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Malonylated flavonoids and blue flower colour in lupin
phytochemistry, Vol. 34, No. 2, pp. 421-423, 1993 printed in Great Britain.
0031-9422/93 $6.00+0.00 © 1993PergamonPress Ltd
MALONYLATED FLAVONOIDS AND BLUE FLOWER COLOUR IN LUPIN KOSAKU TAKEDA,JEFFREY B. HARBORNEand PETER G. WATERMAN* Department of Botany, University of Reading, Reading RG6 2AS, U.K.; *Phytochemistry Research Laboratories, Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow G1 IXW, U.K. (Received 4 March 1993) Key Word Index--Lupinus; Leguminosae; Russell hybrids; blue flower colour; copigmentation; delphinidin 3-(6"-malonylglucoside); apigenin 7-(6"-malonylglucoside).
Abstract--Blue flowers of the garden lupin Russell hybrids contain delphinidin 3-(6"-malonylglueoside), whereas pink flowers contain the corresponding cyanidin and pelargonidin derivatives in a 1 : 3 ratio. Blue flowers also contain large amounts of apigenin 7-(6"-malonylglucoside) and small amounts of the luteolin analogue. The structure of the apigenin conjugate was unambiguously established by 2D N M R techniques. It is proposed that blue flower colour in lupin is due to copigrnentation of the two malonylated glucosides of delphinidin and apigenin, possibly linked in vivo covalently through a common malonic acid residue.
INTRODUCTION Although blue flower colour is a dominant feature of both garden and wild Lupinus species, little is known of the basis of this blue colour. Earlier studies by Bayer [1] suggested that the pigments were delphinidin and cyanidin derivatives, present in vivo as a metal complex with iron. Pink lupins were reported to contain mono- and diglycosides of pelargonidin, while violet forms contained mixtures of all these anthocyanins. There was some uncertainty regarding the sugars present in these pigments. A recent survey of flowering plants for zwitterionic anthocyanins was initiated, following the realization that malonylated and similarly substituted cyanic pigments are widespread in nature [2]. A survey of 15 species of legume flowers showed that such compounds were generally absent from the family, although two samples (Vicia sativa and blue garden lupin) were positive [3]. We therefore decided to reinvestigate the pigments of the garden lupin, in order to determine the form of the conjugation and also to establish the basis of the blue colour. The material studied included blue and pink forms of the well known Russell hybrids, which are derived from L. polyphyllus Lindl. and L. arboreus Sims [4]. The results of this investigation are presented here.
RESULTS
The blue form of the Russell hybrid contained a single anthoeyanin, identified as delphinidin 3-(6"-malonylglucoside), while the pink form contained a 1 : 3 mixture of cyanidin and pelargonidin 3-(6"-malonylglucoside). The presence of malonic acid in all three pigments was established by TLC identification after alkaline hydrolyPlff'l'O 34.'2-H
421
sis, which also yielded the 3-monoglucosides. The attachment of the malonic acid at the 6-position of glucose was determined by hydrogen peroxide oxidation and cochromatography of the resulting 6-malonylglucose with an authentic marker [3, 5]. This assignment was confirmed by co-chromatography of the original three pigments with authentic markers (Table I). All three of these anthocyanins probably occur relatively widely in plants, although they have only been described so far from a few sources. Pelargonidin 3-(6"malonylglucoside) was first reported in 1986 from Callistephus chinensis [5] and the cyanidin analogue in 1984 from Cichorium intybus [6]. The delphinidin pigment has so far been recorded only in flowers of Verbena hybrida [7] and Hibiscus syriacus [8]. Its presence in blue lupin is thus the third report of its occurrence as a flower pigment. The co-occurring flavones were then examined in the blue flowers. Large amounts of apigenin 7-(6"malonylglucoside) were obtained, together with smaller amounts of the luteolin analogue. Identifications were first based on UV spectral analysis, and acid and alkaline hydrolysis, the latter giving malonic acid in each case, together with the corresponding 7-monoglucoside. A search of the literature showed that an apigenin 7-malonylglucoside was isolated from parsley cell cultures in 1973 [9] but that the location of the malonic acid residue on the glucose moiety was not then established. The sample was therefore submitted to a comprehensive NMR analysis. The simple 1H NMR spectrum (Table 2) confirmed the presence of apigenin, glucose and glucose esterified with malonic acid. The glucoside and malonic acid esterified glucoside co-existed in deuterated pyridine in a ratio of 3: 7. 13C N M R data were obtained using the J M O D procedure (Table 2) and direct IH-~ac coupling was identified by ~H -~ 3C-COBIDEC. Coupling patterns
