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Print version ISSN 0102-695X
Rev. bras. farmacogn. vol.21 no.6 Curitiba Nov./Dec. 2011 Epub Aug 26, 2011
Susana A. DandlenI; A. Sofia LimaII; Marta D. MendesII; M. Graça MiguelII,III; M. Leonor Faleiro*,IV; M. João SousaV; Luís G. PedroII; José G. BarrosoII; A. Cristina FigueiredoII
ICDCTPV, Faculdade de Ciências e Tecnologia, Universidade do Algarve, Portugal
IICentro Biotecnologia Vegetal, Instituto de Biotecnologia e Bioengenharia, Faculdade Ciências Lisboa, Universidade de Lisboa, Portugal
IIIDepartamento de Química e Farmácia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, Portugal
IVCentro de Biomedicina Molecular e Estrutural, Instituto de Biotecnologia e Bioengenharia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, Portugal
VDepartamento de Biologia e Biotecnologia, Escola Superior Agrária, Instituto Politécnico de Bragança, Portugal
Thyme essential oils are well recognized by their excellent biological activities and the antimicrobial activity of Portuguese thyme essential oils has been investigated with promising results, particularly against food borne pathogens. In this study the potential antimicrobial activity of the essential oils of five species of Thymus (Lamiaceae), namely Th. caespititius Brot., Th. camphoratus Hoffmanns. & Link, Th. capitellatus Hoffmanns. & Link., Th. carnosus Boiss. and Th. zygis L. was evaluated against Candida albicans, Haemophilus influenza, Helicobacter pylori, Listeria monocytogenes, Salmonella enterica and Streptococcus pneumoniae. H. pylori strains were the most susceptible bacteria, particularly to the essential oils of Th. caespititius (Planalto Central), Th. zygis (Rebordãos) and Th. caespititius (Pico) which minimum inhibitory concentration (MIC) values ranged from 0.05 to 0.08 mg.mL-1. Th. caespititius essential oil from Planalto Central or its main component, carvacrol significantly (p<0.05) inhibited the intracellular growth of H. pylori, and showed no citotoxicity to the gastric cell line. Our results suggest the potential of this essential oil and its main component as a promising tool as anti-Helicobacter agent potentiating the eradication of this important gastroduodenal pathogen.
Keywords: anti-Helicobacter, antimicrobial activity, carvacrol, cytotoxicity, essential oil, ThymeISSN 0102-695X
The genus Thymus L. is a polymorphic taxon, both chemically and morphologically. Species of Thymus are small perennial herbs native from Europe and Asia (Morales, 2002; Sáez and Stahl-Biskup, 2002; Figueiredo et al., 2008; Howath et al., 2008).
In Portugal, eleven species of Thymus (Lamiaceae), totalizing fourteen taxa and five sections can be found: Sect. Mastichina (Mill.) Benth., Sect. Micantes Velen., Sect. Pseudothymbra Benth., Sect. Serpyllum (Mill.) Benth. [subsect. Alternantes Klover and subsect. Pseudomarginati (H. Braun & Borbás) Jalas] and Sect. Thymus [subsect. Thymus and subsect. Thymastra R. Morales]. Thymbra capitata (L.) Cav. [=Thymus capitatus (L.) Hoffmanns. & Link, Thymus creticus DC., Corydothymus capitatus Rechenb. f., Satureja capitata L.] belongs to the genus Thymbra, nevertheless Franco included it in the genus Thymus owing to the similarities between these species (Franco, 1984).
Several aspects of Portuguese thyme species including botany, taxonomy, ethnobotany, phytochemistry, pharmacology, molecular biology, among others, were recently reviewed (Figueiredo et al., 2008; Trindade et al., 2009).
In the present work the evaluation of the antimicrobial activity of several Portuguese thyme essential oils was extended to essential oils from diverse populations from different locations, both on mainland Potugal and on the Azores archipelago. One of the tested essential oils showed a very promising activity against the gastroduodenal pathogen, Helicobacter pylori leading to the determination of the effect of the essential oil on the intracellular viability of H. pylori in a human gastric adenocarcinoma cell type.
