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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Helv Chim Acta. Author manuscript; available in PMC 2016 June 7.
Published in final edited form as:
PMCID: PMC4896482
NIHMSID: NIHMS786724

Phenolic Derivatives from the Root Bark of Oplopanax horridus

Abstract

Four new phenolic derivatives, including two phenylpropanoid glycosides, one benzoate glycoside, and one lignan glycoside, together with one known glyceride, were isolated from the root bark of Oplopanax horridus. The structures of the new compounds were elucidated as 3-{4-[(6-O-acetyl-β-D-glucopyranosyl) oxy]-3,5-dimethoxyphenylpropanoic acid (1), (+)-[5,6,7,8-tetrahydro-7- (hydroxylmethyl)-10,11-dimehoxydibenzo[a,c][8]annulen-6-yl]methyl β-D-glucopyranoside (2), (+)-methyl 4-[6-O-{3-hydroxy-3-methyl-5-(1-methylpropyl)oxy]-5-oxopentanoyl}-4-O-(β-D-glucopyranosyl)-β-D-glucopyranosyl)oxy]-3-methoxybenzoate (3), and 2-methoxy-4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenyl 6-O-{3-hydroxy-3-methyl-5-[(1-methylpropyl)oxy]-5-oxopentanoyl-4-O-β-D-glucopyranosyl-β-D-glucopyranoside (4) on the basis of spectroscopic techniques including NMR and MS analyses. The known compound was identified as glycer-2-yl ferulate (5) by comparing its physical and spectral data with those reported in the literature.

Keywords: Oplopanax, Oplopanax horridus, lignan, phenylpropanoid

Introduction

Phenolic constituents, including phenylpropanoids and lignans, were recently isolated from the genus Oplopanax [14]. Phenylpropanoids have been shown to possess activities in protecting against biotic stress such as infections, wounding, pollutants, and UV irritation [5]. Lignans from natural resources exhibit anticancer [6], antioxidant, antiviral, and anti-inflammatory activities [7] [8]. A previous phytochemical investigation on the root bark of Oplopanax horridus led to the isolation and identification of some polyynes from the hydrophobic extract [9]. As part of our research work on bioactive compounds from O. horridus, we studied the hydrophilic extract and isolated three phenylpropanoid glycosides and one lignan glycoside along with one known glyceride. Herein, we report the isolation and structure elucidation of these phenolic compounds.

Results and Discussion

After a series of column chromatography (silica gel) and preparative HPLC, an 80%-EtOH extract of air-dried root bark of O. horridus yielded four new natural products 14 (for the names, see the Exper. Part) together with one known compound, glycer-2-yl ferulate (5). The structures of 15 (Fig. 1) were elucidated by their physicochemical properties and spectroscopic data. The known compound, glycer-2-yl ferulate (5), was also confirmed by comparing its physical and spectral data with those reported in the literature. Compound 1 was obtained as a white powder. Its molecular formula was deduced to be C19H26O11 from the quasi-molecular-ion peak at m/z 453.1359 ([M + Na]+, C19H26NaO11+; calc. 453.1373) in the HR-ESI mass spectrum. The UV absorption maxima displayed at 228 and 256 nm implied the presence of conjugated double bond system in this compound. The IR spectrum of 1 indicated the presence of an aromatic ring based on the absorptions at 1601, 1512, 1432 and 897 cm−1. Acidic hydrolysis of 1 yielded D-glucose, which was identified by GC-MS analysis. The 1H-NMR spectrum of 1 exhibited signals of two symmetrical aromatic H-atoms (δ(H) 7.34 (s), two symmetrical MeO groups (δ(H) 3.89 (s)), the Me group of AcO (2.50 (s)), as well as an anomeric H-atom (5.04 (d, J = 7.6)). The corresponding coupling constant (J = 7.6) suggested that the D-glucopyranosyl unit possessed β-configuration (Table 1). The 13C-NMR spectrum (Table 1) displayed signals of four different sp2 C-atoms (δ(C) 154.5, 140.0, 127.3, and 105.5) due to a symmetrical 1,3,4,5-tetrasubstituted aromatic ring, of six different O-bearing sp3 C-atoms (101.7, 77.8, 75.7, 75.6, 71.7, and 64.5) assignable to a D-glucosyl moiety, of one MeO group (57.0), of AcO C-atoms (168.1 and 27.0) as well as of a COOH group signal (172.3), and two more sp3 C-atom signals at 46.4 and 46.0.

