Variations among Anthocyanins in the Floral Organs of Seven Cultivars of Hyacinthus orientalis

J Plant Physiol. Vol. 155. pp. 285-287 (1999)

JOUR.ALOF

Plane Pb,s.olo.,

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© 1999 URBAN &. FISCHER

I Short Communication

I

Variations among Anthocyanins in the Floral Organs of Seven Cultivars of Hyacinthus orientalis KEIZO HOSOKAWA

Tsukuba Medicinal Plant Research Station, National Institute of Health Science, 1 Hachimandai, Tsukuba, Ibaraki 305, Japan Received May 5, 1998· Accepted December 8,1998

Summary

The accumulation of seven acylated anthocyanins in four cultivars of Hyacinthus orientalis with blue flowers and of nine acylated anthocyan ins in three cultivars with red or pink flowers was demonstrated by high-performance liquid chromatography. A survey of the anthocyanins in the floral organs (perianth, anthers, ovaries) of these cultivars revealed that the dominant anthocyanin was delphinidin 3-0-(6-0tram-Jrcoumaroyl-j3-D-glucoside)-5-0-(6-O-malonyl-j3-D- glucoside) (4) in the cultivars with blue flowers and cyanidin 3- 0-(6- 0-trans-Jrcoumaroyl-j3-D-glucoside)-5- 0-(6- O-malonyl-j3-D-glucoside) (6) or pelargonidin 3-0-(6-0-tram-Jrcoumaroyl-j3-D-glucoside)-5-0-(6-O-malonyl-j3- D-glucoside) (7) in cultivars with red or pink flowers. Different patterns of anthocyanin were observed in each floral organ. In the cultivars with red or pink flowers, there were remarkable differences in levels of 6 and 7 between the perianth and ovaries and the anthers. By contrast, in two of the four cultivars with blue flowers, little difference between organs was found in relative levels of 4 and 6. Key words: Hyacinthus orientalis, Anthocyanin pattern, Floralorgam.

Introduction

The anthocyan ins in the perianth of Hyacinthus orientalis, which belongs to Liliaceae, have not been thoroughly analyzed, even though this species has been cultivated for a long time around the world. We investigated anthocyanins in the perianth of several cultivars of H orientalis (Hosokawa et al., 1995 a, b, c), and we reported the relative levels of various anthocyanins in the perianth of cv. Delft Blue, which has blue flowers (Hosokawa et al., 1996 a), and in cv. Hollyhock, which has red flowers (Hosokawa et al., 1996 b). In cv. Delft Blue, the dominant anthocyanin is delphinidin 3-0-(6-0trans-p-coumaroyl-j3 -D-glucoside) -5-0- (6-0-malonyl-j3 -Dglucoside) (4), with lower levels of delphinidin 3-0-(6-0-cisp- coumaroyl-j3 -D-glucoside)-5- 0-(6- O-malonyl-j3-D-glucoside) (1), delphinidin 3-0-(6-O-caffeoyl-j3-D-glucoside)-50-(6-O-malonyl-j3-D-glucoside) (2), delphinidin 3-0-(6-0-

trans- Jrcoumaroyl- 13 -D-glucoside)-5-0- 13 -D-glucoside (3), petunidin 3- 0-(6- O-trans-Jrcoumaroyl-j3- D-glucoside)-5- 0(6-0-malonyl-j3-D-glucoside) (5), cyanidin 3-0-(6-0-tramJrcoumaroyl-j3 -D-glucoside)-5- 0-(6- O-malonyl-j3-D-glucoside) (6) and pelargonidin 3-0-(6-0-tram-Jrcoumaroyl-j3-Dglucoside)-5-0-(6-O-malonyl-j3-D-glucoside) (7). In cv. Hollyhock, the dominant anthocyanin is pelargonidin 3- 0-(6- 0trans-p-coumaroyl-j3 -D-glucoside)-5-0-(6-0-malonyl-j3 -Dglucoside) (7), with lower levels of pelargonidin 3-0-j3-D-glucoside-5- 0-(6- O-malonyl-j3-D-glucoside) (8), pelargonidin 3- 0-(6- O-cis-Jrcoumaroyl-j3- D-glucoside)-5- 0-(6- O-malonyl -j3-D-glucoside) (9), pelargonidin 3-0-(6-O-caffeoyl-j3-Dglucoside)-5-0-(6-0-malonyl-j3-D-glucoside) (10), cyanidin 3-0-(6-0-trans-Jrcoumaroyl-j3-D-glucoside)-5-0- j3-D-glucoside (11), cyanidin-3- 0 -(6- O-tram-p -coumaroyl-j3 -Dglucoside)-5- 0-( 6- O-malonyl-j3-D-glucoside) (6), pelargonidin 3- 0-(6- O-tram-p-coumaroyl-j3- D-glucoside)-5- 0-13- D0176-1617/99/155/285 $ 12.00/0

