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Lignan glycosides from the Rhizomes of Smilax trinervula and their Biological activities
Phytochemistry Letters 20 (2017) 1–8
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Lignan glycosides from the Rhizomes of Smilax trinervula and their Biological activities Jicheng Shua , Fang Lianga,b , Genhua Zhua , Xing Liua , Jiangli Yua , Huilian Huanga,* a b
Key Laboratory of Modern Preparation of TCM, Jiangxi University of Traditional Chinese Medicine, Ministry of Education, Nanchang 330004, China Jiangxi Pharmaceutical School, Nanchang 330004, China
A R T I C L E I N F O
Article history: Received 22 July 2016 Received in revised form 5 February 2017 Accepted 1 March 2017 Available online xxx Keywords: Smilax trinervula Smilacaceae Lignan glycosides Cytotoxic activity
A B S T R A C T
Phytochemical investigation of the rhizomes of Smilax trinervula led to isolation and structure elucidation of eight lignan glycosides, including five new lignans, namely, (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 tetramethoxy-7, 90 -epoxylignan-70 -one 40 -O-b-D-glucopyranoside (1), (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 -epoxylignan-70 -one 4-O-b-D- glucopyranoside (2) (7S, 8R)-4, 9, 90 -trihydroxy3, 30 , 5-trimethoxy-40 , 7-epoxy-8, 50 -neolignan 90 -O-b-D-glucopyranoside (3), (7R, 8R)-4, 9, 90 trihydroxy-3, 5-dimethoxy-7.O.40 , 8.O.30 - neolignan 90 -O-b-D-glucopyranoside (4), and (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy-8, 40 -oxy-neolignan 4-O-b-D-glucopyranoside (5), along with three known compounds (6-8). Their structures were established mainly on the basis of 1D and 2D NMR spectral data, ESI–MS and comparison with the literature. Compounds 1-8 were tested in vitro for their cytotoxic activity against four human tumor cell lines (SH-SY5Y, SGC-7901, HCT-116, Lovo). Compounds 3 and 5 exhibited cytotoxic activity against Lovo cells, with IC50 value of 10.4 mM and 8.5 mM, respectively. © 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
1. Introduction The genus Smilax (Smilacaceae) comprises about 370 species, which are mainly distributed in the tropical and temperate zones throughout the world, especially in East Asia and North America (Tsukamoto, 1998). There are about 200 species of the genera in China (Flora of China Editorial Committee, 2004). Smilax is commonly known as “Ba-Qia” or “Jin-Gang-Teng” in Chinese. However, only Smilax china is used to expel wind, drain off dampness and remove carbuncles (Chinese Pharmacopoeia Committee, 2010). In recent years, detailed research on the phytochemistry and pharmacology of this genus focused on Smilax china. Previous phytochemistral investigations have resulted in the isolation and identification of characteristic steroidal saponins, lignans and various phenols (Shao et al., 2007; Huang et al., 2007; Li et al., 2007). Various pharmacological properties, such as antiinflammatory, antimicrobial, antinociceptive, neuroprotective and antitumor activity, have been reported (Huang and Huang, 2000; Shu et al., 2006; Kim et al., 1989; Chen et al., 2001). However, to the best of our knowledge, until now, only two studies have been reported on Smilax trinervula (S. trinervual)
* Corresponding author. E-mail address: [email protected] (H. Huang).
(Liang et al., 2015; Huang et al., 2015). As a member of the Smilax genera, S. trinervula may possess some pharmacological activity and active substances similar to Smilax china. As a part of our ongoing search of potentially bioactive components from the genus Smilax, we have systematically investigated the constituents of S. trinervula. Our previous studies on the EtOAc fraction of the ethanol extract of the rhizomes of S. trinervula have led to the isolation of several new phenylpropanoids (Shu et al., 2015). A further detailed investigation of the n-BuOH fraction of the same extract has resulted in the isolation of 5 new lignan glycosides (15), along with three known lignan glycosides (6-8). 2. Results and discussion The n-BuOH soluble part partitioned from the ethanol extract of S. trinervula was fractionated by a macroporous resin column to give five fractions A, B, C, D and E. Fraction B was subfractionated by a combination of silica gel column chromatography, Sephadex LH20 column chromatography and preparative HPLC to give eight lignan glycosides, including five new lignan glycosides, namely, (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 epoxylignan- 70 -one 40 -O-b-D-glucopyranoside (1), (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 -epoxylignan-70 -one 4-O-b-D-glucopyranoside (2) (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5trimethoxy-40 , 7-epoxy-8, 50 -neolignan 90 -O-b-D-glucopyranoside
http://dx.doi.org/10.1016/j.phytol.2017.03.002 1874-3900/© 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
2
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(3), (7R, 8R)-4, 9, 90 - trihydroxy-3, 5-dimethoxy-7.O.40 , 8.O.30 neolignan 90 -O-b-D-glucopyranoside (4), and (7S, 8R)-4, 9, 90 trihydroxy-3, 30 , 5-trimethoxy-8, 40 -oxy-neolignan 4-O-b-D-glucopyranoside (5), and three known lignan glycosides, (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy-40 , 7-epoxy-8, 50 - neolignan 4-Ob-D-glucopyranoside (6) (Wang et al., 2010), symplocosneolignan (7) (Cai et al., 2011) and rourinoside (8) (He et al., 2006), respectively (Fig. 1). Compound 1 was isolated as an amorphous powder. The molecular formula C28H36O14 of 1 was deduced from HR-ESI–MS spectrometry (see Experimental section). In the 1H NMR and 13C NMR spectrum of 1 (Table 1), two pairs of geminated aromatic proton signals at dH 6.70 (2H, s) and 7.39 (2H, s), assigned to H-2/6 (signals at dc 105.3) and H-20 /60 (signals at dc 107.9), together with the other four quaternary aromatic carbons at dc 133.8, 136.2, 132.9 and 141.0, suggested the presence of two tetrasubstituted symmetrical benzene rings. These substituents were four MeO groups, resonating at dH 3.85 (2 MeO) and 3.92 (2 MeO). Signals at dH 5.11 (1H, d, J = 7.5 Hz) with dC104.4 corresponding to the anomeric proton and anomeric carbon, along with carbon signals at dc 75.8, 78.5, 71.4, 77.9, 62.6 and proton signals in the region of dH
3.20–3.51, suggested 1 possess a b-glucopyranosyl unit (Mohamed, 2001). Furthermore, the sugar was identified by GC– MS analysis after acid hydrolysis (Xin et al., 2009). The glucopyranosyl moiety was embedded at C-40 supported by the HMBC correlation (Fig. 2) between the anomeric proton at dH 5.11 (H-1”) and aromatic carbon at dc 141.0 (C-40 ). In addition, the carbon signals at dc 85.4 (d), 55.1 (d), 61.7 (t), 50.6 (d), 71.2 (t), together with a carbonyl carbon signals at dc 200.6 were assigned to a hydroxymethyl-substituted tetrahydrofuranoid. From the above observations, 1 was identified as 4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 -epoxylignan-70 -one 40 -O-b-D- glucopyranoside (Guan et al., 2008). The absolute stereochemistry was determined by a ROESY experiment and comparison with the literature. Cis and trans orientation of the substituents at C-7 and C8 would give signals of H-7 at ca dH 5.5 and dH 4.7, respectively (Jung et al., 1998). The H-7 signal of 1 was observed at dH 4.68, that C-7 and C-8 were in the trans configuration of substituents. The ROESY spectrum (Fig. 3) displayed the correlation between the proton signals at dH 2.64 (H-8) and dH 4.26 (H-80 ), but was not observed between the proton signals at dH 2.64 (H-8) and dH 4.63 (H-7), indicated the assignments of same ring-side position (cis)
Fig. 1. Chemical structure of compounds 1-8.
J. Shu et al. / Phytochemistry Letters 20 (2017) 1–8 Table 1 NMR of 1 and 2 (400 MHz for 1H, MeOH-d4, 100 MHz for
3
13
C, MeOH-d4).
