New isorhamnetin glycosides and other phenolic compounds from calendula officinalis

Article abstract of DOI:10.1007/s10600-013-0759-x

A total of 39 known compounds and two new flavonoid glycosides that were identified as isorhamnetin-3-O-(2″-acetyl)-β-D-glucopyranoside and isorhamnetin-3-O-(2″,6″-diacetyl)- β-D-glucopyranoside were isolated from florets of Calendula officinalis (Asteraceae). The distribution of phenolic compounds in morphological groups of C. officinalis was studied. It was found that peripheral florets had the highest flavonoid content (36.66 mg/g); tubular florets (9.95 mg/g) and leaves (9.65 mg/g), phenylpropanoids. Anthocyanins, among which cyanidin derivatives dominated, were identified for the first time in C. officinalis florets.

Full text of DOI:10.1007/s10600-013-0759-x

Chemistry of Natural Compounds, Vol. 49, No. 5, November, 2013 [Russian original No. 5, September–October, 2013]
NEW ISORHAMNETIN GLYCOSIDES AND OTHER
PHENOLIC COMPOUNDS FROM Calendula officinalis
D. N. Olennikov* and N. I. Kashchenko
UDC 582.71:547.918
A total of 39 known compounds and two new flavonoid glycosides that were identified as isorhamnetin-3-O-
(2ꢀ-acetyl)-ꢁ-D-glucopyranoside and isorhamnetin-3-O-(2ꢀ,6ꢀ-diacetyl)- ꢁ-D-glucopyranoside were isolated
from florets of Calendula officinalis (Asteraceae). The distribution of phenolic compounds in morphological
groups of C. officinalis was studied. It was found that peripheral florets had the highest flavonoid content
(36.66 mg/g); tubular florets (9.95 mg/g) and leaves (9.65 mg/g), phenylpropanoids. Anthocyanins, among
which cyanidin derivatives dominated, were identified for the first time in C. officinalis florets.
Keywords: Calendula officinalis, Asteraceae, isorhamnetin-3-O-(2ꢀ-acetyl)-ꢁ-D-glucopyranoside, isorhamnetin-3-
O-(2ꢀ,6ꢀ-diacetyl)-ꢁ-D-glucopyranoside, flavonoids, anthocyanins, HPLC.
Calendula officinalis L. is an annual herbaceous plant of the familyAsteraceae that is widely used in medical practice.
Chemical studies found in C. officinalis various classes of compounds including terpenes, phenols, lipids, carbohydrates, etc.
[1]. C. officinalis is a cultivated species that is remarkable for its adaptability that enables it to be grown even in inhospitable
agricultural zones, in particular, in the Transbaikal territory. Several double-flowered varieties (Big Orange, Egypt Sun,
Flame Dancer, Geisha Girl, Indian Prince, Radio, Red Black Centered) were recommended for cultivation of C. officinalis in
the Republic of Buryatiya. These are characterized by high productivity and a prolonged vegetative period. Information on
the chemical compositions of these varieties has not been published. Therefore, the goal of the present work was to study
phenolic compounds from seven C. officinalis varieties introduced into the Republic of Buryatiya.
Preliminary studies of florets of the studied C. officinalis varieties for the presence of the principal compound groups
showed that the contents of essential oil, carotenoids, and water-soluble polysaccharides in them were 1.4–2.9, 5.14–7.59,
and 14.51–28.81 mg/g, respectively (Table 1). The total flavonoid concentration varied from 10.52 (Flame Dancer) to
26.79 mg/g (Big Orange), which was greater than that for raw material grown in the central part of Russia (2.6–9.1 mg/g) [2],
Estonia (2.1–6.8 mg/g) [3], Italy (2.8–7.5 mg/g) [4], and Brazil (14.1–14.4 mg/g) [5]. Then, we studied the phenolic components
from florets of Big Orange variety, from which fractionation and chromatographic separation of the extracted compounds
identified 41 compounds including 39 known compounds and two new flavonoids 1 and 2.
OCH
3
3'
OH
4'
HO
7
O
O
O
3
1''
O
2''
O
5
OH
CH
3
O
OH
OH
6''
RO
1, 2
1: R = H; 2: R = Ac
Institute of General and Experimental Biology, Siberian Branch, Russian Academy of Sciences, 670047, Ulan-Ude,
Ul. Sakhꢂyanovoi, 6, fax: 7 (3012) 43 47 43, e-mail: oldaniil@rambler.ru. Translated from Khimiya Prirodnykh Soedinenii,
No. 5, September–October, 2013, pp. 717–723. Original article submitted May 24, 2013.
©
0009-3130/13/4905-0833 2013 Springer Science+Business Media New York
833

TABLE 1. Chemical Composition of Florets of C. officinalis Varieties, mg/g*
Compound group
Big Orange
Egypt Sun
Flame Dancer
Geisha Girl Indian Prince
Radio
Red Black Centered
Flavonoids
Essential oil
Carotenoids
26.79
2.6
6.56
14.63
19.24
1.8
5.47
17.45
10.52
2.9
7.59
24.48
20.11
2.5
6.97
10.28
17.25
1.4
5.14
28.81
18.42
1.8
5.98
15.51
16.34
2.1
6.77
14.51
Polysaccharides
______
*Of air-dried raw material mass.
