Oestrogen-like Effects of Lignans from Sambucus williamsii Hance in Osteoblastic Cells and Rat Metabolites

1. Lignans from Sambucus williamsii Hance exerts oestrogen-like effects in osteoblastic cells and its metabolites in rats Dr. Hui-Hui Xiao Department of Applied Biology and Chemical Technology The Hong Kong Polytechnic University Hung Hom, Hong Kong Pharmacology 2016

2. Elder Wand Gummies Syrup Eye gel Sambucus williamsii Hance(SWH) is a shrub or small tree growing to a height of 5–6 m that is widely distributed in northeastern China Pharmacology 2016

3. Clinical application history of SWH • The dried stem of Sambucus williamsii Hance (SWH) was first recorded in Tang Materia Medica (A.D. 659) for healing fractures and alleviating pain. Pharmacology 2016

4. Anti-osteoporosis effects of SWH in vivo Bone biomechanics parameter BMD BMD BV/TV Uterus index Phytoestrogen free diet We first demonstrated the anti-osteoporosis effects of SWH in animal model. However, it stimulated the increase of uterus weight at 1 mg/kg in ovariectomized (OVX) mice. Pharmacology 2016

5. Effects of SWC both in vivo and in vitro Uterus index Cell proliferation BMD of tibia ALP Pharmacology 2016

6. What are the bioactive ingredients that account for the protective effects of SWC on bone? Pharmacology 2016

7. 85 compounds were isolated from SWC, including 55 lignans, 6 phenolic acids, 12 triterpenoids and 12 other compounds . Pharmacology 2016

8. Activity screening using cell proliferation assay on UMR 106 cells Pharmacology 2016

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15. 56 compounds(40 lignans, 6phenolic acids, 9 tritepenoids and 1 aliphatic compound ) showed significantly effects on cell proliferation. Lignans were the bioactive ingredients that account for beneficial effects of SWC on bone. Pharmacology 2016 2016/8 Pharmacology 2016 15

16. Ingredients absorbed into bloodstream treated with SWC Blank (n = 2) dd-H2O Administration for 3 days 0.5 h (n = 2) 1.0 h (n = 2) C57BL/6J (~20 g) male, n = 10 SWC (310 mg/kg/d) 1.5 h(n = 2) 2.0 h (n = 2) Blood collection from hepatic portal vein UPLC/QTOFMS Pharmacology 2016

17. (7R,8S)-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxypropanyl)-2-methoxyphenoxyl]-1, 3-propanediol （PPD） (7R,8S)-dihydrodehydrodiconiferyl alcohol（DDA） 0.5 h plasma PPD/DDA pharmacokinetic profiles blank Pharmacology 2016

18. What are the mechanisms underlying the protective effects of SWC? Pharmacology 2016

19. PPD stimulated proliferationanddifferentiation in MC3T3-E1 cellsand UMR 106 cells, and mineralizationin BMSC cells Proliferation ALP activity UMR 106 MC3T3-E1 UMR 106 MC3T3-E1 Results were obtained from three independent experiments and were expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. (A) Cells were treated with vehicle (0.1% ethanol, without inducer); (B) Cells were treated with vehicle (0.1% ethanol, with inducer); (C) Cells were treated with E2 (10-8 M, with inducer); (D) Cells were treated with PPD (10-8 M, with inducer) Mineralization BMSCs Pharmacology 2016 19

20. PPD modulated the osteoblastic and osteoclastic gene expressions in UMR 106 cells Runx2 ALP Osteocalcin OPG/GAPDH RANKL/GAPDH OPG/RANKL Results were obtained from three independent experiments and were expressed as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control. Pharmacology 2016

21. The effects of PPD on osteoblast proliferation and differentiation were totally abolished by co-treatment with ICI182,780 and U0126 PPDmightactthroughestrogenreceptor(ER)andmitogen-activatedproteinkinase(MAPK)signalingpathways. 2016/8 Pharmacology 2016 21

22. PPD failed to bind to estrogen receptor alpha (ERa) and beta (ERb) up to the concentration of 10-6M. PPD did not activate ER or ER-mediated, ERE-dependent luciferase activities in UMR 106 cells Pharmacology 2016 22

23. PPD upregulated the phosphorylation of ERK and ERα (Ser118) at 10 min in UMR106 cells Results were obtained from three independent experiments and were expressed as mean  SEM. *P < 0.05, **P < 0.01 versus control. PPD exerts oestrogen-like actions in osteoblast-like cells via ligand-independent, estrogen response element (ERE)-independent and mitogen-activated protein (MAP) Kinase-mediated rapid nongenomic ER signaling pathway. Pharmacology 2016

24. What is the fate of lignans absorbed in body? How do the lignans been metabolized and excreted ? Pharmacology 2016

25. Plasma (upto8h) (50 mg/kg) Bile (8 h) Urine (24 h) Feces (24 h) Pharmacology 2016

26. Possible metabolic pathway of DDA in rat Pharmacology 2016

27. Summary • 40 lignans, 6phenolic acid, 9 tritepenoids and 1 aliphatic compound showed significantly effects on cell proliferation. Lignans were the bioactive ingredient that account for beneficial effects of SWC on bone • PPD exerts oestrogen-like actions in osteoblast-like cells via ligand-independent, ERE-independent and MAPK-mediated rapid nongenomic ER signaling pathway. • Ten metabolites of DDA were identified in urine, plasma and feces of rats. Oxidation and glucuronidation are the major metabolic reactions in rat. Pharmacology 2016

28. Acknowledgement Dr. Chi-On Chan Dr. Daniel KW Mok Dr. Shun-Wan Chan Dr. Raymond Cooper Prof. Xin-Sheng Yao Dr Fang Xie Dr Xu-Juan Yang Dr Yan Zhang Dr Yi Dai Prof. Man-Sau Wong Dr. Ka-Chun Wong Dr. Quan-Gui Gao Dr. Xiao-Li Dong Min-Xian He Si-Si Cao Li-Ping Zhou Pharmacology 2016