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Des sources du savoir aux médicaments du futur

 | 
Jacques Fleurentin
, 
Jean-Marie Pelt
, 
Guy Mazars

2. Études chimiques et pharmacologiques

Anti-inflammation assays

Marit-Saskia Wahrendorf, F Sporer et M Wink

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

Figure 2. Inhibition of lipoxygenase activity by Harpagophytum procumbens in different extracts of human buffy coat blood

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
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

Auteurs

Institut für Pharmazeutische Biologie, Universität Heidelberg Im Neuenheimer Feld 364 69121 Heidelberg (Deutschland)

Institut für Pharmazeutische Biologie, Universität Heidelberg Im Neuenheimer Feld 364 69121 Heidelberg (Deutschland)

Institut für Pharmazeutische Biologie, Universität Heidelberg Im Neuenheimer Feld 364 69121 Heidelberg (Deutschland)

© IRD Éditions, 2002

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