New Dihydrochalcones and Anti-Platelet Aggregation Constituents from the Leaves of Muntingia calabura Original Paper
Abstract Two new dihydrochalcones, 2,3-dihydroxy-4,3¢,4¢,5¢-tetramethoxydihydrochalcone (1) and 4,2¢,4¢-trihydroxy-3¢-methoxydihydrochalcone (2), and a new flavanone, (2R,3R)-(±)-3,5-dihydroxy-6,7-dimethoxyflavanone (3), together with nineteen known compounds have been isolated from the leaves of Muntingia calabura. The structures of three new compounds were determined through spectral analyses including extensive 2D-NMR data. Among the isolates, 2,3-dihydroxy-4,3¢,4¢,5¢-tetramethoxydihydrochalcone, 5,7-dihydroxy-3-methoxyflavone, 5,7-
Introduction 572
Muntingia calabura L. (Tiliaceae) is an evergreen tree, distributed in tropical America and introduced in southern Taiwan as a cultivated plant [1]. This plant is rich in flavonoids with flavones, flavanones, flavans, and biflavans as the major constituents, some of which have demonstrated cytotoxic activities [2], [3], [4], [5]. In the course of our continuing search for novel biologically active compounds from natural sources, we screened extracts of this plant for thrombolytic activity. The methanol extract of the leaves of M. calabura displayed antiplatelet aggregation activity. Investigation of the CHCl3- and n-BuOH-soluble fractions of the leaves of this plant led to the isolation and characterization of two new dihydrochalcones, 2,3-dihydroxy-4,3¢,4¢,5¢-tetramethoxydihydrochalcone (1) and 4,2¢,4¢-trihydroxy-3¢-methoxydihydrochalcone (2), and a new flavanone, (2R,3R)-(±)-3,5-dihydroxy-6,7-dimethoxyflavanone (3), along with nineteen known
Jih-Jung Chen1 Hsinn-Hsing Lee1 Cheng-Dean Shih1 Chang-Hui Liao2 Ih-Sheng Chen3 Tsung-Hsien Chou1,3
dihydroxy-6-methoxyflavone, 5,4¢-dihydroxy-3,7-dimethoxyflavone, (2S)-7,8,3¢,4¢,5¢-pentamethoxyflavan, (2S)-5¢-hydroxy7,8,3¢,4¢-tetramethoxyflavan, and methyl gallate exhibited significant anti-platelet aggregation activity in vitro. Key words Muntingia calabura ´ Tiliaceae ´ dihydrochalcones ´ 2,3-dihydroxy-4,3¢,4¢,5¢-tetramethoxydihydrochalcone ´ 4,2¢,4¢-trihydroxy3¢-methoxydihydrochalcone ´ flavanone ´ (2R,3R)-(±)-3,5-dihydroxy-6,7-dimethoxyflavanone ´ anti-platelet aggregation activity
compounds. This paper describes the structural elucidation of these new compounds and the anti-platelet aggregation activities of the isolates.
Materials and Methods General experimental procedures All melting points were determined on a Yanaco micro-melting point apparatus and are uncorrected. IR spectra (KBr or neat) were taken on a Perkin Elmer system 2000 FT-IR spectrometer. Optical rotations were measured using a Jasco DIP-370 polarimeter in CHCl3. CD spectra were recorded on a Jasco J-810 spectropolarimeter. UV spectra were obtained on a Jasco UV-240 spectrophotometer. EI-mass spectra were recorded on a VG Biotech Quattro 5022 spectrometer. HR-EI, FAB, and HR-FAB mass spectra were recorded on a JEOL JMX-HX 110 mass spectrometer.
