Bull. Natl. Mus. Nat. Sci., Ser. B, 34(1), pp. 53–62, March 21, 2008

Quantitative Variation of Anthocyanins and Other Flavonoids
in Autumn Leaves of Acer palmatum
Tsukasa Iwashina1,2* and Yoshinori Murai2
1

Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba 305–0005, Japan
2
Graduate School of Agriculture, Ibaraki University, Ami 300–0393, Japan* Corresponding author: E-mail: iwashina@kahaku.go.jp

Abstract Anthocyanins and other flavonoids in autumn leaves of Acer palmatum were qualitatively and quantitatively surveyed. Cyanidin 3-O-glucoside (chrysanthemin) was found in autumn
leaves of Acer palmatum as a major pigment, together with a minor cyanidin glycoside. Six C-glycosylflavones were also isolated as other flavonoids and characterized as isoorientin, orientin, isovitexin, vitexin, and acylated isoorientin and orientin. The anthocyanin content increased with theprogress of coloration of the leaves and decrease of chlorophyll and carotenoid contents. On theother hand, C-glycosylflavones gradually decreased. Thus, it was shown that the anthocyanins andC-glycosylflavones were independently synthesized to each other in autumn leaves, in spite of thesame biosynthetic pathway, i.e., shikimate pathway.
Key words : Acer palmatum, anthocyanins, autumn leaves, chrysanthemin, C-glycosylflavones.

Introduction
The genus Acer is famous for the beautiful
coloring of the leaves in autumn. It has been reported by Hattori and Hayashi (1937) for the firsttime that anthocyanin, cyanidin 3-O-glucoside,i.e. chrysanthemin, is a major pigment in autumn
leaves of Acer circumlobatum Maxim. (A.
japonicum Thunb.) and A. ornatum Carr. var.
matsumurae (Koidz.) Koidz. (A. amoenum
Carr. var. matsumurae (Koidz.) Ogawa). Later, Ji
et al. (1992a) have surveyed the autumn coloredand spring sprouted leaves of 119 Acer taxa by
HPLC, and showed that the major anthocyanin iscyanidin 3-O-glucoside as the cases of theautumn colored leaves, together with cyanidin 3O-rutinoside, 3-O-galloylglucoside and 3,5-di-Oglucoside and 3-O-galloylrutinoside as minor
pigments. Moreover, chrysanthemin has been
proved to be major anthocyanin in autumn leavesof the various plant species (Hayashi and Abe,
1955; Murrell and Wolf, 1969; Ishikura, 1972;
Yoshitama and Ishikura, 1988; Iwashina, 1996).
On the other hand, related cyanidin 3-O-galacto-side, i.e. idaein, was found in some plants, e.g.
Coteneaster horizontalis Decne., Rhaphiolepis
indica (L.) Lindle. ex Ker. var. umbellata (Thunb.
ex Murray) Ohashi (Rosaceae), Stuartia pseudocamellia Maxim. (Theaceae), Elliottia paniculata(Sieb. & Zucc.) Benth. & Hook. (Ericaceae) andso on, as major anthocyanin (Iwashina, 1996).
Twenty-six Acer species are native to Japan(Shimizu, 1989). Of their species, A. palmatum
Thunb. is most popular one and has most beautiful colored leaves in autumn, and many cultivars
have been grown as ornamentals. Two anthocyanins, cyanidin 3-O-glucoside and 3-O-rutinoside, were isolated from the colored leaves ofthe species, together with leucoanthocyanidins(Ishikura, 1972). Two acylated anthocyanins,
cyanidin 3-O-[2-O-(galloyl)-b -D-glucoside] and3-O-[2-O-(galloyl)-6-O-(L-rhamnosyl)-b -D-glucoside], were also isolated from the red springleaves (Ji et al., 1992b). Though the former pigment was also observed in autumn red leaves,major anthocyanin is cyanidin 3-O-glucoside as
well as other Acer species (Iwashina, 1996).
Four C-glycosylflavones, vitexin, isovitexin,

