Glucosyloxybenzyl eucomate derivatives from Vanda teres stimulate HaCaT cytochrome c oxidase

Article abstract of DOI:10.1021/np1006636

Eucomic acid [(2R)-2-(p-hydroxybenzyl)malic acid)] (1) and three new glucopyranosyloxybenzyl eucomate derivatives, vandaterosides I (2), II (3), and III (4), were isolated and identified from the stems of Vanda teres. Their cellular antiaging properties were evaluated in a human immortalized keratinocyte cell line (HaCaT) by monitoring their effect on cytochrome c oxidase activity, implicated in mitochondrial respiratory function and cellular energy production. Eucomic acid (1) and vandateroside II (3) increased cytochrome c oxidase activity and/or expression, without enhancing cellular mitochondrial content. These two V. teres biomarkers apparently contributed to stimulate respiratory functions in keratinocytes. Since aging and its pathologies may be ascribed to a decline in mitochondrial functions, these biomarkers have the potential to become new natural ingredients for antiaging preparations to remedy age-related disorders such as skin aging. (Chemical Equation Presented).

Full text of DOI:10.1021/np1006636

ARTICLE
pubs.acs.org/jnp
Glucosyloxybenzyl Eucomate Derivatives from Vanda teres Stimulate
HaCaT Cytochrome c Oxidase.
†
†,§
‡
‡,^
†
Charlotte Simmler, Cyril Antheaume, Patrice Andr ꢀe , Fr ꢀe d ꢀe ric Bont ꢀe , and Annelise Lobstein
†
Faculty of Pharmacy, Strasbourg University, 67400 Illkirch, France
§
Faculty of Pharmacy, SCAꢀUMR 7200 67400 Illkirch, France
^
Guerlain, 92290 Levallois Perret, France
‡
LVMH Recherche, 45800 Saint Jean de Braye, France
S Supporting Information
b
ABSTRACT: Eucomic acid [(2R)-2-(p-hydroxybenzyl)malic acid)] (1) and
three new glucopyranosyloxybenzyl eucomate derivatives, vandaterosides I
(
2), II (3), and III (4), were isolated and identified from the stems of Vanda
teres. Their cellular antiaging properties were evaluated in a human immorta-
lized keratinocyte cell line (HaCaT) by monitoring their effect on cytochrome
c oxidase activity, implicated in mitochondrial respiratory function and cellular
energy production. Eucomic acid (1) and vandateroside II (3) increased
cytochrome c oxidase activity and/or expression, without enhancing cellular
mitochondrial content. These two V. teres biomarkers apparently contributed
to stimulate respiratory functions in keratinocytes. Since aging and its pathologies may be ascribed to a decline in mitochondrial
functions, these biomarkers have the potential to become new natural ingredients for antiaging preparations to remedy age-related
disorders such as skin aging.
he orchid family is believed to be the second largest family of
The mitochondrial respiratory chain plays a key role in cell
aging processes. Skin epidermal disorders and aging are partly
7
T
flowering plants, with almost 30 000 currently registered
1
species, found in 880 genera. However, only a few of them have
been investigated regarding their chemical composition and
biological activities. Genera from the Epidendroideae subfamily
are widely distributed. Dendrobium, Bletilla, Cymbidium, Gastro-
associated with a decline in cellular energy level linked to mito-
8
chondrial respiratory chain dysfunctions. Mitochondrial mem-
brane potential is created by electron transfer through respiratory
chain enzymatic complexes IꢀIV. This potential is critical for the
1
7,9
dia, and Vanda species occur in tropical areas. The genus Vanda
proper functioning of this organelle and for ATP production.
comprises 50 to 60 species, mostly found in the southeast of Asia:
Thailand, India, Laos, and Burma. Only four Vanda species, V.
At the end of this respiratory chain, cytochrome c oxidase
(complex IV) reduces oxygen to form water. Therefore, cyto-
chrome c oxidase stimulation could be an appropriate target for
any substances that attenuate the cell aging process. Here, the
effect of the major constituents of V. teres stems, compounds 1 to
4, on cytochrome c oxidase isolated from a human keratinocyte
cell line (HaCaT) is reported on.
1
tessellata Hook. ex G.Don., V. parviflora Lindl., V. parishii Rchb.f.,
and V. coerulea Griff. Ex.Lindl, have been chemically investigated,
2
,3
leading to the identification of phenanthropyran derivatives
4
and parishin a glucopyranosyloxybenzyl citrate. This last com-
pound and its derivatives were also identified in Gastrodia elata
5
Blume tubers. Only V. tesselata was tested for its wound-healing
6
properties, whereas phenanthropyrans isolated from V. coerulea
were analyzed for their antioxidant and anti-inflammatory activ3-
ities through an inhibition of PGE-2 production on HaCaT.
