- Email: [email protected]
Knipholone, a selective inhibitor of leukotriene metabolism
ARTICLE IN PRESS
Phytomedicine 13 (2006) 452–456 www.elsevier.de/phymed
SHORT COMMUNICATION
Knipholone, a selective inhibitor of leukotriene metabolism A.A. Wubea, F. Bucara,, K. Asresb, S. Gibbonsc, M. Adamsa, B. Streita, A. Bodensiecka, R. Bauera a
Department of Pharmacognosy, Institute for Pharmaceutical Sciences, Karl-Franzens University Graz, Universitaetsplatz 4/1, A-8010 Graz, Austria b Department of Pharmacognosy, School of Pharmacy, Addis Ababa University, P.O. Box 1176, Ethiopia c Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK
Abstract Inhibition of leukotriene formation is one of the approaches to the treatment of asthma and other inflammatory diseases. We have investigated knipholone, isolated from the roots of Kniphofia foliosa, Hochst (Asphodelaceae), for inhibition of leukotriene biosynthesis in an ex vivo bioassay using activated human neutrophile granulocytes. Moreover, activities on 12-lipoxygenase from human platelets and cycloxygenase (COX)-1 and -2 from sheep cotyledons and seminal vesicles, respectively, have been evaluated. Knipholone was found to be a selective inhibitor of leukotriene metabolism in a human blood assay with an IC50 value of 4.2 mM. However, at a concentration of 10 mg/ ml, the compound showed weak inhibition of 12(S)-HETE production in human platelets and at a concentration of 50 mM it produced no inhibition of COX-1 and -2. In our attempt to explain the mechanism of inhibition, we examined the antioxidant activity of knipholone using various in vitro assay systems including free radical scavenging, nonenzymatic lipid peroxidation, and metal chelation. Knipholone was found to be a weak dose-independent free radical scavenger and lipid peroxidation inhibitor, but not a metal chelator. Therefore, the leukotriene biosynthesis inhibitory effect of knipholone was evident by its ability either to inhibit the 5-lipoxygenase activating protein (FLAP) or as a competitive (non-redox) inhibitor of the enzyme. Cytotoxicity results also provided evidence that knipholone exhibits less toxicity for a mammalian host cell. r 2005 Elsevier GmbH. All rights reserved. Keywords: Kniphofia foliosa; Knipholone; Leukotrienes; 5-Lipoxygenase; 12-Lipoxygenase; Cycloxygenases
Introduction 5-Lipoxygenase (5-LOX) is the key enzyme involved in the first two biosynthesis steps of leukotrienes from arachidonic acid: the stereospecific oxygenation leading to formation of 5(S)-hydroperoxy-6-trans-8,11,14-ciseicosatetraenoic acid (5-HPETE), and further dehydraCorresponding author. Tel.: +43 316 380 5531; fax: +43 316 380 9860. E-mail address: [email protected] (F. Bucar).
0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2005.01.012
tion to leukotriene A4 (Samuelsson, 1983). Leukotrienes play a major role in the inflammatory response to injury and they have been implicated in the pathogenesis of several chronic inflammatory diseases, most notably asthma, psoriasis, rheumatoid arthritis and inflammatory bowel diseases. So far, two therapeutic strategies have been developed to inhibit the enzyme, 5-LOX, involved in leukotrienes biosynthesis: direct 5-LOX inhibitors and indirect inhibitors which interfere with 5-lipoxygenase activating protein (FLAP). The direct 5-LOX inhibitors interact with the enzyme via a redox,
ARTICLE IN PRESS A.A. Wube et al. / Phytomedicine 13 (2006) 452–456
OH
O
OH
CH3 O OH
HO
CH3 OCH3
O
Fig. 1. The structure of knipholone.
a nonredox or an iron chelating mechanism (McMillan and Walker, 1992), while FLAP inhibitors block the association of the enzyme with the cellular membrane (Dixon et al., 1990). Knipholone, a binary compound composed of the anthraquinone chrysophanol and an acetylphloroglucinol (Fig. 1), was originally isolated from the roots of Kniphofia foliosa, Hochst (Asphodelaceae) together with chrysophanol (Dagne and Steglich, 1984) and evaluated for its antiprotozoal properties (Bringmann et al., 1999, 2003). In the present study, we evaluated the leukotriene inhibitory activity and mechanism of inhibition by knipholone isolated from the roots of K. foliosa. The perennial herb K. foliosa grows wild in the central and southern highlands of Ethiopia. The roots have long been used in traditional medicine for the treatment of abdominal cramps and wound healing (Abate, 1989).
