Bull. Natl. Mus. Nat. Sci., Ser. B, 45(4), pp. 165–168, November 22, 2019

Reconstruction of Flower Color of Amherstia nobilis
by in vitro Examination
Takayuki Mizuno1, Nobuyuki Tanaka1, Mu Mu Aung2,Tomohisa Yukawa1 and Tsukasa Iwashina1,*1

Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan2
Forest Research Institute, Forest Department, Ministry of Natural Resources andEnvironmental Conservation, Yezin, Nay Pyi Taw, Myanmar* E-mail: iwashina@kahaku.go.jp(Received 17 July 2019; accepted 25 September 2019)

Abstract Flower anthocyanin of Amherstia nobilis has been reported as pelargonidin 3-O-pentoside. However, they were more recently identiﬁed as cyanidin 3-O-glucoside and 3-O-xyloside,and peonidin 3-O-glucoside, together with eight ﬂavonol glycosides, i.e. isorhamnetin 3-O-glucoside, 7-O-glucoside, 3,7-di-O-glucoside and 3-O-rutinoside, quercetin 3-O-rutinoside and 3-O-glucoside, and kaempferol 3-O-rutinoside and 3-O-glucoside. Color of cyanidin 3-O-glucoside which
is major anthocyanin in the ﬂowers is originally red but not reddish orange. We presumed thatcolor of anthocyanins is hypsochromically shifted under lower pH, and measured pH of thepressed juice of the petals. As the results, it was proved that pH of the petals is lower (pH 3.3) andin spite of ﬂower pigments are cyanidin glycosides, the ﬂowers of A. nobilis express reddishorange. This was followed by in vitro examination.
Keywords : Amherstia nobilis, cell sap pH, cyanidin 3-O-glucoside, in vitro examination, reddish
orange ﬂower color.

Introduction
The genus Amherstia (Leguminosae) consistsof only one species, A. nobilis Wall. and is
endemic to Myanmar. Although the species is
growing in some botanical gardens in tropicalarea and greenhouses, its wild population inMyanmar is not known. A. nobilis is generally
regarded as one of the most beautiful of all ﬂowering trees (Fig. 1) (Bisby et al., 1994). Its ﬂower
anthocyanin has been reported to be pelargonidin
3-O-pentoside without chemical data (Beale etal., 1941). More recently, three anthocyanins andeight ﬂavonols were isolated from the reddish
orange ﬂowers of this species. However, anthocyanins were identiﬁed as cyanidin 3-O-gluco© 2019 National Museum of Nature and Scienceside and 3-O-xyloside, and peonidin 3-O-glucoside (Iwashina et al., 2019). On the other hand,
ﬂavonols were characterized as isorhamnetin
3-O-glucoside, 7-O-glucoside, 3,7-di-O-glucoside and 3-O-rutinoside, quercetin 3-O-rutinosideand 3-O-glucoside, and kaempferol 3-O-rutinoside and 3-O-glucoside (Iwashina et al., 2019).
Although major anthocyanin was cyanidin
3-O-glucoside, its ﬂower color was reddish
orange which is such as due to pelargonidin and/or carotenoids. In this survey, we clariﬁed thecoloring mechanism of reddish orange ﬂowers bypH measurement of the petals and in vitro examination.

Takayuki Mizuno et al.

166

Fig. 1. Flowers of Amherstia nobilis (photographed in Bogor Botanic Garden, Indonesia by one of the author,
T. Iwashina in 1 July 2016).

Materials and Methods
Plant materials
The ﬂowers were collected in the greenhouseof the Tsukuba Botanical Garden, National
Museum of Nature and Science, Tsukuba, Japanin 21 January 2016.
Isolation and identiﬁcation
Flowers were extracted with MeOH/HCOOH(92 : 8). Anthocyanins and ﬂavonols in crude
extracts were characterized by HPLC comparisons with authentic samples which were obtained
from the ﬂowers of this species (Iwashina et al.,2019). HPLC measurement was performed using
Shimadzu HPLC system (Shimadzu Co., Ltd.,Kyoto,), Inertsil ODS-4 column (I.D. 6.0 ×150 mm, GL Sciences Inc., Tokyo), at ﬂow-rateof 1.0 ml min-1, detection wave length of 530 nm
for anthocyanins and 350 nm for ﬂavonols,and eluents were MeCN/HOAc/H2O/H3PO4(10 : 8 : 79 : 3) for anthocyanins and MeCN/H2O/
H3PO4 (20 : 80 : 0.2) for ﬂavonols.

pH measurement
The pH values of the pressed juice from theﬂower tissues were measured using a handhold
pH meter (Twin pH; AS-212, Horiba Instruments, Kyoto). The reddish orange epidermis
was carefully peeled and placed between glasses,
and pressed to get the pressed juice. The pH of
the pressed juice prepared was immediately measured. Measurement were triplicated.
In vitro examination
The pH effect of cyanidin 3-O-glucoside (5 ×10−4 M) was determined at different pH values(pH 2.6, 3.3, 4.3, 5.3 and 6.3) of Mellvaine
buffer prepared by mixing 0.1 M citric acid and
0.2 M Na2HPO4. The absorption spectra of thesolution were measured immediately (0 min)and after 30 min.
Results and Discussion
Flower color of Amherstia nobilis was reddish
orange, presumed that it is due to carotenoid and/