Thus, considering their biological activities on human cells and
their chemical composition, Orchidaceae from the genus Vanda
have not been well studied. These observations led to a focus on
V. teres (Roxb.) Lindl., also known as Papilionanthe teres (Roxb.)
Schltr. The isolation, structural elucidation, and first biological
investigations of four compounds, namely, eucomic acid (1)
and three new glycopyranosyloxybenzyl eucomate derivatives,
vandaterosides I (2), II (3), and III (4), are herein reported
’ RESULTS AND DISCUSSION
Isolation and Structural Elucidation of Vandaterosides.
The methanolic extract of V. teres stems was subjected to
Sephadex LH-20 column chromatography eluted successively
with H O, H O/MeOH, and MeOH. Fractions with similar
2 2
TLC profiles were pooled. Fractions 1, 2, 3, and 5 were subjected
to semipreparative RPHPLC to furnish vandaterosides I (0.65%
Received: September 17, 2010
Published: April 21, 2011
(Scheme 1).
Copyright r 2011 American Chemical Society and
American Society of Pharmacognosy
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Journal of Natural Products
ARTICLE
Scheme 1. Structures of Eucomic Acid (1) and Vandatero-
sides I (2), II (3), and III (4)
the para hydroxybenzyl unit was confirmed, and conse-
quently, C-3 (δ 42.4) was located between the two carbonyl
C
functions.
The vandateroside I structure (2) shared characteristic fea-
tures with eucomic acid (1) (Scheme 1). The HSQC spectrum
clearly showed one glucopyranosyl pattern and one correlation
0
between the anomeric proton (δ 4.84) [(1H, d, J = 7.0 Hz), H-4 -
O-Glc-1] and the anomeric carbon (δ 100.5). Furthermore, the
C
0
0
aromatic protons H-2 /6 (δ 7.22) correlated with the aromatic
C-4 (δ 157.5) and with C-7 (δ 65.8). The H -7 (δ 4.98)
0
0
0
C
C
2
protons correlated with C-4, indicating that the glucosyloxyben-
zyl moiety was attached to this carbonyl function. Thus, the
positions of the β-glucopyranosyl and p-hydroxybenzyl alcohol
moieties in 2 were defined. On the basis of these findings, the
structure of the new compound vandateroside I (2) was defined
0
as 4-(4 -β-D-glucopyranosyloxybenzyl)-2-(p-hydroxybenzyl)malate,
0
also corresponding to 4-(4 -β-D-glucopyranosyloxybenzyl)-
eucomate.
Vandateroside II (3) was obtained as a white, amorphous
2
0
powder and exhibited a negative specific rotation, [R]
ꢀ56
D
(
c 0.1, MeOH). In the UV spectrum, absorption maxima were
observed at 203 (log ε 4.52), 225 (log ε 4.53), and 278 nm (log ε
.55), characteristic of a hydroxybenzyl skeleton. The IR spec-
3
trum showed absorption bands similar to those of 2. Positive
þ
HRESIMS revealeda quasi-molecular ion[(Mþ NH )] at m/z =
4
7
94.28867, and thus the molecular formula of 3 was calculated
for C H O . The acid hydrolysis of vandateroside II (3) gave
37 44 18
D-glucose, which was identified after derivatization by GC-EIMS
1
13
and comparison with a D-glucose reference. The 1D ( H,
C
(
Table 1)) and 2D NMR spectra of 3 shared common chemical
1
shifts with those of 2. The H NMR spectrum and HSQC
correlations displayed unambiguously a third A B system [δ
7.22, 7.00 (2H each, both d, J = 8.6 Hz) H-2 /6 , H-5 /3 ]
linked with a second glucopyranosyl moiety [δ 4.84 (1H, d, J =
2
2
00
0
0
00 00
yield, m/m dry material), II (1.33%), and III (0.23%) together
with eucomic acid (0.51%).
0
0
Vandateroside I (2) was obtained as a yellow resin and
6.8 Hz) H-4 -O-Glc-1], which was confirmed by the HMBC
20
exhibited a negative specific rotation, [R] ꢀ50 (c 0.1, MeOH).
experiment. The HMBC spectrum showed long-range correla-
D
00
In the UV spectrum, absorption maxima were observed at 203
tions between the following proton and carbon pairs: H-4 -O-
0
0
00
00
00 00
00
(
log ε 4.08), 225 (log ε 4.04), and 278 nm (log ε 3.16), charac-
Glc, H-2 /6 and C-4 and between H-2 /6 and C-7 . The
teristic of a hydroxybenzyl skeleton. The IR spectrum showed
position of the second glucosyloxybenzyl moiety was determined
ꢀ
1
00
absorption bands at 2512 and 1727 cm , corresponding to ester
carbonyl and carboxyl functions, and at 1612, 1512, and
through long-range correlations observed from H -7 (δ 4.96),
2
H -3, and H -5 to C-1 (δ 169.4) (Supporting Information).
