Bull. Natl. Mus. Nat. Sci., Ser. B, 41(1), pp. 33–44, February 20, 2015

Anthocyanins and Other Flavonoids from Amorphophallus titanum
Having Largest Inﬂorescence in Plant Kingdom, and Other Two Species
Tsukasa Iwashina1,*, Ayumi Uehara1,2, Junichi Kitajima3 and Tomohisa Yukawa1
1

Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan2
Department of Chemistry, Hiyoshi Campus, Keio University,
Hiyoshi 4–1–1, Kohoku-ku, Yokohama, Kanagawa 223–8521, Japan
3
Laboratory of Pharmacognosy, Showa Pharmaceutical University,
Higashi-tamagawagakuen 3, Machida, Tokyo 194–8543, Japan* E-mail: iwashina@kahaku.go.jp(Received 8 November 2014; accepted 24 December 2014)

Abstracts Amorphophallus titanum has a largest inﬂorescence of the plant kingdom. In this survey, ﬁve anthocyanins and 14 other ﬂavonoids were isolated from the spathe and spadix appendix
of A. titanum. Anthocyanins were identiﬁed as cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside,peonidin 3-O-glucoside, peonidin 3-O-rutinoside and pelargonidin 3-O-rhamnosylglucoside. On
the other hand, other ﬂavonoids were characterized as vitexin, vitexin 2″-O-glucoside, isovitexin,
isovitexin 2″-O-glucoside, orientin, isoorientin, vicenin-2, lucenin-2, schaftoside, isoschaftoside,
isovitexin X″-O-rhamnoside, isoscoparin X″-O-glucoside (C-glycosylﬂavones), chrysoeriol
7-O-glucoside (ﬂavone) and kaempferol 3-O-robinobioside (ﬂavonol). Anthocyanins and other ﬂavonoids in the inﬂorescences of other two Amorphophallus species, A. rivieri and A. paeoniifolius
were also isolated and identiﬁed.
Key words : Amorphophallus paeoniifolius, Amorphophallus rivieri, Amorphophallus titanum,
Anthocyanins, Flavonols, C-Glycosylﬂavones.

Introduction
The genus Amorphophallus is mainly distributed in the tropical zone of the Old World andconsists of 80–90 species (Ohashi, 1982). Of
their species, A. titanum is endemic to Sumatra
Island and has the largest inﬂorescence, ca. 3 mhigh and 1 m wide, of the plant kingdom. Its
ﬂowering in the botanical gardens is very rare. In
2012 and 2014, the ﬂowers of the species cameout at the Tsukuba Botanical Garden, National
Museum of Nature and Science, Japan. The
spathe and spadix appendix of the species aredark purple and their anthocyanins have beenreported to be cyanidin 3-O-glucoside and3-O-rutinoside, and peonidin and pelargonidin
3-O-(p-coumaloylglucosides) (Gallori et al.,2004).
Amorphophallus paeoniifolius grows in Sri
Lanka, the Philippines, Malaysia, Indonesia, andother Southeast Asia countries. Flavonol, quercetin, has been isolated from the corm of the species (Sharstry et al., 2010). Amorphophallus rivieri is native to Indochina and southern China
and cultivated in Japan for edible. However,polyphenols including ﬂavonoids are notreported until now.
As the ﬂavonoids of other Amorphophallus
species, anthocyanin, cyanidin 3-O-rutinosidehas been found in the spathe of A. abyssinicus(A.Rich.) N.E.Brown and A. stuhlmannii (Engl.)
Engl. & Gehrm. (Williams et al., 1981). A rare
ﬂavonol, 3,5-diacetyltambulin (7,8,4′-trimethoxy3,5-diacetoxyﬂavone), has been isolated from theTsukasa Iwashina et al.