Fig. 1
Structures of 1 – 5
Table 1
1H- and 13C-NMR Data (500 and 125 MHz, resp., in CD3OD) of 1a). δ in ppm, J in Hz

Based on HMQC and HMBC experiments, the downfield chemical-shift value of C(6′) from δ(C) 62.6 to 64.5 suggested that the AcO group was attached to C(6′) of the glucopyranosyl group, on the basis of the comparison with the chemical-shift value of C(6) of a glucopyranosyl moiety without any substituent [10]. However, the correlation between CH2(6′) (δ(H) 4.33 and 4.18) and the MeC=O C-atom (δ(C) 168.1) was not observed in the HMBC spectra. Even so, the experimental data were sufficient to elucidate the structure of 1, as depicted in Fig. 1.

Compound 2 was obtained as an amorphous powder with an optical rotation value of [α]D20=+18.1 (c = 0.5, MeOH), and its IR spectrum exhibited strong absorption bands due to a OH group at 3210 cm−1 and aromatic rings at 1622, 1570 and 1220 cm−1. Compound 2 displayed a [M + H]+ ion peak at m/z 491.2274 (C26H35O9+; calc. 491.2281) in the HR-ESI-MS spectra, indicating the molecular formula C26H34O9. Acidic hydrolysis of 2 also yielded D-glucose identified by GC/MS analysis. The 1H-NMR spectrum (Table 2) showed the signals of H-atoms at δ(H) 7.03 (dd, J = 8.1, 8.1, 1 H), 6.69 (dd, J = 8.0, 1.4, 1 H), 6.61 (ddd, J = 8.0, 8.0, 1.2, 1 H), and 6.53 (dd, J = 8.0, 2.0, 1 H), which were ascribable to an ortho-substituted benzene ring, of two aromatic H-atoms at 6.67 and 6.65, (2s) assignable to a 1,2,4,5-tetrasubstituted benzene ring. The H-atom signals of a glucosyl moiety were detected at δ(H) 3.30 – 3.80. Additionally, two more MeO signals at δ(H) 3.81 (s) and 3.82 (s) overlapped with these signals.

Table 2
1H- and 13C-NMR Data (500 and 125 MHz, resp., in CD3OD) of 2a). δ in ppm, J in Hz

The 13C-NMR profile displayed 26 signals, including those assignable to two aromatic rings (δ(C) 150.6, 148.8, 146.2, 145.5, 137.5, 133.9, 122.9, 122.8, 117.9, 115.9, 114.4, and 113.6), a glucose moiety (δ(C) 103.2, 78.2, 77.9, 75.0, 71.4, and 62.6), and two MeO groups at 56.4 and 56.6; Table 2). Based on the 1H-, 1H-COSY, HMQC, DEPT, and HMBC spectra analysis (Fig. 2), the aglycone was similar to 1,4-dibenzyl butane [11]. The coupling constant between the anomeric H-atom H-C(1″) and H-C(2″) (J = 7.8) indicated that the β-configuration of the glucose. The 1H-,1H-COSY spectrum provided the correlations CH2(7)/H-C(8) as well as H-C(8) and H-C(8′), indicated that C(8) and C(8′) were connected. Thus, structure of 2 was determined as shown in Fig. 1. The absolute configuration of this compound could not be determined due to the shortage of material.

