Antiestrogenic constituents of the Thai medicinal plants Capparis flavicans and Vitex glabrata

Article abstract of DOI:10.1021/np9006298

Antiestrogenic compounds were investigated from Thai indigenous plants for galactogogues since estrogen is reported to suppress lactation in breastfeeding women. The aerial parts of the Thai medicinal plant Capparis flavicans, which has traditionally been

Full text of DOI:10.1021/np9006298

1
954
J. Nat. Prod. 2009, 72, 1954–1959
Antiestrogenic Constituents of the Thai Medicinal Plants Capparis flaWicans and Vitex glabrata
Prathan Luecha, Kaoru Umehara,* Toshio Miyase, and Hiroshi Noguchi
School of Pharmaceutical Sciences, UniVersity of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
ReceiVed October 6, 2009
Antiestrogenic compounds were investigated from Thai indigenous plants for galactogogues since estrogen is reported
to suppress lactation in breastfeeding women. The aerial parts of the Thai medicinal plant Capparis flaVicans, which
has traditionally been used to promote lactation, gave the new compound capparoside A (1), along with 28 known
compounds. The leaves of Vitex glabrata belong to the same genus as the chaste tree (Vitex agnus-castus), which is
used traditionally to support lactation, and afforded the new compounds khainaoside A (14), khainaoside B (15), and
khainaoside C (16), together with six known compounds. The isolates were tested for their biological activity using the
estrogen-responsive human breast cancer cell lines MCF-7 and T47D. Syringaresinol (3) and principin (6), from C.
flaVicans, and khainaoside A (14) showed the most potent inhibitory effects on estrogen-enhanced cell proliferation
among all compounds isolated. These results suggest that the lactation-promoting properties of C. flaVicans might be
related to the inhibitory effect on excess estrogen of women who experience insufficient breastfeeding and highlight the
possibility of using V. glabrata leaves for their antiestrogenic properties.
Estrogen acts as a regulator in the milk production process during
components were subjected to HP20 resin column chromatography
by elution with H O, 50% MeOH, and MeOH to give H O (60 g),
1
,2
pregnancy. While breastfeeding, women have high levels of
2
2
3
prolactin and relatively low levels of estradiol. High levels of
50% MeOH (18 g), and MeOH eluates (3 g), respectively. The
ethyl acetate-soluble fraction and 50% MeOH eluate suppressed
the estrogen-enhanced proliferation in MCF-7 and T47D cells at a
concentration of 0.2 µg/mL. Thus, these two fractions were
subjected to silica gel column chromatography and HPLC to give
29 pure compounds, including a new compound (1). Twenty-eight
known compounds were identified as (7R,8R)-threo-guaiacylglyc-
estradiol can inhibit lactation due to reduced milk secretion and
4
,5
changes in mammary secretory epithelial morphology.
An inadequate milk production has often been observed in
6
,7
mothers who deliver preterm infants.
Other factors such as
diabetes, hypothyroidism, obesity, thecalutein cysts, and polycystic
8
ovarian syndrome may also lead to insufficient milk production.
1
5
Herbal galactogogues, such as fenugreek (Trigonella foenum-
graecum) seed, goat’s rue (Galega officinalis), fennel (Foeniculum
Vulgare) seed, and chaste tree (Vitex agnus-castus) seed are used
erol 8-O-4′-sinapyl ether 7-O-ꢀ-D-glucopyranoside (2), (()-
16 17
syringaresinol (3), (()-3′,3′′-bisdemethylpinoresinol (4), isoamer-
1
8
19
icanol A (5), princepin (6), (-)-dehydrodiconiferyl alcohol 4-O-
9
20
widely today in an attempt to overcome this problem.
ꢀ-D-glucopyranoside
glucopyranosyl) secoisolariciresinol (8), spionoside B (9),
tachioside (10), koaburaside (11), 4-hydroxy-2,6-dimethox-
yphenol 1-O-ꢀ-D-glucopyranoside, leonuriside (12), adenosine
(7),
(()-8,8′-dimethoxy-1-O-(ꢀ-D-
21 22
While there are indigenous herbal galactogogues available in
Thailand, there is limited scientific evidence supporting their use.
In a continuation of work on the bioactive constituents of Thai
medicinal plants, Capparis flaVicans and Vitex glabrata were
investigated as potential herbal galactogogues.
2
3
24
2
5
26
27
18
(13), tamarixetin 3-O-ꢀ-D-galactoside, americanol A, isoprin-
1
9
cepin, (-)-5,5′-dimethoxy-4-O-(ꢀ-D-glucopyranosyl) laricires-
28
29
29
29
Capparis flaVicans Kurz. (Capparidaceae), which is known as
inol, alangilignoside B, alangilignoside C, alangilignoside D,
30
10
“
kra-chick” or “ngua-lea” in Thailand, is used as a folk medicine
(-)-lariciresinol 4-O-ꢀ-D-glucopyranoside, 3R-O-ꢀ-D-glucopy-
31 32 33 23
in northeast Thailand for promoting lactation in breastfeeding
ranosyl lyoniresinol, icariside B
1
,
roseoside, isotachioside,
1
1
28
women. The plant grows in mixed deciduous forests at 40-350
m above sea level in Thailand and Myanmar. As no research on
the chemical constituents of this plant has been carried out
previously, we have investigated an extract from the aerial parts
of C. flaVicans.
vanilloloside, 4-hydroxy-3-methoxyphenyl 1-O-ꢀ-D-xylopyrano-
syl-(1f6)-ꢀ-D-glucopyranoside, alangionoside C, and 4-(hy-
droxymethyl)-2,6-dimethoxyphenyl 1-O-ꢀ-D-glucopyranoside, re-
3
4
35
36
spectively, by comparison of their spectroscopic data (especially
NMR) and optical rotation with the reported values in the literature.
