Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol28.Issue9.Nov2000:

Biochemical Systematics and Ecology 28 (2000) 887}902

Anthocyanins in #owers of genus Rosa, sections
Cinnamomeae ("Rosa), Chinenses, Gallicanae and
some modern garden roses
Yuki Mikanagi!,*, Norio Saito", Masato Yokoi#,
Fumi Tatsuzawa#
!Natural History Museum and Institute, Chiba, 955-2 Aoba-cho, Chuo-ku, Chiba, 260-8682 Japan
"Meiji-gakuin University, Totsuka-ku, Yokohama, Kanagawa, 244-8539 Japan
#Faculty of Horticulture, Chiba University, 648 Matsudo, Chiba, 271-8510 Japan
Received 17 March 1999; accepted 15 November 1999

Abstract
Forty-four taxa of three sections (Cinnamomeae ("Rosa) 26, Chinenses 8 and Gallicanae 10)
and eight modern garden roses in the genus Rosa were surveyed for their #oral anthocyanins.
Eleven anthocyanins: 3-glucosides and 3,5-diglucosides of cyanidin (Cy), pelargonidin (Pg) and
peonidin (Pn), 3-rutinosides and 3-o-coumaroylglucoside-5-glucosides of Cy and Pn, and Cy
3-sophoroside, were isolated from #owers of these taxa and identi"ed by chemical and
spectroscopic techniques. Four anthocyanins: Cy 3-rutinoside, Pn 3-rutinoside, Pn 3-ocoumaroylglucoside-5-glucoside and Cy 3-sophoroside were found for the "rst time in Rosa
#owers.
Investigated sections of wild roses showed characteristic distribution of anthocyanins. Cy

3,5-diglucoside was the dominant anthocyanin detected in all three sections, but accumulation
of Pn 3,5-diglucoside distinguished sections Cinnamomeae from other sections, and the occurrence of Cy 3-glucoside separates section Chinenses from others.
Cy 3-sophoroside was detected in large amount in some taxa of section Cinnamomeae: e.g., R.
moyesii and its related cultivars, and R. rugosa cv. Salmon Pink. The acylated Cy glycoside was
found in all sections and also in some modern garden roses, while the acylated Pn glycoside was
detected in the section Cinnamomeae, but not in sections Chinenses and Gallicanae. According
to anthocyanin distribution patterns, eight groups were classi"ed chemotaxonomically in genus
Rosa. ( 2000 Elsevier Science Ltd. All rights reserved.

* Corresponding author. Tel.: #81-43-265-3111; fax: #81-43-266-2481.
E-mail address: mikanagi@chiba-muse.or.jp (Y. Mikanagi)
0305-1978/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 9 9 ) 0 0 1 2 7 - 1

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Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

Keywords: Rosa; Rosaceae; Floral anthocyanins; Cyanidin 3-sophoroside; Cyanidin 3-rutinoside; Peonidin
3-rutinoside; Acylated anthocyanins; Chemotaxonomy

1. Introduction
Flower colour investigation of roses so far have shown that four anthocyanins,
3-glucosides and 3,5-diglucosides of cyanidin (Cy) and peonidin (Pn), can be detected
in #owers of wild Rosa species, and also pelargonidin (Pg) 3-glucoside and Pg
3,5-diglucoside are detected in Rosa cultivars (WillstaK tter and Nolan, 1915; Harborne,
1961, 1967; Arisumi, 1963, 1967; Yokoi, 1974, 1975; Yokoi et al., 1979; Saito et al.,
1982; Mikanagi et al., 1990,1994,1995; Biolley et al., 1992, 1994a,b; Raimond et al.,
1995). In addition, one acylated anthocyanin, o-coumaroylcyanidin 3,5-diglucoside,
was reported to be present in R. cv. Frensham (Arisumi, 1967). To our knowledge, only
one paper has reported that Pg derivatives were found in some wild Rosa taxa
(Eugster and MaK rki-Fischer, 1991).
In our previous investigations we could not "nd Pg derivatives in wild taxa
(Mikanagi et al., 1995). We have already surveyed over 200 taxa of Rosa including
some old garden roses, and about 120 taxa contained anthocyanins. Wild taxa in
section Cinnamomeae seem to contain more unknown anthocyanins than other
sections, and we consider that a study of anthocyanins of section Cinnamomeae is
necessary. Therefore, in the present study, we have investigated the anthocyanins in
Rosa, the main purpose being the structural elucidation of these unknown compounds. In particular, we have examined the wild roses of section Cinnamomeae in
Japan, and compared them with those in other countries, as well as old garden roses

and some modern garden roses.
In the study, Cy 3-rutinoside, Pn 3-rutinoside, Pn 3-o-coumaroylglucoside-5glucoside and Cy 3-sophoroside were found for the "rst time in Rosa #owers, and in
total, eleven anthocyanins were detected. We wish to report the structure and
distribution of these pigments.

2. Materials and methods
2.1. Plant materials
Fresh or dried petals of 26 taxa in section Cinnamomeae: eight taxa in section
Chinenses, 10 taxa in section Gallicanae and eight modern garden roses in genus Rosa
were collected from various sources as listed in Table 1, arranged by sections and
groups. Names of species, varieties, hybrids and cultivars are those by Rehder (1949),
Hara (1957), Satake et al. (1989) and Cairns (1993), and they were con"rmed by YM.
Voucher specimens of wild roses were deposited at the Natural History Museum and
Institute, Chiba (CBM).

Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

889

Table 1

Plant materials (wild taxa, old garden roses and modern garden roses)
Taxa
Wild taxa and old garden roses
Section Cinnamomeae ("section Rosa)
(No. in Table 5)
Rosa acicularis Lindl. (R01)
R. arkansana Porter (R02)
R. bella Rehder and Wilson (R03)
R. cinnamomea L. (R04)
R. forrestiana Boulenger (R05)
R. marretii LeH v. (R06)

R. moyesii Hemsl. & Wilson (R07)
R. moyesii cv. Arthur Hillier (R08)
R. moyesii cv. Eddie's Crimson (R09)
R. moyesii cv. Fargesii (R10)
R. moyesii cv. Geranium (R11)
R. moyesii cv. Highdownensis (R12)
R. moyesii cv. Hillieri (R13)
R. nipponensis CreH p. (R14)

R. nutkana Presl. (R15)
R. pendulina var. oxyodon (Boissier)
Rehder (R16)
R. rugosa Thunb. ex Murray (R17)

R. rugosa var. plena Regel (R18)
R. rugosa cv. Maikwai (R19)
R. rugosa cv. Rosaraie de l'Hay (R20)
R. rugosa cv. Salmon Pink (R21)
R. rugosa cv. Scabrosa (R22)
R. rugosa cv. Scarlet (R23)
R. sweginzowii Koehne (R24)
R. willmottiae Hemsl. (R25)
R. x iwara Sieb. (R26)!
Section Chinenses
R. chinensis Jacq.
R. chinensis var. minima (Sims) Voss

Sources

Monbetsu, Hokkaido, Japan (wild)
Keisei Rose Nursery, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)
Monbetsu, Hokkaido, Japan (wild)
Sugadaira, Japan (wild)
Teshikaga, Japan (wild)
Keisei Rose Nursery, Japan (cultivated)
The Garden of Roses, UK (cultivated)
Keisei Rose Nursery, Japan (cultivated)
The Garden of Roses, UK (cultivated)
Keisei Rose Nursery, Japan (cultivated)
The Garden of Roses, UK (cultivated)
Keisei Rose Nursery, Japan (cultivated)
The Garden of Roses, UK (cultivated)
The Garden of Roses, UK (cultivated)
Keisei Rose Nursery, Japan (cultivated)
The Garden of Roses, UK (cultivated)

The Garden of Roses, UK (cultivated)
Mt. Fuji, Japan (wild)
Mt. Haku-san, Japan (wild)
Mt. Tsurugi-san, Japan (wild)
Mt. Shibutsu-san, Japan (wild)
Keisei Rose Nursery, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)
Sado Isl., Japan (wild)
Abashiri, Hokkaido, Japan (wild)
Chiba University, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)
The Garden of Roses, UK (cultivated)
Keisei Rose Nursery, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)
The Garden of Roses, UK (cultivated)
Keisei Rose Nursery, Japan (cultivated)
Sado Isl., Japan (wild)

Keisei Rose Nursery, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)
(continued on next page)

890

Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

Table 1 (continued)
Taxa
R.
R.
R.
R.
R.
R.

chinensis
chinensis

chinensis
chinensis
chinensis
chinensis

Sources
var. spontanea Rehder & Wilson
cv. Fabvier
cv. Miss Lowe
cv. Mutabilis
cv. Pompon de Paris
cv. Slater's Crimson China

Section Gallicanae
R. gallica L.
R. gallica cv. Cardinal de Rich "
: lieu
R. gallica cv. Rosa Mundi
R. gallica cv. Shigyoku
R. gallica cv. Violacea

R. x centifolia cv. Bullata
R. x centifolia cv. Muscosa
R. x damascena Miller
R. x damascena cv. Bifera
R. x damascena cv. Gloire de Guilan
Modern garden roses
Hybrid Tea
R. cv. La France
R. cv. OleH
R. cv. Papa Meilland

Keisei
Keisei
Keisei
Keisei
Keisei
Keisei

Rose
Rose

Rose
Rose
Rose
Rose

Nursery,
Nursery,
Nursery,
Nursery,
Nursery,
Nursery,

Japan
Japan
Japan
Japan
Japan
Japan

(cultivated)
(cultivated)
(cultivated)
(cultivated)
(cultivated)
(cultivated)

Keisei
Keisei
Keisei
Keisei
Keisei
Keisei
Keisei
Keisei
Keisei
Keisei

Rose
Rose
Rose
Rose
Rose
Rose
Rose
Rose
Rose
Rose

Nursery,
Nursery,
Nursery,
Nursery,
Nursery,
Nursery,
Nursery,
Nursery,
Nursery,
Nursery,

Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan

(cultivated)
(cultivated)
(cultivated)
(cultivated)
(cultivated)
(cultivated)
(cultivated)
(cultivated)
(cultivated)
(cultivated)

R. cv. Seika

Keisei Rose Nursery, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)
Tokyo Metropolitan Jindai Botanical Park (cultivated)
Tokyo Metropolitan Jindai Botanical Park (cultivated)

Floribunda
R. cv. Frensham
R. cv. Orange Bunny

Keisei Rose Nursery, Japan (cultivated)
Tokyo Metropolitan Jindai Botanical Park (cultivated)

Miniature and Polyantha
R. cv. Red Meillandina
R. cv. The Fairy

Keisei Rose Nursery, Japan (cultivated)
Keisei Rose Nursery, Japan (cultivated)

!R. x iwara is considered to be a natural hybrid between R. rugosa and R. multiyora Thunb. ex Murray
(section Synstylae)

2.2. Isolation of acylated anthocyanins
Fresh #owers (ca. 1.0 kg) of Rosa cv. Red Meillandina were extracted with MAW
(10l; MeOH}HOAc}H O, 9 : 1 : 10). The extract was concentrated to 500 ml. The
2
concentrated extract was puri"ed by Diaion HP-20 CC, PC, TLC and HPLC as
previously reported (Saito et al., 1995). Solvents used were 15% HOAc, BAW
(n-BuOH}HOAc}H O, 4 : 1 : 5), 5% HOAc}MeOH and MAW for CC, PC and
2
TLC. Prep. HPLC was run on a Waters C (19/]150 mm) column at 403C with
18
a #ow rate of 4 ml min~1 monitoring at 530 nm. Solvent system used a linear gradient
elution for 40 min from 40 to 85% solvent B (1.5% H PO , 20% HOAc, 25% MeCN
3
4
in H O) in solvent A (1.5% H PO in H O). The evaporated residues were dissolved
2
3
4
2

Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

891

in a small volume of 5% HOAc}EtOH followed by the addition of excess of Et O,
2
and then dryed to give pigment powder: Cy 3-o-coumaroylglucoside-5-glucoside ca.
10 mg, Pn 3-o-coumaroylglucoside-5-glucoside ca. 5 mg.
2.3. Preparation of other anthocyanins
Three anthocyanins were also puri"ed from R. cv. Red Meillandina extracts as
follows; Cy 3,5-diglucoside ca. 30 mg, Cy 3-glucoside ca. 5 mg and Pn 3,5-diglucoside
ca. 6 mg. The other six pigments were extracted from dry or fresh petals of R. moyesii
and its cultivars and R. cv. Orange Bunny (ca. 30}80 g) with MAW at room temperature and "ltered. After concentration the extact, the pigments were puri"ed by
preparative PC (BAW and 15% HOAc) and TLC (BAW and 15% HOAc). Then, six
pigments (ca. 0.1}10 mg) were obtained as follows; Cy 3-sophoroside and Pn 3glucoside were obtained from the mixed red #owers of R. moyesii and its cultivars. Cy
3-rutinoside and Pn 3-rutinoside were obtained from R. moyesii cv. Arthur Hillier. Pg
3,5-diglucoside and Pg 3-glucoside were obtained from orange-red #owers of R. cv.
Orange Bunny.
2.4. Analyses of anthocyanins
Fresh or dried Rosa petals (fresh weight of each taxon ca. 1}3 g) were extracted with
HOAc}MeOH-H O (1 : 10 : 9) and the extracts were analyzed for the anthocyanin
2
distribution. The anthocyanin distribution in each taxon was examined by HPLC and
2D-TLC with comparison to authentic anthocyanins (Mikanagi et al., 1995).
HPLC was run on a Waters C18 (4.6/]250 mm) column at 403C with a #ow rate
of 1 ml min~1 monitoring at 530 nm for anthocyanins. Solvent system used were as
follows: a linear gradient elution for 85 min from 20 to 80% solvent B (1.5% H PO ,
3
4
20% HOAc, 25% MeCN in H O) in solvent A (1.5% H PO in H O). Retention
2
3 4
2
times of anthocyanins detected in this study are shown in Table 2.
2D-TLC was carried out on Avicel cellulose plates (Funakoshi) in BuHCl (nBuOH}2M-HCl 1 : 1, top layer) for the "rst direction and AHW (HOAc}HCl}H O
2
15 : 3 : 82) for the second. Identi"cation of the structure of these anthocyanins was
based on the standard methods of TLC, HPLC, UV spectroscopy and NMR spectroscopy (Harborne, 1967, 1984; Saito and Harborne, 1992; Saito et al., 1995; Tatsuzawa
et al., 1996.).
2.5. Reference anthocyanins
Reference samples of Cy 3,5-diglucoside, Cy 3-sophoroside, Cy 3-rutinoside, Cy
3-glucoside, Cy 3-o-coumaroylglucoside-5-glucoside, Pn 3,5-diglucoside, Pn 3-rutinoside, Pn 3-glucoside, Pn 3-o-coumaroylglucoside-5-glucoside, Pg 3,5-diglucoside and
Pg 3-glucoside were available to use, and were fully identi"ed by spectroscopic and
chemical methods. Cis-isomers of the acylated anthocyanins were detected by the
process of Saito and Harborne (1992).

892

Anthocyanins!

2
5
7
9
10"
3
6
8
11#
1
4

Rf values (]100)

Spectral data in 0.1% HCl}MeOH

BAW

BuHCl

1% HCl

AHW

j max (nm)

E /E
(%)
440 .!9

AlCl
3

24
10
23
19
31
27
20
24
33
30
23

15
4
15
14
22
20
5
9
25
30
7

4
7
5
20
6
5
10
11
8
7
17

14
22
33
40
23
18
30
33
28
22
36

269,
270,
282,
283,
280,
280,
278,
279,
270,
269,
267,

24
16
24
29
19
28
13
29
15
43
21

#
#
#
#
#
0
0
0
0
0
0

528
526
531
526
310, 525
527
524
528
310, 525
509
507

HPLC
Rt (min)

FAB-MS
[M]`

37.4
33.1
39.6
35.0
49.2, 56.0
43.5
19.4
37.8
54.0, 60.9
41.8
35.4

449
611
*
611
757
463
625
*
771
433
595

!For key to abbreviation, see Materials and Methods: (1) Pg 3-glucoside; (2) Cy 3-glucoside; (3) Pn 3-glucoside; (4) Pg 3,5-diglucoside; (5) Cy 3,5-diglucoside; (6)
Pn 3,5-diglucoside; (7) Cy 3-rutinoside; (8) Pn 3-rutinoside; (9) Cy 3-sophoroside; (10) Cy 3-o-coumaroylglucoside-5-glucoside; (11) Pn 3-o-coumaroylglucoside-5-glucoside.
"HPLC Rt 49.2 min"cis-form, 56.0"trans-form.
#HPLC Rt 54.0 min"cis-form, 60.9"trans-form.

Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

Table 2
Chromatographic and spectral properties of anthocyanins in #owers of genus Rosa

Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

893

2.6. Mass and NMR spectroscopy
Positive and negative ion FAB mass spectra of pigments were measured by the
JEOL JMS SX-102 mass spectrometer, positive FAB-MS in HCl}MeOH#glycerol
and negative FAB-MS in glycerol. The detailed structures of Cy 3-sophoroside and
3-o-coumaroylglucoside-5-glucoside of Cy and Pn were determined by the analysis of
1H-NMR and 1H-1H COSY spectra. 1H-NMR spectra of pigments were measured by
JEOL FX-400 spectrometer in 10% TFA-90% DMSO-d .
6
3. Results and discussion
In the survey of anthocyanins in Rosa #owers of 52 taxa by HPLC analysis, 11
major anthocyanin peaks were observed as shown in Fig. 1, and Tables 2 and
4 (pigments 1}11). Among them, the 3-glucosides and 3,5-diglucosides of Cy, Pn and
Pg (1, 2, 3, 4, 5 and 6) were con"rmed with authentic samples and con"rmed the
former studies (Arisumi, 1963, 1967; Yokoi, 1974, 1975; Yokoi et al., 1979; Saito et al.,
1982 and Mikanagi et al., 1994). In most taxa, Cy 3,5-diglucoside (5) was detected as
the major constituent. However, some taxa in section Cinnamomeae contained larger
amounts of Pn 3,5-diglucoside (6) than 5, and some taxa in section Chinenses
contained larger amounts of Cy 3-glucoside (2) than 5.
The detailed structure of Cy 3-o-coumaroylglucoside-5-glucoside (10) which was
reported as o-coumaroylcyanin by Arisumi (1967) was determined unambiguously in

Fig. 1. Rosa anthocyanins.
1 pelargonidin 3-glucoside, R
"H
1,2,3,4
2 cyanidin 3-glucoside, R
"H, R "OH
2,3,4
1
3 peonidin 3-glucoside, R
"H, R "OCH
2,3,4
1
3
4 pelargonidin 3,5-diglucoside, R
"H, R "glucosyl
1,3,4
2
5 cyanidin 3,5-diglucoside, R "H, R "OH, R "glucosyl
3,4
1
2
6 peonidin 3,5-diglucoside, R "H, R "OCH , R "glucosyl
3,4
1
3 2
(Additional Rosa anthocyanins)
7 cyanidin 3-rutinoside, R "H, R "OH, R "rhamnosyl
2,4
1
3
8 peonidin 3-rutinoside, R "H, R "OCH , R "rhamnosyl
2,4
1
3 3
9 cyanidin 3-sophoroside, R "H, R "OH, R "glucosyl
2,3
1
4
10 cyanidin 3-o-coumarylglucoside-5-glucoside, R "H, R "OH, R "glucosyl, R "o-coumaryl
4
1
2
3
11 peonidin 3-o-coumarylglucoside-5-glucoside, R "H, R "OCH , R "glucosyl, R "o4
1
3
2
3
coumaryl

894

Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

this study. Furthermore, the occurrence of four anthocyanins, Cy 3-rutinoside (7), Pn
3-rutinoside (8), Cy 3-sophoroside (9) and Pn 3-o-coumaroylglucoside-5-glucoside
(11), are recorded for the "rst time in genus Rosa (Fig. 1). Contents of 7, 8, 10 and 11
were small, but 9 accumulated in large amounts in some taxa in section Cinnamomeae.
The distribution of anthocyanins in each section is described in detail.
3.1. Structural elucidation of anthocyanins
Eleven anthocyanins were isolated in pure form from MAW extracts of the #owers
of Rosa, by repeated chromatography on Diaion HP-20, preparative PC, TLC and
HPLC. On acid hydrolysis, 2, 5, 7, 9 and 10 produced cyanidin as the aglycone, 3, 6,
8 and 11 produced peonidin, and 1 and 4 produced pelargonidin. As sugar moieties,
all pigments contained glucose, and additionally, 7 and 8 contained rhamnose with
glucose, 10 and 11 contained o-coumaric acid as the acyl component. Their chromatographic and spectral properties are shown in Table 2. Pigments 1}6 were easily
identi"ed by the analysis of the chromatographic and spectral data with authentic
anthocyanins as follows: Pg 3-glucoside (1), Cy 3-glucoside (2), Pn 3-glucoside (3), Pg

Table 3
1H-NMR data of o-coumaroylanthocyanins and cyanidin 3-sophoroside in #owers of genus Rosa using
DMSO-d
6
H
Anthocyanin
4
6
8
2@
5@
6@
-OCH
3
o-coumaric acid
2.6
3.5
a
b
Glucose"
1
2
3
4
5
6a
6b

10!

11!

8.79
6.97
7.00
8.00
7.04
8.22

s
brs
brs
brs
d
brd

7.37
6.57
6.26
7.36
[A]
5.48
3.61
3.50
3.24
3.37
3.93
4.33

d
d
d
d

(8.6)
(8.6)

(8.2)
(8.2)
(16.2)
(16.2)
[B]
5.06
3.49
3.37
3.22
3.49
3.68
3.76

9!

8.89
7.03
7.16
8.19
7.13
8.32
3.96

s
brs
brs
brs
d
brd
s

7.41
6.78
6.30
7.41
[A]
5.54
3.58
3.51
3.30
3.76
4.26
4.48

d
d
d
d

(8.1)
(8.1)

(8.1)
(8.1)
(15.0)
(15.0)
[B]
5.13
3.54
3.40
3.38
3.54
3.73
3.80

8.90
6.77
6.94
8.06
7.10
8.22

s
d
d
d
d
dd

[A]
5.63
4.00
3.67
3.11
3.48
3.89}3.72

(2.0)
(2.0)
(2.4)
(8.7)
(2.4, 8.7)

[C]
4.68
3.01
3.13
3.50
3.79}3.63

!(10) cyanidin 3-o-coumaroylglucoside-5-glucoside; (11) peonidin 3-o-coumaroylglucoside-5-glucoside;
(9) cyanidin 3-sophoroside.
"All the observed vicinal coupling constants were ca. 7.0}10.0 Hz.

Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

895

3,5-diglucoside (4), Cy 3,5-diglucoside (5) and Pn 3,5-diglucoside (6). Pigments 7 and
8 were identi"ed by HPLC only as Cy 3-rutinoside (7) and Pn 3-rutinoside (8). Among
these pigments, the 3-rutinosides of Cy (7) and Pn (8) were found for the "rst time in
the #owers of Rosa. Pigments 9, 10, 11 were identi"ed to be Cy 3-sophoroside (9), Cy
3-o-coumaroylglucoside-5-glucoside (10) and Pn 3-o-coumaroylglucoside-5-glucoside
(11) by similar process of pigments 1}8. Pigments 9 and 11 have not been reported in
Rosa previously.
Furthermore, the detailed structures of these three pigments (9, 10 and 11) were
con"rmed by the analysis of 1H-NMR and 1H-1H COSY spectra (Table 3). The
FAB-mass spectrum of 9 gave its molecular ion [M]` at 611 m/z C H O
25 27 16
(calculated, 611.529) indicating one molecule of cyanidin and two molecules of
glucose. In the 1H-NMR spectrum, the signals of six protons in the cyanidin moiety
were easily assigned by the analysis of the 1H-1H COSY spectrum of 9 (Table 3), and
those of the sugar moiety appeared in the region 3.13}5.63 ppm. Two typical doublets
were found to be two anomeric protons at d 5.63 (d, J"7.5 Hz, Glc A) and d 4.68 (d,
J"7.5 Hz, Glc C). Also, a proton at d 4.00 (t, J"8.3 Hz) being shifted to a lower "eld
was directly correlated to the H-1A of glucose A by analysis of its 1H-1H COSY
spectrum. Thus, this proton was assigned to the H-2A of Glc A. Therefore, Glc C is
attached to the OH-2 of Glc A through a glucosidic bond. Consequently, 9 was
determined to be Cy 3-[2-O-(b-D-glucopyranosyl)-b-D-glucopyranoside], which has
not been reported in Rosa.
The FAB-mass spectra of 10 and 11 gave their molecular ions [M]` at 757 and
771 m/z which correspond to C H O (calculated, 757.674) and C H O
36 37 18
37 39 18
(calculated, 771.701), respectively, indicating 10 to be composed of Cy, two molecules
of glucose and one molecule of o-coumaric acid, and also 11 to be composed of Pn,
two glucose and one o-coumaric acid. In the 1H-NMR spectra, both signals of
pigments 10 and 11 were superimposable except signals of the 3@-OCH of 11 as
3
shown in Table 3. Signals from the sugar moieties of 10 and 11 were observed in the
region of d 3.22}5.54, and all vicinal coupling constants of four glucose moieties were
at 7.0}10.0 Hz. The chemical shifts and the large coupling constants of four anomeric
protons appeared at d 5.48 (J"7.4 Hz, Glc A of 10), d 5.06 (J"7.4 Hz, Glc B of 10),
d 5.54 (J"7.4 Hz, Glc A of 11) and d 5.13 (J"7.3 Hz, Glc B of 11), showing four
glucose units to be b-D-glucopyranosides. The down"eld shift of the methylenes of Glc
A of 10 (d 3.93 and 4.33) and 11 (d 4.26 and 4.48) indicated the o-coumaroyl moieties to
be attached to the OH-6 of glucose A in both pigments. In the chemical shifts of
o-coumaroyl moieties of 10 and 11, the two pairs of ole"nic protons (10, d 6.26 and
7.36; 11 d 6.30 and 7.41) had large coupling constants (J"16.2, 10 and J"15.0, 11).
Thus, the ole"nic parts of both of the o-coumaric acid moieties have trans con"gurations. By H2O2 degradation of 10 and 11, o-coumaroylglucose was also detected by
TLC. Therefore, the structure of 10 was determined to be Cy 3-O-[6-O-(trans-ocoumaroyl)-b-D-glucopyranoside]-5-O-[b-D-glucopyranoside], and also the structure
of 11 was Pn 3-O-[6-O-(trans-o-coumaroyl)-b-D-glucopyranoside]-5-O-[b-D-glucopyranoside]. This paper reports for the "rst time that the o-coumaric acid groups in
acylated Rosa anthocyanins (10 and 11) are located at the OH-6 of their 3-glucose
residues.

