Bull. Natl. Mus. Nat. Sci., Ser. B, 43(3), pp. 87–91, August 22, 2017

Flower Pigments of Black Pea Thermopsis barbata (Fabaceae) in Bhutan
Yoshinori Murai1, *, Rinchen Yangzom2, Choki Gyeltshen2, Kencho Dorji2,
Choki Wangmo2 and Tsukasa Iwashina1
1

Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan2
National Biodiversity Centre, Ministry of Agriculture and Forests,
Post Box 875 Serbithang, Thimphu, Bhutan*E-mail: murai@kahaku.go.jp(Received 14, May 2017; accepted 28, June 2017)

Abstract Flower pigments of Black Pea (Thermopsis barbata) sampled from alpine zone inwestern Bhutan were surveyed. Major anthocyanins of the blackish ﬂowers were delphinidin
3-O-glucoside, cyanidin 3-O-glucoside and petunidin 3-O-glucoside together with some minor
anthocyanins and other ﬂavonoids. Interestingly, chlorogenic acid was also found from the ﬂowers. Contribution of each compound to ﬂower coloration and other ecological functions are discussed.
Key words : anthocyanins, black ﬂower, chlorogenic acid, Thermopsis barbata, Fabaceae.

IntroductionThe genera Thermopsis belongs to the family
Fabaceae (Leguminosae) and consists of 23 species in East Asia and North America (Mabberley,2008). Thermopsis barbata Royle (Fig. 1) is distributed from south-western China to Kashmir inhigh montane meadows at ca. 3000–4250 m
above sea level. The ﬂowers is 2–3 cm length andunique blackish purple color and the leaves arecovered with silvery hairs.
There are several ﬂowers called as ʻblack
ﬂowerʼ in plant kingdoms. Some studies onblackish (dark purple or dark red) ﬂower coloration have been carried out using some ornamental cultivars of pansy (Viola tricolor L.) (Violaceae), tulip (Tulipa gesneriana L.) (Liliaceae),hollyhock (Alcea rosea L.) (Malvaceae), dahlia(Dahlia variabilis Hort.) (Asteraceae), chocolate
cosmos (Cosmos atrosanguineus (Hook.) Voss)(Asteraceae) (Shibata and Ishikura, 1960; Takedaand Hayashi, 1965; Hosaka et al., 2012; Ama-miya and Iwashina, 2016; Deguchi et al., 2016).
However, the number of related studies on blackish ﬂowers of wild species is limited, and noinformation is available for phenolic compounds
in the dark purple ﬂowers of T. barbata.
On the other hand, alpine plants grow under
severe environmental conditions (e.g., intense
UV-B and cold) and they tend to accumulate efﬁcient antioxidant and UV-absorbing compounds
in their ﬂowers and leaves (Spitaler et al., 2006;
Murai and Iwashina, 2010, 2015, 2016; Murai etal., 2014).
The aim of this study was to reveal the phenolic compounds in the ﬂowers of T. barbata, anddiscuss the ﬂower coloration and chemical property related to environmental conditions.
Materials and Methods
Plant materials
Thermopsis barbata was collected fromJomolhari Trek near Jangothang (3708 m a.s.l.),Yoshinori Murai et al.

88Thimphu, in early July 2015. Voucher specimen
was deposited in the National Herbarium(THIM) of National Biodiversity Centre, Bhutan.
Extraction and separation
Dried ﬂowers of T. barbata (1.2 g) wereextracted with MAW (MeOH/HOAc/HCOOH ＝75 : 5 : 20). The concentrated extracts wereapplied to preparative paper chromatography(PPC) using solvent systems: BAW (n-BuOH/HOAc/H2O ＝ 4 : 1 : 5, upper phase) and 15%HOAc.
The compounds were puriﬁed by Sephadex
LH-20 column chromatography eluted withMAW2 (MeOH/HOAc/H2O ＝ 70 : 5 : 25).
HPLC analysis
High performance liquid chromatography(HPLC) separation of the extracts ﬁltered with
GL Chromatodisk 13N (0.45 μm pore size, GLSciences, Inc., Japan) was performed with a Shimadzu Prominence HPLC system using aSunShell C18 column [2.6 μm, I.D. 4.6 mm ×100 mm (ChromaNik Technologies Inc., Japan)]at a ﬂow rate of 0.4 mL min−1, detection wavelength at 190–700 nm and H3PO4/HOAc/MeCN/Fig. 1.

Thermopsis barbata.

