Petasites hybridus Extracts in vitro Inhibit COX-2 and PGE₂ Release by Direct Interaction with the Enzyme and by Preventing p42/44 MAP Kinase Activation in Rat Primary Microglial Cells

Abstract

Rhizomes of butterbur, Petasites hybridus L. (Asteraceae), have been used since ancient times for the treatment of inflammatory diseases. In the present study, the effects of lipophilic extracts from rhizomes of Petasites hybridus on the formation and release of prostaglandin E₂ were investigated. The extracts had different contents of petasin and isopetasin: A: 2.1 % and 0.4 %, B: 0.2 % and 0.1 %, C: 12.1 % and 6.1 % and D: 21.9 % and 9.4 %, respectively. Direct inhibition of cyclooxygenase (COX) -1 and -2 isoenzymes and inhibition of the expression of COX-2 and p42/44 MAP kinase in rat primary microglial cells were tested. All extracts were found to be only weak direct inhibitors of COX-1 (IC₅₀ > 400 μg/mL). However, most extracts revealed a strong inhibitory activity against the inducible isoform COX-2 (A: IC₅₀ = 30.4 μg/mL; B: IC₅₀ = 60.6 μg/mL; C: IC₅₀ = 22.6 μg/mL; D: IC₅₀ = 20.0 μg/mL). This activity was not correlated to the content of petasin and isopetasin. Pure petasin and isopetasin neither inhibited COX-1 nor COX-2 (IC₅₀ > 400 μM for both compounds and enzymes). Petasites extracts dose-dependently inhibited LPS-induced and thus COX-2-mediated PGE₂ release in primary rat microglial cells (A: IC₅₀ = 2.4 μg/mL; C: IC₅₀ = 5.8 μg/mL and D: IC₅₀ = 4.6 μg/mL). Also this effect was independent from the petasin and isopetasin content. COX-2 synthesis in microglia was totally blocked with 5 μg/mL of C whereas COX-1 synthesis was not influenced. C and D did not affect the LPS-induced activation of p38 MAPK and IκBα, but they prevented the LPS-induced activation of p42/44 MAPK. Therefore, these Petasites hybridus extracts can be regarded as natural selective inhibitors of COX-2 and its expression, an effect which is independent from the petasin content.

Abbreviations

COX:cyclooxygenase

IκBα:inhibitory protein kappaBalpha

iNOS:inducible nitric oxide synthase

LOX:lipoxygenase

NF-κB:nuclear factor-kappa B

PG:prostaglandins

MAPK:mitogen activated protein kinase

Key words

Inflammation - microglia - Petasites hybridus - Asteraceae - cyclooxygenase - petasin - isopetasin

