Oral Metabolism and Efficacy of Kalanchoe pinnata Flavonoids in a Murine Model of Cutaneous Leishmaniasis

Abstract

Leishmaniasis is a parasitic disease that threatens 350 million people worldwide. In a search for new antileishmanial drugs, the in vitro activity of flavonoids from Kalanchoe pinnata (Crassulaceae) was previously demonstrated in infected cells. In order to demonstrate the safety and oral activity of K. pinnata, flavonoids were evaluated in vivo in a murine model of cutaneous leishmaniasis. Daily oral doses of quercetin 3-O-α-L-arabinopyranosyl (1→2)-α-L-rhamnopyranoside, quercetin 3-O-α-L-rhamnopyranoside, and free quercetin (16 mg/kg body weight) all were able to control the lesion growth caused by Leishmania amazonensis and to significantly reduce parasite load. These flavonoids were as effective as the crude K. pinnata aqueous extract given at 320 mg/kg body weight. HPLC-DAD-MS analysis of the plasma of extract-treated mice suggested that quercetin and quercetin glucuronides are the main metabolites of K. pinnata quercetin glycosides. Our results indicate that K. pinnata quercetin glycosides are important active components of the aqueous extract and that they possess potent oral efficacy against cutaneous leishmaniasis.

Key words

Kalanchoe pinnata - Crassulaceae - quercetin glycosides - HPLC-DAD-MS - leishmaniasis - metabolism

