Anti-Inflammatory Activity of Leontopodium alpinum and its Constituents

• Abstract
• Introduction
• Materials and Methods
• Extract preparation and purification of compounds
• Croton oil-induced mouse ear dermatitis
• Preparation of human polymorphonuclear leukocytes (PMNL) and chemotaxis assay
• Results
• Discussion
• Conclusions
• Acknowledgements
• References

Abstract

The aerial parts and roots of Leontopodium alpinum Cass. (Asteraceae) were investigated for their in vivo topical anti-inflammatory activity using the inhibition of Croton oil-induced ear dermatitis in mice. For both of the plant parts, the dichloromethane extract induced a dose-dependent oedema reduction, being more active than the methanol and 70 % aqueous methanol extracts. Moreover, the dichloromethane extract of the aerial parts was more active than that of the roots (ID₅₀ = 221 and 338 μg/cm², respectively). Fatty acids make a significant contribution to the anti-oedema activity of the dichloromethane extract of the aerial parts, whereas bisabolane sesquiterpenes, tricyclic sesquiterpenes, coumarins and lignans are involved in the activity of the root extract. Two bisabolane derivatives reduced also the polymorphonuclear neutrophil leukocytes accumulation in the inflamed tissue, while a 7α-silphiperfol-5-ene type sesquiterpene and a coumarin derivative inhibited the in vitro chemotaxis of these inflammatory cells.

Introduction

Leontopodium alpinum Cass. (Asteraceae), widely known as ‘Edelweiss’, grows on rocky and grassy slopes, mostly on limestone, at altitudes from 220 - 3140 m in the mountainous regions of Europe, especially in the Alps, Carpatians, the mountains of the Balkan peninsula, the Tatra and the Pyrennees. In alpine folk medicine, extracts of this plant are used as therapy for abdominal aches, tonsillitis, bronchitis, cancer, diarrhoea, dysentery and fever [1]. More than 100 years ago, when tourism started in the Alps and newly built railroads provided easy access, the existence of L. alpinum became endangered and it was necessary to protect this plant. Edelweiss was the first plant protected by law in Austria in 1886. In the meantime the situation has changed and the survival of this plant seems to be guaranteed. Additionally, L. alpinum is cultured in large quantities (e. g., in Switzerland) allowing the harvesting of tons of dried material per year.

Recently we reported on the isolation of new bisabolane type sesquiterpenes (1R*,2S*,4R*,5S*)-4-(acetyloxy)-2-[3-(acetyloxy)-1,5-dimethyl-4-hexenyl]-5-ethylcyclohexyl (Z)-2-methyl-2-butenoate (1), its cyclohexenyl derivative (2) and 3-methyl-1-{2-[(1R*,2S*,5R*,6R*)-2,5,6-tri(acetyloxy)-4-methyl-3-cyclohexenyl]propyl}-2-butenyl (Z)-2-methyl-2-butenoate (3a, 3b), two stereoisomers which differ from each other by the relative configurations of the chiral centres of the hexenyl side chain [2]. Additionally, the coumarin derivative obliquin (4), the lignan [(2R*,3S,4S)-4-(3,4-dimethoxybenzyl)-2-(3,4-dimethoxy-phenyl)tetrahydro-3-furanyl] methyl (Z)-2-methyl-2-butenoate (5), the benzofuran glucoside (2R*,3S*)-1-(2-[1-(hydroxymethyl)vinyl]-3-[β-D-glucosyloxy]-2,3-dihydro-benzo[b]furan-5-yl)-1-ethanone (6) and the tricyclic sesquiterpene [(1R*,3aS,6R)-1,3a,6-trimethyl-1,3a,4,5,5a,6,7,8-octahydrocyclopenta[c]pentalen-2-yl]methyl acetate (7) as well as modhephene (9), silphinene (8) and isocomene (10) were isolated [1], [3] (Fig. [1]).

