A novel norneolignan glycoside and four new phenolic glycosides from the stems of Viburnum fordiae Hance

Introduction

Some trees, including their stems and branches, have been considered to be a rich source of bioactive secondary metabolites. Lignans and phenolics, as the more abundant constituents, have attracted increasing attention due to their health-promoting effects and diverse biological activities including antihyperglycemic, anti-inflammatory, antioxidant, antitumor, antimicrobial and neuroprotective properties (Willför et al. 2003, 2004a,b; Iwai et al. 2006; Gao et al. 2008; Donoso-Fierro et al. 2009; Liimatainen et al. 2012; Smeds et al. 2012; Si et al. 2013; In et al. 2014; Latva-Mäenpää et al. 2014; Si et al. 2016, 2017). These constituents have been reported in the chemical investigations on Viburnum (Adoxaceae) plants (Zhu et al. 2006; In et al. 2015). It is remarkable that a novel phenolic glycoside, exhibiting potent antioxidant activity, has been isolated from the leaves of Viburnum fordiae Hance (Wu et al. 2008).

Viburnum fordiae, a small deciduous tree that can grow up to 5 m, is widely distributed in the south of China. It is a highly ornamental tree because of its spherical crown, dense branches and leaves, showy white tufted flowers and crimson drupe-type fruits. The fruits can be either eaten raw or used for making wine in China (Wang 2009; Wu et al. 2012; Yang 2016). The roots and leaves, as well-known traditional Chinese medicine, are for the treatment of rheumatic arthralgia and allergic dermatitis (Zhonghuabencao Editorial Board 1999). Also, we know that the stems are employed to cure acute toothache in folk medicine (Cai et al. 2004). Due to the lack of relevant reports on their biologically or pharmacologically active substances, V. fordiae has not been explored commercially in the healthcare, food and drug development fields. Therefore, the aim of the present study was to investigate the chemical constituents from the stems of V. fordiae. The isolation and structural elucidation of a novel norneolignan glycoside (1), categorized as an unusual 7-noraryl-4′,7-epoxy-8,5′-neolignan glycoside (Zhuang et al. 2014), four new phenolic glycosides (2–5) and a known neolignan glycoside (6), which was found for the first time in Viburnum, will be described.

Materials and methods

Extraction and isolation:

The extraction and fractionation procedures are described in Figure 1. Air-dried and finely pulverized stems of V. fordiae Hance (22.9 kg) were extracted with EtOH (95% v/v) three times, 2 h for each. The filtrated and concentrated (in vacuo) extract was dissolved in water and then successively partitioned with petroleum ether, ethylacetate and n-butanol to yield four main fractions. The n-butanol fraction (711.0 g) was subjected to the macroporous resin HPD-100 column to obtain two corresponding portions with a successive elution using H₂O and 95% EtOH. The portion (474.2 g) eluted by 95% EtOH was then applied to silica gel CC (ø 12×100 cm), eluting with a gradient of CHCl₃-MeOH (100:0→100:3→100:8→100:12 →100:20→100:35→100:50, v/v, 30 l of each), to give seven fractions (F₁–F₇).

Figure 1:

Extraction and fractionation procedures of chemical constituents from the stems of Viburnum fordiae.

Fraction F₄ (17.4 g) was chromatographed over silica gel (ø 4.2×55 cm) and eluted with CHCl₃-MeOH (20:1→15:1→10:1→5:1→1:1, v/v, 2.5 l of each). The eluted solutions were monitored by TLC and integrated as three fractions (F_4-1–F_4-3). F_4-2 (9.1 g) was subjected to the open column (ø 5.2×55 cm) packed with MCI gel, eluting with MeOH-H₂O (10:90→20:80→40:60→60:40→80:20, v/v, 2 l of each), to yield five fractions (F_4-2-1–F_4-2-5). F_4-2-3 (1.5 g) was applied to Sephadex LH-20 CC (ø 2.2×150 cm) and eluted with CHCl₃-MeOH (2:1, v/v, 0.6 l) 3 times. After the combination, the concentrated portion was reapplied to Sephadex LH-20 CC (ø 4.0×150 cm, MeOH-H₂O, 20:80, v/v, 3.6 l) to yield four fractions (F_4-2-3-1–F_4-2-3-4). F_4-2-3-2 (516 mg) was chromatographed over Sephadex LH-20 (ø 2.2×150 cm, CHCl₃-MeOH, 2:1, v/v, 0.6 l) and ODS (ø 3.2×50 cm, MeOH-H₂O, 10:90→20:80→40:60→55:45→70:30, v/v, 0.8 l of each), respectively, to give the mixtures (3 and 4). The mixtures were separated by preparative HPLC (MeOH-H₂O, 25:75, v/v, flow rate of 2 ml min⁻¹) to afford compounds 3 (19 mg, t_R 53.7 min) and 4 (12 mg, t_R 51.8 min). From F_4-2-3-3 (312 mg), compound 2 (24 mg, t_R 59.9 min) was obtained by preparative HPLC (MeOH-H₂O, 25:75, v/v, flow rate of 2 ml min⁻¹) after being subjected to Sephadex LH-20 (ø 2.2×150 cm, CHCl₃-MeOH, 2:1, v/v, 0.6 l) and ODS CC (ø 3.2×50 cm, MeOH-H₂O, 10:90→20:80→40:60→55:45→70:30, v/v, 0.8 l of each), respectively.

