Study of skin anti-ageing and anti-inflammatory effects of dihydroquercetin, natural triterpenoinds, and their synthetic derivatives1

Article abstract of DOI:10.1134/S1068162012030028

Accessible triterpenoids of ursane and lupane series, the flavonoid dihydroquercetin and their syn-thetic derivatives with polar substituents were tested in vitro for inhibition of collagenase 1 (MMP-1) in UVB irradiation assay. Ursolic acid and uvaol disuccinate were the most active inhibitors in the ursane series. In the lupane series, the best inhibition was manifested by carboxymethyl ester of betulonic acid and betulin succi-nates. Down-regulation of MMP-1 by dihydroquercetin and its synthetic derivatives surpassed the activity of a standard (retinoic acid). Pleiades Publishing, Ltd., 2012.

Full text of DOI:10.1134/S1068162012030028

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2012, Vol. 38, No. 3, pp. 328–334. © Pleiades Publishing, Ltd., 2012.
Study of Skin AntiꢀAgeing and AntiꢀInflammatory Effects
of Dihydroquercetin, Natural Triterpenoinds,
and Their Synthetic Derivatives1
Chan Woo Leea, Nok Hyun Parka, Jin Woong Kimb, Byung Hun Umc, A. V. Shpatovd,
, 2
E. E. Shultsd, I. V. Sorokinad, and S. A. Popovd
aAmoreꢀPacific Co. R&D Center, 314ꢀ1, Boraꢀdong, Giheungꢀgu, Yonginꢀsi, GyeonggiꢀDo, 446ꢀ729, Republic of Korea
bDept. of Applied Chemistry, Hanyang University,
55 Hanyangdaehakꢀro, Sangnokꢀgu, Ansan, Kyeonggiꢀdo, 426ꢀ791, Republic of Korea
cNatural Products Research Center, Korea Institute of Science and Technology (KIST) Gangneung Institute,
Gangneung 210ꢀ340, Gangwonꢀdo, Republic of Korea
dNovosibirsk Institute of Organic Chemistry, Lavrentyev Ave. 9, Novosibirsk, 630090 Russian Federation
Received May 16, 2011; in final form, June 1, 2011
Abstract—Accessible triterpenoids of ursane and lupane series, the flavonoid dihydroquercetin and their synꢀ
thetic derivatives with polar substituents were tested in vitro for inhibition of collagenase 1 (MMPꢀ1) in UVB
irradiation assay. Ursolic acid and uvaol disuccinate were the most active inhibitors in the ursane series. In the
lupane series, the best inhibition was manifested by carboxymethyl ester of betulonic acid and betulin succiꢀ
nates. Downꢀ regulation of MMPꢀ1 by dihydroquercetin and its synthetic derivatives surpassed the activity
of a standard (retinoic acid).
Keywords: collagenaseꢀ1, triterpenoids, dihydroquercetin, photoageing, interleukinꢀ8
DOI: 10.1134/S1068162012030028
2
1
INTRODUCTION
aging the skin, collagen synthesis becomes lower and
MMPꢀ1 levels become higher even in sunꢀprotected
human skin [6].
Human skin photoageing caused by exposure to
sun results in origination of coarse wrinkles, discolorꢀ
ation, abnormal dermal pigmentation and loss of skin
elasticity. Tissue connective components are degradꢀ
able by the family of proteolytic enzymes, matrix metꢀ
alloproteinases including MMPꢀ1, interstitial collageꢀ
nase. MMPꢀ1 is found in a variety of cells, including
fibroblasts, endothelial cells, smooth muscle cells,
chondrocytes, and multiple tumor types [1]. The synꢀ
ergistic action of MMPꢀ1 with other MMP enzymes is
responsible for degrading of skin collagen and elastin.
In human skin, MMPꢀ1, but not MMPꢀ14 or MMPꢀ
15, is highly induced by UV irradiation and appears to
be responsible for collagen fragmentation associated
with chronic sun exposure [2].
Treatment of photo damaged skin with certain natꢀ
ural chemicals, e.g., topical retinoids improves skin
clinical appearance [7]. Retinoic acid (RA) is known
to be a good suppressor of MMPꢀ1 [8]. Terpenoids,
flavonoids, and their accessible synthetic derivatives
are of interest as components for human skin protecꢀ
tion. Derivatives of the lupane (betulin, betulinic
acid), ursane (ursolic acid, UA), and oleanane series
[9] were tested as suppressors of proteinases. Certain
synthetic derivatives of triterpenoids were also examꢀ
ined for MMPꢀ1 inhibition [10]. Cytostatic and antiꢀ
proliferative activity was discovered for ursolic acid
[11, 12], and more interestingly, it showed a memꢀ
brane stabilizing effect [13]. Betulin derivatives were
reported to have an ability to suppress UVBꢀinduced
MMPꢀ1 expression [14].
