Vascular barrier protective effects of eckol and its derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.04.026

In this Letter, we first investigated the barrier protective effects of eckol and its derivatives against pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) and in mice. Data showed that eckol (1) and dieckol (2) inhibited lipop

Full text of DOI:10.1016/j.bmcl.2012.04.026

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3710–3712
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Vascular barrier protective effects of eckol and its derivatives
Tae Hoon Kim a, , Taeho Lee b, , Sae-Kwang Ku c, , Jong-Sup Bae b,
⇑
a Department of Herbal Medicinal Pharmacology, Daegu Haany University, Gyeongsan 712-715, Republic of Korea
b College of Pharmacy, Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701, Republic of Korea
c Department of Anatomy and Histology, College of Oriental Medicine, Daegu Haany University, Gyeongsan 712-715, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In this Letter, we first investigated the barrier protective effects of eckol and its derivatives against pro-
inflammatory responses in human umbilical vein endothelial cells (HUVECs) and in mice. Data showed
that eckol (1) and dieckol (2) inhibited lipopolysaccharide (LPS)-mediated barrier disruption and trans-
endothelial migration of leukocytes to human endothelial cells. Eckol (1) also suppressed acetic acid
induced-hyperpermeability and carboxymethylcellulose-induced leukocytes migration in vivo. Interest-
ingly, the barrier protective effects of dieckol (2) were better than those of eckol (1) and hydroxyl groups
in dieckol (2) positively regulate protective effects.
Received 2 March 2012
Revised 2 April 2012
Accepted 5 April 2012
Available online 12 April 2012
Keywords:
Eckol
Dieckol
Ó 2012 Elsevier Ltd. All rights reserved.
Vascular barrier integrity
Endothelium
LPS
Vascular endothelial barrier serves to separate the inner space
of the blood vessel from the surrounding tissue and to control
the exchange of cells and fluids between the two.1,2 This barrier
exhibits a variety of important functions, including control of coag-
ulation, fibrinolysis, vascular tone, growth and immune response.3
And it is dynamic and highly susceptible to the regulation by var-
ious stimuli of physiological and pathological origin.1,2 It is well
recognized that disruption of vascular barrier integrity results in
marked increases in permeability to fluid and solute and is neces-
sary to provide the access of leukocytes to the inflamed tissues and
the central pathophysiologic mechanism of many vascular inflam-
matory disease processes such as sepsis and atherosclerosis.2,4,5
Thus, altered permeability of the endothelial barrier is a character-
istic hallmark of inflammatory responses which contributes to the
morbidity and mortality in several inflammatory diseases such as
sepsis, acute lung injury and anaphylaxis.6,7 In addition, as an ini-
tial event of inflammation, leukocytes adhere to the vascular lining
and migrate into the inflamed tissue.8 The adhesion of circulating
leukocytes to the vascular endothelium is a fundamental step in
leukocyte extravasation during inflammation.9,10 In particular,
endothelial dysfunction is related to leukocyte recruitment during
the formation of the inflammatory lesion.11,12 Therefore, inhibition
of leukocytes migration to vascular endothelium and barrier
permeability as a therapeutic approach is an attractive way to
potentially prevent early inflammatory injury. Therefore, agents
that inhibit the leukocyte migration to vascular endothelium and
enhance endothelial cell barrier function are desirable for a variety
of inflammatory diseases.2,13
The search for anticancer drugs and anti-inflammatory agents
from natural products represents an area of great interest world-
wide.14 Eisenia bicyclis is a common perennial brown alga of the
family Laminariaceae that inhabits the middle Pacific coast around
Korea and Japan.15 This seaweed is consumed as appetisers, casse-
roles, muffins, pilafs, soups, toasted dishes and many other types of
food. The known biological activities of this brown alga15 include
effects on skin disease, Alzheimer’s disease, allergies, diabetes
and cancer.15–17 Previously, antioxidant activity of E. bicyclis
phlorotannins such as eckol (1) and dieckol (2) has been re-
ported.15 However, the effect of eckol (1) and its derivatives (2–
4) on vascular barrier integrity in both cellular system and animal
model have not yet been elucidated (Fig. 1). The objective of the
present study was to test naturally occurring anti-inflammatory
agent from E. bicyclis16 for its vascular barrier protective effect on
endothelial cells and mice.