54

Tsukasa Iwashina and Yoshinori Murai

orientin and isoorientin, have been found ingreen leaves of A. palmatum as other flavonoid
compounds (Aritomi, 1962, 1963). A flavonol,
kaempferol was also detected from the leaves,together with ellagic acid (Bate-Smith, 1978).
The mechanism of autumn leaf coloration has
been reviewed by some authors (e.g. Hayashi,
1968; Takeda, 1983; Lee, 2002). However, qualitative and quantitative variation of otherflavonoids, e.g. flavones, flavonols, flavanones or
dihydroflavonols, which are synthesized on thesame biosynthetic pathway, i.e. shikimate pathway, was hardly surveyed, in relation with
autumn coloration of the leaves.
In this paper, we describe quantitative variation of anthocyanin, other flavonoids, and total
chlorophylls and carotenoids along with theprogress of autumn leaves coloration of Acerpalmatum.
Materials and Methods
Plant Materials
Acer palmatum Thunb., which was used in thisstudy (acc. No. TBG50382), is growing in the
Tsukuba Botanical Garden, National Museum of
Nature and Science, Tsukuba, Japan.
Isolation of flavonoids
Fresh leaves (12.5 g) of A. palmatum were collected in 26 Oct. 2006 and extracted with MeOH.
The concentrated extracts were applied to preparative paper chromatography using solvent systems: BAW (n-BuOH/HOAc/H2O  4 : 1 : 5,
upper phase), 15% HOAc and then BEW (nBuOH/EtOH/H2O  4 : 1 : 2.2). The isolated
flavonoids were purified by Sephadex LH-20 column chromatography using solvent system: 70%
MeOH. Of their flavonoids, four ones, F1 (ca.
50 mg), F3 (ca. 5 mg), F4 (ca. 10 mg) and F5(trace amount), were obtained as pale yellow
powders.
Quantitative HPLC analysis of anthocyanins andother flavonoids
Fresh leaves (each 0.2 g) of A. palmatum (each5 samples) were collected in 26 Oct., 2 Nov., 13
Nov., 18 Nov., 22 Nov, 25 Nov. and 29 Nov.,
2006, along with the progress of autumn coloration, and were extracted with 0.1% HCl
in MeOH (3 ml) for anthocyanins and MeOH
(3 ml) for other flavonoids. After filtration with
Maisyori-disc H-13-5 (Tosoh), the extracts wereanalysed by HPLC using Pegasil ODS (I.D.
6.0150 mm, Senshu Scientific Co. Ltd.), atflow-rate: 1.0 ml min1, detection: 190–700
nm, and eluent: MeCN/HOAc/H2O/H3PO4(6 : 8 : 83 : 3) for anthocyanins and MeCN/H2O/
H3PO4 (22 : 78 : 0.2) (Sol. I) and (15 : 85 : 0.2)
(Sol. II) for other flavonoids. Relative anthocyanin and other flavonoid contents were determined from the peak area of each compounds on
the HPLC chromatograms.
Quantitative visible spectral analysis of total
chlorophyll and carotenoid contents
The crude MeOH extracts, which were obtained for quantitative HPLC analysis (seeabove), were directly measured visible spectra(400–700 nm) by Shimadzu Multipurpose Spectrophotometer MPS-2000. Relative chlorophylland carotenoid contents were determined fromthe absorbance of l max 663 nm (chlorophylls) orthat of l max 446 nm (carotenoids).
Liquid chromatograph-mass spectra (LC-MS)
LC-MS were measured using a Pegasil-ODS(I.D. 2.0150 mm, Senshu Scientific Co. Ltd.),
at a flow-rate of 0.2 ml min1, eluting with
HCOOH/MeCN/H2O (5 : 15 : 80 or 22 : 5 : 73),
injection: 10 m l, ESI 4.5 kV, ESI 3.5 kV, 250°C.
Identification of anthocyanin and other flavonoids
Major anthocyanin from the leaves of A.
palmatum was identified as cyanidin 3-O-glucoside by direct HPLC comparison with authentic
chrysanthemin from Acer japonicum (Hattori and
Hayashi, 1937). Six flavonoid glycosides wereidentified by UV spectroscopy according to
Mabry et al. (1970), LC-MS, characterization ofacid hydrolysates in 12% HCl, 100°C, 30 min,

Anthocyanins and flavonoids in autumn leaves of Acer palmatum

26 Oct.

2 Nov.

13 Nov.

18 Nov.

22 Nov.

25 Nov.

29 Nov.

Fig. 1.

