Fitoterapia 82 (2011) 676681

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Fitoterapia
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e

Malaysianol A, a new trimer resveratrol oligomer from the stem bark of Dryobalanops aromatica
A. Wibowo a, N. Ahmat a,, A.S. Hamzah a, A.S. Suan b, N.H. Ismail a, R. Ahmad a, F.M. Jaafar a, H. Takayama c
a b c

Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor Darul Ehsan, Malaysia Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Kuala Selangor, Selangor, Malaysia Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan

a r t i c l e

i n f o

a b s t r a c t
A new resveratrol trimer, malaysianol A (1), ve known resveratrol oligomers: laevifonol (2), ampelopsin E (3), -viniferin (4), -viniferin (5), diptoindonesin A (6), and bergenin (7) have been isolated from the acetone extract of the stem bark of Dryobalanops aromatica by combination of vacuum and radial chromatography techniques. Their structures were established on the basis of their spectroscopic evidence and comparison with the published data. The cytotoxic activity of the compounds was tested against several cell lines in which compound 4 was found to inhibit strongly the growth of HL-60 cell line. 2011 Elsevier B.V. All rights reserved.

Article history: Received 9 December 2010 Accepted in revised form 9 February 2011 Available online 19 February 2011 Keywords: Dipterocarpaceae Dryobalanops aromatica Resveratrol oligomers Malaysianol A Cytotoxic

1. Introduction Dryobalanops is a genus of owering plants from Dipterocarpaceae family. The genus is very unique, as there are only seven species worldwide, conned to the tropical forests of West Malesia (Sumatra, Peninsular Malaysia and Borneo) [1]. Like other Dipterocarpaceae genera, this genus is a good source of resveratrol oligomers (trans-3,4,5-trihydroxystilbene) [2,3] which possess various bioactivities including antioxidant [48], antifungal [9], anti-HIV-1 and cytotoxic effects [10], cyclooxygenase-I and -II-inhibitory [11], antiplatelet-aggregation [12,13], and tyrosinase-inhibitory [14]. Previous study on Dryobalanops genus reported only two resveratrol oligomers, cis- and trans-diptoindonesin B from the tree bark of D. oblongifolia [15]. These compounds are unique, as they are of different oxidative types as compared to other resveratrol oligomers commonly found in Dipterocarpaceae family. In a continuing search for resveratrol oligomers, we report here the structure of a new resveratrol oligomer derivative, malaysianol
Corresponding author. Tel.: +60 3 55444619; fax: +60 3 55444562. E-mail address: noriz118@salam.uitm.edu.my (N. Ahmat). 0367-326X/$ see front matter 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.tote.2011.02.006

A (1) and its proposed biogenetic pathway. The structural elucidation of the compound was done on the basis of spectroscopic data, including UV, IR, MS, and 1D and 2D NMR. This paper also reports the cytotoxic property of the resveratrol oligomers against several cancer cell lines. 2. Experimental 2.1. General experimental procedures The following instruments were used: CD spectrum was recorded on a spectropolarimeter (JASCO J-720WI). UV and IR spectra were measured with a Varian Conc. 100 instruments and a Perkin Elmer Spectrum One FTIR spectrometer, respectively. HRESI-MS were obtained with an Agilent TOFLC/MS G6224A mass spectrometer. The 1H and 13C NMR spectra were recorded with a Bruker Advance Model [300 MHz (1H) and 75 MHz (13C)], and the melting points were measured on a Melting-Point Apparatus with microscope JM628. The following adsorbents were used for purication: vacuum liquid chromatography with Merck Si-gel 60 (540 m, cat. no. 1.07747), radial chromatography with Merck Si-gel 60

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GF254 containing gypsum (540 m, cat. no. 1.07749), and TLC analysis with Merck Kieselgel 60 F254 0.25 mm (cat. no. 1.05554). Solvents used in this research are analytical grade and technical grade that were distilled before used. 2.2. Plant material The stem bark of Dryobalanops aromatica was collected from Balok Reserve Forest, Pahang, Malaysia, and a voucher specimen (SK1762/08) was deposited at the herbarium of Universiti Putra Malaysia, Malaysia. 2.3. Extraction and isolation The dried powder of the stem bark of D. aromatica (4 kg) was macerated with acetone (3x10 L), and evaporated under reduced pressure to give a dark brown residue (400 g). The dried acetone extract was dissolved in a small volume of MeOH (300 mL), and added with diethyl ether to a volume 2 L to give a MeOH-diethyl ether soluble fraction (80 g) after decantation and evaporation. Part of the fraction (60 g) was subjected to vacuum liquid chromatography (VLC), (silica gel, 400 g; eluted with mixtures of n-hexane/EtOAc 5:5, 4:6, 3:7, 2:8 and 1:9, and EtOAc/MeOH 9:1 and 8:2) to give 10 major fractions (DR1DR10). Fraction DR6 (4 g) was refractionated
Table 1 1 H NMR spectroscopy data of 1. No 1a 2a/6a 3a/5a 4a 7a 8a 9a 10a 11a 12a 13a 14a 1b 2b/6b 3b/5b 4b 7b 8b 9b 10b 11b 12b 13b 14b 1c 2c/6c 3c/5c 4c 7c 8c 9c 10c 11c 12c 13c 14c H (mul., J in Hz) 7.25 6.86 5.46 4.57 6.25 6.17 6.25 6.98 6.63 6.76 6.64 6.52 7.05 6.65 4.71 5.91 5.86 5.97 5.86
1

