Antioxidative flavonol glycosides from the flowers of Abelmouschus manihot

Article abstract of DOI:10.1007/s11418-012-0651-1

Bioassay-guided fractionation led to the isolation of six new flavonol glycosides, floramanosides A (1), B (2), C (3), D (4), E (5), and F (6), from the flowers of Abelmouschus manihot. Their structures were elucidated by chemical and spectroscopic methods (UV, IR, HRESI-TOF-MS, 1D- and 2D-NMR). Activity screening results showed that the compounds had 2,2-diphenyl-1- picrylhydrazyl scavenging and aldose reductase inhibitory activities.

Full text of DOI:10.1007/s11418-012-0651-1

J Nat Med (2013) 67:78–85
DOI 10.1007/s11418-012-0651-1
ORIGINAL PAPER
Antioxidative flavonol glycosides from the flowers
of Abelmouschus manihot
Yi Zhang
Lifeng Han
•
Wei He
•
•
Chunmei Li
•
•
Qiu Chen
•
Erwei Liu
Tao Wang
Received: 30 January 2012 / Accepted: 24 February 2012 / Published online: 10 March 2012
Ó The Japanese Society of Pharmacognosy and Springer 2012
Abstract Bioassay-guided fractionation led to the isola-
tion of six new flavonol glycosides, floramanosides A (1),
B (2), C (3), D (4), E (5), and F (6), from the flowers of
Abelmouschus manihot. Their structures were elucidated
by chemical and spectroscopic methods (UV, IR, HRESI–
TOF-MS, 1D- and 2D-NMR). Activity screening results
showed that the compounds had 2,2-diphenyl-1-picrylhy-
drazyl scavenging and aldose reductase inhibitory
activities.
constituents from A. manihot flowers, we have reported the
isolation and structure elucidation of 25 flavones [3–5], and
some of them significantly inhibited triglyceride accumula-
tion in mature 3T3-L1 adipocytes, which potential mecha-
nism was also discussed [6]. In a continuing study on this
herbal medicine, the 95% EtOH extract and its 70% eluted
fraction of D101 resin of A. manihot flowers showed strong
activity on radical scavenging and aldose reductase inhibi-
tion. From the active fraction, six new flavonol glycosides,
floramanosides A (1), B (2), C (3), D (4), E (5), and F (6) were
obtained. This paper deals with the isolation of these com-
pounds and the elucidation of their antioxidant effects.
Keywords Abelmouschus manihot ꢀ Flavonol glycosides
ꢀ
Antioxidative activity ꢀ Aldose reductase inhibitor ꢀ DPPH
scavenging activity
Results and discussion
Introduction
The dried flowers of A. manihot (2.8 kg) were extracted with
95% EtOH under reflux three times, to give the 95% EtOH
extract (648 g, 23.14%). This extract (500 g) was subjected to
Abelmoschus manihot (L.) Medic, family Malvaceae, is an
annual herbal plant widely distributed in China. Its flowers
have been used as a traditional Chinese medicine for the
treatment of chronic renal disease and diabetic nephropa-
thy, and several flavonoids (isoquercitrin, hyperoside,
hibifolin, quercetin, gossypetin, and myricetin, etc.) have
previously been isolated from the flowers [1, 2]. During the
course of our characterization studies on the bioactive
D101 column chromatography (H O ? 70% EtOH ? 95%
2
EtOH) to give the water-, 70% EtOH-, and 95% EtOH-eluted
fractions (10.21, 5.55, and 1.02%, respectively).
The above-mentioned fractions were assessed for radical
scavenging activity of 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical and inhibition activity of aldose reductase
(AR). The 95% EtOH extract (DPPH radical: SC50
34.7 lg/mL; AR inhibition: IC50 10.0 lg/mL) and 70%
EtOH-eluted fraction (DPPH radical: SC50 8.0 lg/mL; AR
inhibition: IC50 8.3 lg/mL) of D101 resin from the 95%
EtOH extract of A. manihot flowers showed strong activi-
ties for the two assay systems.
Y. Zhang ꢀ T. Wang (&)
Tianjin State Key Laboratory of Modern Chinese Medicine,
312 Anshanxi Road, Nankai District, Tianjin 300193, China
e-mail: wangt@263.net
The 70% EtOH-eluted fraction was subjected to normal-,
reversed-phase silica gel and Sephadex LH-20 column
chromatographies, and finally HPLC to afford six new
compounds (1–6) (Fig. 1).
W. He ꢀ C. Li ꢀ Q. Chen ꢀ L. Han ꢀ E. Liu ꢀ T. Wang
Institute of Traditional Chinese Medicine, Tianjin University
of Traditional Chinese Medicine, 312 Anshan Road, Nankai
District, Tianjin 300193, China
1
23

J Nat Med (2013) 67:78–85
79
OH
OR
4
spectra of 2 indicated the presence of a b-D-glucopyranosyl
OH
OH
R2
R3
00
moiety [d : 4.80 (1H, d, J = 7.2 Hz, H-1 ) and 3.35–3.80
HO
O
O
HO
O
O
H
OR
3
00
(
6H, m, H-2–6 ); d : 60.2, 69.2, 73.2, 75.9, 76.7, 102.3].