1
2
C
1
13
1 2 3 4 5 6 7 8 9
– 6.70 (s) – – – 6.70 (s) 4.63 (d, J = 7.9 Hz) 2.64 (dddd, 2.7, 4.7, 5.6, 7.9 Hz) 3.73 (dd, J = 4.7, 11.4 Hz) 3.66 (dd, J = 5.6, 11.4 Hz) – 7.39 (s) – – – 7.39 (s) – 4.26 (m) 4.26 (m) 4.18 (d, J = 9.6 Hz) 5.11 (d, J = 7.5 Hz) 3.47–3.51 (m) 3.39–3.44 (m) 3.39–3.44 (m) 3.20–3.24 (m) 3.76 (dd, J = 2.3, 12 Hz) 3.63 (dd, J = 5.3, 12 Hz) 3.85 (s) 3.92 (s)
133.8 105.3 149.3 136.2 149.3 105.3 85.4 55.1 61.7
– 6.78 (s) – – – 6.78 (s) 4.68 (d, J = 7.9 Hz) 2.64 (m) 3.69–3.71 (2H, m)
139.4 105.6 149.2 135.7 149.2 105.6 85.1 55.2 61.6
132.9 107.9 154.4 141.0 154.4 107.9 200.6 50.6 71.2
– 7.39 (s) – – – 7.39 (s) – 4.23–4.28 (m) 4.23-4.28 (m) 4.21 (dd, J = 2.9, 9.5 Hz) 4.85 (d, J = 7.5 Hz) 3.45–3.49 (m) 3.39–3.42 (m) 3.39–3.42 (m) 3.18–3.22 (m) 3.76 (dd, J = 2.4, 12 Hz) 3.65 (dd, J = 5.2, 12 Hz) 3.86 (s) 3.92 (s)
128.8 107.8 154.3 143 154.3 107.8 200.1 50.3 71.7
C-10 C-20 C-30 C-40 C-50 C-60 C-70 C-80 C-90 C-1” C-2” C-3” C-4” C-5” C-6” 3,5-OMe 30 ,50 -OMe
H
and trans orientations for H-8/H-80 and H-7/H-8, respectively. In addition, the characteristic coupling constant of H-7 and H-8 (7.9 Hz) in 1 and the negative [a]25 D of 1 measured at 31.41 (0.191, MeOH) were also in accordance with the known compound mentioned (Guan et al., 2008; Qin et al., 2013). Therefore, 1 was determined as (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 tetramethoxy-7, 90 -epoxylignan-70 -one 40 -O-b-D- glucopyranoside. However, Guan et al. (Guan et al., 2008) reported one analogous lignan glycoside from Akebia trifoliate. The authors proposed the wrong conclusion because of the trans orientations for H-7/H-8. By reconsidering structure and data afforded by authors, the compound 2 reported in the literature should be (7S, 8R,80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 -epoxylignan-70 -one 9-O-b-D- glucopyranoside, not be (7S, 8S, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 - epoxylignan-70 -one 9-O-b-D- glucopyranoside. Compound 2 was isolated as an amorphous powder. HR-ESI–MS spectrometry showed the same molecular formula as that of 1 [595.2035 [M] (calcd for C28H35O14)]. The 1H NMR and 13C NMR spectrum of 2 (Table 1) were very similar with those of 1. The above information suggested 2 was an isomer of 1. By comparing and analyzing their NMR data, the obvious differences were the chemical shifts of C-1, C-4, C-10, C-40 , H-1”, C-1”. The difference was the location of glucopyranosyl unit. 2 was placed at C-4. This was deduced by an HMBC experiment (Fig. 2), which showed a correlation between the anomeric proton at dH 4.85 and C-4 at dC 135.7. Thus, 2 was identified as (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 -epoxylignan-70 -one 4-O-b-D- glucopyranoside. Compound 3 was obtained as an amorphous powder. HR-ESI– MS spectrometry (see Experimental part) showed the molecular formula C27H36O12. In the 1H NMR and 13C NMR spectrum of 3
C
104.4 75.8 77.9 71.4 78.5 62.6 57.2 56.8
1
H
13
C
105.4 75.7 77.8 71.4 78.3 62.6 56.9 57
(Table 2), 18 signals for 12 aromatic carbons at dc 134.1 (s), 104.2 (2*s), 149.4 (2*s), 137.0 (s), 136.4 (s), 114.3 (s), 145.2 (s), 147.5 (s), 129.8 (s), 118.0 (s), and 6 alphatic carbons at dc 89.1 (d), 55.6 (d), 65.0 (t), 32.94 (t), 32.98 (t) and 69.9 (t) were observed. This suggested that this compound might be a dihydrobenzofuran neolignan (García-Muñoz et al., 2006; Chin et al., 2008). In addition, signals at dH 4.25 (1H, d, J = 7.8 Hz) with dC 104.5 corresponding to the anomeric proton and anomeric carbon, along with carbon signals at dc 75.2, 78.2, 71.7, 77.9 62.8 and proton signals in the region of dH 3.17–3.32, suggested that 3 had a b-glucopyranosyl unit (Mohamed, 2001). Furthermore, the sugar was identified by GC–MS analysis after acid hydrolysis (Xin et al., 2009). The above observaions together with careful comparison of the NMR data of 3 with those of the known neolignan compound 6 indicated that 3 was 4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy-40 , 7epoxy-8, 50 -neolignan 90 -O-b-D- glucopyranoside. It was confirmed by the HMBC spectrum (Fig. 2). The HMBC correlation peaks MeO and C-3, MeO and C-5, MeO and C-30 , and the anomeric proton resonance of glucose H-1” to C-90 supported the above observations. The stereochemistry of C-7 and C-8 was assigned as trans by the ROESY correlations of H-7/H-9. One the other hand, a significant difference in chemical shifts of C-8 in 9-hydroxydihydrobenzofuran neolignans, appearing at dc ca. 54 in the trans isomers and at dc ca. 49 in the cis ones (García-Muñoz et al., 2006, Jiménez-González et al., 2005), is a reliable criterion to distinguish both diastereoisomers. The C-8 signal of 3 was observed at dc 55.6, that implied C-7 and C-8 had a trans configuration of substituents. In addition, the characteristic coupling constant of H-7 and H-8 (6.3 Hz) in 3 and the negative [a]25 D of 3 measured at 5.41 (0.185, MeOH) were also in accordance with the known compound mentioned (Wang et al., 2010). Therefore, 3 was determined as (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy-40 , 7-epoxy-8, 50 -neolignan 90 -O-b-D- glucopyranoside.
4
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Fig. 2. Key HMBC of Compounds 1-5.
Compound 4 was obtained as an amorphous powder. HR-ESI– MS spectrometry (see Experimental part) showed the molecular formula C26H34O13. In the 1H NMR and 13C NMR spectrum of 4 (Table 3), a pairs of geminated aromatic proton signals at dH 6.71 (2H, s), assigned to H-2/6 (signals at dc 105.9), two meta-coupled aromatic proton signals at dH 6.68 (1H, d, J = 1.9 Hz) and 6.50 (1H, d, J = 1.9 Hz), together with the other seven quaternary aromatic carbons at dc 128.7, 149.5, 137.3, 135.7, 147.5, 133.3 and 145.7, suggested the presence of two tetrasubstituted symmetrical and unsymmetrical benzene rings. On the other hand, the 13C NMR spectrum showed 6 alphatic carbons at dc 77.9 (d), 79.8 (d), 62.2 (t), 32.7 (t), 35.3 (t) and 61.9 (t) suggesting that this compound might be a neolignan. In addition, the 1H NMR spectrum showed a resonance at dH 4.91 (d, J = 7.6 Hz) corresponding to the anomeric proton of a b-D- glucopyranose moiety, and the 13C NMR spectrum also exhibited the corresponding carbon resonances at dc 103.2 (d), 75.0 (d), 77.9 (d), 71.5 (d), 78.3 (d) and 62.6 (t). Furthermore, the sugar was identified by GC–MS analysis after acid hydrolysis (Xin et al., 2009). The above observations together with careful comparison of the NMR data of 3 indicated that 4 was a lignan glucoside, with great similarity to the known compound eusiderin N (Yang Kuo et al., 2013). The obvious difference was the chemical shift of C-9. A resonance at dH 1.19 (3H, d, J = 6.6 Hz) in eusiderin N was replaced by a signal at dH 3.72 (2H, m) in 4 in the 1H NMR, and in the corresponding 13C NMR spectrum, a signal at dc 17.4 (q) in eusiderin N was substituted for a signal at dc 62.2 (t), which
suggested that C-9 was substituted by a hydroxyl group. This was supported by the analysis of the molecular formula and the HMBC correlation between the anomeric proton at dH 3.72 (H-9) and the alphatic carbon at dc 79.8 (C-8) and 77.9 (C-7) (Fig. 2). The stereochemistry of C-7 and C-8 was assigned as trans by the ROESY correlations of H-7/H-9 (Fig. 3). One the other hand, the characteristic coupling constant of H-7 and H-8 (8.0 Hz) in 4 and the negative [a]25 D of 4 measured at 34.34 (0.182, MeOH) were also in accordance with the known compound mentioned (Yang Kuo et al., 2013; Da Silva et al., 1989). Therefore, 4 was determined as (7R, 8R)-4, 9, 90 -trihydroxy-3, 5-dimethoxy-7.O.40 , 8. O.30 -neolignan 90 -O-b-D- glucopyranoside. Compound 5 was isolated as an amorphous powder and its molecular formula was determined to be C27H38O13 by HR-ESI–MS (see Experimental section). In the 1H NMR spectrum (Table 3), a singlet signal for two H-atoms in the aromatic region at dH 6.76 (2H, s) assigned to H-2/6, suggested the presence of a 1, 3, 4, 5tetrasubstituted symmetrical benzene ring, and three aromatic protons signals at dH 6.79(d, J = 8.2 Hz), 6.77(d, J = 1.9 Hz) and 6.65 (dd, J = 2.0, 8.2 Hz), indicated that this compound possessed a 1, 3, 4-trisubstituted benzene ring. These substituents were three MeO groups, resonating at dH 3.80 (2 MeO) and 3.76 (MeO). In addition, the 1H NMR and 13C NMR displayed signals at dH 4.78 (1H, d, J = 7.4 Hz) with dC 105.6 corresponding to the anomeric proton and anomeric carbon of a b-glucopyranosyl unit (Mohamed, 2001). Furthermore, the sugar was identified by GC–MS analysis
J. Shu et al. / Phytochemistry Letters 20 (2017) 1–8
5
Fig. 3. Key NOESY correlations of Compounds 1-4.
after acid hydrolysis (Xin et al., 2009). Moreover, the 1H NMR spectrum showed ten proton signals at dH 4.82 (1H, d, J = 6.1 Hz), 4.30 (1H, ddd, J = 3.9, 5.4, 6.1 Hz), 3.85 (1H, dd, J = 5.4, 12.0 Hz), 3.72– 3.82 (1H, m), 2.59 (2H, t, J = 7.5 Hz), 1.78 (2H, m), 3.54 (2H, t, J = 6.5 Hz), suggesting the presence of 1, 2, 3-propanetriol moiety and a 1-propanol moiety. The above evidence suggested the presence of two C6-C3 units arising from both a neolignan and a glucose moiety, which was supported by analysis of the NMR and HMBC spectra (Fig. 2). According to the above information, the NMR spectrum of compound 5 was identical with that of a known compound symplocosneolignan (Cai et al., 2011), but obvious differences were seen in three aromatic protons with ABX coupling system, which suggested that 5 had one less MeO group than symplocosneolignan. This was supported by the analysis of its molecular formula. Judging from the coupling constant (J = 6.0 Hz) for H-7 and H-8, 5 was concluded to possess the erythro relative stereochemistry (Braga et al., 1984). Judging from the negative cotton effect at 240 nm, the absolute configuration of 5 was determined to be 7S, 8R (Cai et al., 2011; Arnoldi and Merlini, 1985; Huo et al., 2008; Kónya et al., 2004; Ishikawa et al., 2002). One the other hand, the negative [a]25 D of 5 measured at 30.56 (0.319, MeOH) were also in accordance with the known compound mentioned (Cai et al., 2011). Therefore, compound 5 was concluded to be (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy- 8, 40 -oxyneolignan 4-O-b-D- glucopyranoside.