Fig. 2
Key HMBCs (H →C) of 2 – 4

Compound 3 was obtained as a colorless gum with an optical rotation of [α]D20=+9.4 (c = 0.5, MeOH). The UV spectrum showed absorption maxima at 220 and 265 nm. The IR spectrum displayed absorption bands for OH (3409 cm−1), C=O (1720 cm−1) and aromatic ring (1597 and 1513 cm−1). The molecular-ion peak at m/z 707.2446 ([M + H]+, C31H47O18+, calc. 707.2762) in HR-ESI mass spectrum provided the molecular formula C31H46O18. Acidic hydrolysis of 3 gave D-glucose. The 1H-NMR spectrum of 3 displayed signals of three aromatic H-atoms at (δ(H) 7.63 (dd, J = 8.5, 2.0), 7.60 (d, J = 2.0), 7.21 (d, J = 8.5)) suggesting the presence of a 1,3,4-trisubstituted aromatic ring, and of a MeO group (3.91). Compared to oplopanpheside A [2], the NMR data of 3 revealed the presence of an additional sugar moiety (δ(C) 103.9, 75.1, 78.3, 75.3, 71.8, and 62.5), and of a sec-Bu group (δ(H) 3.80 – 3.83 (m, 1 H), 1.58 – 1.61 (m, 1 H), 1.44 – 1.47 (m, 1 H), 1.20 (d, J = 6.3, 3 H); and 0.94 (t, J = 7.5, 3 H); and δ(C) 79.1, 30.3, 21.5, and 9.9; Table 3). The sec-Bu group was linked via an O-atom to C(5″) of the 3-hydroxy-3-methylglutaryl moiety on the basis of the HMBC H-C(2[triple prime]′)/C(5″). The chemical shift and coupling constant values (δ(H) 4.33 (d, J = 7.8)) indicated the relative configuration of the additional sugar moiety as β. The HMBCs (Fig. 2) showed correlations between CH2(6′) of glucosyl (δ(H) 4.20, 4.45) and C(1″) (δ(C) 172.4) of the sec-butyl 3-hydroxy-3-methylglutaryl moiety revealed that C(6′) and C(1″) was connected via a glycosidic bond. Though a few relevant HMBCs were not observed, the downfield shift of C(6′) resonance of the glucosyl moiety from δ(C) 62.6 to 64.8 suggested that the sec-butyl 3-hydroxy-3-methylglutaryl moiety was attached to C(6′) of the glucopyranosyl group [11]. The downfield shift of C(4′) resonance of the glucosyl moiety from δ(C) 71.5 to 74.6 evidenced that one more sugar moiety was connected with C(4′). Accordingly, the structure of 3 was identified as depicted in Fig. 1.

Table 3
1H- and 13C-NMR Data (500 and 125 MHz, resp., in CD3OD) of 3 and 4a). δ in ppm, J in Hz

Compound 4 was purified as a colorless with a specific rotation [α]D20=+7.5 (c = 0.4, MeOH). The HR-ESI-MS displayed the [M + Na]+ ion peak at m/z 755.2755 (C33H48NaO18+, calc. 755.2738), indicating that molecular formula C33H48O18. The UV spectrum showed absorption maxima at 207 and 213 nm. The IR spectrum of 4 evidenced the presence of OH (3418 cm−1) and conjugated C=O (1711 cm−1) groups, and an aromatic ring (1601 and 1514 cm−1) groups. Compared with 3, signals of an additional pair of (E)-olefinic H-atoms (δ(H) 7.19 (d, J = 16.0), and 6.78 (d, J = 16.0)) were observed in the 1H-NMR spectrum of 4 (Table 3). Based on the combination of 1H-, 1H-COSY, HSQC, HMBC, and ROESY experiments, the NMR signals of 4 were assigned as compiled in Table 3. The HMBCs (Fig. 2) of H-C(7)/C(2), C(6), and C(9) indicated the presence of a ferulic acid moiety. The 3-hydroxy-3-methylglutaryl moiety was at C(6′) of the glucopyranosyl group based on downfield shift of C(6′) resonance of the glucosyl moiety from δ(C) 62.6 to 64.7 [11]. The connection of another sugar moiety and a sec-Bu group could be deduced as for compound 3. Thus, the structure of 4 was elucidated as shown in Fig. 1.