Fraction F₅ (112.8 g) was further divided into five fractions (F_5-1–F_5-5) through MCI gel CC (ø 5.2×55 cm, MeOH-H₂O, 5:95→ 15:85→35:65→55:45→70:30, v/v, 3 l of each). F_5-1 (3.4 g) was applied to ODS CC (ø 3.2×50 cm, MeOH-H₂O, 5:95→10:90→ 20:80→30:70→40:60, v/v, 0.8 l of each) to give fractions F_5-1-1–F_5-1-4. F_5-1-2 (581 mg) was purified over Sephadex LH-20 (ø 2.2×150 cm) with MeOH (0.4 l) 3 times to yield compound 6 (135 mg). F_5-3 (42.0 g) was subjected to MCI gel (ø 5.2×55 cm, MeOH-H₂O, 15:85→25:75→40:60→60:40→80:20, v/v, 2.5 l of each) and Sephadex LH-20 (ø 2.2×150 cm, CHCl₃-MeOH 2:1, v/v, 0.6 1), respectively, to afford two disrelated portions F_5-3-1 and F_5-3-2. From F_5-3-2 (5.6 g), compound 5 (31 mg) was purified by Sephadex LH-20 (ø 1.5×150 cm, MeOH, 0.2 l) after being chromatographed over ODS (ø 3.2×50 cm, MeOH-H₂O, 5:95→10:90→20:80→30:70→40:60, v/v, 0.8 l of each) and Sephadex LH-20 (ø 2.2×150 cm, MeOH-H₂O, 25:75, v/v, 1.2 l), respectively. Another fraction F_5-5 (17.4 g) was firstly chromatographed over ODS CC (ø 3.2×50 cm, MeOH-H₂O, 5:95→10:90→15:85→20:80→40:60, v/v, 0.8 l of each) to give F_5-5-1–F_5-5-3. The main fraction F_5-5-2 was then subjected to Sephadex LH-20 (ø 2.2×150 cm, MeOH, 0.4 l) and ODS CC (ø 3.2×50 cm, MeOH-H₂O, 5:95→10:90→15:85→20:80, v/v, 0.8 l of each), respectively, to produce five fractions (F_5-5-2-1–F_5-5-2-5) . Compound 1 (61 mg) was finally obtained from the fraction F_5-5-2-2 through preparative thin-layer chromatography (PTLC) combined with Sephadex LH-20 CC (ø 1.5×150 cm, MeOH, 0.2 l).

Compound 1, white amorphous powder; [α]D25–14.0 (c 0.2, MeOH); UV (MeOH) λ_max nm (log ε) 237 (4.51); HRESIMS m/z 565.1901 [M+Na]⁺ (calcd for C₂₅H₃₄O₁₃Na, 565.1892); IR (KBr) v_max 3391, 2921, 2851, 1648, 1467, 1383, 1143, 1049, 980, 810, 618 cm⁻¹; ¹H NMR (DMSO-d₆, 600 MHz) and ¹³C NMR (DMSO-d₆, 150 MHz) data, see Table 1.

Table 1:

NMR spectroscopic data for compound 1 in DMSO-d₆.

