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Arch Pharm Res Vol 35, No 6, 1021-1035, 2012
DOI 10.1007/s12272-012-0610-0
1021
Inhibitory Activity of Coumarins from Artemisia capillaris against
Advanced Glycation Endproduct Formation
Hyun Ah Jung1, Jin Ju Park2, Md. Nurul Islam2, Seung Eun Jin2, Byung-Sun Min3, Je-Hyun Lee4, Hee Sook Sohn1,
and Jae Sue Choi2
1Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju 561-756, Korea, 2Department
of Food Science and Nutrition, Pukyong National University, Busan 608-737, Korea, 3College of Pharmacy, Catholic Uni-
versity of Daegu, Gyeongbuk 712-702, Korea, and 4College of Oriental Medicine, Dongguk University, Gyeongju 780-
714, Korea
(Received February 25, 2012/Revised March 20, 2012/Accepted March 22, 2012)
Since glycation can lead to the onset of diabetic complications due to chronic hyperglycemia,
several indigenous
Artemisia
species were evaluated as potential inhibitors of advanced glyca-
tion endproducts (AGE). Among them, the
Artemisia capillaris
plant demonstrated the high-
est AGE inhibitory activity. Repeated column chromatography was performed to isolate a new
acylated flavonoid glycoside, acacetin-7-
O
-(6''-
O
-acetyl)-
β
-
D
-glucopyranosyl-(1
→
2)[
α
-
L
-rham-
nopyranosyl]-(1
→
6)-
β
-
D
-glucopyranoside, along with 11 known flavonoids (acacetin-7-
O
-
β
-
D
-
glucopyranosyl-(1
→
2)[
α
-
L
-rhamnopyranosyl]-(1
→
6)-
β
-
D
-glucopyranoside, linarin, quercetin, hyper-
oside, isorhamnetin, isorhamnetin 3-galactoside, isorhamnetin 3-glucoside, isorhamnetin 3-
arabinoside, isorhamnetin 3-robinobioside, arcapillin, and cirsilineol), six coumarins (umbellif-
erone, esculetin, scopoletin, scopolin, isoscopolin, and scoparone), and two phenolic derivatives
(4,5-di-
O
-caffeoylquinic acid and chlorogenic acid). In determining the structure-activity rela-
tionship (SAR), it was found that the presence and position of hydroxyl group of test coumarins
(coumarin, esculin, isoscopoletin, daphnetin, 4-methylcoumarin, and six isolated coumarins)
may play a crucial role in AGE inhibition. A free hydroxyl group at C-7 and a glucosyl group
instead of a methoxyl group at C-6 are two important parameters for the inhibitory potential
of coumarins on AGE formation.
A. capillaris
and five key AGE inhibitors, including 4,5-di-
O
-
caffeoylquinic acid, umbelliferone, esculetin, esculin, and scopoletin, were identified as poten-
tial candidates for use as therapeutic or preventive agents for diabetic complications and oxi-
dative stress-related diseases. We understand this to be the first detailed study on the SAR of
coumarins in AGE inhibition.
Key words:
Artemisia capillaris
, Compositae, Coumarin, Advanced glycation endproduct, 4,5-
Di-
O
-caffeoylquinic acid
INTRODUCTION
Advanced glycation endproducts (AGEs) are known
contributors to excessive oxidative stress and inflam-
mation, which have been linked to recent epidemics of
diabetes and cardiovascular disease. A major conse-
quence of hyperglycemia in long-term diabetes mellitus
is the excessive nonenzymatic glycosylation of proteins,
which is primarily a result of long-term exposure to
elevated glucose concentrations (Kochakian, 1996). In
particular, surging AGE formation and increased pro-
tein glycation are strongly implicated in diabetic com-
plications and Alzheimer’s disease (Sasaki et al., 1998).
During chronic hyperglycemia, the non-enzymatic gly-
cation process between a reducing sugar and a free
amino group of proteins, also known as a Millard reac-
tion, leads to the formation of complex protein adducts,
Correspondence to:
Jae Sue Choi, Department of Food Science
and Nutrition, Pukyong National University, Busan 608-737,
Korea
Tel: 82-51-629-5845, Fax: 82-51-629-5842
E-mail: choijs@pknu.ac.kr
Hyun Ah Jung, Department of Food Science and Human Nutri-
tion, Chonbuk National University, Jeonju 561-756, Korea
Tel: 82-63-270-4882, Fax: 82-63-270-3854
E-mail: jungha@jbnu.ac.kr
1022 H. A. Jung et al.
or AGEs. This includes the formation of pentosidine,
carboxymethyllysine, crossline, and pyralline via con-
secutive rearrangement, oxidation, and reduction of
Amadori products (Altan, 2003; Ahmed, 2005). Since
some of these products are highly reactive substances,
excessive glycation and further crosslinking between
proteins and reactive dicarbonyl species can occur
(Peyrou and Sternberg, 2006; Ahmed and Thornalley,
2007). Thus, agents that inhibit the formation of AGEs
have emerged as being potentially therapeutic in patients
with diabetes and age-related diseases. In particular,
the oxidation process is believed to play an important
role in AGE formation. A series of oxidation of Amadori
products leads to the formation of dicarbonyl interme-
diates that can react with nearby lysine or arginine resi-
dues to form protein crosslinks and AGEs. Reactive
carbonyl compounds may also be generated from the
metal ion-catalyzed autoxidation of glucose (Rahbar
and Figarola, 2003; Peyrou and Sternberg, 2006).
Therefore, promising AGE inhibitors with antioxidant
properties may effectively block AGE formation by pre-
venting further oxidation of Amadori products and
metal-catalyzed glucose oxidation.
The
Artemisia
genus has recently emerged as a source
of natural occurring therapeutic agents for diabetes
and diabetic complications due to the bioactive compo-
nents it possesses, including caffeoylquinic acids, fla-
vonoids, and coumarins (Chen et al., 1994; Logendra
et al., 2006; Cui et al., 2009; Hong et al., 2009). The anti-
diabetic effects of
Artemisia princeps
and
A. dracun-
culus
were recently observed in an animal model (Jung
et al., 2007; Ribnicky et al., 2009; Kheterpal et al., 2010).
A large number of species from this family have long
been used in traditional herbal medicines due to their
anti-malarial, anti-viral, anti-tumor, anti-pyretic, anti-
hemorrhagic, anti-coagulant, antifungal, anti-anginal,
antioxidant, hepatoprotective, anti-ulcerogenic, anti-
spasmodic, anti-complementary, and interferon-inducing
properties (Tan et al., 1998; Kordali et al., 2005; Wright,
2005).
Artemisia
plants, particularly
A. capillaris
,
A.
iwayomogi
,
A. princeps
, and
A. argyi
, are important
medicinal materials utilized in traditional Asian medi-
cines, including traditional Chinese medicine (TCM)
and traditional Japanese medicine (Kampo).
Artemisia
iwayomogi
Kitamura and
A. capillaris
Thunb., referred
to in Korea as Haninjin and Injinho, have been tradi-
tionally prescribed in the treatment of diuresis and as
anti-inflammatory and hepatotherapeutic drugs (Yook,
1989; Lee et al., 2011).
Artemisia capillaris
is commonly distributed in sandy
areas along the Korean coastline and is known as
halophyte. Similar to the
Artemisia
species, this plant
has been frequently used in the treatment of liver
disease, including hepatitis, jaundice, fatty liver, and
bilious disorder. Infusions of the buds, stems and leaves
of
A. capillaris
have been used in TCM primarily as a
choleric, anti-inflammatory, antipyretic, and diuretic
agent for treating epidemic hepatitis (Tang and
Eisenbrand, 1992). A wide variety of pharmacological
and biological activities of
A. capillaris
have been re-
ported including antioxidant (Hong et al., 2007; Seo
and Yun, 2008), cytoprotective (Hong et al., 2007),
hepatoprotective (Lee et al., 2000; Choi et al., 2011), anti-
inflammatory (Hong et al., 2004; Kwon et al., 2011),
antimicrobial (Seo et al., 2010), anticancer (Cha et al.,
2009), anti-obesity (Hong et al., 2009), and choleretic
effects (Okuno et al., 1981). The phytochemical pro-
perties of
A. capillaris
have been widely investigated.
