Chemical Constituents from the Roots and Stems of Erycibe obtusifolia and Their In Vitro Antiviral Activity

• Introduction
• Results and Discussion
• Materials and Methods
• General experimental procedures
• Plant material
• Extraction and isolation
• Isolates
• Acid hydrolysis and sugar analysis of 12–15
• Antiviral assay
• Supporting information
• Acknowledgements
• References

Abstract

Three new quinic acid derivatives, 4-O-caffeoyl-3-O-sinapoylquinic acid methyl ester (1), 5-O-caffeoyl-4-O-syringoylquinic acid methyl ester (2), and 4-O-caffeoyl-3-O-syringoylquinic acid methyl ester (3), as well as four new coumarin glycosides, 7-O-(3-O-sinapoyl-β-D-glucopyranosyl)-6-methoxycoumarin (12), 7-O-(6-O-sinapoyl-β-D-glucopyranosyl)-6-methoxycoumarin (13), 7-O-(2-O-sinapoyl-β-D-glucopyranosyl)-6-methoxycoumarin (14), and 7-O-(6-O-syringoyl-β-D-glucopyranosyl)-6-methoxycoumarin (15), together with eight known compounds (4–11) were isolated from the roots and stems of Erycibe obtusifolia. Their structures were elucidated on the basis of spectroscopic analysis and chemical evidence. All the compounds were screened for their in vitro antiviral activity against respiratory syncytial virus with a cytopathic effect reduction assay. Among them, the di-O-caffeoyl quinates 8–11 displayed a potent in vitro anti-respiratory syncytial virus effect.

Introduction

Erycibe obtusifolia Benth. (Convolvulaceae) is mainly distributed in southern China such as Guangdong and Hainan provinces. The roots and stems of E. obtusifolia, named “Ding Gong Teng” in Chinese, are commonly used as a traditional Chinese medicine for the treatment of various rheumatoid diseases [1], [2]. Phytochemical investigations have revealed that the main chemical constituents of this plant are coumarins, quinic acid derivatives [3], and alkaloids [4]. Among them, scopolin exhibited significant anti-inflammatory activity [5] and baogongteng A showed muscarinic agonistic activity [6].

During the course of our ongoing program to search for natural antiviral agents from medicinal plants growing in southern China [7], [8], [9], [10], the n-BuOH soluble fraction of the ethanol extract from the roots and stems of E. obtusifolia was found to show an in vitro antiviral effect against respiratory syncytial virus (RSV) with an IC₅₀ value of 12.5 µg/mL. The result inspired us to investigate the chemical constituents of the active fraction, which led to the isolation of three new quinic acid derivatives (1–3) and four new coumarin glycosides (12–15), together with eight known compounds [methyl 3-O-(4″-hydroxy-3″,5″-dimethoxybenzoyl)-chlorogenate (4), methyl 5-O-caffeoyl-3-O-sinapoylquinate (5), methyl 5-O-caffeoyl-4-O-sinapoylquinate (6), 5-O-caffeoylquinic acid methyl ester (7), methyl 3,4-di-O-caffeoyl quinate (8), methyl 3,5-di-O-caffeoyl quinate (9), methyl 4,5-di-O-caffeoyl quinate (10), and 4,5-dicaffeoylquinic acid (11)] ([Fig. 1]). Furthermore, all the isolated compounds were evaluated for their in vitro anti-RSV activity. Herein, we report the isolation and structural elucidation of these new compounds, as well as the in vitro anti-RSV activity of all the isolated compounds.