896

Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

3.2. Distribution of Cy 3-sophoroside, Cy 3-rutinoside and Pn 3-rutinoside
In this survey 26 taxa of section Cinnamomeae, 24 taxa (92%) contained Cy
3-sophoroside (9), 14 taxa (54%) contained Cy 3-rutinoside (7), and 19 taxa (73%)
contained Pn 3-rutinoside (8) (Table 4). R. moyesii and its cultivars contained large
amounts of 9. In particular, the blood-red coloured #owers of R. moyesii cv. Geranium
contained 9 as 56% of total anthocyanins. This pigment is probably associated with
the special colour of that cultivar. One more taxa in section Cinnamomeae, R. rugosa
cv. Salmon Pink, contained 9 (8%). This cultivar is a wild mutant of R. rugosa which
was found in east Hokkaido, Japan, and identi"ed by Suzuki. We suspect that the
glycosylation enzyme for the 5-position of this mutant is lacking, and consequently it
accumulates 9 and Cy 3-glucoside (2) instead of Cy 3,5-diglucoside (5). The occurrence
of 9 was mainly restricted to section Cinnamomeae, but some old garden roses in
section Gallicanae also contained this pigment in small amounts.
Pigment 7 was detected in sections Cinnamomeae and Chinenses in small amounts.
Pigment 8 was found in almost every taxa we analyzed, but the content was small and
except for some taxa in section Cinnamomeae, could be detected by HPLC only. The
occurrences of these pigments 7, 8 and 9 is new for Rosa (Eugster and MaK rki-Fischer,
1991; Biolley et al., 1994a,b; Mikanagi et al., 1995). It is supposed that the reason these
pigments were not distinguished in earlier studies is due to the very close Rf values of
7 and 8 to 5 on TLC (Table 2) and the limited distribution of 9 in section Cinnamomeae
(Table 4).
In our previous studies (Mikanagi et al., 1990, 1995), it was shown that the major
#ower #avonols of section Cinnamomeae were the 3-sophorosides of kaempferol (K)
and quercetin (Q), whereas those of sections Chinenses and Gallicanae were 3glucosides of K and Q. In this study, Cy 3-sophoroside (9) was detected in section
Cinnamomeae, but hardly at all in sections Chinenses and Gallicanae. This suggests
that the same glycosylation process occurs for #avonols and anthocyanins in Rosa.
The restriction of distribution of 9 to section Cinnamomeae suggests the possibility
that it can be used as a chemical marker for section Cinnamomeae identi"cation.
In addition, there was a pigment which seems to be Pn 3-sophoroside because the
Rt. 41.2 min on HPLC agrees with that of the authentic anthocyanin (Table 4,
Pigment No. ]). The amount of this anthocyanin is very small, but its distribution is
mainly in section Cinnamomeae.
3.3. Distribution of acylated anthocyanins
The occurrence of o-coumaroylcyanins was reported in the #owers of R. cv.
Frensham by Arisumi (1967). The detailed structure determination and distribution of
these pigments has not been previously reported. In this study we found acylated
anthocyanins in some Rosa #owers, and determined unambiguously both acylated
anthocyanins to be the 3-O-(6-O-trans-o-coumaroyl-b-D-glucopyranoside)-5-O-(b-Dglucopyranoside) of Cy (10) and Pn (11) (Tables 2 and 4). Also, we con"rmed a small
amount of cis-isomers of these anthocyanins by HPLC with the methods of Saito and
Harborne (1992). The occurrence of 10 has been widely observed in Rosa (Table 4), it

Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

897

being the major pigment in the #owers of R. moyesii and its cultivars, R. pendulina var.
oxyodon, R. sweginzowii, R. willmottiae (section Cinnamomeae), R. chinensis cv. Fabvier,
cv. Slater's Crimson China (section Chinenses), R. gallica cv. Violacea, R. x centifolia
cv. Muscosa (section Gallicanae), R. cv. Frensham, R. cv. Red Meillandina and R. cv.
The Fairy (modern garden roses). On the other hand, 11 has been found only in the
#owers of section Cinnamomeae, mainly in R. acicularis, R. arkansana, R. cinnamomea,
R. moyesii and its cultivars, R. rugosa cv. Maikwai and cv. Roseraie de l'Hay, R.
sweginzowii, R. willmottiae (section Cinnamomeae) and a modern garden rose, R. cv.
Red Meillandina.
In the #owers of R. acicularis, R. bella, R. moyesii cv. Arthur Hillier and R. nutkana
two unknown anthocyanins (UK1 and UK2 in Table 4) were detected. From the Rts of
both pigments, they are suspected to be polyacylated anthocyanins (Strack and Wray,
1994). We are attempting to resolve the chemical structure of these anthocyanins.
As the occurrence of acylated anthocyanins is considered to be important for the
stability and bluing e!ect of #ower colour (Lu et al., 1992; Dangles et al., 1993; Saito
et al., 1995; Figueiredo et al., 1996), the accumulation of acylated anthocyanins in
#owers of Rosa is expected to be important for new #ower colour creation. Further
studies of the newly discovered Rosa anthocyanins (7, 8, 9, ], 10, 11, UK1 and UK2)
are necessary for rose breeding in the future.
In summary, the taxa investigated in this study can be divided into eight groups on
the basis of their hydroxylation, methylation, glycosylation and acylation patterns as
shown in Table 5. Modern garden roses reveal heterogeneity in anthocyanin patterns
which re#ect their complicated origins, but we place them in group VIII for the sake of
convenience. Taxa in sections Chinenses and Gallicanae were placed in groups VI and
VII, respectively by patterns of anthocyanins as shown in Table 5. Group VI is
characterized with much accumulation of Cy 3-glucoside (2) and VII is characterized
with remarkable concentration of Cy 3,5-diglucoside (5) in the petals.
Most taxa of section Cinnamomeae are grouped as a homogeneous unit by morphological characters, but anthocyanin constituents of taxa in this section show
a range of patterns of methylation, glycosylation and acylation, and are divided into
"ve groups. Grossi et al. (1998) wrote that through the analyses of #ower #avonols
and leaf enzymes, three evolutionary trends could be recognized, corresponding to the
sections Synstylae, Pimpinellifolliae and Cinnamomeae pro parte. The half of section
Cinnamomeae are the most advanced and constitute the nucleus of the evolution.
Unfortunately, we have no data about sections Synstylae and Pimpinelifoliae which
contain only a few cyanic species. However, our results showed that section Cinnamomeae has a very diverse anthocyanin distribution, and may support Grossi's
hypothesis for the evolution of roses.
On the Japanese wild roses of this section, R. marretii (R06) and R. nipponensis (R14)
are classi"ed in Nipponensis group (group IV in Table 5) with two other taxa: R.
forrestiana (R05) from W-China and R. pendulina var. oxyodon (R16) from Caucasus.
In their anthocyanin constituents, Cy 3,5-diglucoside is absolutely dominant and
other anthocyanins are absent. R. rugosa cv. Salmon Pink (R21) in group III is also
a wild taxa in the "eld of Japan, but it seems a special case of mutation of R. rugosa
(R17). The rest of Japanese cyanic roses, R. acicularis (R01), R. rugosa and R. ] iwara

898

Table 4
Anthocyanin distribution in #owers of genus Rosa
Anthocyanins (as %)!