Vitex glabrata R.Br. (Verbenaceae), which is called “khai-nao”
25
Capparoside A (1), [R]
formula C18
57.1331 in the HRFABMS. Its H NMR spectrum indicated the
D
-62.8, was assigned the molecular
1
0
in the Thai language, is used for its bark and roots as a folk
medicine in Thailand as a tonic, an antidiarrheal agent, and an
+
26
H O12, from its molecular ion [M + Na] peak at m/z
1
4
1
2
antipyretic. In previous studies, three ecdysteroids were isolated
presence of a trisubstituted aromatic ring (ABX system) from signals
at δ 7.02 (d, J ) 9 Hz), 6.96 (d, J ) 3 Hz), and 6.31 (dd, J ) 9,
1
3,14
from the stem bark and root bark of this species.
This paper deals with the isolation of constituents from these
plants, their structure characterization by spectroscopic methods,
and the assessment of antiestrogenic activity of the isolates as an
index for their lactation-promoting activity using estrogen-
responsive MCF-7 and T47D cell lines.
3
4
Hz). Two anomeric proton signals at δ 4.68 (d, J ) 8.5 Hz) and
.30 (d, J ) 7.5 Hz) and a methoxy group signal at δ 3.80 were
also observed. On acid hydrolysis, 1 gave 1,4-dihydroxy-2-
methoxybenzene as its aglycon and two sugar units. The sugar
components were identified by GC analysis as D-glucose and
D-xylose. The HMBC spectrum revealed connectivities between (i)
δ 4.68 (H-1′) and 141.0 (C-1), (ii) δ 4.30 (H-1′′) and 69.7 (C-6′),
and (iii) δ 3.80 (MeO-2) and 152.1 (C-2). Consequently, the
structure of capparoside A (1) was established as 2-methoxy-4-
hydroxyphenol 1-O-ꢀ-D-xylopyranosyl-(1f6)-ꢀ-D-glucopyranoside.
Results and Discussion
A methanolic extract of the aerial parts of C. flaVicans was
partitioned with ethyl acetate and H
2
O to yield ethyl acetate- (22
2
O-soluble fractions (90 g). Then, the H O-soluble
g) and H
2
The methanolic extract of the leaves of V. glabrata was
partitioned with ethyl acetate and H
2
O to yield ethyl acetate- (76
O-soluble fraction
*
To whom correspondence should be addressed. Tel and Fax: + 81-
5
4-264-5661. E-mail: umehara@mail.u-shizuoka-ken.ac.jp.
g) and H O-soluble fractions (124 g). Next, the H
2
2
1
0.1021/np9006298 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/28/2009

Antiestrogenic Constituents of Thai Medicinal Plants
Journal of Natural Products, 2009, Vol. 72, No. 11 1955
Chart 1. Compounds from C. flaVicans and V. glabrata
was subjected to HP20 resin column chromatography by elution
with H O, 50% MeOH, and MeOH to give H O- (57 g), 50%
2 2
lignan glycoside with a dioxabicyclooctyl unit, and its structure
was similar to that of prinsepiol, which was first isolated from
Prinsepia utilus. Acid hydrolysis of 14 gave 3,4-methylenediox-
yprinsepiol as the aglycon and a sugar moiety, which was identified
by GC analysis as D-glucose. The HMBC spectrum showed
41
MeOH- (49 g), and MeOH-soluble fractions (19 g), respectively.
The MeOH eluate was subjected to silica gel column chromatog-
raphy and HPLC to give nine pure compounds, including three new
compounds (14-16). Six known compounds were identified as (-)-
2
connectivities of five partial structures, namely, (i) δ 5.91 (O-CH -
3
7
syringaresinol 4-O-ꢀ-D-glucopyranoside (17), (+) eucommin A
O) to δ 148.7, 148.8 (C-3, C-4); (ii) δ 6.93 (H-2) to δ 91.0 (C-7);
(iii) δ 7.28 (H-2′) to δ 88.6 (C-7′); and (iv) δ 4.78 (H-1′′) to δ
146.3 (C-3′). The attachment position of the sugar was also
confirmed by the NOE enhancement of H-2′ (δ 7.28), when the
anomeric proton (δ 4.78) was irradiated. The absolute configuration
of the dioxabicyclooctyl ring was determined to be 7R, 8S, 7′R,
and 8′S from the negative Cotton effect at 227 nm in the CD
spectrum, with reference to the CD data for princepiol and its
3
8
37
(
18), (()-isoeucommin A (19), (+) pinoresinol 4-O-ꢀ-D-
38
glucopyranoside (20), (-)-apigenin 7-O-ꢀ-D-glucopyranoside
3
9
40
(
21), and (-)-isocitruscin C (22), respectively, by comparison
of their spectroscopic data with literature values.
