Iridoids from the roots of Valeriana jatamansi Jones

Phytochemistry 141 (2017) 156e161

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Iridoids from the roots of Valeriana jatamansi Jones Ru-Jing Wang a, b, Hai-Mei Chen a, b, Fan Yang b, Yun Deng b, Hui AO c, Xiao-Fang Xie a, b, Hong-Xiang Li c, Hai Zhang a b, Zhi-Xing Cao a, b, Li-Xia Zhu b, Yin Chen b, Cheng Peng a, b, **, Yu-Zhu Tan a, b, * a State Key Laboratory Breeding Base of Systematic Research Development and Utilization of Chinese Medicine Resources, Sichuan Province and Ministry of Science and Technology, Chengdu, 611137, China b Pharmacy College, Chengdu University of Traditional Chinese Medicine, Chengdu, 611137, China c Aanalysis and Test Center, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 December 2016 Received in revised form 26 March 2017 Accepted 21 May 2017

Five iridoids, named as chlorovaltrate P-T, together with six known analogues, (4b,8b)-8-methoxy-3methoxy-10-methylene-2,9-dioxatricyclo[4.3.1.03,7]decan-4-ol, chlorovaltrate A, (1R,3R,5R,7S,8R,9S)3,8-epoxy-1-O-ethyl-5-hydroxyvalechlorine, 8-methoxy-4-acetoxy-3-chlormethyl-10-methylen-2,9(1S,3R,5R,7S,8R,9S)-3,8-epoxy-1-O-ethyl-5-hydroxyvalechlorine, dioxa-tricyclo[4.3.1.03,7]decan, (1R,3R,5R,7S,8R,9S)-3,8-epoxy-1-O-methyl-5-hydroxyvalechlorine were isolated from the roots of Valeriana jatamansi (syn. Valeriana wallichii). Their structures were elucidated by extensive analysis of 1D, 2D NMR and HRESIMS spectroscopic. The absolute configuration of chlorovaltrate P-T were established by comparing their experimental and calculated electronic circular dichroism (ECD) spectra. 3,8-epoxy iridoids exhibited weak cytotoxicity against the lung adenocarcinoma (A 549) and gastric carcinoma cells (SGC 7901). Some also showed moderate neuroprotective effects against CoCl2-induced neuronal cell death in PC12 cells. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Valeriana jatamansi Caprifoliaceae Iridoids Cytotoxicity Neuroprotective activity

1. Introduction Valeriana jatamansi Jones belongs to the family Caprifoliaceae, an annual herb mainly distributed in China and India (Mathela et al., 2005). It is well known as traditional Chinese medicine with tranquilizing hypnotic, nervous disorders, epilepsy, insanity, snake poisoning and skin diseases (Ming et al., 1997; Mathela et al.,
ndez et al., 2004). Its root, as an important substitute of 2005; Ferna European V. officinalis, has been used to treat nervous disorders
ndez et al., 2004). Previous chemical (Mathela et al., 2005; Ferna investigation of V. jatamansi revealed the presence of iridoids, sesquiterpenoids, essential oil, flavone and lignans (Ming et al., 1997; Tang et al., 2002; Verma et al., 2011; Lin et al., 2010a,b; Tan

et al., 2016; Xu et al., 2011a,b). Valepotriates, a family of iridoid esters exhibiting potent cytotoxic and antitumor activities, have been attracting great interest in natural products (Yu et al., 2005; Tang et al., 2002; Becker et al., 1984; Bounthanh et al., 1981). What is worth mentioning, volvaltrate B induced a significant percentage of definitive remissions of ovarian tumors in the female mice (Zhang et al., 2010). As a part of our ongoing efforts to search for bioactive natural products on antineoplastic and neuroprotective agent from Valeriana genus, we herein report five iridoids, named chlorovaltrate P-T (1e5) and six known analogues (6e11) (see Fig. 1). Reported herein are the isolation, structure elucidation and biological activities including cytotoxicity and neuroprotective effects of the isolates. 2. Results and discussion

