Acylated pelargonidin 3-sambubioside-5-glucosides in Matthiola incana

0031-9422(95)00824-1

Pergamon

Phytochemistry, Vol.41, No. 6, pp. 1613-1620, 1996 Copyright © 1996ElsevierScience Ltd Printed in Great Britain. All rights reserved 0031 9422/96$15.00 + 0.00

ACYLATED PELARGONIDIN 3-SAMBUBIOSIDE-5-GLUCOSIDES IN M A T THIOLA INCANA NORIO SAITO, FUMI TATSUZAWA,*t AKI HONGO,* KHIN WLA WIN,* MASATO YOKOI,* ATSUSHI SHIGIHARA~ TOSHIO HONDA~

and

Chemical Laboratory, Meiji-Gakuin University, Totsuka, Yokohama, Japan; *Faculty of Horticulture, Chiba University, Matsudo, Chiba, Japan; :~Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo, Japan (Received in revisedform 25 September 1995)

Key Word Index--Matthiola incana; Cruciferae; red and red-purple flower colour; acylated pelargonidin 3-sambubioside-5-glucoside; malonic acid; sinapic acid; ferulic acid; p-coumaric acid.

Al~traet--Ten acylated pelargonidin 3-sambubioside-5-glucosides were isolated from the red-purple flowers of Matthiola incana, and also pelargonidin 3-glucoside was isolated from the brownish-red flowers of this plant. FAB mass measurements of 10 acylated anthocyanins gave their molecular ions I-M] ÷ at 903-1195 m/z, which were based on acylated pelargonidin 3-sambubioside-5-glucosides with malonic acid, sinapic acid, p-coumaric acid, caffeic acid and/or ferulic acid. This was confirmed by the analysis of NMR spectra and the experiments of acid and alkaline hydrolysis. By spectral and chemical methods, seven of the 10 pigments were determined to be pelargonidin 3-0[2-•-(2-•-(acy•-•)-•-D-xy••pyran•sy•)-6-•-(acy•-••)-•-D-g•uc•pyran•side]-5-•-[6-•-(ma••ny•)-•-D-g•uc•pyran•side-l• in which acyl moieties varied between sinapic, ferulic, caffeic and p-coumaric acids. The occurrence of these pigments was examined in 10 red-purple, 10 salmon-pink, three apricot and three copper colour cultivars of M. incana by HPLC. The acylated pelargonidin 3-sambubioside-5-glucosides were present as the dominant pigments in the red-purple, salmon-pink and apricot colour cultivars. On the other hand, pelargonidin 3-glucoside was present as a dominant anthocyanin in the copper colour cultivars and also pelargonidin 3-sambubioside-5-glucoside was confirmed by HPLC as a minor pigment in the copper colour flowers.

INTRODUCTION

In continuing work on flower colour variation due to acylated anthocyanins in the various color cultivars of Matthiola incana, we have already reported the occurrence of four new acylated cyanidin 3-sambubioside-5glucosides in purple-violet flower cultivars [1]. Earlier, Seyffert reported the presence of acylated pelargonidin 3-glycoside and 3,5-diglycoside in the red flowers of this plant [2-1, and Harborne reported the presence of pelargonidin 3-glucoside in the brownish-red flowers and also pelargonidin 3-ferulyl-p-coumarylsambubioside-5-glucoside in the red or red-purple flowers of this plant [3, 4]. In this paper we report the presence of 15 pelargonidin glycosides in 26 flowers of red-purple, salmon-pink, apricot or copper coiour cultivars, and also the structural determination of seven novel acylated pelargonidin 3sambubioside-5-glucosides.

RESULTSAND DISCUSSION In a survey of 26 reddish cultivars of M. incana by HPLC analysis, 15 anthocyanin peaks were observed in the reddish flowers of these cultivars (Table 1). Among ~'Author to whom correspondence should be addressed.

