An Anti-Inflammatory ent-Kaurane from the Stems of Annona squamosa that Inhibits Various Human Neutrophil Functions

Abstract

We have previously shown that 11 ent-kauranes isolated from the stems of Annona squamosa exhibited immunomodulating effects in leukocytes. In this study, a cellular model using isolated human neutrophils, which are important in the pathogenesis of rheumatoid arthritis, ischemia-reperfusion injury, chronic obstructive pulmonary disease, asthma and other inflammatory diseases, was established in order to elucidate the anti-inflammatory functions of 16β,17-dihydroxy-ent-kauran-19-oic acid (1). Reactive oxygen species (ROS) and granule proteases produced by neutrophils contribute to the pathogenesis of inflammatory diseases. Compound 1 inhibited the generation of superoxide anion, the formation of ROS, and the release of elastase in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils in a concentration-dependent manner with IC₅₀ values of 3.95 ± 0.68, 12.20 ± 2.16, and 12.52 ± 2.26 μM, respectively. The anti-inflammatory actions were not attributable to cytotoxicity because incubation of the neutrophils with 1 did not result in lactate dehydrogenase release. Compound 1 did not display antioxidant or superoxide anion-scavenging activity. Furthermore, neither subcellular NADPH oxidase activity nor cAMP-dependent pathways were altered by 1. Compound 1 significantly inhibited rapid calcium release from internal calcium stores induced by FMLP but not by thapsigargin. In summary, the presented results indicate that the inhibitory effects of 1 on respiratory burst and degranulation of human neutrophils are through the inhibition of cytosolic calcium mobilization, but not via the cAMP-dependent pathways.

Abbreviations

AC:adenylate cyclase

cAMP:cyclic adenosine 3′,5′-monophosphate

DPPH:1,1-diphenyl-2-picrylhydrazyl

FMLP:formyl-L-methionyl-L-leucyl-L-phenylalanine

O₂ ^·-:superoxide anion

PDE:phosphodiesterase

PKA:protein kinase A

LDH:lactate dehydrogenase

SOD:superoxide dismutase

WST-1:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt

Annona squamosa - Annonaceae - elastase - ent-kaurane - neutrophil - superoxide anion

References

• 1 Kan W S. Pharmaceutical Botany. National Research Institute of Chinese Medicine Taiwan; 1979: p 246
• 2 Fujita E, Node M. Diterpenoids of Rabdosia species.  Fortschr Chem Org Naturst. 1984;  46 78-157
• 3 Wu Y C, Hung Y C, Chang F R, Cosentino M, Wang H K, Lee K H. Identification of ent-16β,17-dihydroxykauran-19-oic acid as an anti-HIV principle and isolation of the new diterpenoids annosquamosins A and B from Annona squamosa .  J Nat Prod. 1996;  59 635-7
• 4 Somova L I, Shode F O, Moodley K, Govender Y. Cardiovascular and diuretic activity of kaurene derivatives of Xylopia aethiopica and Alepidea amatymbica .  J Ethnopharmcol. 2001;  77 165-74
• 5 Barton D, Nakanishi K, Meth-Cohn O. Comprehensive Natural Products Chemistry. Vol. 2 Oxford; Elsevier Science 1999: pp 231-4
• 6 Chang F R, Yang P Y, Lin J Y, Lee K H, Wu Y C. Bioactive kaurane diterpenoids from Annona glabra .  J Nat Prod. 1998;  61 437-9
• 7 Chen C Y, Chang F R, Cho C P, Wu Y C. ent-Kaurane diterpenoids from Annona glabra .  J Nat Prod. 2000;  63 1000-3
• 8 Yang Y L, Chang F R, Wu C C, Wang W Y, Wu Y C. New ent-kaurane diterpenoids with anti-platelet aggregation activity from Annona squamosa .  J Nat Prod. 2002;  65 1462-7
• 9 Chen C Y, Chang F R, Wu Y C. The constituents from the stems of Annona cherimola .  J Chin Chem Soc. 1997;  44 313-9
• 10 Chen C Y, Wu D Y, Chang F R, Wu Y C. Lignans and kauranes from the stems of Annona cherimola .  J Chin Chem Soc. 1998;  45 629-34
• 11 Yang Y L, Chang F R, Hwang T L, Chang W T, Wu Y C. Inhibitory effects of ent-kauranes from the stems of Annona squamosa on superoxide anion generation by human neutrophils.  Planta Med. 2004;  70 256-8
• 12 Witko-Sarsat V, Rieu P, Descamps-Latscha B, Lesavre P, Halbwachs-Mecarelli L. Neutrophils: molecules, functions and pathophysiological aspects.  Lab Investig. 2000;  80 617-53
• 13 Ennis M. Neutrophils in asthma pathophysiology.  Curr Allergy Asthma Rep. 2003;  3 159-65
• 14 Borregaard N. The human neutrophil. Function and dysfunction.  Eur J Haematol. 1998;  41 401-13
• 15 Roos D, van Bruggen R, Meischl C. Oxidative killing of microbes by neutrophils.  Microbes Infect. 2003;  5 1307-15
• 16 Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g.  Scand J Clin Lab Invest. 1968;  97 77-89
• 17 Hwang T L, Hung H W, Kao S H, Teng C M, Wu C C, Cheng S J. Soluble guanylyl cyclase activator YC-1 inhibits human neutrophil functions through a cGMP-independent but cAMP-dependent pathway.  Mol Pharmacol. 2003;  64 1419-27
• 18 Cross A R, Parkinson J F, Jones O T. The superoxide-generating oxidase of leucocytes. NADPH-dependent reduction of flavin and cytochrome b in solubilized preparations.  Biochem J. 1984;  223 337-44
• 19 Dahlgren C, Karlsson A. Respiratory burst in human neutrophils.  J Immunol Methods. 1999;  232 3-14
• 20 Sklar L A, McNeil V M, Jesaitis A J, Painter R G, Cochrane C G. A continuous, spectroscopic analysis of the kinetics of elastase secretion by neutrophils. The dependence of secretion upon receptor occupancy.  J Biol Chem. 1982;  257 5471-5
• 21 Dahlgren C, Karlsson A. Respiratory burst in human neutrophils.  J Immunol Methods. 1999;  232 3-14
• 22 Faurschou M, Borregaard N. Neutrophil granules and secretory vesicles in inflammation.  Microbes Infect. 2003;  5 1317-27
• 23 Berridge M J. Inositol trisphosphate and calcium signalling.  Nature. 1993;  361 315-25
• 24 Castrillo A, de Las Heras B, Hortelano S, Rodriguez B, Villar A, Bosca L. Inhibition of the nuclear factor kappa B (NF-kappa B) pathway by tetracyclic kaurene diterpenes in macrophages. Specific effects on NF-kappa B-inducing kinase activity and on the coordinate activation of ERK and p38 MAPK.  J Biol Chem. 2001;  276 15 854-60
• 25 Wong K L, Yang H Y, Chan P, Cheng T H, Liu J C, Hsu F L, Liu I M, Cheng Y W, Cheng J T. Isosteviol as a potassium channel opener to lower intracellular calcium concentrations in cultured aortic smooth muscle cells.  Planta Med. 2004;  70 108-12