C
OR1
OR1
OH
OR2
Except for the above-mentioned carbons of the glycoside,
13
the C-NMR spectrum of 2 exhibited fifteen carbons in the
Floramanoside A (1): R
Floramanoside B (2): R
1
1
1
= Gal → Xyl, R
2
1
2
= R
= Glc
3
= H
Floramanoside D (4): R1 = Gal → Rha, R = CH , R3 = R4 = H
= H, R = 6-acetyl Glc
Floramanoside F (6): R1 = R = R = H, R3 = 6-ethyl GlcA
6
1
2
3
2
3
1
2
3
4
= H, R
= OH, R
Floramanoside E (5): R
= R
= R
Floramanoside C (3): R = R3 = H, R2 = OGlcA
2
4
fields of d 98.5–176.5, which indicated that its aglycone
was a flavonol. In the H-NMR spectrum of 2, the proton
Gal: β-D-galatopyranosyl; Glc: β-D-glucopyranosyl; Glu: β-D-glucuronopyranosyl; Xyl: β-D-xylopyranosyl;
Rha: α-
L
-rhamnopyranosyl
1
signal of d 6.29 (1H, s), 11.92 (1H, s) indicated the pres-
ence of a penta-substitution benzyl and 5-OH of flavonol,
respectively. d 7.62 (1H, d, J = 2.0 Hz) and 7.67 (1H, d,
Fig. 1 Chemical structures of compounds 1–6
Floramanoside A (1) was isolated as a yellow amor-
J = 2.0 Hz) suggested there was
a
1,2,3,5-tetra-
2
5
phous powder with negative rotation [[a]D -40.68
c = 1.51, MeOH)]. Its molecular formula was determined
to be C H O by HRESI–TOF-MS (m/z 613.1398
(
1
3
Table 1 C-NMR data (d) for compounds 1–6 (measured in DMSO-
2
6 28 17
?
6
d )
[
M?H] , calcd for C H O 613.1399). The IR spectrum
26 29 17
of it showed absorption bands ascribable to hydroxyl
Position
1
2
3
4
5
6
-
1
-1
3301 cm ), unsaturated ketone (1657 cm ) functions,
(
2
155.2
133.1
177.2
161.1
98.4
145.6
135.7
176.0
152.3
97.9
147.9
135.5
175.6
156.3
98.2
153.3
135.9
172.4
160.6
96.2
146.1
135.9
175.8
160.6
98.2
146.8
135.4
175.6
156.4
98.4
-
1
and aromatic ring (1607, 1504, 1454 cm ). The UV
spectrum indicated the presence of a flavanol skeleton
3
4
(
360, 255 nm). Acid hydrolysis of 1 with 5% aqueous
H SO –1,4-dioxane (1:1, v/v) afforded D-xylose and
5
2
4
6
D-galactose, whose absolute configurations (D-) were
7
163.9
93.1
153.0
124.4
145.0
102.6
121.2
107.6
145.4
137.2
146.0
110.5
156.2
125.0
148.2
102.7
120.4
107.7
145.5
135.9
145.5
107.7
162.7
94.6
163.9
93.5
156.4
124.9
148.2
102.4
121.9
116.0
144.4
147.5
115.5
119.7
determined by GC analysis of their trimethysilyl thiazoli-
1
dine derivatives [9, 10]. The H- (DMSO-d ) and
8
6
1
3
9
156.0
103.7
120.0
108.6
145.3
136.7
145.3
108.6
158.0
106.7
121.0
115.7
144.6
147.8
115.0
121.4
156.0
102.9
122.0
115.4
144.8
148.6
116.0
123.1
C-NMR (Table 1) spectra of 1, which were assigned by
1
1
10
0
various NMR experiments including H– H COSY,
HMQC, and HMBC spectra, indicated there was a my-
ricetin part [d 6.19 (1H, d, J = 2.0 Hz, H-6), 6.37 (1H, d,
1
0
2
0
0
0
3
J = 2.0 Hz, H-8), 7.24 (2H, s, H-2 ,6 ), 12.71 (1H, br. s,
-OH)], together with b-D-xylopyranosyl and b-D-galacto-
0
4
5
0
0
00
5
pyranosyl moieties [d 4.57 (1H, d, J = 7.2 Hz, H-1 ), 5.72
(1H, d, J = 7.6 Hz, H-1 )] in the structure. Furthermore,
the assignment of glycoside protons was determined by
0
00
6
Gal/Glc/GlcA
00
1
1
1
98.3
79.9
73.6
67.6
75.8
59.8
102.3
73.2
75.9
69.2
76.7
60.2
106.0
73.9
75.5
71.6
75.9
171.3
103.3
71.1
101.5
73.0
75.6
69.6
73.7
63.3
106.2
73.7
75.1
71.3
75.5
168.6
proton–proton correlations observed from the H– H
COSY experiment. Finally, the linkages of the b-D-galac-
topyranosyl and b-D-xylopyranosyl moieties were clarified
on the basis of the HMBC experiment, which showed long-
0
0
0
0
0
0
0
0
2
3
4
5
6
73.28
68.0
range correlations between the proton d 5.72 (1H, d,
73.25
65.2
H
0
0
00
J = 7.6 Hz, H-1 ) and carbon d 133.1 (C-3) and between
C
000
the proton d 4.57 (1H, d, J = 7.2 Hz, H-1 ) and carbon
d 79.9 (C-2 ). Consequently, the structure of floramano-
Xyl/Rha
000
H
0
0
1
104.5
73.9
76.0
69.3
65.3
99.9
70.3
70.5
71.8
68.2
17.8
55.8
C
0
0
0
0
00
00
00
00
side A was determined as myricetin-3-O-b-D-xylopyrano-
syl(1?2)-b-D-galactopyranoside (1).
Floramanoside B (2) was obtained as a yellow amor-
2
3
4
5
6
2
D
5
phous powder and exhibited a negative rotation [[a]
–
4
5.48 (c = 0.91, MeOH)]. HRESI–TOF-MS analysis
revealed its molecular formula to be C H O (m/z
000
2
1
20 14
CH O–
3
?
