CHAPTER RESULTS 3.1 Measurement of combinatorial estrogenic activity using ultrasensitive, cell-based ER-driven reporter gene assays 85 Responses of ERα- and β-driven reporter gene assays to estradiol and its metabolites 85 3.1.1 3.1.2 Specificity of ER-driven reporter gene assays 3.1.3 Validation of ERα and ERβ cell-based bioassays 90 3.1.4 Isoform selectivity of DPN, PPT and genistein 93 3.1.5 Estrogenicity of common flavonoids and Epimedium prenylflavonoids 95 3.1.6 Anti-estrogenic activities of SERMs and ER-antagonist in ERα and ERβ cell lines 99 3.1.7 3.2 88 Combinatorial effects of binary mixtures of estradiol with genistein and Epimedium prenylflavonoids 101 Combinatorial cell proliferative effects on estrogen-responsive MCF-7 breast cancer cells 104 3.2.1 MCF-7 breast cancer cell proliferative effects of estradiol and its metabolites 104 3.2.2 Effects on estradiol-induced breast cancer cell growth by endogenous steroids 107 3.2.3 Validation of MCF-7 breast cancer cell proliferation assay 109 3.2.4 Induction of MCF-7 breast cancer cell growth by Epimedium prenylflavonoids and genistein 111 3.2.5 Effects on cell proliferation of estrogen-induced of MCF-7 breast cancer cells by Epimedium prenylflavonoids, genistein and 4-hydroxytamoxifen 113 82 Traditionally prepared aqueous Epimedium decoction does not exert significant estrogencity in humans revealed using a panel of sensitive, cell-based estrogen-responsive bioassays 116 ERα bioactivity and concentration of estrogenic aglycones in a traditionally prepared Epimedium decoction 116 3.3.2 Estrogenicity of serum before and following ingestion of estradiol valerate and Epimedium pubescens decoction 118 3.3.3 Correlations of estrogen-responsive bioassays with estrone and estradiol levels in human sera 3.3 123 Lack of estrogenicity of a traditionally prepared Epimedium decoction was due to low bioavailability of estrogenic Epimedium aglycones in humans 126 3.4.1 GC-MS method development 126 3.4.1.1 Sample extraction and derivatization 126 3.4.1.2 GC-MS characterization of derivatized genistein, icaritin and desmethylicarition 128 3.4.1.3 Validation of GC-MS method 132 3.3.1 3.4 3.4.2 Concentrations of icaritin and desmethylicarition in human sera obtained after oral ingestion of a traditionally prepared Epimedium decoction 134 83 Vast majority of Epimedium prenylflavonoids were conjugated and rendered non-estrogenic following oral ingestion of a prenylflavonoid-enriched, standardized Epimedium extract in rats 137 3.5.1 LC–MS/MS method development 137 3.5.1.1 Ion pair selection 137 3.5.1.2 Linearity 144 3.5.1.3 3.5 Matrix effects and stability 147 3.5.2 Unconjugated flavonoid content in rat sera following administration of Epimedium 3.5.3 Total flavonoid content in rat sera after digestion with glucuronidase/sulfatase 157 3.5.5 Estrogenic activity in glucuronidase/sulfatase with 159 Convergence of global gene expression profiles of MCF-7 breast cancer cells treated with estradiol, genistein, Epimedium extract and its compounds 160 3.6.1 Global gene expression profiles of estradiol, genistein, 4hydroxytamoxifen, Epimedium extract and its compounds in human MCF-7 breast cancer cells 160 3.6.2 Comparison of gene expression profiles of genistein, Epimedium extract, icariside I, icaritin, desmethylicaritin to