Compound 5 was isolated as a light yellowish gum and deduced as glycer-2-yl ferulate by spectroscopic analyses. The NMR (in (D6)DMSO) data were similar to those (in C5D5N and CD3OD) reported in [12] [13] because different solvents were used for NMR experiments [13].

Experimental part

General

HPLC-Grade MeOH and MeCN were purchased from Merck (Germany). The deionized H2O used for HPLC was purified by a Milli-Q purification system (Millipore, USA). All anal.-grade org. solvents were purchased from Uni-Chem (USA). The glassware was from Büchi (Switzerland). The glass columns packed with separation materials were purchased from Xiamei (Shanghai, P. R. China). Macroporous resin (pre-treated type, D-101) was purchased from Haiguang Chemical Industrial Company (Tianjin, P. R. China). Supercritical fluid extraction: supercritical fluid extractor SFT-250 (Supercritical Fluid Technologies, Inc., USA). TLC: Precoated silica gel GF254 plates (SiO2; Qingdao Haiyang Chemical Co., Ltd., Qingdao, P. R. China). Column chromatograpy (CC): SiO2 (100 – 200 and 200 – 300 mesh; Qingdao Haiyang Chemical Co., Ltd.) and reversed-phase C18 (RP-C18) SiO2 (40–63 mm; Alltech, USA). Anal. HPLC: Agilent 1100 liquid chromatograph with an Agilent Zorbax SB RP-C18 column (250 mm × 4.6 mm i.d., 5 μm; Alltech, USA). Prep. HPLC: Agilent 1100 liquid chromatograph with a Phenomenex Luna RP-C18 column (250 mm × 22 mm i.d., 5 μm). Optical rotations: PerkinElmer Model 341 polarimeter. UV Spectra: Beckman Coulter DU 640 spectrophotometer; λmax (log ε) in nm. IR Spectra: PerkinElmer Spectrum 100 FT-IR spectrometer; KBr pellets; V in cm−1. 1H- and 13C-NMR spectra: Bruker AV- 500 spectrometer (Bruker, Germany); δ in ppm rel. to Me4Si as internal standard, J in Hz. ESI-MS: Agilent 1100 series LC/MSD Trap VL mass spectrometer (Agilent, USA); in m/z. HR-ESI-MS: Agilent time-of-flight (TOF) mass spectrometer; in m/z.

Plant material

The dried root bark of O. horridus was collected and authenticated by C.-Z. W. from Chicago, IL, USA, in March, 2009. A voucher specimen (OH-20090312-1) has been deposited with the Laboratory of Quality Control, State Key Laboratory for Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao, P. R. China.