2
2
C
ꢀ
1
1
226 cm , assignable to an aromatic ring, in addition to strong
Thus, the positions of the β-glucopyranosyloxybenzyl and
p-hydroxybenzyl alcohol moieties in 3 were defined. On the basis
of these findings, the structure of vandateroside II (3) was defined
ꢀ
1
absorption bands at 3340 and 1074 cm , suggestive of a glyco-
side moiety. Positive HRESIMS revealed a quasi-molecular ion
þ
0 00
[
(M þ NH )] at m/z = 526.19236 and revealed the molecular
as 1,4-bis(4 ,4 -β-D-glucopyranosyloxybenzyl)-2-(p-hydroxybenzyl)
4
0
00
formula of 2 to be C H O . The acid hydrolysis of 2 gave
malate, also corresponding to 1,4-bis(4 ,4 -β-D-glucopyrano-
24 28 12
D-glucose, which was identified after derivatization by GC-EIMS.
syloxybenzyl)eucomate.
Vandateroside III (4) was obtained as a yellow resin and
1
13
The H and C NMR ((DMSO-d ) Table 1) spectra of 2,
6
20
D
which were assigned by 2D gradient NMR experiments
exhibited a negative specific rotation, [R] ꢀ37 (c 0.1, MeOH).
(
[
(
HSQC, HMBC, COSY), showed methoxybenzyl protons
In the UV spectrum, absorption maxima were observed at 203
(log ε 4.52) and 276 nm (log ε 3.56). The IR spectrum showed
absorption bands similar to those of 2 and 3. The molecular
0
δ 4.98 (2H, s) H -7 ], two methylene protons [δ 2.41, 2.74
2
1H each, both d, J = 16.0 Hz) H -3; δ 2.77 (2H, m) H -5],
2
2
and two A B systems specific to a para-substituted aromatic
formula of 4, C H O , was determined by the quasi-molecular
2
2
52 60 24
0
0
þ
pattern [δ 7.22, 6.98 (2H each, both d, J = 8.5 Hz) H-2 /6 ,
ion in the positive HRESIMS, [(M þ NH )] at m/z =
4
0
0
H-5 /3 ; δ 6.90, 6.59 (2H each, both d, J = 8.5 Hz) H-7/11,
1086.38484. Its acid hydrolysis liberated D-glucose, which was
identified after derivatization by GC-EIMS and comparison with
a D-glucose reference. The 1D (Table 1) and 2D NMR spectra
showed chemical shifts similar to those of compound 3. New
additional signals corresponding to a third glucopyranosyl moi-
ety [δ 4.82 (1H, d, J = 7.8 Hz) H-2-O-Glc-1], linked to a trans-
cinnamate ester [δ 6.67, 7.68 (1H each, both d, J = 15.7 Hz), 7.74
(2H, dd, J = 2.8, 6.8 Hz), 7.44 (3H, m)], were observed. The
H-8/10]. In the HMBC experiment of 2, H -5 correlated with
2
the C-1 (δC 171.6) carbonyl carbon and with quaternary
carbons C-2 (δC 75.3) and aromatic C-6 (δC 125.6). The
chemically equivalent H-7/11 aromatic protons correlated
with the oxygenated C-9 (δ 156.0) and with C-5 (δ 43.6).
C
C
In addition H -3 gave correlations with C-2, C-1, and C-4
2
(
δC 173.7) (Supporting Information). Thus the position of
9
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Journal of Natural Products
ARTICLE
1
13
Table 1. H and C NMR Spectroscopic Data (400 MHz, 100 MHz, DMSO-d ) for Vandaterosides I (2), II (3), and III (4)
6
vandateroside I (2)
(J in Hz)
vandateroside II (3)
(J in Hz)
vandateroside III (4)
(J in Hz)
position
mult.
