Bioactivity of Lignans from Taxus baccata Nurgun Erdemoglua,*, Bilge Senera, and M. Iqbal Choudharyb a Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey. Fax: +90-312-2235018. E-mail: [email protected] b HEJ Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan * Author for correspondence and reprint requests Z. Naturforsch. 59c, 494Ð498 (2004); received December 29, 2003/March 15, 2004 Three lignan derivatives, (Ð)-taxiresinol (1), (Ð)-3Ј-demethylisolariciresinol-9Ј-hydroxyiso- propylether (2) and (Ð)-3-demethylisolariciresinol (3), previously isolated from the heart- wood of Taxus baccata L. (Taxaceae) were investigated for cytotoxicity against the Oncology Cell Line Panel (breast, colon, ovary, prostate, lung and a normal adult bovine aortic endo- thelial cell line) as well as for antimicrobial activities. Besides, a chloroform-soluble portion of the ethanol extract from the heartwood of T. baccata was also tested for antimicrobial activities. Compounds 1 Ð 3 did not demonstrate much cytotoxic potency according to the reference drug etoposide. With the exception of compound 2, the other two lignans (1 and 3) and the chloroform extract were shown to possess antifungal activity, whereas only the chloroform extract exhibited antibacterial activity against Pseudomonas aeruginosa. Key words: Taxus baccata, Lignans, Antimicrobial Activity, Cytotoxicity Introduction cata resulted in the isolation of taxoids and lignans The genus Taxus L. (Taxaceae), yew, is widely (Erdemoglu, 1999; Erdemoglu et al., 2001, 2003). distributed in Europe, North America, Eastern Lignans are known to possess various biological Asia and Asia Minor. There are eight Taxus spe- activities such as antibacterial, antifungal, antiviral, cies and two hybrids worldwide and Taxus baccata antioxidant, anticancer and anti-inflammatory ef- L. is the single representative in Turkey. T. baccata, fects (MacRae and Towers, 1984). We previously English yew, is an evergreen and widespread shrub tested the ethanol extract of T. baccata heartwood commonly used for ornamental landscaping for antimicrobial activity. The extract showed sig- (Davis and Cullen, 1965; Van Rozendall et al., nificant activity against gram negative bacteria and 1999). However, due to its poisonous properties, against fungi (Erdemoglu and Sener, 2001). In our only few records document the plant as folk medi- previous studies, in vivo anti-inflammatory and cine, i.e. as an abortifacient, antimalarial, and anti- antinociceptive activity and in vivo anti-ulcerogenic rheumatic and against bronchitis (Bryan-Brown, potency of the isolated compounds from T. baccata 1932; Appendino, 1993) as well as against asthma were investigated (Kupeli et al., 2003; Gurbuz et al., (Singh, 1995). 2004). All compounds, taxoids and lignans, were The genus Taxus L. has interested many re- shown to possess significant antinociceptive activity searchers since the discovery of the anticancer against p-benzoquinone induced abdominal con- agent paclitaxel (TaxolTM), a diterpenoid alkaloid tractions, while only lignan derivatives significantly originally isolated from the bark of the Pacific yew, inhibited carrageenan-induced hind paw edema in T. brevifolia (Wani et al., 1971). The drug is the mice (Kupeli et al., 2003). Besides, all compounds first natural product described that stabilized were shown to possess significant anti-ulcerogenic microtubules and has been approved by the FDA activity, and the effect of taxiresinol was found to be for the treatment of ovarian, breast and non-small- the most prominent (Gurbuz et al., 2004). cell lung carcinomas (Rowinsky, 1997). So far, sev- There are a number of reports on lignan deriva- eral hundred different taxoids, lignans, flavonoids, tives with cytotoxic activity (Habtemariam, 2003; steroids and sugar derivatives have been isolated Chang et al., 2000). The aryltetralin lignan (Ð)-po- from different parts of various Taxus species dophyllotoxin occupies an unique position among (Parmar et al., 1999). Our previous phytochemical the lignan natural products since a glucopyrano- investigations on the chloroform-soluble portion side derivative was recognized as a potent antitu- of the ethanol extract of the heartwood of T. bac- mor factor (Jardine, 1980). The cytotoxicity and 0939Ð5075/2004/0700Ð0494 $ 06.00 ” 2004 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D N. Erdemoglu et al. · Bioactivity of Lignans 495 antimicrobial activity of these lignans have not Antibacterial assay been much evaluated so far. This prompted us to To test for antibacterial activity, the agar-well investigate cytotoxic and antimicrobial activities of diffusion method (Cayaugh, 1963) was used. A the isolated lignans (Ð)-taxiresinol (1), (Ð)-3Ј- Ј bacterial lawn was prepared on a nutrient agar demethylisolariciresinol-9 -hydroxyisopropylether plate by dispensing soft agar containing 100 µl cul- (2) and (Ð)-3-demethylisolariciresinol (3)(Fig.1) tures. 