Eucalmaidins A-E, (+)-oleuropeic acid derivatives from the fresh leaves of Eucalyptus maideni

Article abstract of DOI:10.1021/np900290s

Five new (+)-oleuropeic acid derivatives, eucalmaidins A-E (1-5), together with 12 known compounds (6-17), were isolated from the fresh leaves of Eucalyptus maideni. Structures of the new compounds were determined on the basis of spectroscopic analyses (H

Full text of DOI:10.1021/np900290s

1608
J. Nat. Prod. 2009, 72, 1608–1611
Eucalmaidins A-E, (+)-Oleuropeic Acid Derivatives from the Fresh Leaves of Eucalyptus
maideni
Li-Wen Tian,†,‡ Ying-Jun Zhang,*,† Yi-Fei Wang,§ Chi-Choi Lai,§ and Chong-Ren Yang†,⊥
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming 650204, People’s Republic of China, Graduate UniVersity of Chinese Academy of Sciences, Beijing 10049, People’s Republic of
China, Jinan UniVersity, Guangzhou 510632, People’s Republic of China, and Weihe Biotech Research and DeVelopment Center,
Yuxi 653101, People’s Republic of China
ReceiVed May 14, 2009
Five new (+)-oleuropeic acid derivatives, eucalmaidins A-E (1-5), together with 12 known compounds (6-17), were
isolated from the fresh leaves of Eucalyptus maideni. Structures of the new compounds were determined on the basis
of spectroscopic analyses (HSQC, HMBC, and 1H-1H COSY), chemical degradation, and enzymatic hydrolysis. Of the
tested compounds, only quercetin showed slight anti-herpes simplex virus 1 (HSV-1) activity in vitro.
The genus Eucalyptus (Myrtaceae), containing about 945 species,
mainly grows in the tropical and subtropical areas of the world,
and most of them are native to Australia.1 It was introduced into
China in 1890, and currently about 300 species are distributed there.
This genus is known to be rich sources of bioactive secondary
metabolites. A number of unusual phloroglucinol-coupled terpe-
noids, named euglobals, macrocarpals,2 and eucalyptals,3 have been
isolated in the past decades. In addition, a series of phenolic
glycosides conjugated with oleuropeic acid were also reported.4-6
Some of them displayed antitumor-promoting4 and antioxidant5
activities.
Eucalyptus maideni F. Muell is a tall timber tree growing widely
in the southern parts of China.7 The trunks are commonly used in
forestry, while its leaves are extracted for essential oil. Our detailed
study led to the isolation of five new (+)-oleuropeic acid derivatives,
Figure 1. New compounds 1-5 isolated from Eucalyptus maideni.
together with 12 known compounds from the fresh leaves of this
plant. Their structures were determined by extensive spectroscopic
HRESIMS (m/z 381.1326 [M + Cl]-, calcd 381.1316). The 13C
NMR spectrum of 1 showed 10 carbon signals comprising one
carboxylic (δ 169.0), one trisubstituted double bond (δ 141.3,
131.3), two methyl (δ 27.0, 26.6), three methylene (δ 28.5, 24.6,
26.3), one methine (δ 45.6), and one oxygen-bearing quaternary
carbon (δ 72.8). These observations suggested the presence of an
oleuropeic acid unit, also found in cypellocarpins A-C from E.
