CHAPTER CONCLUSION 5.1 Significance of findings 5.2 Limitations 218 5.3 Will Epimedium exert any beneficial effects in-vivo? 223 5.4 Future work 226 215 214 5.1 Significance of findings This work and the bioassay tools developed in this thesis can serve as a model framework for future projects which focus on development of estrogenic botanicals as drugs for menopause, maintenance of bone health and other conditions requiring estrogenic action in humans, from the lab bench to bedside This thesis reported the importance and value of the development and validation of appropriate tools with high throughput, in this case, were cell-based assays that were needed to rapidly track and measure global estrogenic activity in complex mixtures, from herbal preparations, formulations to ex-vivo serum samples, which contain a myriad of bioactive compounds, known and unknown These bioassays can also be used to rapidly screen and select compounds with optimal ERα- or ERβ-selective properties for estrogen replacement therapy that has lower adverse effects such as breast cancer cell growth, thereby reducing the number of expensive, larger-scale studies with negative clinical outcomes Although a compound or preparation is shown to be estrogenic when tested directly in the bioassays, it cannot be assumed that it would exert similar activity when consumed Rigorous pharmacokinetics and pharmacodynamics studies on complex botanical extracts are have been seriously constrained by the lack of suitable tools that can simultaneously track serum levels and bioactivities of the many compounds, known and unknown, that exert pharmacological effects in both animal models and humans The development of these cell-based assays enabled new insights into the pharmacokinetic and pharmacodynamic interactions underlying the biological effects of a botanical preparation like Epimedium Two notable experiments were reported in this thesis include a clinical trial where human volunteers ingested a traditional aqueous preparation of Epimedium pubescens and another female ovariectomized rats being fed with an enriched extract made from Epimedium brevicornu Although they are not directly comparable due 215 to differences in their experimental design, it was found that only trace amounts of unconjugated prenylflavonoids were absorbed into the bloodstream From the rat study, it was found that the vast majority of prenylflavonoids absorbed were conjugated which were rendered relatively non-estrogenic during first pass metabolism The appearance of total amount of bioactive prenylflavonoids in sera corresponded broadly with estrogenic activity that was measured after treatment of serum samples with β-glucuronidase and sulfatase Non-linear pharmacokinetics and prolonged effects for up to 72 h following administration of a single dose of Epimedium extract were also observed Coupled processes of enteric and enterohepatic recycling may allow different prenylflavonoids to be reabsorbed which resulted in longer than expected apparent plasma half-lives for some Epimedium compounds and their conjugates β-glucuronidases present in many tissues such as bone, brain and mammary glands may convert conjugated prenylflavonoids in these tissues into their bioactive form if they become deconjugated Gene expression profiling revealed the ability of estrogenic Epimedium prenylflavonoids to induce the transcription of CYP1A1, which is a gene mediated by AhR AhR has been regarded as an inducible transcription factor that controls the expression of enzymes that metabolize potentially dangerous xenobiotic chemicals However, there are AhR ligands, such as icaritin found in Epimedium, which have been reported to be able to initiate the degradation of the ER and suppress estrogen signaling and proliferation of breast cancer cells By mediating with these pathways, these beneficial AhR ligands can be used to influence breast cancer initiation and progression The ability of Epimedium compounds to act as dual activators for ERα and AhR mechanistically represent a new class of SERMs These compounds should be further examined for development of a new family of drugs that can be co-administered during hormone replacement therapy to reduce associated breast cancer risk 216 Lastly, it is important to take note of the some experimental design pit-falls presented in this thesis which were discussed at length in Section 5.2 – Limitations Due to a range of differences in experimental design, the results from the clinical trial where human volunteers ingested a traditional aqueous preparation of Epimedium pubescens and another where rats were fed with an enriched extract made from Epimedium brevicornu are best interpreted in isolation as they were not easily and cannot be directly compared Two major differences in experimental design include the use of different botanical extracts and model organisms for the two studies Notably, it is vital to ensure consistency in the range of parameters that are to be measured and conditions that need