estradiol 166 3.6.3 Gene functions specific to genistein, Epimedium extract, icariside I, icaritin and desmethylicaritin 168 3.6.4 Effects of estradiol, genistein, 4-hydroxytamoxifen, Epimedium extract and its compounds on GREB1 expression in human MCF-7 breast cancer cells 3.7 154 3.5.4 Measurement of estrogenic effects in rat sera using the ERα bioassay 3.6 150 179 Summary of findings 181 sera after digestion 84 3.1 Measurement of combinatorial estrogenic activity using ultra-sensitive, cell-based ER-driven reporter gene assays 3.1.1 Responses of ERα- and β-driven reporter gene assays to estradiol and its metabolites To examine their response to natural steroidal estrogens, ERα and ERβ stable cells that the bioassays were based on were exposed separately to increasing doses of estradiol and its metabolites, namely, estrone and estriol, which were dissolved in Eagle’s minimum essential medium supplemented with 10% charcoal-stripped fetal calf serum for 24 h All three estrogenic ligands exhibited a sigmoidal dose-response behavior in both cell lines (Fig 10) Estrone and estriol were full agonists towards both ER isoforms as they elicited the same maximum activation level of the luciferase reporter gene as estradiol Dose-response experiments with the standard estrogen, estradiol, indicated that the ERα stable cell line had an EC50 value of 85 pM for this compound (Table 4) Estradiol was a less potent activator of the ERβ cell line and its EC50 value was about 3-fold higher than that observed with ERα (232 and 85 pM for ERβ and ERα respectively) (Table 4) Estriol and estrone were less potent agonists for both ERα and ERβ compared to estradiol (Table 4) The rank order in terms of descending estrogenic potencies for these three natural steroidal estrogenic ligands in both cell lines is estradiol > estriol > estrone 85 Figure 10: Dose-dependent activation of the luciferase reporter gene in ERα and ERβ cells by estradiol and its metabolites ERα (A) or ERβ (B) cells were exposed to increasing doses of estrone (─■─), estradiol (─○─) and estriol (─×─) dissolved in Eagle’s minimum essential medium supplemented with 10% charcoal-stripped fetal bovine serum for 24 h Results are expressed as a percentage relative to saturation dose of estradiol and presented as mean ± SEM Estrogenic Activity (% of 10 nM E2) 120 100 (A) ERα 80 60 40 20 Estrogenic Activity (% of 10 nM E2) 120 100 (B) ERβ 80 60 40 20 -14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 log[ligand] 86 Table 4: Estrogenic effects of natural estrogens, ER-isoform selective agents, common flavonoids and Epimedium prenylflavonoids measured in-vitro with ERdriven luciferase reporter genes in HeLa cells stably expressing ERα or ERβ Emax (% of 10 nM estradiol) EC50 (nM) ERα ERβ ERα ERβ Estradiol 0.09±0.01 0.23±0.04 100 100 Estrone 6.9±1.1 86±1.1 100 100 Estriol 0.7±1.1 6.4±1.1 100 100 PPT 0.16±1.16 NSA 100 NSA DPN 101±1.2 2.2±1.2 100 100 Genistein 921±1.4 137±1.2 461 544 Apigenin 2463±1.1 868±1.1 323 187 Kaempferol 3898±1.1 429±1.2 175 73.9 Luteolin 4769±1.1 2464±1.0 181 98.7 Quercetin 8124±1.1 2946±1.1 53.6 9.90 NSA NSA NSA NSA Icariside I 5457±1.8 NSA 43.9 NSA Icariside II 13070±1.8 6770±1.0 43.3 22.8 Icaritin 839±1.2 NSA 68.8 NSA Desmethylicaritin 71±1.6 79±7.2 44.7 14.3 Compound Natural estrogens