Affiliation 1 Graduate Institute of Pharmaceutical Technology, Tajen University, Pingtung, Taiwan 2 Graduate Institute of Natural Products, Chang Gung University, Taoyuan, Taiwan 3 Graduate Institute of Pharmaceutical Sciences, Kaohsiung Medical University, Kaohsiung, Taiwan Correspondence Dr. J. J. Chen ´ Department of Pharmacy ´ Tajen University ´ Pingtung 907 ´ Taiwan ´ Phone: +886-8-762-4002 ext. 332 ´ Fax: +886-8-7625308 ´ E-mail:
[email protected] Received November 1, 2006 ´ Revised January 8, 2007 ´ Accepted April 10, 2007 Bibliography Planta Med 2007; 73: 572±577 Georg Thieme Verlag KG Stuttgart ´ New York DOI 10.1055/s-2007-967196 ´ Published online May 22, 2007 ISSN 0032-0943
Plant material The leaves of M. calabura were collected from Kaohsiung City, Taiwan, in June 2001 and identified by Dr. I. S. Chen. A voucher specimen (Chen 6103) was deposited in the herbarium of the Faculty of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan. Extraction and isolation The dried leaves (5 kg) was extracted with cold MeOH and the extract concentrated under reduced pressure. The MeOH extract (290 g), when partitioned between H2O-CHCl3 (1 : 1), afforded a CHCl3-soluble fraction (fr. A, 70 g). The H2O-soluble fraction was partitioned between H2O and n-BuOH (1 : 1) to afford an nBuOH-soluble fraction (fr. B, 90 g) and an H2O-soluble fraction (fr. C, 112 g). Fr. A (70 g) was chromatographed on silica gel (2.35
Anti-platelet aggregation test Blood was collected from the rabbit marginal vein, anticoagulated with EDTA (6 mM) and centrifuged for 10 min at 90 g and Chen J-J et al. New Dihydrochalcones and ¼ Planta Med 2007; 73: 572 ± 577
Original Paper
NMR spectra, including COSY, NOESY, HMBC, and HSQC experiments, were recorded on a Varian Unity 400 or a Varian Inova 500 spectrometer operating at 400 and 500 MHz (1H) and 100 and 125 MHz (13C), respectively, with chemical shifts given in ppm (d) using TMS as an internal standard. Silica gel (70 ± 230, 230 ± 400 mesh) (Merck; Darmstadt, Germany) was used for CC. Silica gel 60 F-254 (Merck) was used for TLC and preparative TLC.
kg), eluting with CH2Cl2, gradually increasing the polarity with MeOH to give 15 frs: fr. A1 ± A9 (each 4 L, CH2Cl2), fr. A10 (4 L, CH2Cl2-MeOH, 20 : 1), fr. A11 (4 L, CH2Cl2-MeOH, 10 : 1), fr. A12 ± A13 (each 4 L, CH2Cl2-MeOH, 5 : 1), fr. A14 (4 L, CH2Cl2-MeOH, 2 : 1) and fr. A15 (4 L, MeOH). Fr. A3 (2.1 g) was chromatographed on silica gel (82 g) eluting with n-hexane, gradually increasing the polarity with EtOAc to give 5 frs (each 2 L, fr. A3 ± 1 ± fr. A3 ± 5). Fr. A3 ± 1 (252 mg) was further purified by preparative TLC (nhexane-EtOAc) to obtain 18 (2.8 mg) (Rf = 0.31). Fr. A4 (2.6 g) was chromatographed on silica gel (90 g) eluting with CH2Cl2MeOH (20 : 1) to give 10 frs (each 800 mL, fr. A4 ± 1 ± fr. A4 ± 10). Fr. A4 ± 1 (143 mg) was further purified by preparative TLC (CH2Cl2) to obtain 5 (3.3 mg) (Rf = 0.51) and 20 (2.7 mg) (Rf = 0.47). Fr. A4 ± 2 (210 mg) was further purified by preparative TLC (n-hexane-acetone, 7: 1) to obtain 22 (2.7 mg) (Rf = 0.24). Fr. A4 ± 3 (168 mg) was further purified by preparative TLC (n-hexane-acetone, 8 : 1) to obtain 21 (2.5 mg) (Rf = 0.23). Fr. A4 ± 4 (172 mg) was further purified by preparative TLC (CHCl3-EtOAc, 30 : 1) to obtain 19 (3.7 mg) (Rf = 0.07). Fr. A4 ± 5 (220 mg) was further purified by preparative TLC (nhexane-acetone, 7: 1) to obtain 11 (3.1 mg) (Rf = 0.43). Fr. A4 ± 6 (320 mg) was further purified by preparative TLC (CH2Cl2-EtOAc, 20 : 1) to obtain 2 (2.3 mg) (Rf = 0.49) and 6 (5.2 mg) (Rf = 0.48). Fr. A6 (1.5 g) was chromatographed on silica gel (70 g) eluting with n-hexane-EtOAc (7: 1) to obtain 10 frs (each 800 mL, fr. A6 ± 1 ± fr. A6 ± 10). Fr. A6 ± 2 (242 mg) was further purified by preparative TLC (n-hexane-acetone, 7: 1) to give 15 (33 mg) (Rf = 0.36). Fr. A6 ± 6 (205 mg) was further purified by preparative TLC (CH2Cl2-MeOH, 20 : 1) to obtain 16 and 17 (59.5 mg) (Rf = 0.69). Fr. A8 (5.6 g) was chromatographed on silica gel (175 g), eluting with CH2Cl2, gradually increasing the polarity with MeOH to give 15 frs (each 800 mL, fr. A8 ± 1 ± fr. A8 ± 15). Fr. A8 ± 1 (186 mg) was further purified by preparative TLC (CHCl3acetone, 20 : 1) to obtain 1 (1.5 mg) (Rf = 0.49) and 8 (2.1 mg) (Rf = 0.46). Fr. A8 ± 3 (196 mg) was further purified by preparative TLC (CHCl3-MeOH, 20 : 1) to obtain 10 (4.5 mg) (Rf = 0.48). Fr. A8 ± 10 (142 mg) was further purified by preparative TLC (CHCl3-MeOH, 20 : 1) to obtain 7 (3.2 mg) (Rf = 0.60) and 9 (3.8 mg) (Rf = 0.45). Fr. A8 ± 11 (152 mg) was further purified by preparative TLC (CHCl3-acetone, 10 : 1) to obtain 14 (3.5 mg) (Rf = 0.48). Fr. A8 ± 12 (110 mg) was further purified by preparative TLC (n-hexane-acetone, 2 : 1) to obtain 4 (3.3 mg) (Rf = 0.36). Fr. B (50 g) was chromatographed on silica gel (1650 g), eluting with CH2Cl2, gradually increasing the polarity with MeOH to give 15 frs: fr. B1 ± B2 (each 3 L, CH2Cl2-MeOH, 10 : 1), fr. B3 ± B4 (each 3 L, CH2Cl2-MeOH, 5 : 1), fr. B5 ± B9 (each 3 L, CH2Cl2MeOH, 2 : 1), fr. B10 ± B13 (each 3 L, CH2Cl2-MeOH, 1 : 1), fr. A14 ± B15 (each 2 L, MeOH). Fr. B1 (1.8 g) was chromatographed on silica gel (62 g) eluting with CH2Cl2-MeOH (8 : 1) to obtain 7 frs (each 250 mL, fr. B1 ± 1 ± fr. B1 ± 7). Fr. B1 ± 3 (187 mg) was further purified by preparative TLC (CHCl3-MeOH, 2 : 1) to obtain 12 (4.1 mg) (Rf = 0.71). Fr. B3 (1.65 g) was chromatographed on silica gel (60 g) eluting with CHCl3-MeOH (6 : 1) to obtain 10 frs (each 700 mL, fr. B3 ± 1 ± fr. B3 ± 10). Fr. B3 ± 5 (142 mg) was further purified by preparative TLC (CHCl3-MeOH, 5 : 1) to obtain 13 (3.7 mg) (Rf = 0.76).
573
Original Paper 574
room temperature to obtain platelet-rich plasma (PRP). Platelet suspension was prepared from this EDTA-anticoagulated PRP according to the washing procedures described previously [6]. Platelet numbers were counted by a Coulter counter (Model ZM; Coulter; Luton, UK) and adjusted to 4.5 108 platelets/mL. The platelet pellets were finally suspended in Tyrode's solution of the following composition (mM): NaCl (136.8), KCl (2.8), NaHCO3 (11.9), MgCl2 (2.1), NaH2PO4 (0.33), CaCl2 (1.0) and glucose (11.2), containing bovine serum albumin (0.35 %). Platelet aggregation was measured at 37 8C by the turbidimetric method as described by O'Brien [7] using a Chrono-Log Lumi-aggregometer (ChronoLog; Havertown, PA, USA). The platelet suspensions were stirred at 1200 rpm. All the tested compounds were dissolved in dimethyl sulfoxide (DMSO). In order to eliminate the effect of the solvent on the aggregation, the final concentration of DMSO was fixed at 0.5 %, which did not affect the aggregation measured. Percentages of aggregation were calculated using the absorbance of