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BioMed Central
Page 1 of 11
(page number not for citation purposes)
BMC Complementary and
Alternative Medicine
Open Access
Research article
Novel phytoandrogens and lipidic augmenters from Eucommia
ulmoides
Victor YC Ong
1,2
and Benny KH Tan*
1
Address:
1
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, 10 Kent Ridge Crescent, 119260,
Singapore and
2
Faculty of Medicine, Nursing and Health Sciences, Monash University, Wellington Road, Victoria 3800, Australia
Email: Victor YC Ong - yekcheng@yahoo.com; Benny KH Tan* - phctankh@nus.edu.sg
* Corresponding author
Abstract
Background: Plants containing compounds such as the isoflavonoids, with female hormone-like
effects that bind to human estrogen receptors, are known. But none has been previously shown to
have corresponding male hormone-like effects that interact with the human androgen receptor.
Here, we report that the tree bark (cortex) of the Gutta-Percha tree Eucommia ulmoides possesses
bimodal phytoandrogenic and hormone potentiating effects by lipidic components.
Methods: The extracts of E. ulmoides were tested using in-vitro reporter gene bioassays and in-vivo
animal studies. Key compounds responsible for the steroidogenic effects were isolated and
identified using solid phase extraction (SPE), high performance liquid chromatography (HPLC), thin
layer chromatography (TLC), gas chromatography-mass spectroscopy (GC-MS), electron spray
ionisation-mass spectroscopy (ESI-MS) and nuclear magnetic resonance (NMR).
Results: The following bioactivities of E. ulmoides were found: (1) a phenomenal tripartite
synergism exists between the sex steroid receptors (androgen and estrogen receptors), their
cognate steroidal ligands and lipidic augmenters isolated from E. ulmoides, (2) phytoandrogenic
activity of E. ulmoides was mediated by plant triterpenoids binding cognately to the androgen
receptor (AR) ligand binding domain.
Conclusion: In addition to well-known phytoestrogens, the existence of phytoandrogens is
reported in this study. Furthermore, a form of tripartite synergism between sex steroid receptors,
sex hormones and plant-derived lipids is described for the first time. This could have contrasting
clinical applications for hypogonadal- and hyperlipidaemic-related disorders.
Background
The androgen receptor (AR) plays a pivotal role in human
(both male and female) physiology such as skeletal mus-
cle development, bone density, fertility and sex drive
[1,2]. The α and β estrogen receptors (ERs), likewise, have
fundamental impact on the sex hormone-mediated phys-
iological milieu. Conversely, over-active sex steroid
(androgen and estrogen) receptors have been linked to
increased risks of hormone-sensitive tumours such as
prostate and breast cancers. Availability and binding of
cognate ligands to the ligand binding domain (LBD) of
the sex steroid receptors are required for the proportionate
expression of specific genes responsible for such sex hor-
mone-mediated processes [3,4].
Published: 29 January 2007
BMC Complementary and Alternative Medicine 2007, 7:3 doi:10.1186/1472-6882-7-3
Received: 8 August 2006
Accepted: 29 January 2007
This article is available from: http://www.biomedcentral.com/1472-6882/7/3
© 2007 Ong and Tan; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
BMC Complementary and Alternative Medicine 2007, 7:3 http://www.biomedcentral.com/1472-6882/7/3
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Vegetative foods such as the legumes, particularly soybean
(Glycine max), contain phytoestrogens that modulate the
transcriptional activities of the estrogen receptor isoforms,
α and β. The former has been linked to the chemopreven-
tion of specific cancers in the breast and prostate gland
[5].
Here, we report that the tree bark (cortex) of the Gutta-
Percha tree Eucommia ulmoides OLIVER possesses novel
bimodal phytoandrogenic and synergistic augmentation
of hormone-dependent receptor activity. E. ulmoides is
also known variously as the Gutta Percha Tree, the Rubber
Bark Tree or Du-Zhong [6,7]. The toothed elliptic leaves
and tree bark of E. ulmoides (figure 1) are used medicinally
in herbal pharmacopoeias such as Kampo (traditional
Japanese medicine) and Zhong-Yao (traditional Chinese
medicine) for indications such as the relief of back pain,
to increase stamina, to make bones and muscles 'strong'
and to hasten recovery from fatigue. It is noted that these
are male hormone-related pharmacological effects.