to be employed throughout the project Lessons learnt from these two studies would greatly enhance the design and comparison of future similar studies on Epimedium and similar herbal drugs A good starting point for a pharmacokinetic and pharmacodynamic study on a similar estrogenic botanical preparation would be to refer to the rat experiment reported in this thesis which used the appropriate animal model, that is, female ovariectomized rats, which were fed separately with increasing doses of an enriched extract made from Epimedium brevicornu and treated over a period of 72 hours, with an inclusion of a positive control of rats fed with a standard estrogen prodrug, such as, estradiol benzoate Chemical analytical methods coupled with cell-based bioassays that can measure global estrogenicity of sera of animals were employed to understand the levels and bioactivity of conjugated and unconjugated active compounds respectively 217 5.2 Limitations The first study, which involved a human clinical trial, was a pilot project to validate the cell-based bioassays in a clinical setting These bioassays were developed for use to measure global activity in serum and to evaluate the effects of ligands in complex mixtures on ERα, ERβ bioactivity and MCF-7 breast cancer cell growth In this human study, healthy male subjects, instead of females, were enrolled to reduce interference from endogenously estrogens encountered in the latter group, which may mask the effects of administered estrogenic drugs The subjects were administered separately with estradiol valerate or Epimedium pubescens decoction Serum samples were obtained and assayed ex-vivo for levels of estrone and estradiol by tandem mass spectrometry, for ERα and ERβ bioactivity and MCF-7 breast cancer cell proliferative effects The concentrations of icaritin and desmethylicaritin and bioactivity in sera after ingestion of Epimedium pubescens decoction by human subjects were also measured as they are prenylflavonoids unique to Epimedium which exerted stronger estrogenic activities than their glycosides and flavonoids such as apigenin, kaempferol, luteolin and quercetin In the view of the low bioactivity measured in a traditionally prepared, aqueous extract, a brand new study was done in collaboration with Dr Willmar Schwabe Pharmaceuticals (Karlsruhe, Germany) A new standardized, prenylflavonoid-enriched extract based on Epimedium brevicornu was formulated and fed in three increasing doses to female ovariectomized Sprague–Dawley rats, with an additional group of rats which are fed with estradiol benzoate included that served as the positive control The use of female ovariectomized Sprague–Dawley rats is a more appropriate model for testing drugs for use to enhance post-menopausal bone health 218 In contrast to the earlier study, the serum bioactivity and concentrations of both unconjugated and conjugated levels of ingested prenylflavonids were monitored The levels of conjugated prenylflavonoids were included to see whether lack of estrogenic activity of Epimedium was due to conjugation of active compounds during first pass metabolism The quantification of prenylflavonoid glycosides, such as, icariin, icariside I and icariside II was also undertaken to understand the metabolism of Epimedium glycosides Although one may want to compare whether the prenylflavonoid-enriched extract had greater bioavailability compared to the aqueous decoction due to higher amounts of compounds present, meaningful comparison is hampered by the lack of consistency in the design of the two experiments Table 22 lists these differences Table 22: Methodological differences present in the human and rat studies Reference Li et al., 2009 Wong et al., 2009 Model organism Human males Ovariectomized female rats Sample size rats per time-point Herb species Epimedium pubescens Epimedium brevicornu Extraction method Dose(s) mg/kg body weight Study Duration (h) Water 70% EtOH 90.1 100, 300 & 600 48 72 Unconjugated icaritin & desmethylicaritin Unconjugated Icariin, icariside I, icariside II, icaritin, desmethylicaritin Compounds analyzed Total Icariside II, icaritin, desmethylicaritin 219 In the human clinical trial, male human subjects were recruited Male subjects, instead of females, were used so as to reduce interference from endogenously produced estrogens, which may mask the effects of administered estrogenic drugs With the intention to develop Epimedium as a botanical drug for use to maintain bone health after menopause, the more ideal candidates to use for such a study would be post-menopausal women as they will be a more relevant physiological model Differences in metabolism and plasma transport of steroids between men and women would likely to also influence the results of the study Results obtained from this human study may not be appropriate for comparison with those obtained from ovariectomized Sprague–Dawley rat study due to species differences in the model organism used Both studies also suffered from small sample sizes as shown