New bisabolane sesquiterpenes from the rhizomes of Curcuma xanthorrhiza Roxb. and their inhibitory effects on UVB-induced MMP-1 expression in human keratinocytes

Article abstract of DOI:10.1002/hlca.201300372

Two new bisabolane sesquiterpenoids, 1 and 2, along with five known ones, 13-hydroxyxanthorrhizol (3), 12,13-epoxyxanthorrhizol (4), xanthorrhizol (5), β-curcumene (6), and β-bisabolol (7), were isolated from the rhizomes of Curcuma xanthorrhiza Roxb. The

Full text of DOI:10.1002/hlca.201300372

4
38
Helvetica Chimica Acta – Vol. 97 (2014)
New Bisabolane Sesquiterpenes from the Rhizomes of Curcuma xanthorrhiza
Roxb. and Their Inhibitory Effects on UVB-Induced MMP-1 Expression in
Human Keratinocytes
a
a
a
a
by Ji-Hae Park ), Ye-Jin Jung ), Mohamed Antar Aziz Mohamed ), Tae Hoon Lee ),
b
b
c
a
a
Chang-Ho Lee ), Daeseok Han ), Myung-Chong Song ), Jiyoung Kim ), and Nam-In Baek* )
a) Graduate School of Biotechnology and Research Institute of Life Sciences & Resources, Kyung Hee
University, Yongin 446-701, Korea
(
phone: þ 82-31-201-2610; fax: þ 82-31-204-8116; e-mail: nibaek@khu.ac.kr)
b) Division of Matabolism and Functionality Research, Korea Food Research Institute, Sungnam 463-
7
46, Korea
c) Intelligent Synthetic Biology Center, KAIST, Daejeon 305-701, Korea
Two new bisabolane sesquiterpenoids, 1 and 2, along with five known ones, 13-hydroxyxanthorrhizol
3), 12,13-epoxyxanthorrhizol (4), xanthorrhizol (5), b-curcumene (6), and b-bisabolol (7), were
(
isolated from the rhizomes of Curcuma xanthorrhiza Roxb. The chemical structures of the new
compounds were determined to be (7R,10R)-10,11-dihydro-10,11-dihydroxyxanthorrhizol 3-O-b-d-
glucopyranoside (1) and (ꢀ)-curcuhydroquinone 2,5-di-O-b-d-glucopyranoside (2) on the basis of 1D-
and 2D-NMR spectroscopic analyses and optical-rotation characteristics. Compounds 2 and 3 decreased
MMP-1 expression in UVB-treated human keratinocytes by ca. 8.9- and 7.6-fold at the mRNA level, and
by ca. 9.2- and 6.6-fold at the protein level, respectively. The results indicate that the isolated compounds
may have anti-aging effects through inhibition of MMP-1 expression in skin cells.
Introduction. – The rhizomes of Curcuma xanthorrhiza (turmeric spices, temu
lawaq) of the Zingeberaceae have been used for medicinal purposes mainly in
Southeast Asia. In Indonesia, the rhizome is widely used as a tonic and cholagogue [1],
and in Europe it is also employed in choleretic drug preparations [2]. Curcuminoids
(diarylheptanoids) and essential oils are major bioactive ingredients exhibiting
stimulant, aspirant, carminative, cordeal, emenagogue, astringent, detergent, anti-
cancer, antinociceptive, diuretic, and martirnet bioactivities [3 – 6]. Xanthorrhizol is a
phenolic bisabolane sesquiterpene obtained as the main constituent of rhizome oil from
C. xanthorrhiza [7]. Previously, more than 50 bisabolane sesquiterpenes have been
identified from the turmeric rhizomes of the genus Curcuma [8 – 20]. The current study
was initiated to search for other active compounds from the rhizomes of C.
xanthorrhiza and led to the isolation of seven bisabolane sesquiterpenoids, among
which two were new compounds and two were compounds isolated for the first time
from this source. 13-Hydroxyxanthorrhizol (3) and 12,13-epoxyxanthorrhizol (4) were
previously isolated from the rhizomes of C. longa, and xanthorrhizol (5), b-curcumene
(6), and b-bisabolol (7) were previously isolated from the rhizomes of C. xanthorrhiza.
The two new compounds were identified as (7R,10R)-10,11-dihydro-10,11-dihydroxy-
xanthorrhizol 3-O-b-d-glucopyranoside (1) and (ꢀ)-curcuhydroquinone 2,5-di-O-b-d-
glucopyranoside (2; Fig. 1).
ꢀ
2014 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 97 (2014)
439
Fig. 1. Compounds 1 and 2, isolated from Curcuma xanthorrhiza Roxb., and compounds 3 – 7
Recently, a variety of herbs and plants have been widely used in applications for the
treatment of collagen synthesis and collagenase inhibition [21]. UV Irradiation causes
various adverse effects on human skin such as skin cancer and premature skin aging
(
photoaging) [22]. Type 1 collagen accounts for 70 – 90% of all collagen, which is
synthesized by fibroblasts as a soluble precursor, type 1 procollagen, which is secreted
and processed to form insoluble collagen fibers in the dermis. The production of
collagen in the skin is reduced by UV irradiation, whereas gene expression of matrix
metalloproteinases (MMPs) is increased. MMP-1 is the protein most responsible for
the degradation of type 1 collagen in the skin [23]. It has been reported that natural
compounds from plants are potential sources of MMP inhibitors, which may protect
from UV-induced skin aging [24]. Herein, we describe the isolation of seven bisabolane
sesquiterpenoids, 1 – 7, and the structure determination of the two new ones, 1 and 2.