MMPꢀ1 treatment of young skin in organ culture
causes fragmentation of collagen fibrils, which reduces
fibroblast stretch, consistent with reduced mechanical
tension, as observed in aged human skin [3–5]. With
Betulinic acid was found to inhibit stromelysin and
collagenase effectively, while its analog, Cꢀ28 alcohol
betulin, demonstrated lesser inhibition potency
against these proteases [15]. Low toxicity and cytotoxꢀ
icity levels, characteristic of betulin and its esters
[16, 17], make them prospective as additives for skin
protection. Certain flavonoids (sumaflavone and
amentoflavone) have been demonstrated to reduce
effectively the MMPꢀ1 levels [18].
1
The article is published in the original.
Abbreviations: MMPꢀ1, matrix metalloproteinaseꢀ1; collageꢀ
naseꢀ1; ILꢀ8, Interleukinꢀ8; DMEM, Dulbecco’s modified
Eagle’s medium; FBS, fetal bovine serum; PBS, phosphate
buffered saline; HaCaT cells, Immortalized human Keratiꢀ
nocytes.
Corresponding author: phone: 8ꢀ383ꢀ330ꢀ67ꢀ48; eꢀmail:
kjwoong@hanyang.ac.kr, spopov@niboch.nsc.ru.
2
328

STUDY OF SKIN ANTIꢀAGEING AND ANTIꢀINFLAMMATORY EFFECTS
329
To develop the derivatives of natural products with leukinꢀ8 inhibition tests. Interleukinꢀ8, causing
effective suppression of MMPꢀ1 expression, in this recruitment of inflammatory cells and inducing a furꢀ
study we examined the accessible triterpenoids and ther increase in oxidant stress mediators, is known as a
their derivatives as potential inhibitors of UVꢀinduced key parameter in localized inflammation [20].
damage of human fibroblasts. Modification of triterꢀ
penoids, achieved by introduction of polar functions
RESULTS AND DISCUSSION
to their molecules, can enhance the efficacy of prepaꢀ
rations (which can be due to, e.g., the increase in solꢀ
ubility). Betulonic acid, the component of Betula speꢀ
cies extracts, which is available as betulin oxidation
product, is also of interest as a suppressor of MMPs.
We were interested in preparation of polar nonꢀ
toxic derivatives of accessible triterpenoids and dihydꢀ
roquercetin (Schemes 1–3). Treatment of triterpeꢀ
noids (
presence of pyridine resulted in hydrogen succinates
of ursolic acid (II), uvaol ( ), and betulin (VII VIII).
Alkylation of ursolic acid ( ) and betulonic acid (IX
with ethyl 2ꢀchloroacetate followed with saponificaꢀ
tion with ethanolic KOH afforded the corresponding
carboxymethyl esters (III), ( ). Accessible Mannich
adducts (XII), (XIII) were prepared by reaction of
I), (IV), (VI) with succinic anhydride in the
We prepared several polar esters of betulin, uvaol,
ursolic acid, and betulonic acid and tested their inhiꢀ
bition effect on collagenaseꢀ1 (MMPꢀ1). The flaꢀ
vonoid dihydroquercetin and its Mannich adducts
[19] were also examined as possible inhibitors of
MMPꢀ1 (we did not succeed to find the data on downꢀ
regulation of MMPꢀ1 with dihydroquercetin).
V
I
,
)
X
Antiꢀinflammatory activity of natural compounds dihydroquercetin (XI) with formaldehyde and morꢀ
and their synthetic derivatives were evaluated in interꢀ pholine or diethylamine [19].
30
29
20
19
H
H
12
26
18
17
R2
R2
25
1
28
14
9
O
8
H
H
3
5
27
R1
R1
H
24
23
(I) R1, R2=OH
(IV) R1, R2=OH
O
O
O
O
O
(II) R1=
R2=OH
O
(V) R1, R2= O
OH
OH
OH
Py
O
O
O
(II) R1=H, R2=
O
Scheme 1. Derivatives with ursane skeleton for MMPꢀ1 and ILꢀ8 tests.
