Abstract
Naphthoquinones and triterpenes isolated from the roots of Euclea natalensis. A.DC (Ebenaceae) were evaluated for their inhibitory activity against Mycobacterium tuberculosis.. Crude extract, diospyrin and 7-methyljuglone isolated from the plant, exhibited minimum inhibitory concentrations of 8.0, 8.0, and 0.5 µg ml−1, respectively, against M. tuberculosis. H37 Rv (ATCC 27294), a drug-sensitive strain. Minimum inhibitory concentrations (MICs) of 7- methyljuglone against a panel of clinical pan-sensitive and drug-resistant strains of M. tuberculosis. ranged from 0.32 to 1.25 µg/ml. The concentration of 7-methyljuglone that effected a 90% reduction of growth of M. tuberculosis. Erdman within J774.1 macrophages was 0.57 µg/ml. The superior intracellular and extracellular inhibition of M. tuberculosis. by 7-methyljuglone relative to that of the antituberculosis drugs streptomycin and ethambutol suggests that this compound be considered as a lead for further investigations.
Introduction
Tuberculosis (TB) continues to be an enormous global concern as it infects millions of people annually. With the emergence of multidrug-resistant strains of Mycobacterium tuberculosis., the need to search for new anti-TB drugs has become urgent. Euclea natalensis. A.DC, a tree belonging to the family Ebenaceae, commonly occurs in the east coast of southern Africa and also grows widely in eastern Mozambique (Van Wyk & Van Wyk, Citation1997). The plant has many uses in traditional medicine, including as a treatment for chest complaints, bronchitis, pleurisy, chronic asthma, urinary tract infections, and venereal diseases (Pujol, Citation1990).
In our continued search for biologically active natural products from E. natalensis., we investigated the roots of the plant extensively. A previous phytochemical study of E. natalensis. has afforded a bioactive naphthoquinone, diospyrin, from the roots of the plant, which has been found to have good inhibitory activity against M. tuberculosis. and a few Gram-positive and Gram-negative bacterial species (Lall & Meyer, Citation2001). We describe below the isolation of another naphthoquinone (7-methyljuglone), triterpenes (lupeol and betulin) and their bioactivity against M. tuberculosis.. Because in human tuberculosis, M. tuberculosis. resides in both intracellular and extracellular environments, in this study the activity of the crude extract of the roots of E. natalensis., diospyrin and recently isolated 7-methyljuglone, against M. tuberculosis. was determined both in axenic medium and in a macrophage cell line.
Materials and Methods
Plant Collection
Roots of E. natalensis. were collected in October 2002 from Gaza district in Mozambique. A voucher specimen (FK 3) was deposited and identified at the H.G.W.J. Schweickerdt Herbarium (PRU), Pretoria.
Extraction and Isolation
Ground root powder (200 g)of E. natalensis. was extracted using a Soxhlet apparatus with 1.5 l of chloroform. Evaporation of the solvent in vacuo. provided 10.2 g. of crude extract. The extract was subjected to silica gel column chromatography (gradient elution from hexane to chloroform to ethyl acetate) to give eight fractions () The nonpolar fractions 3–6 of the crude root extract exhibited the highest inhibitory activity. On the basis of these results, fractions 4 and 5 + 6 were used for further isolation of the active constituents.
Table 1.. Inhibitory activity of Euclea natalensis. fractions (roots, chloroform extract) and phytochemicals against Mycobacterium tuberculosis. (H37Rv).
Fraction 4 (A) (1.5 g) and fraction 5 + 6 (B) (2.5 g) were applied separately onto a glass column (3 cm in diameter and 5 cm long) packed with 40 g of silica gel and chromatographed using 6 × 100 ml hexane-ethylacetate mixtures of increasing polarity. Altogether, 21 fractions (20 ml each) were collected. Fractions 11–14 (hexane-Et OAc, 80:20) were further subjected to a Sephadex LH 20 column using ethanol as an eluent to provide 60 mg of crystalline 7-methyljuglone (0.03%) (). Crystallization of fractions 17–19 obtained from the silica gel column, with ethyl acetate resulted in the triterpene lupeol (150 mg, 0.07%) ().
Figure 1 Isolated compounds from the roots of Euclea natalensis.
Altogether 30 fractions were collected from fraction 5 + 6. Fractions 21–23 (hexane-EtOAc, 75:25) were further chromatographed on a silica gel column eluted with (CHCl3/EtOAc 3:1), which yielded 130 mg of powdered diospyrin (0.065%) (). Another triterpene, betulin (44.5 mg, 0.02%) (), was obtained from fractions 24–30 using preparative thin-layer chromatography. 1H NMR and 13C NMR spectra of all the isolated compounds were recorded in CDCl3 on a Bruker AM 300 MHz spectrometer. The 1H NMR and 13C NMR shifts of compounds matched the previously reported literature values (Vijver & Gerritsma, Citation1974; Khan et al., Citation1978). Copies of the original spectra are obtainable from the author of correspondence.