these peaks, 12 anthocyanins were isolated and identified as 1 (frequency 9.0%), 2 (55.1%), 3 (27.0%), 4 (60.0%), 5 plus 6 (31.2%), 7 (4.4%), g (7.2%), 9 (5.7%), 10 (11.9%), 11 (78.9%) and 12 (5.8%). These anthocyanins (1-10) were isolated from the redpurple flowers of these cultivars with MAW (methanol-acetic acid-water, 9: 1:10) solvent, and also an anthocyanin (11) was isolated from the brownish-red cultivars. These pigments were purified using Diaion HP-20 column chromatography, paper chromatography and HPLC. The R I values, Rt(min) and spectral data are shown in Table 2, and the pigment 11 from the brownishred flowers was easily identified to be pelargonidin 3glucoside. This structure was confirmed by the analysis of 1HNMR and FAB mass spectra (Tables 2 and 3). The anthocyanins of 1-10 yielded only one pigment (12) as their deacyl anthocyanins by alkaline hydrolysis with NaOH under nitrogen. As acyl moieties, these pigments yield four hydroxycinnamic acids and malonic acid, by hydrolysis; malonic acid (1-5), sinapic acid (2, 3, 6, 7, g and 10), ferulic acid (1, 2, 4 and 6), p-coumaric acid (3, 5 and 7) and caffeic acid (g and 9). Deacylanthocyanin (12) The R I values, Rt and spectral data for deacyl anthocyanin are shown in Table 2. On acid hydrolysis 12

1613

1614

N. SAITO et al.

,.Q

"0 oo (.;.~

°~

o

0 "0 +~

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r~

0 .~, ..~ ,.~

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I

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i

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Acylated pelargonidin glucosides in Matthiola incana

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gave pelargonidin, glucose and xylose. Partial acid hydrolysis gave rise to 3- and 5-glucoside, 3-sambubioside and 3,5-diglucoside. Fast atom bombardment (FAB) mass spectrum of 12 indicates the molecular ion peak at m/z 727. The 1 H N M R of 12 shows the presence of pelargonidin and two hexosides and one pentoside, all of which are deduced to be fl-D-pyranosides from their (vicinal) coupling constants (J = 7.7-9.5 Hz). The H-4 of pelargonidin appears at the lowest magnetic field (8.98 ppm). A proton at 67.20 having a long-range coupling to H-4 (J = ca 0.5 Hz) can be assigned to H-8, which is further spin-coupled with H-6 (67.04, J = 1.8 Hz). The positions of the glycosidic links were determined as follows: irradiations at each of H-1 (65.63) of Glc A, H-1 (65.15) ofGlc B and H-1 (64.81) of xylose caused negative NOEs to H-4, H-6 of pelargonidin and H-2 of Glc A, respectively. The H202 oxidation of 12 gave sambubiose [1]. Thus, the structure of 12 is pelargonidin 3-0-[2-0(fl-D-xylopyranosyl)-fl-D-glucopyranoside]-5-O-[fl-Dglucopyranoside].

~ ' ~ ' t ' ~

Pioment 2, 6 (demalonylated 2) and 4 (desinapylated 2) O

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The FAB mass spectra of 2, 6 and 4 gave their molecular ions at 1195, 1109 and 989m/z, respectively, in good agreement with the masses calculated for C56H59029, C53H57026 and C45H49025. Analysis of the 1H NMR spectra of 2, 6 and 4 revealed the presence of one molecule of pelargonidin, two molecules of glucose, each of one molecule of xylose in 2, 6 and 4, and furthermore the additional presence of each of one of sinapic, ferulic and malonic acids in 2, each of one of sinapic and ferulic acids in 6, and also each of one of ferulic and malonic acids in 4. The aromatic proton signals of pelargonidin and hydroxycinnamic acids of 2, 6 and 4 were assigned by I H - 1H COSY and negative N O E difference (DIFNOE) spectra as shown in Table 3. All 10 olefinic proton signals of sinapic and ferulic acids of 2, 6 and 4 had large coupling constants (J = 15.7 15.gHz), indicating these five hydroxycinnamic acids to have the trans configurations. The signals of the sugar moieties of 2 were observed in the region of 65.71-3.22 (Table 3). The signals of three anomeric protons appeared at 65.71 (d, J = 7.4 Hz, Glc A), 5.20 (d, J = 7.5 Hz, Glc B) and 5.22 (d, J = 7.5 Hz, xylose), and the assigned sugar protons had coupling constants J = 7.2-11.3 Hz. Therefore, these two glucose, and one xylose residues of 2 must be of the fl-Dglucopyranose and fl-D-xylopyranose forms. The H-2 of Glc A (64.02 t, J = 8.9 Hz) being shifted to a lower magnetic field was assigned and correlated to H-I of Glc A by the analysis of XH-XH COSY spectrum of 2, indicating that xylose was attached to the OH-2 of Glc A through a glycosyl bond and formed a sambubiose unit. Observed D I F N O E s between H-1 ofGlc A and H-4 of pelargonidin, and between H-1 of Glc B and H-6 of pelargonidin indicated that GIc A and Glc B are attached to the 3-OH and 5-OH of pelargonidin, respectively, through the glycosidic bonds. By the irradiation of H-4 of pelargonidin, NOEs were observed at H-a, -fl, -2 and -6

N . S A I T O et al.

