Chemical Composition of Hexane Extract of Citrus aurantifolia and Anti-Mycobacterium tuberculosis Activity of Some of Its Constituents

The main aim of this study was to isolate and characterize the active compounds from the hexane extract of the fruit peels of Citrus aurantiifolia, which showed activity against one sensitive and three monoresistant (isoniazid, streptomycin or ethambutol) strains of Mycobacterium tuberculosis H37Rv. The active extract was fractionated by column chromatography, yielding the following major compounds: 5-geranyloxypsoralen (1); 5-geranyloxy-7-methoxycoumarin (2); 5,7-dimethoxycoumarin (3); 5-methoxypsoralen (4); and 5,8-dimethoxypsoralen (5). The structures of these compounds were elucidated by 1D and 2D NMR spectroscopy. In addition, GC-MS analysis of the hexane extract allowed the identification of 44 volatile compounds, being 5,7-dimethoxycoumarin (15.79%), 3-methyl-1,2-cyclopentanedione (8.27%), 1-methoxy-ciclohexene (8.0%), corylone (6.93%), palmitic acid (6.89%), 5,8-dimethoxypsoralen (6.08%), α-terpineol (5.97%), and umbelliferone (4.36%), the major constituents. Four isolated coumarins and 16 commercial compounds identified by GC-MS were tested against M. tuberculosis H37Rv and three multidrug-resistant M. tuberculosis strains using the Microplate Alamar Blue Assay. The constituents that showed activity against all strains were 5 (MICs = 25–50 μg/mL), 1 (MICs = 50–100 μg/mL), palmitic acid (MICs = 25–50 μg/mL), linoleic acid (MICs = 50–100 μg/mL), oleic acid (MICs = 100 μg/mL), 4-hexen-3-one (MICs = 50–100 μg/mL), and citral (MICs = 50–100 μg/mL). Compound 5 and palmitic acid were the most active ones. The antimycobacterial activity of the hexane extract of C. aurantifolia could be attributed to these compounds.

A chemical reinvestigation of Mexican lime oil of C. aurantifolia led to the identification of 98 compounds, suggesting that during distillation, lime oil undergoes modifications in its chemical composition because heating of the juice-oil emulsion and an acidic environment provoke transformations that generate more stable compounds. Authors provided insights into the acid catalyzed hydrolysis and rearrangement reactions of the bicyclic hydrocarbons named and -terpinene, sabinene, and -thujene, which generate alcohols (-terpineol, terpinen-4-ol, endo-fenchol, borneol, isoborneol) and hydrocarbons (terpinolene, limonene, fenchene, camphene, -terpinene, -terpinene) [33]. These insights could explain at some point the chemo-qualitative and chemo-quantitative differences from one sample to another. It is worth mention that the extraction processes of oils accounted for those chemical differences. Chemo-qualitative analysis of the volatile components exhibited substantial differences between the hexane extract of this research and the distilled oil of C. aurantifolia of previous reports, detecting 5,7-dimethoxycoumarin, 5,8-dimethoxypsoralen, and -terpineol as the most abundant constituents, in contrast to limonene and -terpineol of the distilled oil. Previous studies of essential lime oils reported limonene as the most abundant component, but in this work limonene is found in trace amounts in the hexane extract.
Biological results showed that 5,8-dimethoxypsoralen (5) inhibited cellular growth of multidrug-resistant M. tuberculosis strains with MIC values in the range of 25-50 g/mL. Analysis of the structure-activity relationship between coumarins and furanocoumarins indicates that furan moiety contributes to potency, as can be observed for furanocoumarins 5 (MICs = 25 and 50 g/mL) and 1 (MICs = 50 and 100 g/mL) in comparison to coumarins 2 and 3 (MICs > 200 g/mL) ( Table 2). The possible mechanism of action could be related to previous reports in which certain furanocoumarins can not only intercalate into deoxyribonucleic acid (DNA) and create cross-links with thymidine residues, but also can form covalent links to apoproteins and permanently inactivate cytochrome P450 enzymes [34]. It has been reported in the literature that compound 5 possesses antimicrobial [35], spasmogenic [36], cardiovascular [37], cancer chemopreventive [38], vasorelaxing [39], and non-phototoxic effects [40,41]. Additionally, compound 1 possesses antimutagenic effects [42] and it is a cancer chemopreventive agent [43]. The saturated fatty acid palmitic acid exhibited higher activity against multidrug-resistant M. tuberculosis strains (MICs = 50 g/mL) than the unsaturated fatty acids oleic acid and linoleic acid, which showed less activity (MICs = 100 g/mL). Saravanakumar's research group reported the activity of oleic acid (MIC 25 µg/mL), linoleic acid (MIC 50 µg/mL), and palmitic acid (no significant) against M. tuberculosis H37Rv using the Bactec-460 method [44]. For both studies, there was agreement for linoleic acid (MIC 50 µg/mL). However, in this study, the results for oleic acid (MIC 100 µg/mL) and palmitic acid (MIC 25 µg/mL) against H37Rv using the Microplate Alamar Blue Assay were the opposite of those reported by Saravanakumar. On the other hand, Hirsch and Barchet published the bacteriostatic activity of saturated fatty acids C 10 -C 16 against M. tuberculosis and those results are in agreement with the antimycobacterial activity of palmitic acid (C16:0) found in our study [45]. Reports in the literature have shown that long-chain unsaturated fatty acids such as oleic and linoleic acid are selective inhibitors of the enoyl-acyl carrier protein reductase (FabI), which account for their antibacterial activities through the inhibition of fatty acid synthesis [46]. Therefore, these experimental findings could be considered to explain the antimycobacterial activities of oleic and linoleic acids.

