Triterpene Acids from Frankincense and Semi-Synthetic Derivatives That Inhibit 5-Lipoxygenase and Cathepsin G.

Age-related diseases, such as osteoarthritis, Alzheimer's disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) pathway. Boswellic acids (BAs) are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging.

Tetra- and pentacyclic triterpene acids from the ancient anti-inflammatory remedy frankincense as inhibitors of microsomal prostaglandin E(2) synthase-1.

The microsomal prostaglandin E2 synthase (mPGES)-1 is the terminal enzyme in the biosynthesis of prostaglandin (PG)E2 from cyclooxygenase (COX)-derived PGH2. We previously found that mPGES-1 is inhibited by boswellic acids (IC50 = 3-30 µM), which are bioactive triterpene acids present in the anti-inflammatory remedy frankincense. Here we show that besides boswellic acids, additional known triterpene acids (i.e., tircuallic, lupeolic, and roburic acids) isolated from frankincense suppress mPGES-1 with increased potencies. In particular, 3α-acetoxy-8,24-dienetirucallic acid (6) and 3α-acetoxy-7,24-dienetirucallic acid (10) inhibited mPGES-1 activity in a cell-free assay with IC50 = 0.4 µM, each. Structure-activity relationship studies and docking simulations revealed concrete structure-related interactions with mPGES-1 and its cosubstrate glutathione. COX-1 and -2 were hardly affected by the triterpene acids (IC50 > 10 µM). Given the crucial role of mPGES-1 in inflammation and the abundance of highly active triterpene acids in frankincence extracts, our findings provide further evidence of the anti-inflammatory potential of frankincense preparations and reveal novel, potent bioactivities of tirucallic acids, roburic acids, and lupeolic acids.

Interference of boswellic acids with the ligand binding domain of the glucocorticoid receptor.

Boswellic acids (BAs) possess anti-inflammatory properties in various biological models with similar features to those of glucocorticoids (GCs), such as suppression of the release of pro-inflammatory cytokines. Hence, the molecular mechanism of BAs responsible for their anti-inflammatory features might be attributable to interference with the human glucocorticoid receptor (GR). Due to obvious structural similarities with GCs, we conducted pharmacophore studies as well as molecular docking simulations of BAs as putative ligands at the ligand binding site (LBS) of the GR in distinct functional states. In order to verify receptor binding and functional activation of the GR by BAs, radiometric binding assays as well as GR response element-dependent luciferase reporter assay were performed with dexamethasone (DEX) as a functional positive control. With respect to the observed position of GCs in GR crystal complexes in the active antagonist state, BAs docked in a flipped orientation with estimated binding constants reflecting nanomolar affinities. For validation, DEX and other steroids were successfully redocked into their crystal poses in similar ranges as reported in the literature. In line with the pharmacophore and docking models, the BAs were strong GR binders (radiometric binding assay), albeit none of the BAs activated the GR in the reporter gene assay, when compared to the GC agonist DEX. The flipped scaffolds of all BAs dislodge the known C-11 function from its receiving amino acid (Asn564), which may explain the silencing effects of receptor-bound BAs in the reporter gene assay. Together, our results constitute a compelling example of rigid keys acting in an adaptable lock qualifying as a reversed induced fit mechanism, thereby extending the hitherto published knowledge about molecular target interactions of BAs.

A novel C(28)-hydroxylated lupeolic acid suppresses the biosynthesis of eicosanoids through inhibition of cytosolic phospholipase A(2).