We also investigated the anti-aging protective effects of these compounds against UV
irradiation.
Results and Discussion. – Compound 1 was isolated as a yellow amorphous powder
ꢀ
1
ꢀ1
and showed IR absorbance bands of OH (3374 cm ) and aromatic (1578 cm ) groups.
The molecular weight was determined to be 414 from the molecular-ion peak at m/z
þ
4
15 ([M þ H] ) in the FAB mass spectrum (positive-ion mode), and a molecular
formula of C H O was deduced from the molecular-ion peak (m/z 415.2307 ([M þ
2
1
34
8
þ
1
H] ; C H O ; calc. 415.2333) in the HR-FAB-MS. The H-NMR spectrum (Table)
21
35
8
2
exhibited three HꢀC (sp ) signals of a 1,2,4-trisubstituted benzene ring at d(H) 7.01 (d,
J ¼ 8.0, HꢀC(5)), 6.98 (d, J ¼ 1.2, HꢀC(2)), and 6.75 (dd, J ¼ 8.0, 1.2, HꢀC(6)). An O-
bearing CH signal at d(H) 3.18 (dd, J ¼ 10.8, 2.0, HꢀC(10)) was also observed. In the
high magnetic field region, a CH signal at d(H) 2.67 – 2.61 (m, HꢀC(7)), two CH
2
signals at d(H) 1.84 – 1.81, 1.60 – 1.58 (CH (8)) and 1.48 – 1.45, 1.28 – 1.25 (CH (9)), an
2
2
allyl-Me signal at d(H) 2.22 (s, Me(14)), a Me signal at d(H) 1.22 (d, J ¼ 6.8, Me(15)),
and two Me signals at d(H) 1.09 (s, Me(12)) and 1.05 (s, Me(13)) were observed. In

4
40
Helvetica Chimica Acta – Vol. 97 (2014)
Table. 1H- and 13C-NMR Data (400 and 100 MHz, resp.; CD OD) of Compounds 1 and 2. d in ppm,
3
J in Hz. Atom numbering as indicated in Fig. 1.
Position
1
2
d(H)
d(C) d(H)
d(C)
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
1
2
3
4
5
6
1
2
3
4
5
6
148.0
115.0
157.2 6.97 (s)
126.2
131.4
121.9 6.99 (s)
41.3 3.50 – 3.34 (m)
36.9 1.63 – 1.59 (m), 1.49 – 1.43 (m)
30.6 1.94 – 1.87 (m), 1.84 – 1.78 (m)
79.8 5.12 (t-like, J ¼ 6.4)
73.8
136.9
151.7
120.0
127.3
152.9
115.6
32.4
38.6
27.3
126.1
131.7
17.8
25.9
16.3
22.1
103.9
75.0
78.2
71.4
78.0
62.5
6.98 (d, J ¼ 1.2)
7.01 (d, J ¼ 8.0)
6.75 (dd, J ¼ 8.0, 1.2)
2.67 – 2.61 (m)
1.84 – 1.81 (m), 1.60 – 1.58 (m)
1.48 – 1.45 (m), 1.28 – 1.25 (m)
3.18 (dd, J ¼ 10.8, 2.0)
1
1
1
1
1
1
1.09 (s)
1.05 (s)
2.22 (s)
25.6 1.51 (s)
24.9 1.63 (s)
16.1 2.23 (s)
1.22 (d, J ¼ 6.8)
22.8 1.15 (d, J ¼ 6.8)
’
’
’
’
’
’
’’
’’
’’
’’
’’
’’
4.85 (d, J ¼ 7.6)
102.8 4.72 (d, J ¼ 7.6)
a
a
3.47 )
75.0 3.46 )
a
a
3.40 )
78.3 3.42 )
71.5 3.32 )
78.1 3.36 )
62.6 3.87 ), 3.72 )
a
a
3.34 )
a
a
3.38 )
a
a
3.87 (dd, J ¼ 12.0, 2.0), 3.69 (d, J ¼ 12.0, 6.4)
4.73 (d, J ¼ 7.2)
104.1
75.1
78.8
71.4
78.1
a
3.45 )
a
3.41 )
a
3.33 )
a
3.35 )
a
3.86 )
62.6
a
3
.72 )
a) Overlapped signals, reported without multiplicity.
addition, signals of a hemiacetal H-atom (d(H) 4.85 (d, J ¼ 7.6, HꢀC(1’))), O-bearing
CH H-atoms (d(H) 3.87 (dd, J ¼ 12.0, 2.0, H ꢀC(6’)) and 3.69 (dd, J ¼ 12.0, 6.4,
2
a
H ꢀC(6’)), and four O-bearing CH H-atoms (d(H) 3.48 – 3.46 (m, HꢀC(2’)), 3.40 – 3.36
b
(
overlapped, HꢀC(3’) and HꢀC(5’)), and 3.31 – 3.30 (m, HꢀC(4’)) were detected due to
a hexose moiety.
