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Anthocyanins in Fruits ofPassiflora edulisandP. suberosa
JOURNAL OF FOOD COMPOSITION AND ANALYSIS ARTICLE NO.
10, 49–54 (1997)
FC960514
Anthocyanins in Fruits of Passiflora edulis and P. suberosa LINDA KIDØY,* ANNE METTE NYGA˚RD,* ØYVIND M. ANDERSEN,*,1 ATLE T. PEDERSEN,* DAGFINN W. AKSNES,* AND BERNARD T. KIREMIRE† *Department of Chemistry, University of Bergen, N-5007 Bergen, Norway; and †Department of Chemistry, Makerere University, Kampala, Uganda Received June 10, 1996, and in revised form October 11, 1996 The analysis of anthocyanin pigments in the passion fruit Passiflora edulis and in the highly colored fruits of P. suberosa was performed using combinations of chromatographic and spectroscopic techniques. In addition to cyanidin 3-glucoside (97%), small amounts of cyanidin 3-(69malonylglucoside) (2%) and pelargonidin 3-glucoside (1%) were found in the rind of the passion fruit. The 3-(69-malonylglucoside) and 3-glucoside of cyanidin, delphinidin, petunidin, and pelargonidin were identified in extracts of P. suberosa. Anthocyanins acylated with malonic acid constituted 27% of the total anthocyanin content in this latter species. q 1997 Academic Press
INTRODUCTION
In the genus Passiflora the anthocyanins contribute to the spectacular color patterns of the flowers and the deep purple color of some of the fruits. In the purple passion fruit (P. edulis) Pruthi (1961) found pelargonidin 3-diglucoside in the fruit rind, while Harborne (1967) only reported delphinidin 3-glucoside and Ishikura and Sugahara (1979) cyanidin 3-glucoside in the fruits of this species. From other species in Passiflora Halim and Collins (1970) identified the 3,5-diglucosides of delphinidin, petunidin, and malvidin in addition to cyanidin 3-glucoside in the giant granadilla (P. quadrangularis). Billot (1974) reported the same anthocyanidin 3,5-diglucosides in addition to the 3-glucosides of delphinidin, petunidin, and malvidin to occur in flowers of this plant. The anthocyanins of the passion fruit rind have been suggested as a natural colorant (Pruthi, 1961). Because of divergent reports, we decided to reexamine the anthocyanin content of the purple passion fruit as well as to identify the anthocyanin content of the highly colored fruits of its relative, P. suberosa. MATERIALS AND METHODS
Plant materials. The fruits of P. suberosa (200 g each in two samples) were collected from the same bush in September 1994 at Makerere University, Kampala, Uganda. The identification of the plant was carried out in the Botany Department, Makerere University. A voucher specimen has been deposited in the herbarium of the Department of Chemistry, Makerere University. The purple passion fruits (300 g in one sample) were purchased. The samples were extracted with methanol acidified with 2% trifluoroacetic acid. One P. suberosa sample was extracted with methanol containing concen1
To whom correspondence should be addressed. Fax: /47 55 589490. 49
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trated HCl (0.1%, v/v). The filtered anthocyanin extracts were concentrated under reduced pressure. Standards. The 3-glucosides of delphinidin and cyanidin were isolated from black currant (Ribes nigrum), while petunidin 3-glucoside and pelargonidin 3-glucoside were isolated from whortleberry (Vaccinium myrtillus) and strawberry (Fragaria ananassa), respectively. Chromatographic analysis of the anthocyanins. The extracts were purified by partition against ethyl acetate before application on an Amberlite XAD-7 column (Andersen, 