Through the combined use of varied technologies –
recombinant DNA constructs, reporter gene assays, ani-
mal studies and separation chemistry, extracts of E. ulmo-
ides were shown for the first time, to specifically activate
the tranactivational capacity of the sex steroid receptors in
both in-vitro and in-vivo settings. A series of bioassay-
guided fractionation showed that the phytoandrogenic
and hormone potentiating effects of E. ulmoides were
mediated by distinct groups of phytocompounds; triterpe-
noids and short-chain lipids respectively.
Methods
Cell culture
The mammalian COS-7 cell line was maintained in
DMEM cell culture medium with 10% fetal bovine serum
(FBS) and 1% of each amino acids (glutamine, arginine
and lysine). Hela cells were maintained in RPMI 1640 cell
culture medium (with 10% FBS, 1% L-glutamine, 1% L-
lysine and 1% arginine). Both cell lines were incubated at
37°C in 5% CO
2
gaseous environment.
Transient transfection of mammalian cell lines
Mutant and wild-type (WT) chimeric constructs were
transfected into Hela cells, using the lipofection tech-
nique. Both are as described in [8]. Hela cells, which are a
homologous cell line, was utilised for the transactivation
studies. These Hela cells were cotransfected with a luci-
ferase reporter gene containing two AREs [p(ARE)
2
-Tata-
Luc]. Transfected cells were exposed to androgens for 48
hr before harvesting and quantification of luciferase activ-
ity. The androgens used were testosterone and dihydrotes-
tosterone (DHT).
Binding site of phytoandrogens
Radioligand displacement assays were carried out using
tritiated testosterone to determine binding site of the phy-
toandrogens in E. ulmoides. The COS-7 cells were trans-
fected with AR and then exposed to 3 nM of tritiated
testosterone and the indicated amounts of DHT (nM),
cortisol (nM) or ethanolic E. ulmoides (EU) extract (1 con-
centration factor (c.f.) = 50 ng dry weight/ml treatment
medium) for 2 hr at 37°C. The treated cells were harvested
and the amount of tritiated testosterone bound to AR was
then measured by scintillation counting. Specific binding
is expressed as percent tritium bound to AR, where 100%
is the amount of specific tritiated-testosterone bound in
the absence of competing cold ligand minus background
(non-specific binding to substrate and proteins). Each
data point, the mean of quadruplicates, represents the
amount of radiolabelled testosterone specifically bound
on exposure to indicated doses of DHT, ethanolic EU
extract or cortisol.
Luciferase assay
Luciferase assay was performed with the TD-20/20 Lumi-
nometer, following the instructions provided by the man-
ufacturer (Promega, USA). Briefly, the transfected cells
were lysed in 1× Reporter Lysis Buffer (RLB). The cell
lysate was mixed with Luciferase Assay Substrate immedi-
ately before measuring. The luciferase activity was meas-
ured in relative light units (RLU).
Plant material
The dried cortex of Eucommia ulmoides OLIVER was pur-
chased from the local wholesaler dealing exclusively in
Chinese medicinal herbs. Dr Ruth Kiew, Keeper of Her-
barium & Library, National Parks Board, Singapore,
authenticated the plant material. A voucher specimen (BT-
4) was deposited with the Singapore Botanic Gardens
Herbarium.
Processed bark (cortex) from the evergreen tree Eucommia ulmoidesFigure 1
Processed bark (cortex) from the evergreen tree Eucommia
ulmoides. Originating from temperate regions of China,
botanical parts of E. ulmoides such as the leaves and bark are
used medicinally in the Chinese and Japanese Pharmacopia.
Note the silvery threads of resin (Gutta Percha) in between
the sliced portion of the specimen (green arrow).
