Antimycobacterial Activity of Geranylated Furocoumarins from Tetradium daniellii

• Abstract
• Introduction
• Materials and Methods
• Plant material
• General experimental
• NMR spectroscopy
• In vitro antimycobacterial assay
• Isolation
• Results and Discussion
• Acknowledgements
• References

Abstract

Seven geranylated furocoumarins were isolated from the fruits of Tetradium daniellii (Benn.) T.G. Hartley (Rutaceae) and tested for their antimycobacterial activity against Mycobacterium fortuitum, M. smegmatis and M. phlei. They were shown to be highly active, with minimal inhibitory concentrations (MICs) ranging from 8 to 64 μg/mL. Xanthotoxin and xanthotoxol, representing furocoumarins lacking the lipophilic geranyl side chain, were tested similarly and were shown to be inactive. Geraniol alone was inactive as well. The antimycobacterial activity of the substances was dependent on the position and polarity of the geranyl moiety. The compounds were purified using chromatographic methods. Structure elucidation was achieved with 1D and 2D NMR experiments.

Introduction

Tuberculosis (TB) is the second leading infectious cause of death in the world. It has been estimated that 7 to 8 million new cases of active TB and 2 to 3 million deaths occur each year [1]. The ever-increasing occurrence of multidrug-resistant strains demands new classes of potent antimycobacterial compounds with novel modes of action. Experience has shown plant secondary metabolites to be a rewarding field for the discovery of new antimycobacterial leads [2]. Due to very slow growth rates and pathogenicity of M. tuberculosis, a number of assays, employed in the search for novel antimycobaterial leads, use related species such as M. smegmatis and M. fortuitum as a test model [3]. Here, we report the antimycobacterial properties of seven geranylated furocoumarins (1 - 7) isolated from the fruits of Tetradium daniellii against Mycobacterium fortuitum, M. smegmatis and M. phlei. []

Materials and Methods

Plant material

Unripe fruits of T. daniellii were collected in the Botanical Garden of the University of Graz, Austria. Voucher specimens are deposited at the Institute of Pharmaceutical Sciences, Department of Pharmacognosy, Graz, Austria.

General experimental

All chemicals were purchased from Merck (VWR International; Darmstadt, Germany) if not stated otherwise. Mueller-Hinton broth, Columbia blood agar and defibrinated horse blood were obtained from Oxoid Ltd. (Hampshire, England); tetrazolium bromide, isoniazid and ethambutol were purchased from Sigma (St. Louis, MO, USA). Xanthotoxin was obtained from Fluka (Buchs, Switzerland), xanthotoxol from Roth (Karlsruhe, Germany). Geraniol was obtained from Dragoco (Vienna, Austria).

TLC: Merck Kieselgel 60, n-hexane:ethyl-acetate 1 : 1, sprayed with anisealdehyde sulphuric acid reagent. Analytical HPLC: Merck-Hitachi, L7100 pump, L7200 Autosampler, L7455 DAD used at 230 nm and D7000 interface using a Merck Lichrospher® 100, RP 18 (5 μm), 125 × 4 mm column. Semi-preparative HPLC: Merck L6200 A Intelligent Pump, Merck L4500 DAD used at 230 nm, Merck, Lichrospher® 100, RP 18 (10μm), 250 × 10 mm column. HPLC-MS: Thermo Finnigan Surveyor liquid chromatograph equipped with a Merck Lichrospher® 100, RP 18 (5 μm), 125 × 4 mm column. The LC was interfaced with a Finnigan LCQ Deca XP Plus mass detector operating in the ESI positive mode. The mass spectra were recorded in scan mode. Mobile phase in all HPLC systems: acetonitrile:water from 5 : 95 to 95 : 5 in 30 min, PDA detection at 230 nm. Optical rotation: polarimeter 241 MC (Perkin Elmer; Wellesley, MA, USA).