1988). The anthocyanins were separated on a Sephadex LH-20 column (100 1 2.6 cm; Pharmacia) using methanol/trifluororacetic acid/water (49.5/1/49.5, v/v) as eluent. High-performance liquid chromatography (HPLC) was performed on an HP1050 module system (Hewlett–Packard) using an ODS Hypersil column (20 1 0.5 cm, 5 mm). Two solvents were used for elution: A, formic acid/water (1/9, v/v) and B, methanol/formic acid/water (5/1/4, v/v). The elution profile was 0–4 min, 10% B in A (isocratic); 4–21 min, 10–100% B in A (linear gradient). The flow rate was 1.2 mL min01. Prior to injection, all samples were filtered through a 0.45-mm Millipore membrane filter. The chromatograms were recorded as the average values of the absorptions on every second nanometer between 500 and 540 nm using a photodiode array detector (HP 1050). The relative amounts of each anthocyanin are reported as percentages of total peak area in each chromatogram without taking into account the different molar absorption coefficients of the pigments. UV/Vis absorption spectra were recorded on-line during HPLC analysis, and spectral measurements were made over the wavelength range 210–600 nm in steps of 2 nm. Nuclear magnetic resonance spectroscopy. The NMR experiments were obtained at 400.13 and 100.62 MHz for 1H and 13C, respectively, on a Bruker AM 400 instrument at 257C. The 13C and residual 1H methanol resonances of the solvent CF3COOD/ CD3OD (1/9, v/v) were used as secondary references (d49.0 and d3.4 from tetramethylsilane, respectively). The one-dimensional 1H and 13C (spin echo Fourier transform) and the two-dimensional one-bond heteronuclear shift correlation (Bax and Morris, 1981) experiments were performed on a 5-mm 1H– 13C dual probe. The inverse twodimensional long-range heteronuclear shift correlation experiment (Bax and Summers, 1986) was run on a 5-mm inverse probe. The NMR simulations were performed by means of the PANIC program available in the Bruker Software Library. Petunidin 3-O-b-D-glucopyranoside (3). 1H NMR: d 9.05 (H-4), 6.72 (H-6), 6.96 (H-8), 8.02 (H-2*), 7.82 (H-6*), 4.07 (OMe), 5.42 (H-1 glc), 3.76 (H-2 glc), 3.63 (H3 glc), 3.54 (H-4 glc), 3.67 (H-5 glc), 4.03 (H-6A glc), 3.81 (H-6B glc). 13C NMR: d 163.81 (C-2), 145.18 (C-3), 136.54 (C-4), 159.26 (C-5), 103.34 (C-6), 170.53 (C7), 95.17 (C-8), 157.67 (C-9), 113.39 (C-10), 119.91 (C-1*), 109.34 (C-2*), 149.74 (C-3*), 147.45 (C-4*), 145.75 (C-5*), 113.68 (C-6*), 57.16 (OMe), 103.74 (C-1 glc), 74.95 (C-2 glc), 78.21 (C-3 glc), 71.14 (C-4 glc), 78.67 (C-5 glc), 62.38 (C-6 glc). Delphinidin 3-O-(69-O-malonyl-b-D-glucopyranoside) (5). 1H NMR: d 8.94 (H-4), 6.75 (H-6), 6.93 (H-8), 7.81 (H-2*,H-6*), 5.38 (H-1 glc), 3.80 (H-2 glc), 3.66 (H-3 glc), 3.52 (H-4 glc), 3.91 (H-5 glc), 4.65 (H-6A glc), 4.38 (H-6B glc), 3.44 (malonyl methylene). 13C NMR: d 164.01 (C-2), 145.65 (C-3), 135.82 (C-4), 158.76 (C-5), 103.60 (C-6), 170.21 (C-7), 95.19 (C-8), 157.52 (C-9), 113.10 (C-10), 119.93 (C-1*), 112.64 (C-2*,C-6*), 147.47 (C-3*,C-5*), 145.75 (C-4*), 103.33 (C-1 glc), 74.60 (C-2 glc), 77.84 (C-3 glc), 71.32 (C-4 glc), 75.91 (C-5 glc), 65.46 (C-6 glc), 168.64 and 170.39 (malonyl carbonyls).
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FIG. 1. HPLC chromatogram of the crude extract of P. suberosa. The identity of the chromatographic peaks are: (1, delphinidin 3-glucoside; 2, cyanidin 3-glucoside; 3, petunidin 3-glucoside; 4, pelargonidin 3-glucoside; 5, delphinidin 3-(69-malonylglucoside); 6, cyanidin 3-(69-malonylglucoside); 7, petunidin 3(69-malonylglucoside); and 8, pelargonidin 3-(69-malonylglucoside).