13
TABLE 2. PMR (500 MHz) and C NMR (125 MHz) Spectra of 1 and 2 (DMSO-d , ꢃ, ppm, J/Hz)
6
1
2
C atom
DEPT
ꢃH
ꢃC
ꢃH
ꢃC
Isorhamnetin
2
3
4
5
6
7
8
9
10
1ꢂ
2ꢂ
3ꢂ
4ꢂ
5ꢂ
C
C
C
C
CH
C
CH
C
C
C
CH
C
156.4
133.9
178.3
161.0
99.9
164.2
93.8
156.1
104.1
121.7
112.9
149.2
146.4
115.7
122.5
55.2
156.4
134.0
178.2
161.2
99.8
164.3
93.4
156.1
104.2
121.9
112.9
149.0
146.3
115.9
122.4
55.3
6.15 (1Í, d, J = 2.2)
6.33 (1Í, d, J = 2.2)
6.14 (1Í, d, J = 2.2)
6.33 (1Í, d, J = 2.2)
7.86 (1Í, d, J = 2.0)
7.85 (1Í, d, J = 2.0)
C
CH
CH
CH3
6.89 (1Í, d, J = 9.0)
7.45 (1Í, d, J = 9.0, 2.0)
3.81 (3Í, s)
6.87 (1Í, d, J = 9.0)
7.44 (1Í, dd, J = 9.0, 2.0)
3.82 (3Í, s)
ꢂ
6
ꢂ
3 -ÎÑÍ3
ꢁ
-D-Glucopyranose
CH
CH
CH
CH
CH
CH2
5.81 (1Í, d, J = 7.7)
4.58 (1Í, dd, J = 8.1, 8.6)
101.7
75.9
77.9
70.2
76.3
61.1
5.83 (1Í, d, J = 7.7)
4.60 (1Í, dd, J = 8.1, 8.6)
101.8
76.0
78.0
70.2
76.7
63.9
1ꢂꢂ
2ꢂꢂ
3ꢂꢂ
4ꢂꢂ
5ꢂꢂ
3.11–3.35 (3Í, m)
3.15–3.36 (3Í, m)
3.75 (1Í, m, ÍA),
3.51 (1Í, m, ÍB)
1.59 (3Í, s)
4.25 (1Í, dd, J = 2.0, 12.0, ÍA),
4.09 (1Í, m, ÍB)
ꢂꢂ
6
CH3
C
CH3
C
19.6
170.2
1.57 (3Í, s)
19.7
170.2
20.3
2ꢂꢂ- ÑÎÑÍ3
2ꢂꢂ- ÑÎÑÍ3
6 - ÑÎÑÍ3
1.63 (3Í, s)
ꢂꢂ
171.0
6ꢂꢂ- ÑÎÑÍ3
Compound 1 was a yellow amorphous powder of formula C H O according to HR-ESI-MS data {m/z 543.421
24 24 13
+
–1
([M + Na] ; calcd 543.441)}. The IR spectrum contained bands for ester carbonyl (1725 cm ), which indicated that 1 was
acylated. Isorhamnetin and glucose were identified in the acid hydrolysis products. Alkaline hydrolysis produced isorhamnetin-
3-O-ꢁ-D-glucopyranoside and acetate, which was identified by HPLC. Isorhamnetin was detected after enzymatic hydrolysis
of 1 by ꢁ-glucosidase. This was consistent with the presence of an acetyl in the carbohydrate part of the glycoside. PMR and
C NMR spectroscopic data confirmed the ꢁ-configuration of the anomeric center of the glucopyranose. The PMR spectrum
contained a resonance for an anomeric proton of glucopyranose as a doublet at 5.81 ppm (1H, J = 7.7 Hz); the C NMR
13
13
spectrum, a resonance at 101.7 ppm (Table 2). Also, the PMR spectrum exhibited a singlet for acetyl at 1.59 ppm. The
presence of a 1H doublet of doublets at 4.58 ppm indicated that a glucopyranose secondary hydroxyl was acetylated (C-2ꢀ,
C-3ꢀ, C-4ꢀ) because the resonance would have appeared at stronger field if the C-6ꢀ primary hydroxyl was acetylated, like for
isorhamnetin-3-O-(6ꢀ-acetyl-)-ꢁ-D-glucopyranoside (30) [6]. The C-2ꢀ resonance of the carbohydrate of 1 underwent a weak-
13
field shift (+2.8 ppm) according to C NMR spectroscopy. This was due to acylation at this position. The resonance of the
acetyl carbonyl (170.2 ppm) in the HMBC spectrum correlated with proton H-2ꢀ of the glucopyranose (4.58 ppm), confirming
that C-2ꢀ was acetylated. The studies established the structure of 1 as isorhamnetin-3-O-(2ꢀ-acetyl-)-ꢁ-D-glucopyranoside.