Reconstruction of Amherstia nobilis Flowers

or anthocyanin, especially pelargonidin. Practically, pelargonidin 3-O-pentoside has beenreported as ﬂower pigment without chemical data(Beale et al., 1941). However, cyanidin 3-O-glucoside was found in the ﬂowers of this species asa major pigment, together with minor cyanidin
3-O-xyloside and peonidin 3-O-glucoside, andeight ﬂavonol glycosides, instead of pelargonidin
glycoside and carotenoids (Iwashina et al.,2019). Although cyanidin glycosides commonly
express red color in almost ﬂowers except for a
few plants, e.g. Meconopsis spp. (Takeda et al.,
1996; Tanaka et al., 2001; Yoshida et al., 2006)
and Centaurea cyanus L. (Takeda et al., 2005;
Shiono et al., 2005), reddish orange color was
expressed by cyanidin 3-O-glucoside in A. nobilis ﬂowers. Such the fact was presumed that
other factors are changed the pigment color (red)to reddish orange in this ﬂowers. Generally,
because color of anthocyanins is hypsochromically shifted under lower pH (acidity), pH of thepressed juice of the ﬂowers was measured. As
the results, it was shown that pH of the petals islower (pH 3.3) and in spite of major ﬂower pigment is cyanidin 3-O-glucoside, the ﬂowers of A.
nobilis expressed reddish orange. It was followed
by in vitro examination (Table 1 and Fig. 2).
Absorption maxima of intact petals was λmax
511 nm (Fig. 2A). In in vitro examination,absorption maxima of cyanidin 3-O-glucosidewere measured in conditions of pH 2.6, 3.3, 4.3,

167

5.3 and 6.3 (Table 1 and Fig. 2). In lower acidic
solutions (pH 2.6 and 3.3), their absorption maxima showed λmax 511 and 512 nm without additional absorption peaks and shoulders, which was
the same with that of intact petals. On the otherhand, absorption maxima, λmax 519 nm wasshown in pH 4.3. Moreover, absorption maxima
appeared as two peaks (434 and 529 nm, and 439and 535 nm, respectively) in pH 5.3 and 6.3 (Fig.
2B). Although absorption maxima were measured after 30 min, those of pH 2.6 and 3.3 werevery stable. However, those of pH 5.3 and 6.3
were unstable (Fig. 2C). Thus, it was proved that
reddish orange color of A. nobilis ﬂowers is dueto lower acidic condition in cell sap. Accompanyingly, although absorption maxima of cyanidin3-O-glucoside were measured in addition to isorhamnetin 3-O-glucoside which was one of the
major ﬂavonols in the ﬂowers of A. nobilis, thebathochromic and also hypsochromic shifts wereTable 1. Absorption maxima of cyanidin 3-Oglucoside with various pH
pH
2.6
3.3
4.3
5.3
6.3

λ max (nm)
0 min

30 min511
512
519
434, 529
439, 535

511
511
510
514
438, 533

λmax of fresh petal of Amherstia nobilis was shown at511 nm.

Fig. 2. Absorption spectra of intact petal (A), cyanidin 3-O-glucoside with various pH (0 min, B, and after 30 minC).

Takayuki Mizuno et al.

168

hardly occurred (data not shown).
The pH of ﬂower cell sap is generally slightly
acidic (Shibata et al., 1949). However, pH of thepigmented epidermal cell sap of A. nobilis wasfound to be extremely lower. The pH of epidermis of the blue and greenish blue petals ofmorning glory cv. Heavenly Blue and jade vain
(Strongylodon macrobotrys A. Gray) werereported to be slightly alkali (pH 7.5) (Stewart etal., 1975) and pH 7.9 (Takeda et al., 2010),respectively. By contraries, pH of the ﬂower cellsof Begonia cv. Orange Schwabenland and Azalea
cv. Gloria were lower pH (2.5 and 3.1, respectively). Their ﬂower colors were strong redorange and light yellowish pink in spite of theiranthocyanins are cyanidin glycosides (Stewart etal., 1975). Although major pigment of A. nobilis
was cyanidin 3-O-glucoside, its ﬂower color ischanged to reddish orange due to lower acidity ofcell saps. There is close correlation between
ﬂower color and pollinators (Harborne andGrayer, 1994). Flavonol glycosides directly do
not inﬂuence the ﬂower color of A. nobilis. However, since insects and birds can discriminate UVrange, ﬂavonol glycosides may be useful as pollinator attractants. Although wild population of A.
nobilis in Myanmar is unknown, we presume thatunknown insects, birds or animals, which weretempted to reddish orange color, visit to thisﬂowers.
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