Anti-inflammatory properties have been reported for L. leontopodioides: In in vivo experiments on rats, an extract of this Asian species suppressed reversed passive arthus-induced swelling of rat hind paws, inhibited cutaneous haemorrhage and leukocyte migration [4].

Illnesses and complaints against which L. alpinum is traditionally used are linked to inflammatory pathologies. This and observations of Li et al. [4] prompted us to investigate extracts and single constituents of L. alpinum for their ability to inhibit the Croton oil-induced ear dermatitis in mice, after topical application [5]. To get first clues on the possible mechanisms of anti-inflammatory action, fractions and pure constituents were tested for inhibition of chemotaxis of human polymorphonuclear neutrophil leukocytes (PMNL).

Materials and Methods

Extract preparation and purification of compounds

Dried aerial parts and roots of L. alpinum were obtained from cultured sources as a gift from Pentapharm Ltd., Basel, Switzerland. Voucher specimens are deposited in the herbarium (CH 02 - 0603 and CH 4002) of the Institute of Pharmacy. Ground roots (10.0 g) were extracted successively with dichloromethane, methanol and 70 % of aqueous methanol (three times for 5 min with 45 mL of each solvent) using an Ultra-Turrax (Janke & Kunkel T25; Staufen, Germany). Extracts were evaporated to dryness yielding 79 mg of the dichloromethane, 230 mg of the methanol and 211 mg of the aqueous methanol extract. Aerial parts of L. alpinum were extracted in the same way using 150 mL instead of 45 mL of each solvent (yields: 81 mg of the dichloromethane extract, 1127 mg of the methanol extract and 1811 mg of the aqueous methanolic extract). A chlorophyll-free extract was prepared by filtering the crude dichloromethane extract through a 1 : 2 mixture of Cellite® and activated charcoal, using dichloromethane as solvent.

Compounds 1 - 7 were obtained from the roots as described previously [1], [2], [3]. Purities of compounds 1 - 7 were determined by HPLC analysis: 1 - 5 showed a minimum purity of 95 %, 6 and 7 of 90 %, respectively. The stereoisomers 3a and 3b were tested as mixture in a ratio of 1 : 1. Silphinene (8), modhephene (9) and isocomene (10) were also tested as a mixture (8 - 10). According to GC analysis this mixture comprised 49.1 % isocomene, 31.3 % modhephene, 8.3 % silphinene and 11.3 % of an unknown compound.

The dichloromethane (DCM) extract of the aerial parts was fractionated by Sephadex® LH-20 (Pharmacia Biotech, Sweden) column chromatography using an 85 : 15 (v/v) mixture of dichloromethane and acetone. Fractions were combined according to their TLC and HPLC pattern to give f11 - f14.

Analysis of pure compounds, extracts and fractions was done by TLC, HPLC/UV and GC in comparison with authentic reference substances according to the literature [1], [2], [3].

Croton oil-induced mouse ear dermatitis

Topical anti-inflammatory activity was evaluated as inhibition of the Croton oil-induced ear dermatitis in mice [5]. Animal experiments complied with the Italian D.L. no. 116 of 27 January 1992 and associated guidelines in the European Communities Council Directive of 24 November 1986 (86/609 ECC), concerning animal welfare. Animals were kept for one week before the experiment, at constant conditions of temperature (21 ± 1 °C) and humidity (60 - 70 %), with a fixed artificial light cycle (7.00 - 19.00 h). All experiments were uniformly started between 10.00 a. m. and 12.00 a. m., in order to avoid variations in the inflammatory response due to circadian fluctuations in the levels of corticosteroids.