4
97.0929 [M?H] , calcd for C H O 497.0926). The IR
21 21 14
CH CO–
3
20.2
spectrum of 2 showed absorption bands at 1655 and
-
CH
CH
CH
3
3
3
CO–
170.1
1
1
572 cm
ascribable to c-pyrone function, and broad
bands at 3341 and 1075 cm suggestive of a glycoside
CH
CH
2
O–
O–
13.7
60.5
-
1
2
1 13
structure. The H- (DMSO-d ) and C-NMR (Table 1)
6
123

8
0
J Nat Med (2013) 67:78–85
substitution benzyl in 2. Its aglycone and glycoside posi-
tions were determined by the HMBC experiment (Fig. 2),
(1:1, v/v) afforded D-glucuronic acid, whose absolute
configuration (D-) was determined by GC analysis of its
trimethysilyl thiazolidine derivative [9, 10]. Finally, the
linkage position of the b-D-glucuronoyl moiety was deter-
mined by the HMBC experiment, which showed long-
which showed long-range correlations between d 4.80
H
00
0
(
1H, d, J = 7.2 Hz, H-1 ) and d 145.4 (C-3 ); dH 6.29
C
(
1H, s, H-6) and d 102.6 (C-10), 124.4 (C-8), 152.3 (C-5)
C
0
and 153.0 (C-7); d 7.62 (1H, d, J = 2.0 Hz, H-6 ) and d
range correlation between dH 4.71 (1H, d, J = 7.6 Hz,
H
C
0
0
0
0
0
00
1
1
1
1
1
07.6 (C-2 ), 121.2 (C-1 ), 137.2 (C-4 ), 145.6 (C-2), and
0
H-1 ) and d 125.0 (C-8) (Fig. 2). Consequently, the
C
46.0 (C-5 ); d 7.67 (1H, d, J = 2.0 Hz, H-2 ) and d
structure of floramanoside C was determined to be
0
H
C
0
0
0
0
0
10.5 (C-6 ), 121.2 (C-1 ), 137.2 (C-4 ), 146.0 (C-5 ) and
3,3 ,4 ,5,5 ,7,8-heptahydroxyl flavone-8-O-b-D-glucurono-
pyranoside (3).
Floramanoside D (4) was isolated as a yellow amor-
45.6 (C-2); dH 11.92 (1H, s, 5-OH) and d 97.9 (C-6),
C
02.6 (C-10) and 152.3 (C-5). Finally, the presence of a D-
2
5
glucopyranosyl moiety in 2 was demonstrated by the acid
hydrolysis, derivative and GC analysis processes like those
of 1 [9, 10]. On the basis of the above-mentioned evidence,
the structure of floramanoside B was clarified to be
phous powder, which exhibited negative rotation [[a]D –
14.5° (c = 0.46, MeOH)]. Its molecular formula was
determined to be C H O by negative-ion HRESI–TOF-
-
2
8 32 16
MS (m/z 623.1628 [M–H] , calcd for C H O
8 31 16
2
0
0
0
0
3
,3 ,4 ,5 ,5,7,8-heptahydroxyl flavone-3 -O-b-D-glucopy-
ranoside (2).
613.1618). Acid hydrolysis of 4 with 5% aqueous H SO –
2 4
1,4-dioxane (1:1, v/v) afforded L-rhamnose and D-galact-
ose, whose absolute configurations (L-, D-) were determined
by GC analysis of their trimethysilyl thiazolidine deriva-
Floramanoside C (3) was obtained with negative rota-
2
5
tion [[a] ?49.0° (c = 0.31, MeOH)]. The molecular
D
1
13
formula, C H O , of 3 was determined from negative-
-
tives [9, 10]. The H- DMSO-d ) and C-NMR (Table 1)
6
2
1
18 15
ion HRESI–TOF-MS (m/z 509.0554 [M–H] , calcd for
C H O 509.0552). The IR spectrum of 3 indicated the
spectra of 4, which were assigned by various NMR
1
1
experiments including H– H COSY, HMQC, and HMBC
spectra, indicated the presence of a 5-methyl quercetin part
2
1 17 15
-
1
presence of carboxyl (1721 cm ), c-pyrone (1652 and
-
568 cm ) functions, and a glycoside structure (3342,
1
1
[d 3.82 (3H, s, 5-OCH ), 6.38 (1H, d, J = 1.6 Hz, H-6),
3
-
1
1
072 cm ). The H- (DMSO-d ) and C-NMR (Table 1)
13
1
6.45 (1H, d, J = 1.6 Hz, H-8), 6.81 (1H, d, J = 8.4 Hz,
0
6
0
spectra, which were assigned by various NMR experiments
H-5 ), 7.52 (1H, d, J = 2.4 Hz, H-2 ), 7.62 (1H, dd,
0
1
including H– H COSY, HMQC, and HMBC spectra,
1
J = 2.4, 8.4 Hz, H-6 )], together with a-L-rhamnopyrano-
indicated that its aglycone was the same as 2,
syl and b-D-galactopyranosyl moieties [d 1.07 (3H, d,
0
0
0
000
000
3
7
,3 ,4 ,5 ,5,7,8-heptahydroxyl flavone [d 6.25 (1H, s, H-6),
.44 (2H, s, H-2 ,6 ), 12.44 (1H, br. s, 5-OH)]. The proton
1
J = 6.4 Hz, 6 -CH ), 4.44 (1H, d, J = 1.6 Hz, H-1 );
3
0
0
00
5.04 (1H, d, J = 8.0 Hz, H-1 )]. The methyl and glyco-
sides linkage positions were determined by the long-range
signals in H-NMR spectrum [d 4.71 (1H, d, J = 7.6 Hz,
0
0
00
H-1 ), 3.30–3.60 (4H, m, H-2–5 )] and carbon signals in
1
correlations between d 3.82 (3H, s, 5-OCH ) and d 160.6
H
3
C
3
000 00
C-NMR spectrum (d : 71.6, 73.9, 75.5, 75.9, 106.0,
(C-5), 4.44 (1H, d, J = 1.6 Hz, H-1 ) and d 65.2 (C-6 ),
C
C
00
1
71.3) exhibited a b-D-glucuronoyl moiety. Furthermore,
acid hydrolysis of 3 with 5% aqueous H SO –1,4-dioxane
together with dH 5.04 (1H, d, J = 8.0 Hz, H-1 ) and d
135.9 (C-3) observed in the HMBC experiment (Fig. 3).