(
VI) R1=OH, R2=OH
O
O
O
O
VII) R1, R2=
O
H
(
H
(VI)
(VII)
OH
R1
Py, 100°C
H
O
O
O
O
O
(
VIII) R2=OH, R1=
O
R2
OH
Py, 30°C
(VIII)
H
29
20
O
O
30
(
IX)
O
R1=OH
19
17
1
K2CO3
H
Cl
H
18
O
25H
13
O
23
14
2
2
EtOH, KOH
H+
R1
9
(
X
) R1=
O
10
6
OH
5
3
27
26
O
H
Scheme 2. Derivatives with lupane skeleton for MMPꢀ1 and ILꢀ8 tests.
24 23
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330
CHAN WOO LEE et al.
The effect of compounds (
I
)–(XIII) on the production of MMPꢀ1 by human dermal fibroblasts
Conꢀ UVB
trol (40 mJ)
RA
(
I
)
(
II
)
(
III
)
(IV)
Concentraꢀ
tion of tested
compound,
ppm
–
–
0.3 0.75
1.5
3.0
0.1
1
0.1
1
0.1
1
0.1
1
MMPꢀ1, %
to control
Standard
100.0 189.8 145.0 118.3 103.1 95.2 125.9 84.8 147.0 127.4 125.9 142.4 129.6 119.5
11.31 17.36 5.81 14.85 4.62 21.52 12.81 26.04 8.88 17.60 17.01 8.73 14.08 11.59
deviation, %
(V
)
(VI
)
(VII
)
(
VIII
)
(IX)
Concentraꢀ
tion of tested
compound,
ppm
0.1
1
0.1
1
10
0.1
1
10
0.1
1
10
0.1
1
10
MMPꢀ1, %
to control
Standard
140.3 91.2 193.8 142.9 139.2 173.2 95.7
8.77 15.10 16.14 9.79
3.0 113.3 110.6 17.6 92.1 97.1 73.9
5.9 10.52 22.05 16.2
8.8 19.61 35.71 2.5 17.94 5.08
deviation, %
(X
)
(XI
)
(XII
)
(XIII)
Concentraꢀ
tion of tested
compound,
ppm
0.1
1
0.1
1
10
0.1
1
10
0.1
1
10
MMPꢀ1, %
to control
Standard
88.1
9.85
41.9
3.87
106.3
8.92
94.7
9.69
31.0
18.7
124.3
6.17
94.0
4.26
6.9
6.9
105.2
13.37
88.4
2.19
13.9
11.1
deviation, %
The results are obtained in three independent experiments.
OH
OH
OH
OH
HO
O
HO
R
O
O
CH2O, RH
OH
OH
O
OH
OH
(XI)
(XII) R=
N
O
(XIII) R=NEt2
Scheme 3. Dihydroquercetin and its derivatives for MMPꢀ1 and ILꢀ8 tests.
The effects of compounds (
mal fibroblasts are given in table.
I
–
XIII) on human derꢀ tives in concentrations of 0.1 ppm decreased MMPꢀ1
to 128–149%.
The best indices were found for ursolic acid (
uvaol bis(hydrogen succinate) ( ). Uvaol (IV) activity
I) and
V
MMPꢀ1 Regulation by Ursane Derivatives (Fig. 1)
(concentration ~1.0 ppm) was slightly inferior to that
of RA.
At low concentration levels of UA derivatives
(0.1 ppm), MMPꢀ1 inhibition was comparable or
slightly exceeded that of the standard (RA).
UVB irradiation caused MMPꢀ1 expression
growth up to 189% as compared to control. The addiꢀ
tion of retinoic acid (RA) (0.3 ppm) downꢀregulated
MMPꢀ1 expression to 145%.
Among UA derivatives, the best results were
The increase in RA concentration caused the furꢀ observed for ursolic acid (
ther reduction of MMPꢀ1 expression. Ursane derivaꢀ (I) at 0.1 and 1 ppm was superior over the standard
I): the inhibition activity of
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STUDY OF SKIN ANTIꢀAGEING AND ANTIꢀINFLAMMATORY EFFECTS
331
190
170
150
130
110
90
RA
I
II
IV
V
III
10
70
0.1
1
Conc., ppm
Fig. 1. Effect of ursane derivatives on MMPꢀ1 expression.
250
200
150
100
50
RA
VI
VII
VIII
IX
X
0
0.1
1
Conc., ppm
10
Fig. 2. Effect of lupane derivatives on MMPꢀ1 expression.
(RA) at 0.3 and 1.5–3.0 ppm, respectively, and signifꢀ
icantly better than the corresponding results obtained
for uvaol. Ursolic acid hydrogen succinate (II) and
carboxymethyl ester (III) were inferior in MMPꢀ1
Betulin (VI) demonstrated insignificant inhibition
level, inferior to RA.