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
1
2
3
3
4
5
6
7
8
9
1
7
2
5
4
4
2
3
4
5
6
C
C
171.6
75.3
42.4
169.4
75.3
42.3
170.0
80.4
39.5
a
CH
2
2.74, d (16.0)
2.41, d (16.0)
2.84, d (16.7)
2.48, d (16.7)
2.95, d (17.7)
2.81, d (17.7)
b
C
173.7
43.6
173.4
43.5
169
CH
C
2
2.77, m
2.81, m
40.8
2.99, m
125.7
131.5
114.9
156.0
129.1
65.8
125.4
131.2
114.5
156.0
129.0
65.1
125.3
131.5
114.4
156.1
130.3
66.2
, 11
, 10
CH
CH
C
6.90, d (8.5)
6.59, d (8.5)
6.91, d (8.5)
6.60, d (8.5)
6.96, d (8.5)
6.58, d (8.5)
0
0
C
CH
CH
CH
C
2
4.98, s
4.96, s
5.01, d (6.6)
7.26, d (8.6)
7.03, d (8.6)
0
0
0
0
0
0
, 6
, 3
129.4
116.2
157.5
100.5
73.1
7.22, d (8.5)
6.98, d (8.5)
129.4
116.0
157.1
100.2
73.0
7.25, d (8.6)
7.01, d (8.6)
129.6
116.2
157.4
100.1
73.2
-O-Glc-1
CH
CH
CH
CH
CH
CH
4.84, d (7.0)
3.23, m
4.86, d (6.8)
3.23, m
4.88, d (7.2)
3.23, m
77.0
3.35, m
76.8
3.30, m
76.6
3.28, m
69.7
3.15, m
69.5
3.15, m
69.7
3.15, m
76.6
3.28, m
76.4
3.28, m
77.0
3.33, m
2
60.7
3.68, dd (1.5,12.5)
60.5
3.68, dd (4.9,11)
3.45, dd (5.3,11)
4.84, d (6.8)
3.23, m
60.7
3.69, dd (1.7,11.7)
3.45, dd (6.8,11.7)
4.87, d (7.2)
3.23, m
3
.45, dd (6.1,12.5)
0
0
4
2
3
4
5
6
-O-Glc-1
CH
CH
CH
CH
CH
CH
100.2
73.0
76.8
69.5
76.4
60.5
100.1
73.2
76.6
69.7
77.0
60.7
3.30, m
3.28, m
3.15, m
3.15, m
3.28, m
3.33, m
2
3.68, dd (4.9,11)
3.69, dd (1.7,11.7)
3.45, dd (6.8,11.7)
3.45, dd (5.3,11)
0
0
0
0
0
0
0
1
7
7
2
5
4
2
2
3
4
5
6
C
128.9
65.6
128.7
65.6
0
a
CH
2
4.96, s
4.97, d (5)
4.93, d (5)
7.27, d (8.6)
7.03, d (8.6)
0
b
0
0
0
00
00
, 6
, 3
CH
CH
C
129.4
115.9
157.1
7.22, d (8.6)
7.00, d (8.6)
129.7
116.1
157.3
98.0
71.6
78.0
67.4
76.5
60.3
-O-Glc-1
CH
CH
CH
CH
CH
CH
4.82, d (7.8)
3.26, m
4.92, t (9.5)
3.39, m
3.10, m
2
3.49, dd (4.5,11.8)
3.68, dd (1.7,11.8)
Cin-1
Cin-2
Cin-3
Cin-4
Cin-5,9
Cin-6,8
Cin-7
C
165.7
118.4
144.2
134.2
128.3
128.9
128.7
CH
CH
C
6.67, d (15.7)
7.68, d (15.7)
CH
CH
CH
7.74, dd (2.8, 6.8)
7.44, m
7.44, m
HMBC spectrum shared similar long-range correlations with the
HMBC spectrum of 3. A long-range correlation was observed
between the third anomeric proton H-2-O-Glc-1 and C-2
(δ 80.4), revealing the position of the third glucosyl unit.
C
9
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Journal of Natural Products
ARTICLE
Long-range correlations were also observed between the proton
and carbon pairs of the cinnamyl moiety: H-Cin-6/Cin-8 and
C-Cin-4, H-Cin-9 and C-Cin-3, H-Cin-3 and C-Cin-1, and
H-Cin-2 and C-Cin-4. In addition, HMBC correlations were
observed between H-3 of the 2-O-glucosyl unit [δ 4.92 (1H, t, J =
9
.5 Hz)] and the anomeric carbon (δ 98.0) as well as C-Cin-1
C
(
δC 165.7), indicating that the cinnamyl moiety was attached to
C-3 of this 2-O-glucosyl unit (Supporting Information). Thus,
the position of the β-glucopyranosyl-trans-cinnamyl ester moiety
in 4 was defined. On the basis of these findings, vandateroside III
0
00
(
4) was defined as 1,4-bis(4 ,4 -β-D-glucopyranosyloxybenzyl)-
2
-(β-D-glucopyranosyl-3-trans-cinnamyl ester)-2-(p-hydroxybenzyl)-
0
00
malate, also corresponding to 1,4-bis(4 ,4 -β-D-glucopyrano-
syloxybenzyl)-2-(β-D-glucopyranosyl-3-trans-cinnamyl ester)
eucomate.