In particular,
A. capillaris
contains an essential oil with
polyacetylene derivatives, such as capillene, capillone,
and capillin, as well as mono- and sesquiterpenes (
α
-
and
β
-pinene,
p
-cymene,
α
-terpineol, bornyl acetate,
methyleugenol,
β
-elemene, and
β
-caryophyllene) (Wright,
2005). Recently, the main components of
A. capillaris
essential oil were determined to be 1,8-cineole, germa-
crene D, and camphor (Liu et al., 2010). Capillarisin
(flavone), the major constituent of
A. capillaris
, along
with capillartemisin A and B, two new stereoisomeric
constituents, demonstrated choleretic effects in exper-
imental studies (Wright, 2005). The scoparone, a cou-
marin derivative isolated from this species, had a pre-
ventative effect on carbon tetrachloride or galactosa-
mine-induced hepatotoxicity in hepatocyte cell cultures
(Kiso et al., 1984), as well as anti-inflammatory and
analgesic effects (Yamahara et al., 1989; Jang et al.,
2005).
Regarding the use of
Artemisia
species and its
derived compounds for their anti-diabetic complication
effects, the majority of studies have focused on aldose
reductase inhibition of coumarins, flavonoids, and phe-
nolic compounds isolated from this species. Further-
more, few studies have focused on the AGE inhibition
of coumarins. The present study was designed to evalu-
ate the AGE inhibitory activities of coumarin and its
derivatives as well as phenolic compounds and flavo-
noids from
A. capillaris
. This study was conducted with
this scope of information because
A. capillaris
and its
derivatives were identified as potential potent degraders
or inhibitors against AGE generation.
MATERIALS AND METHODS
General
Melting points were measured on a Mitamura-Riken
apparatus and were uncorrected. The EI-MS was re-
corded on a Hewlett-Packard 5989B spectrometer (Agilent
AGE Inhibitory Coumarins from
Artemisia capillaris
1023
Technologies) and a JEOL JMS-700 spectrometer. The
ESI-MS was obtained on an Applied Biosystems Mariner
time-of-flight mass spectrometer with an electrospray
interface. The
1
H- and
13
C-NMR spectra were acquired
using a JEOL JNM ECP-400 spectrometer at 400 and
100 MHz, as well as a Bruker AVANCE II 900 spec-
trometer at 900 and 225 MHz in deuterated solvents
[methanol (MeOH)-
d
4
, dimethylsulfoxide (DMSO)-
d
6
,
chloroform (CDCl
3
)]. Column chromatography was con-
ducted using silica (Si) gel 60 (70-230 mesh, Merck),
Sephadex LH20 (20-100
µ
m, Sigma), and Diaion HP20
(250-850
µ
m, Sigma). All TLC was conducted on pre-
coated Merck Kieselgel 60 F
254
plates (20
×
20 cm, 0.25
mm, Merck) using 50% H
2
SO
4
as a spray reagent.
Chemicals and reagents
1,1-Diphenyl-2-picrylhydrazyl (DPPH),
L
-ascorbic acid,
2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) di-
ammonium salt (ABTS), bovine serum albumin (BSA),
D-(
−
)-fructose, D-(+)-glucose, aminoguanidine hydrochlo-
ride, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic
acid (trolox),
DL
-glyceraldehyde dimer, nicotinamide
adenine dinucleotide phosphate (NADPH), quercetin,
coumarin, caffeic acid, ferulic acid, dihydrocaffeic acid,
p
-coumaric acid, 3,4-dimethoxycinnamic acid, diethylene-
triaminepentaacetic acid (DTPA),
L
-2-amino-3-mercapto-
3-methylbutanoic acid (
L
-penicillamine), and dimethyl
sulfoxide (DMSO) were purchased from Sigma-Aldrich
Co. Peroxynitrite (ONOO
−
) was purchased from Cayman
Chemicals Co. Dihydrorhodamine 123 (DHR 123) was
purchased from Molecular Probes. Sodium azide was
obtained from Junsei Chemical Co. Anti-nitrotyrosine
(clone 1A6, mouse-monoclonal primary antibody, IgG2b),
horseradish peroxide-conjugated anti-rabbit, and anti-
mouse antibodies were purchased from Millipore Co.
Polyvinylidenefluoride (PVDF) membrane (Immobilon-
P) was obtained from Millipore Co. Supersignal
®
West
Pico Chemiluminescent Substrate was obtained from
Pierce Biotechnology, Inc. All reagents were purchased
from Duksan Pure Chemical Co., E. Merck, Fluka, or
Sigma & Aldrich Company, unless otherwise stated.
Plant materials
The
A. capillaris
whole plants were collected from
Yeongcheon Province, Korea in August 2009, and con-
firmed by Prof. Je-Hyun Lee at College of Oriental
Medicine, Dongguk University (Kyeongju). A voucher
specimen (no. 20093008) was deposited in the author’s
laboratory (J. S. Choi). The MeOH extracts of 11 species
(
A. apiacea
Hance,
A. rubripes
Nakai,
A. japonica
Thunb.,
A. sylvatica
Max.,
A. iwayomogi
Kitamura,
A. stolonifera
Max.,
A. argyi
Levi. et Vaniot,
A. princeps
var.
orientalis
Pampan,
A. selengensis
Turcz,
A. capillaris
Thunb.,
A.
keiskeana
Miq.) were purchased from the Korean Plant
Extract Bank under the Korea Research Institute of
Bioscience and Biotechnology in February 2006.
Extraction, fractionation, and isolation
A whole
A. capillaris
plant was dried and ground to
a powder. The dried powder (19.0 kg) was extracted
with hot MeOH (50.0 L × 3 times) for 3 h. After filtra-
tion, the total filtrate was concentrated
in vacuo
at
40°C to yield MeOH extract (1.81 kg). The MeOH extract
was suspended in a mixture of distilled water-MeOH
(9:1) and successively partitioned with dichloromethane
(CH
2
Cl
2
), ethyl acetate (EtOAc), and
n
-butanol (
n
-
BuOH) to yield CH
2
Cl
2
(707 g), EtOAc (282 g), and
n
-
BuOH (387 g) fractions respectively, as well as H
2
O
residue (435 g).
Initially, about half of the CH
2
Cl
2
fraction (350 g)
was subjected to silica gel column chromatography
eluted with CH
2
Cl
2
. This was followed by the addition
of gradually increasing amounts of MeOH to yield 20
subfractions (CF01 to CF20). Fraction 6 (CF06) was fur-
ther chromatographed on the silica gel using CH
2
Cl
2
-
MeOH to obtain four subfractions (CF06F1 to CF06F4).
Fraction 7 (CF07, 113 g) was then subjected to silica
gel chromatography using
n
-hexane-EtOAc (10:1
→
0:1)
to yield 12 subfractions (CF07F01 to CF07F12). From
the subfractions, CF07F07 was filtered to separate the
crystal, which was later identified as scoparone (2.70
g). The CF07F07 filtrate was further chromatographed
over a Sephadex LH20 column using CH
2
Cl
2
to obtain
two subfractions (CF07F07-1 to CF07F07-2). Repeated
chromatography of CF07F07-1 in the silica gel column
using CH
2
Cl
2
-
n
-hexane-MeOH (30:10:1) yielded umbel-
liferone (20 mg) and capillarisin (330 mg). The CF07F09
precipitate was separated after filtration and further
chromatographed on the silica gel column using CH
2
Cl
2
-
n
-hexane-MeOH (30:10:1) to obtain cirsilineol (57 mg)
and isorhamnetin (60 mg). The mother liquor of CF07F09
was chromatographed on silica gel using
n
-hexane-
EtOAc to obtain scopoletin (33 mg). Fraction 11 (CF07F11)
was filtered to yield arcapillin (61 mg).