Results and Discussion

Compound 1 was obtained as yellow oil. The HR-ESI-MS spectrum of 1 showed an [M – H]⁻ ion at m/z 573.1617 (calcd. for C₂₈H₂₉O₁₃, 573.1614), consistent with the molecular formula C₂₈H₃₀O₁₃. The UV spectrum of 1 showed the absorptions maxima at 204, 221, 242, and 328 nm. The IR spectrum implied the presence of hydroxyl (3423 cm⁻¹) and carbonyl (1702 cm⁻¹) groups, as well as an aromatic ring (1604 and 1516 cm⁻¹). The ¹H NMR spectrum of 1 showed proton signals due to a 1,3,4-trisubstituted benzene ring at δ _H 7.04 (1H, d, J = 1.9 Hz), 6.98 (1H, dd, J = 8.3, 1.9 Hz), and 6.74 (1H, d, J = 8.3 Hz), and a trans-disubstituted double bond at δ _H 7.48 (1H, d, J = 15.9 Hz) and 6.26 (1H, d, J = 15.9 Hz), suggesting the presence of a caffeoyl moiety. In addition, the ¹H NMR spectrum showed two aromatic protons at δ _H 6.95 (2H, s), two olefinic protons due to a trans-disubstituted double bond at δ _H 7.50 (1H, d, J = 15.9 Hz) and 6.46 (1H, d, J = 15.9 Hz), as well as proton signals due to two methoxy groups at δ _H 3.76 (6H, s), indicating the presence of a sinapoyl moiety in 1. The remaining signals for three oxygenated methine protons at δ _H 5.43 (1H, m), 5.02 (1H, dd, J = 6.2, 3.1 Hz), and 4.05 (1H, m), and four methylene protons at δ _H 2.25 (1H, dd, J = 13.1, 3.7 Hz), 2.12 (1H, dd, J = 13.3, 6.2 Hz), 1.97 (1H, dd, J = 13.1, 8.7 Hz), and 1.93 (1H, dd, J = 13.3, 2.3 Hz) indicated the existence of a quinic acid moiety. The ¹³C NMR spectrum of 1 also exhibited the characteristic signals due to caffeoyl, sinapoyl, and quinic acid moieties, respectively. Interpretation of the ¹H-¹H COSY, HSQC, and HMBC spectra of 1 led to the unambiguous assignment of all proton and carbon resonances ([Table 1]).

In the HMBC spectrum of 1, the correlations between H-3 (δ _H 5.43) of the quinic acid moiety and C-9′ (δ _C 165.8) of the sinapoyl moiety, and between H-4 (δ _H 5.02) of the quinic acid and C-9″ (δ _C 165.9) of the caffeoyl moiety were observed, which indicated that the sinapoyl and caffeoyl moieties were attached to the C-3 and C-4 positions of the quinic acid moiety, respectively. The additional methoxy group (δ _H 3.64; δ _C 51.7) was located at the C-7 position of the quinic acid based on the HMBC correlation between the methoxy protons (δ _H 3.64) and C-7 (δ _C 174.0) of quinic acid. Thus, the structure of 1 was determined to be 4-O-caffeoyl-3-O-sinapoyl quinic acid methyl ester.

Compound 2 was isolated as yellow oil. The molecular formula of 2 was established as C₂₆H₂₈O₁₃ on the basis of the [M – H]⁻ ion at m/z 547.1460 (calcd. for C₂₆H₂₇O₁₃, 547.1457) in its HR-ESI-MS spectrum. The UV and IR spectra of 2 also showed the characteristic absorptions for a quinic acid derivative. Similar to 1, the ¹H NMR spectrum of 2 showed the characteristic proton signals due to caffeoyl and quinic acid moieties. In addition, the ¹H NMR spectrum of 2 showed the signals for two aromatic protons at δ _H 7.24 (2H, s), and two methoxy protons at δ _H 3.80 (6H, s), which indicated the presence of a syringoyl moiety. The ¹H-¹H COSY, HSQC, and HMBC spectra of 2 allowed the assignment of all proton and carbon signals ([Table 1]). In the HMBC spectrum of 2, the correlations between H-4 (δ _H 5.02) of quinic acid and C-7′ (δ _C 165.1) of syringoyl, as well as between H-5 (δ _H 5.50) of quinic acid and C-9″ (δ _C 165.5) of the caffeoyl moiety suggested that the syringoyl and caffeoyl moieties were attached to the C-4 and C-5 positions of the quinic acid, respectively. Moreover, the HMBC correlation between the methoxy protons (δ _H 3.63) and C-7 (δ _C 173.4) of quinic acid indicated that the additional methoxy group was located at the C-7 position of the quinic acid. Hence, compound 2 was elucidated as 5-O-caffeoyl-4-O-syringoyl quinic acid methyl ester.

