Phyto-SERM Constitutes from Flemingia macrophylla

Abstract

The methanolic extract of Flemingia macrophylla roots exhibited significant estrogenic activity in the transgenic plant assay system which was comparable to the activity of soybean extract. Utilizing estrogenic activity-guided fractionation, one new compound, fleminigin, together with 23 known compounds were isolated from F. macrophylla roots' methanolic extract. The structure of the new compound was identified based on intensive spectroscopic analysis and the full spectral data for one of the isolated compounds, flemichin E, was introduced for the first time in the current investigation. The estrogenic and anti-estrogenic activities of the isolated compounds were evaluated revealing that the isolated isoflavonoids may act as partial estrogen agonists, as well as antagonists. Additionally, the anti-inflammatory and the cytotoxic activities of the isolated compounds were studied. These results suggested the potential applications of F. macrophylla extract and its isolated compounds as selective estrogen receptor modulators (SERMs).

Full text

Int. J. Mol. Sci. 2013, 14, 15578-15594; doi:10.3390/ijms140815578
International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Article
Phyto-SERM Constitutes from Flemingia macrophylla
Wan-Chun Lai
1
, Ya-Ting Tsui
1
, Abdel Nasser B. Singab
2
, Mohamed El-Shazly
1,2
,
Ying-Chi Du
1
, Tsong-Long Hwang
3
, Chin-Chung Wu
1
, Ming-Hong Yen
1
, Ching-Kuo Lee
4
,
Ming-Feng Hou
5
, Yang-Chang Wu
6,7,8,
* and Fang-Rong Chang
1,5,
*
1
Graduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University,
Kaohsiung 80708, Taiwan; E-Mails: stellapple7@gmail.com (W.-C.L.);
shinygirl1983@hotmail.com (Y.-T.T.); ycdu0626@gmail.com (Y.-C.D.);
ccwu@kmu.edu.tw (C.-C.W.); yen@kmu.edu.tw (M.-H.Y.)
2
Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy,
Ain-Shams University, Organization of African Unity Street, Abassia, Cairo 11566, Egypt;
E-Mails: dean@pharma.asu.edu.eg (A.N.B.S.); elshazly444@googlemail.com (M.E.-S.)
3
Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan;
E-Mail: htl@mail.cgu.edu.tw
4
Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei 110, Taiwan;
E-Mail: cklee@tmu.edu.tw
5
Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan;
E-Mail: mifeho@kmu.edu.tw
6
School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan
7
Chinese Medicine Research and Development Center, China Medical University Hospital,
Taichung 404, Taiwan
8
Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan
* Authors to whom correspondence should be addressed;
E-Mails: yachwu@mail.cmu.edu.tw (Y.-C.W.); aaronfrc@kmu.edu.tw (F.-R.C.);
Tel.: +886-4-2205-3366-1012 (Y.-C.W.); +886-7-3121-101-2162 (F.-R.C.);
Fax: +886-4-2206-0248 (Y.-C.W.); +886-7-3114-773 (F.-R.C.).
Received: 15 March 2013; in revised form: 6 July 2013 / Accepted: 15 July 2013 /
Published: 26 July 2013
Abstract: The methanolic extract of Flemingia macrophylla roots exhibited significant
estrogenic activity in the transgenic plant assay system which was comparable to the
activity of soybean extract. Utilizing estrogenic activity-guided fractionation, one new
compound, fleminigin, together with 23 known compounds were isolated from
OPEN ACCESS

Int. J. Mol. Sci. 2013, 14 15579
F. macrophylla roots’ methanolic extract. The structure of the new compound was
identified based on intensive spectroscopic analysis and the full spectral data for one of the
isolated compounds, flemichin E, was introduced for the first time in the current
investigation. The estrogenic and anti-estrogenic activities of the isolated compounds were
evaluated revealing that the isolated isoflavonoids may act as partial estrogen agonists, as
well as antagonists. Additionally, the anti-inflammatory and the cytotoxic activities of the
isolated compounds were studied. These results suggested the potential applications of
F. macrophylla extract and its isolated compounds as selective estrogen receptor
modulators (SERMs).
Keywords: Flemingia macrophylla; menopausal; phytoestrogen; fleminigin; flemichin E;
pER8:GUS
1. Introduction
Plants with estrogenic properties have been widely used as dietary supplements for postmenopausal
women in the last few decades [1]. They possess a myriad of protective physiological activities against
neurological diseases [2], hypertension [3], cardiovascular diseases [4] and excessive oxidative
reactions [5,6]. Identification of phytoestrogenic natural sources from thousands of terrestrial plants is
a challenging task, which represents the bottle neck in developing new dietary supplements and
pharmaceutical lead drugs. This stern challenge was tackled by several research groups in the past few
decades developing in vitro techniques for their detection. One of the highly efficient detection
techniques is the transgenic plant system, pER8:GUS, which contains a human estrogen receptor (ER)
and β-glucuronidase (GUS) reporter genes [7]. The transgenic plant system can be easily utilized to
screen the estrogenic activity of extracts and pure compounds with high efficiency and versatility [8].
In our ongoing research to discover new sources of phytoestrogens, 22 herbal (folk) medicines used in
the treatment of fractures, osteoporosis, and related bone diseases were selected for evaluating their
estrogenic activity. Among these herbs, the methanolic (MeOH) extract of Flemingia macrophylla
(Willd.) Kuntze ex Prain roots showed significant estrogen–like activity with a minimum active
concentration (MAC) of 0.78 μg/mL, which was higher but still comparable to soybean extract, a
standard phytoestrogenic extract with a MAC of 0.25 μg/mL.
F. macrophylla is a shrubby herb which is widely distributed in southern China, India, Indonesia,
and Taiwan [9]. The roots of this plant have been used in folk medicine for the treatment of fractures,
trauma, arthritis, rheumatism, and influenza. Previous studies reported that F. macrophylla crude
extract exhibited neuroprotective [10], analgesic [11], and anti–inflammatory activities [11]. It was
also found to be effective in the treatment of osteoporosis [12]. Recent reports have suggested a close
relationship between F. macrophylla estrogen-like activity and its neuroprotective [10] and
anti-osteoporotic activities [12]. Despite these findings, the effect of F. macrophylla extracts and its
purified compounds on estrogen receptors has never been investigated.
In this study, the estrogenic/antiestrogenic effects of F. macrophylla extract and its constituents
were investigated using a transgenic plant assay system as well as an in vitro reporter assay in human