in Table 22 Blood at the various time-points was taken from the same human subjects over time whereas four rats were sacrificed at each time interval Inter-individual differences may affect the results of both studies more significantly due to small sample sizes used Further difficulties arise when different species of Epimedium and extraction methods employed in the two studies, which result in two vastly different herbal extracts From chemical profiles obtained from quantitative analysis via chromatographic tools used in this study, the dried residue from the aqueous decoction made from Epimedium pubescens was found to contain mg/g icariin, 0.119 mg/g icaritin and 0.031 mg/g desmethylicaritin The amounts of icariside I and icariside II were not determined then The enriched Epimedium brevicornu extract generally comprised more prenylflavonoids, especially in terms of icariin but it had a lesser amount of icaritin - 143.3 mg/g icariin, 0.009 mg/g icaritin and 0.122 mg/g desmethylicaritin The amounts of icariside I and icarside II were determined to be 0.157 mg/g and 18.68 mg/g respectively The increased 220 amount of prenylflavonoids in the latter extract was due to the increased solubility of the compounds in 70% ethanol that was used for extraction Table 23: Comparison of extract dosages and amounts of prenylflavonoids ingested from traditionally prepared aqueous Epimedium pubescens decoction and prenylflavonoid-enriched extract from Epimedium brevicornu Extract dosage Aqueous Epimedium pubescens decoction 90.1 Icariin 0.180 14.3 42.9 85.8 Icariside I Not determined 0.0157 0.0471 0.0942 Icariside II Not determined 1.9 5.6 11.2 Icaritin 0.0107 0.0009 0.0027 0.0054 Desmethylicaritin 0.00279 0.0122 0.0366 0.0732 Preparation Prenylflavonoid-enriched Epimedium brevicornu extract (70% ethanol extracted) 100 300 600 Decoction was prepared from 50 g of dried leaves from Epimedium pubescens Yield after extraction was 12.6% and mean weight of human subjects was 70 kg Amount of extract and compounds ingested are expressed in mg / kg body weight On a dry weight basis, the lowest dose of Epimedium brevicornu employed in the rat study (100 mg/kg) closely approximates the amount of Epimedium pubescens extract (90.1 mg/kg) that was ingested by human subjects (Table 23) As a result of the use of different extraction methods, the profiles of prenylflavonoids ingested were markedly different, especially for icariin, which was nearly 80 times more abundant in the prenylflavonoid-enriched Epimedium brevicornu extract (see Table 23) With reference to the pharmacokinetic data from both studies, unconjugated desmethylicaritin was not detected in human sera in all time-points whereas it could be detected in low levels in sera from rats that were fed with Epimedium extract at a dose of 100 mg/kg The in-vivo 221 hydrolysis of glycosides like icariin can lead to the production and absorption of aglycones like icaritin and desmethylicaritin into the blood stream and this could have occurred more significantly rats which were fed with the Epimedium brevicornu extract due to a greater amount of icariin and similar precursor glycosides that were present As a result of such differences in starting composition of constituents, the bioavailability of Epimedium compounds cannot be accurately ascertained from the results from both studies The duration of two studies and the number of metabolites that were examined also differed The rat study reported the levels of total amounts of prenylflavonoids absorbed (sum of conjugated and unconjugated metabolites) and a depot effect was observed as the duration of study was prolonged to 72 h On the other hand, the human study ended at 48 h and the total amounts of prenylflavonoids absorbed were not determined It is also not certain whether a similar depot effect would occur in humans The results from both studies are hence best interpreted in isolation due to the differences present in their design as discussed above Lessons learnt from these two studies would greatly enhance the design and comparison of future similar studies on Epimedium and similar herbal drugs 222 5.3 Will Epimedium exert any beneficial effects in-vivo? Sera samples from human subjects who ingested the aqueous decoction contained low amounts of unconjugated icaritin and no desmethylicaritin The dose was likely to be too low and any absorbed aglycones would have been conjugated during extensive first pass metabolism Any unconjugated compounds would be present in trace levels and become bound tightly to albumin in sera during circulation This could be the reason why estrogenicity of sera was not detected in human serum samples as levels of unconjugated prenylflavonoids that were present in nanomolar quantities that are well below the detection limits of the panel of cell-based bioassays (see dose-response curves in Fig 15) Although the amount of prenylflavonoids was enriched in the alcoholic Epimedium brevicornu extract that was fed in three increasing doses in the