Boswellic acids are specific, non-reducing type (nonredox) inhibitors of 5-lipoxygenase1 , one of the two key enzymes in arachidonic acid metabolism and eicosanoid synthesis. 5-LO catalyzes the first two steps in the biosynthesis of leukotrienes (LTs) from arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5(S)-HETE) and subsequently to LTA4, which is further converted to LTB4, or LTC4 depending on the enzymes present. These bioactive lipids have pivotal roles in the pathophysiology of inflammation and allergy2. They also regulate carcinogenesis and survival of various cell types and tissues3,4,5 as well as metabolism of the bone6,7.
The activity of 5-LO is regulated in a highly complex manner. The compartmentalization of 5-LO (cytosolic, intracellular or membrane-bound) is cell-type-specific and is a dynamic process. Subcellular distribution and redistribution with or without product formation vary with the stimulator and duration of stimulation. In intact cells, enzymatic activity of 5-LO requires an increase in [Ca2+]I, substrate release by cytosolic or secretory phospholipase A2, ATP, translocation of 5-LO to membrane protein FLAP (a 5-LO activating protein), lipid peroxides8. Endogenous or exogenous ligands of its allosteric site can further modify 5-LO actions. 5-LO can also be activated via phosphorylation by p38 mitogen-activated protein kinase (MAPK)-regulated MAPK-activated protein kinases (MEKs) or extracellular signal-regulated kinases. There is increasing evidence that MEK 1/2 and/or p38 signaling pathways in particular directly participate in 5-LO activation in neutrophils9 and that 5-LO phosphorylation has an impact on the regulation of its protein-protein interactions and compartmentalization, and thus on the catalytic and signaling activities.
LTB4 is one the most potent chemoattractants and activators of leukocytes and has been shown to be involved in inflammatory processes10. The cysteinyl-LTs C4, D4, and E4 cause broncho-constriction and mucus secretion and induce plasma exudation.
Thus these cys-LTs play an important role in asthma11. 5-LO inhibitors revealed therapeutic value in asthma and some benefit in other inflammatory diseases12. Based on the recently discovered effects of 5-LO metabolites to promote proliferation and survival of malignant cells, bone resorption and atherosclerosis, 5-LO products may also possess potential for prevention and therapy of cancer13,14,15, osteoporosis16 and vascular diseases17.
The search for potent orally active and specific 5-LO inhibitors led to the development of synthetic nonredox-type 5-LO inhibitors such as ZD2138 and ZM230487 which were shown to possess high efficiency in several in vitro and ex vivo systems. However, in in vivo assays, significantly higher doses were required for equivalent effects and ZD2138 failed to strongly suppress LT formation at sites of chronic inflammation18 and require glutathione peroxidase activity for efficient 5-LO inhibition. No such redox-dependent effects were observed in 5-LO inhibition by AKBA19
For its anti-inflammatory action, only those boswellic acids having a keto function and/or carboxyl function on ring A were active as 5-lipoxygenase inhibitors.
More importantly, it has been shown that other pentacyclic terpenes lacking these functional groups actually reversed and diluted the lipoxygenase inhibition afforded by AKBA20. Hence, it is desirable to remove these inactive terpenes from the boswellia extract. AKBA has been shown to be a novel, specific, nonredox inhibitor of 5-lipoxygenase, either interacting directly with the 5-LO or blocking its translocation.
Similarly, it was recently shown21 that β-boswellic acid caused a pronounced mobilization of Ca2+ from internal stores and induced the phosphorylation of p38 MAPK, extracellular signal-regulated kinase-2 (ERK-2) and Akt. These effects were concentration dependent, and the magnitude of the response was comparable to those obtained after platelet stimulation with thrombin or collagen. Investigation of platelet functions revealed that β-boswellic acid strongly stimulates the platelet-induced generation of thrombin in an ex-vivo model, the liberation of arachidonic acid, and induces platelet aggregation in a Ca2+ dependent manner. In contrast, AKBA or KBA did not cause aggregation or significant generation of thrombin. Considering that β-boswellic acid is the major component of commercial boswellia extract, it is imperative that this component be removed from the extract to prevent the above-mentioned undesirable effect.
