Cytotoxic Effects of Taxodium distichum. Oils

Abstract

The leaves and fruits of Taxodium distichum. (L.) L. C. Rich. (Taxodiaceae) collected from Nigeria were subjected to hydrodistillation. The essential oils obtained were analyzed by gas chromatography–mass spectrometry (GC-MS). The main compounds were α-pinene (60.5%) and thujopsene (17.6%) from the fruits and thujopsene (27.7%), pimara-8(14),15-diene (13.1%), widdrol (12.8%), and β.-caryophyllene (11.4%) from the leaves. The oils exhibited pronounced cytotoxic activities against PC-3, Hep G2, and Hs 578T human tumor cell lines at tested concentrations. Only the fruit oil displayed a promising antifungal effect (MIC 19.5 µg/mL) against Aspergillus niger..

Keywords:

• α-Pinene
• antimicrobial activity
• cytotoxic activity
• essential oil
• pimara-8(14)
• 15-diene
• Taxodium distichum
• thujopsene
• widdrol

Introduction

Taxodium distichum. (L.) L. C. Rich. (Taxodiaceae), commonly referred to as bald cypress, is an unusual and interesting tree, often growing over 25 m in height and over 300 cm d.b.h. (diameter at breast height) The leaves are small, 5–20 mm long, green to yellow-green and appearing two-ranked. Young trees have a pyramid shape but eventually form an irregular flattened canopy. The fruits are cones and are composed of pellate scales forming a woody, brown sphere with rough surface 1.5 to 4 cm in diameter. The cones disintegrate into irregular seeds.

A number of reports concerning the isolation and characterization of bioactive compounds from various parts of the plant have appeared in the literature. Such compounds include cytotoxic diterpenoid quinone methides, 2-furaldehyde, tannins, flavone and its derivatives, sterols and fatty acids, and proanthocyanidin (Kupchan et al., 19681969; Gieger & Pfleiderer, 1979; Tones et al., 1981). To the best of our knowledge, there are few reports on the chemical constituents of the volatile oil of the plant. El Tantawy et al. (1999) characterized the fruit oil of Taxodium distichum. growing in Egypt to be composed mainly of α.-pinene (87.3%). The observed antimicrobial, antispasmodic, and anti-inflammatory effects of the oil were attributed to its high α.-pinene content. Flamini et al. (2000) has also shown that the feminine cones, leaves, and branches of the plant are rich in α.-pinene (53.7–79.7%) and limonene (3.7–18.7%), while the sample analyzed from China (Liangfeng et al., 1995) contained caryophyllene oxide (41.67%) as the singly abundant constituent with sizeable proportion of bornyl acetate (6.24%), perilla ketone (5.45%), and α.-asarone (5.39%).

In continuation of our study of Nigerian medicinal plants and herbs (Jimoh et al., 2005; Ogunwande et al., 2005), we report herein the results of the study carried out on the chemical constituents, antibacterial, antifungal, and cytotoxic activities of the leaf and fruit oils of Taxodium distichum..

Materials and Methods

Plant samples

Mature leaves and fruits of Taxodium distichum. were collected from a single tree cultivated at the Department of Forestry Resources Management, Faculty of Agriculture and Fisheries, University of Ibadan, Ibadan, Nigeria, in March 2003. The samples were authenticated by Prof. M.A. Bada, of the same department and by Mr. U. Felix of the Herbarium Headquarters, Forestry Research Institute of Nigeria (FRIN), Ibadan, Nigeria, where a voucher specimen (FHI 106568) has been deposited.

Isolation and analysis of the essential oils

The oils of the leaves and fruits of T. distichum. were obtained by separate hydrodistillation of the pulverized fresh plant materials (350 g each) in an all-glass Clevenger-type apparatus for 4 h. The oils collected were separated from water by decantation and dried over anhydrous sodium sulfate and kept under refrigeration until the time of analyses.