Position	δH	δC	DEPT
7	7.94 (s)	144.2	CH
8		117.7	C
9	4.87 (d, 12.3) 4.66 (d, 12.3)	60.2	CH2
1′		133.1	C
2′	7.04 (s)	104.9	CH
3′		144.9	C
4′		143.5	C
5′		128.7	C
6′	7.31 (s)	110.7	CH
7′	6.61 (br.d, 15.9)	129.0	CH
8′	6.39 (dd, 15.9, 5.2)	129.8	CH
9′	4.14 (br.d, 5.2)	61.6	CH2
Glc-1″	4.29 (d, 7.8)	101.8	CH
2″	3.03 (m)	73.3	CH
3″	3.15 (m)	76.6	CH
4″	3.47 (m)	70.7	CH
5″	3.32 (m)	75.6	CH
6″	3.88 (br.d, 10.4) 3.49 (dd, 10.4, 3.3)	67.2	CH2
Rha-1″′	4.67 (br.s)	101.0	CH
2″′	3.03 (m)	70.3	CH
3″′	3.67 (m)	70.5	CH
4″′	3.20 (m)	72.0	CH
5″′	3.50 (m)	68.3	CH
6″′	1.16 (d, 6.2)	17.9	CH3
3′-OCH3	3.95 (s)	55.8	CH3

1. The assignments were based on TOCSY, HSQC, HMBC and distortionless enhancement by polarization transfer (DEPT) experiments.

Compound 2, white amorphous powder; [α]D25–31.7 (c 0.2, MeOH); UV (MeOH) λ_max nm (log ε) 203 (4.60), 218 (4.33), 264 (3.93); HRESIMS m/z 549.1592 [M+Na]⁺ (calcd for C₂₄H₃₀O₁₃Na, 549.1579); IR (KBr) v_max 3354, 1707, 1596, 1514, 1426, 1284, 1222, 1071, 1027, 878, 764, 638 cm⁻¹; ¹H NMR (DMSO-d₆, 600 MHz) data, see Table 2; ¹³C NMR (DMSO-d₆, 150 MHz) data, see Table 3.

Table 2:

¹H NMR spectroscopic data for compounds 2–5.

Position	2	3	4	5
2	6.95 (d, 2.2)	6.95 (d, 1.7)	6.96 (br.s)	7.04 (d, 1.7)
5	7.01 (d, 8.2)	7.02 (d, 8.3)	7.01 (br.d, 8.2)	7.00 (d, 8.5)
6	6.70 (dd, 8.2, 2.2)	6.71 (dd, 8.3, 1.7)	6.69 (br.d, 8.2)	6.69 (dd, 8.5, 1.7)
7	4.42 (d, 4.8)	4.43 (d, 5.0)	4.35 (d, 5.6)	6.44 (d, 15.9)
8	3.44 (m)	3.45 (m)	3.48 (m)	6.24 (dd, 15.9, 5.2)
9	3.31 (m) 3.15 (dd, 11.0, 6.4)	3.31 (m) 3.15 (dd, 10.9, 4.6)	3.43 (m) 3.39 (m)	4.10 (d, 5.2)
Glc1-1′	4.93 (d, 7.2)	4.93 (d, 7.3)	4.92 (d, 7.1)	4.98 (d, 7.3)
2′	3.31 (m)	3.31 (m)	3.31 (m)	3.31 (m)
3′	3.31 (m)	3.31 (m)	3.31 (m)	3.31 (m)
4′	3.26 (m)	3.26 (m)	3.25 (m)	3.26 (m)
5′	3.70 (m)	3.70 (m)	3.70 (m)	3.74 (m)
6′	4.57 (dd, 11.8, 2.1) 4.14 (dd, 11.8, 7.1)	4.58 (dd, 11.8, 1.7) 4.16 (dd, 11.8, 7.1)	4.59 (br.d, 11.8) 4.16 (dd, 11.8, 7.2)	4.57 (dd, 11.8, 2.1) 4.23 (dd, 11.8, 7.3)
Vanilloyl-2″	7.42 (d, 2.0)	7.44 (d, 1.8)	7.44 (d, 1.6)	7.46 (d, 1.9)
5″	6.89 (d, 8.4)	6.92 (d, 8.2)	6.90 (d, 8.3)	7.19 (d, 8.6)
6″	7.47 (dd, 8.4, 2.0)	7.48 (dd, 8.2, 1.8)	7.49 (dd, 8.3, 1.6)	7.54 (dd, 8.6, 1.9)
Glc 2-1″′				5.05 (d, 7.4)
2″′				3.31 (m)
3″′				3.36 (m)
4″′				3.20 (m)
5″′				3.31 (m)
6″′				3.67 (m) 3.48 (m)
3-OCH3	3.73 (s)	3.74 (s)	3.74 (s)	3.76 (s)
3″-OCH3	3.80 (s)	3.81 (s)	3.81 (s)	3.78 (s)

