Hepatoprotective hemiterpene glycosides from the rhizome of Cibotium barometz (L.) J. Sm

Hepatoprotective hemiterpene glycosides from the rhizome of Cibotium barometz (L.) J. Sm

Phytochemistry xxx (2017) 1e6 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Hepatopr...

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Phytochemistry xxx (2017) 1e6

Contents lists available at ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Hepatoprotective hemiterpene glycosides from the rhizome of Cibotium barometz (L.) J. Sm Mei-Ping Xie, Lan Li, Hua Sun, An-Qi Lu, Bo Zhang, Jian-Gong Shi, Dan Zhang, Su-Juan Wang* State Key Laboratory of Active Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 November 2016 Received in revised form 12 February 2017 Accepted 22 February 2017 Available online xxx

Five previously undescribed hemiterpene glycosides, cibotiumbarosides EI, and two known hemiterpene glucosides, were isolated from the rhizome of Cibotium barometz (L.) J. Sm. The structures of cibotiumbarosides EI were established by 1D and 2D NMR spectroscopic analyses and HRMS. The absolute configuration of the aglycone of cibotiumbaroside E was assigned by calculated ECD with the TDDFT method. Cibotiumbarosides F and I both exhibited remarkable hepatoprotective activity against APAP-induced acute liver damage in vitro, which were more effective than the positive control, bicyclol. On the other hand, seven hemiterpene glycosides were all inactive in assays of cytotoxicity, neuroprotection, antidiabetes and anti-inflammation. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Cibotium barometz (L.) J. Sm. Dicksoniaceae Hemiterpene glycosides Hepatoprotection

1. Introduction The rhizome of Cibotium barometz (L.) J. Sm, popularly known as “gou-ji” in traditional Chinese medicine, is used to treat lumbago, rheumatism, polyuria and leucorrhoea in Chinese folk medicine. Its cytotoxicity (Bobach et al., 2014), and anti-osteoporosis (Cuong et al., 2009; Xu et al., 2014; Zhao et al., 2011) activities are widely reported. With respect to phytochemistry, in addition to some commonly known compounds, such as phenolic acids (Xie et al., 2016), g-pyrones (Xu et al., 2013), cerebrosides (Cuong et al., 2009), and ontins (Murakami et al., 1980; Saito et al., 1989; Wu and Yang, 2009), two hemiterpene glucosides (Cuong et al., 2009) were also identified from the C. barometz (L.) J. Sm. Among them, cibotiumbaroside B showed inhibition of osteoclast formation with no effect on BMM cell viability (Cuong et al., 2009). In our preliminary study, the 50% EtOH SP700-resin elution of C. barometz (L.) J. Sm. extracts showed remarkable hepatoprotective activities (37.01% of cell viability) against APAP-induced HepG2 cell damage, which is as effective as the positive control, bicyclol (36.27% of cell viability). Our ongoing research on the discovery of hepatoprotective natural products led to the findings of five

* Corresponding author. E-mail address: [email protected] (S.-J. Wang).

previously undescribed hemiterpene glycosides (1e5), along with two known glycosides, from the hepatoprotective fractions of C. barometz (L.) J. Sm. Among the seven hemiterpene glycosides, two compounds (2 and 5) exhibited remarkable hepatoprotective activities against APAP-induced HepG2 cell damage, which were more effective than the positive control, bicyclol. In this study, the isolation, structural identification and hepatoprotective activities of these hemiterpene glycosides were reported. 2. Results and discussions In this study, 50% EtOH resin elution of extracts of C. barometz (L.) J. Sm. yielded five previously undescribed hemiterpene glycosides, cibotiumbarosides EI (1e5). Their structures were elucidated on the basis of NMR, HRMS and CD spectroscopic analyses. In addition, two known compounds, cibotiumbaroside B (6) and cibotiumbaroside A (7), were identified by comparing their NMR spectroscopic data in the literature (Cuong et al., 2009) (see Fig. 1). Compound 1, obtained as a pale yellow powder, had the molecular formula C20H24O11, as established by HRAPIMS at m/z 463.1216 [MþNa]þ (calcd 463.1211 for C20H24O11Naþ). The 1H NMR spectrum established a caffeoyl moiety by three ABX-type aromatic protons at dH 6.72 (1H, d, J ¼ 8.0 Hz), 6.92 (1H, dd, J ¼ 8.0, 2.0 Hz), 6.99 (1H, d, J ¼ 2.0 Hz) and two trans olefinic protons at dH 6.24 and 7.54 (each d, J ¼ 15.5 Hz). The anomeric proton at dH 4.31 (1H, d,

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Please cite this article in press as: Xie, M.-P., et al., Hepatoprotective hemiterpene glycosides from the rhizome of Cibotium barometz (L.) J. Sm, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.023

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M.-P. Xie et al. / Phytochemistry xxx (2017) 1e6

Fig. 3. Calculated and experimental ECD of aglycone 1a.