To determine the effects of purified eckol or dieckol18 on the
HMGB1-mediated hyperpermeability primary HUVECs, the flux of
albumin in a dual chamber system was monitored as described pre-
viously.19 The inhibitory effect of dieckol (2) was better than that of
eckol (1) (Table 1). The possible explanation for these results is the
number and position of hydrogen donating hydroxyl groups in eckol
(1) or dieckol (2). To verify this hypothesis, the hydroxyl groups of
dieckol (2) were changed to methyl groups or acetyl groups. To do
⇑
Corresponding author. Address: College of Pharmacy, Research Institute of
Pharmaceutical Sciences, Kyungpook National University, 80 Daehak-ro, Buk-gu,
Daegu 702-701, Republic of Korea. Tel.: +82 53 950 8570; fax: +82 53 950 8557.
E-mail address: baejs@knu.ac.kr (J.-S. Bae).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.026

T. Hoon Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3710–3712
3711
Table 2
Effects of different eckols on acetic acid-mediated hyperpermeability in mice
Compound
Dose
lg/mouse (n = 5)
Inhibition (%)
(À) Control (PBS)
(+) Control (acetic acid)
Eckol (1)
Dieckol (2)
Me-dieckol (3)
0.46 0.07
5.54 0.42
0.7%
10
10
10
10
10
l
l
l
l
l
M
M
M
M
M
2.65 0.09**
1.41 0.23**
3.61 0.08*
2.24 0.04**
1.45 0.18**
52.0
74.4
34.7
59.5
73.9
Ac-dieckol (4)
Kaempferol-3-O-sophorosidea
Each data represent the mean S.D. of three different experiments.
*
<0.05 significantly different from the acetic acid.
Figure 1. Structures of eckol and its derivatives.
**
<0.01 significantly different from the acetic acid.
a
Used as positive control.
Table 1
Effects of different eckols on LPS-mediated hyperpermeability in HUVECs
Table 3
Compound
Dose
ELISA OD650
0.096 0.013
Inhibition (%)
Effects of different eckols on LPS-mediated monocyte migration on HUVECs.
(À) Control (PBS)
(+) Control (LPS)
Eckol (1)
Compound
Dose
Migration Index Inhibition (%)
100 ng/ml 0.554 0.042
0.242 0.025*
56.5
(À) Control (PBS)
27.9 4.7
163.6 6.7
10
10
10
10
l
l
l
l
l
M
M
M
M
M
0.138 0.014** 74.9
(+) Control (LPS)
100 ng/
ml
Dieckol (2)
Me-dieckol (3)
Ac-dieckol (4)
0.371 0.012*
0.278 0.028*
33.0
49.5
Eckol (1)
Dieckol (2)
Me-dieckol (3)
Ac-dieckol (4)
Kaempferol-3-O-
sophorosidea
10
10
10
10
10
l
l
l
l
l
M
M
M
M
M
65.9 6.5**
46.8 8.4**
101.9 5.1*
81.0 9.1**
49.8 3.7**
59.6
71.3
37.6
50.3
69.5
Kaempferol-3-O-sophorosidea 10
0.129 0.019** 76.6
Each data represent the mean S.D. of three different experiments.
*
<0.05 significantly different from the LPS.
**
<0.01 significantly different from the LPS.
a
Used as positive control.
Each data represent the mean S.D. of three different experiments.
*
<0.05 significantly different from the LPS.
