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Introduction
Obesity usually results from impaired energy balance caused by
either increased caloric intake and/or decreased energy expendi-
ture. Worldwide increases in the population of obese people are
most likely to be due to eating habits and lack of exercise. Cur-
rently, there are two known types of obesity: visceral and subcu-
taneous. The former type is characterized by marked fat accumu-
lation in the abdominal cavity and is frequently associated with
metabolic aberrations, such as glucose intolerance and hyperlipi-
demia [1]. Recently, much attention has been focused upon var-
ious nutritional factors that may be beneficial for preventing
body fat accumulation and possibly reducing the risk of obesity-
related diseases [2].
The lipolytic action in fat cells plays an important role in the en-
ergy metabolism of mammals. It is well known that lipolytic ac-
tion in fat cells is stimulated by various pharmacological lipoly-
tic hormones, such as catecholamines, norepinephrine and
epinephrine [3]. Catecholamines are also known to stimulate li-
polysis via the
b
-adrenergic pathway, which is involved in ener-
gy expenditure and in the prevention of diet-induced obesity
[4], [5], [6].
Constituents from the Leaves of Nelumbo nucifera
Stimulate Lipolysis in the White Adipose Tissue of Mice
Emika Ohkoshi
1, 3
Hiromi Miyazaki
1
Kazutoshi Shindo
2
Hiroyuki Watanabe
1
Aruto Yoshida
1
Hiroaki Yajima
1
Affiliation
1
KIRIN Brewery Co., Ltd., Central Laboratories for Frontier Technology, Kanagawa, Japan
2
Department of Food and Nutrition, Japan Women's University, Tokyo, Japan
3
Present address: The School of Pharmaceutical Sciences, Ohu University, Fukushima, Japan
Correspondence
Dr. Hiroaki Yajima ´ Central Laboratories for Frontier Technology ´ 1±13±5 Fukuura Kanazawa-ku ´
Yokohama-shi ´ Kanagawa 236±0004 ´ Japan ´ Phone: +81-45-330-9004 ´ Fax: +81-45-88-4047 ´
E-mail: hyajima@kirin.co.jp
Received November 20, 2006 ´ Revised July 25, 2007 ´ Accepted August 20, 2007
Bibliography
Planta Med 2007; 73: 1±5 Georg Thieme Verlag KG Stuttgart ´ New York
DOI 10.1055/s-2007-990223 ´ Published online n
ISSN 0032-0943
Abstract
Nelumbo nucifera Gaertn. (Nymphaceae) has been used for var-
ious medicinal purposes as in Chinese herbal medicine. In partic-
ular, the leaves are known for diuretic and astringent properties,
and are used to treat obesity. During our search for a plant-de-
rived anti-obesity agent from natural products, we have found
that a 50 % ethanol (EtOH) extract prepared from the leaves of N.
nucifera (NN) stimulated lipolysis in the white adipose tissue
(WAT) of mice and that the
b
-adrenergic receptor (
b
-AR) path-
way was involved in this effect. In subsequent experiments, die-
tary supplementation of NN resulted in a significant suppression
of body weight gain in A/J mice fed a high-fat diet. Bioassay-
guided fractionation and repeated chromatography of NN has
led to the isolation and identification of quercetin 3-O-
a
-arabi-
nopyranosyl-(1
®
2)-
b
-galactopyranoside (1), rutin (2), (+)-cate-
chin (3), hyperoside (4), isoquercitrin (5), quercetin (6) and astra-
galin (7). Of these, compounds 1, 3, 4, 5 and 7 exhibited lipolytic
activity, especially in visceral adipose tissue. Our results indicate
that the effects of NN in preventing diet-induced obesity appear
to be due to various flavonoids and that the activation of
b
-AR
pathway was involved, at least in part.