rat study, due to extensive first pass metabolism, the peak serum levels of both unconjugated aglycones, namely, icaritin and desmethylicaritin, as well as, two estrogenic monoglucosides, icariside I and icariside II, were in the low nanomolar levels, even at the highest dose of Epimedium pubescens extract in rats The estrogenicity of rat serum samples for 100 and 200 mg/kg dosages were also not significant when measured the ERα cell-based bioassay Interestingly, estrogenicity equivalent to 10 pM of estradiol was detected in sera at the h time-point from rats fed with the highest dose of Epimedium brevicornu extract (600 mg/kg dose) (Fig 39) which was likely to be conferred by estrogenic metabolites not studied in this work This is indicative that Epimedium can exert estrogenicity in-vivo and future work entails optimization of the human equivalent dosage of extract that need to be taken to ensure efficacy Although the results for a single dose of Epimedium extract in this study may imply that the preparations would not exert significant estrogenicity in-vivo due to the low 223 amounts of unconjugated prenylflavonoids in circulation, it is important to note that in Traditional Chinese Medicine, medicinal herbs are rarely taken via a single dose In the Bencao Gangmu (本草纲目), a Chinese materia medica work written by Li Shizhen (李 时珍) in the Ming dynasty, Epimedium formulations are largely noted to require repeated dosings (Li, circa 1500) As a result of repeated dosings, Epimedium prenylflavonoids and their conjugates may be accumulated in the body over time and these may potentially be bioactive if they reach sufficiently high levels Conjugates can get hydrolyzed into bioactive aglycones in target organs and tissues (Penza et al., 2007) Epimedium is used as part of complicated formulations with other herbs that include rehmania root, curculigo rhizome and dogwood and wolfberry fruits In this study, Epimedium is administered as a single herb preparation and the bioavailability of prenylflavonoids in it may be enhanced when taken in combination with phytochemicals from other plants Lambert et al (2004) reported the co-treatment with piperine from black pepper, enhanced the bioavailability of (-)-epigallocatechin-3-gallate found in green tea in mice Moon & Morris (2007) reported that the co-administration of quercetin and (-)-epigallocatechin-3-gallate significantly increased the biochanin A area under the plasma concentration versus time curve in rats in both intravenous and oral administration of biochanin A According to the ancient text, Shen Nong Ben Cao Jing (神农本草经), Epimedium is documented to strengthen the bones and tendons, rheumatic conditions, impotence, seminal emission, weakness of the limbs, rheumatoid arthralgia with numbness and muscle contracture and climacteric hypertension In particular, much work has been done on the effects of Epimedium on bone health and these have been reviewed in the Introduction of this thesis Like many flavonoids, prenylflavonoids in Epimedium that 224 have been absorbed after ingestion are largely present in the conjugated form in circulation as observed in the rat study The conjugated forms that are present in large amounts in circulation, although rendered non-estrogenic, may still be able to exert beneficial effects on the bone This is supported by the observation that hesperetin-7-Oglucuronide that was reported to enhance primary rat osteoblast proliferation and differentiation, and down-regulated the expression of receptor activator of nuclear factorkappa B with no change in osteoprotegerin expression (Trzeciakiewicz et al., 2010) Hesperetin-7-O-glucuronide may be able to limit the activation of osteoclasts, similar to what have been reported for isoflavones such as daidzein and genistein (Chen et al., 2002) Osteoblasts express receptor activator of nuclear factor-kappa B ligand and osteoprotegerin In this case, osteoprotegerin can act as a decoy receptor for receptor activator of nuclear factor-kappa B ligand which then neutralizes its function in osteoclastogenesis Hesperetin, like prenylflavonoids in Epimedium, is conjugated mainly into glucuronides when ingested 225 5.4 Future work In this study, ultra-sensitive, high throughput, cell-based bioassays have been developed that can be used to distinguish the ERα- or ERβ-selective properties, as well as, cell proliferative effects on MCF-7 breast cancer cells of pure compounds and for measurement of global estrogenicity of complex mixtures To enable the study of the whole spectrum of SERM activities of Epimedium compounds on critical estrogenresponsive tissues such as bone, endometrium and breast, the next step is to develop and validate a panel of such cell-based bioassays Even within the same cell line, differences exist in the ability of different compounds to induce estrogen-regulated gene expression as seen in Ishikawa cells where different levels of cofactors were induced when exposed to tamoxifen, ICI 182,780 and LY 117,018 (Bramlett & Burris, 