Bull. Natl. Mus. Nat. Sci., Ser. B, 36(2), pp. 61–64, May 22, 2010

Luteolinidin 5-O-glucoside from Azolla as a Stable Taxonomic MarkerTsukasa Iwashina1,*, Junichi Kitajima2 and Sadamu Matsumoto1
1

Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, 305–0005 Japan
2
Laboratory of Pharmacognosy, Showa Pharmaceutical University,
Higashi-Tamagawagakuen 3, Machida, Tokyo, 194–8543 Japan* E-mail: iwashina@kahaku.go.jp(Received 23 February 2010; accepted 24 March 2010)

Abstract A red pigment was isolated from the whole plants of Azolla cristata and identified as
3-deoxyanthocyanin, luteolinidin 5-O-glucoside by LC-MS, and 1H and 13C NMR spectra. It wasalso detected from two specimens of Azolla pinnata, which were collected in New Caledonia andAustralia, by HPLC survey for the first time. Five Azolla species, including two species whichwere surveyed in this experiment, were surveyed for 3-deoxyanthocyanins until now, and luteolinidin 5-O-glucoside has been found in all species. Thus, 3-deoxyanthocyanin, luteolinidin 5-O-glucoside, was recognized as a stable taxonomic chemical marker of the genus Azolla.
Key words : Azolla, chemotaxonomy, 3-deoxyanthocyanin, luteolinidin 5-O-glucoside, NMR.

Introduction
The genus Azolla (Salviniaceae) consists of 6or 7 species and is especially distributed from thetropical to temperate zone in the world (Iwatsuki,1992; Mabberley, 1997). Though the wholeplants are usually reddish green, they change tobright red in autumn and winter season. The red
pigments have been surveyed in a few species.
Two 3-deoxyanthocyanins have been isolated
from Azolla filiculoides Lam. and A. caroliniana
Willd. and partially characterized as luteolinidin
and apigeninidin glycosides (Pieterse et al.,1977). As other flavonoids, the presence of
proanthocyanidins has also been reported(Markham, 1988). A 3-deoxyanthocyanin has
been isolated from Azolla mexicana Presl andidentified as luteolinidin 5-O-glucoside by paper
chromatography and UV-visible spectral survey(Holst, 1977). 3-Deoxyanthocyanin in Azolla imbricata Nakai and A. japonica Franch et Savat.
has been surveyed by Ishikura (1982) and luteolinidin 5-O-glucoside was isolated together withseveral phenolic compounds, chlorogenic acid,aesculetin, caffeic acid 3,4-diglucoside, 6-(3glucosylcaffeoyl)-aesculetin, p-coumaroyl glucosyl ester, and 1,6-diesters of caffeic and chlorogenic acids. Thus, it has been shown that luteolinidin 5-O-glucoside is a red pigment in A.
mexicana, A. imbricata and A. japonica (Holst,1977; Ishikura, 1982). However, a major pigment
in A. cristata has not been characterized. In thisexperiment, the red pigment in A. cristata wasisolated and completely identified by LC-MS and1
H and 13C NMR. Moreover, the presence of luteolinidin 5-O-glucoside in A. pinnata R. Br.,
which is not surveyed for 3-deoxyanthocyanin,
was shown by HPLC analysis.
Materials and Methods
Plant materials
Azolla cristata Kaulf. is natively growing inTsukuba Botanical Garden, National Museum of
Nature and Science, Tsukuba, Japan (TNS9546219,TNS9546462-9546464). The species was identified by one of the authors (S. Matsumoto). Azolla
pinnata R. Br. was collected in Mts. Kogis, north62

Tsukasa Iwashina et al.