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Extractions
Dried cortical barks of E. ulmoides were washed with
deionised water to remove contaminants. In each batch
extraction, damp E. ulmoides barks were then macerated
using a commercial grinder (Bosch, Germany). The result-
ing cortical mash was then blended with 100% ethanol
solvent. Using the soak method, the blended ethanolic
mash (100 g/1L) was incubated in a shaken (25 r.p.m.)
water bath (Buchi Labortechnik AG, Switzerland) for 12
hr at 40°C to allow the active compounds to dissolve into
the solvent system. After 12 hr, the menstrum was
decanted and filtered through Whatman No.1 using a
Buchner funnel in vacuo. The resulting filtrate was concen-
trated by evaporation under vacuum at 40°C, using a rota-
tory evaporator (Buchi Labortechnik AG, Switzerland).
The extraction procedure was repeated 8 to 10 times until
the fibrous residue was exhausted. The menstrums col-
lected from each sequential re-extraction were concen-
trated and blended into a semi-solid paste. This paste was
then aliquoted and resuspended in pure ethanol to obtain
stock solutions (50 mg/ml) of tertiary crude extracts for
downstream experimental work.
Animal housing
Male juvenile (prepubertal) WISTAR rats weighing
between 48–64 g (average weight 53 g) were obtained
from the Laboratory Animal Centre, National University
of Singapore. The animals were fed on commercial pel-
leted chow and water was given ad libitum. The rats were
housed individually in wide-bottomed Perspex cages. The
light cycle was diurnal with 12 hr daylight. The animals
were treated in accordance with the "guidelines for the
care and use of laboratory animals for scientific purposes"
issued by the local authority.
Animal studies
To establish the dose response relationship between intra-
muscular (IM) testosterone (Sustanon 5000) injections
and ventral prostate weight (as a measure of prostate
development), prepubertal male WISTAR rats (2 weeks
old) were given IM testosterone injections of 500 µg, 2500
µg and 5000 µg while control animals were given IM
injections of vehicle (olive oil) only. After 5 days, the WIS-
TAR rats were sacrificed by decapitation. The abdominal
flap of each rat was dissected to recover the prostate gland.
The ventral prostate gland was removed and the wet
weight determined. Androgen-mediated prostatic growth
was expressed as the weight of the ventral prostate gland
normalized to 100 g body weight of the individual rats.
To study the androgenic effect of oral E. ulmoides liquid
formulation on ventral prostate growth, prepubertal male
WISTAR rats were administered E. ulmoides extracts via
gavaging with doses of 1 mg, 5 mg, 10 mg and 50 mg in
aliquots of 1 ml vehicle (25% hydroethanolic solution).
Gavaging (gastric intubation) were carried out with gauge
18 feeding needle. Negative control animals were given
oral doses of vehicle (25% hydroethanolic solution) only.
To test the potentiating effect of E. ulmoides extract on tes-
tosterone-mediated prostatic growth, prepubertal male
WISTAR rats were given 5000 µg IM testosterone injec-
tions in conjunction with 50 mg of E. ulmoides extract by
oral gavage. Baseline control animals were given IM injec-
tions of 5000 µg IM testosterone injections alone. Nega-
tive control animals were given IM injections of olive oil
plus concurrent oral gavaging with 25% hydroethanolic
solution.
Fractionations
Semi-preparative open column chromatography was car-
ried out using diol stationary phase as previously
described [10]. The chromatographic column with the E.
ulmoides extract loaded on top was influxed with 50 ml of
each mobile phase in increasing order of polarity –
dichloromethane (DCM), ethyl acetate (EA), ethanol
(EtOH) and water (H
2
O). The mobile phases eluting from
the chromatographic column were collected sequentially
into individual fractions – fraction A, fraction B, fraction
C and fraction D. These fractions were dried in a rotary
evaporator at 40°C and resuspended in ethanol for bio-
assays.
