Structural Studies on Stilbene Oligomers Isolated from the Seeds of Melinjo (Gnetum gnemon L.).

This paper describes the isolation and structural determination of a new stilbene dimer, named 7a-epi-gnetin C, from melinjo (Gnetum gnemon L.) seed extract. The relative structure was elucidated based on NMR spectroscopic evidence, while the absolute configuration was assigned by a combination of NMR and electronic circular dichroism spectroscopic analysis and chemical conversion. 7a-epi-Gnetin C was evaluated as an antioxidant and was shown to have a comparable activity to the known stilbene oligomers. In addition, the structural revision of gnetin L, a known stilbene dimer, was also discussed.

Introduction

Gnetum gnemon L. is a species of the Gnetaceae family commonly called melinjo in Indonesia, known to be rich in resveratrol derivatives. Its fruit and seeds are used as ordinary food in Indonesia and their extracts have been reported to show several pharmacological activities from in vitro to human studies.[1−6] A stilbene monomer, trans-resveratrol (8) and stilbene oligomers such as gnetin C (4), gnemonoside A (5), gnemonoside C (6), gnemonoside D (7), gnetin L, and gnetin E (9) have been isolated from the fruit and/or seeds (Figure ).[1,2] The oligomers possess a 1,2-diaryldihydrobenzofuran skeleton as a basic structural motif. The relative stereochemistry of two chiral centers in the core has been assigned by NOE experiment,[1,2,7−9] whereas the absolute configuration has been traditionally elucidated by comparison of its electronic circular dichroism (ECD) spectrum with that of known structures.[10−12] In our ongoing search for biologically active components from melinjo seed extract (MSE), we isolated a new minor epimer of 4, termed 7a-epi-gnetin C (1) along with known compounds as shown in Figure . In this paper, we report the elucidation of structure 1 and structural revision of gnetin L (2) to the regioisomer 3. In addition, a series of resveratrol derivatives shown in Figure were submitted to the oxygen radical absorbance capacity (ORAC) assay and evaluated as an antioxidant.

Figure 1

Structures of natural stilbene derivatives from G. gnemon L.

Results and Discussion

The seeds (endosperms) of melinjo (G. gnemon L.), collected in Indonesia, were powdered and extracted with 55% aqueous EtOH. MSE with enzymatic hydrolysis of glucosides was successively subjected to ODS column chromatography, silica gel column chromatography, and reversed phase HPLC to give 1. The existence of 1 in the MSE was confirmed by analytical HPLC analysis before being treated with the enzyme. The molecular formula of 1 was established as C28H22O6 by HR-ESI-MS spectrum (m/z: 477.1313 [M + Na]+; calcd for C28H22O6Na, 477.1314). The 1H and 13C NMR data ensured a set of 2,3-diaryl-dihydrobenzofuran ring and the connectivity of units A and B supported by HMBC correlations from H-8a to C-11b and C-12b, which indicated that 1 possessed the same planar structure as that of 4 (Table ). The coupling constant between H-7a and H-8a for 1 is 8.3 Hz, whereas for 4 is reported to be 4.5 Hz.[7] These values were diagnostic and the same as the values reported for cis-dihydrobenzofuran and its trans isomer derived from (−)-ε-viniferin,[13] and the cis- and trans-dihydrobenzofuran moieties included in suffruticosol D from Paeonia suffruticosa,[14] indicating that 1 is in a cis-form on the dihydrofuran ring. In comparison between 1H NMR chemical shifts of 1 (Table ) and those of 4 (Table S3), the cis orientation of A1 and A2 rings in 1 causes up-field shifts for aromatic ring signals of H-2a (Δ −0.22 ppm), H-3a (Δ −0.23 ppm), H-10a (Δ −0.44 ppm), and H-12a (Δ −0.22 ppm) by stacking effects. In addition, the NOESY correlations between vicinal H-7a and H-8a and between two aromatic groups H-2a (6a) and H-10a (14a) further supported a cis-orientation.