Int. J. Mol. Sci. 2013, 14 15580
breast cancer MCF-7 cell line. Bioactivity-guided fractionation of the extract led to the isolation of
24 compounds. Moreover, the anti–inflammatory and cytotoxic activities of the isolated compounds
were also evaluated.
2. Results and Discussion
2.1. Structure Elucidation
Using bioactivity-guided fractionation, the MeOH extract of F. macrophylla roots was fractionated
by column chromatography over silica gel and purified by HPLC. Purification of the extract led to the
isolation of one new compound, fleminigin (1) and 23 known compounds, flemichin E (2) [13],
flemiphilippinin F (3) [14], genistin (4) [15], genistein (5) [16], 2'-hydroxygenistein (6) [17],
cajanin (7) [18], 3'-isoprenylgenistein (8) [19], 7-(3,3-dimethylallyl)genistein (9) [20], prunetin (10) [21],
olmelin (11) [22], erythrinin B (12) [23], 5,2',4'-trihydroxy-7-(3-methylbut-2-enyloxy)isoflavone (13) [24],
neoraufurane (14) [25], isoderrone (15) [17], fleminone (16) [26], an enantiomer of 5,2',4'-trihydroxy-
8,5'-di-(3-methylbut-2-enyl)-6,7-3,3-dimethylpyrano)flavanone (17 and 18) [20], flemiphilippinin D
(19) [27,28], flemiflavanone A (20) [29], flemichin-D (21) [27], 4'-O-methylgallocatechin (22) [30],
β-sitosterol (23) [31], and stigmasterol (24) [31] were obtained (Figure 1). Among the known
compounds, compound 2 was identified in a previous report using mass spectrometry, but its complete
1D and 2D NMR data have never been reported. Compounds 7–10 and 13–15 were isolated from this
species for the first time. Additionally, compound 9 was isolated for the first time from natural sources
(Figure 2).
Compound 1 was obtained as a yellow amorphous solid. The HRESIMS of 1 showed a molecular
ion at m/z 377.1001 [M + Na]
+
, indicating a molecular formula of C
20
H
18
O
6
(calculated 377.1005). UV
absorptions at 263, 295, and 335 nm suggested the presence of an isoflavone skeleton. The IR
spectrum showed characteristic absorptions for a hydroxy (3377 cm
−1
), carbonyl (1651 cm
−1
), and
aromatic (1615 and 1506 cm
−1
) functionalities. The
1
H NMR of 1 (in acetone-d
6
) showed a signal at
δ
H
8.19 (1H, s), which represented a characteristic signal of H–2 in the isoflavone nucleus (Table 1).
From the COSY spectrum, the correlation of one oxymethine (δ
H
4.57, 1H, q, J = 6.4) with one methyl
(δ
H
1.4, 3H, d, J = 6.4), as well as the observed HMBC correlations (Figure 2), suggested that a furan
moiety is fused to ring A in 1. The
1
H and
13
C NMR data of 1 (acetone-d
6
, Table 1) were similar to
those of a previously identified isoflavone, known as 5,7,4'-trihydroxy-4'',4'',5''(ξ)-trimethyl-4'',5''-
dihydro-furano-(7,6,2'',3'')isoflavone [32]. Guided by the reported data of the known compound, we
were able to elucidate the structure of compound 1. Acetone-d
6
and pyridine-d
5
were used as solvents
for
1
H and
13
C NMR analysis of 1 and the spectra were compared to those of the known compound [32].
For example, in compound 1 we noticed an ABX spin systems at δ
H
6.49 (1H, d, J = 2.2), 6.43 (1H,
dd, J = 8.4, 2.2), and 7.12 (1H, d, J = 8.4) indicating the presence of unusual 1',2',4'-trisubstitutions on
ring B as well as an aromatic proton on ring A at δ
H
6.22 (1H, s). These data were similar to those
reported for the known compound [32].