1616

o 6 ~ o 6 ~

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1617

N. SAITOet al.

1618

of ferulic acid as well as protons of Glc A, indicating ferulic acid was attached to the 6-OH of Glc A through an ester bond in 2 (see Fig. 1). The characteristic five protons of 2 shifted in the lower magnetic field were assigned to methylenes of Glc A (t54.20 and 4.42) and Glc B (63.90 and 4.40), and also H-2 of xylose (64.70). Therefore, it was revealed that both the OH-6 of Glc A and B, and also the OH-2 of xylose were acylated by three acid residues in 2. On the other hand, 6 showed three protons shifted to lower magnetic field and 4 also exhibited four protons shifted, and these protons were assigned to methylene protons of Glc A (64.26 and 4.84) and H-2 of xylose (63.06) in 6 and to methylene protons of Glc A (64.33 and 4.41) and Glc B (63.80 and 3.87) by analysis of 1H - ~H COSY spectra of 6 and 4. In order to determine the attachment of malonic acid in 2 and 4, the demalonylated pigments of 2 and 4 were prepared by treatment of 2 and 4 with 1 N H C I - H 2 0 according to the previous procedure [5]. The demalonylated pigment of 2 was found to be identical with 6 on analysis by HPLC and from the spectral properties (Table 2). Thus, the structure of 6 was elucidated by analysis of the ~ H N M R spectra including ~H-~H COSY and D I F N O E spectra instead of that of the demalonylated pigment of 2. The proton chemical shifts of 6 were in good agreement with those of 2 without the proton signals of Glc B and malonic acid moieties (Table 3). By analysis of the IH-~H COSY spectrum of 6 the proton signals of methylene of Glc B were clearly shifted to the upper magnetic field from 64.40 and 3.90 (Glc B of 2) to 63.87 and 3.80, indicating that the malonyl group

was free from the OH-6 of Glc B in 6. Therefore, the structure of 2 is pelargonidin 3-O-[6-O-(trans-ferulyl)-2-

O-(2-O-(trans-sinapyl)-fl-D-xylopyranosyl)-fl-Dglucopyranoside]- 5-O-[6-O-(malon yl)-fl-D-glucopyranoside], which is a new pigment [6, 7]. We have already reported the structural determination of an analogous pigment, present in the purple-violet cultivars of this plant as a major anthocyanin, which is composed of cyanidin 3-sambubioside-5-glucoside, malonic acid, ferulic acid and sinapic acid [1]. The structure of 6 was determined as pelargonidin 3-0-[6-0-( trans-ferulyl)-2-O-

(2-O-(trans-sinapyl)-fl-D-xylopyranosyl)-fl-Dglucopyranoside]-5-O-[fl-D-glucopyranoside], which is a new pigment l-6, 7]. The IH N M R spectrum of 4 was superimposed on that of 2 except for the signals of the sinapyl and xylose moieties (Table 3). Also, the demalonylated pigment of 4 showed that the proton signals of methylene of Glc B were shifted to the upper magnetic field from 64.41 and 3.96 (Glc B of 4) to 63.94 and 3.78. Thus, the structure of 4 is pelargonidin 3-O-[6-O-(trans-ferulyl)-2-O-(fl-D-

xylopyranosyl)-fl-D-glucopyranoside]-5-O-[6-O-(malonyl)-fl-D-glucopyranoside], which is a new pigment 1-6, 7].