6 mm diameter wells were made in each from T. baccata. As related with the above-pre- plate using a sterile metallic borer. The concentra- sented data, this study is designed to investigate in tion of the sample was 1 mg/ml of DMSO. 100 µl vitro cytotoxicity and antimicrobial activity of samples were added into the wells using sterilized three lignans isolated from the chloroform-soluble pipettes. The plates were incubated at 37 ∞C for portion of the ethanol extract of the heartwood of 24 h. Imipenum (N-formimidoylthienamycin mo- T. baccata growing in Turkey. nohydrate) was the antibacterial reference sub- stance at 10 µg/disc. The antibacterial activity was Materials and Methods evaluated by measuring the diameter of the inhibi- Plant materials tion zones (mm). Taxus baccata L. (Taxaceae) was collected from the vicinity of Camlihemsin, Rize, in June 1995. A Antifungal assay voucher specimen (GUE 1560) is kept in the Her- To test for antifungal activity, the agar tube dilu- barium of the Faculty of Pharmacy, Gazi University. tion method, a modification of the agar dilution method of Washington and Sutter (1980), was fol- Chemical procedures lowed. A 1% Saboraud dextrose agar (SDA) was Column chromatography (CC) was performed used in the antifungal assay. Test tubes having ster- µ on silica gel (Kieselgel 60, 0.063Ð0.200 mm, Art. ile SDA were inoculated with test samples (200 g/ ml) and kept in a slanting position at room tem- 7734, Merck) and Kieselgel 60 F254 (0.5 mm thick- ness, Art. 5554, Merck) was used for preparative perature. The fungal culture was inoculated on the slant and growth inhibitions were observed after TLC. Precoated TLC plates (Kieselgel 60 F254) were employed for chromatographic analysis. incubation at 27 ∞C for 7 d. Control agar tubes The heartwood of the plant was dried under were made in parallel and treated similarly except shade and powdered to a fine grade. The material for the presence of test samples. Amphotericin B µ (3078 g) was extracted with 95% EtOH at room (10 g/ml) was the antifungal reference substance µ temperature. The ethanolic extract was evapo- against Aspergillus flavus. Miconazole (10 g/ml) rated under reduced pressure to give a reddish res- was used as the reference compound for the other idue. The residue (308.91 g) was partitioned be- pathogen fungi. The antifungal activity was eval- uated by measuring the inhibition of growth (mm) tween CHCl3 and H2O. The CHCl3 phase was concentrated (63.54 g). A portion (49 g) of the in percent. CHCl3 extract was chromatographed on silica gel and elution was carried out with increasing polari- Cytotoxicity assay ties of different solvents (hexane 5 acetone 5 The bioassay for cytotoxicity was performed by CHCl3 5 CH3OH) to give seven main fractions MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte- (IÐVII). Each fraction was further purified by CC, trazolium bromide] assay (Twentyman and Lus- preparative TLC or recrystallisation. Detailed iso- combe, 1987). Compounds 1Ð3 were evaluated for lation procedures of compounds 1Ð3 were de- in vitro cytotoxicity against a 9-cell line panel. The scribed in our previous studies (Erdemoglu, 1999; panel is made up of eight human tumor cell lines Erdemoglu et al., 2003). (breast, colon, ovary, prostate, and lung) and a normal adult bovine aortic endothelial cell line Microorganisms (ABAE). The cells were seeded in microtiter All microorganism strains used in bioactivity as- plates and the test compounds were added for says were obtained from the microbiological unit 24 h. Cell proliferation was determined by a tetra- of H. E. J. Research Institute of Chemistry, Uni- zolium dye conversion assay after a 72-h drug versity of Karachi, Karachi, Pakistan. exposure and IC50 values for each cell line were determined. Etoposide (4Ј-demethylepipodophyl- 496 N. Erdemoglu et al. · Bioactivity of Lignans lotoxin 9-[4,6-O-(R)-ethylidine--d-glycopyrano- Table I. In vitro cytotoxicity of compounds 1Ð3 against side]) was used as a cytotoxic standard agent along the Oncology Cell Line Panela. with this assay. Compound Mean Min IC50 Max IC50 Max/Min µ µ IC50 [ m][m]IC50 ratio Results and Discussion [µm] Chromatographic separation of the chloroform- soluble portion of the ethanol extract from the 1 > 114 99 > 129 > 1.2 2 > 59 23 > 112 > 5 heartwood of T. baccata collected from Camlihem- 3 > 107 79 > 128 > 1.6 sin, Rize, Turkey yielded lignan type compounds Etoposide 0.623 0.133 9.1 68 (Fig. 1), a well-known furanoid lignan, (Ð)-taxire- (VP-16) sinol (1), and two