The oil samples were subjected to GC-MS analyses using an Agilent system consisting of a model 6890 gas chromatograph, a model 5973 mass selective detector (MSD), and an Agilent ChemStation data system (Asilent Technologies, Santa Clara, CA, USA). The GC column was an HP-5 ms fused silica capillary with a (5% phenyl)-methylpolysiloxane stationary phase, film thickness of 0.25 µm, a length of 30 m, and an internal diameter of 0.25 mm. The carrier gas was helium with a column head pressure of 7.07 PSI and flow rate of 1.0 mL/min. Inlet temperature was 200°C and MSD detector temperature was 280°C. The GC oven temperature program was used as follows: 40°C initial temperature, hold for 10 min; increased at 3°C/min to 200°C; increased 2°C/min to 220°C. The sample was dissolved in CH₂Cl₂, and a split injection technique was used.

Identification of each individual constituent of the essential oils was achieved based on their retention indices (determined with a reference to a homologous series of normal alkanes), and by comparison of their mass spectral fragmentation patterns (NIST database/ChemStation data system) (Adams, 2001).

Cytotoxicity screening

Human Hep G2 hepatocellular carcinoma cells (ATCC no. HB-8065) (Knowles et al., 1980) were grown in an air environment at 37°C in Dulbecco's Modified Eagle's Medium (DMEM) with L-glutamine and 1000 mg glucose per liter of medium, supplemented with 100,000 units penicillin and 10.0 mg streptomycin per liter of medium, and buffered with 30 mM N.-(2-hydroxyethyl) piperazine-N.′-2-ethanesulfonic acid (Hepes), pH 7.35. Cells were plated using medium supplemented with 10% fetal bovine serum and maintained between passaging using medium supplemented with 10% horse serum and 5% fetal bovine serum.

Human Hs 578 T breast ductal carcinoma cells (ATCC no. HTB-129) (Hackett et al., 1977) were grown in a 3% CO₂ environment at 37°C in DMEM with 4500 mg glucose per liter of medium, supplemented with 10% fetal bovine serum, 10 μg bovine insulin, 100,000 units penicillin, and 10.0 mg streptomycin per liter of medium, and buffered with 44 mM NaHCO₃, pH 7.35.

Human PC-3 prostatic carcinoma cells (ATCC no. CRL-1435) (Kaighn et al., 1979) were grown in a 3% CO₂ environment at 37°C in RPMI-1640 medium with L-glutamine, supplemented with 10% fetal bovine serum, 100,000 units penicillin, and 10.0 mg streptomycin per liter of medium and buffered with 15 mM Hepes and 23.6 mM NaHCO₃, pH 7.30.

Hep G2 cells were plated into 96-well cell culture plates at 1.8 × 10⁴ cells per well, Hs 578T cells at 1.0 × 10⁵ cells per well, and PC-3 cells at 1.9 × 10⁴ cells per well. The volume in each well was 100 µL for all cell types. After 48 h, supernatant fluid was removed by suction and replaced with 100 µL growth medium containing either 2.5 or 1.0 µL of DMSO solution of oils or compounds (1% w/w in DMSO), giving a final concentration of 250 or 100 µg/mL, respectively, for each oil or compound. Hep G2 and Hs 578T cells were tested with final concentrations at 250 µg/mL and PC-3 at final concentration of 100 µg/mL. Solutions were added to wells in four replicates. Medium controls and DMSO controls (25 or 10 µL DMSO/mL) were used. Tingenone (250 or 100 µg/mL) was used as a positive control (Setzer et al., 1998). After the addition of compounds, plates were incubated for 48 h at 37°C; medium was then removed by suction, and 100 µL of fresh medium was added to each well. In order to establish percent kill rates, the CellTiter 96 AQueous Non-Radioactive Cell Proliferation assay was performed (Promega, 1996). After colorimetric readings were recorded (using a Molecular Devices SpectraMAX Plus microplate reader, 490 nm), (Molecular Devices Corp., Sunnyvale, CA, USA) average absorbances, standard deviations, and percent kill ratios (% kill compound/% kill DMSO) were calculated.

