514 Reviews New Concepts, Experimental Approaches, and Dereplication Strategies for the Discovery of Novel Phytoestrogens from Natural Sources Authors Thomas Michel, Maria Halabalaki, Alexios-Leandros Skaltsounis Affiliation Division of Pharmacognosy and Natural Products Chemistry, Department of Pharmacy, National and Kapodistrian University of Athens, Athens, Greece Key words Abstract phy-biochemical detection, frontal affinity chro- l" phytoestrogens ! matography-mass spectrometry and pulsed ul- l" plant extract Phytoestrogens constitute an attractive research trafiltration-MS will also be presented. Finally, l" dereplication topic due to their estrogenic profile and their bio- miniaturized methods (microchip and biosensor) l" extraction logical involvement in womanʼs health. Therefore, will be also discussed. l" isolation l" identification numerous studies are currently performed in nat- With the current review, we attempt to give a ural products chemistry area aiming at the dis- wide and holistic overview of the different ap- covery of novel phytoestrogens. The main classes proaches which could be employed in the discov- of phytoestrogens are flavonoids (flavonols, flava- ery of new phytoestrogens. On the other hand, we nones), isoflavonoids (isoflavones, coumestans), anticipate to attract more scientists to the area of lignans, stilbenoids as well as miscellaneous phytoestrogens and to indicate the need of multi- chemical groups abundant in several edible and/ disciplinary concepts. or medicinal plants, belonging mostly to the Le- guminosae family. As for other bioactives, the de- tection of new structures and more potent plant- Abbreviations derived phytoestrogens typically follows the gen- ! eral approaches currently available in the natural ACN: acetonitrile product discovery process. Plant-based ap- ASE: accelerated solvent extraction proaches selected from traditional medicine BCD: biochemical detection knowledge and bioguided concepts are routinely BuOH: butanol employed. However, these approaches are associ- CCC: countercurrent chromatography received February 3, 2013 ated with serious disadvantages such as time- Co(II)-PAB: cobalt(II)-coated paramagnetic revised February 3, 2013 consuming, repeated, and labor intensive pro- affinity beads accepted February 5, 2013 cesses as well as lack of specificity and reproduci- CPC: centrifugal partition chromatography Bibliography bility. In recent years, the natural products chem- DAD: diode array detector DOI http://dx.doi.org/ istry became more technology-driven, and sev- DPPH: 2,2-diphenyl-1-picrylhydrazyl 10.1055/s-0032-1328300 eral different strategies have been developed. EA: ethyl acetate This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. Published online March 11, 2013 Structure-oriented procedures and miniaturized ER: estrogen receptor Planta Med 2013; 79: 514–532 approaches employing advanced hyphenated an- ERE: estrogen response elements © Georg Thieme Verlag KG alytical platforms have recently emerged. They fa- EtOH: ethanol Stuttgart · New York · cilitate significantly not only the discovery of nov- FAC: frontal affinity chromatography ISSN 0032‑0943 el phytoestrogens but also the dereplication pro- FRET: fluorescence resonance energy Correspondence cedure leading to the anticipation of major draw- transfer Dr. Maria Halabalaki backs in natural products discovery. In this re- FTICR: Fourier transform ion cyclotron Laboratory of Pharmacognosy and Natural Products Chemistry view, apart from the traditional concepts followed resonance School of Pharmacy in phytochemistry for the discovery of novel bio- GC‑MS: gas chromatography-MS National and Kapodistrian logically active compounds, recent applications in Hept: heptane University of Athens Panepistimioupoli, Zografou the field of extraction, analysis, fractionation, and Hex: hexane 15771 Athens identification of phytoestrogens will be dis- HPLC: high-performance liquid Greece cussed. Moreover, specific methodologies com- chromatography Phone: + 302107754032 Fax:+302107754594 bining identification of actives and biological HRMS: high-resolution mass spectrometry [email protected] evaluation in parallel, such as liquid chromatogra- HSCCC: high-speed CCC Michel T et al. New Concepts, Experimental … Planta Med 2013; 79: 514–532 Reviews 515 HTS: high-throughput screening PUF: pulsed ultrafiltration LC‑BCD: online LC-based biochemical detection QTOF: quadrupole time-of-flight MAE: microwave-assisted extraction RP: reversed phase MAS: microwave-assisted sonication SERM: selective estrogen receptor modulator MeOH: methanol SFE: supercritical fluid extraction MS: mass spectrometry SHBG: sex hormone binding globulin MSPD: matrix solid phase dispersion SPE: solid phase extraction MtBE: methyl tert-butyl ether SWE: subcritical water extraction NMR: nuclear magnetic resonance TFA: trifluoroacetic acid PCA: principal component analysis UAE: ultrasound-assisted extraction PDMS: polydimethylsiloxane UHPLC: ultra high-performance liquid chromatography PLE: pressurized liquid extraction UMAE: ultrasound- and microwave-assisted extraction Introduction properties (selectivity) depending on the receptor content of spe- ! cific tissues (ERα and ERβ populations) and the concentration of Phytoestrogens are nonsteroidal natural compounds, usually the endogenous estrogens. Finally, phytoestrogens may also have plant constituents or their metabolites, which induce biological no affinity to ER (iv); in this case they act through alternative responses in mammals mimicking or modulating the action of mechanisms such as inhibition or activation of specific enzymes endogenous estrogens (i.e., 17β-estradiol, estriol, estrone). Semi- implied in an estrogenic response [2,10] or altering the total synthetic products and synthetic analogues of natural phytoes- amount of free estrogens in the organism. For instance, it has trogens are also sporadically included [1,2]. The role of phytoes- been reported that phytoestrogens interfere with the synthesis trogens and their possible protective effect in hormone-related of steroid hormones by inhibiting specific enzymes such as aro- disorders and diseases such as postmenopausal syndrome, osteo- matase (responsible for the conversion of androgens to estro- porosis, breast and prostate cancer, and cardiovascular complica- gens) [11,12] and 17β-hydroxysteroid oxydoreductase (respon- tions has been extensively investigated [3–6]. sible for the conversion of estrone to estradiol) [13]. Furthermore, The biological action of phytoestrogens is mainly implemented it has been suggested that phytoestrogens are able to stimulate, through their ability to bind to the ER which belongs to the nu- in the liver, the synthesis of proteins which are capable to bind clear receptors superfamily. To date, two main isoforms, ERα and estrogens (SHBG), affecting the overall percentage of free estro- ERβ, encoded by two different genes, have been described [7]. gens in the blood circulation [14,15] and thereby exhibiting an The primary structure of ER is characterized by three main func- indirect estrogenic effect. tional domains: the area responsible for the binding with the li- On the basis of their chemical structure and in respect to biosyn- gand (hormone, phytoestrogens), the region responsible for the thesis patterns, phytoestrogens may be divided in chalcones, fla- interaction with DNA, and the region regulating transcription [8, vonoids (flavones, flavonols, flavanones, isoflavonoids), lignans, 9]. The two isoforms are differently distributed to organs and tis- stilbenoids, and miscellaneous classes. Particular attention sues and present a different affinity to ligands. should be given to isoflavonoids, the subgroup of flavonoids Phytoestrogens can act either as estrogen agonists or antagonists which includes amongst others the chemical groups of isofla- since they may trigger (i) an estrogenic response, thereby binding vones, isoflavanones, pterocarpanes, and coumestans (l" Fig. 1) and activating the ER, which ultimately induces an estrogenic ef- [16,17]. fect or (ii) an antiestrogenic response, blocking or altering the re- These phytochemicals belong to the wide class of polyphenols ceptor activation by endogenous estrogens and thereby inhib- which are ubiquitous in the plant kingdom and characterized by iting an estrogenic effect. Furthermore, phytoestrogens have also nonsteroidal structures (l" Fig. 1). Specific structural characteris- been classified as SERM. Thus, phytoestrogens can act as estrogen tics resembling those of endogenous estrogens and consequently agonists and antagonists at the same time usually in an organ-de- demonstrating an estrogenic profile are responsible for their af- pendent manner resulting in (iii) mixed agonistic/antagonistic finity to the ER receptor. This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. Fig. 1 Chemical structures of representative phy- toestrogens. Michel T et al. New Concepts, Experimental … Planta Med 2013; 79: 514–532 516 Reviews In particular, the estrogenicity of a molecule which is imple- tive. Chemically, they are compounds consisting of two phenyl- mented through ER is related to specific structural requirements propanoid units linked via a carbon–carbon single bond. The such as the presence of hydroxyl groups able to form hydrogen most active lignans are enterodiol and enterolactone which orig- bonds with key functional amino acids of the binding pocket of