* Corresponding author. Pharmacy College, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China. Tel./fax: þ86 028 61800018. ** Corresponding author. State Key Laboratory Breeding Base of Systematic Research Development and Utilization of Chinese Medicine Resources, Sichuan Province and Ministry of Science and Technology, Chengdu, 611137, China. Tel./ fax: þ86 028 61800237. E-mail addresses: [email protected] (C. Peng), tanyuzhu@cdutcm. edu.cn (Y.-Z. Tan). http://dx.doi.org/10.1016/j.phytochem.2017.05.010 0031-9422/© 2017 Elsevier Ltd. All rights reserved.

Chlorovaltrate P (1), molecular formula C11H15ClO4 by HRESIMS (m/z 269.0499 [M (35Cl)þNa]þ, calcd for C11H15ClO4Na, 269.0557). The 1D NMR spectrum in combination with HSQC spectroscopic data of 1 showed one methoxyl, three methylenes, five methines (three oxygenated) and two quaternary carbons. The NMR spectroscopic data suggested that compound 1 possessed an iridoid-

R.-J. Wang et al. / Phytochemistry 141 (2017) 156e161

157

H-9/H2-10, indicated a b-configuration for the hydroxyl at C-7 and a-configuration for the methoxyl group at C-1. Therefore, the

Fig. 1. Structures of compounds 1e11.

Fig. 2. Selected COSY, HMBC and NOESY correlations of compound 1.

type skeleton with a methoxyl group. The NMR data of 1 were identical to those of (1S,3R,5R,7S,8R,9S)-3,8-epoxy-1-O-methyl-5hydroxyvalechlorine (Lin et al., 2010b). The only differences that could be discerned was the absence of a hydroxyl group at C-5 and hydroxyl group at C-7 instead of acetoxy in 1, which was supported by the HRESIMS. Interpretation of their 2D NMR data established the same overall structure. According to the HMBC spectrum (see Fig. 2), the correlations from H-1' (dH 3.34, s) to C-1(d 98.0) indicated that methoxyl was attached at C-1. Further analysis of the 2D NMR data led to the assignment of all the proton and carbon signals for 1 (Table 1). The relative configuration of 1 was established by the NOESY experiment (see Fig. 2). Generally, naturally occurring iridoids display the oxo-bridge from C-3 to C-8 could only be aoriented, and the H-5 and H-9 could only be b-oriented (Xu et al., 2011a; Li et al., 2013; Lin et al., 2013). NOESY correlations observed for H-7/H-6a, H-9/H-6b, H-9/H-5, H-9/H-1, H-5/H-1 and

Table 1 1 H NMR (400 MHz) and No.