Color variation of the leaves of Acer palmatum with the progress of autumn coloration.

55

56

Tsukasa Iwashina and Yoshinori Murai

and direct TLC and HPLC comparisons withauthentic samples. TLC, UV, acid hydrolysis and
LC-MS data of the isolated flavonoids were as
follows.
Luteolin 6-C-glucoside (isoorientin, F1).
TLC: Rf 0.39 (BAW), 0.47 (BEW), 0.23 (15%
HOAc); UV — dark purple, UV/NH3 — yellow.
UV: l max (nm) MeOH 257 sh, 270, 347;
NaOMe 275, 330 sh, 406 (inc.); AlCl3 276,
422; AlCl3/HCl 262 sh, 278, 297, 358, 384 sh;NaOAc 268 sh, 277, 330 sh, 399; NaOAc/
H3BO3 268, 373. Acid hydrolysis: unhydrolyzable. LC-MS: m/z 449 [MH], 447 [MH]
(luteolin1 mol glucose).
Apigenin 6-C-glucoside (isovitexin, F2).
TLC: Rf 0.63 (BAW), 0.74 (BEW), 0.35 (15%
HOAc); UV — dark purple, UV/NH3 — dark
yellow. UV: l max (nm) MeOH 271, 334;
NaOMe 278, 331, 398 (inc.); AlCl3 278, 303,
354, 383 sh; AlCl3/HCl 278, 302, 347, 378 sh;
NaOAc 278, 311, 393; NaOAc/H3BO3 273,
345. Acid hydrolysis: unhydrolyzable. LC-MS:
m/z 433 [MH], 431 [MH] (apigenin
1 mol glucose).
Luteolin 8-C-glucoside (orientin, F3). TLC:
Rf 0.24 (BAW), 0.27 (BEW), 0.08 (15% HOAc);
UV — dark purple, UV/NH3 — bright yellow.
UV: l max (nm) MeOH 256, 267 sh, 349;
NaOMe 267 sh, 277, 328 sh, 407 (inc.);
AlCl3 271, 422; AlCl3/HCl 259, 297, 359,
386 sh; NaOAc 268 sh, 278, 326 sh, 397;NaOAc/H3BO3 266, 373. Acid hydrolysis:
unhydrolyzable. LC-MS: m/z 449 [MH], 447[MH] (luteolin1 mol glucose).
Apigenin 8-C-glucoside (vitexin, F4). TLC:
Rf 0.39 (BAW), 0.50 (BEW), 0.13 (15% HOAc);
UV — dark purple, UV/NH3 — dark yellow. UV:l max (nm) MeOH 270, 337; NaOMe 279,
329, 395 (inc.); AlCl3 276, 304, 353, 384 sh;AlCl3/HCl 277, 302, 350, 380 sh; NaOAc
279, 312, 392; NaOAc/H3BO3 271, 348. Acidhydrolysis: unhydrolyzable. LC-MS: m/z 433[MH], 431 [MH] (apigenin1 mol glucose).
Acylated orientin (F5). TLC: Rf 0.37
(BAW), 0.49 (BEW), 0.09 (15% HOAc); UV —dark purple, UV/NH3 — yellow. UV: l max (nm)
MeOH 270, 287 sh, 347; NaOMe 278, 320 sh,
407 (inc.); AlCl3 273, 302, 366, 426; AlCl3/
HCl 268, 296 sh, 359, 393 sh; NaOAc 277,
324, 394; NaOAc/H3BO3 271, 296 sh, 375.
Acid hydrolysis: orientin (major) and isoorientin(minor). LC-MS: m/z 601 [MH], 599[MH] (luteolineach 1mol glucose and unknown compound having molecular weight 152).
Acylated isoorientin (F6). TLC: Rf 0.49
(BAW), 0.61 (BEW), 0.06 (15% HOAc); UV —dark purple, UV/NH3 — yellow. UV: l max (nm)
MeOH 257 sh, 271, 350; NaOMe 277, 339,
415 (inc.); AlCl3 277, 300 sh, 365 sh, 422;
AlCl3/HCl 264 sh, 276, 296 sh, 361, 387 sh;
NaOAc 276, 322, 401; NaOAc/H3BO3 271,
300 sh, 378. Acid hydrolysis: isoorientin (major)
and orientin (minor). LC-MS: m/z 601 [MH],599 [MH] (luteolineach 1 mol glucose andunknown compound having molecular weight152).
Results and Discussion
Anthocyanins and other flavonoids in autumn
leaves of A. palmatum
In this survey, two anthocyanin peaks (A1 and
A2) appeared on the HPLC chromatograms (Fig.
2). Of their anthocyanins, A1 was the major pigment and identified as cyanidin 3-O-glucoside(Fig. 3), as reported by Iwashina (1996). Thoughminor A2 was not isolated, it may be cyanidin 3O-galloylglucoside, which has been isolated fromthis plant by Ji et al. (1992b). Cyanidin 3-O-glucoside was contained in 99.3% of total anthocyanin in the leaves of A. palmatum collected in
22 Nov. 2006 and recorded highest anthocyanin
content (Table 1). On the other hand, sevenflavonoid peaks appeared on the HPLC chromatograms (Fig. 4). Of their flavonoids, six ones
were isolated and four major ones were characterized as C-glycosylflavones, isoorientin (F1),
isovitexin (F2), orientin (F3) and vitexin (F4)(Fig. 3), which have already been reported fromthis species (Aritomi, 1962, 1963). Two minor
flavonoids (F5 and F6) were characterized as ori-