834

a
TABLE 3. Antioxidant Activity of Quercetin and Isorhamnetin Glycosides, IC , ꢄg/mL
50
DPPHb
CBAc
Glycoside part
Quercetin
Isorhamnetin
Quercetin
Isorhamnetin
–
9.5
14.31
62.14
> 100
> 100
> 100
6.02
9.95
37.14
85.84
80.23
> 100
3- -Glc
Î
21.29
63.12
57.82
71.39
17.24
35.28
36.02
42.11
ꢂꢂ
3- -(2 -Acetyl-)-Glc
3- -(6ꢂꢂ-Acetyl-)-Glc
ꢂꢂ ꢂꢂ
3- -(2 ,6 -Diacetyl-)-Glc
Î
Î
Î
______
a
b
4 c
n = 5; antiradical activity relative to DPPH ; peroxide degradation of ꢁ-carotene.
a,b
TABLE 4. Contents of Phenolic Compounds in Morphological Groups of C. officinalis, mg/g
Peripheral
florets
Tubular
florets
Compound, compound group
Spathes
Seeds
Leaves
Stems
0.05
0.23
0.03
0.05
–
0.01
0.02
0.11
0.65
0.10
–
0.45
0.11
0.13
0.19
0.05
–
Roots
Caffeic acid (
)
12
0.09
1.34
–
Tr.
Tr.
0.54
0.07
1.22
0.90
2.28
Tr.
0.49
6.91
0.31
Tr.
0.09
0.89
0.13
0.10
–
0.45
0.11
–
–
–
–
–
–
0.06
–
–
–
–
–
1.83
1.77
0.06
–
Tr.
2.11
–
Tr.
0.16
0.82
0.21
–
–
–
–
–
–
Tr.
–
–
–
–
–
3.30
3.30
Tr.
–
0.42
8.46
0.77
Tr.
–
Tr.
–
–
1.42
–
Tr.
3.28
3.04
–
–
–
0.31
0.28
0.83
1.36
0.12
0.22
3.34
–
–
–
–
–
–
–
–
–
–
–
–
3- -Caffeylquinic acid (13)
O
4- -Caffeylquinic acid (31)
O
5- -Caffeylquinic acid (
)
O
14
1,3-di- -Caffeylquinic acid (15)
Tr.
O
3,5-di- -Caffeylquinic acid (17)
1.78
0.46
0.29
0.35
0.34
0.46
0.24
1.04
7.85
1.63
4.53
0.61
2.14
4.09
33.52
9.95
23.57
2.72
20.85
O
4,5-di- -Caffeylquinic acid (18)
O
Manghaslin (37)
Calendoflavobioside (36)
Rutin (33)
Quercetin-3- -(2ꢂꢂ-Rha-)-Rha (32)
O
Tr.
Isoquercitrin (23)
0.63
8.95
1.29
14.62
0.43
1.71
4.63
38.70
2.04
36.66
5.03
31.63
Quercetin-3- -(6ꢂꢂ-acetyl)-Glc (
)
24
O
Typhaneoside (38)
Calendoflavoside (35)
Narcissin (34)
–
Calendoflaside (29)
0.38
1.04
18.81
9.65
9.16
7.74
1.42
0.16
0.04
2.38
0.39
1.99
1.42
0.57
Isorhamnetin-3- -Glc (27)
O
Isorhamnetin-3- -(6ꢂꢂ-acetyl)-Glc (30)
O
6.46
6.46
–
–
–
Identified, including:
phenylpropanoids
flavonoids, including:
quercetin derivatives
isorhamnetin derivatives
0.06
Tr.
______
a
b
Of air-dried raw material mass; variety Big Orange. Tr.: traces.
Compound 2 was a yellow amorphous powder of molecular formula C H
([M + Na] ; calcd 585.479)}. Chemical transformations and UV, IR and NMR spectroscopy were similar to those of 1 and
O
{HR-ESI-MS m/z 585.263
26 26 14
+
isorhamnetin-3-O-(6ꢀ-acetyl-)-ꢁ-D-glucopyranoside (30) with the exceptions that the PMR spectrum contained two acetyl
13
resonances at 1.57 and 1.63 ppm and the C NMR spectrum exhibited chemical shifts for carbonyl at 170.2 and 171.0 ppm
and methyl at 19.7 and 20.3 ppm (Table 2). These facts indicated that two acetyls were present in the structure of 2. Correlations
between the acetyl carbonyls at 170.2 and 171.0 ppm and resonances of protons H-2ꢀ (4.60 ppm) and H-6ꢀ (4.09 and
4.25 ppm) were found. This established that the acetyls were located on C-2ꢀ and C-6ꢀ of ꢁ-glucopyranose. Thus, the
structure of 2 was determined as isorhamnetin-3-O-(2ꢀ,6ꢀ-diacetyl-)-ꢁ-D-glucopyranoside.
Two acetylated derivatives of isorhamnetin-3-O-ꢁ-D-glucoside, i.e., isorhamnetin-3-O-(6ꢀ-acetyl-)-ꢁ-D-
glucopyranoside, which was isolated from Pinus silvestris L. (Pinaceae) [6], and isorhamnetin-3-O-(2ꢀ,3ꢀ,4ꢀ-triacetyl-)-ꢁ-D-
glucopyranoside from leaves of Warburgia stuhlmanii Engl. (Canellaceae) [7], are known. Compounds 1 and 2 were new
natural compounds.
A comparison of the antioxidant activity of 3-O-glucosides of quercetin and isorhamnetin showed that introducing an
acetyl into the glycoside part of the flavonoid reduced the activity (Table 3). The activities of the isorhamnetin derivatives
were significantly less than those of the quercetin derivatives.
