TY - JOUR
AU - Hammami,, Saoussen
AB - Abstract Chemical composition and antimicrobial activity of Teucrium capitatum L. subsp. lusitanicum essential oil was investigated for the first time in the present study. Qualitative and quantitative analyses of the chemical composition by gas chromatography and mass spectrometry (GC–FID and GC–MS) revealed the presence of 60 compounds representing 97.6% of the whole constituents. The main compounds were germacrene D (47.1%), spathulenol (5.8%), α-selinene (5.3%), germacrene A (2.9%), δ-cadinene (2.8%) and cubenol (2.7%). In vitro, the antimicrobial activity was investigated against five bacterial strains along with the yeast Candida albicans using broth microdilution assay. T. capitatum subsp. lusitanicum essential oil showed significant activity against the gram-positive bacteria Staphylococcus aureus (MIC = MBC = 78 μg mL−1), Bacillus subtilis (MIC = MBC = 156 μg mL−1) and the yeast C. albicans (MIC = MFC = 156 μg mL−1). The great potential of antimicrobial effects is most likely due to the very high percentage of sesquiterpene hydrocarbons particularly to germacrene D, for which the antimicrobial properties have been previously reported. Introduction Aromatic plants are used in medicine, perfumes, cosmetics and culinary aromatization. They were regularly employed in several cultures for their unbelievable properties and their usages to treat or prevent several chronic or serious diseases. Over the past several decades, many bacteria have become increasingly immune to several antibiotics of the pharmaceutical arsenal. Therefore, to better control microbial infections and within the objective to reduce the frequency of multiresistant strains, many research groups have been engaged in a continuous struggle against bacterial resistance. Consequently, a particular attention was given to unexplored medicinal plants and marine organisms which are still considered as rich sources of novel antimicrobials that have almost no effects. Essential oils, generally constituted of a complex mixture of hydrophobic terpenes and their oxygenated derivatives, are being screened on a global scale as powerful agents to reduce antimicrobial resistance. Thus, research on the antimicrobial activities, the mode of action and the chemical composition of medicinal plants, more specifically essential oils, is gaining a renewed interest. Teucrium L. is a large genus belonging to the Lamiaceae family. This genus contains around 300 species widespread all over the world, most are concentrated around the Mediterranean basin (1). In Tunisia, Teucrium genus comprises about 19 species in which Teucrium capitatum L. (≡ T. polium subsp. capitatum (L.) Arcang.) belonging to the section Polium and possesses two subspecies T. capitatum L. subsp. capitatum and T. capitatum subsp. lusitanicum (Schreb.) T. Navarro & Rosua (2,3). Several species of this genus have been reported to possess medicinal properties, such as diuretic, hypoglycemic, antipyretic, antiseptic, antiulcer, antispasmodic, antitumor, antirheumatic, antihelmintic, anti-inflammatory and antibacterial effects. Some Teucrium species are also used as a source of condiments for flavoring beer or the seasoning of herbal teas (1,4). In Iranian folk medicine, Teucrium polium tea is used to treat abdominal pain, indigestion, common cold and type 2 diabetes (5). Teucrium species have been extensively investigated as a source of natural products with potential anti-inflammatory, antitumor, antimicrobial, antioxidant, cytotoxic and insecticidal properties (1, 4, 6–8). Previous phytochemical analyses of crude extracts revealed the