Compound 3 showed the same molecular formula as 2 by its HR-ESI-MS data (m/z 547.1460 [M – H]⁻, calcd. for C₂₆H₂₇O₁₃, 547.1457). The UV and IR spectra of 3 were very similar to those of 2, indicating that 3 was also a quinic acid derivative. Similar to 2, the ¹H and ¹³C NMR spectra of 3 showed characteristic proton and carbon signals due to the syringoyl, caffeoyl, and quinic acid moieties. Comparison of the NMR data of 3 with those of 2 revealed that they were almost identical, except for some difference observed at the C-3 and C-5 positions of the quinic acid moiety. In the HMBC spectrum of 3, correlations between H-3 (δ _H 5.55) of quinic acid and C-7′ (δ _C 164.8) of syringoyl, and between H-4 (δ _H 5.01) of quinic acid and C-9″ (δ _C 165.9) of the caffeoyl moiety were observed, which indicated that the syringoyl and caffeoyl moieties were attached to the C-3 and C-4 positions of quinic acid, respectively. The location of the additional methoxy group was also determined to be at the C-7 position of quinic acid by the same method. Therefore, the structure of 3 was identified as 4-O-caffeoyl-3-O-syringoyl quinic acid methyl ester.

Compound 12 was obtained as yellow oil. The HR-ESI-MS spectrum of 12 exhibited an [M – H]⁻ ion at m/z 559.1456 (calcd. for C₂₇H₂₇O₁₃, 559.1457), corresponding to the molecular formula C₂₇H₂₈O₁₃. The UV spectrum of 12 showed the absorptions maxima at 205, 227, and 334 nm. The IR spectrum showed the presence of hydroxyl (3444 cm⁻¹) and carbonyl (1701 cm⁻¹) groups, as well as an aromatic ring (1635, 1558, and 1515 cm⁻¹). The ¹H NMR spectrum of 12 revealed the presence of signals due to a cis-disubstituted double bond at δ _H 7.95 (1H, d, J = 9.5 Hz) and 6.33 (1H, d, J = 9.5 Hz), and two aromatic proton signals at δ _H 7.29 (1H, s) and 7.24 (1H, s), suggesting the existence of a 6,7-disubstituted coumarin core skeleton in 12 [12]. Furthermore, the ¹H NMR spectrum of 12 displayed two olefinic proton signals due to a trans-disubstituted double bond at δ _H 7.60 (1H, d, J = 15.9 Hz) and 6.58 (1H, d, J = 15.9 Hz), two aromatic protons at δ _H 7.04 (2H, s), and two methoxy protons at δ _H 3.82 (6H, s), indicating the presence of a sinapoyl moiety. In addition, the ¹H NMR spectrum displayed the signal of an anomeric proton [δ _H 5.31 (1H, d, J = 7.6 Hz)] for the sugar unit.

Acid hydrolysis of 12 afforded D-glucose, which was identified by HPLC analysis using an authentic sample as a reference. The β-configuration of D-glucose was determined based on the large ³ J _H1,H2 coupling constant (J = 7.6 Hz) of the anomeric proton. The ¹³C NMR and DEPT spectra of 12 revealed the presence of 27 carbon signals, including signals due to a 6,7-disubstituted coumarin, a sinapoyl, as well as a β-D-glucopyranosyl unit. Besides the above assigned signals, there is a methoxy group (δ _H 3.82; δ _C 56.1) in 12. With the aid of 2D NMR experiments, all the ¹H and ¹³C NMR signals of 12 were assigned as shown in [Table 2]. In the HMBC spectrum, the correlation between H-3′ (δ _H 5.09) of glucose and C-9″ (δ _C 166.3) of the sinapoyl moiety was observed, which indicated that the sinapoyl moiety was attached to the C-3′ position of glucose. Furthermore, the HMBC correlations between H-1′ (δ _H 5.31) of glucose and C-7 (δ _C 149.7) of coumarin, and between the methoxy protons (δ _H 3.82) and C-6 (δ _C 146.1) of coumarin were observed, suggesting that the glucose and methoxy groups were located at the C-7 and C-6 positions of the coumarin moiety, respectively. Thus, the structure of 12 was identified as 7-O-(3-O-sinapoyl- β-D-glucopyranosyl)-6-methoxycoumarin.

