ABSTRACT
In this study, branchlets, fruit, or bark of Taxus baccata. L. as well as branchlets or fruits of two other species of Iranian conifers, namely, Platycladus orientalis. France and Cupressus sempervirens. L. var. horizentalis. (Mill) Gunde were collected, identified, and the cytotoxic effects of hydroalcoholic extracts on three human tumor cell lines were determined. Different concentrations of extracts were added to cultured cells and incubated for 72 h. Cell survival was evaluated using the MTT assay. Extracts from bark of female Taxus baccata. showed inhibitory activities against Hela cells. The extracts of the branchlets of male and female T. baccata. as well as obtained extract from fruits of P. orientalis. showed inhibitory activities against MDA-MB-468 cells, whereas the extracts of branchlets of female T. baccata. showed inhibitory activities against KB cells. In conclusion, obtained extract from bark of T. baccata. showed comparable cytotoxic effect to doxorubicin against Hela cells.
Introduction
Isolation and identification of some potent antitumor compounds from plants has encouraged scientists to screen different parts of plant species against cancer cell lines. Biological methods (due to reliability, simplicity, and sensitivity) have been extensively used for evaluation of natural products in the past three decades. There are 10 species of Iranian conifers found in various parts of the country.
One is Taxus baccata. L. (Taxaceae), a dioecious and evergreen shrub or tree growing up to 20 m, with a wide pyramidal crown, leaves 10–30 mm, dark, glossy green above, with two green stomatiferous bands beneath, flowers enclosed in 2–4 membranous perianth segments, fruit a globose syncarp formed from 1 or 2 seeds, surrounded by 2 pairs of red or yellow, fleshy bracts. The tree is distributed in Europe, northern Africa, and in the western region of Asia (Parsa, Citation1949; Franco, Citation1964; Komarov, Citation1965; Riedl, Citation1968; Sabeti, Citation1975; Assadi, Citation1998). Its Persian name is sorkhdar. (Parsa, Citation1949; Sabeti, Citation1975; Assadi, Citation1998).
The dried bark of this plant is used for warmth (Shah & Jain, Citation1988) and its leaves are used for asthma, bronchical disorders (Singh et al., Citation1990), epilepsy (Kapur, Citation1983), and as an emmenagogue (Kamboj, Citation1988). Different parts of this plant are used as an emmenagogue, carminative, cardiotonic, antispasmodic, expectorant, diuretic, external antiseptic (Vohora & Kumar, Citation1971), and abortifacient (Jamwal & Anand, Citation1962), but the plant is highly toxic and its use is not recommended (PDR for Herbal Medicines., Citation2000).
Another Iranian conifer is Platycladus orientalis. Franco [Thuja. orientalis L., P. stricta. Spach, Biota orientalis. (L.) Endl.] (Cupressaceae), a monoecious and evergreen shrub or tree with scale leaves minute, closely imbricate, of two types in opposite and decussate pairs, composed of an inner, median facial pair and an outer, lateral pair, agnate for much of their length and small, free, obtuse tips. Male cones terminal and females are ovoid-pyriform with 8–10 thick, valuate scales and only the central 4 usually fully fertile. The plant is indigenous to Iran, Korea, Manchuria, and northern China (Boissier, Citation1884; Parsa, Citation1949; Riedl, Citation1968; Sabeti, Citation1975; Assadi, Citation1998). Its Persian names are nush., sarve-khomrehi., and sarve-tabari. (Parsa, Citation1949; Sabeti, Citation1975; Assadi, Citation1998).
Fresh leaves of this tree are used as an anti-inflammatory (Panthong et al., Citation1986), and dried leaves are used for flu and cough (Comford, Citation1996), high blood pressure (Panthong et al., Citation1986), bleeding arthralgia (Mikage et al., Citation1984), cancer (Sharma et al., Citation1993), and as a hemostatic (Kosuge et al., Citation1981). Seeds of this plant are used for bronchitis, amnesia, asthma, constipation, spermathorrhea (Nishiama et al., Citation1995), insomnia, and as an antitussive (Nishiama et al., Citation1995).
