Journal of Ethnopharmacology 89 (2003) 265–270 Anti-inflammatory and antinociceptive activity of taxoids and lignans from the heartwood of Taxus baccata L. Esra Küpeli, Nurgün Erdemoglu,˘ Erdem Ye¸silada∗, BilgeSener ¸ Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler 6330, Ankara, Turkey Received 20 November 2002; received in revised form 27 August 2003; accepted 1 September 2003 Abstract Four taxoids (taxusin, baccatin VI, baccatin III and 1␤-hydroxybaccatin I) and five lignans (lariciresinol, taxiresinol, 3-demethylisolaricire- sinol-9-hydroxyisopropylether, isolariciresinol and 3-demethylisolariciresinol) were isolated from the heartwood of Taxus baccata L. (Tax- aceae) growing in Turkey through chromatographic techniques. In vivo anti-inflammatory and antinociceptive activity of these compounds were investigated. All the compounds were shown to possess significant antinociceptive activity against p-benzoquinone-induced abdominal contractions, while only lignan derivatives significantly inhibited carrageenan-induced hind paw edema in mice. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Anti-inflammatory; Antinociceptive; Lignans; Taxaceae; Taxoids; Taxus spec 1. Introduction L. (European yew) is the single representative in Turkey (Davis and Cullen, 1965). Until now, a large number of Genus Taxus L. (Taxaceae), yew, is widely distributed in taxoids possessing different skeleton systems, as well as the northern hemisphere, and has recently attracted a great lignans, flavonoids, steroids and sugar derivatives have been deal of attention as sources for an anticancer agent, pacli- isolated from various Taxus species (Baloglu˘ and Kingston, taxel (Taxol®), a unique diterpene taxoid originally extracted 1999; Parmar et al., 1999). During our course of studies on from the bark of the Pacific yew, Taxus brevifolia (Wani the bioactive components, the chloroform-soluble portion et al., 1971; Baloglu˘ and Kingston, 1999; Parmar et al., of ethanolic extract of the heartwood of Taxus baccata af- 1999). Paclitaxel has been approved for the treatment of forded four taxoids; taxusin (1), baccatin VI (2), baccatin ovarian and breast cancers as well as Kaposi’s sarcoma and III (3) and 1␤-hydroxybaccatin I (4), along with five lig- non-small-cell lung cancers. It is also under clinical trial for nans; lariciresinol (5), taxiresinol (3-demethyllariciresinol) treatment of several other cancers in combination with other (6), 3-demethylisolariciresinol-9-hydroxyisopropylether chemotherapeutic agents (Rowinsky, 1997; Eisenhauer and (7), isolariciresinol (8) and 3-demethyl isolariciresinol (9) Vermorken, 1998). (Erdemoglu,˘ 1999). However, due to the poisonous properties of yew, few Lignans are known to possess various biological activ- records have been encountered as traditional medicine in ities, including antibacterial, antifungal, antiviral, antioxi- the literature. Yew leaves are reported to be used in tradi- dant, anticancer and anti-inflammatory effects (Cho et al., tional medicine as abortifacient, antimalarial, antirheumatic 2001a). Two of the isolated lignans from Taxus baccata and for bronchitis (Bryan-Brown, 1932; Appendino, 1993; heartwood mentioned above; lariciresinol (5) and isolari- Ballero and Fresu, 1993), while dried leaves and barks were ciresinol (8) were shown to possess potent in vitro inhibitory used against asthma (Singh, 1995). It was also listed in effect on tumor necrosis factor-␣ (TNF-␣) production (Cho Avicenna’s cardiac drugs (Tekol, 1989). et al., 2001b). Since TNF-␣ is known as one of the main There are eight Taxus species and two hybrids in the pro-inflammatory cytokines secreted during the early phase world (Van Rozendall et al., 1999) and Taxus baccata of acute and chronic inflammatory diseases such as asthma, rheumatoid arthritis, septic shock, etc. (Cho et al., 2001a), the above-mentioned traditional use of Taxus species in in- ∗ Corresponding author. Fax: +90-312-2235018. flammatory diseases, i.e. asthma and rheumatism may be at- E-mail address: [email protected] (E. Ye¸silada). tributed to its lignan derivatives. On the other hand, a lignan 0378-8741/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2003.09.005 266 E. Küpeli et al. / Journal of Ethnopharmacology 89 (2003) 265–270 fraction possessing 5 and 8 from Fagraea racemosa were 2.2.4. Isolation of lignans also shown to possess analgesic effect on writhing symp- Fraction VII (5.65 g) was rechromatographed on a silica toms in mice (Okuyama et al., 1995). gel column eluting with CHCl3:CH3OH (100:0 → 92:8, As related with the above-presented data, this study is de- v/v) to give 64 subfractions. The subfrs. 11–13 were sub- signed to investigate in vivo anti-inflammatory and antinoci- jected to prep.