C
2
4
Fig. 2 The main HMBC
correlations of compounds 1–3
OH
OH
O
O
OH
OH
OH
OH
O
OH
HO
HO
O
O
HO
O
O
2
HO
HO
OH
6
10
8
O
H
OH
O
5
OH
HO
HO
OH
OH
O
O
OH
HO
HOOC
O
O
O
OH
OH
HO
OH
HO
H
H
HO
H
OH
OH
OH
1
HMBC :
2
3
1
23

J Nat Med (2013) 67:78–85
81
Furthermore, in the NOESY experiment (Fig. 3), the NOE
521.0937). Acid hydrolysis of 6 with 5% aqueous H SO –
2
4
correlation was observed between dH 3.82 (5-OCH ) and
1,4-dioxane (1:1, v/v) yielded D-glucuronic acid, deter-
mined by GC analysis of its trimethysilyl thiazolidine
derivative [9, 10]. The IR spectrum of 6 showed absorption
3
dH 6.38 (H-6), which clarified that the methyoxy was
linked with the carbon at the 5-position. On the basis of the
above-mentioned evidence, the structure of floramanoside
-
1
bands at 1731, 1652 and 1567 cm ascribable to ester
0
0
D was elucidated to be 3,3 ,4 ,7-tetrahydroxyl-5-methoxyl
flavone-3-O-robinoside (4).
Floramanoside E (5) was obtained as a yellow amor-
carboxyl and c-pyrone functions, and broad bands at 3378
and 1075 cm suggestive of a glycoside structure. The
-
1
1
13
H-(DMSO-d ) and C-NMR (Table 1) spectra of 6,
6
2
5
phous powder with negative rotation [[a]D –54.0°
c = 0.75, MeOH)]. The molecular formula, C H O , of
which were assigned by various NMR experiments
1
including H– H COSY, HMQC, and HMBC spectra,
1
(
2
3 22 13
5
5
was determined by negative-ion HRESI–TOF-MS (m/z
-
05.0959 [M–H] , calcd for C H O 509.0988). The IR
exhibited the presence of a gossypetin-8-O-b-D-glucuron-
00
opyranoside part [d 4.76 (1H, d, J = 8.0 Hz, H-1 ), 6.21
2
3 21 13
0
spectrum of 5 indicated the presences of ester carboxyl
-
(1H, s, H-6), 6.83 (1H, d, J = 8.4 Hz, H-5 ), 7.58 (1H, dd,
0
1
-1
0
(1723 cm ) and c-pyrone (1654 and 1570 cm ) func-
tions and a glycoside structure (3346, 1074 cm ). The H-
J = 2.0, 8.4 Hz, H-6 ), 7.90 (1H, d, J = 2.0 Hz, H-2 ),
12.31 (1H, s, 5-OH)], together with an ethoxyl group [d
-
1
1
1
3
00 00
1.02 (3H, t, J = 7.2 Hz, 6 -OCH CH ), 4.02 (2H, m, 6 -
2 3
(DMSO-d6),
experiments including H– H COSY, HMQC, and HMBC
C-NMR (Table 1), and various NMR
1
1
OCH CH )]. The position of the ethoxyl group was
2 3
spectra (Fig. 3), revealed the presence of an acetyl group
0
determined by the HMBC experiment, which exhibited
00
0
0
[
d 1.82 (3H, s, 6 -OOCCH )], together with a quercetin 3 -
long-range correlation between dH 4.02 (2H, m, 6 -
3
O-b-D-glucopyranoside part [d 4.17 (1H, dd, J = 6.0,
OCH CH ) and d 168.6 (C-6’’). On the basis of the above-
mentioned evidence, the structure of floramanoside F was
2
3
C
0
0
1
2.0 Hz), 4.32 (1H, dd, J = 2.0, 12.0 Hz), H -6 , 4.88
2
00
(
1H, d, J = 7.6 Hz, H-1 ), 6.20 (1H, d, J = 1.6 Hz, H-6),
.47 (1H, d, J = 2.0 Hz, H-8), 7.00 (1H, d, J = 8.4 Hz,
elucidated as shown in Fig. 3.
6
Oxidative stress has been implicated in the possible
pathophysiology of different ocular diseases, such as
cerebral ischemia, atherosclerosis, inflammation, diabetes,
and cancer [11]. Aldose reductase, which catalyzes
NADPH-dependent reduction of glucose to sorbitol, has
been proposed to be a key factor for the pathogenesis of
diabetic complications. Free radicals, especially hydroxyl
radicals, are indirectly inhibited by aldose reductase
inhibitor resulting from decreasing polyol levels and
hydroxyl radical formation, via the Fenton reaction
involving H O produced from the activated polyol path-
0
H-5 ), 7.81 (1H, dd, J = 2.0, 8.4 Hz, H-6), 7.95 (1H, d,
0
J = 2.0 Hz, H-2 ), 12.47 (1H, br. s, 5-OH)]. The position
of the acetyl group was determined from the HMBC
experiment, which showed long-range correlations
00
between the protons of position-6 [dH 4.17 (1H, dd,
J = 6.0, 12.0 Hz), 4.34 (1H, dd, J = 2.0, 12.0 Hz)] and
ester carboxyl carbon [dC 170.1 (6’’-CH CO)]. Finally,
3
acid hydrolysis of 5 with 5% aqueous H SO –1,4-dioxane
2
4
(
1:1, v/v) afforded D-glucose, determined by GC analysis
of its trimethysilyl thiazolidine derivative [9, 10]. Conse-
quently, the structure of floramanoside E was determined to
2
2
way [12]. In this study, the antioxidative effects of the six
new compounds against radical scavenging activities of
DPPH radical and inhibition activities of AR were asses-
sed. As shown in Table 2, all of the compounds showed
DPPH scavenging activity, and 1–5 showed AR inhibition
activity. This result indicated that A. manihot flowers may
be useful for oxidant stress-related disease.