Int. J. Mol. Sci. 2013, 14 15581
Figure 1. Structures of the isolated compounds from F. macrophylla.

Int. J. Mol. Sci. 2013, 14 15582
Figure 2.
1
H-
1
H COSY (correlation spectroscopy) and HMBC (heteronuclear multiple
bond correlation) correlations of fleminigin (1).
Table 1.
13
C,
1
H and HMBC data for fleminigin (1).
Position
δ
C
(mult.)
a
δ
H
(mult., J in Hz)
b
HMBC (
1
H→
13
C)
2
155.1
8.33 (s)
3, 4, 9, 1'
3
121.7
4
181.9
5
165.2
6
94.7
6.53 (s)
5, 7, 8, 10
7
164.0
8
112.8
9
153.5
10
106.8
1'
110.0
2'
158.3
3'
104.4
7.08 (d, 2.4)
2', 4', 1', 5'
4'
160.7
5'
107.8
6.94 (dd, 8.4, 2.4)
1', 3'
6'
133.4
7.62 (d, 8.4)
3, 2', 4'
1''
90.9
4.43 (q, 6.4)
4'', 5''
2''
43.8
3''
14.2
1.28 (d, 6.4)
1'', 2'', 4'', 5''
4''
25.5
1.37 (s)
8, 1'', 2'', 5''
5''
21.3
1.14 (s)
8, 1'', 2'', 3'', 4''
a
13
C NMR (acetone-d
6
, 100 MHz) for 1;
b
1
H NMR (acetone-d
6
, 400 MHz) for 1.
The HMBC spectrum of the known compound showed correlations between the singlet aromatic
proton (H-8, δ
H
6.54) and the two carbons attached to the oxygen atom in ring A (C-7 and C-9)
(δ
C
153.9 and 164.4) [32] indicating that the furan moiety is fused to C-6 and C-7 of ring A. On the
other hand, the HMBC spectrum of 1 showed that the singlet aromatic proton (H-6, δ
H
6.22) is
correlated to two different carbons in ring A (C-5 and C-7) (δ
C
166.7 and 164.8), suggesting that the
furan moiety is linked to C-7 and C-8 of ring A (Figure 2). Based on the aforementioned data, the

Int. J. Mol. Sci. 2013, 14 15583
structure of the new compound 1 was identified as 5,2',4'-trihydroxy-7,8-(1,2,2-trimethyl-
dihydrofurano)isoflavone, and was named fleminigin (1).
Table 2.
13
C,
1
H and HMBC data for flemichin E (2).
Position
δ
C
(mult.)
a
δ
H
(mult., J in Hz)
b
HMBC (
1
H→
13
C)
2
77.2
5.52 (dd, 13.2, 3.0)
3
42.0
3.13 (dd, 17.1, 3.0)
2, 4
2.82 (dd, 17.1, 13.2)
4
4
196.5
5
156.7
5-OH
12.9 (s)
5, 10
6
103.2
7
158.7
8
108.7
9
159.7
10
102.6
1'
117.5
2'
153.6
3'
105.3
6.39 (s)
1', 4', 5'
4'
153.9
5'
111.0
6'
128.3
6.94 (s)
2, 4', 3''''
1''
115.6
6.64 (d, 10.2)
7, 3''
2''
126.2
5.51 (d, 10.2)
6, 3''
3''
78.2
4''-CH
3
28.3
1.43 (s)
2'', 3'', 5''
5''-CH
3
28.4
1.45 (s)
2'', 3'', 4''
1'''
21.4
3.20 (2H, d, 7.8)
7, 8, 9, 2''', 3'''
2'''
122.3
5.10 (td, 7.8, 1.8)
4''', 5'''
3'''
131.7
4'''-CH
3
25.8
1.67 (d, 1.8)
2''', 3''', 5'''
5'''-CH
3
17.8
1.68 (s)
2''', 3''', 4'''
1''''
77.1
2''''
69.7
3.80 (dd, 5.4, 4.8)
3''''
30.7
3.01 (dd,16.8, 4.8)
4', 5', 6', 2''''
2.71 (dd,16.8, 5.4)
4', 5', 6', 1'''', 2''''
4''''-CH
3
22.2
1.37 (s)
1'''', 2'''', 5''''
5''''-CH
3
24.8
1.33 (s)
1'''', 2'''', 4''''
a
13
C NMR (chloroform-d
1
, 150 MHz) for 2;
b
1
H NMR (chloroform-d
1
, 600 MHz) for 2.
Compound 2 was obtained as a yellow amorphous solid. The HRESIMS of 2 showed a molecular
ion peak at m/z 529.2202 [M + Na]
+
, corresponding to the molecular formula C
30
H
34
O
7
(calculated
529.2199). Full
1
H and
13
C NMR data are summarized in Table 2 as well as the 2D NMR spectra,
which were introduced for this compound for the first time. The planar structure of 2 was confirmed by
1
H and
13
C NMR data which were compared to the incomplete NMR data previously reported for this
compound [13]. In the NOESY spectrum, H–2'''' (δ
H
3.80, 1H, dd, J = 5.4, 4.8) showed a correlation