Materials and Methods
Plants of the different Thymus species (Th. caespititius Brot., Th. camphoratus Hoffmanns. & Link, Th. capitellatus Hoffmanns. & Link., Th. carnosus Boiss. and Th. zygis L.), family Lamiaceae, were collected, during the flowering period (May-July, 2007-2009), on mainland Portugal and on the Azores islands Pico, S. Jorge and Terceira, totalizing 28 population samples. After collection, the plant material was kept at -20 °C until extraction. For each species, voucher specimens have been deposited in the Herbarium of the Museu, Laboratório e Jardim Botânico de Lisboa (Th. caespititius LISU 173798; Th. camphoratus LISU 237718; Th. capitellatus LISU 237722; Th. carnosus LISU 237727 and Th. zygis subsp. sylvestris LISU 237738) and in the Herbarium of the Departamento de Biologia e Biotecnologia, Escola Superior Agrária de Bragança (Th. zygis subsp. zygis BREZA, number not yet assigned).
Isolation of the essential oils
The essential oils were isolated from the aerial part of the plants by hydrodistillation during three hours, using a Clevenger-type apparatus according to the European Pharmacopoeia method (Council of Europe, 2007) in order to estimate oil yield and to obtain pure essential oils. The essential oils were stored at -20 ºC in the dark prior to analysis. For the antimicrobial activity determination, the pure isolated essential oils were solved in 2-propanol (1:5, v/v).
Chemical composition of the essential oils
Gas chromatographic analyses were performed using a Perkin Elmer Autosystem XL gas chromatograph equipped with two flame ionization detectors (FID), a data handling system and a vaporizing injector port into which two columns of different polarities were installed: a DB-1 fused-silica column (polydimethylsiloxane, 30 m x 0.25 mm i.d., film thickness 0.25 µm; J & W Scientific Inc., Rancho Cordova, CA, USA) and a DB-17HT fused-silica column [(50% phenyl)-methylpolysiloxane, 30 m x 0.25 mm i.d., film thickness 0.15 µm; J & W Scientific Inc.]. Oven temperature was programmed, 45-175 °C, at 3 °C.min-1, subsequently at 15 °C.min-1 up to 300 °C, and then held isothermal for 10 min; injector and detector temperatures, 280 °C and 300 °C, respectively; carrier gas, hydrogen, adjusted to a linear velocity of 30 cm.s-1. The samples were injected using split sampling technique, ratio 1:50. The volume of injection was 0.1 µL of a pentane-oil solution (1:1). The percentage composition of the oils was computed by the normalization method from the GC peak areas, calculated as mean values of two injections from each oil, without using correction factors.
The gas chromatography-mass spectrometry unit consisted on a Perkin Elmer Autosystem XL gas chromatograph, equipped with DB-1 fused-silica column (30 m x 0.25 mm i.d., film thickness 0.25 µm; J & W Scientific, Inc.), and interfaced with a Perkin-Elmer Turbomass mass spectrometer (software version 4.1, Perkin Elmer, Shelton, CT, USA). Injector and oven temperatures were as above; transfer line temperature, 280 °C; ion source temperature, 220 °C; carrier gas, helium, adjusted to a linear velocity of 30 cm.s-1; split ratio, 1:40; ionization energy, 70 eV; scan range, 40-300 u; scan time, 1 s. The identity of the components was assigned by comparison of their retention indices, relative to C9-C22 n-alkane indices and GC-MS spectra from a home-made library, constructed based on the analyses of reference oils, laboratory-synthesised components and commercial available standards.
The microorganisms used in this study are listed in Table 1. The microbial cultures were maintained at -80 °C until use. The recovery and maintenance of the microbial strains were as previously described (Faleiro et al., 2003; Faleiro et al., 2005; Hazzit et al. 2009).
The antimicrobial activity of the tested essential oils was evaluated by agar diffusion as previously described (Faleiro et al., 2003; Faleiro et al., 2005, Hazzit et al. 2009). The antibiotic chloramphenicol (30 μg/disc) and amphotericin B (10 μg/disc) were used as positive reference antimicrobial agents and 2-propanol as negative. The assays were done in triplicate.
Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) of the essential oils of Th. caespititius from Pico, Th. caespititius from Planalto Central and Th. zygis from Rebordãos for Helicobacter pylori 26695 was determined. Each essential oil, at 0.03 mg.mL-1, 0.05 mg.mL-1 and 0 08 mg.mL-1, was added to Columbia agar medium (supplemented with 10%, v/v blood). The bacterial viability was determined as described by Miguel et al. (2008). The assay was done in triplicate.
The cell line 23132/87 DSMZ nº ACC201, human gastric adenocarcinoma cell type, was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). ACC201 cells were grown in RPMI 1640 medium (Gibco-Invitrogen, USA) supplemented with 10% Fetal bovine serum (Gibco-Invitrogen, USA) Penicillium- Streptomycin Solution (100 U mL-1 penicillin and 100 μg mL-1 streptomycin (Gibco-Invitrogen, USA) at 37 ºC in the presence of 5% (v/v) CO2. The cells were grown until they reached 90% of confluence and were removed by trypsin/EDTA 0.25 % (Sigma, Madrid, Spain).
The cytotoxicity of the essential oil of Th. caespititius from Planalto Central against cell line 23132/87 DSMZ nº ACC201 was determined by the MTT method (Vibrant MTT Cell Proliferation assay kit, Molecular Probes inc., Invitrogen US) using 96-well microplate (Greiner Bio-One GmbH). Four essential oil concentrations were tested (0.08 mg.mL-1, 0.50 mg.mL-1, 1.00 mg.mL-1 and 2.00 mg.mL-1). The tested essential oils concentrations were selected according the obtained MIC value for H. pylori and the higher values in the base of the IC50 value that corresponds to different antioxidant activities (Dandlen et al., 2010). All determinations were done in triplicate and, for control, the RPMI 1640 culture medium with no essential oil added, supplemented with 2- propanol and 5% H2O2 (v/v) (cytotoxic agent, positive control), was used. Carvacrol was tested at 61.9% (v/v) concentration, which corresponds to its percentage amount in the pure essential oil. The cell line viability was determined by absorbance (A540 nm) using a microplate reader (TECAN Infinite M200) after 30 min, 1, 4 and 8 h after essential oil contact.
The effect of the essential oil on intracellular Helicobacter pylori viability
Intracellular viability of H. pylori 26695 in the presence of the essential oil was determined on the human gastric adenocarcinoma cell line 23132/87 (DSMZ nº ACC201) following the procedure described by Fahey et al. (2002). The cells were cultured in RPMI 1640 medium and after trypsinization the cells were washed with Hank's balanced salt solution. A cell suspension of 8 x 105 cell mL-1 was used to inoculate each well of a 96-well microplate. The plates were incubated overnight at 37 °C in the presence of 5% CO2. The culture monolayers were inoculated with H. pylori 26695 at 8 x 107. The incubation of the inoculated cell monolayers occurred during 12 h at 37 °C. The cells were then washed 6x with Hank's solution to eliminate nonadherent bacteria and incubated with 100 μg.mL-1 of gentamicin during 1.5 h to kill extracellular bacteria. Before addition of the essential oil and control compounds the monolayers were washed 6x with Hank's solution. The monolayers were subjected to addition of 1) culture medium with no agents added 2) culture medium with 30 μg.mL-1 chloramphenicol (MIC value for H. pylori), 3) culture medium supplemented with 0.08 mg.mL-1 of Th. caespititius (Planalto Central) essential oil and 4) 61.9% of carvacrol (percentage of the compound in the essential oil sample). The microplates were incubated at 37 °C. The culture medium with no supplement was used as a negative control. To determine the intracellular bacterial viability the cell line was washed 6x with Hank's solution and lysed with distilled water. The recovered bacteria were inoculated in Columbia agar supplemented with 10% (v/v) blood.
The percentage composition of the essential oils was used to determine the relationship between the different Portuguese thyme samples by principal components analysis (PCA) using the NTSYS-pc software (Rohlf, 1992). The results on the antimicrobial activity were used to perform a cluster analysis. A dendrogram was generated to illustrate the overall relationships between the tested essential oils. The dendrogram was constructed using UPGMA clustering using again the NTSYS software.