1. ¹H NMR data (δ) were measured at 600 MHz in DMSO-d₆. Proton coupling constants (J) in Hz were given in parentheses. The assignments were based on TOCSY, HSQC and HMBC experiments.

Table 3:

¹³C NMR spectroscopic data for compounds 2–5.

Position	2	3	4	5
1	137.4	137.4	137.4	131.1
2	111.3	111.4	111.6	109.8
3	148.3	148.3	148.2	148.9
4	145.2	145.3	145.3	145.7
5	114.6	114.7	114.2	115.0
6	118.5	118.5	119.1	118.8
7	72.6	72.6	73.7	128.4
8	75.8	75.8	75.4	129.0
9	62.6	62.6	63.0	61.6
Glc 1-1′	100.1	100.2	100.0	99.5
2′	73.2	73.1	73.2	73.1
3′	76.6	76.6	76.7	76.6
4′	70.0	70.0	70.1	70.1
5′	73.9	73.9	73.9	73.8
6′	63.8	63.9	63.9	64.1
Vanilloyl-1″	120.0	120.5	120.5	122.9
2″	112.6	112.7	112.8	112.7
3″	147.5	147.4	147.4	148.6
4″	151.9	151.6	151.6	150.7
5″	115.3	115.2	115.2	114.4
6″	123.6	123.5	123.5	122.9
7″	165.5	165.4	165.4	165.2
Glc 2-1″′				99.7
2″′				73.1
3″′				77.1
4″′				69.5
5″′				76.8
6″′				60.5
3-OCH3	55.6	55.7	55.6	55.6
3″-OCH3	55.7	55.7	55.7	55.7

1. ¹³C NMR data (δ) were measured at 150 MHz in DMSO-d₆. The assignments were based on TOCSY, HSQC and HMBC experiments.

Compound 3, white amorphous powder; [α]D25–33.4 (c 0.2, MeOH); UV (MeOH) λ_max nm (log ε) 202 (4.75), 265 (3.93); HRESIMS m/z 549.1592 [M+Na]⁺ (calcd for C₂₄H₃₀O₁₃Na, 549.1579); IR (KBr) v_max 3383, 1707, 1598, 1515, 1427, 1285, 1223, 1072, 1028, 764, 637 cm⁻¹; ¹H NMR (DMSO-d₆, 600 MHz) data, see Table 2; ¹³C NMR (DMSO-d₆, 150 MHz) data, see Table 3.

Compound 4, white amorphous powder; [α]D25–29.5 (c 0.2, MeOH); UV (MeOH) λ_max nm (log ε) 206 (4.54), 219 (4.46), 265 (4.08); HRESIMS m/z 549.1590 [M+Na]⁺ (calcd for C₂₄H₃₀O₁₃Na, 549.1579); IR (KBr) v_max 3372, 1708, 1597, 1515, 1427, 1286, 1222, 1072, 1028, 876, 764, 626 cm⁻¹; ¹H NMR (DMSO-d₆, 600 MHz) data, see Table 2; ¹³C NMR (DMSO-d₆, 150 MHz) data, see Table 3.

Compound 5, white amorphous powder; [α]D25–24.5 (c 0.2, MeOH); UV (MeOH) λ_max nm (log ε) 206 (4.59), 257 (4.14), 291 (3.71); HRESIMS m/z 677.2062 [M+Na]⁺ (calcd for C₃₀H₃₈O₁₆Na, 677.2052); IR (KBr) v_max 3370, 1715, 1512, 1417, 1274, 1219, 1027, 955, 764 cm⁻¹; ¹H NMR (DMSO-d₆, 600 MHz) data, see Table 2; ¹³C NMR (DMSO-d₆, 150 MHz) data, see Table 3.