Fig. 1. Structures of compounds 1e7 isolated from the rhizome of Cibotium barometz.

J ¼ 8.0 Hz) as well as acid hydrolysis and HPLC analysis indicated the presence a b-D-glucosyl moiety. There were five-carbon aglycones signals, including one carbonyl (dC 180.0), two oxymethylenes (dC 69.6 and 69.1), one methine (dC 42.2), and one methylene (dC 27.1), in addition to the caffeoyl and glucosyl moiety in 13C NMR. The HMBC correlations (Fig. 2) from H-5 (dH 4.35) and H-1' (dH 4.10, 3.83) to C-2 (dC 180.0) and C-3 (dC 42.2), H-1' (dH 4.10, 3.83) to C-4 (dC 27.1), and H-4 (dH 2.35) to C-5 (dC 69.1) established this aglycone structure as 3-hydroxymethyltetrahydrofuran-2-one, based on the similarity of the NMR data to that previously reported (Larsen et al., 1996). The downfield shifted H-4'' (dH 4.76) suggested that the caffeoyl moiety was connected to C-400 of the glucose, which was further supported by the HMBC cross-peak from H-4'' (dH 4.76) to C-9000 (dC 168.9). The H-1' (dH 4.10 and 3.83) was correlated to anomeric C-1'' (dC 105.0) and confirmed the linkage of glucose moiety at C-1' (Fig. 2). The experimental ECD of the aglycone 1a, afforded by enzyme hydrolysis of 1, was in accordance with the calculated ECD of (3S)-3-hydroxymethyltetrahydrofuran2-one (Fig. 3). Therefore, compound 1, cibotiumbaroside E, was established as (3S)-3-methylenetetrahydrofuran-2-one (4-O-

Fig. 2. Key correlations of compounds 1¡5.

caffeoyl)-b-D-glucopyranoside. Compound 2 was obtained as a yellow amorphous powder with the molecular formula C18H22O11 established from HRAPIMS at m/z 437.1067 [MþNa]þ (calcd 437.1054 for C18H22O11Naþ). The 1H NMR and 13C NMR spectra of 2 were very similar to those of 1, except for the aromatic moiety. The proton signals at dH 6.75 (1H, d, J ¼ 8.0 Hz), 7.38 (1H, dd, J ¼ 8.0, 2.0 Hz), and 7.39 (1H, d, J ¼ 2.0 Hz) and the seven carbon signals at dC 166.8, 150.4, 144.8, 122.3, 121.1, 116.0, and 114.5 indicated the presence of a protocatechuoyl moiety (Ozipek et al., 1999). The five-carbon aglycone of 2 was same as that of 1 and confirmed by HMBC correlations (Fig. 2) and enzyme hydrolysis. The H-1' (dH 3.96 and 3.83) was correlated to anomeric C1'' (dC 103.4), which confirmed the linkage of the glucose moiety at C-1' (Fig. 2). The downfield shifted H-6'' (dH 4.54 and 4.34) indicated that the protocatechuoyl moiety was attached to C-600 , which is supported by HMBC cross-peaks from H-6'' (dH 4.54 and 4.34) to the 000 C-7 (dC 166.8). The anomeric proton dH 4.28 (1H, d, J ¼ 8.0 Hz), together with the acid hydrolysis and HPLC analysis, identified the glucose as b-D-glucose. Thus, compound 2, cibotiumbaroside F, was elucidated as (3S)-3-methylenetetrahydrofuran-2-one (6-O-protocatechuoyl)-b-D-glucopyranoside. Compound 3, pale yellow powder, had a molecular formula C18H20O11 as determined by HRAPIMS at m/z 411.0922 [M-H] 1 13 (calcd 411.0933 for C18H19O 11). The H and C NMR data identified 3 as a protocatechuoyl hemiterpene glucoside similar with those of cibotiumbaroside A (7) (Cuong et al., 2009), except for the sugar moiety. The HMBC correlations from H-10 (dH 4.44, 4.35) to C-3 (dC 122.4) and C-4 (dC 149.4), H-4 (dH 7.58) to C-2 (dC 174.0) and C-5 (dC 71.3) confirmed the aglycone structure as 3-hydroxymethylfuran2(5H)-one. All glycosyl protons were completely attributed by 1 H-1H COSY analysis (Fig. 2). The triplet H-300 (4.04, t, J ¼ 3.0 Hz), together with the H-200 (3.34, dd, J ¼ 8.0, 3.0 Hz) and H-400 (3.56, dd, J ¼ 10.0, 3.0 Hz), indicated that H-300 of the sugar was equatorial, due to its small coupling constants with adjacent protons. Combined with the anomeric proton at dH 4.69 (1H, d, J ¼ 8.0 Hz) and followed by acid hydrolysis, the sugar in 3 was identified as b-Dallose (Seigler et al., 2002). The downfield shifted H-600 (dH 4.51 and 4.31) suggested that the protocatechuoyl moiety was connected to C-600 of the glucose, which was further supported by the HMBC cross-peaks from H-600 (dH 4.51 and 4.31) to C-7000 (dC 167.2). Correlation from H-10 (dH 4.44 and 4.35) to C-100 (dC 100.9) identified the linkage of the alloyl moiety at C-10. Hence, compound 3, cibotiumbaroside G, was elucidated as 3-methylenefuran-2(5H)-one (6O-protocatechuoyl)-b-D-allopyranoside. Compound 4 was obtained as a pale yellow powder with the