13
The C-NMR spectrum (Table) showed signals corresponding to 21 C-atoms: an O-
bearing olefin quaternary, two olefin quaternary, three olefinic CH C-atoms, an O-
bearing quaternary and an O-bearing CH C-atom, a CH, two CH , and four Me group,
2
and hexose signals, indicating that compound 1 was a sesquiterpene monoglycoside.
The sugar was identified as a b-glucopyranose from the chemical shifts observed,
including a hemiacetal signal at d(C) 102.8 (C(1’)), four O-bearing CH C-atom signals
at d(C) 78.3 (C(3’)), 78.1 (C(5’)), 75.0 (C(2’)), and 71.5 (C(4’)), and of an O-bearing
CH C-atom at d(C) 62.6 (C(6’)). The olefinic region displayed six aromatic signals,
2

Helvetica Chimica Acta – Vol. 97 (2014)
441
including an O-bearing olefin quaternary signal (d(C) 157.2 (C(3)), two olefin
quaternary signals (d(C) 148.0 (C(1)) and 126.2 (C(4))), and three olefin CH signals
(d(C) 131.4 (C(5)), 121.9 (C(6)), and 115.0 (C(2))). Furthermore, the oxygenated
region showed an O-bearing quaternary signal (d(C) 73.8 (C(11))) and an O-bearing
CH signal (d(C) 79.8 (C(10))). The Me signals at d(C) 25.6, 24.9, 22.8, and 16.1 were
assigned to C(12), C(13), C(15), and C(14), respectively. The unsaturation degree was
five as deduced from the molecular formula C H O , indicating that aglycone was
21
34
8
composed of one benzene ring moiety with an aliphatic side chain. Consequently,
compound 1 was identified as a bisabolane sesquiterpene glucopyranoside. The relative
configuration of the anomeric C-atom was confirmed to be b from the coupling
1
constant of the hemiacetal H-atom signal (J ¼ 7.6 Hz) in the H-NMR spectrum. To
determine the locations of the glucose moiety, aliphatic chain, and OH and Me groups
in compound 1, 2D-NMR experiments such as gHSQC and gHMBC were conducted.
In the gHMBC spectrum, two olefinic CH H-atom signals at d(H) 6.98 (HꢀC(2)) and
6
.75 (HꢀC(6)) showed cross-peaks with CH C-atom signals at d(C) 41.3 (C(7)) through
3
J correlation. Also, the anomeric H-atom signal at d(H) 4.85 (HꢀC(1’)) showed a
3
correlation with the O-bearing olefin quaternary signal at d(C) 157.2 (C(3)) through J
correlation. Based on these results, the aliphatic chain and the b-d-glucose moiety were
determined to be located at C(1) and C(3), respectively. The O-bearing CH signal at
d(H) 3.18 (HꢀC(10)) of the aliphatic chain showed correlations with the O-bearing
quaternary signal at d(C) 73.8 (C(11)) and CH signal at d(C) 36.9 (C(9)), and the O-
2
bearing quaternary signal at d(C) 73.8 (C(11)) showed cross-peaks with the Me signals
at d(H) 1.09 (Me(12)) and 1.05 (Me(13)). From these results, two OH groups were
determined to be located at the terminal end of the aliphatic chain. As a result, the new
compound 1 was determined as a 10,11-dihydro-10,11-dihydroxyxanthorrhizol 3-O-b-
d-glucopyranoside. The configurations of both of C(7) and C(10) were determined as
(
R) by the comparison of the specific rotation value of the aglycone 1a with that
20
reported in the literature [7]. The specific rotation value ([a] ) of the aglycone of
D
compound 1, which was obtained through acid hydrolysis of compound 1, was as ꢀ 34.
Hence, compound 1 was (7R,10R)-10,11-dihydro-10,11-dihydroxyxanthorrhizol 3-O-b-
d-glucopyranoside, a new compound.