Results and discussion

A novel norneolignan glycoside (1), four new phenolic glycosides (2–5) and a known neolignan glycoside (6) were isolated from the ethanolic extract of the stems of V. fordiae Hance shown in Figure 2. The chemical structures of previously undescribed compounds were determined by spectroscopic data [¹H and ¹³C NMR, total correlation spectroscopy (TOCSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple-bond correlation (HMBC) and optical rotatory dispersion (ORD)] and chemical methods. The known neolignan glycoside, found for the first time in Viburnum, was determined as dehydrodiconiferyl alcohol 9′-O-β-d-glucopyranoside (6) by comparing spectroscopic data with those reported in the literature (Jiang et al. 2001). In previous biological and pharmaceutical investigations, compound 6 exhibited anti-inflammatory and antinociceptive potential (Tatli et al. 2008).

Figure 2:

Chemical structures of isolated compounds 1–6 from the stems of Viburnum fordiae.

Compound 1, a white amorphous powder, had a molecular formula C₂₅H₃₄O₁₃ as deduced by analysis of positive HRESIMS (m/z 565.1901 [M+Na]⁺, calcd for C₂₅H₃₄O₁₃Na, 565.1892). The ¹H NMR spectrum of 1 (Table 1) was interpreted containing a 1,3,4,5-tetrasubstituted benzene ring [δ_H 7.31 (1H, s) and 7.04 (1H, s)]. An isolated olefinic proton at δ_H 7.94 (1H, s), a methylene group at δ_H 4.87 (1H, d, J=12.3 Hz), 4.66 (1H, d, J=12.3 Hz), and a methoxy signal at δ_H 3.95 (3H, s) were also observed. Besides, a 3-(E)-hydroxypropenyl unit [δ_H 6.61 (1H, br.d, J=15.9 Hz), 6.39 (1H, dd, J=15.9, 5.2 Hz), and 4.14 (2H, br.d, J=5.2 Hz)] was determined according to the TOCSY correlations (Figure 3). In the HMBC spectrum of 1, the correlations from H-7 (δ_H 7.94) to C-4′ (δ_C 143.5), C-5′ (δ_C 128.7), C-8 (δ_C 117.7), and C-9 (δ_C 60.2), from H-6′ (δ_H 7.31) to C-8 (δ_C 117.7), from H-9 (δ_H 4.87, 4.66) to C-5′ (δ_C 128.7), C-7 (δ_C 144.2) and C-8 (δ_C 117.7), and taking into consideration of the chemical shifts of C-7 (δ_C 144.2) and C-4′ (δ_C 143.5), suggested that C-7 was connected to C-4′ via an oxygen bridge, thus furnishing a benzofuran skeleton in 1. The ¹H NMR spectrum of 1 showed two anomeric protons at δ_H 4.67 (1H, br.s) and 4.29 (1H, d, J=7.8 Hz), corresponding to δ_C 101.0 and 101.8. An HMBC correlation between the anomeric proton H-1″′ (δ_H 4.67) and C-6″ (δ_C 67.2) identified a rhamnosyl (1→6) glucopyranosyl linkage. The glucopyranosyl unit was elucidated as β-configuration by the relatively large coupling constant (J=7.8 Hz) of the anomeric proton (Hudson and Dale 1917). The α-configuration of the rhamnopyranosyl moiety was deduced by the chemical shifts of C-3″′ at δ_C 70.5 and C-5″′ at δ_C 68.3 (Kasai et al. 1979). d-Glucose and l-rhamnose, derivatized to acetylated aldononitriles before determined by gas chromatography-mass spectrometry (GC-MS) analysis, were yielded through acid hydrolysis of 1 (Fu et al. 2010). The C-9 location of the sugar chain in the aglycone was elucidated by the HMBC correlation of the anomeric proton H-1″ (δ_H 4.29) of the glucopyranosyl unit with C-9 (δ_C 60.2). Accordingly, the structure of 1 was established as (7′E)-9′-hydroxy-3′-methoxy-7-noraryl-4′,7-epoxy-8,5′-neolignan-7′-ene-9-O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside.

Figure 3:

Key TOCSY and HMBC correlations for compounds 1, 2, and 5.