Materials and methods Analytical TLC was performed on Merck silica gel 60 F254 and RP-18 F254s plates. Column chromatography (CC) was performed on Merck silica gel 60 (70–240 mesh) and size exclusion chromatography on Sephadex LH-20. Solid phase separation was conducted using Isolute C18 EC (10 g) columns. Semi-preparative HPLC was performed using LiChrosphers RP-18 (10 mm, 250 10 mm i.d.) column. NMR spectra were recorded at 500 MHz for 1H and 125 MHz for 13C on a Bruker AVANCE 500 spectrometer. Mass spectra were determined by LC–ESI–MS analysis on a Thermo Finnigan LCQ Deca XP Plus mass spectrometer connected to a Surveyer LC-system (Thermo Finnigan). Absorbances for antioxidant tests were determined with a WALLAC 1420 Multilable Counter, Perkin ElmerTM Life Sciences. UV–visible spectra were recorded using a SPECTROD
453
50 spectrophotometer (Zeiss). The absorbance for LTB4 quantification was conducted using the photometric ELISA plate reader, Tecan RAIN BOW. Trypan blue solution, eicosatetraenoic acid, type VII Folch bovine brain extract, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 1-butanol and glutathion were purchased from Sigma Chemicals. Ca ionophor A 23187, KH2PO4–K2HPO4 buffer pH 7.413 (25 1C) (PBS), Tween 80R, trichloroacetic acid (TCA), thiobarbituric acid (TBA), butylated hydroxyltoluene (BHT), NaOH and epinephrin-hydrogentartrate were obtained from Fluka. Ethanol p.a., formic acid, citric acid solution, CaCl2 2H2O p.a., anhydrous D-glucose, MgCl2 6H2O. KCl, TRIS p.a., HCl solution, NH4Cl solution, NaCl, TU¨RKS solution, FeCl3 6H2O, CuSO4 5H2O, FeSO4 7H2O, dimethyl sulfoxide (DMSO) and Na2EDTA were bought from Merck. Ascorbic acid and baicalein were obtained from Aldrich. Quercetin and Tris/HCl buffer (pH 8.0) were obtained from Roth. LTB4 EIA Kit, purified PGHS-1 and -2, indomethacin and NS-398 were obtained from Cayman Chemical, Ann Arbor, MI, USA. Arachidonic acid and dextran were purchased from Amersham Pharmacia Biotech AB. 12(S)-HETE correlated-EIA kit was purchased from Assay Designs, Ann Arbor, MI, USA. PGE2-EIA kit was obtained from R & D systems, Minneapolis, MN, USA. Zileuton was purchased from Sequoia Research Products Ltd., Oxford, UK.
Plant materials The roots of K. foliosa were collected in April 2001 from Grassland very close to Dinsho, Bale, Ethiopia (alt. 2700–2850 m) and identified by Mr. Melaku Wondafrash, the National Herbarium, Department of Biology, Addis Ababa University. A voucher specimen (collection number 1482) has been deposited in the National Herbarium for future reference.
Extraction and isolation Air-dried ground roots (500 g) of K. foliosa were extracted successively by petroleum ether, dichloromethane and methanol using a Soxhlet apparatus for 24 h. The dichloromethane extract was evaporated under reduced pressure to yield 5.5 g (1.1%) residue and subjected to CC on silica gel eluting with petroleum ether and a 10% stepwise gradient with dichloromethane and methanol to afford 20 fractions. Fractions 12–13 (1.2 g) from CH2Cl2/CH3OH (9:1–8:2) were combined and subjected to solid phase separation using a CH3OH/H2O gradient elution followed by Sephadex LH-20, CH2Cl2/CH3OH (1:1) to yield a reddish fraction (720 mg), which was purified further by semi-preparative RP-18 HPLC with CH3CN/H2O (6:4) isocratic system
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to afford knipholone (1) (97 mg). The structure was identified by analysis of the spectral data and by comparison with literature values (Dagne and Steglich, 1984).
