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Galloylcyanidin glycosides from acer
Phytochemistry, Vol. 31, No. 2. pp. 655~657, 1992 Printedin Great Britain.
‘(
GALLOYLCYANIDIN SHI-BAO
Faculty
of Horticulture,
JI, NORIO SAITO,*
GLYCOSIDES
MASATO YOKOI,
Chiba University, Matsudo, Yokohama, Japan; tHoshi
ATSUSHI SHIGIHARA~
and
ACER TOSHIO HONDAS
Chiba, Japan; *Chemical Laboratory, Meiji-Gakuin College of Pharmacy, Shinagawa, Tokyo, Japan (Received 12 March
Key Word
cyanidin
FROM
003 1 9422/92 55.00 + 0.00 1992 PergamonPressplc
University,
Totsuka,
1991)
Index-dcer; Aceraceae; maple; red spring leaves; anthocyanin; galloylcyanidin glycoside; 3-0-[2”-O-(galloyl)-fi-D-glucoside]; cyanidin 3-0-[2”-0-(galloyl)-6”-0-(a-~-rhamnosyl)-~-~-glucoside].
gallic acid;
Abstract-Two novel anthocyanins, cyanidin 3-O-[2”-O-(galloyl)-b-D-glucoside] and 3-O-[2”-O-(galloyl)-6”-0-(E-Lrhamnosyl)-p-D-glucoside] were isolated as common anthocyanins from red spring leaves of Acer. Cyanidin 3-0-[2”0-(galloyl)-/?-D-glucoside] was also observed in several autumn red leaves of Acer, but its presence was rather sporadic and minor.
INTRODUCTION
The anthocyanin component of red autumn leaves in Acer dissectum var. atropurpureum, and var. versicolor, A. palmatum var. atropurpureum, and A. campestre was first reported to be cyanidin 3-monoside by Robinson and Robinson (1932) [l]. Hattori and Hayashi (1937) also isolated a crystalline cyanidin glycoside as a major red leaf pigment from autumn leaves of A. ornatum var. matsumurae, A. circumlobatum, and A. sieboldianum, and identified it as cyanidin 3-glucoside [2]. Furthermore, Ishikura (1972) found two anthocyanins, cyanidin 3glucoside and cyanidin 3-rutinoside, as major anthocyanins of red spring leaves of Acer palmatum var. amoeum, and var. palmatum, and A. buergerianum [3]. Hitherto, these two anthocyanins were considered as major anthocyanins of Acer red leaves. In the course of our preliminary investigations of anthocyanins in leaves of 89 species and 204 cultivars of Acer [unpublished data], we found two novel acylated anthocyanins in red spring leaves as additional common anthocyanins as well as cyanidin 3-glucoside and 3rutinoside. We now report the isolation and structure determination of these two novel anthocyanins by spectral methods. For the sake of convenience, these two pigments are numbered 1 and 2.
RESULTS AND DISCUSSION
Pigments 1 and 2 were widely distributed in red leaves of Acer as common anthocyanins, particularly, in spring. As shown in Table 1, they exhibited higher Rr values than cyanidin 3-glucoside and 3-rutinoside, but identical values of absorption maxima in the visible region. Pigment 1 was extracted from red fresh spring leaves of A. platanoides L. cv Crimson Sentry and 2 was extracted from A. palmatum Thunb. cv Beni-komachi with methanol-acetic acid-water (9: 1: 10) or 0.1% HCl-methanol. These pigments were purified by Sephadex column (LH-20), PC and TLC on cellulose (solvents BAW 4: 1: 2 or 4: 1:5, AHW 15: 3 : 82), together with cyanidin 3-glucoside and 3-rutinoside.