Male CD-1 mice (28 - 32 g; Harlan Italy, S. Pietro al Natisone, Italy) were anaesthetised with ketamine hydrochloride (145 mg/kg, intraperitoneally) before the induction of phlogosis. Cutaneous inflammation was induced by applying 80 μg of Croton oil dissolved in 15 μL of an appropriate vehicle to the inner surface of the right ear (surface: about 1 cm²). The left ear remained untreated. Control animals received only the irritant solution, while the other animals received both the irritant and the substances under testing, dissolved in the relevant vehicle. The following vehicles were used: acetone (for DCM extracts, fractions, compounds, indomethacin and the relevant controls), 42 % aqueous ethanol for methanol and 70 % aqueous methanol extracts and their controls). Indomethacin served as a positive control. Six hours after application, mice were sacrificed and a plug (6 mm) from both the treated and the untreated control ear was taken. Two experimental groups of five animals were used for each tested dose level. Inflammation was measured as oedematous response and infiltration of granulocytes.

Evaluation of the oedematous response: Oedema was quantified by the differences in weight between the treated and the untreated (opposite) ear plugs. The antioedema activity was expressed as percentage of oedema reduction in treated mice with regard to control mice [5].

Evaluation of the granulocyte infiltration: The granulocytes infiltrate was quantified in the treated ears by measuring myeloperoxidase activity in the same plug used for the determination of the oedematous response [5]. Peroxidase was extracted using hexadecyltrimethylammonium bromide (HTAB; Eastmann Kodak Co., N.Y., USA) according to Bradley et al. [6] and the enzyme activity was measured by a colorimetric assay using tetramethylbenzidine (TMB; Sigma, St. Louis, Missouri, USA) as chromogen [7]. Ear plugs, suspended in 1 mL buffered saline (0.1 M sodium acetate buffer, pH 4.2) containing 0.1 % HTAB, were homogenized by Ultra-Turrax (Janke & Kunkel T25; Staufen, Germany) for 5 sec at 20,000 r. p. m. The homogenates were centrifuged at 15,000 g for 5 min and the supernatants were used for the peroxidase assay. In each well of a 96-multiwell plate, 25 μL of supernatant were mixed with 50 μL of the chromogen solution [2.83 mM TMB dissolved in 0.1 M sodium acetate buffer pH 4.2, containing 0.1 % (w/v) HTAB]. The enzyme reaction was started by the addition of 75 μL of 0.7 mM hydrogen peroxide. After 5 min incubation at room temperature, the peroxidase reaction was stopped by 50 μL of 4 M acetic acid, containing 10 mM sodium azide. The absorbance was then determined at 620 nm using an Automated Microplate Reader (Spectra & Rainbow Reader; Tecan Italia S.r. l., Cologno Monzese, Italy). The peroxidase activity was expressed as enzyme units in 1 mL of supernatant. One unit of peroxidase activity was defined as the amount of enzyme oxidizing 1 nmol of TMB per minute. The enzyme activity of each sample was determined in duplicate.

Data evaluation: Data, expressed as mean ± standard error of the mean, were analysed by one-way analysis of variance followed by the Dunnett’s test for multiple comparisons of unpaired data. A probability level lower than 0.05 was considered as statistically significant. ID₅₀ values (dose giving 50 % oedema inhibition) were calculated by graphic interpolation of the dose-effect curves.

Preparation of human polymorphonuclear leukocytes (PMNL) and chemotaxis assay

Chemotaxis and cytotoxicity studies with human polymorphonuclear leukocytes (PMNL) were performed according to Kaneider et al. [8].

PMNL were obtained from the peripheral blood of healthy volunteers (anticoagulated with EDTA) after Lymphoprep® (Nycomed, Denmark) density gradient centrifugation, dextran sedimentation and hypotonic lysis of contaminating erythrocytes using sodium chloride solution. This procedure yielded > 95 % neutrophils (determined by morphology in GIEMSA stains) and > 99 % viability (established by trypan dye exclusion). For chemotaxis experiments the cell preparations were resuspended in RPMI 1640 without L-glutamine (Biological Industries, Israel) containing 0.5 % bovine serum albumin (BSA; 5 % g/w solution of bovine serum albumin, Behring, Germany, in distilled water) or in phenol red-free Hanks balanced salt solution (HBSS) (Gibco BRL Life Technologies, Land) with 1 % BSA for respiratory burst measurements.