34

tuberous roots of A. campanulatus Blume ex
Decne (＝A. paeoniifolius) (Khan et al., 2008).
Unknwon C-glycosylﬂavone was detected fromthe leaves of A. stuhlmannii (Williams et al.,1981). In this paper, we describe the isolationand identiﬁcation of the anthocyanins, ﬂavone,ﬂavonols and C-glycosylﬂavones from the inﬂorescences of three Amorphophallus species, A.
titanum, A. paeoniifolius and A. rivieri.
Materials and Methods
General
UV spectra were recorded on a Shimadzu
MPS-2000 Multipurpose recording spectrophotometer (Shimadzu, Japan) according to Mabry
et al. (1970). LC-MS were measured on Shimadzu LC-MS systems using Inertsil ODS-4 column [I.D. 2.1×100 mm (GL Sciences, Japan)],at a ﬂow-rate of 0.1 ml min−1, eluting with
MeCN/H2O/HCOOH (5 : 90 : 5) for anthocyaninsand (12 : 83 : 5 or 15 : 80 : 5) for other ﬂavonoids,
ESI＋ 4.5 kV and ESI− 3.5 kV, 250˚C. HPLC survey of the isolated compounds and crude extracts
was performed with a Shimadzu HPLC systems
using Inertsil ODS-4 column (I.D. 6.0×150 mm)
at a ﬂow-rate of 1.0 ml min−1, eluting with
MeCN/HOAc/H2O/H3PO4 (10 : 8 : 79 : 3) (Solv. I)
for anthocyanins, a Senshu Pak Pegasil ODS column (I.D. 6.0×150 mm, Senshu Scientiﬁc Co.
Ltd., Japan) at a ﬂow-rate of 1.0 ml min−1, eluting with MeCN/H2O/H3PO4 (15 : 85 : 0.2) (Solv.
II) and (18 : 82 : 0.2) (Solv. III) for other ﬂavonoids. Detection wave length was 530 nm foranthocyanins and 350 nm for other ﬂavonoids.
Acid hydrolyses of the anthocyanins and otherﬂavonoids were performed in 12% HCl, 100°C,
30 min. After reaction, isoamyl alcohol (anthocyanins) or diethyl ether (other ﬂavonoids) was
added in the solution. Aglycones migrated toorganic layer, and glycosidic sugars and C-glycosylﬂavones remained in aqueous layer. Aglycones and C-glycosylﬂavones were identiﬁed by
HPLC comparisons with authentic samples, andsugars were compared with authentic sugars by
paper chromatography or TLC using solvent sys-tems, BBPW (n-BuOH/benzene/pyridine/H2O＝
5 : 1 : 3 : 3) and BTPW (n-BuOH/toluene/pyridine/
H2O＝5 : 1 : 3 : 3). The solvent systems of qualitative TLC (Merck, Germany) and preparative
paper chromatography (PPC) are as follows;
BAW (n-BuOH/HOAc/H2O＝4 : 1 : 5, upper phase),
BEW (n-BuOH/EtOH/H2O＝4 : 1 : 2.2) and 15%
HOAc. NMR spectra were measured in pyridined5 at 600 MHz (1H NMR) and 150 MHz (13C
NMR).
Plant materials
Three Amorphophallus species, A. titanum(Becc.) Becc. ex Arcangeli (TBG157495), A.
paeoniifolius (Dennst.) Nicolson (TBG84663)
and A. rivieri Durieu (＝A. konjac K.Koch)(TBG157597) (Fig. 1) were used as plant materials. They are cultivated in the Tsukuba Botanical
Garden, National Museum of Nature and Science, Tsukuba, Japan.
Extraction and separation
Fresh spathe (454 g) and spadix appendix(945 g) of A. titanum, and fresh inﬂorescences(138 g and 300 g) of A. paeoniifolius and A. rivieri
were extracted with MeOH : HCOOH (92 : 8).
After ﬁltration, the concentrated extracts wereapplied to PPC using solvent systems, BAW and
15% HOAc. Other ﬂavonoids were further separated with solvent system, BEW. The isolated
anthocyanins and other ﬂavonoids were puriﬁed
by Sephadex LH-20 column chromatographyusing solvent systems, MeOH/H2O/HCOOH(70 : 25 : 5) and 70% MeOH, respectively.
Identiﬁcation
The isolated compounds were identiﬁed by
UV spectroscopy, LC-MS, characterization ofacid hydrolysates, 1H and 13C NMR, and TLC
and HPLC comparisons with authentic samples.
TLC, HPLC, LC-MS and NMR data of the isolated compounds are as follow.
Cyanidin 3-O-glucoside (chrysanthemin, 1).
HPLC: tR (min) 8.22 (solv. I). UV: λmax (nm)0.01%MeOH-HCl 282, 529; ＋AlCl3 556; E440/
Emax 28%. LC-MS: m/z 449 [M]＋ (molecular ion

Anthocyanins and Flavonoids from Amorphophallus titanum

35

Fig. 1. Three Amorphophallus species used as plant materials.

peak, cyanidin＋1 mol glucose), m/z 287[M−162]＋ (fragment ion peak, cyanidin).
Cyanidin 3-O-rutinoside (keracyanin, 2).
HPLC: tR (min) 9.22 (solv. I). UV: λmax (nm)0.01%MeOH-HCl 280, 527; ＋AlCl3 546; E440/
Emax 29%. LC-MS: m/z 595 [M]＋ (molecular ion
peak, cyanidin＋each 1 mol rhamnose and glucose), m/z 287 [M−308]＋ (fragment ion peak,cyanidin).
Peonidin 3-O-glucoside (oxycoccicyanin, 3).
HPLC: tR (min) 16.81 (solv. I). LC-MS: m/z 463[M]＋ (molecular ion peak, peonidin＋1 mol glucose), m/z 301 [M−162]＋ (fragment ion peak,peonidin).
Peonidin 3-O-rutinoside (4). HPLC: tR (min)17.82 (solv. I). UV: λmax (nm) 0.01%MeOHHCl 280, 529; ＋AlCl3 528; E440/Emax 30%.
LC-MS: m/z 609 [M]＋ (molecular ion peak,peonidin＋each 1 mol rhamnose and glucose),m/z 301 [M−308]＋ (fragment ion peak, peonidin).
Pelargonidin 3-O-rhamnosylglucoside (5).
HPLC: tR (min) 13.86 (solv. I). UV: λmax (nm)0.01%MeOH-HCl 277, 512; ＋AlCl3 512.
LC-MS: m/z 579 [M]＋ (molecular ion peak,pelargonidin＋each 1 mol rhamnose and glucose), m/z 271 [M−308]＋ (fragment ion peak,pelargonidin).
Cyanidin 3-O-diglucoside (6). HPLC: tR (min)6.18 (solv. I). UV: λmax (nm) 0.01%MeOH-HCl
280, 527; ＋AlCl3 546; E440/Emax 25%. LC-MS:
m/z 611 [M]＋ (molecular ion peak, cyanidin＋2 mol glucose), m/z 287 [M−324]＋ (fragment ion peak, cyanidin).
Pelargonidin 3-O-glucoside (callistephin, 7).
HPLC: tR (min) 11.72 (solv. I). UV: λmax (nm)0.01%MeOH-HCl 272, 512; ＋AlCl3 513; E440/
Emax 42%. LC-MS: m/z 433 [M]＋ (molecular ion
peak, pelargonidin＋1 mol glucose).
Vitexin (8). TLC: Rf 0.48 (BAW), 0.44
(BEW), 0.16 (15%HOAc); color UV (365 nm)
dark purple, UV/NH3 dark greenish yellow.
HPLC: tR (min) 6.11 (solv. II). UV: λmax (nm)
MeOH 270, 329; ＋NaOMe 278, 326, 394 (inc.);＋AlCl3 275, 304, 346, 377sh; ＋AlCl3/HCl 274,
303, 344, 376sh; ＋NaOAc 279, 310, 390;
＋NaOAc/H3BO3 271, 344. LC-MS: m/z 431[M−H]– (molecular ion peak, apigenin＋1 mol
glucose).
Vitexin 2″-O-glucoside (9). TLC: Rf 0.36
(BAW), 0.40 (BEW), 0.59 (15%HOAc); color
UV (365 nm) dark purple, UV/NH3 dark greenish
yellow. HPLC: tR (min) 6.37 (solv. II). UV:
λmax (nm) MeOH 270, 334; ＋NaOMe 280,

36

Tsukasa Iwashina et al.

