1. LOGO Anti-cancer effect and structural characterization of endo-polysaccharide from cultivated mycelia of Inonotus obliquus Life Sciences 79 (2006) 72–80 2008/05/27

2. www.themegallery.com LOGO Outline  Introduction  Materials and methods  Results  Discussion  Conclusion

3. www.themegallery.com LOGO Introduction  白樺茸是一種生長於白樺樹上的藥用真菌，其子實體 呈現類似炭的黑色塊狀型態主要是受到內部茶褐色色 素沈澱的影響﹔這種真菌的活性極強，會不斷吸取白樺 樹的養分，大約十至十五年之後就會把白樺樹的精華 吸收殆盡，使得白樺樹枯死。由於這種特殊的活性， 被認為具有藥用價值，因此受到重視。

4. www.themegallery.com LOGO 分類學  真菌界  擔子菌門  層菌綱  非褐菌目  多孔菌科  褐臥孔菌屬

5. www.themegallery.com LOGO 文獻回顧  白樺茸含有大量的植物纖維類多醣體，具有良好 的抗癌、降血糖、免疫提升作用。  其中的葡聚糖、異多醣體、果膠 等物質，還可以 作為細胞膜成分之一，可以提升免疫細胞的活力， 抑制癌細胞擴散、防止癌症復發。  白樺茸抗癌機制並非直接攻擊癌細胞，而是藉著 活化免疫細胞、強化全身的防禦機制，而達到預 防癌症或抑制癌細胞增殖的目的。

6. www.themegallery.com LOGO Materials and methods

7. www.themegallery.com LOGO 實驗流程

8. www.themegallery.com LOGO Cancer cell  Hur7 (human hepatoma)  HEC-1B (human endometrial epithelial cells)  B16F10 (murine melanoma)  A549 (human lung carcinoma)  KATO-III (human stomach carcinoma)  SW156 (kidney adenocarcinoma)  MCF-7 (human breast adenocarcinoma)  SK-OV3 (human ovary adenocarcinoma)

9. www.themegallery.com LOGO Normal cell  RAW 264.7 (murine macrophage)  HUVEC (human umbilical vein endothelial cells)  HEK293T (human embryo kidney cells)

10. www.themegallery.com LOGO Measurement of the nitric oxide producing activity of macrophages 5×10 5 cells/ml of RAW 264.7cell on 24-well plates containing either endo-polysaccharide or lipopolysaccharide 24 h at 37 °C equal volume of Griess reagent absorbance at 540 nm

11. www.themegallery.com LOGO BDF1 0.2 ml of 1×10 5 B16F10 intraperitoneally endo-polysaccharide 1, 3, 10, 30 mg/kg/day Control 0.85%NaCl orally endo-polysaccharide 30, 100, or 300 mg/kg/day Control distilled water Survival rate (%)=(MST of treated group/MST of control group)×100

12. www.themegallery.com LOGO Cytotoxicity test 1×10 5 cells/ml 100 μ l cell 10, 50, 100, and 200 μ g/ml polysaccharide solutions 50 μl MTT 48h 4h 37 ℃ measurement at 540

13. www.themegallery.com LOGO NMR spectroscopy  Bruker Advance 600 and Bruker Advance DPX-300  Trace amounts of water in the purified polysaccharide were eliminated using pure D 2 O

14. www.themegallery.com LOGO Determination of the sugar content and molecular weight 10 μl of 1% purified endo-polysaccharide 2 M trifluoroacetic acid (TFA) or 6 N HCl for 4 h at 100 °C centrifugation for 30 min at 32,600×g high pH anion exchange chromatograph (HPAEC-PAD)

15. www.themegallery.com LOGO HPAEC-PAD  醣類在高鹼性溶液中 (pH 12-14) 會易於離子化 (ionization) ，利用每一種單醣分子有各自離子化的 pH 範圍，調整溶液的的 pH 質，我們即可利用陰離 子交換樹酯來分離不同的單醣分子。  再利用 pulse amperometric detector (PAD) 來偵測醣 類因離子化而造成的氧化電位，並判斷流析物成分。 也因此特性， HPAEC-PAD 除了可分析單醣，亦可 分析寡醣及多醣類。

