Chemical and Antimicrobial Studies of Cupressus sempervirens L. and C. horizentalis Mill. Essential Oils

1. Introduction

     Cupressus sempervirens L. [C. fastigiata DC., C. sempervirens L. var. fastigiata Hansen], family Cupressaceae, is a monoecious and evergreen tree, up to 20-30 m high which grows in south and east of Europe, west part of Asia including various parts of Iran. Its Persian name is “Sarve Shirazi” or “Sarve Kashi” [1-8]. C. horizentalis Mill. [C.  sempervirens  L. var. horizentali (Mill.) Loud.] family Cupressaceae, is a monoecious and evergreen aromatic tree, up to 25 m high, broad and pyramidal growing; branches horizontal. This plant is widely grown from Crete to Iran. Its Persian name is “Zarbin” [1, 6-8]. C. sempervirens L. is a medicinal plant and act as expectorant and used externally for treating coughs and bronchitis [9]. There is no previous report on phytochemical characteristics and biological effects of these plants in Iran. However, a few phytochemical and biological studies on C. sempervirens L. in other parts of the world have been reported [10 -14].

     As a part of our systematic research on the chemical composition and antimicrobial properties of Iranian conifers, in this investigation, the essential oils content of fruits and terminal branches with adherent leaves (leaves) of both C. sempervirens and C. horizentalis was evaluated. In addition, the defatted ethanolic extracts of these organs were investigated qualitatively for the presence of alkaloids, flavonoids, saponins and tannins.

2. Materials and methods

2.1. Plant material

     The fruits and leaves of C. horizentalis Mill. were collected from Sorkesh forest at the height of 1000 m from the sea. This place is in province of Golestan which is located in the north of Iran. The fruits and leaves of C. sempervirens L. were collected from the National Botanical Garden of Iran (Tehran). The collected materials were stored at -20 °C in order to prevent changes in the chemical components [15].

      The plant material was identified by Dr. M. Assadi, Research Institute of Forest and Rangelands, Ministry of Jahad Keshavarzi, Iran. Voucher specimens have been deposited in the Herbarium of National Botanical Garden of Iran.

2.2. Isolation and analysis of non-volatile components

     The fruits and leaves of each plant (500 g) were dried at 50 °C and powdered separately. Each powder was defatted with petroleum ether (b.p. 40-60 °C) using Soxhlet apparatus (6 h). The chemical components of defatted powders were extracted by maceration with 70% ethanol (×4). The ethanolic extracts were concentrated at reduced pressure and analyzed for the presence of alkaloids [16], flavonoids [17], saponins [18] and tannins [19].

2.3. Isolation and analysis of volatile components

     Volatile oils were isolated from fresh plant material by wet steam distillation for 4 h [20]. The essential oils were separated from the aqueous layer and dried over anhydrous sodium sulfate and stored under nitrogen gas in a sealed vial and -20 °C until analyzed.

2.4. Gas chromatography-mass spectrometric analysis

     The  GC-MS  apparatus  consisted  of  a Varian 3400 GC equipped with a fused-silica column (DB-5, 30 m × 0.25 mm i.d., film thickness 0.25 mm; J&W Scientific Inc), and interfaced with a quadruple mass spectrometric detector (Incos50, Finnigan). The operating conditions were: Oven temperature 60-280 °C with the rate of 3 °C /min., injector mode: split injection; with the carrier gas, He; flow rate 2 ml/min.; ion source, 70 eV; ionization current, 750 mA; scan range, 40-300 u.

     The oil components were identified from their retention indices (RI) obtained with reference to n-alkane series (Sigma, UK) on DB-5 column, mass spectra with those of authentic samples, composition of their mass spectra and fragmentation patterns reported in literature [21], computer matching with MS-data bank (Saturn version 4). The peak area method was followed for quantitative determination of different constituents; the percentages were calculated relatively [21].

2.5. Antimicrobial activity determination

      The antimicrobial activity of each essential oil obtained from different parts of the plant was determined using five strains of microorganisms from the Persian Type Culture Collection (PTCC) [22]. The following microbial strains were used: Bacillus subtilis (PTCC 1023), Staphylococcus aureus (PTCC 1112), Escherichia coli (PTCC 1038), Pseudomonas aeroginosa (PTCC 1074) and Candida albicans (PTCC 5027). Minimum inhibitory concentrations (MICs) were determined using the agar dilution method [23]. Muller Hinton Agar medium (Oxoid, France) was prepared and sterilized by autoclaving for 20 min. at 121 °C. The medium was cooled to 50 °C and two-fold serial dilutions of the parent solution (essential oil plus 30% DMSO v/v) in adequate melted (50 °C) agar medium (1 ml) were prepared and added to 24-well plates (Greiner, France). The medium was allowed to solidify and then inoculated with the previously prepared microorganism suspension using the quadran streak method. For each strain tested, the adequacy of growth conditions, the effect of positive controls (gentamycin for bacteria and clotrimazole for fungus) and the sterility of the medium were tested in two wells. Plates were incubated for 24 h at 37 °C for the bacteria and 48 h at 25 °C for C. albicans.

3. Results

     The amount of non-volatile components in defatted ethanolic extracts of the fruits and leaves of C. sempervirens and C. horizentalis are shown in Table 1.

Table 1. Major non-volatile components of fruits and leaves of Cupressus sempervirens L. and C. horizentalis Mill.

      The essential oils isolated separately from fruits and leaves of C. sempervirens and C. horizentalis were colorless with a strong odor. The fruits and leaves of C. sempervirens yielded 0.1% and 0.3% v/w of volatile oil while the fruits and leaves of C. horizentalis yielded 0.4% and 0.5% (v/w) of volatile oil, respectively.

     GC-MS analysis of leaves and fruits volatile oils of C. sempervirens led to the identification of 13 and 9 components, respectively (Table 2). However, for C. horizentalis leaves and fruits volatile oils 10 and 9 components were identified, respectively (Table 3).

Table 2. Chemical composition of the volatile oil of Cupressus sempervirens L. fruits and leaves.

t = trace (< 0.1 %)

* The retention indices were determined on DB-5 capillary column.

Table 3. Chemical composition of the volatile oil of Cupressus horizentalis. Mill. fruits and leaves.