Abstract
In this research, extracts prepared from plant materials were tested against Mycobacterium tuberculosis. H37Rv (ATCC 27294). Of the 107 plants tested, five exhibited more than 90% inhibition of growth of Mycobacterium tuberculosis., at concentrations lower than 100 µg/ml. Jervin from Veratrum album. L., usnic acid from Usnea barbata. (L.) Mott, and alantolactone from Inula helenium. L. subsp. turcoracemosa. were isolated and their minimum inhibitory concentrations (MICs) were determined as 25, 12.5, and 3.125 µg/ml, respectively.
Introduction
Tuberculosis is one of the important diseases worldwide with about 7.3 million new cases and 3 million deaths per year (World Health Organization, Citation1998). The problems of tuberculosis are related by the spread of HIV infection and the increases in prevalence of drug resistance as well as multidrug-resistant strains (Cohn et al., Citation1997). In order to control tuberculosis in the long term, new drugs are certainly required. One research area relative to tuberculosis is the development of new antituberculosis drugs through screening of natural products. In recent years, a large number of reports on the antimycobacterial activity of natural products from plants have appeared. In this research, 107 different plant species belonging to 39 families from Turkey were evaluated for antimycobacterial activity. Jervin from Veratrum album. L. (Liliaceae), usnic acid from Usnea barbata. (L.) Mott (Usneaceae), and alantolactone from Inula helenium. L. subsp. turcoracemosa. Grierson (Asteraceae) were isolated and identified as principal active components.
Materials and Methods
General experimental procedures
1H NMR and 13C NMR spectra were recorded on a Bruker 400 MHz spectrometer (Karlsruhe, Germany). Mass spectra were obtained on a VG-Platform Micromass (EI+) (Manchester, UK) instrument. Melting points were determined on an Electrothermal 9200 Digital Melting Point Apparatus and are uncorrected. Gas chromatography-mass spectrometry (GC-MS) analysis was carried out on a Hewlett-Packard model 6890 gas chromatograph (Valbron, Germany) combined with a Hewlett-Packard model 5972 A MS detector. The column was HP-5MS 5% phenylmethyl siloxane (30 m × 0.25 mm ID, 0.25-µm film thickness). Carrier gas was helium with a 1.3 ml/min flow rate. Injection temperature was 250°C. Column temperature was programmed from 120°C (3 min) to 230°C (8 min) at 20°C/min to 290°C (2 min) at 10°C/min; split ratio 1/100. Electron ionization (EI) data was acquired using the following conditions: 70 eV electron energy, 190°C source temperature.
Plant material
Studied plants were collected between May and July 1998 and 1999. Collecting regions and parts used are given in . All voucher specimens were deposited at the Gazi Universitesi Fen Fakültesi Herbaryumu-GAZİ in Ankara, Turkey. Plants were identified by our co-author Prof. M. Vural.
Table 1.. Antimycobacterial activities of the ethanol extracts against Mycobacterium tuberculosis. H37Rv.
Preparation of extracts and fractions
Air-dried and powdered plant materials were extracted with 70% ethanol by maceration. For the fractions, air-dried and powdered plant material was sequentially extracted at room temperature with petroleum ether, chloroform, methanol and water. The extracts were separately evaporated under vacuum to dryness.
Antimycobacterial assay
Extracts, fractions, and isolated compounds were tested for inhibition of Mycobacterium tuberculosis. H37Rv (ATCC 27294) by the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) (Birmingham, Alabama, USA) (Collins & Franzblau, Citation1997; Cantrell et al., Citation1998).
Isolation of the compounds
Jervine (24.5 mg), a major alkaloid, was isolated from the alkaloid fraction of the air-dried and powdered leaves of Veratrum album. (100 g) as previously reported, and its physical and spectral data were in agreement with those published in the literature (Atta-ur-Rahman et al., 1991).