H2O (3 : 8 : 3 : 86) as eluent. Injection volume was
1 μL. HPLC chromatograms of anthocyanins andother compounds are shown in Fig. 2. To identify
each compound, HPLC comparison with authentic samples of delphinidin 3-O-glucoside, petunidin 3-O-glucoside (Tokiwa Phytochemical Co.,
Ltd., Japan), cyanidin 3-O-glucoside (Murai etal., 2008), quercetin 3-O-glucoside (Murai et al.,2014), luteolin 7-O-rutinoside (Iwashina andKadota, 1999) and chlorogenic acid (MP Biomedicals, LLC, France) were carried out. The
retention times and UV-Vis spectra of A1–A3,O1, O3 and O4 were identical with those ofauthentic specimens.
LC-MS analysis
Liquid chromatography-mass spectrometry(LC-MS) was performed with the Shimadzu
LCMS-2010EV system using Inertsil ODS-4 column [HP 3 μm, I.D. 2.1 mm× 100 mm (GL Sci-Fig. 2. HPLC chromatogram of MAW extracts
from Thermopsis barbata.
(a) A1: Delphinidin 3-O-glucoside, A2: Cyanidin 3-O-glucoside, A3: Petunidin 3-O-glucoside. Detection: 530 nm.
(b) O1: Chlorogenic acid, O2: Caffeic acidderivative, O3: Quercetin 3-O-glucoside, O4:Luteolin 7-O-rutinoside. Detection: 350 nm.

Flower Pigments of Thermopsis barbataences Inc., Japan)] at a ﬂow rate of 0.2 mL
min−1, detection wavelength at 190–700 nm, andHCOOH/MeCN/H2O (5 : 10 : 80) as eluent. Injection volume was 3 μL. ESI＋ 4.5 kV, ESI− 3.5 kV,
250°C.
UV-Vis absorption spectra
UV-Vis absorption spectra of the isolated compounds were measured by a Shimadzu UV-2600
UV-Vis Spectrophotometer (Shimadzu Corporation, Japan) (anthocyanins: 220–700 nm, otherphenolics: 220–500 nm).
Identiﬁcation of compounds
The isolated compounds were identiﬁed by
UV-Vis spectra, LC-MS, characterization of acidhydrolysates (anthocyanidins) and HPLC comparisons with authentic specimens. Caffeic acidderivative (O2) was extrapolated from its UV
spectra by UV-Vis spectrophotometer and HPLC
photodiode array detector.
In this survey, three anthocyanins (A1–A3),
two phenolic acids (O1 and O2) and two ﬂavonoids (O3 and O4) were found in the ﬂowers ofT. barbata. Chemical data of the isolated compounds are as follows.
Delphinidin 3-O-glucoside (A1). Purple solution. UV: λmax (nm) 0.01%HCl–MeOH 277,540; ＋AlCl3 323, 580. LC-MS: m/z 465 [M]＋(delphinidin ＋ 1 mol glucose), 303 [M-162]＋(delphinidin). HPLC: Rt 5.0 min. Acid hydroly-89

sate: delphinidin.
Cyanidin 3-O-glucoside (A2). Red solution.
UV: λmax (nm) 0.01%HCl–MeOH 281, 528;
＋AlCl3 313, 569. LC-MS: m/z 449 [M]＋ (cyanidin ＋ 1 mol glucose), 287 [M-162]＋ (cyanidin).
HPLC: Rt 7.6 min. Acid hydrolysate: cyanidin.
Petunidin 3-O-glucoside (A3). Purple solution. UV: λmax (nm) 0.01%HCl–MeOH 278,538; ＋AlCl3 313, 586. LC-MS: m/z 479 [M]＋(petunidin ＋ 1 mol glucose), 317 [M-162]＋(petunidin). HPLC: Rt 10.5 min. Acid hydrolysate: petunidin.
Chlorogenic acid (O1). UV: λmax (nm) MeOH299sh, 325. HPLC: Rt 5.3 min. LC-MS: m/z 355[M＋H]＋, 353 [M−H]−.
Caffeic acid derivative (O2). UV: λmax (nm)
MeOH 298sh, 326. HPLC: Rt 23.2 min. LC-MS:m/z 367 [M−H]−.
Quercetin 3-O-glucoside (O3). Pale yellowsolution. UV: λmax (nm) MeOH 255, 268sh,355. HPLC: Rt 32.1 min. LC-MS: m/z 463[M−H]− (quercetin ＋ 1 mol glucose) and 303[M−162＋H]＋ (quercetin).

Fig. 4. Chemical structure of chlorogenic acid
(O1) from Thermopsis barbata.