For solid phase extraction (SPE), the SPE C-18 cartridge
was flushed with ethanol to activate the C-18 hydrocar-
bon moieties. This was followed by water to displace the
ethanol phase and equilibrate the solid phase in an aque-
ous environment. Using SPE method described in Ong
[10], fraction A was further resolved into four purer frac-
tions AA, AB, AC and AD. Fraction AB with dominant phy-
toandrogenic activity was subsequently re-
chromatographed in C-18 matrix using high performance
liquid chromatography (HPLC) to obtain a phytoandro-
genic fraction (AB-P1) of even greater purity. AB-P1 frac-
tion was subjected to thin layer chromatography (TLC)
and a phytoandrogenic triterpenoid fraction TLC4-5 was
recovered for molecular mass measurement using electron
spray ionization-mass spectroscopy (ESI-MS). The TLC
chromatogram of fraction AB-P1 was subjected to vanil-
lin-sulphuric acid visualization reaction to detect presence
of triterpenoids by spraying 5% ethanolic sulphuric acid.
This was followed by 1% ethanolic vanillin. Colorimetric
development was completed by heating the treated TLC
plate at 120°C for 15 min in a thermostat oven.
Fraction C was similarly subjected to SPE using C-18 SPE
cartridge and sequential fractions recovered were fractions
CA and CB. The recovered fraction CB was subjected to
1
H
NMR and GC-MS [10]. The overall scheme, from raw herb
to confirmatory bioassays, is detailed in figure 2.
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Schematics of research process flowFigure 2
Schematics of research process flow. E. ulmoides cortex was macerated and crude extracts obtained using solvents. These sol-
vents were dichloromethane (DCM), ethyl acetate (EA), ethanol (EtOH) and water (H
2
O). The crude extracts were subjected
to steroidogenic reporter gene and binding assays. Bioactive fractions were subsequently obtained through bioassay-guided
fractionations using column chromatography, thin layer chromatography and high performance liquid chromatography (HPLC).
Pharmacologically active phytocompounds purified from the bioactive fractions were identified through spectroscopic and
nuclear magnetic resonance (NMR) studies. Confirmatory reporter gene assays were then carried out using pure compounds
to reproduce steroidogenic effects.
EUCOMMIA MACERATED CORTEX
EXTRACTION with
solvents
CRUDE EXTRACTS
DCM EA EtOH H
2
O
REPORTER GENE,
BINDING ASSAYS AND
PURIFICATION OF
BIOACTIVE FRACTIONS BY:
column chromatography,
thin layer BIOACTIVE FRACTIONS
chromatography,
HPLC
CHARACTERISATION AND
SPECTROSCOPIC STRUCTURAL ELUCIDATION OF
and NMR studies PURE COMPOUNDS
CONFIRMATORY REPORTER GENE ASSAYS
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Statistical analyses
All in-vitro reporter gene results were expressed as means ±
SE from triplicate assays. Results of the radioligand dis-
placement assays were expressed as means ± SE from
quadruplicate assays. One-way ANOVA analyses were car-
ried out to assess statistical significance for in-vivo animal
experiments. Differences with p-value < 0.001 were con-
sidered statistically significant.
Results and discussion
In Luc reporter gene bioassays, E. ulmoides cortical extract
demonstrated androgenic and estrogenic activities by
weakly activating AR and ER transactivational function in
a dose-dependent manner (figures 3 and 4). Highly spe-
cific radioisotopic ligand displacement bioassay showed
that phytocompounds in the E. ulmoides extract were able
to compete with and displace bound
3
H-labelled testo-
sterone from the AR ligand binding domain (figure 5).
Subsequent bioassay-guided purification of the andro-
genic extract using chromatographic and ESI-MS tech-
niques revealed that this phytoandrogenic activity was
being mediated by triterpenoids, which differs from the
phytoestrogenic effect exerted by isoflavonoids [5] (fig-
ures 6 and 7).
Remarkably, combination of DHT and the E. ulmoides
extract in the presence of the AR led to increases in AR-
mediated reporter gene expression ranging from 112% to
204%, even at saturating levels of DHT (figure 8). This
potentiating effect was a tripartite synergism between the
Radioligand displacement assay of E. ulmoides (EU) extractFigure 5
Radioligand displacement assay of E. ulmoides (EU) extract.
