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 Erdemoğlu, Erdem Yeşilada∗ , Bilge Şener 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 -demethylisolariciresinol-9 -hydroxyisopropylether, isolariciresinol and 3-demethylisolariciresinol) were isolated from the heartwood of Taxus baccata L. (Taxaceae) 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 Genus Taxus L. (Taxaceae), yew, is widely distributed in the northern hemisphere, and has recently attracted a great deal of attention as sources for an anticancer agent, paclitaxel (Taxol® ), a unique diterpene taxoid originally extracted from the bark of the Pacific yew, Taxus brevifolia (Wani et al., 1971; Baloğlu and Kingston, 1999; Parmar et al., 1999). Paclitaxel has been approved for the treatment of ovarian and breast cancers as well as Kaposi’s sarcoma and non-small-cell lung cancers. It is also under clinical trial for treatment of several other cancers in combination with other chemotherapeutic agents (Rowinsky, 1997; Eisenhauer and Vermorken, 1998). However, due to the poisonous properties of yew, few records have been encountered as traditional medicine in the literature. Yew leaves are reported to be used in traditional medicine as abortifacient, antimalarial, antirheumatic and for bronchitis (Bryan-Brown, 1932; Appendino, 1993; Ballero and Fresu, 1993), while dried leaves and barks were used against asthma (Singh, 1995). It was also listed in Avicenna’s cardiac drugs (Tekol, 1989). There are eight Taxus species and two hybrids in the world (Van Rozendall et al., 1999) and Taxus baccata ∗ Corresponding author. Fax: +90-312-2235018. E-mail address: yesilada@pharmacy.gazi.edu.tr (E. Yeşilada). L. (European yew) is the single representative in Turkey (Davis and Cullen, 1965). Until now, a large number of taxoids possessing different skeleton systems, as well as lignans, flavonoids, steroids and sugar derivatives have been isolated from various Taxus species (Baloğlu and Kingston, 1999; Parmar et al., 1999). During our course of studies on the bioactive components, the chloroform-soluble portion of ethanolic extract of the heartwood of Taxus baccata afforded four taxoids; taxusin (1), baccatin VI (2), baccatin III (3) and 1-hydroxybaccatin I (4), along with five lignans; lariciresinol (5), taxiresinol (3 -demethyllariciresinol) (6), 3 -demethylisolariciresinol-9 -hydroxyisopropylether (7), isolariciresinol (8) and 3-demethyl isolariciresinol (9) (Erdemoğlu, 1999). Lignans are known to possess various biological activities, including antibacterial, antifungal, antiviral, antioxidant, anticancer and anti-inflammatory effects (Cho et al., 2001a). Two of the isolated lignans from Taxus baccata heartwood mentioned above; lariciresinol (5) and isolariciresinol (8) were shown to possess potent in vitro inhibitory effect on tumor necrosis factor-␣ (TNF-␣) production (Cho et al., 2001b). Since TNF-␣ is known as one of the main pro-inflammatory cytokines secreted during the early phase 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 inflammatory diseases, i.e. asthma and rheumatism may be attributed 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 also shown to possess analgesic effect on writhing symptoms in mice (Okuyama et al., 1995). As related with the above-presented data, this study is designed to investigate in vivo anti-inflammatory and antinociceptive activity of lignans and taxoids isolated from the chloroform-soluble portion of ethanolic extract of the heartwood of Taxus baccata. 2. Experimental 2.1. Plant material The heartwood of Taxus baccata L. (Taxaceae) was collected from the vicinity of Camlihemsin, Rize, in June 1995, from wild trees (altitude 1400 m). An authenticated voucher specimen (GUE 1560) was kept in the Herbarium of Faculty of Pharmacy, Gazi University. 2.2. Chemical procedures 2.2.1. General Column chromatography was performed by using Silica gel (Kieselgel 60, 0.063–0.200 mm, Art. 7734, Merck) and Kieselgel 60 F254 (0.5 mm thickness, Art. 5554, Merck) was used for prep.