Isoform-selective agents Common flavonoids Epimedium prenylflavonoids Icariin EC50: Half-maximal activity extrapolated from dose-response curves; numbers are given in presented as the mean ± SEM from three determinations Emax: Maximum estrogenic effect extrapolated from dose-response curves NSA: No significant activity 87 3.1.2 Specificity of ER-driven reporter gene assays In addition to estrogens, there are other endogeneous steroids present in the blood in-vivo, for example, testosterone, cortisol, dihydrotestosterone and progesterone Hence, it is necessary to test for cross reactivity between these endogeneous steroids with estrogens To this, ERα and ERβ stable cells were incubated with increasing doses of steroidal hormones in the presence of 50 pM estradiol and their effects on estradiol’s estrogenicity Addition of dihydrotestosterone did not change the estrogenicity of estradiol as measured by ERα and ERβ bioassays (Fig 11) Similar addition of cortisol or progesterone also did not change the estrogenicity of the added estradiol However, dose-dependent increases in serum estrogenicity were detected when cells were treated with increasing doses of testosterone in the presence of estradiol (Fig 11) This increase was due to the conversion of testosterone to estradiol by aromatase in the cells, which was prevented by the addition of 50 µM of DL-aminoglutethimide, an aromatase inhibitor, to all samples during the assay DL-aminoglutethimide also inhibits the transformation of androstenedione to estrone (Mak et al., 1999) 88 Figure 11: Effects of endogenous steroids on the estrogenicity of 50 pM of estradiol in ER-driven reporter gene assays ERα (A, B) and ERβ (C, D) stable cells were incubated with increasing doses of progesterone (―■―), dihydrotestosterone (―□―), hydroxycortisone (―•―) and testosterone (―○―) in the presence of 50 pM estradiol Dose-response curves were obtained in the absence (A, C) and presence of 50 µM of DL-aminoglutethimide, AG (B, D) Results are expressed as fold induction relative to vehicle treated control and presented as the mean ± SEM, (* p 0.95) A representative ERβ calibration curve within the clinical range was best fitted with a exponential curve with a correlation coefficient of R2 > 0.95 (Fig 12B) 91 Table 16: Regulated genes involved in enzymatic processes according to annotations by gene ontology in the Entrez Gene database Microarray data were normalized and filtered using Genesifter software Values represent mean fold changes of transcripts that were significantly induced in the range of treatments (mean values of triplicate experiments) compared to the vehicle control UniGene Name Gene Identifier E2 GEN EPIM ICAR I ICT DICT ICA ICAR II 4OHT CYP1A1 NM_000499 0.44 0.50 1.76 2.59 2.25 3.14 1.13 2.71 0.93 PPP2R2B NM_181676 0.54 0.37 0.47 0.31 0.56 0.81 1.31 1.20 0.87 TGM1 NM_000359 0.56 0.37 0.54 0.41 0.53 0.58 1.03 1.07 0.67 ENO3 NM_001976 0.88 0.58 0.59 0.45 0.49 1.27 1.18 1.46 0.97 TGM2 NM_004613 1.05 2.27 2.01 2.30 1.70 0.53 0.82 0.57 0.92 DHRS2 NM_182908 1.74 2.33 1.97 2.07 1.78 2.01 0.98 1.10 1.24 B3GALNT1 NM_033167 1.76 2.11 1.75 1.89 1.81 1.37 1.01 0.91 1.15 ABAT NM_000663 1.77 2.63 2.26 2.74 2.00 1.45 0.81 0.87 1.19 AMD1 NM_001634 1.81 2.79 2.21 2.03 2.26 1.36 0.81 0.90 1.10 GALNT4 NM_003774 2.15 2.50 1.68 2.08 1.77 