Khainaoside A (14), [R]
25
D
-40.0, was assigned the molecular
+
formula C25 13, as a result of its molecular ion [M + Na] peak
at m/z 559.1447 in the HRFABMS. The H NMR spectrum showed
two sets of trisubstituted aromatic rings from the signals at δ 7.28
28
H O
1
42
glucoside. Consequently, the structure of khainaoside A (14) was
assigned as 3,4-methylenedioxyprinsepiol 3′-O-ꢀ-D-glucopyrano-
side.
(
d, J ) 2 Hz), 6.96 (dd, J ) 8.5, 2 Hz), and 6.82 (d, J ) 8.5 Hz)
and δ 6.93 (d, J ) 2 Hz), 6.85 (dd, J ) 8, 2 Hz), and 6.77 (d, J )
Hz), and two pairs of methylene protons at δ 3.96 (d, J ) 10
Hz) and 4.07 (d, J ) 10 Hz) and δ 3.98 (d, J ) 10 Hz) and 4.07
2
5
8
Khainaoside B (15), [R]
32
molecular formula C26H O13 from its molecular ion [M + Na]
1
D
-63.8, was confirmed to have the
+
(
(
d, J ) 10 Hz). Three singlet signals at δ 4.93, 4.95, and 5.91
2H) were also observed together with an anomeric proton at δ
peak at m/z 575.1768 in HRFABMS. The H NMR spectrum
indicated trans olefinic protons at δ 7.58 (d, J ) 16 Hz) and 6.34
(d, J ) 16 Hz), ABX-type coupled aromatic protons at δ 7.17 (d,
4
.78 (d, J ) 7.5 Hz). The NMR spectra of 14 suggested it to be a

1
956 Journal of Natural Products, 2009, Vol. 72, No. 11
Luecha et al.
(1H and 13
C
J ) 2 Hz), 7.06 (dd, J ) 8.5, 2 Hz), and 6.80 (d, J ) 8.5 Hz), and
two hemiacetal protons at δ 4.69 (d, J ) 8 Hz) and 5.54 (d, J ) 5
Hz). The H NMR spectrum also suggested the presence of a
Table 1. NMR Data of Compound 1 in MeOH-d
at 400 and 100.4 MHz, respectively)
4
1
position
δ
H
(J in Hz)
δ
C
feruloyl moiety and a sugar in the structure of this compound. The
aglycon of 15 was suspected to be a monoterpene from its molecular
formula, and the NMR data indicated it to be an iridoid. The NMR
data of 15 were similar to those of 7-O-E-feruloylloganic acid
except for downfield shifts for H-9 (+0.5 ppm) and C-7 (+4.2 ppm)
1
2
3
4
5
6
141.0
152.1
102.1
154.9
107.8
120.7
104.2
74.9
77.8
71.5
77.3
69.7
6.96, d (3)
6.31, dd (9,3)
7.02, d (9)
4
3
and upfield shifts for H-7 (-0.35 ppm) and C-9 (-3.9 ppm).
These effects were due to the configuration of the methyl group at
1
′
4.68, d (8.5)
3.41, t (8.5)
3.42, t (8.5)
3.39, t (8.5)
3.48, m
4
4
2′
3
4
5′
6′
C-8 as found in 8-epiloganin. The HMBC spectrum gave
′
′
information of connectivities of three partial structures as (i) δ 5.54
(
H-1) to δ 99.8, 152.6 (C-1′, C-3); (ii) δ 7.17 (H-2′) to δ 146.7
(
C-7′); (iii) δ 7.58 (H-7′) to δ 169.0 (C-9′); and (iv) δ 4.69 (H-1′′)
3.75, dd (11.5,6)
4
.06, dd (11.5,2)
to δ 96.0 (C-1). A sugar moiety was identified by GC analysis as
D-glucose after an acid hydrolysis of 15. From this evidence, the
structure of khainaoside B (15) was assigned as 7-O-E-feruloyl-
1
2
3
4
′′
4.30, d (7.5)
3.19, dd (9,7.5)
3.30, t (9)
105.3
75.1
77.6
71.2
66.8
′′
′′
′′
8
-epiloganic acid.
3.48, m
25
Khainaoside C (16), [R]
D
-20.8, was assigned the molecular
5′′
3.15,
dd (11.5,10)
+
3.84, dd (11.5,5.5)
3.80, s
formula C25H O11, due to its molecular ion [M + Na] peak at
28
1
MeO
56.6
m/z 527.1514 in the HRFABMS. The H NMR spectrum showed
two sets of trisubstituted aromatic rings from the signals at δ 7.02
(
d, J ) 8.5 Hz), 6.99 (d, J ) 2 Hz), and 6.78 (dd, J ) 8.5, 2 Hz)
and δ 7.04 (d, J ) 2 Hz), 6.93 (dd, J ) 8.5, 2 Hz), and 6.79 (d, J
8.5 Hz). Two pairs of trans-olefinic protons at δ 6.40 (brd, J )
6 Hz) and 6.07 (dt, J ) 16, 6 Hz) and δ 7.53 (d, J ) 16 Hz) and
.24 (d, J ) 16 Hz) were observed, together with an anomeric
level less than 10 or 50 pM at the concentrations tested and were
expressed as strong (S) or mild (M), respectively.