Cyanidin 3-O-(69-O-malonyl-b-D-glucopyranoside) (6). 1H NMR: d 9.03 (H-4), 6.76 (H-6), 7.00 (H-8), 8.12 (H-2*), 7.12 (H-5*), 8.37 (H-6*), 5.37 (H-1 glc), 3.76 (H-2 glc), 3.63 (H-3 glc), 3.52 (H-4 glc), 3.88 (H-5 glc), 4.63 (H-6A glc), 4.37 (H-6B glc), 3.48 (malonyl methylene). Pelargonidin 3-O-(69-O-malonyl-b-D-glucopyranoside) (8). 1H NMR: d 9.09 (H4), 6.78 (H-6), 7.01 (H-8), 8.68 (H-2*,H-6*), 7.14 (H-3*,H-5*), 5.34 (H-1 glc), 3.72 (H-2 glc), 3.61 (H-3 glc), 3.50 (H-4 glc), 3.89 (H-5 glc), 4.53 (H-6A glc), 4.36 (H6B glc), 3.49 (malonyl). Mass spectrometry systems. The samples were evaporated to dryness, dissolved in 5% trifluoroacetic acid (in methanol) and subjected to LC–MS analysis on a VG platform LC–MS system with electrospray interface (positive mode) and acetonitrile/ water (75/25, v/v) as mobile phase. Fast atomic bombardement mass spectra were obtained on a VG TRIO-2 mass spectrometer using a gun with xenon gas. The samples were mixed with glycerol. RESULTS AND DISCUSSION
The HPLC chromatogram of the crude extract of the fruits of P. suberosa showed eight anthocyanins (1–8) (Fig. 1), while the extract of the passion fruit rind showed one major anthocyanin (2) in addition to two minor pigments (4 and 6). The UV/Vis spectrum of 5 taken during on-line HPLC showed a visible maximum at 527 nm with A440/A527 of 29% (Table 1) indicating a 3-glycoside with a delphinidin nucleus (Ramstad et al., 1995). The aromatic region in the one-dimensional proton NMR spectrum of 5 showed a 1H singlet at d 8.94 (H-4), a 2H singlet at d 7.81 ( H-
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KIDØY ET AL. TABLE 1 CHROMATOGRAPHIC (HPLC) AND SPECTROSCOPIC (VISIBLE) DATA AND THE RELATIVE PROPORTIONS OF THE ANTHOCYANINS IDENTIFIED IN FRUITS OF PASSIFLORA SUBEROSA AND P. EDULIS
a b
glc, glucoside; mal, malonyl. See Fig. 2 for general structure. Relative amounts of the total anthocyanin content.
2*,6*), and a 2H AX system at d 6.75 (H-6, broad singlet) and 6.93 (H-8) in accordance with a delphinidin nucleus (Ramstad et al., 1995). The spectral region d 60–80 in the 13 C spin echo Fourier transform NMR spectrum contained five resonances which together with the anomeric carbon were in agreement with one hexose. The 1H and 13 C shift values, assigned by the one-bond heteronuclear shift correlation NMR experiment and the large vicinal 1H– 1H coupling constants of the sugar ring protons (7– 10 Hz) revealed by iterative spin simulation (PANIC) were in agreement with those of b-D-glucopyranoside with a 4C1 chair conformation. The binding site of the sugar was confirmed through a long-range correlation peak between the anomeric proton and the aglycone C-3 in the HMBC experiment to be the 3 position of the aglycone. The 2H singlet at d 3.44 in the 1H NMR spectrum together with the two carbonyl carbon signals (d 168.6 and 170.4) found in the spin echo Fourier transform NMR spectrum of 5 identified the acyl group to be malonyl (Andersen and Fossen, 1995). The downfield shift of the C-69 carbon and the two H-69 protons showed the acyl group to be attached to the 69-position of the glucose unit. We observed that the malonyl protons of 5 disappeared rather rapidly due to exchange with deuterium in the NMR solvent. This deuterium exchange was also observed during recording of the NMR spectra of 6 and 8. The NMR data together with the molecular ion peak [MH/] of m/z 551 and the fragment ion at m/z 465 found in the electrospray mass spectrum identified 5 to be delphinidin 3-O-(69-malonyl-b-D-glucopyranoside). Pigments 1–4 were cochromatographed (HPLC) and showed similar UV/Vis spectra (Table 1) with the 3-glucosides of delphinidin, cyanidin, petunidin, and pelargonidin, respectively. The UV/Vis spectra of 6–8 were similar to those of 2–4, respectively (Table 1). The prolonged retention times of 6–8 and the lack of absorption due to aromatic acids in the UV/Vis spectra indicated these anthocyanins to be the malonylated analogues of 2–4, respectively (Andersen and Fossen, 1995). The identities of 3, 6, and 8 were confirmed by NMR data (see Materials and Methods). Anthocyanins acylated with aliphatic acids are known to be sensitive to acids (Harborne, 1986). When fruits of P. suberosa were extracted with methanol containing
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ANTHOCYANINS IN FRUITS OF Passiflora
FIG. 2. General structure for the anthocyanins in P. edulis and P. suberosa. See Table 1 for details.