422
K. TAKEDAet al.
Table 1. Co-chromatography of lupin pigments with authentic anthocyanins Rs
( x 100) in
Pigment
BAW
BuHC1
1% HCl
HOAc-HCI
Lupin P-1 Cy 3-(6"-malonyl Glc) Deacylated P-1 Cy 3-Glc Lupin P-2 Pg 3-(6"-malonyl Glc) Deacylated P-2 Pg 3-Glc Lupin B Dp 3-(6"-malonyl Glc)* Deacytated B Dp 3-GIc
35 35 27 27 44 44 42 42 25 25 14 14
21. 21 10 10 36 36 25 25 10 10 04 04
05 05 03 03 11 11 06 06 03 03 01 01
17 17 11 11 31 31 23 23 12 12 07 07
Lupin P-1 and P-2 from pink flowers; Lupin B from blue flowers. *The sample was kindly donated by Terahara et al. I-7]; the other two authentic malonylated anthocyanins were from our own collection.
Table 2. Chemical shift data for apigenin 7-(6"-malonylglucoside) and apigenin 7glucoside
C/H 2 3 4 4a 5 6 7 8 8a 1' 2'/6' 3'/5' 4' 1" 2" 3" 4" 5" 6"
IH
Malonylglucoside 13C
6.92 s
6.88 d (2.2) 7.07 d (2.2)
7.99 d (8.8) 7.30 d (8.8) 5.76 d (7.5) 4.25 t (7.5) 4.40 m 4.40 m 4.25 m 4.88 dd (11.9, 4.8) 5.17 dd (11.9, 1.7)
1'"
2"' 3"'
3.90 ABq (15.5)
165.4 104.4 183.2 107.0 163.0 101.0 164.3 95.8 158.2 122.2 129.4 117.3 163.2 102.2 78.6 75.0 71.5 76.0 65.7
1H
Glucoside t3C
6.93 s
6.88 d (2.2) 7.13 d (2.2)
7.91 d (8.8) 7.22 d (8.8) 5.86 d (7.5) 4.25 t (7.5) 4.40 m 4.40 m 4.40 m 4.40 m 4.61 dd (12.0, 2.0)
165.3 104.3 183.3 106.9 162.9 101.0 164.4 95.7 158.2 122.41 29.411 7.2 163.2 102.1 78.8* 75.1 71.5 79.5* 62.7
168.4 43.1 169.9
*Interchangeable.
for the flavone and glucose protons were obtained by use of COSY-45, C O S Y - L R and T O C S Y experiments. Two problems remained, the position of esterification of the malonic acid and the point of attachment of the glucose to the apigenin. The relative deshielding of the resonances for H2-6 of the glucose suggested esterification at that position and this was confirmed by a study
of long-range heteronuclear coupling, using the H M B C procedure [10], which revealed 3J-coupling between H 26 and the malonyl carbonyl carbon resonating at 168.4ppm. Similarly, a 3j-coupling was observed between the anomeric proton of the glucose and a resonance at 164.25 ppm attributable to the apigenin A-ring. As the 164.25 ppm resonance also showed H M B C correlations
Malonylated flavonoids in lupin with both H-6 and H-8, and the only oxygen-bearing carbon with which both H-6 and H-8 can show coupling (2j) is C-7, then assignment of the glycosidic linkage through C-7 is unambiguous. Further evidence for placing the glucose at C-7 came from the NOESY spectrum which showed NOE interactions between the anomeric proton, and H-6 and H-8. The related luteolin 7-(6"-malonylglucoside) was recently obtained from carrot leaf as an oviposition stimulant to the female Black Swallowtail butterfly [11]. It was analysed by 1H NMR spectroscopy but our analysis of the apigenin analogue is more rigorous in establishing the position of the malonic acid at C-6". We assume that our luteolin analogue from lupin is identical to the 6"-malonic ester from its co-occurrence with the apigenin derivative. Blue flower colour in lupin is thus based on the presence of delphinidin 3-(6"-malonylglucoside) as the chromophore and apigenin 7-(6"-malonylglucoside) as the copigment, together with the presence of iron, as earlier reported by Bayer [1]. This co-pigment complex appears to be similar to that of the blue cornflower, Centaurea cyanus (Compositae) where the components are cyanidin 