835

a
TABLE 5. Contents of Phenolic Compounds in Florets of C. officinalis Varieties, mg/g
Compound,
compound group
Flame
Dancer
Indian
Prince
Red Black
Centered
Big Orange
Egypt Sun
Geisha Girl
Radio
0.17
2.09
1.80
1.96
3.72
0.29
0.30
0.61
5.01
1.27
8.52
0.15
0.98
0.69
27.56
2.26
25.30
8.68
16.62
0.06
0.63
1.14
1.10
1.23
0.28
0.06
0.33
4.84
1.10
4.60
0.17
0.59
1.80
17.93
0.69
17.24
4.14
13.10
0.20
5.77
1.12
1.26
0.66
0.06
0.11
Tr.
2.22
0.46
2.10
0.12
0.42
1.22
15.72
5.97
9.75
3.21
6.54
0.14
3.00
0.96
1.32
1.49
0.32
0.20
0.62
4.74
1.05
4.57
0.25
0.85
1.64
21.24
3.14
18.10
4.91
13.10
0.06
1.81
0.66
0.98
0.95
0.23
0.15
0.23
3.76
0.82
3.53
0.23
0.79
3.27
17.47
1.87
15.60
3.20
12.40
0.11
1.66
1.29
1.61
1.52
0.24
0.11
0.18
4.06
0.94
5.04
0.18
0.62
1.62
19.18
1.77
17.41
4.95
12.46
0.08
2.49
0.83
1.12
1.15
0.21
0.22
0.46
2.93
0.67
4.13
0.23
0.81
1.59
16.92
2.57
14.35
3.99
10.36
12
13
37
36
33
32
23
24
38
35
34
29
27
30
Identified, including:
phenylpropanoids
flavonoids, including:
quercetin derivatives
isorhamnetin derivatives
Total contentb:
flavonoids
26.79
–
19.24
2.29
10.52
3.12
20.11
–
17.25
0.79
18.42
–
16.34
2.48
anthocyanins
______
a
b
Of air-dried raw material mass; spectrophotometric method. Tr.: traces.
The known compounds that were isolated from florets of C. officinalis also included phenolic acids such as vanillic
(3) and syringic (4); phenylpropanoids such as cinnamic (5), p- (6) and o-coumaric (7), ferulic (8), isoferulic (9), caffeic acids
(12), 1-O- (40), 3-O- (13), 4-O- (31), 5-O- (14), 1,3-di-O- (15), 3,4-di-O- (16), 3,5-di-O- (17), 4,5-di-O- (18), 1,3,5-tri-O-
(19), and 3,4,5-tri-O-caffeylquinic acids (20); 3-O-p-coumarylquinic acid (41), 5-O-ferulylquinic acid (21), 1-O-caffeylglucose
(39); flavonoids such as quercetin (10), isorhamnetin (11), quercitrin (22), isoquercitrin (23), quercetin-3-O-(2ꢀ-acetyl-)-ꢁ-D-
glucopyranoside (24), quercetin-3-O-(6ꢀ-acetyl-)-ꢁ-D-glucopyranoside (25), quercetin-3-O-(2ꢀ,6ꢀ-diacetyl-)-ꢁ-D-
glucopyranoside (26), isorhamnetin-3-O-ꢁ-D-glucopyranoside (27), isorhamnetin-3-O-ꢅ-L-rhamnopyranoside (28),
calendoflaside (29), isorhamnetin-3-O-(6ꢀ-acetyl-)-ꢁ-D-glucopyranoside (30), quercetin-3-O-(2ꢀ-ꢅ-L-rhamnopyranosyl)-ꢅ-
L-rhamnopyranoside (32), rutin (33), narcissin (34), calendoflavoside (35), calendoflavobioside (36), manghaslin (37), and
typhaneoside (38). Compounds 3, 4, 6, 8, 10–13, 22, 27, 29, and 33–38 were identified earlier in C. officinalis [8–11].
Compounds 5, 7, 9, 14–21, 23–26, 28, 30–32, and 39–41 were detected in it for the first time.
According to HPLC data, peripheral (36.66 mg/g) and tubular florets (23.57 mg/g) of C. officinalis typically had the
greatest contents of flavonoids. These morphological groups were dominated by isorhamnetin derivatives narcissin (34),
typhaneoside (38), and isorhamnetin-3-O-(6ꢀ-acetyl-)-ꢁ-D-glucopyranoside (30). The contents of quercetin glycosides were
less than 15% of the total flavonoids (Table 4). Trace concentrations of flavonoids were found in spathes and seeds. Quercetin
derivatives isoquercitrin (23), quercetin-3-O-(2ꢀ-acetyl-)-ꢁ-D-glucopyranoside (24), and calendoflavobioside (36) predominated
in flavonoids from leaves and stems. This group of compounds was not observed in roots.
Phenylpropanoids were represented by free caffeic acid (12) in addition to mono- and dicaffeylquinic acids, which
were detected in all morphological groups. A feature of the aerial organs was the dominance of monocaffeylquinic acids
(1.34–9.23 mg/g) and 3-O-caffeylquinic acid (13) as the principal compound (0.23–8.46 mg/g). A high concentration of
dicaffeylquinic acids (3.68 mg/g) was detected in roots. The dominant component was 4,5-di-O-caffeylquinic acid (18, 3.34 mg/g).