cypellocarpa.4 The 1H NMR spectrum also showed typical signals
of an olefinic proton at δ 7.02 (m) and two tertiary methyls at δ
1.19 and 1.16 (each 3H, s). In addition, the complex proton signal
patterns arising from the sugar moiety, as well as the appearance
of the anomeric proton signals at δ 5.10 (1/2 H, d, J ) 3.7 Hz)
(R-form) and 4.47 (1/2 H, d, J ) 7.8 Hz) (ꢀ-form) in the ratio of
1:1 and the anomeric carbon signals at δ 94.0 and 98.3, indicated
that 1 is an anomeric mixture. Acidic hydrolysis of 1 liberated
D-glucose, which was determined by GC analysis of its correspond-
ing trimethylsilylated L-cysteine adduct. The presence of the (+)-
oleuropeic acid moiety was confirmed by the methanolysis of 1
with NaOMe in MeOH, which gave the (+)-oleuropeic acid methyl
ester {[R]D +60 (CHCl3)}.3 The linkage of the (+)-oleuropeoyl
moiety at C-6 of the D-glucose unit was established by an HMBC
experiment, which showed correlations of H-6 of glucose [δ 4.43,
4.27 (ꢀ-form), δ 4.38, 4.23 (R-form)] with the carboxylic carbon
at δ 169.0 (C-7′) of the oleuropeoyl moiety. On the basis of the
above evidence, the structure of 1 was determined to be 6-O-
oleuropeoyl-D-glucopyranose and named eucalmaidin A.
analyses, including HSQC, HMBC, 1H-1H COSY NMR, chemical
methods, and enzymatic hydrolysis. In addition, the in vitro anti
HSV-1 activity of the isolated compounds is also described.
Results and Discussion
The 80% aqueous acetone extract of the fresh leaves of E.
maideni was partitioned between CHCl3 and H2O. The H2O portion
was subjected to repeated column chromatography over Diaion
HP20SS, Sephadex LH-20, Toyopearl HW-40, and MCI-gel CHP-
20P to yield 17 compounds (1-17). Compounds 6-17 were
determined to be the known compounds (()-oleuropeic acid (6),8
cypellocarpins A-C (7-9),4 cypellogin B (10),5 quercetin (11),9
quercetin 3-O-R-L-rhamnopyranoside (12),9 quercetin 3-O-ꢀ-D-
glucopyranoside (13),9 quercetin 3-O-ꢀ-D-(6′′-feruloyl)galactopy-
ranoside (14),10 syringetin 3-O-ꢀ-D-glucopyranoside (15),11 (-)-
dihydrodehydrodiconiferyl alcohol 9-O-ꢀ-D-glucopyranoside (16),12
and (+)-isolariciresinol 3R-O-ꢀ-D-glucopyranoside (17),13 on the
basis of detailed spectroscopic analyses, together with comparison
of their spectroscopic and physical data with reported data. The
known compounds 6 and 14-17 were reported from the genus
Eucalyptus for the first time.
Eucalmaidin A (1) was obtained as a pale, amorphous powder.
Its molecular formula, C16H26O8, was established on the basis of
* To whom correspondence should be addressed. Tel: + 86-871-522-
3235. Fax: + 86-871-515-0124. E-mail: zhangyj@mail.kib.ac.cn.
† Kunming Institute of Botany.
Eucalmaidin B (2), a pale, amorphous powder, had a molecular
formula of C23H30O12 deduced from the HRESIMS (m/z 497.1675
[M - H]-, calcd 497.1659), which was the same as that of
cypellocarpin A (7) (see Supporting Information). Comparison of
‡ Graduate University of the Chinese Academy of Sciences.
§ Jinan University.
⊥ Weihe Biotech Research and Development Center.