330, 397 (inc.); ＋AlCl3 277, 303, 349, 383;
＋AlCl3/HCl 278, 302, 343, 382sh; ＋NaOAc
280, 312, 392; ＋NaOAc/H3BO3 271, 345.
LC-MS: m/z 593 [M−H]– (molecular ion peak,
apigenin＋2 mol glucose).
Isovitexin (10). TLC: Rf 0.71 (BAW), 0.68
(BEW), 0.42 (15%HOAc); color UV (365 nm)
dark purple, UV/NH3 dark greenish yellow.
HPLC: tR (min) 8.02 (solv. II). UV: λmax (nm)
MeOH 271, 334; ＋NaOMe 279, 327, 399 (inc.);＋AlCl3 278, 303, 350, 377sh; ＋AlCl3/HCl 279,
303, 346, 376sh; ＋NaOAc 278, 313, 394;
＋NaOAc/H3BO3 272, 347. LC-MS: m/z 433[M＋H]＋, 431 [M−H]– (molecular ion peaks,
apigenin＋1 mol glucose).
Isovitexin 2″-O-glucoside (11). TLC: Rf 0.45
(BAW), 0.51 (BEW), 0.69 (15%HOAc); color
UV (365 nm) dark purple, UV/NH3 dark greenish
yellow. HPLC: tR (min) 6.52 (solv. II). UV:
λmax (nm) MeOH 270, 334; ＋NaOMe 280,
330, 397 (inc.); ＋AlCl3 277, 303, 349, 383;
＋AlCl3/HCl 278, 302, 343, 382sh; ＋NaOAc
280, 312, 392; ＋NaOAc/H3BO3 271, 345.
LC-MS: m/z 593 [M−H]– (molecular ion peak,
apigenin＋2 mol glucose).
Orientin (12). TLC: Rf 0.31 (BAW), 0.27
(BEW), 0.11 (15%HOAc); color UV (365 nm)
dark purple, UV/NH3 dark yellow. HPLC: tR(min) 5.27 (solv. II). UV: λmax (nm) MeOH 257,
269, 349; ＋NaOMe 270, 327sh, 407 (inc.);＋AlCl3 274, 422; ＋AlCl3/HCl 263sh, 276, 296,
358, 385sh; ＋NaOAc 272, 276, 327sh, 400;
＋NaOAc/H3BO3 266, 376, 427sh. LC-MS: m/z
449 [M＋H]＋, 447 [M−H]– (molecular ion
peaks, luteolin＋1 mol glucose).
Isoorientin (13). TLC: Rf 0.47 (BAW), 0.45
(BEW), 0.28 (15%HOAc); color UV (365 nm)
dark purple, UV/NH3 dark yellow. HPLC: tR(min) 6.01 (solv. II). UV: λmax (nm) MeOH 258,
270, 350; ＋NaOMe 270, 337sh, 411 (inc.);＋AlCl3 276, 422; ＋AlCl3/HCl 262sh, 278,296sh, 357, 384sh; ＋NaOAc 270, 402;
＋NaOAc/H3BO3 267, 378, 426sh. LC-MS: m/z
449 [M＋H]＋, 447 [M−H]– (molecular ion
peaks, luteolin＋1 mol glucose).
Vicenin-2 (14). TLC: Rf 0.17 (BAW), 0.19

(BEW), 0.40 (15%HOAc); color UV (365 nm)
dark purple, UV/NH3 dark greenish yellow.
HPLC: tR (min) 4.73 (solv. II). UV: λmax (nm)
MeOH 272, 334; ＋NaOMe 282, 335, 399 (inc.);＋AlCl3 279, 303, 353, 384sh; ＋AlCl3/HCl 279,
303, 347, 383sh; ＋NaOAc 282, 398; ＋NaOAc/
H3BO3 278, 283sh, 323, 349, 410sh. LC-MS: m/z
595 [M＋H]＋, 593 [M−H]– (molecular ion
peaks, apigenin＋2 mol glucose).
Lucenin-2 (15). TLC: Rf 0.09 (BAW), 0.09
(BEW), 0.31 (15%HOAc); color UV (365 nm)
dark purple, UV/NH3 dark yellow. HPLC: tR(min) 4.14 (solv. II). UV: λmax (nm) MeOH
255sh, 272, 344; ＋NaOMe 268, 280, 340sh, 411(inc.); ＋AlCl3 279, 402; ＋AlCl3/HCl 260sh,
279, 296sh, 356, 385sh; ＋NaOAc 268sh, 281,
327sh, 404; ＋NaOAc/H3BO3 275, 285sh, 361,
422. LC-MS: m/z 611 [M＋H]＋, 609 [M−H]–(molecular ion peaks, luteolin＋2 mol glucose).
Schaftoside (16). TLC: Rf 0.21 (BAW), 0.20
(BEW), 0.40 (15%HOAc); color UV (365 nm)
dark purple, UV/NH3 dark greenish yellow.
HPLC: tR (min) 5.63 (solv. II). UV: λmax (nm)
MeOH 272, 333; ＋NaOMe 283, 334, 399 (inc.);＋AlCl3 279, 305, 350, 384sh; ＋AlCl3/HCl 279,
303, 345, 384sh; ＋NaOAc 282, 314sh, 397;
＋NaOAc/H3BO3 276sh, 284, 322, 350sh, 410.
LC-MS: m/z 565 [M＋H]＋, 563 [M−H]– (molecular ion peaks, apigenin＋each 1 mol arabinose
and glucose).
Isoschaftoside (17). TLC: Rf 0.32 (BAW),
0.35 (BEW), 0.40 (15%HOAc); color UV(365 nm) dark purple, UV/NH3 dark greenish
yellow. HPLC: tR (min) 5.63 (solv. II). UV:
λmax (nm) MeOH 272, 333; ＋NaOMe 283,
334, 399 (inc.); ＋AlCl3 279, 305, 350, 384sh;＋AlCl3/HCl 279, 303, 345, 384sh; ＋NaOAc
282, 314sh, 397; ＋NaOAc/H3BO3 276sh, 284,
322, 350sh, 410. LC-MS: m/z 565 [M＋H]＋, 563[M−H]– (molecular ion peaks, apigenin＋each
1 mol arabinose and glucose).
Isovitexin X″-O-rhamnoside (18). TLC: Rf
0.53 (BAW), 0.53 (BEW), 0.75 (15%HOAc);
color UV (365 nm) dark purple, UV/NH3 darkgreenish yellow. HPLC: tR (min) 6.75 (solv. II).
UV: λmax (nm) MeOH 270, 329; ＋NaOMe 280,