References

• 1 World Health Organization, Division of Control of Tropical Diseases. http://www.who.int/tdr/diseases/leish/diseaseinfo. (accessed in 2008)
• 2 Croft S L, Yardley V. Chemotherapy of leishmaniasis.  Curr Pharm Des. 2002;  8 319-42
• 3 Da Silva S AG, Costa S S, Rossi-Bergmann B. The anti-leishmanial effect of Kalanchoe is mediated by nitric oxide intermediates.  Parasitology. 1999;  118 575-82
• 4 Da Silva S AG, Costa S S, Mendonça S CF, Silva E M, Moraes V LG, Rossi-Bergmann B. Therapeutic effect of oral Kalanchoe pinnata leaf extract in murine leishmaniasis.  Acta Trop. 1995;  60 201-5
• 5 Torres-Santos E C, Da Silva S AG, Costa S S, Santos A PPT, Almeida A P, Rossi-Bergmann B. Toxicological analysis and effectiveness of oral Kalanchoe pinnata on a human case of cutaneous leishmaniasis.  Phytother Res. 2003;  17 801-3
• 6 Muzitano M F, Cruz E A, Almeida A P, Silva S AG, Kaiser C R, Guette C. et al . Quercitrin: An antileishmanial flavonoid glycoside from Kalanchoe pinnata.  Planta Med. 2006;  72 81-3
• 7 Muzitano M F, Tinoco L W, Guette C, Kaiser C R, Rossi-Bergmann B, Costa S S. Assessment of antileishmanial activity of new and unusual flavonoids from Kalanchoe pinnata. .  Phytochemistry. 2006;  67 2071-7
• 8 Demicheli C, Ochoa R, Silva J BB, Falcão C AB, Rossi-Bergmann B, Melo A L. et al . Oral delivery of meglumine antimoniate using beta-cyclodextrin for the treatment of leishmaniasis.  Antimicrob Agents Chemother. 2004;  48 100-3
• 9 Rossi-Bergmann B, Falcão C AB, Lenglet A, Santos C RB, Pinto D C, Traub-Cseko Y. A simple fluorimetric method for assessing drug and vaccine efficacy in cutaneous leishmaniasis using Leishmania amazonensis expressing green fluorescence protein.  Mem Inst Oswaldo Cruz. 1999;  94 74
• 10 Rossi-Bergmann B, Costa S S, Borges M BS, Da Silva S AG, Noleto G R, Souza M LM. et al . Immunosuppressive effect of the aqueous extract of Kalanchoe pinnata in mice.  Phytother Res. 1994;  8 399-402
• 11 Pal S, Chaudhuri A KN. Anti-inflammatory action of Bryophyllum pinnatum leaf extract.  Fitoterapia. 1990;  6 527-33
• 12 Middleton E, Kandaswami C, Theoharides T C. The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer.  Pharmacol Rev. 2000;  52 673-751
• 13 Mittra B, Saha A, Chowdhury A R, Pal C, Mandal S, Mukhopadhyay S. et al . Luteolin, an abundant dietary component is a potent antileishmanial agent that acts by inducing topoisomerase II-mediated kinetoplast DNA cleavage leading to apoptosis.  Mol Med. 2000;  6 527-41
• 14 Sen G, Mandal S, Roy S S, Mukhopadhyay S, Biswas T. Therapeutic use of quercetin in the control of infection and anemia associated with visceral leishmaniasis.  Free Radic Biol Med. 2005;  38 1257-64
• 15 Sarkar S, Mandal S, Sinha J, Mukhopadhyay S, Das N, Basu M K. Quercetin: critical evaluation as an antileishmanial agent in vivo in hamsters using different vesicular delivery modes.  J Drug Target. 2002;  10 573-8
• 16 Sen G, Mukhopadhyay S, Ray M, Biswas T. Quercetin interferes with iron metabolism in Leishmania donovani and targets ribonucleotide reductase to exert leishmanicidal activity.  J Antimicrob Chemother. 2008;  61 1066-75
• 17 Jean-Moreno V, Rojas R, Goyeneche D, Coombs G H, Walker J. Leishmania donovani: Differential activities of classical topoisomerase inhibitors and antileishmanials against parasite and host cells at the level of DNA topoisomerase I and in cytotoxicity assays.  Exp Parasitol. 2006;  112 21-30
• 18 Tasdemir D, Kaiser M, Brun R, Yardley V, Schmidt T J, Tosun F. et al . Antitrypanosomal and antileishmanial activities of flavonoids and their analogues: in vitro, in vivo, structure-activity relationship, and quantitative structure-activity relationship studies.  Antimicrob Agents Chemother. 2006;  50 1352-64
• 19 Walle T. Absorption and metabolism of flavonoids.  Free Radic Biol Med. 2004;  36 829 -37
• 20 O’Leary K A, Day A J, Needs P W, Mellon F A, O’Brien N M, Williamson G. Metabolism of quercetin-7- and quercetin-3-glucuronides by an in vitro hepatic model: the role of human β–glucuronidase, sulfotransferase, catechol-O-methyltransferase and multi-resistant protein 2 (MRP2) in flavonoid metabolism.  Biochem Pharmacol. 2003;  65 479-91
• 21 Day A J, Bao Y, Morgan M RA, Williamson G. Conjugation position of quercetin glucuronides and effect on biological activity.  Free Radic Biol Med. 2000;  29 1234-43
• 22 Day A J, Gee J M, Dupont M S, Johnson I T, Williamson G. Absorption of quercetin-3-glucoside and quercetin-4’-glucoside in the rat small intestine: the role of lactase phlorizin hydrolase and the sodium-dependent glucose transporter.  Biochem Pharmacol. 2003;  65 1199-206
• 23 Erlund I, Kosonen T, Alfthan G, Maenpaa J, Perttunen K, Kenraali J. et al . Pharmacokinetics of quercetin from quercetin aglycone and rutin in healthy volunteers.  Eur J Clin Pharmacol. 2000;  56 545-53
• 24 Prasain J K, Wang C C, Barnes S. Mass spectrometric methods for the determination of flavonoids in biological samples.  Free Radic Biol Med. 2004;  37 1324-50
• 25 Wang L, Morris M E. Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine.  J Chromatogr B. 2005;  821 194-201
• 26 Spencer J PE, El Mohsen M MA, Rice-Evans C. Cellular uptake and metabolism of flavonoids and their metabolites: implications for their bioactivity.  Arch Biochem Biophys. 2004;  423 148-61

1 Current address: Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brazil