Article pubs.acs.org/jnp
Airborne Antituberculosis Activity of Eucalyptus citriodora Essential Oil René F. Ramos Alvarenga,‡ Baojie Wan,† Taichi Inui,† Scott G. Franzblau,† Guido F. Pauli,†,‡ and Birgit U. Jaki*,†,‡ †
Institute for Tuberculosis Research and ‡Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, United States S Supporting Information *
ABSTRACT: The rapid emergence of multi- and extensively drug-resistant tuberculosis (MDR/XDR-TB) has created a pressing public health problem, which mostly affects regions with HIV/AIDS prevalence and represents a new constraint in the already challenging disease management of tuberculosis (TB). The present work responds to the need to reduce the number of contagious MDR/XRD-TB patients, protect their immediate environment, and interrupt the rapid spread by laying the groundwork for an inhalation therapy based on anti-TB-active constituents of the essential oil (EO) of Eucalyptus citriodora. In order to address the metabolomic complexity of EO constituents and active principles in botanicals, this study applied biochemometrics, a 3-D analytical approach that involves highresolution CCC fractionation, GC-MS analysis, bioactivity measurements, and chemometric analysis. Thus, 32 airborne anti-TB-active compounds were identified in E. citriodora EO: the monoterpenes citronellol (1), linalool (3), isopulegol (5), and α-terpineol (7) and the sesquiterpenoids spathulenol (11), β-eudesmol (23), and τ-cadinol (25). The impact of the interaction of multiple components in EOs was studied using various artificial mixtures (AMxs) of the active monoterpenes 1, 2, and 5 and the inactive eucalyptol (33). Both neat 1 and the AMx containing 1, 2, and 33 showed airborne TB inhibition of >90%, while the major E. citriodora EO component, 2, was only weakly active, at 18% inhibition.
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global burden has been attributed to India, the Russian Confederation, and China.2 MDR-TB refers to M.tb strains resistant to at least isoniazid and rifampicin, and XDR-TB to the strains resistant to isoniazid, rifampicin, any of the aforementioned fluoroquinolones, and either kanamycin, capreomycin, or amikacin.2,8 Owing to the increasing abundance of MDR-/XDR-TB, for which the current contemporary drug regimen lacks efficacy, there is a particular need to break the vicious cycle of spreading MDR/XDR-TB infections in the immediate environment of infected patients. In addition, a highly desirable property of any new intervention would be to replace and/or supplement current chemotherapy and reduce the length of treatment. Few reports of the use of essential oils (EOs) in anti-TB inhalation therapy have been published. In 1899, cinnamon and peppermint EOs were already used to treat TB.9 Although the initial results showed no growth inhibitory activity, the importance of treating TB through inhalation, as well as the screening of EOs in the gaseous phase, was stated.9 Two other studies using Mentha piperita L. EO for inhalation therapy in combination with multidrug therapy suggested that the EO was
uberculosis (TB) is a chronic disease, acquired mostly by inhalation of the pathogen Mycobacterium tuberculosis (M.tb), which establishes itself in the macrophages and causes lung disruption.1 TB is the second major cause of mortality among infectious diseases after HIV. In recent years, there were approximately 9 million new TB cases and 1.4 million TB deaths annually, among which 990 000 were HIV-negative patients and 430 000 were HIV-related.2 The reactivation of dormant or latent TB in asymptomatic patients, the coinfection with HIV or otherwise immunocompromised patients, and the emergence of multi-drug- and extensively drug-resistant (MDR/XDR-TB) strains have prioritized the need of discovering new scaffolds/drugs with antimycobacterial activity.3−5 The priorities include the search for alternative therapies in order to target MDR/XDR-TB strains and to shorten the treatment length and the bactericidal/bacteriostatic drug intake. MDR/XDR-TB treatments are more toxic, longer, and less effective and involve the use of second-line drugs, such as amikacin, kanamycin, capreomycin, and the fluoroquinolones levofloxacin, moxifloxacin, gatifloxacin, ethionamide, protionamide, cycloserine, and aminosalicylic acid,6 and they are less tolerable and present more drug−drug interactions in cases of patients co-infected with HIV.7,8 According to the WHO,2 XDR-TB has been identified in 84 countries, and an estimate of 310 000 MDR-TB cases among pulmonary TB patients were reported in 2011; a contribution of approximately 60% to the © 2014 American Chemical Society and American Society of Pharmacognosy
Special Issue: Special Issue in Honor of Otto Sticher Received: October 15, 2013 Published: March 18, 2014 603
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more effective in infiltrative pulmonary tuberculosis and may act as a TB recurrence prevention agent.10,11 In another study, a Eucalyptus globulus EO preparation was used to treat pulmonary TB in one patient where sputum conversion was observed after inhalation therapy.12 In the last three cases, conventional treatment was followed. Biochemometric methodology and its application in drug discovery has been recently proposed.13 The approach involves the combination of high-resolution preparative fractionation (e.g., countercurrent separation methods), high-resolution analysis for chemical identification (e.g., GC-MS or LC-MS), and bioassay evaluation of all fractions, as well as chemometric evaluation of the resulting physicochemical and biological data by means of Pearson’s correlation. Biochemometric analysis allows the assignment and identification of biologically active components in complex mixtures without further fractionation and isolation of the bioactive principles.13 Using this method, the anti-TB bioactive compounds from Oplopanax horridus and Dracaena angustifolia have been identified and reported.13,14 The volatile monoterpenoids responsible for anti-TB activity do not show a specific mechanism of action, but rather a cell surface/membrane-related “sanitizing” effect.15−29 Therefore, development of resistance30 is not expected, and for safety reasons, a virulent pan-sensitive strain of M.tb. H37Rv (ATCC 27294) was chosen for this initial study. The goal of the present work was to identify the optimal composition of the airborne anti-TB-active fraction(s) contained in the essential oil of the lemon-scented gum tree, Eucalyptus citriodora Hook. (Myrtaceae), through the application of the biochemometric approach, and to investigate synergy in artificial mixtures of EO components. The long-term aims are the possible use of E. citriodora EO preparations for inhalation treatment of TB in infected patients, to interrupt their contagiousness, and/or to treat healthy persons to prevent them from contracting TB.
