Lupinalbin A as the most potent estrogen receptor α- and aryl hydrocarbon receptor agonist in Eriosema laurentii de Wild. (Leguminosae)
© Ateba et al.; licensee BioMed Central Ltd. 2014
Received: 2 April 2014
Accepted: 5 August 2014
Published: 9 August 2014
Eriosema laurentii De Wild. (Leguminosae) is a plant used in Cameroon against infertility and gynecological or menopausal complaints. In our previous report, a methanol extract of its aerial parts was shown to exhibit estrogenic and aryl hydrocarbon receptor agonistic activities in vitro and to prevent menopausal symptoms in ovariectomized Wistar rats.
In order to determine the major estrogen receptor α (ERα) agonists in the extract, an activity-guided fractionation was performed using the ERα yeast screen. To check whether the ERα active fractions/compounds also accounted for the aryl hydrocarbon receptor (AhR) agonistic activity of the crude methanol extract, they were further tested on the AhR yeast screen.
This study led to the identification of 2′-hydroxygenistein, lupinalbin A and genistein as major estrogenic principles of the extract. 2′-hydroxygenistein and lupinalbin A were, for the first time, also shown to possess an AhR agonistic activity, whereas genistein was not active in this assay. In addition, it was possible to deduce structure-activity relationships.
These results suggest that the identified compounds are the major active principles responsible for the estrogenic and AhR agonistic activities of the crude methanol extract of the aerial parts of Eriosema laurentii.
KeywordsEriosema laurentii de Wild. Yeast transactivation assays Estrogen receptor α Aryl hydrocarbon receptor Genistein 2′-Hydroxygenistein Lupinalbin A
Plants from the Leguminosae family are well known for their medicinal properties and are used for different purposes in connection with women’s reproductive function, bone density, cardiovascular health and cancer prevention. Their therapeutic effects are very often attributed to the estrogen-like phytoconstituents known as phytoestrogens. For instance, the health benefits of soybeans (Glycine max L.) and red clover (Trifolium pratense L.) on menopause-related health problems have frequently been ascribed to their high isoflavone content [1–3]. Phytoestrogen exposure occurs primarily through dietary intake of foods or food supplements  and these compounds display estrogenic properties due to the binding to estrogen receptors in target cells. Beside estrogen receptors, phytoestrogens can also directly or indirectly modulate a wide range of signaling pathways, including the aryl hydrocarbon receptor (AhR) [5, 6]. Although the effect of this receptor on the reproductive physiology is not yet fully understood, studies have reported its involvement in several antiestrogenic activities [7–9].
Eriosema laurentii De Wild. (Leguminosae) is widely distributed in West and Central Africa where it is used as traditional remedy and food . In Cameroon, E. laurentii preparations are used for the treatment of infertility and various gynecological and menopausal complaints. Our previous study showed that the methanol extract of the aerial parts of E. laurentii exhibited agonistic activities at the estrogen receptor α and the aryl hydrocarbon receptor in yeast transactivation assays and prevented menopause-related symptoms induced by ovariectomy in rats . In addition, the safety profile of this extract indicated a broad safety margin following acute and subchronic oral administration . Therefore, in order to identify the chemical constituents responsible for the observed estrogenic and AhR agonistic activities, an activity-guided fractionation was performed using the ERα- and AhR-yeast assays on recombinant Saccharomyces cerevisiae strains.
General experimental procedures
Solvents for extraction and fractionation (analytical grade) as well as HPLC-grade acetonitrile (Chromanorm) and methanol (LiChrosolv) were obtained from VWR International (West Chester, Pennsylvania, USA). Glacial acetic acid (Rotichrom) was purchased from Carl Roth (Karlsruhe, Germany). Genistein (HPLC quality) for the dereplication approach was purchased from Sigma-Aldrich (St. Louis, Missouri, USA).