Anthocyanins and Flavonoids from Amorphophallus titanum

331, 393 (inc.); ＋AlCl3 273, 304, 349, 377sh;＋AlCl3/HCl 278, 303, 339, 380sh; ＋NaOAc
280, 313, 389; ＋NaOAc/H3BO3 270, 347.
LC-MS: m/z 579 [M＋H]＋, 577 [M−H]–(molecular ion peaks, apigenin＋each 1 molrhamnose and glucose).
Isoscoparin X″-O-glucoside (19). TLC: Rf
0.14 (BAW), 0.12 (BEW), 0.36 (15%HOAc);
color UV (365 nm) dark purple, UV/NH3 darkgreenish yellow. HPLC: tR (min) 5.25 (solv. II).
UV: λmax (nm) MeOH 273, 345; ＋NaOMe 282,
341sh, 411 (inc.); ＋AlCl3 262sh, 277, 300sh,
360, 387sh; ＋AlCl3/HCl 257sh, 277, 298sh,
358, 387sh; ＋NaOAc 282, 337sh, 409;
＋NaOAc/H3BO3 276, 285sh, 344, 412. LC-MS:
m/z 625 [M＋H]＋ (molecular ion peak, chrysoeriol＋2 mol glucose), m/z 461 [M−162−H]–(fragment ion peak, chrysoeriol＋1 mol glucose).
Chrysoeriol 7-O-glucoside (20). TLC: Rf 0.48
(BAW), 0.41 (BEW), 0.09 (15%HOAc); color
UV (365 nm) dark purple, UV/NH3 yellow.
HPLC: tR (min) 16.16 (solv. II). UV: λmax (nm)
MeOH 252, 269, 342; ＋NaOMe 264, 276sh,
391 (inc.); ＋AlCl3 262sh, 274, 296, 358, 385sh;＋AlCl3/HCl 259sh, 276, 297, 354, 385sh;＋NaOAc 258, 265sh, 404; ＋NaOAc/H3BO3
269, 348. LC-MS: m/z 463 [M＋H]＋, 461[M−H]– (molecular ion peaks, chrysoeriol＋
1 mol glucose).
Kaempferol 3-O-robinobioside (21). TLC: Rf
0.69 (BAW), 0.63 (BEW), 0.75 (15%HOAc);
color UV (365 nm) dark purple, UV/NH3 darkgreenish yellow. HPLC: tR (min) 9.33 (solv. II).
UV: λmax (nm) MeOH 267, 343; ＋NaOMe 276,
327, 400 (inc.); ＋AlCl3 275, 303, 352, 384;
＋AlCl3/HCl 276, 302, 347, 387sh; ＋NaOAc
275, 316, 394; ＋NaOAc/H3BO3 267, 353.
LC-MS: m/z 593 [M−H]– (molecular ion peak,kaempferol＋each 1 mol rhamnose and galactose), m/z 287 [M−308＋H]＋ (fragment ion
peak, kaempferol).
Quercetin 3-O-glucoside (isoquercitrin, 22).
TLC: Rf 0.65 (BAW), 0.65 (BEW), 0.31
(15%HOAc); color UV (365 nm) dark purple,
UV/NH3 yellow. HPLC: tR (min) 7.33 (solv. II).
UV: λmax (nm) MeOH 257, 265sh, 358;

37

＋NaOMe 274, 331, 407 (inc.); ＋AlCl3 274,
431; ＋AlCl3/HCl 268, 300, 359, 394sh;＋NaOAc 273, 327, 395; ＋NaOAc/H3BO3 261,
380. LC-MS: m/z 463 [M−H]– (molecular ion
peak, quercetin＋1 mol glucose), m/z 302[M−162＋H]＋ (fragment ion peak, quercetin).
Vitexin 2″-β-O-xylopyranoside (23). TLC: Rf
0.47 (BAW), 0.55 (BEW), 0.73 (15%HOAc);
color UV (365 nm) dark purple, UV/NH3 darkgreenish yellow. HPLC: tR (min) 6.22 (solv. II).
UV: λmax (nm) MeOH 270, 330; ＋NaOMe 279,
331, 394 (inc.); ＋AlCl3 276, 304, 348, 384sh;＋AlCl3/HCl 277, 302, 343, 383sh; ＋NaOAc
279, 311, 390; ＋NaOAc/H3BO3 271, 345.
LC-MS: m/z 565 [M＋H]＋, 563 [M−H]–(molecular ion peaks, apigenin＋each 1 mol
xylose and glucose). 1H NMR (600 MHz, pyridine-d5): δ 13.85 (1H, s, 5-OH), 8.25 (2H, d,
J＝6.7 Hz, H-2′,6′), 7.24 (2H, d, J＝6.7 Hz,
H-3′,5′), 6.82 (1H, s, H-3), 6.74 (1H, s, H-6),
5.84 (1H, d, J＝9.2 Hz, glucosyl H-1), 5.01 (1H,
brs, xylosyl H-1), 4.95 (1H, m, glucosyl H-2),
4.53 (1H, m, glucosyl H-4), 4.52 (1H, m, glucosyl H-6a), 4.42 (1H, m, glucosyl H-6b), 4.41
(1H, m, glucosyl H-3), 4.08 (1H, m, glucosylH-5), 3.83 (1H, m, xylosyl H-3), 3.82 (1H, m,xylosyl H-5a), 3.82 (1H, m, xylosyl H-2), 3.76
(1H, m, xylosyl H-4), 3.18 (1H, m, xylosylH-5b). 13C NMR (150 MHz, pyridine-d5): (apigenin) δ 165.1 (C-2), 103.5 (C-3), 183.2 (C-4),
162.5 (C-5), 99.4 (C-6), 164.7 (C-7), 105.4(C-8), 158.3 (C-9), 105.4 (C-10), 123.2 (C-1′),
129.5 (C-2′,6′), 117.0 (C-3′,5′), 162.6 (C-4′);(C-glucose) δ 73.5 (C-1), 82.0 (C-2), 80.6 (C-3),
72.2 (C-4), 83.5 (C-5), 62.9 (C-6); (xylose) δ
106.7 (C-1), 75.0 (C-2), 77.4 (C-3), 70.8 (C-4),
66.5 (C-5).
Kaempferol 3-O-glucoside (astragalin, 24).
TLC: Rf 0.81 (BAW), 0.77 (BEW), 0.39
(15%HOAc); color UV (365 nm) dark purple,
UV/NH3 dark greenish yellow. HPLC: tR (min)8.34 (solv. III). UV: λmax (nm) MeOH 266, 349;
＋NaOMe 275, 325, 398 (inc.); ＋AlCl3 274,
303, 351, 391; ＋AlCl3/HCl 274, 302, 348, 389;
＋NaOAc 275, 313, 392; ＋NaOAc/H3BO3 267,
353. LC-MS: m/z 447 [M−H]– (molecular ion