Similarly, the EtOAc fraction was also subjected to sil-
ica gel column chromatography using
n
-hexane-EtOAc
(10:1
→
0:1) to obtain 15 subfractions (EF01 to EF15).
Fraction 3 (EF03) was further chromatographed on a
silica gel column eluted with
n
-hexane-EtOAc to obtain
esculetin (10 mg). Repeated chromatography of EF05
using
n
-hexane-acetone (10:1
→
0:1) yielded quercetin
(100 mg). EF-8 was chromatographed using hexane-
EtOAc (10:1
→
0:1) to obtain chlorogenic acid (50 mg).
The
n-
BuOH fraction was chromatographed over
Diaion HP-20 using the H
2
O-MeOH (1:0
→
6:4
→
4:6
→
0:1)
solvent system to yield four major subfractions, BF01
1024 H. A. Jung et al.
(H
2
O, 264.17 g), BF02 (40% MeOH, 60.82 g), BF03
(60% MeOH, 40.95 g), and BF04 (100% MeOH, 12.76
g). Fraction 2 (BF02, 40% MeOH subfraction) was then
chromatographed on a silica gel column using CH
2
Cl
2
-
MeOH (10:1) with gradually increasing MeOH concen-
trations to yield 15 subfractions (BF02F01 to BF02F15).
Following the filtration of combined fractions BF02F04
to BF02F08, the precipitate was recrystallized using
MeOH to yield hyperoside (3.523 g). Repeated chro-
matography of BF02F09 in the silica gel column using
CH
2
Cl
2
-MeOH (10:1
→
1:1) yielded four subfractions
(BF02F09-1 to BF02F09-4). Fraction 4 (BF02F09-4) was
then chromatographed over the silica gel column using
EtOAc-MeOH-H
2
O (24:1:1) to obtain isorhamnetin-3-
robinoside (267 mg). Repeated chromatography of
BF02F05 over the silica gel using CH
2
Cl
2
-MeOH (10:1)
yielded scopolin (43 mg) and isoscopolin (40 mg). On the
other hand, BF03 (60% MeOH) was chromatographed
over the silica gel column using CH
2
Cl
2
-MeOH (20:1)
with gradually increasing MeOH concentrations to
obtain 17 subfractions (BF03F01 to BF03F17). The de-
cantation of BF03F03 yielded isorhamnetin-3-galacto-
side (510 mg) and linarin (acacetin 7-rutinoside, 64
mg). Fraction 2 (BF03F02) was subjected to silica gel
chromatography in a solvent system containing CH
2
Cl
2
-
MeOH (15:1) which yielded four subfractions (BF03F02-
1 to BF03F02-4). Fraction 3 (BF03F02-3) was chroma-
tographed over the silica gel column using EtOAc-
MeOH-H
2
O (50:1:1) to yield isorhamnetin-3-glucopyr-
anoside (62 mg). All subfractions of BF03F02 were com-
bined and further chromatographed over the silica gel
column using CH
2
Cl
2
-MeOH-H
2
O (7:1:0.1), followed by
purification of the crystal over a Sephadex LH20 col-
umn to yield isorhamnetin-3-arabinopyranoside (disti-
chin) (8 mg). Fraction 6 (BF03F06) was chromatographed
over the silica gel column using solvent EtOAc-MeOH-
H
2
O (24:1:1) to obtain acacetin-7-
O
-(6''-
O
-acetyl)-7-
β
-
D
-
glucopyranosyl)-(1
→
2)[
α
-
L
-rhamnopyranosyl-(1
→
6)]-
β
-
D
-glucopyranoside (11 mg) and acacetin-7-
O
-
β
-
D
-gluco-
pyranosyl-(1
→
2)[
α
-
L
-rhamnopyranosyl]-(1
→
6)-
β
-
D
-gluco-
pyranoside (7.0 mg).
The isolated compounds were identified and charac-
terized by spectroscopic methods, including
1
H- and
13
C-
NMR and through comparison with published spectral
data and TLC analysis (Kariyone et al., 1960; Markham
et al., 1978; Numata et al., 1980; Tsukamoto et al., 1985;
Nakatani et al., 2000; Wawer and Zielinska, 2001;
Nazaruk and Gudej, 2003; Lee et al., 2004; Jeong et al.,
2006; Park et al., 2009; Bayoumi et al., 2010; Veitch et
al., 2010). Spectral data for all isolated compounds can
be obtained from the corresponding authors. The struc-
tures of all isolated compounds and test compounds
are shown in Fig. 1.
Acacetin-7-
O
-(6''-
O
-acetyl)-
β
-
D
-glucopyranosyl-
(1
→
2)[
α
-
L
-rhamnopyranosyl-(1
→
6)-
β
-
D
-gluco-
pyranoside
ESI/MS (positive); 797.1, ESI/MS (negative); 795.2;
1
H-NMR (DMSO-
d
6
):
δ
12.91 (1H, brs, 5-OH), 8.05 (2H,
d,
J
= 9.0 Hz, H-2'/H-6'), 7.16 (2H, d,
J
= 9.0 Hz, H-3'/H-
5'), 6.95 (1H, s, H-3), 6.75 (1H, d,
J
= 1.8 Hz, H-8), 6.48
(1H, d,
J
= 1.8 Hz, H-6), 3.87 (3H, s, 4'-OCH
3
);
β
-Glc
(primary): 5.23 (1H, d,
J
= 7.4 Hz, Glc H-1), 3.53 (1H,
m, Glc H-2), 3.52 (1H, m, Glc H-3), 3.22 (1H, m, Glc H-
4), 3.68 (1H, m, Glc H-5), 3.86 (1H, dd,
J
= 11.8, 1.9 Hz,
Glc H-6a), 3.46 (1H, dd,
J
= 11.8, 6.3 Hz, Glc H-6b);
α
-
Rha: 4.56 (1H, br d,
J
= 1.6 Hz, Rha H-1), 3.68 (1H, m,
Rha H-2), 3.48 (1H, m, Rha H-3), 3.17 (1H, m, Rha H-
4), 3.42 (1H, dd,
J
= 9.4, 6.3 Hz, Rha H-5), 1.08 (3H, d,
J
= 6.2 Hz, Rha H-6);
β
-Glc (terminal): 4.52 (1H, d,
J
=
7.8 Hz, t-Glc H-1), 3.02 (1H, br t,
J
= 8.5 Hz, t-Glc H-2),
3.22 (1H, m, t-Glc H-3), 3.08 (1H, m, t-Glc H-4), 3.42(1H,
m, t-Glc H-5), 4.16 (1H, dd,
J
= 10.8, 1.9 Hz, t-Glc H-6a),
4.00 (1H, dd,
J
= 10.8, 6.3 Hz, t-Glc H-6b), 1.96 (3H, s,
-OCOCH
3
);
13
C NMR (DMSO-
d
6
):
δ
163.95 (C-2), 103.84
(C-3), 182.06 (C-4), 170.31 (-OCOCH
3
), 161.2 (C-5), 99.74
(C-6), 162.84 (C-7), 94.78 (C-8), 156.90 (C-9), 105.51 (C-
10), 122.66 (C-1’), 128.40 (C-2'/C-6'), 114.73 (C-3'/C-5'),
162.50 (C-4'), 55.58 (4'-OCH
3
), 20.57 (-OCOCH
3
);
β
-Glc
(primary); 98.23 (Glc C-1), 83.05 (Glc C-2), 75.58 (Glc C-
3), 69.11 (Glc C-4), 75.44 (Glc C-5), 65.92 (Glc C-6);
α
-
Rha: 100.50 (Rha C-1), 70.37 (Rha C-2), 70.75 (Rha C-3),
72.05 (Rha C-4), 68.35 (Rha C-5), 17.81 (Rha C-6);
β
-Glc
(terminal); 104.75 (Glc C-1), 74.60 (Glc C-2), 75.89 (Glc
C-3), 69.67 (Glc C-4), 73.72 (Glc C-5), 63.60 (Glc C-6).