Betulin bis(hydrogen succinate) and mono(hydroꢀ
gen succinate) (VII, VIII) were found comparable with
the standard (RA) showing similiar inhibition at 1 ppm,
and closeꢀup to that of RA at 0.75–3 ppm. At 10 ppm
inhibition to UA (
I).
Uvaol 3,28ꢀbis(hydrogen succinate) (
V) demonꢀ
strated better activity than the parent uvaol (IV),
whereas for (IV) the notable MMPꢀ1 upꢀregulation at
(
VII, VIII) showed very good MMPꢀ1 suppression
10 ppm was observed, while inhibition at 1 ppm for (
was comparable with that of UA ( ).
V)
(about 90 and 80% downꢀregulation, respectively.
I
Betulonic acid ( ), which is closely structurally
X
related to betulinic acid, known for its good collagenase
inhibition [15], was comparable or superior to standard
(RA) within the test concentration range. Surprisingly,
betulonic acid carboxymethyl ester (X) manifested
~60% inhibition of MMPꢀ1 at 1 ppm concentration—
MMPꢀ1 Regulation by Lupane Derivatives (Fig. 2)
The betulin esterification products with succinic
anhydride demonstrated MMPꢀ1 suppression enhanceꢀ
ment (the similar pattern was observed for uvaol (IV) and
its bis(hydrogen succinate) (V), (see above).
the best results among the tested compounds.
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332
CHAN WOO LEE et al.
160
140
120
100
80
RA
XI
XII
XIII
60
40
20
0
0.1
1
Conc., ppm
10
Fig. 3. Effect of dihydroquercetin derivatives on MMPꢀ1 expression.
MMPꢀ1 Regulation by Dihydroquercetin Derivatives
and in 13C NMR spectra, δC 76.90 (CDCl3). Signals in
the NMR spectra were assigned by correlation with
those of ursolic acid ( ) [21, 22], ursolic acid 3ꢀacetate
II) [23]; values are given in Hertz.
Melting points were measured on a Kofler melting
(Fig. 3)
I
Dihydroquercetin (XI) and its Mannich condensaꢀ
(
J
tion products (XII, XIII) downꢀregulated MMPꢀ1 activꢀ
ity better than the standard. Dihydroquercetin (XI) and
point apparatus and are uncorrected. Optical rotation
was measured on a PolAAR 3005 polarimeter (Great
Britain) at 589 nm in a tube 5 cm long. TLC was carꢀ
ried out on Sorbfil plates (Russia). Purity of the tested
its synthetic derivatives (XII, XIII) possessed similar
activity level within the range of tested concentrations.
compounds was
≥
90% (by 1H NMR).
Results of ILꢀ8 Experiments
None of the tested compounds demonstrated notaꢀ
ble levels of activity in ILꢀ8 experiments.
Reagents
To summarize, the flavonoid dihydroquercetin,
triterpenoids of the ursane and lupane series and their
synthetic derivatives with polar substituents were studꢀ
ied as potential inhibitors of MMPꢀ1 in UVB irradiaꢀ
tion tests. Antiinflammatory activity was studied in
ILꢀ8 experiments, Ursolic acid and uvaol bis(hydroꢀ
gen succinate) were the most active inhibitors of
MMPꢀ1 in the ursane series. In the lupane series, the
best inhibition of MMPꢀ1 was manifested by carꢀ
boxymethyl ester of betulonic acid and betulin
bis(hydrogen succinate). The activity of dihydroquerꢀ
cetin derivatives was similar to those of the parent
compound and exceeded the activity of retinoic acid.
None of the studied compounds showed notable
antiꢀinflammatory activity in ILꢀ8 experiments.
Dulbecco’s modified Eagle’s medium (DMEM),
Penicillin/Streptomycin, fetal bovine serum were purꢀ
chased from Lonza (Switzerland), phosphateꢀbuffered
saline (PBS) was from Welgene (South Korea).
Ursolic acid (
vitis idaea L. peels extract according to the method
[24]. Ursolic acid 3ꢀ(hydrogen succinate) (II) was
synthesized as described in [25], mp 243–244 (lit.
I) 95%+ was prepared from Vaccinium
°
C
23
mp 248–249°C), αD = +46.8 (
c
1, MeOH). Alkylaꢀ
) with ethyl 2ꢀchloroacetate and
subsequent hydrolysis afforded 3ꢀhydroxyursanꢀ12ꢀ
eneꢀ28ꢀoic acid carboxymethyl ester (III) [26]. Uvaol
IV) was synthesized from methyl ursolate according
to the published method [27], mp 228–229
(MeOH), (lit. mp 223–224 ).