0.1% HCl, the proportions of 5–8 decreased while the proportions of 1–4 increased accordingly. The anthocyanin proportions were, however, not changed when HCl was substituted with trifluoroacetic acid. Anthocyanins acylated with aliphatic acids have previously not been found in the genus Passiflora; however, the use of mild extraction solvents and sensitive detection methods (on-line HPLC) show that acylated anthocyanins may be more common in this genus than indicated in earlier reports. The highly colored fruits of P. suberosa (Fig. 2) are a good source for anthocyanidin 3-(69-malonylglucosides) (Table 1). The anthocyanin petunidin 3-(69-malonylglucoside) has previously been identified only in Hibiscus syriacus (Kim et al., 1989). ACKNOWLEDGMENTS ˚ se Raknes for running the mass spectrometry analysis, and to Ms. Hope We are grateful to Mrs. A Kamusiime for providing fruits of P. suberosa. We acknowledge the Norwegian Universities’ Committee for Development, Research and Education for financial support. A.T.P. thanks The Research Council of Norway for a fellowship.
REFERENCES ANDERSEN, Ø. M. (1988). Semipreparative isolation and structure determination of pelargonidin 3-O-aL-rhamnopyranosyl-(1 r 2)-b-D-glucopyranoside and other anthocyanins from the tree Dacrycarpus dacrydioides. Acta Chem. Scand. B 42, 462–468. ANDERSEN, Ø. M., AND FOSSEN, T. (1995). Anthocyanins with an unusual acylation pattern from stem of Allium victorialis. Phytochemistry 40, 1809–1812. BAX, A., AND MORRIS, G. (1981). An improved method for heteronuclear chemical shift corrrelation by two-dimensional NMR. J. Magn. Reson. 42, 501–505. BAX, A., AND SUMMERS, M. F. (1986). 1H and 13C assignments from sensitivity-enhanced detection of heteronuclear multiple-bond connectivity by 2D multiple quantum NMR. J. Am. Chem. Soc. 108, 2094– 2095. BILLOT, J. (1974). Pigments anthocyaniques des fleurs de Passiflora quadrangularis. Phytochemistry 13, 2886. HALIM, M. M., AND COLLINS, R. P. (1970). Anthocyanins of Passiflora quadrangularis. Bull. Torrey Bot. Club 97, 247–248. HARBORNE, J. B. (1967). Comparative Biochemistry of the Flavonoids, p. 132. Academic Press, London.
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HARBORNE, J. B. (1986). The natural distribution in angiosperms of anthocyanins acylated with aliphatic dicarboxylic acid. Phytochemistry 25, 1887–1894. ISHIKURA, N., AND SUGAHARA, K. (1979). A survey of anthocyanins in fruits of some angiosperms. II. Bot. Mag. Tokyo 92, 157–161. KIM, J. H., NONAKA, G.-I., FUJIEDA, K., AND UEMOTO, S. (1989). Anthocyanidin malonylglucosides in flowers of Hibiscus syriacus. Phytochemistry 28, 1503–1506. PRUTHI, J. S., SUSHEELA, R., AND LAL, G. (1961). Anthocyanin pigment in passion fruit rind. J. Food Sci. 26, 385–388. RAMSTAD, B., PEDERSEN, A. T., AND ANDERSEN, Ø. M. (1995). Delphinidin 3-a-arabinopyranoside and other anthocyanins from flowers of Rhododendron cv. Lems Stormcloud. J. Hortic. Sci. 70, 637–642.