1 3 4 5 6a 6b 7 8 9 10a 10b 11a 11b 10 1

1

relative configuration of 1 was assigned as 1S*,3R*,5R*,7S*,8R* and 9S*. The absolute configuration of 1 was determined by the experimental and calculated ECD data. Comparison of the experimental ECD data of 1 with the calculated data for the model molecules indicated 1 be in agreement with the 1S, 3R,5R,7S,8R and 9S configuration (see Fig. 3). Chlorovaltrate Q (2), molecular formula C11H15ClO5 by HRESIMS (m/z 285.0490 [M (35Cl)þNa]þ, calcd for C11H15ClO5Na 285.0506). Analysis of its 1D and 2D NMR indicated that 2 was similar to those of 1 (Table 1), suggesting that they were structural analogues except for the presence of hydroxyl group in 2. This assumption was corroborated by the interpretation of 2D NMR spectra (Fig. S2 in Supplementary Information). The NOESY correlations of H-7/H-6a, HO-5b/H-6b, H-9/H-1 and HO-5b/H-1 indicated a b-configuration for the hydroxyl at C-7 and a-configuration for the methoxyl group at C-1 (Fig. S3 in Supplementary Information), which was the same as compound 1. The calculated ECD spectrum of 2 showed a weak negative Cotton effect at 213 nm, but no conspicuous negative Cotton effect at 218 nm in experimental ECD spectrum (Fig. S4 in Supplementary Information). The similar NOESY correlations and biogenetic considerations assumed the assignment of the absolute configuration of 2 to be the same as that of 1. Chlorovaltrate R (3), molecular formula C11H15ClO4 by HRESIMS (m/z 269.0486 [MþNa]þ, calcd for C11H15ClO4Na, 269.0557), showed NMR data similar to those of compound 1 (Table 1). Comparison of the spectroscopic data and key NOESY correlations with compound 1, the relative configurations at C-1, C-3, C-5, C-8 and C-9 in compound 3 were same to 1 (see Fig. 4). Moreover, the NOESY cross peaks of H-7/H-9 and H-7/H-5, but not of H-7/H-9 and H-7/H-5 for compound 1 indicated b-orientation of H-7 in compound 3, which was unambiguous difference between compound 1 and 3. In regard to the absolute configuration of the hydroxyl group at C-7, the ECD spectra of the molecules for (1S, 3R, 5R, 7R, 8R and 9S)-3 and its enantiomer were calculated using the quantum chemical TDDFT method. Comparison of the experimental cotton effect (CE) of 3 (positive CE at 208 nm and negative CE at 226 nm) with the calculated data for the model molecule assigned 3 have the 1S, 3R, 5R, 7R, 8R and 9S configuration (see Fig. 5). Chlorovaltrate S (4) was assigned as C23H34O10 on the basis of HRESIMS (m/z 493.2050 [MþNa]þ, calcd for C23H34O10Na, 493.2050). The 1D NMR spectrum in combination with HSQC exhibited twenty three carbon signals, including one methoxyl, five

13

C NMR (100 MHz) data of compounds 1e3 (d in ppm, J in Hz).

a

2a

3b

dC

dH

dC

dH

dC

dH

98.0 CH 92.1 CH 148.7 C 38.9 CH 42.6 CH2

4.93 br.s 5.14 s

96.7 CH 93.2 CH 152.2 C 76.1 C 49.7 CH2

5.00 br.s 5.19 s

96.3 CH 93.9 CH 148.3 C 37.0 CH 41.4 CH2

4.92 d (2.3) 5.13 s

75.1 82.9 41.7 47.7

CH C CH CH2

107.7 CH2 55.5 CH3

2.97 1.84 1.94 3.81

m ddd (13.7, 7.3, 2.9) m s

2.27 3.79 4.13 4.81 4.91 3.34

br.d (5.0) d (11.0) d (11.0) br.s br.s s

The H NMR data of the hydroxy at C-5 of 2: dH 5.69 (1H, br.s, H-HO-5). a Recorded in DMSO-d6. b Recorded in CDCl3.

71.6 83.2 47.9 47.7

CH C CH CH2

107.2 CH2 55.8 CH3

1.85 dd (13.4, 3.0) 2.25 dd (13.4, 7.1) 3.65 m 2.22 3.82 4.09 5.03 5.13 3.34

br.s d (11.1) d (11.1) br.s br.s s

77.1 83.1 42.2 47.5

CH C CH CH2

108.1 CH2 55.3 CH3

3.21 1.90 2.14 4.26

m ddd (14.0, 7.4, 3.1) dd (14.0, 7.4) dd (7.3, 2.9)

2.44 3.85 3.92 4.83 4.92 3.39

dd (4.8, 3.1) d (11.2) d (11.2) d (1.0) d (1.0) s

158

R.-J. Wang et al. / Phytochemistry 141 (2017) 156e161 Table 2 1 H NMR (400 MHz) and 13C NMR (100 MHz) data of compounds 4e5 (d in ppm, J in Hz). No.

1 3 4 5 6a 6b 7 8 9 10a 10b 11a 11b 10 20 Fig. 3. Experimental and calculated ECD spectra of 1,4 and 5.