Chemotaxis of PMNL into cellulose nitrate towards soluble attractants was measured using a 48-well microchemotaxis chamber (Neuro Probe, Gaithersburg, MD), in which a 5 μm-pore-sized filter (Sartorius, Göttingen, Germany) separated the upper and lower chambers. Before testing chemotaxis cells were pre-incubated for 30 min with various concentrations of test substances or medium, washed twice with phosphate-buffered saline and resuspended in medium. PMNL then were allowed to migrate towards the chemoattractants formyl-methionyl-leucyl-phenylalanine (fMLP; 10 nM) or interleukin-8 (1 nM) to measure directed migration. Both chemicals were purchased from Sigma Chemical Corp. (St. Louis, MO). Undirected migration was determined by using medium instead of a specific chemoattractant.

After 30 min at 37 °C in humidified atmosphere (5 % CO₂), the nitrocellulose filters were dehydrated, fixed, and stained with haematoxylin-eosin. Migration depth of PMNL into the filter was quantified by microscopy, measuring the distance (μm) from the surface of the filter to the leading front of three cells. Data are expressed as chemotaxis index (mean ± SD, n = 4), which is the ratio between the distances of directed and undirected migration of PMNL into the nitrocellulose filters. Tests on statistical significance were carried out with the Mann-Whitney U test.

For detection of cytotoxicity the MTT test [9] was used. This colorimetric assay is based on the conversion of the tetrazolium salt MTT to formazan crystals by intact mitochondria. The measured absorbance at 570 nm is therefore directly proportional to the number of vital cells. MTT was dissolved in PBS to a final concentration of 5 mg/mL. PMNL were incubated with test compounds [2 × 10 ^{- 5} M] for 30 min, and washed twice. Then MTT was added for further 3 h before formazan crystals were dissolved with DMSO. The plates were agitated for 5 min on a plate shaker and absorbance was measured with an ELISA reader at a wavelength of 570 nm.

Results

The DCM extract of the aerial parts of L. alpinum exerted a dose-dependent anti-inflammatory activity, inducing from 36 % to 85 % oedema inhibition at doses ranging from 100 to 1000 μg/cm². Methanol and 70 % aqueous methanol extracts were less active and, at the dose of 1000 μg/cm², they induced 54 % and 33 % oedema reduction, respectively. Extracts from the roots showed a similar activity rank: at the doses ranging from 100 to 1000 μg/cm², the DCM extract induced from 39 % to 49 % oedema reduction, while 1000 μg/cm² of the methanol and 70 % aqueous methanol extracts provoked 25 % and 15 % oedema reduction, respectively.

As reference, at doses ranging from 30 to 300 μg/cm², the non steroidal anti-inflammatory drug indomethacin induced from 18 % to 79 % oedema inhibition. The activity is correlated to the polarity of the extracts: lipophilic extracts, the DCM ones, were more active than hydrophilic ones. Furthermore, the DCM extract of the aerial parts was more active than that obtained from the plant root: their ID₅₀ values (dose giving 50 % oedema inhibition) were 221 and 338 μg/cm², respectively. The two extracts were only about two and four times less active than the reference drug indomethacin (ID₅₀ = 93 μg/cm²) (Table [1]).

In order to identify the constituents responsible for the anti-inflammatory activity, the crude DCM extract of the aerial parts was sequentially separated by Sephadex^® LH-20 column chromatography into four fractions (f11 - f14). Their anti-oedema activity was evaluated (Table [2]) and compared to that of the DCM extract and of the chlorophyll-free DCM extract. At the dose of 100 μg/cm², the DCM extract induced 36 % oedema inhibition, similarly to the chlorophyll-free extract (39 % oedema inhibition) and to fractions f12 and f14 (36 % and 38 % inhibition, respectively). The main constituents of f14 were linolic and linoleic acid. Fraction f11, containing mainly chlorophyll and triterpenes, exhibited a lower effect (27 % inhibition). The most potent fraction was f13, which significantly exceeded the activity of the crude DCM extract (54 % oedema inhibition). At present the composition of the fractions f12 and f13 is unknown.

