E.L. Yong

Pharmacokinetics of prenylflavonoids and correlations with the dynamics of estrogen action in sera following ingestion of a standardized Epimedium extract.

To explore pharmacokinetic properties of prenylflavonoids from the Traditional Chinese Medicinal plant Epimedium, three doses of a standardized extract (100, 300 and 600 mg/kg body weight), were administered to ovariectomized rats and serial blood samples were obtained. Serum concentrations of the Epimedium prenylflavonoids icariin, icariside I, icariside II, icaritin and desmethylicaritin were determined by LC-MS/MS. Aliquots of sera were also applied to human cell lines that permanently express ERalpha and ERbeta proteins for the ex vivo measurement of estrogenic activity. All five prenylflavonoids exhibit non-linear dose-dependent increases in the area under concentration versus time curves. Two distinct pharmacokinetic patterns were evident, an early phase wherein icariin and icariside II reached t(max) 0.5-1 h, and a late phase wherein icariside I, icaritin and desmethylicaritin peaked at t(max) 8h. Total concentrations of icaritin and desmethylicaritin reached C(max) approximately 2 microM and approximately 0.25 microM respectively. Estrogenic activity in Epimedium-treated rat sera lagged by several hours compared to animals treated with control drug estradiol benzoate, corresponding to the appearance of bioactive metabolites desmethylicaritin, icaritin and icariside I. Following glucuronidase/sulphatase treatment, prolonged estrogenic activity at higher Epimedium doses (300 and 600 mg/kg of body weight) was evident, and correlated with the persistence of micromolar levels of icaritin at the 48-72 h sampling period. The depot effect resulted in time-concentration bioactivity profiles at the three Epimedium doses (area under curve 374, 543, and 771pME2 h(-1)) that exceeded that observed for estradiol benzoate (148pME2 h(-1)). Our study correlated the pharmacokinetics of prenylflavonoids with the dynamics of their estrogenic effects and reveals the potential estrogenicity of this Epimedium extract. This study may aid the development of prenylflavonoids as drugs for menopause and other conditions requiring estrogenic action.

Simple and sensitive liquid chromatography–tandem mass spectrometry assay for simultaneous measurement of five Epimedium prenylflavonoids in rat sera

A rapid and sensitive method to separate and quantify icariin, icariside I, icariside II, icaritin and desmethylicaritin in rat sera was developed using liquid chromatography-tandem mass spectrometry. Serum samples were extracted with ethyl acetate without further derivatization. Using coumestrol as an internal standard, calibration curves with good linearity (r(2)>0.99) within the concentration range of 0.78-12.5 nM for icariin, icaritin and desmethylicaritin, and 0.78-100 nM for icariside I and II, were obtained in the multiple reaction monitoring mode. For all analytes, the limits of detection and quantification were <1 nM and 1-2 nM, respectively. Inter- and intra-assay variabilities were <15% and accuracies were between 94% and 114%, respectively. This method was successfully applied to quantify levels of icariin, icariside I, icariside II, icaritin and desmethylicaritin in rat sera after oral administration of an Epimedium preparation.

Preparation and characterization of 4-isopropylcalix[4]arene-capped (3-(2-O-β-cyclodextrin)-2-hydroxypropoxy)-propylsilyl-appended silica particles as chiral stationary phase for high-performance liquid chromatography

A new type of 4-isopropylcalix[4]arene-capped (3-(2-O-β-cyclodextrin)-2-hydroxypropoxy)propylsilyl-appended silica particles (IPC4CD-HPS) has been prepared by treatment of bromoacetate-substituted (3-(2-O-β-cyclodextrin)-2-hydroxypropoxy)propylsilyl-appended silica particles (BACD-HPS) with 4-isopropylcalix[4]arene oxyanions in anhydrous N-methyl-2-pyrrolidone. The bonded silica IPC4CD-HPS has been successfully used as chiral stationary phase (CSP) in high-performance liquid chromatography (HPLC) for the first time. The synthetic stationary phase was characterized by means of elemental analysis and Fourier transform infrared spectroscopy. This new CSP has a chiral selector with two anchored functional moieties: 4-isopropylcalix[4]arene and β-cyclodextrin. The chromatographic performance of IPC4CD-HPS was investigated by separation of positional isomers of several disubstituted benzenes and enantiomers of some chiral drug compounds under reversed-phase conditions. The results showed that IPC4CD-HPS had excellent selectivity for the separation of aromatic positional isomers and enantiomers of chiral compounds due to the cooperative functioning of the anchored 4-isopropylcalix[4]arenes and β-cyclodextrins.