Compound 2 was isolated as a brown amorphous powder and showed IR
ꢀ
1
ꢀ1
absorbance bands of OH (3374 cm ) and aromatic (1595 cm ) groups. The molecular
þ
weight was determined as 558 from the molecular-ion peak at m/z 559 ([M þ H] ) in
the FAB-MS (positive-ion mode), and a molecular formula of C H O was deduced
27
42 12
þ
from the molecular-ion peak (m/z 559.2730 ([M þ H] , C H O ; calc. 559.2755) in
27
43 12
1
the HR-FAB-MS. In the H-NMR (400 MHz, CD OD) spectrum of compound 2, most
3
signals were similar to those of compound 1, with the exception of the signals of an
additional sugar moiety, an olefinic H-atom (d(H) 5.12 (t-like, J ¼ 6.4)), and two
aromatic H-atom singlets (d(H) 6.99 and 6.97) due to a 1,2,4,6-tetrasubstituted benzene
13
ring. The C-NMR (100 MHz, CD OD) spectrum of 2 exhibited signals corresponding
3
to 27 C-atoms, including those of two hexoses and a benzene moiety, two O-bearing
olefinic quaternary C-atom signals (d(C) 152.9 (C(5)) and 151.7 (C(2))), two olefinic
quaternary C-atom signals (d(C) 136.9 (C(1)) and 127.3 (C(4))), two olefinic CH
signals (d(C) 120.0 (C(3)) and 115.6 (C(6))), and two olefinic signals (d(C) 131.7
(C(11)) and 126.1 (C(10))). The two sugar moieties were identified as b-glucopyranoses

4
42
Helvetica Chimica Acta – Vol. 97 (2014)
from their chemical shifts. The 2D-NMR experiment confirmed the location of two
glucopyranoses and other key functional groups in 2. In the gHMBC spectrum, the
correlations of the olefin CH signal at d(H) 6.99 (HꢀC(6)) showed a cross-peak with
the C-signal at d(C) 32.4 (C(7)). The signals of two anomeric H-atoms at d(H) 4.73
(
HꢀC(1’’)) and 4.72 (HꢀC(1’)) showed correlations with the O-bearing olefinic
quaternary C-atom signals at d(C) 151.7 (C(2)) and 152.9 (C(5)). Based on these
results, two b-d-glucose moieties were determined to be located at C(2) and C(5). As a
result, compound 2 was determined to be a curcuhydroquinone 2,5-di-O-b-d-
glucopyranoside. The configuration of C(7) was determined as (R) by the comparison
of the specific rotation value of the aglycone with that in the literature [25]. The
aglycone of compound 2 was determined to be (7R)-configured from the specific
20
rotation value ([a] ¼ ꢀ28) of the aglycone obtained through acid hydrolysis of
D
compound 2. As a result, compound 2 was determined to be a new compound (ꢀ)-
curcuhydroquinone 2,5-di-O-b-d-glucopyranoside.
The known compounds were identified as 13-hydroxyxanthorrhizol (3) [26], 12,13-
epoxyxanthorrhizol (4) [7], xanthorrhizol (5) [27], b-curcumene (6) [28], and b-
bisabolol (7) [29] by comparing their spectroscopic data with those reported in the
literature.
The compounds isolated from C. xanthorrhiza Roxb. were evaluated for their
effects on viability of HaCaT keratinocyte cells. None of the phytochemicals exhibited
inhibition of cell viability up to a 20 mm concentration (Fig. 2,a), indicating that they
are not toxic to cells in the range of concentrations used in this study. Each compound
was also evaluated for its effects on the expression of MMP-1 in UVB-treated HaCaT
Fig. 2. Effects of the isolated compounds on cell viability and MMP-1 mRNA expression levels: a) cells
grown in the culture medium in the presence of various concentrations of isolated compounds for 48 h. Cell
viability was measured by MTT assays. All data are given as means ꢁ SD of at least three independent
experiments with triplicate samples. b) Levels of MMP-1 mRNA determined by RT-PCR analysis. b-
Actin mRNA was used as an internal control.

Helvetica Chimica Acta – Vol. 97 (2014)
443
cells. The keratinocyte cells were treated with each of the compounds at 5 mm
2
concentrations for 24 h, exposed to UVB for 15 s (20 mJ/cm ) and incubated for
24 h. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that
the new compound, (ꢀ)-curcuhydroquinone 2,5-di-O-b-d-glucopyranoside (2) and 13-
hydroxyxanthorrhizol (3) strongly suppressed MMP-1 mRNA expression in UVB-
treated HaCaT cells. The inhibitory effects of the two compounds on MMP-1
expression were further determined at the protein level, as well as at mRNA levels in
UVB-irradiated cells in the presence of various concentrations of the compounds. RT-
PCR analysis revealed that treatment of cells with 2 or 3 resulted in inhibition of the
elevated MMP-1 mRNA expression in UVB-stimulated HaCaT cells in a concen-
tration-dependent manner, without affecting the mRNA levels of b-actin (Fig. 3,a and
b). Compounds 2 and 3 reduced the MMP-1 mRNA expression in keratinocytes by ca.
8.9- and 7.6-fold, respectively. The protein levels of MMP-1 were analyzed by Western
blot analysis, which evidenced that the protein levels of MMP-1 were decreased in
HaCaT cells treated with 2 or 3 by ca. 9.2- and 6.6-fold, respectively (Fig. 3,c and d).
Taken together, our results demonstrated that 2 and 3 from C. xanthorrhiza Roxb.
inhibit production of MMP-1 in UV-irradiated HaCaT cells. These findings suggest that
C. xanthorrhiza Roxb. could be utilized as an effective agent to prevent the UVB-
mediated cutaneous alterations and photoaging.
Fig. 3. Effect of (ꢀ)-curcuhydroquinone 2,5-di-O-b-d-glucopyranoside (2) and 13-hydroxyxanthorrhizol
(
3) on expression of matrix metalloproteinase-1 (MMP-1). HaCaT Cells were pretreated with various
2
concentrations of 2 or 3 for 24 h and exposed to UVB (20 mJ/cm ). The UV-treated cells were then
cultured in serum-free medium for 24 h. a) and b) Levels of MMP-1 mRNA determined by RT-PCR
analysis; quantification by densitometry. b-Actin mRNA was used as an internal control. c) Levels of
MMP-1 protein measured by Western blot analysis using a monoclonal antibody against human MMP-1.