In a bioassay-guided approach six pure compounds (1, 2, 4 - 7), a mixture of the stereoisomers 3a and 3b as well as a mixture of three sesquiterpenes (8 - 10) were isolated from the crude DCM extract of the roots. The pure compounds as well as the mixtures (100 μg/cm²), exhibited an oedema inhibition ranging from 28 % to 50 %, comparable to that of indomethacin (59 % oedema reduction) (Table [3]).

Leukocyte infiltration in the mouse ear tissue was assayed as myeloperoxidase activity. Since oedema reduction and assembling of leukocytes follow different time curves [9], ear samples were taken at the maximum of oedema formation (6 h) as well as at the maximum of infiltration of leukocytes (24 h). At 100 μg/cm², only compounds 1 and the mixture of 3a and 3b induced a significant reduction of leukocyte recruitment in the inflamed tissue: after 6 h they provoked 21 % and 26 % reduction, respectively, an effect similar to that observed 24 h after the induction of inflammation (34 % and 25 % inhibition, respectively) (Table [4]).

Compounds 1 - 7 were assayed for their effects on fMLP or IL-8 induced chemotaxis. After incubation with test compounds cells were washed in order to prevent unintentional scavenging of fMLP or IL-8 during the chemotaxis experiment. The fMLP induced chemotactic response of PMNL (Fig. [2]) was significantly inhibited by compound 7 at a concentration of 2 × 10 ^{- 9} M (23 %; p = 0.047; n = 5) and by compound 4 at 2 × 10 ^{- 5} M (36 %; p = 0.016; n = 5). Both compounds also inhibited the IL-8 induced chemotaxis, compound 7 by 49 % at 2 × 10 ^{- 9} M (p = 0.021; n = 4) and compound 4 by 58 % at 2 × 10 ^{- 7} M (p = 0.021; n = 4). Dose-dependencies of 4 and 7 show a U-shaped graph, which is a common phenomenon in cell functional assays due to receptor down-regulation at high agonist concentrations and possible counteracting effects on intracellular signal transduction at different concentrations [8]. No activities at all were observed for 1, 3a,b, 5 and 6. The crude dichloromethane extract of the roots exhibited necrotic alterations of PMNL observed by means of microscopic analysis. This effect might be due to cytotoxic effects of the extract as confirmed by the MTT assay. In contrast, none of the pure compounds exerted any cytotoxic activity (data not shown).

Discussion

Topically administered extracts, fractions and pure constituents of L. alpinum significantly inhibited the Croton oil-induced mouse ear oedema. Activities of the pure compounds 1 - 7 and of fraction f13 were comparable to that of the NSAID indomethacin (Tables [2] and [3]) and to those previously observed, using the same experimental model, for other natural plant constituents, e. g., sesquiterpene lactones from Leontodon hispidus [10] and triterpenes from Calendula officinalis [11] and Salvia officinalis [12]. The bisabolanes 1 - 3 isolated from the root extract of L. alpinum inhibited the oedema formation by 33 - 46 % at the dose of 100 μg/cm². Furthermore, compounds 1 and the mixture of the stereoisomers 3a and 3b reduced the leukocytes accumulation in the inflamed tissue. Their activity is not surprising, as a similar compound, (-)-α-bisabolol, a constituent of chamomile [Chamomilla recutita (L.) Rauschert] possesses strong anti-phlogistic effects, as was shown using different animal models of inflammation, e. g., the carrageenin rat paw oedema, the cotton pellet granuloma, the adjuvant arthritis in the rat and the UV-erythema of the guinea pig [13].