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FC960514
Anthocyanins in Fruits of Passiflora edulis and P. suberosa LINDA KIDØY,* ANNE METTE NYGA˚RD,* ØYVIND M. ANDERSEN,*,1 ATLE T. PEDERSEN,* DAGFINN W. AKSNES,* AND BERNARD T. KIREMIRE† *Department of Chemistry, University of Bergen, N-5007 Bergen, Norway; and †Department of Chemistry, Makerere University, Kampala, Uganda Received June 10, 1996, and in revised form October 11, 1996 The analysis of anthocyanin pigments in the passion fruit Passiflora edulis and in the highly colored fruits of P. suberosa was performed using combinations of chromatographic and spectroscopic techniques. In addition to cyanidin 3-glucoside (97%), small amounts of cyanidin 3-(69malonylglucoside) (2%) and pelargonidin 3-glucoside (1%) were found in the rind of the passion fruit. The 3-(69-malonylglucoside) and 3-glucoside of cyanidin, delphinidin, petunidin, and pelargonidin were identified in extracts of P. suberosa. Anthocyanins acylated with malonic acid constituted 27% of the total anthocyanin content in this latter species. q 1997 Academic Press
INTRODUCTION
In the genus Passiflora the anthocyanins contribute to the spectacular color patterns of the flowers and the deep purple color of some of the fruits. In the purple passion fruit (P. edulis) Pruthi (1961) found pelargonidin 3-diglucoside in the fruit rind, while Harborne (1967) only reported delphinidin 3-glucoside and Ishikura and Sugahara (1979) cyanidin 3-glucoside in the fruits of this species. From other species in Passiflora Halim and Collins (1970) identified the 3,5-diglucosides of delphinidin, petunidin, and malvidin in addition to cyanidin 3-glucoside in the giant granadilla (P. quadrangularis). Billot (1974) reported the same anthocyanidin 3,5-diglucosides in addition to the 3-glucosides of delphinidin, petunidin, and malvidin to occur in flowers of this plant. The anthocyanins of the passion fruit rind have been suggested as a natural colorant (Pruthi, 1961). Because of divergent reports, we decided to reexamine the anthocyanin content of the purple passion fruit as well as to identify the anthocyanin content of the highly colored fruits of its relative, P. suberosa. MATERIALS AND METHODS
Plant materials. The fruits of P. suberosa (200 g each in two samples) were collected from the same bush in September 1994 at Makerere University, Kampala, Uganda. The identification of the plant was carried out in the Botany Department, Makerere University. A voucher specimen has been deposited in the herbarium of the Department of Chemistry, Makerere University. The purple passion fruits (300 g in one sample) were purchased. The samples were extracted with methanol acidified with 2% trifluoroacetic acid. One P. suberosa sample was extracted with methanol containing concen1
To whom correspondence should be addressed. Fax: /47 55 589490. 49
0889-1575/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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trated HCl (0.1%, v/v). The filtered anthocyanin extracts were concentrated under reduced pressure. Standards. The 3-glucosides of delphinidin and cyanidin were isolated from black currant (Ribes nigrum), while petunidin 3-glucoside and pelargonidin 3-glucoside were isolated from whortleberry (Vaccinium myrtillus) and strawberry (Fragaria ananassa), respectively. Chromatographic analysis of the anthocyanins. The extracts were purified by partition against ethyl acetate before application on an Amberlite XAD-7 column (Andersen, 1988). The anthocyanins were