Acacetin-7-
O
-
β
-
D
-glucopyranosyl-(1
→
2)[
α
-
L
-rham-
nopyranosyl]-(1
→
6)-
β
-
D
-glucopyranoside
1
H-NMR (DMSO-
d
6
):
δ
8.05 (2H, d,
J
= 9.0 Hz, H-2'/
6'), 7.15 (2H, d,
J
= 9.0 Hz, H-3'/5'), 6.95 (1H, s, H-3),
6.85 (1H, s, H-8), 6.51 (1H, s, H-6), 3.86 (3H, s, 4'-OCH
3
);
β
-Glc (primary): 5.20 (1H, d,
J
= 6.4 Hz, Glc H-1);
α
-
Rha: 4.56 (1H, s, Rha H-1);
β
-Glc (terminal): 4.48 (1H,
d,
J
= 8.1 Hz, t-Glc H-1);
13
C-NMR (DMSO-
d
6
):
δ
163.6
(C-2), 103.4 (C-3), 181.7 (C-4), 160.8 (C-5), 99.5 (C-6),
162.5 (C-7), 94.8 (C-8), 156.5 (C-9), 105.1 (C-10), 122.3
(C-1'), 128.1 (C-2'/C-6'), 114.4 (C-3'/C-5'), 162.1 (C-4'),
55.2 (4'-OCH
3
);
β
-Glc (primary): 98.1 (Glc C-1), 82.3
(Glc C-2), 75.2 (Glc C-3), 68.9 (Glc C-4), 75.0 (Glc C-5),
65.6 (Glc C-6);
α
-Rha: 100.2 (Rha C-1), 70.0 (Rha C-2),
70.4 (Rha C-3), 71.7 (Rha C-4), 68.0 (Rha C-5), 17.5
(Rha C-6);
β
-Glc (terminal): 104.4 (t-Glc C-1), 74.4 (t-
Glc C-2), 75.8 (t-Glc C-3), 69.3 (t-Glc C-4), 76.7 (t-Glc
C-5), 60.3 (t-Glc C-6)
Inhibition of AGE formation
The inhibition of AGE formation was modified from
AGE Inhibitory Coumarins from
Artemisia capillaris
1025
methods outlined in Vinson and Howard (1996). To
prepare the AGE reaction solution, 10 mg/mL bovine
serum albumin in 50 mM sodium phosphate buffer
(pH 7.4), along with 0.02% sodium azide to prevent
bacterial growth, was added to 0.2 M fructose and 0.2
M glucose. The reaction mixture (950
µ
L) was then
combined with various concentrations of the samples
(50
µ
L, final concentration: 200
µ
g/mL for the extracts,
fractions, and compounds) dissolved in 10% DMSO.
Following incubation at 37
o
C for 7 day, the fluorescence
intensity of the reaction products was determined using
a spectrofluorometric detector (FL × 800 microplate
fluorescence reader, Bio-Tek Instruments, Inc.), with
excitation and emission wavelengths at 350 and 450
nm, respectively. The percentage of AGE inhibition was
determined from a graphical plot of the data and is ex-
pressed as the mean ± S.E.M. of triplicate experiments.
The nucleophilic hydrazine compound aminoguanidine
was used as a reference in the AGE assay.
Fig. 1.
Structures of compounds isolated from
A. capillaris
and test compounds.
1026 H. A. Jung et al.
Assay for rat lens aldose reductase (RLAR) in-
hibitory activity
This study adhered to the Guidelines for the Care
and Use of Laboratory Animals approved by Pukyong
National University. The rat lens homogenate was
prepared according to a slightly modified method from
Hayman and Kinoshita (1965). The lenses were removed
from the eyes of Sprague-Dawley rats (Samtako Bio-
Korea, Inc.) weighing 250-280 g. The lenses were homo-
genized in sodium phosphate buffer (pH 6.2), which was
prepared from sodium phosphate dibasic (Na
2
HPO
4
·
H
2
O,
0.66 g) and sodium phosphate monobasic (NaH
2
PO
4
·
H
2
O,
1.27 g) in 100 mL of double distilled H
2
O. The super-
natant was obtained by centrifugation of the homoge-
nate at 10,000 rpm and 4
o
C for 20 min and was frozen
until use. A crude AR homogenate with a specific activity
of 6.5 U/mg was used in the enzyme inhibition evalua-
tions. The reaction solution consisted of 620
µ
L of 100
mM sodium phosphate buffer (pH 6.2), 90
µ
L of AR
homogenate, 90
µ
L of 1.6 mM NADPH, and 9
µ
L of the
samples (final concentration: 100
µ
g/mL for the extracts
and fractions; 10
µ
g/mL and 100
µ
g/mL for the com-
pounds dissolved in 100% DMSO) or 9
µ
L of 100% DMSO.
The substrate included 90
µ
L of 50 mM of
DL
-glyceral-
dehyde. The AR activity was determined by measuring
the decrease in NADPH absorption at 340 nm over a
4 min period on an Ultrospec
®
2100pro UV/Visible spec-
trophotometer with SWIFT II Applications software
(Amersham Biosciences). Quercetin, a well-known AR
inhibitor was used as a reference. The inhibition per-
centage (%) was calculated as [1
−
(
∆
A sample/min
−
∆
A blank/min)/(
∆
A control/min
−
∆
A blank/min)]
×
100,
where
∆
A sample/min represents the reduction in ab-
sorbance over 4 min for the test sample and substrate,
and
∆
A control/min represents the same, but with 100%
DMSO instead of the test sample. The 50% inhibition
concentration is expressed as the mean ± S.E.M.
Assay for DPPH radical scavenging activity
The DPPH radical scavenging effect was evaluated
using methods outlined in Blois (1958) with slight modi-
fications. One hundred sixty microliters (
µ
L) of MeOH
solution at various concentrations (final concentration
(f.c.) 320
µ
g/mL for the extracts, fractions, and the com-
pounds) were added to 40
µ
L DPPH methanol solution
(1.5 × 10
−
4
M). After gently mixing the sample gently and
letting it stand at them at room temperature for 30 min,
the optical density was measured at 520 nm using a
VERSAmax microplate spectrophotometer (Molecular
Devices). The antioxidant activity of the samples was
expressed as IC
50
values (
µ
g/mL or
µ
M required to in-
hibit DPPH radical formation by 50%), which were cal-
culated from the log-dose inhibition curve.
L
-Ascorbic
acid was used as the positive control. The IC
50
values are
expressed as the mean ± S.E.M. of triplicate experi-
ments.
Trolox equivalent antioxidant capacity (TEAC)
This assay was based on the ability of different sub-
stances to scavenge the ABTS radical cation (ABTS
•
+
)
as compared with the positive control trolox (Re et al.,
1999). To oxidize colorless ABTS to blue-green ABTS
•
+
,
a 7 mM ABTS stock solution was mixed with 2.45 mM
potassium persulfate (1:1, v/v) and left at room tem-
perature in the dark for 12-16 h until the reaction was
complete and the absorbance was stable. The blue/
green ABTS
•
+
solution was diluted in ethanol (EtOH)
to an absorbance of 0.70 ± 0.02 at 734 nm for meas-
urement. The photometric assay was conducted with
180
µ
L of the ABTS
•
+
solution and 20
µ
L of test sample
dissolved in the EtOH solution (f.c. 100
µ
g/mL) that
was stirred for 30 sec. The optical density was meas-
ured at 734 nm after 2 min using a VERSAmax micro-
plate spectrophotometer (Molecular Devices). The anti-
oxidant activities of the samples were calculated by
determining the decrease in absorbance at different
concentrations using the following equation: E = [(A
c
−
A
t
)/A
c
] × 100, where A
t
and A
c
are the absorbance with
and without samples, respectively. Trolox and L-ascorbic
acid were used as the positive controls. The TEAC
results are expressed as IC
50
values (mean ± S.E.M. of
triplicate experiments).