presence of several bioactive phytochemicals such as iridoids, flavonoids and terpenoids (1). To our knowledge, the chemical composition and antimicrobial activity of the essential oil obtained from T. capitatum subsp. lusitanicum (≡T. lusitanicum Schreb.) have never been investigated. Therefore, and as a continuity of our study on the valorization of Tunisian Teucrium species (9,10), the present study aimed to investigate for the first time, the chemical composition and the antimicrobial properties of T. capitatum subsp. lusitanicum essential oils. Experimental Plant material and extraction of essential oil Fresh aerial parts of samples of T. capitatum subsp. lusitanicum were harvested from Nebeur, North-West of Tunisia during the blooming-fruiting season (June 2014). Herbal material was then dried under ambient conditions. Selection, collection and identification of uninfected material were carried out by one of the authors (REM), a researcher–teacher in the Laboratory of Botany, Cryptogamy and Plant Biology at the Faculty of Pharmacy of Monastir-Tunisia, where voucher specimens have been deposited ([LAM/Teu.capit/lus; 2406/2014]) in the personal herbarium of REM, at the Service of Botany and Plant Biology, Faculty of Pharmacy of Monastir, Tunisia. Identified species were then kept in a dark place for later use. Extraction of the essential oils One hundred grams of the collected material was powdered then submitted to 3 h hydro-distillation with a Clevenger-type apparatus. The essential oil was collected, dried over anhydrous sodium sulphate, filtered and stored in sealed brown glass vials at 0°C until analysis. GC–FID and GC–MS analysis Gas chromatography–flame ionization detector (GC–FID) The gas chromatographic analysis of the essential oil was carried out on a Varian 450 gas chromatograph equipped with flame ionization detector (FID), using a stationary phase ZB-5 (30 m × 0.25 mm i.d., 0.25 μm film thickness) column under the reported experimental conditions (11–13), nitrogen was a carrier gas at 1 mL min-1 flow rate. The temperature program was 60–220°C at 3°C min-1; for the injector and detector, temperatures were 230 and 250°C, respectively. The injection volume was 1.0 μL of 1% solution diluted in n-hexane; the split ratio was 50:1. Gas chromatography–mass spectrometry (GC–MS) The gas chromatographic and mass spectrometric analysis of the essential oil was carried out on a Thermo Scientific Trace Ultra gas chromatograph interfaced with a Thermo Scientific ITQ 1100 Mass Spectrometer fitted with ZB-5 (30 m × 0.25 mm i.d., 0.25 μm film thicknesses) column. The oven temperature was programmed from 60–220°C at 3°C min-1 using helium as a carrier gas at 1 mL min-1. The injector temperature was 230°C; the injection volume 0.1 μL of 1% solution prepared in n-hexane, split ratio 50:1. The mass spectrometry (MS) spectrum was taken at 70 eV with a mass scan range of 30–480 amu. All of the experimental parameters were applied based on those reported earlier (14–16). Identification of the organic volatiles The essential oil constituents were identified by retention index (RI), determined using linear temperature program-based analysis, with n-alkanes C8-C25 as a reference, and with similar chromatographic settings on the ZB-5 column. A mass spectral computer library search (NIST 08 Version 2.0 f) was also performed with WILEY MS 9th Edition, and the mass spectral data were compared along with the co-injection of marketable samples purchased from Sigma-Aldrich (St. Louis, MO, USA), with ≥98% purity (17). The peak area of gas chromatography (GC)–FID response was used to determine the percentage