separated on a Sephadex LH-20 column (100 1 2.6 cm; Pharmacia) using methanol/trifluororacetic acid/water (49.5/1/49.5, v/v) as eluent. High-performance liquid chromatography (HPLC) was performed on an HP1050 module system (Hewlett–Packard) using an ODS Hypersil column (20 1 0.5 cm, 5 mm). Two solvents were used for elution: A, formic acid/water (1/9, v/v) and B, methanol/formic acid/water (5/1/4, v/v). The elution profile was 0–4 min, 10% B in A (isocratic); 4–21 min, 10–100% B in A (linear gradient). The flow rate was 1.2 mL min01. Prior to injection, all samples were filtered through a 0.45-mm Millipore membrane filter. The chromatograms were recorded as the average values of the absorptions on every second nanometer between 500 and 540 nm using a photodiode array detector (HP 1050). The relative amounts of each anthocyanin are reported as percentages of total peak area in each chromatogram without taking into account the different molar absorption coefficients of the pigments. UV/Vis absorption spectra were recorded on-line during HPLC analysis, and spectral measurements were made over the wavelength range 210–600 nm in steps of 2 nm. Nuclear magnetic resonance spectroscopy. The NMR experiments were obtained at 400.13 and 100.62 MHz for 1H and 13C, respectively, on a Bruker AM 400 instrument at 257C. The 13C and residual 1H methanol resonances of the solvent CF3COOD/ CD3OD (1/9, v/v) were used as secondary references (d49.0 and d3.4 from tetramethylsilane, respectively). The one-dimensional 1H and 13C (spin echo Fourier transform) and the two-dimensional one-bond heteronuclear shift correlation (Bax and Morris, 1981) experiments were performed on a 5-mm 1H– 13C dual probe. The inverse twodimensional long-range heteronuclear shift correlation experiment (Bax and Summers, 1986) was run on a 5-mm inverse probe. The NMR simulations were performed by means of the PANIC program available in the Bruker Software Library. Petunidin 3-O-b-D-glucopyranoside (3). 1H NMR: d 9.05 (H-4), 6.72 (H-6), 6.96 (H-8), 8.02 (H-2*), 7.82 (H-6*), 4.07 (OMe), 5.42 (H-1 glc), 3.76 (H-2 glc), 3.63 (H3 glc), 3.54 (H-4 glc), 3.67 (H-5 glc), 4.03 (H-6A glc), 3.81 (H-6B glc). 13C NMR: d 163.81 (C-2), 145.18 (C-3), 136.54 (C-4), 159.26 (C-5), 103.34 (C-6), 170.53 (C7), 95.17 (C-8), 157.67 (C-9), 113.39 (C-10), 119.91 (C-1*), 109.34 (C-2*), 149.74 (C-3*), 147.45 (C-4*), 145.75 (C-5*), 113.68 (C-6*), 57.16 (OMe), 103.74 (C-1 glc), 74.95 (C-2 glc), 78.21 (C-3 glc), 71.14 (C-4 glc), 78.67 (C-5 glc), 62.38 (C-6 glc). Delphinidin 3-O-(69-O-malonyl-b-D-glucopyranoside) (5). 1H NMR: d 8.94 (H-4), 6.75 (H-6), 6.93 (H-8), 7.81 (H-2*,H-6*), 5.38 (H-1 glc), 3.80 (H-2 glc), 3.66 (H-3 glc), 3.52 (H-4 glc), 3.91 (H-5 glc), 4.65 (H-6A glc), 4.38 (H-6B glc), 3.44 (malonyl methylene). 13C NMR: d 164.01 (C-2), 145.65 (C-3), 135.82 (C-4), 158.76 (C-5), 103.60 (C-6), 170.21 (C-7), 95.19 (C-8), 157.52 (C-9), 113.10 (C-10), 119.93 (C-1*), 112.64 (C-2*,C-6*), 147.47 (C-3*,C-5*), 145.75 (C-4*), 103.33 (C-1 glc), 74.60 (C-2 glc), 77.84 (C-3 glc), 71.32 (C-4 glc), 75.91 (C-5 glc), 65.46 (C-6 glc), 168.64 and 170.39 (malonyl carbonyls).
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FIG. 1. HPLC chromatogram of the crude extract of P. suberosa. The identity of the chromatographic peaks are: (1, delphinidin 3-glucoside; 2, cyanidin 3-glucoside; 3, petunidin 3-glucoside; 4, pelargonidin 3-glucoside; 5, delphinidin 3-(69-malonylglucoside); 6, cyanidin 3-(69-malonylglucoside); 7, petunidin 3(69-malonylglucoside); and 8, pelargonidin 3-(69-malonylglucoside).