Characterisation of the ex vivo leukotriene bioassay The bioassay for inhibition of leukotriene biosynthesis was performed as described before (Adams et al., 2004) using human neutrophile granulocytes. Knipholone was tested at the concentrations 20, 10, 5 and 1 mM and quantified in duplicate. Mean values and the standard deviations were determined (Microsoft, Excel). IC50 values with fiducial limits (FL) were calculated by probit-log analysis as described for quantitative bioassays (Finney, 1978) using SPSS 6.0 for MS Windows.
12(S)-Lipoxygenase assay The 12(S)-LOX inhibitory assay was conducted ex vivo using human platelets as reported previously (Schneider et al., 2004). Baicalein was used as a positive control. The mean values of two measurements were taken and tests were conducted three times.
Metal chelating activity The metal chelating capacity of knipholone was measured with FeSO4, CuSO4 and FeCl3 solutions by the method described in the literature (Brown et al., 1998) with some modification. The compound tested was dissolved in ethanol at a concentration of 1 mM. From the stock solution, a solution of 50 mM was prepared in a cuvette with PBS (10 mM, pH 7.4) and the absorption spectra were recorded from 200 to 600 nm. Scans with 50 and 100 mM FeSO4, CuSO4 and FeCl3 were taken after 10 s and compared with the spectrum of knipholone alone. The effect of 2.5-fold EDTA concentration (125 mM) was also examined. Quercetin was used as a standard.
Brine shrimp cytotoxicity assay The test was microplate (Solis concentration of brine shrimps in analysis.
performed in triplicate in 96-well et al., 1993). The ED50 value, the knipholone effective to kill 50% of the test, was determined by probit
Results and discussion Cycloxygenase-1 and -2 assays The assays were performed with purified PGHS-1 from ram seminal vesicles for cycloxygenase (COX)-1 and purified PGHS-2 from sheep placental cotyledons for COX-2 as previously described (Fiebich et al., 2005). The percent inhibitions of 50 mM concentration of knipholone were determined for both enzymes. Indomethacin and NS-398 were used as a positive control for COX-1 and -2, respectively.
Free radical scavenging activity using DPPH assay The DPPH assay was done as described before (Schneider et al., 2004). Quercetin and ethanol were used as a positive control and blank, respectively. The test was performed in triplicate and the absorbances were averaged before calculation.
Iron/ascorbate-induced non-enzymatic lipid peroxidation assay This assay was performed as previously described (Burits and Bucar, 2000) using phospholipid liposomes prepared from Type VII Folch bovine brain extract dissolved in PBS.
In our ex vivo leukotriene biosynthesis inhibition assay knipholone showed a high dose-dependent activity (Fig. 2) with an IC50 value of 4.2 mM (3.6–4.9, 95% FL) being approximately twice as active as the commercial 5LOX inhibitor zileuton (IC50 ¼ 10.4 mM, 9.0–11.7, 95% FL). Knipholone was tested for inhibition of 12(S)HETE production using human platelets at 10 mg/ml concentration which resulted in 28.6% inhibition. Baicalein was used as a positive control and exhibited 52% inhibition at 5 mg/ml. However, at concentrations up to 50 mg/ml, the compound produced no inhibition of other enzymes related to inflammation, such as COX-1 and -2 compared to the positive controls indomethacin which inhibits COX-1 with an IC50 value of 0.9 mM and NS-398 which inhibits COX-2 with an IC50 value of 2.6 mM. Therefore, knipholone showed higher affinity for the 5-LOX pathway than for cycloxygenases and 12LOX. In this study we examined whether redox and chelating mechanisms might be involved in the 5-LOX inhibitory effect of knipholone via antioxidant tests, such as free radical scavenging using DPPH, lipid peroxidation inhibitory activity on bovine brain liposomes, and metal chelation with Cu2+, Fe2+ and Fe3+. The radical scavenging activity of knipholone tested on the stable DPPH radical showed a very weak antioxidant property with an IC50 value of 355 mM
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120 Knipholone Zileuton
100
% Inhibition
80 60 40 20 0 0
5
10
15
20
Concentration µM
Fig. 2. Inhibition of leukotriene metabolism by knipholone and zileuton at the test concentrations 20, 10, 5 and 1 mM. The mean values from three tests are shown with their standard deviations.
