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

Pharmakologische Untersuchung und Identifizierung von Wirkmechanismen definierter Extrakte und Komponenten des Weihrauchharzes

Extracts from Boswellia species are used for therapy of diverse diseases since antiquity (1). Recent clinical studies with pilot character and experiments in animal models confirm the therapeutic efficacy in inflammatory diseases like osteoarthritis (2-5), inflammatory bowel diseases (6-9), asthma bronchiale (10), and different cancers (11-13). A central role in the anti-inflammatory efficiency is attributed to the species-specific boswellic acids (BAs), as different target structures were described for them. Even though further components are present in the oleo-gum resin with similar quantity as BAs, they were rather neglected in previous studies. This thesis highlights the pharmacologic action of extracts from the oleo-gum resin of different Boswellia species and the triterpenic acids isolated thereof. By activity-guided fractionation of extracts, several triterpenic acids could be identified as potent inhibitors of the microsomal Prostaglandin E2 synthase-1 (mPGES-1). The inhibitory effect of some quantitatively relevant tirucallic acids even exceeded that of the BAs. With minor impact on enzymes upstream of PGE2 biosynthesis (cyclooxygenases (COX), cPLA2alpha), this action suggests effective inhibition of inflammation with reduced risk of adverse effects. Moreover it could be shown that the recently identified triterpene 3-O-acetyl-28-hydroxy-lupeolic acid (Ac-OH-LA) is a direct inhibitor of the activity of the cytosolic phospholipase A2alpha (cPLA2alpha) (14). The inhibition of cPLA2alpha effected reduced release of arachidonic acid in cell-free systems as well as in different A23187-stimulated blood cells. The reduced formation of arachidonic acid metabolites in the cell model could partially be reversed by addition of exogenous arachidonic acid. Functionally, Ac-OH-LA reduced the cPLA2alpha-dependant platelet aggregation induced by collagen. In all target structures investigated in this thesis (5-lipoxygenase, mPGES-1, COX, cPLA2alpha, cathepsin G, human leukocyte elastase), further triterpenic acids showed similar or superior potency compared to BAs. This suggests a pivotal role of triterpenic acids besides BAs in the anti-inflammatory activity of frankincense extracts. 1. H. P. Ammon, Planta Med 72, 1100 (Oct, 2006). 2. N. Kimmatkar, V. Thawani, L. Hingorani, R. Khiyani, Phytomedicine 10, 3 (Jan, 2003). 3. S. Sontakke et al., Indian J Pharmacol 39, 27 (Feb, 2007). 4. K. Sengupta et al., Arthritis Research & Therapy 10, (2008). 5. K. Sengupta et al., Int J Med Sci 7, 366 (2010). 6. H. Gerhardt, F. Seifert, P. Buvari, H. Vogelsang, R. Repges, Z Gastroenterol 39, 11 (Jan, 2001). 7. I. Gupta et al., Planta Med 67, 391 (Jul, 2001). 8. I. Gupta et al., Eur J Med Res 2, 37 (Jan, 1997). 9. A. Madisch et al., Int J Colorectal Dis 22, 1445 (Dec, 2007). 10. I. Gupta et al., Eur J Med Res 3, 511 (Nov 17, 1998). 11. G. Janssen et al., Klin Padiatr 212, 189 (Jul-Aug, 2000). 12. J. R. Streffer, M. Bitzer, M. Schabet, J. Dichgans, M. Weller, Neurology 56, 1219 (May 8, 2001). 13. S. Kirste et al., Cancer, (Feb 1, 2011). 