The coumarin obliquin (4), the lariciresinol derivative 5 and the tricyclic sesquiterpene 7 (all administered at 100 μg/cm²), provoked an oedema inhibition ranging from 45 % to 50 % (Table [1]). Coumarins are known to exhibit a wide range of biological activities, but there is a controversy about their anti-inflammatory activity [14]. There are only few reports on anti-inflammatory activity of lignans [15] and none of them deals with in vivo effects of lariciresinol derivatives, as compound 5. In vitro inhibitory effects of lariciresinols on TNF-α production have been described by Cho et al. [16].

Literature data concerning the anti-inflammatory activity of 7α-silphiperfol-5-ene type sesquiterpenes (7) are completely lacking according to our knowledge.

Oedema inhibitory activity of the linolic and linolenic acids-rich fraction f14, obtained from the aerial parts of L. alpinum, is in agreement with data obtained by Singh [17]. He recently reported on the anti-inflammatory activity of linolenic acid in the rat paw oedema assay. As mechanism of action inhibition of the cyclooxygenase/lipoxygenase pathway of arachidonic acid metabolism was discussed. Indeed, like other n-3 polyunsaturated fatty acids [18], linolenic acid could inhibit the production of pro-inflammatory eicosanoids from arachidonic acid, a possible mechanism at the basis of its anti-inflammatory action as well as of that of fraction f14 containing this compound. Studies to identify the pharmacologically active constituents of the fractions f12 and f13, which also exhibited rather high effects in the mouse ear oedema assay, are still in progress.

Although chemotaxis of PMNL is a crucial step in the pathophysiology of inflammation, only few plant constituents are known to be inhibitors: diphenyl thiosulfinate, a constituent from onions, is reported to inhibit the fMLP induced-chemotaxis in agarose gel by 25 % (concentration: 0.1 μM) [19]; acetophenones from Picrorhiza kurroa Royle ex Benth., the plants anti-inflammatory and anti-asthmatic principles, inhibited the fMLP-induced chemotaxis by 50 - 60 % at 100 μM, depending on their structural properties [20]; sanguiin H-11, a tannin isolated from Sanguisorba officinalis L., inhibited the CINC-1 induced chemotaxis of neutrophils by 10.7 % at 10 μM as well as the fMLP- and PAF-induced chemotaxis to a similar extent [21]. The coumarin 4 and the 7α-silphiperfol-5-ene type sequiterpene 7 significantly inhibited the IL-8-induced chemotaxis by 58 % (concentration: 2 × 10 ^{- 7} M) and 49 % (concentration: 2 × 10 ^{- 9} M), respectively. Effects on the fMLP-induced chemotaxis were still significant, but not as remarkable as those obtained in the former experiment.

Conclusions

Among the Leontopodium constituents tested, bisabolane sesquiterpenes, tricyclic sesquiterpenes, coumarins and lignans are considered to contribute equally to the oedema inhibitory activity of the crude root extract. Fatty acids (with other compounds) strongly contribute to the anti-inflammatory effect of the aerial parts. The anti-inflammatory activities of the extracts and pure constituents of L. alpinum were similar to that of the standard drug indomethacin and comparable to constituents of other anti-inflammatory herbal drugs. Evaluation of the anti-inflammatory potency of L. alpinum extracts, fractions and compounds in other in vivo models as well as elucidation of their possible mechanisms of action is in progress.

Acknowledgements

We wish to thank Pentapharm Ltd. (Basel, CH) for providing plant material.