composition of each individual compound without using a correction factor. The identified constituents of the essential oil are listed in Table 1. Antimicrobial activity Microbial strains In vitro, the essential oil was tested against six references microorganisms: three gram-negative bacteria: Escherichia coli (ATCC 8739), Salmonella enterica (CIP 8039), Pseudomonas aeruginosa (ATCC 9027), two gram-positive bacteria; Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) and the yeast Candida albicans (ATCC 30031). Microorganisms were preserved in nutrient agar (Sigma) at 37°C, except the yeast C. albicans which was kept at 28°C. Figure 1 Open in new tabDownload slide GC-TIC of the essential oil of T. capitatum subsp. lusitanicum. Figure 1 Open in new tabDownload slide GC-TIC of the essential oil of T. capitatum subsp. lusitanicum. Microwell plate dilution assay The antimicrobial activity was performed through the microwell plate dilution assay using sterile 96-well plates (18). This method aims to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal and fungicidal concentrations (MBC and MFC). The essential oil was dissolved in dimethyl sulfoxide (5%) supplemented with Tween 80 (Sigma) at a final concentration of 5 mg mL−1. 200 μL of the essential oil dilution was dispensed into the wells of the first row of the microtiter plate. All other wells were then filled with 100 μL of Mueller Hinton nutrient broth for microorganisms with the exception of the yeast, for which Sabouraud broth was used as a culture medium. Serial doubling dilutions of the oil were prepared over the range 2.5-4 × 10−3 mg mL−1. Fresh overnight cultures were prepared in Mueller Hinton Broth (Sigma) and adjusted to 0.5 McFarland standards turbidity. Each well was then inoculated with 20 μL of the suspension (106 CFU mL-1). Finally, 10 μL of Resazurin solution as an indicator of growth was added to each well. Gentamicine (40 mg mL−1) and Amphotericin B served as positive controls for bacteria strains and the yeast, respectively. Each assay included a negative control with no essential oil. Plates were incubated during 24 h at 37°C for bacteria and at 28°C for the yeast. After incubation, the wells were examined for the growth of microorganisms and the MICs were determined. Microbial growth results in the appearance of pink color of the solution indicating the reduction or precipitation of the dye. The MIC is defined as the lowest concentration of the essential oil at which the microorganism does not demonstrate visible growth. To confirm MBC, 10 μL of broth was taken from the wells containing the MIC and twice the MIC (not showing bacterial growth) and inoculated in Mueller Hinton Agar during 24 h at 37°C for bacteria or in Sabouraud dextrose agar for 24 h at 28°C for the yeast C. albicans. The MBC is defined as the lowest concentration of the essential oil at which 99.9% of incubated microorganisms are completely killed (19). Results The light-yellow essential oil was obtained by hydrodistillation from T. capitatum subsp. lusitanicum aerial parts with a yield of 0.4%. The chemical composition of the essential oil was analyzed by GC–FID and GC–MS and the components were identified on the basis of comparison of their RI values with those reported in the literature (20) (Figure 1). In total, 60 organic volatiles were detected, they accounted for 97.6% of the whole constituents. Sesquiterpene hydrocarbons were shown to be the largest group of the essential oil sample with a percentage of 71.3%, followed by oxygenated sesquiterpenes 14.2%. However, monoterpene hydrocarbons and oxygenated monoterpenes were detected at low and comparative percentages of 5.9% and 5.2%, respectively, as highlighted in Table I. According to the obtained data, T. capitatum subsp. lusitanicum is a germacrene D rich chemotype (47.1%). In addition, other compounds including spathulenol (5.8%), α-selinene (5.3%), germacrene A (2.9%), δ-cadinene (2.8%) and cubenol (2.7%) were identified in relatively small quantities. Table I Chemical Composition of T. capitatum subsp. Lusitanicum Essential Oil Number . Compounds . RI . Content (%) . Identification . 