10.1021/np900290s CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/09/2009

(+)-Oleuropeic Acid DeriVatiVes from Eucalyptus maideni
Journal of Natural Products, 2009, Vol. 72, No. 9 1609
Table 1. 1H and 13C NMR Data of Compounds 1 and 5 in Methanol-d4 (δ values; J in Hz, in parentheses)
1
δH
δC
5
position
Glc-1
2
3
4
5
6
ꢀ
R
ꢀ
R
δH
δC
4.47 d (7.8)
3.13 t (8.4)
3.33 m
3.34 m
3.49 m
5.10 d (3.7)
3.33 m
3.66 t (9.2)
3.33 m
3.98 m
4.38 m
98.3
76.3
78.1
71.8
75.6
64.8
94.0
73.9
74.9
72.1
70.9
64.9
5.49 d (7.5)
3.40 m
3.45 t (8.9)
3.38 t (8.5)
3.60 m
95.7
73.9
77.9
71.3
76.2
64.3
4.43 m
4.27 m
4.40 dd (2, 11)
4.24 dd (6, 11)
4.23 m
oleuropeoyl-1′
131.3
141.3
28.5
131.1
143.1
28.7a
28.6a
45.5
2′
7.02 m
2.02 m
2.31 m
1.52 m
1.99 m
1.21 m
2.14 m
2.49 m
7.15 m
2.34 m
2.02 m
1.53 m
2.02 m
1.21 m
2.50 m
2.16 m
3′,3′′
4′,4′′
5′,5′′
45.6
24.6
24.5
6′, 6′′
26.3
26.4b
26.2b
167.2
72.8
27.0
26.5
130.6
141.6
168.8
7′
169.0
72.8
27.0
26.6
8′,8′′
9′,9′′
1.19 s
1.16 s
1.17 s
1.17 s
10′,10′′
oleuropeoyl-1′′
2′′
7′′
7.02 m
a,b Assignments for the same corresponding position of different moieties may be interchanged.
Table 2. 1H NMR Data of Compounds 2 and 3 in Methanol-d4
(δ values; J in Hz, in parentheses)
the 1D NMR data with those of cypellocarpin A suggested that
the structure of 2 was composed of an oleuropeoyl, a glucosyl,
and a gallic acid moiety. However, instead of the two m-coupled
protons at δ 7.32, 7.22 (d, 2.6 Hz) from the gallic acid moiety of
7, compound 2 showed one two-proton singlet at δ 7.05 (2H, s)
arising from a symmetrically substituted gallic acid moiety. This
suggested C-4 substitution of the gallic acid moiety in 2, relative
to C-3 substitution in 7. In the HMBC spectrum of 2, the anomeric
proton [δ 4.64 (d, 7.7 Hz)] of glucose was correlated with the C-4
(δ 135.7) of gallic acid, confirming the linkage between the gallic
acid and glucosyl moieties. In addition, HMBC correlation of the
glucosyl H-6′ (δ 4.54, 4.24) with the oleuropeoyl carboxylic carbon
at δ 168.8 was also observed. Methanolysis of 2 with NaOMe in
MeOH gave (+)-oleuropeic acid methyl ester {[R]D +56.7
(CHCl3)}. Acidic hydrolysis of 2 gave D-glucose as a sugar residue.
Consequently, the structure of eucalmaidin B was assigned as shown
in 2.
2
3
position
δH
δC
δH
7.40 s
δC
aglycone-1
136.5
110.1
150.8
135.7
168.8
135.7
108.3
153.7
137.6
174.3
56.9
104.6
75.6
77.9
2,6
3,5
4
7.05 s
7
OMe
Glc-1′
2′
3.91 s
4.88 d (7.7)
3.48 m
3.52 t (9.0)
3.45 t (9.3)
3.58 m
4.64 d (7.7)
107.4
74.9
77.4
71.5
76.3
64.4
3.52 dd (8.0, 8.9)
3.46 t (8.8)
3.40 t (9.3)
3.62 m
3′
4′
72.0
75.7
64.6
5′
6′
4.54 m
4.24 m
4.42 m
4.27 m
oleuropeoyl-1′′
130.9
142.1
28.6
131.1
141.5
28.6
Eucalmaidin C (3) was obtained as a pale, amorphous powder.