Table 1

1H and 13C NMR Data for 1 in Acetone-d6

position	δC	δH (J in Hz)	HMBC	NOE
1a	129.05
2a (6a)	129.05	7.00 (2H, d, J = 8.7 Hz)	1a, 4a, 6a, 7a	3a (5a), 7a, 10a (14a)
3a (5a)	115.1	6.61 (2H, d, J = 8.7 Hz)	1a, 5a	2a (6a)
4a	157.4
7a	90.3	5.85 (1H, d, J = 8.3 Hz)	1a, 2a (6a), 9a	2a (6a), 8a
8a	51.7	4.56 (1H, d, J = 8.3 Hz)	9a, 10a (14a), 11b, 12b	7a, 10a (14a)
9a	142.2
10a (14a)	108.6	5.73 (2H, d, J = 2.3 Hz)	8a, 12a	2a (6a), 8a
11a (13a)	158.7		10a (14a), 12a
12a	101.6	5.99 (1H, t, J = 2.3 Hz)	10a (14a), 11a (13a)
1b	130.0
2b (6b)	128.7	7.45 (2H, d, J = 8.2 Hz)	4b, 6b, 7b	7b, 8b
3b (5b)	116.4	6.86 (1H, d, J = 8.2 Hz)	1b, 4b, 5b
4b	158.3
7b	129.13	7.11 (1H, d, J = 16.5 Hz)	2b (6b), 8b, 9b	2b (6b), 10b, 14b
8b	126.8	6.99 (1H, d, J = 16.5 Hz)	7b, 10b, 14b	2b (6b), 10b, 14b
9b	140.8
10b	99.3	6.72 (2H, d, J = 0.9 Hz)	8b, 11b, 12b, 14b	7b, 8b
11b	162.9
12b	117.1
13b	155.3
14b	108.3	6.62 (2H, d, J = 0.9 Hz)	8b, 10b, 12b, 13b	7b, 8b
11a, 13a-OH		7.85 (2H, s)	10a (14a), 12a

The absolute configuration of 1 was determined by the optical data comparison of a phenol derivative 10 derived from both 1 and 4. Namely, treatment of 1 with a Pd/C catalyst under hydrogen atmosphere at room temperature underwent a reductive opening of the tetrahydrofuran ring at C-7a to give 10 {[α]D25 −60.8 (c 0.02, MeOH)} (Scheme ). Its 1H and 13C NMR data were identical with those of the reduction product prepared[15] from 4, and its optical rotation was also nearly equal in sign and magnitude[16] to that of 10 {[α]D26 −66.8 (c 0.11, MeOH)} derived from 4 {[α]D25 −18.5 (c 0.1, MeOH)}, indicating that the configuration of C-8a of 1 was the same as 4. Therefore, 1 was determined to be the C-7a epimer of 4.

Scheme 1

Preparation of 10 by Hydrogenation of 1 or 4

The absolute configuration of (−)-gnetin C (shown as 4 in Figure ) was reported to be 7aS,8aS by a vibrational circular dichroism (VCD) study.[17] We independently ensured this assignment using a fully methylated benzaldehyde derivative, 12 prepared from 4 via 11 (Scheme ). This compound was designed in order to suppress the effect of hydrogen bonding[18] and the free rotation of a terminal styryl unit. As expected, the experimental ECD spectrum of 12 showed good agreement with the calculated one for (7aS,8aS)-12 at ca. 210 nm, a strong positive band at ca. 245 nm, a broad negative band at ca. 280 nm, and a broad positive band at ca. 325 nm (Figure ). Meanwhile, the theoretical ECD spectrum calculated for (7aR,8aS)-12 exhibited a different feature to that of the experimental one. From these results, the absolute configuration of the parent compound 4 was unambiguously assigned as 7aS,8aS, which is consistent with the previous result.[17] Thus, the absolute configuration of the new compound 1 was determined as 7aR,8aS.

Scheme 2

Chemical Conversion of 4 into a Benzaldehyde Derivative 12

Figure 2

Calculated and experimental ECD spectra of 12.

