSY, the 1H–1H TOCSY et al., 2000; Toki et al., 1994, 2004). and the 1H–13C HSQC-TOCSY spectra of 1 (Fig. 5) were In this paper, we report the isolation and identification in agreement with four b-glucopyranoses and one a- of a novel covalently linked anthocyanin–flavone C-glyco- rhamnopyranose (Fossen and Andersen, 2006)(Table 2).