Key words
Nymphaceae ´ Nelumbo nucifera ´ lipolysis ´ visceral ´ subcuta-
neous ´ obesity ´ flavonoid glycosides
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/plantamedica
P
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Original Paper
1
N. nucifera (Nymphaceae) has been used for various medicinal
purposes in Chinese herbal medicine. In particular, the leaves
are known for diuretic and astringent properties, and are used
to treat obesity, sweating, and as a hemostyptic [7]. Recently, it
was reported that ICR mice fed a high-fat diet and treated with
N. nucifera leaf extract and exercise regimes exhibited a reduced
body weight gain and demonstrated activated lipolysis in 3T3-L1
adipocytes, probably via the
b
-adrenergic pathway [8]. In the
present study, we demonstrate how a 50% ethanol (EtOH) ex-
tract prepared from the leaves of N. nucifera (NN) can exert an ef-
fect on lipolysis in visceral and subcutaneous adipose tissues in
mice. We additionally show how this extract affects the body
weight gain in the absence of exercise in A/J mice, a strain of
mice that is sensitive to
b
-AR agonists [9]. Furthermore, we
isolated the active ingredients from NN and determined their re-
spective structures.
Material and Methods
Materials
Bovine serum albumin (fraction V, essentially fatty acid-free)
was purchased from Calbiochem (San Diego, CA, USA). The Glyc-
erol measurement kit was provided by Zen-Bio (Research Trian-
gle Park, NC, USA). Krebs-Ringer bicarbonate buffer (KRB), (+)-ca-
techin, rutin and quercetin were obtained from Sigma-Aldrich
(St. Louis, MO, USA; purity > 90% by HPLC). Isoquercitrin, hypero-
side and astragalin were provided by Extrasynthese (Genay,
France; purity > 90 % by HPLC). All other chemicals were pur-
chased from Wako Pure Chemicals (Osaka, Japan).
Extraction and isolation
Powdered dry leaves (165 g) prepared from N. nucifera were pur-
chased from Nanjo Lotus Productive Cooperation (Fukui, Japan),
and were extracted by exhaustive percolation with 50% EtOH.
The extract was concentrated under reduced pressure to give a
50% EtOH extract (NN; 44.6 g, yield 27.0%). A voucher specimen
(KIRIN0105) has been deposited in the KIRIN Brewery Co. Ltd.,
Central Laboratories for Frontier Technology, Kanagawa, Japan.
NN was then dissolved in H
2
O, and was applied onto a Diaion HP-
20 column, which was then eluted with H
2
O (18.1 g, Fraction I),
50% EtOH (20.5 g, Fraction II) and MeOH (1.8 g, Fraction III) in
turn. Since Fraction II caused increased lipolysis in adipose tis-
sues, this fraction was subsequently subjected to an additional
procedure. Fraction II was subjected to column chromatography
on a Toyopearl HW 40 column chromatograph (832 cm, H
2
O-
MeOH gradient, 1: 0
®
0: 1) and produced 14 separate fractions
(II-1 to II-14) according to their TLC patterns. TLC was performed
on silica gel plates (60 F-254, Merck; Darmstadt, Germany). Frac-
tions were visualized by spraying the plates with ferric chloride,
Dragendorff reagent or thymol-10% sulfuric acid solution fol-
lowed by heating. Fraction II-2 was separated by HPLC (Phenom-
enex Gemini C18 110 5
m
m, 10 i. d.250 mm, TFA/CH
3
CN/
H
2
O = 0.5/15/85, flow rate; 3 mL/min) to give compounds 1
(5.0 mg, t
R
; 28 min) and 2 (2.0 mg, t
R
= 42 min). Fraction II-12
was separated by HPLC (Shiseido Capcell Pak C18 MGII 5
m
m, 4.6
i.d. 250 mm, CH
3
CN/H
2
O = 10/90, flow rate; 1 mL/min) to give
3 (6.0 mg, t
R
= 14 min). Fraction II-7 was further separated by
HPLC YMC-Pak ODS-AQ S-5
m
m, 20 i.d. 250 mm, CH
3
COOH/
CH
3
CN/H
2
O = 0.1/15/85, flow rate; 8 mL/min) to give 4
(23.9 mg, t
R
= 69 min) and 5 (23.0 mg, t
R
= 77 min). Fraction II-
8 was separated by HPLC (Phenomenex Gemini C18 110 5
m
m,
10 i.d.250 mm, TFA/CH
3
CN/H
2
O = 0.5/20/80, flow rate; 3 mL/
min) to give 7 (15.0 mg, t
R
= 27 min) and 6 (2.8 mg, t
R
= 93 min).