Anthocyanins and flavonoids in autumn leaves of Acer palmatum

57

Fig. 2. HPLC chromatogram of the anthocyanins in autumn leaves of Acer palmatum (22 Nov. 2006). Flow-rate:
1.0 ml min1; Detection: 530 nm; and Eluent: MeCN/HOAc/H2O/H3PO4 (6 : 8 : 83 : 3). A1Cyanidin 3-Oglucoside and A2Cyanidin glycoside.
Table 1.

Quantitative variations of anthocyanins, and total chlorophylls and carotenoids in Acer palmatum leaves.
Anthocyanins

Date

Total
chlorophylls

Totalcarotenoids

26 Oct.

1.231

2.401

2 Nov.

(1.05)
1.17(1.12)
2.15

13 Nov.

(1.00)
0.73(1.00)
1.68

18 Nov.

(0.62)
0.61(0.78)
1.46

22 Nov.

(0.52)
0.08(0.68)
0.99

25 Nov.

(0.07)
0.03(0.46)
0.79

29 Nov.

(0.03)
0.02(0.37)
0.67(0.02)(0.31)

A1

A2

Total
anthocyanins

138,8492
100.0%(0.24)
589,996
79.1%3(1.00)
2,738,880
95.4%(4.64)
3,240,981
99.0%(5.49)
5,396,960
99.3%(9.15)
4,739,052
99.6%(8.03)
3,944,404
99.5%(6.69)—

138,849

156,065
20.9%(1.00)
132,8302
4.6%(0.85)
32,9331.0%(0.21)
39,8290.7%(0.26)
19,8860.4%(0.13)
19,5410.5%(0.13)(0.19)
746,061(1.00)
2,871,710(3.85)
3,273,914(4.39)
5,436,789(7.29)
4,758,938(6.38)
3,963,945(5.31)

Each fresh leaves (0.2 g) was extracted with 0.1% MeOH–HCl (3 ml) for anthocyanins or MeOH (3 ml) for chlorophylls and carotenoids; measurement was at 530 nm (anthocyanins), 663 nm (chlorophylls) or 446 nm (carotenoids).
1
Absorbance at 663 nm (chlorophylls) or 446 nm (carotenoids).
2
Peak area at 530 nm.
( )Relative amounts of chlorophylls, carotenoids and anthocyanins as absorbance or peak area of the samples
collected in 2 Nov, 2006 is 1.00.
3Each anthocyanin percentage.
A1Cyanidin 3-O-glucoside, A2cyanidin glycoside.