Extraction and Isolation

After remsoval of the volatile oil from the plant material by supercritical fluid extraction, pressurized liquid extraction and HPLC methods were applied to analyze the main components in order to optimize the best ratio between EtOH and H2O as the extraction solvent. The results suggested that the most effective solvent, 80% EtOH/H2O, should be selected as extraction solvent with pressure refluxing. Then, the 80%-EtOH/H2O extract was diffused into pure H2O (5 l) and extracted with petroleum ether (PE; 60 – 90 °C), AcOEt, and BuOH. The three org. solvents used for extraction were saturated with H2O, and the extractions were carried out three times with a volume of 5 l for each solvent. The BuOH extract was subjected to a macroporous resin column (EtOH/H2O 0:100, 40:60 and 95:5) to afford Fr. 1 (210 g), Fr. 2 (160g) and Fr. 3 (80g). A 105-g portion of Fr. 2 was then subjected to CC (SiO2; 100–200 mesh; CHCl3/MeOH 10:1 to 0:1) to give ten subfractions, Fr. 2.1–2.10. Fr. 2.3 (13 g) was separated by CC (RP-C18; SiO2; MeOH/H2O 40:60), then further by prep. HPLC (MeCN/H2O 25:75) to afford 1 (8 mg). Fr. 2.5 (18 g) was subjected to CC (SiO2; 200–300 mesh; CHCl3/MeOH 5:1), to afford five subfractions, Frs. 2.5.12.5.5. Fr. 2.5.2 (2 g) was purified by CC (RP-C18; SiO2; MeOH/H2O 40:60), then by prep. HPLC (MeCN/H2O 22:78) to afford 2 (12 mg). Fr. 2.5.3 (1.7 g) was separated by CC (RP-C18; SiO2; MeOH/H2O 38:62), then by prep. HPLC (MeCN/H2O 18:82) to afford 3 (5 mg) and 4 (4 mg). Fr. 2.5.4 (1.9 g) was subjected to CC (RP-C18; SiO2; MeOH/H2O 35:65), then to prep. HPLC (MeCN/H2O 16:84) to afford 5 (11 mg).

3-{4-[(6-O-Acetyl-β-D-glucopyranosyl)oxy]-3,5-dimethoxyphenyl}propanoic Acid (1)

Amorphous powder. [α]D20=+8.5 (c = 0.6, MeOH). UV (MeOH): 256 (2.76), 228 (2.21), 210 (1.43). IR: 3252, 2876, 1740, 1601, 1512, 1432, 897. 1H- and 13C-NMR: see Table 1. ESI-MS: 453 [M + Na]+. HR-ESI-MS: 453.1359 ([M + Na]+, C19H26NaO11+; calc. 453.1373).

[7-(Hydroxymethyl)-10,11-dimethoxy-5,6,7,8-tetrahydrodibenzo[a,c][8] annulen-6-yl]methyl β-D-Glucopyranoside (2)

Amorphous powder. [α]D20=+18.1 (c = 0.5, MeOH). UV (MeOH): 228 (1.88), 216 (1.43), 208 (0.78). IR: 3408, 2936, 1597, 1509, 1463, 1422, 1075, 827, 625. 1H- and 13C-NMR: see Table 2. ESI-MS: 491 [M + H]+. HR-ESI-MS: 491.2274 ([M + H]+, C26H35O9+; calc. 491.2281).

Methyl 4-[(6-O-{3-Hydroxy-3-methyl-5-[(1-methylpropyl)oxy]-5-oxopentanoyl}-4-O-(β-D-glucopyranosyl)-β-D-glucopyranosyl)oxy]-3-methoxybenzoate (3)

Colorless gum, [α]D20=+9.4 (c = 0.5, MeOH). UV (MeOH): 194 (0.40), 220 (2.67), 265 (1.43), 358 (0.03). IR: 3459, 2926, 1710, 1597, 1519, 1468, 1430, 1383, 1239, 1128, 829, 667. 1H- and 13C-NMR: see Table 3. ESI-MS: 729 [M + Na]+. HR-ESI- MS: 707.2446 ([M + H]+, C31H47O18+; calc. 707.2762).

2-Methoxy-4-[(1E)-3-methoxy-3-oxoprop-1-en-1-yl]phenyl 6-O-{3-Hydroxy-3- methyl-5-[(1-methylpropyl) oxy]-5-oxopentanoyl}-4-O-β-D-glucopyranosyl-β-D- glucopyranoside (4)

Colorless gum, [α]D25=+7.5 (c = 0.4, MeOH). UV (MeOH): 193 (0.90), 207 (2.63), 213 (2.72), 218 (2.65), 244 (1.56), 298(1.74), 327 (1.94). IR: 3418, 2930, 1711, 1631, 1601, 1514, 1435, 1384, 1275, 1219, 1181, 1128, 1075, 1027, 893, 765. 1H- and 13C-NMR: see Table 3. ESI-MS: 755 [M + Na]+. HR-ESI-MS: 755.2755 ([M + Na]+, C33H48NaO18+; calc. 755.2738).