The chromatographic data and spectral properties of four purified anthocyanins are shown in Table 1. The are 0.25 for 1, and 0.3 1 for 2. These valuesof E440/Evis,max data indicated that both anthocyanins are typical 3glycosides of cyanidin. Moreover, these pigments showed higher absorbances at 280 nm than those of simple cyanidin 3-glycosides, indicating the presence of aromatic acyl groups such as p-hydroxybenzoic acid [4]. Acid hydrolysis of 1 yielded cyanidin, glucose and gallic acid, and the same treatment of 2 produced cyanidin, glucose, rhamnose and gallic acid. Partial acid hydrolysis of 1 gave only cyanidin 3-glucoside as an intermediate, and also this hydrolysis of 2 gave 1, cyanidin 3-rutinoside, and cyanidin 3-glucoside as three intermediates. Mild alkaline hydrolysis of 1 with 0.1 N NaOH under N, produced cyanidin 3-glucoside, and similar hydrolysis of 2 afforded cyanidin 3-rutinoside, respectively. Also both pigments produced gallic acid. To confirm the presence of gallic acid, IR of 2 was recorded as a KBr disk, and an absorption band of carbonyl group was observed at 1695-1685 cm-‘. Oxidation with alkaline hydrogen peroxide of both 1 and 2 produced galloylglucose and galloylrutinose, respectively. Both galloylsugars showed blue fluorescence after ammonia fuming under UV-light and a brown-red colour with aniline hydrogen phthalate. The R, values are described in the and &,,, of both galloylsugars experimental. The FAB mass spectrum of 1 gave a molecular ion WI’ at 601 m/z and that of 2 [M]’ at 747 m/z, respectively. Both were in agreement with the masses (m/z 601.493) and C34H35019 calculated for C,,H,,O,, (m/z 747.635). To confirm these structures, ‘H NMR and ‘H-‘H COSY spectra were measured in CF,CO,DDMSO-d, (1: 9) (Table 2). The proton signals of the sugar moiety of 1 appeared in the region 63.1-5.9. An anomeric proton was observed at 65.89 (J= 8.1 Hz), and all vicinal coupling constants of glucose were 8.1-9.2 Hz. Therefore, this glucose must be a P-D-glucopyranose. By analysis of the ‘H-‘H COSY spectrum of this pigment, the peak at 65.28 (dd, 5=8.1, 9.2 Hz) was assigned to the proton of glucose H-2 and 655
PINTO31:2-s
656
S.-B. Jr et al. Table
2. ‘HNMR spectral data of compounds (in CF,CO,D-DMSO-d,, 1:9) 1
H cyanidin 4 6 8 2 5’ 6
1 and 2
2
moiety 8.97 6.84 6.94 7.84 6.89 8.04
s d (1.8) br s d (2.2) d (8.8) dd (8.8, 2.2)
8.88 6.89 6.93 7.82 6.87 8.03
s br s br s d (2.2) d (8.6) dd (8.6, 2.2)
gallic acid moiety 236 glucose 1 2 3 4 5 6a 6b rhamnose 1 2 3 4 5
7.08 s
7.08 s
moiety 5.89 d (8.1) 5.28 dd (8.1, 9.2) 3.75 t (9.2) 3.13* t (9.0) 3.48* dt (9.0, 9.0) 3.74 dd (9.0, 11.0) 3.81 d (11.0)
5.91 5.28 3.76 3.42 3.95 3.53 4.00
d (8.1) dd (8.1, 9.2) t (9.2) t (9.0) dt (8.4, 9.0) (13.7, 8.4) d (13.7)
moiety 4.58 br s 3.67 br s 3.55* brs 3.21 t (9.5) 3.46* dd (9.5, 5.9)
*Assigned by ‘H-‘H COSY Coupling constants (J in Hz) in parentheses.