Crystallisation of the extract of Betula pendula bark
gave betulin 95%+ [28], mp 255–257 (EtOH), (lit.
mp 256–258 ).
(3,28ꢀDihydroxylupꢀ20ꢀene)ꢀbis(hydrogen succiꢀ
(EtOAcꢀhexane), (lit.
) and 3,28ꢀdihydroxylupaneꢀ20ꢀene
ppm relative to signals of the solvents used as the interꢀ 28ꢀ(hydrogen succinate) (VIII), mp 240–242
tion of ursolic acid (
I
(
°
C
°
C
EXPERIMENTAL
°
C
1H and 13C NMR spectra were recorded on a
Bruker AVꢀ300 (300.13 MHz for 1Н, 75.48 MHz for
13C) and Bruker AMꢀ400 (400.13 MHz for 1Н,
°
C
100.62 MHz for 13C) at room temperature in CDCl3 or nate) (VII), mp 112–114
C
°
CDCl3ꢀpyridineꢀd5. The chemical shifts are given in mp 110–112 C
°
°
C
1
nal standards: in H NMR spectra, δH 7.24 (CHCl3)
,
(iꢀPrOH), (lit. mp 242–244 C) were prepared by
°
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
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2012

STUDY OF SKIN ANTIꢀAGEING AND ANTIꢀINFLAMMATORY EFFECTS
333
reaction of betulin with succinic anhydride in the presꢀ reaction mixture was poured to water and the precipiꢀ
ence of pyridine as described in [29, 30]. tate was filtered and washed with water ( 20 mL).
Betulonic acid (IX), mp 249–252 (lit. mp 250–
254°C) was prepared by oxidation of betulin (VI) with
chromium trioxide [31]. Alkylation of betulonic acid
IX) with ethyl 2ꢀchloroacetate and subsequent
hydrolysis afforded 3ꢀoxoꢀlupaneꢀ20ꢀeneꢀ28ꢀoic acid
carboxymethyl ester ( ).
3
×
The wet residue was added to a solution of KOH (0.4 g,
7.2 mmol) in EtOH (10 mL), refluxed for 1 h and
cooled to ambient temperature. The mixture was
poured into 5% aq. HCl (50 mL), filtered, washed with
water and dried on the open air. Yield 0.98 g (87%) as
23
°
C
(
X
white crystals, mp ~110
°
C (dec), αD = +17.8 (c 1.0,
Dihydroquercetin (XI) 95%+ was purchased from
VedaBio (Russia). The Mannich reaction of (XI) with
amines and formaldehyde was the route to 6ꢀ(morꢀ
pholinomethyl)ꢀdihydroquercetin (XII), mp 163–
MeOH)
.
1
Signals in the NMR H and 13C spectra were
assigned by correlation with those of betulonic acid
(in CDCl3) [33].
165
thyl)ꢀdihydroquercetin
(lit. mp 200–202 ) [18].
°
C
(lit. mp 160–162
°
C
(
) and 6ꢀ(diethylaminomeꢀ
XIII), mp 199–203
°
C
1
NMR H: 9.22 (br.s, COOH), 4.69 (br.s, H29a),
°
C
4.60 (AB, ΔνAB = 11.7 Hz, JAB 15.8 Hz, OCH2COOH
(2 H)), 4.56 (br.s., H29b), 2.95 (ddd, J 10.5, 10.5,
4.5 Hz, H19), 1.64 (s, 3 H30), 1.03 (s, 3 H23§), 0.98
(s, 3 H24§), 0.94 (s, 3 H27§), 0.91 (s, 3 H25§), 0.87
(s, 3 H26§).
Syntheses
(31,28ꢀDihydroxyursꢀ12ꢀen)ꢀbis(hydrogen succiꢀ
nate) (V). A mixture of uvaol (IV) (0.44 g, 1 mmol) and
succinic anhydride (1 g, 10 mmol) was refluxed in a
mixture of pyridine (1 mL) and toluene (10 mL) for
10 h. The cooled reaction mixture was washed with
water (50 mL), 10% H2SO4 (20 mL) and evaporated.