Lignans having a dioxabicyclooctyl ring, such as syringaresinol
)
1
6
(
3) and pinoresinol, have been reported to be transformed to the
mammalian lignans enterodiol and enterolactone by human gut
46
proton at δ 4.85 (d, J ) 7 Hz). A singlet methoxy signal was also
found at δ 3.85. The NMR spectra of 16 were similar to those of
dracunculifoside D isolated from Baccharis dracunculifolia except
for the loss of one methoxy signal. The HMBC spectrum showed
connectivities of four partial structures, namely, (i) δ 4.85 (H-1′)
to δ 147.3 (C-4); (ii) δ 3.85 (OMe-3) to δ 151.0 (C-3); (iii) δ 6.99
microflora. These metabolites were recognized by their suppress-
4
7
2
ing effect on E -enhanced MCF-7 proliferation in nude mice.
Recently, a clear positive association between lignans and plasma
sex hormone-binding globulin (SHBG) levels was found by using
urinary enterodiol and enterolactone. The urinary lignans were also
negatively associated with plasma testosterone levels. These findings
2
45
(
H-2) to δ 131.0 (C-7); (iv) δ 7.04 (H-2′′) to δ 147.1 (C-7′′); (v)
δ 7.53 (H-7′′) to δ 168.9 (C-9′′); and (vi) δ 4.36, 4.50 (H-6′) to δ
68.9 (C-9′′). On acid hydrolysis, 16 gave caffeic acid and coniferyl
suggested that lignans promote greater binding of E to SHBG and
4
8
reduce the amount of free E2.
1
This is the first phytochemical study of any plant part of C.
flaVicans and of the leaves of V. glabrata and also the first
investigation of the estrogenic/antiestrogenic activities of isolates
from these species. These results highlight potential new uses for
these taxa. On the basis of the effects of the compounds found in
these assays, it is possible that C. flaVicans and V. glabrata promote
lactation by inhibiting the effect of estradiol in breastfeeding
women. However, a number of hormones and mechanisms are also
involved in lactation such as the dopaminergic system, thyroid
alcohol as aglycons and a sugar unit, which was identified by GC
analysis as D-glucose. Consequently, the structure of khainaoside
C (16) was assigned as {3-methoxy-4-[(6-O-(E)-caffeoyl)-ꢀ-D-
glucopyranosyl]}phenyl-1-propen-3-ol.
Estrogenic activity based on stimulatory effects on MCF-7 and
T47D cell proliferation was tested with increasing concentrations
of all 38 compounds obtained in this study at 0.1, 1, 10, and 100
µM, and their stimulatory activity on cell proliferation was
4
9
determined by comparison with a positive control, estradiol (E
2
),
hormones, and prolactin. Other mechanisms might promote such
activity, and detailed pharmacological and toxicological experiments
would be needed to confirm this hypothesis.
at concentrations ranging from 1 to 100 pM. The bioassay results
indicated that no compound exhibited significant stimulatory effects
on MCF-7 and T47D cell proliferation (data not shown).
Experimental Section
Antiestrogenic activities based on MCF-7 and T47D cell
proliferation were also investigated for all 38 compounds. Estradiol
(E ) at 100 pM was used initially to enhance cell proliferation, and
2
each compound was tested at 0.1, 1, 10, and 100 µM. Twenty-two
compounds showed significant antiestrogenic effects in this assay
General Experimental Procedures. Optical rotations were measured
on a JASCO DIP-360 digital polarimeter. UV spectra were obtained
using a Hitachi U2010 spectrophotometer, and CD spectra were
1
recorded on a JASCO J-20A spectropolarimeter. H NMR (400 MHz)
1
3
and C NMR (100 MHz) spectra were recorded on a JEOL JNM-R
00 instrument with chemical shifts in δ (ppm) using tetramethylsilane
TMS) as an internal standard, and coupling constants, J, are in hertz.
(
Table 3). Lignans 3, 6, and 14 exhibited strong inhibitory activity
on E -enhanced cell proliferation for both cell lines, and low
concentrations were found for their iEqE values (3, 5 and 7 µM;
, 7 and 6 µM; 14, <0.1 and 3 µM for MCF-7 and T47D,
4
(
2
1
Mass spectra were recorded using JEOL JMS 700 mass spectrometers.
HPLC was carried out with a JASCO model 887-PU pump and an
875-UV variable-wavelength detector with reversed-phase columns
(TSK-GEL ODS, 5 µm, 2 × 25 cm, Tosoh Chemicals Co., Ltd., at 9
mL/min with detection at 205 nm; Develosil-Lop-ODS, 12-20 µm, 5
6
respectively). Those compounds showing antiestrogenic activity
were observed to commonly bear a dioxabicyclooctyl ring in their
structure. Although 4 has an analogous structure to 3, the two
catechol groups in the structure led to a lower activity when
compared to two 3,5-dimethoxypyrogalloyl groups. The analogous
lignan glycosides, 17-20, showed high activity particularly against
×
50 cm × 2, Nomura Chemical Co., Ltd., at 45 mL/min with detection
at 205 nm; and Ascentis Phenyl, 5 µm, 2.2 × 25 cm, Supelco at 9
mL/min with detection at 205 nm). Gas chromatography (GC) was
performed on a Hitachi G-3000 gas chromatograph.