1 α-Pinene 941 0.5 RI, MS, CI 2 Camphene 956 0.1 RI, MS, CI 3 Thuja-2,4(10)-diene 961 0.1 RI, MS 4 Sabinene 979 t RI, MS 5 β-Pinene 982 1.5 RI, MS, CI 6 Myrcene 993 0.1 RI, MS, CI 7 α-Terpinene 1,018 0.2 RI, MS, CI 8 p-Cymene 1,027 1.3 RI, MS 9 Limonene 1,034 0.5 RI, MS, CI 10 1,8-Cineole 1,037 0.3 RI, MS, CI 11 γ-Terpinene 1,063 1.6 RI, MS, CI 12 cis-Sabinene hydrate 1,072 0.8 RI, MS 13 trans-Sabinene hydrate 1,102 0.5 RI, MS 14 α-Campholenal 1,130 0.2 RI, MS 15 Sabina ketone 1,163 0.2 RI, MS 16 Borneol 1,170 0.4 RI, MS, CI 17 Terpin-4-ol 1,181 0.4 RI, MS, CI 18 α-Terpineol 1,193 0.2 RI, MS, CI 19 Verbenone 1,212 0.7 RI, MS 20 trans-Carveol 1,222 0.4 RI, MS 21 Cumin aldehyde 1,244 0.1 RI, MS 22 Carvone 1,248 0.6 RI, MS, CI 23 (E)-Anethole 1,289 0.3 RI, MS, CI 24 Thymol 1,295 0.7 RI, MS, CI 25 Carvacrol 1,304 0.2 RI, MS, CI 26 δ-Elemene 1,342 t RI, MS 27 α-Cubebene 1,354 0.2 RI, MS 28 Eugenol 1,362 0.2 RI, MS, CI 29 cis-Carvyl acetate 1,364 t RI, MS 30 α-Ylangene 1,376 t RI, MS 31 α-Copaene 1,380 0.8 RI, MS 32 β-Bourbonene 1,389 2.2 RI, MS 33 β-Cubebene 1,393 0.5 RI, MS 34 β-Elemene 1,395 t RI, MS 35 β-Caryophyllene 1,424 2.5 RI, MS, CI 36 β-Copaene 1,440 0.4 RI, MS 37 trans-α-Bergamotene 1,442 0.3 RI, MS 38 α-Guaiene 1,444 0.2 RI, MS 39 α-Humulene 1,458 0.5 RI, MS, CI 40 Seychellene 1,466 0.7 RI, MS 41 cis-Muurola-4(14),5-diene 1,468 1.0 RI, MS 42 Dauca-5,8-diene 1,473 0.1 RI, MS 43 Germacrene D 1,485 47.1 RI, MS 44 cis-β-Guaiene 1,495 0.5 RI, MS 45 α-Selinene 1,500 5.3 RI, MS 46 α-Muurolene 1,503 1.1 RI, MS 47 γ-Cadinene 1,518 1.5 RI, MS 48 δ-Cadinene 1,528 2.8 RI, MS 49 trans-Cadina-1,4-diene 1,536 0.2 RI, MS 50 α-Cadinene 1,542 0.2 RI, MS 51 α-Calacorene 1,548 0.3 RI, MS 52 Elemol 1,554 0.2 RI, MS 53 Germacrene A 1,562 2.9 RI, MS 54 11-Norbourbonan-1-one 1,566 0.3 RI, MS 55 Spathulenol 1,583 5.8 RI, MS 56 Caryophyllene oxide 1,588 1.8 RI, MS, CI 57 1,10-di-epi-Cubenol 1,623 0.6 RI, MS 58 1-epi-Cubenol 1,633 0.3 RI, MS 59 Cubenol 1,646 2.7 RI, MS 60 Valerianol 1,660 2.5 RI, MS Monoterpene hydrocarbons 5.9 Oxygenated monoterpenes 5.2 Sesquiterpene hydrocarbons 71.3 Oxygenated sesquiterpenes 14.2 Phenyl derivatives 1.0 Total identified 97.6 Number . Compounds . RI . Content (%) . Identification . 1 α-Pinene 941 0.5 RI, MS, CI 2 Camphene 956 0.1 RI, MS, CI 3 Thuja-2,4(10)-diene 961 0.1 RI, MS 4 Sabinene 979 t RI, MS 5 β-Pinene 982 1.5 RI, MS, CI 6 Myrcene 993 0.1 RI, MS, CI 7 α-Terpinene 1,018 0.2 RI, MS, CI 8 p-Cymene 1,027 1.3 RI, MS 9 Limonene 1,034 0.5 RI, MS, CI 10 1,8-Cineole 1,037 0.3 RI, MS, CI 11 γ-Terpinene 1,063 1.6 RI, MS, CI 12 cis-Sabinene hydrate 1,072 0.8 RI, MS 13 trans-Sabinene hydrate 1,102 0.5 RI, MS 14 α-Campholenal 1,130 0.2 RI, MS 15 Sabina ketone 1,163 0.2 RI, MS 16 Borneol 1,170 0.4 RI, MS, CI 17 Terpin-4-ol 1,181 0.4 RI, MS, CI 18 α-Terpineol 1,193 0.2 RI, MS, CI 19 Verbenone 1,212 0.7 RI, MS 20 trans-Carveol 1,222 0.4 RI, MS 21 Cumin aldehyde 1,244 0.1 RI, MS 22 Carvone 1,248 0.6 RI, MS, CI 23 (E)-Anethole 1,289 0.3 RI, MS, CI 24 Thymol 1,295 0.7 RI, MS, CI 25 Carvacrol 1,304 0.2 RI, MS, CI 26 δ-Elemene 1,342 t RI, MS 27 α-Cubebene 1,354 0.2 RI, MS 28 Eugenol 