Compounds 1-8 were evaluated for cytotoxic activities against four human cell lines (SH-SY5Y, SGC-7901, HCT-116 and Lovo) with Vero as a positive control. Compounds 1-8 were inactive (IC50> 100 mM) to SH-SY5Y, SGC-7901, and HCT-116 cell lines. Compounds 3 and 5 exhibited cytotoxic activity against Lovo cells, with IC50 value of 10.4 mM and 8.5 mM, respectively. 3. Experimental 3.1. General procedure and regents 1
H (400 MHz), 13C (100 MHz), and 2D NMR spectra were obtained on Bruker AV-400 with TMS as internal reference, and using methanol-d4 as solvents. Electrospray ionisation (ESI) mass spectra were acquired in the positive ion mode on a LCQ DECAXP instrument (Thermo Finnigan, San Jose, CA, USA) equipped with an ion trap mass analyzer. HR-ESI–MS were obtained in the positive ion mode on Waters UPLC Premir Q-TOF (Waters Inc., Milford, MA, USA). CD spectra were obtained on an Olis DSM 1000 spectrometer (Olis Inc., Augusta, GA, USA). Optical rotations were acquired on Shenguang SGW-1 digital polarimeter (Jingke, Shanghai, China). GC–MS were obtained on Thermo Finnigan Trace DSQ (TR-5MS column: 60 m 0.25 mm 2.5 mm). TLC plates were HSGF254 SiO2 from Yantai Jiangyou Silica Gel Development Co., Ltd., China. Column chromatography (CC) silica gel (SiO2; 200–300 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex
6
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Table 2 NMR of 3 and 6 (400 MHz for 1H, MeOH-d4, 100 MHz for
13
C, MeOH-d4).
3 C
1
1 2 3 4 5 6 7 8 9
– 6.67(1H, s) – – – 6.67(1H, s) 5.49(1H, d, J = 6.3 Hz) 3.51–3.56(1H, m) 3.80–3.84(1H, m) 3.74–3.76(1H, m) – 6.74(1H, d, J = 3.7 Hz) – – – 6.74(1H, d, J = 3.7 Hz) 2.67(2H, t, J = 7.6 Hz) 1.86–1.93(2H, m) 3.91–3.95(1H, m) 3.51–3.56(1H, m) 4.25(1H, d, J = 7.8 Hz) 3.17–3.32(1H, m) 3.17–3.32(1H, m) 3.17–3.32(1H, m) 3.17–3.32(1H, m) 3.87(1H, dd, J = 1.6, 12.0 Hz) 3.65(1H, dd, J = 5.0, 12.0 Hz) 3.80(6H, s) 3.86(3H, s)
C-10 C-20 C-30 C-40 C-50 C-60 C-70 C-80 C-90 C-1” C-2” C-3” C-4” C-5” C-6” 3,5-OMe 30 -OMe
H
LH-20 (GE-Healthcare Bio-Sciences AB, Uppsala, Sweden), ODS (Grace C18, Grace Davison Discovery Sciences, Columbia, MD, USA) were employed as packing materials, semi-preparative HPLC (Grace Prevail C18 column, 5 mm, 10.0 mm I.D x 250 mm, Grace Davison Discovery Sciences, Columbia, MD, USA). All other chemicals were of analytical reagent grade. 3.2. Plant material The rhizomes of S. trinervula were collected from Yichun City, Jiangxi Province, China in May 2011, and identified by Professor Chengxin Fu (Laboratory of Systematic and Evolutionary Botany and Biodiversity, College of Life Sciences, Zhejiang University). A voucher specimen (No.20110502) was deposited at the Key Laboratory of Modern Preparation of TCM, Jiangxi University of Traditional Chinese Medicine, China. 3.3. Extraction and isolation The rhizomes of S. trinervula (30 kg) were extracted with 70% (v/v) aqueous ethanol. The 70% EtOH extract was concentrated under reduced pressure to give a residue (2705 g), which was extracted with EtOAc and n-BuOH, respectively. The EtOAc extract was concentrated to yield to the EtOAc fraction 249.2 g, and the nBuOH extract was concentrated to get the n-BuOH fraction 470.3 g. Part of the dried n-BuOH extract (about 470 g) was subjected to column chromatography using macroporous resins column chromatography (6 kg, 20*50 cm) with gradient mixtures of H2O/EtOH. Starting the elution with H2O (Fraction A, 3 l), and reducing polarity by adding EtOH (30%-95%, Fraction B-D, 2 l, each). Fractions were evaporated, the weight of Fractions A-E were 203 g, 65 g, 106 g, 61 g and 20 g, respectively. Fr. B (60 g) was further divided into five parts by MCI GEL CHP20P column chromatography (500 ml, 6*26 cm). Starting the elution with 20% MeOH (Fr.B1,
6 13
1
C
134.1 104.2 149.4 137.0 149.4 104.2 89.1 55.6 65.0 136.4 114.3 145.2 147.5 129.8 118.0 32.94 32.98 69.9 104.5 75.2 77.9 71.7 78.2 62.8 56.84 56.78
H
– 6.73(1H, s) – – – 6.73(1H, s) 5.56(1H, d, J = 5.8 Hz) 3.60–3.67 (1H, m) 3.84–3.89(1H, m) 3.72–3.79(1H, m) – 6.73(1H, s) – – – 6.71(1H, s) 2.62(2H, t, J = 8.0 Hz) 1.77–1.84(2H, m) 3.56(2H, t, J = 6.5 Hz)
3.39–3.49(1H, m) 3.39–3.49(1H, m) 3.39–3.49(1H, m) 3.17–3.21(1H, m) 3.72–3.79(1H, m) 3.64(1H, dd, J = 5.0, 11.9 Hz) 3.81(6H, s) 3.87(3H, s)
13
C
140.3 104.5 154.4 135.7 154.4 104.5 88.5 55.8 65.1 137.2 114.3 145.3 147.5 129.5 118.0 32.9 35.8 62.2 105.3 75.7 77.8 71.4 78.3 62.6 57.0 56.8
600 ml), and reducing polarity by adding MeOH (40%-100%, Fr.B25, 600 ml, each). Fractions were evaporated. Fr.B-4 (80% MeOH, 5 g) and Fr.B5 (100% MeOH, 3 g) were combined after inspection of analytical TLC (CHCl3/MeOH 20:1). Fr.B-2 (7.9 g) was further subjected to silica gel column chromatography (200 g, 5* 50 cm) with gradient mixtures CHCl3/MeOH (15:1, 10:1, 5:1, 1:1, 800 ml, each). Each 100 ml elution was collected and evaporated, and some elution was combined by inspection of analytical TLC (CHCl3/ MeOH 8:1). Finally, ten fractions (Fr.B2–1 10) were got. Fr.B2-2 (235 mg) was further purified by semipreparative HPLC (11% Acetonitrile/H2O, 3.0 ml/min) to yield 1 (8.2 mg, tR17.2 min), 2 (11 mg, tR23.7 min), 3 (8.7 mg, tR29.5 min). Fr.B2-3 (905 mg) was further purified by Sephadex LH-20 (MeOH). Each 20 ml elution was collected, and the same elution was combined by the TLC inspection. Two main fractions (Fr.B2-3-4, 97 mg, and Fr.B-2-3-8, 59 mg) were got. Fr.B2-3-4 (97 mg) was subjected to semipreparative HPLC (12% Acetonitrile/H2O, 3.0 ml/min) to yield 4 (5.1 mg, tR21.2 min), 5 (6.4 mg, tR27.6 min), 6 (9 mg, tR31.1 min). Fr. B2-3-8 (59 mg) was further purified by semipreparative H PLC (12.5% Acetonitrile/H2O, 3.0 ml/min) to yield 7 (9.2 mg, tR15.8 min), and 8 (8 mg, tR22.4 min). 3.3.1. (7S, 8R,80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 epoxylignan-70 -one 40 -O-b-D- glucopyranoside (1) White powder (MeOH); [a]25 D 31.41 (c 0.191, MeOH); UV (MeOH) lmax (loge) 280 (4.94) nm; HR-ESI–MS m/z 595.2023 [M] (calcd for C28H35O14); 1H NMR and 13C NMR see Table 1. 3.3.2. (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 epoxylignan-70 -one 4-O-b-D- glucopyranoside (2) White powder (MeOH); [a]25 D 61.73 (c 0.243, MeOH); UV (MeOH) lmax (loge) 225 (4.95), 305 (4.89) nm; HR-ESI–MS m/z 595.2035 [M] (calcd for C28H35O14); 1H NMR and 13C NMR see Table 1.
J. Shu et al. / Phytochemistry Letters 20 (2017) 1–8 Table 3 NMR of 4 and 5 (400 MHz for 1H, MeOH-d4, 100 MHz for
13
C, MeOH-d4).