was shifted to a lower field, suggesting that gallic acid is bonded to the OH-2 of the glucose moiety. Thus 1 is cyanidin 3-O-[2”-O-(galloyl)$-D-pyranoglucoside]. ‘HNMR signals of 2 due to glucose and rhamnose protons appeared in the region 63.2-5.9. The singlet at 67.08 was assigned to H-2 and H-6 of gallic acid. The anomeric proton of glucose was assigned at 65.91 (d, J =8.1 Hz) and all vicinal coupling constants of glucose were 8.1-9.2 Hz. Therefore, the stereochemistry of glucose was confirmed to be P-D-glucopyranose. The anomeric proton of rhamnose was observed at 64.58 as a singlet. The ‘HNMR spectrum of vicinal coupling constants (5+ 5 = 9.5) in the rhamnose moiety showed it to have the cc-L-pyranorhamnose configuration (Table 2). By analysis of the ‘H-‘H COSY spectrum at the sugar moieties, the downfield shift of H-2 of glucose (6 5.28, dd, J = 8.1, 9.2 Hz) indicated that the gallic acid was attached to OH-2 as in 1. Thus 2 is cyanidin 3-O-[2”-O-(galloyl)6”-O-(L-rhamnosyl)+?-D-glucoside]. Galloyl kaempferol, quercetin and myricetin glycosides have already been reported in plants [S], but galloyl anthocyanins have not been reported to date. In spring, these two pigments were distributed widely in red Acer leaves. In autumn, however, 1 was only found in A. pycnanthum, A. rubrum, A. saccharum ssp. grandidentatum and nine other cultivars. Furthermore, 2 was absent. EXPERIMENTAL Plant materials. The plants used in this work (Acer palmatum Thunb. cv. Beni-komachi and A. platanoides L. cv. Crimson
Galloylcyanidin glycosides from Acer Sentry) were from the Experimental Farm of the Faculty of Horticulture, Chiba University. Extraction and purification of anthocyanins. The fresh red leaves of Acer (ca 750 g) were extracted with MAW (MeOHHOAc-H,O 9: 1: 10) or 0.1% HCl-MeOH (41) at room temp. for 10 hr. The extracts were coned and washed with Et,O. The reddish purple powder of anthocyanin was dissolved in 0.1% HCI-MeOH. After passing through Sephadex (LH-20) with 15% HOAc, the eluate was evapd and purified by PC (No. 51 papers, Toyo) and TLC (Cellulose, Avicel SF, Funakoshi) in BAW (4: 1:2) and AHW (15:3:82). Lastly, the residues were washed with Et,0 from H,O-EtOH, and gave ca 30 mg of 1,ca 45 mg of 2, ca 60 mg of cyanidin 3-glucoside and ca 60 mg of cyanidin 3rutinoside powder, respectively. Standard analysis. Pigment identifications were carried out by standard procedures involving deacylation with alkaline and hydrolysis with acid [6, 73 and H,O, oxidation. Because gallic acid is unstable in alkaline solvents, H,O, oxidation was first carried out in 2 ml H,O with 2 drops of 30% H,O, for 1 hr, and then 2 ml 0.1 M NH,OH in MeOH was added, and kept for ca 40 min. After that the solvents were evapd to dryness in uacuo. Galloylsugars were dissolved with EtOH and purified by TLC (solvent BEW). TLC was carried out on Microcrystalline cellulose (Avicel SF, Funakoshi). Solvents used for anthocyanins were BAW (n-BuOH-HOAc-H,O, 4: 1: 5, upper solvents), AHW (HOAc-HCl-H,O, 15: 3 :82). BuHCl (n-BuOH-HC1 1: 1, upper solvents) and 1% HCl, for gallic acid were 6% aq.-HOAc, BMA (C,H,-MeOH-HOAc, 45: 8:4), and for sugars and galloyl-
657
sugars were BAW (4: 1: 5, upper solvents), PhOH (satd with H,O), BEW (n-BuOH-EtOH-H,O, 20: 5: 1l), and BPW (nBuOH-pyridine-H,O, 6: 3 : 1). Cyanidin and sugars were determined as previously [6,7]. Gallic acid was detected by means of Folin-Ciocalteu method [7]. ‘H NMR (400 MHz) and ‘H-‘H COSY of anthocyanins were recorded on in CF,CO,DDMSOd, (1:9). Galloyl-glucose: blue fluorescence after ammonia fuming, 1,,, 272.6 nm, R, values: 27.0 in BAW, 27.3 in BEW, 34.1 in PhOH. Galloyl-rutinose: blue fluorescence after ammonia fuming, &,,,, 272.6 nm, R, values: 25.8 in BAW, 26.1 in BEW, 34.1 in PhOH. R, values of glucose: 16.9 in BAW, 19.3 in BEW, 27.3 in PhOH. REFERENCES
1. Robinson, G. M. and Robinson, R. (1932) Biochem. J. 26, 1647. 2. Hattori, S. and Hayashi, K. (1937) Acta Phytochem. 10, 129. 3. Ishikura, N. (1973) Kumamoto J. Sci., Biol. 11, 43. 4. Terahara, N., Toki, K., Saito, N., Honda, T., Isono, T., Furumoto, H. and Kontani, Y. (1990) J. Chem. SOL, Perkin Trans. 1, 3327. 5. Harborne, J. B. and Williams, C. A. (1988) in The Flauonoids-Advances in Research since 1980 (Harborne, J. B., ed.),
p. 303. Chapman & Hall, London. 6. Harbome, J. B. (1966) Comparative Biochemistry of the Flavonoids. Academic Press, London. 7. Harborne, J. B. (1973) Phytochemical Methods. Chapman & Hall, London.