Please cite this article in press as: Xie, M.-P., et al., Hepatoprotective hemiterpene glycosides from the rhizome of Cibotium barometz (L.) J. Sm, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.023

M.-P. Xie et al. / Phytochemistry xxx (2017) 1e6

molecular formula C20H26O13, as indicated by the HRAPIMS at m/z 1 13 475.1433 [MþH]þ (calcd 475.1446 for C20H27Oþ C 13). The H and NMR spectra elucidated 4 as a caffeoyl hemiterpene glucoside, similar with those of cibotiumbaroside B (6) (Cuong et al., 2009), except for the absent of two methoxy groups. The HMBC correlations from H-10 (dH 4.00 and 4.39) to C-3 (dC 83.8) and C-4 (dC 39.5), H-4 (dH 2.62 and 2.08) to C-2 (dC 110.0), C-3 (dC 83.3), and C-5 (dC 107.7), H-2 (dH 4.83) to C-5 (dC 107.7) established the aglycone of 5 as 2,3,5-trihydroxy-3-hydroxymethyltetrahydrofuran. The anomeric proton at dH 4.40 (1H, d, J ¼ 8.0 Hz), combined with acid hydrolysis and HPLC analysis, indicated the sugar moiety as b-Dglucose. The downfield shifted H-400 (dH 4.97) suggested that the caffeoyl moiety was connected to C-400 of the glucose, which was further supported by the HMBC cross-peak from H-400 (dH 4.97) to C-9000 (dC 167.7). The correlation from H-10 to C-100 identified the linkage of glucosyl moiety at C-10. An NOE cross-peak was observed between H-2 (dH 4.75) and H-5 (dH 5.21) in NOESY spectrum, suggesting that a cis-2,5-dihydroxy orientation was the major conformation. However, no NOE correlations between H-10 (dH 3.92 and 3.82) and H-2 (dH 4.75) were observed, suggesting the presence of trans-2,3-dihydroxy groups. Consequently, compound 4, cibotiumbaroside H, was established as 2,3,5-trihydroxy-3methylenetetrahydrofuran (4-O-caffeoyl)-b-D-glucopyranoside. Compound 5 was obtained as a white powder with the molecular formula C18H24O10, as determined by HRAPIMS at m/z 399.1304 1 13 [MeH] (calcd 399.1297 for C18H23O C 10). In the H NMR and NMR spectra, the signals at dH 6.79 (1H, d, J ¼ 8.0 Hz), 7.44 (1H, dd, J ¼ 8.0, 2.0 Hz), and 7.43 (1H, d, J ¼ 2.0 Hz) and dC 166.7, 150.3, 145.1, 122.2, 121.2, 116.2 and 114.4 were assigned to a protocatechuoyl moiety. The anomeric proton at dH 4.31 (1H, d, J ¼ 7.8 Hz), together with acid hydrolysis and HPLC analysis, suggested the presence of a b-D-glucosyl moiety. The residual signals in 13C NMR, including two oxymethylenes (dC 68.3, 64.7), one terminal double bond (dC 110.1, 146.7), and one methylene (dC 32.7), were assigned to a five-carbon aglycone. The HMBC correlations from H-4 (dH 2.37) to C-1' (dC 64.7), C-2 (dC 110.1), C-3 (dC 146.7) and C-5 (dC 68.3), H-2 (dH 5.02, 4.86) to C-1' (dC 64.7) and C-4 (dC 32.7) established the aglycone of 5 as 2-hydroxymethyl-4-hydoxylbutene. The downfield shifted H-6'' (dH 4.56 and 4.38) suggested that the protocatechuoyl moiety was connected to C-600 of the glucose, which was further supported by the HMBC cross-peaks from H-6'' (dH 4.56 and 4.38) to C-7000 (dC 166.7). The anomeric proton at dH 4.31 (1H, d, J ¼ 7.8 Hz) was correlated to C-5 at dC 68.3, which confirmed the linkage of the glucose at C-5. Then, compound 5, cibotiumbaroside I, was elucidated as 2-hydroxymethyl-1-butene 4-O-(6-O-protocatechuoyl)-bD-glucopyranoside. Compounds 1e7 were all isolated from the hepatoprotective fractions of C. barometz (L.) J. Sm. Thus, these compounds were tested for hepatoprotective activities against APAP-induced acute liver damage in vitro using the MTT method. The hepatoprotective drug bicyclol was used as the positive control. At 10 mM, compounds 2 and 5 reduced the APAP-induced HepG2 cell death by increasing the cell viability from 29.22% to 38.08% and 45.43%, respectively. These compounds were more effective than the positive control bicyclol (35.12%), as shown in Table 3. The other compounds were inactive in the same assay. The pathophysiology of APAP-induced liver injury was complex. It was reported (Jaeschke et al., 2011) that the reactive metabolite formation and protein binding of APAP, especially to mitochondrial proteins, induced mitochondrial oxidant stress and peroxynitrite formation eventually leading to necrotic cell death, and lipid peroxidation was not a relevant mechanism of APAP-induced liver injury. Although protocatechuic acid (Liu et al., 2002) could reduce oxidative stress induced by t-BHP in rat liver by evaluating malondialdehyde (MDA) and glutathione (GSH). The hepatoprotective activity of compounds