Fig. 3. Chemical structures of anthocyanins fromThermopsis barbata.
A1: Delphinidin 3-O-glucoside (R1 ＝ H, R2 ＝OH), A2: Cyanidin 3-O-glucoside (R1 ＝ R2 ＝H), A3: petunidin 3-O-glucoside (R1 ＝ CH3, R2＝ OH).

Fig. 5. Chemical structure of quercetin 3-O-glucoside (O3) from Thermopsis barbata.

Yoshinori Murai et al.

90

Fig. 6. Chemical structure of luteolin 7-O-rutinoside (O4) from Thermopsis barbata.

Luteolin 7-O-rutinoside (O4). Pale yellowsolution. UV: λmax (nm) MeOH 256, 266sh,349. HPLC: Rt 46.4 min. LC-MS: m/z 595[M＋H]＋, 593 [M−H]− (luteolin ＋ 1 mol glucose ＋ 1 mol rhamnose) and 287 [M−308＋H]＋(luteolin).
Results and Discussion
Blackish coloration and anthocyanins from Thermopsis barbata
The present study simultaneously revealed thecomposition of anthocyanins, other ﬂavonoids
and phenolic acids in the ﬂowers of Thermopsis
barbata for the ﬁrst time. The major anthocyanins responsible for the blackish color were
3-O-glucosides of delphinidin (A1), cyanidin(A2) and petunidin (A3) (Figs. 2 and 3). Whenthe black coloration occurs, there is broad andstrong absorption in the visible region. The combination of delphinidin and cyanidin covers wide
range of visible light. Cyanidin 3-O-rutinosideand delphinidin 3-O-rutinoside were major
anthocyanins in the dark purple perianth-bottoms
of some tulip cultivars (Nakayama et al., 2004).
The glycosylation pattern of each anthocyanin
from T. barbata was simple compared with previous reports. In the case of blackish ﬂowers ofwild species, the dark purple ﬂowers of Lisianthius nigrescens Cham. et Schlecht. (Gentianaceae) contain delphinidin 3-O-rhamnosyl(1→6)-galactoside and delphinidin 3-O-rhamnosyl(1→6)-galactoside-5-O-glucoside (Markham etal., 2004). Furthermore, dark red ﬂowers of Pulsatilla cernua (Thunb.) Bercht. et C.Presl(Ranunculaceae) contained complicatedly acyl-ated anthocyanin, pelargonidin 3-[2″-(2‴-Ecaffeoly-β-D-glucopyranosyl)-β-D-galactopyranoside](Yoshitama et al., 1998).
Other ﬂavonoids sometimes affect ﬂower colorations as co-pigment substances. However, L.
nigrescencs and P. cernua barely contain otherﬂavonoids. T. barbata also does not contain such
substances (Fig. 2b). Though chlorogenic acid
(Fig. 4) was found in the ﬂowers of T. barbata,
the compound has obstructed co-pigmentation in
hydrangea ﬂower (Takeda et al., 1985). These
results suggests that the possibility of co-pigment
effect in the blackish coloration of T. barabata islow. Anthocyanins and other ﬂavonoids, i.e., ﬂavone and ﬂavonol, are share the crucial steps ofbiosynthetic pathway. The synthesis of anthocyanins may have priority over other ﬂavonoids insuch species. Further investigations, e.g., evaluation of color using wild species and the relationship with pollinators, are needed.
Other ecological signiﬁcanceThe ﬂowers of T. barbata contained chlorogenic acid (O1) as major phenolic compound.
Chlorogenic acid has been reported as an efﬁcient antioxidant. In addition, two minor compounds, quercetin 3-O-glucoside (O3) and luteolin 7-O-rutinoside (O4) (Figs. 5 and 6) in theﬂowers of T. barbata, are also thought to bestrong antioxidant (Rice-Evans et al., 1996;Pietta, 2000). Recent numerous studies showedthat several plants growing in higher altitudes
synthesize antioxidant and UV-absorbing substances including phenolic acids and ﬂavonoids(Spitaler et al., 2006; Murai and Iwashina, 2010,2015; Murai et al., 2009, 2014, 2015, 2016). We
preliminary reported chlorogenic acid fromalpine Campanula species (Murai et al. 2014).
UV radiation cases not only direct physiologicaldamages but indirect oxidation damages to plant
cells and tissues, and such oxidation stresses arealso caused by cold and other environmental factors. Flower is the most crucial reproductive
organ of plants. The compound might play animportant role in protection against several environmental stresses in high altitudes.

Flower Pigments of Thermopsis barbata

Acknowledgements
This work was partly supported by JSPS
KAKENHI Grant Numbers JP15H02914,
JP16K18830.
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