The blot underwent rehybridization with antibody against b-actin to verify equal loading of proteins in
each lane. d) Western blot data quantified by densitometry. Significance compared with the control,
*
: P < 0.05. Significance compared with UVB treatment alone, **: P < 0.05. The data are expressed as the
means ꢁ SD of three individual experiments.

4
44
Helvetica Chimica Acta – Vol. 97 (2014)
Experimental Part
General. FAB-MS: JEOL JMSAX-700 (Tokyo, Japan). TLC: Kieselgel 60 F254 and RP-18 F254S
(
Merck) plates; visualization with UV lamp Spectroline model ENF-240 C/F (Spectronics Corporation,
New York, NY, USA) and a 10% H SO soln. SiO (Kieselgel 60, Merck, DE-Darmstadt) and ODS
2
4
2
(
LiChroprep RP-18, Merck) resins. Column chromatography (CC): Optical rotations JASCO P-1010
digital polarimeter (Jasco, Tokyo, Japan). IR Spectra: PerkinElmer Spectrum One FT-IR spectrometer
1
13
(
4
GB-Buckinghamshire). H- and C-NMR (400 and 100 MHz, resp.) spectra: Varian Unity Inova AS-
00 FT-NMR spectrometer (Palo Alto, CA, USA). Antibody against human MMP-1 was obtained from
R&D Systems (Minneapolis, MN, USA). HRP-Conjugated anti-mouse IgG antibody was obtained from
Sigma (St. Louis, MO, USA). Other chemicals were obtained commercially from SigmaꢀAldrich (St.
Louis, MO, USA).
Plant Material. C. xanthorrhiza Roxb. was supplied from the Korea Food Research Institute,
Sungnam, Korea, in January 2012, and was identified by Dae-Keun Kim, College of Pharmacy, Woosuk
University, Jeonju, Korea. A voucher specimen (KHU12-0111) has been deposited with the Laboratory
of Natural Products Chemistry, Kyung Hee University, Yongin, Korea.
Extraction and Isolation. The dried and powdered rhizomes of C. xanthorrhiza were extracted at r.t.
using 80% aq. MeOH (3 ꢂ 36 l) for 24 h, followed by filtration. The filtrates were evaporated in vacuum,
and the obtained MeOH extracts were suspended in H O (3 l) and then extracted successively with
2
AcOEt (2 ꢂ 3 l) and BuOH (2 ꢂ 22.8 l). The org. and aq. layers were concentrated to give the residues of
the AcOEt fraction (CXE; 901 g), the BuOH fraction (CXB; 40 g), and the H O fraction (CXW;
2
1
445 g). The CXE (178 g) was subjected to CC (SiO CC (f12 ꢂ 15 cm); hexane/AcOEt 1:1; 27 l). The
2
eluting solns. were monitored using TLC analysis and ultimately produced 16 fractions, CXE-1 to CXE-
1
1
0
6. Fr. CXE-2 (68 g, elution volume/total volume (V /V ) 0.05 – 0.08) was subjected to CC (SiO (f 8.5 ꢂ
e
t
2
5 cm); hexane/AcOEt 20 :1; 17.5 l) to yield 15 fractions, CXE-2-1 – CXE-2-15. Fr. CXE-2-2 (3.3 g; V /V
e
t
.03 – 0.07) was subjected to CC (ODS (f 4 ꢂ 11 cm); MeOH/H O 5 :1, 5.4 l) to ultimately furnish 19
2
fractions along with a purified compound 6 (CXE-2-2-14; 133 mg; V /V 0.61 – 0.74; TLC (ODS F ;
254S
e
t
acetone/H O 6 :1): R 0.40). Fr. CXE-2-5 (3.2 g, V /V 0.17 – 0.23) was subjected to CC (ODS CC (f 6 ꢂ
2
f
e
t
1
0 cm); MeOH/H O 2 :1; 6.7 l) to yield 13 fractions, CXE-2-5-1 – CXE-2-5-13. Fr. CXE-2-5-12 (648 mg;
2
V /V 0.93 – 1.00) was purified by CC (ODS (f 4.5 ꢂ 7 cm); MeOH/H O 3 :1; 600 ml) to furnish nine
e
t
2
fractions along with a purified compound 5 (CXE-2-5-12-4; 206 mg, V /V 0.34 – 0.44; TLC (ODS F ;
254S
e
t
MeOH/H O 8 :1): R 0.49) and compound 7 (CXE-2-5-12-8; 26 mg; V /V 0.66 – 1.00; TLC (ODS F254S;
2
f
e
t
MeOH/H O 8 :1): R 0.41). Fr. CXE-2-15 (805 mg; V /V 0.98 – 1.00) was subjected to CC (ODS (f 4 ꢂ
2
f
e
t
1
0 cm); MeOH/H O 3 :2; 3 l) to furnish 19 fractions, CXE-2-15-1 – CXE-2-15-19. Fr. CXE-2-15-12
2
(
25 mg; V /V 0.42 – 0.45) was purified by CC (SiO (f 3 ꢂ 10 cm); hexane/AcOEt 2 :1; 215 ml) to
e
t
2
produce six fractions along with a purified compound 4 (CXE-2-15-12-2; 4.6 mg; V
e
t
/V 0.31 – 0.42; TLC
(
(
6
ODS F254S; MeOH/H O 6 :1): R 0.61). Fr. CXE-6 (1.6 g; V /V 0.19 – 0.23) was subjected to CC (ODS
2
f
e
t
f 4.5 ꢂ 8 cm); MeOH/H O (2 :1 ! 5 :1, 2 l of each) to yield 23 fractions, CXE-6-1 – CXE-6-23. Fr. CXE-
2
-7 (260 mg; V /V 0.04 – 0.06) was purified by CC (SiO (f 3 ꢂ 12 cm); CHCl /AcOEt 15 :1; 4.9 l) to
e
t
2
3
afford 18 fractions along with a purified compound 3 (CXE-6-7-15; 34 mg; V /V 0.36 – 0.42; TLC (SiO
2
e
t
F254; CHCl /MeOH 15 :1): R 0.58). The BuOH extract (CXB; 34 g) was submitted to CC (SiO (f 9 ꢂ
3
f
2
1
5 cm); CHCl /MeOH 12 :1 ! 10 :1 ! 8 :1; 8 l of each; and CHCl /MeOH/H O 8 :3 :1; 14 l). The eluting
3
3
2
solns. were monitored using TLC analysis and ultimately 22 fractions, CXB-1 – CXB-22 were obtained.