1
MCF-7 cells, with low iEqE values. The phenylpropanoid gluco-
Chemicals. Fetal bovine serum (FBS) was purchased from Gibco
Grand Island, NY). Eagle’s MEM and RPMI media were purchased
sides (16, 22) also displayed high activity against MCF-7 cells,
whereas the phenol glucosides (1, 10-12) showed lower activity
for the cells. Some samples constantly suppressed E activity to a
2
(
from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Antibiotics were
purchased from Meiji Seika Kaisha Ltd. (Tokyo, Japan). L-Glutamine

Antiestrogenic Constituents of Thai Medicinal Plants
Journal of Natural Products, 2009, Vol. 72, No. 11 1957
(1H and 13C at 400 and 100.4 MHz, respectively)
4
Table 2. NMR Data for Compounds 14-16 in MeOH-d
1
4
15
16
position
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
1
2
3
4
5
6
132.3
109.4
148.7
148.8
108.5
122.0
5.54, d (5)
7.43, s
96.0
133.9
111.8
151.0
147.3
118.4
120.5
6.93, d (2)
6.99, d (2)
b
b
152.6
113.5
31.8
6.77, d (8)
6.85, dd (8,2)
3.10, ddd (8.5,8,7.5)
1.99, dt (15,7.5)
7.02, d (8.5)
6.78, dd (8.5,2)
39.1
2
.25, ddd (15,8.5,3,5)
7
8
9
4.93, s
91.0
89.0
76.6
4.93, ddd (7.5,5,3.5)
2.45, quin,d (8,5)
2.60, ddd (8,8,5)
82.7
43.1
43.3
6.40, br, d (16)
6.07, dt (16,6)
4.14, dd (6,1.5)
131.0
129.0
63.7
3.98, d (10)
a
4
.07, d (10)
1
1
1
2
3
4
5
6
0
1
′
1.12, d (8)
7.17, d (2)
14.3
170.6
127.8
111.9
149.4
150.6
116.5
124.1
130.0
118.6
146.3
148.2
116.5
124.3
4.85, d (7)
3.56, dd (9,7)
3.48, t (9)
3.38, t (9)
3.67, m
102.7
74.9
77.9
72.1
75.6
64.6
′
7.28, d (2)
′
′
′
6.82, d (8.5)
6.96, dd (8.5,2)
6.80, d (8.5)
7.06, dd (8.5,2)
′
4.36, dd (12,7.5)
.50, dd (12,2.5)
4
7′
8′
9′
4.95, s
88.6
89.0
77.0
7.58, d (16)
6.34, d (16)
146.7
115.8
169.0
3.96, d (10)
a
4
.07, d (10)
1′′
2′′
3′′
4′′
5′′
6′′
4.78, d (7.5)
3.47, dd (9,7.5)
3.38, t (9)
104.2
74.9
77.7
71.5
78.4
62.7
4.69, d (8)
3.21, dd (9,8)
3.38, t (9)
3.25, t (9)
3.28, m
99.8
74.8
78.0
71.8
78.4
62.9
127.8
115.2
146.9
149.7
116.6
123.1
7.04, d (2)
3.45, t (9)
6.79, d (8.5)
6.93, dd (8.5,2)
3.68, dd (12,5.5)
.89, dd (12,2)
3.65, dd (12,6.5)
3.90, dd (12,2)
3
7
8
9
′′
′′
′′
7.53, d (16)
6.24, d (16)
147.1
115.1
168.9
OCH
MeO
2
O
5.91, s
102.5
3.88, s
56.5
3.85, s
56.8
a
b
Signals can be interchanged in the same column. Signals can be interchanged in the same column.
a
Table 3. Inhibitory Activities of Isolates against E
2
-Enhanced MCF-7 and T47D Cell Proliferation
MCF-7
T47D
b
b
compound
iEqE50 (µM)
iEqE10 (µM)
iEqE
1
(µM)
IL
iEqE50 (µM)
iEqE10 (µM)
iEqE
1
(µM)
IL
c
1
2
3
4
5
6
7
8
9
<0.1
<0.1
2
0.2
<0.1
0.3
0.3
6
4
31
6
>100
>100
5
<0.1
<0.1
<0.1
0.2
<0.1
0.4
<0.1
0.6
0.2
<0.1
2
>100
>100
1
26
9
1
63
>100
61
10
37
39
69
<0.1
<0.1
<0.1
19
>100
>100
7
M
M
c
94
73
>100
>100
4
7
6
0.3
>100
>100
93
15
>100
>100
>100
>100
70
>100
>100
3
>100
>100
>100
>100
>100
>100
2
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
4
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
0.1
0.8
<0.1
0.2
10
11
12
13
14
15
16
17
18
19
20
21
22
>100
>100
>100
>100
d
<0.1
<0.1
<0.1
<0.1
<0.1
4
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
0.2
S
0.4
2
d
S
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
0.1
d
d
d
>100
S
S
S
5
<0.1
>100
<0.1
<0.1
d
S
2
d
d
<0.1
<0.1
<0.1
>100
0.7
S
S
d
>100
S
c
0.4
0.4
>100
M
e
Tam
9
a
iEqE50, iEqE10, and iEqE
1
represent the concentrations of the compound that inhibited the cell proliferation enhanced by 100 pM of E
2
to levels
equivalent to those induced by 50, 10, and 1 pM of E
2
treatment, respectively. These values were determined by linear regression analysis using four
b
c
different concentrations. IL Inhibitory level of the compound. Mild inhibition (M), more than 50% inhibition through the concentrations tested.
d
e
Strong inhibition (S), more than 90% inhibition through the concentrations tested. Tamoxifen.
was purchased from Wako Pure Chemical Industries Ltd. (Osaka,
Japan). 17ꢀ-Estradiol and dextran-coated charcoal (DCC) were pur-
chased from Sigma Chemicals (St. Louis, MO).