In the course of identifying other isolated compounds, we found that NMR spectral data in acetone-d6 of 3 was almost identical to those of gnetin L (2)[2] with a 3-hydroxy-5-methoxyphenyl group. The 1H NMR spectra of the isolated 3 in acetone-d6, chemical shifts of some aromatic signals were overlapped and second order effects were observed. To clarify the substitution pattern of aromatic rings, we tried to measure 1H NMR spectra in several solvent systems. In the 1H NMR spectra of 3 in CD3CN, the three proton signals in the A1 ring were observed at 6.76, 6.80, and 6.92 ppm as doublet of doublets (J = 7.8, 1.8 Hz), doublet (J = 7.8 Hz), and doublet (J = 1.8 Hz), respectively (Figure ). The ortho-coupling protons at δH 6.76 and 6.80 (J = 7.8 Hz) and meta-coupling protons at δH 6.76 and 6.92 (J = 1.8 Hz) proposed an ABX system of 4-hydroxy-3-methoxyphenyl group for the A1 ring. In addition, the HMBC correlation peak from the hydroxy proton signal at δH 6.57 (1H, s) to δC 115.6 (C-5a) indicated that the hydroxy group was attached to the C-4a position (Figure ). Thus, we concluded that the structure of natural gnetin L should be revised to be the regioisomer 3. This structural revision of gnetin L (3) revealed that gnetin L[2] and the later reported macrostachyol D by Sri-in et al. in 2011 were identical.[19]

Figure 3

Expanded 1H NMR spectra in comparison with spin–spin coupling patterns for 4-hydroxy-3-methoxyphenyl ring portion of 3 in acetone-d6 (a) and CD3CN (b).

Figure 4

HMBC correlations for 3 in CD3CN.

Finally, we have tested the antioxidant activities of the new as well as known compounds, as it is well known that polyphenols have potentially high antioxidant activities.[20] As listed in Table , all tested compounds showed stronger ORAC activity compared with l-ascorbic acid (Table ). Compounds 1 and 3 were less active than 4, indicating that a change in the stereochemistry of C-7a in A1 ring and the methoxy group at C-3a in A1 ring decreased the activity. In addition, compounds 5–7 were less active than 4, indicating that glucosylation of the hydroxyl group at C-4a in A1 ring and at C-4b in B1 ring decreased the activity. Although the glucosyl substitution effects of the hydroxyl group in A1 and B1 ring on DPPH radical scavenging activity reported by Kato et al. in 2009 were very small, the number and position of free phenolic hydroxyl groups in the molecule will contribute to the antioxidant activity as well as other polyphenols. Furthermore, the activity of 9 was 4-fold less active than 4 and 8. This suggested that a molecule larger than a dimer is less effective as an antioxidant.

Table 2

ORAC Antioxidant Capacity per mol of Stilbene Oligomers and l-Ascorbic Acida

compound	typeb	mmol TE/molc
7a-epi-gnetin C (1)	resveratrol dimer	5081 ± 420
gnetin L (3)	a resveratrol unit and an isorhapontigenin unit	5298 ± 414
gnetin C (4)	resveratrol dimer	8597 ± 619
gnemonoside A (5)	resveratrol dimer	4679 ± 586
gnemonoside C (6)	resveratrol dimer	6336 ± 627
gnemonoside D (7)	resveratrol dimer	5663 ± 997
trans-resveratrol (8)	resveratrol monomer	8241 ± 350
gnetin E (9)	resveratrol trimer	2129 ± 237
l-ascorbic acidd	−	638 ± 59

Data are expressed as the mean ± SD of three experiments.

This classification was based on the structure of 8 as a monomer.

Data are expressed as Trolox equivalent per 1 mol sample.

l-Ascorbic acid was used as positive control.[26]