Fr. CXB-21 (1.2 g; V /V 0.68 – 0.71) was subjected to CC (ODS (4 ꢂ 11 cm); MeOH/H O 1:2; 3 l) to
e
t
2
yield nine fractions CXB-21-1 – CXB-21-9. Fr. CXB-21-8 (103 mg; V /V 0.80 – 1.00) was purified by CC
e
t
(
ODS (3 ꢂ 5 cm); MeOH/H O 1:1; 320 ml) to furnish nine fractions, CXB-21-8-1 – CXB-21-8-9, along
2
with the purified compound 1 (CXB-21-8-5; 58 mg; V /V 0.41 – 0.43; TLC (ODS F ; MeOH/H O 3 :2):
e
t
254S
2
R 0.50). Fr. CXB-22 (830 mg; V /V 0.72 – 0.74) was subjected to the CC (ODS (4 ꢂ 6 cm); MeOH/H O
f
e
t
2
1
9
:2; 1.8 l) to give 14 fractions, CXB-22-1 – CXB-22-14, and also yielded a purified compound 2 (CXB-22-
; 24 mg; V /V 0.14 – 0.33; TLC (ODS F ; MeOH/H O 1:1): R 0.40).
e
t
254S
2
f
(
7R,10R)-10,11-Dihydro-10,11-dihydroxyxanthorrhizol 3-O-b-d-Glucopyranoside (¼ 5-[(1R,4R)-
4
,5-Dihydroxy-1,5-dimethylhexyl]-2-methylphenyl b-d-Glucopyranoside; 1). Amorphous powder.

Helvetica Chimica Acta – Vol. 97 (2014)
445
2
0
1
[
a] ¼ ꢀ33.8 (c ¼1.0, MeOH). IR (CaF ): 3374, 2923, 1578, 1451, 1246. H- and 13C-NMR: see the Table.
D
2
þ
þ
8
HR-FAB-MS: 415.2307 ([M þ H] , C H O ; calc. 415.2333).
21
35
(
ꢀ)-Curcuhydroquinone 2,5-Di-O-b-d-glucopyranoside (¼2-[(1R)-1,5-Dimethylhex-4-en-1-yl]-4-
20
(
(
b-d-glucopyranosyloxy)-5-methylphenyl b-d-Glucopyranoside; 2). Amorphous powder. [a]
D
¼ ꢀ40.8
1
13
c ¼ 0.5, MeOH). IR (CaF ): 3374, 2919, 1595, 1500, 1448, 1190. H- and C-NMR: see the Table. HR-
2
þ
þ
43 12
FAB-MS: 559.2730 ([M þ H] , C H O ; calc. 559.2755).
27
Acid Hydrolysis of 1 and 2, and Isolation of the Aglycones. A soln. of 1 (6 mg) in 1m HCl (3 ml) was
heated at 908 in a heating block for 3 h. The mixture was added to 2 ml of MeOH and extracted with
hexane (5 ml). The hexane layer was concentrated and then purified by a Supelclean LC-Si SPE tube
(
hexane/AcOEt 3 :1) to furnish the aglycone (1a; 0.6 mg).
A soln. of compound 2 (8.6 mg) in 1m HCl (3 ml) was treated as described for 1 to yield the aglycone
2a; 0.8 mg).