compared to the positive control quercetin (IC50 ¼ 3.2 mM). We found it beneficial to examine the lipid peroxidation property of knipholone as polyphenols in general can influence the lipid peroxidation process by affecting the activity of enzymes such as lipoxygenases and cycloxygenases (Cao et al., 1997). Knipholone was also found to be a weak inhibitor of phospholipids liposomes peroxidation with an IC50 value of 311 mM compared to the positive control quercetin (IC50 ¼ 1.4 mM). Solutions of knipholone (50 mM) in PBS were scanned with and without FeSO4, CuSO4 and FeCl3 solutions (50 and 100 mM) and the spectra generated were compared. With the addition of the metal ions, neither the absorbance nor the intensity of the UV-bands changed even with a concentration of 100 mM of metal ions. Therefore, there is no complexation between knipholone and the metal ions. Consequently, a knipholone–Fe interaction in the leukotriene metabolism assay is unlikely. Knipholone was screened for cytotoxicity against brine shrimp nauplii in a micro-dilution assay and showed a weak cytotoxic effect with ED50 value of 0.58 mM. Although several classes of compounds have been investigated for their 5-LOX inhibitory activity, relatively few anthraquinones and their derivatives have been examined for their potential anti-inflammatory properties. Naturally occurring plant-derived antioxidants including, flavonoids and coumarins inhibit leukocyte 5-LOX, and greatest potency was found in those phenolics possessing vicinal diol functional groups (Laughton et al., 1991). The inhibitory activity of these compounds could also be due to chelation with a nonhaem iron ion, which is the constituent of 5-LOX, and/
455
or reducing the iron from the active ferric form to the inactive ferrous form (Morel et al., 1994; Kemal et al., 1987). Knipholone failed to scavenge the stable free radical, DPPH, as it is not a hydrogen donating compound, even though the acetylphloroglucinol moiety of knipholone contains two hydroxyl groups. Phenolic compounds could act as antioxidants if substitution at either the ortho or para position increases the electron density of the hydroxyl group and lower the oxygen–hydrogen bond energy (Wang et al., 1999). Knipholone, however, lacks this structural feature (Fig. 1). Therefore, the leukotriene biosynthesis inhibitory effect of knipholone was evident by its ability either to inhibit the FLAP or as a competitive (non-redox) inhibitor of the enzyme. The results of this study clearly indicate that knipholone has powerful ex vivo leukotriene metabolism inhibitory capacity and could be a potential candidate for a new anti-asthma drug.
Acknowledgements A.A. Wube gratefully acknowledges The Austrian Academic Exchange Service (O¨AD) scholarship. We would like to thank Dr. Haselbacher-Marko, Institute of Hygiene, Medical University of Graz, Austria, for preparation of blood samples and Mr. Melaku Wondafrash for identification of the plant material.
References Abate, G., 1989. In: Demissew, S. (Ed.), Etse Debdabe: Ethiopian Traditional Medicine. Addis Ababa University Press, Addis Ababa. Adams, M., Kunert, O., Bauer, R., 2004. Inhibition of leukotriene biosynthesis by quinolone alkaloids from the fruit of Evodia rutaecarpa. Planta Med. 70, 904–908. Bringmann, G., Menche, D., Bezabih, M., Abegaz, B.M., Kaminsky, R., 1999. Antiplasmodial activity of knipholone and related natural phenylanthraquinones. Planta Med. 65, 757–758. Bringmann, G., Hoerr, V., Holzgrabe, U., Stich, A., 2003. Antitrypanosomal naphthylisoquinoline alkaloids and related compounds. Pharmazie 58, 343–346. Brown, J.E., Khodr, H., Hider, R.C., Rice-Evans, C.A., 1998. Structural dependence of flavonoid interactions with Cu2+ ions: implications for their antioxidant properties. Biochem. J. 330, 1173–1178. Burits, M., Bucar, F., 2000. Antioxidant activity of Nigella sativa essential oil. Phytother. Res. 14, 323–328. Cao, G., Sofic, E., Prior, R.L., 1997. Antioxidants and prooxidant behaviour of flavonoids: structure–activity relationships. Free Radic. Biol. Med. 22, 749–760. Dagne, E., Steglich, W., 1984. Knipholone: a unique anthraquinone derivative from Kniphofia foliosa. Phytochemistry 23, 1729–1731.