Structural analysis of isolated compounds
1
H- and
13
C-NMR data (TMS as internal standard) were recorded
in CD
3
OD (compounds 1±3) or pridine-d
5
(compoundS 4±7)as
required, on a Bruker AM-400 spectrometer at 400 and 100
MHz, respectively. MS data were recorded with a Waters
ZQ2000 MS, 2690 Separation system and a 2996 PDA detector
using a reversed-phase column (Phenomenex Gemini 5
m
m,
4.6 mm i.d. 250 mm) and eluted with 14% acetonitrile in 1 %
TFA in water (1.0 mL/min) in the ESI positive mode. Semi-pre-
parative HPLC was performed using a Hitachi pump (model L-
600) with RI (Hitachi L-3300) and UV detector (
l
0 254 nm, Hita-
chi UV-1200). The optical rotation value was recorded with a Jas-
co-P-1020 polarimeter.
Animals and diets
Female C57BL/6J mice (5 weekS old) and male A/J mice (5 weeks
old) were obtained from Charles River Laboratories Japan (Tokyo,
Japan) and CLEA Japan (Tokyo, Japan), respectively. The mice
were housed under a 12 h light/12 h dark cycle in a temperature
and humidity-controlled room. Mice were adapted to their new
housing conditions for one week. Male A/J mice (5 weeks old)
were divided into three groups matched for body weight (control
group; n = 6, NN group; n = 7, and CL316,243 group; n = 4). The
animals were fed ad libitum with a high-fat diet (HFD; D12451M,
Research Diets, Inc; New Brunswick, NJ, USA) for THE experi-
mental period. The NN group and CL316,243 group were fed a
high-fat diet containing 1% (w/w) of NN and 0.001% (w/w)
CL316,243 group, respectively. The food intake and body weight
of each group was recorded twice a week. For the lipolysis ex-
periment, white adipose tissue (WAT) was obtained from 40
week old C57BL/6J female mice that were fed distinct high-fat
diets ad libitum, as described previously [10]. The study was con-
ducted in accordance with the guidelines for animal care, hand-
ling, and termination from Kirin Pharmaceutical, which are in
line with international and Japanese guidelines of animal care
and welfare.
Preparation of isolated adipocytes and lipolysis assays
Adipose fat tissues were isolated from visceral and subcutaneous
fat pads of female C57BL/6J mice (described above) and lipolysis
assays were performed by the Rodbell method with minor mod-
ification [11]. In brief, the fat pads were minced with scissors and
placed in a plastic tube containing 20 mL KRB buffer (pH 7.4)
AND 10 mM Hepes, 6 mM glucose, and 4% bovine serum albumin
(fraction V, essentially fatty acid free). The minced adipose tissue
was subsequently washed three times with the same buffer. Li-
polysis was assayed by measuring glycerol release into the incu-
bation medium. In brief, 0.5 mL of isolated adipose fat tissue was
added to 1.0 mL of KRB buffer containing 10 mM Hepes, 6 mM
glucose, and 4% of bovine serum albumin in the presence or ab-
sence of samples and/or drugs. The drugs used were as follows:
epinephrine (1.0 or 0.5
m
M) and propranolol (non-selective
b
-AR
antagonist; from 0.1 to 10
m
M). Stock solutions of the samples
were prepared in DMSO for the lipolytic assay [final maximal
Ohkoshi E et al. Constituents from the ¼ Planta Med 2007; 73: 1 ± 5
Original Paper
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concentration was 0.1% (v/v)]. After 2 h of incubation at 378Cina
shaking incubator, aliquots (20
m
L) of the cell-free medium were
recovered and the glycerol content, an index of lipolysis, was de-
termined enzymatically with glycerol reagent using a spectro-
photometric method [12].
Fig. 1 The effects of a 50% EtOH extract prepared from N.
nucifera (NN) leaves. A Lipolysis in both visceral and subcuta-
neous WAT was stimulated with NN at various concentra-
tions. Data are expressed as the mean S.D. of triplicate cul-
tures. Statistical significance from vehicle DMSO-treated
control cultures is indicated as * p < 0.05, ** p < 0.01 or
*** p < 0.001. B The
b
-adrenergic antagonist, propranolol
attenuated NN induced lipolysis in both visceral and subcu-
taneous WAT, respectively. Results are mean S.D.
a
p < 0.01
and
b
p < 0.05 versus NN 0.1 mg/mL group. C Effect of lipo-
lytic compounds on body weight gain of A/J mice. Mice were
randomly assigned at 5 weeks of age to a control high fat-
diet (9679;, n = 6), a high-fat diet containing 1 %
NN(9675;, n = 7) or a high-fat diet containing 0.001%
CL316,243 (9650;, n = 4). Results are represented as mean
S.D. of 4 ± 7 mice in each group. * p < 0.05 and ** p < 0.01
versus control group.
