Flavonoids and Stilbenoids with COX-1 and COX-2 Inhibitory Activity from Dracaena loureiri

• Abstract
• Materials and Methods
• References

Abstract

Fom the stem wood of Dracaena loureiri, a new homoisoflavanone named loureiriol (1) and eight known flavonoid and stilbenoid derivatives, including 5,7-dihydroxy-3-(4-hydroxybenzyl)-4-chromanone (2), 4,4′-dihydroxy-2,6-dimethoxydihydrochalcone (3), 2,4′-dihydroxy-4,6-dimethoxydihydrochalcone (4), 4′-hydroxy-2,4,6-trimethoxydihydrochalcone (5), 4,6,4′-trihydroxy-2-methoxydihydrochalcone (6), 4,3′,5′-trihydroxystilbene (7), 4,3′-dihydroxy-5′-methoxystilbene (8) and 4-hydroxy-3′,5′-dimethoxystilbene (9) were isolated. These compounds were evaluated for their inhibitory activity against the enzymes cyclooxygenase-1 and cyclooxygenase-2. Potent but non-selective activity was found for the stilbenoids 7 - 9 (IC₅₀ 1.29 - 4.92 μM) whereas weak or no activity was observed for the flavonoids 1 - 6.

The stem wood of Dracaena loureiri Gagnepain (Agavaceae) has long been used in Thai traditional medicine as an antipyretic and an analgesic [1]. Previous chemical investigations of this plant revealed the presence of some flavonoids [1], [2], [3], [4]. Several studies have been done on the bioactivities of these constituents, including their antibacterial, cytotoxic and estrogenic effects [3], [4]; however, no supporting evidence for the medicinal claim of the plant has been found so far. In an effort to determine the active principles of this plant, and as a continuation of our studies on bioactive compounds of natural origin [5], we examined a hexane, an ethyl acetate and a methanol extract prepared from the stem wood of D. loureiri for their in vitro inhibitory activity against the enzyme cyclooxygenase-2 (COX-2), using an established method [6]. Chromatographic separation of the ethyl acetate fraction resulted in the isolation of a new homoisoflavanone named loureiriol (1) and eight known compounds (2 - 9). These compounds were then assessed for their inhibitory activity against the enzymes COX-1 and COX-2.[]

Compound 1 showed its molecular ion at m/z 302.07997 in the HREIMS, corresponding to the molecular formula C₁₆H₁₄O₆. The UV absorptions at 214, 224, 292 and 330 nm were suggestive of a homoisoflavanone skeleton [3], [4]. The ¹H- and ¹³C-NMR properties of 1 (Table [1]) were similar to those of eucomol (10) [3], except for the absence of the methoxy resonance. The mass difference between 1 and 10 indicated that 1 is the demethyl derivative of 10, and the proposed structure was further confirmed by the ions at m/z 149 and m/z 153 in the EIMS. Fig. [1] shows important HMBC correlations for 1. The stereochemistry at C-3 of 1 was proposed to be R-configuration, similar to that of 10, according to the sign of its specific rotation [3].

Each of the isolates (1-9) was evaluated for its inhibitory activity against the enzymes COX-1 and COX-2 (at 10 μg/ml), using a previously described protocol [6]. Compounds showing > 80 % inhibition were further analyzed for their IC₅₀ values. From Table [2], it can be seen that the stilbenes 7 - 9 were potent inhibitors of both COX enzymes (IC₅₀ 1.29 - 4.92 μM), being more active than aspirin (IC₅₀ 11.41 - 19.80 μM). The data suggest the role of these stilbenes in the fever- and pain-relieving effects of D. loureiri. However, the COX inhibitory activities of 7 - 9 were less than those of indomethacin (IC₅₀ 0.005 - 0.006 μM). It should be noted that no selectivity was observed for these natural compounds, as compared with NS-398, a well-known selective COX-2 inhibitor. With regard to the flavonoids 1-6, weak or no activity was observed at the concentration of 10 μg/ml.

Materials and Methods

General experimental procedures: Optical rotations were measured on a Perkin-Elmer 341 polarimeter. UV spectra were obtained on a Shimadzu UV-160 spectrophotometer, and IR spectra were recorded with a Perkin-Elmer FT-IR 1760X spectrophotometer. EI and HREI mass spectra were obtained with a Finnigan MAT TSQ 700 spectrometer. ¹H (300 MHz) and ¹³C (75 MHz), DEPT, HETCOR, and HMBC spectra were obtained with a Bruker Avance DPX-300 NMR spectrometer. Samples were recorded in acetone-d ₆, or CDCl₃ with solvent signal as internal standard.