Plant Materials. The aerial parts of Capparis flaVicans Kurz. were
collected at Khon Kaen Province, Thailand, in August 2006, and the
leaves of Vitex glabrata R.Br. were collected at Chantaburi Province,

1
958 Journal of Natural Products, 2009, Vol. 72, No. 11
Luecha et al.
Thailand, in November 2007. The plants were identified by Dr. T.
Thitimetharoj, a botanist from the Faculty of Pharmaceutical Sciences,
Khon Kaen University, Thailand, and voucher specimens numbered
PL-001 and PL-003, respectively, were deposited at the herbarium of
the Faculty of Pharmaceutical Sciences, Khon Kaen University.
Extraction and Isolation. The dried aerial parts of C. flaVicans (2.6
kg) were extracted three times with hot MeOH (3 × 20 L) by refluxing
for 6 h. The extracts were combined and concentrated under reduced
pressure at 60 °C to yield 130 g of dried extract. This concentrated
extract was suspended in water (1.5 L) and partitioned with EtOAc (6
EtOAc- (76 g) and H
fraction was subjected to HP20 column chromatography using H
50% MeOH, and MeOH as eluents (5 L each) to yield H O (57 g),
2
O-soluble (124 g) fractions. The H
2
O-soluble
2
O,
2
50% MeOH (49 g), and MeOH extracts (19 g). The MeOH eluate (19
g) was separated by silica gel column chromatography (6 × 50 cm)
and fractionated (300 mL for each fraction) using
a
2
chloroform-MeOH-H O gradient solvent system (9:1:0, 8:2:0, 8:2:
0.2, 8:3:0.3, 65:35:10, 5 L each). Fractions were collected and pooled
by TLC analysis to afford 14 combined fractions. From these combined
fractions, fraction 5 [0.3 g: eluted with chloroform-MeOH-H O (8:
2
2:0)] was subjected to semipreparative HPLC [column: Tosoh, TSKgel
×
1.5 L) to yield dried EtOAc- (22 g) and H
The H O-soluble fraction was subjected to HP20 column chromatog-
raphy using H O, 50% MeOH, and MeOH as eluents (5 L each) to
yield H O (60 g), 50% MeOH (18 g), and MeOH eluates (3 g). The
2
O-soluble (90 g) fractions.
2
ODS-100 V, 2 × 25 cm; solvent: acetonitrile-water (25:75); detector:
2
R R
205 nm] to give 17 (69 mg; t 15 min), 18 (24 mg; t 17 min), and 22
2
(20 mg;
chloroform-MeOH-H
HPLC [column: Tosoh, TSKgel ODS-100 V, 2 × 25 cm; solvent:
MeCN-water (20:80); detector: 205 nm] to give 19 (6 mg; t 32 min)
and 20 (16 mg; t 34 min). Fraction 9 [1.9 g: eluted with
chloroform-MeOH-H O (8:2:0)] was subjected to semipreparative
HPLC [column: GL Sciences, Inertsil ODS-EP, 2 × 25 cm; solvent:
acetonitrile-water (20:80); detector: 205 nm] to give 21 (12 mg; t 44
O (8:2:0)]
t
R
29 min). Fraction
6
[0.5 g: eluted with
EtOAc-soluble fraction (22 g) was chromatographed on a silica gel
column (6 × 50 cm) and fractionated (300 mL for each fraction) using
a chloroform-MeOH gradient solvent system (99:1, 98:2, 95:5, 90:
2
O (8:2:0)] was subjected to semipreparative
R
1
0, 5 L each). Fractions were collected and pooled by TLC analysis to
R
afford 15 combined fractions. From these combined fractions, fraction
2
5
[0.6 g: eluted with chloroform-MeOH (99:1)] was subjected to
semipreparative HPLC [column: Tosoh, TSKgel ODS-100 V, 2 × 25
R
cm; solvent: MeOH-water (40:60); detector: 205 nm] to give 3 (2
min). Fraction 8 [1.2 g: eluted with chloroform-MeOH-H
2
mg; t
R
28 min). Fraction 15 [4.4 g: eluted with chloroform-MeOH
was subjected to preparative HPLC [column: Tosoh, TSKgel ODS-
(
9:1)] was subjected to preparative HPLC [column: Nomura Chemical,
Develosil-Lop-ODS, 5 × 50 cm × 2; solvent: acetonitrile-water (18:
2 f 38:62); detector: UV 205 nm] to afford 20 fractions. From these
fractions, fractions 9 (52 mg; t 190 min), 11 (46 mg; t 230 min), 13
45 mg; t 270 min), 14 (36 mg; t 300 min), 16 (50 mg; t 390 min),
and 17 (53 mg; t 420 min) were subjected to semipreparative HPLC
column: Supelco, Ascentis Phenyl, 2.2 × 25 cm; detector: UV 205
nm] to yield 4 [50 mg; t 32 min with acetonitrile-water (17:83)],
tamarixetin3-O-ꢀ-D-galactoside[2mg;t 110minwithacetonitrile-water
15:85)], 5 [2 mg; t
americanol A [7 mg; t
isoprincepin [5 mg; t
and 6 [7 mg; t
80-TS, 6 × 60 cm × 2; solvent: acetonitrile-water (20:80 f 30:70);
R
detector: UV 205 nm] to afford 15 (38 mg; t 246 min) and 12 fractions.