Figure 1. Deconvoluted biochromatogram of the recombined highresolution countercurrent fractions (HR-HSCCC K-values in HterAc 10:1:10 on x-axis) of E. citriodora, reflecting their % inhibition (y-axis) measured in the gaseous contact assay (GCA) against Mycobacterium tuberculosis H37Rv. The biochromatogram distinguishes a total of 37 biopeaks, eight of which (biopeaks 3, 6, 8−10, 30, 33, and 34) correspond to the fractions (Fr.) with the highest bioactivity (Fr. 6, 9, 11−13, 45, 49, and 51).
sources,31,33−35 and it has become a powerful analytical tool in the drug/scaffold discovery process due to its lack of irreversible adsorption and, therefore, loss of bioactivity during bioassay-guided fractionation procedures.36 For the purpose of studying airborne anti-TB activity, this study employed a refined version of a gaseous contact assay (GCA; see Experimental Section and S2, Supporting Information), which was optimized to assess the susceptibility of virulent pan-sensitive strains of Mycobacterium tuberculosis H37Rv (M.tb) against volatile agents such as EOs. Preliminary studies had shown that the widely used broth and/or agarbased susceptibility assays such as MABA37 fail to detect the (airborne) anti-TB activity of EOs such as those of E. citriodora. In comparison to typical antituberculosis assays, the GCA method provides a means of susceptibility testing for volatile components such as EOs and EO components. The GCA allows the phytoconstituents to be distributed in the microatmosphere and interact with the target microorganism long enough to exert a biological activity. This newly developed antiTB assay is based on the microatmosphere technique,38 which consists of cultivating the target microorganism(s) in Petri dishes on agar medium and incubating the dishes in reversed position after depositing the essential oil/fraction on a filter paper in the middle of the dish cover (Supporting Information, S2). The test EO or fraction evaporates in the atmosphere of the dish, and the volatiles can exert their inhibitory effect on the inoculated microorganism. This method is suitable for EOs and their respective fractions, which usually are chemically complex, volatile without residue, and hydrophobic.39 The complete data set of the GCA results is shown in the Supporting Information, S6. The preparative HSCCC fractionation of E. citriodora EO in the biphasic system of HterAc (n-hexane/methyl tert-butyl ether/acetonitrile) 10:1:10 produced 160 fractions, which were recombined based on their TLC pattern, yielding 60 recombined fractions. From the GCA outcomes, eight of the recombined HR-HSCCC fractions contained the major
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RESULTS AND DISCUSSION In order to identify the composition of the airborne anti-TBactive E. citriodora EO fractions, the biochemometric approach was applied and involved the following four steps: (i) highresolution (HR) preparative fractionation of the complex mixture by means of high-speed countercurrent chromatography (HSCCC); (ii) bioactivity determination of the recombined HR-HSCCC fractions and establishment of a biochromatogram that correlates anti-TB inhibition percentages as the bioassay outcome vs CCC K-values (Figure 1); (iii) GCMS analysis of the HR-HSCCC recombined fractions and generation of a 3-D data set of CCC-GC-MS data (Supporting Information, S5); and (iv) chemometric analysis by application of Pearson’s correlation in order to establish the relationship between biological activity (biochromatogram) and the chemical 3-D matrix.13 HSCCC is a method that like all countercurrent separation (CS) techniques uses immiscible liquids as both mobile and stationary phases. Accordingly, the separation in CS is based on differential solubility and partitioning of the analyte(s) in the biphasic solvent systems, and the analyte(s) concentration in each phase is established by the partition coefficient (K).31−34 HSCCC provides the capability of fractionating crude and complex mixtures containing compounds of a wide range of polarities, molecular weights, functional groups, and chemical structure classes. Furthermore, HSCCC is a capable purification method for chemical constituents derived from natural 604
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Table 1. Individual E. citriodora EO Bioactive Components in the Anti-TB-Active Fractions Identified by the NIST MS Databasea #
compound name
K
calculated PTRI
r
MF
RMF
probability [%]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
citronellol citronellal linalool citronellyl acetate isopulegol p-menthan-3,8-diol α-terpineol (p-menth-1-en-8-ol) isopulegol acetate farnesal trans-farnesol spathulenol viridiflorol ledol globulol aromadendrene β-caryophyllene caryophyllenyl alcohol 1,5,5,8-tetramethyl-12-oxabicycle[9.1.0]dodeca-3,7-diene α-himalachene β-selinene β-cadinene δ-cadinene β-eudesmol α-eudesmol τ-cadinol cubenol geranylgeraniol 6-methyl-2,4-ditert-butylphenol alloaromadendrene 4,4a,5,6,7,8-hexahydro-4a-methyl-2(3H)-naphthalenone neoisopulegol* neoisoisopulegol*
0.69 1.02 0.74 1.55 0.87 0.87 0.82 1.55 1.79 1.79 1.18 1.55 1.18 1.79 2.98 2.98 1.60 1.55 1.60 1.79 2.98 2.98 1.60 1.80 1.55 1.80 1.60 1.79 2.98 1.55 0.82 2.98
