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

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 microM, enhanced 5-LO product formation in ionophore-challenged polymorphonuclear cells (PMNs) (e.g., from 1981 +/- 177 to 3042 +/- 208 pmol at 10 microM 3-oxo-TA), and initiated Ca(2+) mobilization, MEK-1/2 phosphorylation, 5-LO translocation, and 5-LO product formation in resting cells (534 +/- 394 pmol/5 x 10(6) PMNs). In cell-free 5-LO assays, 3-oxo-TA acted only inhibitory (IC(50) value of about 3 microM), 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.

Acetyl-11-keto-beta-boswellic acid, a constituent of a herbal medicine from Boswellia serrata resin, attenuates experimental ileitis.

The gum resin extract from Boswellia serrata (H15), an herbal product, was recently shown to have positive therapeutic effects in inflammatory bowel disease (IBD). However, the mechanisms and constituents responsible for these effects are poorly understood. This study examined the effect of the Boswellia extract and its single constituent acetyl-11-keto-beta-boswellic acid (AKBA) on leukocyte-endothelial cell interactions in an experimental model of IBD. Ileitis was induced by two subcutaneous injections of indomethacin (7.5 mg/kg) in Sprague-Dawley rats 24 h apart. Rats also received oral treatment with the Boswellia extract (H15) or AKBA at two different doses (low and high) equivalent to recommendations in human disease over 2 days. Controls received only the carriers NaHCO3 (subcutaneously) and tylose (orally). Effects of treatment were assessed by intravital microscopy in ileal submucosal venules for changes in the number of rolling and adherent leukocytes and by macroscopic and histological scoring. Increased leukocyte-endothelial cell adhesive interactions and severe tissue injury accompanied indomethacin-induced ileitis. Treatment with the Boswellia extract or AKBA resulted in a dose-dependent decrease in rolling (up to 90%) and adherent (up to 98%) leukocytes. High-dose Boswellia extract as well as both low- and high-dose AKBA significantly attenuated tissue injury scores. Oral therapy with the Boswellia extract or AKBA significantly reduces macroscopic and microcirculatory inflammatory features normally associated with indomethacin administration, indicating that the anti-inflammatory actions of the Boswellia extract in IBD may be due in part to boswellic acids such as AKBA.

MEK-1/2 inhibition prevents 5-lipoxygenase translocation in N-formylpeptide-challenged human neutrophils.

In order to elucidate the role of mitogen-activated protein kinase kinase (MEK-1/2) in 5-lipoxygenase (5-LO) activation we studied the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced 5-LO translocation in human blood neutrophils (PMNs). In non-primed, Ca(2+)-repleted PMNs, fMLP consistently stimulated MEK-1/2 phosphorylation, but induced 5-LO translocation and product formation (430+/-128 pmol; SEM, n=13) only in 13 of 18 PMN preparations from different healthy donors. In fMLP-responsive cells, the MEK-1/2 inhibitor PD098059 (50 microM) attenuated MEK phosphorylation and abolished 5-LO activation at the translocation step. The fMLP-mediated 5-LO product formation was also sensitive to MEK inhibition by U0126 and to p38 inhibition by SB203580. But in contrast to PD098059, U0126 at 10 microM and SB203580 at 20-50 microM impaired 5-LO activity in the cell-free assay setting, suggesting direct actions of higher concentrations of U0126 and SB203580 on 5-LO apart from MEK and p38 inhibition, respectively. These data show that fMLP initiates 5-LO product formation in non-primed, Ca(2+)-repleted human blood PMNs from healthy donors, and that MEK signaling is pivotal, but not sufficient for 5-LO activation in response to the receptor agonist fMLP.

Workup-dependent formation of 5-lipoxygenase inhibitory boswellic acid analogues.