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Dixon, R.A.F., Diehl, R.E., Opas, E., Rands, E., Vickers, P.J., Evans, J.F., Gillard, J.W., Miller, D.K., 1990. Requirement of a 5-lipoxygenase-activating protein for leukotriene synthesis. Nature 343, 282–284. Fiebich, B.L., Grozdeva, M., Hess, S., Hu¨ll, M., Danesch, U., Bodensieck, A., Bauer, R., 2005. 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. Planta Med. 71, 12–19. Finney, J.D., 1978. Statistical Methods in Biological Assay. Charles Griffin, London, pp. 58–65. Kemal, C., Louis-Flamberg, P., Krupinski-Olsen, R., Shorter, A.L., 1987. Reductive inactivation of soybean lipoxygenase 1 by catechols: a possible mechanism for regulation of lipoxygenase activity. Biochemistry 26, 7064–7072. Laughton, M.J., Evans, P.J., Moroney, M.A., Hoult, J.R.S., Halliwell, B., 1991. Inhibition of mammalian 5-lipoxygenase and cyclo-oxygenase by flavonoids and phenolic dietary additives. Relationship to antioxidant activity and to iron ion-reducing ability. Biochem. Pharmacol. 42, 1673–1681.
McMillan, R.M., Walker, E.R.H., 1992. Designing therapeutically effective 5-lipoxygenase inhibitors. Trends Pharmacol. Sci. 13, 323–330. Morel, I., Lescoat, G., Cillard, P., Cillard, J., 1994. Role of flavonoids and iron chelation in antioxidant action. Methods Enzymol. 234, 438–443. Samuelsson, B., 1983. Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science 220, 568–575. Schneider, I., Gibbons, S., Bucar, F., 2004. Inhibitory activity of Juniperus communis on 12(S)-HETE production in human platelets. Planta Med. 70, 471–474. Solis, P.N., Wright, C.W., Anderson, M.M., Gupta, M.P., Phillipson, J.D., 1993. A microwell cytotoxicity assay using Artemia salina (brine shrimp). Planta Med. 59, 250–252. Wang, M., Jin, Y., Ho, C.-T., 1999. Evaluation of resveratrol derivatives as potential antioxidants and identification of a reaction product of resveratrol and 2,2-diphenyl1-picryhydrazyl radical. J. Agric. Food Chem. 47, 3974–3977.
Phytomedicine 13 (2006) 452–456 www.elsevier.de/phymed
SHORT COMMUNICATION
Knipholone, a selective inhibitor of leukotriene metabolism A.A. Wubea, F. Bucara,, K. Asresb, S. Gibbonsc, M. Adamsa, B. Streita, A. Bodensiecka, R. Bauera a
Department of Pharmacognosy, Institute for Pharmaceutical Sciences, Karl-Franzens University Graz, Universitaetsplatz 4/1, A-8010 Graz, Austria b Department of Pharmacognosy, School of Pharmacy, Addis Ababa University, P.O. Box 1176, Ethiopia c Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK
Abstract Inhibition of leukotriene formation is one of the approaches to the treatment of asthma and other inflammatory diseases. We have investigated knipholone, isolated from the roots of Kniphofia foliosa, Hochst (Asphodelaceae), for inhibition of leukotriene biosynthesis in an ex vivo bioassay using activated human neutrophile granulocytes. Moreover, activities on 12-lipoxygenase from human platelets and cycloxygenase (COX)-1 and -2 from sheep cotyledons and seminal vesicles, respectively, have been evaluated. Knipholone was found to be a selective inhibitor of leukotriene metabolism in a human blood assay with an IC50 value of 4.2 mM. However, at a concentration of 10 mg/ ml, the compound showed weak inhibition of 12(S)-HETE production in human platelets and at a concentration of 50 mM it produced no inhibition of COX-1 and -2. In our attempt to explain the mechanism of inhibition, we examined the antioxidant activity of knipholone using various in vitro assay systems including free radical scavenging, nonenzymatic lipid peroxidation, and metal chelation. Knipholone was found to be a weak dose-independent free radical scavenger and lipid peroxidation inhibitor, but not a metal chelator. Therefore, the leukotriene biosynthesis inhibitory effect of knipholone was evident by its ability either to inhibit the 5-lipoxygenase activating protein (FLAP) or as a competitive (non-redox) inhibitor of the enzyme. Cytotoxicity results also provided evidence that knipholone exhibits less toxicity for a mammalian host cell. r 2005 Elsevier GmbH. All rights reserved. Keywords: Kniphofia foliosa; Knipholone; Leukotrienes; 5-Lipoxygenase; 12-Lipoxygenase; Cycloxygenases
Introduction 5-Lipoxygenase (5-LOX) is the key enzyme involved in the first two biosynthesis steps of leukotrienes from arachidonic acid: the stereospecific oxygenation leading to formation of 5(S)-hydroperoxy-6-trans-8,11,14-ciseicosatetraenoic acid (5-HPETE), and further dehydraCorresponding author. Tel.: +43 316 380 5531; fax: +43 316 380 9860. E-mail address: [email protected] (F. Bucar).