(
(
7R,10R)-10,11-Dihydro-10,11-dihydroxyxanthorrhizol (¼(3R,6R)-6-(3-Hydroxy-4-methylphen-
2
0
1
1
yl)-2-methylheptane-2,3-diol; 1a). [a] ¼ ꢀ34 (c ¼ 0.04, CHCl ). H-NMR (400 MHz) ): 7.00 (d, J ¼
D
3
7
2
.6, HꢀC(5)); 6.65 (dd, J ¼ 7.6, 1.6, HꢀC(6)); 6.60 (d, J ¼ 1.6, HꢀC(2)); 3.26 (dd, J ¼ 10.8, 2.0, HꢀC(10));
.61 – 2.57 (m, HꢀC(7)); 2.19 (s, Me(14)); 1.79 – 1.83 (m, CH (8)); 1.32 – 1.36 (m, CH (9)); 1.20 (s,
2
2
Me(15)); 1.17 (s, Me(13)); 1.09 (s, Me(12)).
2
0
(
ꢀ)-Curcuhydroquinone (¼2-Methyl-5-[(2R)-6-methylhept-5-en-2-yl]benzene-1,4-diol; 2a). [a]
¼
D
1
1
ꢀ
28 (c ¼ 0.01, CHCl ). H-NMR (400 MHz) ): 6.52 (s, HꢀC(6)); 6.42 (s, HꢀC(3)); 5.07 (dd, J ¼ 7.2,
3
3
.6, HꢀC(10)); 2.94 – 2.97 (m, HꢀC(7)); 2.21 (s, Me(14)); 1.87 – 1.90 (m, CH (9)); 1.66 (s, Me(13)); 1.49 –
2
1
.52 (m, CH (8)); 1.49 (s, Me(12)); 1.12 (d, J ¼ 7.2, Me(15)).
2
Cell Culture. Human keratinocyte HaCaT cells were grown in Dulbeccoꢂs modified Eagleꢂs medium
(
DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 unit/ml penicillin and
1
00 mg/ml streptomycin) at 378 in a humidified incubator containing 5% CO and 95% air. DMEM, FBS,
2
antibiotics, and trypsin EDTA were obtained from Invitrogen (Carlsbad, CA, USA).
3
Cell Viability Assay. HaCaT Cells were seeded in 96-well plates at a density of 5 ꢂ 10 cells/well and
allowed to attach for 24 h. After discarding the growth medium, cells were treated with various
concentrations of phytochemicals isolated from C. xanthorrhiza Roxb. in serum-free medium for 48 h.
After incubation, cells were treated with 100 mg/ml of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-2H-
tetrazolium bromide (MTT) for 1 h. The formazan precipitate was dissolved in 200 ml of DMSO and the
absorbance at 560 nm was determined spectrophotometrically. Analyses were repeated three times, and
the results are expressed as the means of three independent experiments.
UV Irradiation. In brief, HaCaT cells were pretreated with or without phytochemicals in serum-free
media for 24 h and then rinsed twice with phosphate-buffered saline (PBS). The cells were then
2
irradiated with UVB (20 mJ/cm ) at 312 nm for 15 s using a UVB lamp (Bio-Sun Lamps, Vilber Lourmat,
F-Marine), which provided uniform irradiation at a distance of 15 cm, and radiation intensities were
monitored with a JIC 119 amplifier (BEC, Brookline, MA, USA.). All irradiations were performed
under a thin layer of PBS. After irradiation, cells were treated with or without phytochemicals in serum-
free media for an additional 24 h. Mock-irradiated controls followed the same schedule of medium
changes without UVB irradiation.
Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). Total RNAs were prepared from cells
using a TRIzol Reagent kit (Invitrogen, Carlsbad, CA, USA). Total RNA (2 mg) was reverse-transcribed
using M-MuLV reverse transcriptase (Fermentas Life Science, Glen Burnie, MD, USA). The primers
used for PCR amplification in this study were as follows: MMP-1, forward 5’- AGCGTGTGACAG-
TAAGCTAA-3’ and reverse 5’-GTTTTCCTCAGAAAGAGCAGCAT-3’; b-actin mRNA levels were
used as internal controls.
Western Blot Analysis. Cells were lysed in radio immunoprecipitation assay (RIPA) buffer (150 mm
NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mm Tris · HCl,
pH 7.5, 2 mm ethylenediaminetetraacetic acid (EDTA)-containing proteinase inhibitor cocktail (Roche,
Indianapolis, IN, USA)). Protein concentrations were quantified with a protein assay kit (Bio-Rad
Laboratories, Philadelphia, PA, USA). Proteins (30 mg/lane) were resolved with 8 – 12% SDS-
1)
Atom numbering as indicated in Fig. 1.

4
46
Helvetica Chimica Acta – Vol. 97 (2014)
polyacrylamide gel electrophoresis. Peroxidase-conjugated antibody was used as a secondary antibody.
The membranes were developed with an enhanced chemiluminescence system from GE Healthcare
(
UK-Buckinghamshire) and exposed to X-ray film (Fuji Photo Film Co. Ltd., Japan).
Statistical Analysis. All experiments were performed with triplicate samples and repeated at least
three times. The data are presented as means ꢁ SD, and statistical comparisons between groups were
performed using one-way ANOVA, followed by Studentꢂs t-test.
This study was supported by the Mental Health Food Development Program from the Korea Food
Research Institute (20130352U0054101S000100) and by grants from the National Coordinating Center for
Global Cosmetics R&D (A103017) of the Foundation of Korea Cosmetic Industry Institute.
REFERENCES
[
[
[
1] H. H. Hirschhorn, J. Ethnopharmacol. 1983, 7, 123.