0
be quercetin-3 -O-(6’’-acetyl)-b-D-glucopyranoside (5).
Floramanoside F (6) was also obtained as a yellow
amorphous powder, which exhibited positive rotation
2
5
D
[
[a] ?83.9° (c = 0.62, MeOH)]. Its molecular formula
was revealed to be C H O by negative-ion HRESI–
-
TOF-MS (m/z 521.0917 [M–H] , calcd for C H O
2
3 22 14
23
21 14
Fig. 3 The main HMBC and
NOE correlations of compounds
OH
OH
OH
O
O
O
OH
HO
4
–6
HO
HO
O
O
HO
O
O
HO
HO
OH
8
3
5
O
H
OH
O
O
CH O
OH
3
H C
O
O
3
O
H C
3
HO
O
O
O
O
O
OH
OH
OH
HMBC:
NOE:
H
HO
H
OH
HO
H
OH
HO OH
OH
4
5
6
123

8
2
J Nat Med (2013) 67:78–85
Table 2 Effects of compounds 1–6 on DPPH scavenging and aldose
reductase inhibition activities (ARI)
EtOH extract (648 g, 23.14%). This extract (500 g) was
subjected to D101 resin, and eluted with H O, 70% and
2
95% EtOH successively, to obtain the H O- (220.1 g,
2
Compound
DPPH (SC50, lM)
ARI (IC50, lM)
1
0.21%), 70% EtOH- (120.2 g, 5.55%) and 95% EtOH-
1
2
3
4
5
6
10.1
6.2
17.8
13.7
(22.2 g, 1.02%) eluted fractions, respectively.
The 70% EtOH-eluted fraction (96.0 g) was isolated by
10.4
12.5
24.0
25.1
7.1
silica gel column chromatography [1000 g, CHCl –MeOH
3
2.2
(100:3 ? 10:1, v/v) ? CHCl –MeOH–H O (7:3:1 ?
3
2
8.3
6:4:1, v/v/v, the lower layer) ? MeOH] to afford nine
[100.0
fractions [Fr. 1 (1.0 g), Fr. 2 (2.5 g), Fr. 3 (4.2 g), Fr. 4
(
(
4.7 g), Fr. 5 (16.0 g), Fr. 6 (22.0 g), Fr. 7 (23.2 g), Fr. 8
12.2 g), Fr. 9 (6.2 g)].
Experimental
Fr. 4 (3.5 g) was separated by ODS column chromatog-
raphy [MeOH–H O (30% ? 40% ? 50% ? 60% ?
2
General experimental procedures
100%) ? CHCl –MeOH–H O (6:4:1, v/v/v, the lower
3
2
layer)], and six fractions were obtained [Fr. 4-1 (770.0 mg),
Fr. 4-2 (581.0 mg), Fr. 4-3 (280.0 mg), Fr. 4-4 (408.0 mg),
Fr. 4-5 (915.0 mg), Fr. 4-6 (382.0 mg)]. Fr. 4-5 (915.0 mg)
Ò
Optical rotations were measured on a Rudolph Autopol
IV automatic polarimeter (l = 50 mm), whereas IR spectra
were recorded on a Varian 640-IR FT-IR spectrophotom-
eter and UV spectra on a Varian Cary 50 UV–Vis spec-
was purified by PHPLC [MeOH–H O (50:50, v/v) ? 1%
2
HAc] to give floramanosides E (5, 20.0 mg, 0.0015%) and F
(6, 30.0 mg, 0.0023%). Fr. 7 (12.0 g) was subjected to ODS
1
13
trophotometer. H- and C-NMR spectra were determined
1
on a Varian 400MR spectrometer at 400 MHz for H- and
1
00 MHz for C-NMR, with tetramethylsilane (TMS) as
column chromatography [MeOH–H O (10% ? 20% ?
2
3
1
30% ? 40% ? 50% ? 100%)] to yield seven fractions
[Fr. 7-1 (0.54 g), Fr. 7-2 (0.53 g), Fr. 7-3 (1.03 g), Fr. 7-4
(4.31 g), Fr. 7-5 (4.11 g), Fr. 7-6 (0.55 g), Fr. 7-7 (0.24 g)].
Fr. 7-4(1.78 g)wasseparatedwith Sephadex LH-20(MeOH)
to give 10 fractions [Fr. 7-4-1 (162.0 mg), Fr. 7-4-2
(85.7 mg), Fr. 7-4-3 (441.0 mg), Fr. 7-4-4 (189.5 mg), Fr.
7-4-5 (268.0 mg), Fr. 7-4-6 (454.0 mg), Fr. 7-4-7 (40.0 mg),
Fr. 7-4-8 (92.5 mg), Fr. 7-4-9 (58.0 mg), Fr. 7-4-10
(22.0 mg)]. Fr. 7-4-2 (85.7 mg) was further purified by
an internal standard. Positive- and negative-ion HRESI–
TOF-MS were recorded on an Agilent Technologies 6520
Accurate-Mass Q-TOF LC/MS spectrometer.