3-(6"-succinylglucoside)-5-glucoside, apigenin 7-glucuronide-4'-(6"-malonylglucoside), iron and magnesium [12]. The presence of a malonic acid residue in both pigment and copigment of lupin, and the known instability of this linkage in vitro, does suggest the possibility of an in vivo covalent linkage through malonic acid to the sugar residues of pigment and copigment. Provisional evidence for such a dimer has already been obtained by Strack et al. [13] in orchid flowers, but further work is needed to see whether such a dimer is present in the blue lupin. EXPERIMENTAL
NMR spectra were obtained on Bruker AMX-400 spectrometer using Bruker standard microprograms. All experiments were run in CsDsN using the solvent as int. standard. Isolation of anthocyanins and flavones. Freshly picked pink and blue petals of Russell hybrid iupins, or material stored at - 2 0 °, were used for the extraction of the pigments. The petals were immersed in M e O H H O A c - H 2 0 (10: 1:9, MAW) overnight and the filtered extracts were evapd. The residue was dissolved in H2O and the soln was passed through a column of XAD-7 (1.4 x 24 cm). After washing with H20 , the pigment adsorbed was eluted with M e O H - H O A c - H 2 0 (14: 1:5). The pigment fr. was evapd to dryness in vacuo at 30°. The residue,
423
after dissolution in MAW, was passed through a Sephadex LH-20 column (1.4 x 24 cm). The anthocyanin ft. was evapd to dryness and further purified by prep. PC using the solvent n - B u O H - H O A c - H 2 0 (4:1:5, BAW) and 15% HOAc. The flavone fr. from the Sephadex LH20 sepn crystallized out from aq. MeOH and was then twice recrystallized from aq. EtOH. Pigment identifications. Procedures for identifying malonylated anthocyanins in Compositae were used here [5]. All pigments were mobile electrophoretically'at pH 4.4 and had UV-visible spectra in line with lit. data. Analysis included acid and alkaline hydrolysis, detailed identification of the aglycone, sugar and organic acid [5], and H202 oxidation to give 6-malonylglucose, identified by co-chromatography with authentic material. The 2 flavones, apigenin and luteolin 7-(6"-malonyl)glucoside, were likewise identified on the basis of electrophoretic mobility at pH 4.4, UV spectral shifts, acid hydrolysis to aglycone, sugar and malonic acid, and alkaline hydrolysis to 7glucoside and malonic acid.
Acknowledoement--NMR spectra were run in the University of Strathclyde NMR Laboratory. REFERENCES
1. Bayer, E. (1959) Chem. Ber. 92, 1062. 2. Harborne, J. B. and Boardley, M. (1985) Z. Naturforsch. 40c, 305. 3. Harborne, J. B. (1986) Phytochemistry 25, 1887. 4. Huxley, A. (1992) New Dictionary of Gardening, Vol. 3. Macmillan, London. 5. Takeda, K., Harborne, J. B. and Self, R. (1986) Phytochemistry 25, 1337. 6. Bridle, P., Loeflter, R. S. T., Timberlake, C. F. and Self, R. (1984) Phytochemistry 23, 2968. 7. Terahara, N., Shioji, T., Toki, K., Saito, N. and Honda, T. (1989) Phytochemistry 28, 1507. 8. Kim, J. H., Nonaka, G. I., Fujieda, K. and Uemoto, S. (1989) Phytochemistry 28, 1503. 9. Kreuzaler, F. and Hahlbrock, K. (1973) Phytochemistry 12, 1149. 10. Bax, A. and Summers, M. F. (1986) J. Am. Chem. Soc. 106, 2093. 11. Feeny, P., Sachder, K., Rosenberry, L. and Carter, M. (1988) Phytochemistry 27, 3439. 12. Goto, T. (1987) Fortschr. Chemic Organ. Naturst. 52, 113. 13. Strack, D., Busch, E. and Klein, E. (1989) Phytochemistry 28, 2127.