(2R)-2-(p-Hydroxybenzyl)malic acid (1) was first isolated
from the bulbs of Eucomis punctata L’H ꢀe r. (Liliaceae) and then
10
described as eucomic acid. Eucomic acid (1) was also found in
Lycoris radiata Herb. (Amaryllidaceae), in Lotus japonicus
(
Regel.) K. Larsen (Fabaceae), and in orchid species such as
11
Cattleya trianaei Linden & Rchb.f and Encylica michuacana Schltr.
Eucomic acid, considered as an auxin,
10,11
is consequently not a
chemotaxonomic marker of the Orchidaceae family. This auxin
shares a common central structure with vandaterosides (Scheme 1)
and may be considered as their biosynthetic precursor: vanda-
terosides could be regarded as glucopyranosyloxybenzyl euco-
mate derivatives. In addition, the vandateroside glucopyrano-
syloxybenzyl moiety corresponds to gastrodin, which was first
4
12
described in V. parishii and was also found in G. elata tubers.
Thus, vandaterosides I (2) and II (3) can be considered as euco-
mic acid mono- and digastrodin esters, while vandateroside III (4) is
a β-3-trans-cinnamylglucopyranoside of vandateroside II (3).
10
Figure 1. (A) Effects of vandaterosides IꢀIII and eucomic acid on
cytochrome c oxidase activity. Untreated HaCaT cells were used as
negative control and indicated a basal enzyme activity. Cytochrome c
oxidase activity was expressed as percentage (mean ( SD for three
independent experiments) and compared to the basal enzyme activity
set at 100 ( 3%. A ratio > 1 indicated a stimulation of cytochrome c
oxidase. (B) Effects of vandaterosides I and II and eucomic acid on
mitochondrial biogenesis. The total quantity of cox2 mitochondrial gene
was compared to the total quantity of ucp-2 nuclear gene. A ratio cox2/
ucp-2 > 1 indicated a stimulation of cox2 replication, consequently
representing mitochondrial biogenesis. Basal mitochondrial biogenesis
measured with untreated HaCaT cells was expressed as 100 ( 5% and
used as the negative control. Resveratrol (50 μM) was used as the
positive control in both experiments. *p < 0.05 compared to the negative
control.
According to Heller et al., the absolute configuration of
eucomic acid has been determined after comparison with piscidic
acid. Eucomic acid displayed a negative specific rotation indica-
tive of 2R absolute configuration by comparison with piscidic
acid. We observed that our isolated eucomic acid gave a negative
2
0
specific rotation ([R]
ꢀ24 in MeOH), in accordance with
D
1
0,11
published data.
All vandaterosides, considered as eucomic
acid mono- and digastrodin esters, displayed negative specific
2
0
rotation ([R] ꢀ50, ꢀ56, and ꢀ37, in MeOH respectively for
D
vandaterosides I, II, and III). Consequently, after comparison
with eucomic acid, the 2R absolute configuration of the three
vandaterosides was established.
Further phytochemical analyses revealed that vandaterosides
IꢀIII were found not only in the stems but also in the leaves and
roots of V. teres.
Skin aging has been ascribed to mitochondrial dysfunctions
and to a subsequent increase of oxidative stress and decline in
cellular energy level. Cytochrome c oxidase plays a key role in
c oxidase with 60-fold lower concentrations than resveratrol
14
(at 50 μM) used as the positive control. For all concentrations
tested, a tendency for vandateroside II (3) to enhance cyto-
chrome c oxidase activity (158 ( 33%) more than resveratrol
(138 ( 8%) was observed (Figure 1A).
8
the respiratory chain maintaining mitochondrial membrane poten-
7,9
tial and participating in cellular energy metabolism. On the
basis of these premises, it was reasonable to determine whether
eucomic acid and vandaterosides IꢀIII could stimulate cyto-
An increase in cytochrome c oxidase synthesis or in mitochon-
drial biogenesis could also influence the global stimulation effect
measured with eucomic acid and vandateroside II. The mito-
chondrial biogenesis was evaluated through the quantitation of
intracellular mitochondrial DNA. Cox2 mitochondrial gene en-
13
chrome c oxidase activity in HaCaT cells.