NMR spectroscopy

NMR data were recorded using a Varian Unity lnova 600 MHz spectrometer operating at a proton frequency of 599 MHz with a 5 mm triple-resonance probe equipped with a pulsed field z-gradient. The samples containing a solution of 1 - 2 mg of substance in 0.75 mL of CDCl₃ were measured at 298 K with TMS as internal standard. The performed 1D and 2D experiments are described by Seebacher et al. [4].

In vitro antimycobacterial assay

Strains of M. fortuitum (ATCC 6841), M. smegmatis (ATCC 19 429) and M. phlei (ATCC 19 249) were from American Type Culture Collection and kindly provided by Dr. Simon Gibbons (Centre of Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London).

The antimycobacterial activities of the test compounds were evaluated by the minimum inhibitory concentration assay reported by Schinkovitz et al. [5] in 96-well microtiter plates. Two-fold serial dilution of test solutions were prepared to get final concentrations ranging from 128 to 0.25 μg/mL. Mycobacterial strains were cultivated on Columbia blood agar, supplemented with 7 % defibrinated horse blood agar. The lowest assay concentrations of the test compounds that produced complete inhibition of the macroscopic growth (MIC) were detected by addition of 20 μL of a methanolic solution of tetrazolium redox dye (MTT, 3.5 mg/ mL) followed by incubation at 37 °C for 20 min and visual evaluation. All tests were performed three times in duplicate and ethambutol and isoniazid were used as positive controls.

Isolation

120 g of lyophilized fruits of T. daniellii were extracted with n-hexane for 24 hours using a Soxhlet apparatus. The extract (6.29 g) was further fractioned by Sephadex column chromatography, with methanol as a mobile phase resulting in ninety-one fractions of 10 mL each which were analysed by TLC and HPLC. The similar fractions 16 - 21 were combined (220 mg) and subjected to semi-preparative HPLC, yielding the furocoumarins 9-[(6′,7′-dihydroxy-3′,7′-dimethyl-2′-octenyl)oxy]-7H-furo[3,2-g][1]benzopyran7-one (1), 9-{[(2E′,5E′)-7′-hydroxy-3′,7′-dimethyl-2′,5′-octadienyl]oxy}-7H-furo[3,2-g][1]benzopyran-7-one (2), 9-[(6′-hydroxy-3′,7′-dimethyl-2′,7′-octadienyl)oxy]-(E)-(+)-7H-furo[3,2-g][1]benzopyran-7-one (3 = lansiumarin C), 9-{[5′-(3′′,3′′-dimethyloxiranyl)-3′-methyl-2′-pentenyl]oxy}-(E)-7H-furo[3,2-g][1]benzopyran-7-one (4), 4-{[(2′E)-5′-(3′′,3′′-dimethyloxiranyl)-3′-methyl-2′-pentenyl]oxy}-7H-furo[3,2-g][1]benzopyran-7-one (5= epoxybergamottin), 9-{[(2′E)-3′,7′-dimethyl-2′,6′-octadienyl]oxy}-7H-furo[3,2-g][1]benzopyran-7-one (6) and 4-{[(2′E)-3′,7′-dimethyl-2′,6′-octadienyl]oxy}-7H-furo[3,2-g][1]benzopyran-7-one (7 = bergamottin). The two isomers 2 and 3 could not be separated with the methods employed here. They were isolated and tested as a 60 : 40 mixture. ESI-LC-MS was used to determine molecular masses. Structure elucidation for all the isolated coumarins was achieved by 1D and 2D NMR spectroscopy. Spectral data were assigned (Table [1] , Table [2]). The numbering of the C atoms follows Stevenson et al. [6]. The purity of all isolated substances was determined by HPLC-DAD peak integration and was shown to be: 1 : 97.4 %, 2 + 3 : 84.3 %, 4 : 93.6 %, 5 : 97.8 %, 6 : 99.8 % and 7 : 94.3 %, respectively. The yield of the substances was: 1 : 3.9 mg, 2 + 3 : 10.8 mg, 4 : 51.8 mg, 5 : 40.0 mg, 6 : 33.7 mg and 7 : 9.0 mg. The UV spectra were recorded using HPLC-DAD. All the substances had practically identical spectra with maxima at 220, 250 and 305 nm. The substances showed no optical rotation.

