| Abstract | Primjena fitoestrogena se kontinuirano povećava zbog njihove biološke aktivnosti i potencijalno korisnih učinaka. Na tržištu se
nalazi sve veći broj pripravaka koji sadrže različite fitoestrogene namijenjene ženama u postmenopauzi kao posebno rizičnoj
skupini. Osim što se razlikuju kvantitativno s obzirom na ukupni sadržaj fitoestrogena, dodatno se razlikuju i kvalitativno s
obzirom na zastupljenost pojedinih fitoestrogena.
Cilj je ovog rada odrediti farmakokinetičke parametre skupini 15 strukturno sličnih fitoestrogena (genistein, biokanin A,
daidzein, formononetin, genistin, sisotrin, daidzin, ononin, prunetin, 3,4,7-trihidroksi izoflavon, 4,6,7-trihidroksi izoflavon,
4,6,7-trimetoksi izoflavon, genistein 4,7-dimetil eter, kumestrol, kumestrol dimetil eter) u svrhu boljeg razumijevanja njihove
bioraspoloživosti i učinaka na organizam. Zatim, na temelju kromatografski određene lipofilnosti razviti novu, brzu, selektivnu
i osjetljivu analitičku metodu za određivanje 8 najznačajnijih fitoestrogena iz skupine izoflavona u obliku aglikona i njihovih
glikozida.
Određeni su parametri koji opisuju lipofilnost skupine strukturno sličnih fitoestrogena primjenom tekućinske kromatografije
ultravisoke djelotvornosti obrnutih faza (RP-UHPLC). Ispitane su njihove interakcije s fosfolipidima staničnih membrana
primjenom tekućinske kromatografije visoke djelotvornosti i kolona s vezanim umjetnim membranama. Određeni su udjeli
vezanja fitoestrogena za najzastupljenije proteine plazme primjenom biomimetičke kromatografije. Permeabilnost fitoestrogena
i način prijenosa kroz sluznicu gastrointestinalnog trakta određeni su primjenom modela Caco-2 stanica. Dobiveni rezultati
diferenciraju fitoestrogene sukladno njihovoj strukturi, gdje su aglikoni pokazali značajno višu lipofilnost, jaču interakciju s
fosfolipidima, viši udio vezanja za alfa-1-kiseli glikoprotein i višu permeabilnost u usporedbi s glikozidima. Međutim, udio
vezanja za humani serumski albumin bio je visok za sve ispitivane fitoestrogene. Eksperimentalno određeni farmakokinetički
parametri uspoređeni su s izračunatim različitim računalnim programima.
Razvijena je nova RP-UHPLC metoda primjenom QbD pristupa za istovremenu kvntifikaciju najznačajnijih fitoestrogena iz
skupine izoflavona u obliku glikozida (genistin, sisotrin, daidzin, ononin) i aglikona (genistein, biokanin A, daidzein,
formononetin) bez prethodno provedenog dugotrajnog postupka hidrolize. Metoda je validirana prema ICH smjernicama i
primijenjena u analizi pripravaka različitih dozirnih oblika Razvijena je i dvodimenzionalna metoda tekućinske kromatografije
ultravisoke djelotvornosti spregnute s masenom spektrometrijom (2D-UHPLC-MS) za pouzdanu identifikaciju fitoestrogena iz
skupine izoflavona u složenom matriksu ispitivanih uzoraka. Analiza pripravaka s fitoestrogenima namijenjenim ženama u
postmenopauzi novom metodom pokazala je velika odstupanja između kromatografski određenog i deklariranog sadržaja
ispitivanih izoflavona. |
| Abstract (english) | The use of phytoestrogens has increased remarkably due to their biological activity and potential beneficial effects. An increasing
number of dietary supplements containing different phytoestrogens are widely present in the market targeting postmenopausal
women as a particular risk group. The content of isoflavones in dietary supplements not only differ quantitatively concerning
their total isoflavone content, but also qualitatively concerning their isoflavone spectrum.
The aim of the present study was to obtain data on pharmacokinetic properties for a set of 15 structurally related phytoestrogens
(biochanin A, daidzein, formononetin, genistein, genistein-4,7-dimethylether, prunetin, 3,4,7-trihydroxyisoflavon, 4,6,7-
trihydroxyisoflavon, 4,6,7-trimethoxyisoflavon, daidzin, genistin, ononin, sissotrin, coumestrol, coumestrol dimethylether) for
the purpose of better understanding their biological activity and effects. Thereafter, based on the chromatographically determined
lipophilicity, a novel, fast, selective and sensitive analytical method for simultaneous quantification of the 8 most important
isoflavones in the aglycone and glycoside forms was developed.
Lipophilicity of structurally related phytoestrogens was measured using reversed phase ultra-high performance liquid
chromatography (RP-UHPLC). The phospholipid binding measurements were carried out using high performance liquid
chromatography (HPLC) on a column with a stationary phase that contains phosphatidylcholine. The protein binding
measurements were carried out using biomimetic chromatography. Caco-2 cells were used to estimate the intestinal permeability
and the way of transport across the membrane of phytoestrogens test compounds. Obtained results differentiate phytoestrogens
according to their structure where aglycones show significantly higher lipophilicity, IAM partitioning, AGP binding and Caco-
2 permeability comparing to glucosides. However, HSA binding was very high for all investigated compounds. Experimentally
obtained pharmacokinetic parameters were compared with in silico predicted data by different computer platforms.
A new RP-UHPLC method was developed for simultaneous determination of the most significant phytoestrogens from
isoflavone family in aglycone (genistein, daidzein, biochanin A and formononetin) and glycoside (genistin, daidzin, sissotrin,
ononin) forms without prior time-consuming process of hydrolysis. The method was validated as per ICH recommendations and
was successfully applied to the analysis of various dietary supplements. Additionally to identification using external standard,
the identity of selected compounds in samples was confirmed by using two-dimensional ultra-high performance liquid
chromatography-tandem mass spectrometry (2D-UHPLC-MS). Analysis of dietary supplements containing phytoestrogens
intended for use in postmenopausal women, using newly developed method, revealed significant deviations between measured
and labelled content of investigated isoflavones. |