3

2 and 5 still need for further research because some protocatechuic derivatives 3 and 7 were inactive in the APAP-induced liver injury assay. In addition, compounds 1e7 were also assessed for their inhibitory activity against PTP1B and a-glucosidase, LPS-induced NO production of BV2 cell, glutamate-induced neuron death (SKN-SH cell), serum deprivation-induced PC12 cell death, cytotoxicity of several human cancer cell lines (HCT-116, HepG2, BGC-823, NCIH1650, A2780), but they all were inactive at the concentration of 10 mM. 3. Conclusion In summary, five previously undescribed hemiterpene glycosides, along with two knowns, were isolated from the rhizome of C. barometz (L.) J. Sm. The absolute configuration of aglycone 1a was established by calculated ECD based on the TDDFT method. Furthermore, compounds 2 and 5 exhibited remarkable hepatoprotective activities against APAP-induced HepG2 cell death, which were more effective than the positive control, bicyclol. This is the first report of the hepatoprotective activities of the chemical constituents and extracts of C. barometz (L.) J. Sm., indicating their potential value for further research. 4. Experimental 4.1. General Melting points were recorded on an XT5B micromelting point apparatus. UV Spectra were recorded on a J-810 spectrophotometer (JASCO, Japan). IR spectra were recorded on a Nicolet impact 400 spectrometer (Thermo Fisher, USA) by an FT-IR microscope transmission method. Optical rotations were measured on a model 343 polarimeter (Perkin-Elmer, USA) in methanol. Mass spectra were recorded on 1100 Series Trap-SL LC-MSD (Agilent, USA) or APITOFMS 10000 (Hexin, China). The NMR spectra were recorded in an Inova 500 (Varian, USA) or AVIIIHD 600 (Bruker, Germany) spectrometer. Open column chromatography was carried out using silica gel (100e160 or 200e300 mesh, Qingdao Marine Chemical Co., Qingdao, China) or Sephadex LH-20 (GE, USA) as the stationary phase. TLC was conducted on silica gel GF254 plates. Preparative HPLC was performed on an Agilent 1100 instrument (USA) with columns RP C18 (10 m or 5 m). Snailase was bought from Beijing Biodee Biotechnology Co. (China). Other chemicals were of analytical or HPLC grade. 4.2. Plant material The rhizome of Cibotium barometz (L.) J. Sm. were obtained from Anguo herbal medicine market (Hebei, China), and collected from the Pingxiang (Guangxi, China). A voucher specimen (No. ID-S2606) was identified by Prof. Lin Ma and deposited at the Institute of Materia Medica, Chinese Academy of Medical Sciences. 4.3. Extraction and isolation The dried rhizomes of Cibotium barometz (L.) J. Sm. (60 kg) were powdered and extracted with 50% aqueous ethanol (240 L  2 h, 180 L  1.5 h, 180 L  1.5 h) under reflux. The concentrated extracts (7.76 kg) were dissolved in water (60 L), then conducted on SP-700 macroporous resin adsorption chromatography, and eluted by H2O (120 L), 30% EtOH (160 L), 50% EtOH (160 L), 95% EtOH (160 L). The 50% EtOH elutions (1098 g) were subjected to CC over an MCI CHP20P and eluted successively with H2O and 10%e82% EtOH to give seven fractions (fr.1e7). Of the fractions, fr.2 (123.5 g) was