4
5
C
1
13
1 2 3 4 5 6 7 8 9
– 6.71(1H, s) – – – 6.71(1H, s) 4.83(1H, d, J = 8.0 Hz) 4.04(1H,ddd, J = 1.8, 4.3, 8.0 Hz) 3.70-3.74 (2H, m)
128.7 105.9 149.5 137.3 149.5 105.9 77.9 79.8 62.2
C-10 C-20 C-30 C-40 C-50 C-60 C-70 C-80 C-90 C-1” C-2” C-3” C-4” C-5” C-6”
– 6.68(1H, d, J = 1.9 Hz) – – – 6.50(1H, d, J = 1.9 Hz) 2.59(2H, t, J = 7.5 Hz) 1.76-1.83(2H, m) 3.55(2H, t, J = 6.5 Hz) 4.91(1H, d, J = 7.6 Hz) 3.36–3.53 (1H, m) 3.36–3.53 (1H, m) 3.36–3.53 (1H, m) 3.36–3.53 (1H, m) 3.90 (1H, dd, J = 2.1,12.0 Hz) 3.70 (1H, dd, J = 5.5, 12.0 Hz) 3.86 (6H, s) –
135.7 111.1 147.5 133.3 145.7 112.1 32.7 35.3 61.9 103.2 75.0 77.9 71.5 78.3 62.6
3,5-OMe 30 - OMe
7
H
3.3.3. (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy-40 , 7-epoxy-8, 50 neolignan 90 -O-b-D- glucopyranoside (3) White powder (MeOH); [a]25 D 5.41 (0.185, MeOH); UV (MeOH) lmax (loge) 230(4.98), 280(4.35) nm; HR-ESI–MS m/z 551.2131 [M] (calcd for C27H35O12); 1H NMR and 13C NMR see Table 2. 3.3.4. (7R, 8R)-4, 9, 90 -trihydroxy-3, 5-dimethoxy-7.O.40 , 8.O.30 neolignan 90 -O-b-D- glucopyranoside (4) 34.34 (0.182, MeOH); UV White powder (MeOH); [a]25 D (MeOH) lmax (loge) 225 (4.90), 275 (3.85) nm; HR-ESI–MS m/z 553.1918 [M] (calcd for C26H34O13); 1H NMR and 13C NMR see Table 3. 3.3.5. (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy-8, 40 -oxyneolignan 4-O-b-D- glucopyranoside (5) White powder (MeOH); [a]25 D 30.56 (c 0.319, MeOH); UV (MeOH) lmax (loge) 220 (4.25), 280 (3.55) nm; HR-ESI–MS m/z 593.1836 [M + Na]+ (calcd for C27H38O13Na); 1H NMR and 13C NMR see Table 3. 3.4. Cytotoxicity activity The cytotoxicity of the isolated compounds (The purity of compounds 1–8 were 95.5%, 93.5%, 91.7%, 94.5%, 98.6%, 97.2%, 96.6% and 95.0%, respectively.) 2 against 4 human tumor cell lines (SH-SY5Y, SGC-7901, HCT-116, Lovo) was evaluated using the MTT method. Cells were seeded in 96-well microplates at a density of 150 per well and were cultured in cell culture medium (RPMI1640 medium supplemented with 10% FBS, 100 U/mL penicillin and 100 g/mL streptomycin) for 12 h, then treated with the test compounds added from DMSO dissolved stock solution. The final DMSO concentration never exceeded 0.2% (v/v). Previous experiments showed that DMSO at this concentration did not modify the cell activities. After 48 h in culture, cells were incubated with MTT (0.5 mg/mL, 4 h) and subsequently resolved in DMSO. The
1
C
56.9 –
H
– 6.76(1H, s) – – – 6.76(1H, s) 4.82(1H, d, J = 6.0 Hz) 4.30(1H, ddd, J = 3.9, 5.4, 6.0 Hz) 3.85 (1H, dd, J = 5.4, 12.0 Hz) 3.72-3.82 (1H, m) – 6.77(1H, d, J = 1.9 Hz) – – 6.79(1H, d, J = 8.2 Hz) 6.65(1H, dd, J = 2.0, 8.2 Hz) 2.59 (2H, t, J = 7.5 Hz) 1.78 (2H, m) 3.54 (2H, t, J = 6.5 Hz) 4.78(1H, d, J = 7.4 Hz) 3.39–3.46(1H, m) 3.39–3.46(1H, m) 3.39–3.46(1H, m) 3.15–3.20 (1H, m) 3.72-3.82 (1H, m) 3.66 (1H, dd, J = 5.1, 12.0 Hz) 3.80(6H, s) 3.76(3H, s)
13
C
139.6 106.3 153.9 135.6 153.9 106.3 74.1 86.1 62.2 138.0 114.0 151.7 147.2 119.2 121.7 32.7 35.5 62.2 105.6 75.7 77.8 71.3 78.4 62.6 57.0 56.5
absorbance in control and drug treated wells was measured in an automated microplate reader at 570/630 nm. All experiments were carried out in triplicate and repeated twice. The cytotoxicity was expressed as IC50 values (50% inhibitory concentration). 3.5. Acid hydrolysis of 1-5 The absolute configuration of glucose was determined as described by Xin et al. Compounds 1-5 (respectively, 2 mg) were heated with 1 mol/L HCl (2 ml) for 4 h at 105 C. The mixture was cooled, neutralized, and partitioned between AcOEt (2 ml) and H2O (2 ml). The aq. layer was evaporated and contained in a vial. The following solns. were added: a) (2S)-1-aminopropan-2-ol/MeOH 1:8 (20 ml); b) AcOH/MeOH 1:4 (17 ml); c) 3% Na[BH3CN] in MeOH (13 ml). The vial was capped, and the mixture was allowed to react for 2 h at 65 C. After cooling, 3 mol/L aq. CF3COOH was added dropwise until the pH dropped to pH 1–2. The mixture was evaporated and co-evaporated with H2O (3 0.5 ml) and MeOH (5 0.5 ml). The residue was dried overnight in a vacuum desiccator and treated with pyridine/Ac2O 1:1 for 1 h at 100 C. After cooling, the mixture was extracted with CHCl3 and the extract washed with H2O (3 1 ml) and sat. NaHCO3 soln. (3 1 ml). The org. phase was dried (Na2SO4) and subjected to GC/MS. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (NSFC) (Nos. 31370376, 81360630 and 81360628) and Natural Science Foundation of Jiangxi Province (No. 20142BCB23023 and 20161BAB205219). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2017.03.002.
8
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Reference: Arnoldi, A., Merlini, L., 1985. Asymmetric synthesis of 3-methyl-2-pheyl-1, 4benzodioxanes. Absolute configuration of the neolignans eusiderin and eusiderin C and D. J. Chem. Soc. Perkin Trans. I 6, 2555–2557. Braga, A.C.H., Zacchina, S., Badano, H., Sierra, M.G., Rúveda, E.A., 1984. 13C NMR spectral and conformational analysis of 8-O-4' neolignans. Phytochemistry 23, 2025–2028. Cai, W., Matsunami, K., Otsuka, H., Takeda, Y., 2011. Symplocosionosides A-C, Three megastigmane glycosides, a neolignan glucoside, and symplocosins A and B, Two triterpene glycosyl esters from the leaves of Symplocos cochinchinensis var. philippinensis. AJPS 2, 609–618. Chen, D.S., Liu, Y.N., Fu, L., 2001. Study on the pharmacologicalaction of the extracts from Smilax china L. on promoting blood circulation of remove blood stasis. J. China Pharm. Univ. 21, 448–450. Chin, Y.W., Chai, H.B., Keller, W.J., Kinghorn, A.D., 2008. Lignans and other consituents of the fruits of Euterpe oleracea (Acai) with antioxidant and cytoprotective activities. J. Agric. Food Chem. 56, 7759–7764. Chinese Pharmacopoeia Committee, 2010. Chinese Pharmacopoeia, vol. 1. China Medical Science Press, Beijing, pp. 290. Da Silva, M.S., Barbosa-Filho, J.M., Yoshida, M., Gottlieb, O.R., 1989. Benzodioxane and b–arylpropane type neolignans from licaria chrysophylla. Phytochemistry 28, 3477–3482. Flora of China Editorial Committee, 2004. Flora of China, vol. 15. Science Press, Beijing, pp. 196. García-Muñoz, S., Álvarez-Corral, M., Jiménez-González, L., López-Sánchez, C., Rosales, A., Muñoz-Dorado, M., Rodríguez-García, I., 2006. Synthesis of dihydrodehydrodiconiferyl alcohol and derivatives through intramolecular CH insertion. Tetrahedron 62, 12182–12190. Guan, S.H., Xia, J.M., Lu, Z.Q., Chen, G.T., Jiang, B.H., Liu, X., Guo, D.A., 2008. Structure elucidation and NMR spectral assignments of three new lignan glycosides from Akebia trifoliate. Magn. Reson. Chem. 46, 186–190. He, Z.D., Ma, C.Y., Tan, G.T., Sydara, K., Tamez, P., Southavong, B., Bouamanivong, S., Soejarto, D.D., Pezzuto, J.M., Fong, H.H., Zhang, H.J., 2006. Rourinoside and rouremin, antimalarial constituents from Rourea minor. Phytochemistry 67, 1378–1384. Huang, Z.Q., Huang, Y.R., 2000. Clinical application of single Smilax china L. on prolong life of the patients suffer from liver neoplasms. Chin. Natl. Folk Med. J. 45, 212–213. Huang, H.L., Lu, Z.Q., Chen, G.T., Zhang, J.Q., Wang, W., Yang, M., Guo, D.A., 2007. Phenylpropanoid subsitituted catechins and epicatechins from Smilax china. Helv. Chim. Acta 90, 1751–1757. Huang, H.L., Liang, J., Shu, J.C., Liang, Y.H., Li, FQ., 2015. Use of steroidal saponins compounds isolated from Smilax trinervula for anti-tumor. Chinese patent, NO. 2015101757780.
