Chemosystematic Value of the Essential Oil Composition of Thuja species Cultivated in Poland—Antimicrobial Activity

In the framework of the correlation between chemotaxonomy and chemical analysis studies, the chemical composition of the essential oils of four varieties of Thuja species cultivated in Poland − T. occidentalis ‘globosa’, T. occidentalis ‘aurea’, T. plicata and T. plicata ‘gracialis’ − were investigated by GC and GC-MS. Thirty-one compounds were identified from T. occidentalis ‘globosa’, representing 96.92% of the total oil; twenty-seven from T. occidentalis ‘aurea’ (94.34%); thirty-one from T. plicata (94.75%); and thirty compounds from T. plicata ‘gracialis’ (96.36%). The main constituents in all samples were the monoterpene ketones α- and β-thujone, fenchone and sabinene, as well as the diterpenes beyerene and rimuene. The chemosystematic value of the total ketone content of all samples (which varied from 54.30–69.18%) has been discussed and investigated. The constituents, beyerene and the mixture of α- and β-thujone, were isolated from the oils and tested against six Gram-positive and -negative bacteria and three pathogenic fungi. The oils of the two T. plicata species exhibited significant antimicrobial activity, while the mixture of α- and β-thujone showed very strong activity as well.


Introduction
Thuja is a small genus of the Cupressaceae family comprising five extant species, of which T. occidentalis L. and T. plicata Donn ex D. Don are widely grown in western and eastern North America and Europe [1]. T. occidentalis (Eastern arborvitae, Atlantic red cedar) which comprises shrubs or trees up to 15-35 meters tall, with spreading crowded branches, occurs in eastern North America, occupying a geographical range between the sub Arctic taiga-tundra interface in the north and the belt of deciduous angiosperm forests in the south, while in Europe it is grown as an ornamental tree [2]. Leaves are evergreen, scale-like, bright green above and pale green below. T. plicata (Western arborvitae, Pacific red cedar), a tree 50-70 meters tall with arching branches and leaves that are glossy green above and white-striped on the lower surface, is distributed in the Pacific Coastal Mountains and the Rocky Mountains of western North America [1]. Both species are used as a commercial crop and are managed for timber in Europe and North America. In folk medicine, Thuja occidentalis has been used to treat bronchial catarrh, enuresis, cystitis, psoriasis, uterine carcinomas, amenorrhea and rheumatism [1]. Cedar leaf oil can be obtained by steam distillation or hydrodistillation of the foliage and is used for the production of perfumes, insecticides, soaps and deodorants [3,4] The major constituent of the oil, the monoterpene thujone, is used pharmacologically as an active ingredient in the production of nasal decongestants and cough suppressants, perfumes, shoe polishes and soaps, while many cultivars are grown for ornamental purposes [5].
The oil of western red cedar leaves (T. plicata) was first investigated by Brandel, who reported the presence of thujone, fenchone, and esters of borneol [6]. In 1912, Rose and Livingston confirmed the major constituent to be Z-thujone (80-85%), and identified α-pinene, thujyl alcohol and thujyl acetate but could not isolate fenchone or bornyl esters [6]. Guenther points out that thujone occurs in nature as a mixture of the stereoisomers Z-thujone and E-isothujone, which has been confirmed by Rudloff [6] for T. plicata (Z-thujone 77.5% and E-isothujone 7.8%) and later [7] for 29 populations from the USA.
Regarding Thuja species growing in Europe, the essential oils from the wood from various Thuja species occurring in Czechoslovakia have been studied before [9], as well as the leaf oil from two different harvests of Slovakian T. occidentalis L. 'malonyana' ( Table 2) [10]. The aim of this study was the investigation of the chemical composition (Table 1) of the essential oil of the leaves of two Thuja species cultivated in Poland (T. occidentalis and T. plicata). The four different varieties are T. occidentalis "aurea", T. occidentalis "globosa", T. plicata and T. plicata "gracialis". We also report the isolation of their abundant terpenes (beyerene and α-and β-thujone) and the antimicrobial assay of the oils and the isolated terpenes (Table 3).

Results and Discussion
The oils from dried Thuja leaves were light yellow, with distinct sharp odors and the yield varied from 0.58% v/w to 0.87% v/w. The main components of the essential oils of each variety of the two studied species with their percentages and their retention indices are listed in Table 1, while the results of the antibacterial and antifungal activities of the essential oils and their main components are presented in Table 3. Furthermore, a comparison of the previous reported data concerning the most abundant compounds in T. occidentalis and T. plicata leaf oils of different origin [2,[6][7][8]10,11] with our data from Polish species is given in Table 2.
The study of the four samples resulted in the identification of thirty-one compounds in the oil of T. occidentalis "globosa" (area 96.92%) while in the oil of and T. occidentalis "aurea" twenty-seven constituents have been identified (area 94.34%), having both as major constituents α-thujone (50.14 and 51.60%, respectively), beyerene (8.54% and 11.28%, respectively), sabinene (4.55% and 3.43% respectively) and camphor (4.47 and 3.09 % respectively). The characteristic difference between them is that T. occidentalis "globosa" has a high content of the ketones β-thujone and fenchone, while T. occidentalis "aurea" has high levels of the diterpene rimuene. Regarding the reported leaf oil of eastern white cedar (T. occidentalis) [11], the main terpene components are almost the same in comparison with our results, but the content of fenchone is much higher (12.80%).
The chemical profiles of the oils of the two T. plicata were also comparable, as thirty-two compounds have been identified in T. plicata ( 94.75%) and thirty in the oil of T. plicata "gracialis" (96.36%) having also α-thujone (62.12% and 54.48%, respectively), β-thujone (7.06% and 6.39%), terpinen-4-ol (4.66% and 3.11%) and sabinene (6.00% and 2.94%) among the most abundant compounds. On the other hand, T. plicata shows higher content of the ketone fenchone, while T. plicata "gracialis" has high levels of the diterpene beyerene. According to our results, the analysis of the leaf essential oil of Canadian red cedar (T. plicata) [7], where thujone is the abundant compound (73.16%), but also the content of sabinene, terpinen-4-ol and isothujone was considerably high, was similar to this report. Notable is the absence of fenchone, which has a high percentage in our analysis (7.06%).
Leaf oil analysis for Thuja genus has been proved as a safe chemosystematic tool in the studies on different species and subspecies [12]. The chemosystematic value of the total ketone content, especially of thujone isomers and fenchone, is confirmed from this study, which is in accordance with the previous published data, for Thuja from different origin, as is shown in Table 2. It may, therefore be concluded that leaf oil analysis can be of considerable help, providing basic information needed for the chemosystematic approach of a genus.