2] M. Wichtl, F. C. Czygan, ꢃTeedrogenꢂ, Wissenschaftliche Verlagsgesellschaft, Stuttgart, 1989.
3] R. Remadevi, E. Surendran, T. Kimura, ꢃTurmeric in Traditional medicineꢂ, Ed. P. N. Ravindran,
K. K. Nirmal Babu, K. Sivaraman, CRC Publishers, New York, 2007, p. 409.
4] B. Sasikumar, Plant Gen. Resour. 2005, 3, 230.
5] H.-S. Jeon, M.-H. Jo, H.-J. Kim, M.-H. Lee, S.-K. Yu, C. S. Kim, S.-Y. Lee, S.-G. Kim, H. S. Chun, E.
Park, D. K. Kim, J. Korean Soc. Appl. Biol. Chem. 2012, 55, 451.
[
[
[
6] S.-H. Park, Y.-B. Sim, S.-M. Kim, Y.-J. Kang, J.-K. Lee, H.-W. Suh, J. Korean Soc. Appl. Biol. Chem.
2012, 55, 57.
[
[
7] M. I. Aguilar, G. Delgado, M. D. L. Hern a´ ndez, M. L. Villarreal, Nat. Prod. Lett. 2001, 15, 93.
8] J. L. Funk, J. B. Frye, J. N. Oyarzo, N. Kuscuoglu, J. Wilson, G. McCaffrey, G. Stafford, G. Chen, R. C.
Lantz, S. D. Jolad, A. M. S o´ lyom, P. R. Kiela, B. N. Timmermann, Arthritis Rheum. 2006, 54, 3452.
9] G. Bar-Sela, R. Epelbaum, M. Schaffer, Curr. Med. Chem. 2010, 17, 190.
[
[
[
[
10] J.-J. Chen, C.-S. Tsai, T.-L. Hwang, P.-C. Shieh, J.-F. Chen, P.-J. Sung, Food Chem. 2010, 119, 974.
11] Y. C. Zeng, J. M. Liang, G. X. Qu, F. Qiu, Acta. Pharm. Sin. 2007, 17, 738.
12] W. Li, J.-T. Feng, Y.-S. Xiao, Y.-Q. Wang, X.-Y. Xue, X.-M. Liang, J. Asian Nat. Prod. Res. 2009, 11,
569.
[
[
[
[
13] B. T. Golding, E. Pombo, C. J. Samuel, J. Chem. Soc., Chem. Commun. 1982, 363.
14] S. Imai, M. Morikiyo, K. Furihata, Y. Hayakawa, H. Seto, Agric. Biol. Chem. 1990, 54, 2367.
15] A. C. C. M. Manzan, F. S. Toniolo, E. Bredow, N. P. Povh, J. Agric. Food Chem. 2003, 51, 6802.
16] T. Nishiyama, T. Mae, H. Kishida, M. Tsukagawa, Y. Mimaki, M. Kuroda, Y. Sashida, K. Takahashi,
T. Kawada, K. Nakagawa, M. Kitahara, J. Agric. Food Chem. 2005, 53, 959.
17] M. Ohshiro, M. Kuroyanagi, A. Ueno, Phytochemistry 1990, 29, 2201.
[
[
[
[
[
[
[
18] L. Y. Wang, M. Zhang, C. F. Zhang, Z. T. Wang, Acta. Pharm. Sin. 2008, 43, 724.
19] M. E. M. Braga, P. F. Leal, J. E. Carvalho, M. A. A. Meireles, J. Agric. Food Chem. 2003, 51, 6604.
20] P. K. Awasthi, S. C. Dixit, Pharmacognosy 2009, 1, 312.
21] H.-M. Chiang, H.-C. Chen, T.-J. Lin, I-C. Shih, K.-C. Wen, Food Chem. Toxicol. 2012, 50, 4260.
22] D. L. Narayanan, R. N. Saladi, J. L. Fox, Int. J. Dermatol. 2010, 49, 978.
23] H. J. Moon, S. H. Lee, M. J. Ku, B. C. Yu, M. J. Jeon, S. H. Jeong, V. A. Stonik, T. N. Zvyagintseva,
S. P. Ermakova, Y. H. Lee, Eur. J. Dermatol. 2009, 19, 129.
[
24] Y. P. Hwang, J. H. Choi, H. J. Yun, E. H. Han, H. G. Kim, J. Y. Kim, B. H. Park, T. Khanal, J. M.
Choi, Y. C. Chung, H. G. Jeong, Food Chem. Toxicol. 2011, 49, 93.
[
[
[
[
[
25] F. J. McEnroe, W. Fenical, Tetrahedron 1978, 34, 1661.
26] M. I. Aguilar, G. Delgado, R. Bye, E. Linares, Pytochemistry 1993, 33, 1161.
27] Z.-T. Du, S. Zheng, G. Chen, D. Lv, Molecules 2011, 16, 8053.
28] T. Massmune, K. Orito, A. Murai, Bull. Chem. Soc. Jpn. 1966, 39, 2503.
29] J. P. Minyard, A. C. Thompson, P. A. Hedin, J. Org. Chem. 1968, 33, 909.
Receceived October 14, 2013