Huo, C., Liang, H., Zhao, Y., Wang, B., Zhang, Q., 2008. Neolignan glycosides from symplocos caudata. Phytochemistry 69, 788–795. Ishikawa, T., Fujimatu, E., Kitajima, J., 2002. Water-soluble constituents of anise: new glucoside of anethole glycol and its related compounds. Chem. Pharm. Bull. 50, 1460–1466. Jiménez-González, L., Álvarez-Corral, M., Muñoz-Dorado, M., Rodríguez-García, I., 2005. A concise and diasteroselective total synthesis of cis and transpterocarpans. Chem. Commun. 2689–2691. Jung, K.Y., Kim, D.S., Oh, S.Y., Park, S.H., Lee, I.S., Lee, J.J., Shin, D.H., Lee, H.K., 1998. Magnones A and B, novel anti-PAF tetrahydrofuran lignans from the flower buds of Magnolia fargesii. J. Nat. Prod. 61, 808–811. Kónya, K., Kurtán, T., Kiss-Szikszai, A., Juhász, L., Antus, S., 2004. A general CDmethod for the configurational assignment of erythro-8, 4'-oxyneolign-8'-enes. Arkat: Arkivoc 8, 72–78. Kim, S.W., Son, K.H., Chung, K.C., 1989. Mutagenic effect of steroidal saponins from Smilax china rhizomes. Yakhak Hoechi 33, 285–289. Li, Y.L., Gan, G.P., Zhang, H.Z., Wu, H.Z., Li, C.L., Huang, Y.P., Liu, Y.W., Liu, J.W., 2007. A flavonoid glycoside isolated from Smilax china L. rhizome in vitro anticancer effects on human cancer cell lines. J. Ethnopharmacol. 113, 115–124. Liang, Y., Yao, S., Liang, F., Liang, J., Huang, G., Liu, M., Huang, H., 2015. Structural characterization of steroidal saponins from Smilax trinervula using ultra highperformance liquid chromatography coupled with LTQ-Orbitrap mass spectrometry. J. Chromatogr. B 974, 75–82. Mohamed, K.M., 2001. Phenylpropanoid glucosides from chrozophora obliqua. Phytochemistry 58, 615–618. Qin, Y., Yin, C., Cheng, Z., 2013. A new tetrahydrofuran lignan diglycoside from Viola tianshanica. Maxim. Molecules 18, 13636–13644. Shao, B., Guo, H., Cui, Y., Ye, M., Han, J., Guo, D., 2007. Steroidal saponins from Smilax china and their anti-inflammatory activities. Phytochemistry 68, 623–630. Shu, X.S., Gao, Z.H., Yang, X.L., 2006. Anti-inflammatory and antinociceptive activities of Smilax china L. aqueous extract. J. Ethnopharmacol. 103, 327–332. Shu, J., Liang, F., Liang, J., Liang, Y., Li, F., Shao, F., Liu, R., Huang, H., 2015. Phenylpropanoids and neolignans from Smilax trinervula. Fitoterapia 104, 64– 68. Tsukamoto, Y., 1998. The Grand Dictionary of Horticulture, vol. 2. Shogakukan, Tokyo, pp. 507. Wang, Y.H., Sun, Q.Y., Yang, F.M., Long, C.L., Zhao, F.W., Tang, G.H., Niu, H.M., Wang, H., Huang, Q.Q., Xu, J.J., Ma, L.J., 2010. Neolignans and caffeoyl derivatives from selaginella moellendorffii. Helv. Chim. Acta 93, 2467–2477. Xin, W.B., Chou, G.X., Wang, Z.T., 2009. Triterpenoids and saponins from leaves of Uncaria hirsuta. Helv. Chim. Acta 92, 638–644. Yang Kuo, L.M., Zhang, L.J., Huang, H.T., Lin, Z.H., Liaw, C.C., Cheng, H.L., Lee, K.H., Morris- Natschke, S.L., Kuo, Y.H., Ho, H.O., 2013. Antioxidant lignans and chromone glycosides from Eurya japonica. J. Nat. Prod. 76, 580–587.
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Lignan glycosides from the Rhizomes of Smilax trinervula and their Biological activities Jicheng Shua , Fang Lianga,b , Genhua Zhua , Xing Liua , Jiangli Yua , Huilian Huanga,* a b
Key Laboratory of Modern Preparation of TCM, Jiangxi University of Traditional Chinese Medicine, Ministry of Education, Nanchang 330004, China Jiangxi Pharmaceutical School, Nanchang 330004, China
A R T I C L E I N F O
Article history: Received 22 July 2016 Received in revised form 5 February 2017 Accepted 1 March 2017 Available online xxx Keywords: Smilax trinervula Smilacaceae Lignan glycosides Cytotoxic activity
A B S T R A C T
Phytochemical investigation of the rhizomes of Smilax trinervula led to isolation and structure elucidation of eight lignan glycosides, including five new lignans, namely, (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 tetramethoxy-7, 90 -epoxylignan-70 -one 40 -O-b-D-glucopyranoside (1), (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 -epoxylignan-70 -one 4-O-b-D- glucopyranoside (2) (7S, 8R)-4, 9, 90 -trihydroxy3, 30 , 5-trimethoxy-40 , 7-epoxy-8, 50 -neolignan 90 -O-b-D-glucopyranoside (3), (7R, 8R)-4, 9, 90 trihydroxy-3, 5-dimethoxy-7.O.40 , 8.O.30 - neolignan 90 -O-b-D-glucopyranoside (4), and (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy-8, 40 -oxy-neolignan 4-O-b-D-glucopyranoside (5), along with three known compounds (6-8). Their structures were established mainly on the basis of 1D and 2D NMR spectral data, ESI–MS and comparison with the literature. Compounds 1-8 were tested in vitro for their cytotoxic activity against four human tumor cell lines (SH-SY5Y, SGC-7901, HCT-116, Lovo). Compounds 3 and 5 exhibited cytotoxic activity against Lovo cells, with IC50 value of 10.4 mM and 8.5 mM, respectively. © 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
1. Introduction The genus Smilax (Smilacaceae) comprises about 370 species, which are mainly distributed in the tropical and temperate zones throughout the world, especially in East Asia and North America (Tsukamoto, 1998). There are about 200 species of the genera in China (Flora of China Editorial Committee, 2004). Smilax is commonly known as “Ba-Qia” or “Jin-Gang-Teng” in Chinese. However, only Smilax china is used to expel wind, drain off dampness and remove carbuncles (Chinese Pharmacopoeia Committee, 2010). In recent years, detailed research on the phytochemistry and pharmacology of this genus focused on Smilax china. Previous phytochemistral investigations have resulted in the isolation and identification of characteristic steroidal saponins, lignans and various phenols (Shao et al., 2007; Huang et al., 2007; Li et al., 2007). Various pharmacological properties, such as antiinflammatory, antimicrobial, antinociceptive, neuroprotective and antitumor activity, have been reported (Huang and Huang, 2000; Shu et al., 2006; Kim et al., 1989; Chen et al., 2001). However, to the best of our knowledge, until now, only two studies have been reported on Smilax trinervula (S. trinervual)
* Corresponding author. E-mail address: [email protected] (H. Huang).
(Liang et al., 2015; Huang et al., 2015). As a member of the Smilax genera, S. trinervula may possess some pharmacological activity and active substances similar to Smilax china. As a part of our ongoing search of potentially bioactive components from the genus Smilax, we have systematically investigated the constituents of S. trinervula. Our previous studies on the EtOAc fraction of the ethanol extract of the rhizomes of S. trinervula have led to the isolation of several new phenylpropanoids (Shu et al., 2015). A further detailed investigation of the n-BuOH fraction of the same extract has resulted in the isolation of 5 new lignan glycosides (15), along with three known lignan glycosides (6-8). 2. Results and discussion The n-BuOH soluble part partitioned from the ethanol extract of S. trinervula was fractionated by a macroporous resin column to give five fractions A, B, C, D and E. Fraction B was subfractionated by a combination of silica gel column chromatography, Sephadex LH20 column chromatography and preparative HPLC to give eight lignan glycosides, including five new lignan glycosides, namely, (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 epoxylignan- 70 -one 40 -O-b-D-glucopyranoside (1), (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 -epoxylignan-70 -one 4-O-b-D-glucopyranoside (2) (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5trimethoxy-40 , 7-epoxy-8, 50 -neolignan 90 -O-b-D-glucopyranoside
http://dx.doi.org/10.1016/j.phytol.2017.03.002 1874-3900/© 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
2
J. Shu et al. / Phytochemistry Letters 20 (2017) 1–8
(3), (7R, 8R)-4, 9, 90 - trihydroxy-3, 5-dimethoxy-7.O.40 , 8.O.30 neolignan 90 -O-b-D-glucopyranoside (4), and (7S, 8R)-4, 9, 90 trihydroxy-3, 30 , 5-trimethoxy-8, 40 -oxy-neolignan 4-O-b-D-glucopyranoside (5), and three known lignan glycosides, (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy-40 , 7-epoxy-8, 50 - neolignan 4-Ob-D-glucopyranoside (6) (Wang et al., 2010), symplocosneolignan (7) (Cai et al., 2011) and rourinoside (8) (He et al., 2006), respectively (Fig. 1). Compound 1 was isolated as an amorphous powder. The molecular formula C28H36O14 of 1 was deduced from HR-ESI–MS spectrometry (see Experimental section). In the 1H NMR and 13C NMR spectrum of 1 (Table 1), two pairs of geminated aromatic proton signals at dH 6.70 (2H, s) and 7.39 (2H, s), assigned to H-2/6 (signals at dc 105.3) and H-20 /60 (signals at dc 107.9), together with the other four quaternary aromatic carbons at dc 133.8, 136.2, 132.9 and 141.0, suggested the presence of two tetrasubstituted symmetrical benzene rings. These substituents were four MeO groups, resonating at dH 3.85 (2 MeO) and 3.92 (2 MeO). Signals at dH 5.11 (1H, d, J = 7.5 Hz) with dC104.4 corresponding to the anomeric proton and anomeric carbon, along with carbon signals at dc 75.8, 78.5, 71.4, 77.9, 62.6 and proton signals in the region of dH
3.20–3.51, suggested 1 possess a b-glucopyranosyl unit (Mohamed, 2001). Furthermore, the sugar was identified by GC– MS analysis after acid hydrolysis (Xin et al., 2009). The glucopyranosyl moiety was embedded at C-40 supported by the HMBC correlation (Fig. 2) between the anomeric proton at dH 5.11 (H-1”) and aromatic carbon at dc 141.0 (C-40 ). In addition, the carbon signals at dc 85.4 (d), 55.1 (d), 61.7 (t), 50.6 (d), 71.2 (t), together with a carbonyl carbon signals at dc 200.6 were assigned to a hydroxymethyl-substituted tetrahydrofuranoid. From the above observations, 1 was identified as 4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 -epoxylignan-70 -one 40 -O-b-D- glucopyranoside (Guan et al., 2008). The absolute stereochemistry was determined by a ROESY experiment and comparison with the literature. Cis and trans orientation of the substituents at C-7 and C8 would give signals of H-7 at ca dH 5.5 and dH 4.7, respectively (Jung et al., 1998). The H-7 signal of 1 was observed at dH 4.68, that C-7 and C-8 were in the trans configuration of substituents. The ROESY spectrum (Fig. 3) displayed the correlation between the proton signals at dH 2.64 (H-8) and dH 4.26 (H-80 ), but was not observed between the proton signals at dH 2.64 (H-8) and dH 4.63 (H-7), indicated the assignments of same ring-side position (cis)
Fig. 1. Chemical structure of compounds 1-8.
