A New Neolignan Glycoside from the Leaves of Acer truncatum

Abstract

A new neolignan glycoside, (7R,8R)-7,8-dihydro-9′-hydroxyl-3′-methoxyl- 8-hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′-benzofuranpropanol 9′-O-β-D- glucopyranoside (1) was isolated from the leaves of Acer truncatum along with (7R,8R)-7,8-dihydro-9′-hydroxyl-3′-methoxyl-8-hydroxymethyl-7-(4-O-α-L-rhamno- pyranosyloxy-3-methoxyphenyl)-1′-benzofuranpropanol (2), schizandriside (3), lyoniresinol (4), berchemol (5), (-)-pinoresinol-4-O-β-D-glucopyranoside (6), hecogenin (7), chlorogenic acid (8) and neochlorogenic acid (9). Their structures were elucidated on the basis of extensive spectroscopic data. The absolute configuration of compounds 1 was established by its CD spectrum. The antibacterial activities of compounds 1-7 were evaluated.

Introduction

The genus Acer belongs to the family Aceraceae and there are more than 150 Acer flora species in China [1]. The roots of Acer truncatum (also known by the common names Shantung, Painted or Purple-blow Maple) have been used as folk medicine to treat lumbago and its leaves have been used to prepare a sanitary tea [2]. In our previous reports on the phytochemical investigation of A. truncatum,we have described flavonoid glycosides [3], which had strong activity in thrombus, phenylpropanoids [4], egastigmanes [4] and sesquiterpenes [4]. During our ongoing investigations into the chemical constituents of this plant, the new neolignan glycoside (7R,8R)-7,8-dihydro-9′-hydroxyl- 3′-methoxyl-8-hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′-benzofuranpropanol 9′-O-β-D- glucopyranoside (1) and eight known compounds: (7R,8R)-7,8-dihydro-9′-hydroxyl-3′-methoxyl-8- hydroxymethyl-7-(4-O-α-L-rhamnopyranosyloxy-3-methoxyphenyl)-1′-benzofuranpropanol (2), schi-

zandriside (3), lyoniresinol (4), berchemol (5), (-)-pinoresinol-4-O- β-D-glucopyranoside (6), hecogenin (7), chlorogenic acid (8) and neochlorogenic acid (9) were isolated from a water extract of A. truncatum leaves. The identification of the known compounds was supported by comparison with published data of related compounds [5,6,7,8,9,10,11,12]. Compounds 1-7 were evaluated for their antibacterial activities against Escherichia coli, Staphylococcus aureus, Micrococcus luteus and Bacillus cereus.

Results and Discussion

Compound 1, a colorless amorphous powder, showed a [M-H]⁻ ion peak at m/z 521.2039 in the negative ion HRESI-MS, indicating the molecular formula C₂₆H₃₄O₁₁. Its ¹H-NMR spectrum showed three ABX type phenyl protons at δ_H 6.76 (1H, d, J = 8.2 Hz, H-5), 6.81 (1H, d, J = 8.5 Hz, H-6), 6.93 (1H, d, J = 1.6 Hz, H-2)], two singlet signals at δ_H 6.72 (1H, s, H-2′) and 6.75 (1H, s, H-6′), two methoxy signals at δ_H 3.79 (3H, s) and 3.82 (3H, s), and two C₃ units at δ_H 5.48 (1H, d, J = 6.3 Hz, H-7), 3.45 (1H, m, H-8), 3.74 (1H, m, Ha-9), 3.80 (1H, m, Hb-9), and at δ_H 2.65 (2H, br t, J = 7.4 Hz, H-7′), 1.88 (2H, m, H-8′), 3.51 (1H, m, Ha-9′) and 3.92 (1H, m, Hb-9′). Furthermore, the ¹H- and ¹³C-NMR spectral data (Table 1) indicated the presence of a β-glucopyranosyl moiety (J_{1″, 2′′} = 7.8 Hz), which was in accordance with an [M -H -162]^- peak observed at m/z 359 in the negative FAB-MS spectrum. In addition to two methoxyl carbons and the glucopyranosyl group signals, 18 skeletal carbon resonances appeared in the ¹³C-NMR spectrum (Table 1). Significant HMBC correlations were also observed between H-7/C-4′ and H-8/C-5′. These spectral features indicated that 1 was a 7-aryl-8-hydroxymethyl-7,8-dihydrobenzofuranoid-type neolignan formed by two phenylpropanoid units [13,14,15,16,17]. The two methoxyl groups were located at C-3 and C-3′ and the β -glucopyranosyl group was connected at C-9′, based on the HMBC and ROESY correlations (Figure 1). The ¹H- and ¹³C-NMR data of 1 were almost equivalent to those of glochidioboside [13], however, an obvious NOE was observed for H-7 [δ_H 5.48 (d, J = 6.3 Hz)] on irradiation at H-8 [δ_H 3.45 (m)] in the NOE experiment of 1, which suggested that the substituents at C-7 and C-8 were is a cis- relative configuration. The absolute stereochemistry at C-7 and C-8 were assigned to be both R, on the basis of position Cotton effects at 243 nm and 294 nm and negative ones at 260 nm in its CD spectrum [17], Consequently, the structure of 1 was elucidated as (7R,8R)-7,8-dihydro-9′-hydroxyl-3′-methoxyl-8- hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′-benzofuranpropanol 9′-O-β-D-glucopyranoside (1, Figure 1).

