Inhibitory effects of 16-hydroxycleroda-3,13(14)E-dien-15-oic acid on superoxide anion and elastase release in human neutrophils through multiple mechanisms Han-Lin Chang a , Fang-Rong Chang b , Jin-Shan Chen c , Hui-Po Wang d , Yi-Hsiu Wu a , Chien-Chiao Wang a , Yang-Chang Wu b , Tsong-Long Hwang a, ⁎ a Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan b Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan c Department of Anatomy, College of Medicine, Taipei Medical University, Taipei, Taiwan d Department of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan Received 3 October 2007; received in revised form 29 January 2008; accepted 14 February 2008 Available online 4 March 2008 Abstract Reactive oxygen species and granule proteases produced by neutrophils contribute to the pathogenesis of inflammatory diseases. In this study, a cellular model in isolated human neutrophils was established to elucidate the anti-inflammatory functions of 16-hydroxycleroda-3,13(14)E-dien- 15-oic acid (PL3S), a clerodane diterpenoid from Formosan Polyalthia longifolia var. pendula. PL3S significantly inhibited the generation of superoxide anion and the release of elastase in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils in a concentration-dependent fashion with IC 50 values of 3.06 ± 0.20 and 3.30 ± 0.48 μM, respectively. PL3S did not affect cAMP-dependent pathway, and the inhibitory effect of PL3S was not reversed by protein kinase A inhibitor. PL3S did not display antioxidant or superoxide anion-scavenging ability, and it failed to alter the subcellular NADPH oxidase activity. PL3S concentration-dependently inhibited calcium mobilization caused by FMLP but not thapsigargin. Furthermore, PL3S attenuated the FMLP-induced protein kinase B (AKT) and p38 mitogen-activated protein kinase phosphorylation. However, PL3S had no effect on FMLP-induced phosphorylation of extracellular regulated kinase and c-Jun N-terminal kinase. In summary, these results indicate that the suppressive effects of PL3S on human neutrophil respiratory burst and degranulation are at least partly mediated by inhibition of calcium, AKT, and p38 signaling pathways. © 2008 Elsevier B.V. All rights reserved. Keywords: Clerodane diterpenoid; Elastase; Neutrophil; Polyalthia longifolia; Superoxide anion 1. Introduction Polyalthia longifolia var. pendula (Annonaceae) is native to the drier regions of Sri Lanka and is locally known as ‘Ulta Ashok’ and is commonly cultivated in Pakistan and India. This plant is used as an antipyretic agent in indigenous systems of medicine (Saleem et al., 2005). Today, P. longifolia var. pendula is in large-scale cultivation in southern Taiwan as a landscape plant. Pharmacological studies on the bark and leaves of this plant display effective antimicrobial activity (Faizi et al., 2003a,b), cytotoxic function (Chang et al., 2006; Chen et al., 2000), and hypotensive effect (Saleem et al., 2005). In spite of this, the anti-inflammatory effect of P. longifolia var. pendula remains to be established. Neutrophils are active phagocytes that act as a crucial component of innate immunity. Although antimicrobial func- tions of neutrophils are essential to host defense, their extensive or inappropriate activation often causes unwanted tissue damage, such as rheumatoid arthritis, ischemia-reperfusion Available online at www.sciencedirect.com European Journal of Pharmacology 586 (2008) 332 – 339 www.elsevier.com/locate/ejphar ⁎ Corresponding author. Graduate Institute of Natural Products, College of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kweishan 333, Taoyuan, Taiwan. Tel./fax: +886 3 2118506. E-mail address: htl@mail.cgu.edu.tw (T.-L. Hwang). 0014-2999/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.02.041