Anthocyanidin 3-Galactosides from Flowers of Abrus Canescens Bak
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African Journal of Pure and Applied Chemistry Vol. 5(10), pp. 356-360, 30 September, 2011 Available online at http://www.academicjournals.org/AJPAC ISSN 1996 - 0840 ©2011 Academic Journals Full Length Research Paper Anthocyanidin 3-galactosides from flowers of Abrus canescens Bak Robert Byamukama1, George Ogweng2, Monica Jordheim3, Øyvind M. Andersen3 and Bernard T. Kiremire1* 1Chemistry Department, Makerere University, P. O. Box 7062, Kampala, Uganda. 2Chemistry Department, Kyambogo University, P. O. Box 1, Kyambogo- Kampala, Uganda. 3Department of Chemistry, University of Bergen, Allegt.41, 5007 Bergen, Norway. Accepted 26 July, 2011 Three anthocyanins, the 3-galactoside of delphinidin (1) (8.5%), petunidin (2) (9.2%) and malvidin (3) (73.8%) were isolated from the deep red-purple flowers of Abrus canescens by a combination of chromatographic techniques. Their structures were elucidated by on-line diode array detection chromatography and homo- and hetero-nuclear magnetic resonance (NMR) techniques. Key words: Abrus canescens, Fabaceae, anthocyanin, malvidin 3-galactoside, petunidin 3-galactoside, delphinidin 3-galactoside, taxonomy. INTRODUCTION The genus Abrus belongs to the family Fabaceae MATERIALS AND METHODS (Leguminosae), and consists of about 13 species distributed in the tropic regions worldwide (Hutchinson Plant material and Dalziel, 1958; Verdcout, 1971; Labat, 1991; Thulin, Flowers of Abrus canescens were collected from the outskirts of 1994). In Africa, Abrus gawenensis Thulin., Abrus Budongo National Forest Reserve in Western Uganda in canescens Bak., Abrus pulchellus Thw. and Abrus September 2008. The flowers were stored in a deep freezer before precatorius L. are the most common species (Agbagwa, extraction. The identification of the plant was carried out in the 2007; Thulin, 1994; Hutchinson and Dalziel, 1958). Abrus Botany Department at Makerere University and voucher specimen (No.GO22/2008) has been deposited in the herbarium of that canescens is a climbing shrub that grows to a height of Department. 0.5 to 3 m at the edges of rivers or swamps in the altitude range of 600 to 1500 m above sea level (Lock, 1989). Root extracts from A. canescens have been used as a Isolation of anthocyanins genital stimulant (Burkill, 1995) and roots macerated in palm wine are considered as an aphrodisiac (Bouquet, The flowers (80 g) were weighed and extracted with 600 mL of methanol containing 0.5% (v/v) trifluoroacetic acid (TFA) for 8 h. 1969). The filtered extract was concentrated under reduced pressure at During our bioprospecting survey of anthocyanins in 30°C, purified by partition (several times) against ethyl acetate the East-African flora, we came across anthocyanidin 3- before application to an Amberlite XAD-7 column. After washing the galactosides to be responsible for the colour of A. column with H2O, the anthocyanins adsorbed to the column were canescens flowers. The taxonomical potential of the eluted with methanol containing 0.5% (v/v) TFA. The concentrated glycosidic pattern of the anthocyanins in A. canescens is anthocyanin extract was further purified by Sephadex LH-20 chromatography using H2O-MeOH-TFA (80:19.5:0.5, v/v/v) as discussed. eluent. The individual anthocyanins were separated using preparative HPLC (Gilson 305/306 pump equipped with a UV 6000LP detector) equipped with an ODS Hypersil column (25 × 2.2 cm; i.d.; 5 mm). Two solvents were used for elution: A = formic *Corresponding author. E-mail: [email protected]. acid-water (1:9, v/v) and B = formic acid- water- methanol (1:4:5; Tel: +256-772-589-313. Fax: +256-41-531 061. v/v/v). The elution profile consisted of a linear gradient from 10 to Byamukama et al. 357 mAU Min Figure 1. HPLC chromatogram of anthocyanin 1-3 in an acidified extract of flowers of Abrus canescens Bak. Detected at 520 ± 20 nm. 100% B for 30 min, isocratic elution (100% B) for the next 10 min, wavelength range 200 to 600 nm in steps of 2 nm. The NMR followed by a linear gradient from 100 to 10% B for 1 min. The flow experiments were obtained at 600.13 and 150.92 MHz for 1H and rate was 14 ml/min for 41 min and aliquots of 500 l were injected. 