(2, at 2 g) with radial chromatography (silica gel, plate 4 mm, eluted with mixtures of n-hexane/EtOAc/MeOH 4.5:5:0.5) to give six fractions (DR6.1DR6.6). Purication of fraction DR6.2 (800 mg) with radial chromatography (plate 2 mm, CHCl3/ acetone 6:4) afforded compounds 1 (107 mg) and 3 (397 mg). Further purication of fraction DR6.4 (200 mg) with radial chromatography (plate 1 mm, CHCl3/MeOH 8.5:1.5) yielded compound 2 (93 mg). Using the same methodology, purication of fraction DR5 gave compounds 4 (91 mg) and 5 (20 mg) while fraction DR10 yielded compounds 6 (30 mg) and 7 (100 mg). Malaysianol A (1): dark brown solid; m.p. 225; UV (MeOH) max nm (log ): 204 (0.95), 226 (1.16), 286 (1.72), and 322 (1.64); IR (KBr) max cm 1 : 3400, 2920, 1605, 1514, 1449, 1339, 1242, 1159, 1084, 999, and 832; 1HNMR (d6-acetone) see Table 1; 13C NMR (d6-acetone) see Table 1; and HRESI-MS m/z: [M + H]+ 681.2120 (calc. for C42H33O9, 681.2125). CD (c = 0.125 mM, MeOH): 204.8 [ = 46.2], 222.6 [ 5.7], 238.6 [ 12.2], 273.8 [ 1.4], 307.8 [ 3.3], and 358.6 [0] nm. 3. Results and discussion The Phytochemical investigation of the acetone extract of the stem bark of D. aromatica yielded a new compound 1 together with six known compounds which were identied

C 134.9 128.8 117.1 159.2 95.0 58.8 148.3 107.9 160.8 103.0 160.8 107.9 130.1 129.4 117.1 159.2 123.4 134.7 131.1 121.4 163.0 92.8 163.4 122.9 134.1 130.0 116.1 158.4 54.9 91.8 144.0 109.6 159.8 102.7 159.8 109.6
13

HMBC (1H 13C) C-4a, C-3a/5a, C-7a C-1a, C-2a/6a, C-4a C-1a, C-2a/6a, C-8a, C-9a, C-14b, C-13b C-1a, C-7a, C-9a, C-10a, C-14a, C-13b, C-14b C-8a, C-9a, C-12a, C-13a, C-14a C-10a, C-11a, C-13a, C-14a C-8a,C- 9a, C-10a, C-12a, C-13a C-1b, C-3b/5b, C-4b C-1b, C-2b/6b, C-4b C-1b, C-8b C-1b, C-7b, C-9b, C-10b, C-14b C-10b, C-11b, C-13b C-3c/5c, C-4c, C-7c C-1c, C-2c/6c, C-4c C-1c, C-2c/6c, C-8c, C-9c, C-10b, C-11b, C-10c/14c, C-7c, C-9c C-8c, C-12c, C-11c, C-13c, C-14c C-10c C-11c, C-13c, C-14c C-8c, C-10c, C-12c, C-11c, C-13c

(d, 8.5) (d, 8.6) (d, 5.0) (d, 5.0) (d, 2.2) (t, 2.2) (d, 2.2) (d, 8.7) (d, 8.7) (d, 16.9) (d, 16.9)

(1H, s)

(d, 8.6) (d, 8.6) (d, 7.9) (d, 7.9) (d, 2.2) (t, 2.2) (d, 2.2)

Measured in acetone-d6 at 300 MHz ( H) and 75 MHz ( C).

