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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-hexa3,5-dien-2-one
• 分子式: C12H12O4
• 分子量: 220.22
 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.
Hispolon induces apoptosis in cancer cells :
treatment of oral cancer
• That hispolon could induce human epidermoid
KB cell apoptosis accompanied by the collapse of
mitochondrial membrane potential.
treatment of gastric 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

Other studies also showed that hispolon has
•
•
•
•
antiviral
antiproliferative
immunomodulatory activity
antioxidant activity
antiviral of Hispolon :
• hispolon showed considerable antiviral activity
against influenza viruses type A and B.
Received 8 November 2002; accepted 8 April 2003
antiproliferative of Hispolon :
• Hispolon also suppress mitogen induced
proliferation of spleen lymphocytes of mice.
Pharmazie. 1996 Sep;51(9):667-70
Isolation and purification of hispolon
1.5kg fruiting body of PL
fraction 3 (3.37 g)
95%EtOH
Etopside
removal of the EtOH under
reduced pressure at 40 ◦C, a dark
yellow residue (64.5 g)
silica gel column chromatography
silica gel column chromatography
reversed-phase HPLC
red crystals (65 mg)
hispolon
eight major fractions
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:5mg/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.
con
2.5mg/ml 5mg/ml 10mg/ml
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:
2.5mg/ml; lane3: 5mg/ml; lane4: 10mg/ml.
1.04%
24.52%
11.29%
67.90%
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.
rhodamine 123 (Rh123)
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. LanBo 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。
粒線體膜電位
Cyt c
Fig. 5. Measurements of mitochondrial membrane potential and Cyt C release in KB cells during the hispolon
treatment. (A). KB cells were treated with 10mM Rh123 for 10 min, and then incubated with 2.5, 5 and 10
mg/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 mg/ml hispolon for the
indicated time. Cyt C release was determined by immunoassay as described in the text. C: control; H2.5: 2.5
mg/ml; H5: 5 mg/ml; H10: 10 mg/ml.
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
Thank you for your attention