Int. J. Mol. Sci. 2013, 14 15584
with H-5'''' (1.33, s), confirming the relative configuration of H-2'''' and the methyl group (H-5'''') as
cis. However, the absolute configuration of C-2'''' is still uncertain. By comparing the CD values of 2
with the reported data of similar structures [28], a negative cotton effect at 300 nm and a positive
cotton effect at 340 nm indicated that the stereo configuration of C-2 is S. Thus, compound 2 was
identified as (2S)-5,2'-dihydroxy-6,7-(1,1-dimethylpyrano)-8-(3-methylbut-2-enyl)-4',5'-(2-hydroxy-
1,1-dimethylpyrano)flavanone, or flemichin E (2).
2.2. Estrogenic and Anti-Estrogenic Activities
The isolated compounds were subjected to the estrogen receptor (ER) binding affinity evaluation
assay utilizing an efficient transgenic plant assay system [8]. The results showed that compounds 4, 6,
7, and the reference drug, genistein (5), exhibited a significant estrogenic activity (MAC < 5 μM) in
the transgenic plant assay system (Table 3). Isoflavone derivatives, compounds 1, 3, 8, 12, and 13,
showed a moderate estrogenic activity. However, the flavone derivatives, compounds 19 and 21,
exhibited weak estrogenic activity only at high doses (MAC > 50 μM). Compound 22 even showed
weaker estrogenic activity exclusively at higher concentration (MAC > 300 μM).
Table 3. Estrogenic activity of F. macrophylla isolated compounds.
Compounds
MAC (μM)
17β-estradiol
5.93 × 10
−4
1
35.31
3
36.98
4
0.0139
5
0.0037
6
0.042
7
2.6
8
36.98
12
59.17
13
28.25
16
>500
19
58.96
21
118.48
22
312.50
Furthermore, the estrogenic activity of the isolated compounds was confirmed by the transcription
of an estrogen responsive element (ERE) utilizing human breast cancer ER–positive MCF-7 cells. The
activity of the isolated compounds on the ERE was determined by the detection of the secreted alkaline
phosphatase (SEAP) reporter protein. The results showed that compounds 3, 4, 6, 7, 9, and 10
significantly increased the estrogenic activity (increase in SEAP% by 500%–700%) at a concentration
of 10 μM (Figure 3), which was comparable to those of genistein (5) at 10 μM (Figure 3). Compounds
1, 8, 19, and 21 showed a slight increase in the estrogenic activity (increase in SEAP% < 500%)
compared to the basal activity (SEAP% 100%). However, compounds 16, 18, 20, and 22 did not show
any estrogenic activity (Figure 3).

Int. J. Mol. Sci. 2013, 14 15585
Figure 3. Estrogenic activity in MCF-7 cells of pure components from F. macrophylla.
The SEAP activity induced by the positive control, E2 (0.1 nM). Cells were treated by the
target compounds (10 μM) and the SEAP activity was compared to B (Blank), which was
set to 100% SEAP activity. Each column represents a percentage of SEAP activity and is
expressed as mean ± SEM (n = 4).
Figure 4. Antiestrogenic activity in MCF-7 cells of pure components from F. macrophylla.
The SEAP activity induced by E2 (0.1 nM) was set to 100%. Cells treated with E2 (0.1 nM)
and tamoxifen (TAM, 2 μM) or raloxifene (RAL, 20 nM), were selected as positive
controls. Pure compounds (10 μM) were treated in the presence of E2 (0.1 nM). Each
column represents a percentage of SEAP activity and is expressed as mean ± SEM (n = 4).
Certain phytoestrogens and estrogenic active compounds may possess counter pharmacological
activity through acting as antiestrogenic agents, and they are known as selective estrogen receptor
modulators (SERMs) [33]. Therefore, the isolated compounds were treated with E2 (0.1 nM) to