4a

5b

dC

dH

dC

dH

96.3 CH 92.9 CH 152.2 C 76.3 C 47.6 CH2

5.08 d (3.1) 5.30 s

96.4 CH 94.3 CH 152.9 C 77.0 C 48.3 CH2

5.26 d (3.2) 5.20 s

73.3 81.8 44.9 64.8

CH C CH CH2

107.2 CH2 54.8 CH3

1.85 dd (14.2, 2.9) 2.30 dd (14.3, 7.3) 4.65 dd (7.3, 2.9) 2.52 4.24 4.37 5.07 5.14 3.27

d (3.0) d (11.7) d (11.7) br.s br.s s

74.9 84.2 44.7 64.3

CH C CH CH2

108.1 CH2 63.6 CH3 15.3

2.01 dd (13.7, 3.3) 2.45 dd (13.7, 7.4) 4.03 dd (7.4, 3.3) 2.57 3.82 3.89 5.05 5.32 3.50 1.18

d (3.1) d (11.4) d (11.4) br.s br.s m 3.81 m t (7.1)

The signals of the substituent at C-7 and C-10 in 1HNMR. For 4 (the acetoxyl group):

dH 1.99 (1H, s, H-200 ); (the a-isovaleroxyisovaleroxy group): dH 4.69 (1H, d, J ¼ 4.7 Hz,

H-2000 ), 2.11 (1H, m, H-3000 ), 0.90 (3H, d, J ¼ 6.9 Hz, H-4000 ), 0.92 (3H, d, J ¼ 6.9 Hz, H-5000 ), 2.24 (2H, m, H-2000 '), 1.99 (1H, m, H-3000 '), 0.92 (3H, J ¼ 6.6 Hz, H-4000 '), 0.92(3H, J ¼ 6.6 Hz, H-5000 '). In 13C NMR. For 4 (the acetoxyl group): dc 169.4 (C, C-100 ), 20.8 (CH3, C-200 ); (the a-isovaleroxyisovaleroxy group): dC 168.8 (C, C-1000 ), 76.1 (CH, C-2000 ), 29.5 (CH, C-3000 ), 17.2 (CH3, C-4000 ), 18.4 (CH3, C-5000 ), 172.0 (C, C-1000 '), 42.3 (CH2, C-2000 '), 25.3 (CH, C-3000 '), 22.1 (CH3, C-4000 ', 5000 '). a Recorded in DMSO-d6. b Recorded in CDCl3.

methyls, four methylenes (an O-bearing and an exo-methylene), seven methines (four oxygenated), as well as six quaternary carbons. Based on the reported compounds from V. jatamansi, the NMR spectroscopic data (Table 2) suggested that compound 4 had an iridoid-type skeleton with an acetoxy and an a-[(isovaleryloxy) isovaleryloxy] group (Lin et al., 2009, 2010b). Detailed comparison of the NMR spectroscopic data with jatairidoid C indicated that the

two compounds have a similar structure, with an oxo-bridge between C-3 and C-8 (Table 2). The only difference between 4 and jatairidoid C was the hydroxy group at C-1 in jatairidoid C was replaced by a methoxyl group in 4 (Xu et al., 2012). The iridoid-type skeleton, an acetoxy, and an a-[(isovaleryloxy)isovaleryloxy] in 4 were confirmed by the interpretation of 2D NMR spectra (see Fig. 6). Sequentially, the acetoxy located at C-7, methoxyl at C-1 and the a-[(isovaleryloxy)isovaleryloxy] at C-10, were fully supported by the HMBC correlations of H-7 to C-100, H-1'to C-1 and H-10 to C1000. By further analysing of the 2D NMR spectra, all the proton and carbon signals for 4 were unambiguously assigned (Table 2). Thus, the planar structure of 4 was disclosed. The NOESY correlations of H-7/H-6a, H-9/H-6b, H-9/H2-10, H-1/H-9 and H-1/H2-10 suggested a b-configuration for the acetoxy at C-7 and a-configuration for the methoxyl group at C-1. Moreover, the NOESY cross peaks of H-2000 / H-7a, but not of H-2000 /H-9b indicated a-orientation of H-2000 for compound 4. The comparable cotton effects of 1 and 4 allowed the assignment of the absolute configuration of 4 to be the same as that of 1(see Fig. 3). Chlorovaltrate T (5) possessed a molecular formula C12H18O6, as determined by HRESIMS (m/z 281.1002 [MþNa]þ, calcd for C12H18O6Na, 281.1001). The 1D NMR data of 5 (Table 1) was similar