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OH OH
12b

H O HO
A1
1a 7a 8a 10b 9b 9a

O H
8c 14b 7c 9c

H HO
C2
12c

O
B2

OH HO
A1

H O
B2

H
C2

A1

C1

OH

B2

OH H O H
C2

H H
A2

H
7b

8b

1c

OH

HO

H
C1

HO
C1

HO

A2
1b 12a

OH H OH
B1

OH

A2

OH
B2

O H H HO H O
A2 A1

OH

B1

O HO H O O H

OH

B1

HO

OH
A. Wibowo et al. / Fitoterapia 82 (2011) 676681

OH OH
B1

OH

OH

HO

2
OH OH HO

H HO
A1

O
B2

OH H HO
A1

O OH O
B2

H O HO HO OH OH MeO O H OH H O OH
B1 A1 B2

H
C2

OH

OH

H HO
A2

H
C1

HO

A2

OH

OH
B1

HO

A2

HO

OH
B1

HO O OH

OH OH

8 = cis 9 = trans

Fig. 1. Compounds isolated from D. aromatica (17) and cis- and trans-suffruticosol D (8, 9).

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as laevifonol (2) [16], ampelopsin E (3) [17], -viniferin (4) [18], -viniferin (5) [19], diptoindonesin A (6) [20], and bergenin (7) [21] by analysis of the spectral data and comparison with the literature (Fig. 1). A molecular formula of C42H32O9 was deduced for 1 from its [M+ H]+ ion at m/z 681.2120 in the HRESI-MS, a formula characteristic for a resveratrol trimer. The UV absorptions at 204, 286 and 286 nm were typical for an oligostilbene skeleton [2], while the IR spectrum showed the presence of aromatic structures (1605 and 1514 cm 1) which contain hydroxyl (3400 cm 1) and alkyl groups (2920 cm 1). The 13C NMR (APT, Attached Proton Test) spectrum of 1 showed 30 signals representing 42 carbon atoms that are distributed into nine oxyaryl, eight quarternary aromatic, 19 methine aromatic, four methine alkyl, and two olenic carbons. The 1H NMR spectrum (Table 1) showed the presence of six doublets of ortho-coupled aromatic signals at H 7.25/6.86, 6.98/6.63 and 7.05/6.65 from three para-hydroxyphenyl groups (A1-, B1- and C1-rings). Signals of two symmetrical doublets of meta-coupled aromatic protons belonging to two 3,5-dioxygenated aromatic rings were seen at H 6.25 and 5.86 while signals at H 6.17 and 5.97 are attributable to two triplets from meta-coupled aromatic protons (A2- and C2-rings). In addition, a singlet observed at H 6.52 is for 3,5-dioxygenated-2,6-disubstituted aromatic ring (B2-ring). Furthermore, two pairs of mutually coupled aliphatic methine proton at H 5.46/4.57 (H7a/H8a), and 4.71/5.91 (H7c/H8c) can be assigned to two 1,2-diaryldihydrobenzofuran moieties, while two olenic protons at H 6.76 and 6.64 belong to trans1,2-disubstituted vinyl group. The above evidence suggested that 1 could be a resveratrol trimer with one free stilbene unit and two 1,2-dihydrobenzofuran rings. From the analysis of the NMR data, we can conclude that the structure units of A- and B-rings of 1 are very similar to ampelopsin E (3) [17], and the only difference is on the C-ring structure unit. These can only happen when compound 3 loses its symmetricity. Recently, stereoisomers of 3, cis- and trans-suffruticosol D (8, 9) have been reported from the seeds of Paeonia suffruticosa [22]. Observation on the 1H NMR spectrum of 1 suggests that 1 is another stereoisomer similar to 3, 8 and 9. To prove our observation, we further analyzed the HMBC and NOE correlations of 1. The HMBC spectrums of 1 (Table 1), in particular, revealed 1 H13C long range correlations between oxygenated methine aliphatic proton at H 5.91 with carbon at C 144.0 (C9c)/109.6 (C10c/C14c) indicating that C2-ring was unambiguously at oxygenated carbon (C8c) which is part of the dihydrobenzofuran ring. Whereas position of C1-ring at non-oxygenated carbon (C7c) was shown by correlations between proton at H 4.71 (H7c) with carbon at C 134.1 (C1c)/130.0 (C2c/C6c)/122.9 (C14b)/163.4 (C13b). These correlations distinguished the structure of 1 from 3 and suffruticosol D (8, 9), where the position of C1- and C2-rings is opposite. HMBC spectrum also revealed correlations between the aromatic singlet proton at H 6.25 with carbon signal at C 122.9 (C14b), 163.4 (C13b), 163.0 (C11b), and 121.4 (C10b) indicating the attachment of proton at H 6.25 to C12b of B-ring. Two units of 1,2-dihydrobenzofuran ring that attached to B-ring were conrmed with the correlations of carbon C10b/C11b and C13b/C14b with proton at H 5.46 (H7a)/4.57 (H8a) and 4.71 (H7c)/5.91 (H8c), respectively. Besides that, the position of trans1,2-disubstituted vinyl group that attached to B2-ring was also observed with the correlations between the olenic protons at