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KEIZO HOSOKAWA

glucoside (12), pelargonidin 3-0-(6-0-trans-p-coumaroyl- /3D-glucoside)-5-0-(4-O-malonyl-/3-D-glucoside) (13) and pelargonidin 3-0-(6-O-feruloyl-/3-D-glucoside)-5-0-(6-O-malonyl-/3-D-glucoside) (14). To continue the analysis of patterns of anthocyanins in the perianth, in this study, the profiles of anthocyan ins in the perianth of the other cultivars were determined by high-performance liquid chromatography (HPLC). A survey was also made of patterns of anthocyanins in various floral organs (perianth, anthers and ovaries) to provide insight into the organspecific expression and biosynthesis of anthocyanins.

Materials and Methods Plant materials

Bulbs (ca. 6 cm in diameter) of H. orientalis cv. Delft Blue, cv. Blue Orchid, cv. King Codro and cv. Ostara, which are blue-flower cuItivars (BFCs); cv. Hollyhock and cv. Jan Bos, which are redflower cultivars (RFCs); and cv. Lady Derby, which is a pink-flower cuItivar (PFC) were obtained from Heiwaen Co. (Tenri, Nara, Japan) in September 1991 and planted in an experimental field at Chigasaki in Kanagawa, Japan. Floral organs (perianth, anthers and ovaries) were harvested at anthesis of flowers during the last ten days of March and the first ten days of April 1992. They were freeze-dried for subsequent analysis. Extraction and identification ofacylated anthocyanins in each organ

Each freeze-dried organ (perianth, anthers and ovaries) of each cultivar was extracted three times with a mixture of ethanol, water and acetic acid (lO: 9: 1, v/v) at 4°C, except in the case of ovaries of the double-flowered cv. Hollyhock since ovaries are absent in this cultivar. Extracts were analyzed by HPLC. Anthocyanins were separated on a Chromatorex-ODS column (5 ~m; 4.6 mm i.d. x 150 mm; Fuji Silysia Chern. Co., Aichi, Japan) by elution with a mixture of acetonitrile, acetic acid, water and trifluoroacetic acid (9: 11.25: 79.25:0.5, v/v) at a flow rate of 1.0 mLmin- l . Elution was monitored at 533 nm for BFCs and at 5lO nm for RFCs and the PFC. Results and Discussion To date, it has been generally accepted that the main anthocyanin pigments of hyacinth are pelargonidin, cyanidin and delphinidin pigments, namely, the 3-(p-coumaroylglucoside)-5-glucosides (Harborne 1967). However, the dominant anthocyanins were 4 in the perianths of BFCs and 7 in those of RFCs and the PFC (Tables 1 and 2). The dominant hyacinth pigments were pelargonidin, cyanidin and delphinidin pigments that were 3-0-(trans-p-coumaroyl-/3-D-glucoside)-5-0-(malonyl-/3-D-glucoside) derivatives. It is possible that malonic acid derivatives of anthocyanins were previously overlooked because of their lability during extraction procedures with solvents that contained HC!. Indeed, van Sumere et a!. (1985) noted that extraction with solvents that contained HCl resulted in degradation of pigment, the extract of which was related to the concentration ofHC!. In the case of anthocyanins in the perianth, similar patterns were obtained from BFCs, RFCs and the PFC (Tables 1 and 2). However, there were slight differences in patterns be-

Table 1: Relative levels of anthocyanins from the floral organs of four cultivars of H. orientalis with blue flowers, as determined by HPLC.

Cultivar and organ

Relative level of anthocyanin (area % at 533 nm) 2

3

4

5

6

7

Delft Blue perianth anthers ovarIes

5.4 1.3 0.8

0.2 0.1 0.2

2.1 8.2 2.1

83.4 67.4 90.1

5.6 2.5 3.1

2.1 18.4 3.1

1.2 2.1 0.6

Blue Orchid perianth anthers ovarIes

6.8 2.3 5.8

0.3 0.2 0.2

2.7 6.0 2.6

78.4 75.5 87.1

7.5 2.6 2.2

2.1 11.3 2.1

2.2 2.1 nd l

KingCodro perianth anthers ovarIes

6.0 2.5 2.9

0.2 0.2 0.4

3.6 7.9 4.1

77.6 80.6 82.2

7.0 3.5 4.7

3.7 3.5 4.2

1.9 1.8 1.5

6.1

0.3 0.2 0.2

3.1 9.9 3.1

82.3 80.5 87.4

4.5 3.2 4.0

2.2 2.9 1.5

1.5 2.2 0.6

Ostara perianth anthers ovarIes I

1.1

3.2

nd: Not detected.