Int. J. Mol. Sci. 2013, 14 15586
determine their antiestrogenic activity in the ER–positive MCF-7 cell line. Results showed that
compounds 4–8 reduced the ERE transcriptional activity of MCF-7 cells in the presence of E2
(Figure 4), which suggests that 4 and 6–8 may act as partial estrogen antagonists similar to genistein
(5) [34]. Interestingly, 3'-isoprenylgenistein (8) showed a more potent anti-estrogenic effect compared
to genistein (5). This dual activity opens a new avenue for selecting and designing a perfect SERM,
acting preferentially on the nervous and cardiovascular systems, with minimum activity on mammary
glands and uterus.
Several research groups have shown that isoflavonoids exhibited more potent estrogenic activity
compared to flavonoids [35]. Certain structural features in isoflavonoids seemed to affect their
phytoestrogenic activity. The parent skeleton should have at least a single hydroxy group in ring A or
B. The highest estrogenic activity resulted from the presence of two hydroxy groups at the longitudinal
extremities of the skeleton at C-7 and C-4' [36]. Through studying the estrogenic activity of the
isolated compounds, we found that the most active compound was genistein (5) with two hydroxy
groups at C-7 and C-4'. It has an additional hydroxy group at C-5 which forms an intramolecular
hydrogen bond to the carbonyl group improving the hydrogen bond formation ability of 7-OH to the
ER binding site [37,38].
On the other hand, the presence of 2′-OH as in 6 resulted in an 11–fold reduction of the estrogenic
activity (Table 3). The activity was even more dramatically reduced when 7-OH was replaced by
O-isoprenyl substitution as in 13. Glucosylation of the hydroxy group at C-7 as in 4 resulted in a 3–4
fold decrease in activity compared to genistein (5) implying the importance of a free hydroxy group at
C-7 [37]. This observation was also supported by the marked decrease in the estrogenic activity
(60 folds) of compound 7 possessing a methoxy group at C-7 (Table 3). Compound 12 showed only
weak estrogenic activity suggesting that the presence of 6-isoprenyl moiety reduced the ER binding
affinity. Moreover, the presence of 3'-isoprenyl unit (8) or 7,8-furan ring (1 and 3) reduced the
estrogenic activity (MAC 35.31-37.98 μM) (Table 3). The estrogenic activity of the prenylated
flavonoids (16, 19, and 21) was lower compared to isoflavonoids. However, the data of the in vitro
reporter assay (Figure 4) revealed that compounds 3–7 exhibited comparable estrogenic activity. The
data collected from both assays suggested that the estrogenic activity of the isoflavonoids may be
attributed to the ER-dependent or ER-independent pathways [39].
2.3. Anti–Inflammatory and Cytotoxic Activities
The anti-inflammatory activity of F. macrophylla crude extract has been recently reported [11]. The
authors utilized λ-carrageenan to induce inflammation and paw edema in mice. The inflammation
response has been linked to the neutrophils’ infiltration and the production of neutrophil-derived free
radicals [11].There is considerable evidence from clinical and experimental studies that the recruitment
of neutrophils into joints is a critical hallmark of rheumatoid arthritis [40,41].
The anti-inflammatory activity was measured through testing the inhibition of superoxide anion
generation and elastase release by neutrophils after induction with formyl-methionyl-leucyl-phenylalanine
(fMLP) and cytochalasin B (CB). Compounds 3, 6, 7, and 8 showed antioxidant activity against the
production of superoxide anions (Table 4). Compounds 6 and 7 were able to compete in the inhibition
of elastase release (IC
50
4.32 and 4.33 μg/mL) to the same level demonstrated by the positive control,

Int. J. Mol. Sci. 2013, 14 15587
genistein (5) (IC
50
4.25 μg/mL). A recent report [42] has suggested the therapeutic potential of
genistein in targeting rheumatoid arthritis through its anti-inflammatory effect. The structural
similarity between compounds 6, 7, as well as 8 and genistein (5) suggests a similar anti-inflammatory
effect for these compounds which should be confirmed with further in vitro and in vivo studies.
We also evaluated the cytotoxic activity of the isolated compounds against different cancer lines. In
the MTT cytotoxicity assay, the crude extract was inactive at 20 μg/mL. The tested compounds
showed insignificant cytotoxicity and only compounds 19 and 21 exhibited marginal cytotoxicity
against liver, breast, lung, and oral cancer cell lines.
Table 4. Anti-inflammatory activity of pure compounds isolated from F. macrophylla.
Compounds
IC
50
(g/mL) or Inh%
Superoxide anion
Elastase release
3
3.74 ± 0.30
a,
***
23.31 ± 3.82
c,
***
4
6.82 ± 1.41 **
11.9 ± 5.02
5
0.66 ± 0.06
a
4.25 ± 1.23
a
6
1.89 ± 0.05
a
4.32 ± 0.14
a
7
8.86 ± 1.34
a
4.22 ± 0.72
a
8
2.18 ± 0.24
a
10.31 ± 3.82
b,
***
16
10.31 ± 3.82
40.92 ± 11.08
b
19
73.08 ± 4.89 **
55.26 ± 5.94
c,
**
21
ND
92.87 ± 2.27
b,
**
22
29.37 ± 4.42 **
−4.34 ± 5.95
ND: not determined. Percentage of inhibition (Inh%) at 10 μg/mL. Results are presented as the mean ± S.E.M.
(n = 3–4). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control value.
a
Concentration necessary
for 50% inhibition (IC
50
).
b
These compounds alone enhanced superoxide generation or elastase release by
human neutrophils in the absence of formyl-methionyl-leucyl-phenylalanine (fMLP) and cytochalasin B
(CB).
c
This compound could induce superoxide generation or elastase release by human neutrophils in the
presence of CB.
3. Experimental Section
3.1. General Experimental Procedures
The used HPLC was composed of dual Shimadzu LC-10AT pumps and a Shimadzu SPD-10A
UV-vis detector (Shimadzu Inc., Kyoto, Japan), as well as a Waters Atlantis T3 RP 150 × 4.6 mm, 5 µm
preparative column (Waters Corp., Milford, CT, USA) or a Thermo ODS Hypersil 250 × 4.6 mm,
5 µm preparative column (Thermo Fisher Scientific Inc., Rockford, IL, USA). UV spectra were
obtained using a JASCO UV-530 ultraviolet spectrophotometer (JASCO Inc., Tokyo, Japan). IR
spectra were obtained on a Mattson Genesis II infrared spectrophotometer (Thermo Fisher Scientific
Inc., Tokyo, Japan). Optical rotations were measured with JASCO DIP 370 and P-1020 digital
polarimeters (JASCO Inc., Tokyo, Japan). NMR (400 MHz and 600 MHz) spectra were obtained on a
Varian Unity 400 MHz FT-NMR and a Varian Unity 600 MHz FT-NMR (Varian Inc., Palo Alto, CA,
USA). ESI-MS data were collected on a VG Biotech Quattro 5022 mass spectrometer (VG Biotech,