Tritiated testosterone was incubated with transfected Hela
cells transiently expressing AR protein. Concentration-
dependent competitive displacement of tritiated testoster-
one by androgenic E. ulmoides (EU) extract (1c.f. = 50 ng/ml
treatment medium) was measured by the scintillation count
of the bound radioisotopic testosterone to AR protein. DHT
(nM) and cortisol (nM) acted as positive and negative con-
trols respectively. Data are mean ± SE of four replicates.
0
20
40
60
80
100
120
00.31 3 10
C oncentration of ethanolic EU extract (cf*), DH T (nM ) and C ortisol (nM )
Percentage of tritiated testosterone remainin
g
Ethanolic EU extract (cf*) (1cf=50ng dry w eight/ml treatment m edium )
DHT (nM)
C ortisol (nM )
Phytoandrogenicity of E. ulmoides (EU) extractFigure 3
Phytoandrogenicity of E. ulmoides (EU) extract. The dose
response curve of an E. ulmoides extract with concentrations
ranging from 0.25 ng/ml treatment medium to 50 ng/ml treat-
ment medium. Hela cells transiently expressing androgen
receptor (AR) in the presence of AR-responsive luciferase
reporter gene (Luc) were exposed to increasing doses of the
extract. Comparatively, testosterone (1 nM) has 100-fold Luc
activity relative to the maximal E. ulmoides 6.4-fold Luc activ-
ity. Data are mean ± SE of three replicates.
0
1
2
3
4
5
6
7
8
0.25 0.5 2.5 5 25 50
Ethanolic EU extract (ng/ml treatment medium)
Phytoestrogenicity of E ulmoides (EU) extractFigure 4
Phytoestrogenicity of E ulmoides (EU) extract. Phytoestro-
genic effect of E ulmoides (EU). Hela cells were transfected
with human estrogen receptor and estrogenic effect of an
ethanolic EU extract measured with MMTV-ERE-Luc
reporter gene. Data are mean ± SE of three replicates.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
control 5 25 50
Ethanolic EU extract (ng dry weight/ml treatment medium)
Estrogenic activity (fold induction)
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AR protein, its cognate steroidal ligand and E. ulmoides's
extract. This is highly unusual as normally, androgen-
mediated AR transcriptional capacity, akin to all ligand-
dependent steroid receptors, plateaus at saturating doses
of its cognate ligand. A similar synergistic effect was
observed when E. ulmoides extract was tested in combina-
tion with estradiol in the presence of the estrogen receptor
(ER) α (figure 9).
To confirm the specificity of these agonistic and synergis-
tic interactions between the sex steroid receptors and E.
ulmoides cortical extract, the progesterone (PR) and gluco-
corticoid (GR) receptors (which belong to the same super-
family of steroid receptors) were also subjected to the
same series of reporter gene bioassays (data not shown).
The cortical extract did not exert any activating effects,
alone or in combination with progesterone and cortisol
on the PR and GR respectively. This validates the specifi-
city of E. ulmoides's sex steroidogenic effects on the holo-
androgen and estrogen receptors.
In-vivo animal studies were then carried out to investigate
the androgenic and hormone potentiating effects of E.
ulmoides extract when it is administered orally. At baseline
(without testosterone), the mean ventral prostate weight
of the male Wistar rats was 45 mg/100 g body weight.
With a saturating dose of 5000 µg IM testosterone injec-
tion, the androgen-mediated ventral prostate growth
steadied at 85 mg prostate weight/100 g body weight; one-
way ANOVA showing statistically insignificant difference
(p-value = 0.0562) (figure 10). Administration of E. ulmo-
ides oral formulations at dry weight doses of 1 mg, 5 mg,
10 mg and 50 mg (n = 5 for each dosage) similarly
brought about a statistically insignificant increase over
baseline (p-value = 0.9319) at the maximum dose of 50
mg (figure 11).
However, a combined administration of 5000 µg IM tes-
tosterone injection and E. ulmoides by oral gavaging (N =
15) augmented androgen-mediated ventral prostate
growth (figure 12). When an oral dose of 50 mg E. ulmo-
ides extract was co-administered together with 5000 µg of
IM testosterone (n = 5 for each combination treatment),
the mean ventral prostate weight increased to 93 mg/100
g body weight, demonstrating a highly significant δ differ-
ence over baseline of 53 mg/100 g body weight (p-value <
0.001). The outcome of these animal studies further indi-
cates that E. ulmoides potentiates the effect of testosterone
on the androgen receptor (figure 12).