Its molecular formula C25H34O12 was elucidated from the HRESIMS
2′′
3′′
7.03 m
2.01 m
2.32 m
1.54 m
1.98 m
2.01 m
2.15 m
2.49 m
6.89 m
2.02 m
2.35 m
1.55 m
1.20 m
2.05 m
2.10 m
2.42 m
1
(m/z 525.1978 [M - H]-, calcd 525.1972). The H and 13C NMR
4′′
5′′
45.5
24.5
45.5
24.5
spectra of 3 were similar to those of 2, except for the appearance
of two methoxy groups [δH 3.91 (6H, s) and δC 56.9 (2 × C)],
suggesting that the C-3 and C-5 hydroxy groups in 2 were
methylated. This was further confirmed by the HMBC correlation
of the O-methyl protons (δ 3.91) with C-3 and C-5 of the gallic
acid moiety at δ 153.7. Correlations of H-1′ [δ 4.88, (d, J ) 7.7
Hz)] with C-4 (δ 137.6) and H-6′ (δ 4.42, 4.27 m) with C-7′′ (δ
168.7) were also observed in the HMBC experiment. Methanolysis
of 3 with NaOMe gave (+)-oleuropeic acid methyl ester {[R]D
+42.5 (CHCl3)}, while acidic hydrolysis of 3 yielded D-glucose as
a sugar residue. On the basis of the above evidence, the structure
of compound 3 was determined to be 3,5-dimethyleucalmaidin B
and named eucalmaidin C.
6′′
26.2
26.3
7′′
168.8
72.9
27.1
26.4
168.7
72.9
27.1
26.5
8′′
9′′
10′′
1.18 s
1.17 s
1.24 s
1.23 s
L-cysteine adduct. In addition, the 15 aromatic carbons (Table 3)
and five typical aromatic proton signals [δ 6.18, 6.43 (each 1H, s,
H-6, 8), 7.68 (brs, H-2′), 7.17 (1H, d, J ) 8.7 Hz, H-5′), 7.55 (1H,
d, J ) 8.7 Hz, H-6′)] revealed the existence of a quercetin moiety.
The connectivity of the glucosyl unit with oleuropeoyl and quercetin
units was further confirmed by the HMBC spectrum, in which
correlations of the anomeric proton (δ 5.07) with C-4′ (δ 145.4)
and the glucosyl H-6′′ (δ 4.42, 4.04) with the oleuropeoyl C-7′′′
(δ 165.2) were observed. Thus, the structure of compound 4 was
determined as quercetin 4′-O-(6-O-oleuropeoyl)-R-D-glucopyrano-
side and named eucalmaidin D.
Eucalmaidin D (4), a yellowish, amorphous powder, showed a
quasi-molecular ion peak at m/z 629.1852 in the HRESIMS,
corresponding to the molecular formula C31H34O14. The 1H and 13
C
NMR spectra showed the presence of an oleuropeoyl and a glucosyl
moiety. The small coupling constant (3.7 Hz) as well as the 13C
NMR data assigned an R configuration of the glucosyl anomeric
center.14 Acidic hydrolysis of 4 liberated D-glucose, which was
determined by GC analysis of its corresponding trimethylsilylated

1610 Journal of Natural Products, 2009, Vol. 72, No. 9
Tian et al.
Table 3. 1H and 13C NMR Data of Compound 4 in DMSO-d6
(δ values; J in Hz, in parentheses)
Table 4. Anti-HSV-1 Activity of Compounds 1-3, 5-9, 11,
12, 16, and 17
a
b
position
δH
δC
position
δH
δC
compound
MNCC (mM)
TIC (mM)
d
aglycone-2
3
4
5
6
7
8
9
10
1′
2′
3′
4′
5′
6′
Glc-1′′
2′′
3′′
145.5 4′′
135.5 5′′
175.0 6′′
159.7
97.3 oleuropeoyl-1′′′
163.1 2′′′
92.3 3′′′
155.2
102.1 4′′′
124.1 5′′′
114.1
144.7 6′′′
145.4
3.20 m
3.70 m
4.42 m
4.04 m
69.3
72.9
62.6
1
2
3
5
6
7
8
9
11
12
16
17
>0.58
0.40
0.38
0.20
>1.09
0.03
0.02
>0.38
<0.02
0.01
0.39
0.38
1.11
-
-
-
-
6.18 s
128.5
-
6.93 m 139.3
-
6.43 s
2.20 m
1.95 m
1.37 m
1.90 m
1.07 m
2.37 m
2.04 m
26.1
-
-
42.7
0.33
-
7.68 s
21.9
24.0
-
-
aciclovir
0.0043c
7.17 d (8.7) 114.6 7′′′
7.55 d (8.7) 118.3 8′′′
165.2
69.4
25.9
25.3
a MNCC: maximal noncytotoxic concentration against Vero cells.
b TIC: total inhibitory concentration against HSV-1. c IC50 value
(concentration required to reduce 50% of cytopathic effect). d -: no
activity.