Pigment 3, 7 (demalonylated 3) and 5 (desinapylated 3) The FAB mass spectra of 3, 7 and 5 gave their molecular ions at 1165, 1079 and 959 m/z, respectively, in good agreement with the masses calculated for CssHsTOEa, C52H55025 and C44H47024. Analysis of the 1 H N M R spectra of 3, 7 and 5 revealed the presence of pelargonidin

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Fig. 1. Acylated pelargonidin glycosides 1-7 isolated from the red flowers of Matthiola incana. 1: R 1 = H , R 2 = O M e ; 2: R 1 = R 2 = O M e , 3: R I = O M e , R 2 = H; 4: R 2 = O M e , 5: R 2 = H; 6: R t = R 2 = O M e ; 7:

R~ = OMe, R 2 = H. Observed NOEs are indicated by arrows.

Acylated pelargonidin glucosides in Matthiola incana 3-sambubioside-5-glucoside and, furthermore, the additional presence of each of one molecule of sinapic and p-coumaric acids in 7 and also each of one molecule of p-coumaric and malonic acids in 5. The aromatic proton signals of pelargonidin, sinapic acid and p-coumaric acid of 3, 7 and 5 were assigned by ~H-~HCOSY and D I F N O E spectra as shown in Table 3. All 10 olefinic proton signals of sinapic and p-coumaric acids of 3, 7 and 5 had large coupling constants (J = 15.8-15.9 Hz), indicating these five hydroxycinnamic acids to have the trans configurations (Table 3). These three pigments were glucosylated with sambubiose at OH-3 and glucose at OH-5 of pelargonidin, and these three sugar units were confirmed to be of the fl-D-glucopyranose and fl-D-xylopyranose forms by the observed vicinal coupling constants (J = 7.2-11.3 Hz) of the proton signals. The characteristic five protons of 3 shifted in the lower magnetic field were assigned to methylenes of Glc A (64.26 and 4.35) and Glc B (64.05 and 4.38), and H-2 of xylose (64.69) by analysis of the ~H-1H COSY spectrum. Compound 7 showed three protons shifted in the lower magnetic field, and also 5 exhibited four protons shifted in the lower magnetic field. These protons were assigned to methylene protons of Glc A (64.30 and 4.36) and H-2 of xylose (64.71) in 7, and to methylene protons of Glc A (64.27 and 4.42) and Glc B (64.07 and 4.39) in 5 (Table 3). Therefore, the OH-6 groups of Glc A and B, and the OH-2 of xylose were acylated with sinapic, p-coumaric and malonic acids in 3, the OH-6 of Glc A and OH-2 of xylose were acylated with sinapic and p-coumaric acids in 7, and also the OH-6 groups of Glc A and Glc B were acylated with p-coumaric and malonic acids in 5. In order to determine the attachment of malonic acid in 3 and 5, the demalonylated pigments of 3 and 5 were prepared by the process described before for the demalonylated 2 and 4. The demalonylated pigment of 3 was found to be identical with 7 on analysis by HPLC and from the spectral properties (Table 2). Therefore, the attachment of the position of malonic acid was found to be the OH-6 of Glc B in 3. Furthermore, by irradiation at H-4 of pelargonidin, rather weak NOEs were observed at the signals of H-~, -/L and 3, -5 of p-coumaric acid, indicating that p-coumaric acid is bonded with Glc A at OH-6 of Glc A. Thus, pigment 3 is pelargonidin 3-0-[2-

O-(2-O-(trans-sinapyl)-fl-D-xylopyranosyl)-6-O-(trans-pcoumaryl)-fl-D-glucopyranoside]-5-O-[6-O-(malonyl)-flD-glucopyranoside], which is a new pigment I-6,7], and 7 is pelargonidin 3-O-[2-O-(2-O-(trans-sinapyl)-fl-D-

xylopyranosyl)-6-O-(trans-p-coumaryl)-fl-D-glucopyranoside]-5-O-[fl-D-glucopyranoside], also a new pigment [6, 7]. The tH NMR spectrum of 5 was superimposed on that of 3 except for the signals of the sinapyl moiety (Table 3). Also, the demalonylated pigment 5 gave its molecular ion at 873 m/z. Thus, the structure of 5 was assumed to be pelargonidin 3-O-[6-O-(trans-p-coumaryl)-2-O-(fl-D-

xylopyranosyl)-fl-D-glucopyranoside]-5-O-[ 6-O-(malonyl)-fl-D-glucopyranoside], which is a new pigment [6, 7].