1,362 0.2 RI, MS, CI 29 cis-Carvyl acetate 1,364 t RI, MS 30 α-Ylangene 1,376 t RI, MS 31 α-Copaene 1,380 0.8 RI, MS 32 β-Bourbonene 1,389 2.2 RI, MS 33 β-Cubebene 1,393 0.5 RI, MS 34 β-Elemene 1,395 t RI, MS 35 β-Caryophyllene 1,424 2.5 RI, MS, CI 36 β-Copaene 1,440 0.4 RI, MS 37 trans-α-Bergamotene 1,442 0.3 RI, MS 38 α-Guaiene 1,444 0.2 RI, MS 39 α-Humulene 1,458 0.5 RI, MS, CI 40 Seychellene 1,466 0.7 RI, MS 41 cis-Muurola-4(14),5-diene 1,468 1.0 RI, MS 42 Dauca-5,8-diene 1,473 0.1 RI, MS 43 Germacrene D 1,485 47.1 RI, MS 44 cis-β-Guaiene 1,495 0.5 RI, MS 45 α-Selinene 1,500 5.3 RI, MS 46 α-Muurolene 1,503 1.1 RI, MS 47 γ-Cadinene 1,518 1.5 RI, MS 48 δ-Cadinene 1,528 2.8 RI, MS 49 trans-Cadina-1,4-diene 1,536 0.2 RI, MS 50 α-Cadinene 1,542 0.2 RI, MS 51 α-Calacorene 1,548 0.3 RI, MS 52 Elemol 1,554 0.2 RI, MS 53 Germacrene A 1,562 2.9 RI, MS 54 11-Norbourbonan-1-one 1,566 0.3 RI, MS 55 Spathulenol 1,583 5.8 RI, MS 56 Caryophyllene oxide 1,588 1.8 RI, MS, CI 57 1,10-di-epi-Cubenol 1,623 0.6 RI, MS 58 1-epi-Cubenol 1,633 0.3 RI, MS 59 Cubenol 1,646 2.7 RI, MS 60 Valerianol 1,660 2.5 RI, MS Monoterpene hydrocarbons 5.9 Oxygenated monoterpenes 5.2 Sesquiterpene hydrocarbons 71.3 Oxygenated sesquiterpenes 14.2 Phenyl derivatives 1.0 Total identified 97.6 RI: Retention Index relative to C8-C30 n-alkanes on the ZB-5 column, MS: NIST and Wiley library and the literature, CI: Co-injection of commercial samples, t: trace (<0.1%). Open in new tab Table I Chemical Composition of T. capitatum subsp. Lusitanicum Essential Oil Number . Compounds . RI . Content (%) . Identification . 1 α-Pinene 941 0.5 RI, MS, CI 2 Camphene 956 0.1 RI, MS, CI 3 Thuja-2,4(10)-diene 961 0.1 RI, MS 4 Sabinene 979 t RI, MS 5 β-Pinene 982 1.5 RI, MS, CI 6 Myrcene 993 0.1 RI, MS, CI 7 α-Terpinene 1,018 0.2 RI, MS, CI 8 p-Cymene 1,027 1.3 RI, MS 9 Limonene 1,034 0.5 RI, MS, CI 10 1,8-Cineole 1,037 0.3 RI, MS, CI 11 γ-Terpinene 1,063 1.6 RI, MS, CI 12 cis-Sabinene hydrate 1,072 0.8 RI, MS 13 trans-Sabinene hydrate 1,102 0.5 RI, MS 14 α-Campholenal 1,130 0.2 RI, MS 15 Sabina ketone 1,163 0.2 RI, MS 16 Borneol 1,170 0.4 RI, MS, CI 17 Terpin-4-ol 1,181 0.4 RI, MS, CI 18 α-Terpineol 1,193 0.2 RI, MS, CI 19 Verbenone 1,212 0.7 RI, MS 20 trans-Carveol 1,222 0.4 RI, MS 21 Cumin aldehyde 1,244 0.1 RI, MS 22 Carvone 1,248 0.6 RI, MS, CI 23 (E)-Anethole 1,289 0.3 RI, MS, CI 24 Thymol 1,295 0.7 RI, MS, CI 25 Carvacrol 1,304 0.2 RI, MS, CI 26 δ-Elemene 1,342 t RI, MS 27 α-Cubebene 1,354 0.2 RI, MS 28 Eugenol 1,362 0.2 RI, MS, CI 29 cis-Carvyl acetate 1,364 t RI, MS 30 α-Ylangene 1,376 t RI, MS 31 α-Copaene 1,380 0.8 RI, MS 32 β-Bourbonene 1,389 2.2 RI, MS 33 β-Cubebene 1,393 0.5 RI, MS 34 β-Elemene 1,395 t RI, MS 35 β-Caryophyllene 1,424 2.5 RI, MS, CI 36 β-Copaene 1,440 0.4 RI, MS 37 trans-α-Bergamotene 1,442 0.3 RI, MS 38 α-Guaiene 1,444 0.2 RI, MS 39 α-Humulene 1,458 0.5 RI, MS, CI 40 Seychellene 1,466 0.7 RI, MS 41 cis-Muurola-4(14),5-diene 1,468 1.0 RI, MS 42 Dauca-5,8-diene 1,473 0.1 RI, MS 43 Germacrene D 1,485 47.1 RI, MS 44 cis-β-Guaiene 1,495 0.5 RI, MS 45 α-Selinene 1,500 5.3 RI, MS 46 α-Muurolene 1,503 1.1 RI, MS 47 γ-Cadinene 1,518 1.5 RI, MS 48 δ-Cadinene 1,528 2.8 RI, MS 49 trans-Cadina-1,4-diene 1,536 0.2 RI, MS 50 α-Cadinene 1,542 0.2 RI, MS 51 α-Calacorene 1,548 0.3 RI, MS 52 Elemol 1,554 0.2 RI, MS 53 Germacrene A 1,562 2.9 RI, MS 54 11-Norbourbonan-1-one 1,566 0.3 RI, MS 55 Spathulenol 1,583 5.8 RI, MS 56 Caryophyllene oxide 1,588 1.8 RI, MS, CI 57 1,10-di-epi-Cubenol 1,623 0.6 RI, MS 58 1-epi-Cubenol 1,633 0.3 RI, MS 59 Cubenol 1,646 2.7 RI, MS 60 Valerianol 1,660 2.5 RI, MS Monoterpene hydrocarbons 5.9 Oxygenated monoterpenes 5.2 Sesquiterpene hydrocarbons 71.3 Oxygenated sesquiterpenes 14.2 Phenyl derivatives 1.0 Total identified 97.6 Number . Compounds . RI . Content (%) . Identification . 