Aerial parts of E. laurentii were collected at July 10th, 2010 in Bazou, West Region of Cameroon. The plant was identified and authenticated by Mr. Victor Nana, botanist at the Cameroon National Herbarium, where a voucher specimen has been deposited under the number 24480/SRF/Cam.
Preparation of the extract and fractionation
Identification of the major active compounds
Gradient 1: 5% B (0 min), 5% B (5 min), 20% B (15 min), and 20% B (30 min)
Gradient 2: 20% B (0 min), 20% B (5 min), 34% B (10 min), 34% B (30 min), and 36.4% B (37.5 min)
Each gradient was followed by a column cleaning and re-equilibration step. The eluent flow was split in a 1:4 ratio before the ESI ion source, which was operated as follows: capillary voltage: ±3.7 kV, nebulizer: 26 psi (N2), dry gas flow: 9 L/min (N2), and dry temperature: 340°C. MS2, MS3, and MS4 spectra were obtained in an automated data-dependent acquisition mode (collision gas: He, isolation window: 4 Th, fragmentation amplitude: 1.0 V).
1H (ppm) JH,H(Hz)
13C (ppm) 
1H (ppm) JH,H(Hz)
13C (ppm) 
6.281 (d 2.2)
6.258 (d 2.2)
6.483 (d 2.2)
6.335 (d 2.2)
7.007 (d 2.0)
6.424 (d 2.0)
6.903 (dd 2.0/8.5)
6.412 (dd 2.0/8.5)
7.780 (d 8.5)
6.955 (d 8.5)
2′-Hydroxygenistein: +ESIMS m/z 287.0 [M + H]+; +ESIMS2 (287.0 →) m/z 271.0 (8), 268.9 (8), 259.0 (27), 244.9 (35), 230.9 (19), 216.9 (100), 202.9 (11), 189.0 (16), 175.0 (34), 161.0 (23), 153.0 (95), 149.0 (34), 137.0 (11); -ESIMS m/z 284.9 [M-H]-; -ESIMS2 (284.9 →) m/z 240.9 (6), 216.9 (100), 198.9 (8), 174.9 (6).
Lupinalbin A: +ESIMS m/z 285.0 [M + H]+; +ESIMS2 (285.0 →) m/z 256.9 (100), 228.9 (23), 212.9 (10), 185.0 (12), 173.0 (6); +ESIMS3 (285.0 → 256.9 →) m/z 228.9 (100), 173.2 (7); -ESIMS m/z 282.9 [M-H]-; -ESIMS2 (282.9 →) m/z 265.8 (8), 264.8 (17), 254.9 (100), 238.9 (24), 236.9 (7), 226.9 (10), 214.9 (8), 210.9 (8), 172.9 (7); -ESIMS3 (282.9 → 254.9 →) m/z 236.8 (6), 226.9 (100), 210.9 (14), 182.9 (10).
Compounds assigned in the extracts of E. laurentii by HPLC-DAD and HPLC/ESI-MS
257sh, 269, 349
254, 266sh, 349
Yeast transactivation screens
Disodium hydrogen phosphate dihydrate (Na2HPO4 · 2H2O), potassium chloride (KCl), magnesium sulfate heptahydrate (MgSO4 · 7H2O), sodium carbonate (Na2CO3) and ammonium sulfate ((NH4)2SO4) were obtained from Merck (Darmstadt, Germany). Sodium dihydrogen phosphate dihydrate (NaH2PO4 · 2H2O), N-lauroylsarcosine (Sarkosyl), o-nitrophenol-β-galactopyranoside (ONPG), DL-dithiothreitol (DTT), dimethylsulfoxide (DMSO), D-(+) glucose, estradiol, 5α-dihydroxytestosterone, genistein, progesterone, and β-naphthoflavone were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Yeast nitrogen base was obtained from Difco (Franklin Lakes, New Jersey, USA), amino acids from Serva Feinbiochemica (Heidelberg, Germany), dropout medium without tryptophan (DO-trp) from Sigma-Aldrich, and dropout supplement media without tryptophan and uracile (CSM-trp-ura) from MP Biomedicals.