1.76 0.90 0.77 1.10 PPM1K NM_152542 2.42 3.13 2.26 2.12 2.08 1.79 1.01 1.03 0.72 TH NM_199293 2.47 3.20 1.81 2.73 1.50 2.18 1.00 1.06 1.26 HS3ST3A1 NM_006042 2.67 4.52 3.25 2.47 3.17 1.90 1.12 1.34 1.40 CA2 NM_000067 2.89 5.67 4.47 4.22 4.15 1.16 0.72 0.62 1.50 E2: estradiol; GEN: genistein, EPIM: Epimedium extract, ICAR I: icariside I; ICARII: icariside II; ICT: icaritin; DICT: desmethylicaritin, 4OHT: 4-hydroxytamoxifen 170 Table 17: Regulated genes involved in signal transduction according to annotations by gene ontology in the Entrez Gene database Microarray data were normalized and filtered using Genesifter software Values represent mean fold changes of transcripts that were significantly induced in the range of treatments (mean values of triplicate experiments) compared to the vehicle control UniGene Name IL1R1 Gene Identifier NM_000877 E2 GEN EPIM ICAR I ICT DICT ICA ICAR II 4OHT 0.41 0.20 0.35 0.41 0.49 0.49 1.05 0.99 0.73 GPR81 NM_032554 0.43 0.51 0.67 0.60 0.78 0.51 0.91 0.97 0.88 BLNK NM_013314 0.46 0.51 0.71 0.69 0.85 0.55 0.98 0.98 0.89 EDARADD NM_145861 0.47 0.50 0.67 0.49 0.69 0.63 1.16 1.10 0.88 IGFBP3 NM_000598 0.57 0.38 0.46 0.43 0.57 0.71 1.01 1.00 0.88 DOCK11 NM_144658 0.59 0.60 0.82 0.71 0.97 0.37 0.78 0.72 0.74 EDN2 NM_001956 0.66 0.36 0.47 0.40 0.55 0.70 1.26 1.28 0.98 GUCY1A3 NM_000856 1.39 1.81 1.80 2.44 1.79 1.58 1.11 0.97 1.08 IGFBP4 NM_001552 1.51 4.83 4.38 6.05 4.02 0.66 0.88 0.86 1.21 OPN3 NM_014322 1.71 2.37 1.64 1.74 1.70 1.44 1.06 0.93 1.22 ASB13 NM_024701 1.75 1.97 1.77 1.74 2.04 1.40 1.02 1.09 1.03 PEX11A NM_003847 1.76 2.22 2.17 2.31 2.09 1.57 0.94 1.04 1.45 STC1 NM_003155 1.87 2.79 2.54 2.59 2.28 1.73 0.77 1.05 1.45 CELSR2 NM_001408 1.88 2.14 1.68 2.35 1.38 1.38 0.96 0.83 1.23 171 UniGene Name MICA Gene Identifier NM_005931 E2 GEN EPIM ICAR I ICT DICT ICA ICAR II 4OHT 1.96 1.70 1.46 1.32 1.71 2.18 1.18 1.26 1.15 RASGRP1 NM_005739 2.13 1.86 1.94 2.00 2.37 1.72 0.98 0.96 1.56 RET NM_020630 2.58 5.57 3.49 3.94 2.88 1.49 0.72 0.73 1.11 DEPDC6 NM_022783 2.60 4.39 3.48 4.36 2.76 1.67 0.78 0.85 1.22 RAPGEFL1 NM_016339 2.77 3.77 2.69 3.09 2.29 1.82 0.94 1.04 1.41 SGK1 NM_005627 3.51 3.93 2.49 2.83 2.92 2.64 0.84 0.95 1.16 PKIB NM_181794 3.63 3.19 2.19 2.52 1.82 2.81 0.81 0.77 1.28 HSPB8 NM_014365 3.81 5.56 5.22 4.56 4.96 3.24 1.04 1.19 2.04 STC2 NM_003714 4.38 4.95 4.56 4.81 4.80 3.92 0.86 1.12 1.97 RERG NM_032918 4.41 3.77 2.84 3.57 2.25 4.37 1.05 1.36 1.71 SGK3 NM_001033578 4.68 4.89 3.43 3.77 2.95 1.75 0.99 0.97 1.09 CALCR NM_001742 9.35 8.98 6.26 7.89 5.01 7.23 0.96 1.17 2.49 E2: estradiol; GEN: genistein, EPIM: Epimedium extract, ICAR I: icariside I; ICARII: icariside II; ICT: icaritin; DICT: desmethylicaritin, 4OHT: 4-hydroxytamoxifen 172 Table 18: Regulated genes involved in cell proliferation according to annotations by gene ontology in the Entrez Gene database Microarray data were normalized and filtered using Genesifter software Values represent mean fold changes of transcripts that were significantly induced in the range of treatments (mean values of triplicate experiments) compared to the vehicle control UniGene Name BMF Gene Identifier NM_033503 E2 GEN EPIM ICAR I ICT DICT ICA ICAR II 4OHT 0.41 0.34 0.58 0.64 0.69 0.73 0.96 1.18 0.83 TP53INP1 NM_033285 0.43 0.45 0.58 0.60 0.69 0.35 0.89 0.70 0.68 CTGF NM_001901 