Cytotoxic Activity and Essential Oil Composition of Leaves and Berries of Juniperus excelsa.

ABSTRACT

The composition of the water-distilled essential oils of the berries and leaves of Juniperus excelsa. M. Bieb. were analyzed by gas chromatography-mass spectrometry (GC-MS). The main components in the berries of J. excelsa., accounting for 56.1% of the oil, were determined as α-pinene (34.0%), cedrol (12.3%), L-verbenol (5.4%), and D-verbenol (4.4%) while in the leaves of J. excelsa., accounting for 63.2% of the oil, were found to be α-pinen (29.7%), cedrol (25.3%), α-muurolene (4.4%), and 3-carene (3.8%). Cytotoxic and antimicrobial potential of the berries and leaves of J. excelsa. were investigated.

Keywords:

• Biological activity
• Cupressaceae
• essential oil
• Juniperus excelsa. M. Bieb

Introduction

Although 60 Juniperus. L. species grow in the world (Welch, 1986), the Juniperus. L. species is represented by only 8 species in Turkey (Coode & Cullen, 1982); it is mainly distributed from Balkan Peninsula, Turkey, Saudi Arabia, Yemen, Oman, Iran, Crimea, Afghanistan (Kerfoot & Lavronos, 1984) to Taiwan (Coode & Cullen, 1982). They are known to be used as a traditional local remedy for tuberculosis and jaundice in Saudi Arabia (Muhammad et al., 1992). In Turkey, Juniperus. species, including J. excelsa. M. Bieb, J. communis. subsp. nana., J. drupacea. Lab., J. foetidissima. Wild., J. sabina. L., and J. oxycedrus. L. have been used as a treatment for cold cough as well as bronchitis and hemorrhoids. J. oxycedrus. is also used for many purposes including the treatment of urinary infections, hemorrhoidal and rheumatismal pains (Sezik & Yeşilada, 1999), and has also shown some hypotensive effects (Bello et al., 1997). Alcohol extract of J. excelsa. displayed acaricidal activity (Tsitsin et al., 1973), and sandracopimaric acid, isolated from J. excelsa., showed antibacterial activity against Bacillus subtilis., Staphylococcus aureus., and Streptococcus durans. (Muhammad et al., 1992). In addition, in our previous study, hexane and methanol extracts of J. excelsa. and a new labdane juniperexcelsic acid, isolated from its hexane extract, were found to be moderately active against Mycobacterium tuberculosis. (Topçu et al., 1999), and both extracts also showed some activity against standard bacteria. Furthermore, the hexane extract of the berries tested against a panel cell line showed high activity against KB (human epidermoid carcinoma) (ED₅₀ = 1.9 µg/ml) and LNCaP (hormone-dependent human prostate cancer cell line) (ED₅₀ = 1.3 µg/ml), whereas methanol extract was found weakly active against KB (ED₅₀ = 1.3 µg/ml). The berries hexane extract was found more or less active against COL2 (human colon cancer) and gave a positive response to ASK 9 glioma cells. In the current study, both berries and leaf methanol extracts of J. excelsa. have been investigated in a yeast-based microtiter assay for their DNA-damaging properties (Schwikkard et al., 2000): they were not found to be DNA-damaging agents, however the leaf extract can be considered as a general antifungal agent. The leaf methanol extract was also tested against A2780 (human ovarian cancer cell line) and found to be weakly active (IC₅₀ = 17.7 µg/ml), and the leaf essential oil extract was found to be moderately active (IC₅₀ = 13.7 µg/ml) against LU1 (human lung cancer) cell line.

The only naturally growing species in the southern hemisphere, J. procera. Hochst. ex. Endl. from Africa, probably a sibling species of J. excelsa., has previously been investigated for its essential oil, and marked differences were found between this species and Grecian J. excelsa.. In the current study, Turkish J. excelsa. leaf oil showed the main difference from African J. procera. was having cedrol in fairly high quantity that had similar composition to J. excelsa. essential oil from Greece.

Materials and Methods

Plant material

The berries and leaves of Juniperus excelsa. M. Bieb. were collected from Isparta, Southwestern Turkey, in June 2001. A voucher specimen was deposited at the Herbarium of the Faculty of Pharmacy, University of Ankara (AEF 19787).

Isolation of the essential oil

The essential oil of leaves (173 g) and berries (430 g) of J. excelsa. were obtained by hydrodistillation for 3 h in a Clevenger-type apparatus. The yield of the leaves oil was 1.72 ml [1.0% (v/w)] and of the berries oil was 13.74 ml [3.2% (v/w)].