Assay for ONOO
−
scavenging activity
The ONOO
−
scavenging activity was assessed using
a modified method from Kooy et al. (1994), which in-
volved monitoring highly fluorescent rhodamine 123
that was rapidly produced from non-fluorescent DHR
123 in the presence of ONOO
−
. The rhodamine buffer
(pH 7.4) consisted of 50 mM sodium phosphate dibasic,
50 mM sodium phosphate monobasic, 90 mM sodium
chloride, 5.0 mM potassium chloride, and 100
µ
M DTPA.
The final DHR 123 concentration was 5.0
µ
M. The
assay buffer was prepared prior to use and placed on
ice. The test sample was dissolved in 10% DMSO (f.c.
100
µ
M). The background and final fluorescent inten-
sities of the samples were measured five minutes after
treatment with and without the addition of authentic
ONOO
−
(10
µ
M) dissolved in 0.3 N sodium hydroxide.
The fluorescence intensity of the oxidized DHR 123
was evaluated using a fluorescence microplate reader
(Bio-Tek Instruments Inc., FL×800) at excitation and
emission wavelengths of 480 and 530 nm, respectively.
Values of ONOO
−
scavenging activity were calculated
as the final fluorescence intensity minus the background
fluorescence determined via the detection of DHR 123
AGE Inhibitory Coumarins from
Artemisia capillaris
1027
oxidation.
L
-Penicillamine was used as the positive
control.
Inhibition of ONOO
-
-mediated tyrosine nitra-
tion
In order to examine the inhibition of ONOO
−
-induced
BSA nitration, 2.5
µ
L of various concentrations of each
test sample dissolved in 10% EtOH (v/v) were added
to 95
µ
L of BSA (0.5 mg protein/mL). The mixtures
were incubated at room temperature (25
o
C) for 10 min
and then mixed with 2.5
µ
L of ONOO
−
(200
µ
M). Fol-
lowing incubation at 25
o
C for 10 min, the mixed sam-
ple was added to a Bio-Rad gel buffer at a ratio of 1:1
and boiled for 5 min to denature the proteins. The
total protein equivalent for the reactant was separated
on a 10% SDS-polyacrylamide minigel at 80 V for 30
min, followed by 100 V for 1 h, and then transferred to
a PVDF membrane at 80 V for 120 min in a wet transfer
system (Bio-Rad). The membrane was immediately
placed in a blocking solution (5% nonfat dry milk in
Tris-buffered saline containing 0.1% Tween-20 (TBST)
buffer (pH 7.4) at room temperature for 1 h. The mem-
brane was washed three times for 10 min in TBST
buffer and then incubated overnight with a monoclo-
nal anti-nitrotyrosine antibody (5% nonfat dry milk)
diluted 1:2000 in TBST buffer at 4
o
C. After another
three washings in TBST buffer for 10 min, the mem-
brane was incubated with HRP-conjugated sheep anti-
mouse secondary antibody and diluted 1:2000 in TBST
buffer at room temperature for 1 h. The membrane
was then washed with TBST buffer three times for 10
min, and the antibody labeling was visualized using a
Supersignal West Pico Chemiluminescent Substrate
(Pierce) according to the manufacturer’s instructions
after exposure to X-ray film (GE Healthcare Ltd.). Pre-
stained blue protein markers were used for molecular-
weight determination.
Statistics
Statistical significance was analyzed using one-way
ANOVA and Student’s t-test (Systat In.) with
p
< 0.01
considered statistically significant. All results are ex-
pressed as the mean ± S.E.M. from three experiments.
RESULTS AND DISCUSSION
Isolation of a new acetyl flavone glycoside
Acacetin-7-
O
-
β
-
D
-glucopyranosyl-(1
→
2)[
α
-
L
-rhamno-
pyranosyl]-(1
→
6)-
β
-
D
-glucopyranoside was identified
from its respective NMR spectra and confirmed by
comparison with those of published data (Veitch et al.,
2010). A new acylated flavonoid glycoside was isolated
as a white amorphous powder with molecular formula
C
36
H
44
O
20
deduced from the [M + H]
+
and [M - H]
−
peaks
at
m/z
797.1 and 795.2 in combinations of positive
and negative ESI-MS that were supported by the
13
C-
NMR spectrum. Its UV spectrum exhibited character-
istic absorbance bands of flavones at 268 nm and 334
nm. The
13
C-NMR spectrum of the new acylated flavo-
noid glycoside was very similar to acacetin-7-
O
-
β
-
D
-
glucopyranosyl-(1
→
2)[
α
-
L
-rhamnopyranosyl]-(1
→
6)-
β
-
D
-glucopyranoside with the exception of two addition-
al carbon signals arising from an aliphatic carbon
(20.57 ppm) and a carbonyl carbon (170.31 ppm). The
aliphatic carbon was identified as a methyl carbon
based on the integration of the corresponding proton
resonances thus indicating the presence of an acetyl
moiety. Comparison of the carbon chemical shift values
of a new compound
with the values of the parent gly-
coside showed that the C-6 carbon signal of the terminal
glucopyranoside moiety had shifted downfield by about
3.3 ppm, indicating that the acetyl group was attached
to this carbon atom. This was further supported by the
HMBC correlation between the acetyl carbonyl group
and methylene protons (H-6'') of the terminal glucose.
Therefore, the structure of the new compound was
determined to be a new acylated flavonoid glycoside,
acacetin-7-
O
-(6''-
O
-acetyl)-
β
-
D
-glucopyranosyl-(1
→
2)[
α
-
L
-rhamnopyranosyl]-(1
→
6)-
β
-
D
-glucopyranoside. Fur-
ther confirmation of the chemical structure was deter-
mined through mild alkaline hydrolysis with 2 N sodium
hydroxide at ambient temperature to yield the respect-
ive deacylated flavonoid glycoside, which was then
identified based on TLC or HPLC compared to an au-
thentic sample. To the best of the authors’ knowledge,
this is the first report of acacetin-7-
O
-(6''-
O
-acetyl)-
β
-
D
-glucopyranosyl-(1
→
2)[
α
-
L
-rhamnopyranosyl]-(1
→
6)-
β
-
D
-glucopyranoside from natural sources.
Advanced glycation endproduct inhibitory ac-
tivity of MeOH extracts from selected
Artemisia
species
The MeOH extracts of different species were evalu-
ated based on their inhibitory activity against AGE
formation at a concentration of 10
µ
g/mL. The AGE
inhibitory activities of the 12 selected species of the
genus
Artemisia
are summarized in Table I. Whole
A.
capillaris
and
A. japonica
plants exhibited the strong-
est AGE inhibitory activities with inhibition percent-
ages (%) of 87.47 ± 0.76 and 81.52 ± 0.97, respectively,
as compared to aminoguanidine (67.66 ± 0.37%), a well-
known AGE inhibitor. The aerial part of
A. capillaris
and the whole
A. sylvatica
plant exhibited inhibition
values of 67.34 ± 4.43% and 62.07 ± 2.09%, respect-
ively, followed by
A. stolonifera
with 41.14 ± 2.46%.
Three
Artemisia
species, including whole
A. princeps
1028 H. A. Jung et al.
var.
orientalis
,
A. iwayomogi
, and
A. rubripes
plants
exhibited moderate AGE inhibitory activity with in-
hibition % of 24.41 ± 1.94, 24.06 ± 1.69, and 21.40 ± 2.88,
respectively. However,
A. montana
,
A. apiacea
,
A. argyi
,
A. selengenis
, and
A. keiskeana
did not exhibit any in-
hibitory activity at the test concentrations. To evaluate
the potential and efficacy of the
Artemisia
species as
anti-diabetic complications drugs, RLAR inhibition and
AGE inhibition were used as representative mechani-
sms for addressing anti-diabetic complications. Inter-
estingly, differences were noted in the anti-diabetic com-
plication potentials of the
Artemisia
species.