Rt. (min)

Species, varieties and cultivars

Section Cinnamomeae ("section Rosa)
Rosa acicularis (R01)
Rosa arkansana (R02)
Rosa bella (R03)
Rosa cinnamomea (R04)
Rosa forrestiana (R05)
Rosa marretii (R06)
Rosa moyesii (R07)
Rosa moyesii cv. Arthur Hillier (R08)
Rosa moyesii cv. Eddie's Crimson (R09)
Rosa moyesii cv. Fargesii (R10)
Rosa moyesii cv. Geranium (R11)
Rosa moyesii cv. Highdownensis (R12)
Rosa moyesii cv. Hillieri (R13)
Rosa nipponensis (R14)
Rosa nutkana (R15)
Rosa pendulina var. oxyodon (R16)
Rosa rugosa (R17)
Rosa rugosa var. plena (R18)
Rosa rugosa cv. Maikwai (R19)
Rosa rugosa cv. Roseraie del'Hay (R20)
Rosa rugosa cv. Salmon Pink (R21)
Rosa rugosa cv. Scabrosa (R22)
Rosa rugosa cv. Scarlet (R23)

5

9

7

2

10

33.1

35.0

39.6

37.4

49.2

56.0

6

x

8

3

11

37.8

41.2

44.6

43.5

54.0

60.9

4

1

UK1 UK2

35.4

41.8

55.0

Cy

Pn

Pg

3,5- 3-sop 3-rut 3-glu 3,5diglu
diglu-oC

3,5- 3-sop? 3-rut 3-glu 3,5diglu
diglu-oC

3,5- 3-glu ?
diglu

26
61
80
33
97
98
50
38
57
23
45
41
74
94
30
95
32
35
39
16
]
26
79

0
]
]
]
]
]
17
4
]
16
6
2
6
]
]
]
1
2
]
0
8
]
]

1
0
3
0
]
0
]
1
0
]
]
0
]
]
1
]
]
0
0
]
2
0
0

0
0
1
0
]
]
28
7
]
13
35
1
9
1
0
]
1
1
1
0
24
0
0

cis

trans

]
]
]
]
]
0
]
]
]
]
]
]
1
]
]
]
]
]
]
]
0
]
]

1
1
1
]
1
0
2
2
2
]
]
1
4
]
]
2
]
]
]
]
0
]
1

44
32
8
62
0
0
0
23
34
34
0
49
0
1
24
]
63
60
53
76
0
70
17

]
]
0
]
]
0
]
]
0
1
]
0
]
1
0
0
]
]
0
]
]
0
0

9
0
]
0
]
0
0
2
]
]
0
]
]
]
6
]
]
]
]
]
1
]
]

0
0
0
0
]
0
0
4
]
4
0
1
]
0
]
]
]
1
]
1
61
]
]

cis

trans

1
1
]
1
0
]
0
]
1
]
0
1
0
0
]
0
]
]
]
]
0
]
]

5
3
]
2
0
1
0
2
4
2
0
4
0
0
1
0
]
1
3
2
0
0
]

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

58.5

?

2
0
3
0
0
0
0
3
0
0
0
0
0
0
4
0
0
0
0
0
0
0
0

8
0
1
0
0
0
0
8
0
0
0
0
0
0
30
0
0
0
0
0
0
0
0

Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

Pigment No. 5

74
47
52

]
]
]

0
0
0

0
0
]

1
]
]

6
2
]

14
39
46

0
0
0

0
]
0

0
1
]

]
1
]

2
6
1

0
0
0

0
0
0

0
0
0

0
0
0

Section Chinenses
Rosa chinensis Jacq.
Rosa chinensis var. minima
Rosa chinensis var. spontanea
Rosa chinensis cv. Fabvier
Rosa chinensis cv. Miss Lowe
Rosa chinensis cv. Mutabilis
Rosa chinensis cv. Pomponde de Paris
Rosa chinensis cv. Slater's Crim China

85
87
28
82
43
23
71
58

0
0
0
0
]
0
0
0

]
]
2
]
1
]
0
]

13
11
56
10
53
73
27
33

]
]
1
]
]
0
]
]

1
]
1
5
]
0
]
4

0
0
0
2
0
0
]
0

0
0
0
0
0
0
0
]

]
]
1
]
]
]
]
]

]
]
0
]
]
2
]
1

0
0
0
0
0
0
0
]

0
0
0
0
0
]
0
]

0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0

Section Gallicanae
Rosa gallica
Rosa gallica cv. Cardinal de Richelieu
Rosa gallica cv. Rosa Mundi
Rosa gallica cv. Shigyoku
Rosa gallica cv. Violacea
Rosa]centifolia cv. Bullata
Rosa]centifolia cv. Muscosa
Rosa]damascena
Rosa]damascena cv. Bifera
Rosa]damascena cv. Gloire de Guilan

92
97
98
98
96
98
96
97
97
97

0
]
]
]
]
1
1
1
]
1

0
0
0
0
0
0
0
0
0
0

6
1
]
]
]
]
1
1
1
1

]
]
0
]
]
0
]
]
]
]

1
1
]
1
2
0
2
1
]
1

0
]
0
]
]
0
]
0
0
0

0
0
0
0
0
0
0
0
0
0

]
0
0
]
]
0
]
]
]
]

]
]
0
]
]
]
]
]
]
]

0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0

Modern
Rosa cv.
Rosa cv.
Rosa cv.
Rosa cv.
Rosa cv.
Rosa cv.
Rosa cv.
Rosa cv.