References

• 1 Lipton R B, Göbel H, Einhäupl K M, Wilks K, Mauskop K. Petasites hybridus root (Butterbur) is an effective preventive treatment for migraine. Neurology, in press (2004)
• 2 Grossmann W, Schmidramsl H. An extract of Petasites hybridus is effective in the prophylaxis of migraine.  Int J Pharmacol Ther. 2000;  38 430-5
• 3 Bauer H W, Kuehne P. Therapie von Harnleiterkoliken mit einem neuen Spasmoanalgetikum.  Therapiewoche. 1986;  36 3756-9
• 4 Kälin P, Sulger Büel E. Gemeine Pestwurz (Petasites hybridus). Portrait einer Arzneipflanze: Geschichte, Pharmakologie und Klinik.  Schweiz Zschr GanzheitsMedizin. 2002;  14 267-74
• 5 Bucher K. Über ein antispastisches Prinzip in Petasites officinalis Moench.  Arch Exper Path Pharmakol. 1950;  213 69-71
• 6 Brune K, Bickel D, Peskar B A. Gastro-protective effects by extracts of Petasites hybridus: The role of inhibition of peptido-leukotriene synthesis.  Planta Medica. 1993;  59 494-6
• 7 Bickel D, Röder T, Bestmann H J, Brune K. Identification and characterization of inhibitors of peptido-leukotriene-synthesis from Petasites hybridus .  Planta Medica. 1994;  60 318-22
• 8 Scheidegger C, Dahinden C, Wiesmann U. Effects of extracts and of individual components from Petasites on prostaglandin synthesis in cultured skin fibroblasts and on leucotriene synthesis in isolated human peripheral leucocytes.  Pharm Acta Helv. 1998;  72 376-80
• 9 Thomet O AR, Wiesmann U N, Blaser K, Simon H U. Differential inhibition of inflammatory effector functions by petasin, isopetasin and neopetasin in human eosinophils.  Clin Exp Allergy. 2001;  31 1-12
• 10 Thomet O AR, Wiesmann U N, Schapowal A, Bizer C, Simon H -U. Role of petasin in the potential anti-inflammatory activity of a plant extract of Petasites hybridus .  Biochem Pharmacol. 2001;  61 1041-7
• 11 Ko W -C, Lei C -B, Lin Y -L, Chen C -F. Relaxant effects of petasins in isolated guinea pig trachea and their structure-activity relationships.  Planta Medica. 2000;  66 650-2
• 12 Ko W -C, Lei C -B, Lin Y, Chen C -F. Mechanisms of relaxant action of S-petasin and S-isopetasin, sesquiterpenes of Petasites formosanus, in isolated guinea pig trachea.  Planta Medica. 2001;  67 224-9
• 13 Martelletti P, Zicari A, Realacci M, Fiore G, De Filippis S, Stirparo G. et al . Expression of NOS-2, COX-2 and Th1/Th2 cytokines in migraine.  J Headache Pain. 2001;  2 S51-6
• 14 Fiebich B L, Lieb K, Hull M, Aicher B, van Ryn J, Pairet M. et al . Effects of caffeine and paracetamol alone or in combination with acetylsalicylic acid on prostaglandin E (2) synthesis in rat microglial cells.  Neuropharmacology. 2000;  39 2205-13
• 15 Fiebich B L, Lieb K, Engels S, Heinrich M. Inhibition of LPS-induced p42/44 MAP kinase activation and iNOS/NO synthesis by parthenolide in rat primary microglial cells.  J Neuroimmunol. 2002;  132 18-24
• 16 Debrunner B. Untersuchungen zur Struktur und Analytik der Inhaltsstoffe von Petasites hybridus [Petasin-Chemovarietät]. PhD thesis Bern; 1994
• 17 Reininger E, Bauer R. New PGHS-1 and -2 microtiter assays for the screening of herbal drugs and in vitro inhibition of PGHS-isoenzyms by polyunsaturated fatty acids from Platycodi radix. Phytomedicine 2004 submitted
• 18 Reininger E. Vergleichende phytochemische und pharmakologische Untersuchungen zur Hemmung der Prostaglandin-H-Synthase isoenzyme mit Arzneidrogen der chinesischen Medizin, insbesondere Platycodi radix und Chaenomelis fructus. PhD thesis, University Düsseldorf 2001
• 19 Bauer M KA, Lieb K, Schulze-Osthoff K, Berger M, Gebicke-Haerter P J, Bauer J. et al . Expression and regulation of cyclooxygenase-2 in rat microglia.  Eur J Biochem. 1997;  243 726-31
• 20 Fiebich B L, Lieb K, Kammerer N, Hull M. Synergistic inhibitory effect of ascorbic acid and acetylsalicylic acid on prostaglandin E2 release in primary rat microglia.  J Neurochem. 2003;  86 173-8
• 21 Fiebich B L, Mueksch B, Boehringer M, Hull M. Interleukin-1beta induces cyclooxygenase-2 and prostaglandin E2 synthesis in human neuroblastoma cells: involvement of p38 mitogen-activated protein kinase and nuclear factor-kappaB.  J Neurochem. 2000;  75 2020-8
• 22 Ramer R, Brune K, Pahl A, Hinz B. R(+)-methanandamide induces cyclooxygenase-2 expression in human neuroglioma cells via a non-cannabinoid receptor-mediated mechanism.  Biochem Biophys Res Commun. 2001;  286 1144-52
• 23 Nelson P T, Soma L A, Lavi E. Microglia in diseases of the central nervous system.  Ann Med. 2002;  34 491-500
• 24 Mitchell J A, Akarasereenont P, Thiemermann C, Flower R J, Vane J R. Selectivity of nonsteroidal antiinflammatory drugs as inhibitors of constitutive and inducible cyclooxygenase.  Proc Natl Acad Sci USA. 1994;  90 11 693-7
• 25 Hinz B, Brune K. Spezifische COX-2-Inhibitoren: Perspektiven einer Therapie mit neuen analgetischen und antiinflammatorischen Wirkstoffen.  Wien Klin Wochenschr. 1999;  111/3 103-12
• 26 Vane J R, Bakhle Y S, Botting R M. Cyclooxygenases 1 and 2.  Ann Rev Pharmacol Toxicol. 1998;  38 97-120
• 27 Wang G -J, Shum A Y-C, Lin Y -L, Liao J -F, Wu X -C, Ren J et a l. Calcium channel blockade in vascular smooth muscle cells: major hypotensive mechanism of S-petasin, a hypotensive sesquiterpene from Petasites formosanus .  J Pharm Exp Therapeutics. 2001;  297 240-6
• 28 Wang G -J, Wu X -C, Lin Y -L, Ren J, Shum A Y -C, Wu Y -Y, Chen C -F. Ca²⁺ channel blockading effect of iso-S-petasin in rat aortic smooth muscle cells.  Eur J Pharmacol. 2002;  445 239-45
• 29 Wang G -J, Liao J -F, Hintz K K, Chen W -P, Su M -J, Lin Y -L, Shi C -C, Chen C -F, Ren J. Calcium-antagonizing activity of S-petasin, a hypotensive sesquiterpene from Petasites formosanus, on inotropic and chronotropic responses in isolated rat atria and cardiac myocytes.  Naunyn-Schmiedeberg’s Arch Pharmacol. 2004;  369 322-9
• 30 Wu S -N, Chen H, Lin Y -L. The mechanism of inhibitory actions of S-petasin, a sesquiterpene of Petasites formosanus, on L-type calcium current in NG108 - 15 neuronal cells.  Planta Medica. 2003;  69 118-24
• 31 Ko W -C, Wang S -H, Chen M -C, Lin Y -L, Chen C -F. S-isopetasin, a sesquiterpene of Petasites formosanus, allosterically antagonized carbachol in isolated guinea pig atria.  Planta Medica. 2002;  68 652-5
• 32 Berger D, Burkhard W, Schaffner W. Influence of Petasites hybridus on dopamine-D₂ and histamine-H₁ receptors.  Pharm Acta Helv. 1998;  72 373-80