2006 World Congress on Chinese Medicine in HK

1. Session CS 12.5 2006 World Congress on Chinese Medicine in HK Inhibition of twenty-six natural polyphenols in lipid peroxidation on rat liver mitochondria and human liver cancer cell  Hui-Fang Chang and Ling-Ling Yang Department of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan.

2. Introduction • Liver disease is one of important health problems in Asia. The prevention and treatment of liver disease is a necessary target for drug development in clinic of Eastern countries.

3. (http://www.doh.gov.tw/statistic/index.htm) The top ten factors of death in Taiwan

4. (http://www.doh.gov.tw/statistic/index.htm) Trend of mortality caused by hepatic cancer in Taiwan

5. Part-ⅠAnti-lipid peroxidative effects on rat mitochondria of natural polyphenols Part-Ⅱ The antiproliferation activities of natural polyphenols on human hepatocarcinoma

6. Part-Ⅰ Anti-lipid peroxidative effects on rat mitochondria of natural polyphenols • Compounds: 26 of natural polyphenols (Yang’s Lab.) -- 14 of Flavonoids -- 10 of Anthraquinones -- 2 of Stilbenes • Positive control：Silymarin

7. Anti-lipid peroxidation assay SD mice were killed and liver mitochondria 1ml (10%,W/V) were prepared in PBS under 4℃ Protein contain 20 mg/ml Addition of FeCl2incubated at 37℃ for 1hr 1. Centrifugation, 50 ml aliquots of the super fluids were added to thiobarbituric acid 2.Incubated in 90 ℃water bath for 1 hr 3.Add 5% NaOH/ MeOH under ice bath TBARS assay Quantitative analysis of MDA(TBA)2 by HPLC Instrument: Shimadzu 10Avp -- Column: LiChroCART RP-18 -- Detector: UV 532 nm -- Temp: 25 ℃ -- Mobile Phase: 60 % KH2PO4：40 % methanol -- Flow: 1.0 mL/min

8. Figure 1. FeCl2 inducedLPO on rat liver mitochondria**P＜0.01 vs. control, n=3

9. Figure 2. Effect of natural polyphenols on MDA(TBA)2 formation in SD rat liver mitochondria *P＜0.05, **P＜0.01 vs. control, n=3

10. Figure 3. LPO inhibition of natural polyphenols on rat liver mitochondria which is induced by FeCl2 **P＜0.01 vs. control, n=3

11. Table 1. IC50 of natural polyphenols on rat liver mitochondria against LPO

12. Free radical scavenge • DPPH free radical scavenge assay ELISA reader wave length: 517 nm Positive control: Gallic acid • Superoxide anion free radical scavenge assay ELISA reader wave length: 560 nm Positive control : Superoxide dismutase (SOD) • Hydrogen peroxide (H2O2) free radical scavenge assay ELISA reader wave length: 610 nm Positive control : Catalase

13. Inhibition of superoxide anion formation ELISA reader wave length: 295 nm Positive control: Allopurinol • Iron(Ⅱ)Chelation ELISA reader wave length: 295 nm Positive control: Ethylenediaminetetraacetic acid (EDTA) • The ferric reducing ability of plasma (FRAP) assay ELISA reader wave length: 593 nm Standard: Trolox

14. Figure 4. DPPH free radical scavenging ability (%) of natural polyphenols Gallic acid 8.51 mg/ml, *P＜0.05, **P＜0.01 vs. control, n=3

15. Table 2. IC50 of natural polyphenols scavenging DPPH free radical

16. Figure 5. Superoxide anion free radical scavenging ability (%) of natural polyphenols SOD 39.70 mg/ml,*P＜0.05, **P＜0.01 vs. control, n=3

17. Figure 6. Hydrogen peroxide free radical scavenging ability (%) of natural polyphenols Catalase 1.84 mg/ml, *P＜0.05, **P＜0.01 vs. control, n=3

18. Figure 7. Inhibition (%) of superoxide anion formation by natural polyphenols *P＜0.05, **P＜0.01 vs. control, n=3

19. Figure 8. Chelated (%) of Fe2+ by natural polyphenol EDTA 250 mg/ml,*P＜0.05, **P＜0.01 vs. control, n=3

20. Figure 9. Reductive ability from natural polyphenols as measured by FRAP method *P＜0.05, **P＜0.01 vs. control, n=3 #TEAC: Trolox equipment antioxidant capacity

21. Part-Ⅱ The antiproliferation activities of natural polyphenols on human hepatocarcinoma • Chang liver obtained from BCRC • Hep G2 obtained from BCRC • Positive control: Adriamycin (ADR) Negative control: Triton • [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay • Morphology assay -- Giemsa stain

22. Figure 10. Cytotoxicity of natural polyphenols on Chang liver cell line Triton 0.01%,**P＜0.01 vs. control, n=3

23. Figure 11. Cytotoxicity of natural polyphenols on Hep G2 cell line Triton 0.01%,*P＜0.05, **P＜0.01 vs. control, n=3

24. Table 3. IC50 of FON-7g on Chang liver and Hep G2 Table 4. Time dependent of FON-7g on Hep G2

25. Control ADR FON-7g 0.1 (mg/ml) FON-7g 1 (mg/ml) FON-7g 10 (mg/ml) FON-7g100 (mg/ml) Figure 12. FON-7g induced apoptosis in human Hep G2 cells

26. M C A 0.1 1 10 100 Bax C A 0.1 1 10 100 M Caspase-3 Figure 13.Activation of bax and caspase-3 during apoptosis induced by FON-7g in human Hep G2 cells

27. Conclusion • 9 of 26 natural polyphenols (50 mg/ml) exerted significant inhibitory effects (＞50%) on SD rat mitochondria. 10 of 26 natural polyphenols (50 mg/ml) could scavenge DPPH free radical. • 1 of 26 natural polyphenols exhibited have no cytotoxicity on Chang liver, and which have cytotoxicity on Hep G2 that is FON-7g. • The target antiproliferation comopound is high toxicities to cancer cell and low toxicity to normal cell which is a necessary requirement in antiproliferation drug’s development. Therefore, FON-7g is a potential antiproliferation drug in the future.

28. Thank you for your attention