Compound 1 was isolated as a yellow amorphous solid, mp 186–188 °C, –42° (c 0.00024, MeOH), which exhibited a molecular ion peak in the negative-ion high resolution ESIMS at [M−H]^–m/z 469.1275 (calcd. 469.1293) attributable to the molecular formula C₂₈H₂₂O₇, which corresponded to a resveratrol dimer. This assumption was reinforced by the UV and IR absorption data, together with the ¹H- and ¹³C-APT NMR data, which was assigned by the interpretation of the HMQC, HMBC, ¹H-¹H COSY and NOESY spectra (Table 1). UV absorption λ_max nm (MeOH): 282; IR (KBr) ν_max cm^–1: 3366 (OH), 2922 (C–H aliphatic), 1602, 1516 and 1449 (C=C aromatic), 1249 and 1175 (C–O oxyaryl), and 835 (p-disubstituted benzene).

The ¹H-NMR spectral data of 1 exhibited the presence of two sets of ortho-coupled aromatic protons which can be assigned to two 4-hydroxyphenyl groups [ring A1: δ 7.09 (2H, d, J = 8.6 Hz, H-2a and 6a), δ 6.76 (2H, d, J = 8.6 Hz, H-3a and 5a), and ring B1: δ 7.13 (2H, d, J = 8.2 Hz, H-2b and 6b), δ 6.57 (2H, d, J = 8.2 Hz, H-3b and 5b)], two sets of meta-coupled aromatic protons on two 1,2,3,5-tetrasubstituted benzene rings [ring A2: δ 6.18 (1H, br s, H-14a), δ 6.41 (1H, d, J = 1.5 Hz, H-12a), and ring B2: δ 6.94 (1H, d, J = 1.8 Hz, H-14b), δ 6.11 (1H, d, J = 1.8 Hz, H-12b)], five phenolic hydroxyl groups (δ 8.05, 8.22, 8.23, 8.54 and 8.57), and one aliphatic hydroxyl group (δ 3.63). The ¹H-NMR spectrum also showed two pairs of aliphatic methine protons coupled successively: H-7a (δ 5.67, d, J = 11.7) and H-8a (δ 4.19, d, J = 11.7); H-7b (δ 5.46, br s) and H-8b (δ 5.09, br s) as shown in Table 2. The large coupling constant of H-7a and H-8a (11.7 Hz) in compound 1 indicated that the protons were in trans orientation [21], which is also exhibited by its stereoisomers, namely (+)-ampelopsin A (11.7 Hz) [22], (−)-hemsleyanol A (9.8 Hz) [17] and (+)-balanocarpol (9.3 Hz) [23]. On the other hand, H-7b and H-8b of 1 gave two broad singlets as those in (+)-balanocarpol [23], which proved that the two protons were in cis orientation. However, trans–H-7b~H-8b in (+)-ampelopsin A [22] and (−)-hemsleyanol A [17] gave coupling constants of 5.0 and 5.9 Hz for H-7b whereas H-8b produced broad singlet and broad doublet respectively.

Relative configuration of methine protons at C-8a, C-7b and C-8b on a cycloheptane ring in compound 1 can be further verified by the results of NOESY experiment. The absence of NOEs between H-8a and either H-7b or H-8b revealed that H-8a and H-7b~H-8b were in opposite sides of the cycloheptane ring. This also confirmed that both H-7b and H-8b were in the cis position. (+)-Balanocarpol where all three hydrogens were on the same side of the cycloheptane ring had significant NOEs between H-8a/H-7b and H-8a/H-8b [23]. Since H-7a and H-8a of 1 were trans based on the above ¹H-NMR spectral data, the relative configuration of methine protons at C-7a, C-8a, C-7b and C-8b were β, α, β and β. Other significant NOEs in support of these observations were between β H-7a/H-14a, β H-7b/β H-8b and β H-8b/H-14b. In the HMBC spectrum (Table 1, Figure 2a), significant correlations were observed between C-2a(6a)/H-7a, C-8a/H-7a, C-9a/H-7a, C-9a/H-8a and C-10b/H-8a, indicated that a pair of benzylic methine protons of β H-7a and α H-8a was assigned to the protons on a dihydrofuran ring.

There were significant correlations observed between C-9a/H-8b, C-9b/H-7b, C-1b/H-7b, C-11a/H-7b, C-10a/H-7b and C-8b/H-7b. These correlations proved that there was one more pair of methine protons attached to C-7b (β H-7b) and C-8b (β H-8b). The relative stereo structure for compound 1 was confirmed as shown in Figure 1. It should be noted that the above three resveratrol dimers, which were stereoisomers to compound 1, were previously isolated from Ampelopsis brevipedunculata var. hancei (Vitaceae) [(+)-ampelopsin A] [22], H. parvifolia (Dipterocarpaceae) [(+)-balanocarpol] [23] and Shorea hemsleyana (Dipterocarpaceae) [(−)-hemsleyanol A] [17].