Subsequent
1
H NMR and GC analyses of active fraction
CB showed the major presence of the 8-carbon polysatu-
rated fatty acid, caprylic acid, along with other lipids (fig-
ure 13 and table 1). Bioassays using pure caprylic acid and
other polysaturated fatty acids (PFAs) correlated with the
augmenting effect of E. ulmoides on the AR (figure 14) in
varying degrees. Ethanolic extract of coconut (Cocos nuci-
fera) flesh, rich in C-8 caprylic acid and other polysatu-
rated fatty acids [11], replicated the hormone potentiating
effect of both E. ulmoides extract and pure caprylic acid in
AR bioassays (data not shown).
Okadaic acid, a known phosphorylation promoter, is able
to strongly augment androgen-dependent AR activity
[12]. Interestingly, fatty acids can also promote phospho-
rylation. One instance is oleic acid, a C-18 cis-monosatu-
rated fatty acid [13]. It is possible that AR and ER
augmentation by both E. ulmoides extract and caprylic
acid arise from a common tripartite synergism between
the steroid receptors, sex steroids and fats, based on a
phosphorylation mechanism.
Our studies suggest intrinsic hormone potentiating effect
in PFAs per se; in addition to the calorific and structural
roles that have previously been identified (for e.g. cell
membrane components or structural precursors of pro-
inflammatory cytokines). The tripartite synergism demon-
strated in this study may also be helpful in clarifying the
known epidemiological link between high dietary satu-
rated fat intake (rich in PFAs), obesity and increased risks
TLC silica fractionation of fraction AB-P1 and triterpenoid detectionFigure 6
TLC silica fractionation of fraction AB-P1 and triterpenoid
detection. Lane 1 (DHT) = 20 µl of 1 mM dihydrotestoster-
one spotted on, band-wise. Lane 2 (Aucubin): 20 µl of 10 uM
aucubin spotted on, band-wise. Lane 3 (AB-P1): 20 µl of frac-
tion AB-P1 (1c.f.) spotted on, band-wise. Colorimetric devel-
opment was carried out using vanillin-sulphuric acid reaction
to detect presence of triterpenoids.
4-5
3-4
2-3
2
5
4
3
1-2
1
AB-P1AucubinDHT
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of hormone-related diseases such as prostate or breast
cancer [14].
Conclusion
The novel discoveries reported in this study add phytoan-
drogens and lipidic augmenters to the emerging list of
hormomimetics (such as phytoestrogens) known to exist
in plants. Pharmaceutical utility of lipidic augmenters in
the treatment of hypogonadal conditions such as meno-
pause or andropause could be exploited based on this
mechanism of tripartite synergism. The link between
excess dietary lipids, hyperandrogenism and hormone-
related disorders should also be further explored in the
light of these findings.
Competing interests
The author(s) declare that they have no competing inter-
ests.
Authors' contributions
OYC: Designed and performed the study along with draft-
ing the manuscript.
BTKH: Supervised and guided aspects of the study along
with the drafting of the manuscript.
Both authors have read and approved the contents of this
paper.
Modeled fragmentation pattern of phytoandrogen structure using Euphol (MW 426), a plant triterpenoidFigure 7
Modeled fragmentation pattern of phytoandrogen structure using Euphol (MW 426), a plant triterpenoid. The ESI-MS spectra
of phytoandrogen fraction TLC4-5 indicated a MW of 426. Molecular ions – 1) MS1 = 425, MS2 = 365, MS3 = 310. Euphol
(MW 426) was used to model the ESI-MS spectra of the phytoandrogen TLC4-5.
