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should certainly help in interdisciplinary discussions of somewhat fuzzy notions such as research material
specification, molecular similarity, antioxidant properties, estrogenicity, chemoprevention, etc. Despite the
constant refinement of scientific information through the exchange of peer opinion, some statements that
lack factual support can persist in literature for decades. Nearly all papers tackling the subject of isoflavone
activity underline genistein similarity to 17-β-estradiol (E2), offering only a single parameter - distance
of ca. 12Å between two hydroxyl groups in both ER ligands for support of that opinion. Such superficial
statements disregard the fact that molecular similarity is an important issue in current cheminformatics as
well as medicinal chemistry, which calls for quantification in reference to a selected model [49-51] . It should
not be overlooked that molecular parametrization systems, which are compatible with AI, ML, and neural
networks, must take over to be able to cope with big data. Coping with expanding databases, which store
astronomical quantities of information on the structures of chemical and biological objects and their
parameters and interrelations, quickly become essential for in silico drug design and initial evaluation [52-54] .

Natural isoflavones belong to the phenylpropanoid (flavonoid) category through a common biogenetic
pathway and share a considerable part of chemical and biochemical characteristics with the large group of
plant polyphenols, also these being from other branches of aromatic secondary metabolite origin. Their
fundamental structural difference from other subcategories of flavonoids (which are 2-aryl chromones)
resides in the phenylpropanoid ABC ring system connectivity [Figure 2], which stems from isoflavone
synthase action, transferring the aromatic B ring to the C-3 atom of the AC chromenone system. Unlike
ubiquitous flavones, flavonols and anthocyanidins which are widespread throughout the plant kingdom,
isoflavones occur mainly in the family Fabaceae, which is particularly important for animal feed and
[55]
human nutrition .
An interest in biological activity of isoflavones initiated when seasonal intake of Trifolium subterraneum,
containing an isoflavone fraction rich in formononetin, was identified as the cause of sheep fertility
problems (called “clover disease”) in Western Australia around 1940 [56,57] . After thorough veterinarian
investigation which followed, isoflavones were classified as phytoestrogens and occasionally even included
in a category of endocrine disruptors (negative classification of environmental industrial pollutants with
phenolic characteristics). Later, this seemingly local problem, seriously affected the perception of soy as
principal agricultural crop and important source of animal feed and human food [58,59] . Soybeans contain on
average more than one mg/g of isoflavones (genistein, daidzein, and glycitein and their glycosides; Figure 2)
which during regular oil separation-oriented industrial process end up in the protein fraction and further
in soy flour-derived products [60,61] . Phytoestrogenic food components may be considered beneficial for
some consumer segments (such as women in their post-menopausal period of life) but may cause serious
concerns for others (infants fed with soy-based formula, prepubertal youth, cancer patients, etc.) [62-64] . This
warranted basic pharmacological research which started soon after the discovery of estrogen receptors (ERs),
and its results are a matter of continuous reassessment, critical review and constant debate. Isoflavones
such as the soy constituent genistein are proven ER ligand subtypes, although with considerably lower
affinity than the natural substrate: 17-β-estradiol. Because they can attain much higher concentrations
than natural estrogens, competition for the ER binding site is possible, as proven by radioisotope-labeling
experiments. However, the ligand-receptor affinity issue is only a minute part of the estrogenic effect
complexity. Bioavailability, pharmacokinetics and metabolism (including microbiome biotransformation)
can make a dramatic difference on a systemic level. Pharmacodynamically, phytoestrogens can exert their
effects via different mechanisms and pathways. Ligands that can directly enter cells and internalize into
the nucleus bind to ER and initiate ERE (estrogen response elements) responses effects via interactions
with DNA (genomic mechanism). Alternatively, non-genomic signaling takes place when ligands bind to
membrane receptors and start intracellular kinase cascades. Genistein (4’, 5, 7-trihydroxyisoflavone), one of
the most studied NPs, is a good example of a partially agonistic ligand of both ERs, which are differentially
expressed in various tissues and can regulate antagonistically such basic processes as cell cycle, proliferation