Compound 13 showed the identical molecular formula as 12 by its HR-ESI-MS data (m/z 559.1449 [M – H]⁻, calcd. for C₂₇H₂₇O₁₃, 559.1457). The UV and IR spectra of 13 also showed the typical absorptions of a coumarin skeleton. Acid hydrolysis of 13 also afforded D-glucose. Similar to 12, the ¹H and ¹³C NMR spectra of 13 also revealed the presence of sinapoyl, glucose, and 6-methoxycoumarin moieties. With the aid of ¹H-¹H COSY, HSQC, HMBC, and ROESY experiments, all the ¹H and ¹³C NMR signals of 13 were assigned as shown in [Table 2]. The HMBC correlations between Ha-6′ (δ _H 4.38)/Hb-6′ (δ _H 4.23) of glucose and C-9″ (δ _C 166.5) of the sinapoyl moiety suggested that the sinapoyl was located at the C-6′ position of glucose in 13. In addition, the HMBC correlation between H-1′ (δ _H 5.20) of glucose and C-7 (δ _C 149.5) of the 6-methoxycoumarin moiety were observed, which indicated that the glucose in 13 was also attached to the C-7 position of 6-methoxycoumarin. Therefore, the structure of 13 was determined to be 7-O-(6-O-sinapoyl-β-D-glucopyranosyl)-6-methoxycoumarin.

Compound 14 showed the same molecular formula as 12 and 13 by its HR-ESI-MS data. Comparison of the ¹H and ¹³C NMR data of 14 with those of 12 and 13 indicated that 14 possessed the same 6-methoxycoumarin, sinapoyl, and glucose moieties. The existence of D-glucose was further confirmed by HPLC analysis of the acid hydrolyzate. The linkages of different moieties could be assigned by an HMBC experiment. Thus, in the HMBC spectrum of 14, the correlation between H-2′ (δ _H 4.96) of glucose and C-9″ (δ _C 165.6) of the sinapoyl moiety was observed, suggesting the sinapoyl unit was attached to the C-2′ position of glucose. In addition, the location of the glucose was also determined by the HMBC correlation between H-1′ and C-7. Thus, the structure of 14 was identified as 7-O-(2-O-sinapoyl-β-D-glucopyranosyl)-6-methoxycoumarin.

The molecular formula of 15 was established as C₂₅H₂₆O₁₃ by its HRESIMS (m/z 533.1304 [M – H]⁻, calcd. for C₂₅H₂₅O₁₃, 533.1301). Similar to compounds 12–14, the UV and IR spectra of 15 showed the characteristic absorptions corresponding to a coumarin core skeleton. Acid hydrolysis of 15 also afforded D-glucose. Besides the proton signals due to a 6-methoxycoumarin moiety, the ¹H NMR spectrum of 15 displayed signals for two aromatic protons at δ _H 7.12 (2H, s) and two methoxy groups at δ _H 3.70 (6H, s), which indicated the presence of a syringoyl moiety. With the aid of 1D and 2D NMR experiments, all the ¹H and ¹³C NMR signals of 15 were assigned as shown in [Table 2]. The HMBC correlation between Ha-6′ (δ _H 4.62) of glucose and C-7″ (δ _C 165.5) of the syringoyl moiety indicated that the syringoyl was located at the C-6′ position of glucose. In the ROESY spectrum of 15, the correlation between H-1′ (δ _H 5.24) of glucose and H-8 (δ _H 7.16) of the 6-methoxycoumarin moiety was observed, which suggested that the glucose was attached to the C-7 position of the 6-methoxycoumarin moiety. Consequently, the structure of 15 was determined to be 7-O-(6-O-syringoyl-β-D-glucopyranosyl)-6-methoxycoumarin.