0944-7113/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2005.01.012
tion to leukotriene A4 (Samuelsson, 1983). Leukotrienes play a major role in the inflammatory response to injury and they have been implicated in the pathogenesis of several chronic inflammatory diseases, most notably asthma, psoriasis, rheumatoid arthritis and inflammatory bowel diseases. So far, two therapeutic strategies have been developed to inhibit the enzyme, 5-LOX, involved in leukotrienes biosynthesis: direct 5-LOX inhibitors and indirect inhibitors which interfere with 5-lipoxygenase activating protein (FLAP). The direct 5-LOX inhibitors interact with the enzyme via a redox,
ARTICLE IN PRESS A.A. Wube et al. / Phytomedicine 13 (2006) 452–456
OH
O
OH
CH3 O OH
HO
CH3 OCH3
O
Fig. 1. The structure of knipholone.
a nonredox or an iron chelating mechanism (McMillan and Walker, 1992), while FLAP inhibitors block the association of the enzyme with the cellular membrane (Dixon et al., 1990). Knipholone, a binary compound composed of the anthraquinone chrysophanol and an acetylphloroglucinol (Fig. 1), was originally isolated from the roots of Kniphofia foliosa, Hochst (Asphodelaceae) together with chrysophanol (Dagne and Steglich, 1984) and evaluated for its antiprotozoal properties (Bringmann et al., 1999, 2003). In the present study, we evaluated the leukotriene inhibitory activity and mechanism of inhibition by knipholone isolated from the roots of K. foliosa. The perennial herb K. foliosa grows wild in the central and southern highlands of Ethiopia. The roots have long been used in traditional medicine for the treatment of abdominal cramps and wound healing (Abate, 1989).
Materials and methods Analytical TLC was performed on Merck silica gel 60 F254 and RP-18 F254s plates. Column chromatography (CC) was performed on Merck silica gel 60 (70–240 mesh) and size exclusion chromatography on Sephadex LH-20. Solid phase separation was conducted using Isolute C18 EC (10 g) columns. Semi-preparative HPLC was performed using LiChrosphers RP-18 (10 mm, 250 10 mm i.d.) column. NMR spectra were recorded at 500 MHz for 1H and 125 MHz for 13C on a Bruker AVANCE 500 spectrometer. Mass spectra were determined by LC–ESI–MS analysis on a Thermo Finnigan LCQ Deca XP Plus mass spectrometer connected to a Surveyer LC-system (Thermo Finnigan). Absorbances for antioxidant tests were determined with a WALLAC 1420 Multilable Counter, Perkin ElmerTM Life Sciences. UV–visible spectra were recorded using a SPECTROD
453
50 spectrophotometer (Zeiss). The absorbance for LTB4 quantification was conducted using the photometric ELISA plate reader, Tecan RAIN BOW. Trypan blue solution, eicosatetraenoic acid, type VII Folch bovine brain extract, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 1-butanol and glutathion were purchased from Sigma Chemicals. Ca ionophor A 23187, KH2PO4–K2HPO4 buffer pH 7.413 (25 1C) (PBS), Tween 80R, trichloroacetic acid (TCA), thiobarbituric acid (TBA), butylated hydroxyltoluene (BHT), NaOH and epinephrin-hydrogentartrate were obtained from Fluka. Ethanol p.a., formic acid, citric acid solution, CaCl2 2H2O p.a., anhydrous D-glucose, MgCl2 6H2O. KCl, TRIS p.a., HCl solution, NH4Cl solution, NaCl, TU¨RKS solution, FeCl3 6H2O, CuSO4 5H2O, FeSO4 7H2O, dimethyl sulfoxide (DMSO) and Na2EDTA were bought from Merck. Ascorbic acid and baicalein were obtained from Aldrich. Quercetin and Tris/HCl buffer (pH 8.0) were obtained from Roth. LTB4 EIA Kit, purified PGHS-1 and -2, indomethacin and NS-398 were obtained from Cayman Chemical, Ann Arbor, MI, USA. Arachidonic acid and dextran were purchased from Amersham Pharmacia Biotech AB. 12(S)-HETE correlated-EIA kit was purchased from Assay Designs, Ann Arbor, MI, USA. PGE2-EIA kit was obtained from R & D systems, Minneapolis, MN, USA. Zileuton was purchased from Sequoia Research Products Ltd., Oxford, UK.