58

Tsukasa Iwashina and Yoshinori Murai

Fig. 3. Chemical structures of the anthocyanin and C-glycosylflavones isolated from the leaves of Acerpalmatum.

entin and isoorientin, which were acylated by an
unknown substance having molecular weight
152, such as monohydroxymethyl benzoic acid,by LC-MS survey.
Anthocyanin and other flavonoid compositionin the leaves was qualitatively the same during all
sampling period.

Quantitative variation of anthocyanin and otherflavonoids with the progress of autumn leaf coloration
The variation of total anthocyanins, C-glycosylflavones, chlorophylls and carotenoids wasshown in Tables 1–3, and Figs. 5 and 6. Anthocyanin content increased with the progress ofautumn leaf coloration, and reached to maximum
amount (7.29 times as compared with that of theAnthocyanins and flavonoids in autumn leaves of Acer palmatum

59

Fig. 4. HPLC chromatogram of C-glycosylflavones in autumn leaves of Acer palmatum (22 Nov. 2006). Flowrate: 1.0 ml min1; Detection: 350 nm; and Eluent: MeCN/H2O/H3PO4 (22 : 78 : 0.2). F1Isoorientin,
F2Isovitexin, F3Orientin, F4Vitexin, F5Acyalted orientin, F6Acylated isoorientin and F7
Unknown flavonoid.

Fig. 5. Qualitative variations of total anthocyanins, other flavonoids, chlorophylls and carotenoids in autumn
leaves of Acer palmatum. Left and right axes show the relative level of anthocyanin and other flavonoid
contents.

leaves in 2 Nov. 2006) in 22 Nov. 2006 (Table 1
and Fig. 5). Thereafter, anthocyanin content
gradually decreased. Total chlorophyll andcarotenoid contents were also decreased. Especially, they were rapidly decreased in 22 Nov.
with increase of anthocyanin. On the other hand,
total C-glycosylflavone content gradually decreased, though it temporarily increased in 22

Nov. with increase of anthocyanin content. However, they ephemerally increased in the samples
collected in 22 Nov. 2006, in which anthocyanin
content maximally increased. Relative amount ofeach C-glycosylflavones did not basically varied
during the collection period. Seasonal variationof anthocyanins and other flavonoids in autumn
leaves has been investigated on aquatic plant,

F23,001,959
27.3%(0.48)
6,281,028
38.2%(1.00)
4,408,14030.9%(0.70)
3,681,992
29.4%(0.59)
4,823,225
29.6%(0.77)
3,221,10628.6%(0.51)
2,712,609
28.3%(0.43)

F1

5,054,5381
46.0%2(0.72)
7,041,040
42.8%(1.00)
6,263,892
44.0%(0.89)
5,542,055
44.3%(0.79)
7,077,709
43.4%(1.01)
5,209,621
46.2%(0.74)
4,163,022
43.4%(0.59)
1,351,310
12.3%(0.54)
2,521,542
15.3%(1.00)
2,094,037
14.7%(0.83)
1,745,989
14.0%(0.69)
2,255,561
13.8%(0.89)
1,348,229
12.0%(0.53)
1,312,825
13.7%(0.52)

F3
635,166
5.8%(13.00)48,8640.3%(1.00)
664,984
4.7%(13.61)
686,9075.5%(14.06)855,6945.2%(17.51)
613,585
5.4%(12.56)632,278
6.6%(12.94)F4F5495,615
4.5%(1.86)
267,045
1.6%(1.00)
385,203
2.7%(1.44)
402,756
3.2%(1.51)
591,201
3.6%(2.21)
439,4523.9%(1.64)
370,586
3.9%(1.38)

Other flavonoids

291,233
2.6%(1.76)
165,558
1.0%(1.00)
280,592
2.0%(1.69)
285,190
2.3%(1.72)
446,5072.7%(2.70)
298,747
2.6%(1.80)
263,971
2.7%(1.59)

F6

Quantitative variations of other flavonoids in Acer palmatum leaves.