Figure 1. Selected HMBC and ROESY correlations of 1.

Figure 1. Selected HMBC and ROESY correlations of 1.

Table 1. The ¹H- and ¹³C-NMR Data of 1 (in CD₃OD).

Position	δH (J in Hz)	δC	Position	δH (J in Hz)	δC
1	-	134.8s	5′	-	129.9s
2	6.93 (d, 1.6)	110.6d	6′	6.75 (s)	118.0d
3	-	149.0s	7′	2.65 (t, 7.4)	32.9t
4	-	147.4s	8′	1.88 (m)	32.9t
5	6.76 (d, 8.2)	116.1d	9′	3.51 (m) 3.92 (m)	70.0t
6	6.81 (dd, 1.6, 8.2)	119.7d	1″	4.25 (d, 7.8)	104.4d
7	5.48 (d, 6.3)	88.9d	2″	3.23 (m)	75.1d
8	3.45 (m)	55.3d	3″	3.34 (m)	78.1d
9	3.80, 3.74 (2H, m)	65.0t	4″	3.32 (m)	71.6d
1′	-	136.8s	5″	3.26 (m)	77.8d
2′	6.72 (s)	114.2d	6″	3.67 (dd,11.7, 3.9) 3.87 (br.s)	62.7t
3′	-	145.1s	3-OMe	3.79 (s)	56.4q
4′	-	147.4s	3′-OMe	3.82 (s)	56.8q

Table 1. The ¹H- and ¹³C-NMR Data of 1 (in CD₃OD).

Position	δH (J in Hz)	δC	Position	δH (J in Hz)	δC
1	-	134.8s	5′	-	129.9s
2	6.93 (d, 1.6)	110.6d	6′	6.75 (s)	118.0d
3	-	149.0s	7′	2.65 (t, 7.4)	32.9t
4	-	147.4s	8′	1.88 (m)	32.9t
5	6.76 (d, 8.2)	116.1d	9′	3.51 (m) 3.92 (m)	70.0t
6	6.81 (dd, 1.6, 8.2)	119.7d	1″	4.25 (d, 7.8)	104.4d
7	5.48 (d, 6.3)	88.9d	2″	3.23 (m)	75.1d
8	3.45 (m)	55.3d	3″	3.34 (m)	78.1d
9	3.80, 3.74 (2H, m)	65.0t	4″	3.32 (m)	71.6d
1′	-	136.8s	5″	3.26 (m)	77.8d
2′	6.72 (s)	114.2d	6″	3.67 (dd,11.7, 3.9) 3.87 (br.s)	62.7t
3′	-	145.1s	3-OMe	3.79 (s)	56.4q
4′	-	147.4s	3′-OMe	3.82 (s)	56.8q

Biological activity

Compounds 1-7 were tested for their antibacterial activities against Escherichia coli, Staphylococcus aureus, Micrococcus luteus and Bacillus cereus using the paper disk method. All stock cultures were grown on tryptic soy agar plates. Test strains were transferred to fresh tryptic soy broth before use and a disk containing only DMSO was used as negative control. The compounds were found to be inactive at concentrations of up to 50 µg/disk, except for schizandriside (3), which showed moderate antibacterial activity, affording inhibitory zone sizes of 11 mm against Staphylococcus aureus at a concentration of 2 μg/disk.

Conclusions

Nine phenolic constituents including a new neolignan glycoside were isolated from the leaves of Acer truncatum. Their structures were established on the basis of 1D- and 2D-NMR experiments, CD data and comparison with literature values. The antibacterial activities of pure compounds 1-7 was tested against four microbial species. Only schizandriside (3) showed moderate antibacterial activity against S. aureus.

