Planta Med 2005; 71(12): 1106-1111
DOI: 10.1055/s-2005-873128
Original Paper
© Georg Thieme Verlag KG Stuttgart · New York

Andrographolide Isolated from Andrographis paniculata Induces Cell Cycle Arrest and Mitochondrial-Mediated Apoptosis in Human Leukemic HL-60 Cells

Hon-Yeung Cheung1 , 2 , Sau-Ha Cheung2 , Jieliang Li1 , Chui-Shan Cheung1 , Wai-Ping Lai2 , Wang-Fun Fong1 , Fo-Man Leung1
  • 1Department of Biology & Chemistry, City University of Hong Kong, Hong Kong SAR, China
  • 2Pharmaceutical & Chemical Technology Center Ltd., City University of Hong Kong, Hong Kong SAR, China
Further Information

Publication History

Received: November 8, 2004

Accepted: June 24, 2005

Publication Date:
25 October 2005 (online)


The in vitro cytotoxicities of the ethanol extract of Andrographis paniculata (APE) and its main diterpenoid components were evaluated in various cancer cells. APE was found to be significantly growth inhibitory to human acute myeloid leukemic HL-60 cells with an IC50 value of 14.01 μg/mL after 24 h of treatment. Among the three main diterpenoids in A. paniculata, andrographolide exhibited the highest degree of cytotoxicity followed by deoxyandrographolide while neoandrographolide was the least effective. Laser confocal microscopy and gel electrophoresis studies revealed chromosomal DNA fragmentations suggesting the occurrence of apoptosis. An increase of G0/G1 phase cells from 51.88 % to 78.69 % was noted after andrographolide treatment for 36 h. The G0/G1 phase arrest and apoptosis were associated with disappearance of mitochondrial cytochrome c and increased expression of Bax but decreased expression of Bcl-2 proteins in the inhibited cells. Although the order of all these events has not been determined, it is concluded that APE and andrographolide induce cell cycle arrest and affect an intrinsic mitochondria-dependent pathway of apoptosis by regulating the expression of some pro-apoptotic markers in HL-60 cells.


APE:extract of Andrographis paniculata

DSAM:deoxyandrographolide succinic acid monoester

HIV:human immunodeficiency virus

MTT:3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyl-tetrazolium bromide

AO/EB:acridine orange/ethidium bromide

PBS:phosphate buffer saline

FACS:fluorescence-activated Cell Sorting system

PI:propidium iodide

SDS-PAGE:sodium dodecyl polyacrylamide gel electrophoresis

ECL:enhance dchemiluminescence


  • 1 Kapil A, Koul I B, Banerjee S K, Gupta B D. Antihepatotoxic effects of major diterpenoid constituents of A. paniculata .  Biochem Pharmacol. 1993;  46 182-5
  • 2 Puri A, Saxena R, Saxena R P, Saxena K C. Immunostimulant agent from A. paniculata .  J Nat Prod. 1993;  56 995-9
  • 3 Zhang C Y, Tan B KH. Mechanism of cardiovascular activity of A. paniculata in the anaesthetized rat.  J Ethnopharmacol. 1997;  56 97-101
  • 4 Gupta S, Choudhry M A, Yadava J NS, Srivastava V, Tandon J S. Antidiarreheal activity of diterpenes of Andrographis (Kalmegh) against Escherichia coli enterotoxin in in vivo modes.  Int J Crude Drug Res. 1990;  28 273-83
  • 5 Shukla B, Visen P KS, Patnaik G K, Dhawan B N. Choleretic effect of andrographolide in rats and guinea pigs.  Planta Med. 1992;  58 146-8
  • 6 Chiou W F, Lin J J, Chen C F. Andrographolide suppresses the expression of inducible nitric oxide synthase in macrophage and restores the vasoconstriction in rat aorta treated with lipopolysaccharide.  Br J Pharmacol. 1998;  125 327-34
  • 7 Matsuda T, Kuroyanagi M, Sugiyama S, Umehara K, Ueno A, Nishi K. Cell differentiation-inducing diterpenes from A. paniculata Nees.  Chem Pharm Bull. 1994;  42 1216-25
  • 8 Kumar R A, Sridevi K, Kumar N V, Nanduri S, Rajagopal S. Anticancer and immunostimulatory compounds from Andrographis paniculata .  J Ethnopharmacol. 2004;  92 291-5
  • 9 Holt S, Comac L. Miracle herbs: How herbs combine with modern medicine to treat cancer, heart disease, AIDS and more. New York; Carol Publishing 1998
  • 10 Cheung H Y, Cheung C S, Kong C K. Detection of bioactive diterpenoids from Andrographis paniculata by micellar electrokinetic chromatography.  J Chromatogr A. 2001;  930 171-6
  • 11 Chiu L CM, Wan J MF. Induction of apoptosis in HL-60 cells by eicosapentaenoic acid (EPA) is associated with downregulation of bcl-2 expression.  Cancer Let. 1999;  145 17-27
  • 12 Franky L C, Choi H L, Chen Z Y, Peter S F, Huang Y. Induction of apoptosis in prostate cancer cell lines by a flavonoid, baicalin.  Cancer Let. 2000;  160 219-28
  • 13 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.  J Immunol Methods. 1983;  65 55-63
  • 14 Lin Y S, Lei H Y, Low T L, Shen C L, Chou L J, Jan M S. In vivo induction of apoptosis in immature thymocytes by Staphylococcal enterotoxin B.  J Immunol. 1992;  149 1156-63
  • 15 Wyllie A H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation.  Nature. 1980;  284 555-6
  • 16 Liu X, Kim C N, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c.  Cell. 1996;  86 147-57
  • 17 Bradford M M. A rapid and sensitive method for the quantitations of microgram quantities of protein utilizing the principle of protein-dye binding.  Anal Biochem. 1976;  72 248-54
  • 18 Alley M C, Scudiero D A, Monks P A, Hursey M L, Czerwinski M J, Fine D L. et al . Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay.  Cancer Res. 1988;  48 589-601
  • 19 Lily I H, Wendy A B, Annika M, Martin H NT. Cell death by apoptosis following anticancer drug treatment in vitro .  Inter J Oncol. 1995;  6 585-93
  • 20 Tsujimoto Y, Shimizu S. The voltage-dependent anion channel: an essential player in apoptosis.  Biochimie. 2002;  84 187-93
  • 21 Shih S C, Stutman O. Cell cycle-dependent tumor necrosis factor apoptosis.  Cancer Res. 1996;  56 1591-8
  • 22 Chen J H, Hsiao G, Lee A R, Wu C C, Yen M H. Andrographolide suppresses endothelial cell apoptosis via activation of phosphatidyl inositol-3-kinase/Akt pathway.  Biochem Pharmacol. 2004;  67 1337-45
  • 23 Jin Y H, Yim H, Park J H, Lee S K. Cdk2 activity is associated with depolarization of mitochondrial membrane potential during apoptosis.  Biochem Biophys Res Commun. 2003;  305 974-80

Dr H.-Y. Cheung

Department of Biology & Chemistry

City University of Hong Kong

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Hong Kong SAR

People’s Republic of China

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