Glycer-2-yl Ferulate (=1,3-Dihydroxypropan-2-yl (2E)-3-(4-Hydroxy-3- methoxyphenyl)prop-2-enoate; 5)

Light yellowish gum. [α]D20=+52.1 (c = 0.5, MeOH). UV (MeOH): 264 (2.75), 226 (1.84), 210 (0.91). IR: 3396, 2965, 1695, 1634, 1593, 1518, 1376, 1273, 1180, 1127, 1033, 975, 841. 1H-NMR ((D6)DMSO, 500 MHz): 7.52 (d, J = 15.8, H-C(7)); 7.29 (d, J = 1.5, H-C(2)); 7.07 (dd, J = 7.8, 1.5, H-C(6)); 6.80 (d, J = 7.8, H-C(5)); 6.43 (d, J = 15.8, H-C(7)); 5.20~5.22 (m, H-C(2′)); 3.80 (s, MeO); 3.50 (d, J = 6.0, H-C(1′, 3′)). 13C-NMR ((D6)DMSO, 125 MHz): 166.3 (C(9)); 149.3 (C(3)); 147.9 (C(4)); 144.5 (C(7)); 125.6 (C(1)); 122.9 (C(8)); 115.5 (C(5)); 115.2 (C(6)); 111.1 (C(2)); 73.1 (C(2′)); 71.3 (C(1′, 3′)); 56.4 (MeO). ESI-MS: 269 ([M + H]+, C13H17O6+).

Acidic Hydrolysis

Compounds 1 (1.4 mg), 2 (1.6 mg), and 3 (1.1 mg) were hydrolyzed with 2M CF3COOH (1 ml) in a sealed glass tube with screw cap, which was filled with N2 at 100° for 2 h, resp. The hydrolyzed soln. was evaporated to dryness under 50°, and then MeOH (2 ml) was added for further evaporation and complete removal of CF3COOH. The hydrolysate was used for derivatization.

Sugar analysis

The stock soln. of standard monosaccharides (1 ml) was treated with NH2OH·HCl/pyridine soln. (1 ml) in a sealed glass tube at 90° for 30 min. Ac2O (1 ml) was added, and heating was continued for another 30 min before the soln. was cooled to r.t. The cooled soln. was evaporated to dryness under reduced pressure at 50°. The residue was dissolved in dry MeOH (2 ml). The mixture was filtered through a 0.45-μm syringe filter (Agilent Technologies) prior to injection into the GC/MS system. The hydrolysate was reacted with NH2OH·HCl and Ac2O to form the derivatives by the procedures mentioned above for sugar determination. GC/MS was performed on an Agilent 6890 gas chromatograph coupled with an Agilent 5973 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). A HP-5MS capillary column (30 m × 0.25 mm, i.d.) coated with 0.25 μm film 5% phenylmethylsiloxane was used for separation. The column temp. was set at 1758 and held for 7 min, then programmed at 5°/min to 1858, held for 5 min, and then at 4°/min to 230°. Split injection (2 ml) with a split ratio of 1:50 was applied. High purity He was used as carrier gas with a flow rate of 1.0 ml min−1. The mass spectrometer was operated in electron-impact (EI) mode, the scan range was 40–550 amu, the ionization energy was 70 eV, and the scan rate was 2.89 s per scan. The inlet and ionization source temp. were 250 and 280°, resp.

Acknowledgments

This work was supported by the NIH/NCCAM (AT004418 and AT005362 to C.-S. Y.), the University of Macau (UL015/09-Y1 to S.-P. L), the Fundamental Research Funds for the Central Universities of Central South University (1681–7608040003 to W.-H. H.) grants and the National Natural Scientific Foundation of China (No. 31400306 to W.-H. H.).

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