All of the four stereoisomers shared a same basic planar structure. However, the chemical shifts for aliphatic protons of H-7a, H-8a, H-7b and H-8b of the four stereoisomers were markedly different as shown in Table 2. These differences have been suggested to be mainly due to the A1 and B1 rings in their energy-optimized conformations as in Figure 3.

The NOE interactions between H-8a/H-2a(6a), H-7a/H-14a, H-8b/H-14b and H-8a/H-2b(6b) in compound 1 (Figure 3a) and (+)-ampelopsin A (Figure 3b) [22] lead to cis and skew-cis conformations, while the absence of NOE interaction between H-8a/H-2b(6b) in (+)-balanocarpol (Figure 3d) [23] would suggest that it was in a trans conformation. The absence of NOE interaction between H-8a/H-2b(6b) in the skew-cis conformation of (−)-hemsleyanol A (Figure 3c) [17], unlike that in the skew-cis conformation of (+)-ampelopsin A, was due to the relative positions of H-8a and B1 ring which were farther apart in the actual energy-optimized 3D-model of the molecule. It should be noted that the NOE correlations between H-2a(6a)/H-14a and H-7a/H-8a in compound 1 seem to suggest that the H-7a and H-8a were cis to each other with H-7a in α position. However, this contradicted with the large coupling constants between H-7a and H-8a of the trans in compound 1 (11.7 Hz) as well as in its stereoisomers of (+)-ampelopsin A (11.7 Hz) [22], (−)-hemsleyanol A (9.8 Hz) [17] and (+)-balanocarpol (9.3 Hz) [23]. The chemical shift for H-8a in compound 1 was more upfield (δ 4.19) like the one found in (+)-ampelopsin A (δ 4.17) [22] when compared to that in (−)-hemsleyanol A (δ 5.41) [17] and (+)-balanocarpol (δ 5.16) [23]. This strongly suggested that the A1 and B1 rings in compound 1 and (+)-ampelopsin A were in cis configuration. The chemical shifts for H-8a in (−)-hemsleyanol A and (+)-balanocarpol which appeared 1.23 and 0.98 ppm more downfield indicated that the A1 and B1 rings for the latter were in trans configuration (Table 2). Although the A1 and B1 rings for (−)-hemsleyanol A were in cis configuration, its B1 ring did not give any deshielding effect like in (+)-balanocarpol since those rings were in the same orientation as shown in Figure 3c,d. The H-8a in (−)-hemsleyanol A was more deshielded than that in (+)-balanocarpol because the former lay within the deshielding area up front to the A1 ring whereas this ring in the latter was a little tilted. Moreover NOE correlations [Table 1, Figure 2b] between H-8a/H-2a(6a) and H-8a/H-2b(6b) in compound 1 positively suggested that its A1, H-8a and B1 were all on the same side of the plane. The results from the energy-optimized conformations as calculated by GaussView 5.0 and Gaussian 09W using DFT-6-31G-(d,p) method, have confirmed the above deductions (Figure 3). Therefore, 1 is a new stereoisomer of these three compounds.

The results of cytotoxicity evaluation of compounds 1 to 6 as LC₅₀ (mM) are shown in Table 3. The compounds were considered safe when their LC₅₀s are higher than 100 μM [25]. All compounds tested possessed LC₅₀ values more than 100 μM (161 to 830 μM).

IR spectra were recorded on a Perkin Elmer GX FT-IR spectrophotometer (Waltham, MA, USA). UV spectra were measured on Shimadzu UV-160 (200–400 nm, Kyoto, Japan). ¹H and ¹³C-APT NMR spectra were recorded in acetone-d₆ using JEOL ECP400 spectrometer (400 and 100 MHz for ¹H and ¹³C; Akishima, Japan). Mass spectra were measured in electron spray ionization mode on Bruker (micro TOF-Q; Bremen, Germany) LC-MS spectrometer (ESI-MS in negative mode, Dionex, Sunnyvale, CA, USA). Melting points were determined by Stuart SMP10 melting point apparatus (Burlington, VT, USA) and were uncorrected. Optical rotations were recorded on Jasco Polarimeter P-1020 (Easton, MD, USA) in MeOH. Vacuum liquid chromatography (VLC) was carried out on Si-gel 60 GF₂₅₄ (Merck, Damstadt, Germany), radial chromatography (RC) was done on Si-gel 60 PF₂₅₄ (Merck) and TLC was performed on pre-coated silica gel (Merck, Kieselgel 60 F₂₅₄ 0.25 mm), and detected by UV light (254 nm) or by CeSO₄ spraying reagent followed by heating. All solvents used were of analytical grades. Absorbance values for BCLA and cytotoxic assays were measured on microplate reader (Labsystem Multiskan Multisoft, Basingstoke, UK) and DPPH assay on Shimadzu UVmini-1240 spectrophotometer (Kyoto, Japan). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) used to maintain and supplement the cell, respectively, were from Flowlab (North Ryde, Australia). Samples emulsifying was done on Branson 5200 (Los Angeles, CA, USA) sonicator.