Eicosanoids are potent lipid mediators derived from phospholipase (PL)-released arachidonic acid (AA) coupled to subsequent metabolism by cyclooxygenase (COX)-1/2 or lipoxygenases (LO) which are involved in a variety of homeostatic biological functions and inflammation. We have investigated three lupeolic acids (LA) from the gum resin of Boswellia carterii for their ability to interfere with eicosanoid biosynthesis in human blood cells. A novel, yet unknown C(28)-hydroxylated LA, that is, 3α-acetoxy-28-hydroxylup-20(29)-en-4ß-oic acid (Ac-OH-LA) was found to inhibit the biosynthesis of COX-, 5-LO- and 12-LO-derived eicosanoids from endogenous AA in activated platelets, neutrophils, and monocytes from human blood with consistent IC(50) values of 2.3-6.9 µM. In contrast, two other LAs lacking the C(28)-OH moiety were essentially inactive in this respect. Inhibition of eicosanoids by Ac-OH-LA correlated with reduced release of AA in intact cells. When AA was exogenously provided as substrate for cellular eicosanoid biosynthesis the inhibitory effects of Ac-OH-LA were essentially reversed, even though some inhibition of 5-LO and COX-1 product formation still remained. Finally, by means of a cell-free phospholipid hydrolysis assay using human recombinant cytosolic PLA(2)α, we show that Ac-OH-LA may directly interfere with cPLA(2)α activity (IC(50) = 3.6 µM). Together, we identified a novel, naturally occuring C(28)-hydroxylated LA which acts as efficient inhibitor of cPLA(2)α and consequently suppresses eicosanoid biosynthesis in intact cells.

Green tea epigallocatechin-3-gallate inhibits microsomal prostaglandin E(2) synthase-1.

Prostaglandin (PG)E(2) is a critical lipid mediator connecting chronic inflammation to cancer. The anti-carcinogenic epigallocatechin-3-gallate (EGCG) from green tea (Camellia sinensis) suppresses cellular PGE(2) biosynthesis, but the underlying molecular mechanisms are unclear. Here, we investigated the interference of EGCG with enzymes involved in PGE(2) biosynthesis, namely cytosolic phospholipase (cPL)A(2), cyclooxygenase (COX)-1 and -2, and microsomal prostaglandin E(2) synthase-1 (mPGES-1). EGCG failed to significantly inhibit isolated COX-2 and cPLA(2) up to 30 microM and moderately blocked isolated COX-1 (IC(50)>30 microM). However, EGCG efficiently inhibited the transformation of PGH(2) to PGE(2) catalyzed by mPGES-1 (IC(50)=1.8 microM). In lipopolysaccharide-stimulated human whole blood, EGCG significantly inhibited PGE(2) generation, whereas the concomitant synthesis of other prostanoids (i.e., 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-keto PGF(1alpha)) was not suppressed. Conclusively, mPGES-1 is a molecular target of EGCG, and inhibition of mPGES-1 is seemingly the predominant mechanism underlying suppression of cellular PGE(2) biosynthesis by EGCG.

Carnosic acid and carnosol potently inhibit human 5-lipoxygenase and suppress pro-inflammatory responses of stimulated human polymorphonuclear leukocytes.

Carnosic acid (CA) and carnosol (CS) are phenolic diterpenes present in several labiate herbs like Rosmarinus officinalis (Rosemary) and Salvia officinalis (Sage). Extracts of these plants exhibit anti-inflammatory properties, but the underlying mechanisms are largely undefined. Recently, we found that CA and CS activate the peroxisome proliferator-activated receptor gamma, implying an anti-inflammatory potential on the level of gene regulation. Here we address short-term effects of CA and CS on typical functions of human polymorphonuclear leukocytes (PMNL). We found that (I), CA and CS inhibit the formation of pro-inflammatory leukotrienes in intact PMNL (IC(50)=15-20 microM [CA] and 7 microM [CS], respectively) as well as purified recombinant 5-lipoxygenase (EC number 1.13.11.34, IC(50)=1 microM [CA] and 0.1 microM [CS], respectively), (II) both CA and CS potently antagonise intracellular Ca(2+) mobilisation induced by a chemotactic stimulus, and (III) CA and CS attenuate formation of reactive oxygen species and the secretion of human leukocyte elastase (EC number 3.4.21.37). Together, our findings provide a pharmacological basis for the anti-inflammatory properties reported for CS- and CA-containing extracts.