Plant material: Dracaena loureiri stem wood was purchased in 1999 from a drugstore in Bangkok [4]. Authentication was done by comparison with specimens at the Museum of Natural Medicine, Chulalongkorn University. A voucher specimen (KL 062 542) is on deposit at the Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.

Extraction and isolation: Dried powdered stem wood of D. loureiri (6 kg) was extracted successively with hexane, EtOAc and MeOH to give a hexane (50 g), an EtOAc (450 g) and a MeOH (950 g) extract, respectively. The EtOAc fraction showed 97 % inhibition against COX-2 at 100 μg/ml and was further investigated. A portion of EtOAc extract (100 g) was subjected to vacuum liquid chromatography on silica gel (400 g) using a hexane-EtOAc-MeOH step-gradient (hexane-EtOAc 4 : 1, 1 L; 7 : 3, 2.5 L; 3 : 2, 2.5 L; 1 : 1, 12 L; EtOAc, 4 L; MeOH, 2 L) to give 9 fractions (Fractions A-I). Fraction E (8 g) was further separated on a vacuum column on silica gel (100 g) with a CHCl₃-MeOH step-gradient (CHCl₃, 300 mL; CHCl₃-MeOH 97 : 3, 2500 mL; 94 : 6, 1700 mL; 92 : 8, 500 mL; 9 : 1, 1000 mL; 4 : 1, 200 mL; 3 : 2, 200 mL; 1 : 4, 200 mL; MeOH, 400 mL) to give 17 fractions (Fractions I - XVII). Recrystallization of fraction V from hexane-CHCl₃ gave pure 4 (381 mg; R_f 0.36, silica gel, CHCl₃-MeOH 9 : 1), and a mother liquor which was further separated on a column (silica gel, 240 g; CHCl₃-MeOH step-gradient: CHCl₃, 6.2 L; CHCl₃-MeOH 99 : 1, 5 L; MeOH, 0.25 L) to give 12 fractions. Fraction 3 from this column was subjected to HPLC (Prep-sil column, 22 × 250 mm; CHCl₃-MeOH 96 : 4; 3 ml/min; UV 365 nm) to give 9 (11 mg; t_R 19.7 min; R_f 0.40, silica gel, CHCl₃-MeOH 96 : 4). Fraction 8 was separated on a silica gel column (10 g) with CHCl₃-MeOH 99 : 1 (720 mL) to give 7 fractions (fractions a-g), and fraction e from this column was further purified on a column (Sephadex LH-20, 2.5 × 83 cm; CHCl₃-MeOH 1 : 1, 300 m) to furnish 5 (40 mg; R_f 0.34, silica gel, hexane-EtOAc 1 : 1). Separation of fraction X by gel filtration (Sephadex LH-20, 2.5 × 71 cm; MeOH, 2 L) yielded 2 (756 mg; [α]_D ²⁵ -25°, c 0.4, MeOH; R_f 0.23, silica gel, CHCl₃-MeOH 9 : 1) and 8 (170 mg; R_f 0.24, silica gel, CHCl₃-MeOH 9 : 1) and a fraction which was re-chromatographed (Sephadex LH-20, 2.5 × 67 cm; MeOH, 300 mLl) to give 3 (112 mg; R_f 0.26, silica gel, CHCl₃-MeOH 9 : 1). Fraction XI was separated on a silica gel (220 g) column with CHCl₃-MeOH 93 : 7 (5 L) to give 13 fractions. Fraction 5 from this column was subjected to molecular exclusion chromatography (Sephadex LH-20, 2.5 × 67 cm; CHCl₃-MeOH 1 : 1, 400 mL) to give 1 (29 mg; R_f 0.20, silica gel, CHCl₃-MeOH 9 : 1) while fraction 7 was passed through a Sephadex LH-20 column (2.5 × 67 cm; MeOH, 300 mL) to give 6 (78 mg; R_f 0.18, silica gel, CHCl₃-MeOH 9 : 1). Fraction XV was separated on a Sephadex LH-20 column (2.5 × 55 cm; acetone, 210 mL) to give 7 (30 mg; R _f 0.32, silica gel, CHCl₃-MeOH 85 : 15).