0031-9422/93 $6.00+0.00 © 1993PergamonPress Ltd
MALONYLATED FLAVONOIDS AND BLUE FLOWER COLOUR IN LUPIN KOSAKU TAKEDA,JEFFREY B. HARBORNEand PETER G. WATERMAN* Department of Botany, University of Reading, Reading RG6 2AS, U.K.; *Phytochemistry Research Laboratories, Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow G1 IXW, U.K. (Received 4 March 1993) Key Word Index--Lupinus; Leguminosae; Russell hybrids; blue flower colour; copigmentation; delphinidin 3-(6"-malonylglucoside); apigenin 7-(6"-malonylglucoside).
Abstract--Blue flowers of the garden lupin Russell hybrids contain delphinidin 3-(6"-malonylglueoside), whereas pink flowers contain the corresponding cyanidin and pelargonidin derivatives in a 1 : 3 ratio. Blue flowers also contain large amounts of apigenin 7-(6"-malonylglucoside) and small amounts of the luteolin analogue. The structure of the apigenin conjugate was unambiguously established by 2D N M R techniques. It is proposed that blue flower colour in lupin is due to copigrnentation of the two malonylated glucosides of delphinidin and apigenin, possibly linked in vivo covalently through a common malonic acid residue.
INTRODUCTION Although blue flower colour is a dominant feature of both garden and wild Lupinus species, little is known of the basis of this blue colour. Earlier studies by Bayer [1] suggested that the pigments were delphinidin and cyanidin derivatives, present in vivo as a metal complex with iron. Pink lupins were reported to contain mono- and diglycosides of pelargonidin, while violet forms contained mixtures of all these anthocyanins. There was some uncertainty regarding the sugars present in these pigments. A recent survey of flowering plants for zwitterionic anthocyanins was initiated, following the realization that malonylated and similarly substituted cyanic pigments are widespread in nature [2]. A survey of 15 species of legume flowers showed that such compounds were generally absent from the family, although two samples (Vicia sativa and blue garden lupin) were positive [3]. We therefore decided to reinvestigate the pigments of the garden lupin, in order to determine the form of the conjugation and also to establish the basis of the blue colour. The material studied included blue and pink forms of the well known Russell hybrids, which are derived from L. polyphyllus Lindl. and L. arboreus Sims [4]. The results of this investigation are presented here.
RESULTS
The blue form of the Russell hybrid contained a single anthoeyanin, identified as delphinidin 3-(6"-malonylglucoside), while the pink form contained a 1 : 3 mixture of cyanidin and pelargonidin 3-(6"-malonylglucoside). The presence of malonic acid in all three pigments was established by TLC identification after alkaline hydrolyPlff'l'O 34.'2-H
421
sis, which also yielded the 3-monoglucosides. The attachment of the malonic acid at the 6-position of glucose was determined by hydrogen peroxide oxidation and cochromatography of the resulting 6-malonylglucose with an authentic marker [3, 5]. This assignment was confirmed by co-chromatography of the original three pigments with authentic markers (Table I). All three of these anthocyanins probably occur relatively widely in plants, although they have only been described so far from a few sources. Pelargonidin 3-(6"malonylglucoside) was first reported in 1986 from Callistephus chinensis [5] and the cyanidin analogue in 1984 from Cichorium intybus [6]. The delphinidin pigment has so far been recorded only in flowers of Verbena hybrida [7] and Hibiscus syriacus [8]. Its presence in blue lupin is thus the third report of its occurrence as a flower pigment. The co-occurring flavones were then examined in the blue flowers. Large amounts of apigenin 7-(6"malonylglucoside) were obtained, together with smaller amounts of the luteolin analogue. Identifications were first based on UV spectral analysis, and acid and alkaline hydrolysis, the latter giving malonic acid in each case, together with the corresponding 7-monoglucoside. A search of the literature showed that an apigenin 7-malonylglucoside was isolated from parsley cell cultures in 1973 [9] but that the location of the malonic acid residue on the glucose moiety was not then established. The sample was therefore submitted to a comprehensive NMR analysis. The simple 1H NMR spectrum (Table 2) confirmed the presence of apigenin, glucose and glucose esterified with malonic acid. The glucoside and malonic acid esterified glucoside co-existed in deuterated pyridine in a ratio of 3: 7. 13C N M R data were obtained using the J M O D procedure (Table 2) and direct IH-~ac coupling was identified by ~H -~ 3C-COBIDEC. Coupling patterns