Table 2: Relative levels of anthocyanins from the floral organs of three cuItivars of H. orientalis with red or pink flowers, as determined by HPLC.

CuItivar and organ

Relative level of anthocanin (area % at 510 nm) 8

9

Hollyhock perianth anthers . I ovarIes

3.0 0.6 2.0 6.6

Jan Bos perianth anthers ovarIes

2.0

Lady Derby perianth anthers ovarIes I

2

10

11

1.4 6.7 nd 2 2.4

6

12

2.3 57.1

6.5 5.4

0.2 0.7 1.8 nd 4.7 0.3 0.2

4.0 2.4 4.8 77.2 2.0 12.3

10.0 2.2 5.9

0.4 0.8 1.2 nd 12.6 0.2 nd

8.9 2.5 4.9 80.9 1.0 9.3

1.1

3.5 1.1

13

7

14

1.0 73.7 4.8 nd 24.1 2.4

nd nd nd

74.3 6.4 12.9 nd 70.5 4.1

7.1 nd 1.0 nd 4.0 nd

76.2 0.6 10.6 0.3 68.9 4.0

Ovaries are absent in cv. Hollyhock, which is double-flowered. nd: Not detected.

tween the perianth and ovaries and the anthers of cv. Delft Blue and cv. Blue Orchid. The relative level of 6, as compared to that of 4, was somewhat higher in anthers than that in the perianth and ovaries. In the case of cv. King Codro and cv. Ostara, however, the relative levels of respective anthocyanins were almost the same in all four floral organs. In the various floral organs of RFCs and PFC, the main difference was observed in relative levels of 6 and 7. Anthocyanin 7 was the dominant anthocyanin in the perianth and ovaries, while 6 was dominant in anthers. Thus, the dominant anthocyanin, 6 or 7, differed between the perianth and

Anthocyanins of Hyacinthus orientalis ovaries and the anthers. The difference was associated with levels of anthocyanidins, namely, pelargonidin and cyanidin. It is likely that the difference is related to the step at which the B-ring of anthocyanidin is hydroxylated during the biosynthesis of anthocyanin and might be due to differences in activity of flavonoid 3'-hydroxylase or flavonoid 3', 5'hydroxylase in the various floral organs. There is a report of variation in levels of flavonol among different organs of Vigna mungo and these variations were explained in terms of flavonoid 3'-hydroxylase in the tissues (Mato and Ishikura, 1993). This is the first report of patterns of anthocyanins in the floral organs of a large number of cultivars of H. orientalis. It is evident that there are significant differences in anthocyanin patterns among the floral organs of RFCs and PFC but not in those of BFCs. It is postulated that the anthocyanin pattern in each floral organ changes depending on whether the dominant anthocyanidin is pelargonidin and/or cyanidin. The observation that the anthocyanin profile of each floral organ is quite distinct is of interest. Further surveys of the an-

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thocyanin patterns in the floral organs of other cultivars must be made before firm conclusions can be drawn. References HARBORNE, J. B.: Flavonoids of the Monocotyledoneae. In: Comparative Biochemistty of the Flavonoids. pp. 233-249. Academic Press, London (1967). HOSOKAWA, K., Y. FUKUNAGA, E. FUKUSHI, and J. KAWABATA: Phytochemistry 38, 1293-1298 (1995 a). - - - - Phytochemistry 39, 1437-1441 (1995 b). - - - - Phytochemistry 40, 567- 571 (1995 c). - - - - Phytochemistry 41,1531-1533 (1996 a). - - - - Phytochemistry 42, 671-672 (1996 b). MATO, M. and N. ISHIKURA: J. Plant Physiol. 142,647-650 (1993). VAN SUMERE, c. E, K. V. CASTEELE, R. DE LoosE, and J. HEURSEL: The Biochemistry of Plant Phenolics. In: VAN SUMERE, C. E and P. L. LEA (eds.): Annual Proceedings of the Phytochemical Society of Europe, vol. 1, pp. 17-43. Clarendon Press, Oxford (1985).