1234 1159 1102 1356 1148 1341 1196 1280 1537 1620 1592 1643 1675 1583 1463 1431 1598 1627 1640 1608 1518 1535 1618 1641 1672 1675 2072 1522 1475 1568 1173 1184
0.90 0.51 0.60 0.69 0.73 0.48 0.92 0.75 0.70 0.80 0.83 0.68 0.78 0.84 0.73 0.61 0.69 0.53 0.69 0.70 0.69 0.54 0.77 0.74 0.83 0.76 0.78 0.70 0.53 0.78 0.46 0.70
935 939 870 924 900 860 886 897 924 822 900 919 803 891 902 929
935 940 872 925 904 862 915 897 941 826 900 932 804 892 913 931 808 867 954 832 898 873 840 863 910 824 858 884 909 822
39.2 51.3 59.3 35.8 36.1 49.7 42.4 88.1 84.1 95.4 55.0 82.0 72.8 73.6 55.9 55.4 45.9 53.8 56.7 73.6 80.1 79.6 95.4 96.5 80.8 74.5 76.6 90.4 54.9 86.1
844 953 823 892 832 840 840 907 800 856 883 902 821
a
Probability was set to 35% (ρ = 0.35) and spectral matching factor (MF) to 800. Listed parameters are the partition coefficients, K, in the HterAc 10:1:10 solvent system, determined experimentally from the HSCCC/EECCC chromatograms; calculated programmed temperature retention indices (PTRI); Pearson’s coefficient correlation factor (r); spectral matching factor (MF), and reversed matching factor (RMF). Compounds annotated with * were identified only by PTRI using a series of n-alkanes from C8 to C40.51,52,67,68
the presence of synergistic, potentiating, and/or antagonistic effects among components. The results of the biochemometric analysis are shown in Table 1. A high Pearson’s coefficient correlation value (r) is indicative of a highly probable biological active compound,13 as r is a measure of peak shape similarity (biopeak from deconvoluted biochromatogram vs GC peak) and, thereby, links chemical to biological data.13 Accordingly, the r correlation values can be used to locate bioactives in (bio)chromatograms, and the consistency of the relationship between bioactivity and the chemical entities is reflected by the r value. An r value higher than 0.70 is considered to indicate significance. The chemical structures of the bioactives assigned by this procedure are shown in Chart 1. A considerable number of positive coefficient correlations were obtained from the rather polar (0.6 ≤ K ≥ 1.2) and lipophilic (2.9 ≤ K ≥ 3.3) ranges of the HterAc 10:1:10 solvent system. At K = 0.69, corresponding to biopeak 3 and 92% inhibition in the GCA (Figure 1), a GC peak with a correlation of r = 0.90 was matched to the acyclic alcoholic monoterpene 1. At K = 0.81, biopeak 5 (76% inhibition), a GC peak corresponding to 7, showed a high correlation of r = 0.92. The guaiene-type
portions of the active principles, showing 92−100% airborne inhibition the M.tb. The GCA (% inhibition) and chromatographic (K values) data derived from the HSCCC fractionation were combined and plotted in order to produce the anti-TB biochromatogram shown in Figure 1. This biochromatogram is key to the biochemometric analysis13 by providing information about the distribution of the bioactive(s) after CS fractionation.40 In order to match the lipophilicity of the EO, the HSCCC separation was carried out with a nonaqueous solvent system, and elution-extrusion mode (EECCC) was employed to optimized the width of the sweet spot while minimizing solvent consumption.31 Our results indicated that the metabolites contained in the complex EO mixture form a multicomponent system in which all components “coexist”. Once this balanced system is disrupted, e.g., by performing HSCCC fractionation, the separated metabolites display different volatilities and diffusion rates in the microenvironment under GCA conditions compared to the original EO. The airborne M.tb growth inhibitory activity measured in the GCA is a function of the presence and diffusion speed of the bioactives,41 their concentration in the fractions and EOs, and 605
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Chart 1. Structures of the Anti-TB-Active Terpenoids Identified in the Essential Oil of E. citriodora Hook. by Biochemometric Analysisa
Monoterpenoids: β-citronellol (1), (S)-citronellal (2), linalool (3), citronellyl acetate (4), isopulegol (5), p-menthan-3,8-diol (6a/6b), α-terpineol (7), isopulegyl acetate (8). Sesquiterpenoids: farnesal (9), farnesol (10), spathulenol (11), viridiflorol (12), ledol (13), globulol (14), aromadendrene (15), β-caryophyllene (16), β-caryophyllene alcohol (17), α-humulene epoxide II (18), α-himalachene (19), β-selinene (20), βcadinene (21), δ-cadinene (22), β-eudesmol (23), α-eudesmol (24), τ-cadinol (25), cubenol (26). a
sesquiterpenoid, 11 (K = 1.18), showed an r value of 0.83. This compound has previously been reported as anti-TB bioactive from Oplopanax horridus, where it had exhibited a correlation value of 0.88 using biochemometric analysis.13 The guaienetype stereoisomers 13 and 14 showed r values of 0.78 and 0.84 (K = 1.18 and 1.79), respectively. The eudesmane-type sesquiterpenoid alcohols 23 and 24 (K = 1.80, and 1.60) correlated with their respective biopeaks with r values of 0.77 and 0.74, respectively. Finally, the cadinene-type alcohols 25 and 26, corresponding to biopeaks at K = 1.55 and K = 1.80, showed significant correlations with r values of 0.83 and 0.76, respectively. The analysis of the composition of the individual bioactive fractions based on relative GC abundance (AUC %) is shown in Figure 2. The bioactivity of the fractions F6, F7, and F8 can be attributed to the presence of the highly active compound 1. The biological response decreases from F6 to F7 and F8 due to the presence of inactive compounds, and this decline reflects the concentration drop of 1 across the fractions. However, the 20% points equivalent to 22% decrease in activity from F6 to F7 cannot be explained with the mere concentration decrease of 1, which still counts for more than 90% of the fraction, but more likely is due to a major reduction in the content of other active and/or synergistic constituents. This can be addressed