8
R R
From these fractions, fractions 6 (26 mg; t 146 min) and 7 (33 mg; t
160 min) were subjected to semipreparative HPLC [column: Tosoh,
R
R
(
R
R
R
TSKgel ODS-100 V, 2 × 25 cm; detector: 205 nm] to yield 14 [8 mg;
R
t
R
R
22 min with acetonitrile-water (22.5:77.5)] and 16 [3 mg; t 22
[
min with acetonitrile-water (20:80)]. All known compounds were
identified by comparison with published data.
Acid Hydrolysis of Glycosides (1 and 14-16). Identification of
37–40
R
R
5
0
(
R
145 min with acetonitrile-water (15:85)],
R
sugars was conducted according to a previously reported procedure.
Compound 1 (1 mg) was heated to 100 °C with dioxane (0.05 mL)
and 5% H SO (0.05 mL) for 1 h. After dilution with H O, the reaction
mixture was extracted twice with EtOAc, and the H O layer was passed
through an Amberlite IRA-60E column. The H O eluate was concen-
trated, and the residue was treated with D-cysteine (0.05 mg) in H
72 min with acetonitrile-water (20:80)],
100 min with acetonitrile-water (22.5:77.5)],
R
2
4
2
R
56 min with MeOH-water (50:50)], respectively.
The 50% MeOH-soluble (18 g) fraction was chromatographed by
2
2
silica gel column chromatography (6 × 50 cm) and fractionated (300
2
O
mL for each fraction) using a chloroform-MeOH-H
2
O gradient
(0.03 mL) and pyridine (0.015 mL) to 60 °C for 1 h with stirring. After
the solution had been concentrated and the reaction mixture dried,
pyridine (0.015 mL), hexamethyldisilazane (0.015 mL), and trimeth-
ylsilyl chloride (0.015 mL) were added to the residue. The reaction
mixture was then heated at 60 °C for 30 min, and the supernatant was
analyzed by GC. GC conditions: column Supelco SPB-l, 0.25 mm ×
solvent system (80:20:0, 80:20:0.2, 7:13:8, 65:35:10, 5 L each).
Fractions were collected and pooled by TLC analysis to afford 13
combined fractions. From these combined fractions, fraction 3 [0.4 g:
eluted with chloroform-MeOH (8:2)] was subjected to semipreparative
HPLC [column: Supelco, Ascentis Phenyl, 2.2 × 25 cm; solvent:
acetonitrile-water (15:85); detector: UV 205 nm] to give 5,5′-
27 m; column temp: 230 °C; carrier gas: N
2
; t
R
(min): D-glucose (18.8),
dimethoxy-4-O-(ꢀ-D-glucopyranosyl)lariciresinol (2 mg; t
R
58 min),
L-glucose (17.7), D-xylose (10.4), and L-xylose (9.7). The t of D-glucose
R
alangilignoside C (20 mg; t 81 min), and alangilignoside D (8 mg; t
R
R
and D-xylose were detected from 1. Acid hydrolysis of 14-16 was
performed by the same manner used for 1, and the t of D-glucose was
R
89 min). Fraction 4 [1.5 g: eluted with chloroform-MeOH-water (8:
2:0.2)] was subjected to semipreparative HPLC (column: Supelco,
detected in these compounds.
2
5
Ascentis Phenyl, 2.2 × 25 cm; detector: UV 205 nm) with various
Capparoside A (1): colorless, amorphous powder; [R]
D
-62.8 (c
solvent systems to afford pure compounds as follows: acetonitrile-water
0.2, MeOH); UV (MeOH) λmax (log ε) 222 (sh) (2.76), 285 (2.49), 308
1
13
(
12.5:87.5) solvent system gave 3R-O-(ꢀ-D-glucopyranosyl) lyoniresinol
(sh) (2.13) nm; H and C NMR, Table 1; HRFABMS m/z 457.1331
+
(
88 mg; t
4 min), 2 (2 mg; t
6 mg; t 96 min), 7 (8 mg; t
130 min); acetonitrile-water (10:90) yielded icariside B
6 min) and roseoside (24 mg; t 38 min); acetonitrile-water (5:95)
26 min), isotachioside (12 mg; t 35 min),
48 min), 11 (11 mg; t 35 min), 12 (25 mg; t
56 min), and 4-(hydroxymethyl)-2,6-dimethox-
yphenyl 1-O-ꢀ-D-glucopyranoside (20 mg; t 80 min). In addition, on
R
64 min), alangionoside C (6 mg; t
94 min), lariciresinol 4-O-ꢀ-D-glucopyranoside
104 min), and alangilignoside B (4 mg;
(9 mg; t
R
54 min), 8 (10 mg; t
R
[M + Na] (calcd for C18
Khainaoside A (14): brown, amorphous powder; [R]
0.2, MeOH); UV (MeOH) λmax (log ε) 230 (4.26), 282 (3.96), 317 (sh)
26
H O12 Na, 457.1322).