Tsukasa Iwashina et al.

38

peak, kaempferol＋1 mol glucose), m/z 287[M−162＋H]＋ (fragment ion peak, kaempferol).
Authentic samples
Authentic samples, which were used in thissurvey, and their origin were as follows: cyanidin
3-O-glucoside, cyanidin 3-O-rutinoside, peonidin
3-O-glucoside, peonidin 3-O-rutinoside and pelargonidin 3-O-glucoside from Extra Synthese,
France, vitexin from the fronds of Adiantum
venustum Don (Parkeriaceae) (Iwashina et al.,1995), isovitexin from the ﬂowers of Iris ensata
Thunb. (Iridaceae) (Iwashina et al., 1996), isovitexin 2″-O-glucoside and vitexin 2″-O-glucosidefrom the fronds of Cyrtomium falcatum (L.f.) C.
Presl (Dryopteridaceae) (Iwashina et al., 2006),
orientin from the leaves of Begonia formosana(Hayata) Masam. (Begoniaceae) (Iwashina et al.,2008a), isoorientin from the leaves of Vitex
rotundifolia L.ﬁl. (Verbenaceae) (Iwashina et al.,2011), vicenin-2 and lucenin-2 from the frondsof Asplenium normale D.Don (Aspleniaceae)(Iwashina et al., 2010), schaftoside and isoschaftoside from the aerial parts of Osyris alba L.
(Santalaceae) (Iwashina et al., 2008b), kaempferol 3-O-robinobioside from the aerial parts of
Cassytha ﬁliformis L. (Lauraceae) (Murai et al.,2008), kaempferol 3-O-glucoside from the ﬂowersof Glycine max (L.) Merr. (Leguminosae) (Iwashina et al., 2007), and quercetin 3-O-glucosidefrom the leaves of Calystegia sordanella (L.)
Roem. et Schult. (Convolvulaceae) (Murai et al.,2014).

Acid hydrolysis of 1 and 2 produced cyanidin
as anthocyanidin. And also, glucose, and glucose
and rhamnose were detected as glycosidic sugars, respectively. As the results, they were identiﬁed as cyanidin 3-O-glucoside (chrysanthemin,
1, Fig. 3) and cyanidin 3-O-rutinoside (keracyanin, 2, Fig. 3) by HPLC comparisons withauthentic samples. Similarly, 3 and 4 were identiﬁed as peonidin 3-O-glucoside (oxycoccicyanin,
Fig. 3) and peonidin 3-O-rutinoside (Fig. 3).
Anthocyanin 5 was obtained as a minor compo-

Results and Discussion
Anthocyanins from Amorphophallus titanum, A.
rivieri and A. paeoniifolius
Five anthocyanins (1–5) were isolated fromthe spathe and spadix appendix of Amorphophallus titanum and the inﬂorescence of A. rivieri.
Their anthocyanin composition was the samewith each other. On the other hand, three anthocyanins (1, 6 and 7) were found in the inﬂorescence of A. paeoniifolius (Fig. 2).

Fig. 2. HPLC patterns of the anthocyanins in theinﬂorescences of three Amorphophallus species.
1: cyanidin 3-O-glucoside, 2: cyanidin 3-Orutinoside, 3: peonidin 3-O-glucoside, 4: peonidin 3-O-rutinoside, 5: pelargonidin 3-O-rhamnosylglucoside, 6: cyanidin 3-O-diglucoside and 7:
palergonidin 3-O-glucoside.

Anthocyanins and Flavonoids from Amorphophallus titanum

39

Fig. 3. Anthocyanins isolated from the inﬂorescences of three Amorphophallus species.

nent from A. titanum and A. rivieri. Pelargonidin,
glucose and rhamnose were liberated by acidhydrolysis. Attachment of each 1 mol glucose
and rhamnose to pelargonidin was shown by
appearance of molecular ion peak, m/z 579 [M]＋of LC-MS. Thus, 5 was characterized as pelargonidin 3-O-rhamnosylglucoside. Anthocyanins 6
and 7 were isolated from A. paeoniifolius. They
produced cyanidin and pelargonidin by acidhydrolysis, respectively. Attachment of 2 mol
glucose to 3-position of cyanidin was shown by
LC-MS survey, i.e. appearance of molecular ion

peak, m/z 611 [M]＋, and UV spectral survey.
From the results described above, 6 was characterized as cyanidin 3-O-diglucoside. On the other
hand, 7 was identiﬁed as pelargonidin 3-O-glucoside (callistephin) (Fig. 3) by HPLC comparison
with authentic sample.
In this survey, ﬁve anthocyanins, cyanidin
3-O-glucoside (1), cyanidin 3-O-rutinoside (2),peonidin 3-O-glucoside (3), peonidin 3-O-rutinoside (4) and pelargonidin 3-O-rhamnosylglucoside (perhaps 3-O-rutinoside, 5), were isolated
and characterized from A. titanum and A. rivieri.