14. M. Verhoff et al., Biochem Pharmacol, (Jun 23, 2012).Extrakte aus Boswellia-Arten werden seit der Antike in der Behandlung verschiedener Krankheiten eingesetzt (1). Neuere klinische Pilotstudien und Experimente im Tiermodell bestätigten die therapeutische Wirksamkeit bei entzündlichen Erkrankungen wie Osteoarthritis (2-5), entzündlichen Darmerkrankungen (6-9), Asthma bronchiale (10) und bestimmten Krebserkrankungen (11-13). Für die Art-spezifischen Boswelliasäuren (BAs) wurden mehrere Zielstrukturen beschrieben, weshalb ihnen eine zentrale Rolle in der anti-entzündlichen Wirkung zugeschrieben wird. Doch obwohl einige der übrigen Inhaltsstoffe ähnliche Anteile des Harzes ausmachen wie BAs, wurden sie in bisherigen Untersuchungen eher vernachlässigt. In dieser Arbeit wird die pharmakologische Wirkung von Harz-Extrakten aus verschiedenen Boswellia-Arten und von daraus isolierten Triterpensäuren untersucht. So konnte durch aktivitätsgeleitete Fraktionierung von Extrakten eine Reihe von Triterpensäuren als potente Hemmstoffe der mikrosomalen Prostaglandin E2 Synthase-1 (mPGES-1) identifiziert werden. Die Hemmwirkung durch verschiedene quantitativ bedeutsame Tirucallensäuren überstieg dabei die der BAs. Bei geringem Einfluss auf übergeordnete Enzyme der PGE2-Biosynthese (Cyclooxygenasen (COX), cPLA2alpha) verspricht diese Wirkung eine effektive Hemmung von Entzündungsvorgängen bei geringem Risiko unerwünschter Effekte. Des Weiteren konnte die direkte Aktivitätshemmung der zytosolischen Phospholipase A2alpha (cPLA2alpha) durch das kürzlich identifizierte Triterpen 3-O-Acetyl-28-hydroxy-Lupansäure (Ac-OH-LA) nachgewiesen werden (14). Sowohl im zellfreien System als auch in verschiedenen A23187-stimulierten Blutzellen bewirkte die cPLA2alpha-Hemmung eine reduzierte Arachidonsäurefreisetzung. Im Zellmodell konnte die herabgesetzte Bildung daraus abgeleiteter Metabolite durch Zugabe von Arachidonsäure teilweise umgekehrt werden. Funktionell reduzierte Ac-OH-LA die cPLA2alpha-abhängige Thrombozytenaggregation nach Stimulation mit Kollagen. An allen in dieser Arbeit untersuchten Zielstrukturen (5-Lipoxygenase, mPGES-1, COX, cPLA2alpha, Cathepsin G, humane Leukozytenelastase) zeigten weitere Triterpensäuren ähnliche oder höhere Potenz als BAs. Dies legt eine wesentliche Rolle der Triterpensäuren neben BAs in den anti-entzündlichen Wirkungen von Weihrauchextrakten nahe. 1. H. P. Ammon, Planta Med 72, 1100 (Oct, 2006). 2. N. Kimmatkar, V. Thawani, L. Hingorani, R. Khiyani, Phytomedicine 10, 3 (Jan, 2003). 3. S. Sontakke et al., Indian J Pharmacol 39, 27 (Feb, 2007). 4. K. Sengupta et al., Arthritis Research & Therapy 10, (2008). 5. K. Sengupta et al., Int J Med Sci 7, 366 (2010). 6. H. Gerhardt, F. Seifert, P. Buvari, H. Vogelsang, R. Repges, Z Gastroenterol 39, 11 (Jan, 2001). 7. I. Gupta et al., Planta Med 67, 391 (Jul, 2001). 8. I. Gupta et al., Eur J Med Res 2, 37 (Jan, 1997). 9. A. Madisch et al., Int J Colorectal Dis 22, 1445 (Dec, 2007). 10. I. Gupta et al., Eur J Med Res 3, 511 (Nov 17, 1998). 11. G. Janssen et al., Klin Padiatr 212, 189 (Jul-Aug, 2000). 12. J. R. Streffer, M. Bitzer, M. Schabet, J. Dichgans, M. Weller, Neurology 56, 1219 (May 8, 2001). 13. S. Kirste et al., Cancer, (Feb 1, 2011). 14. M. Verhoff et al., Biochem Pharmacol, (Jun 23, 2012)

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

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

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

Tetra- and Pentacyclic Triterpene Acids from the Ancient Anti-inflammatory Remedy Frankincense as Inhibitors of Microsomal Prostaglandin E₂ 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 (<b>6</b>) and 3α-acetoxy-7,24-dienetirucallic acid (<b>10</b>) 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