The stem bark of Shorea acuminata was collected from Universiti Kebangsaan Malaysia (UKM) Forest Reserve, Bangi, in September 2009. A voucher specimen (UKMB 23520) has been deposited in the UKM Herbarium, and identified by Mr. Sani Miran.

The dried powder of stem bark of Shorea acuminata (1 kg) was macerated with acetone (3 × 5 L, 3 days each) at room temperature. The extract was concentrated using rotary evaporator to yield a brownish acetone extract (49.6 g, 4.96%) that was fractionated by VLC eluted with mixtures of n-hexane-EtOAc of increasing polarity. The eluates that showed similar profile on TLC chromatogram were combined to give five fractions A–E. Fraction C (1.5 g) was subjected to RC by eluting with CHCl₃-MeOH (9:1) to afford compound 3 (4.9 mg). Purification of fraction D (400 mg) by RC using CHCl₃-MeOH (8.4:1.6) followed by preparative TLC (CHCl₃-MeOH, 8.2:1.8) gave compounds 1 (12.0 mg), 2 (4.0 mg) and 4 (7.6 mg). Purification of fraction E (600 mg) by RC using CHCl₃-MeOH (8:2) followed by preparative TLC afforded compounds 5 (22.5 mg) and 6 (5.2 mg).

Compound 1: Yellow amorphous solid, m.p. 186–188 °C, –42° (c 0.00024, MeOH), UV (MeOH) λ_max 282 nm; IR (KBr) ν_max cm^–1: 3366, 2922, 1602, 1516 and 1449, 1249 and 1175, 835. ESIMS (neg): m/z 469.1275 [M−H]^–, calcd: 469.1293; ¹H- and ¹³C-NMR data, see Table 1.

The isolated compounds were tested for in-vitro cytotoxicity using Vero cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [28]. The Vero cell line was initiated from kidney of a normal adult African green monkey, Cercopitheus aethiops, obtained from Virology Laboratory, School of Biosciences and Biotechnology, Faculty of Science and Technology, UKM. Vero cells were maintained in DMEM, supplemented with 10% FBS and cultured at 37 °C in a humidified 5% CO₂ incubator. The concentration of stock compound solution was 1.0 mg/mL prepared by dissolving 1 mg compound in 50 μL methanol and 950 μL 5% FBS-DMEM. Sonicator was used to emulsify the compounds for 40 min before the two-fold dilutions made in 5% FBS-DMEM to produce a compound solution at concentrations of 0.5, 0.25, 0125 and 0.0625 mg/mL. Briefly, a total of 50 mL of cell suspension with different concentrations were added to each well in the 96-well micro-titer plates. As a positive control, each well from first to third wells was added with cells of 2.5 × 10⁵, 1.25 × 10⁵ and 0.625 × 10⁵ cells/mL. For the negative control (100% cell death, LC₁₀₀), 50 mL of DMEM without cells were added to the twelfth well. Cells of 2.5 × 10⁵ cells/mL were added into the fourth to eleventh wells. The plates were incubated for 48 h at 37 °C with 5% CO₂ until a monolayer is formed. A total of 50 μL of test compound solution were placed in each well containing the monolayer cells and 50 μL of phosphate buffered saline was added to each control cell well and negative control well (LC₁₀₀), and incubated for 24 h at 37 °C with 5% CO₂. After the incubation period, MTT (20 μL, 5 mg/mL) was added into each well and the cells incubated for 2 to 4 h, until a purple precipitate was clearly visible under a microscope, the medium together with MTT (190 μL) was aspirated off from the wells, DMSO (100 μL) was added and the plate shaken for 5 min. The absorbance for each well was measured at 540 nm in a micro-titer plate reader and percentage of cell viability (CV) was calculated manually using the formula: CV = [(Abs_sample − Abs_{negative control})/(Abs_cell − Abs_{negative control})] × 100. A dose-response curve was plotted to enable the calculation of the concentration that kills 50% of the Vero cells (LC₅₀).

Values expressed are means of the three replicate determinations ± standard deviation. All statistical analyses were carried out using SPSS 16.00 for Windows. To determine whether there were any differences between activities of samples, variance analysis (one-way ANOVA) was applied to the results. Values of p < 0.05 were considered as significant different (α = 0.05).