Antimicrobial screening

Both the leaf and fruit oils were screened for antibacterial activities against Gram-positive bacteria Bacillus cereus. (ATCC no. 14579) and Staphylococcus aureus. (ATCC no. 29213) and Gram-negative bacteria Pseudomonas aeruginosa. (ATCC no. 27853) and Escherichia coli. (ATCC no. 25922). Minimum inhibitory concentrations (MICs) were determined using the microbroth Dilution technique (Sahm & Washington, 1991). Dilutions of the oils were prepared in cation-adjusted Mueller-Hinton broth (CAMHB) beginning with 50 µL of 1% w/w solutions of the oils in DMSO plus 50 µL CAMHB. The oil solutions were serially diluted (1:1) in CAMHB in 96-well plates. Organisms at a concentration of approximately 1.5 × 10⁸ colony-forming units (CFU)/mL were added to each well. Plates were incubated at 37°C for 24 h; the final MIC was determined as the lowest concentration without turbidity. Gentamicin was used as a positive antibiotic control and DMSO was used as a negative control. Antifungal activity was determined as described using Candida albicans. (ATCC no. 10231) in yeast-nitrogen base growth medium with approximately 7.5 × 10⁷ CFU/mL. Antifungal activity against Aspergillus niger. (ATCC no. 16401) was determined as above using potato dextrose broth inoculated with A. niger. hyphal culture diluted to a McFarland turbidity of 1.0. Amphotericin B was the positive control.

Results and Discussion

The oils, separately obtained from the mature leaves and fruits by hydrodistillation procedure were separated from water by decantation. Both oils were yellowish-pink in color and at the yields of 1.5% and 1.3% v/w, respectively. After the GC-MS analyses, 30 compounds were identified, 15 of which were common to both oils (Table 1). The fruit oil was composed predominately of monoterpenoids (67.7%), with α.-pinene (60.5%) as the major compound. The minor monoterpenoid hydrocarbons detected were α.-pinene (2.3%), myrcene (2.2%), and limonene (1.7%). Sesquiterpenes were also abundant constituents (27.8%) with thujopsene (17.6%) being the main representative. The high content of α.-pinene among the monoterpenoids, and thujopsene among the sesquiterpenoids in the fruit oil, is in agreement with the earlier report of sample analyzed in Egypt (El Tantawy et al., 1999).

Table 1. Constituents of the volatile oils of Taxodium distichum.

		Area percentage (%)
Compounds.^a.	RI^b.	Fruits	Leaves
α.-Pinene	938	60.5	0.9
Camphene	951	0.1	–
β.-Pinene	979	2.3	Tr
Myrcene	991	2.2	Tr
δ.-3-Carene	1010	Tr	–
Limonene	1027	1.7	–
Terpinolene	1085	Tr	–
4-Terpineol	1180	Tr	–
α.-Terpineol	1190	–	2.6
Bornyl acetate	1284	0.9	1.8
Isolongifolene	1385	Tr	–
β.-longipinene	1395	0.5	Tr
α.-Cedrene	1410	0.8	Tr
β.-Caryophyllene	1419	1.6	11.4
Thujopsene	1432	17.6	27.7
α.-Humulene	1453	–	1.7
trans.-β.-Farnesene	1459	0.4	Tr
β.-Chamigrene	1475	0.6	1.3
β.-Himachalene	1500	0.8	–
α.-Chamigrene	1502	–	Tr
Cuparene	1506	1.8	6.3
δ.-Cadinene	1524	–	Tr
Cadina-1,4-diene	1532	0.3	–
C15H24O	1584	4.0	9.2
Widdrol	1599	1.9	12.8
Humulene epoxide II	1608	0.4	–
3-Thujopsanone	1647	0.3	1.2
α.-Bisabolol	1682	0.3	2.8
Pimara-8(14),15-diene	1937	0.5	13.1
Abietatriene	2046	–	2.7
Abietadiene	2069	–	1.7
Total		94.6	88.0

^a.Order of elution on HP-5ms fused silica capillary column.

^b.Retention index on HP-5ms fused silica capillary column.

−, not detected; Tr, trace amount < 0.1%.

The compositional profiles of the leaf oil showed a markedly qualitative and quantitative variation with those of the fruit oil. It was characterized by a high proportion of sesquiterpenes (78.3%), represented by thujopsene (27.7%), pimara-8(14),15-diene (13.1%), widdrol (12.8%), β.-caryophyllene (11.4%), and cuparene (6.3%). Monoterpenes and diterpenes were the other constituents present in the leaf oil. The major oxygenated monoterpenes were α.-terpineol (2.6%) and bornyl acetate (1.8%), while the diterpenes were abietatriene (2.7%) and abietadiene (1.7%). The principal compounds in this study, thujopsene, pimara-8(14),15-diene, widdrol, and cuparene, were not identified in the leaf oil sample from Italy (Flamini et al., 2000) or in that of China (Liangfeng et al., 1995). Also, we could not detect caryophyllene oxide and myristicin, which were the representative components of the Chinese sample of T. distichum., in the Nigerian grown sample. Oxygen containing sesquiterpenoid compound present in a significant quantity (4.0% and 9.2% in the fruit and leaf oil, respectively) remained unidentified.