of Noumea, New Caledonia, 480 m alt., 4 Nov.
1997 (TNS9508992) by S. Matsumoto, and near
Brisbane, Australia, 8 Aug. 1997 (TNS9527088)
by Takehisa Nakamura. Voucher specimens weredeposited in the herbarium of National Museumof Nature and Science, Tokyo (TNS).
Extraction and isolation of anthocyanin
Fresh whole plants (ca. 1 kg) of A. cristatawere extracted with HCOOH/MeOH (8 : 92). The
concentrated extracts were applied to preparative
paper chromatography using solvent systems:
BAW (n-BuOH/HOAc/H2O4 : 1 : 5, upperphase) and 15% HOAc. The anthocyanin bands
was eluted with HCOOH/MeOH (8 : 92) and purified by Sephadex LH-20 column chromatography using solvent system: MeOH/H2O/HCOOH(70 : 25 : 5).
Dry specimens of A. pinnata were extractedwith HCOOH/MeOH (8 : 92). After concentration, the extracts were surveyed by HPLC foranthocyanin identification.
High performance liquid chromatography(HPLC)
HPLC was performed with Shimadzu HPLC
systems using a Senshu Pak PEGASIL ODS column (I.D. 6.0150 mm, Senshu Scientific Co.
Ltd.), at a flow-rate of 1.0 ml min1. Detection
was 500 nm and eluent was H3PO4/HOAc/
MeCN/H2O (3 : 8 : 6 : 83).
Liquid chromatograph-mass spectra (LC-MS)
LC-MS was performed with Shimadzu LCMS systems using Senshu Pak PEGASIL ODS
column (I.D. 2.0150 mm, Senshu Scientific Co.
Ltd.), at a flow-rate of 0.1 ml min1, ESI 4.5 kV
and ESI 3.5 kV, 250°C. The eluent was
MeCN/H2O/HCOOH (3 : 92 : 5).
Identification of anthocyanin
The isolated anthocyanin was identified byTLC, HPLC, UV-visible spectral survey, LC-MS,
and 1H and 13C NMR spectra. Their data are asfollows.
TLC: Rf 0.18 (BAW), 0.06 (1%HCl); visi-ble—reddish orange, UV—dark purple. HPLC:tR 8.34 min. UV-visible spectra: l max
0.1%MeOH-HCl 277, 496 nm; AlCl3 276sh,512, 545sh nm; E440/Emax 20%. LC-MS: m/z 433[M] (luteolinidin1 mol glucose), m/z 271 [M162] (luteolinidin). 1H NMR (600 MHz,DMSO-d6TFA): d 9.29 (1H, d, J8.8 Hz, H4), 8.35 (1H, d, J8.9 Hz, H-3), 8.07 (1H, dd,J2.1 and 8.7 Hz, H-6), 7.94 (1H, d, J1.3 Hz,H-2), 7.27 (1H, s, H-8), 7.18 (1H, d, J8.5 Hz,H-5), 7.07 (1H, d, J1.3 Hz, H-6), 5.19 (1H, d,J7.7 Hz, glucosyl H-1), 3.81 (1H, d, J10.5
Hz, glucosyl H-6a), 3.61 (1H, dd, J5.3 and12.0 Hz, glucosyl H-6b), 3.56 (1H, m, glucosylH-5), 3.50 (1H, m, glucosyl H-2), 3.46 (1H, m,glucosyl H-3), 3.34 (1H, m, glucosyl H-4). 13C
NMR (150 MHz, DMSO-d6TFA): (luteolinidin) d 171.4 (C-2), 111.4 (C-3), 148.1 (C-4),
156.5 (C-5), 103.7 (C-6), 169.9 (C-7), 96.9 (C8), 158.0 (C-9), 112.0 (C-10), 120.2 (C-1),115.7 (C-2), 147.2 (C-3), 155.4 (C-4), 117.3(C-5), 125.0 (C-6); (glucose) d 101.4 (C-1),
73.3 (C-2), 76.0 (C-3), 69.7 (C-4), 77.6 (C-5),
60.5 (C-6).
Results
A reddish orange pigment was obtained fromthe whole plants of Azolla cristata. The absorption maxima of the compound were 495 (Band I)
and 277 (Band II) nm, showing that the pigmentis 3-deoxyanthocyanin. In addition to AlCl3,
Band I bathochromically shifted, indicating thepresence of B-ring ortho-dihydroxyl groups.
Since the molecular ion peak, m/z 433 [M] anda fragment ion peak, m/z 271 [M-162] appeared
by LC-MS, it was shown that the original glycoside is tetrahydroxyanthocyanin monohexoside.
The presence of seven aromatic protons (H-3, H4, H-6, H-8, H-2, H-5 and H-6) was shown by1
H NMR spectra. The coupling constant of theglucosyl anomeric proton signal (d 5.19) wasJ7.7 Hz, showing that the glucose is b -linkage
to luteolinidin nucleus. Since HMBC correlation
between glucosyl anomeric proton signal at d5.19 and C-5 carbon signal at d 156.5 was recog-

Luteolinidin 5-glucoside from Azolla

Fig. 1.