98
69
94
96
85
37
73
95

0
0
0
0
0
0
0
0

0
]
0
0
]
0
]
0

]
3
1
3
]
]
1
]

0
]
]
0
]
0
1
]

0
]
]
]
8
]
5
3

0
]
1
0
3
0
13
0

0
0
0
0
0
0
0
0

0
]
]
]
]
]
]
]

0
]
]
0
]
0
]
0

0
0
0
0
0
0
]
0

0
0
0
0
]
0
2
0

1
22
1
0
0
58
2
1

0
2
0
0
]
3
]
0

0
0
0
0
]
0
]
0

0
0
0
0
]
0
]
0

garden roses
La France
Ole
Papa Meilland
Seika
Frensham
Orange Bunny
Red Meillandina
The Fairy

Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

Rosa sweginzowii (R24)
Rosa willmottiae (R25)
Rosa]iwara (R26)

!Percent of total absorbance of all detected anthocyanins at 530 nm by HPLC analysis. value (0.1"0, 0.1(value(0.5"], 0.5(value(1.5"1.
Rts of &]' pigments are identical to each pigments, but chemical structures are not yet con"rmed.
899

900
Table 5
Hydroxylation, methylation, glycosylation and acylation patterns of anthocyanins in #owers of genus Rosa (value(0.5%"`!a, 0.5%(
value(1%"`#a, 1%(value(10%"`La, 10%(value"`Ua)

Hydroxylation

B-ring

Section Cinnamomeae
(section Rosa)

Section
Chinenses

Section
Gallicanae

Modern
garden roses

I!

II!

III!

IV!

V!

VI!

VII!

VIII!

U

U

U

U

U

U

U

U

4@-OH

(Cyanidin
& peonidin)
(Pelargonidin)-

!

!

!

!

!

!

!

U, !

3@- & 4@-OH

Methylation

Aglycone

3@-O-Me

(Peonidin)

U

U

U

!

!

#

!

U, !

Glycosylation

Aglycone

3-O-monogly
3-O-digly
5-O-gly

(3-glu)
(3-rut & 3-sop)
(3,5-diglu)

!
L
U

L
L
U

U
L
!

!
!
U

U
U
U

U
#
U

#
#
U

L
!
U

Sugar

2-O-gly

(sop; 2-glucosylglucose)
(rut; 6-rhamnosylglucose)

!

L

L

!

U

!

#

!

L

L

L

!

!

!

!

!

L

L

!

#

L

L

#

L, !

6-O-gly

Acylation

Sugar

6-O-Ac

(3-o-coumaroylglu-5-glu)

!Patterns of anthocyanins were divided into eight groups as follows, R-numbers indicate Rosa taxa of section Cinnamomeae shown in Table 1.
I Acicularis group - R01, R02, R03, R04, R09, R15, R17, R18, R19, R20, R22, R23, R24, R25 and R26;
II Moyesii Arthur Hillier group - R08, R10 and R12;
III Rugosa Salmon Pink group - R21;
IV Nipponensis group - R05, R06, R14 and R16;
V Moyesii group - R07, R11 and R13;
VI Chinenses group; VII Gallicanae group; VIII Modern garden roses group.

Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

Hydroxylation, methylation, glycosylation and acylation patterns

Y. Mikanagi et al. / Biochemical Systematics and Ecology 28 (2000) 887}902

901

(R26) belong to the Acicularis group (group I in Table 5). This group has the largest
number of taxa, and they contain mainly 3,5-diglucosides of Cy and Pn and lack the
3-glucoside and 3-sophoroside of Cy and Pn. Although R. nipponensis is sometimes
treated as a variety of R. acicularis (R. acicularis Lindl. var. nipponensis Hook. "l.), the
di!erence of anthocyanin distribution between these species is clearly shown in this
result.

Acknowledgements
We are grateful to the secretary general of the Royal National Rose Society, UK,
Mr. S. Suzuki and Mr. H. Hirabayashi in Keisei Rose Nursery, Japan, Mr. Ichikawa
in Tokyo Metropolitan Jindai Botanical Park, Japan for provision of plant materials.
We also thank Miss M. Yoshikawa and Miss F. Hayano for their help in collecting
plant materials and chemical analysis. The authors greatfully acknowledge Dr. N.B.
Clark and Dr. Y. Yazaki at CSIRO Forestry and Forest Products for his assistance
with the English expression in this manuscript. Financial support by &Fujiwara
Natural History' is greatly acknowledged.

References
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genetic analyses and to the application of those results to the practical breeding. I. Sci. Bull. Fac. Agric.
Kyushu 20 (2), 131}149.
Arisumi, K., 1967. Studies on the #ower colours in Rosa, with special references to the biochemical and
genetic analyses and to the application of those results to the practical breeding. III. Bull. Fac. Agr.
Yamaguchi University No. 18, 1077}1089.
Biolley, J.-P., Jay, M., Barbe, J.-P., 1992. Chemometric Approach (Flavonoids) in an automatic recognition
of modern rose cultivars. Biochem. Syst. Ecol. 20, 697}705.
Biolley, J.-P., Jay, M., Forkmann, G., 1994a. Pigmentation patterns of modern rose mutants throw light on
the #avonoid pathway in Rosa]hybrida. Phytochemistry 36, 1189}1196.
Biolley, J.-P., Jay, M., Viricel, M.-R., 1994b. Flavonoid diversity and metabolism in 100 Rosa]hybrida
cultivars. Phytochemistry. 35, 413}419.
Cairns, T. (Ed.), 1993. Modern Roses 10. The American Rose Society, Louisiana.
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34, 119}124.
Eugster, C.H., MaK rki-Fischer, E., 1991. The Chemistry of rose pigments. Angew Chem. Int. Ed. Engl. 30,
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