A study of the accumulation of pure phenolic compounds of C. officinalis in different varieties showed that marker
components (12, 13, 23, 24, 27, 29, 30, 32–38) were typically present for all studied varieties (Table 5). The ratios of the
contents of individual compounds for different varieties were similar. However, it should be noted that the principal flavonoid
of varieties Big Orange, Radio, and Red Black Centered was narcissin (34, 4.13–8.52 mg/g); for Egypt Sun, Flame Dancer,
Geisha Girl, and Indian Prince, typhaneoside (38, 2.22–4.84 mg/g). The dominance of 34 was found earlier in cultivars of
C. officinalis from Italy [12]; 38, from Serbia [13].
836

a
TABLE 6. Contents of Anthocyanins in Tubular (T) and Peripheral (P) Florets of C. officinalis Varieties, mg/g
Compound,b
Egypt Sun
Flame Dancer
Indian Prince
Red Black Centered
compound group
T
P
T
P
T
P
T
P
Cy-3- -Glc
2.41
0.52
1.05
0.89
0.65
0.61
1.12
1.59
8.84
9.45
0.97
Tr.
0.30
–
0.38
Tr.
Tr.
0.41
2.06
3.57
3.01
0.83
1.49
0.55
0.56
0.89
1.31
2.32
10.96
12.61
1.24
Tr.
0.56
Tr.
0.20
Tr.
0.39
0.63
3.02
3.92
0.85
–
–
Tr.
–
–
–
–
–
–
–
Tr.
Tr.
1.57
0.44
0.89
0.41
0.21
0.28
0.64
0.99
5.43
6.24
1.15
Tr.
0.64
–
0.16
Tr.
0.48
0.83
3.26
3.47
O
Cy-3,5- -Glc
O
Cy-3- -Rut
O
Dp-3- -Glc
–
O
Mv-3- -Glc
0.39
0.35
–
–
1.59
2.35
O
Pn-3- -Glc
O
Pg-3,5- -Glc
O
Pt-3- -Glc
O
Identified
Total contentc
______
a
c
b
Of air-dried raw material mass; Cy, cyanidine; Dp, delphinidin; Mv, malvidin; Pn, peonidin; Pg, pelargonidin; Pt, petunidin;
spectrophotometric method. Tr.: traces.
Anthocyanins (0.79–3.12 mg/g) were found in four varieties of C. officinalis (Egypt Sun, Flame Dancer, Indian
Prince, and Red Black Centered). A distinguishing feature of these varieties was intense raspberry-red pigmentation in the
central flower part. Chromatographic (HPLC) separation showed the presence of eight compounds that were identified as
3-O-glucopyranosides of cyanidine, delphinidin, malvidin, peonidin, pelargonidin, and petunidin; 3,5-di-O-glucopyranosides
of cyanidine and pelargonidin; and cyanidine-3-O-rutinoside (Table 6). The dominant compound was cyanidine-3-O-
glucopyranoside, the contents of which in tubular and peripheral florets were 0.85–3.01 and 0.97–1.24 mg/g, respectively.
Tubular florets were the main location of this compound group with the greatest (12.61 mg/g) concentration found for variety
Flame Dancer. Anthocyanins were found in C. officinalis for the first time.
EXPERIMENTAL
Varieties Big Orange, Egypt Sun, Flame Dancer, Geisha Girl, Indian Prince, Radio, and Red Black Centered were
grown in the open at experimental plantations of the IGEB, SB, RAS (2011) from authenticated seeds obtained at the
N. V. Tsitsin Main Botanical Garden, RAS (Moscow, Russia). The agrotechnical conditions recommended for C. officinalis
were used for cultivation [14]. Florets and morphological groups were collected during full flowering (June-July); seeds and
roots, during fruiting (August). Samples of raw materials are preserved in the Herbarium of the IGEB, SB, RAS
(No. FAs/to-15/04-02/0811i, FAs/to-15/05-02/0811i, FAs/to-15/06-02/0811i, FAs/to-15/07-02/0811i, FAs/to-15/08-02/0811i,
FAs-to-15/09-02/0811i, and FAs/to-15/10-02/0811i).
Column chromatography (CC) used silica gel (SiO , Sigma), reversed-phase silica gel (RP-SiO , Sigma), Sephadex
2
2
LH-20 (Pharmacia), polyamide (Woelm), and Amberlite XAD7HP (Sigma). Spectrophotometric studies were performed on
an SF-2000 spectrophotometer (OKB Spektr); MS analysis, on an MAT 8200 high-resolution mass spectrometer (Finnigan).
IR spectra were recorded in KBr pellets (1:100) on an FT-801 IR-Fourier spectrometer (HPF Simeks); NMR spectra, on a
VXR 500S NMR spectrometer (Varian). Total contents of flavonoids were determined by differential spectrophotometry
calculated as narcissin [15]; carotenoids and anthocyanins, by spectrophotometry calculated as ꢁ-carotene and cyanidine-3-O-
glucopyranoside, respectively [16]; essential oil, gravimetrically after steam distillation; water-soluble polysaccharides, by
the anthrone–H SO method [17]. Antiradical activity with respect to the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was
2
4
determined by the Seyoum method [18]; peroxide degradation of ꢁ-carotene (CBA), in the DMSO–H O –linoleic acid system
2
2
[19].