1619

Pigment 1 The FAB mass spectrum of I gave its molecular ion at 1165 m/z, corresponding to C55Hs7028, pelargonidin 3diferulylsambubioside-5-malonylglucoside. The detailed structure of 1 was elucidated by 1H NMR measurement using the 1H-1H COSY spectrum. The 13 aromatic proton signals were observed, and assigned to be the protons of one molecule of pelargonidin and two molecules of ferulic acid in the low magnetic region as shown in Table 3. In the sugar region, the three anomeric proton signals of two glucosyl and one xylosyl residues were assigned at the low magnetic field (65.68 Glc A, 5.18 Glc B and 5.19, respectively) with large coupling constants (d = 7-8 Hz). Thus, these three sugar parts are of the fl-anomers. On the glucosyl residue (Glc A) the low-field shift of H-2 (63.98) indicated that the OH-2 of Gic A is glycosylated with xylose. The characteristic five protons shifted in the low field were assigned to be methylene protons of Glc A (64.20 and 4.39) and Glc B (63.88 and 4.37) and also H-2 of xylose (64.67), indicating that the OH-6 groups of Glc A and Glc B and also the OH-2 of xyiose were acylated with acids. In order to determine the position of the attachment of malonic acid in 1, the demalonylated pigment of 1 was prepared by the process described before for those of 2-5. The OH-6 of Glc B of this demalonyl 1 was found to be free from malonic acid by analysis of the tH-~H COSY spectrum, because of the methylene protons of Glc B being shifted from 63.88 and 4.37 (1) to a higher magnetic field (6 3.90-3.80). Therefore, the structure of 1 is pelargonidin 3-O-[2-O-(2-O-(trans-

ferulyl)-fl-D-xylopyranosyl)-6-O-(trans-ferulyl)-fl-Dglucopyranoside]- 5-O-[ 6-O-(malon yl)-fl-D-glucopyranoside], which is a new pigment [6, 7].

Other anthocyanins (8-10) These pigments were composed of pelargonidin 3-sambubioside-5-glucosides, confirmed by alkaline hydrolysis, and their spectral and chromatographic data are shown in Table 1. By alkaline hydrolysis, 8 produced ferulic acid, 9 produced sinapic, caffeic and malonic acids, and 10 produced caffeic and malonic acids. By HPLC analysis, 8 was identical with the demalonylated 4. From the analytical results and FAB mass spectral data, the structures of 8-10 were tentatively estimated to be pelargonidin 3-ferulylsambubioside-5-glucoside as 8, pelargonidin 3-sinapylcaffeylsambubioside-5-malonylglucoside as 9 and pelargonidin 3-caffeylsambubioside-5-malonylglucoside as 10. As shown in Table 1, there were three pelargonidin glycoside types to produce the reddish flower colours in these cultivars. The first type was mainly composed of triacylated anthocyanins such as 2 and 3, and exhibited the red-purple or salmon-pink flower colour. The second type was composed of diacylated anthocyanins such as 4 and 5, and showed the same flower colours. However, the last one was composed of nonacylated anthocyanins such as 11 and 12, and exhibited the copper flower colour. The amounts of hydroxycinnamic residues in the pigments might be considered to

1620

N. SAITOet al.

assist in the production of the bluish flower colour in these cultivars [1-3].