Luteolinidin 5-O-glucoside.

nized, it was indicated that the glucose is attached to 5-position of luteolinidin. Thus, the 3deoxyanthocyanin was identified as luteolinidin
5-O-b -glucopyranoside (Fig. 1).
Only one peak appeared by HPLC survey of A.
pinnata extract and its retention time and UV
spectral properties agreed with those of luteolinidin 5-O-glucoside from A. cristata. The presenceof luteolinidin 5-O-glucoside in A. cristata andA. pinnata was reported in this paper for the firsttime.
Discussion
Almost anthocyanins of flowering plants arecommon anthocyanins, i.e. 3-oxyanthocyaninssuch as pelargonidin, cyanidin and delphinidin(Iwashina, 2000). On the other hand, the distribution of 3-deoxyanthocyanins such as apigeninidin
and luteolinidin is extremely limited in flowering
plants. They have been reported as flower pigments of bird-pollinated species in the family
Gesneriaceae, e.g. Alloplectus vittatus, Gesneria
cuneifolia,
Rechsteineria
cardinalis,R.
macrorhiza, Chrysothemis pulchella andHypocyrta glabra (Harborne, 1966, 1967). Apigeninidin and luteolinidin glycosides were also
isolated from Sorghum spp. (Gramineae) as phytoalexins (Stafford, 1968; Nip and Burns, 1969,
1971; Nicholson et al., 1987, 1988; Hipskind
et al., 1990; Snyder et al., 1991; Kouda-Bonafoset al., 1994; Pale et al., 1997).
Recently, the biosynthetic pathway of 3-de-63

oxyanthocyanins was shown that lack of F3H
activity allows action of the DFR/FNR on substrates and production of flavan-4-ols, and is thenlikely converted to 3-deoxyanthocyanins throughthe action of the ANS and subsequent glucosylation, by the examination of Sinningia cardinalis(Gesneriaceae) which produces luteolinidin andapigeninidin 5-O-glucosides (Winefield et al.,2005).
The occurrence of 3-deoxyanthocyanins has
been reported from comparatively many species
in ferns and bryophytes than in flowering plants.
Luteolinidin 5-O-glucoside and 5-O-diglucosidewere isolated from musci, Bryum spp. (Bendzand Mårtensson, 1961, 1963; Bendz et al., 1962).
In ferns, luteolinidin 5-O-glucoside and otherglycosides were found in Adiantum veitchianum
and A. pedatum cv. “Klondyke”, Dryopteris
erythrosora, Blechnum brasiliense var. corcovadense, and three Pteris species, P. longipinnula,
P. quadriauria and P. vittata (Harborne, 1966).
Six luteolinidin and six apigeninidin glycosides,
and acetylated luteolinidin 5-O-laminaribiosidewere isolated from two Blechnum species, B. procerum and B. novae-zelandicae (Crowden andJarman, 1974; Swinny, 2001). Though their distribution is sporadic in ferns and bryophytes, theoccurrence of 3-deoxyanthocyanins, especiallyluteolinidin 5-O-glucoside, in the genus Azolla islikely stable taxonomic chemical marker.
References
Bendz, G. and Mårtensson, O. 1961. Moss anthocyanins.
Acta Chemica Scandinavica 15: 1185.
Bendz, G., Mårtensson, O. and Terenius, L. 1962. Mosspigments I. The anthocyanins of Bryum cryophilum O.
Mårt. Acta Chemica Scandinavica 16: 1183–1190.
Bendz, G. and Mårtensson, O. 1963. Moss pigments II.
The anthocyanins of Bryum rutilans Brid. and Bryum
weigelii Spreng. Acta Chemica Scandinavica 17: 266.
Crowden, R. K. and Jarman, S. J. 1974. 3-Deoxyanthocyanins from the fern Blechnum procerum. Phytochemistry 13: 1947–1948.
Harborne, J. B. 1966. Comparative biochemistry offlavonoids-II. 3-Desoxyanthocyanins and their systematic distribution in ferns and gesnerads. Phytochemistry
5: 589–600.

64

Tsukasa Iwashina et al.