Compound 2, white amorphous powder, was assigned to a molecular formula C₂₄H₃₀O₁₃ due to analysis of positive HRESIMS (m/z 549.1592 [M+Na]⁺, calcd for C₂₄H₃₀O₁₃Na, 549.1579). Acid hydrolysis of 2 yielded β-d-glucose, which was concluded by the positive optical rotation [α]D25=+40.1 (c 0.10, H₂O) and the coupling constant of an anomeric proton (J=7.2 Hz), vanillic acid identified by co-TLC with authentic standard sample, and aglycone 2a with [α]D25=–17.1 (c 0.15, MeOH). In the ¹H NMR spectrum of 2 (Table 2), two sets of ABX proton signals [δ_H 6.95 (1H, d, J=2.2 Hz), 7.01 (1H, d, J=8.2 Hz), 6.70 (1H, dd, J=8.2, 2.2 Hz) and 7.42 (1H, d, J=2.0 Hz), 6.89 (1H, d, J=8.4 Hz), 7.47 (1H, dd, J=8.4, 2.0 Hz)] were attributed to two 1,3,4-trisubstituted benzene rings, and two methoxy signals at δ_H 3.73 (3H, s) and 3.80 (3H, s) were also observed. Besides, a C3-unit of C-7–C-8–C-9 [δ_H 4.42 (1H, d, J=4.8 Hz), 3.44 (1H, m), 3.31 (1H, m) and 3.15 (1H, dd, J=11.0, 6.4 Hz)] was determined according to the TOCSY experiment (Figure 3). In the HMBC spectrum of 2, long-range correlations from H-7 (δ_H 4.42) to C-1 (δ_C 137.4), C-2 (δ_C 111.3), C-6 (δ_C 118.5), C-8 (δ_C 75.8), and C-9 (δ_C 62.6), from H-2″ (δ_H 7.42) to C-3″ (δ_C 147.5) and C-7″ (δ_C 165.5), from H-6″ (δ_H 7.47) to C-4″ (δ_C 151.9) and C-7″ (δ_C 165.5), as well as from methoxy protons at δ_H 3.73 and δ_H 3.80 to C-3 (δ_C 148.3) and C-3″ (δ_C 147.5), respectively, confirmed the presence of a guaiacylglycerol unit and a vanilloyl residue. The chemical shift of glucosyl C-6′ (δ_C 63.8) indicated that the vanilloyl residue was located at C-6′ in 2. This was supported by the observed cross-peaks between H-6′ (δ_H 4.57, 4.14) and C-7″ (δ_C 165.5) in the HMBC spectrum. The HMBC correlation between the anomeric proton H-1′ (δ_H 4.93) and C-4 (δ_C 145.2) was interpreted that β-d-glucosyl group is linked at C-4. According to the literature (Lin et al. 2007; Kim et al. 2009), in the same solvent, the values of Δδ_C8−C7 were different due to the threo and erythro isomers. The Δδ_C8−C7 values of 2 (3.2) and 3 (3.2) were larger than that of 4 (1.7). This indicates that the glycerol moieties of 2 and 3 possess a threo relative configuration, while 4 should be the erythro form.

The ¹H NMR data of 2a are in good agreement with those of threo-guaiacylglycerol, indicating that 2 is threo-guaiacylglycerol 4-O-β-d-(6-O-vanilloyl)glucopyranoside. As threo-guaiacylglycerol with 7R,8R configuration was reported to have a negative [α]D25 value (Ishikawa et al. 2002), the absolute configuration at C-7 and C-8 of 2a was assigned to 7R,8R. Thus, the structure of 2 was determined as (7R,8R)-guaiacylglycerol 4-O-β-d-(6-O-vanilloyl)glucopyranoside.

Compounds 3 and 4, white amorphous powder, were found to have the same formulae C₂₄H₃₀O₁₃ from positive HRESIMS m/z 549.1592 [M+Na]⁺ (calcd 549.1579) and 549.1590 [M+Na]⁺ (calcd 549.1579), respectively. The spectroscopic data of 3 and 4 (Tables 2 and 3), completely identical to those of 2, indicates that they are isomers of 2. However, 2, 3 and 4 were separated by preparative HPLC with retention times of 59.9, 53.7 and 51.8 min, respectively, under the same chromatographic conditions. Acid hydrolysis of 3 and 4 yielded β-d-glucose, vanillic acid and aglycones (3a and 4a, respectively). The positive optical rotation [α]D25=+17.1 (c 0.15, MeOH) of 3a confirmed that its configuration was 7S and 8S (Warashina et al. 2005). Accordingly, 3 was confined to be (7S,8S)-guaiacylglycerol 4-O-β-d-(6-O-vanilloyl)glucopyranoside. The configuration of 4a was confirmed to be 7S and 8R by [α]D25=+10.3 (c 0.15, MeOH) (Ishikawa et al. 2002). Thus, 4 was assigned as (7S,8R)-guaiacylglycerol 4-O-β-d-(6-O-vanilloyl)glucopyranoside.