NMR 13C: 218.89 (s, C3), 175.23 (s, C28§), 173.23
(s, COOH§), 150.13 (s, C20), 109.60 (t, C29), 59.74
(t, OCH2COOH), 56.35 (s, C17), 54.76 (d, C5), 49.74
(d, C9), 49.16 (d, C18), 47.20 (s, C4), 46.59 (d, C19),
42.33 (s, C14), 40.49 (s, C8), 39.45 (t, C1), 38.10 (d,
C13), 36.73 (t, C22), 36.73 (s, C10), 33.95 (t, C2),
33.47 (t, C7), 31.69 (t, C16), 30.24 (t, C21), 29.35
(t, C15), 26.49 (q, C23), 25.38 (t, C12), 21.25
(t, C11), 20.88 (q, C24), 19.50 (t, C6), 19.20 (q, C30),
15.82 (q, C26#), 15.51 (q, C25#), 14.44 (q, C27).
The residue was washed with water and filtered to give
the title compound (85%) as white crystals, m.p.
23
~95
°
C
(dec) (EtOAc) αD = +32.6 (
c
1.0, MeOH)
.
1
NMR H: 10.05 (br.s, 2 CH2CH2COOH (2 H)),
5.10 (dd,
H3), 4.06 (d,
H28b), 2.62 (m, 2CH2CH2COOH (8 H)), 1.05 (s, 3
J
3.0, 3.0 Hz, H12), 4.50 (dd,
J
8.0, 8.0 Hz,
J
10.9 Hz, H28a), 3.62 (d,
J
10.9 Hz,
,#
The assignments marked with the same symbols §
,
could be interchanged.
H27), 0.94 (s, 3 H25§), 0.93 (s, 3 H26§), 0.90 (d,
J 5.7
Hz, 3 H30#), 0.82 (s, 3 H23§), 0.82 (s, 3 H24§), 0.78
(m, 3H 29#).
Methods (MMPꢀ1). Cell culture for MMPꢀ1. Priꢀ
mary cultures of dermal fibroblasts were established
from human adult foreskins of healthy volunteers in
(DMEM) supplemented with 1% Penicillin/Streptoꢀ
NMR 13C: 178.00 and 178.00 (s, 2 COOH), 171.97
(s, COO(C3)§), 171.73 (s, COO(C28)§), 137.97 (s,
C13), 125.38 (d, C12), 81.34 (d, C3), 71.45 (t, C28),
55.10 (d, C5), 54.11 (d, C18), 47.40 (d, C9), 41.78
(s, C14), 39.80 (s, C8), 39.16 (d, C19#), 39.04
(d, C20#), 38.28 (t, C1), 37.58 (s, C4), 36.79 (s, C10‡),
36.58 (s, C17‡), 35.52 (t, C22), 32.49 (t, C7), 30.32
(t, C21), 29.23 and 29.00 (t, 2CH2CH2COOH¶),
mycin, and 10% fetal bovine serum (Lonza) at 37 C in
°
a humidified atmosphere of 5% CO2. Cells were culꢀ
tured until they were 90% confluent before being pasꢀ
saged. Dermal fibroblasts from passages 4 to 10 were
used for the experiments.
UV irradiation and MMPꢀ1 assay. For each experꢀ
iment, cells were maintained in the culture medium
without FBS for 24 h. Serumꢀstarved cells were
washed twice with PBS and exposed to UVB irradiaꢀ
tion (UVB lamp: G15TBE, Sankyo Denki, Japan).
The total energy dose of UVB irradiation was
40 mJ/cm2. After UVB exposure, serumꢀfree DMEM
containing each RA and the tested reagents at the
indicated concentration was added to the cells. The
culture medium was harvested 24 h later. MMPꢀ1
sandwich ELISA assays were performed according to
the manufacturer’s protocols (RPN 2610, Amersham
Bioscience) using precoated 96ꢀwell immunoplates,
rabbit antiꢀhuman MMPꢀ1 antibodies, and antiꢀrabꢀ
bit horseradish peroxidase conjugate. 3,3',5,5'ꢀTetꢀ
ramethylbenzidine(TMB) was used as a peroxidase
29.00 and 28.93 (t, 2CH2CH2COOH¶), 27.84
(q, C23), 25.82 (t, C15), 23.33 (t, C2†), 23.25
(t, C16†), 23.18 (t, Cꢀ11†), 23.18 (q, C27), 21.12
(q, C30), 18.01 (t, C6), 17.15 (q, C29 ), 16.60
(q, C26 ), 16.50 (q, Cꢀ24 ), 15.55 (q, C25).