platelet suspension to represent 0 % aggregation and the absorbance of Tyrode's solution as 100 % aggregation. Aspirin (Bayer Vital; Leverkusen, Germany) was used as a positive control. Data were analyzed using Student's t test. Isolated compounds 2,3-Dihydroxy-4,3¢,4¢,5¢-tetramethoxydihydrochalcone (1): Colorless amorphous solid (CH2Cl2-MeOH), m. p. 71 ± 73 8C; IR: nmax = 3426 (OH), 1672 (C = O), 1585, 1504, 1462 cm±1 (aromatic ring C = C stretch); EI-MS: m/z (rel. int.) = 362 (M+, 75), 347 (13), 332 (12), 195 (100), 167 (11), 153 (36); HR-EI-MS: C19H22O7, found: 362.1365 [M]+, calcd.: 362.1366; 1H-NMR (CDCl3, 400 MHz): d = 3.00 (2H, t, J = 7.6 Hz, H-b), 3.25 (2H, t, J = 7.6 Hz, Ha), 3.84 (3H, s, OMe-4), 3.91 (6H, s, OMe-3¢ and OMe-5¢), 3.92 (3H, s, OMe-4¢), 5.82 (1H, br s, D2O exchangeable, OH-3), 6.14 (1H, br s, D2O exchangeable, OH-2), 6.42 (1H, d, J = 8.6 Hz, H-5), 6.86 (1H, d, J = 8.6 Hz, H-6), 7.26 (2H, s, H-2¢ and H-6¢); 13C-NMR (CDCl3, 100 MHz): d = 25.4 (C-b), 39.0 (C-a), 55.8 (OMe-4), 56.3 (OMe-3¢), 56.3 (OMe-5¢), 60.9 (OMe-4¢), 103.5 (C-5), 105.6 (C-2¢), 105.6 (C-6¢), 120.5 (C-1), 124.5 (C-6), 131.0 (C-1¢), 134.4 (C-3), 142.6 (C-4¢), 146.7 (C-2), 151.5 (C-4), 153.2 (C-3¢), 153.2 (C-5¢), 199.3 (C = O). 4,2¢,4¢-Trihydroxy-3¢-methoxydihydrochalcone (2): Colorless needles (n-hexane-EtOAc), m. p. 161 ± 163 8C; UV (MeOH): lmax (log e) = 209 (4.08), 228 (sh, 3.75), 285 nm (3.81); (MeOH + KOH): 212 (4.10), 257 (3.53), 338 nm (3.87); IR: nmax = 3350 (OH), 1632 cm±1 (C = O); EI-MS: m/z (rel. int.) = 288 (M+, 32), 273 (11), 167 (12), 166 (56), 121 (33), 107 (100); HR-EI-MS: C16H16O5, found: 288.0994 [M]+, calcd: 288.0998; 1H-NMR (CDCl3, 400 MHz): d = 2.95 (2H, t, J = 7.6 Hz, H-b), 3.21 (2H, t, J = 7.6 Hz, H-a), 3.99 (3H, s, OMe-3¢), 6.30 (1H, s, D2O exchangeable, OH-4¢), 6.49 (1H, d, J = 9.0 Hz, H-5¢), 6.70 (2H, d, J = 8.8 Hz, H-3 and H-5), 7.12 (2H, d, J = 8.8 Hz, H-2 and H-6), 7.44 (1H, d, J = 9.0 Hz, H-6¢), 12.89 (1H, s, D2O exchangeable, OH-2¢); 13C-NMR (CDCl3, 100 MHz): d = 29.5 (C-b), 39.9 (C-a), 60.7 (OMe-3¢), 106.5 (C-5¢), 114.3 (C-1¢), 115.6 (C3 and C-5), 126.3 (C-6¢), 129.6 (C-2 and C-6), 132.9 (C-1), 134.1 (C3¢), 155.0 (C-4¢), 156.6 (C-2¢), 157.6 (C-4), 204.4 (C = O). (2R,3R)-(±)-3,5-Dihydroxy-6,7-dimethoxyflavanone (3): Yellow amorphous powder (CHCl3-MeOH), m. p. 189 ± 190 8C; [a]25 D : ±19.4 (c 0.1, MeOH); CD (MeOH): nm (De) = 324 (+ 1.7), 285 (±6.4); UV (MeOH): lmax (log e) = 218 (4.41), 234 (4.21), 293 (4.12), 341 Chen J-J et al. New Dihydrochalcones and ¼ Planta Med 2007; 73: 572 ± 577
nm (3.85); IR: nmax = 3413 (br. OH), 1665 (C = O), 1593, 1501, 1452 cm±1 (aromatic ring C = C stretch); EI-MS: m/z (rel. int.) = 316 (M+, 81), 287 (9), 197 (100), 181 (89), 170 (13), 153 (18), 91 (24), 69 (20); HR-EI-MS: C17H16O6, found: 316.0938 [M]+, calcd.: 316.0947; 1H-NMR (CDCl3, 400 MHz): d = 3.47 (1H, br s, D2O exchangeable, OH-3b), 3.86 (3H, s, OMe-6), 3.89 (3H, s, OMe-7), 4.59 (1H, d, J = 12.0 Hz, H-3a), 5.10 (1H, d, J = 12.0 Hz, H-2), 6.14 (1H, s, H-8), 7.47 (3H, m, H-3¢, H-4¢ and H-5¢), 7.56 (2H, br.d, J = 7.0 Hz, H-2¢ and H-6¢), 11.04 (1H, s, D2O exchangeable, OH-5); 13C-NMR (CDCl3, 100 MHz): d = 56.4 (OMe-7), 61.0 (OMe-6), 72.5 (C-3), 83.8 (C-2), 92.2 (C-8), 100.9 (C-4a), 127.4 (C-2¢ and C-6¢), 128.8 (C-3¢ and C-5¢), 129.5 (C-4¢), 130.9 (C-6), 136.0 (C-1¢), 154.3 (C-5), 158.8 (C-8a), 161.7 (C-7), 196.4 (C-4).