Please cite this article in press as: Xie, M.-P., et al., Hepatoprotective hemiterpene glycosides from the rhizome of Cibotium barometz (L.) J. Sm, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.023

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applied to a Sephadex LH-20 column with H2O, 50% MeOH, 100% MeOH to give three fractions (fr. 2.1e3). Fr. 2.1 was loaded on a Sephadex LH-20, eluted with 30% MeOH, then subjected to midpressure RP-LC eluted by MeOHeH2O (0e70%), producing three fractions (fr.2.1.1e3). Fr. 2.1.1 was then loaded on a Sephadex LH-20, producing 4 fractions (fr. 2.1.1.1e4). From this series, fr.2.1.1.3 was subjected to CC over a silica gel (CHCl3eMeOH, 100:11:100) and was finally purified by RP-HPLC (27% MeOH) to obtain compound 5 (2 mg). Fr.2.1.3 was loaded on a Sephadex LH-20 with an elution of 50% MeOH, then purified by RP-HPLC (25% MeOH) to obtain compound 2 (40 mg). Fr. 2.2 was separated by silica gel CC (CHCl3MeOH, 100:11:100) to give five fractions (fr. 2.2.1e5). Fr.2.2.1 was loaded on a Sephadex LH-20, and finally purified by RPHPLC (35% MeOH) to produce compound 3 (4.5 mg) and compound 7 (8 mg). Fr.2.2.2 and fr.2.2.3 were combined based on TLC similarities. These fractions were then subjected to mid-pressure RP-LC eluted by MeOHeH2O (0e70%), resulting in 11 fractions (fr. 2.2.2.1e11). Fr. 2.2.2.5e7 was subjected to CC on silica gel (CHCl3eMeOH, 100:11:100). The CHCl3eMeOH (10:1) fractions were concentrated and loaded on a Sephadex LH-20, then finally purified by RP-HPLC (40% MeOH) to obtain compound 1 (20 mg). Fr.2.2.4 was separated by silica gel CC (CHCl3eMeOH, 100:11:100) to give 11 fractions (fr. 2.2.4.1e11). Fr. 2.2.4.3 was purified by Sephadex LH-20 and eluted with 50% MeOH to produce compound 4 (2 mg) and compound 6 (50 mg).

Table 2 13 C NMR spectroscopic data of compounds 1e7 in methanol-d6. NO.

1b

2b

3a

4a

5a

6b

7c

2 3 4 5 10 100 200 300 400 500 600 1000 2000 3000 4000 5000 6000 7000 8000 9000 2-OCH3 5-OCH3

180.0 42.2 27.1 69.1 69.6 105.0 75.5 76.6 72.8 76.1 62.8 128.0 115.5 147.2 150.1 116.9 123.4 148.0 115.1 168.9

178.9 40.5 25.4 67.2 68.2 103.4 73.6 76.4 70.3 74.1 63.2 121.1 114.5 144.8 150.4 116.0 122.3 166.8

174.0 122.4 149.4 71.3 63.7 100.9 71.0 71.9 71.5 67.8 62.7 130.3 116.1 144.9 150.5 114.7 122.9 167.2

110.0 83.8 39.5 107.7 70.3 100.1 75.5 72.8 72.3 77.9 61.9 127.8 114.4 147.7 149.6 116.4 123.0 146.6 115.1 167.7

110.1 146.7 32.7 68.3 64.7 102.9 73.7 76.4 70.4 74.0 63.4 114.4 116.2 145.1 150.3 121.2 122.2 166.7

110.8 84.4 40.2 108.3 71.0 100.2 76.1 73.1 72.8 78.2 62.5 127.9 115.5 147.2 150.0 116.8 123.4 148.1 114.8 168.6 56.0 56.3

175.4 131.5 150.9 72.7 64.1 104.5 75.1 77.9 71.7 75.6 64.7 122.6 117.1 146.3 151.9 116.1 123.9 168.2

Measured at a150 MHz or b125 MHz. Referred from the literature (Cuong et al., 2009).