-TLC. Analytical TLC was performed on precoated plates (Kieselgel 60 F254 , Art. 5554, Merck) and visualized under UV254 light, and then sprayed with anisaldehyde reagent and heated. 2.2.2. Extraction, isolation and purification The air-dried and powdered heartwood (3078 g) was extracted with 95% EtOH at room temperature. The ethanolic extract was evaporated to dryness in vacuo and a reddish residue was obtained. The residue was diluted with H2 O and then extracted with CHCl3 . The CHCl3 -soluble portion was evaporated under reduced pressure to give a residue (49 g), which was subjected to column chromatography eluted with increasing polarities of different solvents (hexane → acetone → CHCl3 → CH3 OH) to give seven main fractions (frs. I–VII) according to TLC. Each fraction was further purified by CC, prep.-TLC or recrystallisation. 2.2.3. Isolation of taxoids Compound 1 was obtained from the fr. II by CC eluting with hexane:acetone (100:0 → 60:40, v/v) mixture to give seven subfractions (subfrs.). The subfrs. 5–7 was crystallized from acetone to afford 1 (171 mg, 0.011%). Fraction III was chromatographed on prep.-TLC with hexane:acetone (70:30, v/v) to give 2 (40.5 mg, 0.0025%). Fraction IV was crystallized from hexane:acetone (1:1) mixture to afford 3 (155.4 mg, 0.0096%). Fraction V was subjected to CC over a silica gel column eluting with CHCl3 :CH3 OH (100:0 → 90:10, v/v) mixture and then prep.-TLC with CHCl3 :CH3 OH (80:20, v/v) to provide 4 (76.4 mg, 0.0047%). 2.2.4. Isolation of lignans Fraction VII (5.65 g) was rechromatographed on a silica gel column eluting with CHCl3 :CH3 OH (100:0 → 92:8, v/v) to give 64 subfractions. The subfrs. 11–13 were subjected to prep.-TLC with CHCl3 :CH3 OH (90:10, v/v) to give 5 (45.7 mg, yield: 0.0028%) and 7 (20.2 mg). From subfrs. 14–22, 7 (42.6 mg) was also obtained through recrystallization 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 : CH3 OH (80:20, v/v) system to afford 6 (84.6 mg, 0.0052%). The subfrs. 28–33 was crystallized from CHCl3 to give 8 (41.3 mg, 0.0026%). The subfrs. 58–64 was subjected to a silica gel CC eluting with CHCl3 :CH3 OH (100:0 → 80:20, v/v) and then prep.-TLC using CHCl3 :CH3 OH (80:20, v/v) to afford 9 (71.4 mg, 0.0044%). 2.3. Pharmacological procedures 2.3.1. Animals Male Swiss albino mice (20–25 g) were purchased from the animal breeding laboratories of Refik Saydam Central Institute of Health (Ankara, Turkey). The animals left for 2 days for acclimatization to animal room conditions were maintained on standard pellet diet and water ad libitum. The food was withdrawn on the day before the experiment, but allowed free access of water. A minimum of six animals was used in each group. Throughout the experiments, animals were processed according to the suggested ethical guidelines for the care of laboratory animals. 2.3.2. Preparation of test samples for bioassay Test samples were given orally to test animals after suspending in a mixture of distilled H2 O and 0.5% sodium carboxymethyl cellulose (CMC). The control group animals received the same experimental handling as those of the test groups except that the drug treatment was replaced with appropriate volumes of the dosing vehicle. Either indomethacin (10 mg/kg) or acetyl salicylic acid (ASA) (100 mg/kg) in 0.5% CMC was used as reference drug. 2.3.3. Antinociceptive activity 2.3.3.1. p-Benzoquinone-induced abdominal constriction test in mice (Okun et al., 1963). Sixty minutes after the oral administration of test samples, the mice were intraperitoneally injected with 0.1 ml/10 g body weight of 2.5% (v/v) p-benzoquinone (PBQ; Merck) solution in distilled H2 O. Control animals received an appropriate volume of dosing vehicle. The mice were then kept individually for observation and the total number of abdominal contractions (writhing movements) was counted for the next 15 min, starting on the fifth minute after the PBQ injection. The data represent average of the total number of writhes observed. The antinociceptive activity was expressed as percentage change from writhing controls. Aspirin (ASA) (100 mg/kg) was used as reference drug. E. Küpeli et al. / Journal of Ethnopharmacology 89 (2003) 265–270 2.3.4. Carrageenan-induced hind paw oedema The modified method of Kasahara et al. (1985) was used with modifications in measuring periods (Yeşilada and Küpeli, 2002). The difference in footpad thickness between the right and left foot was measured with a pair of dial thickness gauge calipers (Ozaki Co., Tokyo, Japan). Mean values of treated groups were compared with mean values of a control group and analyzed using statistical methods. Sixty minutes after the oral administration of test sample or dosing vehicle each mice was injected with freshly prepared (0.5 mg/25 l) suspension of carrageenan (Sigma, St. Louis, MO, USA) in physiological saline (154 nM NaCl) into subplantar tissue of the right hind paw. As the control, 25 l saline solution was injected into that of the left hind paw. The difference in footpad thickness between treated and untreated paws was accepted a paw edema was measured in every 90 min during 6 h after induction of inflammation by a gauge calipers (Ozaki Co.). Mean values of treated groups were compared with mean values of a control group and analyzed using statistical methods. Indomethacin (10 mg/kg) was used as reference drug. 267 OCOC H3 CH3 OCO 18 19 10 CH 3 OCO 12 13 11 9 16 8 17 3 15 14 1 7 6 5 4 OCOC H3 H 2 H 20 1. Taxusin 18 12 13 14 19 10 11 R2 O R1 C H 3 OC O 16 8 17 3 15 1 OR 9 2 7 4 H OH 6 5 20 O OC OC H 3 O C =O 1' 6' 2' 5' 3' 4' 2.3.5. Acute toxicity Animals employed in the carragenan-induced paw edema experiment were observed during 24 h and mortality was recorded, if happens, for each group at the end of observation period. 2.3.6. Gastric–ulcerogenic effect After the analgesic activity experiment mice were killed under deep ether anesthesia and stomachs were removed. Then the abdomen of each mouse was opened through the great curvature and examined under dissecting microscope for lesions or bleedings. 2.3.7. Statistical analysis of data Data obtained from animal experiments were expressed as mean standard error (±S.E.M.). Statistical differences between the treatments and the control were evaluated by ANOVA and Student–Newman–Keuls post hoc tests. P < 0.05 was considered to be significant (∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001). 3. Results and discussion In our continuing research for bioactive components of Taxus baccata growing in Turkey, the heartwood of the plant was found to be richer than the other parts for taxoids and lignans (Erdemoğlu, 1993). According to the procedure described in the experimental section, four taxoids (1–4), representing skeletally three different groups, and five lignans (5–9), belonged to two different groups, have been isolated using column chromatography and prep.-TLC. Spectral techniques employed in order to elucidate the structures were described previously (Erdemoğlu, 1999). The structures are summarized as follows (Figs. 1 and 2): R 2. Baccatin VI 10 11 CH3OCO 9 1 8 17 3 OH 2 H OCOCH 3 19 OCOCH 3 16 15 2 C O CH 3 =O CH3OCO 12 13 14 R 1 O CO CH 3 H 3. Baccatin III 18 R COCH 3 7 6 OCOCH 3 4 H O 20 OCOCH 3 4. 1β-Hydroxybaccatin I Fig. 1. Taxoids (1–4) isolated from the heartwood of T. baccata. (a) Taxusin (1) was identified as a exocyclic methylene containing taxoid, (b) Baccatin VI (2) and baccatin III (3) were determined as an oxetane ring possessing taxoids, (c) 1-Hydroxybaccatin I (4) was elucidated as an epoxide ring possessing taxoid, (d) Lariciresinol (5) and taxiresinol (6) were furanoid type lignans, (e) 3 -Demethylisolariciresinol-9 -hydroxyisopropylether (7), isolariciresinol (8) and 3-demethylisolariciresinol (9) were dibenzylbutane type lignans. The isolated taxoids are known as specific compounds for Taxus genus (Erdemoğlu and Şener, 2000). Among the lignan derivatives, although lariciresinol (5) and taxiresinol 268 E. Küpeli et al. / Journal of Ethnopharmacology 89 (2003) 265–270 CH3O 7 2 1 4 HO 9 8 3 6 5 R O 8' 9' 7' OH 1' 5. (-)-Lariciresinol 6. (-)-Taxiresinol 2' 6' CH3 