Results and Discussion 2,3-Dihydroxy-4,3¢,4¢,5¢-tetramethoxydihydrochalcone (1) was isolated as a colorless amorphous solid. The HR-EI-MS gave an [M]+ ion at m/z = 362.1365, consistent with a molecular formula of C19H22O7. A conjugated carbonyl group was revealed by the IR absorption at 1672 cm±1, along with a resonance signal in the 13CNMR spectrum at d = 199.3. The presence of a hydroxy group in the molecule was revealed by the band at 3426 (br) cm±1 in the IR spectrum, which was confirmed by the signals at d = 5.82 and 6.14 (each br s, D2O exchangeable, OH-3 and OH-2) in the 1HNMR spectrum. The 1H-NMR spectrum of 1 showed the presence of four methoxy groups, four methylene protons, and four aromatic protons. Two two-proton triplets at d = 3.25 and 3.00 (J = 7.6 Hz) were typical of H2-a and H2-b of a dihydrochalcone derivative [8]. Two lower field aromatic protons at d = 7.26 (2H, s, H-2¢ and H-6¢) suggested that one of the aryl groups was present as an aroyl unit; this was supported by the EI-mass spectrum which gave the ion m/z = 195 (1a) as base peak. The EI-mass spectrum also showed the significant fragmentions 1b (m/z = 153), indicating the presence of a 2,3-dihydroxy-4-methoxyphenyl substituent. On the basis of the evidence above, the structure of 1 was elucidated as 2,3-dihydroxy-4,3¢,4¢,5¢-tetramethoxydihydrochalcone. This was further confirmed by 1H-1H COSY and NOESY experiments (Fig. 1). The assignment of 13C-NMR resonances was confirmed by the HSQC and HMBC techniques (Fig. 1), which also supported the structure of 1. 4,2¢,4¢-Trihydroxy-3¢-methoxydihydrochalcone (2) was isolated as colorless needles. The EI-MS afforded the molecular ion [M]+ at m/z = 288, implying a molecular formula of C16H16O5, which was confirmed by the HR-EI-MS (m/z = 288.0994). The UV absorptions of 2 at 209, 228 sh, and 285 nm suggested the presence of a p-hydroxyacetophenone nucleus [9]. A conjugated carbonyl group was revealed by the IR absorption at 1632 cm±1, along with a resonance signal in the 13C-NMR spectrum at d = 204.4. The presence of hydroxy groups in the molecule was revealed by the band at 3350 (br) cm±1 in the IR spectrum, which was confirmed by the signals at d = 5.82, 6.30, and 12.89 (each 1H, each s, D2O exchangeable, OH-4, 4¢, and 2¢, respectively) in the 1H-NMR spectrum. The 1H-NMR spectrum of 2 was similar to that of lusianin [9], except for that OH-4 [d = 5.82 (1H, br s)] of 2 replaced OMe4 [d = 3.71 (3H, s)] of lusianin [9]. Analysis of the 1H-NMR spectrum of 2 showed signals for six aromatic protons, an aromatic methoxy group, a chelated and two normal phenolic hydroxy
Fig. 1 Significant NOESY (A) and HMBC (B) correlations of 1.
Fig. 2 Significant NOESY (A) and HMBC (B) correlations of 2.
Original Paper Fig. 3 Significant NOESY (A) and HMBC (B) correlations of 3.
groups, and four methylene protons. Four of these aromatic protons appeared as a pair of doublets [d = 7.12 and 6.70 (each 2H, d, J = 8.8 Hz)] representing an A2B2 system of a p-disubstituted benzene derivative. The remaining two aromatic protons of 2 also appeared as a pair of doublets [d = 6.49 and 7.44 (J = 9.0 Hz)] corresponding to two ortho-coupled protons, which resembled those of H-5¢ and H-6¢ of lusianin [9]. Two two-proton triplets at d = 3.21 and 2.95 (J = 7.6 Hz) were typical of H2-a and H2-b of a dihydrochalcone derivative [8]. The position of the methoxy group was confirmed through observation of the prominent fragment peaks at m/z = 166 (2a) and 107 (2b) in the EI-mass spectrum. Based on the above data, the structure of 2 was elucidated as 4,2¢,4¢-trihydroxy-3¢-methoxydihydrochalcone; this was further confirmed by COSY and NOESY (Fig. 2) experiments. The assignment of 13C-NMR resonances was confirmed by DEPT, HSQC and HMBC (Fig. 2) techniques, which also supported the structure of 2.