5.07 d (3.7)
3.36 m
3.36 m
99.6 9′′′
72.2 10′′′
74.8
0.98 s
0.96 s
Column chromatography (CC) was performed on Diaion HP20SS
(Mitsubishi Chemical Co.), MCI-gel CHP-20P (75-150 µm, Mitsubishi
Chemical Co.), Sephadex LH-20 (25-100 µm, Pharmacia Fine
Chemical Co.), RP-8 (40-63 µm, Merck), and Toyopearl HW-40 (fine
grade) (TOSOH, Japan). Precoated silica gel plates (Qingdao Haiyang
Chemical Co.) were used for TLC. Detection was done by spraying
the plates with anisaldehyde-sulfuric acid, followed by heating.
Sustainable medium used for assays was Dulbecco’s modified Eagle’s
medium with 2% fetal bovine serum, whose pH value was adjusted to
7.2 by 0.75% NaHCO3 and Hepes buffer (47.6 g of Hepes was dissolved
in H2O (200 mL) and its pH adjusted to 7.5-8.0 by 1 N NaOH).
Plant Material. The fresh leaves of E. maideni were collected in
the Botanical Garden of Kunming Institute of Botany, Chinese Academy
of Sciences, Yunnan, China, during May 2007. A voucher specimen
(KIB-ZL2007001) has been deposited in the State Key Laboratory of
Phytochemistry and Plant Resources in West China, Kunming Institute
of Botany, Chinese Academy of Sciences.
Eucalmaidin E (5) had a molecular formula of C26H40O10,
which was established from the HRESIMS (m/z 547.2321 [M
+ Cl]-, calcd 547.2310). The additional 166 mass units
compared to 1 corresponded to the presence of a second
oleuropeoyl unit. The 1D NMR spectra of 5 were similar to those
of 1. However, instead of an anomeric free glucose moiety in 1,
1
the H NMR spectrum of compound 5 showed the presence of
1
a ꢀ-glucosyl unit [δ 5.49 (d, J ) 7.5 Hz)]. In addition, the H
and 13C NMR spectra displayed two sets of signals assignable
to two oleuropeoyl moieties [δ 7.15, 7.02 (each 1H, m, H-2′,
2′′), 1.17 (12H, s, CH3-9′, 9′′, 10′, 10′′)]. Connectivities of the
two oleuropeoyl moieties with the glucosyl unit were revealed
by the HMBC experiment, in which the glucosyl anomeric proton
(δ 5.49) and H-6 (δ 4.40 and 4.24) were correlated with C-7′ (δ
167.2) and C-7′′ (δ 168.8), respectively. Enzymatic hydrolysis
of 5 yielded (+)-oleuropeic acid and eucalmaidin A. Thus, the
structure of eucalmaidin E was constructed as shown in 5.
Eucalmaidin D (4) represents a rare example of an R-configured
glucoside in nature. Instead of the quercetin C-3 substitution with
the 6-O-oleuropeoyl-ꢀ-D-glucosyl moiety in cypellogins,5 eucal-
maidin D (4) has an 6-O-oleuropeoyl-R-D-glucosyl moiety linked
at C-4′ of the quercetin moiety.
Extraction and Isolation. The fresh leaves of E. maideni (3.5 kg)
were extracted with 80% aqueous acetone at room temperature (3 ×
10 L, each 1 week). The extracts were concentrated under reduced
pressure and then partitioned with CHCl3 (6 × 2 L) after filtration of
the precipitate. The water portion was concentrated to a small volume
(200 mL) and subjected to Diaion HP20SS column chromatography
(CC), eluting with MeOH-H2O (0:1-1:0) to afford seven fractions.