Preparation and application of methylcalix[4]resorcinarene-bonded silica particles as chiral stationary phase in high-performance liquid chromatography†‡

Two new types of methylcalix[4]resorcinarene-bonded stationary phases, (3-(C-methylcalix[4]resorcinarene)-2-hydroxypropoxy)-propylsilyl-appended silica particles (MCR-HPS) and bromoacetate-substituted MCR-HPS particles (BAMCR-HPS), have been synthesized and used as chiral stationary phases for high-performance liquid chromatography (HPLC) for the first time. The synthetic stationary phases are characterized by means of elemental analysis and Fourier-transform infrared spectroscopy. The chromatographic behavior of MCR-HPS and BAMCR-HPS was studied with several disubstituted benzenes and some chiral drug compounds under both normal phase and reversed-phase conditions. The results show that MCR-HPS has excellent selectivity for the separation of aromatic positional isomers and BAMCR-HPS exhibits excellent performance for separation of enantiomers of chiral compounds.

Determination of breviflavone A and B in Epimedium herbs with liquid chromatography-tandem mass spectrometry.

Two new types of minor flavonoids, breviflavone A and B, have been recently isolated and identified from Epimedium brevicornu in our previous research. Breviflavone B is a novel flavonoid with potent and specific estrogen receptor (ER) bioactivity. Its positional isomer, breviflavone A, is not ER active. Therefore, it is important to determine the two minor components, breviflavone A and B, in Epimedium herbs. In this report, a robust method for measurement of the two breviflavones in Epimedium ethanolic extracts has been developed by using liquid chromatography tandem mass spectrometry via selected-reaction monitoring (m/z 437-->m/z 367 for breviflavone A and m/z 437-->m/z 351 for breviflavone B) under negative electrospray ionization mode. This method has been successfully used to determine the two breviflavones in ethanolic herbal extracts of five major Epimedium species (E. brevicornu, E. koreanum, E. pubescens, E. sagittatum, and E. wushanese) from various sources. The contents of the two breviflavones range from 0.0181 to 0.1791% for breviflavone A and 0.0026 to 0.0252% for breviflavone B in the dried ethanolic extracts of those Epimedium herbal samples.

Preparation and application of rifamycin-capped (3-(2-O-β-cyclodextrin)-2-hydroxypropoxy)-propylsilyl-appended silica particles as chiral stationary phase for high-performance liquid chromatography

Rifamycin-capped (3-(2-O-β-cyclodextrin)-2-hydroxypropoxy)-propylsilyl-appended silica particles (RCD-HPS), a new type of substituted β-cyclodextrin-bonded chiral stationary phase (CSP) for high-performance liquid chromatography (HPLC), have been synthesized by the treatment of bromoacetate-substituted-(3-(2-O-β-cyclodextrin)-2-hydroxypropoxy)-propylsilyl-appended silica particles (BACD-HPS) with rifamycin SV in anhydrous acetonitrile. The stationary phase is characterized by means of elemental analysis and Fourier-transform infrared spectroscopy. This new CSP has a chiral selector with two recognition sites: rifamycin and β-cyclodextrin (β-CD). The chromatographic behavior of RCD-HPS was studied with several disubstituted benzenes and some chiral drug compounds under reversed-phase HPLC mobile phase conditions. The results show that RCD-HPS has excellent selectivity for the separation of aromatic positional isomers and enantiomers of chiral compounds due to the cooperative functioning of rifamycin and β-CD.

Serum free estradiol and estrogen receptor-α mediated activity are related to decreased incident hip fractures in older women.

There is paucity of data from Asian women on the association between serum estrogens and osteoporotic hip fracture risk. We conducted a case-control study nested within a population-based prospective cohort, The Singapore Chinese Health Study, to evaluate serum estrogens levels, ERα-mediated estrogenic activity and hip fracture risk in postmenopausal Asian women. Among 35,298 women who were recruited between 1993 and 1998, 15,410 women donated blood for research between 1999 and 2004. From this subcohort, we identified 140 cases who subsequently suffered hip fracture after blood donation, and 278 age-matched controls. Serum levels of total estrone, estradiol and sex hormone binding globulin levels were measured in a blinded fashion among cases and controls. ERα-mediated estrogenic activity of serum samples was quantified using a sensitive ERα-driven cell bioassay. Women with hip fracture had lower serum estrogens than control women. Compared to the lowest quintile, women in the highest quintile of free estradiol exhibited a statistically significant 57% reduction in risk of hip fracture (95% confidence interval (CI), 6-80%), with a dose-dependent relationship (p for trend=0.021). High levels of ERα-mediated estrogenic activity were also associated with decreased risk of hip fracture (p for trend=0.048). Overall, women with relatively high levels of both free estradiol and ERα-mediated estrogenic activity had a 55% reduction in hip fracture risk (95% CI, 17-76%) compared to women with low levels of both. High levels of free estradiol and ERα-mediated estrogen activity in sera were associated with reduced hip fracture risk in Chinese postmenopausal women.