13C, respectively, on a Bruker Biospin AV-600 MHz instrument 13 equipped with a cryogenic probe at 298 K. The deuteriomethyl C 1 signal and the residual H signal of the solvent, CF3COOD–CD3OD Co-chromatography (TLC, On-line HPLC) (95:5; v/v), were used as secondary references (δ 49.0 and δ 3.4 1 13 ppm from TMS for H and C, respectively) (Andersen and Fossen, 1 1 13 1 13 1 Co-chromatographic methods included TLC and on-line HPLC. The 2003). The NMR experiments H, H- C HSQC, H- C HMBC, H- 1 1 1 TLC was carried out on microcrystalline cellulose (F 5556, Merck) H DQF-COSY and H- H TOCSY were recorded. with the solvent HCO2H-HCl-H2O (1:1:2, v/v/v). The Agilent 1100 HPLC system was equipped with a HP 1050 diode-array detector and a 200 × 4.6 mm i.d., 5 μm ODS Hypersil column, (Supelco, Bellefonte, USA). Two solvents; C, water (0.5% TFA) and D, RESULTS AND DISCUSSION acetonitrile (0.5% TFA) were used for elution. The elution profile for HPLC consisted of initial conditions with 90 C and 10% D followed The HPLC chromatogram (Figure 1) of a weakly acidified by linear gradient elution for the next 10 min to 14% D, isocratic methanol extract of the flowers of Abrus canescens elution (10 to 14 min) and the subsequent linear gradient conditions; 14 to 18 (to 16% D), 18 to 22 (to 18% D), 22 to 26 (to revealed the presence of three major (1-3) anthocyanins, 23% D), 26 to 31 (to 28% D) and 31 to 32 min (to 40% D), isocratic 8.5, 9.2 and 73.8%, respectively. The anthocyanins in the elution 32 to 40 min (40% D) and final linear gradient elution 43 to extracts were purified by partition against ethyl acetate 46 min (to 10% B). The flow-rate was 1.0 ml/min and aliquots of 15 followed by Amberlite XAD-7 column chromatography μL were injected with an Agilent 1100 Series, Micro Autosampler. and separated by Sephadex LH-20 chromatography and Before injection, all samples were filtered through a 0.45 mm Millipore membrane filter. Anthocyanins from crowberries, preparative HPLC. The isolated anthocyanins were Empetrum nigrum Coll. (Käppä et al., 1984; Ogawa et al., 2008) checked for homogeneity by analytical HPLC. The UV- were used as references. These TLC and HPLC methods have visible spectra of 1-3 recorded on-line during HPLC been earlier described by Byamukama et al. (2006). analysis showed visible maxima around 532 nm, and A440/Avis-max of about 29% (Table 1), in accordance with 3- glycosides of either delphinidin, petunidin or malvidin Spectroscopy (Andersen, 1987; Guisti and Wrolstad, 2001). 1 The UV–visible absorption spectra were recorded on-line during The aromatic region of the H NMR spectrum of 3 HPLC analysis and the spectral measurements were made over the showed a singlet at δ 9.16 (br, H-4), a 2H singlet at δ 358 Afr. J. Pure. Appl. Chem. Table 1. Chromatographic and on-line spectral data of the anthocyanidin 3-galactosides (1-3) isolated from flowers of Abrus canescens Bak. On-line HPLC Rf (TLC) Compound Relative amount (%) Vismax (nm) A440/AVis-max (%) tR (min) 1 8.5 530 29 16.32 0.11 2 9.2 530 29 22.93 0.19 3 73.8 532 29 27.60 0.27 Table 2. 1H and 13C NMR spectral data for malvidin 3-O-β-galactopyranoside (3) isolated from flowers of Abrus canescens Bak. 1H δ (ppm), J (Hz) 13C δ (ppm) 2 164.0 3 145.6 4 9.15 d 0.6 137.3 5 159.1 6 6.75 d 1.9 103.4 7 170.2 8 7.05 dd 0.6, 1.9 96.9 9 157.8 10 113.3 1' 119.7 2' 8.12 s 110.6 3' 149.7 4' 147.5 5' 149.7 6' 8.12 s 110.6 OCH3 4.10 s (∫6 H) 57.2 3-O-β-galactopyranoside 1'' 5.39 d 7.8 104.6 2'' 4.08 dd 7.8, 9.8 72.2 3'' 3.77 dd 3.3, 9.8 75.0 4'' 4.03 dd 1.1, 3.3 70.2 5'' 3.89 m 77.8 6A'' 3.85 m 62.3 6B'' 3.85 m 8.12 (H-2′/6′) and an AX system at δ 7.04 (dd, J = 0.9, 1.9 sequentially coupled protons, H-3'', H-4'', H-5'' and H- Hz, H-8) and δ 6.76 (d, J = 1.9 Hz, H-6) indicating an 6A''/H-6B'', were thereafter assigned from other cross anthocyanidin with a symmetrically substituted B-ring. peaks in the COSY spectrum (Table 2). The carbon The chemical shifts of the corresponding carbons of the atoms of the sugar of Pigment 3 were assigned from the aglycone were assigned from the HSQC experiment HSQC spectrum. The chemical shifts, coupling constants while the remaining quaternary carbon atoms were and crosspeaks of the sugar moiety (Table 2) were in assigned from the HMBC resonances (Table 2). The accordance with a β-galactopyranosyl. The cross peak at singlet at δ 4.10 (2 × OCH3) and the crosspeak at δ δ 5.40/ 146.67 (H-1''/C-3) in the HMBC spectrum 4.10/149.9 (OCH3/ C-3′, 5′) in the HMBC spectrum confirmed the linkage of the β-galactopyranose to the 3- showed that the aglycone was malvidin.