Loureiriol (1): White powder; [α]_D ²⁵ -128° (c 0.1, MeOH); UV (MeOH): λ_max (log ε) = 214 (4.46), 224 (4.41), 292 (4.32), 330 (3.73) nm; IR (KBr): ν = 3416, 1643, 1593, 1515, 1275, 1163, 835 cm^-1; ¹H- (300 MHz) and ¹³C- (75 MHz) NMR see Table [1]; EIMS: m/z (rel. int.) = 302 (M⁺, 41), 196 (100), 195 (82), 153 (87), 149 (20), 107 (90), 77 (31), 69 (25), 43 (15); HREIMS 302. 07 997 (calcd for C₁₆H₁₄O₆, 302.07904).

Known compounds (2 - 9) were identified by comparison of their physical and spectral (UV, IR ¹H- and ¹³C-NMR, and MS) properties with published values [3], [4], [7]. Copies of the original spectra are obtainable from the author of correspondence.

Evaluation of cyclooxygenase inhibitory activity: Assays were conducted in triplicate according to an established protocol [6]. First, each of the test samples was evaluated at 10 μg/mL, and its percent inhibition was determined. Compounds exhibiting more than 80 % inhibition at this concentration were further analyzed for their IC₅₀ values. Aspirin, NS-398 and indomethacin were used as positive controls.

References

• 1 Meksuriyen D, Cordell G A, Ruangrungsi N, Tantivatana P. Traditional medicinal plants of Thailand, IX. 10-Hydroxy-11-methoxydracaenone and 7,10-dihydroxy-11-methoxydracaenone from Dracaena loureiri .  Journal of Natural Products. 1987;  50 1118-25
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• 6 Kirtikara K, Swangkul S, Ballou L R. The analysis of nonsteroidal antiinflammatory drug selectivity in prostaglandin G/H synthase (PGHS)-null cells.  Inflammation Research. 2001;  50 327-32
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Kittisak Likhitwitayawuid, Ph. D.

Department of Pharmacognosy

Faculty of Pharmaceutical Sciences

Chulalongkorn University

Bangkok 10330, Thailand

Email:

Fax: +66-2255-8227

References

• 1 Meksuriyen D, Cordell G A, Ruangrungsi N, Tantivatana P. Traditional medicinal plants of Thailand, IX. 10-Hydroxy-11-methoxydracaenone and 7,10-dihydroxy-11-methoxydracaenone from Dracaena loureiri .  Journal of Natural Products. 1987;  50 1118-25
  CrossrefPubMedSearch in Google Scholar
• 2 Meksuriyen D, Cordell G A. Retrodihydrochalcones from Dracaena loureiri .  Journal of Natural Products. 1988;  51 1129-35
  CrossrefPubMedSearch in Google Scholar
• 3 Meksuriyen D, Cordell G A. Traditional medicinal plants of Thailand, XIII. Flavonoids derivatives from Dracaena loureiri (Agavaceae).  Journal of the Science Society of Thailand. 1988;  14 3-24
  PubMedSearch in Google Scholar
• 4 Ichikawa K, Kitaoka M, Taki M, Takaishi S, Iijima Y, Boriboon M, Akiyama T. Retrodihydrochalcones and homoisoflavones isolated from Thai medicinal plant Dracaena loureiri and their estrogenic agonist activity.  Planta Medica. 1997;  63 540-3
  PubMedSearch in Google Scholar
• 5 Likhitwitayawuid K, Sritularak B. A new dimeric stilbene with tyrosinase inhibitory activity from Artocarpus gomezianus .  Journal of Natural Products. 2001;  64 1457-9
  CrossrefPubMedSearch in Google Scholar
• 6 Kirtikara K, Swangkul S, Ballou L R. The analysis of nonsteroidal antiinflammatory drug selectivity in prostaglandin G/H synthase (PGHS)-null cells.  Inflammation Research. 2001;  50 327-32
  PubMedSearch in Google Scholar
• 7 Adinolfi M, Barone G, Lanzetta R, Laonigro G, Mangoni L, Parrilli M. Three 3-benzyl-4-chromones from Muscari comosum .  Phytochemistry. 1985;  24 624-6
  CrossrefPubMedSearch in Google Scholar

Kittisak Likhitwitayawuid, Ph. D.

Department of Pharmacognosy

Faculty of Pharmaceutical Sciences

Chulalongkorn University

Bangkok 10330, Thailand

Email:

Fax: +66-2255-8227