Screening of Antitubercular Constituents
Isolates and Drug Preparation
M..tuberculosis. H37Rv, ATCC 27294 (America Type Culture Collection, Rockville, MD, USA), drug-resistant strains DP 481/01, DP 482/01, DP 492/01 and two clinical isolates, WC 64/02 and WH 51/02, obtained from patients' sputa, were subcultured on Middlebrook 7H11 agar (Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). These strains are routinely tested for their susceptibility against existing TB drugs at the Medical Research Council, Pretoria, South Africa. Suspensions were prepared in 0.04% (v/v) Tween 80–0.2% bovine serum albumin (Sigma Chemical Co., St Louis, MO, USA.) so that their turbidities matched that of a McFarland no. 1 turbidity standard. Suspensions were further diluted 1:25 in 7H9GC broth (4.7 g of Middlebrook 7H9 broth base [Difco, Detroit, MI, USA], 20 ml of 10% [v/v] glycerol, 1 g of Bacto Casitone [Difco], 880 ml of distilled water, 100 ml of oleic acid, albumin, dextrose, and catalase [Remel, Lenexa, KS, USA]. Rifampin (RMP), streptomycin (SM), and ethambutol (EMB) were obtained from Sigma. Stock solutions of SM and EMB were prepared in deionized water, and RMP was prepared in dimethylsulfoxide (DMSO). Stock solutions were diluted in 7H9GC broth to two times the maximum desired final testing concentrations prior to their addition to microplates.
MABA
The microplate Alamar blue Assay (MABA) was used to determine the minimum inhibitory concentrations (MICs) of drugs and compounds against M. tuberculosis. strains (Collins & Franzblau, Citation1997). The highest concentration of DMSO was 1% for testing against drug-sensitive strains. Final drug concentration ranges were as follows: for RMP, 0.0156 to 1.0 µg ml−1, for SM, 0.125 to 32.0 µg mL−1and for EMB, 0.5 to 128.0 µg mL−1.
Cytotoxicity assay
Crude chloroform extract of the roots of E. natalensis., diospyrin and 7-methyljuglone, were selected for further tests such as cytotoxicity and intracellular activity. Cytotoxicity was determined by exposing different concentrations of samples to green monkey kidney cells (Vero) and a mouse macrophage cell line, J774A.1. Samples were dissolved at 20–40 mg ml−1 in DMSO. Geometric threefold dilutions were performed in growth medium MEM (Gibco, Grand Island, NY, USA) + 10% fetal bovine serum (HyClone, Logan, UT, USA) + 25 mM N.-(2-hydroxyethyl)-piperazine-N.′-2-ethanesulfonic acid (HEPES, Gibco) + 0.2% NaHCO3 (Gibco) + 2 mM glutamine (Irvine Scientific, Santa Ana, CA, USA). Final DMSO concentrations did not exceed 1% v/v. Drug dilutions were distributed in duplicate in 96-well tissue culture plates (Becton Dickinson Labware, Lincoln Park, NJ, USA) at a volume of 50 µl/well. An equal volume containing either 5 × 105 log phase Vero cells (CCL-81; American Type Culture Collection) or mouse macrophage cells (J774A.1) (Ralph et al., Citation1975) was added to each well and the cultures were incubated at 37°C in an atmosphere of 5% CO2 in air. After 72 h, cell viability was measured using the CellTiter 96 aqueous nonradioactive cell proliferation assay (Promega Corp., Madison, WI, USA) according to the manufacturer's instructions. Absorbance at 490 nm was read in a Victor2 multilabel reader (Perkin Elmer). The IC50 was determined using a curve-fitting program. Maximum cytotoxicity (100%) was determined by lysing the cells with sodium dodecyl sulfate (Sigma, St. Louis, MO).
Intracellular Activity
The mouse macrophage cell line J774A.1 (ATCC TIB-67) was used to study the activity of samples against intracellular M. tuberculosis.. Cells were cultured in Dulbecco's modified Eagle medium (D-MEM) supplemented with 10% FBS, 1% glutamine, at 37°C and 5% CO2. Twenty millimeter of media (D-MEM) were dispensed into a 75 cm3 flask. Cells were detached by scraper from the flask, centrifuged at 1000 rpm for 5 min, suspended in 10 ml media and counted using Trypan blue, after which the cell suspension was adjusted to 1 to 3 × 105 cells ml−1. Coverslips (Fisher Scientific) were placed in the 24-well plates, and 1 ml cells were added to each well. Plates were incubated at 37°C, 5% CO2 for 24 h until confluent
M. tuberculosis. Erdman (ATCC 35801) was diluted with the cell culture media to a final concentration or 1 to 3 × 105 CFU ml−1 and 500 ul was added to each well of another 24-well plate. The coverslips were transferred with the cells from the old plates to the new 24-well plates and incubated for 2–3 h at 37°C under 5% CO2. Extracellular bacteria were removed by washing the coverslips with HBSS (Fisher Scientific). The coverslips were transferred to new 24-well plates, and 1 ml of fresh media was added to each well. Cultures were incubated at 37°C, under 5% CO2 for 7 days.