Gas chromatography-mass spectrometry (GC-MS)

GC-MS qualitative and quantitative analyses were carried out with a HP 5890 GC, Micromass Zabspec system (double-focusing magnetic sector, Manchester, UK). DB-5 (5% diphenyl-95% dimethyl siloxane) fused silica column (60 m × 0.25 mm, Ø with 0.5-µm film thickness) was used with helium at 1 ml/min as a carrier gas. GC oven temperature was kept at 40°C for 4 min, programmed to 280°C at rate of 5°C/min, and kept constant at 280°C for 10 min. Split ratio was adjusted to 1:50. The injection volume was 0.1 µl. The MS were taken at 70 eV ionization energy. The mass range was from m./z. 50–700 amu, scan time 2 s. Library search was carried out using the NIST, Wiley GC/MS, and TÜBİTAK, MRC, Gebze-Kocaeli libraries of essential oil constituents. Relative percentage amounts of the separated compounds were calculated from the total ion chromatography by the computerized integrator.

Activity tests

A yeast-based dose-response microtiter assay was carried out according to McBrien et al. (1995). The bioactivity of the samples was evaluated throughout the fractionation using RS321N, pRAD52, and RS321NYCp50 genetically engineered Saccharomyces cerevisae. yeast strains. Growth inhibition was determined using a microplate assay in which the RS321NpRAD52 strain was seeded individually in minimal media (Difco) plus glucose and galactose (respectively), and RS321NYCp50 was seeded in minimal media plus galactose. Samples were dissolved in 10% DMSO and transferred to the seeded microtiter wells at a 1:10 dilution, for a final testing concentration of 100 µg/ml. Microtiter plates were incubated at 28°C for 48–72 h or until an optimum optical density of 0.15–0.25 was reached. Growth inhibition was elucidated using a linear regression analysis of the dose-response scheme, and activity was reported in terms of an IC₅₀ value, which is the concentration (µg/ml) necessary to produce 50% cell inhibition. Streptonigrin at 0.001 µg/ml and etoposide at 20 µg/ml were both used as positive controls for the RS321NpRAD52 and RS321NYCp50 strains.

A panel cytotoxicity assay was carried out according to Likhitwitayawuid et al. (1993). The used cell lines were cultured KB (human epidermoid carcinoma), BC1 (human breast cancer), LU1 (human lung cancer), COL-2 (human colon cancer), KB-V (+VLB) (drug resistant KB), P-388 (mouse leukemia), and LNCaP (hormone-dependent human prostate cancer), and ellipticine was used as a positive control.

A2780 ovarian cancer cytotoxicity assay was carried out according to Schwikkard et al. (2000), and actinomycin D was used as a positive control.

Results and Discussion

The results from GC-MS analyses are presented in Table 1. The oil of the berries and leaves of J. excelsa. contain 67 compounds represented by 93.7% and 92.80% of the total oil, respectively. The main compounds from both berries and leaves of J. excelsa. were characterized as α-pinene and cedrol with different percentages. α-Pinene was 34.0% and cedrol 12.3% of the berries oil, whereas they were 29.7% and 25.3%, respectively, of the leaf oil. The other major compounds were L-verbenol (5.4%), D-verbenol (4.4%), eucarvone (3.8%), p.-cymene (3.0%), limonene (2.3%), and α-muurolene (2.2%) in J. excelsa. berries oil, whereas in the leaves oil of J. excelsa. were α-muurolene (4.4%), 3-carene (3.8%), β-myrcene (2.5%), limonene (2.4%), and terpinolene (1.9%) in addition to the minor components (Table 1).

Table 1. The percentage composition of the total oil from the leaves and berries of Juniperus excelsa. M. Bieb.