Fraction 4 (22 g) was chromatographed over Sephadex LH-20
(MeOH-H2O, 0:1-1:0) to yield five subfractions (A-E). Subfraction
A was chromatographed over MCI-gel CHP-20P (0-80% MeOH) to
yield 3 (21 mg) and 6 (25 mg). Subfraction B was subjected to CC
over MCI-gel CHP-20P (30%-100% MeOH) and silica gel
(CHCl3-MeOH-H2O, 8.5:1.5:0.1-8:2:0.2) to yield 16 (7 mg) and 17
(18 mg). Subfraction C was applied to MCI-gel CHP-20P (20%-100%,
MeOH) and Rp-8 (50%-100%, MeOH) CC, as well as preparative
TLC of silica gel (C6H6-HCOOEt-HCOOH, 2:7:1), to yield 1 (91
mg), 2 (30 mg), and 7 (98 mg). Subfraction E was chromatographed
over silica gel (CHCl3-MeOH-H2O, 8.5:1.5:0.1-7:3:0.5) and MCI-
gel CHP-20P (20%-100% MeOH) to afford 12 (18 mg), 13 (7 mg),
and 15 (33 mg). Fraction 5 (4 g) was successively chromatographed
over Sephadex LH-20 (0-60% MeOH), MCI-gel CHP-20P (40%-100%
MeOH), and Toyopearl HW-40 (0-40% MeOH) to yield 1 (16 mg)
and 5 (12 mg). Fraction 6 (1.92 g) was subjected to CC over Sephadex
LH-20 (0-100%, MeOH), MCI-gel CHP-20P (40%-100% MeOH),
and silica gel (CHCl3-MeOH-H2O, 9:1:0.1- 8:2:0.2) to yield 4 (10
mg), 8 (18 mg), 9 (70 mg), 10 (6 mg), 11 (70 mg), and 14 (7 mg).
Eucalmaidin A (1): pale, amorphous powder; [R]20D +26.7 (c 0.3,
MeOH); UV (MeOH), λmax (log ε) 225 (3.54) nm; IR (KBr) νmax 3413,
2930, 2886, 1696, 1258, 1073, 1059, 1035 cm-1; 1H NMR (methanol-
d4, 500 MHz), see Table 1; 13C NMR (methanol-d4, 125 MHz), see
Table 1; FABMS (negative ion mode) m/z 345 [M - H]-; HRESIMS
m/z 381.1326 [M + Cl]- (calcd for C16H26O8Cl, 381.1316).
Compounds 1-3, 5-9, 11, 12, 16, and 17 were evaluated for
their in vitro anti-herpes simplex virus type 1 (HSV-1) activity using
a cytopathic effect (CPE) assay and cytotoxicity on African green
monkey kidney cells (Vero cells) by the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The maximal
noncytotoxic concentrations (MNCC) against Vero cells and total
inhibitory concentrations (TIC) against HSV-1 of compounds 1-3,
5-9, 11, 12, 16, and 17 are shown in Table 4. Of all the tested
compounds, only quercetin (11) showed slight anti-HSV-1 activity.
Among the (+)-oleuropeic acid derivatives, compounds 2, 3, 5,
and 7-9 showed stronger cytotoxicity than 6-O-oleuropeoyl-D-
glucopyranose (1).
Experimental Section
General Experimental Procedures. Optical rotations were measured
with a HORIBA SEPA-300 high-sensitive polarimeter. IR spectra were
measured on a Bio-Rad FTS-135 spectrophotometer. NMR spectra were
measured in methanol-d4 or DMSO-d6 solution on a Bruker DRX-500
instrument (500 MHz for 1H NMR and 125 MHz for 13C NMR) at 25
°C, using TMS as an internal standard. FABMS were recorded on a
VG Auto Spec-3000 mass spectrometer using glycerol as matrix.
ESIMS and HRESIMS were recorded on an API QSTAR Pular-1 mass
spectrometer (for compounds 1 and 5, one drop of 0.01% aqueous NaCl
was added while measuring the HRESIMS). The GC was performed
on an HP5890 gas chromatograph (Agilent, America) with a quartz
capillary column (30 mm × 0.32 mm × 0.25 µm); detection, FID.
