Anthocyanidin 3-Galactosides from Flowers of Abrus Canescens Bak

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 canescens is a climbing shrub that grows to a height of (No.GO22/2008) has been deposited in the herbarium of that 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 equipped with a cryogenic probe at 298 K. The deuteriomethyl 13C 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 ppm from TMS for 1H and 13C, 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. The sugar region position of the aglycone. The major pigment 3 was of the 1H NMR spectrum indicated one anomeric signal therefore confirmed as malvidin 3-O-β-galactopyranoside at δ 5.40 with a coupling constant of 7.7 Hz indicating (Figure 2). presence of one monosaccharide with a β-configuration. Pigments 1-3 co-chromatographed (TLC and HPLC) Starting from the anomeric proton at δ 5.40 (d, 7.7 Hz, H- with the 3-galactoside of delphinidin, petunidin and 1''), the observed cross peak at δ 4.09 in the DQF-COSY malvidin, respectively, from crowberry, Empetrum nigrum spectrum permitted the assignment of H-2′′. The chain of Coll. (Käppä et al., 1984; Ogawa et al., 2008). Pigments Byamukama et al. 359

Figure 2. The structure of the three anthocyanidin 3-galactosides isolated from flowers of Abrus canescens Bak.: R1= R2 = OH (1), delphinidin 3-galactoside; R1= OCH3, R2 = OH (2), petunidin 3-galactoside and R1= R2 = OCH3 (3), malvidin 3-galactoside.

1 and 2 were hence identified as delphinidin 3- financial support. GO gratefully acknowledges Kyambogo galactoside and petunidin 3-galactoside, respectively, University for tuition fees. using chromatographic methods (Table 1). All the reported anthocyanins (1-3) isolated from flowers of A. canescens have 3’,4’,5’-tri-O-substitutions REFERENCES on their anthocyanidin B-rings. This pattern is similar to Agbagwa IO (2007). Evaluation of Diagnostic Vegetative and the pattern found for most other species in the subfamily Reproductive Characters among Abrus species in Nigeria. Aust. J. Faboideae; with malvidin glycosides as main pigments Basic Appl. Sci., 1(4): 841-852. with petunidin glycosides and delphinidin glycosides and Andersen ØM (1987). Anthocyanins in fruits of Vaccinium uliginosum L. other type of anthocyanins present in decreasing order (Bog whortleberry). J. Food Sci., 52: 665-666 and 680. Andersen ØM, Fossen T (2003). Characterisation of anthocyanins by (Harborne, 1967; Ishikura, 1978). This observation may NMR, In: Current protocols in Food analytical Chemistry, Wrolstad, R. be the prototype of a situation in the subfamily E., ed. John Wiley, New York Chapter 1. Faboideae, where some anthocyanidins (malvidin and Bouquet AJ (1969). Natural products as an alternative remedy. . Royal petunidin) are formed as a result of modifications of other Botanic Gardens. Kew 2(4): 166-179. Brugliera F, Holton TA, Stevenson TW, Farcy E, Lu CY and Cornish EC anthocyanidins (delphinidin) and not by the activity of (1994). Isolation and characterisation of cDNA clone corresponding anthocyanidin synthase on flavan-3,4-cis-diol during the to the Rt locus of Petunia hybrida. Plant J., 5: 81-92. biosynthetic process. In Petunia hybrida, for example, Burkill HM (1995). The Useful Plants of West Tropical Africa. 3. 2nd Ed. peonidin, petunidin and malvidin have been reported to Families J-L. Roy. Bot. Gardens Kew, 3(2): 610. Byamukama R, Jordheim M, Kiremire B, Namukobe J, Andersen ØM be formed from modification of their respective cyanidin (2006). Anthocyanins from flowers of Hippeastrum cultivars. Sci. and delphinidin B-rings by methyltransferases (Brugliera Hort., 109: 262-266. et al., 1994; Jonsson et al., 1984). However, although the Guisti MM, Wrolstad RE (2001). Characterization and measurement of aglycone pattern seem to be consistent within the family anthocyanins by UV-visible spectroscopy, in Current protocols in Food Analytical Chemistry, Wrolstad RE and Schwartz RE, Eds., Fabaceae, the glycosidic patterns varies a lot with the John Wily, New York. main being the 3-rhamnoside, 3-rhamnoside-5-glucoside, Harborne J (1967). Comparative Biochemistry of the Flavonoids. 3-glucoside, 3-(2-xylosylgalactoside) and 3-glucoside-5- Academic Press, London Chapter 5. glucoside (Harborne, 1967; Ishikura, 1978). In this Hutchinson J, Dalziel JM (1958). Flora of West Tropical Africa, Part 2. Crown Agents for Oversea Governments and Administrations, context the 3-galactosides of the anthocyanins (1-3) of A. London. canescens in the genus Abrus may have taxonomical Ishikura N, Ito S, Shibata M (1978). Paper chromatographic survey of potential. anthocyanins in Leguminosae. III. Identification and distribution pattern of anthocyanins in twenty-two legumes. Bot. Mag. Tokyo, 91: 25-30.

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