Compound	RT (min)	Juniperus excelsa. (Berries)	Juniperus excelsa. (Leaves)
Toluene	15.35	t	t
Tricyclene	22.40	t	0.4
α-Pinene	23.24	34.0	29.7
Fenchene	23.43	0.4	0.5
Camphene	23.52	0.8	0.8
Sabinene	24.28	0.1	0.3
m.-Cymene	24.34	0.4	—
β-Myrcene	24.52	1.5	2.5
1,3,8-p.-Menthatriene	25.21	1.1	0.1
3-Carene	26.04	0.8	3.8
p.-Cymene	26.32	3.0	1.3
α-Terpinene	26.38	—	0.2
Limonene	26.46	2.3	2.4
Thujene	26.51	—	1.4
o.-Cresol	27.22	0.2	—
Terpinen-4-acetate	27.45	—	0.8
Unidentified	28.15	0.2	0.1
Isopropenyl toluene	28.38	0.1	t
Terpinolene	28.56	—	1.9
Fenchone	29.11	0.3	t
Mentha-2,8-dien-1-ol	29.21	0.7	0.1
3-Thujanone	29.40	1.8	0.1
L-Verbenol	29.59	4.4	t
α-Pyronene	30.00	—	0.2
Fenchyl alcohol	30.07	—	0.2
α-Campholenal	30.25	1.7	0.5
D-Verbenol	31.10	5.4	0.9
p.-Mentha-1,5-dien-8-ol	31.22	0.5	—
Alcamphor	31.24	—	0.4
Camphor	31.28	0.7	t
β-Phellandrene-8-ol	31.34	—	0.2
Camphene hydrate	31.38	0.2	—
Isopinacamphone	31.52	0.6	—
1-Pinocarvone	31.56	0.6	—
Umbellulone	31.60	0.9	—
Borneol	32.07	0.9	0.3
1-Terpinene-4-ol	32.19	0.2	0.6
Thymol	32.24	0.9	—
Methyl tolyl ketone	32.30	0.3	0.1
α-Terpineol	32.42	0.6	0.4
Naphthalene	32.58	—	0.2
Myrtenol	33.02	1.1	—
Myrtenal	33.08	1.4	0.2
Verbenone	33.31	0.2	0.7
Eucarvone	33.38	3.8	—
Pulegone	34.23	0.2	—
L-Carvone	34.29	0.3	—
Bornyl acetate	35.48	1.6	1.3
Isobornyl acetate	35.58	0.1	—
D-Ledol	37.21	0.1	t
Piperitonene	37.44	0.1	—
α-Cububene	37.52	0.1	0.1
Unidentified	38.04	0.1	0.5
Z.-Cinerone	38.26	0.1	0.1
Sorberol	38.42	0.2	—
Himachalene	38.47	0.2	0.1
Copaene	38.55	0.2	0.1
α-Cedrene	39.21	0.1	0.5
α-Longipinene	39.29	0.1	0.1
Germacrene D	40.19	0.1	0.1
α-Muurolene	40.27	2.2	4.4
α-Cadinene	40.37	—	0.4
γ-Muurolene	40.45	—	1.8
Thujopsene	40.57	0.2	0.9
α-Curcumene	41.27	0.1	0.6
Acoradien	41.38	0.2	—
Aristolene	41.45	0.8	—
Chamaecynone	41.58	0.1	—
β-Cadinene	42.08	—	1.2
Dysoxylonene	42.19	0.2	0.3
Caryophyllene	42.33	—	0.2
Bornyl isobutanoic ester	42.38	0.1	—
Unidentified	42.41	—	0.2
γ-Cadinene	42.52	0.7	1.9
Calamenene	43.02	0.1	0.2
Caryophyllene oxide	45.11	0.2	0.2
Cedrol	45.59	12.3	25.3
α-Cadinol	46.19	0.4	0.5
Nootkatone	46.59	0.2	—
Manoyloxide	55.25	0.1	t
Ağabeyetatrien	56.46	0.1	0.1
8,14-Pimaradien	56.60	—	t
Manool	56.92	—	0.1
Kaur-16-ene	57.50	—	0.3

t = trace (<0.05%); RT = retention time on the DB-5 column. Unidentified compounds are not reported < 0.5%.

In both oils, the high percentage of α-pinene and cedrol was the most characteristic feature, besides a large amount of D- and L-verbenol 9.77% in the berries oil, whereas the leaves had D- and L-verbenol only 0.9%. In literature, essential oil of J. excelsa. from Jamma (Thappa et al., 1987) and essential oil of J. excelsa. from Greece have been reported (Adams, 1990ab), and the main compounds were cedrol (28.0%), limonene (22.7%), and α-pinene (22.5%) of the Grecian J. excelsa. leaf essential oil. In contrast, in the leaves of J. procera., the sibling species of J. excelsa., cedrol was not found: however, the wood of J. procera. is known as a rich source for cedrol (Adams, 1990b). As a result, Turkish J. excelsa. leaves oil was fairly similar to Grecian J. excelsa. oil, and particularly the berries of Turkish J. excelsa. can be considered as a rich source of α-pinene with 34.0% yield.

Both berries and leaves methanol extracts of J. excelsa. have been investigated in a yeast-based microtiter assay for their DNA-damaging properties (Schwikkard et al., 2000). In this yeast-based microtiter assay, any compound or extract that was cytotoxic to the yeast in the YCp50 galactose and pRAD52 glucose plates (>65% inhibition) but was not cytotoxic in the pRAD52 galactose plate (<35% inhibition) was considered as an active agent. The IC₅₀ difference between pRAD52 galactose and YCp50 galactose was 2.097 and between pRAD52 galactose and pRAD52 glucose was 0.593, however no hit. Therefore, the leaf extract was considered at least as a general antifungal agent and possibly a cytotoxic agent. It was then tested against A2780 ovarian cell line and showed moderate activity with an IC₅₀ value of 17.7 µg/ml (McBrien et al., 1995). Furthermore, the leaf essential oil extract was tested against four cell lines (LU1, COL2, KB, LNCaP) and showed a marginal activity only against LU1 with an IC₅₀ value of 13.7 µg/ml (Likhitwitayawuid et al., 1993).

Acknowledgments

The authors are grateful to Prof. Dr. J.M. Pezzuto (Purdue University, School of Pharmacy, West Lafayette, Indiana) for a panel of cytotoxic activity test, except A2780. We thank the NSF-TÜBİTAK Research Fund (Project No. 0002071-TBAG 53), which is supportive of this project.