Cyanidin 3-O-(69-O-malonyl-b-D-glucopyranoside) (6). 1H NMR: d 9.03 (H-4), 6.76 (H-6), 7.00 (H-8), 8.12 (H-2*), 7.12 (H-5*), 8.37 (H-6*), 5.37 (H-1 glc), 3.76 (H-2 glc), 3.63 (H-3 glc), 3.52 (H-4 glc), 3.88 (H-5 glc), 4.63 (H-6A glc), 4.37 (H-6B glc), 3.48 (malonyl methylene). Pelargonidin 3-O-(69-O-malonyl-b-D-glucopyranoside) (8). 1H NMR: d 9.09 (H4), 6.78 (H-6), 7.01 (H-8), 8.68 (H-2*,H-6*), 7.14 (H-3*,H-5*), 5.34 (H-1 glc), 3.72 (H-2 glc), 3.61 (H-3 glc), 3.50 (H-4 glc), 3.89 (H-5 glc), 4.53 (H-6A glc), 4.36 (H6B glc), 3.49 (malonyl). Mass spectrometry systems. The samples were evaporated to dryness, dissolved in 5% trifluoroacetic acid (in methanol) and subjected to LC–MS analysis on a VG platform LC–MS system with electrospray interface (positive mode) and acetonitrile/ water (75/25, v/v) as mobile phase. Fast atomic bombardement mass spectra were obtained on a VG TRIO-2 mass spectrometer using a gun with xenon gas. The samples were mixed with glycerol. RESULTS AND DISCUSSION
The HPLC chromatogram of the crude extract of the fruits of P. suberosa showed eight anthocyanins (1–8) (Fig. 1), while the extract of the passion fruit rind showed one major anthocyanin (2) in addition to two minor pigments (4 and 6). The UV/Vis spectrum of 5 taken during on-line HPLC showed a visible maximum at 527 nm with A440/A527 of 29% (Table 1) indicating a 3-glycoside with a delphinidin nucleus (Ramstad et al., 1995). The aromatic region in the one-dimensional proton NMR spectrum of 5 showed a 1H singlet at d 8.94 (H-4), a 2H singlet at d 7.81 ( H-
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KIDØY ET AL. TABLE 1 CHROMATOGRAPHIC (HPLC) AND SPECTROSCOPIC (VISIBLE) DATA AND THE RELATIVE PROPORTIONS OF THE ANTHOCYANINS IDENTIFIED IN FRUITS OF PASSIFLORA SUBEROSA AND P. EDULIS
a b
glc, glucoside; mal, malonyl. See Fig. 2 for general structure. Relative amounts of the total anthocyanin content.
2*,6*), and a 2H AX system at d 6.75 (H-6, broad singlet) and 6.93 (H-8) in accordance with a delphinidin nucleus (Ramstad et al., 1995). The spectral region d 60–80 in the 13 C spin echo Fourier transform NMR spectrum contained five resonances which together with the anomeric carbon were in agreement with one hexose. The 1H and 13 C shift values, assigned by the one-bond heteronuclear shift correlation NMR experiment and the large vicinal 1H– 1H coupling constants of the sugar ring protons (7– 10 Hz) revealed by iterative spin simulation (PANIC) were in agreement with those of b-D-glucopyranoside with a 4C1 chair conformation. The binding site of the sugar was confirmed through a long-range correlation peak between the anomeric proton and the aglycone C-3 in the HMBC experiment to be the 3 position of the aglycone. The 2H singlet at d 3.44 in the 1H NMR spectrum together with the two carbonyl carbon signals (d 168.6 and 170.4) found in the spin echo Fourier transform NMR spectrum of 5 identified the acyl group to be malonyl (Andersen and Fossen, 1995). The downfield shift of the C-69 carbon and the two H-69 protons showed the acyl group to be attached to the 69-position of the glucose unit. We observed that the malonyl protons of 5 disappeared rather rapidly due to exchange with deuterium in the NMR solvent. This deuterium exchange was also observed during recording of the NMR spectra of 6 and 8. The NMR data together with the molecular ion peak [MH/] of m/z 551 and the fragment ion at m/z 465 found in the electrospray mass spectrum identified 5 to be delphinidin 3-O-(69-malonyl-b-D-glucopyranoside). Pigments 1–4 were cochromatographed (HPLC) and showed similar UV/Vis spectra (Table 1) with the 3-glucosides of delphinidin, cyanidin, petunidin, and pelargonidin, respectively. The UV/Vis spectra of 6–8 were similar to those of 2–4, respectively (Table 1). The prolonged retention times of 6–8 and the lack of absorption due to aromatic acids in the UV/Vis spectra indicated these anthocyanins to be the malonylated analogues of 2–4, respectively (Andersen and Fossen, 1995). The identities of 3, 6, and 8 were confirmed by NMR data (see Materials and Methods). Anthocyanins acylated with aliphatic acids are known to be sensitive to acids (Harborne, 1986). When fruits of P. suberosa were extracted with methanol containing
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ANTHOCYANINS IN FRUITS OF Passiflora
FIG. 2. General structure for the anthocyanins in P. edulis and P. suberosa. See Table 1 for details.