more specifically in fraction 8, which contains at least one additional active component (potential candidates are linalool, α-terpineol, neoisopulegol, and p-menthan-3,8-diol) and explains the relatively moderate activity loss of 16% points (≡ 17.4% decrease) compared to fraction 1 and relative to the almost 50% points (equivalent to 48.4%) decrease of 1. In the case of the anti-TB-bioactive fraction F9, the combination of 1 (11%), 2 (2%), 5 (60%), and 7 (0.61%) can be made responsible for the high M.tb growth inhibition. A significant change in bioactivity is seen in F10, where 1 is decreased (1%) and the active compound 5 is increased (51%), while the moderately active compound 2 rises in concentration to 45%. The activity of F11 and F12, in which 1 is almost absent at 90%, is another strong indication of a synergistic effect among 2 and the combination of 1, 5, 6, 11, 31, and 32. The bioactivity of F13 responds to the presence and dynamic interactions of active compounds in the mixture, such as compounds 1, 5, 11, and 13. The single compound and artificial mixture (AMxs) analysis by GCA is summarized in Figure 3. Four single entities, 1, 2, 5, and 33, and their combinations were tested under identical GCA conditions. Compound 2, the major constituent of E. citriodora EO, showed a low growth inhibition of M.tb H37Rv (18%), while compound 1 exerted a 100% inhibition. 606
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Figure 2. GC relative area % (AUC) of anti-TB bioactive fractions (Fr.6 to Fr.13). The graph represents the relative composition of monoterpenoids, sesquiterpenoids, and miscellaneous for each anti-TB bioactive fraction in the K value range from 0.60 to 1.25. Compounds annotated with * were identified by PTRI.51,52,67,68
possible interpretation is that 33 might increase the penetration of the (other) bioactives by enhancing cell-membrane permeation.42 Similar results related to relative compositions have been reported for Staphylococcus aureus and Salmonella typhi as target microorganisms when analyzing inhibition in a contact test in solid agar media.43 This report described activity for the combination of the compounds 2 + 1 + 33 in the ratio 90:7.5:2.5, suggesting a synergistic effect between these three terpenoids. Nevertheless, isobolographic analysis would have to be carried out to prove synergy in strictu sensu. A similar analogy could be applied to AMx4, which exhibited a high airborne TB inhibition at 86%, while the single constituents 5 and 2 only exerted 64% and 18% inhibition, respectively. The antimicrobial properties of EOs and their chemical constituents have been studied extensively, and it has been outlined that those containing aldehydes and/or phenols as major components often showed the highest antibacterial activity, followed by those containing terpene alcohols.44 EOs composed of terpene ketones or ethers generally showed weaker activity, and those containing terpene hydrocarbons no activity.44 One earlier study screened the antimycobacterial activity of several Eucalyptus species (E. citriodora Hook., E. botryoides Sm., E. camaldulensis Dehn, E. deglupta Blume, E. globules Labill., E. grandis W. Hill ex Maiden, E. maculata Hook., and E. tereticornis Sm.) against Mycobacterium avium.45 This mycobacterial strain was found to be sensitive to all the EOs in which the major components were citronellal and/or citronellol, including E. citriodora Hook.45 According to another study,43 E. citriodora exerts its antibacterial activity against Staphylococcus aureus and Salmonella typhi through the synergy of citronellal and citronellol, while 1,8-cineole is active only when combined with a 90:7.5:2.5 mixture of citronellal, citronellol, and cineole, the natural blend of these three compounds in the native EO.43,46 Such terpenoids and their lipophilic nature may be a key feature in the penetration and disruption of the Mycobacterium cell wall.47 The minimum inhibitory concentrations (MICs) of both volatile and non-
Figure 3. Comparison of the airborne anti-TB activity (inhibition %) against M.tb H37Rv in the GCA of neat monoterpenes and their artificial mixtures (AMxs) supports the conclusion of the involvement of synergistic activity among EO constituents. While citronellal (2) shows low anti-TB activity, citronellol (1) shows high inhibition (100%). Eucalyptol (33) shows no activity, and isopulegol (5) exhibits a 64% inhibition. Considering the activities of the neat constituents, AMx1 (2 + 1 in 1:1 ratio) and AMx2 (2 + 1 in 9:1 ratio) exert anti-TB activity in a citronellol (1)-dose-dependent manner. However, AMx3 (2 + 1 + 33 in 8:1:1 ratio) and AMx4 (2 + 1 + 5 in 8:1:1 ratio) show 96% and 86% inhibition, respectively, and exemplify the overadditive and/or synergistic effects.