A. capillaris
exhibited the most significant inhibitory activity against
AGE formation and
A. montana
exhibited no inhibi-
tory activity. On the other hand, the inhibitory activity
of
A. montana
on RLAR was eight times greater than
that of
A. capillaris
in our previous study (Jung et al.,
2011). Although more research is needed to fully un-
derstand this difference, the slight difference in compo-
sition and concentration of bioactive compounds in the
selected assays may play a role. Despite the contrast-
ing results of the two assays, these identical species
demonstrated marked inhibitory activity against RLAR
activity and AGE formation, indicating
A. capillaris
has potential as a therapeutic or preventive agent for
diabetic complications.
Advanced glycation endproduct and rat lens
aldose reductase inhibitory activities by the
A.
capillari
s MeOH extract and its solvent soluble
fractions
Since the whole
A. capillaris
plant exhibited the
most potent inhibitory of AGE activity among the tested
Artemisia
species, this plant was further investigated
in order to identify the active compounds responsible
for the AGE inhibition. The MeOH extract of
A. capil-
laris
was partitioned with CH
2
Cl
2
, EtOAc, and
n
-BuOH,
respectively. The AGE inhibitory effect of individual
fractions was then evaluated. As shown in
Table II,
the MeOH extract and
n
-BuOH fraction exhibited sig-
nificant AGE inhibitory activity with IC
50
values of
2.22 ± 0.05 and 2.46 ± 0.14
µ
g/mL, followed by the
n
-
BuOH and CH
2
Cl
2
fractions with IC
50
values of 3.69 ±
0.16 and 5.13 ± 0.02
µ
g/mL, as compared to amino-
guanidine with an IC
50
value of 86.84 ± 1.09
µ
g/mL.
Thus, bioactive-guided fractionation of the MeOH ex-
tract of
A. capillaris
revealed that the EtOAc and
n
-
BuOH fractions were expected to harbor a variety of
potent AGE inhibitors. In addition, most flavonoids that
are known AGE inhibitors were expected to be present
in large amounts in the polar EtOAc and
n
-BuOH
fractions (Muthenna et al., 2011; Nicolle et al., 2011).
In vitro
antioxidant activities of the
A. capillaris
MeOH extract and its solvent soluble fractions
During AGE generation, highly reactive oxygen/nitro-
gen species and dicarbonyl intermediates are produced,
which then induce the oxidation and degradation of
numerous biomolecules (Rahbar, 2007). Since the
oxidation process is believed to play an important role
in AGEs formation, further antioxidant capacities
were investigated in the DPPH, TEAC, and ONOO
−
assays. As demonstrated in Table III, most fractions of
Table I.
Inhibitory activity of the MeOH extracts from
selected
Artemisia
species on advanced glycation endpro-
ducts
Test samples Parts
*
Inhibition
(%)
a
Artemisia capillaris
Thunb. WP 87.47 ± 0.76
Artemisia japonica
Thunb. WP 81.52 ± 0.97
Artemisia capillaris
Thunb. AR 67.34 ± 4.43
Artemisia sylvatica
Max. WP 62.07 ± 2.09
Artemisia stolonifera
Max. WP 41.14 ± 2.46
Artemisia princeps
var
. orientalis
Pampan WP 24.41 ± 1.94
Artemisia iwayomogis
Kitamura WP 24.06 ± 1.69
Artemisia rubripes
Nakai WP 21.40 ± 2.88
Artemisia montana
Pampan WP -7.46 ± 0.76
Artemisia apiacea
Hance WP -9.39 ± 0.02
Artemisia argyi
Levi. et Vaniot LF -12.23 ± 4.31
Artemisia selengensis
Turcz WP -12.89 ± 0.92
Artemisia keiskeana
Miq. WP -20.30 ± 0.62
Aminoguanidine
b
67.66 ± 0.32
a
The inhibition percentage is expressed as the mean ±
S.E.M. of triplicate experiments at 10
µ
g/mL;
b
Aminogua-
nidine was used as a positive control.
*Abbreviations: Whole plant (WP), Leaf (LF), Aerial part
(AR).
Table II.
Advanced glycation endproduct and rat lens
aldose reductase inhibitory activities by the
A. capillaris
MeOH extract and its solvent soluble fractions
Test samples AGE
a
RLAR
b
MeOH extract 2.22 ± 0.05 0.46 ± 0.01
CH
2
Cl
2
fraction 5.13 ± 0.02 3.13 ± 0.28
EtOAc fraction 2.46 ± 0.14 0.16 ± 0.05
n
-BuOH fraction 3.69 ± 0.16 0.20 ± 0.00
H
2
O fraction 35.63 ± 0.93 3.98 ± 0.07
Aminoguanidine
c
86.84 ± 1.09
Quercetin
d
0.10 ± 0.01
a,b
The concentration yielding 50% inhibition (IC
50
,
µ
g/mL)
was calculated from the log dose inhibition curve and is
expressed as the mean ± S.E.M. of triplicate experiments;
c
Aminoguanidine was used as a positive control for AGE
inhibitors;
d
Quercetin was used as a positive control for
RLAR inhibitors.
AGE Inhibitory Coumarins from
Artemisia capillaris
1029
A. capillaris
exhibited marked scavenging activity in
three selected antioxidant assays, with the exception
of the nonpolar CH
2
Cl
2
fraction. Among the various
fractions of
A. capillaris
, the EtOAc fraction exerted
the most potent radical scavenging activity, with an
IC
50
value of 3.16 ± 0.07
µ
g/mL, as compared with the
positive control, L-ascorbic acid (IC
50
= 2.10 ± 0.41
µ
g/
mL) in the DPPH assay; an IC
50
value of 5.46 ± 0.18
µ
g/mL, as compared with the positive control, trolox
(IC
50
= 2.10 ± 0.41
µ
g/mL) in the TEAC assay; and an
IC
50
value of 0.55 ± 0.02
µ
g/mL, as compared with the
positive control,
L
-penicillamine (IC
50
= 0.26 ± 0.03
µ
g/
mL) in the ONOO
−
assay. These results correspond
well with those of previous radical scavenging studies
(Jung et al., 2004; Hong et al., 2007). Recently, the
EtOAc fraction of
A. capillaris
has been reported to
enhance the antioxidant defense system by reducing
reactive oxygen species generation and oxidative de-
generative substances as well as increasing the gluta-
thione (GSH) content and cellular antioxidant enzyme
activity in high-fat diet-induced obese mice and type 2
diabetic mice (Hong and Lee, 2009a, 2009b). The
n
-
BuOH fraction exhibited significant scavenging activ-
ity, with IC
50
values of 15.27 ± 0.62, 12.30 ± 0.25, and
1.46 ± 0.04
µ
g/mL in the DPPH, TEAC, and ONOO
−
assays, respectively. Since two active fractions harbored
high flavonoid content and flavonoids are well-known
free radical scavengers and metal ion chelators that par-
ticipate in the inhibition of AGE formation (Muthenna et
al., 2011), further bioactive-guided isolation of EtOAc
and
n
-BuOH fractions was performed.