Compounds 4–11 were identified as methyl 3-O-(4″-hydroxy-3″,5″-di-methoxybenzoyl)-chlorogenate (4), methyl 5-O-caffeoyl-3-O-sinapoylquinate (5), methyl 5-O-caffeoyl-4-O-sinapoylquinate (6), 5-O-caffeoylquinic acid methyl ester (7), methyl 3,4-di-O-caffeoyl quinate (8), methyl 3,5-di-O-caffeoyl quinate (9), methyl 4,5-di-O-caffeoyl quinate (10), and 4,5-dicaffeoylquinic acid (11), respectively, by comparison of their spectroscopic data with those reported in the literature [3], [13], [14], [15], [16].

All the isolated compounds including quinic acid derivatives and coumarin glycosides were tested for their in vitro anti-RSV activity using the cytopathic effect (CPE) reduction assay. The MTT method was applied to measure the cytotoxicity of the compounds on HEp-2 cells. The selectivity index (SI) value calculated from the ratio of CC₅₀/IC₅₀ was used as an important parameter to evaluate the antiviral activity of the active compounds. As a result ([Table 3]), all the quinic acid derivatives 1–11 showed anti-RSV activity to a different extent, whereas coumarin glycosides 12–15 did not show an anti-RSV effect under the higher concentration of 50 µg/mL. Among the active compounds, di-O-caffeoyl quinates 8–11 displayed the strongest anti-RSV activity with much higher SI values than that of ribavirin. Although some studies concerned the anti-RSV activity of quinic acid derivatives, they only focused on the caffeoylquinic acids [7], [10], [17]. The present study provided the structural diversity of quinic acid derivatives, and the antiviral activity of sinapoylquinic acids and syringoylquinic acids were investigated for the first time.

Materials and Methods

General experimental procedures

Optical rotations were measured on a JASCO P-1020 polarimeter. UV spectra were measured on a JASCO V-550 UV/VIS spectrophotometer with a 1 cm length cell. IR spectra were recorded on a JASCO FT/IR-480 plus FT-IR spectrometer. ¹H, ¹³C, and 2D NMR spectra were recorded on Bruker AV-400 and AV-300 spectrometers. HR-ESI-MS data in the negative ion mode were obtained on an Agilent 6210 ESI/TOF mass spectrometer. For column chromatography, macroporous resin Diaion HP-20 (Mitsubishi Chemical Corporation), MCI gel (75–150 µm; Mitsubishi), ODS (50 µm; YMC), silica gel (300–400 mesh; Qingdao Marine Chemical Group Corporation), and Sephadex LH-20 (Pharmacia) were used. TLC analyses were carried out using precoated silica gel GF₂₅₄ plates (Yantai Chemical Industry Research Institute). Analytic high-performance liquid chromatography (HPLC) was performed on an Agilent chromatography equipped with a G1311C pump and a G1315D diode-array detector (DAD) with a Cosmosil 5C₁₈-MS-II column (4.6 × 250 mm, 5 µm). Preparative HPLC was carried out on an Agilent instrument equipped with a G1310B pump and a G1365D detector with a Cosmosil 5C₁₈-MS-II Waters column (20 × 250 mm, 5 µm).

Plant material

The roots and stems of Erycibe obtusifolia were collected in Haikou city, Hainan province of P. R. China, in June of 2010 and authenticated by Prof. Guang-Xiong Zhou (Institute of Traditional Chinese Medicine & Natural Products, Jinan University). A voucher specimen (No. 100605) was deposited at the Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou, P. R. China.