A highly-porous synthetic resin (D101) was purchased
from Haiguang Chemical Co., Ltd. (Tianjin, China). Silica
gel CC was obtained from Qingdao Haiyang Chemical Co.,
Ltd., (48–75 lm, Qingdao, China). Sephadex LH-20 (GE
Healthcare Bio-Sciences, Switzerland) were used to purify
the total flavonoids from the whole residue. HPLC was
performed on ODS (Cosmosil 5C18-MS-II, Tokyo, Japan;
U = 20 mm, L = 250 mm, flow rate: 9.0 ml/min), and the
eluate was monitored with a UV detector (Shimadzu RID-
PHPLC [MeOH–H O (40:60, v/v) ? 1% HAc] to yield
2
floramanoside D (4, 14.5 mg, 0.0039%). Fr. 8 (8.0 g) was
subjected to ODS column chromatography [MeOH–H O
2
(10% ? 20% ? 30% ? 40% ? 50% ? 70% ? 100%-
) ? CHCl –MeOH–H O (6:4:1, v/v/v, thelower layer)], and
1
0A UV–VIS, Japan). Pre-coated TLC plates with Silica
3
2
gel GF254 (Tianjin Silida Technology Co., Ltd., Tianjin,
China) were used to detect the purity of isolate achieved by
spraying with 10% aqueous H SO –EtOH, following by
10 fractions [Fr. 8-1 (0.32 g), Fr. 8-2 (0.23 g), Fr. 8-3
(0.32 g), Fr. 8-4(0.21 g), Fr. 8-5 (1.46 g), Fr. 8-6(3.41 g), Fr.
8-7 (0.15 g), Fr. 8-8 (0.20 g), Fr. 8-9 (1.05 g), Fr. 8-10
(0.08 g)] were obtained. Fr. 8-6 (2.59 g) was separated with
2
4
heating.
Sephadex LH-20 [MeOH-H O (50:50, v/v)] to give six
2
Plant material
fractions [Fr. 8-6-1 (597.4 mg), Fr. 8-6-2 (376.7 mg), Fr. 8-6-
3
(573.7 mg), Fr. 8-6-4 (367.4 mg), Fr. 8-6-5 (520.0 mg), Fr.
The flowers of A. manihot (L.) Medic were collected from
Luoshan county, Henan province, China and identified by
Dr. Li Tianxiang. The voucher specimen was deposited at
the Academy of Traditional Chinese Medicine of Tianjin
University of TCM (No. 20081112).
8-6-6 (34.6 mg)]. Fr. 8-6-2 (376.7 mg) was finally purified by
HPLC [MeOH–H O (35:65, v/v) ? 1% HAC] to afford
2
floramanoside D (4, 20.1 mg, 0.0017%). Separated by the
same HPLC condition as Fr. 8-6-2, floramanosides A (1,
12.0 mg, 0.0010%) and B (2, 52.7 mg, 0.0046%) were iso-
lated from Fr. 8-6-3 (573.7 mg) and Fr. 8-6-4 (367.4 mg),
respectively. Fr. 9 (5.0 g) was separated by Sephadex LH-20,
Extraction and isolation
eluted with MeOH–H O (1:1, v/v), and finally purified by
2
The dried flowers of A. manihot (2.8 kg) were extracted
with 95% EtOH under reflux three times to give the 95%
HPLC [MeOH–H O (35:65, v/v) ? 1% HAC] to afford
2
floramanoside C (3, 15.0 mg, 0.0011%).
1
23

J Nat Med (2013) 67:78–85
Characterization data
Floramanoside A (1)
83
(100 MHz, DMSO-d ) spectroscopic data, see Table 1.
6
HRESI–TOF-MS: negative-ion mode m/z 509.0554 [M–
-
H] (calcd for C H O 509.0552); positive-ion mode m/z
2
1 17 15
?
45.0342 [M?Cl] (calcd for C H O Cl 545.0340).
5
21 18 15
2
5
Yellow powder: [a] –40.6° (c = 1.51, MeOH); IR mmax
D
-
KBr) cm : 3301, 2907, 2725, 1657, 1607, 1504, 1454,
1
(
Floramanoside D (4)