Plant materials The roots of K. foliosa were collected in April 2001 from Grassland very close to Dinsho, Bale, Ethiopia (alt. 2700–2850 m) and identified by Mr. Melaku Wondafrash, the National Herbarium, Department of Biology, Addis Ababa University. A voucher specimen (collection number 1482) has been deposited in the National Herbarium for future reference.
Extraction and isolation Air-dried ground roots (500 g) of K. foliosa were extracted successively by petroleum ether, dichloromethane and methanol using a Soxhlet apparatus for 24 h. The dichloromethane extract was evaporated under reduced pressure to yield 5.5 g (1.1%) residue and subjected to CC on silica gel eluting with petroleum ether and a 10% stepwise gradient with dichloromethane and methanol to afford 20 fractions. Fractions 12–13 (1.2 g) from CH2Cl2/CH3OH (9:1–8:2) were combined and subjected to solid phase separation using a CH3OH/H2O gradient elution followed by Sephadex LH-20, CH2Cl2/CH3OH (1:1) to yield a reddish fraction (720 mg), which was purified further by semi-preparative RP-18 HPLC with CH3CN/H2O (6:4) isocratic system
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to afford knipholone (1) (97 mg). The structure was identified by analysis of the spectral data and by comparison with literature values (Dagne and Steglich, 1984).
Characterisation of the ex vivo leukotriene bioassay The bioassay for inhibition of leukotriene biosynthesis was performed as described before (Adams et al., 2004) using human neutrophile granulocytes. Knipholone was tested at the concentrations 20, 10, 5 and 1 mM and quantified in duplicate. Mean values and the standard deviations were determined (Microsoft, Excel). IC50 values with fiducial limits (FL) were calculated by probit-log analysis as described for quantitative bioassays (Finney, 1978) using SPSS 6.0 for MS Windows.
12(S)-Lipoxygenase assay The 12(S)-LOX inhibitory assay was conducted ex vivo using human platelets as reported previously (Schneider et al., 2004). Baicalein was used as a positive control. The mean values of two measurements were taken and tests were conducted three times.
Metal chelating activity The metal chelating capacity of knipholone was measured with FeSO4, CuSO4 and FeCl3 solutions by the method described in the literature (Brown et al., 1998) with some modification. The compound tested was dissolved in ethanol at a concentration of 1 mM. From the stock solution, a solution of 50 mM was prepared in a cuvette with PBS (10 mM, pH 7.4) and the absorption spectra were recorded from 200 to 600 nm. Scans with 50 and 100 mM FeSO4, CuSO4 and FeCl3 were taken after 10 s and compared with the spectrum of knipholone alone. The effect of 2.5-fold EDTA concentration (125 mM) was also examined. Quercetin was used as a standard.
Brine shrimp cytotoxicity assay The test was microplate (Solis concentration of brine shrimps in analysis.
performed in triplicate in 96-well et al., 1993). The ED50 value, the knipholone effective to kill 50% of the test, was determined by probit
Results and discussion Cycloxygenase-1 and -2 assays The assays were performed with purified PGHS-1 from ram seminal vesicles for cycloxygenase (COX)-1 and purified PGHS-2 from sheep placental cotyledons for COX-2 as previously described (Fiebich et al., 2005). The percent inhibitions of 50 mM concentration of knipholone were determined for both enzymes. Indomethacin and NS-398 were used as a positive control for COX-1 and -2, respectively.
Free radical scavenging activity using DPPH assay The DPPH assay was done as described before (Schneider et al., 2004). Quercetin and ethanol were used as a positive control and blank, respectively. The test was performed in triplicate and the absorbances were averaged before calculation.
Iron/ascorbate-induced non-enzymatic lipid peroxidation assay This assay was performed as previously described (Burits and Bucar, 2000) using phospholipid liposomes prepared from Type VII Folch bovine brain extract dissolved in PBS.