Extraction and isolation

The air-dried roots and stems of E. obtusifolia (20.0 kg) were powdered and extracted with 95 % (v/v) EtOH under reflux (2 × 200 L) twice (2 h each). The EtOH extract was concentrated under vacuum to afford a residue (963 g), which was suspended in H₂O and then partitioned successively with EtOAc and n-BuOH. After removing the solvent, the n-BuOH soluble fraction (273 g) was subjected to macroporous resin HP-20 column (15 × 60 cm) eluted with EtOH-H₂O mixtures (10 : 90; 30 : 70; 50 : 50; 70 : 30; 95 : 5, v/v, each 30 L). The 30 % EtOH eluate (77 g) was subjected to an MCI gel column (7 × 60 cm) using gradient mixtures of MeOH-H₂O (0 : 100; 10 : 90; 15 : 85; 20 : 80; 25 : 75; 30 : 70; 40 : 60; 50 : 50; 100 : 0, each 15 L) as the eluent to afford twelve fractions (Fr. 1–Fr. 12). Fr. 4 (8 g) was separated by a Sephadex LH-20 column (3 × 80 cm, MeOH) to yield five subfractions (Fr. 4-1–Fr. 4-5). Fr. 4-2 (3 g) was then subjected to an ODS column (4 × 30 cm) eluted with gradient mixtures of MeOH-H₂O (25 : 75; 30 : 70; 35 : 65, each 2 L) to afford five subfractions (Fr. 4-2-1–Fr. 4-2-5). Fr. 4-2-3 (820 mg) was then purified by preparative HPLC on a reversed-phase C₁₈ column (20 × 250 mm, 5 µm) using MeOH-H₂O (52 : 48, 6 mL/min) as the eluent to yield compounds 2 (22 mg), 4 (11 mg), and 5 (14 mg). Compounds 1 (24 mg), 3 (12 mg), and 6 (11 mg) were afforded from Fr. 4-2-4 (560 mg) by preparative HPLC using MeOH-H₂O (45 : 55, 6 mL/min) as the mobile phase. Fr. 4-4 (2 g) was separated by an ODS column (4 × 30 cm) with MeOH-H₂O (25 : 75; 30 : 70; 35 : 65, each 1.5 L) as the eluent to give four subfractions (Fr. 4-4-1–Fr. 4-4-4). Fr. 4-4-3 (312 mg) was subsequently purified by preparative HPLC on a reversed-phase C₁₈ column (20 × 250 mm, 5 µm) using MeOH-H₂O (45 : 55, 6 mL/min) as the eluent to yield compound 7 (10 mg). Compounds 8 (36 mg), 9 (47 mg), 10 (58 mg), and a mixture (108 mg) were obtained from Fr. 4-4-1 (660 mg) by preparative HPLC using MeOH-H₂O (47 : 53, 6 mL/min) as the mobile phase. The mixture was further purified by a silica gel column (2 × 45 cm) using CHCl₃-MeOH-H₂O (80 : 20 : 2) as the eluent to yield compound 11 (50 mg). Fr. 3 (6 g) was separated by a Sephadex LH-20 column (3 × 80 cm, MeOH) to give four subfractions (Fr. 3-1–Fr. 3-4). Fr. 3-3 (520 mg) was further separated by preparative HPLC on a reversed-phase C₁₈ column (20 × 250 mm, 5 µm) eluted with CH₃CN-H₂O (25 : 75, 6 mL/min) to yield compounds 12 (11 mg), 13 (10 mg), 14 (16 mg), and 15 (5 mg), respectively.

Isolates

4-O-caffeoyl-3-O-sinapoylquinic acid methyl ester (1): yellow oil; [α]_D ¹⁷: – 44.5 (c 0.20, MeOH); UV (MeOH) λ _max (log ε): 204 (4.01), 221 (4.07), 242 (4.03), 328 (4.26) nm; IR (KBr): ν _max 3423, 1702, 1604, 1516, 1457, 1283, 1158, 1118, 1029, 984, 809 cm⁻¹; HR-ESI-MS m/z: 573.1617 [M – H]⁻ (calcd. for C₂₈H₂₉O₁₃, 573.1614); ¹H NMR (DMSO-d ₆, 400 MHz) and ¹³C NMR (DMSO-d ₆, 100 MHz) data, see [Table 1].