Results and Discussion

The main constituents found in the fruit of T. daniellii were substituted furocoumarins. They differed by the position of their side chain, which was attached via an ether bridge to position 5 or 8 of the 7H-furo[3,2-g]benzopyran-7-one ground structure. The side chains showed oxidation on positions 6′′ and 7′′. Compounds 6 and 7 had previously been known from T. daniellii [6]. Compounds 1 - 5 had been known from other rutaeceaous species [7], [8], [9], [10], [11], [12], [13]. The pure substances of which more than 2 mg (2 + 3, 4, 5, 6, 7) were available were then tested for their antimycobacterial activity against Mycobacterium fortuitum, M. smegmatis and M. phlei. They were shown to be highly active, with minimal inhibitory concentrations (MICs) ranging from 8 to 64 μg/mL (Table [3]). Xanthotoxin and xanthotoxol, representing furocoumarins lacking the lipophilic geranyl side chain, were tested similarly and were shown to be inactive. Geraniol, representing the free geranyl side chain showed no activity either. The MICs of the standard antibiotic drugs ethambutol and isoniazid ranged from 1 - 16 μg/mL.

The antimycobacterial activity seems to depend on the position and polarity of the geranyl side chain. Compound 5 was more active against M. smegmatis and M. phlei than its C-8 substituted equivalent 4, and 7 was more active than 6 against M. phlei. Compounds 6 and 7 were more active than their epoxides (4 and 5). Xanthotoxin and xanthotoxol, both lacking the side chain, were inactive. It can therefore be concluded that the lipophilic side chain enhances the antimycobacterial activity of furocoumarins. Geraniol, the free geranyl alcohol, however, showed no activity whatsoever.

Presumably, all the derivatives 1 - 5 are non-enzymatically formed oxidation products of 6 and 7, as none of the substances with asymmetric carbons showed optical rotation. Biosynthetically, it seems that a prenyl transferase unspecifically binds an O-geranyl moiety to either C-5 or C-8. Linear furocoumarins, known to be photoreactive agents per se, may enhance radical formation, which causes the various oxidation products. Coumarins have previously been reported to show activity against rapidly growing strains of mycobacteria. Schinkovitz et al. [5], reported osthrutin from Peucedanum ostruthium to have an MIC of 3.4 - 6.7 μg/mL. Stavri et al. [14] reported MICs of 64 - 128 μg/mL for pangelin, a 5-prenylated furocoumarin from Ducrosia anethifolia (Apiaceae).

Acknowledgements

The authors are grateful to Dr. Simon Gibbons for kindly providing us with the mycobacterial strains. Abraham Abebe Wube gratefully acknowledges a scholarship from the Austrian Academic Exchange Service (ÖAD).

References

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• 2 Okunade A L, Elvin-Lewis M PF, Lewis W H. Natural antimycobacterial metabolites: current status.  Phytochemistry. 2004;  65 1017-32
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• 10 Ziegler H, Spiteller G. Coumarins and psoralens from Sicilian lemon oil (Citrus limon (L.) Burm. f.)  Flavour Fragr J. 1992;  7 129-39
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• 11 Ohta T, Maruyama T, Nagahashi M, Miyamoto Y, Hosoi S, Kiuchi F. et al . Paradisidin C: a new CYP3A4 inhibitor from grapefruit juice.  Tetrahedron. 2002;  58 6631-5
  CrossrefPubMedSearch in Google Scholar
• 12 Stanley W L, Vannier S H. Chemical composition of lemon oil. I. Isolation of a series of substituted coumarins.  JACS. 1957;  79 3488-91
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• 13 Spath E, Kainrath P. Berichte der Universität Wien. 1937 70B: 2272-6
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• 14 Stavri M, Mathew K T, Bucar F, Gibbons S. Pangelin, an antimycobacterial coumarin from Ducrosia anethifolia .  Planta Med. 2003;  69 956-9
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Prof. Dr. Rudolf Bauer