Stimulation of Cytochrome c Oxidase and Mitochondrial
Biogenesis. For each concentration tested, vandaterosides I and
III did not display any significant stimulation of global cyto-
chrome c oxidase activity (Figure 1A). Compared to the control
enzyme activity, eucomic acid and vandateroside II showed a
significant stimulation of cytochrome c oxidase. Eucomic acid (1)
and vandateroside II (3) (at 0.81 μM) activated cytochrome
1
5
codes expression of cytochrome c oxidase subunit II. Thus,
whether eucomic acid and vandateroside II could enhance cox2
quantity and to a lesser extent HaCaT mitochondrial content was
examined (Figure 1B). The total quantity of mitochondrial cox2
gene was compared to the total quantity of ucp-2 nuclear gene
9
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Journal of Natural Products
ARTICLE
1
6
encoding the uncoupling protein 2. The negative control was
untreated HaCaT cells and reflected a basal cox2 replication and
mitochondrial biogenesis (set at 100 ( 5% for a cox2/ucp-2 ratio
normalized to 1). All results were compared to the negative
The column was eluted with MeOH/(H
2
O þ 0.1% HCO
2
H) using a
linear gradient starting from 20% up to 47% MeOH in 13.5 min,
maintaining 47% MeOH during 10 min, and finally from 47% to 100%
MeOHin 10 min. The fractions were monitoredbetween 200 and 400 nm.
Reagents for Biological Analyses. Resveratrol, DMSO, all other
chemicals, cytochrome c(from equine heart), Bradford solution, and bovine
serum albumin (BSA) were obtained from Sigma (Lyon, France). Cell
proliferation kit II reagent (XTT) was provided by Roche Diagnostic
14
control. Resveratrol (50 μM) was used as the positive control.
Eucomic acid and vandaterosides I and II did not display any
increase in cox2 mitochondrial gene quantity compared to ucp-2
quantity. Consequently, they did not enhance the HaCaT
mitochondrial biogenesis and cox2 quantity.
(Meylan, France). Bicinchoninic acid (BCA) assay kit for protein dosage
was obtained from Uptima Interchim (Montlu c- on, France). Dulbecco’s
minimum essential medium (DMEM), fetal bovine serum (FBS) for
HaCaT culture, penicillin/streptomycin, and glutamine were purchased
from Invitrogen (Cergy-Pontoise, France), and MIX-PCR SYBR Green
was obtained from Applied Biosystems (Courtaboeuf, France).
Plant Material. Vanda teres was identified by Dr. Josef Margraf in
Thailand. A voucher specimen (No P-th-1004) was deposited at the
quarantine office of the Bangkok Agricultural Department. Stems from
flowering specimens were collected in October 2006.
Cut stems were dried in the sunshine in hanging baskets, so as to
avoid contamination by soil and to prevent fungus development. After
arrival in France, dried, cut stems were pulverized in an ultracentrifuge
crusher (Retsch ZM 200) just before extraction. The collection of stems
was carried out in accordance with CITES (Convention on International
Trade in Endangered Species of Wild Fauna and Flora) regulations.
Extraction and Isolation. Dried V. teres stems (100 g) were
The same kind of results were observed with the mitochon-
drial 16S rRNA gene coding 16S rRNA, a component of the
1
5
ribosomal protein 30S subunit (data not shown). Conse-
quently, the total level of mitochondria in HaCaT cells was not
modified either by eucomic acid (1) or by vandaterosides II (3)
or I (2), suggesting that enhancement of cytochrome c oxidase
activity previously measured was not ascribed to a modification
of intracellular cox2 gene level. Subsequently, vandateroside II
(
3) and eucomic acid (1) may increase cytochrome c oxidase
synthesis or directly stimulate cytochrome c oxidase activity.
Interestingly, these glucopyranosyloxybenzyl eucomate deri-
vatives, vandaterosides IꢀIII, are closely related to glucopyrano-
syloxybenzyl tartrate, malate, and citrate derivatives described in
other orchids such as Coeloglossum viride (L.) Hartm. var.
1
7
18
bracteatum (Willd.), Gymnadenia conopsea (L.) R. Br., and
G. elata,
5
,12
respectively. Dactylorhine B, a glucopyranosyloxy-
successively extracted by cyclohexane, CH
automatic Soxhlet (Avanti 2055) to yield cyclohexane (1.8 g), CH
(
2
Cl
2
, and MeOH using an
Cl
1.4 g), and MeOH (6.9 g) extracts, respectively. An aliquot of the
benzyl-2-isobutyl tartrate derivative, was shown to display a
2
2
stimulating effect on cytochrome c oxidase activity and ATP
19
production on rat brain mitochondria. It was demonstrated
herein that eucomic acid (1) and vandateroside II (3) could also
stimulate cytochrome c oxidase activity and/or expression in the
human keratinocyte cell line without enhancing cellular mito-
chondrial content. Further analyses should be performed to
determine whether eucomic acid (1) and vandateroside II (3)
could potentially increase cytochrome c oxidase expression.