Advanced glycation endproduct inhibitory ac-
tivity of coumarin derivatives
With the exception of coumarin, scoparone, and 7-
methoxy coumarin, all other coumarins demonstrated
significant a concentration-dependent inhibitory activ-
ity against AGE formation (Table IV). Esculin, the 6-
O
-glucoside of esculetin, demonstrated the most potent
AGE inhibitory activity with an IC
50
value of 0.85 ± 0.01
µ
M, as compared with the positive control, aminogua-
nidine (IC
50
= 932.66 ± 4.94
µ
M), followed by umbelli-
ferone, scopoletin, and esculetin with IC
50
values of 2.95
± 0.02, 3.90 ± 0.05, and 5.02 ± 0.02
µ
M, respectively. Re-
garding the above results, the genin of esculin, escule-
tin, which has two hydroxyl groups at the C-6 and C-
7 positions, exhibited approximately six-fold weaker
activity than esculin. Umbelliferone, which has one
hydroxyl group at the C-7 position, and scopoletin,
which has an identical structure to esculetin except
for an additional methoxyl group at C-6 instead of a
hydroxyl group, also demonstrated significant inhibi-
tory activity with IC
50
values of 2.95 ± 0.02 and 3.90 ±
0.05
µ
M, respectively. However, replacement of the C-
7 hydroxyl group of umbelliferone and scopoletin with
a methoxyl group (i.e., 7-methoxy coumarin and sco-
parone) or a glucosyl group (i.e., scopolin), or the re-
Table III.
In vitro
antioxidant activities of the
A. capillaris
MeOH extract and its solvent soluble fractions
Test samples DPPH
a
TEAC (ABTS)
b
ONOO¯
c
MeOH extract 33.95 ± 2.30 17.96 ± 0.40 0.67 ± 0.08
CH
2
Cl
2
fraction 110.04 ± 1.42 45.63 ± 0.50 5.88 ± 0.10
EtOAc fraction 3.16 ± 0.07 5.46 ± 0.18 0.55 ± 0.02
n
-BuOH fraction 15.27 ± 0.62 12.30 ± 0.25 1.46 ± 0.04
H
2
O fraction 81.55 ± 1.84 >100 >10
L
-Ascorbic acid
d
2.10 ± 0.41
Trolox
e
9.53 ± 0.02
L
-Penicillamine
f
0.26 ± 0.03
a-c
The concentration yielding 50% inhibition (IC
50
,
µ
M) was calculated from the log dose inhibition curve and is expressed
as the mean ± S.E.M. of triplicate experiments;
d-f
Compounds were used as positive controls.
Table IV.
Inhibitory activity of coumarin derivatives on
advanced glycation endproducts
Test compounds IC
50
value
a
Coumarin NA
Umbelliferone 2.95 ± 0.02
Esculetin 5.02 ± 0.02
Esculin 0.85 ± 0.01
Scopoletin 3.90 ± 0.05
Isoscopoletin 18.78 ± 0.50
Scopolin 60.10 ± 0.11
Isoscopolin 41.04 ± 2.71
Scoparone 204.97 ± 1.34
Daphnetin 43.21 ± 1.31
7-Methoxycoumarin 490.20 ± 9.32
Aminoguanidine
b
932.66 ± 4.94
a
The concentration yielding 50% inhibition (IC
50
,
µ
M) was
calculated from the log dose inhibition curve and is
expressed as the mean ± S.E.M. of triplicate experiments;
b
Aminoguanidine was used as a positive control. NA: No
activity at the tested concentrations.
1030 H. A. Jung et al.
moval of hydroxyl groups at the C-6 and C-7 positions
(i.e., coumarin), significantly decreased AGE inhibition.
Additionally, daphnetin with a 7,8-dihydroxyl group
was found to exhibit less inhibitory activity than the
6,7-dihydroxylated coumarin (i.e., esculetin). These re-
sults suggest that the inhibitory activity of coumarins
on AGE formation is largely dependent on the presence
and position of a hydroxyl group in their structure.
Taking into account the detailed structure-activity
relationship (SAR) features of coumarin derivatives, it
is clear that the hydroxyl group at the C-7 position
plays a pivotal role in the observed inhibitory activity.
Furthermore, the addition of a hydroxyl group in the
ortho
position to the C-7 hydroxyl group of the umbeli-
ferone skeleton (i.e., esculetin or daphnetin) signifi-
cantly decreased the inhibitory activity of these cou-
marins. The addition of a methoxyl group at C-6 (i.e.,
scopoletin) did not alter the potency, but the addition
of a glucosyl moiety (i.e., esculin) greatly increased the
inhibitory activity, suggesting that a free hydroxyl
group at C-7 as well as a glucosyl group instead of a
methoxyl group at C-6 are two important parameters
in the inhibitory potential of these coumarins on AGE
formation. Similarly, isoscopolin (7-methoxylated cou-
marin) exhibited greater inhibitory activity on AGE
than scopolin (7-glucosylated coumarin). Unlike the
SAR of most test coumarins, isoscopoletin (6-hydroxyl-
7-methoxyl coumarin) exhibited three times as much
inhibitory activity as scopoletin (7-hydroxyl-6-methoxy-
lated coumarin) in AGE inhibition.
Previous studies indicate scopoletin has demonstrated
a remarkable inhibition of advanced glycation endpro-
ducts formation (Lee et al., 2010). Ramesh and Pugalendi
(2007) revealed that umbelliferone, a natural antioxi-
dant and benzopyrone, exerted anti-diabetic effects by
controlling glycemia. Coumarin’s anti-diabetic effects
have been reported to include a significant protective
effect on glycoprotein metabolism (Pari and Rajarajeswari,
2010; Rajarajeswari and Pari, 2011). The inhibitory
activity of coumarin against carbohydrate metabolic
key enzymes resulted in alterations to glucose metab-
olism (Pari and Rajarajeswari, 2009). Regarding the
AR inhibition of coumarins, many studies indicate that
simple coumarins possess a hydroxyl group at either
the C-6 or C-7 position (Park et al., 2011). Additionally,
substitutions in the hydroxyl group at C-7 enhance
potency toward AR, while the hydroxyl group at C-7
interferes with aldehyde reductase inhibition activity
(Kato et al., 2010). Umbelliferone and esculetin could
also significantly inhibit the accumulation of sorbitol
in human erythrocytes (Kato et al., 2008). A conjugate
between coumarin and aspirin, nicousamide, was pro-
posed to delay diabetic nephropathy by inhibiting AGE
formation and crosslinking AGEs in renal cells and a
streptozotocin rat model of diabetes (Li et al., 2010,
2012). Although the inhibitory activity of coumarin
and aspirin conjugate as well as several coumarins on
AGE has been reported, the present study is the first
to report the detailed SAR of coumarin derivatives.
In vitro
antioxidant activities of coumarin
derivatives
Recognizing the relationship between AGE inhibi-
tion and antioxidant activity, several selected coumarins
were also evaluated by
in vitro
radical scavenging
(Table V) and ONOO
−
-mediated tyrosine nitration
assays (Fig. 2). Esculetin with a dihydroxyl group ex-
hibited the most potent radical scavenging activity,
with IC
50
values of 4.46 ± 0.15, 3.07 ± 0.14, and 0.42 ±
0.01
µ
M, in the DPPH, TEAC, and ONOO
−
assays, re-
spectively. On the other hand, scopoletin with a 7-
hydroxyl group demonstrated antioxidant activities in
both TEAC and ONOO
−
assays with respective IC
50
values of 26.91 ± 0.00, 6.35 ± 0.63, and 0.42 ± 0.01
µ
M.
Esculin and scopolin demonstrated limited and moder-
ate antioxidant activities in the TEAC and ONOO
−
assays, respectively, and did not exhibit scavenging
activities in other assays. Considering the antioxidant
activities of scopletin, there are slight differences be-
tween these results and those reported by Lee et al.
(2007), who indicated that esculetin demonstrated anti-
oxidant activity whereas coumarin, umbelliferone, es-
culin, and scopoletin demonstrated weak antioxidant
activity in radical scavenging assays.
Since the mechanisms of AGE formation may be
similar to protein nitration, additional research on the
effect of coumarins on protein nitration was performed.
As demonstrated in Fig. 2, esculetin, scopoletin, and
scopolin exerted marked concentration-dependent in-
hibition, while scoparone, umbelliferone, and coumarin
did not exhibit inhibition at the tested concentrations.
Interestingly, the results from the ONOO
−
-mediated
tyrosine nitration assay coincided with those of the
in
Table V.