Experimental

General

FAB mass spectra were obtained on a VG Auto spec-3000 spectrometer and high-resolution ESI mass spectra were recorded on an API Qstar Pulsar instrument. 1D- and 2D-NMR experiments were performed on Bruker AM-400 and DRX-500 instruments with TMS as internal standard. Chemical shifts (δ ) were expressed in ppm with reference to the solvent signals; coupling constants (J) are given in Hertz (Hz). IR spectra were taken in KBr on a Bio-Rad FTS-135 infrared spectrophotometer. Optical rotations were measured in a JASCO DIP-370 digital polarimeter. UV spectra were measured using a Shimadzu UV-2401PC spectrophotometer. CD spectra were run on a JASCO J-810 instrument. Column chromatography (CC) was performed using 200-300 mesh silica gel (Qingdao Marine Chemical Inc., Qingdao, P.R. China), on silica gel H (10-40 μm, Qingdao Marine Chemical Inc.) and Lichroprep RP-18 (43-63 μm, Merck).

Plant material

Leaves of A. truncatum were collected in Kunming, Yunnan province, P. R. China, in August 2004. The plants were identified by Prof. Ting-Zhi Xu, Kunming Institute of Botany, Chinese Academy of Science.

Extraction and isolation

Air-dried leaves of A. truncatum (20 kg) were extracted with H₂O. The extract was evaporated in vacuo to give a black-brown gum, which was applied to ADS-7 porous resin and divided into four fractions: H₂O fraction, 30% EtOH fraction, 70% EtOH fraction, and 90% EtOH fraction. The 30% EtOH fraction (256 g) was subjected to CC (SiO₂, CHCl₃ /MeOH 9:1→7:3) to afford fractions Fr.1-10, as judged by TLC. Fr. 2 (20 g) was further purified by CC (first SiO₂, petroleum ether/AcOEt, then RP-18 gel) to afford 7 (135 mg). Fr. 3 (12 g) was subjected to CC (SiO₂, petroleum ether/AcOEt) to afford 5 (20 mg). Fr. 4 (18 g) was subjected to CC (SiO₂, petroleum ether/AcOEt ) to yield 4 (37 mg). Fr. 5 (19 g) was subjected to CC (SiO₂, CHCl₃/AcOEt) to yield 8 (13 mg) and 9 (10 mg). Fr. 7 (23 g) was subjected to CC (SiO_2, petroleum ether/AcOEt) to afford 6 (226 mg). Fr. 9 (20 g) was subjected to CC (first, SiO₂; CHCl₃/MeOH, then RP-18 gel) to afford 1 (37 mg), 2 (65 mg) and 3 (171 mg).

(7R,8R)-7,8-dihydro-9′-hydroxyl-3′-methoxyl-8-hydroxymethyl-7-(4-hydroxy-3-methoxyphenyl)-1′- benzofuranpropanol 9′-O-β-D-glucopyranoside (1): Colorless amorphous powder; ＝ –9.99 (MeOH, c 0.69); UV λ_max (log ε): 206 (4.76), 281 (3.82) nm; Negative FAB-MS: m/z 521 [M-H]^-, 359 [M-H-162]^-; HR-ESI-MS: m/z 521.2039 [M-1]^- (calcd for C₂₆H₃₃O₁₁ 521.2022); IR ν_max (KBr): 3422, 2935, 2880, 1608, 1518, 1499 cm^-1; CD (c = 2.08′10^-5 mol·L^-1 in MeOH): [θ]₂₉₄ + 1.7 × 10³, [θ]₂₆₀ – 0.7 × 10³, [θ]₂₄₃ + 6.7 × 10³.; ¹H- and ¹³C- NMR data see Table 1.

Antibacterial and antifungal activity [4]

Antibacterial activity was tested by the disk diffusion method with minor modifications. E. coli, S. aureus, M. luteus and B. cereus were subcultured in tryptic soy broth (TSB), incubated for 18 h at 37 ′C and then the bacterial cells were suspended, according to the McFarland protocol, in saline solution to produce a suspension of about 10^-5 CFU·mL^-1. An aliquot of this suspension (15 µL) was mixed with sterile tryptic soy agar (TSA, 15 mL) at 40 ′C and poured onto an agar plate in a laminar flow cabinet. Each tested compound was dissolved in DMSO and added to a paper disk (6 mm diameter) that was dried and placed on the agar plate containing the bacterial cells (5 samples/disk plus control). A disk containing only DMSO was used as negative control. The susceptibility of the bacteria to the test compounds was determined by the formation of an inhibitory zone after 18 h of incubation at 37 ′C. Experiments were run in triplicate, and the results were determined as mean values of the three measurements.