‘(
GALLOYLCYANIDIN SHI-BAO
Faculty
of Horticulture,
JI, NORIO SAITO,*
GLYCOSIDES
MASATO YOKOI,
Chiba University, Matsudo, Yokohama, Japan; tHoshi
ATSUSHI SHIGIHARA~
and
ACER TOSHIO HONDAS
Chiba, Japan; *Chemical Laboratory, Meiji-Gakuin College of Pharmacy, Shinagawa, Tokyo, Japan (Received 12 March
Key Word
cyanidin
FROM
003 1 9422/92 55.00 + 0.00 1992 PergamonPressplc
University,
Totsuka,
1991)
Index-dcer; Aceraceae; maple; red spring leaves; anthocyanin; galloylcyanidin glycoside; 3-0-[2”-O-(galloyl)-fi-D-glucoside]; cyanidin 3-0-[2”-0-(galloyl)-6”-0-(a-~-rhamnosyl)-~-~-glucoside].
gallic acid;
Abstract-Two novel anthocyanins, cyanidin 3-O-[2”-O-(galloyl)-b-D-glucoside] and 3-O-[2”-O-(galloyl)-6”-0-(E-Lrhamnosyl)-p-D-glucoside] were isolated as common anthocyanins from red spring leaves of Acer. Cyanidin 3-0-[2”0-(galloyl)-/?-D-glucoside] was also observed in several autumn red leaves of Acer, but its presence was rather sporadic and minor.
INTRODUCTION
The anthocyanin component of red autumn leaves in Acer dissectum var. atropurpureum, and var. versicolor, A. palmatum var. atropurpureum, and A. campestre was first reported to be cyanidin 3-monoside by Robinson and Robinson (1932) [l]. Hattori and Hayashi (1937) also isolated a crystalline cyanidin glycoside as a major red leaf pigment from autumn leaves of A. ornatum var. matsumurae, A. circumlobatum, and A. sieboldianum, and identified it as cyanidin 3-glucoside [2]. Furthermore, Ishikura (1972) found two anthocyanins, cyanidin 3glucoside and cyanidin 3-rutinoside, as major anthocyanins of red spring leaves of Acer palmatum var. amoeum, and var. palmatum, and A. buergerianum [3]. Hitherto, these two anthocyanins were considered as major anthocyanins of Acer red leaves. In the course of our preliminary investigations of anthocyanins in leaves of 89 species and 204 cultivars of Acer [unpublished data], we found two novel acylated anthocyanins in red spring leaves as additional common anthocyanins as well as cyanidin 3-glucoside and 3rutinoside. We now report the isolation and structure determination of these two novel anthocyanins by spectral methods. For the sake of convenience, these two pigments are numbered 1 and 2.
RESULTS AND DISCUSSION
Pigments 1 and 2 were widely distributed in red leaves of Acer as common anthocyanins, particularly, in spring. As shown in Table 1, they exhibited higher Rr values than cyanidin 3-glucoside and 3-rutinoside, but identical values of absorption maxima in the visible region. Pigment 1 was extracted from red fresh spring leaves of A. platanoides L. cv Crimson Sentry and 2 was extracted from A. palmatum Thunb. cv Beni-komachi with methanol-acetic acid-water (9: 1: 10) or 0.1% HCl-methanol. These pigments were purified by Sephadex column (LH-20), PC and TLC on cellulose (solvents BAW 4: 1: 2 or 4: 1:5, AHW 15: 3 : 82), together with cyanidin 3-glucoside and 3-rutinoside.