The petroleum ether extract of 10 g of air-dried and powdered radix of Inula helenium. subsp. turcoracemosa. was evaporated and subjected to preparative thin-layer chromatography (TLC) using hexane:AcOEt (85:15) to afford colorless needles (55.8 mg). It was identified as alantolactone by comparison of its melting point and NMR spectra with published data (Marshall & Cohen, Citation1964; Okunade & Wiemer, Citation1985). Mass spectral data of alantolactone are reported for the first time. EI-MS (70 eV), m/z.: 234 (M + 2, 2.98), 233 (M + 1, 11.91), 232 (M+, 20.84), 217 (32.74), 203 (2.98), 187 (6.55), 171 (39.28), 91 (26.19), 83 (100).
The petroleum ether extract of 10 g of air-dried and powdered Usnea barbata. was evaporated. On standing at room temperature; yellow crystals separated (105.2 mg), which were identified as (+)-usnic acid by comparison of its physical (Correché et al., Citation1998) and spectral data (Ingólfsdóttir et al., Citation1998; König & Wright, Citation1999) with previously reported values. This is the first report of mass spectral assignment for usnic acid. EI-MS (70 eV), m/z.: 346 (M + 2, 4.32), 345 (M + 1, 19.18), 344 (M+, 78.24), 329 (3.61), 301 (2.86), 260 (75.87), 233 (100), 217 (26.33), 214 (5.13), 161 (3.92), 83 (7.43).
Results and Discussion
Antimycobacterial activities of the ethanol extracts against Mycobacterium tuberculosis. H37Rv are shown in . At concentrations below 100 µg/ml, five of the extracts caused more than 90% inhibition of growth of M. tuberculosis.. Fractions prepared by using solvents in the order of increasing polarity from Veratrum album, Inula helenium. subsp. turcoracemosa., and Usnea barbata. were tested. Results from screening fractions of these plants against Mycobacterium tuberculosis. H37Rv are given in . Active fractions were chemically investigated for their active constituents. The pure compounds were identified using physical and spectroscopic methods (MS, 1H NMR, and 13C NMR), and the activities were confirmed. TLC analysis of the chloroform fraction of V. album. found alkaloids as the major compound in this fraction. On the basis of this result, the alkaloid fraction was obtained from the leaves of V. album., and jervine was isolated, and both were tested against M. tuberculosis. separately. Their MIC values were determined as 50 and 25 µg/ml, respectively. This is the first report for antimycobacterial activity of V. album.. Alantolactone and usnic acid were obtained as major compounds from I. helenium. subsp. turcoracemosa. and U. barbata., respectively. Minimum inhibitory concentrations (MICs) of alantolactone and usnic acid were determined as 3.125 and 12.5 µg/ml, respectively. The MIC value of usnic acid was determined as 32 µg/ml against M. aurum. by Ingólfsdóttir et al. (Citation1998). It is shown that usnic acid exhibited inhibitory activity against M. tuberculosis. at a lower concentration than M. aurum.. Chloroform and methanol fractions of I. helenium. subsp. turcoracemosa. were analysed by GC-MS together with alantolactone isolated from this plant, and GC-MS analysis of the fractions was performed using the conditions indicated in general experimental procedures. The major compound was alantolactone, and a small amount of isoalantolactone was identified in these two fractions by comparison of its mass spectra with those at Wiley GC-MS Library. Other active plant extracts have been worked out to determine the compounds responsible for this activity.
Table 2.. Antimycobacterial activities of the fractions against Mycobacterium tuberculosis. H37Rv.
Acknowledgments
Antimycobacterial data were provided by the Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) through a research and development contract with the U.S. National Institute of Allergy and Infectious Diseases (contract no. N01-AI-45246). This research was financially supported by TÜBİTAK (research grant no. SBAG-1681). Inula helenium. subsp. turcoracemosa. was collected by Canan Karamenderes (Ege University, Faculty of Pharmacy, Department of Pharmaceutical Botany, İzmir, Turkey).
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