Chemical composition, antimicrobial properties and toxicity evaluation of the essential oil of Cupressus lusitanica Mill. leaves from Cameroon

Plant material and extraction of essential oil

Fresh leaves were collected from non-flowering Cupressus lusitanica Mill. plants in the campus of the University of Dschang in August 2010. The plant material was identified at the Cameroon National Herbarium in Yaounde where a voucher specimen is deposited under the code number HNC 66102. The leaves were subjected to hydrodistillation in batches of 2 kg in 3 L of water using a Clevenger-type apparatus. The obtained essential oil was dried over anhydrous sodium sulphate and stored at + 4°C until tested and analyzed.

Fractionation of essential oil

Five grams of the essential oil was subjected to column chromatography on silica gel (20 g, silica gel 40G, column 30 × 300 mm) with a gradient of hexane-acetyl acetate (95:5, 90:10, 85:15, 80:20, 70:30, 50:50, 25:75), 100% acetyl acetate and methanol. 80 fractions were collected. The band patterns of the fractions were examined on silica gel plates (silice 60G, type F₂₅₄, Alugram® SIL) with hexane-acetyl acetate (8:2 v/v) as the developing reagent. The plates were sprayed with 50% of sulphuric acid and heated at 100°C for band visualization. Fractions showing similar band patterns were combined and a total of seven fractions (F₁-F₇) were obtained.

GC-MS analysis conditions

The GC/MS analysis of the essential oil was performed using a Hewett-Packard 5890II GC, equipped with a HP-5 MS capillary spectrometer (30 m × 0.25 mm i.d., 0.25 μm film thickness) and a mass system with ionization energy of 70 ev. Helium was the carrier gas at a flow rate of 1 ml/min. Injector and MS transfer line temperatures were set at 220 and 290°C, respectively. Column temperature was gradually increased from 50 to 150°C at a rate of 3°C/min, and then held for 10 min. Finally, it was raised to 250°C at 10°C/min. Diluted samples (1:100 v/v in hexane) of 1.0 μl were injected manually in the split-less mode. The components were identified by comparing their relative retention times and mass spectra with those of standards, NBS75K library data of the GC/MS system, and literature data [15]. The results were further confirmed by comparing the elution order of the compounds with their relative retention indices on non-polar phases as reported by Adams [15].

Microorganisms and growth conditions

The microorganisms used in this study consisted of two Gram (+) bacteria- Enterococcus faecalis ATCC 10541 and Staphylococcus aureus ATCC 25922; six Gram (−) bacteria- Escherichia coli ATCC 11775, Klebsiella pneumoniae ATCC13883, Proteus mirabilis, Pseudomonas aeruginosa ATCC 27853, Salmonella typhi ATCC 6539 and Shigella flexneri; and 6 fungi- Candida albicans ATCC 9002, C. glabrata IP 35, C. krusei ATCC 6258, C. lusitaniae ATCC 200950, C. parapsilosis ATCC 22019 and C. tropicalis ATCC 750. The reference strains (ATCC) were obtained from American Type Culture Collection. The two clinical bacterial isolates were collected from Pasteur Center (Yaounde-Cameroon) and the candidal strain was obtained from Pasteur institute (IP, Paris-France). The bacterial and candidal strains were grown at 35°C and maintained on nutrient agar (NA, Conda, Madrid, Spain) and Sabouraud Dextrose Agar (SDA, Conda) slants respectively.

Antimicrobial assay

The essential oil and its fractions from C. lusitanica were tested for antimicrobial activities using agar disc diffusion technique to determine the diameter of growth inhibition zones while broth macrodilution method was used to determine the MIC and MBC/MFC.

Agar diffusion test

The disc diffusion method [16] was employed for the determination of antimicrobial activities of essential oil and its fractions (F1, F3, F4 and F5). Briefly, 0.1 ml of suspension of the test microorganisms containing 1.5 × 10⁷ cfu/ml of bacteria or 1.5 × 10⁶ spores/ml was spread on Mueller Hinton agar or Sabouraud dextrose agar medium respectively. Filter paper discs (Whattmann n°3, 6 mm in diameter) were soaked with 10 μl of the different concentrations of oil (100%, 10%, 0.1%v/v) and fractions (10%, 1%, 0.1%v/v), and placed on the seeded plates. A negative control was prepared using the solvent (10% DMSO) employed to dissolve the oil. Gentamicin and nystatin (10 μg/disc) were used as positive reference drugs for bacteria and fungi respectively. The fractions F2, F6 and F7 were not tested in this assay due to insufficient quantities. The plates were incubated at 37°C (24 hrs) for bacterial strains, and 35°C (48 hrs) for yeast. Antimicrobial activity was evaluated by measuring the diameters of inhibition zones. Each assay in this experiment was repeated three times.