Results and Discussion
The identified compounds in the 28 essentials oils from Portuguese Thymus species analysed, from mainland Portugal and three Azores islands, are indicated in Table 2, as well as their essential oil yields. All essential oils are clearly dominated by monoterpenes (64-99%) with oxygen-containing monoterpenes (40-82%) being the major group in almost all samples (23) whereas monoterpene hydrocarbons (8-59%) appeared as the major group only in five samples. The essential oil yield (0.4-3.6% v.w-1) shows some variation even within each species: Th. caespititius (0.4-2.3%), Th. camphoratus (0.7-1.9%), Th. capitellatus (1.7-3.6%), Th. carnosus (0.6-1.2%) and Th. zygis (0.5-1.0%).
In Th. caespititius essential oils, the five major components are carvacrol (t-62%), α-terpineol (4-50%), thymol (t-35%), carvacryl acetate (n.d.-19%) and p-cymene (2-14%). The variation of the major components in each oil allows to define three chemotypes: α-terpineol type (all samples from plants collected on mainland Portugal), carvacrol type (two samples from Pico island) and thymol type (samples from S. Jorge and Terceira islands). These results are according to previous studies on Th caespititius collected in the Azorean archipelago (Pereira et al., 2000; Pereira et al., 2003; Santos et al., 2005)
The five major components in the essential oils isolated from Th. camphoratus are 1,8-cineole (1-47%), borneol (1-23%), camphor (0.4-19%), α-pinene (4-12%) and terpinen-4-ol (0.4-10%). According to the dominant component in each sample, three are 1,8-cineole-rich essential oils whereas the fourth is borneol-rich. In Th. capitellatus essential oils, the five major components are 1,8-cineole (7-35%), borneol (16-22%), camphene (11-18%), camphor (5-18%) and α-pinene (9-14%). All the analysed essential oils from Th. carnosus are dominated by borneol (20-31%) and camphene (15-23%), being terpinen-4-ol (8-14%), α-pinene (4-10%) and bornyl acetate (4-10%) the other three major components.
In the case of Th. zygis, p-cymene (24-40%) is present in considerable amounts in all essential oil samples, whereas carvacrol (1-35%) dominates in three samples, one of which is from subsp. zygis, and thymol (1-24%) appears as the second major component in the other two samples. γ-Terpinene (5-11%) and camphene (2-6%) are the other two major components in these essential oils.
The scatter plot, obtained by PCA (Figure 1), showed four groups of individuals (A, B, C and D), along axis PC1 (44% of explained variance) and PC2 (18% of explained variance), according to their major volatile components. Borneol and camphene were the main compounds in the essential oils from the individuals in group A, the same for 1,8-cineole and borneol in group B and for carvacrol in group C. For individuals in group D, α-terpineol or thymol were the major compounds in their essential oils.
The results obtained on the chemical characterization of the essential oils from the Portuguese thyme species used in this study, are according to the large chemical polymorphism for the majority of Portuguese Thymus taxa (Figueiredo et al., 2008).
The inhibitory activities of the tested Thymus essential oils are indicated in Table 3. The different microorganisms tested showed different susceptibilities to the diverse Thymus essential oils. The most susceptible to the majority of Thymus essential oil was H. pylori (both strains, J99 and 26695) followed by Streptococcus pneumoniae D39. S. aureus, Salmonella Thyphimurium and Candida albicans showed to be resistant to the majority of the tested Thymus essential oils. L. monocytogenes strains showed an intermediate susceptibility, in particular to Th. capitellatus from Tróia and Th. caespititius from Pico (Table 3).
Besides both H. pylori strains could be considered the most susceptible bacteria it was evident that they display a different susceptibility to the same essential oil, namely H. pylori J99 showed a very low inhibition zone (8.17±2.25 mm) to the essential oil of Th. capitellatus (Santiago de Cacém) but the strain H. pylori 26695 showed a higher inhibition zone (14.50±0.87 mm). The same behaviour can be seen for the essential oils of Th. caespititius (Óbidos), Th. zygis subsp. sylvestris (Duas Igrejas and Alcanena) and Th. carnosus (Tróia). The essential oils of Th. caespititius (Planalto Central), Th. zygis subsp. zygis (Rebordãos) and Th. caespititius (Pico) produced the highest inhibition zone when tested against H. pylori 26695, namely 22.50±4.33 mm, 20.83±1.44 mm and 21.67±2.89 mm, respectively. The MIC value varied from 0.05 to 0.08 mg.mL-1 for Th. caespititius (Planalto Central), Th. zygis Bragança (Rebordãos) and Th. caespititius (Pico) essential oils determined for H. pylori 26695. Carvacrol, a phenolic monoterpene, is the main component of these essential oils.