Signals in the NMR H and 13C spectra were
assigned by correlation with those of uvaolꢀ
feate (in CDCl3) [32]. The assignments marked with
1
3
β
ꢀyl cafꢀ
the same symbols §
3ꢀOxoꢀlupꢀ20ꢀenꢀ28ꢀoic acid carboxymethyl ester
(X). To a suspension of betulonic acid ( ) (1.0 g,
2.2 mmol) in DMF (10 mL), anhydrous powdered
K2CO3 (0.45 g, 3.3 mmol) and ethylꢀ2ꢀchloroacetate
could be interchanged.
, #,‡,¶,†,
I
(0.27 g, 2.2 mmol) were added. The mixture was stirred substrate. Absorbances were read at 450 nm using a
at ambient temperature for 4 h (TLC monitoring). The microtiter plate reader.
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CHAN WOO LEE et al.
11. Cha, H.J., Park, M.T., and Chung, H.Y., Oncogene
1998, vol. 6, pp. 771–778.
,
Methods (ILꢀ8)
(1) Cell cultures. Immortalized human Keratiꢀ
nocytes (HaCaT cells) were maintained at 37 C in a
humidified 5% CO2 atmosphere in DMEM medium
that contained 10% FBS, and Penicillin/Streptomyꢀ
cin. The cells were plated in the previous day to treatꢀ
ment in the following day.
12. EsꢀSaady, D., Simon, A., JayatꢀVignoles, C., et al.,
°
Anticancer Res., 1996, vol. 16, pp. 481–486.
13. Han, S.K., Yi, K.O., and Park, S.J., Lipids, 1997,
vol. 32, pp. 769–773.
14. Lin, W.ꢀY., Sadhasivam, S., and Lin, F.ꢀH., Proc. Bioꢀ
chem., 2009, vol. 44, no. 6, pp. 678–684.
(2) Measurement of ILꢀ8 (interleukinꢀ8) secretion.
Overnight plated HaCaT cells in 24ꢀwell plates were
washed two times with PBS and exposed to UVB radiꢀ
ation (30 mJ/cm2). Then the medium was exchanged
for Phenol redꢀfree DMEM that contained 2% FBS,
and Penicillin/Streptomycin in the absence or presꢀ
ence of test substances for 24 h. To determine the levels
of ILꢀ8 secreted into cultured media, the supernatants
were collected and analyzed for the presence of ILꢀ8
using commercial available ELISA kits according to
the instructions from the manufacturer (R&D Sysꢀ
tems). At the same time, cell proliferation assay was
performed with WSTꢀ1 reagent (Roche) and used to
compensate for the substance toxicity.
15. Sun, H.H., Kaplita, P.V., Houck, D.R., Stawicki, M.B.,
McGarry, R., Wahl, R.C., Gillum, A., and Cooper, R.,
Phytother. Res., 1996, vol. 10, pp. 194–197.
16. Zhang, Y.Wu., Ren, Y., Fu, J., and Zhang, Y., Food
Chem. Toxicol., 2004, vol. 42, pp. 1867–1875.
17. Chaturvedula, V.S.P., Schilling, J.K., Johnson, R.K.,
and Kingston, D.G.I., J. Nat. Prod., 2003, vol. 66,
pp. 419–422.
18. Lee, C.ꢀW., Choi, H.ꢀJ., Kim, H.ꢀS., Kim, D.ꢀH.,
Chang, I.ꢀS., Moon, H.T., Lee, S.ꢀY., Oh, W.K., and
Woo, E.ꢀR., Bioorg. Med. Chem., 2008, vol. 16,
pp. 732–738.
19. Kuhareva, T.S., Krasnov, V.A., Koroteev, M.P.,
Kaziev, G.Z., Kuleshova, L.N., Korlyukov, A.A.,
Antipin, M.Yu., and Nifantiev, E.E., Russ. J. Org.
Chem., 2004, vol. 40, pp. 1237–1240.
(3) Statistical analysis. Experimental data were
analyzed by pairedꢀsamples
t test [34] using
20. Vlahopoulos, S., Boldogh, I., Casola, A., and Brasier, A.R.,
MINITAB® statistical software to compare the differꢀ
ence between each of the treatment groups and the
control group.
Blood, 1999, vol. 94, pp. 1878–1889.
21. Sang, S., Lapsley, K., Rosen, R.T., and Ho, C.ꢀT.,
J. Agric. Food Chem., 2002, vol. 50, pp. 607–609.
22. Xu, H.ꢀX., Sim, K.ꢀYe., Zeng, F.ꢀQ., and Wan, M.,
US Patent No. 5916919, Chem. Abstr., vol. 131, p. 80.