Ohkoshi E et al. Constituents from the ¼ Planta Med 2007; 73: 1 ± 5
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Original Paper
3
Statistical analysis
Results are expressed as mean S.D. of triplicate cultures of two
experiments (for in vitro experiments) or of 4±7 mice in each
group (for in vivo experiments). Statistical significance was de-
termined by the Student's t-test and the Mann-Whitney U-test
following ANOVA, and the differences were considered signifi-
cant if p < 0.05.
Results and Discussion
The effects of NN on lipolysis were examined in vitro by measur-
ing the amount of glycerol released from WAT, which was pre-
pared from C57BL/6J mice with significant amounts of body fat.
NN-stimulated glycerol release, both in visceral and subcuta-
neous WAT, occurred in a dose-dependent manner and the effect
was approximately equal in both cases (Fig.1A). To assess the in-
volvement of the
b
-ARs pathway in the lipolysis stimulated by
NN, a
b
-ARs antagonist, propranolol, was used in the same assay.
As shown in Fig.1B, stimulation of lipolysis by NN in both viscer-
al and subcutaneous WAT was dose-dependently inhibited and
almost completely abolished by 10
m
M propranolol (99.8% and
99.6%, respectively), suggesting the involvement of
b
-AR activa-
tion. In addition, no significant difference was observed in the in-
hibition rate of the two different adipose tissues. These data sup-
port the fact that the NN extract prepared by ourselves increased
lipolytic activity in mouse adipose tissue via the stimulation of
the
b
-AR signaling pathway, which has been reported to be in-
volved in the lipolysis in 3T3-L1 cells induced by a disparate ex-
tract prepared from N. nucifera leaves [8]. Subsequently, the ef-
fect of dietary supplementation with 1 % (w/w) NN was exam-
ined in A/J mice, which are sensitive to
b
-ARs agonists, fed a
high-fat diet [9]. As shown in Fig. 1C, treatment of A/J mice with
a
b
3-AR agonist, CL316,243 prevented progression of obesity in-
duced by high-fat diet feeding (p < 0.01). Meanwhile, treatment
of the mice with NN tended to reduce the body weight gain, but
no significant difference was observed. In this experiment, there
was no change in food intake observed throughout the 12-week
treatment period (see Fig. S1, Supporting Information).
We then tried to separate and isolate the active compounds from
NN using an in vitro lipolysis assay. Of three fractions eluted from
Diaion HP-20 chromatography, fraction II, as a brown mass,
showed TLC chromatographic properties characteristic of major
phenolic glycosides and minor alkaloids. The fraction II then exhib-
ited the highest lipolytic activity in both subcutaneous and visceral
adipose tissues whilst the other two fractions exhibited only a
trace amount of the activity (Fig. 2). Therefore, fraction II was re-
peatedly applied to preparative HPLC to isolate compounds 1±7.