Deficiencies in cutaneous energy capacity are closely related to
alterations in the structure of human skin, especially in the
epidermis. Impairment in keratinocyte energy metabolism con-
tributes to a slowdown in skin rejuvenation and wound-healing
processes, leading to a thinner epidermis with a reduced protec-
2
MeOH extract (1 g) was suspended in H O/MeOH (95:5) and
subjected to a Sephadex LH-20 column (30 g) with a H O/MeOH
2
elution gradient (from 100% H O to 100% MeOH) to yield 20 fractions
2
of 10 mL each. Fractions with similar TLC profiles were pooled to give
eight fractions. Fractions 1 (424 mg), 2 (178 mg), 3 (65 mg), and 5 (40
mg) were further purified by semipreparative RPHPLC with the same
ꢀ
1
gradient elution, a flow rate of 14 mL min , and detection at 205 nm.
3
Compound 1 (60 mg) was obtained from fractions 1 and 2, and
compounds 2 (68 mg), 3 (125 mg), and 4 (20 mg) were obtained from
fractions 2, 3, and 5, respectively. Compound 1 was identified as (2R)-
2
-(hydroxybenzyl)malic acid, also known as eucomic acid, by physical
1
13
D
data comparison ([R] , UV, H and C NMR, MS) with reported
data.
1
0,11
8
tive function. Our results reveal that eucomic acid and vanda-
Acid Hydrolysis of Compounds 2ꢀ4. A solution of 2ꢀ4 (each
teroside II stimulate cellular respiratory functions through cyto-
chrome c oxidase and consequently have the potential to fight
signs of epidermal aging.
2
mg) in 2 M HCl (0.5 mL) was heated under reflux for 3 h. After
cooling, the solution was extracted with n-BuOH. The dried residue of
the aqueous layer was derivatized by pyridine and 1-(trimethylsilyl)-
imidazole (4:1 v/v) during 1 h at 60 ꢀC. The mixture was analyzed
by GC-MS (Trace GS Ultra) with a capillary TR-5MS SQC column
’
EXPERIMENTAL SECTION
(
0.25 μm, 15 m ꢁ 0.25 mm) under the following conditions: 1 min at
General Experimental Procedures. The following instruments
40 ꢀC, increase of temperature with a thermal ramp of 10 ꢀC/min until
250 ꢀC (helium flow rate 1 mL min , injector temperature 250 ꢀC,
transfer temperature 285 ꢀC). Impact electronic detection was carried
out with a DSQII Thermo Scientific mass spectrometer, mass detection
ranges from 0 to 500. Identification of D-glucose was performed by
comparison of its retention time and spectroscopic data with those of an
ꢀ1
were used to obtain physical data: optical rotations, Perkin-Elmer 341
polarimeter (l = 1 cm); UV spectra, Shimadzu UV-2401PC spectro-
meter; IR spectra, Nicolet 380 FT-IR (Thermo Electron Corporation);
3
1
13
H and C NMR spectra, Bruker (400 MHz-100 MHz) Avance III
spectrometer with TMS as internal standard; all the chemical shifts (δ)
were reported in parts per million (ppm) relative to TMS (δ = 0);
ESIMS and HRESIMS, HPLC Agilent 1200RRLC equipped with an
Agilent 6520 Accurate Mass QTOF spectrometer; GC-MS and EIMS,
Trace GS Ultra equipped with a capillary TR-5MS SQC column
authentic sample of D-glucose (Merck K3398837), t
Eucomic acid (1): white needles (MeOH), [R]
MeOH); UV (in MeOH) λ (log ε) 203 (3.9), 224 (3.9), and 278
15.6 min.
R
2
0
D
ꢀ24 (c 0.1,
max
1
(3.2) nm; H NMR (MeOH-d
4
, 400 MHz) δ 4.00 (1H, d, J = 16 Hz,
(
0.25 μm, 15 m ꢁ 0.25 mm i.d.) and DSQII Thermo Scientific mass
H-3a), 4.33 (1H, d, J = 16 Hz, H-3b), 4.29 (1H, d, J = 13.7 Hz, H-5a),
4.38 (1H, d, J = 13.7 Hz, H-5b), 8.50 (2H, d, J = 8.5 Hz, H-7/11), 8.11
spectrometer; HPLC detector, Prostar 330 diode-array detector and
UVꢀvis 151 Gilson wavelength detector; HPLC column, Nucleodur
C18ec column (5 μm, 250 mm ꢁ 4.6 mm i.d.) and Nucleodur C18ec
column (5 μm, 250 mm ꢁ 21 mm i.d.) used for analytical (flow rate
1
3
(2H, d, J = 8.5 Hz, H-8/10); C NMR (MeOH-d , 100 MHz) δ 174.9
4
(C-1), 77.0 (C-2), 44.1 (C-3), 178.3 (C-4), 45.5 (C-5), 127.9 (C-6),
132.8 (C-7 and C-11), 115.9 (C-8 and C-10), 157.5 (C-9); HRESIMS
m/z 239.05549 (calcd for C H O , 239.05611, Δ 2.6 ppm).