Pentacyclic triterpenes from the 11-keto-boswellic acid series were identified as the active principal ingredients of Boswellia resin, inhibiting the key enzyme of leukotriene biosynthesis, 5-lipoxygenase (5-LO). Of the genuine boswellic acids hitherto characterized, 3-O-acetyl-11-keto-beta-boswellic acid, AKBA (1), proved to be the most potent inhibitor of 5-LO. In the course of purification of further boswellic acid derivatives from Boswellia resin, we observed the degradation of the natural compound 3-O-acetyl-11-hydroxy-beta-boswellic acid (2) to the thermodynamically more stable product 3-O-acetyl-9, 11-dehydro-beta-boswellic acid (4). The metastable intermediate of this conversion, under moderate conditions of workup in methanolic solutions, was identified as 3-O-acetyl-11-methoxy-beta-boswellic acid (3). The novel artifactual boswellic acid derivatives inhibited 5-LO product formation in intact cells with different characteristics: 4 almost totally abolished 5-LO activity, with an IC(50) of 0.75 microM, whereas 3 and 9,11-dehydro-beta-boswellic acid (5), the deacetylated analogue of 4, were incomplete inhibitors. The data suggest that the conditions chosen for the workup of Boswellia extracts could significantly influence the potency of their biological actions and their potential therapeutic effectiveness.

Concentration-dependent potentiating and inhibitory effects of Boswellia extracts on 5-lipoxygenase product formation in stimulated PMNL.

Preparations from the gum of Boswellia spec. have been used in the traditional medicine for the treatment of inflammatory diseases. Extracts from B. serrata gum were shown to inhibit leukotriene biosynthesis by impairing the 5-lipoxygenase (5-LO) activity. In order to identify the minimal effective concentrations of extracts in vitro we studied the effects of ethanolic extracts from commercially available resins from two regions (B. serrata gum from India and Olibanum in granis from Arabia) on the 5-LO product formation from endogenous substrate in calcium and ionophore stimulated neutrophils in a defined concentration range. Both extracts inhibited 5-LO product formation in vitro in concentrations greater than 10 to 15 micrograms/ml as reported previously for an ethanolic B. serrata extract. In contrast, lower concentrations of extracts (1 to 10 micrograms/ml) even potentiated 5-LO product formation, especially the biosynthesis of 5(S)-HETE. The in vitro data underline the major importance of drug standardization when Boswellia resin containing preparations are used for the treatment of diseases.

Characterization of an acetyl-11-keto-beta-boswellic acid and arachidonate-binding regulatory site of 5-lipoxygenase using photoaffinity labeling.

AKBA (acetyl-11-keto-beta-boswellic acid), a natural pentacyclic triterpene, is an orally active leukotriene-synthesis inhibitor, which acts by a 5-lipoxygenase-directed, non-redox, non-competitive mechanism. It is the only leukotriene-synthesis inhibitor so far identified that inhibits 5-lipoxygenase activity as an allosteric regulator and not by a reducing or competitive mechanism. To characterize AKBA's effector site we prepared azido125I-KBA (4-azido-5-125iodo-salicyloyl-beta-alanyl-11-keto-beta-bo swellic acid) as a photoaffinity analogue, which inhibited 5-lipoxygenase activity as efficiently as the lead compound and specifically labeled human 5-lipoxygenase protein. The labeling of 5-lipoxygenase by azido-125I-KBA strictly depended on the presence of calcium ([Ca2+]free > 500 nM) and was abolished by heat denaturation or by prior incubation with a series of pentacyclic triterpenes (e.g., amyrin, beta-boswellic acid, AKBA and 18a-glycyrrhetinic acid). In contrast, 18-beta-glycyrrhetinic acid and competitive 5-lipoxygenase inhibitors (e.g., ZM-230,487 and L-739,010) did not affect labeling. Arachidonic acid, in enzyme-activity-inhibiting concentrations, reduced photoincorporation (IC50 about 10 microM), whereas a variety of other long-chain fatty acids and their derivatives (e.g., arachidinic acid, arachidonic acid methyl ester, lipoxins A4 and B4) had no effect. The inhibitory arachidonate action on labeling was not affected by blocking the substrate-binding site by micromolar amounts of the competitive inhibitor L-739,010. Therefore, we suggest that AKBA binds in presence of calcium to a site which is distinct from the substrate binding site of 5-lipoxygenase. The AKBA-binding site is likely to be identical with a regulatory, second arachidonate binding site of the enzyme.