H 3' 5' OR 4' OH RO 2 9 7 8 1 3 OH 4 HO 6 5 7' 1 ' OR2 8' 9' 2' 6' 3' 5' 4' OH OR1 R2 R R1 7. (-)-3'-Demethylisolariciresinol9'-hydroxyisopropylether CH3 H 8. (-)-Isolariciresinol CH3 CH3 H H CH3 H OH C CH3 9. (-)-3-Demethylisolariciresinol CH3 Fig. 2. Lignans (5–9) isolated from the heartwood of T. baccata. (6) were also previously reported from various other Taxus species (Mujumdar et al., 1972; Chattopadhyay et al., 1997; Kawamura et al., 2000) along with other plant species (Raju and Pillai, 1989; Ullah et al., 1999; Okunishi et al., 2001), 7 and 9 were identified as new lignans of isolariciresinol derivatives. Moreover, 7 is the first example of a lignan attached a hydroxyisopropyl group at C-9 (Erdemoğlu et al., 2003). Isolariciresinol (8) was previously isolated from other Taxus species (Erdtman and Tsuno, 1969; Das et al., 1993) as well as other plants (Weinges, 1961; Raju and Pillai, 1989). As shown in Tables 1 and 2, chloroform-soluble portion of the ethanolic extract from the heartwood of Taxus baccata exhibited significant antinociceptive and anti-inflammatory Table 1 Effect of taxoids (1–4) and lignans (5–9) isolated from Taxus baccata against p-benzoquinone-induced writhings in mice Material Control Number of writhings ± S.E.M. Dose (mg/ml) 41.5 ± 4.7 – CHCl3 extract 1 2 3 4 5 6 7 8 9 300 30 30 30 30 100 100 100 100 100 ASA 100 Values are expressed in mean ± S.E.M., 24.5 30.3 28.2 25.8 26.3 23.8 25.8 26.8 28.5 27.8 ± ± ± ± ± ± ± ± ± ± – 2.9 2.8 4.5 2.1 4.3 3.0 3.1 3.4 2.6 2.6 18.2 ± 1.6 ∗ P < 0.05; ∗∗ P < 0.01; Inhibitory ratio (%) ∗∗∗ Ratio of ulceration 0/6 40.9∗∗ 26.9∗ 32∗ 37.8∗ 36.6∗ 42.7∗∗ 37.8∗ 35.4∗ 31.3∗ 33∗ 0/6 1/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 56.1∗∗∗ 1/6 P < 0.001 when compared with control group. E. Küpeli et al. / Journal of Ethnopharmacology 89 (2003) 265–270 269 Table 2 Effects of taxoids (1–4) and lignans (5–9) isolated from Taxus baccata against carrageenan-induced paw edema in mice Material Control Swelling thickness (×10−2 mm) ± S.E.M. (percent inhibition) Dose (mg/kg) – CHCl3 extract 1 2 3 4 5 6 7 8 9 300 30 30 30 30 100 100 100 100 100 Indomethacin 10 Values are expressed in mean ± S.E.M., 90 min 180 min 270 min 45.5 ± 4.7 52.7 ± 4.6 59.7 ± 4.7 37.7 41.2 44.2 39.3 43.2 40.0 37.2 40.5 41.0 35.3 ± ± ± ± ± ± ± ± ± ± 4.7 4.5 5.5 4.5 5.6 4.0 3.6 3.2 3.9 4.5 (17.1) (9.5) (2.9) (13.6) (5.1) (12.1) (18.2) (10.9) (9.9) (22.4) 42.5 46.8 45.2 43.5 45.7 45.7 42.2 45.7 43.2 40.0 P < 0.05; ∗∗ P < 0.01; 4.9 4.1 5.9 4.7 4.7 3.4 3.4 3.3 5.5 3.9 (19.4) (11.2) (14.2) (17.5) (13.3) (13.3) (19.9) (13.3) (18.0) (24.1) 36.8 ± 3.3 (30.2)∗ 33.7 ± 4.4 (25.9) ∗ ± ± ± ± ± ± ± ± ± ± ∗∗∗ 46.8 51.8 51.3 48.2 48.5 46.3 47.0 46.8 49.2 45.2 ± ± ± ± ± ± ± ± ± ± 360 min 3.2 4.2 6.3 4.6 4.2 3.7 3.7 3.9 4.9 4.6 67.2 ± 4.7 (21.6)∗ (13.2) (14.1) (19.3) (18.8) (22.4) (21.3) (21.6) (17.6) (24.3) 39.5 ± 3.1 (33.8)∗ 45.3 56.8 56.7 53.2 54.0 49.2 49.3 49.7 51.0 50.3 ± ± ± ± ± ± ± ± ± ± 3.0 3.9 6.1 4.6 4.9 3.2 2.8 3.0 3.4 3.1 (32.6)∗ (15.5) (15.6) (20.8) (19.6) (26.8)∗ (26.6)∗ (26.0)∗ (24.1) (25.1)∗ 40.8 ± 3.3 (39.3)∗∗ P < 0.001 when compared with control group. activity. Isolated compounds were then administered in doses estimated according to their ratio in the chloroform extract; i.e. taxoids were given in 30 mg/kg and lignans in 100 mg/kg. All the compounds were found to possess a significant antinociceptive activity, but not as potent as ASA. However, except 1, all were found safe from the view point of gastric damage. On the other hand, only lignan derivatives were shown to possess anti-inflammatory activity (8 was not significant but near to the limit). These results were in accordance with the previous studies. Two of the studied lignans; lariciresinol (5) and isolariciresinol (8), were reported to possess potent in vitro inhibitory effect on TNF-␣ production, a pro-inflammatory cytokine (Cho et al., 2001b); and thus lignan derivatives in the present study might show their anti-inflammatory effects via the same mode. 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