Tsukasa Iwashina et al.

40

Though the spathe and spadix appendix of A.
titanum were separately extracted, their anthocyanin composition was qualitatively the same. On
the other hand, three anthocyanins, cyanidin
3-O-glucoside (1), cyanidin 3-O-diglucoside (6)and pelargonidin 3-O-glucoside (7), were isolated from the inﬂorescence of A. paeoniifolius.
A. titanum and A. paeoniifolius, and A. rivieri arenative to tropical Asia, and temperate zones ofAsia, respectively. However, anthocyanin pattern
was the same between A. titanum and A. rivieri,
but not A. paeoniifolius. Anthocyanins in theinﬂorescence of A. titanum have been surveyed,and 3-O-glucoside and 3-O-rutinoside of cyanidin, and peonidin and pelargonidin 3-O-(pcoumaroylglucosides) were reported (Gallori etal., 2004). Of their anthocyanins, though the former two were found in this survey, the latter two
acylated anthocyanins were not detected. Instead,three anthocyanins, peonidin 3-O-glucoside (3),peonidin 3-O-rutinoside (4) and pelargonidin
3-O-rhamnosylglucoside (5) were newly isolated. The anthocyanins in the inﬂorescences ofA. rivieri and A. paeoniifolius were reported forthe ﬁrst time.
Flavonoids in the spathe and spadix appendix ofAmorphophallus titanum
Fourteen ﬂavonoids were isolated from thespathe and spadix appendix of A. titanum. UV
spectral properties of ﬂavonoids 8 and 10
showed that they are ﬂavones having 5-, 7- and4′-hydroxyl groups (Mabry et al., 1970). Since
they were unhydrolyzable by hot acid treatment,
it was shown that they are C-glycosylﬂavones.
Attachment of 1 mol hexose to apigenin was
cleared by LC-MS, i.e. appearance of molecularFig. 4.

ion peaks, m/z 433 [M＋H]＋ and 431 [M−H]–.
Finally, they were identiﬁed as vitexin (5,7,4′-trihydroxyﬂavone 8-C-glucoside, 8) and isovitexin(5,7,4′-trihydroxyﬂavone 6-C-glucoside, 10)(Fig. 4) by TLC and HPLC comparisons withauthentic samples. UV spectral properties of 9
and 11 were also the same with those of 8 and10, showing that they are 5,7,4′-trihydroxyﬂavone derivatives. Vitexin and isovitexin were liberated by acid hydrolysis of 9 and 11, respectively. Attachment of additional 1 mol glucose tovitexin and isovitexin was proved by LC-MS, i.e.
appearance of molecular ion peak, m/z 593[M−H]–. Finally, 9 and 11 were identiﬁed asvitexin 2″-O-glucoside and isovitexin 2″-Oglucoside (Fig. 5) by TLC and HPLC comparisons with authentic standards. It was shown by
UV, hot acid treatment and LC-MS that 12
and 13 are 5,7,3′,4′-tetrahydroxylated ﬂavone
C-monohexosides. Thus, they were identiﬁed asorientin (5,7,3′,4′-tetrahydroxyflavone 8-Cglucoside, 12) and isoorientin (5,7,3′,4′-tetrahydroxyﬂavone 6-C-glucoside, 13) (Fig. 6) by TLC
and HPLC comparisons with authentic samples.
Flavonoid 14 was unhydrolyzable by hot acidtreatment, showing that it is C-glycosylﬂavone.
Attachment of 2 mol hexose to 5,7,4′-trihydroxyﬂavone was shown by LC-MS, i.e. appearance ofmolecular ion peaks, m/z 595 [M＋H]＋ and 593[M−H]–. Finally, 14 was identiﬁed as vicenin-2(5,7,4′-trihydroxyﬂavone 6,8-di-C-glucoside) (Fig.
7) by TLC and HPLC comparison with authentic
sample. Similarly, 15 was estimated as lucenin-2(5,7,3′,4′-tetrahydroxyﬂavone 6,8-di-C-glucoside)(Fig. 7). UV spectral survey and hot acid treatment of 16 and 17 showed that they are C-glycosylﬂavones having free 5-, 7- and 4′-hydroxyl

Chemical structures of vitexin (left, 8) and isovitexin (right, 10).

Anthocyanins and Flavonoids from Amorphophallus titanum

Fig. 5.

41

Chemical structures of vitexin 2″-O-glucoside (left, 9) and isovitexin 2″-O-glucoside (right, 11).

Fig. 6.

Chemical structures of orientin (left, 12) and isoorientin (right, 13).

Fig. 7.

Chemical structures of vicenin-2 (left, 14) and lucenin-2 (right, 15).

groups. By LC-MS survey of their compounds,
molecular ion peaks, m/z 565 [M＋H]＋ and 563[M−H]–, appeared on the chromatograms, showing that they are apigenin monopentosylhexoside. It was cleared that they are schaftoside (apigenin 6-C-glucoside-8-C-arabinoside, 16) andisoschaftoside (apigenin 6-C-arabinoside-8-Cglucoside, 17) by TLC and HPLC comparisonswith authentic samples (Fig. 8). Flavonoid 18
produced isovitexin and rhamnose by acid hydrolysis. Since UV spectral properties of originalcompound were essentially the same with those ofisovitexin, rhamnosyl moiety was considered to be
attached to 6-C-glucosyl group of isovitexin.
From the results described above, 18 was characterized as isovitexin X″-O-rhamnoside. However,interglycosidic linkage could not be determined.

To ﬂavonoid 19 is trihydroxy-monomethoxyﬂavone dihexoside proved by LC-MS, i.e. appearance of molecular ion peak, m/z 625 [M＋H]＋.
Isoscoparin (5,7,4′-trihydroxy-3′-methoxyﬂavone
6-C-glucoside) and glucose were liberated byacid hydrolysis of 19. Since UV spectral properties of 19 were the same with those of isoscoparin, it was shown that O-glucose is attached to
6-C-glucosyl group. Thus, 19 was characterized
as isoscoparin X″-O-glucoside. UV spectral survey in addition of various shift reagents showedthat 20 is 7,3′-substituted luteolin (Mabry etal., 1970). Chrysoeriol (5,7,4′-trihydroxy-3′methoxyﬂavone) and glucose were produced byacid hydrolysis. Since molecular ion peaks, m/z
463 [M＋H]＋ and 461 [M−H]–, appeared on thechromatogram of LC-MS, it was shown that

Tsukasa Iwashina et al.