Int. J. Mol. Sci. 2013, 14 15588
Altrincham, UK). High-resolution ESI-MS data were obtained on a Bruker APEX II spectrometer
(FT-ICR/MS, FTMS) (Bruker Daltonics Inc., Billerica, MA, USA).
Column chromatography (CC) was performed on silica gel (40–63 and 63–200 µm, Merck KGaA,
Darmstadt, Germany). Preparative TLC and TLC analyses were performed on silica gel 60 F254 plates
(Merck KGaA) and spots were visualized by UV and by spraying 50% H
2
SO
4
-ethanol solution
followed by heating for 5 min.
Eagle’s phenol-red free minimum essential medium, fetal bovine serum, L-glutamine, and the
antibiotic mixture (penicillin-streptomycin) were purchased from Invitrogen Co. (Invitrogen Co.,
Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Na
3
PO
4
,
EDTA, X-Gluc, and Triton X-100, and doxorubicin [(D1515-98.0%–102.0% (HPLC)] were purchased
from Sigma (Sigma Chemical Co., St. Louis, MO, USA). Dimethyl sulfoxide (DMSO), K
3
Fe(CN)
6
,
K
4
Fe(CN)
6
, were purchased from Merck (Merck KGaA). The purity of the isolated compounds used
for biological assays was determined by HPLC (>95%). 17β-Estradiol (E2) was purchased from TCI
(Tokyo Chemical Industry Co. Ltd., Tokyo, Japan).
3.2. Plant Material
The dried roots of Flemingia macrophylla (Willd.) Kuntze ex Prain (Flemingia-01) were collected
from Taichung City, Taiwan, in February 2008. The material was identified by Dr. Ming-Hong Yen
and deposited in the Graduate Institute of Natural Products, Kaohsiung, Taiwan.
3.3. Extraction and Isolation
The dried roots of F. macrophylla (7.7 kg) were extracted with 95% aqueous MeOH (10 L × 5) at
room temperature and then concentrated under reduced pressure. The crude extract (654.0 g) was
partitioned with H
2
O (2 L × 3) and EtOAc (2 L × 3) to yield a H
2
O layer (524.0 g), an insoluble
portion (1.7 g) and an EtOAc layer (127.4 g). The insoluble portion was recrystallized from MeOH to
yield genistin (4, 1.2 g). The EtOAc layer was then partitioned between n-hexane (2 L) and 90%
aqueous MeOH solution (2 L × 3) to provide an n-hexane layer (35.5 g) and a MeOH layer (91.9 g).
The MeOH layer exhibited the most potent estrogenic activity (Figure S1) and was selected for further
bioactivity-guided fractionation (Figure S2). The MeOH layer was isolated by silica gel column
chromatography (63–200 μM, 8.5 × 35 cm) under a gradient elution of n-hexane:EtOAc:MeOH
(1:0:0→0:0:1) to yield 14 fractions.
Fraction one (FM-1, 243.8 mg) was loaded on a silica gel column (40–63 μM, 2.5 × 30 cm), and
eluted with gradient mixtures of n-hexane:CHCl
3
(1:0→5:1→1:1) to yield 11 fractions. A mixture of
β-sitosterol and stigmasterol (23 and 24) (4.7 mg) was separated from FM-1-10 (16.1 mg) by
preparative TLC (CHCl
3
:EtOAc, 25:1).
Fraction two (FM–2, 546.9 mg) was loaded on a silica gel column (40–63 μM, 3.0 × 33 cm) eluted
with gradient solvent mixtures of n-hexane:CHCl
3
:MeOH (3:1:0→0:1:0→0:30:1→0:20:1→0:10:1),
followed by purification using reversed phase solid–phase extraction (RP-SPE, C18 gel) to yield six
fractions and fleminone (16) (20.6 mg). FM-2-3-SP2 (10.8 mg) was selected to be purified on
RP-HPLC (MeOH:H
2
O, 3:1) yielding flemiflavanone A (20) (5.0 mg).