4.1
CH
3
CH
3
H
3
C
H
3
C
CH
3
CH
3
CH
3
H
HO
CH
3
Euphol (plant triterpenoid)
MW 426
CH
3
CH
3
H
3
C
H
3
C
CH
3
CH
3
CH
3
H
O
CH
3
-[CH
3
]
4
MS (Molecular Ion)
MW 425
-
CH
3
CH
3
H
3
C
H
O
CH
3
MS2 (Main Fragment)
MW 365
-
H
3
C
H
O
CH
3
MS3 (Main Fragment)
MW 310
-
-H
MS2 (Fragment)
MW 60
Ionisation (Proton loss)
MW 1
MS3 (Fragment)
MW 55
CH
3
CH
3
HC
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In-vivo E. ulmoides (EU) extract and ventral prostate growth in prepubertal male WISTAR ratsFigure 11
In-vivo E. ulmoides (EU) extract and ventral prostate growth in
prepubertal male WISTAR rats. The androgenic effect of oral
E. ulmoides liquid formulation on ventral prostate growth.
Prepubertal male WISTAR rats were administered ethanolic
E. ulmoides (EU) extracts via gavaging with doses of 1 mg, 5
mg, 10 mg and 50 mg. Control animals were given oral doses
of vehicle (ethanolic water) only. Prostatic growth is
expressed as the weight of the ventral prostate gland normal-
ized to 100 g body weight of the individual rats. Each data
point is mean ± SE of five animals' ventral prostate weights.
0
10
20
30
40
50
60
70
80
0151050
Ethanolic EU extract alone (mg dry w eight)
Mean prostatic weight (mg/100g body
weight)
p-value = 0.9319
Supra-hormonal properties of E ulmoides (EU) extract on estrogen receptor and estradiolFigure 9
Supra-hormonal properties of E ulmoides (EU) extract on
estrogen receptor and estradiol. The augmenting effect of a
fixed dose of E ulmoides (EU) extract on ER. Cells were
exposed to increasing doses of estradiol, with or without, 50
ng/mL treatment medium of the ethanolic E ulmoides extract,
as indicated. Control cells were not exposed to the ethanolic
E ulmoides extract or estradiol. Estrogenic activity is
expressed as fold increase in reporter gene activity com-
pared to control. Data are mean ± SE of three replicates.
0
1
2
3
4
5
6
7
8
9
10
11
12
control 0.1 1 10
Estradiol (nM)
Estrogenic activity (fold increase)
Estradiol alone Estradiol + ethanolic EU extract (50ng dry weight/ml treatment medium)
Supra-hormonal effect of E. ulmoides (EU) extract on andro-gen receptor and testosteroneFigure 8
Supra-hormonal effect of E. ulmoides (EU) extract on andro-
gen receptor and testosterone. Testosterone-augmenting
activity of a fixed dose of ethanolic EU extract (1c.f. = 50 ng/
ml treatment medium) with increasing doses of testosterone.
Hela cells transiently expressing androgen receptor (AR) in
the presence of AR-responsive luciferase reporter gene (Luc)
were exposed to increasing doses of EU extract. Data are
mean ± SE of three replicates.
0
50
100
150
200
250
300
0.1 1 10 100
Testosterone (nM)
Androgenic activity (fold increase)
Testosterone alone
Testosterone+ethanolic EU extract (1 c.f.)
In-vivo testosterone and ventral prostate growth in prepuber-tal male WISTAR ratsFigure 10
In-vivo testosterone and ventral prostate growth in prepuber-
tal male WISTAR rats. The establishment of a dose response
relationship between IM testosterone injections and ventral
prostatic weight (as a measure of prostate development).
Prepubertal male WISTAR rats were given IM testosterone
injections of 500 µg, 2500 µg and 5000 µg. Control animals
were given IM injections of vehicle (olive oil) only. Prostatic
growth is expressed as the weight of the ventral prostate
gland normalized to 100 g body weight of the individual rats.
Each data point is mean ± SE of five animals' ventral prostate
weights.
0
20
40
60
80
100
120
0 500 2500 5000
Testosterone alone (ug)
Mean prostatic weight (mg/100g body
weight)
p-value = 0.0562
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Table 1: Composition of major fatty acids in Fraction CB by standard gas chromatography (GC) analysis.