5-O-caffeoyl-4-O-syringoylquinic acid methyl ester (2): yellow oil; [α]_D ¹⁷: – 89.4 (c 0.29, MeOH); UV (MeOH) λ _max (log ε): 218 (4.34), 293 (4.11), 332 (4.06) nm; IR (KBr): ν _max 3423, 1705, 1607, 1517, 1458, 1427, 1334, 1281, 1224, 1157, 1102, 1017, 979, 817, 763 cm⁻¹; HR-ESI-MS m/z: 547.1460 [M – H]⁻ (calcd. for C₂₆H₂₇O₁₃, 547.1457); ¹H NMR (DMSO-d ₆, 400 MHz) and ¹³C NMR (DMSO-d ₆, 100 MHz) data, see [Table 1].

4-O-caffeoyl-3-O-syringoylquinic acid methyl ester (3): yellow oil; [α]_D ¹⁷: – 32.8 (c 0.46, MeOH); UV (MeOH) λ _max (log ε): 218 (3.95), 290 (3.76), 333 (3.61) nm; IR (KBr): ν _max 3423, 1702, 1608, 1517, 1466, 1327, 1280, 1227, 1117, 1025, 986, 824, 755 cm⁻¹; HR-ESI-MS m/z: 547.1460 [M – H]⁻ (calcd. for C₂₆H₂₇O₁₃, 547.1457); ¹H NMR (DMSO-d ₆, 400 MHz) and ¹³C NMR (DMSO-d ₆, 100 MHz) data, see [Table 1].

7-O-(3-O-sinapoyl-β-D-glucopyranosyl)-6-methoxycoumarin (12): yellow oil; [α]_D ¹⁷: + 34.1 (c 0.27, MeOH); UV (MeOH) λ _max (log ε): 205 (4.32), 227 (4.16), 334 (4.17) nm; IR (KBr): ν _max 3444, 1701, 1635, 1558, 1515, 1458, 1427, 1281, 1157, 1118, 824, 586 cm⁻¹; HR-ESI-MS m/z: 559.1456 [M – H]⁻ (calcd. for C₂₇H₂₇O₁₃, 559.1457); ¹H NMR (DMSO-d ₆, 400 MHz) and ¹³C NMR (DMSO-d ₆, 100 MHz) data, see [Table 2].

7-O-(6-O-sinapoyl-β-D-glucopyranosyl)-6-methoxycoumarin (13): yellow oil; [α]_D ¹⁷: – 109.5 (c 0.23, MeOH); UV (MeOH) λ _max (log ε): 205 (4.25), 227 (4.07), 330 (4.09) nm; IR (KBr): ν _max 3421, 1702, 1620, 1563, 1513, 1459, 1424, 1381, 1335, 1281, 1157, 1115, 1064, 825, 585 cm⁻¹; HR-ESI-MS m/z: 559.1449 [M – H]⁻ (calcd. for C₂₇H₂₇O₁₃, 559.1457); ¹H NMR (DMSO-d ₆, 400 MHz) and ¹³C NMR (DMSO-d ₆, 100 MHz) data, see [Table 2].

7-O-(2-O-sinapoyl-β-D-glucopyranosyl)-6-methoxycoumarin (14): yellow oil; [α]_D ¹⁷: + 99.9 (c 0.14, MeOH); UV (MeOH) λ _max (log ε): 204 (4.09), 229 (3.94), 333 (4.02) nm; IR (KBr): ν _max 3432, 1720, 1696, 1643, 1562, 1516, 1459, 1423, 1342, 1284, 1257, 1122, 1076, 1017, 824, 593 cm⁻¹; HR-ESI-MS m/z: 559.1456 [M – H]⁻ (calcd. for C₂₇H₂₇O₁₃, 559.1457); ¹H NMR (DMSO-d ₆, 400 MHz) and ¹³C NMR (DMSO-d ₆, 100 MHz) data, see [Table 2].