Harborne, J. B. 1967. Comparative biochemistry offlavonoids-VI. Flavonoid patterns in the Bignoniaceae
and the Gesneriaceae. Phytochemistry 6: 1643–1651.
Hipskind, J. D., Hanau, R., Leite, B. and Nicholson, R. L.
1990. Phytoalexin accumulation in sorghum: identification of an apigeninidin acyl ester. Physiological andMolecular Plant Pathology 36: 381–396.
Holst, R. W. 1977. Anthocyanins of Azolla. American
Fern Journal 67: 99–100.
Ishikura, N. 1982. 3-Desoxyanthocyanin and other phenolics in the water fern Azolla. The Botanical Magazine,
Tokyo 95: 303–308.
Iwashina, T. 2000. The structure and distribution of theflavonoids in plants. Journal of Plant Research 113:
287–299.
Iwatsuki, K. 1992. Ferns and Fern Allies of Japan. pp.
285. Heibonsha, Tokyo (in Japanese).
Kouda-Bonafos, M., Czyzewska, E., Nacro, M. andOehlschlager, A. C. 1994. Isolation of apigeninidinfrom leaf sheaths of Sorghum caudatum. Journal ofChemical Ecology 20: 2123–2125.
Pale, E., Kouda-Bonafos, M., Nacro, M., Vanhaelen, M.,
Vanhaelen-Fastré, R. and Ottinger, R. 1997. 7-OMethyl-apigeninidin, an anthocyanidin from Sorghum
caudatum. Phytochemistry 45: 1091–1092.
Pieterse, A. H., Delange, L. and Vanvliet, J. P. 1977. A
comparative study of Azolla in the Netherlands. Acta
Botanica Neerlandica 26: 433–449.
Mabberley, D. J. 1997. The Plant-Book 2nd. ed. pp. 70.
Cambridge University Press, Cambridge.
Markham, K. R. 1988. Distribution of flavonoids in thelower plants and its evolutionary significance. In: Harborne, J. B. (ed.), The Flavonoids: Advances in Research since 1980. pp. 427–468. Chapman and Hall,London.
Nicholson, R. L., Kollipara, S. S., Vincent, J. R., Lyons, P.
C. and Cadena-Gomez, G. 1987. Phytoalexin synthesisby the sorghum mesocotyl in response to infection bypathogenic and nonpathogenic fungi. Proceedings ofthe National Academy of Sciences of the United Statesof America 84: 5520–5524.
Nicholson, R. L., Jamil, F. F., Snyder, B. A., Lue, W. L.
and Hipskind, J. 1988. Phytoalexin synthesis in the juvenile sorghum leaf. Physiological and Molecular Plant
Pathology 33: 271–278.
Nip, W. K. and Burns, E. E. 1969. Pigment characterization in grain sorghum I. Red varieties. Cereal Chemistry 46: 490–495.
Nip, W. K. and Burns, E. E. 1971. Pigment characterization in grain sorghum II. White varieties. Cereal Chemistry 48: 74–80.
Snyder, B. A., Leite, B., Hipskind, J., Butler, L. G. andNicholson, R. L. 1991. Accumulation of sorghum phytoalexins induced by Colletotrichum graminicola at theinfection site. Physiological and Molecular Plant
Pathology 39: 463–470.
Stafford, H. A. 1968. Relationships between the development of adventitious roots and the biosynthesis of anthocyanins in first internodes of sorghum. Plant Physiology 43: 318–326.
Swinny, E. E., Bloor, S. J. and Wong, H. 2000. 1H and 13C
NMR assignments for the 3-deoxyanthocyanins, luteolinidin 5-glucoside and apigeninidin 5-glucoside.
Magnetic Resonance in Chemistry 38: 1031–1033.
Swinny, E. E. 2001. A novel acetylated 3-deoxyanthocyanidin laminaribioside from the fern Blechnum
novae-zelandiae. Zeitschrift für Naturforshung 56c:177–180.
Winefield, C. S., Lewis, D. H., Swinny, E. E., Zhang, H.,
Arathoon, H. S., Fischer, T. C., Halbwirth, H., Stich,K., Gosch, C., Forkmann, G. and Davies, K. M. 2005.
Investigation of the biosynthesis of 3-deoxyanthocyanins in Sinningia cardinalis. Physiologia Plantarum
124: 419–430.