1
7
2
357, 1303, 1236, 1200, 1167, 1066, 1041, 998, 975, 840,
2
D
5
65, 645, 571; UV k (MeOH) nm (log e): 360 (4.08),
Yellow powder: [a] –14.5° (c = 0.46, MeOH); IR kmax
max
1
-1
(KBr) cm : 3385, 3266, 2981, 2908, 1629, 1605, 1501,
55 (4.12). H-NMR (400 MHz, DMSO-d ): d [3.03 (1H,
6
dd, J = 5.2, 11.6 Hz), 3.67 (1H, dd, J = 4.8, 11.6 Hz), H2-
0
1443, 1355, 1333, 1296, 1272, 1205, 1171, 1137, 1101,
00
000
5
], 3.06 (1H, dd, J = 7.2, 8.8 Hz, H-2 ), 3.13 (1H, dd,
000
1058, 1035, 1006, 978, 827, 788; UV m
(MeOH) nm
max
000
1
J = 8.4, 8.8 Hz, H-3 ), 3.26 (1H, m, overlapped, H-4 ),
3.34 (1H, m, overlapped), 3.47 (1H, br. d, ca. J = 12 Hz),
(log e): 350 (4.25), 255 (4.31). H-NMR (400 MHz,
000
DMSO-d ): d 1.07 (3H, d, J = 6.4 Hz, 6 -CH ), 3.12 (1H,
[
6
3
0
0
00
000
H -6 ], 3.36 (1H, m, overlapped, H-5 ), 3.63 (1H, dd,
2
dd, J = 9.2, 9.2 Hz, H-4 ), [3.22 (1H, dd, J = 6.0,
00
00
J = 2.8, 9.2 Hz, H-3 ), 3.68 (1H, br. d, ca. J = 3 Hz,
0
9.6 Hz), 3.63 (1H, dd, J = 6.0, 9.6 Hz), H -6 ], 3.34 (1H,
2
0
00
000
00
H-4 ), 3.83 (1H, dd, J = 7.6, 9.2 Hz, H-2 ), 4.57 (1H, d,
0
dd, J = 3.2, 9.2 Hz, H-3 ), 3.40 (2H, m, overlapped, H-3
and 5 ), 3.44 (1H, m, overlapped, H-2 ), 3.55 (1H, br. d,
00
00
000 000
J = 7.2 Hz, H-1 ), 5.72 (1H, d, J = 7.6 Hz, H-1 ), 6.19
(
0
0
00
1H, d, J = 2.0 Hz, H-6), 6.37 (1H, d, J = 2.0 Hz, H-8),
ca. J = 6 Hz, H-5 ), 3.56 (1H, dd, J = 8.0, 9.2 Hz, H-2 ),
00
0
0
13
.24 (2H, s, H-2 ,6 ), 12.71 (1H, br. s, 5-OH); C-NMR
7
3.59 (1H, br. s, H-4 ), 3.82 (3H, s, 5-OCH ), 4.44 (1H, d,
3
0
00
00
(
100 MHz, DMSO-d ) spectroscopic data, see Table 1.
6
J = 1.6 Hz, H-1 ), 5.03 (1H, d, J = 8.0 Hz, H-1 ), 6.38
(1H, d, J = 1.6 Hz, H-6), 6.45 (1H, d, J = 1.6 Hz, H-8),
HRESI–TOF-MS: positive-ion mode m/z 613.1398
?
[
0
M?H] (calcd for C H O 613.1399); 635.1218
6 29 17
6.81 (1H, d, J = 8.4 Hz, H-5 ), 7.52 (1H, d, J = 2.4 Hz,
13
H-2 ), 7.62 (1H, dd, J = 2.4, 8.4 Hz, H-6); C-NMR
100 MHz, DMSO-d ) spectroscopic data, see Table 1.
HRESI–TOF-MS: negative-ion mode m/z 623.1628 [M–
2
?
M?Na] (calcd for C H O Na 635.1219).
0
[
2
6 28 17
(
6
Floramanoside B (2)
-
H] (calcd for C H O 623.1618); positive-ion mode m/z
2
8 31 16
2
5
?
659.1390 [M?Cl] (calcd for C H O Cl 659.1384).
Yellow powder: [a] –45.4° (c = 0.91, MeOH); IR mmax
D
28 32 16
-
KBr) cm : 3341, 2947, 1655, 1620, 1572, 1523, 1451,
1
(
1
343, 1257, 1204, 1167, 1075, 1028, 894, 792; UV k
Floramanoside E (5)
max
1
(
MeOH) nm (log e): 340 (4.05), 275 (4.16), 255 (4.20). H-
2
5
NMR (400 MHz, DMSO-d ): d 3.31 (1H, m, overlapped,
Yellow powder: [a] –54.0° (c = 0.75, MeOH); IR mmax
6
D
0
0
00
-1
(KBr) cm : 3346, 2930, 1723, 1654, 1602, 1570, 1513,
H-5 ), 3.32 (1H, m, overlapped, H-4 ), 3.33 (1H, m,
0
0
00
overlapped, H-3 ), 3.40 (1H, dd, J = 7.2, 8.4 Hz, H-2 ),
3.62 (1H, dd, J = 5.2, 11.6 Hz), 3.78 (1H, br. d, ca.
1443, 1367, 1318, 1288, 1252, 1202, 1167, 1074, 1042,
[
889, 807; UV k
(4.01). H-NMR (400 MHz, DMSO-d ): d 1.82 (3H, s, 6’’-
(MeOH) nm (log e): 320 (3.85), 250
max
0
0
00
1
J = 12 Hz), H -6 ], 4.80 (1H, d, J = 7.2 Hz, H-1 ), 6.29
2
6
0
00
(
1H, s, H-6), 7.62 (1H, d, J = 2.0 Hz, H-6 ), 7.67 (1H, d,
13
OOCCH ), 3.26 (1H, dd, J = 8.8, 9.2 Hz, H-4 ), 3.37 (1H,
3
0
00
J = 2.0 Hz, H-2 ), 11.92 (1H, s, 5-OH); C-NMR
100 MHz, DMSO-d ) spectroscopic data, see Table 1.
dd, J = 7.6, 9.2 Hz, H-2 ), 3.38 (1H, dd, J = 8.8, 9.2 Hz,
H-3 ), 3.66 (1H, m, H-5 ), [4.17 (1H, dd, J = 6.0,
00 00
(
6
0
0
HRESI–TOF-MS: positive-ion mode m/z 497.0929
?
12.0 Hz), 4.32 (1H, dd, J = 2.0, 12.0 Hz), H -6 ], 4.88
2
00
[
M?H] (calcd for C H O 497.0926); 519.0743
1 21 14
(1H, d, J = 7.6 Hz, H-1 ), 6.20 (1H, d, J = 2.0 Hz, H-6),
6.47 (1H, d, J = 2.0 Hz, H-8), 7.00 (1H, d, J = 8.4 Hz,
2
?
M?Na] (calcd for C H O Na 519.0745).
[
2
1 20 14
0
H-5 ), 7.81 (1H, dd, J = 2.0, 8.4 Hz, H-6), 7.95 (1H, d,
13
0
Floramanoside C (3)
J = 2.0 Hz, H-2 ), 12.47 (1H, br. s, 5-OH); C-NMR
100 MHz, DMSO-d ) spectroscopic data, see Table 1.
HRESI–TOF-MS: negative-ion mode m/z 505.0959 [M–
(
6
2
5
Yellow powder: [a] ?49.0° (c = 0.31, MeOH); IR mmax
D
-
KBr) cm : 3342, 2911, 1721, 1652, 1607, 1568, 1511,
1
-
(
H] (calcd for C H O 505.0988); positive-ion mode m/z
3 21 13
2
?
1
418, 1329, 1252, 1202, 1174, 1072, 1023, 979, 839, 794;
1
541.0724 [M?Cl] (calcd for C H O Cl 541.0754).