Broth macrodilution tests

The minimal inhibitory concentration values were determined for all bacterial and candidal strains using the macrowell dilution method as described by Zgoda and Porter [17]. The inocula of the bacterial and fungal strains were cultured 18 hrs at 37°C in nutrient broth (NB) and 48 hrs at 35°C in Sabouraud dextrose broth (SDB) respectively. Tests strains were suspended in sterile saline to give a final density of 1.5 × 10⁶ cfu/ml of bacteria or 1.5 × 10⁵ spores/ml.

The 24 well plates were prepared by dispensing into each well 500 μl of nutrient broth. A 500 μl of C. lusitanica essential oil initially prepared at the concentration of 40% v/v was added into each of the first wells, followed by 2-fold serial dilution to obtain concentration range of 10 to 0.08% v/v. To this was separately added 500 μl of the prepared inoculum. The last well containing 500 μl of nutrient broth/Sabouraud dextrose broth without test substance and 500 μl of the inoculum on each strip was used as the negative control. The final volume in each well was 1 ml. The final concentration of Tween 80 in each medium was 0.5%, which did not affect the growth of the tests microorganisms. Gentamicin and nystatin at concentrations ranging respectively from 250–15 μg/ml, and 16–2 μg/ml were used as positive controls. Culture medium + Tween 80 + test oil was used as the sterility control. The fractions F2, F6 and F7 were not tested in this assay due to insufficient quantities. The plates were covered with a sterile plate sealer and incubated under normal atmospheric pressure at 37°C (24 hrs) for bacterial strains, and 35°C (48 hrs) for yeast. The test samples were screened three times against each microorganism. The bacterial or fungal growth was indicated by the presence of turbidity and/or a white “pellet” at the bottom of the well. The minimum inhibitory concentration was determined as the lowest concentration of the test compound that demonstrated no visible growth. The minimum bactericidal/fungicidal concentration (MBC/MFC) was determined by making subcultures of 10 μl of each well showing no turbidity, and negative control on nutrient agar or sabouraud dextrose agar. Subcultures of bacteria and fungi were incubated at 37°C (24 hrs) and 35°C (48 hrs) respectively. The MBC or MFC was considered as the lowest concentration of oil resulting in no growth (death) of the subcultured inoculum compared to the negative control [18].

Animals used

Male Wistar albino rats (110–120 g) and Swiss mice (22–25 g) were used for the toxicity assay. They were housed in standard plastic cages and provided with food and water ad libitum. The studies were conducted according to the ethical guidelines of the Committee for Control and Supervision of Experiments on Animals (Registration no. 173/CPCSEA, dated 28 January, 2000), Government of India, on the use of animals for scientific research.

Acute toxicity assay

This study was carried out in mice (males) as described by Emerson et al. [19]. Swiss albino mice of age 7–8 weeks were chosen at random and assigned to 5 different groups of 6 mice each (3 per cage). Food and water were withdrawn 18 hrs before the oral administration of 0.8 ml oil solution to the mice at the doses 2 to 8 g/kg of bodyweight (with a constant increment of 2). The fractions were not tested in this assay due to insufficient quantities. The control animals received 0.8 ml solution of 8% DMSO. The animals were observed for mortality and behavioural responses for 48 hrs, after which the number of deaths in each group was noted. The effect of the oil was assessed on the basis of mortality, expressed as median lethal dose (LD₅₀). The LD₅₀ value of the essential oil solution was evaluated by calculation [20].

Subacute toxicity assay

For this assay, 30 Wistar rats (males) aged 8 weeks were used. They were chosen at random and assigned to 5 different groups of 6 rats each (2 per cage). The control group received 0.5 ml of 8% DMSO solution while the test groups received corresponding doses of essential oil solution (from 0.8 to 2 g/kg with an increment of 0.4). All the animals were orally gavaged daily for four weeks and were observed for physiological, behavioural responses and for mortality.

Twenty-four hours after the last administration, the rats were anaesthetized and blood collected by cardiac puncture into heparinised and non-heparinised centrifuge tubes to evaluate some biochemical parameters and estimate blood cells. The heparinised blood was used to verify total red blood cell and white blood cell counts [21], and heamatocrit level [22]. Serum was prepared from non-heparinised blood and assessed for alanine aminotransferase (ALT), asparatate aminotransferase (AST), alkaline phosphatase (ALP) and creatinine levels using the commercial kits (BioSystems reagents and instruments). Protein levels in sera and organ homogenates were evaluated by the Biuret’s method [23].