7-O-(6-O-syringoyl-β-D-glucopyranosyl)-6-methoxycoumarin (15): yellow oil; [α]_D ¹⁷: – 75.7 (c 0.14, MeOH); UV (MeOH) λ _max (log ε): 205 (4.25), 222 (4.13), 283 (3.80), 342 (3.33) nm; IR (KBr): ν _max 3421, 1702, 1616, 1563, 1517, 1460, 1424, 1338, 1280, 1218, 1117, 1072, 862, 761, 583 cm⁻¹; HR-ESI-MS m/z: 533.1304 [M – H]⁻ (calcd. for C₂₅H₂₅O₁₃, 533.1301); ¹H NMR (DMSO-d ₆, 400 MHz) and ¹³C NMR (DMSO-d ₆, 100 MHz) data, see [Table 2].

Acid hydrolysis and sugar analysis of 12–15

Compounds 12–15 (each 2 mg) were hydrolyzed with 2 N HCl (5 mL) at 80 °C for 2 h, respectively. The reaction mixture was neutralized with an Amberlite IRA-400 column, and the eluate was concentrated. The residue was dissolved in pyridine (1 mL) and stirred with L-cysteine methyl ester hydrochloride (2 mg) for 1 h at 60 °C, and then O-tolyl isothiocyanate (5 µL) was added to the mixture and heated to 60 °C for an additional 1 h. The reaction mixture was analyzed by HPLC [column: Cosmosil 5C₁₈-MS-II, 4.6 × 250 mm, 5 µm; mobile phase: CH₃CN-0.05 % CH₃COOH in H₂O (25 : 75), 1.0 mL/min; detector: UV at 250 nm]. D-Glucose (t _R 16.4 min) was identified as the sugar moiety of compounds 12–15 by a comparison with authentic samples of D-glucose (t _R 16.4 min) and L-glucose (t _R 14.9 min) [11].

Antiviral assay

Cell and virus: Human larynx epidermoid carcinoma (HEp-2, ATCC CCL-23) cells, as well as RSV A2 (ATCC-VR-1540) and Long (ATCC-VR-26) strains were purchased from Medicinal Virology Institute, Wuhan University. HEp-2 cells were grown in Dulbecco modified Eagleʼs medium (DMEM; Gibco) supplemented with 10 % fetal bovine serum (FBS; Gibco) and antimicrobials (growth medium, GM), and cultured at 37 °C in a humidified atmosphere supplied with 5 % CO₂. For RSV propagation, RSV stock diluted using DMEM with 2 % FBS and antimicrobials (maintenance medium, MM) was added to the confluent HEp-2 cells and continually cultured in the incubator until the maximal RSV syncytia formation. Virus titers were determined by the 50 % tissue culture-infective dose (TCID₅₀) method. The virus stock was stored at −80 °C until use.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay: The cytotoxicity of the compounds on HEp-2 cells was tested with the MTT assay as described in the previous report [9]. Due to the quantitative limitation of the isolated compounds, the initial concentrations of tested samples were set to be 200 µg/mL.

CPE reduction assay: The CPE reduction assay was adopted to evaluate the antiviral activities of the compounds as described in the previous report [9]. Ribavirin (Sigma) was used as a positive control.

Supporting information

UV, IR, NMR, and HR-ESI-MS spectra of the new compounds are available in Supporting Information.

Acknowledgements

This work was supported financially by the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0965), the Joint Fund of NSFC-Guangdong Province (No. U0932004), the National Natural Science Foundation of China (No. 81072535), the Program for New Century Excellent Talents in University (No. NCET-11-0857), and the Science and Technology Planning Project of Guangzhou (No. 2011Y1-00017-2).

Conflict of Interest

There are no conflicts of interest among all authors.

^* These authors contributed equally to this work.

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Correspondence

• References

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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 14 Li B, Huang MJ, Li YL, Zeng GY, Tan JB, Zhou YJ. Antioxidant constituents of Sarcandra glabra (Thunb.) Nakai. J Shenyang Pharm Univ (Shenyang Yaoke Daxue Xuebao) 2009; 26: 900-903
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar

• Supplementary Material