**
<0.01 significantly different from the LPS.
a
Used as positive control.
these, the methylation and acetylation reactions of hydroxyl groups
in dieckol (2) promoted by treatment with iodomethane and acetic
anhydride furnished the respective Me-dieckol (3) (91%) and Ac-
dieckol (4) (87%) (Scheme 1).20
used (up to 20
lM), each eckol (1–4) did not affect cell viability
As shown in Table 1, the inhibitory effect of dieckol was dimin-
ished when its hydroxyl groups were changed to methyl groups.
However, when the hydroxyl groups of dieckol were changed to
acetyl groups, the inhibitory effect of Ac-dieckol (4) was better
than that of Me-dieckol (3). These results suggest that the exis-
tence of hydrogen bond donors and hydrophilic moieties are
important to inhibitory effects. The Me-dieckol (3) and Ac-dieckol
(4) without the hydroxyl groups as hydrogen bond donors showed
the less inhibitory effects than the dieckol (2). The inhibitory effect
of more hydrophilic acetyl substituted dieckol (4) was higher than
its less hydrophilic methyl substituted dieckol (3) (see Table 1).
To confirm this effect in vivo, acetic acid-induced vascular per-
meability in mice was assessed as described previously.21 As
shown in Table 2, eckol (1–4) markedly inhibited the leakage of
dye into the peritoneum in mice. To exclude the possibility that
the inhibition of permeability was due to cytotoxicity caused by
each eckol (1–4), cellular viability assays were performed in
HUVECs treated with each eckol for 24 h. At the concentrations
(data not shown).
The adhesion of leukocytes to endothelial cells and transendo-
thelial migration (TEM) of leukocytes are important steps in the
pro-inflammatory response.22,23 We conducted studies to deter-
mine whether eckol could block the adhesion of monocytes to
HMGB1-stimulated HUVECs. We demonstrated that eckol effec-
tively inhibited the binding of monocytes to HMGB1-stimulated
endothelial cells (data not shown). Further studies revealed that
the adhesion of monocytes to endothelial cells was associated with
their subsequent TEM and that eckol also effectively inhibited this
step (Table 3). To confirm this effect in vivo, CMC–Na-induced leu-
kocyte migration in mice was examined. CMC–Na significantly
stimulated leukocyte migration into the peritoneal cavity of mice
and eckol at doses of 10 lM significantly decreased leukocytes
counts (Table 4). Kaempferol-3-O-sophoroside was used as a posi-
tive control in biological test.24
Collectively, above data showed that these barrier protective
effects of dieckol (2) were better than eckol (1). Recent studies
OH
OR
HO
OH
OH
RO
OR
OR
O
OH
OH
O
OR
HO
O
O
RO
O
O
MeI, K2CO3, DMF (for 3)
or Ac2O, pyridine (for 4)
OH
O
OR
O
O
O
OH
O
O
O
OR
O
OH
OR
OH
OR
OR
3 (91%): R = Me
4 (87%): R = Ac
2
Scheme 1. Synthesis of dieckol derivatives 3 and 4.

3712
T. Hoon Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3710–3712
Table 4
9. Springer, T. A. Cell 1994, 76, 301.
10. Hogg, N.; Berlin, C. Immunol. Today 1995, 16, 327.
11. Gimbrone, M. A., Jr. Am. J. Cardiol. 1995, 75, 67B.
12. Jerzak, P. Pol. Tyg. Lek. 1994, 49, 357.
13. Dudek, S. M.; Garcia, J. G. J. Appl. Physiol. 2001, 91, 1487.
14. Aggarwal, B. B.; Ichikawa, H.; Garodia, P.; Weerasinghe, P.; Sethi, G.; Bhatt, I. D.;
Pandey, M. K.; Shishodia, S.; Nair, M. G. Expert Opin. Ther. Targets 2006, 10, 87.
15. Okada, Y.; Ishimaru, A.; Suzuki, R.; Okuyama, T. J. Nat. Prod. 2004, 67, 103.
16. Joe, M. J.; Kim, S. N.; Choi, H. Y.; Shin, W. S.; Park, G. M.; Kang, D. W.; Kim, Y. K.
Biol. Pharm. Bull. 2006, 29, 1735.