Extraction and Fractionation. Ground florets of C. officinalis variety Big Orange (1.4 kg) were extracted successively
by 96, 70, and 50% EtOH (ꢆ3, 1:20, 40°C). The combined extracts were concentrated to an aqueous residue and extracted
completely with hexane, CHCl , EtOAc, and BuOH to afford five fractions (mass, yield, % of air-dried raw material mass):
3
hexane (F-1, 72.66 g, 5.19%), CHCl (F-2, 57.68 g, 4.12%), EtOAc (F-3, 14.70 g, 1.05%), BuOH (F-4, 63.14 g, 4.51%), and
3
H O residue (F-5, 60.90 g, 4.35%). Fraction F-2 (25 g) was separated using CC over SiO (3 ꢆ 90 cm, C H –EtOAc eluent,
2
2
6 14
100:0ꢇ70:30) with subsequent rechromatography over RP-SiO (2 ꢆ 50 cm, H O–MeCN eluent, 100:0ꢇ0:100). A total of
2
2
837

nine compounds were identified as vanillic (14 mg, 3), syringic (18 mg, 4), cinnamic (24 mg, 5) [20], p-coumaric (11 mg, 6),
o-coumaric (5 mg, 7) [21], ferulic (14 mg, 8), and isoferulic acids (8 mg, 9) [22]; quercetin (9 mg, 10), and isorhamnetin
(16 mg, 11) [23]. Fractions F-3 (12 g) and F-4 (60 g) were separated by CC over polyamide (4 ꢆ 100 cm, H O–EtOH eluent,
2
100:0ꢇ96:4), SiO (3 ꢆ 60 cm, C H –EtOAc eluent, 100:0ꢇ70:30; EtOAc–EtOH, 100:0ꢇ70:30), Sephadex LH-20
2
6 14
(3 ꢆ 70 cm, EtOH–H O eluent, 96:4ꢇ0:100), RP-SiO (2 ꢆ 50 cm, H O–MeCN eluent, 100:0ꢇ0:100), and preparative TLC
2
2
2
on SiO (toluene–EtOAc–HCOOH, 5:4:1). This resulted in the isolation of 29 compounds including from fraction F-3
2
isorhamnetin-3-O-(2ꢀ-acetyl-)-ꢁ-D-glucopyranoside (18 mg, 1), isorhamnetin-3-O-(2ꢀ,6ꢀ-diacetyl-)-ꢁ-D-glucopyranoside
(12 mg, 2); caffeic acid (27 mg, 12) [21]; 3-O- (68 mg, 13), 5-O- (24 mg, 14), 1,3-di-O- (11 mg, 15), 3,4-di-O- (18 mg, 16),
3,5-di-O- (33 mg, 17), 4,5-di-O- (14 mg, 18), 1,3,5-tri-O- (20 mg, 19), and 3,4,5-tri-O-caffeylquinic acids (7 mg, 20);
5-O-ferulylquinic acid (5 mg, 21) [24], quercitrin (18 mg, 22), isoquercitrin (35 mg, 23) [25], quercetin-3-O-(2ꢀ-acetyl-)-ꢁ-D-
glucopyranoside (12 mg, 24), quercetin-3-O-(6ꢀ-acetyl)-ꢁ-D-glucopyranoside (14 mg, 25), quercetin-3-O-(2ꢀ,6ꢀ-diacetyl)-ꢁ-
D-glucopyranoside (4 mg, 26) [26], isorhamnetin-3-O-ꢁ-D-glucopyranoside (31 mg, 27) [25], isorhamnetin-3-O-ꢅ-L-
rhamnopyranoside (5 mg, 28), calendoflaside [isorhamnetin-3-O-(2ꢀ-ꢅ-L-rhamnopyranosyl)-ꢅ-L-rhamnopyranoside] (37 mg,
29) [9], and isorhamnetin-3-O-(6ꢀ-acetyl)-ꢁ-D-glucopyranoside (16 mg, 30) [6]. Fraction F-4 afforded 4-O-caffeylquinic
acid (12 mg, 31) [24], quercetin-3-O-(2ꢀ-ꢅ-L-rhamnopyranosyl)-ꢅ-L-rhamnopyranoside (32 mg, 32) [10], rutin (212 mg, 33),
narcissin [isorhamnetin-3-O-(6ꢀ-ꢅ-L-rhamnopyranosyl)-ꢁ-D-glucopyranoside] (3.25 g, 34) [25], calendoflavoside
[isorhamnetin-3-O-(2ꢀ-ꢅ-L-rhamnopyranosyl)-ꢁ-D-glucopyranoside] (52 mg, 35), calendoflavobioside [quercetin-3-O-
(2ꢀ-ꢅ-L-rhamnopyranosyl)-ꢁ-D-glucopyranoside] (107 mg, 36) [9], manghaslin [quercetin-3-O-(2ꢀ,6ꢀ-di-ꢅ-L-
rhamnopyranosyl)-ꢁ-D-glucopyranoside] (114 mg, 37) [27], and typhaneoside [isorhamnetin-3-O-(2ꢀ,6ꢀ-di-ꢅ-L-
rhamnopyranosyl)-ꢁ-D-glucopyranoside] (2.28 g, 38) [10]. Fraction F-5 (50 g) was placed on Amberlite XAD7HP (300 g)
and eluted with H O and EtOH (50%). The EtOH eluate was concentrated and chromatographed over RP-SiO (2 ꢆ 50 cm,
2
2
H O–MeCN eluent, 100:0ꢇ0:100) to isolate the three compounds 1-O-caffeylglucose (14 mg, 39), 1-O-caffeylquinic acid
2
(21 mg, 40), and 3-O-p-coumarylquinic acid (9 mg, 41) [24].