ACKNOWLEDGMENTS
23. Tkachev, A.V. and Denisov, A.Yu., Tetrahedron, 1994,
This work was supported by the research fund of
Hanyang University (HYꢀ2011ꢀI).
vol. 50, pp. 2591–2598.
24. Ryabinin, A.A., Zh. Obsch. Khim., 1946, vol. 16,
pp. 771–774.
25. Kashiwada, Y., Nagao, T., Hashimoto, A., Ikeshiro, Y.,
Okabe, H., Cosentino, L. M., and Lee, K.ꢀH., J. Nat.
Prod., 2000, vol. 63, pp. 1619–1622.
26. Lee, E.H., Popov, S.A., Lee, J.Y., Shpatov, A.V.,
Kukina, T.P., Kang, S. W., Pan, C.ꢀH., Um, B.H., and
Jung, S.H., Russ. J. Bioorg. Chem., 2012 (in press).
27. Collins, D.O., Ruddock, P.L.D., Chiverton de Grasse, J.,
Reynolds, W.F., and Reese, P.B., Phytochemistry, 2002,
vol. 59, pp. 479–488.
REFERENCES
1. Vincenti, M.P., White, L.A., Schroen, D.J., Benbow, U.,
and Brinckerhoff, C.E., Crit. Rev. Eukaryot. Gene Expr.
,
1996, vol.6, pp. 391–411.
2. Fisher, G.J., Choi H.C., BataꢀCsorgo, Z., Shao, Y.,
Datta, S., Wang, Z.Q., Kang, S., and Voorhees, J.J.,
J. Invest. Dermatol., 2001, vol. 117, pp. 219–226.
3. Chambers, A.F. and Matrisian, L.M., J. Natl. Cancer
Inst., 1997, vol. 89, 1260–7120.
4. Zhou, L.Z., Johnson, A.P., and Rando, T.A., Free Rad.
Biol. Med., 2001, vol. 31, pp.1405–1416.
5. Onoue, S., Kobayashi, T., Takemoto, Y., Sasaki, I., and
Shinkai, H., J. Dermatol. Sci., 2003, vol. 33, pp. 105–
111.
28. Hayek, E.W.H., Jordis, U., Moche, W., and Sauter, F.,
Phytochemistry, 1989, vol. 28, pp. 2229–2239.
29. Krasutsky, P.A., Carlson, R.M., and Karim, R.,
US Patent No. 6642217, 2003.
30. Sun, I.C., Wang, H.ꢀK., Kashiwada, Y., Shen, J.ꢀK.,
Cosentino, L. M., Chen, C.ꢀH., Yang, L.ꢀM., and
Lee, K.ꢀH., J. Med. Chem., 1998, vol. 41, pp. 4648–
4657.
31. Petrenko, N.I., Elantseva, N.V., Petukhova, V.Z., Shaꢀ
kirov, M.M., Shults, E.E., and Tolstikov, G.A., Chem.
Nat. Comp., 2002, vol. 38, pp. 331–339.
32. Nakagawa, H., Takaishi, Yo., Fujimoto, Yo., Duque, C.,
Garzon, C., Sato, M., Okamoto, M., Oshikawa, T., and
Ahmed, Sh.U., J. Nat. Prod., 2004, vol. 67, pp. 1919–
1924.
6. Varani, J., Warner, R.L., GharaeeꢀKermani, M.,
Phan, S.H., Kang, S., Chung, J.H., Wang, Z.Q.,
Datta, S.C., Fisher, G.J., and Voorhees, J.J., J. Invest.
Dermatol., 2000, vol. 114, pp. 480–486.
7. Kligman, A.M., Grove, G.L., Hirose, R., and Leyden, J.J.,
J. Am. Acad. Dermatol., 1986, vol. 15, pp. 836–859.
8. Lateef, H., Stevens, M.J., and Varani, J., Am. J. Pathol.
2004, vol. 165, pp. 167–174.
,
9. Moon, H.ꢀI., Chung, J.H., Lee, J.ꢀK., and Zee, O.ꢀP.,
Arch. Pharm. Res., 2004, vol. 27, pp. 730–733.
33. Barthel, A., Stark, S., and Csuk, R., Tetrahedron, 2008,
vol. 64, pp. 9225–9229.
10. Mix, K.S., Mengshol, J.A., Benbow, U., Vincenti, M.P.,
Sporn, M.B., and Brinckerhoff, C.E., Arthritis Rheum.
,
34. Zimmerman, D.W., J. Educat. Behav. Stat., 1997,
2001, vol. 44, pp. 1096–1104.
vol. 22, pp. 349–360.
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No. 3
2012