0.59 0.50 0.74 0.55 0.62 0.44 0.97 0.80 0.85 CDK6 NM_001259 0.67 0.48 0.64 0.58 0.59 0.72 1.07 0.98 0.97 TP53 NM_000546 1.26 1.69 1.60 2.14 1.58 1.08 1.00 0.98 1.14 OSGIN1 NM_013370 1.78 2.69 2.32 1.88 2.53 1.60 1.08 1.30 1.11 NOLC1 NM_004741 1.97 1.87 1.57 1.65 1.56 2.09 1.20 1.21 1.32 MREG NM_018000 2.02 2.56 2.20 1.87 2.07 2.02 1.07 1.27 1.14 TPD52L1 NM_001003396 3.75 4.77 3.94 4.36 3.30 3.68 0.99 1.15 1.90 E2: estradiol; GEN: genistein, EPIM: Epimedium extract, ICAR I: icariside I; ICARII: icariside II; ICT: icaritin; DICT: desmethylicaritin, 4OHT: 4-hydroxytamoxifen 173 Table 19: Regulated genes involved in transcription according to annotations by gene ontology in the Entrez Gene database Microarray data were normalized and filtered using Genesifter software Values represent fold changes of transcripts that were significantly induced in the range of treatments (mean values of triplicate experiments) compared to the vehicle control UniGene Name HOPX Gene Identifier NM_032495 E2 GEN EPIM ICAR I ICT DICT ICA ICAR II 4OHT 0.44 0.65 0.76 0.56 0.94 0.50 0.98 0.88 1.12 RCAN1 NM_203418 0.46 0.48 0.61 0.28 0.63 0.63 0.97 1.13 0.89 ZNF467 NM_207336 0.71 0.66 0.75 0.49 0.80 0.76 1.19 1.19 1.00 DLX3 NM_005220 1.00 1.36 1.52 1.41 2.20 1.09 0.70 0.94 1.25 GSC NM_173849 1.57 2.19 2.01 1.75 2.03 1.28 1.17 1.28 1.19 XBP1 NM_005080 1.96 2.09 1.76 1.58 1.94 1.85 1.10 1.28 1.30 FOS NM_005252 2.11 1.96 1.79 2.11 1.99 1.76 0.94 1.00 1.19 SOX3 NM_005634 2.75 3.04 2.37 3.13 1.69 2.89 1.18 1.16 1.14 MYB NM_005375 4.34 5.66 4.82 4.87 5.15 2.75 0.84 0.85 1.82 E2: estradiol; GEN: genistein, EPIM: Epimedium extract, ICAR I: icariside I; ICARII: icariside II; ICT: icaritin; DICT: desmethylicaritin, 4OHT: 4-hydroxytamoxifen 174 Table 20: Regulated genes involved in transport according to annotations by gene ontology in the Entrez Gene database Microarray data were normalized and filtered using Genesifter software Values represent mean fold changes of transcripts that were significantly induced in the range of treatments (mean values of triplicate experiments) compared to the vehicle control UniGene Name SLC31A2 Gene Identifier NM_001860 E2 GEN EPIM ICAR I ICT DICT ICA ICAR II 4OHT 0.44 0.26 0.37 0.29 0.44 0.60 1.18 1.25 0.75 DAB2 NM_001343 0.55 0.46 0.58 0.47 0.59 0.69 1.22 1.19 0.90 KCNJ8 NM_004982 0.59 0.48 0.53 0.47 0.48 0.62 1.14 0.91 0.97 ABCC5 NM_005688 0.68 0.47 0.54 0.45 0.60 0.73 1.01 1.02 0.80 SH3RF2 NM_152550 1.34 2.37 1.70 1.88 1.62 1.07 0.81 0.81 1.01 CDH1 NM_004360 1.54 2.33 1.70 1.58 1.70 0.89 1.03 1.02 0.94 KLF10 NM_005655 1.57 2.20 1.44 1.13 1.52 1.37 1.00 0.89 1.00 SLC24A3 NM_020689 1.84 2.53 2.27 2.95 1.69 1.34 0.69 0.79 0.93 LYAR NM_017816 1.84 1.72 1.72 1.75 1.70 2.07 1.10 1.29 1.32 SLC25A19 NM_021734 1.94 1.85 1.66 1.26 1.60 2.08 1.17 1.36 1.40 TIPARP NM_015508 2.04 1.97 2.02 1.97 1.88 2.11 1.01 1.08 1.09 SLC22A5 NM_003060 2.07 1.87 1.57 1.69 1.61 2.04 1.20 1.26 0.83 SLC9A3R1 NM_004252 2.08 2.23 1.86 1.75 1.83 1.81 1.13 1.19 1.44 SLC29A1 NM_001078174 2.08 2.31 1.62 2.37 1.38 2.16 1.02 1.07 1.09 SLC2A1 NM_006516 2.11 2.16 2.12 1.85 2.50 1.75 0.96 1.02 1.47 KCNK5 NM_003740 4.65 4.19 3.51 3.71 5.45 3.41 1.29 1.17 1.99 E2: estradiol; GEN: genistein, EPIM: Epimedium extract, ICAR I: icariside I; ICARII: icariside II; ICT: icaritin; DICT: desmethylicaritin, 4OHT: 