Considering the activities observed for AMx1 (2 + 1 in 1:1 ratio) and AMx2 (2 + 1 in 9:1 ratio), the single entity 1, and the active fraction 6 (biopeak 3, 92% inhibition), a plausible and linear semiquantitative purity−activity relationship may be derived. Although compound 33 lacks airborne anti-TB activity, when combined with 1 and 2 such as in AMx3 (2 + 1 + 33 in 8:1:1 ratio), the inhibitory activity increased to 96%. One 607
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volatile terpenoids from plant sources against M.tb H37Rv have been previously described using the BACTEC radiometric bioassay,36,37 with the result that the activity increases with the lipophilicity of the molecules.32 This is exemplified in the series of the following compounds, which are sorted by decreasing lipophilicity and decreasing activity (Supporting Information, S3): phytol (C20, MIC 2 μg/mL) > farnesol (C15, MIC 8 μg/ mL) > geraniol (C10, MIC 64 μg/mL) equal to citronellol (C10, MIC 64 μg/mL) > nerol (C10, MIC 128 μg/mL).48 The monoterpenes dissectol A and (+)-bornyl piperate have also been reported as having M.tb growth inhibitory activity.49 In summary, the present in vitro results indicate that an EO such as that of E. citriodora can potentially play an important role as a supplemental TB treatment in the form of inhalation therapy. The outcome of the biochemometric analysis provides preliminary information for a combined biologically and chemical standardization of such E. citriodora EO preparations. Such materials should be rich in citronellol, which is highly active as a single entity, isopulegol, linalool, citronellyl acetate, and isopulegyl acetate, as well as in bioactive sesquiterpenoids such as spathulenol, ledol, and aromadendrene, among others. Moreover, it can be concluded that EOs rich in citronellol, such as those of Cinnamomum citriodorum Thw. (Lauraceae),50 Pelargonium graveolens (Geraniaceae),41 or P. roseum (Geraniaceae),41 as well as artificial mixtures of citronellol, isopulegol, and other bioactives and enhancers including 1,8cineole herein reported and tested for synergy, could also be of potential therapeutic use in the protection against MDR/XDRTB. Studying the behavior of single bioactives in the native unfractionated EO remains a major challenge to the inherent dynamic (volatility) complexity of the multicomponent system, and further separation or fractionation without loss of activity is pivotal in EO research. CS technology, which avoids adsorption and allows for loss-free separation, is currently the only suitable separation method for this kind of bioactivity studies. The finding of fractions that contained small amounts of (presumably highly) active compound(s) that could be statistically correlated in the biochemometric matrix supports the conclusion that low-abundance bioactive compounds are also present and that these constituents can be further characterized, e.g., by GC-HR-MS. With regard to analytical methodology, it is important to point out that the establishment and combination of orthogonal analytical separation (HSCCC, GC-MS) and specific biological screening platforms (GCA) paired with statistical analysis are of crucial importance when unraveling, at least partially, the biological and chemical complexity as well as the dynamic interactions in EOs. It has been stated extensively that EOs exert their antibacterial activities through synergistic effects among their structurally varied components, and in many cases, the biological response has been attributed to the major components. As it has been proven here, the major E. citriodora EO component, citronellal (2), does not exert high inhibition on its own, and its presence in the EO actually masks many minor compounds, which are much more biologically active. Nevertheless, potentiation, additive, and/or synergistic effects among major and minor phytoconstituents cannot be disregarded. These studies were conducted with a virulent pan-sensitive M.tb. strain, but as resistance toward volatile essential oil components is unlikely, the bioactivity results should be transferrable to MDR/XDR-TB strains. For further proof,
follow-up studies with more resistant strains are planned. The results of this project are intended to lay the foundation for an inhalation therapy, and the essential oil components are expected to work on the lung surface, which eliminates bioavailability concerns. An inhalation-based mouse model is currently under development and expected to provide in vivo results in the future.
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EXPERIMENTAL SECTION
Essential Oil. The EO of Eucalyptus citriodora Hook. was purchased at Original Swiss Aromatics (San Rafael, CA, USA) and authenticated by comparison of a GC/MS fingerprint with published data.46,51−58 A characteristic of E. citriodora EO is its high content of citronellal (60−90% w/w),46,51−58 followed by its content of citronellol (4−13% w/w)46,51−54,56,58 and isopulegol (2−8%).52,54 The cyclic monoterpenoid 1,8-cineole or eucalyptol is the characteristic compound of Eucalyptus species such as E. radiata,56 E. camaldulensis,53,57 E. deglupta,53 E. urophylla,53 E. propinqua,53 and E. saligna.53 Aromadendrene, globulol, and 1,8-cineole are typical of E. globulus;46,53,59 β-pinene, p-cymene, and β-terpineol are characteristic of E. alba;46 α-pinene, 1,8-cineole, p-cymene, and cryptone are characteristic of E. tereticornis;53,57 α-pinene, p-cymene, and myrtenal are typical of E. robusta;46,53 p-cymene and 1,8-cineole are typical of E. houseana;53 β-caryophyllene and 1,8-cineole are typical of E. raveretiana;53 α-pinene and caryophyllene oxide are characteristic of E. torelliana;52,57 the monoterpenoids limonene, neral, and geranial are usual of E. staigeriana;55 1,8-cineole and α-pinene (in different ratios) are common of E. dundasii,60 E. salubris,60 E. spathulata,60 and E. brockwayii.60 Solvents and Monoterpenes. The monoterpenes, citronellal (96%), citronellol (98%), isopulegol (99%), and eucalyptol (99%), as well as all analytical grade solvents, DCM, MeCN, MTBE, n-hexane, toluene, and EtOAc, were purchased from Sigma-Aldrich (St. Louis, MO, USA). HSCCC Procedure. The EO of E. citriodora was fractionated by HSCCC in reversed-phase mode, using the nonaqueous solvent system of HterAc 10:1:10. The fractionation was carried out in a J-type instrument (model CCC-1000; Pharma-Tech Research