EXPERIMENTAL

Plant material. Red-purple colour flowers of 10 cvs of M. incana: 'Momochidori', 'Kanchidori', 'Boda Rose', 'Higan Oh', 'Benikou', 'Aki no Hana', 'Souen', 'Kurokawa Pink', 'Spray Salmon Pink' and 'Kurokawa Rose'; salmon-pink colour flowers of 10 cvs of M. incana: 'Toen2', 'Hatsuzakura', 'Spray Pink', 'Awazakura', 'Spray Light Pink', 'Souka', 'Sakura no Mai', 'Aki no Yume', 'Banrei' and 'Spray Cherry Pink'; apricot colour flowers of 3 cvs of M. incana: 'Yunami', 'Yu no Mai' and 'Kurokawa Apricot'; and copper colour flowers of 3 cvs of M. incana: 'Suzuka no Niji', 'Suzuka no Akatsuki' and 'Christmas Apricot', collected from plants at the farm of Mr S. Boda (in spring 1992) and Mr H. Kurokawa (in spring 1993), Tateyama, Chiba, Japan. Fresh flowers of these cvs were dried overnight at 40 °. Distribution of anthocyanins in the flowers of 26 cultivars. Dried petal (ca 0.02 g) of each cv. was extracted with 50% MeOH-solvent containing 1.5% HaPO4. TLC and HPLC of these extracts were carried out [1-1. TLC solvents used were BAW (n-BuOH-HOAc-H20, 4:1:5), BuHCI (n-BuOH-2N HCI, 1:1), 1% HC1 and AHW (HOAc-HC1-H20, 15:3:82). Quantitative anal. of anthocyanins was performed by HPLC on a Waters CIa (4.6 ~bx 250mm) column at 40 ° with a flow rate of 1 ml min- 1 and monitoring at 530 nm. Solvent systems used were; linear gradient elution for 30 min from 40 to 85% solvent B (1.5% H3PO4, 20% HOAc, 25% MeCN in H20) in solvent A (1.5% HaPO4 in H20). Isolation of anthocyanins. Mixed dried red-purple flowers (200 g) were extracted with MAW (MeOHHOAc-H20, 9:1:10) (10 i) at room temp. for 6 hr. The extract was concd to 500 ml. The red concd extract was purified by Diaion HP-20 CC, PC and HPLC as described previously [1, 8, 9]. Solvents used were BAW, 5% HOAc-MeOH, 3 and 15% HOAc for CC and PC. Prep. HPLC was run on a Waters C~a (19~b x 150 mm) column at 40 ° with a flow rate of 4 ml min- ~ and monitoring at 530 nm. Solvent system used were; linear gradient elution for 35 min from 40 to 85% solvent B in solvent A. The pigment frs were evapd in vacuo to dryness. The evapn residues were dissolved in a small vol. of I% H O A c MeOH followed by addition of excess Et20 and then drying to give seven pigment powders; 1, ca 20 mg; 2, ca 40 mg; 3, ca 40 mg; 4, ca 20 mg; 5, ca 20 mg; 6, ca 25 mg

and 7 ca 20 mg. Pelargonidin 3-glucoside and pelargonidin 3-sambubioside, 5-glucoside were obtained from the brown-red flowers of M. incana in a similar process to acylated anthocyanins. Standard analysis. Pigment identifications were carried out by a standard procedure involving H20 2 oxidation, deacylation with alkali and hydrolysis with acid [1, 4, 7, 10]. NMR spectra were measured in CF3CO2DDMSO-d6 (1:9) and recorded at 400 MHz for 1 H NMR. Chemical shifts are given in 6 values relative to TMS. Mass spectra were recorded to obtain the positive mode with a magic bullet matrix and negative mode with a glycerol matrix. Demalonylation of anthocyanins. To determine structures of malonylated anthocyanins in detail, each malonylated anthocyanin was dissolved in 1% HCI-H20 solution and kept for 5-7 days at room temp. The demalonyl anthocyanins were then sepd by prep. HPLC [5, 10]. Acylated sugars. Five pigments (1-5) were dissolved in H20 and oxidized with H202 [1, 5-1.The resulting solns were chromatographed in BAW and bands containing the acylated sugars were cut out, eluted and purified by TLC.

REFERENCES

1. Saito, N., Tatsuzawa, F., Nishiyama, A., Yokoi, M., Shigihara, A. and Honda, T. (1995) Phytochemistry 38, 1027. 2. Seyffert, W. (1960) Z. Pflanzenziicht 44, 4. 3. Harborne, J. B. (1964) Phytochemistry 3, 151. 4. Harborne, J. B. (1967) Comparative Biochemistry of Flavonoids. Academic Press, London. 5. Saito, N., Toki, K., Uesato, K., Shigihara, A. and Honda, T. (1994) Phytochemistry 37, 245. 6. Harborne, J. B. and Grayer, R. J. (1988) in The Flavonoids, Advances in Research Since 1980 (Harborne, J. B., ed.), p. 1. Chapman and Hall, London. 7. Strack, D. and Wray, V. (1994) in The Flavonoids, Advances in Research Since 1986 (Harborne, J. B., ed.), p. 1. Chapman and Hall, London. 8. Lu, T. S., Saito, N., Yokoi, M., Shigihara, A. and Honda, T. (1991) Phytochemistry 30, 2387. 9. Lu, T. S., Saito, N., Yokoi, M., Shigihara, A. and Honda, T. (1992) Phytochemistry 31, 289. 10. Saito, N. and Harborne, J. B. (1992) Phytochemistry 31, 3009.