Planta Med 2006; 72(1): 28-33
DOI: 10.1055/s-2005-873149
Original Paper
Pharmacology
© Georg Thieme Verlag KG Stuttgart · New York

Pheophorbide a, a Major Antitumor Component Purified from Scutellaria barbata, Induces Apoptosis in Human Hepatocellular Carcinoma Cells

Judy Yuet-Wa Chan1 , Patrick Ming-Kuen Tang1 , Po-Ming Hon2 , Shannon Wing-Ngor Au1 , Stephen Kwok-Wing Tsui1 , Mary Mui-Yee Waye1 , Siu-Kai Kong1 , Thomas Chung-Wai Mak2 , 3 , Kwok-Pui Fung1 , 2
  • 1Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
  • 2Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
  • 3Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China
Further Information

Publication History

Received: December 29, 2004

Accepted: June 22, 2005

Publication Date:
10 November 2005 (online)

Abstract

Scutellaria barbata has long been used as a Chinese medicine for the treatment of liver diseases such as hepatitis and hepatocellular carcinoma. In the present study, a bioassay-guided method was used to isolate the most active components from Scutellaria barbata. The anti-proliferative effects on human hepatoma HepG2 and Hep3B cells of each fraction at every stage of the purification were monitored. An active component, which is 97 % pure by high performance liquid chromatographic analysis, was isolated. Based on nuclear magnetic resonance (NMR) and mass spectrophotometric (MS) analysis, this active component was identified to be pheophorbide a (C35H36N4O5). Mechanistic studies showed that pheophorbide a induced apoptosis in Hep3B cells, a viral-induced hepatoma cell line. However, it was found to be non-toxic in normal human liver cells WRL-68. DNA fragmentation, sub-G1 cell cycle arrest, as well as suppression of the anti-apoptotic protein Bcl-2, release of cytochrome c to the cytosol, and activation of pro-caspase 3 and pro-caspase 9 were observed when Hep3B cells were treated with 40 μg/mL (i. e., 67.5 μM) pheophorbide a for 48 hours. In conclusion, this is the first report describing the isolation of pheophorbide a from Scutellaria barbata using a bioassay-guided isolation method. The anti-proliferative activity and possible mechanisms of action of pheophorbide a on hepatoma Hep3B cells are also discussed.

References

  • 1 Omata M, Yoshida H. Prevention and treatment of hepatocellular carcinoma.  Liver Transpl. 2004;  10 S111-4
  • 2 Qu M, Xu H, Li Y, Luo H. An illustrated guide to antineoplastic Chinese herbal medicine. 1st edn Hong Kong; The Commercial Press 1990: pp 10-2
  • 3 Gao D, Gao Y, Bai P. Influence of Scutellaria barbata on calcium beaconage of cervix cancer cells.  Zhong Yao Cai. 2003;  26 730-3
  • 4 Wong B Y, Lau B H, Jia T Y, Wan C P. Oldenlandia diffusa and Scutellaria barbata augment macrophage oxidative burst and inhibit tumor growth.  Cancer Biother Radiopharm. 1996;  11 51-6
  • 5 Qian B. Clinical effect of anticancer Chinese medicine. Shanghai; Shanghai Translation Publishing House 1987: pp 6-7
  • 6 Vogelstein B, Kinzler K W. Cancer genes and the pathways they control.  Nat Med. 2004;  10 789-99
  • 7 Jakobi R. Subcellular targeting regulates the function of caspase-activated protein kinases in apoptosis.  Drug Resist Updat. 2004;  7 11-7
  • 8 Chan K K, Chan J Y, Chung K K, Fung K P. Inhibition of cell proliferation in human breast tumor cells by antisense oligonucleotides against facilitative glucose transporter 5.  J Cell Biochem. 2004;  93 1134-42
  • 9 Siu K P, Chan J Y, Fung K P. Effect of arsenic trioxide on human hepatocellular carcinoma HepG2 cells: inhibition of proliferation and induction of apoptosis.  Life Sci. 2002;  71 275-85
  • 10 Chow S K, Chan J Y, Fung K P. Inhibition of cell proliferation and the action mechanisms of arsenic trioxide (As2O3) on human breast cancer cells.  J Cell Biochem. 2004;  93 173-87
  • 11 Zhang J L, Che Q M, Li S Z, Zhou T H. Study on metabolism of scutellarin in rats by HPLC-MS and HPLC-NMR.  J Asian Nat Prod Res. 2003;  5 249-56
  • 12 Sato Y, Suzaki S, Nishikawa T, Kihara M, Shibata H, Higuti T. Phytochemical flavones isolated from Scutellaria barbata and antibacterial activity against methicillin-resistant Staphylococcus aureus .  J Ethnopharmacol. 2000;  72 483-8
  • 13 Roder B. Photosensitizing properties of phorbides.  J Photochem Photobiol B. 1990;  5 519-21
  • 14 Strain H H, Svec W A. Extraction, separation, estimation and isolation of the chlorophylls. In: Vernon LP, Seely GR, editors The Chlorophylls. New York; Academic Press 1966: pp 22-66
  • 15 Nakamura Y, Murakami A, Koshimizu K, Ohigashi H. Identification of pheophorbide a and its related compounds as possible anti-tumor promoters in the leaves of Neptunia oleracea .  Biosci Biotechnol Biochem. 1996;  60 1028-30
  • 16 Hibasami H, Kyohkon M, Ohwaki S, Katsuzaki H, Imai K, Nakagawa M. et al . Pheophorbide a, a moiety of chlorophyll a, induces apoptosis in human lymphoid leukemia molt 4B cells.  Int J Mol Med. 2000;  6 277-9
  • 17 Wyllie A H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation.  Nature. 1980;  284 555-6
  • 18 Fumarola C, Guidotti G G. Stress-induced apoptosis: toward a symmetry with receptor-mediated cell death.  Apoptosis. 2004;  9 77-82
  • 19 Cory S, Huang D C, Adams J M. The Bcl-2 family: roles in cell survival and oncogenesis.  Oncogene. 2003;  22 8590-607
  • 20 Liu X, Kim C N, Yang Y, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c .  Cell. 1996;  86 147-57

Dr. K. P. Fung

Department of Biochemistry

Faculty of Medicine

Room 603

Mong Man Wai Building

The Chinese University of Hong Kong

Shatin

Hong Kong

People’s Republic of China

Phone: +852-2609-6873

Fax: +852-2603-7732

Email: kpfung@cuhk.edu.hk

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