The apoptosis effect of hispolon from Phellinus linteus (Berkeley & Curtis)Teng on human epidermoid KB cells Journal of Ethnopharmacology 105 (2006) 280–285

Introduction 桑黃（ Phellinus linteus ）為一種寄生於闊葉樹上 的刺革菌科菇類，因其生長緩慢，取得不易，加 上難以人工栽培，故有「夢幻菇蕈」之稱。

Phellinus linteus(PL) 的成分介紹 Polysaccharides Triterpenoids phenolics Meshimakobnol Proteins Mineral

Pharmacological study of Phellinus linteus  Phellinus linteus (PL), a traditional medicinal mushroom, for the treatment of various diseases.  gastroenteric disorder  peptic ulcers  lymphatic disease  various cancers

 Recent studies have shown that PL has  anti-inflammatory  antimutagenicity  antibacterial effects  stimulates immunity  inhibits tumor growth and metastasis

Hispolon 分子結構 : 化學名 : 6-(3,4-dihydroxy-phenyl)-4-hydroxy-hexa- 3,5-dien-2-one 分子式 : C 12 H 12 O 4 分子量 :  Hispolon,a yellow pigment was first found in Inonotus hispidus in 1996

Hispolon, an active polyphenol compound, is known to possess potent induces apoptosis in cancer cells.

That hispolon could induce human epidermoid KB cell apoptosis accompanied by the collapse of mitochondrial membrane potential. Hispolon induces apoptosis in cancer cells : treatment of oral cancer

the efficacy of hispolon in human gastric cancer cells and explored the cell death mechanism. Hispolon induced ROS-mediated apoptosis in gastric cancer cells. Free Radical Biology & Medicine 45 (2008) 60–72 treatment of gastric cancer

antiviral antiproliferative immunomodulatory activity antioxidant activity  Other studies also showed that hispolon has

antiviral of Hispolon : hispolon showed considerable antiviral activity against influenza viruses type A and B. Received 8 November 2002; accepted 8 April 2003

Hispolon also suppress mitogen induced proliferation of spleen lymphocytes of mice. antiproliferative of Hispolon : Pharmazie Sep;51(9):667-70

Isolation and purification of hispolon 1.5kg fruiting body of PL 95%EtOH removal of the EtOH under reduced pressure at 40 ◦ C, a dark yellow residue (64.5 g) silica gel column chromatography eight major fractions fraction 3 (3.37 g) silica gel column chromatography reversed-phase HPLC red crystals (65 mg) hispolon Etopside

Results

Fig. 2. Effect of hispolon on the growth of KB cells. (A)Dose-dependent effects of hispolon on the cell growth inhibition of KB cells (exposure: 24h). (B)Time-Dependent effects of hispolon on the inhibition of KB cell growth (hispolon:5  g/ml). Cell viability was determined by a MTT assay as described in the text. Results are expressed as means ± S.E.M. of data obtained in three independent experiments.

Fig.3. Dose-dependence of DNA fragmentation. KB cells were exposed to the Indicated concentrations of hispolon for 24h. Cells were harvested by centrifugation and DNA was extracted. The DNA fragments was separated on 1.5% agarose gel electrophoresis and visualized under ultraviolet light after staining with ethidium bromide. Lane1: control; lane2: 2.5  g/ml; lane3: 5  g/ml; lane4: 10  g/ml. con2.5mg/ml5mg/ml10mg/ml

Fig.4. Flow cytometric analysis of DNA fragmentation for KB cells after hispolon treatment. KB cells were exposed to the indicated concentrations of hispolon for 24h or not. The harvested cells were fixed in 70% ethanol, andstained with PI, followed by flow cytometric analysis. The percentages of cells with hypodiploid DNA contents(sub-G1) represent the fractions undergoing apoptotic DNA degradation. 1.04% 11.29% 24.52% 67.90%

粒線體在呼吸氧化過程中，將所產生的能量以電化學位能儲存於粒線體 內膜 ( 約 250 mV/5~10 nm) ，稱之為 mitochondrial membrane potential (mmp) ，並以此位能來進行電子傳遞鏈，最終產生 ATP 以供細胞使用。 而此一電位差形成於粒線體內膜，為外正內負的形式。 許多親脂性的陽 離子化合物會結合到粒線體內膜，在雷射激發光下放出螢光，可以非常 方便地使用螢光顯微鏡或共軛焦顯微鏡來觀察粒線體的形態和移動的變 化情形。最早被發現的粒線體染劑為 rhodamine 123 (Rh123) ， Dr. Lan- Bo Chen 的實驗室發現 Rh123 可特異性的染在活細胞的粒線體上，而一 些抑制電子傳遞鏈的藥物 都會降低 mmp ，並會減少粒線體攝入 Rh123 的量。 rhodamine 123 (Rh123)

Fig. 5. Measurements of mitochondrial membrane potential and Cyt C release in KB cells during the hispolon treatment. (A). KB cells were treated with 10  M Rh123 for 10 min, and then incubated with 2.5, 5 and 10  g/ml hispolon for a different period of time. At the end of incubation, the mitochondrial membrane potential was measured as described in the text. (B). KB cells were incubated with 2.5, 5 and 10  g/ml hispolon for the indicated time. Cyt C release was determined by immunoassay as described in the text. C: control; H2.5: 2.5  g/ml; H5: 5  g/ml; H10: 10  g/ml. 粒線體膜電位 Cyt c

1. 桑黃中的活性物質 hispolon 可以誘導 KB cell 細胞凋亡 ， 凋亡路徑是粒線體 2. 作著先確定細胞是否凋亡就是 DNA ladder 跟 sub-G1 期的增加並發現粒線體膜電位的改 變與 cyt C 的釋放有 time 與 dose dependent

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