422
K. TAKEDAet al.
Table 1. Co-chromatography of lupin pigments with authentic anthocyanins Rs
( x 100) in
Pigment
BAW
BuHC1
1% HCl
HOAc-HCI
Lupin P-1 Cy 3-(6"-malonyl Glc) Deacylated P-1 Cy 3-Glc Lupin P-2 Pg 3-(6"-malonyl Glc) Deacylated P-2 Pg 3-Glc Lupin B Dp 3-(6"-malonyl Glc)* Deacytated B Dp 3-GIc
35 35 27 27 44 44 42 42 25 25 14 14
21. 21 10 10 36 36 25 25 10 10 04 04
05 05 03 03 11 11 06 06 03 03 01 01
17 17 11 11 31 31 23 23 12 12 07 07
Lupin P-1 and P-2 from pink flowers; Lupin B from blue flowers. *The sample was kindly donated by Terahara et al. I-7]; the other two authentic malonylated anthocyanins were from our own collection.
Table 2. Chemical shift data for apigenin 7-(6"-malonylglucoside) and apigenin 7glucoside
C/H 2 3 4 4a 5 6 7 8 8a 1' 2'/6' 3'/5' 4' 1" 2" 3" 4" 5" 6"
IH
Malonylglucoside 13C
6.92 s
6.88 d (2.2) 7.07 d (2.2)
7.99 d (8.8) 7.30 d (8.8) 5.76 d (7.5) 4.25 t (7.5) 4.40 m 4.40 m 4.25 m 4.88 dd (11.9, 4.8) 5.17 dd (11.9, 1.7)
1'"
2"' 3"'
3.90 ABq (15.5)
165.4 104.4 183.2 107.0 163.0 101.0 164.3 95.8 158.2 122.2 129.4 117.3 163.2 102.2 78.6 75.0 71.5 76.0 65.7
1H
Glucoside t3C
6.93 s
6.88 d (2.2) 7.13 d (2.2)
7.91 d (8.8) 7.22 d (8.8) 5.86 d (7.5) 4.25 t (7.5) 4.40 m 4.40 m 4.40 m 4.40 m 4.61 dd (12.0, 2.0)
165.3 104.3 183.3 106.9 162.9 101.0 164.4 95.7 158.2 122.41 29.411 7.2 163.2 102.1 78.8* 75.1 71.5 79.5* 62.7
168.4 43.1 169.9
*Interchangeable.
for the flavone and glucose protons were obtained by use of COSY-45, C O S Y - L R and T O C S Y experiments. Two problems remained, the position of esterification of the malonic acid and the point of attachment of the glucose to the apigenin. The relative deshielding of the resonances for H2-6 of the glucose suggested esterification at that position and this was confirmed by a study
of long-range heteronuclear coupling, using the H M B C procedure [10], which revealed 3J-coupling between H 26 and the malonyl carbonyl carbon resonating at 168.4ppm. Similarly, a 3j-coupling was observed between the anomeric proton of the glucose and a resonance at 164.25 ppm attributable to the apigenin A-ring. As the 164.25 ppm resonance also showed H M B C correlations
Malonylated flavonoids in lupin with both H-6 and H-8, and the only oxygen-bearing carbon with which both H-6 and H-8 can show coupling (2j) is C-7, then assignment of the glycosidic linkage through C-7 is unambiguous. Further evidence for placing the glucose at C-7 came from the NOESY spectrum which showed NOE interactions between the anomeric proton, and H-6 and H-8. The related luteolin 7-(6"-malonylglucoside) was recently obtained from carrot leaf as an oviposition stimulant to the female Black Swallowtail butterfly [11]. It was analysed by 1H NMR spectroscopy but our analysis of the apigenin analogue is more rigorous in establishing the position of the malonic acid at C-6". We assume that our luteolin analogue from lupin is identical to the 6"-malonic ester from its co-occurrence with the apigenin derivative. Blue flower colour in lupin is thus based on the presence of delphinidin 3-(6"-malonylglucoside) as the chromophore and apigenin 7-(6"-malonylglucoside) as the copigment, together with the presence of iron, as earlier reported by Bayer [1]. This co-pigment complex appears to be similar to that of the blue cornflower, Centaurea cyanus (Compositae) where the components are cyanidin 