Macrophages were lysed using 0.25% sodium dodecyl sulfate (Sigma) and sonicated at 1.5 W for 15 s. Pretreatment samples (T0) were diluted (1:10, 1:100, and 1:1000), and 0.1 ml of the undiluted suspension and the 3 dilutions were plated on 7H11 agar and incubated for 3 weeks. Crude extract, diospyrin and 7-methyljuglone, did not show cytotoxicity on Vero and J774.1 cell lines at concentrations below their MIC in vitro.. All three samples were tested at concentrations equivalent to ½ MIC, MIC, and 2 × MIC. Stock solutions of each were prepared in DMSO, diluted in D-MEM, and added to each well of new 24-well plates. Coverslip cultures were transferred to the plate and incubated for 7 days in the CO2 incubator.
After a week, cells from control and treated wells were lysed and three dilutions were prepared. One hundred micro liters of dilutions were placed on 7H11 agar plates (T7). Colonies were counted after 16–20 days and compared with the control (T0). By comparing these data with CFU obtained from untreated control macrophages, the concentrations of the test compounds required to achieve a 1 log reduction (EC90) and a 2 log reduction (EC99) in viable M. tuberculosis. relative to untreated controls were determined using CurveExpert 1.3 (Microsoft).
Results and Discussion
The crude extract, diospyrin and 7-methyljuglone, exhibited MICs (minimum inhibitory concentrations) of 8.0, 8.0, and 0.5 µg/mL−1, respectively (). In contrast, the triterpenes, lupeol and betulin, were relatively inactive. Although the MIC of 7-methyljuglone was approximately 4 × higher against drug-resistant isolates relative to pan-sensitive isolates, this increase was markedly lower than that observed for rifampin (). Considering this, as well as the low actual MIC values of 7-methyljuglone versus the drug-resistant isolates (1.25 µg/ml) and the lack of structural similarity to existing TB drugs, it is likely that 7-methyljuglone acts on a target(s) not currently exploited in TB therapy. Cytotoxicity results for the Vero cell line indicate that crude extract and diospyrin have IC50 values of 64.87 and 17.78 µg/mL−1, respectively (). The moderate cytotoxicity of naphthoquinones against Vero cells suggests that 7-methyljuglone and diospyrin are capable of intracellular penetration of eukaryotic cells.
Table 2.. Inhibitory activity of 7-methyljuglone isolated from the roots of Euclea natalensis. against drug-resistant and clinical pan-sensitive isolates of Mycobacterium tuberculosis..
Table 3.. Cytotoxicity and intracellular growthinhibition of Mycobacterium tuberculosis. Erdman by the crude extract and naphthoquinones from the roots of Euclea natalensis..
The EC90and EC99 values of the samples against M. tuberculosis. Erdman growing in macrophage culture were derived from the dose responses (–). The weak bactericidal activity of diospyrin indicated by a higher value for EC99, together with the moderate toxicity to Vero cells, disqualifies diospyrin as a drug per se. However, it might serve as an interesting lead for the design and synthesis of a more active molecule. The calculated EC90and EC99 values of the most potent compound, 7-methyljuglone, were 0.57 and 1.74 µg/mL−1, respectively. Because control cultures showed an increase in CFU of slightly greater than 1 log during the 7 days of incubation, EC90is an approximation of the lowest concentration resulting in microbial stasis, while EC99 represents the lowest concentration resulting in bactericidal activity. Comparison of the in vitro. MIC and the EC90 in macrophage culture suggestseffective intracellular penetration in the macrophage. Superior intracellular and extracellular activity of 7-methyljuglone to that of streptomycin and ethambutol against M. tuberculosis. suggest that this compound be considered worthy of further exploration in new TB drug development.
Figure 2 Dose-responsive activity of E. natalensis. components and antituberculosis drugs versus M. tuberculosis. in J774.1 macrophages. Mean (SD) colony forming units of M. tuberculosis. were 1.28 (0.16) × 104 at day 0 and 1.80 (0.082) × 105 at day 7 for untreated controls.
Acknowledgments
This work was supported by grants from the UNESCO LO'REAL, NIH, NRF, and Medical Research Council, Pretoria.
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