Fatty acid Carbon number and type of bond Abundance (%)
Caprylic acid 8:0 78.1977
Palmitic acid 16:0 9.0703
Stearic acid 18:0 6.4948
Palmitalic acid 16:1ω 9 1.5445
Linoleic acid 18:2ω 6 0.7221
Linolenic acid 18:3ω 3 0.6503
Subtotal 96.6797
Minor fatty acids - 3.3203
Total 100
Caprylic acid, palmitic acid and stearic acid are polysaturated fatty acids (PFA). Palmitalic acid, Linoleic acid and Linolenic acid are omega-9, omega-
6 and omega-3 polyunsaturated fatty acids (PUFA) respectively. These fatty acids share the general empirical formula C
n
H
n
O
n
. Caprylic acid was the
major constituent with 78.2% abundance.
In-vivo testosterone-E. ulmoides (EU) extract and ventral prostate growth in prepubertal male WISTAR ratsFigure 12
In-vivo testosterone-E. ulmoides (EU) extract and ventral
prostate growth in prepubertal male WISTAR rats. The syn-
ergistic augmenting effect of E. ulmoides on testosterone
mediated prostate growth. Prepubertal male WISTAR rats
were given IM testosterone injections of 5000 µg (Lane 2).
To test synergism between E. ulmoides and an androgen,
experimental rats were given saturating IM testosterone
injections of 5000 µg in conjunction with oral gavaging of 50
mg of E. ulmoides (EU) extract (Lane 3). Control animals
were given IM injections of olive oil plus concurrent oral gav-
aging of ethanolic water. Prostatic growth is expressed as the
weight of the ventral prostate gland normalized to 100 g
body weight of the individual rats. Each data point is mean ±
SE of five animals' ventral prostate weights.
30
50
70
90
110
123
Treatm ent
Mean prostatic weight (mg/100g body weight)
Lane 1.Control
Lane 2.Te s tosterone alone (5000ug)
Lane 3. Tes tos terone (5000ug) + Ethanolic EU
e xtract (50m g dry w eight)
p-value <0.001
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The
1
H NMR spectra of supra-hormonal fraction CBFigure 13
The
1
H NMR spectra of supra-hormonal fraction CB. Arrow a shows protons from water (
1
H chemical shift of 4.8 – 5 ppm).
Arrow b shows protons from the solvent methanol (
1
H chemical shift of 3.2 – 3.5 ppm). Arrow c shows protons from fatty
acids (
1
H chemical shift of 0.8 – 1.8) ppm.
a
b
c
Effect of short-chain polysaturated fatty acids (PFAs) homol-ogous series (C3 – C9) on androgen-dependent AR transac-tivational capacityFigure 14
Effect of short-chain polysaturated fatty acids (PFAs) homologous series
(C3 – C9) on androgen-dependent AR transactivational capacity. The
effect of short chain (C3 – C8) polysaturated fatty acids (PFAs) on andro-
gen dependent AR transactivational capacity. Hela cells tranfected with AR
expression plasmid were exposed to 10 µM of different PFAs as indicated,
together with 1 nM DHT. C3 = propanoic acid (3:0). C4 = butanoic acid
(4:0). C5 = pentanoic acid (5:0). C6 = n-caproic acid (6:0). C7 = heptanoic
acid (7:0). C8 = caprylic acid (8:0). C9 = pelargonic acid (9:0). AR activity
was measured with the multimeric AR reporter plasmid (ARE
2
-TATA-Luc)
and luciferase activity in relative light units (RLU) was mean ± SE of 3 rep-
licates.
0
500
1000
1500
2000
2500
3000
DHT 1nM C3 C4 C5 C6 C7 C8 C9
Treatment
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Page 11 of 11
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Acknowledgements
This research was wholly funded by the National University of Singapore.
OYC was the recipient of a doctorate research scholarship awarded by the
same institution. Manuscript preparation, which includes writing, was car-
ried out entirely by the authors.
The authors wish to express their appreciation to the following for techni-
cal assistance and facility access: Drs Heng WS, Yong EL, Jenster G and But-
ler M.
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