2
5
7
(
R
D
-40.0 (c
R
R
1
13
t
R
1
R
(3.18) nm; CD (c 0.2 mM, MeOH) [θ]227 -5798; H and C NMR,
+
3
R
28 13
Table 2; HRFABMS m/z 559.1447 [M + Na] (calcd for C25H O
gave 10 (18 mg; t
vanilloloside (6 mg; t
8 min), 13 (14 mg; t
R
R
Na, 559.1428).
2
5
R
R
R
Khainaoside B (15): brown, amorphous powder; [R]
D
-63.8 (c
5
R
0.2, MeOH); UV (MeOH) λmax (log ε) 234 (4.39), 299 (sh) (4.15), 356
1
13
R
(4.29) nm; CD (c 0.4 mM, MeOH) [θ]235 -18450; H and C NMR,
+
using an ODS column [Tosoh, TSKgel ODS-100 V, 2 × 25 cm; solvent,
32 13
Table 2; HRFABMS m/z 575.1768 [M + Na] (calcd for C26H O
MeOH-water (10:90); detector 205 nm], fraction 4 also gave 9 (26
Na, 575.1741).
2
5
mg; t
R
20 min). Fraction 7 [1.4 g: eluted with chloroform-MeOH-water
Khainaoside C (16): brown, amorphous powder; [R]
D
-20.8 (c
(
7:13:8)] was subjected to semipreparative HPLC [column: Tosoh,
TSKgel ODS-100 V, 2 × 25 cm; solvent: acetonitrile-water (5:95);
detector: 205 nm] to give 1 (2 mg; t 34 min) and 4-hydroxy-3-
methoxyphenyl 1-O-ꢀ-D-xylopyranosyl-(1f6)-ꢀ-D-glucopyranoside (2
mg; t 32 min). All known compounds were identified by comparison
with published data.
0.2, MeOH); UV (MeOH) λmax (log ε) 250 (4.08), 264 (4.03), 298 (sh)
1
13
(4.01), 329 (4.03) nm; CD (c 0.02, MeOH); H and C NMR, Table
+
R
28
2; HRFABMS m/z 527.1514 [M + Na] (calcd for C25H O11 Na,
527.1529).
R
Cell Culture. MCF-7 and T47D human breast cancer cells were
1
5–36
51
purchased and cultured as described in a previous report.
The dried leaves of V. glabrata (3.0 kg) were extracted three times
with hot MeOH using the same method as described above for C.
flaVicans. Extracts were combined and concentrated to give 566 g of a
dried extract. A part of this concentrated extract (200 g) was suspended
in water (1.5 L) and partitioned with EtOAc (6 × 1.5 L) to yield dried
Cell Proliferation Assay. The cell proliferation assay was performed
5
1
as described in a previous report. Fluorescence was measured at 590
nm with excitation at 550 nm using a Wallac 1420 ARVOsx multilabel
counter (Perkin-Elmer Inc., Wellesley, MA) to evaluate cell concentra-
tions. Estrogenic activity of isolates was determined by this bioassay.

Antiestrogenic Constituents of Thai Medicinal Plants
Journal of Natural Products, 2009, Vol. 72, No. 11 1959
Antiestrogenic Assay. Antiestrogenic assay was conducted according
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to the procedure for the cell proliferation assay with minor modifica-
4
tions. MCF-7 or T47D cells were seeded at a density of 1 × 10 cells/
well in 96-well plates in 90 µL of 5% DCC-treated, FBS-supplemented
(
19) Weibel, R.; Benirschke, G.; Benirschke, M.; Achenbach, H. Phy-
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2
RPMI phenol red-free medium. Then, 5 µL of estradiol (E ) at a
concentration of 20 nM was added into each well, and the plates were
incubated at 37 °C with 5% CO for 1 h. A 5 µL portion of each test
compound was added to each well with concentrations ranging from 2
to 2000 µM and incubated in a CO incubator for 96 h. In all
experiments, 5 µL of serially diluted tamoxifen at concentrations from
to 200 µM were used as positive controls. Antagonistic effects of
samples were evaluated from the cell populations, and iEqE values of
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4
each sample (iEqE50, iEqE10, and iEqE
required concentrations to inhibit the E
iEqE , the concentration suppressing the E
level of 50; 10; and 1 pM, respectively). When samples constantly
suppressed E activity to the level less than 10 or 50 pM through the
1
) were determined for the
(
2
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1
1
2
effect to the equivalent
(
(
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2
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Data and Statistical Analysis. Statistical differences were deter-
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comparison test. Statistical significance was established at the p < 0.05
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(
Supporting Information Available: H, 13C, and HMBC spectra
of 1 and 14-16. This material is available free of charge via the Internet
at http://pubs.acs.org.
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NP9006298