42

Fig. 8.

Chemical structures of schaftoside (left, 16) and isoschaftoside (right, 17).

Fig. 9. Chemical structure of chrysoeriol 7-O-glucoside (20).

1 mol glucose is attached to chrysoeriol. Thus, 20
was identiﬁed as chrysoeriol 7-O-glucoside (Fig.
9). Flavonoid 21 was only one ﬂavonol from theinﬂorescence of A. titanum. Kaempferol, rhamnose and galactose were liberated by acid hydrolysis of 21. UV spectral properties of the compound showed that this is 3-substituted
kaempferol. Molecular ion peak, m/z 593[M−H]–, appeared on the chromatogram of
LC-MS, showing the attachment of each 1 molrhamnose and galactose to kaempferol. Finally, 21
was identiﬁed as kaempferol 3-O-robinobioside[＝3-O-rhamnosyl-(1→6)-galactoside] (Fig. 10)
by TLC and HPLC comparison with authentic
sample. Though the presence of other ﬂavonoids
was noticed by HPLC analysis of the crude
extract of A. titanum, they could not be isolated
for small amounts.
Flavonoids in the inﬂorescences of A. rivieri and
A. paeoniifolius
Five ﬂavonoids were isolated from the inﬂorescence of A. rivieri. On the other hand, sevenﬂavonoids were identiﬁed from A. paeoniifolius.
Of the ﬂavonoids in A. rivieri, three were identiﬁed as isovitexin (10), orientin (12) and isoorientin (13). Flavonoid 22 liberated quercetin and

Fig. 10. Chemical structure of kaempferol 3-Orobinobioside (21).

glucose by acid hydrolysis. Since UV spectral
properties of original compound showed that 22
is 3-substituted quercetin, it was proved that glucose is attached to 3-position of quercetin.
Finally, 22 was identiﬁed as quercetin 3-O-glucoside (isoquercitrin) (Fig. 11) by TLC andHPLC comparison with authentic sample. Flavonoid 23 was obtained as pale yellow powder(yield, ca. 30 mg). Vitexin and xylose were foundas acid hydrolysates of 23. Attachment of 1 mol
xylose to 8-C-glucosyl group of vitexin wasshown by m/z 565 [M＋H]＋ and 563 [M−H]– ofLC-MS and UV spectral properties. In 1H NMR,
six aromatic proton signals, δ 6.82, 6.74, 8.25
and 7.24 derived from H-3, H-6, H-2′,6′ andH-3′,5′, and two anomeric proton signals, δ 5.84
(d, J＝9.2 Hz) and 5.01 (brs) derived from glucose and xylose, appeared. The interglycosidic
linkage was determined as xylosyl-(1→2)-glucose
from HMBC correlation between xylosyl anomeric proton at δ 5.01 and C-2 signal of 8-Cglucose at δ 82.0. Thus, 23 was identiﬁed asapigenin 8-C-β-xylopyranosyl-(1→2)-glucopyranoside (vitexin 2″-O-xyloside) (Fig. 12).

Anthocyanins and Flavonoids from Amorphophallus titanum

Fig. 11.

43

Chemical structures of quercetin 3-O-glucoside (left, 22) and kaempferol 3-O-glucoside (light, 24).

Fig. 12. Chemical structure of vitexin 2″-O-xyloside(23).

Of seven ﬂavonoids isolated from the inﬂorescence of A. paeoniifolius, six were identiﬁed asvitexin (8), isovitexin (10), orientin (12),vicenin-2 (14), schaftoside (16) and quercetin
3-O-glucoside (22). Acid hydrolysis of anotherﬂavonoid 24 liberated kaempferol and glucose.
Attachment of 1 mol glucose to 3-position of
kaempferol was determined by LC-MS and UV
spectra. Finally, 24 was identiﬁed as kaempferol
3-O-glucoside (astragalin) (Fig. 11) by TLC andHPLC comparison with authentic sample. In A.
paeoniifolius (＝A. campanulatus), quercetin anda rare ﬂavonol, diacetyltambulin, have been isolated from the corm and tuberous roots (Sharstry
et al., 2010; Khan et al., 2008). However, they
were not found in this survey.
Distribution of anthocyanins and other ﬂavonoids in the inﬂorescences of three Amorphophallus species
The anthocyanins and other ﬂavonoids isolated from three Amorphophallus species areshown in Table 1. In A. titanum, ﬂavonoids wereseparately extracted from the spathe and spadix
appendix. Though anthocyanin composition of
their organs was qualitatively the same, other ﬂa-

vonoid composition was different. In the spadix
appendix, all ﬂavonoids which were found in A.
titanum in this survey were present. However,two ﬂavonoids only, vitexin (8) and orientin (12),were isolated from the spathe. This may bemeans the difference of the function of the ﬂavonoids between the spathe and spadix appendix of
A. titanum. Among the three Amorphophallus
species used as plant materials, their ﬂavonoid
pattern was different to each other. However,their major ﬂavonoids are C-glycosylﬂavones,
together with minor ﬂavonols. Anthocyanins andother ﬂavonoids have been surveyed in many
species of the family Araceae, and it has beenreported that major anthocyanins are cyanidin
3-O-rutinoside and 3-O-glucoside (Williams etal., 1981). Furthermore, C-glycosylﬂavones were
found in many Araceous species with minor ﬂavonols, kaempferol and quercetin glycosides(Williams et al., 1981). In A. titanum, thoughchrysoeriol 7-O-glucoside (20) was found as
only one ﬂavone O-glycoside, another ﬂavone
O-glycoside, luteolin 7-O-glucoside, has beenreported from the leaves of Colocasia esculenta(Iwashina et al., 1999). Thus, the anthocyanin
and other ﬂavonoid characters of three Amorphophallus species, A. titanum, A. rivieri and A. paeoniifolius show the same patterns with those ofmany Araceous species.
References
Gallori, S., Giaccherini, C., Bilia, A. R., Mulinacci, N.
and Vincieri, F. F. 2004. Identiﬁcation of anthocyaninsin Amorphophallus titanum (Becc.) Becc. Chromatographia 59: 647–651.
Iwashina, T., Matsumoto, S. and Nakaike, T. 1995. Flavonoid characters of ﬁve Adiantum species in Pakistan.