Phytoplankton blooms: an overlooked marine source of natural endocrine disrupting chemicals.

BACKGROUNDnWe had previously reported high androgenic and estrogenic activities in seawaters in confined clusters close to Singapore. Further investigations revealed a hitherto unsuspected link between estrogenic/androgenic activity and net phytoplankton count.nnnOBJECTIVEnThe primary objective of this study was to investigate the cause of a correlation between net phytoplankton and endocrine activity, and corroborate this observation, and rule out other possible confounding factors. Our secondary objective was to study if these estrogenic secretions can impact human health.nnnMETHODSnFive species of phytoplankton, Gymnodinium catenatum, Prorocentrum minimum, Alexandrium leei, Chattonella marina, and Fibrocapsa japonica, were isolated from Singapore waters and mass cultured and the cells and culture media screened for estrogenic and androgenic activity using human cell-based bioassays.nnnRESULTSnThe raphidophytes C. marina and F. japonica displayed significant estrogenic activity whilst the dinoflagellates G. catenatum and P. minimum displayed significant androgenic activity in both the cell extracts and the cell culture media extract.nnnCONCLUSIONSnOur data shows that selected phytoplankton isolates are potent secretors of estrogenic and androgenic substances, which are potential endocrine disrupting chemicals (EDCs). As the harmful nature of EDCs is largely due to their bioaccumulation in the aquatic food chain our findings imply that the impact of these phytoplankton secretions needs to be investigated especially for seafoods, which are only a single trophic level away from phytoplankton. Alternatively, should these phytoplankton-origin EDCs not accumulate through marine food chains to significantly impact humans or marine mammals, our results indicate that functional assays could greatly over-estimate the risk from naturally occurring EDCs produced by marine phytoplankton. It remains to be determined if these EDCs affect zooplankton and other organisms that directly feed on marine phytoplankton, or if the secreted EDCs can directly impact other marine fauna.

Enantiomeric Separation of 1-Phenyl-1-Propanol Using Calix[4]Arene-Capped β-Cyclodextrin-Bonded Silica Particles as Chiral Stationary Phase in Capillary Electrochromatography

A new type of calix [ arene-capped (3-(2-O-β-cyclodextrin)-2-hydroxypropoxy) propylsilyl-appended silica particles (C4CD-HPS) has been successfully used as chiral stationary phase (CSP) in capillary electrochromatography (CEC) for separation of enantiomers of 1-phenyl-1-propanol for the first time. C4CD-HPS contains a chiral selector with two recognition sites: calix [arene and β-cyclodextrin. Due to the cooperative functioning of the calix [arene and β-cyclodextrin, C4CD-HPS has exhibited excellent enantioselectivity for the enantiomers of 1-phenyl-1-propanol. After the multiple phenolic hydroxyl groups in the calix [arene moieties are ionized in the running buffer, the bonded stationary phase C4CD-HPS becomes negatively-charged to provide high electroosmotic flow (EOF) in CEC. Fast and high-resolution separation for enantiomers of 1-phenyl-1-propanol has been easily achieved on C4CD-HPS. This new type of chiral stationary phase has exhibited great potential for fast enantiomeric separations in CEC.

PREPARATION AND APPLICATION OF MIXED OCTADECYLSILYL- AND (3-(C-METHYLCALIX[4]RESORCINARENE)-HYDROXYPROPOXY)-PROPYLSILYL–APPENDED SILICA PARTICLES AS STATIONARY PHASE FOR HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

A new type of mixed octadecylsily (ODS)- and (3-(C-methylcalix[4]resorcinarene)-2-hydroxypropoxy)-propylsilyl–appended silica particles (ODS-MCR-HPS) have been prepared and used as stationary phase for high-performance liquid chromatography (HPLC) for the first time. Elemental analysis and Fourier-transform infrared spectroscopy have been applied to characterize the synthetic ODS-MCR-HPS. The new stationary phase contains two types of interaction sites for separation: ODS and MCR. The chromatographic performance of ODS-MCR-HPS was investigated by separating several disubstituted benzenes and some aromatic hydocarbons under both reversed-phase and normal-phase conditions. The results show that ODS-MCR-HPS exhibits excellent selectivity for the separation of positional isomers of disubstituted benzenes and mixtures of some aromatic hydrocarbon compounds due to the cooperative and complementary functioning of the ODS and MCR under different mobile phase conditions.