1 α-Pinene 941 0.5 RI, MS, CI 2 Camphene 956 0.1 RI, MS, CI 3 Thuja-2,4(10)-diene 961 0.1 RI, MS 4 Sabinene 979 t RI, MS 5 β-Pinene 982 1.5 RI, MS, CI 6 Myrcene 993 0.1 RI, MS, CI 7 α-Terpinene 1,018 0.2 RI, MS, CI 8 p-Cymene 1,027 1.3 RI, MS 9 Limonene 1,034 0.5 RI, MS, CI 10 1,8-Cineole 1,037 0.3 RI, MS, CI 11 γ-Terpinene 1,063 1.6 RI, MS, CI 12 cis-Sabinene hydrate 1,072 0.8 RI, MS 13 trans-Sabinene hydrate 1,102 0.5 RI, MS 14 α-Campholenal 1,130 0.2 RI, MS 15 Sabina ketone 1,163 0.2 RI, MS 16 Borneol 1,170 0.4 RI, MS, CI 17 Terpin-4-ol 1,181 0.4 RI, MS, CI 18 α-Terpineol 1,193 0.2 RI, MS, CI 19 Verbenone 1,212 0.7 RI, MS 20 trans-Carveol 1,222 0.4 RI, MS 21 Cumin aldehyde 1,244 0.1 RI, MS 22 Carvone 1,248 0.6 RI, MS, CI 23 (E)-Anethole 1,289 0.3 RI, MS, CI 24 Thymol 1,295 0.7 RI, MS, CI 25 Carvacrol 1,304 0.2 RI, MS, CI 26 δ-Elemene 1,342 t RI, MS 27 α-Cubebene 1,354 0.2 RI, MS 28 Eugenol 1,362 0.2 RI, MS, CI 29 cis-Carvyl acetate 1,364 t RI, MS 30 α-Ylangene 1,376 t RI, MS 31 α-Copaene 1,380 0.8 RI, MS 32 β-Bourbonene 1,389 2.2 RI, MS 33 β-Cubebene 1,393 0.5 RI, MS 34 β-Elemene 1,395 t RI, MS 35 β-Caryophyllene 1,424 2.5 RI, MS, CI 36 β-Copaene 1,440 0.4 RI, MS 37 trans-α-Bergamotene 1,442 0.3 RI, MS 38 α-Guaiene 1,444 0.2 RI, MS 39 α-Humulene 1,458 0.5 RI, MS, CI 40 Seychellene 1,466 0.7 RI, MS 41 cis-Muurola-4(14),5-diene 1,468 1.0 RI, MS 42 Dauca-5,8-diene 1,473 0.1 RI, MS 43 Germacrene D 1,485 47.1 RI, MS 44 cis-β-Guaiene 1,495 0.5 RI, MS 45 α-Selinene 1,500 5.3 RI, MS 46 α-Muurolene 1,503 1.1 RI, MS 47 γ-Cadinene 1,518 1.5 RI, MS 48 δ-Cadinene 1,528 2.8 RI, MS 49 trans-Cadina-1,4-diene 1,536 0.2 RI, MS 50 α-Cadinene 1,542 0.2 RI, MS 51 α-Calacorene 1,548 0.3 RI, MS 52 Elemol 1,554 0.2 RI, MS 53 Germacrene A 1,562 2.9 RI, MS 54 11-Norbourbonan-1-one 1,566 0.3 RI, MS 55 Spathulenol 1,583 5.8 RI, MS 56 Caryophyllene oxide 1,588 1.8 RI, MS, CI 57 1,10-di-epi-Cubenol 1,623 0.6 RI, MS 58 1-epi-Cubenol 1,633 0.3 RI, MS 59 Cubenol 1,646 2.7 RI, MS 60 Valerianol 1,660 2.5 RI, MS Monoterpene hydrocarbons 5.9 Oxygenated monoterpenes 5.2 Sesquiterpene hydrocarbons 71.3 Oxygenated sesquiterpenes 14.2 Phenyl derivatives 1.0 Total identified 97.6 RI: Retention Index relative to C8-C30 n-alkanes on the ZB-5 column, MS: NIST and Wiley library and the literature, CI: Co-injection of commercial samples, t: trace (<0.1%). Open in new tab The antimicrobial effect tested against microbial strains was evaluated in terms of MIC and minimum bactericidal/fungicidal concentrations (MBC/MFC); the broth dilution method in 96-well microplates was used. Overall, the essential oil showed a significant antimicrobial activity against all tested strains. Furthermore, it displayed a higher activity against gram-positive bacteria and the yeast C. albicans than gram-negative strains. As shown in Table II, the most susceptible strains were S. aureus (ATCC 6538) (MIC = MBC = 78 μg mL−1) followed by Bacillus subtilis (ATCC 6633) (MIC = MBC = 156 μg mL−1) and the yeast C. albicans (ATCC 30031) (MIC = MFC = 156 μg mL−1). Table II Minimal Inhibitory (MIC), Bactericide (MBC) and Fungicidal (MFC) Concentration Values (μg mL−1) of Gentamicin and T. capitatum Subsp. Lusitanicum Essential Oil Microorganisms . T. capitatum subsp. lusitanicum . Gentamicin . . MIC . MBC/MFC . MIC . Escherichia coli (ATCC 8739) 625 625 4 Salmonella enterica (CIP 8039) 625 625 3 P. aeruginosa (ATCC 9027) 625 625 4 S. aureus (ATCC 6538) 78 78 3 Bacillus subtilis (ATCC6633) 