+
Isorhamnetin-3-O-(2ꢀ-acetyl-)-ꢁ-D-glucopyranoside (1). C H O . HR-ESI-MS m/z: 543.421 ([M + Na] ;
24 24 13
+
+
calcd 543.441). + FAB-ÌS m/z: 521 [M + H] , 317 [(M – Glc – acetate) + H] . UV spectrum (ÌåÎÍ, ꢈ , nm): 256, 266 sh,
max
359; + AlCl 270, 306, 405; + AlCl /HCl 270, 305, 406; + NaOAc 275, 321, 389; + NaOAc/H BO 257, 266 sh, 360;
3
3
3
3
–1
1
13
+ NaOMe 272, 332, 418. IR spectrum (ꢉ , cm ): 1725, 1652 (ꢉ ). Í NMR (500 MHz, ÌåÎÍ-d , ꢃ, ppm) and Ñ NMR
max
ÑÎ
4
(125 MHz, ÌåÎÍ-d , ꢃ, ppm), see Table 2.
Isorhamnetin-3-O-(2ꢀ,6ꢀ-diacetyl-)-ꢁ-D-glucopyranoside (2). C H O . HR-ESI-MS m/z: 585.263 ([M + Na] ;
calcd 585.479). + FAB-ÌS m/z: 563 [M + H] , 317 [{M – Glc – (2 ꢆ acetate)} + H] . UV spectrum (ÌåÎÍ, ꢈ , nm): 256,
4
+
26 26 14
+
+
max
265 sh, 359; + AlCl 270, 305, 405; + AlCl /HCl 271, 305, 406; + NaOAc 275, 322, 390; + NaOAc/H BO 257, 266 sh, 360;
3
3
3
3
–1
1
13
+ NaOMe 272, 332, 419. IR spectrum (ꢉ , cm ): 1724, 1652 (ꢉ ). Í NMR and Ñ NMR, see Table 2.
max
ÑÎ
+
Isorhamnetin-3-O-(6ꢀ-acetyl-)-ꢁ-D-glucopyranoside (30). C H O . HR-ESI-MS m/z: 543.421 ([M + Na] ;
27 30 16
+
+
calcd 543.441). + FAB-ÌS m/z: 521 [M + H] , 317 [(M – Glc – acetate) + H] . UV spectrum (ÌåÎÍ, ꢈ , nm): 255, 266 sh,
max
360; + AlCl 270, 306, 405; + AlCl /HCl 270, 304, 406; + NaOAc 275, 321, 389; + NaOAc/H BO 257, 266 sh, 361;
3
3
3
3
–1
1
+ NaOMe 272, 332, 420. IR spectrum (ꢉ , cm ): 1722, 1650 (ꢉ ). Í NMR (500 MHz, ÌåÎÍ-d , ꢃ, ppm, J/Hz): 1.64
max
ÑÎ
4
(3Í, s, Glc 6ꢂꢂ-COÑÍ ), 3.11–3.36 (4Í, m, Glc H-2ꢂꢂ-5ꢂꢂ), 3.81 (3Í, s, 3ꢂ-ÎÑÍ ), 4.05 (1Í, m, Glc H-6ꢂꢂ ), 4.21 (1Í, dd,
3
3
B
J = 2.0, 12.0, Glc H-6ꢂꢂ ), 5.81 (1Í, d, J = 7.7, Glc H-1ꢂꢂ), 6.14 (1Í, d, J = 2.2, Í-6), 6.33 (1Í, d, J = 2.2, Í-8), 6.89 (1Í, d,
À
13
J = 9.0, Í-5ꢂ), 7.44 (1Í, dd, J = 9.0, 1.8, Í-6ꢂ), 7.85 (1Í, d, J = 2.0, Í-2ꢂ). Ñ NMR (125 MHz, ÌåÎÍ-d , ꢃ, ppm): 20.1
4
(C, 6ꢂꢂ-ÎÑÑÍ ), 55.1 (CH , 3ꢂ-ÎÑÍ ), 63.7 (CH , Glc C-6ꢂꢂ), 70.1 (CH, Glc C-4ꢂꢂ), 73.3 (CH, Glc C-2ꢂꢂ), 76.6 (CH,
3
3
3
2
Glc C-5ꢂꢂ), 77.7 (CH, Glc C-3ꢂꢂ), 93.4 (CH, C-8), 100.0 (CH, C-6), 101.4 (CH, Glc C-1ꢂꢂ), 104.2 (C, C-10), 112.7 (CH, C-2ꢂ),
115.8 (CH, C-5ꢂ), 121.5 (C, C-1ꢂ), 122.6 (CH, C-6ꢂ), 133.8 (C, C-3), 146.3 (C, C-4ꢂ), 149.1 (C, C-3ꢂ), 156.2 (C, C-9), 156.3
(C, C-2), 161.1 (C, C-5), 164.3 (C, C-7), 170.9 (CH , 6ꢂꢂ-ÎÑÑÍ ), 178.2 (C, C-4).
3
3
Total Hydrolysis of 1 and 2. The compound (2 mg) was dissolved in TFA (5 mL, 5%) in Me CO and heated at
2
100°C (2 h). The hydrolysate was concentrated in vacuo, dissolved in MeOH, and analyzed by HPLC. The presence of
isorhamnetin (t 11.21 min, conditions 1) and glucose (t 14.14 min, conditions 2) was established.