The cluster analysis based on the antimicrobial activity data (Figure 2) apart from showing a low correlation between the diverse tested essential oils, revealed a distinctive grouping pattern from that of the PCA of the composition of the essential oils (Figure 1).
The capacity for preventing H. pylori growth was previously observed, in one of our studies with rich-carvacrol essential oils of T. pallescens from Algeria (Hazzit et al., 2009). Nevertheless and as verified in the present work, other components must have a predominant role, either alone or in combination with carvacrol on the antimicrobial activity, because other oils also with high percentages of carvacrol did not show a significant activity. The complexity of the chemical composition of the essential oils with dozen of compounds makes the process of the identification of the component responsible for the antimicrobial activity very difficult. Often the antimicrobial activity results from the synergism or antagonism between several components. In our study one of the examples is the result obtained for T. zygis subsp. sylvestris from Covão do Coelho and from Alcanena, with relative high amounts of carvacrol, comparable to that of T. zygis subsp. zygis, but not showing similar inhibitory activity. These results, suggest that, despite the high percentage of some compounds, the presence of other minor components or chiral isomers in the essential oils, providing a synergistic or antagonistic effect, can be determinant for their bioactivity.
The cytotoxicity of Th. caespititius (Planalto Central) to adenocarcinoma gastric cells (ACC201) was determined and the results are presented in Figure 3. The essential oil used at the MIC value (0.08 mg.mL-1) on the first 30 min the cell line decrease the viability to 45% but at the end of the first hour recovered the viability to 64% and until the end of the assay (8 h) the viability was maintained at 95%. Higher concentrations of the essential oil, 0.50 and 1.00 mg.mL-1 were detrimental to the viability of the gastric cell line, namely on the first 30 min the viability decrease to 30.00±0.05 and 20.00±0.05%, respectively and maintains this low value after 8 h (Figure 3). Carvacrol (61.9%, v/v) had no effect on the gastric cell viability. The essential oil solvent, 2-propanol caused a slight decrease on the viability of the gastric cell line, the viability was between 90 and 80% during the assay period. The activity of H2O2 (5%, v/v) was injurious to the cell line, as predicted.
The intracellular antibacterial activity of the Th. caespititius essential oil from Planalto Central was determined in the human gastric adenocarcinoma cell line 23132/87 inoculated with H. pylori 26695. The results are indicated in Figure 4. The reduction on the H. pylori viability was significant (p<0.05) for all the tested agents (essential oil, carvacrol and the antibiotic chloramphenicol), but no significantly differences were observed between the activity of the essential oil and chloramphenicol (p>0.05). The highest viability reduction (p<0.05) was achieved when carvacrol was used. This component of Th. caespititius essential oil, when compared to control, caused an inhibition of intracellular growth of H. pylori of about 3 log in 8 h (Figure 4). H. pylori nowadays is considered an intracellular microorganism due to its ability to invade various host cells, namely macrophages, dendritric cells and epithelial cells and undergoes replication inside the autophagosome (Wang et al., 2009) and once inside the infected cells the bacterium increases its resistance to antimicrobial agents (Fahey et al., 2002; Chu et al., 2010). Our results show that both Th. caespititus essential oil from Planalto Central and its main component, carvacrol, are effective inhibitors of H. pylori 26695 intracellular growth. Our findings are in agreement with the work of Bergonzelli et al. (2003). The use of carvacrol rich essential oils or its main component seems to have potential to be used in combination with current treatments potentiating a total eradication.
This study was partially funded by the Fundação para a Ciência e a Tecnologia under the research contract PTDC/AGR-AAM/70136/2006. The authors are also grateful for the support of IBB/CBME/CBV, LA, FEDER/POCI 2010. The authors also acknowledge the contribution of Prof. Ana Isabel Dias Correia (Herbarium of the Museu, Laboratório e Jardim Botânico de Lisboa) in the identification and voucher of the plant material.
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Received 2 Oct 2010
Accepted 2 Feb 2011