J. Shu et al. / Phytochemistry Letters 20 (2017) 1–8 Table 1 NMR of 1 and 2 (400 MHz for 1H, MeOH-d4, 100 MHz for
3
13
C, MeOH-d4).
1
2
C
1
13
1 2 3 4 5 6 7 8 9
– 6.70 (s) – – – 6.70 (s) 4.63 (d, J = 7.9 Hz) 2.64 (dddd, 2.7, 4.7, 5.6, 7.9 Hz) 3.73 (dd, J = 4.7, 11.4 Hz) 3.66 (dd, J = 5.6, 11.4 Hz) – 7.39 (s) – – – 7.39 (s) – 4.26 (m) 4.26 (m) 4.18 (d, J = 9.6 Hz) 5.11 (d, J = 7.5 Hz) 3.47–3.51 (m) 3.39–3.44 (m) 3.39–3.44 (m) 3.20–3.24 (m) 3.76 (dd, J = 2.3, 12 Hz) 3.63 (dd, J = 5.3, 12 Hz) 3.85 (s) 3.92 (s)
133.8 105.3 149.3 136.2 149.3 105.3 85.4 55.1 61.7
– 6.78 (s) – – – 6.78 (s) 4.68 (d, J = 7.9 Hz) 2.64 (m) 3.69–3.71 (2H, m)
139.4 105.6 149.2 135.7 149.2 105.6 85.1 55.2 61.6
132.9 107.9 154.4 141.0 154.4 107.9 200.6 50.6 71.2
– 7.39 (s) – – – 7.39 (s) – 4.23–4.28 (m) 4.23-4.28 (m) 4.21 (dd, J = 2.9, 9.5 Hz) 4.85 (d, J = 7.5 Hz) 3.45–3.49 (m) 3.39–3.42 (m) 3.39–3.42 (m) 3.18–3.22 (m) 3.76 (dd, J = 2.4, 12 Hz) 3.65 (dd, J = 5.2, 12 Hz) 3.86 (s) 3.92 (s)
128.8 107.8 154.3 143 154.3 107.8 200.1 50.3 71.7
C-10 C-20 C-30 C-40 C-50 C-60 C-70 C-80 C-90 C-1” C-2” C-3” C-4” C-5” C-6” 3,5-OMe 30 ,50 -OMe
H
and trans orientations for H-8/H-80 and H-7/H-8, respectively. In addition, the characteristic coupling constant of H-7 and H-8 (7.9 Hz) in 1 and the negative [a]25 D of 1 measured at 31.41 (0.191, MeOH) were also in accordance with the known compound mentioned (Guan et al., 2008; Qin et al., 2013). Therefore, 1 was determined as (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 tetramethoxy-7, 90 -epoxylignan-70 -one 40 -O-b-D- glucopyranoside. However, Guan et al. (Guan et al., 2008) reported one analogous lignan glycoside from Akebia trifoliate. The authors proposed the wrong conclusion because of the trans orientations for H-7/H-8. By reconsidering structure and data afforded by authors, the compound 2 reported in the literature should be (7S, 8R,80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 -epoxylignan-70 -one 9-O-b-D- glucopyranoside, not be (7S, 8S, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 - epoxylignan-70 -one 9-O-b-D- glucopyranoside. Compound 2 was isolated as an amorphous powder. HR-ESI–MS spectrometry showed the same molecular formula as that of 1 [595.2035 [M] (calcd for C28H35O14)]. The 1H NMR and 13C NMR spectrum of 2 (Table 1) were very similar with those of 1. The above information suggested 2 was an isomer of 1. By comparing and analyzing their NMR data, the obvious differences were the chemical shifts of C-1, C-4, C-10, C-40 , H-1”, C-1”. The difference was the location of glucopyranosyl unit. 2 was placed at C-4. This was deduced by an HMBC experiment (Fig. 2), which showed a correlation between the anomeric proton at dH 4.85 and C-4 at dC 135.7. Thus, 2 was identified as (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 -epoxylignan-70 -one 4-O-b-D- glucopyranoside. Compound 3 was obtained as an amorphous powder. HR-ESI– MS spectrometry (see Experimental part) showed the molecular formula C27H36O12. In the 1H NMR and 13C NMR spectrum of 3
C
104.4 75.8 77.9 71.4 78.5 62.6 57.2 56.8
1
H
13
C
105.4 75.7 77.8 71.4 78.3 62.6 56.9 57
(Table 2), 18 signals for 12 aromatic carbons at dc 134.1 (s), 104.2 (2*s), 149.4 (2*s), 137.0 (s), 136.4 (s), 114.3 (s), 145.2 (s), 147.5 (s), 129.8 (s), 118.0 (s), and 6 alphatic carbons at dc 89.1 (d), 55.6 (d), 65.0 (t), 32.94 (t), 32.98 (t) and 69.9 (t) were observed. This suggested that this compound might be a dihydrobenzofuran neolignan (García-Muñoz et al., 2006; Chin et al., 2008). In addition, signals at dH 4.25 (1H, d, J = 7.8 Hz) with dC 104.5 corresponding to the anomeric proton and anomeric carbon, along with carbon signals at dc 75.2, 78.2, 71.7, 77.9 62.8 and proton signals in the region of dH 3.17–3.32, suggested that 3 had a b-glucopyranosyl unit (Mohamed, 2001). Furthermore, the sugar was identified by GC–MS analysis after acid hydrolysis (Xin et al., 2009). The above observaions together with careful comparison of the NMR data of 3 with those of the known neolignan compound 6 indicated that 3 was 4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy-40 , 7epoxy-8, 50 -neolignan 90 -O-b-D- glucopyranoside. It was confirmed by the HMBC spectrum (Fig. 2). The HMBC correlation peaks MeO and C-3, MeO and C-5, MeO and C-30 , and the anomeric proton resonance of glucose H-1” to C-90 supported the above observations. The stereochemistry of C-7 and C-8 was assigned as trans by the ROESY correlations of H-7/H-9. One the other hand, a significant difference in chemical shifts of C-8 in 9-hydroxydihydrobenzofuran neolignans, appearing at dc ca. 54 in the trans isomers and at dc ca. 49 in the cis ones (García-Muñoz et al., 2006, Jiménez-González et al., 2005), is a reliable criterion to distinguish both diastereoisomers. The C-8 signal of 3 was observed at dc 55.6, that implied C-7 and C-8 had a trans configuration of substituents. In addition, the characteristic coupling constant of H-7 and H-8 (6.3 Hz) in 3 and the negative [a]25 D of 3 measured at 5.41 (0.185, MeOH) were also in accordance with the known compound mentioned (Wang et al., 2010). Therefore, 3 was determined as (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy-40 , 7-epoxy-8, 50 -neolignan 90 -O-b-D- glucopyranoside.
4
J. Shu et al. / Phytochemistry Letters 20 (2017) 1–8
Fig. 2. Key HMBC of Compounds 1-5.