156 156 2 C. albicans (ATCC 30031) 156 156 11 Microorganisms . T. capitatum subsp. lusitanicum . Gentamicin . . MIC . MBC/MFC . MIC . Escherichia coli (ATCC 8739) 625 625 4 Salmonella enterica (CIP 8039) 625 625 3 P. aeruginosa (ATCC 9027) 625 625 4 S. aureus (ATCC 6538) 78 78 3 Bacillus subtilis (ATCC6633) 156 156 2 C. albicans (ATCC 30031) 156 156 11 Open in new tab Table II Minimal Inhibitory (MIC), Bactericide (MBC) and Fungicidal (MFC) Concentration Values (μg mL−1) of Gentamicin and T. capitatum Subsp. Lusitanicum Essential Oil Microorganisms . T. capitatum subsp. lusitanicum . Gentamicin . . MIC . MBC/MFC . MIC . Escherichia coli (ATCC 8739) 625 625 4 Salmonella enterica (CIP 8039) 625 625 3 P. aeruginosa (ATCC 9027) 625 625 4 S. aureus (ATCC 6538) 78 78 3 Bacillus subtilis (ATCC6633) 156 156 2 C. albicans (ATCC 30031) 156 156 11 Microorganisms . T. capitatum subsp. lusitanicum . Gentamicin . . MIC . MBC/MFC . MIC . Escherichia coli (ATCC 8739) 625 625 4 Salmonella enterica (CIP 8039) 625 625 3 P. aeruginosa (ATCC 9027) 625 625 4 S. aureus (ATCC 6538) 78 78 3 Bacillus subtilis (ATCC6633) 156 156 2 C. albicans (ATCC 30031) 156 156 11 Open in new tab Discussion Chemical composition Following the performed research and collected information, no investigation exists concerning the constituents of the essential oil of T. capitatum subsp. lusitanicum growing in Tunisia and the mentioned results are the first report with respect to the components of the essential oil of this valuable medicinal plant. The results of this study conformed to those of the other researchers to some extent, as a survey of the literature showed that sesquiterpenes and monoterpenes, either hydrocarbons or oxygenated ones, were the major components of many T. capitatum (T. polium subsp. capitatum) essential oils. In fact, a composition dominated by monoterpenes; α- pinene (28.8%), β-pinene (7.2%) and p-cymene (7.0%) was found in the essential oil of T. capitatum from Corsica (21). Essential oils of five populations of the same plant collected in Portugal, showed that oxygenated monoterpenes (33.0%) were the main group in which isomenthone (7.7%) being the main compound for the first sample. While the oil of the second one was dominated by monoterpene hydrocarbons (43.9%), represented by sabinene (11.2%), β-pinene (10.3%) and α-pinene (7.7%). The oils from the three other samples were characterized by a high content of both sesquiterpene hydrocarbons and oxygenated sesquiterpenes, represented by T-cadinol (24%), α-cadinol (9.8%) and δ-cadinene (7.5 and 9.8%), respectively (2). A study on the oil obtained from T. capitatum grown in Greece revealed that sesquiterpenes (55.8%) constituted the main fraction. The most prominent components were caryophyllene (9.8%), α-humulene (3.8%) and germacrene D (3.1%) (4). Iranian T. capitatum essential oil was found to contain α-cadinol (46.2%) as a major constituent followed by caryophyllene oxide (25.9%) (1). Mitić et al. (22) found that germacrene D (31.8%), trans-caryophyllene (8.8%) and bicyclogermacrene (6.2%) were identified as the major compounds of the essential oil from Serbia. Whereas the essential oil from Bulgaria was characterized by a high percentage of monoterpenes. β-pinene (26.8%), α-pinene (9.3%) and limonene (6.4%) were characterized as the most abundant components. Finally, Sesquiterpenes represented the main group of constituents in Algerian T. capitatum essential oil with T-cadinol (18.3%), germacrene D (15.3%) and β-pinene (10.5%) identified as the major components (6). Thus, as noted by Antunes et al. (2), there is a great polymorphism in the volatile