Eucalmaidin B (2): pale, amorphous powder; [R]27D -17.9 (c 1.2,
MeOH); UV (MeOH), λmax (log ε) 209 (3.83) nm; IR (KBr) νmax 3417,
2926, 2887, 1703, 1562, 1394, 1259, 1062 cm-1; 1H NMR (methanol-
d4, 500 MHz), see Table 2; 13C NMR (methanol-d4, 125 MHz), see

(+)-Oleuropeic Acid DeriVatiVes from Eucalyptus maideni
Journal of Natural Products, 2009, Vol. 72, No. 9 1611
Table 2; FABMS (negative ion mode) m/z 497 [M - H]-; HRESIMS
m/z 497.1675 [M - H]- (calcd for C23H29O12, 497.1659).
used as the control. Then each well was overlaid with medium
containing 1% of methylcellulose, and the plate was incubated for 3
days. Thereafter, the cell monolayer was fixed and stained with formalin
and crystal violet, respectively. The viral plaques were counted under
a binocular microscope. The concentration reducing plaque formation
by 100% relative to control was estimated from graphic plots and
defined as 100% inhibitory concentration.
Cytotoxicity Assays. Cytotoxic activity was performed using the
MTT reduction assay.16 Vero cells were seeded into a 96-well plate.
Different concentrations of samples (100 µL), diluted with cell
sustainable medium, were applied to the wells of a 96-well plate
containing confluent cell monolayer in triplicate, while a dilution
medium without sample was used as the control. After 3 days of
incubation, 12 µL of the MTT solution (5 mg/mL in phosphate-buffered
saline) was added to each well. The plate was further incubated for
4 h to allow for MTT formazan formation. After removing the medium,
100 µL of DMSO was added to dissolve the formazan crystals. The
content in the wells was homogenized on a microplate shake 30 min
later. The OD (optical density) was then read on a microplate
spectrophotometer at double wavelengths of 540 and 630 nm. The
maximal noncytotoxic concentration was defined as the maximal
concentration of the sample that did not exert a cytotoxic effect
evaluated from the OD values of nonviable cells.
Eucalmaidin C (3): pale, amorphous powder; [R]27D -27.5 (c 0.2,
MeOH); UV (MeOH), λmax (log ε) 218 (3.03), 265 (2.85) nm; IR (KBr)
ν
max 3425, 2938, 2880, 1703, 1566, 1385, 1123, 1070 cm-1; 1H NMR
(methanol-d4, 500 MHz), see Table 2; 13C NMR (methanol-d4, 125
MHz), see Table 2; ESIMS (negative ion mode) m/z 525 [M - H]-;
HRESIMS m/z 525.1978 [M - H]- (calcd for C25H33O12, 525.1972).
Eucalmaidin D (4): yellowish, amorphous powder; [R]20 -58.0
D
(c 0.5, MeOH); UV (MeOH), λmax (log ε) 251 (4.91), 367 (4.01) nm;
IR (KBr) νmax 3422, 2967, 2924, 1696, 1650, 1601, 1506, 1253, 1067
cm-1; 1H NMR (DMSO-d6, 500 MHz), see Table 3; 13C NMR (DMSO-
d6, 125 MHz), see Table 3; FABMS (negative ion mode) m/z 629 [M
- H]-, 301 [M - H - Glc - oleuropeoyl]-; HRESIMS m/z 629.1852
[M - H]- (calcd for C31H33O14, 629.1870).
Eucalmaidin E (5): pale, amorphous powder; [R]27 -1.3 (c 0.8,
D
MeOH); UV (MeOH), λmax (log ε) 225 (3.42) nm; IR (KBr) νmax 3471,
2924, 2853, 1673, 1641, 1433, 1286 cm-1; 1H NMR (methanol-d4, 500
MHz), see Table 1; 13C NMR (methanol-d4, 125 MHz), see Table 1;
FABMS (negative ion mode) m/z 511 [M - H]-; HRESIMS m/z
547.2321 [M + Cl]- (calcd for C26H40O15Cl, 547.2310).