17. Jung, H. A.; Oh, S. H.; Choi, J. S. Bioorg. Med. Chem. Lett. 2010, 20, 3211.
18. Fresh E. bicyclis was washed three times with water to remove salt. Lyophilized
E. bicyclis was ground into powder before extraction. The dried E. bicyclis
powder (1.0 kg) was extracted with MeOH (10 L Â 3) at room temperature and
the solvent was evaporated in vacuo. The combined crude MeOH extract
(164.3 g) was suspended in 10% MeOH (1.0 L), and then partitioned in turn
with n-hexane (1.0 L Â 3), CH2Cl2 (1.0 L Â 3), EtOAc (1.0 L Â 3), and n-BuOH
(1.0 L Â 3) to yield dried n-hexane- (42.3 g), CH2Cl2 (2.5 g), EtOAc- (23.0 g), n-
BuOH (26.5 g) and H2O-soluble (69.1 g) residues. A portion (10.0 g) of the
EtOAc extract was chromatographed on a Sephadex LH-20 column (4.0 cm
i.d. Â 50 cm) with MeOH and fractioned into seven subfractions (EB01–EB07).
Subfractions EB02 and EB07 were subjected to column chromatography over a
LiChroprep RP-18 column (1.1 cm i.d. Â 37 cm) with aqueous MeOH to yield
pure eckol (1) (tR 4.0 min, 25.2 mg) and dieckol (2) (tR 8.1 min, 17.2 mg).
19. Bae, J. S.; Rezaie, A. R. Blood 2011, 118, 3952.
Effects of different eckols on CMC–Na-mediated leukocytes migration in mice.
Compound
Dose
 106
Inhibition (%)
(À) Control (PBS)
(+) Control (CMC–Na)
Eckol (1)
Dieckol (2)
Me-dieckol (3)
1.20 0.15
6.00 0.29
2.30 0.28**
1.80 0.14**
4.05 0.07*
2.60 0.14**
1.81 0.23*
1.0%
10
10
10
10
10
l
l
l
l
l
M
M
M
M
M
61.7
69.9
32.4
56.7
69.6
Ac-dieckol (4)
Kaempferol-3-O-sophorosidea
Each data represent the mean S.D. of three different experiments.
*
<0.05 significantly different from the CMC–Na.
**
<0.01 significantly different from the CMC–Na.
a
Used as positive control.
have demonstrated that the protective effect against oxidative
stress induced by ROS and UV radiation is correlated with the
number and position of hydrogen-donating hydroxyl groups on
the aromatic ring of the phonolic molecules, and is also affected
by other factors, such as other H-donating groups (–NH, –SH),
etc.25,26 Our results indicated that dieckol (2) has more functional
hydroxyl groups than other tested eckol, therefore, this study with
dieckol unravels a novel vascular barrier protective functions.
20. Preparation of Me-dieckol (3). To a solution of dieckol (2, 45 mg, 0.061 mmol) in
DMF (5 mL) were added iodomethane (0.10 mL, 1.60 mmol) and potassium
carbonate (276 mg, 2.00 mmol) at 0 °C. The reaction mixture was stirred at
room temperature for 9 h, and then diluted with EtOAc, washed with 1 N HCl
and brine, dried over MgSO4. The solvent was removed, and the residue was
purified by flash silica gel column chromatography (hexane/EtOAc, 1:1) to give
7-[2,6-dimethoxy-4-(2,4,7,9-tetramethoxydibenzo[b,e][1,4]dioxin-1-
yloxy)phenoxy]-1-(3,5-dimethoxyphenoxy)-2,4,9-
Acknowledgment
trimethoxydibenzo[b,e][1,4]dioxine (3, 50 mg, 91%) as a white solid: 1H NMR
(300 MHz, CDCl3) d 3.67 (s, 3H), 3.70 (s, 6H), 3.72 (s, 6H), 3.74 (s, 6H), 3.75 (s,
3H), 3.85 (s, 3H), 3.86 (s, 3H), 3.94 (s, 3H), 5.94 (d, J = 3.0 Hz, 1H), 6.11–6.16 (m,
4H), 6.20–6.22 (m, 2H), 6.30 (s, 1H), 6.32 (s, 2H), 6.34 (d, J = 2.7 Hz, 1H); LC–MS
(ESI) m/z 897 ([M+1]+).