Int. J. Mol. Sci. 2013, 14 15589
Fraction three (FM-3, 397.0 mg) was loaded on a silica gel column (40–63 μM, 3.0 × 32 cm) eluted
with gradient solvent mixtures of CHCl
3
:EtOAc (1:0→20:1→15:1) to yield seven fractions. FM-3-4
(104.6 mg) was purified by RP–HPLC (MeOH:H
2
O, 7:3) to yield flemiphilippinin F (3) (15.9 mg) and
isoderrone (15) (3.1 mg) and also two subfractions (FM-3-4-L1 and FM-3-4-L2) were obtained.
Subfraction FM-3-4-L2 (10.9 mg) was purified by RP-HPLC (MeCN:H
2
O, 7:3) to yield
7-(3,3-dimethylallyl)genistein (9) (6.4 mg).
Fraction six (FM–6, 1.5 g) was chromatographed on a silica gel column (40–63 μM, 2 × 29 cm),
which was eluted with CHCl
3
:EtOAc (8:1) to yield five fractions (FM-6-1 to FM-6-5). FM-6-1 (105.3 mg)
was loaded on a silica gel column eluted with CHCl
3
:MeOH (30:1) to yield prunetin (10) (2.5 mg) and
olmelin (11) (1.0 mg). FM-6-3 (308.1 mg) was selected for a silica gel column chromatography to
yield three fractions. FM-6-3-1 (214.7 mg) was purified by RP-SPE with MeOH:H
2
O (3:2→1:0) to
yield six fractions. FM-6-3-1-SP3 (11.2 mg) was purified by RP-HPLC (MeOH
−
:H
2
O, 4:1) to yield
flemichin E (2) (1.5 mg). FM-6-3-1-SP5 (10.4 mg) was purified by RP-HPLC (MeOH:H
2
O, 4:1) to
yield a mixture of 17 and 18 (2.5:1) (5.5 mg). FM-6-3-2 was purified by RP-SPE with MeOH:H
2
O
(3:2→1:0) to yield six fractions. FM-6-3-2-SP3 (74.0 mg) was similarly purified by RP-HPLC
(Waters, C18, 150 × 4.6 mm; MeOH:H
2
O = 3:1, HPLC–Shimadzu LC-10AT; UV-vis 254 nm; flow
rate: 2 mL/min) to yield flemingin (1) (3.6 mg), 8 (48.0 mg) and 13 (1.2 mg). FM-6-5 (259.4 mg) was
chromatographed on silica gel column chromatography, eluted with CHCl
3
:MeOH (30:1) to yield four
fractions (FM-6-5-1 to FM-6-5-4). From FM-6-5-3, compound 21 (74.1 mg) was separated. FM-6-5-2
(110.9 mg) was isolated using the same solvent system (CHCl
3
:MeOH, 30:1) to yield 21 (92.3 mg).
Fraction seven (FM-7, 7.2 g) was recrystallized from n-hexane–EtOAc to render genistein (5)
(898.0 mg). The remaining material of FM-7 was then separated by silica gel column chromatography
and eluted with CHCl
3
-MeOH (25:1→20:1) to yield seven fractions (FM-7-1 to FM-7-7). From
FM-7-6, compound 6 (2'-hydroxygenistein, 241.4 mg) was separated. FM-7-2 (1.1 g) was
chromatographed on silica gel column (CHCl
3
–MeOH, 35:1) and purified by RP-SPE (MeOH:H
2
O,
40:1→1:0) and RP-HPLC (MeOH:H
2
O, 7:3) to yield cajanin (7) (11.3 mg), neoraufurane (14)
(1.7 mg), erythrinin B (12) (3.2 mg), and flemiphilippinin D (19) (4.0 mg).
Fraction nine (FM–9, 5.2 g) was loaded on a silica gel column (63–200 μM, 4.5 × 21 cm) eluted
with CHCl
3
:MeOH (6:1) to yield eight fractions. FM-9-7 (3.1 g) and FM-9-8 (583.4 mg) were
combined and chromatographed on silica gel column eluted with gradient solvent mixtures of
CHCl
3
:MeOH (10:1→6:1→5:1) to yield 4'-O-methyl-gallocatechin (22) (1.7 g). The isolated compounds
were identified by NMR and the spectra were compared with the previously published data.
Flemingin (1), 5,2',4'-trihydroxy-7,8-(1,2,2-trimethyl-dihydrofurano)isoflavone: yellow amorphous
solid; [α]
26
D
: −13.45° (c 0.4, CHCl
3
); UV λ
max
(MeOH) nm (log ε): 263 (4.41), 295 (3.99), 335 (4.52);
IR (neat) V
max
cm
−1
: 3377, 1651, 1615, 1506; HRESIMS m/z 377.1001 [M + Na]
+
(calculated for
C
20
H
18
O
6
: 377.1005);
1
H and
13
C NMR data: Table 1.
Flemichin E (2), (2S)-(5,2'-dihydroxy-6,7-(1,1-dimethylpyrano)-8-(3-methylbut-2-enyl)-4',5'-(2-
hydroxy-1,1-dimethylpyrano)flavanone: yellow amorphous solid; UV λ
max
(MeOH) nm (log ε): 266
(4.63), 273 (4.65), 292 (4.30), 311 (4.18), 364 (3.65), 335 (4.52); IR(neat) V
max
cm
−1
: 3366, 1642,
1601, 1507; HRESIMS m/z 529.2202 [M+Na]
+
(calculated for C
30
H
34
O
7
: 529.2199).