c

3.80 (1H, dd, J ¼ 11.0, 6.0 Hz, H-10 ). 4.3.1. Cibotiumbaroside E (1) Pale yellow powder; C20H24O11, [a]20 D e12.4 (c 0.1, MeOH); mp 131.1e132.5  C. UV (MeOH) lmax nm (log ε): 323 (3.26), 300 (3.18), 244 (3.04), 219 (3.20). CD (c 0.25, MeOH) [q]219e376, [q]244e265, [q]300e190, [q]329e183, [q]350e116. IR nmax: 3396, 2973, 1752, 1701, 1602, 1040 cm1. For 1H and 13C NMR data see Tables 1 and 2. (þ) ESI-MS m/z: 463 [MþNa]þ, 440 [M-H]. HRAPIMS m/z: 463.1216 [MþNa]þ (calcd 463.1211 for C20H24NaOþ 11). 1a: CD (c 0.25, MeOH) q214 þ75. (þ)ESI-MS m/z: 117 [MþH]þ. 1H NMR (CDCl3, 600 MHz): d 2.78 (1H, dddd, J ¼ 10.0, 9.0, 6.0, 4.5 Hz, H-3), 2.30 (2H, m, H-4), 4.40 (1H, td, J ¼ 9.0, 3.0 Hz, H-5), 4.25 (1H, ddd, J ¼ 10.0, 9.0, 7.0 Hz, H-5), 3.96 (1H, dd, J ¼ 11.0, 4.5 Hz, H-10 ),

4.3.2. Cibotiumbaroside F (2) Yellow amorphous powder; [a]20 D þ2.1 (c 0.1, MeOH). UV (MeOH) lmax nm (log ε): 300 (3.18), 267 (3.00). IR nmax: 3413, 2971, 1744, 1699, 1605, 1080 cm1. For 1H and 13C NMR data see Tables 1 and 2 ESIMS m/z: 437 [MþNa]þ, 413 [M-H]. (þ)HRAPIMS: m/z 437.1054 [MþNa]þ (calcd 437.1060 for C18H22NaOþ 11). 4.3.3. Cibotiumbaroside G (3) þ8.0 (c 0.1, MeOH); mp Pale yellow powder; [a]20 D 126.7e127.4  C. UV (MeOH) lmax nm (log ε): 297 (2.90), 264 (3.00). IR nmax: 3387, 2931, 1736, 1701, 1605, 1078 cm1. For 1H and 13C NMR

Table 1 1 H NMR spectroscopic data of compounds 1e7 in methanol-d6. (J in Hz). NO.

1a

2b

2 3 4

2.90 tt (9.4, 4.5) 2.35 m

2.85 tdd (9.0, 5.0, 4.0) 2.26 m

5 10 100 200 300 400 500 600 2000 5000 6000 7000 8000 2-OCH3

4.35 4.22 4.10 3.83 4.31 3.23 3.56 4.76 3.46 3.57 3.49 6.99 6.72 6.92 7.54 6.24

td (8.5, 3.5) q (8.5) dd (10.0, 3.5) dd (10.0, 5.0) d (8.0) t (9.5)* t (9.0)d t (9.5)* dd (9.0, 8.0)c t (10.5)d dd (12.0, 8.0)c d (2.0) d (8.0) dd (8.0, 2.0) d (15.5) d (15.5)

4.24 4.10 3.96 3.83 4.28 3.13 3.34 3.52 3.35 4.54 4.34 7.39 6.75 7.38

3b

dd (8.5, 4.0) q (8.5) dd (10.0, 4.0) dd (10.0, 5.0) d (8.0) t (8.0) me t (9.0) me dd (12.0, 2.0) dd (12.0, 6.0) d (2.0)f d (8.0) dd (8.0, 2.0)f

7.58 br.s *

4.78 m 4.44 4.35 4.69 3.34 4.04 3.56 3.93 4.51 4.31 7.39 6.75 7.37

d (15.0) d (15.0)g d (8.0) dd (8.0, 3.0) t (3.0) dd (10.0, 3.0) ddd (8.0, 5.5, 2.0) d (12.0) dd (12.0, 5.5)g d (2.0), d (8.0) dd (8.0, 2.0)

4a

5a

6b

4.83 s

5.02 br.s, 4.86 br.s

4.75 s

2.62 dd (14.0, 5.5) 2.08 dd (14.0, 3.5) 5.26 dd (5.5, 3.5)

2.37 t (7.0)

2.54 dd (14.0, 5.5), 2.08 dd (14.0, 3.5) 5.17 dd (5.5, 3.5)

4.00 3.90 4.40 3.28 3.74 4.97 3.79 3.66 3.57 7.07 6.80 6.98 7.62 6.32

d (12.0) d (12.0) d (8.0) m mh dd (10.0, 9.0) mh dd (12.0, 2.0) dd (12.0, 6.0) d (2.0) d (8.0) dd (8.0, 2.0) d (16.0) d (16.0)