163,644
1.5%(1.39)
117,5210.7%(1.00)
153,4941.1%(1.31)
159,959
1.3%(1.36)
252,923
1.6%(2.15)
147,724
1.3%(1.26)
144,561
1.5%(1.23)

F7

Each fresh leaves (0.2 g) was extracted with MeOH (3 ml); measurement was at 350 nm.
1
Peak area at 350 nm.
2Each flavonoid percentage.
( )Relative amounts of other flavonoids as peak area of the samples collected in 2 Nov, 2006 is 1.00.
F1Isoorientin, F2Isovitexin, F3Orientin, F4Vitexin, F5Acylated orientin, F6Acylated isoorientin and F7Unknown flavonoids.

29 Nov.

25 Nov.

22 Nov.

18 Nov.

13 Nov.

2 Nov.

26 Oct.

Date

Table 2.

(0.58)(0.69)
9.599,852(0.99)
11,278,464(0.76)
16,302,820(0.87)
12,504,848(1.00)
14,250,342(0.67)
16,442,598

10,993,465

Total

60
Tsukasa Iwashina and Yoshinori Murai

Anthocyanins and flavonoids in autumn leaves of Acer palmatum

61

Fig. 6. Qualitative variations of each C-glycosylflavones in autumn leaves of Acer palmatum. F1Isoorientin,
F2Isovitexin, F3Orientin, F4Vitexin, F5Acylated orientin, F6Acylated isoorientin and F7
Unknown flavonoid.

Egeria densa Planch. (Momose, 1998). The
leaves of the species are commonly green, but
the detached ones easily induces an rare anthocyanin, cyanidin 5-methyl ether 3-O-glucoside,i.e. erodenin, in sucrose solution under light(Momose et al., 1977; Momose, 1996). Flavone
O-glycoside, luteolin 7-O-diglucuronide, accompanied with erodenin and two luteolin derivatives
in colored leaves as other flavonoids (Momose,
1998). This compound was remained stable during incubation of detached leaves, but two luteolin derivatives and some unknown compounds
including caffeic acid derivatives were newly
synthesized with coloration.
Major aspects of the overall biosynthetic pathway to major groups of the flavonoids have fully
been described, and the main enzymes have been
demonstrated so far in cell-free extracts(Stafford, 1990). In the case of Egeria densa
leaves coloration, flavone O-glycosides wereaccompanied with increase of the anthocyanin(Momose, 1998). On the other hand, C-glycosylflavones were synthesized in the case of Acerpalmatum. Though O-glycosylation of theflavones was clearly proved to occur in the terminal of flavonoid biosynthesis, that of C-glycosy-

lation has hardly been reported (Stafford, 1990).
Kerschr and Franz (1987) have reported that a Cglucosyltransferase from Fagopyrum esculentum
Moench seedlings catalyzed the transfer of glucose from UDP-glucose or 2-hydroxyflavanones,e.g. 2,5,7,4-tetrahydroxyflavanone and 2,5,7-trihydroxyflavanone were appropriate substrates.
Moreover, naringenin, chalcononaringenin, apigenin and chrysin can not act as glucosyl acceptors in C-glycosylflavonoid biosynthesis. This
demonstrates that C-glucosylation occurs after
oxidation of flavanone. If such the case is thefact, C-glycosylation early occurs in flavonoid
biosynthesis. In contrast, anthocyanins are synthesized in later step. As the results, it was presumed that seasonal variation between anthocyanins and C-glycosylflavones does not correlate each other in the case of autumn leaves coloration of A. palmatum. Among the plant species
varied the leaves to red from green in autumn,
some species, e.g. Euonymus alatus (Thunb.)
Sieb. and Parthenocissus tricuspidata (Sieb. &Zucc.) Planch., are occurred flavonols such as
quercetin and kaempferol together with anthocyanin, cyanidin 3-O-glucoside (Ishikura, 1977).
The biosynthetic step of the anthocyanins and62

Tsukasa Iwashina and Yoshinori Murai

flavonols are comparatively near than that of Cglycosylflavones. The seasonal variation between
anthocyanins and flavonols should be surveyed asplant materials which synthesize flavonols but
not C-glycosylflavones in relation with autumn
leaves coloration.

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