Compound 1 had the molecular formula C
26
H
28
O
16
as obtained
from NMR and LC-MS (ESI-positive) spectral data (m/z =597[M
+H]
+
). Comparison with quercetin 3-O-
b
-galactopyranoside (com-
pound 4, hyperoside) showed that they differed only in the addi-
tional pentose moiety in compound 1 that indicated the presence
of one galactopyranosyl moiety with the
b
-configuration at the
anomeric proton (
d
=5.38,d,J = 7.3 Hz), and one arabinopyrano-
syl unit with th
a
-configuration at the anomeric proton (
d
= 4.75,
d, J = 6.5 Hz). In the
13
C-NMR spectrum of compound 1 the C-1
¢¢
and C-2
¢¢
carbon signals of the galactosyl moiety were observed
at
d
= 101.9 and 80.6 ppm, respectively, which proved the sug-
gested interglycosidic bond (1
®
2) by the recognizable downfield
shift of the C-2
¢¢
carbon signal (
Dd
= 7.3 ppm) and upfield shift of
the C-1
¢¢
signal (
Dd
= 3.2 ppm) in comparison with compound 4
(see Table S1, Supporting Information). Compound 1 was identi-
fied as quercetin 3-O-
a
-arabinopyranosyl-(1
®
2)-
b
-galactopyra-
noside by comparison with previously reported spectral data
[13]. However, this is the first report of the isolation of this com-
pound from the genus Nelumbo. In addition, rutin (2) [14], (+)-ca-
techin (3)[15],hyperoside(4), isoquercitrin (5) [16], quercetin (6)
[17] and astragalin (7) [14] were also isolated and identified from
NN by direct comparison of the
1
H- and
13
C-NMR spectral data and
MS of an authentic sample (Fig. 3). Moreover, the structure of
compound 3 was confirmed the optical rotation, [
a
]
D
20
: + 12.6 (c
1.0, CH
3
OH). Table 1 shows the regional lipolytic activity of isolat-
ed compounds 1±7 in visceral and subcutaneous WAT at a final
concentration of 100
m
M. Stimulation was observed by com-
pounds 1, 3, 4, 5 and 7 and was predominantly observed in visceral
WAT. These active compounds are monoglycoside or diglycoside
derivatives containing 1
®
2bonds(Fig.3). Furthermore, procyani-
dins (oligomers of catechins) exhibited lipolytic effects in 3T3-L1
adipocytes, although the monomers had no effect upon lipolysis
[18]. Collectively, flavonoids certainly have an advantage in that
they are responsible for the regulation of adipocyte function [19],
[20]. Such effects might depend upon the structure of each com-
pound. However, additional study will be required to elucidate the
structure-activity relationships among these compounds.
NN fractions exhibit greater responsiveness to lipolysis in viscer-
al WAT than in subcutaneous WAT. If the
b
-AR activity of mouse
adipose tissue is higher in visceral than subcutaneous tissue, as
in the case of the human [21], [22], then it follows that the lipo-
lytic activity of the compounds isolated from NN may be depen-
dent upon the
b
-ARs signaling pathway.
Fig. 2 Effects of each NN HP-20 fraction (Fraction I, II, and
III) on lipolysis in isolated regions of WAT. * p < 0.05, ** p <
0.01 and *** p < 0.001 versus control group.
Ohkoshi E et al. Constituents from the ¼ Planta Med 2007; 73: 1 ± 5
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In summary, we have demonstrated NN-induced lipolysis, in
both visceral and subcutaneous WAT. However, our results sug-
gested that isolated compounds were responsible for lipolysis in
visceral WAT, indicating involvement in the
b
-AR pathway. In ad-
dition, in vivo experiments showed that the administration of NN
tended to suppress body weight gain in mice fed a high-fat diet.
The major compounds isolated and identified from NN were fla-
vonoids. These results indicate that the inhibitory effect of NN
upon diet-induced obesity relies upon various flavonoids and
that activation of the
b
-AR pathway was involved, at least in part.
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Fig. 3 Isolated compounds from NN Fraction II.
Table 1 Effects of isolated compounds on lipolysis in visceral and sub-
cutaneous WAT
Samples Concentration Fold induction
visceral subcutaneous
Control (DMSO) 1 0.11 1 0.05
NN 50
m
g/mL 1.70 0.15 1.74 0.17
Fraction II of NN 50
m
g/mL 1.97 0.09 1.85 0.12
Compound 1 100
m
M 1.46 0.34 1.03 0.11
Compound 2 100
m
M 1.10 0.06 1.06 0.06
Compound 3 100
m
M 2.00 0.09 0.97 0.07
Compound 4 100
m
M 2.13 0.21 1.10 0.08
Compound 5 100
m
M 1.64 0.41 0.96 0.09
Compound 6 100
m
M 1.10 0.11 0.89 0.21
Compound 7 100
m
M 1.72 0.22 1.10 0.16
Epinephrine 0.5
m
M 1.61 0.11 1.55 0.11
Ohkoshi E et al. Constituents from the ¼ Planta Med 2007; 73: 1 ± 5
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Original Paper
5
