Tannins and Related Compounds: Killing of Amastigotes of Leishmania donovani and Release of Nitric Oxide and Tumour Necrosis Factor a in Macrophages in vitro Albrecht F. Kiderlen3, Oliver Kayserb, Daneel Ferreirac and Herbert Kolodziejd* a Robert Koch-Institut, Abteilung für Infektionskrankheiten, Nordufer 20, D-13353 Berlin, Germany b Institut für Pharmazie, Pharmazeutische Technologie, Biopharmazie und Biotechnologie, Freie Universität Berlin, Kelchstraße 31, D-12169 Berlin, Germany c National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, University, MS 38677, USA d Institut für Pharmazie, Pharmazeutische Biologie, Freie Universität Berlin, Königin-Luise-Straße 2+4, D-14195 Berlin, Germany Fax: +49-30-838-53729. E-mail: kolpharm@zedat.fu-berlin.de * Author for correspondence and reprint requests Z. Naturforsch. 56c, 444-454 (2001); received February 9/March 8 , 2001 Polyphenols, Leishmania donovani , Immunomodulation, Tumour Necrosis Factor The antileishmanial and immunomodulatory potencies of a series of 28 polyphenols were evaluated in terms of extra- and intracellular leishmanicidal activity and macrophage activa tion for release of nitric oxide (NO), tumour necrosis factor (TNF) and interferon (IFN)- like properties. For this, several functional bioassays were employed including an in vitro model for leishmaniasis in which murine bone marrow-derived macrophages (BM M $) were infected with the obligate intracellular parasite Leishmania donovani , an extracellular Leish mania proliferation assay, a fibroblast-lysis assay (TNF-activity), and a biochemical assay for NO. Except for gallic acid, its methyl ester, shikimic acid and catechin (EC50 25.8-67.9 nM) all polyphenols tested significantly inhibited the intracellular survival of L. donovani amasti gotes (EC 50 0.4-13.9 nM) when compared with the clinically used agent, sodium stibogluco nate (EC50 10.6 nM). In contrast, none of the samples proved to be directly toxic for the extracellular promastigote form of the parasite. Noteworthy, the phenolic samples showed only moderate or no cytotoxicity against the murine host cells (EC50 10 to >144 nM). Al though NO is an important effector molecule in macrophage microbicidal activity, the induc ing potential of the test compounds for its release was found to be very moderate ranging from 7-54 jam (IFN-y/LPS 119 (.im). On the other hand, inhibition of NO production had no apparent effect on intracellular leishmanicidal activity of polyphenols. Their in vitro TNF- inducing potential producing 50% lysis in murine L929 cells increased in the order of simple phenols and flavanols (34-48 U/ml) < A-type proanthocyanidins (53-80 U/ml) < B-type proanthocyanidins (64-200 U/ml) < hydrolyzable tannins (287-350 U/ml) at the host cell subtoxic concentration of 50 ^g/ml. Furthermore, gallic acid and some hydrolyzable tannins showed appreciable IFN-like activities ( 14-23 U/ml) as reflected by inhibition of the cyto- pathic effect of encephalomyocarditis virus on fibroblast L 929 cells. The results provide a rational basis for the recorded anti-infectious efficacy of traditionally used herbal medicines containing tannins in vivo , in the light of both only moderate direct antimicrobial activities of distinct polyphenols in vitro and the limited knowledge on their uptake in humans. Introduction Leishmaniasis is a major public health problem with many clinical manifestations in humans. In their mammalian hosts, protozoa of the genus Leishmania are obligate intracellular parasites of the monocyte-macrophage system. Species of the parasitic protozoa Leishmania are estimated to threaten some 350 million people with a broad range of diseases and to infect an estimated 1.4 million people annually world-wide (Ashford et a l 1992; WHO Report, 1993). Also noteworthy is the alarming increase in Leishmanial HIV co-infec tion, especially in the Mediterranean area. Leish mania donovani , a haemoflagellate protozoan par asite, is the causative agent of potentially fatal visceral leishmaniasis (VL). Despite the major ad verse impact of Leishmania parasites on human 0939-5075/2001/0500-0444 $ 06.00 © 2001 Verlag der Zeitschrift für Naturforschung, Tübingen • www.znaturforsch.com • D