Institute of Pharmaceutical Sciences

Department of Pharmacognosy

University of Graz

Universitätsplatz 4

8010 Graz

Austria

Phone: +43-316-380-8700

Fax: +43-316-380-9860

Email: rudolf.bauer@uni-graz.at

References

• 1 WHO. Fact sheet No. 104. 2004
  Search in Google Scholar
• 2 Okunade A L, Elvin-Lewis M PF, Lewis W H. Natural antimycobacterial metabolites: current status.  Phytochemistry. 2004;  65 1017-32
  CrossrefPubMedSearch in Google Scholar
• 3 Tims K J, Rathbone D L, Lambert P A, Attkins N, Cann S W, Billington D C. Mycobacterium fortuitum as a screening model for Mycobacterium tuberculosis .  J Pharm Pharmacol. 2000;  52 135
  PubMedSearch in Google Scholar
• 4 Seebacher W, Simic N, Weis R, Saf R, Kunert O. Complete assignments of ¹H- and ¹³C-NMR resonances of oleanic acid, 18-alpha-oleanic acid, ursolic acid and their 11-oxo derivatives.  Magn Reson Chem. 2003;  41 636-8
  CrossrefPubMedSearch in Google Scholar
• 5 Schinkovitz A, Gibbons S, Stavri M, Cocksedge M J, Bucar F. Ostruthin: an antimycobacterial coumarin from the roots of Peucedanum ostruthium .  Planta Med. 2003;  69 369-71
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Stevenson P C, Simmonds M SJ, Yule M A, Veitch N C, Kite G C, Irwin D. et al . Insect antifeedant furanocoumarins from Tetradium daniellii .  Phytochemistry. 2003;  63 41-6
  CrossrefPubMedSearch in Google Scholar
• 7 Ito A, Shamon L A, Yu B, Mata-Greenwood E, Lee S K, Van Breemen R B. et al . Antimutagenic constituents of Casimiroa edulis with potential cancer chemopreventive activity.  J Agric Food Chem. 1998;  46 3509-16
  CrossrefPubMedSearch in Google Scholar
• 8 Colombain M, Girard C, Muyard F, Bevalot F, Tillequin F, Waterman P G. Eight new prenylcoumarins from Phebalium clavatum .  J Nat Prod. 2002;  65 458-61
  CrossrefPubMedSearch in Google Scholar
• 9 Ito C, Katsuno S, Furukawa H. Structures of lansiumarin-A, -B, -C, three new furocoumarins from Clausena lansium .  Chem Pharm Bull. 1998;  46 341-3
  PubMedSearch in Google Scholar
• 10 Ziegler H, Spiteller G. Coumarins and psoralens from Sicilian lemon oil (Citrus limon (L.) Burm. f.)  Flavour Fragr J. 1992;  7 129-39
  PubMedSearch in Google Scholar
• 11 Ohta T, Maruyama T, Nagahashi M, Miyamoto Y, Hosoi S, Kiuchi F. et al . Paradisidin C: a new CYP3A4 inhibitor from grapefruit juice.  Tetrahedron. 2002;  58 6631-5
  CrossrefPubMedSearch in Google Scholar
• 12 Stanley W L, Vannier S H. Chemical composition of lemon oil. I. Isolation of a series of substituted coumarins.  JACS. 1957;  79 3488-91
  CrossrefPubMedSearch in Google Scholar
• 13 Spath E, Kainrath P. Berichte der Universität Wien. 1937 70B: 2272-6
  Search in Google Scholar
• 14 Stavri M, Mathew K T, Bucar F, Gibbons S. Pangelin, an antimycobacterial coumarin from Ducrosia anethifolia .  Planta Med. 2003;  69 956-9
  Thieme ConnectPubMedSearch in Google Scholar

Prof. Dr. Rudolf Bauer

Institute of Pharmaceutical Sciences

Department of Pharmacognosy

University of Graz

Universitätsplatz 4

8010 Graz

Austria

Phone: +43-316-380-8700

Fax: +43-316-380-9860

Email: rudolf.bauer@uni-graz.at