Compound 5 was obtained as a white amorphous powder. The molecular formula of 5 was determined as C₃₀H₃₈O₁₆ from the HRESIMS at m/z 677.2062 [M+Na]⁺ (calcd for C₃₀H₃₈O₁₆Na, 677.2052). Acid hydrolysis of 5 yielded d-glucose, vanillic acid and aglycone. In the ¹H NMR spectrum of 5 (Table 2), two sets of ABX proton signals [δ_H 7.04 (1H, d, J=1.7 Hz), 7.00 (1H, d, J=8.5 Hz), 6.69 (1H, dd, J=8.5, 1.7 Hz) and 7.46 (1H, d, J=1.9 Hz), 7.19 (1H, d, J=8.6 Hz), 7.54 (1H, dd, J=8.6, 1.9 Hz)] were attributed to two 1,3,4-trisubstituted benzene rings. The trans-olefinic protons at δ_H 6.44 (1H, d, J=15.9 Hz) and 6.24 (1H, dd, J=15.9, 5.2 Hz) and two oxymethylene protons at δ_H 4.10 (2H, d, J=5.2 Hz) established the presence of an allyl alcohol moiety. This was confirmed by TOCSY spectrum of 5 (Figure 3). In the HMBC spectrum of 5, the correlations were observed between H-8 (δ_H 6.24) and C-1 (δ_C 131.1), H-7 (δ_H 6.44) and C-1(δ_C 131.1), C-2 (δ_C 109.8) and C-6 (δ_C 118.8), and the methoxy proton (δ_H 3.76) and C-3 (δ_C 148.9), indicating the presence of a coniferyl alcohol residue in 5. Meanwhile, the HMBC cross-peaks from H-2″ (δ_H 7.46) to C-3″ (δ_C 148.6) and C-7″ (δ_C 165.2), from H-6″ (δ_H 7.54) to C-4″ (δ_C 150.7) and C-7″ (δ_C 165.2), and from the methoxy proton (δ_H 3.78) to C-3″ (δ_C 148.6), suggested the presence of a vanilloyl group in 5. The β-linkages of glucose moiety were shown by the coupling constant values (J=7.3 and 7.4 Hz) of two anomeric proton signals at δ_H 4.98 and 5.05. The HMBC correlations from H-1′ (δ_H 4.98) to C-4 (δ_C 145.7), H-1″′ (δ_H 5.05) to C-4″ (δ_C 150.7) and H-6′ (δ_H 4.57, 4.23) to C-7″ (δ_C 165.2) were observed. From the above evidence, compound 5 was determined to be coniferyl alcohol 4-O-[6-O-(4-O-β-d-glucopyranosyl)vanilloyl]-β-d-glucopyranoside.

Conclusions

The stems extractives of V. fordiae Hance were for the first time fractionated and analyzed, from which a novel norneolignan glycoside (1), four new phenolic glycosides (2–5), and a known neolignan glycoside (6) have been obtained. The structures of those secondary metabolites including absolute configurations were determined by spectral data, chemical evidence and the careful comparison with authentic compounds of the literature. The new compounds were elucidated as

• (7′E)-9′-hydroxy-3′-methoxy-7-noraryl-4′,7-epoxy-8,5′-neolignan-7′-ene-9-O-α-l-rhamnopyranosyl-(1→6)-β-d-glucopyranoside (1),

• (7R,8R)-guaiacylglycerol 4-O-β-d-(6-O-vanilloyl)glucopyranoside (2),

• (7S,8S)-guaiacylglycerol 4-O-β-d-(6-O-vanilloyl)glucopyranoside (3),

• (7S,8R)-guaiacylglycerol 4-O-β-d-(6-O-vanilloyl)glucopyranoside (4) and

• coniferyl alcohol 4-O-[6-O-(4-O-β-d-glucopyranosyl)vanilloyl]-β-d-glucopyranoside (5).

Compound 6 was obtained from the genus Viburnum for the first time.