Acid Hydrolysis of Compounds 1-4. Compounds 1-4 (each 6
mg, 4 mg for compound 4) were hydrolyzed with 1.5 N HCl (2 mL)
at 80 °C for 5 h. The mixture was neutralized with NaOH (1 N). The
mixture was passed through MCI-gel CHP-20P (1.5 × 14 cm),
developing with H2O. The H2O eluate was evaporated to dryness. The
dry powders were dissolved in pyridine (2 mL), L-cysteine methyl ester
hydrochloride (1.5 mg) was added, and the mixture was heated at 60
°C for 1 h. Trimethylsilylimidazole (1.5 mL) was added, and the mixture
was heated at 60 °C for another 30 min. An aliquot (4 µL) of the
supernatant was removed and directly subjected to GC analysis under
the following conditions: column temp 180-280 °C at 3 deg/min,
carrier gas N2 (1 mL/min), injector and detector temp 250 °C, split
ratio 1:50. The configurations of D-gluose for compounds 1-4 were
determined by comparison of the retentions times of the corresponding
derivatives with standard D-glucose (retention time: 19.208 min),
respectively.
Acknowledgment. We are grateful to the members of the Analytical
Group in the State Key Laboratory of Phytochemistry and Plant
Resources in West China, Kunming Institute of Botany, for the
measurements of all spectra. This work was supported by the NSFC
U0632010 and the State Key Laboratory of Phytochemistry and Plant
Resources in West China, Kunming Institute of Botany, Chinese
Academy of Sciences (P2008-ZZ08).
Supporting Information Available: This material is available free
of charge via the Internet at http://pubs.acs.org.
References and Notes
Methanolysisof1-3.Asolutionof1(6mg)in0.02MNaOMe-MeOH
(1 mL) was kept standing at room temperature for 12 h. The solution was
then subjected to column chromatography over MCI-gel CHP-20P (1.5
× 14 cm), eluting with H2O, 60% and 100% MeOH to give (+)-oleuropeic
acid methyl ester (1a) (3 mg): colorless oil; [R]D +60 (c 0.2, CHCl3); 1H
NMR (CDCl3) δ 6.99 (1H, m, H-2), 2.33, 2.00 (m, H-3), 1.55 (m, H-4),
2.03, 1.23 (m, H-5), 2.54, 2.17 (m, H-6), 1.20 (3H, s, H-9), 1.21 (3H, s,
H-10), 3.72 (3H, s, OCH3); 13C NMR (CDCl3) δ 130.1 (C-1), 139.5 (C-
2), 27.4 (C-3), 44.1 (C-4), 23.3 (C-5), 25.1 (C-6), 167.8 (C-7), 72.4 (C-8),
27.3 (C-9), 26.6 (C-10), 51.6 (OCH3).4 Similar methanolysis of 2 and 3
also gave 1a {[R]D +56.7 (c 0.25, CHCl3) and [R]D +42.5 (c 0.2, CHCl3),
respectively}.
Enzymatic Hydrolysis of 5. A mixture of 5 (10 mg) and ꢀ-glu-
cosidase (8 mg, Sigma) in H2O (1.5 mL) was kept in a water bath at
37 °C for 8 days. The mixture was subjected to MCI-gel CHP-20P
(50% and 100% MeOH) column chromatography. The 50% and 100%
MeOH eluates were separately chromatographed over silica gel
(CHCl3-MeOH-H2O, 9:1:0.1-8:2:0.2, and CHCl3-MeOH, 15: 1) to
give (+)-oleuropeic acid (2 mg) {[R]D +13.3 (c 0.1, MeOH)} and 1
(4 mg) {[R]D +10.0 (c 0.2, MeOH)}, respectively. Oleuropeic acid
and eucalmaidin A were identified by co-chromatography in TLC with
authentic samples.
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