Corp., Baltimore, MD, USA) containing a self-balancing centrifuge rotor equipped with 3 × 120 mL coils, connected to an HPLC peristaltic pump and an LKB BROMMA 2111 Multirac fraction collector. The rotation speed was set to 800 rpm, and the flow rate to 1.5 mL/min. The system was equilibrated for 2 h under the same conditions of rotation speed and flow rate, to give a stationary phase retention of 93.1% (Sf = 0.931). E. citriodora EO (2 mL) was dissolved in 8 mL of the mobile phase prior to injection (10 mL). Fraction collection started 1 h after loading and was set at 4 min (6 mL) per fraction. Elution was performed for 5 h, followed by sweep elution and extrusion of the stationary phase for another 5 h to afford 160 TLCmonitored fractions. The K values were calculated using volumetric measurements and Microsoft Excel 2010. Thin-Layer Chromatography. All the HSCCC fractions were analyzed by TLC. The solvent system used for developing the TLC plates was toluene/EtOAc (95:05). TLC plates of Alugram silica G/ UV254 10 cm × 20 cm with a thickness of 0.20 mm (Macherey-Nagel, Düren, Germany) were used. The compound bands were detected with vanillin/H2SO4 reagent. The HSCCC fractions were recombined according to their TLC profiles (Supporting Information, S4). GC-MS Procedure. The GC-MS analysis was carried out with an Agilent GC MS system, model 7890A (G3440A), equipped with a high-resolution Agilent column HP-5MS (length 30 mts × i.d. 0.250 mm × film 0.25 μm), an autosampler injector 7683B series, and a GC/ MS triple quad 7000A detector. The oven program was as follows: 40 °C held for 2 min and then increased to 140 °C at 3 °C/min; then the temperature was increased from 140 to 250 °C at a rate of 10 °C/min and held constant for 20 min. Helium was used as carrier gas at a flow rate of 1 mL/min. The injector was maintained at 250 °C. The samples were prepared in DCM (250 μL of EO fraction in 1000 μL of solvent) and injected (1 μL) using a split ratio of 1:75. The mass 608
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spectra were recorded over the m/z range from 35 to 650 at one scan per second, with an ionization energy of 70 eV and the ion source temperature set at 230 °C.61 The GC-MS of the homologous series of n-alkanes for the purpose of calculating the programmed-temperature retention indices (PTRI) was recorded under the same conditions. Identification of volatile components was achieved by three methods: (i) comparison of the mass spectra with the NIST online library through the MassHunter workstation software, version B.03.00 (Agilent Technologies, Inc.); (ii) calculation of the PTRI using the homologous series of n-alkanes (C8−C20 and C10−C40); (iii) peak enrichment/co-injection of authentic standards of terpenoid compounds. For PTRI calculation, the third-order polynomial interpolation was used for calculation of the retention indices, considering that this method provides a good fit to nonlinear GC data sets and smoothness and allows for an easy computability with no introduction of extraneous oscillations.62−64 In the case of the MS library search, the minimum matching factor (MF) required was set to 800 and the probability to 0.35 (35%).65 In the MS library search, a measure of the spectra similarity (unknown vs reference) is known as MF, and a high MF is assigned to an unknown represented in the MS library. MFs rank zero in cases of spectra having no peaks in common to 1000 in cases of perfect matches.65 There is little correlation between high MF values65 and the probability of the hit or unknown being found in the MS library, which is due to a “high average of positional isomers with closely undifferentiated spectra”.65 GCA Procedure. This bioassay was modified from the Inouye method66 and optimized for the assessment of essential oil/fractions activity vs M.tb. A virulent, pan-sensitive strain of M.tb. H37Rv (ATCC 27294) was prepared and stored at −80 °C. Aliquots were thawed and plated to determine the actual number of colony-forming units (cfu) per mL. At the time of assay, cultures were thawed and diluted to obtain a cell density such that plating of 100 μL on Middlebrook 7H11 agar in standard Petri dishes will result in 200−400 colonies per plate following three weeks of incubation at 37 °C. The inoculum was added to the 7H11 agar in 100 μL and allowed to dry at room temperature in the biosafety cabinet for 4−5 h. Essential oils, fractions, and pure compounds were solubilized and diluted if necessary in EtOAc, and 5 μL (100 mg/mL) was applied to sterilized, 42.5 mm diameter Whatman #1 filter paper disks placed in an inverted Petri dish lid (volume of gaseous phase ≈ 70 cm3). The Petri dish bottom, containing the M.tb-inoculated agar, was inverted and placed on top of the essential oil/fraction-containing lid. All plates were immediately sealed with shrink seal and incubated at 37 °C. Colony formation was observed at 14 days, and final counts of cfu’s were made after 21 days of incubation. Biochemometric Analysis. The biochemometric analysis was performed according to the method of Inui.13 A total of 60 recombined HR-HSCCC fractions were submitted to parallel GCMS analysis and GCA, as described above. The biochromatogram was obtained from the HSCCC K values and GCA % inhibition data and subsequently deconvoluted by using OriginPro 9.0 software (Figure 1). The total ion chromatograms from each fraction were baseline corrected by applying the peak analyzer function in OriginPro 9.0. Chemical composition of each fraction was determined using the identification methods described above (NIST library, PTRI, authentic samples). The combination of the HSCCC (K values) and GC-MS data (GC-MS tR and abundance) forms the 3D matrix, which was analyzed by Pearson’s correlation against the deconvoluted biochromatogram (% inhibition vs K values). Pearson’s correlation coefficient, r, was calculated using OriginPro 9.0, with the confidence interval set at 95%. In order to perform the chemometric analysis, each biopeak row was compared against each GC retention time within the same region of K values. The r values were calculated to reflect the similarity of the peak shapes (peak height transition) between the biopeak and chromatographic peak at GC retention time. The GC retention times linked to a high r value (ideally, close to 1) were likely to correspond to the compound underlying the peak exhibiting the activity reflected in the biopeak.