H 6.76/6.64 with carbon signals at C 131.1 (C9b), 121.4 (C10b), and 122.9 (C14b). The relative stereochemistry at the chiral carbons in the oligostilbenoid part was determined from the NOE correlations found in the NOESY spectrum. Signicant NOE correlations between H7a and H10a(14a), H8a and H2a(6a) conrmed trans relationship of H7a/H8a. Furthermore, the NOE correlations displayed between H7c and H8c, H2c(6c) and H10c(14c) established cis relationship of H7c/H8c. The fact that the protons of H2c(6c) and H10c(14c) appeared at relatively higher eld further reinforced the observation of the overlapping of rings C1 and C2 (Fig. 2). The spatial relation between H7a and H8c, H8a and H7b, H8b and H7c, and no correlation between H7a and H8c, suggested cis relationship of H7a/H8c and trans for H8a/H7c. Based on these observations, the relative congurations of H7a, H8a, H7c, and H8c of 1 are suggested as rel-(7aR, 8aR, 7cR, and 8cS) (Fig. 2). The ve resveratrol oligomers (26) isolated from D. aromatica species are commonly found in Dipterocarpaceae, but compound 1 is unique and different as it has different type of coupling. The biogenetic pathway for the formation of 1 is also proposed. The route involves coupling of a dimer of -viniferin (5), which has a dihydrobenzofuran ring, with a resveratrol [23]. Usually, the dihydrobenzofuran ring of resveratol oligomer can be formed via oxidative coupling of radical species at C14 with that at C8 (C14C8 type). For example, formation of ampelopsin E (3) was proposed through the condensation of hypothetical radical b (C14a) and c (C8c) followed by the formation of a dihydrobenzofuran ring in the last step (route ii, Fig. 3) [23]. The biogenetic pathway of 1 was proposed from the oxidative coupling of oxygen at hypothetical radical a (OC13b) and carbon

Fig. 2. Key HMBC and NOESY correlations of 1.

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HO OH OH H O

HO

O 13b 14b HO

O 14b

8c 7c HO

H HO

(i)
HO OH HO OH

H HO O

OH

(i)

OH

OH

OH

OH 8c 7c

Formation of C14-C7 bonding

O H

OH

1
H O

H O HO 14b H HO

(ii)

(ii)

Formation of C14-C8 bonding

H O HO 14b

O HO 7c 8c H HO HO OH

H O

H OH

c
H OH HO

H OH

OH

OH

OH

HO

b
OH OH

e
Fig. 3. Proposed biogenetic pathways for 1 and 3.

Table 2 Cytotoxic activity of compound 14. Cell lines Compounds 1 2 3 4 IC50 value (g/ml) HL-60 39.6 3.5 37.6 4.8 31.2 9.7 2.7 0.5 MCF-7 25.1 0.0 50.6 4.3 14.3 2.7 15.7 8.0 HepG2 29.8 2.1 33.8 4.4 44.9 5.3 32.5 3.4 A-549 59.8 0.8 not detected 60.7 0.8 49.7 0.6 WRL-68 40.9 4.0 41.4 2.4 50.1 2.3 41.5 4.3

IC50 activity (inhibition): very strong b 5 g/ml; strong b 510 g/ml; moderate: 1020 g/ml; weak: 20100 g/ml and not active N 100 g/ml [25].

at hypothetical radical c (C8c), followed by the formation of dihyrobenzofuran ring via intramolecular cyclyzation reaction of C14b and C7c on the intermediate (C14C7 type), see Fig. 3 (route i). The effect of the isolated compounds (14) against HL-60 (human leukemia), MCF-7 (human breast cancer), HepG2 (human hepatocellular liver carcinoma), A-549 (human lung adenocarcinoma epithelial) and WRL-68 (normal liver) cell lines was evaluated by using MTT assay as described by Mosmann with slight modications [24]. The results were expressed as IC50 values and summarized in Table 2. As depicted in Table 2, compound 4 was found to inhibit very strongly the growth of HL-60 cell lines (IC50 2.7 g/ml). Compounds 3 and 4 were also found to display moderate activity against MCF-7 cell line (14.3 and 15.7 g/ml respectively), while weak activity was shown by 1 against MCF-7 and HepG2 (25.1 and 29.8 g/ml respectively).

Fst (12/2010) and Dr. Shamsul Khamis for the identication of sample. References
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Acknowledgments The authors would like to thank The Ministry of Higher Education, Malaysia for the nancial support under the Fundamental Research Grant Scheme; 600-RMI/ST/FRGS/5/3/

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