We analyzed the antimycobacterial activity of the hexane extract of rhizomes from
In this paper, the isolation of (8R,8′R,9R)-cubebin, fargesin, and eupomatenoid-1 from the active Hex extract of
The chemical characterization of the isolated compounds was determined by 1H-NMR (Bruker-Avance F, 300 MHz) and 13C-NMR (Variant Unity, 75.4 MHz) using Tetramethylsilane as an internal standard in CDCl3. Electron impact-mass spectra (EI-MS) were obtained on a Jeol AX-505 HA mass spectrometer at 70 eV. Melting points (m.p.) were determined with a Fisher-Johns apparatus and are uncorrected. Open Column chromatography (CC) was carried out by using silica gel 60 GF254 (70–230 mesh, Merck) as a stationary phase, and silica gel 60 F254 precoated aluminum plates (0.2 mm, Merck) were employed for analytical and preparative Thin Layer Chromatography (TLC) analysis. Hex, chloroform (CHCl3), and MeOH were obtained from Mallinckrodt and J. T. Baker.
The spots were visualized by spraying it with a 10% solution of aqueous H2SO4 followed by heating at 100°C. High Performance Liquid Chromatography (HPLC) analyses were carried out with a Waters 600 system controller connected to a photodiode array detector 996, which was programmed to collect data from 220–380 nm at 2.4-nm resolutions. Control of equipment, data acquisition, and processing and the management of chromatographic information were performed by Millennium 32 software program (Waters). Analyses were accomplished on a Spherisorb S100DS2 RP column (4.6 × 250 mm, 10-
Powdered air-dried rhizome (530 g) was macerated (3 × 48 h) with 5 L Hex at room temperature. The extract obtained was filtered and vacuum concentrated to yield 37 g of the crude extract. The Hex extract (35 g) was subjected to CC in silica gel (150 g) and was eluted with Hex : CHCl3 (100→0) and CHCl3 : MeOH (100→0), and 171 fractions of 125 mL each were obtained. Primary fractions (F1–F15) were combined according to a TLC analysis as follows: F1 (69 mg); F2 (10 mg); F3 (18 mg); F4 (92 mg); F5 (69 mg); F6 (149 mg); F7 (115 mg); F8 (434 mg); F9 (258 mg); F10 (322 mg); F11 (1,816 mg); F12 (1,218 mg); F13 (669 mg); F14 (14,109 mg); F15 (5,870 mg).
Fraction F5–F10 was submitted to preparative TLC employing Hex : CHCl3 70 : 30 as an elution system; after this procedure, 53.5 mg of eupomatenoid-1 (
From secondary fractions FG and FH (2 g), fargesin (
Eupomatenoid-1 (
Fargesin (
(8R,8′R,9R)-cubebin (
The Hex extract and pure compounds were tested using microplate Alamar blue assay (MABA), as previously described [
In this study, we describe the isolation of eupomatenoid-1 (
Chemical structures of isolated compounds from
The antimycobacterial activity of the Hex extract and purified compounds determined by the MABA is depicted in Table
Antimycobacterial and antiprotozoal activities of the hexanic extract and pure compounds isolated from
Sample | MIC ( | IC50 ( | ||||||||
H37Rv | CIBIN/UMF15 : 99 | SIN4 | RIF-R | STR-R | INH-R | EMB-R | ||||
Hexanic extract | >100 | 50 | 50 | ND | ND | ND | ND | 0.235 | 0.315 | |
Eupomatenoid-1 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 0.624 | 0.545 | |
Fargesin | 50 | >100 | 50 | 25 | 25 | 12.5 | >50 | 120.6 | 262.7 | |
(8R,8′R,9R)-Cubebin | 50 | 50 | 50 | 100 | 100 | 100 | 100 | 137.3 | 275.0 | |
Rifampicin | 0.06 | >100 | 100 | >25 | 0.06 | 0.06 | 0.06 | — | — | |
Isoniazid | 0.06 | 3.1 | 3.1 | 0.06 | 0.06 | >25 | 0.06 | — | — | |
Streptomycin | 0.5 | >100 | >4 | 0.5 | >8 | 0.5 | 0.5 | — | — | |
Ethambutol | 2.0 | 8 | >16 | 1.0 | 1.0 | 1.0 | >32 | — | — | |
Ofloxacin | — | 0.5 | 8.0 | — | — | — | — | — | — | |
Metronidazole | — | — | — | — | — | — | — | 0.060 | 0.210 |
H37Rv: sensitive strain to INH, RIF, EMB, STR, and pyrazinamide; CIBIN/UMF15:99: resistant strain to INH, RIF, EMB, STR, and pyrazinamide; SIN4: resistant strain to INH, RIF, EMB, STR, rifabutin, ethionamide, and ofloxacin; RIF-R: rifampicin-resistant; STR-R: streptomycin-resistant; INH-R: isoniazid-resistant and EMB-R: ethambutol-resistant. ND: no determined; MIC: minimum inhibitory concentration; IC50: 50% inhibitory concentration. Data are means of three determinations.
The antiprotozoal activity of the Hex extract and of pure compounds
The presence of the lignans and neolignans in
Compound
Of the three pure compounds, fargesin (
It is noteworthy that fargesin was active against
Current tuberculosis chemotherapy is prolonged (24 months), poorly effective, expensive, and is accompanied by severe side effects. Besides, the presence of MDR
A murine model of tuberculosis previously developed by Hernández-Pando et al. [
The Hex extract and eupomatenoid-1 were the most active against both
Several studies supporting the use of natural products and their purified active compounds are an alternative treatment for gastrointestinal infections. In particular, the antiprotozoal activity of
The inappropriate short-term exposure and exposure to sublethal levels of metronidazole have induced parasite drug resistance. Eupomatenoid-1 may therefore be considered as an active principle or even a prototype molecule for the development of novel antiprotozoal agents with activity against metronidazole resistant parasites.
In this study, the activity of (8R,8′R,9R)-cubebin and fargesin, purified from the Hex extract of
Is currently being evaluated, the acute and subacute toxicity of active compounds in a mouse model. Further
The antiprotozoal activity of neolignans and lignans has scarcely been described in the literature, and our results encourage further studies on this issue.
The authors declare that they have no competing interest. All authors read and approved the final paper.
This study was supported by a Grant from the Instituto Mexicano del Seguro Social, projects FOFOI 2005/1/I/102, FIS 2006/1A/I/053 and from Consejo Nacional de Ciencia y Tecnología (CONACYT) 48339-M.