In our ex vivo leukotriene biosynthesis inhibition assay knipholone showed a high dose-dependent activity (Fig. 2) with an IC50 value of 4.2 mM (3.6–4.9, 95% FL) being approximately twice as active as the commercial 5LOX inhibitor zileuton (IC50 ¼ 10.4 mM, 9.0–11.7, 95% FL). Knipholone was tested for inhibition of 12(S)HETE production using human platelets at 10 mg/ml concentration which resulted in 28.6% inhibition. Baicalein was used as a positive control and exhibited 52% inhibition at 5 mg/ml. However, at concentrations up to 50 mg/ml, the compound produced no inhibition of other enzymes related to inflammation, such as COX-1 and -2 compared to the positive controls indomethacin which inhibits COX-1 with an IC50 value of 0.9 mM and NS-398 which inhibits COX-2 with an IC50 value of 2.6 mM. Therefore, knipholone showed higher affinity for the 5-LOX pathway than for cycloxygenases and 12LOX. In this study we examined whether redox and chelating mechanisms might be involved in the 5-LOX inhibitory effect of knipholone via antioxidant tests, such as free radical scavenging using DPPH, lipid peroxidation inhibitory activity on bovine brain liposomes, and metal chelation with Cu2+, Fe2+ and Fe3+. The radical scavenging activity of knipholone tested on the stable DPPH radical showed a very weak antioxidant property with an IC50 value of 355 mM
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120 Knipholone Zileuton
100
% Inhibition
80 60 40 20 0 0
5
10
15
20
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Fig. 2. Inhibition of leukotriene metabolism by knipholone and zileuton at the test concentrations 20, 10, 5 and 1 mM. The mean values from three tests are shown with their standard deviations.
compared to the positive control quercetin (IC50 ¼ 3.2 mM). We found it beneficial to examine the lipid peroxidation property of knipholone as polyphenols in general can influence the lipid peroxidation process by affecting the activity of enzymes such as lipoxygenases and cycloxygenases (Cao et al., 1997). Knipholone was also found to be a weak inhibitor of phospholipids liposomes peroxidation with an IC50 value of 311 mM compared to the positive control quercetin (IC50 ¼ 1.4 mM). Solutions of knipholone (50 mM) in PBS were scanned with and without FeSO4, CuSO4 and FeCl3 solutions (50 and 100 mM) and the spectra generated were compared. With the addition of the metal ions, neither the absorbance nor the intensity of the UV-bands changed even with a concentration of 100 mM of metal ions. Therefore, there is no complexation between knipholone and the metal ions. Consequently, a knipholone–Fe interaction in the leukotriene metabolism assay is unlikely. Knipholone was screened for cytotoxicity against brine shrimp nauplii in a micro-dilution assay and showed a weak cytotoxic effect with ED50 value of 0.58 mM. Although several classes of compounds have been investigated for their 5-LOX inhibitory activity, relatively few anthraquinones and their derivatives have been examined for their potential anti-inflammatory properties. Naturally occurring plant-derived antioxidants including, flavonoids and coumarins inhibit leukocyte 5-LOX, and greatest potency was found in those phenolics possessing vicinal diol functional groups (Laughton et al., 1991). The inhibitory activity of these compounds could also be due to chelation with a nonhaem iron ion, which is the constituent of 5-LOX, and/
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or reducing the iron from the active ferric form to the inactive ferrous form (Morel et al., 1994; Kemal et al., 1987). Knipholone failed to scavenge the stable free radical, DPPH, as it is not a hydrogen donating compound, even though the acetylphloroglucinol moiety of knipholone contains two hydroxyl groups. Phenolic compounds could act as antioxidants if substitution at either the ortho or para position increases the electron density of the hydroxyl group and lower the oxygen–hydrogen bond energy (Wang et al., 1999). Knipholone, however, lacks this structural feature (Fig. 1). Therefore, the leukotriene biosynthesis inhibitory effect of knipholone was evident by its ability either to inhibit the FLAP or as a competitive (non-redox) inhibitor of the enzyme. The results of this study clearly indicate that knipholone has powerful ex vivo leukotriene metabolism inhibitory capacity and could be a potential candidate for a new anti-asthma drug.
Acknowledgements A.A. Wube gratefully acknowledges The Austrian Academic Exchange Service (O¨AD) scholarship. We would like to thank Dr. Haselbacher-Marko, Institute of Hygiene, Medical University of Graz, Austria, for preparation of blood samples and Mr. Melaku Wondafrash for identification of the plant material.
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