The chromatographic data and spectral properties of four purified anthocyanins are shown in Table 1. The are 0.25 for 1, and 0.3 1 for 2. These valuesof E440/Evis,max data indicated that both anthocyanins are typical 3glycosides of cyanidin. Moreover, these pigments showed higher absorbances at 280 nm than those of simple cyanidin 3-glycosides, indicating the presence of aromatic acyl groups such as p-hydroxybenzoic acid [4]. Acid hydrolysis of 1 yielded cyanidin, glucose and gallic acid, and the same treatment of 2 produced cyanidin, glucose, rhamnose and gallic acid. Partial acid hydrolysis of 1 gave only cyanidin 3-glucoside as an intermediate, and also this hydrolysis of 2 gave 1, cyanidin 3-rutinoside, and cyanidin 3-glucoside as three intermediates. Mild alkaline hydrolysis of 1 with 0.1 N NaOH under N, produced cyanidin 3-glucoside, and similar hydrolysis of 2 afforded cyanidin 3-rutinoside, respectively. Also both pigments produced gallic acid. To confirm the presence of gallic acid, IR of 2 was recorded as a KBr disk, and an absorption band of carbonyl group was observed at 1695-1685 cm-‘. Oxidation with alkaline hydrogen peroxide of both 1 and 2 produced galloylglucose and galloylrutinose, respectively. Both galloylsugars showed blue fluorescence after ammonia fuming under UV-light and a brown-red colour with aniline hydrogen phthalate. The R, values are described in the and &,,, of both galloylsugars experimental. The FAB mass spectrum of 1 gave a molecular ion WI’ at 601 m/z and that of 2 [M]’ at 747 m/z, respectively. Both were in agreement with the masses (m/z 601.493) and C34H35019 calculated for C,,H,,O,, (m/z 747.635). To confirm these structures, ‘H NMR and ‘H-‘H COSY spectra were measured in CF,CO,DDMSO-d, (1: 9) (Table 2). The proton signals of the sugar moiety of 1 appeared in the region 63.1-5.9. An anomeric proton was observed at 65.89 (J= 8.1 Hz), and all vicinal coupling constants of glucose were 8.1-9.2 Hz. Therefore, this glucose must be a P-D-glucopyranose. By analysis of the ‘H-‘H COSY spectrum of this pigment, the peak at 65.28 (dd, 5=8.1, 9.2 Hz) was assigned to the proton of glucose H-2 and 655
PINTO31:2-s
656
S.-B. Jr et al. Table
2. ‘HNMR spectral data of compounds (in CF,CO,D-DMSO-d,, 1:9) 1
H cyanidin 4 6 8 2 5’ 6
1 and 2
2
moiety 8.97 6.84 6.94 7.84 6.89 8.04
s d (1.8) br s d (2.2) d (8.8) dd (8.8, 2.2)
8.88 6.89 6.93 7.82 6.87 8.03
s br s br s d (2.2) d (8.6) dd (8.6, 2.2)
gallic acid moiety 236 glucose 1 2 3 4 5 6a 6b rhamnose 1 2 3 4 5
7.08 s
7.08 s
moiety 5.89 d (8.1) 5.28 dd (8.1, 9.2) 3.75 t (9.2) 3.13* t (9.0) 3.48* dt (9.0, 9.0) 3.74 dd (9.0, 11.0) 3.81 d (11.0)
5.91 5.28 3.76 3.42 3.95 3.53 4.00
d (8.1) dd (8.1, 9.2) t (9.2) t (9.0) dt (8.4, 9.0) (13.7, 8.4) d (13.7)
moiety 4.58 br s 3.67 br s 3.55* brs 3.21 t (9.5) 3.46* dd (9.5, 5.9)
*Assigned by ‘H-‘H COSY Coupling constants (J in Hz) in parentheses.
was shifted to a lower field, suggesting that gallic acid is bonded to the OH-2 of the glucose moiety. Thus 1 is cyanidin 3-O-[2”-O-(galloyl)$-D-pyranoglucoside]. ‘HNMR signals of 2 due to glucose and rhamnose protons appeared in the region 63.2-5.9. The singlet at 67.08 was assigned to H-2 and H-6 of gallic acid. The anomeric proton of glucose was assigned at 65.91 (d, J =8.1 Hz) and all vicinal coupling constants of glucose were 8.1-9.2 Hz. Therefore, the stereochemistry of glucose was confirmed to be P-D-glucopyranose. The anomeric proton of rhamnose was observed at 64.58 as a singlet. The ‘HNMR spectrum of vicinal coupling constants (5+ 5 = 9.5) in the rhamnose moiety showed it to have the cc-L-pyranorhamnose configuration (Table 2). By analysis of the ‘H-‘H COSY spectrum at the sugar moieties, the downfield shift of H-2 of glucose (6 5.28, dd, J = 8.1, 9.2 Hz) indicated that the gallic acid was attached to OH-2 as in 1. Thus 2 is cyanidin 3-O-[2”-O-(galloyl)6”-O-(L-rhamnosyl)+?