16. www.themegallery.com LOGO 30μL 1% purified endo-polysaccharide fraction high performance gel permeation chromatography 188,000, 150,000, 75,000, 40,000, 17,500, and 10,000 Da were used as standards

17. www.themegallery.com LOGO Results  Purification of endo-polysaccharide and sugar content determination.(Fig.2A&B) (Table 1) (Fig.3) (Fig.4A&B).(Fig.2A&B) (Table 1) (Fig.3) (Fig.4A&B)  Structural characterization of endo-polysaccharide. (Fig.5A&B) (Fig.5A&B)  Effect of endo-polysaccharide on the survival rate of mice implanted with cancer cells. (Fig.6A&B) (Table 2) (Fig.7) (Fig.8A&B)(Fig.6A&B) (Table 2) (Fig.7) (Fig.8A&B)  Cytotoxicity of endo-polysaccharide against cancer and normal cells. (Fig.9A&B)(Fig.9A&B)

18. www.themegallery.com LOGO Discussion  Kim et al. (2005) reported that the in vitro immuno-stimulating activity of water-soluble endo-polysaccharide isolated from I. obliquus mycelia is associated with functional stimulation of B-lymphocytes and macrophages.  The nitrite production of purified endo-polysaccharide markedly increased compared to crude endo-polysaccharide, indicating that the purified endo-polysaccharides enhance phagocytosis and nitric oxide production in macrophages, resulting in immuno-stimulating activity.  Thus, the anticancer effect of endo-polysaccharide in tumor- bearing mice is probably related to immuno-stimulation.

19. www.themegallery.com LOGO Discussion  The B16F10 murine melanoma cells which we used were a highly metastatic malignant neoplasm of melanocytes. The endo-polysaccharide suppressed the in vivo growth of melanoma cells in mice after both oral and intraperitoneal administration with intraperitoneal being more effective.  In most cases, intraperitoneal administration is more rapid and effective than oral (Bae et al., 2005).  However, it is unknown how orally administered substances are absorbed into the intestine. The anti-tumor action of endo- polysaccharide via oral administration is likely to involve multiple delivery processes (Carini et al., 2004).

20. www.themegallery.com LOGO Discussion  We therefore tested direct cytotoxicity and/or a proliferation effect of endo-polysaccharide in both tumor and normal cells. Most cancer cells were unaffected by endo-polysaccharide, except for the Hur7 and MCF-7 cell lines.  The endo-polysaccharide did not show any direct cytotoxic effect on melanoma cells at a dose of up to 200 μg/ml, and no cytotoxicity for normal cells.

21. www.themegallery.com LOGO Discussion  On the other hand, we found a proliferation effect for RAW264.7 macrophages at a high dose. The proliferation process might activate in vivo tumor growth.  Despite a marginal proliferation effect in vitro, we observed a strong endo-polysaccharide anti-tumor effect against B16F10 murine melanoma cells in vivo. This discrepancy suggests the existence of another action mode.

22. www.themegallery.com LOGO Discussion  Previously, we reported (Kim et al., 2005) that endo- polysaccharide can induce the macrophage enhanced mRNA expression of some inflammatory cytokines, including IL-1β, IL-6, iNOS and TNF-α. Administration of endo- polysaccharide enhanced the production of NO and tumoricidal cytokines by murine peritoneal macrophages at the initial phase of tumor development. The activated macrophages are believed to be involved in tumor cytotoxicity (Maeda et al., 1984).

23. www.themegallery.com LOGO Discussion  The polysaccharide linkage type is another important factor for anti-tumor or immuno-stimulating activity. Our endo- polysaccharide consists of α-linkages, according to 1 H NMR analysis. This structure is different from other known immuno- stimulating β-glucans (Usui et al., 1981; Mizuno et al., 1992).  Especially, the activity was considered to depend chiefly on the backbone structure of α-(1→3)-D-mannans. A detailed structural analysis is needed.

24. www.themegallery.com LOGO Conclusion  α-Linked fucoglucomannan isolated from cultivated mycelia of I. obliquus can inhibit tumor growth in vivo. The endo-polysaccharide-mediated inhibition of tumor growth is apparently caused by an induced humoral immunity of the host defense system rather than by a direct cytotoxic effect against tumor cells. The endo-polysaccharide from I. obliquus mycelia has a potential for clinical use in cancer prevention and treatment.

25. LOGO www.themegallery.com