4-hydroxytamoxifen 175 Table 21: Regulated genes classified in the ‘others’ category which include membrane-, structure-, and protein folding–related genes and the genes with unknown functions according to annotations by gene ontology in the Entrez Gene database Microarray data were normalized and filtered using Genesifter software Values represent mean fold changes of transcripts that were significantly induced in the range of treatments (mean values of triplicate experiments) compared to the vehicle control UniGene Name VTCN1 Gene Identifier NM_024626 E2 GEN EPIM ICAR I ICT DICT ICA ICAR II 4OHT 0.45 0.23 0.33 0.25 0.44 0.88 1.42 1.66 0.89 TMEM140 NM_018295 0.53 0.39 0.49 0.38 0.49 0.64 1.33 1.24 0.92 N4BP3 NM_015111 0.56 0.43 0.54 0.40 0.62 0.91 1.29 1.30 0.91 EPB41L5 NM_020909 0.58 0.48 0.51 0.57 0.51 0.64 1.05 0.88 0.80 C1orf106 NM_018265 0.64 0.43 0.49 0.43 0.45 0.69 1.10 1.12 0.73 GRAMD1A NM_020895 0.75 0.54 0.56 0.45 0.68 1.06 1.30 1.38 0.92 APCDD1 NM_153000 0.88 0.47 0.62 0.52 0.70 0.96 1.11 1.13 0.92 GAB2 NM_080491 1.46 2.31 2.16 2.39 2.01 0.90 0.76 0.67 0.86 C2orf18 NM_017877 1.58 2.07 1.65 1.56 1.72 1.67 1.03 1.10 1.24 WDR51B NM_172240 1.82 2.05 1.82 2.02 1.54 1.24 0.88 0.82 1.15 PCP4 NM_006198 1.98 2.32 1.94 1.95 1.61 2.07 0.78 1.06 1.21 C1QTNF6 NM_031910 2.00 2.53 2.25 2.14 2.11 1.48 0.84 0.97 1.29 C9orf95 NM_017881 2.08 2.78 2.17 2.24 2.02 1.66 0.93 0.96 1.04 C12orf24 NM_013300 2.51 2.40 1.92 2.22 1.86 2.82 1.01 1.35 1.45 176 UniGene Name MSMB Gene Identifier NM_002443 E2 GEN EPIM ICAR I ICT DICT ICA ICAR II 4OHT 2.57 3.52 2.79 3.93 2.30 1.90 0.70 0.91 1.23 LRRC50 NM_178452 2.70 2.95 2.04 2.77 1.85 3.15 0.90 1.35 1.80 GOLSYN NM_001099743 2.74 5.24 3.38 4.04 3.41 1.41 0.65 0.50 1.32 TSKU NM_015516 2.79 4.44 4.37 4.93 5.46 2.81 0.95 1.09 1.28 KRT15 NM_002275 2.86 4.25 3.06 3.29 2.51 2.69 1.02 1.11 1.81 SLITRK4 NM_173078 2.97 2.34 1.76 2.33 1.93 2.49 1.17 0.86 1.19 PLAC1 NM_021796 4.00 3.36 2.10 2.55 1.46 4.72 1.26 1.25 2.00 VWF NM_000552 4.07 4.29 2.02 3.36 1.87 2.74 1.29 1.25 1.20 RLN2 NM_134441 4.61 4.70 5.68 7.34 3.94 4.07 0.72 1.04 2.87 GREB1 NM_014668 8.66 9.73 6.39 8.81 4.49 6.93 0.94 0.92 0.91 KRT13 NM_002274 10.34 7.74 4.11 5.45 3.03 13.96 1.11 0.93 4.13 E2: estradiol; GEN: genistein, EPIM: Epimedium extract, ICAR I: icariside I; ICARII: icariside II; ICT: icaritin; DICT: desmethylicaritin, 4OHT: 4-hydroxytamoxifen 177 Figure 42: Evaluation of estrogenicity of estradiol, Epimedium extract, icariside I, icaritin and desemthylicaritin based on quantitative profiling of functional groups Bars indicate the correlation coefficients between estradiol and Epimedium extract, icariside I, icaritin and desmethylicaritin for the 99-gene set or for the genes categorized into six groups (enzymes, signaling, proliferation, transcription, transport and others) The genes for signaling include kinase-, signaling- and receptor-related genes; the genes for proliferation include cell cycle-, differentiation-, and proliferation-related genes; the genes for transport include calcium-, transport-, processing-, and translation-related genes; the genes classified as others are membrane-, structure-, and protein folding–related genes and the genes with unknown functions Statistical significance of correlation cofficients (R2 values) were evaluated using * p