Evaluation of the Anti-Inflammatory Activity of Zhankuic Acids Isolated from the Fruiting Bodies of Antrodia camphorata

Abstract

We have previously shown that a concentrated ethanol extract of the fruiting bodies of Antrodia camphorata exhibited immunomodulating effects in human leukocytes and fourteen compounds including zhankuic acids A, B, C, and antcin K were identified in the extract. In this study, an acute cellular model in isolated peripheral human neutrophils was established to elucidate the anti-inflammatory effects of these compounds. Reactive oxygen species (ROS) production and firm adhesion by neutrophils display two important responses during inflammation. To evaluate whether these compounds could prevent inflammatory responses by neutrophils, their effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate-13-acetate (PMA)-activated peripheral human neutrophils were examined. Pretreatment with 1 - 25 μM of zhankuic acids A, B, C, or antcin K concentration-dependently diminished fMLP- or PMA-induced ROS production, as measured by a lucigenin-amplified chemiluminescence, with IC₅₀ (μM) around 5 - 20 μM. Zhankuic acids A, B, C, or antcin K also effectively inhibited the fMLP- or PMA-induced firm adhesion without interfering with the up-expression of surface Mac-1 (CD11b/CD18), a β2 integrin mediating the firm adhesion of neutrophils to endothelium. The anti-inflammatory actions of these drugs were not due to cytotoxic effects because no significant difference in cell viability was observed compared to vehicle control. These data suggest that inhibition of both ROS production and firm adhesion by neutrophils has no significant cytotoxic effect that could give these drugs the potential to be anti-inflammatory agents for the clinical treatment.

Key words

Firm adhesion - N-formyl-methionyl-leucyl-phenylalanine - neutrophil - phorbol 12-myristate-13-acetate - reactive oxygen species - zhankuic acid - Antrodia camphorata - Polyporaceae

References

• 1 Tsai Z T, Liaw S L. The use and the effect of Ganoderma . Taichung, Taiwan; San Yun Press 1985: p 116
• 2 Shen C C, Kuo Y C, Huang R L, Lin L C, Dong M J, Chang T T, Chou C J. New ergostane and lanostane from Antrodia camphorata .  J Chin Med. 2003;  14 247-58
• 3 Chen C H, Yang S W, Shen Y C. New steroids from Antrodia cinnamomea, a fungal parasite of Cinnamomun micranthum .  J Nat Prod. 1995;  58 1655-61
• 4 Casimir C M, Teahan C G. The respiratory burst of neutrophils and its deficiency. In: Hellewell PG, Williams TJ, editors Immunopharmacology of Neutrophils. San Diego; Academic Press 1994: p 27-54
• 5 Wardlaw A J, Walsh G M. Neutrophil adhesion receptor. In: Hellewell PG, Williams TJ, editors Immunopharmacology of Neutrophils. San Diego; Academic Press 1994: p 134-7
• 6 Cuzzocrea S, Riley D P, Caputi A P, Salvemini D. Antioxidant therapy: a new pharmacological approach in shock, inflammation, and ischemia/reperfusion injury.  Pharmacol Rev. 2001;  53 135-59
• 7 Middleton E J r, Kandaswami C, Theoharides T C. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer.  Pharmacol Rev. 2000;  52 673-751
• 8 Shen Y C, Chiou W F, Chou Y C, Chen C F. Mechanisms in mediating the anti-inflammatory effects of baicalin and baicalein in human leukocytes.  Eur J Pharmacol. 2003;  465 171-81
• 9 Parij N, Nagy A -M, Fondu P, Neve J. Effects of non-steroidal anti-inflammatory drugs on the luminol and lucigenin amplified chemiluminescence of human neutrophils.  Eur J Pharmacol. 1998;  352 299-305
• 10 Van den Worm E, Beukelman C J, Van den Berg A J, Kroes B H, Labadie R P, Van Dijk H. Effects of methoxylation of apocynin and analogs on the inhibition of reactive oxygen species production by stimulated human neutrophils.  Eur J Pharmacol. 2001;  433 225-30
• 11 Lafeber F P, Beukelman C J, van den Worm E, van Roy J L, Vianen M E, van Roon J A, van Dijk H, Bijlsma J W. Apocynin, a plant-derived, cartilage-saving drug, might be useful in the treatment of rheumatoid arthritis.  Rheumatology. 1999;  38 1088-93
• 12 Peters E A, Hiltermann J T, Stolk J. Effect of apocynin on ozone-induced airway hyperresponsiveness to methacholine in asthmatics.  Free Radic Biol Med. 2001;  31 1442-7
• 13 Shen Y C, Chen C F, Wang S Y, Sung Y J. Impediment to calcium influx and reactive oxygen production accounts for the inhibition of neutrophil Mac-1 up-regulation and adhesion by tetrandrine.  Mol Pharmacol. 1999;  55 186-93
• 14 Ormerod M G. Further applications to cell biology. In: Flow Cytometry, Ormerod MG, editor Oxford University Press: UK 2000: pp 249-50
• 15 Williams F M. Role of neutrophils in respiratory injury. In: Hellewell PG, Williams TJ, editors Immunopharmacology of Neutrophils. San Diego; Academic Press 1994: pp 245-57
• 16 Nathan C. Points of control in inflammation.  Nature. 2002;  420 846-52
• 17 Simons J M, Hart B A, Ip Vai Ching T R, Van Dijk H, Labadie R P. Metabolic activation of natural phenols into selective oxidative burst agonists by activated human neutrophils.  Free Radical Biol Med. 1990;  8 251-8
• 18 Wang Q, Doerschuk C M. Neutrophil-induced changes in the biomechanical properties of endothelial cells: roles of ICAM-1 and reactive oxygen species.  J Immunol. 2000;  164 6487-94
• 19 Shen Y C, Chen C F, Chiou W F. Andrographolide prevents oxygen radical production by human neutrophils: possible mechanism(s) involved in its anti-inflammatory effect.  Br J Pharmacol. 2002;  135 399-406
• 20 Endemann G, Feng Y, Bryant C M, Hamilton G S, Perumattam J, Mewshaw R E, Liu D Y. Novel anti-inflammatory compounds prevent CD11b/CD18, α_Mβ₂ (Mac-1)-dependent neutrophil adhesion without blocking activation-induced changes in Mac-1.  J Pharmacol Exp Ther. 1996;  276 5-12