Methods

General Procedures

All melting points were determined with a Yanaco MP-500 apparatus and are uncorrected. Optical rotations were measured using a JASCO DIP-370 digital polarimeter. IR spectra were recorded by the attenuated total reflection method using ZnSe prism on a JASCO VALOR-III spectrophotometer. 1H and 13C NMR spectra were recorded at 500 and 125 MHz or 600 and 150 MHz with Agilent Technologies V500 or JEOL ECA600 spectrometers. Chemical shifts were referenced to a residual signal (1H) and a solvent signal (13C) of CD3CN (δH 1.93, δC 118.3), acetone-d6 (δH 2.04, δC 29.8), CD3OD (δH 3.30, δC 49.0), and CDCl3 (δH 7.26, δC 77.0). ESIMS were recorded on a JEOL JMS-T100LC mass spectrometer. Preparative HPLC was conducted on a Waters 600E system with a Cosmosil 5C18-AR-II column (250 mm × 20 mmID, Nacalai Tesque). Medium-pressure liquid chromatography (MPLC) was carried out on a Yamazen MPLC system. Merck precoated silica gel 60 F254 plates, 0.25 mm in thickness, were used for analytical thin-layer chromatography.

Plant Material

The seeds (endosperms) of melinjo were collected in Indonesia (Desa Bangkok, Kecamatan Gurah Kabupaten Kediri, Kediri, Jawa Timur) in July 2009 and have been identified by Dr. Eishin Kato. Voucher specimens (number 090716) have been deposited at Hosoda SHC Co. Ltd., Fukui, Japan.

Extraction and Isolation

The preparation of MSE and isolation of 4–7 were performed based on the modified method reported by Kato et al.[2] In brief, the powder of dried endosperms of melinjo was extracted with 55% aqueous ethanol, and the MSE (23.0 g) was subjected to ODS column chromatography, silica gel column chromatography, and recrystallization and/or preparative HPLC to give 4 (0.19 g), 5 (1.15 g), 6 (62 mg), and 7 (0.23 g).

The MSE (150 g) was treated with 3 g of β-glucosidase in 0.02 M citric acid (12 L) at 40 °C for 24 h. The hydrolyzed residue (120 g) was subjected to Daiso Gel column chromatography (Daiso gel IR-60, 600 mm × 80 mmID) with 9 L (each 1 L/fraction) of 6, 12, and 25% MeOH in CHCl3 as the eluent. The fraction eluted with 25% MeOH in CHCl3 was further purified by MPLC using ODS column chromatography (Hi-Flash Column ODS-SM, 100 mm × 26 mmID, Yamazen) using aqueous MeOH (20–80%, 54 min linear, 100%, 10 min hold) with 0.1% TFA as the eluent at a flow rate of 15 mL/min (15 mL × 64). Fraction 31–33 was then purified by preparative HPLC using aqueous MeOH (48–50%, 60 min linear, 50–55%, 60 min linear, 55%, 30 min hold) with 0.1% TFA at a flow rate of 7.5 mL/min to give 9(21) (tR = 124 min, 1.82 mg ≥ 99% HPLC). Furthermore, the hydrolyzed residue (70 g) was subjected to ODS column chromatography (Cosmosil 75C18-PREP, 400 mm × 80 mmID, Nacalai Tesque) with 6 L (each 1 L/fraction) of 20, 40, 60, and 100% aqueous MeOH. The third and fourth fractions eluted with 40% aqueous MeOH were subjected to MPLC using ultra pack silica gel column (300 mm × 37 mmID, Yamazen) with MeOH (10%, 15 min hold, 10–20%, 45 min linear, 20–50%, 45 min linear, 50%, 60 min hold) in CHCl3 as the eluent at a flow rate of 20 mL/min (20 mL × 120). Fraction 27–30 was further purified by preparative HPLC using aqueous MeOH (43–46%, 30 min linear, 46%, 150 min hold) with 0.1% TFA at a flow rate of 7.5 mL/min to give fractions that eluted at 117 and 133 min to give 1 (1.82 mg ≥ 99% HPLC) and 3 (3.59 mg ≥ 99% HPLC), respectively.

7a-epi-Gnetin C (1): [α]D26 + 201 (c 0.11, MeOH); IR (ZnSe) νmax: 3235, 2932, 2840, 1670, 1595, 1509, 1448, 1429, 1251, 1194, 1142, 998, 821, and 799 cm–1; 1H and 13C NMR (600 and 150 MHz, acetone-d6), see Table ; HRESIMS m/z: 477.1313 [M + Na]+ (calcd for C28H22O6Na, 477.1314).