The volatile oils exhibited weak antimicrobial effects against the tested microorganisms (Table 2). Only the fruit oil showed promising antifungal activity to A. niger. with a minimum inhibitory concentration of 19.5 µg/mL This is contrary to the literature report of El-Tantawy et al. (1999) in which the fruit volatile oil of Egyptian grown T. distichum. exhibited pronounced antibacterial activities against P. aeruginosa. (ATCC no. 27853) and E. coli. (ATCC no. 25922) and anticandidal effect against C. albicans. (ATCC no. 32354). These effects were attributed to the high α.-pinene content, which was also identified as the major compound of the fruit oil under study. We have found α.-pinene to possess, a weak antibacterial effect (MIC ≥ 312 g/mL against B. cereus., S. aureus., E. coli., and P. aeruginosa.) (Setzer et al., 1999). On the other hand, the volatile oils exhibited excellent cytotoxic properties toward the growth of three human tumor cell lines PC-3, Hep G2, and Hs 578T cells, except for the leaf oil, which displayed zero inhibitory activity toward Hs 578T cells (Table 3).

Table 2. Antimicrobial activities of the volatile oils (MIC, µg/mL)

Sample	Bc.	Sa.	Ec.	Pa.	Ca.	An
Fruit oil	156	156	312	625	312	19.5
Leaf oil	156	156	312	625	312	156
Gentamicin
Sulfate	1.22	0.61	2.44	1.22	Nt	Nt
Amphotericin B	Nt	Nt	Nt	Nt	0.61	0.61

Bc., Bacillus cereus. (ATCC no. 14579); Sa., Staphylococcus aureus. (ATCC no. 29213); Ec., Escherichia coli. (ATCC no. 25922); Pa., Pseudomonas aeruginosa. (ATCC no. 27853); Ca., Candida albicans. (ATCC no. 10231); An. = Aspergillus niger. (ATCC no. 16401); N, not tested.

Table 3. Cytotoxic potential of essential oils^a.

Sample	PC-3^b.	Hep G2^c.	Hs 578 T^d.
Fruit oil	99.77 (0.23)	100	100
Leaf oil	97.58 (0.15)	95.19 (1.47)	0

^a.% kill at tested concentrations (standard deviations in parentheses).

^b.Human prostate tumor cells (concentration tested 100 µg/mL).

^c.Human liver tumor cells (concentration tested 250 µg/mL).

^d.Human breast (ductal) tumor cells (concentration tested 250 µg/mL).

It is believed that the quantitative composition and the relative proportions of the oil components are widely influenced by the genotype, ontogenic development, and the environmental and growing conditions (Piccaglia et al., 1991), or by the plant species, the chemotypes, and the climatic conditions (Shu & Lawrence, 1997). It also implies the possibility of different medicinal uses of the plant species grown in different regions. Previously, cytotoxic diterpenoid quinone methides (taxodone and taxodione) had been isolated and characterized from the nonvolatile fractions of the plant (Kupchan et al., 19681969). Although the specific components of the studied oils that elicit cytotoxicity activities remain unclear, our data suggest that the oils offer new possibilities for cytotoxicity chemotherapy. Because these oils contained a large number of compounds, the observed cytotoxic effects may be due to the main compound or some minor compounds or a synergy between the main and minor compounds. These results may provide an insight into the medicinal importance of the plant growing in Nigeria. Further research work is aimed at the identification of the main compound(s), among the volatile constituents, responsible for the cytotoxic activities and to determine the mechanisms by which these compounds exert their biological activities.

Acknowledgment

The authors are grateful to Prof. M.A. Bada, Department of Forestry Resources Management, University of Ibadan, Nigeria, for plant collection and to Prof. Jirovetz (University of Vienna, Austria) for useful information. Mrs. Muslimat Buhari assisted in extraction of the oils. We thank Ms. A.K. Boehme and Ms. A. Bansal for assistance with the bioactivity screening. W.N.S. would like to acknowledge generous financial support from an anonymous private donor.