Inhibiting Activities of the Secondary Metabolites of Phlomis brunneogaleata against Parasitic Protozoa and Plasmodial Enoyl-ACP Reductase, a Crucial Enzyme in Fatty Acid Biosynthesis

Abstract

Anti-plasmodial activity-guided fractionation of Phlomis brunneogaleata (Lamiaceae) led to the isolation of two new metabolites, the iridoid glycoside, brunneogaleatoside and a new pyrrolidinium derivative (2S,4R)-2-carboxy-4-(E)-p-coumaroyloxy-1,1-dimethylpyrrolidinium inner salt [(2S,4R)-1,1-dimethyl-4-(E)-p-coumaroyloxyproline inner salt]. Moreover, a known iridoid glycoside, ipolamiide, six known phenylethanoid glycosides, verbascoside, isoverbascoside, forsythoside B, echinacoside, glucopyranosyl-(1→G_i-6)-martynoside and integrifolioside B, two flavone glycosides, luteolin 7-O-β-D-glucopyranoside (10) and chrysoeriol 7-O-β-D-glucopyranoside (11), a lignan glycoside liriodendrin, an acetophenone glycoside 4-hydroxyacetophenone 4-O-(6′-O-β-D-apiofuranosyl)-β-D-glucopyranoside and three caffeic acid esters, chlorogenic acid, 3-O-caffeoylquinic acid methyl ester and 5-O-caffeoylshikimic acid were isolated. The structures of the pure compounds were elucidated by means of spectroscopic methods (UV, IR, MS, 1D and 2D NMR, [α]_D) and X-ray crystallography. Compounds 10 and 11 were determined to be the major anti-malarial principles of the crude extract (IC₅₀ values of 2.4 and 5.9 μg/mL, respectively). They also exhibited significant leishmanicidal activity (IC₅₀ = 1.1 and 4.1 μg/mL, respectively). The inhibitory potential of the pure metabolites against plasmodial enoyl-ACP reductase (FabI), which is the key regulator of type II fatty acid synthases (FAS-II) in P. falciparum, was also assessed. Compound 10 showed promising FabI inhibiting effect (IC₅₀ = 10 μg/mL) and appears to be the first anti-malarial natural product targeting FabI of P. falciparum.

Abbreviations

ACP:Acyl Carrier Protein

CC:Column Chromatography

FabI:Enoyl-Acyl Carrier Protein Reductase

FAS-I:Type I Fatty Acid Synthase System

FAS-II:Type II Fatty Acid Synthase System

MPLC:Medium Pressure Liquid Chromatography

Phlomis brunneogaleata - Lamiaceae - iridoid - pyrrolidinium derivative - betonicine - luteolin 7-O-β-D-glucopyranoside - Plasmodium falciparum enoyl-acyl carrier protein reductase (FabI) - type II fatty acid synthase (FAS-II)

References

• 1 Waller R F, Keeling P J, Donald R G, Striepen B, Handman E, Lang-Unnasch N. et al . Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum .  Proc Natl Acad Sci USA. 1998;  95 12 352-7
• 2 Perozzo R, Fidock D A, Kuo M, Sidhu A S, Valiyaveettil J T, Bittman R. et al . Structural elucidation of the specificity of the antibacterial agent triclosan for malarial enoyl acyl carrier protein reductase.  J Biol Chem. 2002;  277 13 106-114
• 3 Surolia N, Surolia A. Triclosan offers protection against blood stages of malaria by inhibiting enoyl-ACP reductase of Plasmodium falciparum .  Nature Med. 2001;  7 167-73
• 4 McLeod R, Muench S P, Rafferty J B, Kyle D E, Mui E J, Kirisits M J. et al . Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan FabI.  Int J Parasitol. 2001;  31 109-13
• 5 Sheldrick G M. SHELXL97, Program for the refinement of crystal structures. University of Göttingen Germany; 1997
• 6 Patchett A A, Witkop B. Studies on hydroxyproline.  J Am Chem Soc. 1957;  79 185-92
• 7 Blunden G, Gordon S M, Crabb T A, Roch O G, Rowan M G, Wood B. NMR spectra of betaines from marine algae.  Magn Res Chem. 1986;  24 965-71
• 8 Sperandeo N R, Brun R. Synthesis and biological evaluation of pyrazolylnaphthoquinones as new potential antiprotozoal and cytotoxic agents.  ChemBioChem. 2003;  4 69-72
• 9 Kamel M S, Mohamed K M, Hassanean H A, Ohtani K, Kasai R, Yamasaki K. Iridoid and megastigmane glycosides from Phlomis aurea .  Phytochemistry. 2000;  55 353-7
• 10 Chini C, Bilia A R, Keita A, Morelli I. Protoalkaloids from Boscia angustifolia .  Planta Medica. 1992;  58 476
• 11 Ersöz T, Harput Ü I, Çalıs I, Dönmez A A. Iridoid, phenylethanoid and monoterpene glycosides from Phlomis siehana .  Turk J Chem. 2002;  26 1-8
• 12 Abougazar H, Bedir E, Khan I A, Çalıs I. Wiedemanniosides A - E: New phenylethanoid glycosides from the roots of Verbascum wiedemannianum .  Planta Medica. 2003;  69 814-9
• 13 Otsuka H. Phenylethanoids from Linaria japonica .  Phytochemistry. 1993;  32 979-81
• 14 Saracoglu I, Varel M, Hada J, Hada N, Takeda T, Dönmez A A. et al . Phenylethanoid glycosides from Phlomis integrifolia Hub.-Mor.  Z Naturforsch. 2003;  58c 820-5
• 15 Markham K R, Chari V M. ¹³C NMR Spectroscopy of Flavonoids.  In: The Flavonoids: Advances in Research. (Harborne JB; Mabry TJ eds.) London; Chapman and Hall 1982: pp 19-132
• 16 Chaudhuri R K, Sticher O. New iridoid glycosides and a lignan diglucoside from Globularia alypum L.  Helv Chim Acta. 1981;  64 3-15
• 17 Lu Y, Foo L Y. Flavonoid and phenolic glycosides from Salvia officinalis .  Phytochemistry. 2000;  55 263-7
• 18 Cheminat A, Zawatzky R, Becker H, Brouillard R. Caffeoyl conjugates from Echinacea species: Structures and Biological activity.  Phytochemistry. 1988;  27 2787-94
• 19 Peng L Y, Mei S X, Jiang B, Zhou H, Sun H D. Constituents from Lonicera japonica .  Fitoterapia. 2000;  71 713-5
• 20 Veit M, Weidner C, Strack D, Wray V, Witte L, Czygan F C. The distribution of caffeic acid conjugates in the Equisetaceae and some ferns.  Phytochemistry. 1992;  31 3483-5
• 21 Waller R F, Ralph S A, Reed M B, Su V, Douglas J D, Minnikin D E. et al . A type II pathway for fatty acid biosynthesis present drug targets in Plasmodium falciparum .  Antimicrob Agents Chemother. 2003;  47 297-301
• 22 Blunden G, Yang M -H, Yuan Z -X, Smith B E, Patel A, Cegarra J A. et al . Betaine distribution in the Labiatae.  Biochem Syst Ecol. 1996;  24 71-81