LacZ-buffer was composed of 60mM Na2HPO4 · 2H2O, 40mM NaH2PO4 · 2H2O, 10mM KCl, 1mM MgSO4 · 7H2O, and 1mM DTT. For the Z- sarcosyl-buffer, 0.5% N-lauroyl-sarcosine was dissolved in LacZ-buffer with 2mM DTT.
The estrogenic and aryl hydrocarbon receptor agonistic activities were assessed using recombinant Saccharomyces cerevisiae yeast strains 188R1 and YCM3. The estrogen yeast assay is a two-plasmid system containing an expression plasmid with the human ERα gene and a LacZ reporter plasmid. The construct of the AhR yeast assay contains beside a LacZ reporter plasmid, the human AhR and aryl hydrocarbon receptor nuclear translocator (ARNT) genes integrated in chromosome III. Assay performance and data evaluation have been described previously [5, 15].
Briefly, 1 μL of sample was added in 100 μL of yeast culture (OD600 = 0.4) and incubated at 30°C for 5 h and 17 h for ERα and AhR, respectively. After incubation cells were disintegrated by adding 150 μL 2 mM Z-sarcosyl-buffer, the OD600 measured and the microtiter plate incubated at 30°C for 20 min for the complete disintegration. In each well, 50 μL of o-nitrophenyl β-D-galactopyranoside (4 mg/mL in lacZ-buffer) were added and the plate was incubated at 37°C till the development of yellow color. Afterwards, the reaction was stopped by adding 50 μL 1M Na2CO3, the total reaction time noted and the absorption measured at 405 nm (reference wavelength 620 nm).
Data are expressed as means of three independent duplicate experiments ± standard deviation (SD) and analyzed using Student’s t-test. Results were considered significant when p ≤ 0.05.
Results and discussion
In order to identify the major compounds responsible for the estrogenic activity of the methanol extract of the aerial parts of E. laurentii, an activity-guided fractionation was performed as described in more detail above (Figure 1).
Fraction Bu of W-CMAL also induced a β-galactosidase activity in the ERα yeast assay (Figure 4B) and its fractionation yielded 2′-hydroxygenistein (250 mg) as the major component. Fraction Ci of S-CMAL increased the ERα activity in a significant and dose-dependent manner (p ≤ 0.05) starting from 0.1 μg/mL. Its magnitude of activity at 1 μg/mL equals that of estradiol at 0.5-1 nM. Moreover, the fraction 7 of Ci (F7) was about a factor of ten more potent than fraction Ci (Figure 4C). From this subfraction, lupinalbin A (21 mg) was isolated. The identification of lupinalbin A and 2′-hydroxygenistein was performed by MS- and one and two-dimensional NMR-experiments. Data showed good correlation with literature (see Table 1). Genistein, one of the major constituents of E. laurentii as reported previously , was identified using a dereplication approach by TLC, LC-DAD and MS.
The activity-guided fractionation of the crude methanol extract of aerial parts of Eriosema laurentii lead to the identification of components with estrogenic and aryl hydrocarbon receptor agonistic activities. Genistein, 2′-hydroxygenistein and lupinalbin A, which were identified in or isolated from the most active fractions, were found to be full agonists of ERα. Furthermore, we provide the first report of an agonistic activity of lupinalbin A and 2′-hydroxygenistein towards the AhR, whereas genistein remained without effect at the AhR. The results enabled the deduction of structure-activity relationships for AhR agonistic activity between these compounds for the first time as well. Thus, the data presented in this study confirmed that these phytoconstituents contribute to the estrogenic and aryl hydrocarbon receptor agonistic activities of the extract of Eriosema laurentii and might partly account to the beneficial effects of the methanol extract in vivo.
Estrogen receptor alpha.
Sylvin Benjamin Ateba has received an Ernst-Mach-Stipendium granted by the OeAD-Austrian Agency for International Cooperation in Education & Research, financed by BMWF.
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