2003) This can be done by introducing natural ERα or ERβ-driven promoters in bone, endometrium, and breast cell lines to obtain promoter specific markers of SERM activity Examinations of various end-points in these cells are necessary to fully characterize the activity of compounds that may be SERMs With the intention to develop Epimedium as a botanical drug for use to maintain bone health after menopause, future work can be built on the relatively well-designed study which feature female ovariectomized Sprague–Dawley rats that were fed with increasingly doses of a standardized Epimedium brevicornu extract This model organism is a cheaper, more appropriate and relevant physiological model for evaluating Epimedium as a drug that can be ingested to enhance post-menopausal bone health than to enroll post-menopausal females at this initial stage of research Future investigation would involve the use of analytical methods to quantify Epimedium prenylflavonoids and bioassays can also be used to determine if these 226 compounds accumulate in target tissues such as bone of female ovariectomized Sprague– Dawley rats that were fed with a single dose and repeated doses standardized Epimedium brevicornu extract and whether these conjugated compounds can be deconjugated and become bioactive A similar study has been published on hesperitin, the aglycone of hesperidin, found in citrus, was reported to be unequally distributed and accumulated mostly in the aorta of rats that were orally fed with a 0.2% of the aglycone for weeks (Takumi et al., 2011) This observation is in good agreement with published work that reported hesperitin had the ability to strengthen blood vessels and prevent capillary permeability which also supported the idea that hesperitin effectively accumulates in the aorta to enhance vascular function At the same time, to investigate the preventive effect of Epimedium on osteoporosis induced by ovariectomy in rats, female ovariectomized Sprague–Dawley rats after the induction of osteoporosis are divided into three groups, each being fed with the standardized Epimedium extract, estradiol or the negative control To analyze the osteoprotective effects of the various treatments, bone mineral density, bone turnover biochemical markers, serum estrogen and prenylflavonoid levels and bioactivity, and uterine wet weight will be monitored to assess whether the administration of standardized Epimedium extract could prevent bone loss induced by estrogen deficiency The last parameter will provide insights on whether Epimedium can exert any beneficial effect in preventing bone loss without resulting in hyperplasia of the uterus Another worthwhile work to embark on is to ascertain whether the ingestion of estrogenic prenylflavonoids from Epimedium for the purpose of maintaining bone health can increase risk of ERα-positive breast cancer Interestingly, icaritin, a prenylflavonoid found in Epimedium, stimulated the growth of MCF-7 breast cancer cells at low concentrations like an estrogenic ligand on its own and exerted a growth suppressive 227 effect on estrogen-stimulated breast cancer cell proliferation at higher concentrations (Tiong, 2010) Similar to estradiol, icaritin was also found to dose-dependently destabilize ERα protein (Tiong, 2010) Results for microarray gene expression analyses reported in this thesis implicated the aryl hydrocarbon receptor (AhR) signaling for this suppressive effect of icaritin To find out whether the ingestion of estrogenic prenylflavonoids from Epimedium can increase risk of ERα-positive breast cancer, the athymic nude mouse model can be used where the mouse can be implanted with MCF-7 breast cancer cells and estradiol given to stimulate their growth Icaritin will be administered to see if the compound can restrict estradiol-stimulated breast cancer xenograft growth and whether there is any reduced ERα protein levels and related signaling This work will facilitate the development of dual agonists like icaritin which are estrogenic but yet, through activating AhR-signaling, can destabilize ERα protein to restrict ERα-positive breast cancer cell growth Data obtained from proposed future work above will provide further insights that would be useful for the development of Epimedium extract and prenylflavonoids as drugs for menopause, maintenance of bone health and other conditions requiring estrogenic action in humans 228 ... ascertain whether the ingestion of estrogenic prenylflavonoids from Epimedium for the purpose of maintaining bone health can increase risk of ERα-positive breast cancer Interestingly, icaritin,... and sulfatase Non-linear pharmacokinetics and prolonged effects for up to 72 h following administration of a single dose of Epimedium extract were also observed Coupled processes of enteric and. .. endometrium and breast, the next step is to develop and validate a panel of such cell- based bioassays Even within the same cell line, differences exist in the ability of different compounds to induce estrogen- regulated