Statistical analysis

The data were subjected to one-way analysis of variance, and differences between samples at P ≤ 0.05 were determined by Student-Newmann-Keuls multiple range test using the Statistical Package for the Social Sciences (SPSS) program. Experimental results were expressed (where appropriate) as mean ± SEM (standard error to the mean).

Chemical composition of essential oil and its fractions

GC/MS analysis of the crude oil (yield of 0.32%) resulted in the identification of 49 compounds representing 83.3%. Germacrene D (18.5%) epi-zonarene (8.2%), cis-calamenene (8.2%), terpinen-4-ol (6.3%), linalool (6.0%) and umbellulone (6.0%) were the main components, comprising 53.2% of the oil (Table 1). Fractions F1 and F3 were rich in composition than the other fractions.

Antimicrobial activities

The crude essential oil of C. lusitanica showed antimicrobial activities against all the bacteria and fungi tested. The highest activity was observed on bacteria. The diameters of inhibition zones for bacterial and fungal strains were in the ranges of 11–18 mm and 7–14 mm respectively (Table 2). This activity was highest at 10% v/v of oil and was observed to decrease with concentration. The fractions of this oil were less active and selective compared to the crude oil. These fractions inhibited most the growth of bacteria with diameter of inhibition zones varying from 7–10 mm.

The MICs and MBCs/MFCs for the crude oil (quantity of fractions was not enough for the assay) are given in Table 3. Generally, all the microorganisms were sensitive to the test sample. The MIC values of oil for bacteria ranged from 1.25- 10% v/v, with E. faecalis and P. mirabilis being the most sensitive while P. aeruginosa was the least sensitive. It was observed that the fungal strains were more susceptible to the leave essential oil (MIC range from 0.16-1.25% v/v) than bacteria. C. albicans was the most sensitive fungal strain.

Effects of single oral dose of essential oil on mice

The acute toxicity study of C. lusitanica leaf oil on mice showed a slight tolerance to the oil solution. For doses ≥ 4 g/kg the animals responded in uncontrolled movements; their respiratory rhythm increased progressively with increase in dose. The oil provoked deaths of mice; 2 g/kg (0/6), 4 g/kg (1/6), 6 g/kg (2/6) and 8 g/kg (6/6), within 48 hours observation period. The lethal dose-50 (LD₅₀) of this extract was evaluated as 6.33 g/kg bodyweight.

Subacute toxicity

After subjecting the rats to a daily treatment of C. lusitanica leaf oil for four weeks, the animals expressed some changes in their behavioural profile in the test groups. Decreases in movement, aggressiveness, response to touch and noise, and respiratory rhythm were observed. No death was recorded during this treatment either in the control or in the drug-treated groups.

From haematological and biochemical observations (Table 4), the red blood cells and white blood cells decreased with increase in dose of oil. This decrease was significant at 1.5 and 2 g/kg with respect to the control. In a like manner, the heamatocrit levels also decreased with the rising doses of oil, and were significantly higher in the control animals (45.78 ± 1.61%) with respect to test doses (Table 4). The least level (35.96 ± 1.05%) of heamatocrit was recorded at 2 g/kg dose of oil.

In the study of the effects of C. lusitanica leaf oil on some serum enzymes, it was observed that the oil provoked a significant increase in transaminase (AST and ALT) levels in serum at doses of 1.2, 1.6 and 2 g/kg. Compared to the control values of AST (6.63 ± 0.34 U/l) and ALT (10.11 ± 2.32 U/l), the dose of 2 g/kg of oil provoked the greatest increase of 32.65 ± 3.61 U/l and 19.79 ± 3.14 U/l levels of the respective enzymes.

The activity of alkaline phosphatase (ALP) in the serum was not significantly different (P ≤ 0.05) from the control after the oil treatment at the tested doses (Table 4). The creatinine level in serum decreased progressively with increase in essential oil dose (Table 4). The least value (11.00 ± 3.17 mg/l) observed at 2 g/kg was significantly different from the control (24.75 ± 1.68 mg/l).

The protein levels in the studied organs and serum decreased as the oil dose increased. This decrease was significant for the liver, heart, lungs, kidneys, spleen and serum at the dose 2 g/kg compared to the control (Table 5). The heart (71.66 ± 11.43 mg/g) and kidneys (117.20 ± 5.24 mg/g) were most affected since at 0.8 g/kg (least test dose) these values were significantly lower than the control (81.96 ± 17.32 mg/g and 156.37 ± 4.65 mg/g respectively).