D2O exchangeable, OH-5) in the 1H-NMR spectrum. The 1H-NMR spectrum of 3 was similar to that of (2R,3R)-2,3-dihydro-3,5-dihydroxy-7-methoxyflavone [11], except for that OMe-6 [d = 3.86 (3H, s)] of 3 replaced H-6 [d = 6.05 (d, J = 2.1 Hz)] of (2R,3R)-2,3dihydro-3,5-dihydroxy-7-methoxyflavone [11]. The presence of the OH-3 group was supported by the fragment ions at m/z = 287 (M ± CHO) and 91 (120 ± CHO) which were characteristic of 3-hydroxyflavanonol [12]. The absolute configuration of 3 was assigned as (2R,3R) by the CD absorption maximum at 324 nm (De = + 1.7) and 285 nm (De = ±6.4) in analogy with previous CD spectral observation [13]. Based on the above data, the structure of 3 was elucidated as (2R,3R)-(±)-3,5-dihydroxy-6,7-dimethoxyflavanone; this was further confirmed by COSY and NOESY (Fig. 3) experiments. The assignment of 13C-NMR resonances was confirmed by DEPT, HSQC and HMBC (Fig. 3) techniques, which also supported the structure of 3. This is the first report of the occurrence of 3 in a natural source, although it has been synthesized by Asakawa [10].
(2R,3R)-(±)-3,5-Dihydroxy-6,7-dimethoxyflavanone (3) was isolated as a yellow amorphous powder. The EI-MS afforded the molecular ion [M]+ at m/z = 316, implying a molecular formula of C17H16O6, which was confirmed by the HR-EI-MS. The UV absorptions of 3 at 218, 234, 293, and 341 nm suggested the presence of a flavanone nucleus [10]. A conjugated carbonyl group was revealed by the IR absorption at 1665 cm±1, along with a resonance signal in the 13C-NMR spectrum at d = 196.4. The presence of a hydroxy group in the molecule was revealed by the band at 3413 (br) cm±1 in the IR spectrum, which was confirmed by the signals at d = 3.47 (1H, br s, D2O exchangeable, OH-3b) and 11.04 (1H, s,
The four known flavones, 7-methoxyflavone (4) [14], 5,7-dihydroxy-3-methoxyflavone (5) [15], 5,7-dihydroxy-6-methoxyflavone (6) [16], and 5,4¢-dihydroxy-3,7-dimethoxyflavone (7) [17], two flavans, (2S)-7,8,3¢,4¢,5¢-pentamethoxyflavan (8) [3] and (2S)-5¢-hydroxy-7,8,3¢,4¢-tetramethoxyflavan (9) [3], five benzenoids, methyl 4-hydroxybenzoate (10) [18], isovanillic acid (11) [19], p-nitrophenol (12) [20], methyl gallate (13) [21], and transmethyl p-coumarate (14) [22], three steroids, b-sitostenone (15) [23], mixture of b-sitosterol (16) [24] and stigmasterol (17) [24], a triterpenoid, b-amyrenone (18) [25], a benzoquinone, a-tocopherylquinone (19) [26], d-tocopherol (20) [27], a-tocospiro A Chen J-J et al. New Dihydrochalcones and ¼ Planta Med 2007; 73: 572 ± 577
575
Table 1
Inhibitory effects of compounds on the aggregation of washed rabbit platelets induced by thrombin, arachidonic acid (AA), collagen and PAF (platelet-activating factor)
Compound Conc. (mg/mL)
Inhibition (%) Thrombin AA Collagen PAF (0.1 U/mL) (100 mM) (10 mg/mL) (2 ng/mL)
1
100
12.7
50
48.5
Original Paper
9.8
67.0
44.3
7.8
3
100
22.1
62.4
73.5
11.6
4
100
16.8
33.5
31.0
16.6
5
100
13.6
98.2
96.5
12.5
44.7
38.9
90.3
93.0
41.5
50.8
80.5
82.1
33.6
28.7
85.1
88.5
45.6
53.4
61.4
82.5
80.4
71.6
100
11.8
50 7
100
43.2
50 8
100
33.1
50 23.4
9.9 5.5 90.3
9
100
39.7
47.2
33.5
10
100
2.7
6.3
4.8
3.2
11
100
1.4
10.2
6.5
7.1
12
100
1.8
11.6
6.3
5.5
13
100
16.3
100.0
100.0
11.4
50
100.0
100.0
20
95.2
84.1
10
83.2
66.8
5
73.3
52.5
2
50.4
22.8
50
1
b
88.7
55.8
100
6
a
94.1
57.2
2
50
576
98.2
35.1
10.5
2.3
4.8
5.3
15
100
3.2
4.5
5.5
2.8
Mixture of 16 and 17
100
9.2
5.1
8.5
2.5
18
100
4.0
3.8
6.2
3.6
19
100
12.4
8.5
8.8
10.3
20
100
17.2
22.5
25.8
9.8
21
100
6.6
18.9
20.3
11.4
4.5
22
100
5.8
14.7
18.9
13.0
Aspirina
100
1.5
100.0
5.1
2.5
20
5.6
This work was supported by grants from the Council of Agriculture [93AS-2.3.3-FB-e2(3)], Tajen University (Tajen-93103), and the National Science Council of the Republic of China.