Another taxon of Iranian conifers is Cupressus sempervirens. L. var. horizentalis. (Mill.) Gordon [C. horizentalis. Mill., C. sempervirens. L. f. horizentalis. (Mill.) Voss.] (Cupressaceae), a monoecious and evergreen tree growing to 25 m with a pyramidal crown, leaves 0.5–1 mm, dark green obtuse, male cone 4–8 mm, female 25–40 mm, ellipsoid-oblong, green when young and shining yellowish-gray when ripe, with 8–14 mm on each scale. This tree is distributed in Mediterranean regions of Europe, Russia, Turkey, the northern Iran, Syria, Jordan, Lebanon, and northern Africa (Parsa, Citation1949; Komarov, Citation1965; Riedl, Citation1968; Sabeti, Citation1975; Coode & Cullen, Citation1979; Assadi, Citation1998). Its Persian name is zarbin. (Parsa, Citation1949; Sabeti, Citation1975).
The dried leaves of this plant are used as an emmenagogue and for stomach pain (Boukef et al., Citation1989) as well as for diabetes (Castro, Citation1998). The dried fruit of this plant is used for inflammations (Mascolo et al., Citation1987), toothache, laryngitis (Darias et al., Citation1989), as a contraceptive (Jochle, Citation1962), astringent, and antiphrastic (Ponce-Macotela et al., Citation1994). The dried seed of this tree is used for wounds, ulcers, bruises, sores, pimples, pustules, skin eruptions, and erysipelas (Caceres et al., Citation1987). The essential oil of the plant is used externally for headache, colds, cough, and bronchitis (PDR for Herbal Medicines., Citation2000).
Materials and Methods
Plant material
Different parts of T. baccata., P. orientalis., and C. sempervirens. var. horizentalis. were collected from Ghozlogh and Soorkesh (Golestan province, northern Iran) and Sangdehe (Mazandaran province, northern of Iran) in September 2000. The plants were identified by the Department of Botany, University of Tehran. The plant materials were stored at − 20°C before use (Adams et al., Citation1984). Voucher specimens of the plants were deposited in the herbarium of the Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences (Isfahan, Iran).
Extraction and isolation
Fifty grams of each plant part was crushed and soaked in 75 ml of ethanol (80% v/v) for 24 h and then percolated (5 h, 30 drops/min) (List & Schmidt, Citation1984). The extracts were concentrated by a rotary evaporator and dried in an oven at 40°C to give 0.5–0.8 g of solid residue. These solid residues (0.02 g) were dissolved in 100 ml of water containing 0.1% ethanol, filtered, and sterilized using 0.22 µm microbiological filters. Dilution was continued so that the final concentrations of extracts were 10 and 20 µg/ml.
Cell lines
Hela (human cervix carcinoma), KB (human Caucasian/epidermal carcinoma), and MDA-MB-468 (human breast adenocarcinoma) cell lines were purchased from Pasture Institute (Tehran, Iran).
Maintenance of human cell lines
Cells were grown in RPMI-1640 [each 500 ml of RPMI-1640 was supplemented with 10% fetal calf serum, 5 ml penicillin/streptomycin (50 IU ml−1 and 500 µg ml−1, respectively), 5 ml sodium pyruvate (1 mM), NaHCO3 (1 g), and 5 ml L-glutamine (2 mM)]. Completed media was sterilized by 0.22 µm microbiological filters after preparation and kept at 4°C before using. Cell lines were maintained and grown in RPMI 1640 up to 15 subcultures. A sample of each cell line was frozen and kept under liquid nitrogen for future studies.
MTT-based cytotoxicity assay
The cytotoxic effect of obtained extracts against previously mentioned human tumor cell lines was determined by a rapid colorimetric assay, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and compared with untreated controls (Mosmann, Citation1983). This assay is based on the metabolic reduction of soluble MTT by mitochondrial enzyme activity of viable tumor cells, into an insoluble colored formazan product, which can be measured spectrophotometrically after dissolving in dimethyl sulfoxide (DMSO) (Carmichale et al., Citation1987). Briefly, 200 µl of cells (5 × 104 cells per ml of media) were seeded in 96-well microplates and incubated for 24 h (37°C, 5% CO2 air humidified). Then, 20 µl of prepared concentrations of each extract was added, and microplates containing cells and extracts were incubated for another 72 h in the same condition. Doxorubicin was used as a positive control. The first column of each microplate was assumed as negative control (containing no extracts or doxorubicin). To evaluate cell survival, 20 µl of MTT solution (5 mg/ml in phosphate buffer solution) was added to each well and incubated for 3 h. Then, 150 µl of old medium containing MTT was gently replaced by DMSO and pipetted to dissolve any formed formazan crystals. Absorbance was then determined at 540 nm by ELISA plate reader. Each extract concentration was assayed in 8 wells and repeated 6 times. Standard curves (absorbance against number of cells) for each cell line were plotted. Intraday and interday variations were determined. Based on standard curves, percent cell survival was calculated. Percent of cell survival in negative control was assumed to be 100.