23 22 13
UV kmax (MeOH) nm (log e): 305 (3.79), 255 (4.14). H-
NMR (400 MHz, DMSO-d ): d 3.32 (1H, dd, J = 8.0,
Floramanoside F (6)
6
0
0
0
0
8
.0 Hz, H-3 ), 3.40 (1H, dd, J = 8.0, 9.2 Hz, H-4 ), 3.44
1H, dd, J = 7.6, 8.0 Hz, H-2 ), 3.58 (1H, d, J = 9.2 Hz,
00
25
Yellow powder: [a]D ?83.9° (c = 0.62, MeOH); IR mmax
(KBr) cm : 3378, 2985, 2934, 1731, 1652, 1605, 1567,
(
0
0
00
-1
H-5 ), 4.71 (1H, d, J = 7.6 Hz, H-1 ), 6.25 (1H, s, H-6),
7
0
0
13
.44 (2H, s, H-2 ,6 ), 12.44 (1H, br. s, 5-OH); C-NMR
1512, 1444, 1374, 1324, 1253, 1172, 1075, 1017, 902, 819;
123

8
4
J Nat Med (2013) 67:78–85
1
UV kmax (MeOH) nm (log e): 305 (3.88), 260 (4.26). H-
reaction was initiated by the addition of NADPH at 30°C.
After 30 min, the reaction was stopped by the addition of
150 lL 0.5 M HCl. Then, 0.5 mL 6 M NaOH containing
10 mM imidazole was added, and the solution was heated
at 60°C for 20 min to convert NADPH to a fluorescent
product. Fluorescence was measured using a fluoropho-
tometer (FlexStation 3 multimode benchtop reader,
Molecular Devices Corporation, USA) at an extraction
wavelength of 360 nm and an emission wavelength of
460 nm. Epalrestat was used as positive control (IC :
NMR (400 MHz, DMSO-d ): d 1.02 (3H, t, J = 7.2 Hz,
00
-OCH CH ), 3.32 (1H, dd, J = 8.0, 9.2 Hz, H-3 ), 3.44
2 3
6
0
0
6
00
(
1H, dd, J = 8.0, 9.6 Hz, H-4 ), 3.40 (1H, dd, J = 8.0,
00 00
.2 Hz, H-2 ), 3.83 (1H, d, J = 9.6 Hz, H-5 ), 4.02 (2H,
9
0
0
00
m, 6 -OCH CH ), 4.76 (1H, d, J = 8.0 Hz, H-1 ), 6.21
2
3
0
(
1H, s, H-6), 6.83 (1H, d, J = 8.4 Hz, H-5 ), 7.58 (1H, dd,
0
0
J = 2.0, 8.4 Hz, H-6 ), 7.90 (1H, d, J = 2.0 Hz, H-2 ),
1
1
2.31 (1H, s, 5-OH); C-NMR (100 MHz, DMSO-d6)
3
spectroscopic data, see Table 1. HRESI–TOF-MS: nega-
-
tive-ion mode m/z 521.0917 [M–H] (calcd for C H O
5
0
0.08 lg/ml).
23
21 14
?
5
21.0937); positive-ion mode m/z 557.0687 [M?Cl]
calcd for C H O Cl 557.0693).
(
2
3 22 13
DPPH radical scavenging assay
Acid hydrolysis of 1–6
The free radical scavenging activity was assessed using the
DPPH radical by the method described by Matsuda et al.
Solutions of floramanosides A–F (1–6, each 2.0 mg) in 5%
aqueous H SO –1,4-dioxane (0.5 mL) were heated under
[
8]. An ethanol solution of DPPH (100 lM, 1.0 mL) was
mixed with different concentrations of each test compound
0–200 lM, 0.5 mL) and 0.1 M acetate buffer (pH 5.5,
.0 mL), and the absorbance change at 517 nm was mea-
2
4
reflux for 1 h. After cooling, the reaction mixture was
-
neutralized with Amberlite IRA-400 (OH form) and
(
1
removed by filtration. After removal of the solution from
sured 30 min later. The reaction solution without DPPH
was used as a blank test. Measurements were performed in
duplicate, and the concentration required for 50% reduction
the filtrate in vacuo, the residue was treated with H O and
2
MeOH on an ODS column. The H O-eluted fraction was
2
concentrated and the residue was reacted with L-cysteine
methyl ester hydrochloride (2 mg) in pyridine (0.2 mL) at
(
50% scavenging concentration, SC ) of 40 lM DPPH
5
0
radical solution was determined graphically. a-Tocopherol
was used as positive control (SC : 3.48 lg/ml).
6
0°C for 2 h, then N,O-bis(trimethylsilyl)trifluoroaceta-
5
0
mide (BSTFA, 0.2 mL) was added and stirred under reflux
at 60°C for 2 h. The supernatant was then subjected to GC
analysis under the following conditions: GC conditions,
column: Agilent Technologies INC Catalog 19091 J-413
HP-5, 30 m 9 0.320 mm (i.d.) capillary column; column
Acknowledgments This research was supported by Program for
Changjiang Scholars and Innovative Research Team in University
(
PCSIR IRT0973), Applied Basic and Advanced Research Program of
Tianjin (10SYSYJC28900, 10ZCKFSY09300), and Important Drug
Development Fund, Ministry of Science and Technology of China
(2012ZX09103201-031).
temperature: 230°C; carrier gas: N . The identification of
2
derivatives of D-galactose (iii) and D-xylose (v) from 1;
D-glucose (ii) from 2; D-glucuronic acid (i) from 3; D-gal-
actose (iii) and L-rhamnose (iv) from 4; D-galactose (iii)
from 5; and D-glucuronic acid (i) from 6 present in the
supernatant was carried out by comparison of their reten-
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