Gnetin L (3): 1H and 13C NMR (600 and 150 MHz, CD3CN), see Table S2.

Gnetin E (9): 1H and 13C NMR (600 and 150 MHz, acetone-d6), see Table S2.

Analysis of Resveratrol Derivatives

Analysis of resveratrol derivatives in MSE was performed by the Prominence HPLC system (Shimadzu) with a Cosmosil 5C18-AR-II column (250 mm × 4.6 mmID, Nacalai Tesque), following the analysis conditions previously reported.[22] The contents of dimer derivatives in MSE were as follows: 1.5 mg/g 1, 2.2 mg/g 3, 28 mg/g 4, 101 mg/g 5, 16 mg/g 6, 48 mg/g 7, and 0.3 mg/g 8.

(S)-2-(1-(3,5-Dihydroxyphenyl)-2-(4-hydroxyphenyl)ethyl)-5-(4-hydroxyphenethyl)benzene-1,3-diol (10)

From 4. To a stirred solution of gnetin C (10.2 mg, 22.4 μmol) in ethyl acetate (2.0 mL) was added 10% Pd/C (16.1 mg). The mixture was stirred vigorously under a hydrogen atmosphere at room temperature (rt) for 22 h, filtered through a pad of Celite, and then concentrated. The residue was purified by preparative TLC (dichloromethane/methanol = 10:1, 3 developments) to give 10 (7.0 mg, 68%) as an amorphous solid: [α]D26 −66.8 (c 0.11, MeOH); IR (ZnSe) νmax: 3200, 2920, 2843, 1590, 1510, 1423, 1240, 1219, 1142, 1000, and 818 cm–1; 1H NMR (500 MHz, CD3OD): δ 6.95 (2H, d, J = 8.6 Hz), 6.91 (2H, d, J = 8.6 Hz), 6.65 (2H, d, J = 8.6 Hz), 6.55 (2H, d, J = 8.6 Hz), 6.46 (2H, brd, J = 2.2 Hz), 6.04 (2H, s), 6.02 (1H, t, J = 2.2 Hz), 4.65 (1H, dd, J = 9.5, 6.6 Hz), 3.53 (1H, dd, J = 13.4, 9.5 Hz), 3.26 (1H, dd, J = 13.4, 6.6 Hz), 2.74–2.66 (2H, m), and 2.59–2.55 (2H, m); 13C NMR (150 MHz, CD3OD): δ 158.5, 157.6, 156.3, 155.7, 149.6, 142.2, 134.6, 134.3, 130.9, 130.4, 116.2, 116.0, 115.4, 108.34, 108.26, 100.6, 43.4, 39.3, 38.5, and 38.1; HRESIMS m/z: 481.1620 [M + Na]+ (calcd for C28H26O6Na, 481.1627).

From 1. To a stirred solution of 1 (1.10 mg, 2.42 μmol) in methanol (0.5 mL) was added 10% Pd/C (2.5 mg). The mixture was stirred vigorously under a hydrogen atmosphere at rt for 9 h, filtered through a pad of Celite, and then concentrated. The residue was purified by preparative TLC (dichloromethane/methanol = 10:1, 5 developments) to give 10 (0.61 mg, 55%) as an amorphous solid whose spectral data were identical with those of an authentic sample derived from gnetin C: [α]D25 −60.8 (c 0.02, MeOH); HRESIMS m/z: 481.1627 (calcd for C28H26O6Na [M + Na]+, 481.1627).

Permethylation of Gnetin C (11)