-D-glucoside]. Galloyl kaempferol, quercetin and myricetin glycosides have already been reported in plants [S], but galloyl anthocyanins have not been reported to date. In spring, these two pigments were distributed widely in red Acer leaves. In autumn, however, 1 was only found in A. pycnanthum, A. rubrum, A. saccharum ssp. grandidentatum and nine other cultivars. Furthermore, 2 was absent. EXPERIMENTAL Plant materials. The plants used in this work (Acer palmatum Thunb. cv. Beni-komachi and A. platanoides L. cv. Crimson
Galloylcyanidin glycosides from Acer Sentry) were from the Experimental Farm of the Faculty of Horticulture, Chiba University. Extraction and purification of anthocyanins. The fresh red leaves of Acer (ca 750 g) were extracted with MAW (MeOHHOAc-H,O 9: 1: 10) or 0.1% HCl-MeOH (41) at room temp. for 10 hr. The extracts were coned and washed with Et,O. The reddish purple powder of anthocyanin was dissolved in 0.1% HCI-MeOH. After passing through Sephadex (LH-20) with 15% HOAc, the eluate was evapd and purified by PC (No. 51 papers, Toyo) and TLC (Cellulose, Avicel SF, Funakoshi) in BAW (4: 1:2) and AHW (15:3:82). Lastly, the residues were washed with Et,0 from H,O-EtOH, and gave ca 30 mg of 1,ca 45 mg of 2, ca 60 mg of cyanidin 3-glucoside and ca 60 mg of cyanidin 3rutinoside powder, respectively. Standard analysis. Pigment identifications were carried out by standard procedures involving deacylation with alkaline and hydrolysis with acid [6, 73 and H,O, oxidation. Because gallic acid is unstable in alkaline solvents, H,O, oxidation was first carried out in 2 ml H,O with 2 drops of 30% H,O, for 1 hr, and then 2 ml 0.1 M NH,OH in MeOH was added, and kept for ca 40 min. After that the solvents were evapd to dryness in uacuo. Galloylsugars were dissolved with EtOH and purified by TLC (solvent BEW). TLC was carried out on Microcrystalline cellulose (Avicel SF, Funakoshi). Solvents used for anthocyanins were BAW (n-BuOH-HOAc-H,O, 4: 1: 5, upper solvents), AHW (HOAc-HCl-H,O, 15: 3 :82). BuHCl (n-BuOH-HC1 1: 1, upper solvents) and 1% HCl, for gallic acid were 6% aq.-HOAc, BMA (C,H,-MeOH-HOAc, 45: 8:4), and for sugars and galloyl-
657
sugars were BAW (4: 1: 5, upper solvents), PhOH (satd with H,O), BEW (n-BuOH-EtOH-H,O, 20: 5: 1l), and BPW (nBuOH-pyridine-H,O, 6: 3 : 1). Cyanidin and sugars were determined as previously [6,7]. Gallic acid was detected by means of Folin-Ciocalteu method [7]. ‘H NMR (400 MHz) and ‘H-‘H COSY of anthocyanins were recorded on in CF,CO,DDMSOd, (1:9). Galloyl-glucose: blue fluorescence after ammonia fuming, 1,,, 272.6 nm, R, values: 27.0 in BAW, 27.3 in BEW, 34.1 in PhOH. Galloyl-rutinose: blue fluorescence after ammonia fuming, &,,,, 272.6 nm, R, values: 25.8 in BAW, 26.1 in BEW, 34.1 in PhOH. R, values of glucose: 16.9 in BAW, 19.3 in BEW, 27.3 in PhOH. REFERENCES
1. Robinson, G. M. and Robinson, R. (1932) Biochem. J. 26, 1647. 2. Hattori, S. and Hayashi, K. (1937) Acta Phytochem. 10, 129. 3. Ishikura, N. (1973) Kumamoto J. Sci., Biol. 11, 43. 4. Terahara, N., Toki, K., Saito, N., Honda, T., Isono, T., Furumoto, H. and Kontani, Y. (1990) J. Chem. SOL, Perkin Trans. 1, 3327. 5. Harborne, J. B. and Williams, C. A. (1988) in The Flauonoids-Advances in Research since 1980 (Harborne, J. B., ed.),
p. 303. Chapman & Hall, London. 6. Harbome, J. B. (1966) Comparative Biochemistry of the Flavonoids. Academic Press, London. 7. Harborne, J. B. (1973) Phytochemical Methods. Chapman & Hall, London.