Int. J. Mol. Sci. 2013, 14 15590
3.4. Transgenic Plant Material and Estrogen–Like Reporter Assay
The Arabidopsis pER8:GUS line, with an estrogen receptor-based transactivator XVE (pER8)
system, was developed originally by Brand et al. [7]. Seeds of pER8:GUS were grown on medium
(1/2MS, 1% sucrose, 0.8% phytoagar), incubated at 4 °C for 24–36 h in dark conditions for
vernalization, and then germinated under light for three days [43]. The germinated plants were then
transferred into 24-well microtiter plates in the presence or absence of test samples and incubated at
24 °C for 48 h. Plants cultured with 0.31–10 nM 17β-estradiol were taken as the positive control.
3.5. Histochemical Assay
After incubation in the presence or absence of test samples, the plants were soaked in 0.2 mL per
well of the GUS assay solution [50 mM Na
3
PO
4
buffer (pH 7.0), 10 mM EDTA (pH 8.0), 2 mM
X-Gluc, 0.5 mM K
3
Fe(CN)
6
, 0.5 mM K
4
Fe(CN)
6
, and 0.1% Triton X-100] in 24-well plates and were
kept overnight at 37 °C. The plants were then soaked in 70% aqueous EtOH for 1 h to remove
chlorophyll [44]. A ZEISS Axiovert 200 inverted microscope was used to examine GUS staining and
to capture images with a digital camera. The minimum active concentration (MAC) of each sample
was recorded upon the disappearance of the insoluble blue dye (5,5'-dibromo-4,4'-dichloro-indigo).
3.6. Reporter Gene Assay
Human breast adenocarcinoma cells MCF-7 obtained from the Bioresource Collection and Research
Center (BCRC) were cultured in Eagle’s phenol-red free minimum essential medium (MEM)
supplemented with dextran-charcoal treated serum, 2 mM L-glutamine and 10% fetal bovine serum
(Gibco), penicillin, and streptomycin, in an atmosphere of 5% CO
2
at 37 °C. Transfections were made
using a liposome-based method (Lipofectamine 2000, Invitrogen Co., Carlsbad, CA, USA), according
to the manufacturer’s instructions. Briefly, 0.2 μg of pERE-TA-SEAP plasmid (Clontech Laboratories,
Inc., Mountain View, CA, USA) was transfected into 2 × 10
3
cells in 100 μL of the growth medium
per well for 6 h. Cells were washed and treated with samples of interest in growth medium for 48 h.
Aliquots of culture media were analyzed for secreted alkaline phosphatase activity using the reporter
Chemiluminescence Assay Kit, Phospha–LightTM (Applied Biosystems, Foster City, CA, USA). The
MTT colorimetric assay was performed on the cells for assessing their corresponding cytotoxicity [45,46].
Finally, the estrogenic and antiestrogenic data were determined by the formula (final SEAP% =
(SEAP/cell viability × 100) to avoid false data result from cytotoxicity [8]. Tests were carried out in
triplicate or quadruplicate.
3.7. Human Neutrophil Superoxide Anion Generation and Elastase Release
Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation.
Superoxide generation was carried out according to the procedures described previously [47,48].
Elastase release experiment was performed using MeO-Suc-Ala-Ala-ProValp-nitroanilide as the
elastase substrate. Results of the superoxide anion production were collected by monitoring the
superoxide dismutase–inhibitable reduction of ferricytochrome c.

Int. J. Mol. Sci. 2013, 14 15591
3.8. Cytotoxicity
The following human cancer cell lines: liver (Hep3B and Hep G2), lung (A549), oral (Ca 9-22), and
breast (MCF-7 and MDA-MB-231) cancer cell lines were obtained from the American Type Culture
Collection. Cell viability was measured by the MTT colorimetric method [49]. Doxorubicin was used
as a positive control. Absorbance at 550 nm was measured using a Multiskan Ascent microplate reader
(Thermo Labsystems, Waltham, MA, USA). The mean IC
50
is expressed as the concentration of the
agent that reduced cell growth by 50% under the experimental conditions. The results represent the
mean calculated from at least three independent experiments.
4. Conclusions
One new isoflavone (1) with unusual 2',4'-dihydroxy substitutions and 23 known compounds were
isolated from F. macrophylla. In the current study, the 1D, 2D NMR spectra and the stereochemical
configuration of flemichin E (2) was reported for the first time. The results revealed that
F. macrophylla major components exhibited potent binding affinity to ER, and were involved in ERE
transcriptional activity acting as partial agonist and antagonists. The data collected from both reporter
assays were in alignment with each other, suggesting that the transgenic plant system (pER8:GUS) or
human breast cancer ER-positive MCF-7 cells can be used for screening new phytoestrogenic sources.
Acknowledgments
We gratefully acknowledge the financial support for the projects from the National Science Council
(100-2628-B-037-003-MY3; 102-2911-I-002-303) and the Department of Health (DOH102-TD-C-111-
002), Executive Yuan, Taiwan. This work was also in-kind support by the Cancer Center, Kaohsiung
Medical University Hospital and R&D center of the Chinese Herbal Medicines and New Drug, Kaohsiung
Medical University, Kaohsiung. We thank the Institute of the Plant Biology and the Zurich-Basel Plant
Science Centre, University of Zurich, Switzerland and Ken-Ichiro Hayashi, Department of
Biochemistry, Faculty of Science, Okayama University of Science, Japan for providing seeds of
pER8:GUS. We would like to thank the Center for Research Resources and Development (CRRD),
Kaohsiung Medical University for the technical supports and services in LC-MS and NMR analyses.
Conflict of Interest
The authors declare that they have no conflicts of interest.
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