Compound 4 was obtained as an amorphous powder. HR-ESI– MS spectrometry (see Experimental part) showed the molecular formula C26H34O13. In the 1H NMR and 13C NMR spectrum of 4 (Table 3), a pairs of geminated aromatic proton signals at dH 6.71 (2H, s), assigned to H-2/6 (signals at dc 105.9), two meta-coupled aromatic proton signals at dH 6.68 (1H, d, J = 1.9 Hz) and 6.50 (1H, d, J = 1.9 Hz), together with the other seven quaternary aromatic carbons at dc 128.7, 149.5, 137.3, 135.7, 147.5, 133.3 and 145.7, suggested the presence of two tetrasubstituted symmetrical and unsymmetrical benzene rings. On the other hand, the 13C NMR spectrum showed 6 alphatic carbons at dc 77.9 (d), 79.8 (d), 62.2 (t), 32.7 (t), 35.3 (t) and 61.9 (t) suggesting that this compound might be a neolignan. In addition, the 1H NMR spectrum showed a resonance at dH 4.91 (d, J = 7.6 Hz) corresponding to the anomeric proton of a b-D- glucopyranose moiety, and the 13C NMR spectrum also exhibited the corresponding carbon resonances at dc 103.2 (d), 75.0 (d), 77.9 (d), 71.5 (d), 78.3 (d) and 62.6 (t). Furthermore, the sugar was identified by GC–MS analysis after acid hydrolysis (Xin et al., 2009). The above observations together with careful comparison of the NMR data of 3 indicated that 4 was a lignan glucoside, with great similarity to the known compound eusiderin N (Yang Kuo et al., 2013). The obvious difference was the chemical shift of C-9. A resonance at dH 1.19 (3H, d, J = 6.6 Hz) in eusiderin N was replaced by a signal at dH 3.72 (2H, m) in 4 in the 1H NMR, and in the corresponding 13C NMR spectrum, a signal at dc 17.4 (q) in eusiderin N was substituted for a signal at dc 62.2 (t), which
suggested that C-9 was substituted by a hydroxyl group. This was supported by the analysis of the molecular formula and the HMBC correlation between the anomeric proton at dH 3.72 (H-9) and the alphatic carbon at dc 79.8 (C-8) and 77.9 (C-7) (Fig. 2). The stereochemistry of C-7 and C-8 was assigned as trans by the ROESY correlations of H-7/H-9 (Fig. 3). One the other hand, the characteristic coupling constant of H-7 and H-8 (8.0 Hz) in 4 and the negative [a]25 D of 4 measured at 34.34 (0.182, MeOH) were also in accordance with the known compound mentioned (Yang Kuo et al., 2013; Da Silva et al., 1989). Therefore, 4 was determined as (7R, 8R)-4, 9, 90 -trihydroxy-3, 5-dimethoxy-7.O.40 , 8. O.30 -neolignan 90 -O-b-D- glucopyranoside. Compound 5 was isolated as an amorphous powder and its molecular formula was determined to be C27H38O13 by HR-ESI–MS (see Experimental section). In the 1H NMR spectrum (Table 3), a singlet signal for two H-atoms in the aromatic region at dH 6.76 (2H, s) assigned to H-2/6, suggested the presence of a 1, 3, 4, 5tetrasubstituted symmetrical benzene ring, and three aromatic protons signals at dH 6.79(d, J = 8.2 Hz), 6.77(d, J = 1.9 Hz) and 6.65 (dd, J = 2.0, 8.2 Hz), indicated that this compound possessed a 1, 3, 4-trisubstituted benzene ring. These substituents were three MeO groups, resonating at dH 3.80 (2 MeO) and 3.76 (MeO). In addition, the 1H NMR and 13C NMR displayed signals at dH 4.78 (1H, d, J = 7.4 Hz) with dC 105.6 corresponding to the anomeric proton and anomeric carbon of a b-glucopyranosyl unit (Mohamed, 2001). Furthermore, the sugar was identified by GC–MS analysis
J. Shu et al. / Phytochemistry Letters 20 (2017) 1–8
5
Fig. 3. Key NOESY correlations of Compounds 1-4.
after acid hydrolysis (Xin et al., 2009). Moreover, the 1H NMR spectrum showed ten proton signals at dH 4.82 (1H, d, J = 6.1 Hz), 4.30 (1H, ddd, J = 3.9, 5.4, 6.1 Hz), 3.85 (1H, dd, J = 5.4, 12.0 Hz), 3.72– 3.82 (1H, m), 2.59 (2H, t, J = 7.5 Hz), 1.78 (2H, m), 3.54 (2H, t, J = 6.5 Hz), suggesting the presence of 1, 2, 3-propanetriol moiety and a 1-propanol moiety. The above evidence suggested the presence of two C6-C3 units arising from both a neolignan and a glucose moiety, which was supported by analysis of the NMR and HMBC spectra (Fig. 2). According to the above information, the NMR spectrum of compound 5 was identical with that of a known compound symplocosneolignan (Cai et al., 2011), but obvious differences were seen in three aromatic protons with ABX coupling system, which suggested that 5 had one less MeO group than symplocosneolignan. This was supported by the analysis of its molecular formula. Judging from the coupling constant (J = 6.0 Hz) for H-7 and H-8, 5 was concluded to possess the erythro relative stereochemistry (Braga et al., 1984). Judging from the negative cotton effect at 240 nm, the absolute configuration of 5 was determined to be 7S, 8R (Cai et al., 2011; Arnoldi and Merlini, 1985; Huo et al., 2008; Kónya et al., 2004; Ishikawa et al., 2002). One the other hand, the negative [a]25 D of 5 measured at 30.56 (0.319, MeOH) were also in accordance with the known compound mentioned (Cai et al., 2011). Therefore, compound 5 was concluded to be (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy- 8, 40 -oxyneolignan 4-O-b-D- glucopyranoside.
Compounds 1-8 were evaluated for cytotoxic activities against four human cell lines (SH-SY5Y, SGC-7901, HCT-116 and Lovo) with Vero as a positive control. Compounds 1-8 were inactive (IC50> 100 mM) to SH-SY5Y, SGC-7901, and HCT-116 cell lines. Compounds 3 and 5 exhibited cytotoxic activity against Lovo cells, with IC50 value of 10.4 mM and 8.5 mM, respectively. 3. Experimental 3.1. General procedure and regents 1
H (400 MHz), 13C (100 MHz), and 2D NMR spectra were obtained on Bruker AV-400 with TMS as internal reference, and using methanol-d4 as solvents. Electrospray ionisation (ESI) mass spectra were acquired in the positive ion mode on a LCQ DECAXP instrument (Thermo Finnigan, San Jose, CA, USA) equipped with an ion trap mass analyzer. HR-ESI–MS were obtained in the positive ion mode on Waters UPLC Premir Q-TOF (Waters Inc., Milford, MA, USA). CD spectra were obtained on an Olis DSM 1000 spectrometer (Olis Inc., Augusta, GA, USA). Optical rotations were acquired on Shenguang SGW-1 digital polarimeter (Jingke, Shanghai, China). GC–MS were obtained on Thermo Finnigan Trace DSQ (TR-5MS column: 60 m 0.25 mm 2.5 mm). TLC plates were HSGF254 SiO2 from Yantai Jiangyou Silica Gel Development Co., Ltd., China. Column chromatography (CC) silica gel (SiO2; 200–300 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex
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J. Shu et al. / Phytochemistry Letters 20 (2017) 1–8
Table 2 NMR of 3 and 6 (400 MHz for 1H, MeOH-d4, 100 MHz for
13
C, MeOH-d4).
3 C
1
1 2 3 4 5 6 7 8 9
– 6.67(1H, s) – – – 6.67(1H, s) 5.49(1H, d, J = 6.3 Hz) 3.51–3.56(1H, m) 3.80–3.84(1H, m) 3.74–3.76(1H, m) – 6.74(1H, d, J = 3.7 Hz) – – – 6.74(1H, d, J = 3.7 Hz) 2.67(2H, t, J = 7.6 Hz) 1.86–1.93(2H, m) 3.91–3.95(1H, m) 3.51–3.56(1H, m) 4.25(1H, d, J = 7.8 Hz) 3.17–3.32(1H, m) 3.17–3.32(1H, m) 3.17–3.32(1H, m) 3.17–3.32(1H, m) 3.87(1H, dd, J = 1.6, 12.0 Hz) 3.65(1H, dd, J = 5.0, 12.0 Hz) 3.80(6H, s) 3.86(3H, s)
C-10 C-20 C-30 C-40 C-50 C-60 C-70 C-80 C-90 C-1” C-2” C-3” C-4” C-5” C-6” 3,5-OMe 30 -OMe
H
LH-20 (GE-Healthcare Bio-Sciences AB, Uppsala, Sweden), ODS (Grace C18, Grace Davison Discovery Sciences, Columbia, MD, USA) were employed as packing materials, semi-preparative HPLC (Grace Prevail C18 column, 5 mm, 10.0 mm I.D x 250 mm, Grace Davison Discovery Sciences, Columbia, MD, USA). All other chemicals were of analytical reagent grade. 3.2. Plant material The rhizomes of S. trinervula were collected from Yichun City, Jiangxi Province, China in May 2011, and identified by Professor Chengxin Fu (Laboratory of Systematic and Evolutionary Botany and Biodiversity, College of Life Sciences, Zhejiang University). A voucher specimen (No.20110502) was deposited at the Key Laboratory of Modern Preparation of TCM, Jiangxi University of Traditional Chinese Medicine, China. 3.3. Extraction and isolation The rhizomes of S. trinervula (30 kg) were extracted with 70% (v/v) aqueous ethanol. The 70% EtOH extract was concentrated under reduced pressure to give a residue (2705 g), which was extracted with EtOAc and n-BuOH, respectively. The EtOAc extract was concentrated to yield to the EtOAc fraction 249.2 g, and the nBuOH extract was concentrated to get the n-BuOH fraction 470.3 g. Part of the dried n-BuOH extract (about 470 g) was subjected to column chromatography using macroporous resins column chromatography (6 kg, 20*50 cm) with gradient mixtures of H2O/EtOH. Starting the elution with H2O (Fraction A, 3 l), and reducing polarity by adding EtOH (30%-95%, Fraction B-D, 2 l, each). Fractions were evaporated, the weight of Fractions A-E were 203 g, 65 g, 106 g, 61 g and 20 g, respectively. Fr. B (60 g) was further divided into five parts by MCI GEL CHP20P column chromatography (500 ml, 6*26 cm). Starting the elution with 20% MeOH (Fr.B1,