3.93 dd (17.0, 7.0) 3.68 dd (17.0, 7.0) 3.99 s 4.31 3.20 3.38 3.39 3.56 4.56 4.38 7.43 6.79 7.44

d (7.8) dd (9.0, 8.0) mi mi dd (9.0, 6.0) dd (12.0, 2.0) dd (12.0, 6.0) d (2.0) d (8.0) dd (8.0, 2.0)

3.91 3.82 4.32 3.20 3.71 4.90 3.61 3.58 3.50 6.99 6.72 6.90 7.54 6.23 3.32

7b

d (12.5) d (12.5) d (7.5) dd (9.5, 8.0) t (9.5) t (9.5) m dd (12.0, 2.0) dd (12.0, 6.0) d (2.0) d (8.0) dd (8.0, 2.0) d (15.5) d (15.5) s

7.59 m 4.77 m 4.52 4.45 4.36 3.24 3.35 3.37 3.52 4.52 4.32 7.39 6.74 7.37

dd (13.5, 2.0) dd (13.5, 2.0) d (8.0) dd (9.0, 8.0) t (9.0) t (9.0) m dd (12.0, 2.0) m d (2.0) d (8.0) dd (8.0, 2.0)

3.34 s

5-OCH3 a

b

Measured at 600 MHz or 500 MHz. Signals were overlapped to each other. * Signal was overlapped by water or solvent peak.

ce i

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M.-P. Xie et al. / Phytochemistry xxx (2017) 1e6

5

Table 3 Hepatoprotective activities of compounds 1e7 against APAP-induced HepG2 cell death. Group

O.D. Values

Control APAP Bicyclola 1 4 6

1.571 0.569 0.679 0.570 0.522 0.547

***

± ± ± ± ± ±

0.134 0.045*** 0.074# 0.134 0.141 0.110

Cell viability (%)

Group

O.D. Values

100.00 36.22 43.24 36.30 33.20 34.84

Control APAP Bicyclola 2 3 5 7

2.332 0.681 0.819 0.888 0.558 1.059 0.699

P < 0.001, compared with the blank control group; #P < 0.05, compared with APAP model group; Positive control.

##

± ± ± ± ± ± ±

0.139 0.042*** 0.071# 0.055## 0.077 0.109## 0.075

Cell viability (%) 100.00 29.22 35.12 38.08 23.94 45.43 29.97

P < 0.01, compared with the APAP model group.

a

data see Tables 1 and 2 (þ)ESIMS m/z: 435 [MþNa]þ. ()HRAPIMS m/z: 411.0922 [MH] (calcd 411.0933 for C18H19O 11).

simulated by using SpecDis (V1.64, University of Wuerzburg, Germany, 2015).

4.3.4. Cibotiumbaroside H (4) Pale yellow powder; [a]20 -29.4 (c 0.1, MeOH); mp D 138.3e139.1  C. UV (MeOH) lmax nm (log ε): 327 (3.83), 300 (3.68). IR nmax: 3373, 2978, 1694, 1602, 1032 cm1. For 1H and 13C NMR data see Tables 1 and 2 (þ)HRESIMS at m/z 475.1433 [MþH]þ (calcd 475.1446 for C20H27Oþ 13).

4.6. Hepatoprotective assay

4.3.5. Cibotiumbaroside I (5)  White powder; [a]20 D þ10.6 (c 0.1, MeOH); mp 144.9e145.6 C. UV (MeOH) lmax nm (log ε): 294 (2.85), 265 (2.95); IR nmax: 3405, 2978, 1694, 1605, 1080 cm1; for 1H and13C NMR data, see Tables 1 and 2; ESIMS m/z: 423 [MþNa]þ, 399 [M-H]; ()HRAPIMS at m/z 399.1304 [M-H] (calcd 399.1297 for C18H23O10). 4.4. Absolute configuration of sugars Compounds 1e5 (ca. 0.5 mg) were dissolved in 0.5 N HCl (0.1 mL) and heated at 90  C for 2 h. The solution was neutralized with aqueous ammonia and partitioned with EtOAC (0.2 mL  3). After vacuum drying the water phase, the residue was dissolved in pyridine (0.1 mL) containing L-cysteine methyl ester hydrochloride (0.5 mg) and heated at 60  C for 1 h. A 0.1 mL solution of o-tolylisothiocyanate (0.5 mg) in pyridine was added to the mixture, which was heated at 60  C for 1 h. The reaction mixture was directly analyzed by analytical HPLC under the following conditions: column Grace Smart C18 (4.6  250 mm, 5m); mobile phase acetonitrile: 50 mM H3PO4 (25:75); flow rate 0.8 mL/min; wavelength 250 nm; column temperature 35  C. Under the above conditions, the sugars of the isolates obtained by acid hydrolysis gave corresponded results as those of standard sugars. 4.5. Absolute configuration of aglycone 1a and 2a Compound 1 and 2 (each ca. 6 mg) was dissolved in 2 mL water, and 10 mg Snailase was added. After being warmed at 30  C for 24 h, the mixture solution was partitioned with CH2Cl2 (2 mL  3). Then, the CH2Cl2 was evaporated to obtain 1a and 2a. Calculated ECD was performed on the S configuration of 1a. Conformation search was done with the MMFF94 using the MOE software package (MOE2009.10, Chemical Computing Group, Montreal, QC, Canada). Calculated ECD was performed using TDDFT (Gaussian 09 B.01, Gaussian, Wallingford, CT, 2009) at B3LYP/631 þ G(d,p)//B3LYP/6-311 þ G(d,p) level for the configurations within an energy window of 5 kcal/mol. The conductor-like polarizable continuum model was used with MeOH (ε ¼ 32.613) in order to take the solvent effects into consideration. The Boltzmann distribution was calculated based on the relative free energy (DG) and the final ECD (s ¼ 0.33 eV, UV shift ¼ 25 nm) was

