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Anti-inflammation assays
Entrées d'index
Texte intégral
1The aim was to establish effective systems to measure the antiinflammatory potential of ethnopharmacologically used substances. To avoid animal-experiments a modified HET-CAM-Assay (Hens- Egg-Test at the Chorion-Allantoin-Membane), developed by Theisen and Luepke (0,1) is employed.
2The second assay is targeted to a special enzyme system connected to inflammation, which measures lipoxygenase-activity via HPLC (2).
3Anti-inflammatory activity of Harpargophytum procumbens has been demonstrated in different experiments in rats (3,4,5). In vitro experiments showed that the iridoid glycoside harpagoside inhibits the cyclooxygenase- as well as the lipoxygenasepathway of the arachidonic acid cascade (7). Therefore we have tested extracts of Harpagophytum procumbens in the modified HET and the LOX-enzyme-assay.
4The plant was extracted three times to give a 60 %-, 30 %-, and 0 %-ethanolic extract, dried and weighed. Its components are analysed and quantified via HPLC (RP-18 column, gradient elution, UV-detection at 280nm, external standart, after (6)).
Het-cam assay modified
5Fresh fertile White Legorn eggs (Lohmann Selected Leghorn, LSL) were inoculated on day 9 with the substance supposed to have an antiinflammatory effect. A blank control and a positive (Aspisol) were injected as well. An average of 6 eggs per substance was taken. After further incubation for two hours the eggshell above the cell is removed and the CAM (Chorion-Allantois-Membrane) is exposed.
6After dropping 0.3 ml SDS (sodiumdodecylsulfate) on the CAM, time is measured for certain reactions to appear. These were 1.) injection of blood into the vessels; 2.) haemorraghia and 3.) lysis of the vessels.
7A prolongation period of these events is indicative of anti-inflammatory effect.
8All extracts showed antiinflammatory activity in low concentrations down to 0.001 mg/egg.
9The 30 %-ethanolic extract of Harpargophytum procumbens turned out to be slightly more potent than the 60 %- followed by the 0 %- ethanolic extract. (Figure 1)
Lipoxygenase assay
10Leucocytes were isolated from human Buffy-Coat blood. After addition of the testsubstances the reaction is started by incubating them at 37°C with arachidonic acid and Calcium-lonophore. Each sample contains around 106 Leucocytes.
11The products, LTB4, 15-, 12- and 5-HETEs, were measured in an isocratic HPLC-System using a mixture of water and methanol as solvent-system.
12The hight of the peaks also gives an indication of quantitative effects. Peak areas of the products reflected the degree of enzyme inhibition.
13In this Assay-system the 60 %-ethanolic extract seems to be the most potent, followed by the 30 %- and then the 0 %-extract. (Figure 2)
Conclusion
14Aqueous and hydroalcoholic extracts of Harpargophytum procumbens exhibits a clear anti-inflammatory effect in our bioassa. A 30 %-ethanolic extract is the most potent in the HET-CAM-assay, whereas lipogxygenase activity is inhibited maximally with the 60 %-extract.
15Considering costs and reproducebility in comparison to in vivo animal experiments HET-CAM-assay and lipoxygenase-assay are suitable systems for testing antiinflammatory activities of all sorts of subsances.
Figure 1. Relative delay of injection/haemorrhagia/lysis by 60, 30, 0 % ethanolic extract of Harpagophytum procumbens
Bibliographie
References
1. LUEPKE N.P. (1992) Toxikologisch-pharmakologische Prüfmöglichkeiten am bebrüteten Hühnerei, In Schoeffl, Schulte-Herrmann, Tritthart (eds), Möglichkeiten und Grenzen der Reduktion von Tierversuchen, Springer-Verlag.
2 THEISEN N. (1988) Der modifizierte HET-CAM-Test als ein Modell zur Prüfung von Antiphlogistica, Inaugural-Dissertation an der Universität Osnabrück.
3 Nach Professor Dr.Dieter STEINHILBER, Institut für pharmazeutische Chemie, Universität Frankfurt, Marie-Curie-Str.9, 60439 Frankfurt
4 LANHERS M.C., FLEURENTIN J., MORTIER F. et al (1992) Antiinflammatory and analgesic effects of an aqueous extract of Harpagophytum procumbens, Planta Medica, 58, 117-123.
5 BAGHDIKIAN B., LANHERS M.C., FLEURENTIN J. et al (1997) An analytical study, anti-inflammatory and analgesic effects of Harpagophytum procumbens and Harpagopytum zeyheri, Planta Medica, 63, 171-176.
6 ERDÖS A., FONTAINE R., FRIEHE H. et al (1978) Beitrag zur Pharmakologie und Toxikologie verschiedener Extrakte, sowie des Harpagosids aus Harpagophytum procumbens DC, Planta Medica, 34, 97-108.
7. CHRUBASIK S., SPORER F., WINK M. (1996) Zum Harpagosidgehalt verschiedener Trockenextraktpulver aus Harpagophytum procumbens, Forschende Komplementärmedizin, 3, 6-11.
TIPPLER B., SYROVETS T., LOEW D. et al (1996) Harpagophytum procumbens: Wirkung von Extrakten auf die Eicosanoidbiosynthesein Ionophor A23187-stimuliertem menschlichen Vollblut, In Loew D.Rietbrock N. (eds): Phytopharmaka II, Forschung und klinische Anwendung, Darmstadt, Steinkopf-Verlag, 95-100.
Table des illustrations
Légende | Figure 1. Relative delay of injection/haemorrhagia/lysis by 60, 30, 0 % ethanolic extract of Harpagophytum procumbens |
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URL | http://books.openedition.org/irdeditions/docannexe/image/7266/img-1.jpg |
Fichier | image/jpeg, 323k |
Légende | Figure 2. Inhibition of lipoxygenase activity by Harpagophytum procumbens in different extracts of human buffy coat blood |
URL | http://books.openedition.org/irdeditions/docannexe/image/7266/img-2.jpg |
Fichier | image/jpeg, 195k |
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