6 13
1
C
134.1 104.2 149.4 137.0 149.4 104.2 89.1 55.6 65.0 136.4 114.3 145.2 147.5 129.8 118.0 32.94 32.98 69.9 104.5 75.2 77.9 71.7 78.2 62.8 56.84 56.78
H
– 6.73(1H, s) – – – 6.73(1H, s) 5.56(1H, d, J = 5.8 Hz) 3.60–3.67 (1H, m) 3.84–3.89(1H, m) 3.72–3.79(1H, m) – 6.73(1H, s) – – – 6.71(1H, s) 2.62(2H, t, J = 8.0 Hz) 1.77–1.84(2H, m) 3.56(2H, t, J = 6.5 Hz)
3.39–3.49(1H, m) 3.39–3.49(1H, m) 3.39–3.49(1H, m) 3.17–3.21(1H, m) 3.72–3.79(1H, m) 3.64(1H, dd, J = 5.0, 11.9 Hz) 3.81(6H, s) 3.87(3H, s)
13
C
140.3 104.5 154.4 135.7 154.4 104.5 88.5 55.8 65.1 137.2 114.3 145.3 147.5 129.5 118.0 32.9 35.8 62.2 105.3 75.7 77.8 71.4 78.3 62.6 57.0 56.8
600 ml), and reducing polarity by adding MeOH (40%-100%, Fr.B25, 600 ml, each). Fractions were evaporated. Fr.B-4 (80% MeOH, 5 g) and Fr.B5 (100% MeOH, 3 g) were combined after inspection of analytical TLC (CHCl3/MeOH 20:1). Fr.B-2 (7.9 g) was further subjected to silica gel column chromatography (200 g, 5* 50 cm) with gradient mixtures CHCl3/MeOH (15:1, 10:1, 5:1, 1:1, 800 ml, each). Each 100 ml elution was collected and evaporated, and some elution was combined by inspection of analytical TLC (CHCl3/ MeOH 8:1). Finally, ten fractions (Fr.B2–1 10) were got. Fr.B2-2 (235 mg) was further purified by semipreparative HPLC (11% Acetonitrile/H2O, 3.0 ml/min) to yield 1 (8.2 mg, tR17.2 min), 2 (11 mg, tR23.7 min), 3 (8.7 mg, tR29.5 min). Fr.B2-3 (905 mg) was further purified by Sephadex LH-20 (MeOH). Each 20 ml elution was collected, and the same elution was combined by the TLC inspection. Two main fractions (Fr.B2-3-4, 97 mg, and Fr.B-2-3-8, 59 mg) were got. Fr.B2-3-4 (97 mg) was subjected to semipreparative HPLC (12% Acetonitrile/H2O, 3.0 ml/min) to yield 4 (5.1 mg, tR21.2 min), 5 (6.4 mg, tR27.6 min), 6 (9 mg, tR31.1 min). Fr. B2-3-8 (59 mg) was further purified by semipreparative H PLC (12.5% Acetonitrile/H2O, 3.0 ml/min) to yield 7 (9.2 mg, tR15.8 min), and 8 (8 mg, tR22.4 min). 3.3.1. (7S, 8R,80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 epoxylignan-70 -one 40 -O-b-D- glucopyranoside (1) White powder (MeOH); [a]25 D 31.41 (c 0.191, MeOH); UV (MeOH) lmax (loge) 280 (4.94) nm; HR-ESI–MS m/z 595.2023 [M] (calcd for C28H35O14); 1H NMR and 13C NMR see Table 1. 3.3.2. (7S, 8R, 80 R)-4, 40 , 9-trihydroxy-3, 30 , 5, 50 -tetramethoxy-7, 90 epoxylignan-70 -one 4-O-b-D- glucopyranoside (2) White powder (MeOH); [a]25 D 61.73 (c 0.243, MeOH); UV (MeOH) lmax (loge) 225 (4.95), 305 (4.89) nm; HR-ESI–MS m/z 595.2035 [M] (calcd for C28H35O14); 1H NMR and 13C NMR see Table 1.
J. Shu et al. / Phytochemistry Letters 20 (2017) 1–8 Table 3 NMR of 4 and 5 (400 MHz for 1H, MeOH-d4, 100 MHz for
13
C, MeOH-d4).
4
5
C
1
13
1 2 3 4 5 6 7 8 9
– 6.71(1H, s) – – – 6.71(1H, s) 4.83(1H, d, J = 8.0 Hz) 4.04(1H,ddd, J = 1.8, 4.3, 8.0 Hz) 3.70-3.74 (2H, m)
128.7 105.9 149.5 137.3 149.5 105.9 77.9 79.8 62.2
C-10 C-20 C-30 C-40 C-50 C-60 C-70 C-80 C-90 C-1” C-2” C-3” C-4” C-5” C-6”
– 6.68(1H, d, J = 1.9 Hz) – – – 6.50(1H, d, J = 1.9 Hz) 2.59(2H, t, J = 7.5 Hz) 1.76-1.83(2H, m) 3.55(2H, t, J = 6.5 Hz) 4.91(1H, d, J = 7.6 Hz) 3.36–3.53 (1H, m) 3.36–3.53 (1H, m) 3.36–3.53 (1H, m) 3.36–3.53 (1H, m) 3.90 (1H, dd, J = 2.1,12.0 Hz) 3.70 (1H, dd, J = 5.5, 12.0 Hz) 3.86 (6H, s) –
135.7 111.1 147.5 133.3 145.7 112.1 32.7 35.3 61.9 103.2 75.0 77.9 71.5 78.3 62.6
3,5-OMe 30 - OMe
7
H
3.3.3. (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy-40 , 7-epoxy-8, 50 neolignan 90 -O-b-D- glucopyranoside (3) White powder (MeOH); [a]25 D 5.41 (0.185, MeOH); UV (MeOH) lmax (loge) 230(4.98), 280(4.35) nm; HR-ESI–MS m/z 551.2131 [M] (calcd for C27H35O12); 1H NMR and 13C NMR see Table 2. 3.3.4. (7R, 8R)-4, 9, 90 -trihydroxy-3, 5-dimethoxy-7.O.40 , 8.O.30 neolignan 90 -O-b-D- glucopyranoside (4) 34.34 (0.182, MeOH); UV White powder (MeOH); [a]25 D (MeOH) lmax (loge) 225 (4.90), 275 (3.85) nm; HR-ESI–MS m/z 553.1918 [M] (calcd for C26H34O13); 1H NMR and 13C NMR see Table 3. 3.3.5. (7S, 8R)-4, 9, 90 -trihydroxy-3, 30 , 5-trimethoxy-8, 40 -oxyneolignan 4-O-b-D- glucopyranoside (5) White powder (MeOH); [a]25 D 30.56 (c 0.319, MeOH); UV (MeOH) lmax (loge) 220 (4.25), 280 (3.55) nm; HR-ESI–MS m/z 593.1836 [M + Na]+ (calcd for C27H38O13Na); 1H NMR and 13C NMR see Table 3. 3.4. Cytotoxicity activity The cytotoxicity of the isolated compounds (The purity of compounds 1–8 were 95.5%, 93.5%, 91.7%, 94.5%, 98.6%, 97.2%, 96.6% and 95.0%, respectively.) 2 against 4 human tumor cell lines (SH-SY5Y, SGC-7901, HCT-116, Lovo) was evaluated using the MTT method. Cells were seeded in 96-well microplates at a density of 150 per well and were cultured in cell culture medium (RPMI1640 medium supplemented with 10% FBS, 100 U/mL penicillin and 100 g/mL streptomycin) for 12 h, then treated with the test compounds added from DMSO dissolved stock solution. The final DMSO concentration never exceeded 0.2% (v/v). Previous experiments showed that DMSO at this concentration did not modify the cell activities. After 48 h in culture, cells were incubated with MTT (0.5 mg/mL, 4 h) and subsequently resolved in DMSO. The
1
C
56.9 –
H
– 6.76(1H, s) – – – 6.76(1H, s) 4.82(1H, d, J = 6.0 Hz) 4.30(1H, ddd, J = 3.9, 5.4, 6.0 Hz) 3.85 (1H, dd, J = 5.4, 12.0 Hz) 3.72-3.82 (1H, m) – 6.77(1H, d, J = 1.9 Hz) – – 6.79(1H, d, J = 8.2 Hz) 6.65(1H, dd, J = 2.0, 8.2 Hz) 2.59 (2H, t, J = 7.5 Hz) 1.78 (2H, m) 3.54 (2H, t, J = 6.5 Hz) 4.78(1H, d, J = 7.4 Hz) 3.39–3.46(1H, m) 3.39–3.46(1H, m) 3.39–3.46(1H, m) 3.15–3.20 (1H, m) 3.72-3.82 (1H, m) 3.66 (1H, dd, J = 5.1, 12.0 Hz) 3.80(6H, s) 3.76(3H, s)
13
C
139.6 106.3 153.9 135.6 153.9 106.3 74.1 86.1 62.2 138.0 114.0 151.7 147.2 119.2 121.7 32.7 35.5 62.2 105.6 75.7 77.8 71.3 78.4 62.6 57.0 56.5
absorbance in control and drug treated wells was measured in an automated microplate reader at 570/630 nm. All experiments were carried out in triplicate and repeated twice. The cytotoxicity was expressed as IC50 values (50% inhibitory concentration). 3.5. Acid hydrolysis of 1-5 The absolute configuration of glucose was determined as described by Xin et al. Compounds 1-5 (respectively, 2 mg) were heated with 1 mol/L HCl (2 ml) for 4 h at 105 C. The mixture was cooled, neutralized, and partitioned between AcOEt (2 ml) and H2O (2 ml). The aq. layer was evaporated and contained in a vial. The following solns. were added: a) (2S)-1-aminopropan-2-ol/MeOH 1:8 (20 ml); b) AcOH/MeOH 1:4 (17 ml); c) 3% Na[BH3CN] in MeOH (13 ml). The vial was capped, and the mixture was allowed to react for 2 h at 65 C. After cooling, 3 mol/L aq. CF3COOH was added dropwise until the pH dropped to pH 1–2. The mixture was evaporated and co-evaporated with H2O (3 0.5 ml) and MeOH (5 0.5 ml). The residue was dried overnight in a vacuum desiccator and treated with pyridine/Ac2O 1:1 for 1 h at 100 C. After cooling, the mixture was extracted with CHCl3 and the extract washed with H2O (3 1 ml) and sat. NaHCO3 soln. (3 1 ml). The org. phase was dried (Na2SO4) and subjected to GC/MS. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (NSFC) (Nos. 31370376, 81360630 and 81360628) and Natural Science Foundation of Jiangxi Province (No. 20142BCB23023 and 20161BAB205219). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. phytol.2017.03.002.
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