ꢀ1
ꢀ1
1
mL min ) and semipreparative (14 mL min ) profiles, respectively.
3
3
11 11 6
9
53
dx.doi.org/10.1021/np1006636 |J. Nat. Prod. 2011, 74, 949–955

Journal of Natural Products
ARTICLE
2
0
Vandateroside I (2): yellowish resin, [R]
D
ꢀ50 (c 0.1, MeOH);
Cytotoxicity Assay. Cell viability was previously evaluated by XTT
2
0
UV (in MeOH) λmax (log ε) 203 (4.08), 225 (4.04), and 278 (3.16) nm;
max 3340.2, 2512, 1727, 1612, 1512, 1226 and 1042, 1074 cm ; H and
analysis as described by Roehm et al., and results were interpreted after
comparison of total cell protein measurement by a BCA assay. Serial
dilutions of 1ꢀ3 were prepared in DMSO (0.1% v/v final con-
centration). The first higher concentration tested on HaCaT cells was
not cytotoxic (data not shown).
Statistical Analyses. In vitro experiments were performed three
times. All data were expressed as means ( standard deviations. Com-
parison of results was performed by Student's paired t test analysis.
Statistical significance was set at p < 0.05.
ꢀ
1 1
ν
1
3
C NMR data (DMSO-d
6
, 400 MHz) see Table 1; HRESIMS m/z
5
26.19236 (calcd for C H NO , 526.19190, Δ = ꢀ0.87 ppm).
24
32
12
20
Vandateroside II (3): white, amorphous solid, [R] ꢀ56 (c 0.1,
D
MeOH); UV (in MeOH) λmax (log ε) 203 (4.52), 225 (4.53), and 278
(
1
3.55) nm; νmax 3340.2, 2512, 1727, 1612, 1512, 1074 and 1068,
ꢀ1
1
13
014 cm ; H and C NMR data (DMSO-d , 400 MHz) see Table 1;
6
HRESIMS m/z 794.28867 (calcd for C H NO , 794.28659,
3
7
48
18
Δ = ꢀ2.66 ppm).
2
0
Vandateroside III (4): yellowish resin, [R]
D
ꢀ37 (c 0.1 MeOH);
’
ASSOCIATED CONTENT
UV (in MeOH) λmax (log ε) 203 (4.52), and 276 (3.56) nm; ν
max
ꢀ
1 1
3
340.2, 2359, 23411727, 1612, 1512, 1074 and 1068, 1014 cm ; H
S
Supporting Information. HPLC profile of V. teres stem
13
b
and C NMR data (DMSO-d
086.38484 (calcd for C52
64NO24, 1086.38128, Δ = ꢀ3.28 ppm).
Activation of Mitochondrial Cytochrome c Oxidase in
6
, 400 MHz) see Table 1; HRESIMS m/z
1
13
crude extract, H and C NMR spectra of vandaterosides I, II,
and III, and key HMBC correlations are available free of charge
via the Internet at http://pubs.acs.org.
1
H
HaCaT Cells. HaCaT cells (90% confluent, provided by LVMH
Recherche) cultured in six-well plates were treated for 3 h in DMEM
’
AUTHOR INFORMATION
(4.5 g/L glucose) with 0.1% BSA with serial dilutions of compounds
1
ꢀ4 in DMSO (0.1% v/v final concentration). Resveratrol at 50 μM was
Corresponding Author
Tel: þ033 368 8542 41. Fax: þ033 368 8543 10. E-mail:
14
used as the positive control. HaCaT cells were lysed in a HEPES buffer
20 mM HEPES, 0.1%Triton, 1 mM EDTA). Total proteins were
quantified using a Bradford solution, and samples were adjusted to
(
charlotte.simmler@pharma.u-strasbg.fr.
ꢀ
1
2
mg mL . Each cell lysate was first incubated with the assay buffer
3
0
(Sigma) to determine the v (speed of enzyme without added substrate);
’
ACKNOWLEDGMENT
50 μM reduced cytochrome c (from equine heart) was added to
Dr. R. Saladin and Dr. J. Brent Friesen are gratefully acknow-
ledged for helpful comments during the preparation of the
manuscript.
determine the speed of the enzymatic reaction. The disappearance of
reduced cytochrome c was followed with a spectrophotometer at 550 nm
during 90 s (according to the manufacturer’s instructions, Sigma). The
results were calculated from the slopes and expressed as activity of
cytochrome c oxidase (U/μg). The enzymatic activities measured with
vandaterosides (ACOX sample) were compared to the negative control
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