Antimicrobial activity
The reported essential oils and the isolated compounds have been assayed for their antimicrobial activity (Table 3). Previously, the insecticidal activity of the oil of T. occidentalis has been examined and it was characterized as highly toxic [13]. Also the antifungal activity, in order to control the biocontamination in libraries and archives storage areas, of thuja oil has been reported, showing that this oil had little inhibitory effect on the fungal combination [14].
In the antimicrobial screening, the oil of T. plicata exhibited the highest antibacterial (MIC values 0.50-1.25 mg/mL) as well as antifungal (MIC 0.87-1.12 mg/mL) activity, followed by that of T. plicata "gracialis" (MIC values 0.75-1.24 mg/mL and 1.15-1.45 mg/mL, respectively). This antimicrobial activity is suspected to be associated with the high percentage of α-and β-thujone, which exhibited also strong activities against the assayed microorganisms (MIC 0.09-0.83 mg/mL), and are well known as the main active compounds in many essential oils possessing similar antimicrobial properties [15,16]. The diterpene beyerene, has been isolated in this study, to our knowledge, for the first time from essential oil source, and showed interesting antimicrobial (MIC 0.87-1.37 mg/mL) and antifungal (MIC 1.15-1.50 mg/mL) activity as well. The results of this study, showing such an interesting antimicrobial profile, could support the potential use of plant essential oils, which could be of economical benefit.

Conclusions
The chemosystematic value of the total ketone content, especially of thujone isomers and fenchone, is confirmed from this study, as oil analysis for Thuja genus has been proved as a safe chemosystematic tool in previous studies on different species and subspecies [12]. Therefore, it may be amplified that leaf oil analysis can be of considerable help, providing basic information needed for the chemosystematic approach of a genus.
The antimicrobial study of the essential oils and the isolated compounds showed an interesting antimicrobial profile, which could provide economical benefit with the potential use of plant essential oils.

Experimental
General GC Analysis: Analyses were carried out on a Perkin-Elmer Clarus 500 gas chromatograph with FID, fitted with a DB-5 capillary column (30 m × 0.25 mm.; film thickness 0.25 μm). The column temperature was programmed from 75 °C to 200 °C at a rate of 2.5 °C/min. The injector and detector temperatures were programmed at 230 °C and 300 °C, respectively.
GC/MS Analysis: The GC-MS analysis of the essential oils was performed using a Hewlett Packard 6890 series II gas chromatograph equipped with an HP-5 capillary column (30 m, 0.25 mm i.d., 0.25 μm film thickness) and a mass spectrometer 5973 of the same company which was operated on EI mode. Helium was the carrier gas at a flow rate of 1 mL/min. The injector was operated at 200 °C and the oven temperature was programmed as follows: 60 °C for 5 min, then gradually increased to 280 °C with a 3 °C/min rate. In all experiments the samples have been analysed at least twice and the results have been expressed as average. The identification of the compounds was based on comparison of their retention indices (RI), retention times (RT) and mass spectra with those from Wiley libraries spectra, the NIST/NBS and literature data [17][18][19].

Oil isolation
The plant material of each plant (200 g) was subjected to hydrodistillation on a Clevenger-type apparatus for 2.5 h according to the method described in the literature [20]. The obtained essential oils were dried over anhydrous sodium sulfate and stored at 4 °C in the dark until they were analyzed.

Isolation of terpenoids
Samples from the essential oils of both T. occidentalis "globosa" (480 mg) and T. plicata (502 mg) were subjected to preparative-TLC (eluent system toluene: ethyl acetate 93:7, silica gel 60 F 254 pre-coated glass plates, 1 mm thickness). Different zones were obtained after spraying with vanillin reagent and they were extracted with pentane-ethyl acetate 90:10. The extracts were evaporated under reduced pressure (30 °C) down to 2-3 mL and kept in 4 °C until they were analyzed with chromatographic and spectroscopic methods (GC, GC-MS).

Antimicrobial activity assays
Antimicrobial activity of the essential oils and the isolated terpenoids was determined using the agar dilution technique [21]. For all assays, stock solutions of the tested oils and pure compounds in sterile distilled water with 10% Tween 80 have been prepared at 10 and 1 mg/mL, respectively. Serial dilutions of the stock solutions in broth medium (100 μL of Müller-Hinton broth, Sabouraud broth for fungi and blood agar 10% for oral pathogens) were prepared in a microtiter plate (96 wells). Then 1 μL of the microbial suspension (the inoculum, in sterile distilled water) was added to each well. For each strain, the growth conditions and the sterility of the medium were checked and the plates were incubated at 37 °C and the MICs were determined as the lowest concentrations preventing visible growth.