1
100
100.0
Acknowledgements
References
14
50
used as the positive control. From the results of our anti-platelet aggregation tests, the following conclusions can be drawn regarding these isolates: (a) 2,3-dihydroxy-4,3¢,4¢,5¢-tetramethoxydihydrochalcone (1), 5,7-dihydroxy-3-methoxyflavone (5), 5,7dihydroxy-6-methoxyflavone (6), 5,4¢-dihydroxy-3,7-dimethoxyflavone (7), (2S)-7,8,3¢,4¢,5¢-pentamethoxyflavan (8), (2S)-5¢-hydroxy-7,8,3¢,4¢-tetramethoxyflavan (9), and methyl gallate (13) showed significant anti-platelet aggregation activity in vitro (Table 1). (b) Among the flavone analogues (4 ± 7), 5, 6, and 7 at 100 mg/mL showed marked inhibition of platelet aggregation induced by AA and collagen, and the intensity order of the activity was 5 (with 5,7-dihydroxy-3-methoxy) > 6 (with 5,7-dihydroxy6-methoxy) > 7 (with 5,4¢-dihydroxy-3,7-dimethoxy) > 4 (with 7methoxy). (c) Among the benzenoids (10 ± 14), only methyl gallate (13) showed selective inhibitory activity of platelet aggregation induced by AA and collagen. (d) Methyl gallate (13) showed the strongest anti-platelet aggregation activity induced by AA and collagen with IC50 values of ca. 2 and 5 mg/mL, respectively.
Positive control. Percentage inhibitions were compared with the respective control.
(21) [28], and a-tocospiro B (22) [28], were readily identified by comparison of physical and spectroscopic data (UV, IR, 1H-NMR, and MS) with those of corresponding authentic samples or literature values. The anti-platelet effects of the isolates from the leaves of Formosan M. calabura were tested in vitro using the turbidimetric method [7] in washed rabbit platelets induced by thrombin (0.1 U/mL), arachidonic acid (AA, 100 mM), collagen (10 mg/mL), and platelet-activating factor (PAF, 2 ng/mL), all causing about 85 ± 95 % aggregation. The anti-platelet aggregation data are shown in Table 1. The clinically applied anti-platelet agent, aspirin, was Chen J-J et al. New Dihydrochalcones and ¼ Planta Med 2007; 73: 572 ± 577
Liu TS, Lo HC. Tiliaceae in Flora of Taiwan, 2nd edition, Vol. 3. Taipei: Editorial Committee of the Flora of Taiwan; 1993: 730 ± 2. 2 Chen JJ, Lin RW, Duh CY, Huang HY, Chen IS. Flavones and cytotoxic constituents from the stem bark of Muntingia calabura. J Chin Chem Soc 2004; 51: 665 ± 70. 3 Kaneda N, Pezzuto JM, Soejarto DD, Kinghorn AD, Farnsworth NR, Santisuk T et al. New cytotoxic flavonoids from Muntingia calabura roots. J Nat Prod 1991; 54: 196 ± 206. 4 Nshimo CM, Pezzuto JM, Kinghorn AD, Farnsworth NR. Cytotoxic constituents of Muntingia calabura leaves and stems collected in Thailand. Int J Pharmacognosy 1993; 31: 77 ± 81. 5 Su BN, Park EJ, Vigo JS, Graham JG, Cabieses F, Fong HHS et al. Activityguided isolation of the chemical constituents of Muntingia calabura using a quinone reductase induction assay. Phytochemistry 2003; 63: 335 ± 41. 6 Teng CM, Chen WY, Ko WC, Ouyang C. Antiplatelet effect of butylidenephthalide. Biochem Biophys Acta 1987; 924: 375 ± 82. 7 O'Brien JR. Platelet aggregation II. Some results from a new method of study. J Clin Pathol 1962; 15: 452 ± 5. 