General Experimental Procedures
1D and 2D NMR spectra were recorded on Varian spectrometers at 400 and 700 MHz using CDCl 3 as solvent and TMS as the internal standard. CC was carried out to fractionate the active extract. Fractions were monitored by Si gel thin layer chromatography and observed under UV light at 254 and 364 nm. The analysis of volatile constituents of hexane extract was performed on a HP Agilent Technologies 6890 gas chromatograph equipped with a MSD 5973 quadrupole mass detector (HP Agilent, CA, USA) in electron impact mode at 70 eV. On the other hand, in vitro anti-tuberculosis test was performed using the Alamar Blue microassay.

Mycobacterium Tuberculosis Strains
M. tuberculosis H 37 Rv (27294) sensitive to all five first-line antituberculosis drugs (streptomycin, isoniazid, rifampicin, ethambutol and pirazinamide) was obtained from the American Type Culture Collection (ATCC). Multidrug resistant M. tuberculosis H10, M15, and M26 strains were obtained from sputum of tuberculosis patients. These specimens were kindly provided by Dr. Virgilio Bocanegra-García from the Centro de Biotecnología Genomica del Instituto Politécnico Nacional. The local ethics committee approved all protocols used in this study.

Plant Material
Fruits and flowering branches of Citrus aurantifolia were collected in Montemorelos, Nuevo León, México in May 2009. A voucher specimen (Number: 024769) was deposited at the Herbarium of the Faculty of Biological Sciences of the Autonomous University of Nuevo León.

Extraction and Isolation of Constituents from C. aurantifolia
Peels (1.4 kg) were removed from fresh fruits (7.8 kg) and macerated twice with n-hexane (6 L) for 72 h at room temperature. Solvent was removed under reduced pressure to give a yellowish oily residue (16.05 g). The n-hexane extract (16.

GC-MS Analysis
Chemical composition of volatile compounds from the active hexane extract was analysed on a gas chromatograph equipped with a quadrupole mass detector in electron impact mode at 70 eV. Volatile compounds were separated on a HP 5MS capillary column (25 m long, 0.2 mm i.d., 0.3 m film thickness). The oven temperature was set at 40 °C for 2 min and then programmed from 40 to 260 °C at 10 °C/min, and keep it 20 min at 260 °C. Mass detector conditions were as follows: interphase temperature 200 °C and mass acquisition range 20-550. Temperature of injector and detector were set to 250 °C and 280 °C, respectively. The splitless injection mode was carried out with 1 L of oily extract. The carrier gas was helium at a flow rate of 1 mL/min. Identification of volatiles was performed comparing their mass spectra with those of the National Institute of Standards and Technology NIST 1.7 library. In addition, a standard solution of C7-C40-alkanes was used to obtain the retention index of compounds and comparing them with literature data [47]. Semi-quantitative data were calculated from the GC peak areas without using correction factors and were expressed as relative percentage (peak area %) of the total volatile constituents identified.

Antimycobacterial Activity
The activity of all compounds against the M. tuberculosis strains was determined using the Microplate Alamar Blue Assay (MABA) as previously described in the literature [8,9].