-TLC with CHCl3:CH3OH (90:10, v/v) to ceptive activity of lignans and taxoids isolated from the give 5 (45.7 mg, yield: 0.0028%) and 7 (20.2 mg). From chloroform-soluble portion of ethanolic extract of the heart- subfrs. 14–22, 7 (42.6 mg) was also obtained through re- wood of Taxus baccata. crystallization from CHCl3 and total yield of 7 was 62.8 mg (0.0039%). The subfrs. 47–52 was further separated by prep.-TLC using CHCl3:CH3OH (80:20, v/v) system to 2. Experimental afford 6 (84.6 mg, 0.0052%). The subfrs. 28–33 was crys- tallized from CHCl3 to give 8 (41.3 mg, 0.0026%). The 2.1. Plant material subfrs. 58–64 was subjected to a silica gel CC eluting with CHCl :CH OH (100:0 → 80:20, v/v) and then prep.-TLC The heartwood of Taxus baccata L. (Taxaceae) was col- 3 3 using CHCl3:CH3OH (80:20, v/v) to afford 9 (71.4 mg, lected from the vicinity of Camlihemsin, Rize, in June 1995, 0.0044%). from wild trees (altitude 1400 m). An authenticated voucher specimen (GUE 1560) was kept in the Herbarium of Faculty 2.3. Pharmacological procedures of Pharmacy, Gazi University. 2.3.1. Animals 2.2. Chemical procedures Male Swiss albino mice (20–25 g) were purchased from the animal breeding laboratories of Refik Saydam Central 2.2.1. General Institute of Health (Ankara, Turkey). The animals left for Column chromatography was performed by using Silica 2 days for acclimatization to animal room conditions were gel (Kieselgel 60, 0.063–0.200 mm, Art. 7734, Merck) and maintained on standard pellet diet and water ad libitum. The Kieselgel 60 F254 (0.5 mm thickness, Art. 5554, Merck) food was withdrawn on the day before the experiment, but was used for prep.-TLC. Analytical TLC was performed allowed free access of water. A minimum of six animals was on precoated plates (Kieselgel 60 F254, Art. 5554, Merck) used in each group. Throughout the experiments, animals and visualized under UV254 light, and then sprayed with were processed according to the suggested ethical guidelines anisaldehyde reagent and heated. for the care of laboratory animals. 2.2.2. Extraction, isolation and purification 2.3.2. Preparation of test samples for bioassay The air-dried and powdered heartwood (3078 g) was ex- Test samples were given orally to test animals after sus- tracted with 95% EtOH at room temperature. The ethanolic pending in a mixture of distilled H O and 0.5% sodium extract was evaporated to dryness in vacuo and a red- 2 carboxymethyl cellulose (CMC). The control group animals dish residue was obtained. The residue was diluted with received the same experimental handling as those of the test H O and then extracted with CHCl 2 3. The CHCl3-soluble groups except that the drug treatment was replaced with ap- portion was evaporated under reduced pressure to give a propriate volumes of the dosing vehicle. Either indomethacin residue (49 g), which was subjected to column chromatog- (10 mg/kg) or acetyl salicylic acid (ASA) (100 mg/kg) in raphy eluted with increasing polarities of different solvents 0.5% CMC was used as reference drug. (hexane → acetone → CHCl3 → CH3OH)togiveseven main fractions (frs. I–VII) according to TLC. Each fraction 2.3.3. Antinociceptive activity was further purified by CC, prep.-TLC or recrystallisation. 2.3.3.1. p-Benzoquinone-induced abdominal constriction 2.2.3. Isolation of taxoids test in mice (Okun et al., 1963). Sixty minutes after the Compound 1 was obtained from the fr. II by CC elut- oral administration of test samples, the mice were intraperi- ing with hexane:acetone (100:0 → 60:40, v/v) mixture toneally injected with 0.1 ml/10 g body weight of 2.5% to give seven subfractions (subfrs.). The subfrs. 5–7 was (v/v) p-benzoquinone (PBQ; Merck) solution in distilled crystallized from acetone to afford 1 (171 mg, 0.011%). H2O. Control animals received an appropriate volume of Fraction III was chromatographed on prep.-TLC with hex- dosing vehicle. The mice were then kept individually for ane:acetone (70:30, v/v) to give 2 (40.5 mg, 0.0025%). observation and the total number of abdominal contractions Fraction IV was crystallized from hexane:acetone (1:1) (writhing movements) was counted for the next 15 min, mixture to afford 3 (155.4 mg, 0.0096%). Fraction V was starting on the fifth minute after the PBQ injection. The data subjected to CC over a silica gel column eluting with represent average of the total number of writhes observed. CHCl3:CH3OH (100:0 → 90:10, v/v) mixture and then The antinociceptive activity was expressed as percentage prep.-TLC with CHCl3:CH3OH (80:20, v/v) to provide 4 change from writhing controls. Aspirin (ASA) (100 mg/kg) (76.4 mg, 0.0047%). was used as reference drug. E. Küpeli et al. / Journal of Ethnopharmacology 89 (2003) 265–270 267 2.3.4. Carrageenan-induced hind paw oedema CH3OC O OCOC H3 The modified method of Kasahara et al. (1985) was 18 19 used with modifications in measuring periods (Ye¸silada and 10 9 11 7 Küpeli, 2002).