8 Kawanishi K, Takagaki T, Hashimoto Y. Dihydrochalcones from Iryanthera sagotiana. Phytochemistry 1990; 29: 2735 ± 6. 9 Majumder PL, Lahiri S, Mukhoti N. Chalcone and dihydrochalcone derivatives from the orchid Lusia volucris. Phytochemistry 1995; 40: 271 ± 4. 10 Asakawa Y. Chemical constituents of Alnus sieboldiana (Betulaceae) II, the isolation and structure of flavonoids and stilbenes. Bull Chem Soc Jpn 1971; 44: 2761 ± 6. 11 Rossi MH, Yoshida M, Maia JGS. Neolignans, styrylpyrones and flavonoids from an Aniba species. Phytochemistry 1997; 45: 1263 ± 9. 12 Audier HE. Mass spectrometry of flavone compounds. Bull Soc Chim Fr; 1966: 2892 ± 9. 13 Fang JM, Chang CF, Cheng YS. Flavonoids from Pinus morrisonicola. Phytochemistry 1987; 26: 2559 ± 61. 14 Freeman PW, Murphy ST, Nemorin JE, Taylor WC. The constituents of Australian Pimela species. II. The isolation of unusual flavones from P. simplex and P. decora. Aust J Chem 1981; 34: 1779 ± 84.
15
22
Chiang YM, Liu HK, Lo JM, Chien SC, Chan YF, Lee TH et al. Cytotoxic constituents of the leaves of Calocedrus formosana. J Chin Chem Soc 2003; 50: 161 ± 6. 23 Chen JJ, Duh CY, Chen IS. Cytotoxic chromenes from Myriactis humilis. Planta Med 2005; 71: 370 ± 2. 24 Chen JJ, Duh CY, Chen JF. New cytotoxic biflavonoids from Selaginella delicatula. Planta Med 2005; 71: 659 ± 65. 25 Akihisa T, Yamamoto K, Tamura T, Kimura Y, Iida T, Nambara T et al. Triterpenoid ketones from Lingnania chungii McClure: arborinone, friedelin and glutinone. Chem Pharm Bull 1992; 40: 789 ± 91. 26 Chen JJ, Chou ET, Duh CY, Yang SZ, Chen IS. New cytotoxic tetrahydrofuran- and dihydrofuran-type lignans from the stem of Beilschmiedia tsangii. Planta Med 2006; 72: 351 ± 7. 27 Kuo YH, Lo JM, Chen YF. Cytotoxic components from the leaves of Schefflera taiwaniana. J Chin Chem Soc 2002; 49: 427 ± 31. 28 Chiang YM, Kuo YH. Two novel a-tocopheroids from the aerial roots of Ficus microcarpa. Tetrahedron Lett 2003; 44: 5125 ± 8.
Original Paper
Shin JE, Han MJ, Kim DH. 3-Methylethergalangin isolated from Alpinia officinarum inhibits pancreatic lipase. Biol Pharm Bull 2003; 26: 854 ± 7. 16 Buschi CA, Pomilio AB, Gros EG. 5,6,7-Trisubstituted flavones from Gomphrena martiana. Phytochemistry 1981; 20: 1178 ± 9. 17 Dong H, Gou YL, Cao SG, Chen SX, Sim KY, Goh SH et al. Eicosenoes and methylated flavonols from Amomum koenigii. Phytochemistry 1999; 50: 899 ± 902. 18 Machida K, Kikuchi M. Norisoprenoids from Viburnum dilatatum. Phytochemistry 1996; 41: 1333 ± 6. 19 Lee CK, Lee PH, Kuo YH. The chemical constituents from the aril of Cassia fistula L. J Chin Chem Soc 2001; 48: 1053 ± 8. 20 Wu TS, Chang FC, Wu PL, Kuoh CS, Chen IS. Constituents of leaves of Tetradium glabrifolium. J Chin Chem Soc 1995; 42: 929 ± 34. 21 Hattori M, Shu YZ, Tomimori T, Kobashi K, Namba T. A bacterial cleavage of C-glucosyl bond of mangiferin and bergenin. Phytochemistry 1989; 28: 1289 ± 90.
577
Chen J-J et al. New Dihydrochalcones and ¼ Planta Med 2007; 73: 572 ± 577