In vitro
antioxidant activities of coumarin
derivatives
Compounds DPPH
a
TEAC(ABTS)
b
ONOO
-c
Esculetin 4.46 ± 0.15 3.07 ± 0.14 0.42 ± 0.01
Esculin NA 26.91 ± 0.12 NA
Scopoletin NA 15.17 ± 0.00 6.35± 0.63
Scopolin NA NA 34.34 ± 2.69
L
-Ascorbic acid
d
17.00 ± 0.48 15.76 ± 0.23
Trolox
e
19.21 ± 0.73
L
-Penicillamine
f
4.83 ± 0.24
a-c
IC
50
(
µ
M);
d-f
Positive controls. NA: No activity at the
tested concentrations.
AGE Inhibitory Coumarins from
Artemisia capillaris
1031
vitro
ONOO
−
scavenging assay, indicating that escule-
tin exerted the most potent inhibitory activity on
tyrosine nitration. Although coumarin demonstrated
limited activity in our assay, the oral administration
of coumarin to diabetic rats resulted in significantly
reduced lipid peroxides and significantly increased
antioxidant enzymes as well as decreased thiobarbi-
turic acid reactive substances (TBARS), lipid hydro-
peroxides, and conjugated diene concentration in the
liver and kidney tissues (Rajarajeswari and Pari, 2011).
Based on the current body of literature, coumarins act
as effective antioxidants as well as effective inhibitors
of AGE and RLAR, indicating its potential as anti-
diabetic complication drugs.
Advanced glycation endproduct inhibitory ac-
tivity of phenolic compounds
A series of phenolic compounds, including 4,5-di-
O
-
caffeoylquinic acid, chlorogenic acid, caffeic acid, ferulic
acid, dihydrocaffeic acid,
p
-coumaric acid, and 3,4-di-
methoxycinnamic acid were evaluated to assess their
inhibition of AGE formation and consequently, their
SAR. 4,5-Di-
O
-caffeoylquinic acid and chlorogenic acid
significantly inhibited AGE formation with IC
50
values
of 6.48 ± 0.04 and 21.08 ± 0.34
µ
M, respectively (Table
VI). Supporting the SAR between caffeoylquinic acids,
an additional AGE inhibition experiment was per-
formed using a phenolic moiety (i.e., caffeic acid, ferulic
acid, and dihydrocaffeic acid) and a sugar moiety (i.e.,
quinic acid). Much like previous research (Cui et al.,
2009), results from the caffeoylquinic acids suggested
that the number of caffeoyl groups and their position
in the quinic acid moiety may play an important role
in the inhibition of AGE formation.
In addition to their AGE inhibition properties, most
caffeoylquinic acids, including di-
O
-caffeoylquinic acid,
tri-
O
-caffeoylquinic acid, and chlorogenic acid have
been reported as potent natural AR inhibitors (de la
Fuente and Manzanaro, 2003; Logendra et al., 2006).
Cui et al. (2009) indicated that the number of caffeoyl
groups and their position in the quinic acid moiety
may play a key role in their inhibitory activity against
RLAR. 4,5-Di-
O
-caffeoylquinic acid was also reported
to exhibit AR inhibitory activity (Logendra et al., 2006).
Fig. 2.
Dose-dependent inhibition of ONOO
−
-mediated tyrosine nitration by
A. capillaris
coumarin derivatives. The mixture
of test samples, BSA, and ONOO
−
were incubated with shaking at 37
o
C for 30 min. The reactant was resolved by
electrophoresis in 10% polyacrylamide gel. (
A
) Esculetin; (
B
) Scopoletin; (
C
) Scopolin; (
D
) Scoparone; (
E
) Umbelliferone; (
F
)
Coumarin.
Table VI.
Inhibitory activity of phenolic compounds on
advanced glycation endproducts
Test compounds IC
50
value
a
Caffeic acid 47.13 ± 0.04
Ferulic acid 323.43 ± 8.10
Dihydrocaffeic acid 206.55 ± 3.06
p
-Coumaric acid NA
3,4-Dimethoxycinnamic acid NA
Quinic acid NA
Chlorogenic acid 21.08 ± 0.34
4,5-Dicaffeoylquinic acid 6.48 ± 0.04
Aminoguanidine
b
932.66 ± 4.94
a
The concentration yielding 50% inhibition (IC
50
,
µ
M) was
calculated from the log dose inhibition curve and is expressed
as the mean ± S.E.M. of triplicate experiments;
b
Aminogu-
anidine was used as a positive control.
NA: no activity within test concentrations.
1032 H. A. Jung et al.
Advanced glycation endproduct inhibitory ac-
tivity of flavonoids
All tested flavonoids exhibited significant AGE in-
hibitory activities when compared with the positive
control, aminoguanidine. Quercetin and its galactoside
(hyperoside) exerted the most potent inhibitory activ-
ity on AGE with IC
50
values of 24.35 ± 0.95 and 48.20
± 0.05
µ
M, respectively, as compared with aminogu-
anidine, which had an IC
50
value of 932.66 ± 4.94
µ
M.
There have been numerous reports on the anti-diabetic
complication effects of flavonoids, especially through
AR and AGE inhibition. In particular, it has been sug-
gested that the greater the number of hydroxyl groups
at the 3',4',5, and 7 position in flavonoids, the more
potent it is in AGE inhibition (Matsuda et al., 2003;
Stefek, 2011). Isorhamnetin and its glucosides were
also identified as exhibiting anti-diabetic complication
properties
in vivo
and
in vitro
(Yokozawa et al., 2002;
Lee et al., 2005). However, three acacetin derivatives,
including linarin, acacetin triglycoside, and its new
acylated derivatives, did not demonstrate AGE inhibi-
tion at the tested concentration.
In conclusion, a variety of indigenous
Artemisia
spe-
cies were selected to evaluate their AGE inhibition
properties and develop therapeutic drugs that prevent
diabetic complications. Although several
Artemisia
species significantly inhibited AGE formation,
A. capil-
laris
exhibited the most potent AGE inhibitory activity.
Further bioactivity-guided isolation led to a new
acylated flavonoid glycoside, acacetin-7-
O
-(6''-
O
-acetyl)-
β
-
D
-glucopyranosyl-(1
→
2)[
α
-
L
-rhamnopyranosyl]-(1
→
6)-
β
-
D
-glucopyranoside, together with 11 known flavonoids
(acacetin-7-
O
-
β
-
D
-glucopyranosyl-(1
→
2)[
α
-
L
-rhamnopyr-
anosyl]-(1
→
6)-
β
-
D
-glucopyranoside, linarin, quercetin,
hyperoside, isorhamnetin, isorhamnetin 3-galactoside,
isorhamnetin 3-glucoside, isorhamnetin 3-arabinoside,
isorhamnetin 3-robinobioside, arcapillin), six coumarins
(umbelliferone, esculetin, scopoletin, scopolin, isoscopolin,
and scoparone), and two phenolic derivatives (4,5-di-
O
-caffeoylquinic acid and chlorogenic acid).
Due to limited information in recent studies, we
focused on the detailed SAR between AGE inhibitory
activity and coumarin derivatives. The findings of the
detailed SAR between coumarins and AGE inhibition
demonstrated that a free hydroxyl group at C-7 and a
glucosyl group in place of a methoxyl group at C-6 are
two important parameters in the inhibition potential
of these coumarins on AGE formation. Since antioxi-
dant activity may suppress AGE formation processes
(i.e., glycoxidation) at the level of free radical intermedi-
ates, comprehensive and effective coumarins as AGE
and RLAR inhibitors along with antioxidants may be
beneficial uses in the development of anti-diabetic
complication drugs.
ACKNOWLEDGEMENTS
This paper was supported by research funds from
Chonbuk National University in 2010 (R112182101).
This research was also supported by the Basic Science
Research Program through the National Research
Foundation of Korea (NRF), which is funded by the
Ministry of Education, Science and Technology (2011-
0012539), and by a grant from the Food Drug &
Administration, South Korea (2010).
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