Stimulation of leukotriene synthesis in intact polymorphonuclear cells by the 5-lipoxygenase inhibitor 3-oxo-tirucallic acid.

ABSTRACT Commercially available extracts from Boswellia serrata resin used as anti-inflammatory drugs or phytonutrients show paradoxical concentration-dependent potentiating and inhibitory actions on 5-lipoxygenase (5-LO) product synthesis in stimulated PMNs. In our attempt to characterize the stimulating constituents, we identified the tetracyclic triterpene 3-oxo-tirucallic acid (3-oxo-TA), which, in the range from 2.5 to 15 M, enhanced 5-LO product formation in ionophore-challenged polymorphonuclear cells (PMNs) (e.g., from 1981 Ϯ 177 to 3042 Ϯ 208 pmol at 10 M 3-oxo-TA), and initiated Ca 2ϩ mobilization, MEK-1/2 phosphorylation, 5-LO translocation, and 5-LO product formation in resting cells (534 Ϯ 394 pmol/ 5 ϫ 10 6 PMNs). In cell-free 5-LO assays, 3-oxo-TA acted only inhibitory (IC 50 value of about 3 M), demonstrating the pivotal role of intact cell structure for its activating property. In 3-oxo-TA-challenged PMNs, the mitogen-activated protein kinase kinase (MEK)-1/2 inhibitor PD098059 abolished 5-LO product formation, along with inhibition of MEK-1/2 phosphorylation and 5-LO translocation. The 3-acetoxy derivative of 3-oxo-TA acted like 3-oxo-TA in intact PMNs, whereas 3-hydroxy-TA barely stimulated MEK phosphorylation in resting cells and showed only inhibition on ionophore-induced 5-LO product synthesis. Steroid-type tetracycles neither induced 5-LO activation nor had enhancing or inhibitory effects. In summary, defined natural tetracyclic triterpenes, which act as inhibitors of the 5-LO in the cell-free assay, initiate 5-LO activation by a MEK-inhibitor sensitive mechanism and potentiate stimulated product synthesis in intact cells. Because TAs contribute significantly to the overall biological effects of B. serrata resin extracts, special precaution for standardization is recommended when using B. serrata preparations as drugs or dietary supplements. 5-Lipoxygenase (5-LO; EC 1.13.11.34) catalyzes the first two steps in the biosynthesis of leukotrienes and 5(S)-HETE from arachidonic acid. Leukotrienes and 5-oxo-eicosa-tetraenoic acid, a final metabolite from 5(S)-HETE The enzymatic activity of 5-LO, as well as its binding to other macromolecules, is regulated in a highly complex manner (for concise reviews on many aspects of 5-LO, products, and receptors, se

Inhibition by boswellic acids of human leukocyte elastase.

ABSTRACT Frankincense extracts and boswellic acids, biologically active pentacyclic triterpenes of frankincense, block leukotriene biosynthesis and exert potent anti-inflammatory effects. Screening for additional effects of boswellic acids on further proinflammatory pathways, we observed that acetyl-11-keto-␤-boswellic acid, an established direct, nonredox and noncompetitive 5-lipoxygenase inhibitor, decreased the activity of human leukocyte elastase (HLE) in vitro with an IC 50 value of about 15 M. Among the pentacyclic triterpenes tested in concentrations up to 20 M, we also observed substantial inhibition by ␤-boswellic acid, amyrin and ursolic acid, but not by 18␤-glycyrrhetinic acid. The data show that the dual inhibition of 5-lipoxygenase and HLE is unique to boswellic acids: other pentacyclic triterpenes with HLE inhibitory activities (e.g., ursolic acid and amyrin) do not inhibit 5-lipoxygenase, and leukotriene biosynthesis inhibitors from different chemical classes (e.g., NDGA, MK-886 and ZM-230,487) do not impair HLE activity. Because leukotriene formation and HLE release are increased simultaneously by neutrophil stimulation in a variety of inflammationand hypersensitivity-based human diseases, the reported blockade of two proinflammatory enzymes by boswellic acids might be the rationale for the putative antiphlogistic activity of acetyl-11-keto-␤-boswellic acid and derivatives. Frankincense is a gum resin secreted by trees of the genus Boswellia of Burseraceae. From the very beginning of human civilization, it has been used for therapeutic purposes However, in 1991 we observed that boswellic acids also prevent endotoxin-/galactosamine-induced hepatitis in mice . This observation was intriguing, because it had been reported that protection against endotoxic shock could be achieved only by less selective lipoxygenase blockers, not by site-specific leukotriene biosynthesis inhibitors The aim of this study was to determine whether the established pentacyclic triterpene-type 5-LO inhibitor AKBA also affects the activity of HLE. Here, we report that many pentacyclic triterpenes, including the boswellic acids, block HLE activity in vitro but that the combined inhibition of tw

Acetyl-1 1-keto-fl-boswellic acid (AKBA): structure requirements for binding and 5-lipoxygenase inhibitory activity

1 5-Lipoxygenase (5-LOX) products from endogenous arachidonic acid in ionophore-stimulated peritoneal polymorphonuclear leukocytes (PMNL) and from exogenous substrate (20 gM) in 105,000 g supernatants were measured. 2 The effects of natural pentacyclic triterpenes and their derivatives on 5-LOX activity were compared with the inhibitory action of acetyl-l -keto-p-boswellic acid (AKBA), which has been previously shown to inhibit the 5-LOX by a selective, enzyme-directed, non-redox and non-competitive mechanism. 3 The 5-LOX inhibitory potency of AKBA was only slightly diminished by deacetylation of the acetoxy group or reduction of the carboxyl function to alcohol in intact cells (ICs = 1.5 vs. 3 and 4.5 pM, respectively) and in the cell-free system (8 vs. 20 and 45 gM). 5 fl-Boswellic acid (f-BA), lacking the 1 1-keto function, inhibited 5-LOX only partially and incompletely, whereas the corresponding alcohol from fl-BA, as well as amyrin, acetyl-ll-keto-amyrin, 1 l-keto-f-boswellic acid methyl ester had no 5-LOX inhibitory activity up to 50 pM in either system. 5 fl-BA only partially prevented the AKBA-induced 5-LOX inhibition, whereas the non-inhibitory compounds, amyrin and acetyl-1 l-keto-amyrin, almost totally antagonized the AKBA effect and shifted the concentration-inhibition curve for the incomplete inhibitor fl-BA to the right. In contrast, the noninhibitory 1 1-keto-fl-BA methyl ester exerted no antagonizing effect. 6 The results demonstrate that the pentacyclic triterpene ring system is crucial for binding to the highly selective effector site, whereas functional groups (especially the 11 -keto function in addition to a hydrophilic group on C4 of ring A) are essential for 5-LOX inhibitory activity