R
R
Alkaline Hydrolysis of 1 and 2. The compound (2 mg) was dissolved in NaOH solution (1 mL, 0.4%) and heated
at 50°C (30 min). The hydrolysate was neutralized with HCl (0.4%) and placed on a RP-SiO Diapak C16M cartridge
2
(BioKhimMak) that was eluted with H O and EtOH (70%). Analysis of the resulting eluates by HPLC found isorhamnetin-3-
2
O-ꢁ-D-glucopyranoside in the EtOH eluate (t 6.98 min, conditions 1) and acetate in the H O eluate (t 18.24 min,
R
2
R
conditions 3).
838

Hydrolysis of 1 and 2 by ꢁ-Glucosidase. The compound (3 mg) and ꢁ-glucosidase from Prunus amygdalus (50 U,
Sigma) were suspended in phosphate buffer (Na HPO –NaH PO , pH 7.3), incubated at 37°C (12 h), heated at 100°C (24 h),
2
4
2
4
and centrifuged (6,000 g). The supernatant was extracted with EtOAc (2 ꢆ 3 mL), concentrated in vacuo, and dissolved in
MeOH. HPLC detected isorhamnetin (t 11.21 min, conditions 1).
R
HPLC. We used a Milikhrom A-02 microcolumn liquid chromatograph (EkoNova). Conditions 1: Pronto SIL-120-
5-C18 AQ column (2 ꢆ 75 mm, # 5 ꢄm; Metrohm AG); mobile phase: LiClO (0.2 M) in HClO (0.003 M) (A), MeCN (B);
4
4
gradient mode (%B): linear gradient 0–20 min 5–100%; ꢉ 200 ꢄL/min; column temperature 40°C; ꢈ 360 nm. Conditions 2:
Separon 5-NH column (2 ꢆ 75 mm, # 5 ꢄm; Tessek Ltd.); mobile phase: 75% MeCN; isocratic mode (0–20 min);
2
ꢉ 100 ꢄL/min; column temperature 35°C; ꢈ 190 nm. Conditions 3: Rezex ROA-Organic Acid column (2 ꢆ 75 mm, # 5 ꢄm;
Phenomenex); mobile phase: H SO (0.005 M); isocratic mode (0–30 min); ꢉ 300 ꢄL/min; column temperature 35°C;
2
4
ꢈ 210 nm. Plant raw material was analyzed quantitatively using the microcolumn HPLC-UV, for which raw material (40 mg)
was placed into an Eppendorf tube (2 mL), treated with EtOH (1 mL, 60%), sonicated (50 kHz, 30 min, 40°C), and centrifuged
(6,000 g, 20 min).
The resulting extract (500 ꢄL) was diluted with H O (1:5) and placed onto a polyamide cartridge (1 g) that was eluted
2
with H O (25 mL), EtOH (90%) (eluate 1), and NH solution (0.5%) in EtOH (90%) (eluate 2). Eluates 1 and 2 were
2
3
concentrated to dryness, dissolved in EtOH (1 mL, 70%), filtered through a membrane filter (0.45 ꢄm), and used for the
analysis (1 ꢄL). Eluate 1 was used for analysis of flavonoid glycosides; eluate 2, of acylated flavonoids and phenylpropanoids.
Conditions: Milikhrom A-02 microcolumn liquid chromatograph (EkoNova); Pronto SIL-120-5-C18 AQ column (2 ꢆ 75 mm,
# 5 ꢄm; Metrohm AG); mobile phase: LiClO (0.2 M) in HClO (0.006 M) (A), MeCN (B); gradient mode (%B): 0–7.5 min
4
4
11–18%, 7.5–13.5 min 18%, 13.5–15 min 18–20%, 15–18 min 20–25%, 18–24 min 25%, 24–30 min 25–100%;
ꢉ 100 ꢄL/min; column temperature 35°C, ꢈ 330 and 360 nm. The contents of pure components were calculated from calibration
curves that were constructed using commercial samples of standard compounds (caffeic acid, 3-O-, 4-O-, 5-O-caffeylquinic
acids, 1,3-di-O-caffeylquinic acid, rutin, isoquercitrin, narcissin, isorhamnetin-3-O-ꢁ-D-glucopyranoside, all SigmaAldrich),
isolated samples of compounds with purity ꢊ95% (manghaslin, calendoflavobioside, typhaneoside, calendoflavoside,
calendoflaside), and external reference samples [3,5- and 4,5-di-O-caffeylquinic acids using 1,3-di-O-caffeylquinic acid;
quercetin-3-O-(2ꢀ-ꢅ-L-rhamnopyranosyl)-ꢅ-L-rhamnopyranoside using rutin; quercetin-3-O-(6ꢀ-acetyl-)-ꢁ-D-glucopyranoside
using isoquercitrin; isorhamnetin-3-O-(6ꢀ-acetyl-)-ꢁ-D-glucopyranoside using isorhamnetin-3-O-ꢁ-D-glucopyranoside].
Anthocyanins were analyzed on a Summit HPLC (Dionex); Diasfer-110-C18 column (4 ꢆ 150 mm, # 5 ꢄm; BioKhimMak);
mobile phase: MeCN–HCOOH–H O (4:5:41); isocratic mode (20 min); ꢉ 1 mL/min; column temperature 30°C, ꢈ 530 nm.
2
ACKNOWLEDGMENT
The work was supported financially by the RFBR Project 12-03-31547(mol_a).
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