oils of T. capitatum, probably due to genetic factors. As a matter of fact, even for plants collected at the same developmental stage and in very close localities with similar ecological features, the oils notably differ. The previous results of four samples of Teucrium lusitanicum (≡T. capitatum subsp. Lusitanicum) from Portugal showed that there are obvious differences in components of essential oils between the populations, even among those from the same locality. Cavaleiro et al. (23) found that sesquiterpenes, either hydrocarbons or oxygenated, were the major compounds of both essential oil samples collected in Sagres and Cabo de S. Vicente, respectively. Elemol (11.2 and 12.0%), germacrene D (5.3 and 6.0%), T-cadinol (5.2 and 5.5%) and δ-cadinene (4.1 and 5.3%), being the major compounds. Whereas, monoterpene hydrocarbons constituted the main group of the sample collected from Sagres, with α-pinene (8.2%), sabinene (9.6%), β-pinene (10.5%) and limonene (11.5%) identified as the main constituents. In addition, similar proportions of monoterpene hydrocarbons and sesquiterpenes were present in a forth sample collected from Cabo de S. Vicente, β-pinene (11.9%), α-pinene (8.5%), sabinene (7.7%), α-cadinol (9.1%) and δ-cadinene (7.3%), being the most abundant components. Antimicrobial assays In this study, antimicrobial activity of the essential oil extracted from aerial parts of T. capitatum subsp. lusitanicum was tested for the first time. The higher degree of resistance of gram-negative bacteria against hydrophobic antimicrobial compounds like those found in essential oils is due to the external membranes surrounding the cell wall in the latter ones that are rich in hydrophilic lipopolysaccharides that act, therefore as a barrier against penetration of hydrophobic compounds (24). The effectiveness of T. capitatum subsp. lusitanicum essential oil against susceptible bacteria was higher than those previously reported for other species of T. capitatum subsp. lusitanicum in which P. aeruginosa was resistant to the essential oil of this specie (6). However, our results are similar to those of El Amri et al. (25) and Djabou et al. (7), showing that gram-negative bacteria are more resistant to Teucrium capitaium L. essential oil and that S. aureus strain is very sensitive to the essence of this species. We note that, significant antibacterial and antifungal activities of the major constituents of T. capitatum subsp. lusitanicum oil, namely germacrene D and spathulenol, have been previously reported (26–27), Therefore, the presence of all these bioactive components in the essential oil increases the possibility of additive and synergistic effects. It is also possible that other minor constituents of the oil contribute to the antimicrobial activity (28). Conclusion The chemical constituents of the essential oil from aerial parts of Tunisian spontaneous T. capitatum subsp. lusitanicum was analysed by GC–FID and GC–MS methods. Sixty compounds accounting for 97.6% of the oil constituents were identified. The essential oil showed a considerable antimicrobial effect against all tested strains. S. aureus was the most sensible bacterial pathogen. 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TI - Chemical Composition and Antimicrobial Activity of Teucrium Capitatum L. Subsp. Lusitanicum (Schreb.) T. Navarro & Rosua Essential Oil
JO - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmaa086
DA - 2021-01-14
UR - https://www.deepdyve.com/lp/oxford-university-press/chemical-composition-and-antimicrobial-activity-of-teucrium-capitatum-0P0x9suvj4
SP - 134
EP - 139
VL - 59
IS - 2
DP - DeepDyve
ER -