Mixed acetylboswellic acids significantly inhibited the ionophore-stimulated release of the leukotrienes (LT) B4 and C4 from intact human polymorphonuclear neutrophil leukocytes (PMNLs), with IC50 values of 8.48 μg/ml and 8.43 μg/ml, respectively22. Purified acetyl-11-keto-beta-boswellic acid was about three times more potent as inhibitor of the formation of both LTB4 (IC50 = 2.53 μg/ml) and LTC4 (IC50 = 2.26 μg/ml) from human PMNLs in the same assay. After daily intraperitoneal dosage the extract of mixed acetylboswellic acids (20 mg/kg) significantly reduced the clinical symptoms in guinea pigs with experimental autoimmune encephalomyelitis between days 11 and 21. Safayhi et al23 also observed that acetyl boswellic acids inhibited human leukocyte elastase (HLE). HLE is an aggressively destructive serine protease produced and released by PMNL that may play a role in several diseases, including pulmonary emphysema, cystic fibrosis, chronic bronchitis, acute respiratory distress syndrome, glomerulonephritis and rheumatic arthritis.
Because leukotriene formation and HLE release are increased simultaneously by neutrophil stimulation in a variety of inflammation- and 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-beta-boswellic acid and derivatives.
Inflammatory bowel disease is another inflammatory disease where AKBA affords positive therapeutic effects24. Ileitis was induced by indomethacin injection. Oral therapy with AKBA significantly reduced macroscopic and microcirculatory inflammatory features normally associated with indomethacin administration, and resulted in a dose-dependent decrease in rolling (up to 90%) and adherent (up to 98%) leukocytes.
Expression of proinflammatory cytokines by monocytes is tightly regulated by transcription factors such as nuclear factor KB (NFκB). NFκB activation is tightly regulated by its endogenous inhibitor IκB, which complexes with and sequesters NFκB in the cytoplasm. Following cytokine stimulation, IκBα is phosphorylated which initiates the rapid degradation of this inhibitor by the nonlysosomal, ATP-dependent proteolytic complex. IκBα phosphorylation involves various kinases which converge on NFκB-inducing kinase (NIK). The activated NIK then phosphorylaes and activates the IκB kinase complex (IKK). IKK activation leads to phophorylation/degradation of IκBα and subsequent release of NFκB which then translocates to the nucleus and activates traanscription of multiple κB-dependent genes, including TNF-α, IL-6, IL-8 and other chemokines; major histocompatibility complex (MHC) class II, the adhesion molecule ICAM-1, inducible NO synthase and COX-2. Since NFκB plays a central role in mediating proinflammatory gene expression, there is growing interest in modulating its activity and in a recent study25, acetyl boswellic acids, acetyl-α-boswellic acid and AKBA was shown to inhibit NFκB signaling in lipopolysaccharide (LPS)-stimulated human peripheral monocytes. Both compounds inhibited the LPS-induced phosphorylation and degradation of IκBα, and transloca-tion of NFκB to the nucleus.
A recent study showed that acetyl boswellic acids inhibit constitutively activated NFκB signaling and inhibits proliferation of prostate cancer cells (see below).
In another recent study26, the genetic basis of the anti-inflammatory effects of a standardized boswellia extract (BE), enriched in AKBA, was tested in a system of TNF alpha-induced gene expression in human micro vascular endothelial cells (HMEC). The authors conducted the first whole genome screen for TNF alpha- inducible genes in HMEC. Acutely, TNF alpha induced 522 genes and down regulated 141 genes in nine out of nine pair wise comparisons. Of the 522 genes induced by TNF alpha in HMEC, 113 genes were clearly sensitive to BE treatment. Such genes directly related to inflammation, cell adhesion, and proteolysis. The robust BE-sensitive candidate genes were then subjected to further processing for the identification of BE-sensitive signaling pathways. BE prevented the TNF alpha-induced expression of matrix metalloproteinases. BE also prevented the inducible expression of mediators of apoptosis. Most strikingly, however, TNF alpha-inducible expression of VCAM-1 and ICAM-1 were observed to be sensitive to BE. Real-time PCR studies showed that while TNF alpha potently induced VCAM-1 gene expression, BE completely prevented it. This result confirmed the microarray findings and built a compelling case for the anti-inflammatory property of BE.
1. Safayhi H, Mack T, Sabieraj J, et al. Boswellic acids: novel, specific, nonredox inhibitors of 5-lipoxygenase, Pharmacol. Exp. Ther., 1992, 261:1143-46.
2. Samulsson B, Dahlen SE, Lindgren JA, et al. Leukotrienes and lipoxins: structure, biosynthesis and biological effects, Science, 1987, 237:1171-76.
3. Ohd JF, Wikstrom K, Sjolander A, Leukotrienes induce cell-survival signalling in intestinal epithelial cells, Gatroenterology, 2000, 119:1007-18.
4. Romano M, Catalano A, Nutini M, et al. 5-Lipoxygenase regulates malignant mesothelial cell survival: involvement of vascular endothelial growth factor, FASEB J, 2001, 15:2326-36.