This work was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea government [MEST] (Nos.
2011-0026695, 2011-0030124).
Preparation of Ac-dieckol (4). To a solution of dieckol (2, 18 mg, 0.024 mmol) in
pyridine (4 mL) was added acetic anhydride (0.050 mL, 0.53 mmol) at 0 °C. The
reaction mixture was stirred at room temperature for 6 h, and then diluted
with EtOAc, washed with saturated CuSO4, H2O, and brine, dried over MgSO4.
The solvent was removed, and the residue was purified by flash silica gel
column chromatography (hexane/EtOAc, 1:1) to give 4-{3,5-diacetoxy-4-
[4,7,9-triacetoxy-6-(3,5-diacetoxyphenoxy)dibenzo[b,e][1,4]dioxin-2-
yloxy]phenoxy}dibenzo[b,e][1,4]dioxine-1,3,6,8-tetrayl tetraacetate (4, 25 mg,
87%) as a white solid: 1H NMR (300 MHz, CDCl3) d 1.93 (s, 3H), 2.04 (s, 3H), 2.11
(s, 6H), 2.13 (s, 3H), 2.19 (s, 3H), 2.245 (s, 3H), 2.254 (s, 6H), 2.28 (s, 3H), 2.34 (s,
3H), 6.31 (d, J = 2.7 Hz, 1H), 6.43 (d, J = 3.0 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H), 6.57
(d, J = 2.1 Hz, 2H), 6.62 (s, 1H), 6.63 (d, J = 3.0 Hz, 1H), 6.67 (s, 1H), 6.68 (s, 2H),
6.70 (t, J = 2.0 Hz, 1H).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.04.
026.
References and notes
1. Mehta, D.; Malik, A. B. Physiol. Rev. 2006, 86, 279.
2. Komarova, Y. A.; Mehta, D.; Malik, A. B. Sci. STKE 2007, 2007, re8.
3. Gerritsen, M. E.; Bloor, C. M. FASEB J. 1993, 7, 523.
4. He, P. Cardiovasc. Res. 2010, 87, 281.
5. Majno, G.; Palade, G. E. J. Biophys. Biochem. Cytol. 1961, 11, 571.
6. Ware, L. B.; Matthay, M. A. N. Engl. J. Med. 2000, 342, 1334.
7. Dhillon, S. S.; Mahadevan, K.; Bandi, V.; Zheng, Z.; Smith, C. W.; Rumbaut, R. E.
Chest 2005, 128, 1706.
21. Bae, J.-S.; Lee, W.; Rezaie, A. R. J. Thromb. Haemost. 2012, in press.
22. Hansson, G. K.; Libby, P. Nat. Rev. Immunol. 2006, 6, 508.
23. Bae, J. S.; Yang, L.; Manithody, C.; Rezaie, A. R. J. Biol. Chem. 2007, 282, 9251.
24. Kim, T. H.; Ku, S. K.; Bae, J. S. Food Chem Toxicol 2012, 50, 1118.
25. Rice-Evans, C. A.; Miller, N. J.; Bolwell, P. G.; Bramley, P. M.; Pridham, J. B. Free
Radic. Res. 1995, 22, 375.
26. Lien, E. J.; Ren, S.; Bui, H. H.; Wang, R. Free Radic. Biol. Med. 1999, 26, 285.
8. Harlan, J. M. Blood 1985, 65, 513.