Article
ASSOCIATED CONTENT
S Supporting Information *
The list of the publication series on Residual Complexity and Bioactivity of Natural Products. The gaseous contact assay (GCA) setting for testing EOs. The comparison of the anti-TB MIC (BACTEC) values of terpenoids and other anti-TB natural products. The TLC of the recombined HSCCC fractions of the EO of E. citriodora Hook. The 3D CCC-GCMS matrix. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors wish to express gratitude to Drs. J. B. McAlpine and C. Simmler for their critical comments and helpful suggestions during the preparation of the manuscript, as well as to Mrs. D. Hamm, Olympia Apotheke, Karlsruhe (Germany), for stimulating discussions and inspiration.
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DEDICATION Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich, Switzerland, for his pioneering work in pharmacognosy and phytochemistry.
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REFERENCES
(1) Newton, S. M.; Lau, C.; Wright, C. W. Phyther. Res. 2000, 322, 303−322. (2) WHO. Global Tuberculosis Report 2012; 2012. (3) Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K. Nature 2011, 469, 483−490. (4) Georgiev, V. S. Int. J. Antimicrob. Agents 1994, 157−173. (5) Petrini, B.; Hoffner, S. Int. J. Antimicrob. Agents 1999, 13, 93−97. (6) Jassal, M.; Bishai, W. R. Lancet Infect. Dis. 2009, 9, 19−30. (7) Ma, Z.; Lienhardt, C.; McIlleron, H.; Nunn, A. J.; Wang, X. Lancet 2010, 375, 2100−2109. (8) Gandhi, N. R.; Nunn, P.; Dheda, K.; Schaaf, H. S.; Zignol, M.; van Soolingen, D.; Jensen, P.; Bayona, J. Lancet 2010, 375, 1830− 1843. (9) Murrel, W. Br. Med. J. 1899, 1, 202−204. (10) Shkurupiy, V. A.; Mostovaya, G. V.; Kazarinova, N. V.; Ogirenko, A. P.; Nikonov, S. D.; Tkachev, A. V.; Tkachenko, K. G. Probl. Tuberk. 2002, 36−39. (11) Shkurupiy, V. A.; Odintsova, O. A.; Kazarinova, N. V.; Tkachenko, K. G. Probl. Tuberk. Bolezn. Legk. 2006, 43−45. (12) Sherry, E.; Warnke, P. H. Phytomedicine 2004, 95−97. (13) Inui, T.; Wang, Y.; Pro, S. M.; Franzblau, S. G.; Pauli, G. F. Fitoterapia 2012, 83, 1218−1225. (14) Case, R. J.; Franzblau, S. G.; Wang, Y.; Cho, S. H.; Soejarto, D. D.; Pauli, G. F. J. Ethnopharmacol. 2006, 106, 82−89. (15) Trombetta, D.; Castelli, F.; Sarpietro, M. G.; Venuti, V.; Cristani, M.; Daniele, C.; Saija, A.; Mazzanti, G.; Bisignano, G. Antimicrob. Agents Chemother. 2005, 49, 2474−2478. (16) Cristani, M.; D’Arrigo, M.; Mandalari, G.; Castelli, F.; Sarpietro, M. G.; Micieli, D.; Venuti, V.; Bisignano, G.; Saija, A.; Trombetta, D. J. Agric. Food Chem. 2007, 55, 6300−6308. (17) Devi, K. P.; Nisha, S. A.; Sakthivel, R.; Pandian, S. K. J. Ethnopharmacol. 2010, 130, 107−115. (18) Silva, F.; Ferreira, S.; Queiroz, J. a; Domingues, F. C. J. Med. Microbiol. 2011, 60, 1479−1486. 609
dx.doi.org/10.1021/np400872m | J. Nat. Prod. 2014, 77, 603−610
Journal of Natural Products
Article
(19) Xu, J.; Zhou, F.; Ji, B.-P.; Pei, R.-S.; Xu, N. Lett. Appl. Microbiol. 2008, 47, 174−179. (20) Sharma, A.; Bajpai, V. K.; Baek, K.-H. J. Food Saf. 2013, 33, 197−208. (21) Á lvarez-Ordóñez, A.; Carvajal, A.; Arguello, H.; Martínez-Lobo, F. J.; Naharro, G.; Rubio, P. J. Appl. Microbiol. 2013, 115, 50−60. (22) Diao, W.-R.; Hu, Q.-P.; Zhang, H.; Xu, J.-G. Food Control 2014, 35, 109−116. (23) Saad, N. Y.; Muller, C. D.; Lobstein, A. Flavour Fragr. J. 2013, 28, 269−279. (24) Sikkema, J.; De Bont, J. A. M.; Poolman, B. Microbiol. Rev. 1995, 59, 201−222. (25) Lambert, R. J.; Skandamis, P. N.; Coote, P. J.; Nychas, G. J. J. Appl. Microbiol. 2001, 91, 453−462. (26) Helander, I. M.; Alakomi, H.; Latva-Kala, K.; Mattila-Sandholm, T.; Pol, I.; Smid, E. J.; Gorris, L. G. M.; von Wright, A. J. Agric. Food Chem. 1998, 8561, 3590−3595. (27) Gustafson, J. E.; Liew, Y. C.; Chew, S.; Markham, J.; Bell, H. C.; Wyllie, S. G.; Warmington, J. R. Lett. Appl. Microbiol. 