5. Gupta S, Srivastava M, Ahmad N, et al. Lipoxygenase-5 is overexpressed in prostrate carcinoma, Cancer, 2001, 91:737-43.
6. Chen XS, Sheller JR, Johnson EN, et al. Role of leukotrienes revealed by targeted disruption of 5-lipoxygenase gene, Nature, 1994, 372:179-82.
7. Garcia C, Boyce BF, Gilles J, et al. Leukotriene B$ stimulates osteoclaastic bone resorption both in vitro and in vivo, J. Bone Miner. Res., 1996, 11:1619-27.
8. Radmark O, The molecular biology and regulation of 5-lipoxygenase, Am. J. Rresir. Crit. Care Med., 2000, 161:S11-S15.
9. Werz O, Klemm J, Samuelsson B, Radmaark O, 5-Lipoxygenase is phosphorylated by p38 kinase dependentMAPKAP kinase, Proc. Natl. Acad. Sci. USA, 2000, 97:5261-66.
10. Samuelsson B, Leukotrienes: mediators of immediate hypersensitivity and reactions and inflammations, Science, 1983, 220:568-75.
11. Claesson HE, Dahlen SE, Asthma and leukotrienes: antileukotrienes as noveel anti-asthmatic drugs, J. Intern. Med., 1999, 245:205-27.
12. Steinhilber D, 5-Lipoxygenase: a target for antiinflammatory drugs revisited, Curr. Med. Chem., 1999, 6:69-83.
13. Avis I, Hong SH, Martinez A, et al. Five-lipoxygenase inhibitors can mediate apoptosis in human breast cancer cell lines through complex eicosanoid interactions, FASEB J., 2001, 15:2007-9.
14. Ghosh J, Myers CE, Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in human prostrate cancer cells, Proc. Natl. Acad. Sci.USA, 1998:95:13182-87.
15. Ding XZ, Iversen P, Cluck M, et al. Lip[oxygenase inhibitors abolish proliferation of human pancreaatic cells, Biochem. Biophys. Res. Commun., 1999, 261:218-23.
16. Alanko J, Sievi E, Lahteenmaki T, et al. Catechol estrogens as inhibitors of leukotriene synthesis, Biochem. Pharmacol., 1998, 55:101-04
17. Mehrabian M, Allayee H, Wong J, et al. Identification of 5-lipoxygenase as a major gene contributing to atherosclerosis susceptibility in mice, Circ. Res., 2002, 91:120-26.
18. Werz O, Szellas D, Henseler M, Steinhilber D, Nonredox 5-lipoxygenase inhibitors require glutathione peroxidase for efficient inhibition of 5-lipoxygenase activity, Mol. Pharmacol, 1998, 54, 445-51.
19.Fischer L, Szellas D, Radmark O, et al, Phosphorylation- and stimulation-dependent inhibition of cellular 5-lipoxygenase activity by nonredox-type inhibitors, FASEB J., 2003, 17:949-51.
20. Safayhi H, Salier ER, Ammon HP, Mechanism of 5-lipoxygenase inhibtion by acetyl-11-keto-beta-boswellic acid, Am. Soc.Pharmacol. Exp. Ther., 1995, 47:1212-16
21. Poeckel D, Tausch L, Altmann K, et al. Induction of central signalling pathways and select functional effects in human platelets by beta-boswellic acid, Br. J. Pharmacol., 2005, Aug 8; [Epub ahead of print]
22. Widfeuer A, Neu IS, Safayhi H, et al. Effects of boswellic acids extracted from a herbal medicine on the biosynthesis of leukotrienes and the course of experimental autoimmune encephalomyelitis. Arzneimittelforschung, 1998, 48(6): 668-74.
23. Safayhi H, Rall B, Sailer E-R, Ammon HP, Inhibition by boswellic acids of human leukocyte elastase, J Pharmacol Exp Ther, 1997, 281(1): 460-3.
24. Krieglstein CF, Anthoni C, Rijcken EJ, et al. Acetyl-11-keto-boswellic acid, a constituent of a herbal medicine from Boswellia serrata resin, attenuates experimental ileitis, Int. J. Colorectal Dis., 2001, 16:88-95.
25. Syrovets T, Buchele B, Krauss C, et al. Acetyl boswellic acids inhibit lipopolysaccharide-mediated TNFα induction in monocytes by direct interaction with IκB kinaases, J. Immunol., 2005, 174:498-506.
26. Roy S, Khanna S, Shah H, et al. Human genome screen to identify the genetic basis of the anti-inflammatory effects of Boswellia in microvascular endothelial cells, DNA Cell Biol., 2005, 24:244-55
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