Statistical analysis
SIGMASTAT (Jandel Software, San Raphael, CA, USA) was used to perform statistical tests. Analysis of variance followed by Duncan test was used to see the differences among groups. Significance was assumed at the 5% level.
Results and Discussion
For Hela, KB, and MDA-MB-468 cell lines, a good relationship between number of cells and absorbance was observed (r2 = 0.9879, 0.9967, and 0.9618, respectively) (). Intraday and interday variation for all standard curves was acceptable (%CV < 20). Doxorubicin as a positive control showed significant inhibitory effects (cell survival less than 50%) against all tested cell lines.
Figure 1 Relationship between cell numbers and absorbance using MTT assay. Absorbance was determined at 540 nm. Numbers are shown as arbitrary units. A, Hela; B, KB; C, MDA-MB-468; n = 6.
Obtained extracts from the bark of female trees of T. baccata. (10 and 20 µg/ml) and from terminal branchlets of male and female of this species (20 µg/ml) showed an inhibitory effect against Hela cells. Extracts of the branchlets of female trees of T. baccata. (10 and 20 µg/ml) showed inhibitory effects against KB cells (). Obtained extracts from branchlets of male and female trees of T. baccata. (20 µg/ml) as well as extracts of fruits of P. orientalis. (20 µg/ml) showed an inhibitory effect on MDA-MB-468 cells ().
Table 1. Cytotoxic effects of hydroalcoholic extracts of different parts of three species of Iranian conifers against three cancer cell lines after 72 h continuous exposure to each extract.
Obtained extracts from different parts of C. sempervirens. var. horizentalis. had no effect against various cell lines ().
Obtained extract from the bark of female tree of T. baccata. was found to have the most significant inhibitory effect against Hela cells. This effect was comparable with doxorubicin at the same concentration. The extracts of terminal branchlets of male and female trees of T. baccata. showed an inhibitory effect against Hela cells, KB cells, and DMA-MB-468 cells.
A report (Sauter & Wolfensberger, Citation1989) indicated that the aqueous fruit extract of T. baccata. had no effect against CA-mammary-BT-20 cells. Unpublished data of the National Cancer Institute (NCI) of the United States show that the ED50 of hydroalcoholic extract against CA-9KB cells was less than 20.0 µg/ml (see NAPRALERT).
The obtained extracts from fruits of P. orientalis. show an inhibitory effect on MDA-MB-468 cell line. A report from China (Sato, 1989) indicates that the aqueous extract obtained from dried seeds and leaves of P. orientalis. separately have weak activities against CA-mammary-micro alveolar cells. This report indicated that the aqueous extract from dried twigs of the plant had no effect against the mentioned cell line, but another report (Sato, Citation1990) showed that hot aqueous extract of dried twigs of this plant has no effect on CA-JTC-26 cells. Nam and Lee (Citation2000) showed that the methanol extract obtained from the twigs of this plant was strongly cytotoxic against CA-Colon-2 and CA-AS-4 cell lines. Benzene extract of dried leaves of this species was active against Hela cells (Kosuge et al., Citation1985aCitationb).
Methanol and aqueous extracts of dried different parts of C. sempervirens. var. horizentalis. had no effect against various cell lines. A report from Jordan (Alkofahi et al., Citation1997) indicated that ethanol extracts of C. sempervirens. had no effect against CA-mammary-MCF-7 human breast cancer cell.
Acknowledgments
This study was supported by a grant from the research council of Isfahan University of Medical Sciences, Isfahan, Iran. The authors are thankful to the late Dr. K. Javanshir, University of Tehran, for identification of the plant material.
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