1998, 26, 194− 198. (28) Cox, S. D.; Mann, C. M.; Markham, J. L.; Bell, H. C.; Gustafson, J. E.; Warmington, J. R.; Wyllie, S. G. J. Appl. Microbiol. 2000, 88, 170−175. (29) Harold, F. Adv. Microb. Physiol. 1969, 4, 45−104. (30) Zhang, Y.; Yew, W. W. Int. J. Tuberc. Lung Dis. 2009, 13, 1320− 1330. (31) Berthod, A.; Friesen, J. B.; Inui, T.; Pauli, G. F.; Analytiques, S.; Lyon, D.; Cpe, B. Anal. Chem. 2007, 79, 3371−3382. (32) Friesen, J. B.; Pauli, G. F. J. Chromatogr. A 2007, 1151, 51−59. (33) Pauli, G. F.; Pro, S. M.; Friesen, J. B. J. Nat. Prod. 2008, 71, 1489−1508. (34) Marston, A. Phytochemistry 2007, 68, 2786−2798. (35) Hostettmann, K.; Marston, A. J. Liq. Chromatogr. Relat. Technol. 2001, 24, 1711−1721. (36) Pauli, G. F.; Case, R. J.; Inui, T.; Wang, Y.; Cho, S.; Fischer, N. H.; Franzblau, S. G. Life Sci. 2005, 78, 485−494. (37) Collins, L.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004−1009. (38) Kalemba, D.; Kunicka, A. Curr. Med. Chem. 2003, 10, 813−829. (39) Lahlou, M. Phyther. Res. 2004, 18, 435−448. (40) Case, R. J.; Wang, Y.; Franzblau, S. G.; Soejarto, D. D.; Matainaho, L.; Piskaut, P.; Pauli, G. F. J. Chromatogr. A 2007, 1151, 169−174. (41) Kloucek, P.; Smid, J.; Frankova, A.; Kokoska, L.; Valterova, I.; Pavela, R. Food Res. Int. 2012, 47, 161−165. (42) Hendry, E. R.; Worthington, T.; Conway, B. R.; Lambert, P. A. J. Antimicrob. Chemother. 2009, 64, 1219−1225. (43) Low, D.; Rawal, B.; Griffin, W. Planta Med. 1974, 26, 184−189. (44) Inouye, S.; Takizawa, T.; Yamaguchi, H. J. Antimicrob. Chemother. 2001, 565−573. (45) Leite, C. Q.; Moreira, R. R. D.; Neto, J. J. Fitoterapia 1998, 69, 282−283. (46) Cimanga, K.; Kambu, K.; Tona, L.; Apers, S.; De Bruyne, T.; Hermans, N.; Totté, J.; Pieters, L.; Vlietinck, A. J. J. Ethnopharmacol. 2002, 79, 213−220. (47) Copp, B. R. Nat. Prod. Rep. 2003, 20, 535−557. (48) Cantrell, C. L.; Franzblau, S. G.; Fischer, N. H. Planta Med. 2001, 685−694. (49) Copp, B. R.; Pearce, A. N. Nat. Prod. Rep. 2007, 24, 278−297. (50) Leela, N. K.; Vipin, T. M.; Priyanka, V.; Shafeekh, K. M.; Rema, J. J. Essent. Oil Res. 2012, 24, 507−511. (51) Rao, B. R. R.; Kaul, P. N.; Syamasundar, K. V; Ramesh, S. Flavour Fragr. J. 2003, 1, 133−135. (52) Chalchat, J. C.; Figuérédo, G.; Loumouamou, A. N.; Silou, T.; Mapola, G. J. Essent. Oil Res. 2009, 21, 295−299. (53) Traoré, N.; Sidibé, L.; Figuérédo, G.; Chalchat, J. C. J. Essent. Oil Res. 2010, 22, 510−513. (54) Singh, H. P.; Kaur, S.; Negi, K.; Kumari, S.; Saini, V.; Batish, D. R.; Kohli, R. K. LWT−Food Sci. Technol. 2012, 48, 237−241.
(55) Gusmão, N. M. S.; Oliveira, J. V. De; Daniela, M.; Navarro, A. F.; Dutra, K. A.; Walkiria, A.; Wanderley, M. J. A. J. Stored Prod. Res. 2013, 54, 41−47. (56) Mulyaningsih, S.; Sporer, F.; Reichling, J.; Wink, M. Pharm. Biol. 2011, 49, 893−899. (57) Chalchat, J. C.; Garry, R.-P.; Sibidé, L.; Harama, M. J. Essent. Oil Res. 2000, 12, 695−701. (58) Lee, Y.; Kim, J.; Shin, S.; Lee, S.; Park, I. Flavour Fragr. J. 2008, 23, 23−28. (59) Mulyaningsih, S.; Sporer, F.; Zimmermann, S.; Reichling, J.; Wink, M. Phytomedicine 2010, 17, 1061−1066. (60) Zhang, J.; An, M.; Wu, H. Plant Growth Regul. 2012, 68, 231− 237. (61) Cheng, S.-S.; Huang, C.-G.; Chen, Y.-J.; Yu, J.-J.; Chen, W.-J.; Chang, S.-T. Bioresour. Technol. 2009, 100, 452−456. (62) Sun, Y.; Zhang, R.; Wang, Q.; Xu, B. J. Chromatogr. A 1993, 657, 1−15. (63) Guan, Y.; Zhou, L. J. Chromatogr. 1991, 552, 187−195. (64) Castello, G.; Moretti, P.; Vezzani, S. J. Chromatogr. 1993, 635, 103−111. (65) Stein, S. E. J. Am. Soc. Mass Spectrom. 1994, 5, 316−323. (66) Inouye, S.; Yamaguchi, H.; Takizawa, T. J. Infect. Chemother. 2001, 7, 251−254. (67) Babushok, V. I.; Linstrom, P. J.; Zenkevich, I. G. J. Phys. Chem. Ref. Data 2011, 40, 43101−43147. (68) Gbenou, J. D.; Ahounou, J. F.; Akakpo, H. B.; Laleye, A. Mol. Biol. Rep. 2013, 40, 1127−1134.
610
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