0.1% HCl, the proportions of 5–8 decreased while the proportions of 1–4 increased accordingly. The anthocyanin proportions were, however, not changed when HCl was substituted with trifluoroacetic acid. Anthocyanins acylated with aliphatic acids have previously not been found in the genus Passiflora; however, the use of mild extraction solvents and sensitive detection methods (on-line HPLC) show that acylated anthocyanins may be more common in this genus than indicated in earlier reports. The highly colored fruits of P. suberosa (Fig. 2) are a good source for anthocyanidin 3-(69-malonylglucosides) (Table 1). The anthocyanin petunidin 3-(69-malonylglucoside) has previously been identified only in Hibiscus syriacus (Kim et al., 1989). ACKNOWLEDGMENTS ˚ se Raknes for running the mass spectrometry analysis, and to Ms. Hope We are grateful to Mrs. A Kamusiime for providing fruits of P. suberosa. We acknowledge the Norwegian Universities’ Committee for Development, Research and Education for financial support. A.T.P. thanks The Research Council of Norway for a fellowship.
REFERENCES ANDERSEN, Ø. M. (1988). Semipreparative isolation and structure determination of pelargonidin 3-O-aL-rhamnopyranosyl-(1 r 2)-b-D-glucopyranoside and other anthocyanins from the tree Dacrycarpus dacrydioides. Acta Chem. Scand. B 42, 462–468. ANDERSEN, Ø. M., AND FOSSEN, T. (1995). Anthocyanins with an unusual acylation pattern from stem of Allium victorialis. Phytochemistry 40, 1809–1812. BAX, A., AND MORRIS, G. (1981). An improved method for heteronuclear chemical shift corrrelation by two-dimensional NMR. J. Magn. Reson. 42, 501–505. BAX, A., AND SUMMERS, M. F. (1986). 1H and 13C assignments from sensitivity-enhanced detection of heteronuclear multiple-bond connectivity by 2D multiple quantum NMR. J. Am. Chem. Soc. 108, 2094– 2095. BILLOT, J. (1974). Pigments anthocyaniques des fleurs de Passiflora quadrangularis. Phytochemistry 13, 2886. HALIM, M. M., AND COLLINS, R. P. (1970). Anthocyanins of Passiflora quadrangularis. Bull. Torrey Bot. Club 97, 247–248. HARBORNE, J. B. (1967). Comparative Biochemistry of the Flavonoids, p. 132. Academic Press, London.
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HARBORNE, J. B. (1986). The natural distribution in angiosperms of anthocyanins acylated with aliphatic dicarboxylic acid. Phytochemistry 25, 1887–1894. ISHIKURA, N., AND SUGAHARA, K. (1979). A survey of anthocyanins in fruits of some angiosperms. II. Bot. Mag. Tokyo 92, 157–161. KIM, J. H., NONAKA, G.-I., FUJIEDA, K., AND UEMOTO, S. (1989). Anthocyanidin malonylglucosides in flowers of Hibiscus syriacus. Phytochemistry 28, 1503–1506. PRUTHI, J. S., SUSHEELA, R., AND LAL, G. (1961). Anthocyanin pigment in passion fruit rind. J. Food Sci. 26, 385–388. RAMSTAD, B., PEDERSEN, A. T., AND ANDERSEN, Ø. M. (1995). Delphinidin 3-a-arabinopyranoside and other anthocyanins from flowers of Rhododendron cv. Lems Stormcloud. J. Hortic. Sci. 70, 637–642.
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