To a stirred suspension of 4 (10.4 mg, 22.9 μmol) and potassium carbonate (61 mg, 441 μmol) in acetone (0.5 mL) was added methyl iodide (27.4 μL, 441 μmol), and the mixture was stirred under reflux for 2 h. More methyl iodide (27.4 μL, 441 μmol) and acetone (0.5 mL) were added and the reaction was further continued for 3 h. After cooling to rt, the reaction mixture was concentrated, diluted with dichloromethane/methanol (20:1), filtered through a pad of Celite, and then concentrated. The residue was purified by preparative TLC (n-hexane/ethyl acetate = 2:1, 3 developments) to give 11 (11.2 mg, 97%) as an amorphous solid: [α]D27 + 1.2 (c 0.41, CHCl3); IR (ZnSe) νmax: 2920, 2820, 1583, 1505, 1453, 1420, 1241, 1165, 1150, 1023, 821, and 741 cm–1; 1H NMR (500 MHz, CDCl3), see Table S3; 13C NMR (125 MHz, CDCl3): δ 161.6, 160.8, 159.4, 159.3, 156.9, 145.2, 140.3, 133.9, 130.0, 128.4, 127.8, 126.9, 126.8, 115.2, 114.1, 113.9, 105.5, 102.5, 100.4, 98.6, 92.8, 55.7, 55.4, 55.3, and 55.2; HRESIMS m/z: 547.2095 [M + Na]+ (calcd for C33H32O6Na, 547.2097).

(2S,3S)-3-(3,5-Dimethoxyphenyl)-4-methoxy-2-(4-methoxyphenyl)-2,3-dihydrobenzofuran-6-carbaldehyde (12)

To a stirred solution of 11 (11.0 mg, 21.0 μmol) in tetrahydrofuran (0.5 mL) and water (0.2 mL) was added dropwise a solution of OsO4 (ca. 5 μmol) in 2-methyl-2-propanol (50 μL) at rt. After stirring for few minutes, NaIO4 (43 mg, 0.20 mmol) was added by portions. After being stirred for an additional 3 h, the mixture was filtered through a pad of Celite. The filtrate was diluted with ethyl acetate, washed successively with aqueous Na2S, water, and brine, dried, and concentrated. The residue was purified by preparative TLC (n-hexane/ethyl acetate = 4:1, 3 developments) to give 12 (7.0 mg, 80%) as an amorphous solid: [α]D26 + 106.4 (c 0.23, CHCl3); IR (ZnSe) νmax: 2920, 2808, 1685, 1585, 1508, 1450, 1420, 1195, 1150, 1080, 1060, 820, and 745 cm–1; 1H NMR (500 MHz, CDCl3), see Table S3; 13C NMR (125 MHz, CDCl3): δ 191.6, 161.5, 160.9, 159.7, 157.3, 144.2, 139.1, 133.2, 126.8, 123.0, 114.1, 105.6, 105.3, 104.3, 98.7, 93.2, 55.7, 55.6, and 55.3; HRESIMS m/z: 443.1473 [M + Na]+ (calcd for C25H24O6Na, 443.1471).

ECD Calculation

ECD calculations of (7aS,8aS)-12 were initiated with a preliminary MMFF conformational search on a Spartan’10 program.[23] The lower energy conformers within 2.4 kcal/mol from the most stable were optimized at the DFT/B3LYP/6-31G(d) level using a Gaussian 09 program.[24] The resultant stable conformers within 2.0 kcal/mol, from the most stable ones, were further submitted to a DFT/B3LYP/6-311G(d,p) optimization considering the solvent effects. The ECD spectra of the obtained 10 conformers within 1.0 kcal/mol, from the most stable ones, were calculated at the TDDFT/B3LYP/6-311G(d,p) level. The first 55 singlet → singlet electronic transitions were considered. The calculated ECD spectra in Δε unit were obtained by using Gaussian band shapes with 0.15 eV half-width at 1/e of peak height.

ORAC Assay

The ORAC analysis has been performed following the method[25] reported by Shimizu et al. with minor modifications. In brief, a sample was dissolved directly in an ethanol/water mixture (50:50, v/v) and diluted with phosphate buffer (75 mM, pH 7.4). The sample solution (20 μL) was transferred to a 96-well microplate, and then fluorescein (200 μL, 94.4 nM) and AAPH (75 μL, 63.4 mM) were added and mixed. Phosphate buffer (75 mM, pH 7.4) was used as a blank solution, and Trolox as the standard solution. The assay was carried out on a FLUOstar OPTIMA plate reader utilizing fluorescence filters with 485 nm excitation and 520 nm emission. The antioxidant capacity of the sample was calculated as mmol Trolox equivalent per mol (mmol TE/mol).