44

Tsukasa Iwashina et al.

Table 1. Anthocyanins and other ﬂavonoids isolated from the inﬂorescences of three Amorphophallus species
A. titanum (spathe)
Anthocyanins: cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, peonidin 3-O-glucoside, peonidin 3-O-rutinoside,pelargonidin 3-O-rhamnosylglucoside
C-Glycosylﬂavones: vitexin, orientin
A. titanum (spadix appendix)
Anthocyanins: cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, peonidin 3-O-glucoside, peonidin 3-O-rutinoside,pelargonidin 3-O-rhamnosylglucoside
C-Glycosylﬂavones: vitexin, vitexin 2″-O-glucoside, isovitexin, isovitexin 2″-O-glucoside, orientin, isoorientin,
vicenin-2, lucenin-2, schaftoside, isoschaftoside, isovitexin X″-O-rhamnoside, isoscoparin X″-O-glucoside
Flavone: chrysoeriol 7-O-glucoside
Flavonol: kaempferol 3-O-robinobioside
A. rivieri (inﬂorescence)
Anthocyanins: cyanidin 3-O-glucoside, cyanidin 3-O-rutinoside, peonidin 3-O-glucoside, peonidin 3-O-rutinoside,pelargonidin 3-O-rhamnosylglucoside
C-Glycosylﬂavones: isovitexin, orientin, isoorientin, vitexin 2″-O-xyloside
Flavonol: quercetin 3-O-glucoside
A. paeoniifolius (inﬂorescence)
Anthocyanins: cyanidin 3-O-glucoside, cyanidin 3-O-diglucoside, pelargonidin 3-O-glucoside
C-Glycosylﬂavones: vitexin, isovitexin, orientin, vicenin-2, schaftoside
Flavonols: kaempferol 3-O-glucoside, quercetin 3-O-glucoside

In: Watanabe, M. and Hagiwara, H. (eds.), Cryptogamsof the Himalayas, Vol. 3. Nepal and Pakistan. Department of Botany, National Science Museum, Tsukuba.
pp. 179–191.
Iwashina, T., Kamenosono, K. and Yabuya, T. 1996. Isolation and identiﬁcation of ﬂavonoid and related compounds as co-pigments from the ﬂowers of Iris ensata.
Journal of Japanese Botany 71: 281–287.
Iwashina, T., Konishi, T., Takayama, A., Fukada, M. andOotani, S. 1999. Isolation and identiﬁcation of the ﬂavonoids in the leaves of taro. Annals of the Tsukuba
Botanical Garden 18: 71–74.
Iwashina, T., Kitajima, J. and Matsumoto, S. 2006. Flavonoids in the species of Cyrtomium (Dryopteridaceae)
and related genera. Biochemical Systematics and Ecology 34: 14–24.
Iwashina, T., Githiri, S. M., Benitez, E. R., Takemura, T.,
Kitajima, J. and Takahashi, R. 2007. Analysis of ﬂavonoids in ﬂower petals of soybean near-isogenic lines
for ﬂower and pubescence color genes. Journal of
Heredity 98: 250–257.
Iwashina, T., Saito, Y., Peng, C.-I, Yokota, M. andKokubugata, G. 2008a. Foliar ﬂavonoids from two
Begonia species in Japan. Bulletin of the National
Museum of Nature and Science, Series B 34: 175–181.
Iwashina, T., López-Saez, J. A. and Kitajima, J. 2008b.
Flavonoids from Osyris alba. Biochemical Systematics
and Ecology 36: 146–147.
Iwashina, T., Matsumoto, S., Kitajima, J., Nakamura, T.,
Kokubugata, G., Suleiman, M. and Said, I. M. 2010.
Apigenin di- and trirhamnoside from Asplenium normale in Malaysia. Natural Product Communications 5:
39–42.

Iwashina, T., Setoguchi, H. and Kitajima, J. 2011. Flavonoids from the leaves of Vitex rotundifolia (Verbenaceae), and their qualitative and quantitative comparison
between coastal and inland population. Bulletin of the
National Museum of Nature and Science, Series B 37:
87–94.
Khan, A., Rahman, M. and Islam, M. S. 2008. Antibacterial, antifungal and cytotoxic activities of 3,5-diacetyltambulin isolated from Amorphophallus campanulatus
Blume ex Decne. Daru 16: 239–244.
Mabry, T. J., Markham, K. R. and Thomas, M. B. 1970.
The Systematic Identiﬁcation of Flavonoids. Springer,
Berlin.
Murai, Y., Kokubugata, G., Yokota, M., Kitajima, J. andIwashina, T. 2008. Flavonoids and anthocyanins fromsix Cassytha taxa (Lauraceae) as taxonomic markers.
Biochemical Systematics and Ecology 36: 745–748.
Murai, Y., Setoguchi, H., Ono, E. and Iwashina, T. 2014.
Flavonoids and their qualitative variation in Calystegia
soldanella and related species (Convolvulaceae). Natural Product Communications, in press.
Ohashi, H. 1982. Araceae. In: Satake, Y., Ohwi, J., Kitamura, S., Watari, S. and Tominari, T. (eds.), Wild Flowers of Japan, pp. 127–139. Heibonsha, Tokyo.
Sharstry, R. A., Biradar, S. M., Mahadevan, K. M. andHabbu, P. V. 2010. Isolation and characterization of
secondary metabolite from Amorphophallus paeoniifolius for hepatoprotective activity. Research Journal of
Pharmaceutical, Biological and Chemical Sciences 1:
429–437.
Williams, C. A., Harborne, J. B. and Mayo, S. J. 1981.
Anthocyanin pigments and leaf ﬂavonoids in the family
Araceae. Phytochemistry 20: 217–234.