3-(6"-succinylglucoside)-5-glucoside, apigenin 7-glucuronide-4'-(6"-malonylglucoside), iron and magnesium [12]. The presence of a malonic acid residue in both pigment and copigment of lupin, and the known instability of this linkage in vitro, does suggest the possibility of an in vivo covalent linkage through malonic acid to the sugar residues of pigment and copigment. Provisional evidence for such a dimer has already been obtained by Strack et al. [13] in orchid flowers, but further work is needed to see whether such a dimer is present in the blue lupin. EXPERIMENTAL
NMR spectra were obtained on Bruker AMX-400 spectrometer using Bruker standard microprograms. All experiments were run in CsDsN using the solvent as int. standard. Isolation of anthocyanins and flavones. Freshly picked pink and blue petals of Russell hybrid iupins, or material stored at - 2 0 °, were used for the extraction of the pigments. The petals were immersed in M e O H H O A c - H 2 0 (10: 1:9, MAW) overnight and the filtered extracts were evapd. The residue was dissolved in H2O and the soln was passed through a column of XAD-7 (1.4 x 24 cm). After washing with H20 , the pigment adsorbed was eluted with M e O H - H O A c - H 2 0 (14: 1:5). The pigment fr. was evapd to dryness in vacuo at 30°. The residue,
423
after dissolution in MAW, was passed through a Sephadex LH-20 column (1.4 x 24 cm). The anthocyanin ft. was evapd to dryness and further purified by prep. PC using the solvent n - B u O H - H O A c - H 2 0 (4:1:5, BAW) and 15% HOAc. The flavone fr. from the Sephadex LH20 sepn crystallized out from aq. MeOH and was then twice recrystallized from aq. EtOH. Pigment identifications. Procedures for identifying malonylated anthocyanins in Compositae were used here [5]. All pigments were mobile electrophoretically'at pH 4.4 and had UV-visible spectra in line with lit. data. Analysis included acid and alkaline hydrolysis, detailed identification of the aglycone, sugar and organic acid [5], and H202 oxidation to give 6-malonylglucose, identified by co-chromatography with authentic material. The 2 flavones, apigenin and luteolin 7-(6"-malonyl)glucoside, were likewise identified on the basis of electrophoretic mobility at pH 4.4, UV spectral shifts, acid hydrolysis to aglycone, sugar and malonic acid, and alkaline hydrolysis to 7glucoside and malonic acid.
Acknowledoement--NMR spectra were run in the University of Strathclyde NMR Laboratory. REFERENCES
1. Bayer, E. (1959) Chem. Ber. 92, 1062. 2. Harborne, J. B. and Boardley, M. (1985) Z. Naturforsch. 40c, 305. 3. Harborne, J. B. (1986) Phytochemistry 25, 1887. 4. Huxley, A. (1992) New Dictionary of Gardening, Vol. 3. Macmillan, London. 5. Takeda, K., Harborne, J. B. and Self, R. (1986) Phytochemistry 25, 1337. 6. Bridle, P., Loeflter, R. S. T., Timberlake, C. F. and Self, R. (1984) Phytochemistry 23, 2968. 7. Terahara, N., Shioji, T., Toki, K., Saito, N. and Honda, T. (1989) Phytochemistry 28, 1507. 8. Kim, J. H., Nonaka, G. I., Fujieda, K. and Uemoto, S. (1989) Phytochemistry 28, 1503. 9. Kreuzaler, F. and Hahlbrock, K. (1973) Phytochemistry 12, 1149. 10. Bax, A. and Summers, M. F. (1986) J. Am. Chem. Soc. 106, 2093. 11. Feeny, P., Sachder, K., Rosenberry, L. and Carter, M. (1988) Phytochemistry 27, 3439. 12. Goto, T. (1987) Fortschr. Chemic Organ. Naturst. 52, 113. 13. Strack, D., Busch, E. and Klein, E. (1989) Phytochemistry 28, 2127.