References

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• 11 Zidorn C, Dirsch V M, Rüngeler P, Sosa S, Della Loggia R, Merfort I, Pahl H L, Vollmar A M, Stuppner H. Anti-inflammatory activities of hypocretenolides from Leontodon hispidus .  Planta Med. 1999;  65 704-8
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Univ.-Prof. Dr. Hermann Stuppner

Institut für Pharmazie

Leopold-Franzens-Universität Innsbruck

Innrain 52

6020 Innsbruck

Austria

Telefon: +43-512-507-5300

Fax: +43-512-507-2939

eMail: Hermann.Stuppner@uibk.ac.at

References

• 1 Dobner M J, Schwaiger S, Jenewein I H, Stuppner H. Antibacterial activity of Leontopodium alpinum (Edelweiss).  J Ethnopharm. 2003;  89 301-3
  PubMedSuche in Google Scholar
• 2 Stuppner H, Ellmerer E P, Ongania K H, Dobner M. Bisabolane derivatives from Leontopodium alpinum .  Helv Chim Acta. 2002;  85 2982-9
  CrossrefPubMedSuche in Google Scholar
• 3 Dobner M J, Ellmerer-Müller E P, Schwaiger S, Batsugh O, Narantuya S, Stütz M, Stuppner H. New lignan, benzofuran and sesquiterpene derivatives from the roots of Leontopodium alpinum and L. leontopodioides .  Helv Chim Acta. 2003;  86 733-8
  CrossrefPubMedSuche in Google Scholar
• 4 Li L Y, Ye J M, Yin H, Zhu Y M, Tian J M, Gao F. Effect of Leontopodium leontopodioides (Willd.) Beauv. on inflammation induced by animal reversed passive arthus.  Chung Kuo Chung Yao Tsa Chih. 1994;  19 174-6
  PubMedSuche in Google Scholar
• 5 Tubaro A, Dri P, Delbello G, Zilli C, Della Loggia R. The Croton oil ear test revisited.  Agents Actions. 1985;  17 346-9
  PubMedSuche in Google Scholar
• 6 Bradley P P, Priebat D A, Christensen R D, Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker.  J Invest Dermatol. 1982;  78 206-9
  CrossrefPubMedSuche in Google Scholar
• 7 Andrews P C, Krinsky N I. The reductive cleavage of myeloperoxidase in half, producing enzymatically active hemi-myeloperoxidase.  J Biol Chem. 1981;  256 4211-8
  PubMedSuche in Google Scholar
• 8 Kaneider N C, Reinisch C M, Dunzendorfer S, Meierhofer C, Djanani A, Wiedermann C J. Induction of apoptosis and inhibition of migration of inflammatory and vascular wall cells by cerivastatin.  Atherosclerosis. 2001;  158 23-33
  CrossrefPubMedSuche in Google Scholar
• 9 Campling B G, Pym J, Galbraith P, Cole S. Use of the MTT assay for rapid determination of chemosensitivity of human leukemic blast cells.  Leukemia Res. 1988;  12 823-31
  CrossrefPubMedSuche in Google Scholar
• 10 Haberstock H, Marotti T, Banfic H. Neutrophil signal transduction in met-enkephalin modulated superoxide anion release.  Neuropeptides. 1996;  30 193-201
  CrossrefPubMedSuche in Google Scholar
• 11 Zidorn C, Dirsch V M, Rüngeler P, Sosa S, Della Loggia R, Merfort I, Pahl H L, Vollmar A M, Stuppner H. Anti-inflammatory activities of hypocretenolides from Leontodon hispidus .  Planta Med. 1999;  65 704-8
  Thieme ConnectPubMedSuche in Google Scholar
• 12 Zitterl-Eglseer K, Sosa S, Jurenitsch J, Schubert-Zsilavecz M, Della Loggia R, Tubaro A, Bertoldi M, Franz C. Anti-edematous activities of the main triterpendiol esters of marigold (Calendula officinalis L.)  J Ethnopharm. 1997;  57 139-44
  PubMedSuche in Google Scholar

Univ.-Prof. Dr. Hermann Stuppner

Institut für Pharmazie

Leopold-Franzens-Universität Innsbruck

Innrain 52

6020 Innsbruck

Austria

Telefon: +43-512-507-5300

Fax: +43-512-507-2939

eMail: Hermann.Stuppner@uibk.ac.at