HepG2 cell lines were incubated in DMEM supplied with FBS (10%), penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37  C in 5% CO2. The cell suspension (100 mL) was placed in a 96-well microplate for 24 h. Cells were then treated with test compounds (10 mM) and APAP (8 mM) in three parallel wells for 48 h. Bicyclol (10 mM) was used as the positive control. After incubation, the cells were cultured in 100 mL of medium containing MTT (0.5 mg/mL) for another 4 h. The resulting formazan was dissolved in DMSO and was measured on a microplate reader at a wavelength of 570 nm. Cell viability (%) was calculated as OD(sample)/OD(normal)  100. Acknowledgements This research was supported in part by CAMS Innovation Fund for Medical Sciences (CIFMS) No. 2016-I2M-3-011. The authors are grateful for the pharmacological data shared by the following research groups at the Institute of Materia Medica (Beijing, China): Prof. X. G. Chen for cytotoxicity activity, Dr. D. Zhang for hepatoprotective activity and anti-inflammatory activity, Prof. N. H. Chen and Prof. X. L. Wang for neuroprotective activity, and Prof. F. Ye for antidiabetic activity. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.02.023. References Bobach, C., Schurwanz, J., Franke, K., Denkert, A., Sung, T.V., Kuster, R., Mutiso, P.C., Seliger, B., Wessjohann, L.A., 2014. Multiple readout assay for hormonal (androgenic and antiandrogenic) and cytotoxic activity of plant and fungal extracts based on differential prostate cancer cell line behavior. J. Ethnopharmacol. 155, 721e730. Cuong, N.X., Minh, C.V., Kiem, P.V., Huong, H.T., Ban, N.K., Nhiem, N.X., Tung, N.H., Jung, J.W., Kim, H.J., Kim, S.Y., 2009. Inhibitors of osteoclast formation from rhizomes of Cibotium barometz. J. Nat. Prod. 72, 1673e1677. Jaeschke, H., Mcgill, M.R., Williams, C.D., Ramachandran, A., 2011. Current issues with acetaminophen hepatotoxicityda clinically relevant model to test the efficacy of natural products. Life Sci. 88, 737e745. Larsen, D.S., Schofield, A., Stoodley, R.J., Tiffin, P.D., 1996. Diastereoselective hydrogenations of [small alpha]-alkyl [small alpha]-(2,3,4,6-tetra-O-acetyl-[small beta]-D-glucopyranosyloxy)methylene carbonyl compounds. New route to stereopure [small alpha]-alkyl [small alpha]-oxymethyl carbonyl compounds. J. Chem. Soc. Perkin Trans. 1, 2487e2495. Liu, C.-L., Wang, J.-M., Chu, C.-Y., Cheng, M.-T., Tseng, T.-H., 2002. In vivo protective effect of protocatechuic acid on tert-butyl hydroperoxide-induced rat hepatotoxicity. Food Chem. Toxicol. 40, 635e641. Murakami, T., Satake, T., Ninomiya, K., Iida, H., Yamauchi, K., Tanaka, N., Saiki, Y., Chen, C.M., 1980. Pterosin-derivate aus der familie pteridaceae. Phytochemistry 19, 1743e1746. Ozipek, M., Saracoglu, I., Kojima, K., Ogjhara, Y., Cais, I., 1999. Fuhsioside, a new

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Please cite this article in press as: Xie, M.-P., et al., Hepatoprotective hemiterpene glycosides from the rhizome of Cibotium barometz (L.) J. Sm, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.023