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Novel phytoandrogens and lipidic augmenters from Eucommia ulmoides
© Ong and Tan; licensee BioMed Central Ltd. 2007
Received: 08 August 2006
Accepted: 29 January 2007
Published: 29 January 2007
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.
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).
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.
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.
The androgen receptor (AR) plays a pivotal role in human (both male and female) physiology such as skeletal muscle 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 physiological 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 hormone-mediated processes [3, 4].
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 chemoprevention of specific cancers in the breast and prostate gland .
Through the combined use of varied technologies – recombinant DNA constructs, reporter gene assays, animal studies and separation chemistry, extracts of E. ulmoides 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; triterpenoids and short-chain lipids respectively.
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% CO2 gaseous environment.
Transient transfection of mammalian cell lines
Mutant and wild-type (WT) chimeric constructs were transfected into Hela cells, using the lipofection technique. Both are as described in . Hela cells, which are a homologous cell line, was utilised for the transactivation studies. These Hela cells were cotransfected with a luciferase 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 activity. The androgens used were testosterone and dihydrotestosterone (DHT).
Binding site of phytoandrogens
Radioligand displacement assays were carried out using tritiated testosterone to determine binding site of the phytoandrogens in E. ulmoides. The COS-7 cells were transfected 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 concentration 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 was performed with the TD-20/20 Luminometer, following the instructions provided by the manufacturer (Promega, USA). Briefly, the transfected cells were lysed in 1× Reporter Lysis Buffer (RLB). The cell lysate was mixed with Luciferase Assay Substrate immediately before measuring. The luciferase activity was measured in relative light units (RLU).
The dried cortex of Eucommia ulmoides OLIVER was purchased from the local wholesaler dealing exclusively in Chinese medicinal herbs. Dr Ruth Kiew, Keeper of Herbarium & Library, National Parks Board, Singapore, authenticated the plant material. A voucher specimen (BT-4) was deposited with the Singapore Botanic Gardens Herbarium.
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 resulting 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 concentrated by evaporation under vacuum at 40°C, using a rotatory evaporator (Buchi Labortechnik AG, Switzerland). The extraction procedure was repeated 8 to 10 times until the fibrous residue was exhausted. The menstrums collected from each sequential re-extraction were concentrated 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.
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 pelleted 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.
To establish the dose response relationship between intramuscular (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 WISTAR 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 testosterone-mediated prostatic growth, prepubertal male WISTAR rats were given 5000 μg IM testosterone injections in conjunction with 50 mg of E. ulmoides extract by oral gavage. Baseline control animals were given IM injections of 5000 μg IM testosterone injections alone. Negative control animals were given IM injections of olive oil plus concurrent oral gavaging with 25% hydroethanolic solution.
Semi-preparative open column chromatography was carried out using diol stationary phase as previously described . 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 (H2O). 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 bioassays.
For solid phase extraction (SPE), the SPE C-18 cartridge was flushed with ethanol to activate the C-18 hydrocarbon moieties. This was followed by water to displace the ethanol phase and equilibrate the solid phase in an aqueous environment. Using SPE method described in Ong , fraction A was further resolved into four purer fractions AA, AB, AC and AD. Fraction AB with dominant phytoandrogenic activity was subsequently re-chromatographed in C-18 matrix using high performance liquid chromatography (HPLC) to obtain a phytoandrogenic fraction (AB-P1) of even greater purity. AB-P1 fraction 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 vanillin-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.
All in-vitro reporter gene results were expressed as means ± SE from triplicate assays. Results of the radioligand displacement assays were expressed as means ± SE from quadruplicate assays. One-way ANOVA analyses were carried out to assess statistical significance for in-vivo animal experiments. Differences with p-value < 0.001 were considered statistically significant.
Results and discussion
To confirm the specificity of these agonistic and synergistic interactions between the sex steroid receptors and E. ulmoides cortical extract, the progesterone (PR) and glucocorticoid (GR) receptors (which belong to the same superfamily 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 specificity of E. ulmoides's sex steroidogenic effects on the holo-androgen and estrogen receptors.
Composition of major fatty acids in Fraction CB by standard gas chromatography (GC) analysis.
Carbon number and type of bond
Minor fatty acids
Okadaic acid, a known phosphorylation promoter, is able to strongly augment androgen-dependent AR activity . Interestingly, fatty acids can also promote phosphorylation. One instance is oleic acid, a C-18 cis-monosaturated fatty acid . 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 demonstrated in this study may also be helpful in clarifying the known epidemiological link between high dietary saturated fat intake (rich in PFAs), obesity and increased risks of hormone-related diseases such as prostate or breast cancer .
The novel discoveries reported in this study add phytoandrogens 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 menopause 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.
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 carried out entirely by the authors.
The authors wish to express their appreciation to the following for technical assistance and facility access: Drs Heng WS, Yong EL, Jenster G and Butler M.
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