Fig. 5. Experimental and calculated ECD spectra of 3.

Fig. 6. Selected COSY, HMBC and NOESY correlations of compound 4.

Fig. 4. Selected COSY, HMBC and NOESY correlations of compound 3.

R.-J. Wang et al. / Phytochemistry 141 (2017) 156e161

to compound 2 (Table 2), suggesting that they were structural analogues, except for the presence of a hydroxymethyl at C-8 (dC 84.2) and an oxyethyl group at C-1 (dC 96.4) in 5 rather than one chloroethyl at C-8 and one methoxyl group at C-1 in 2. The HMBC correlations of Ha-10 and Hb-10 with C-1, H-10a and H-10b with C-7 and C-9 indicated the oxyethyl group and hydroxymethyl were located at C-1and C-8, respectively (Fig. S2 in Supplementary Information). The key NOESY correlations showed the relative configurations at C-1, C-3, C-5, C-7, C-8 and C-9 in compound 5 were identical to 2 (Fig. S3 in Supplementary Information). The similar NOESY correlations and the comparable Cotton effects of 1 and 5 allowed the assignment of the absolute configuration of 5 to be the same as that of 1(see Fig. 3). In addition to the five iridoids 1e5, the known analogues were identified by comparison of spectroscopic data with those reported in the literature as (4b,8b)-8-methoxy-3-methoxy-10-methylene2,9-dioxatricyclo[4.3.1.03,7]decan-4-ol (6) (Li et al., 2013), chlorovaltrate A (7) (Lin et al., 2013), (1R,3R,5R,7S,8R,9S)-3,8-epoxy-1-Oethyl-5-hydroxyvalechlorine (8) (Lin et al., 2010b), 8-methoxy-4acetoxy-3-chlormethyl-10-methylen-2,9-dioxa-tricyclo[4.3.1.03,7] decan (9) (Thies and Asai, 1972), (1S,3R,5R,7S,8R,9S)-3,8-epoxy-1O-ethyl-5-hydroxyvalechlorine (10) (Lin et al., 2010b), (1R,3R,5R,7S,8R,9S)-3,8-epoxy-1-O-methyl-5-hydroxyvalechlorine (11) (Lin et al., 2010b). The main issue arises as to whether these compounds are real natural products or are artefacts of extraction. To solve this problem, a simulated extraction protocol was then undertaken. The crude material and some compounds especially with the ethyl and acetyl groups were determined by LC-MS (Figs. S5eS11 in Supplementary Information). This result indicated that 5 and 9 are likely to extraction artifacts. In fact, the reaction between the hemiacetalic hydroxyl at C-1 and alcoholic solvents may not easily occur under a simple extraction. This result was supported by the reported literature (Lin et al., 2010b). According to iridoids with oxo-bridge between C-3 and C-8 recently isolated from V. jatamansi and biogenetic consideration, the other compounds are real natural products. Previous bioassay has reported that valepotriates isolated from Valeriana wallichii D. C. and V. jatamansi. exhibited potent cytotoxic and antitumor activities (Yu et al., 2005; Tang et al., 2002; Becker et al., 1984; Bounthanh et al., 1981; Zhang et al., 2010; Lin et al., 2013). This thus extended our attention to the large numbers of iridoids of V. jatamansi. in the discovery of anticancer as drug-lead compounds. Compounds 1e11 were evaluated for cytotoxicity against two human cancer cell lines including lung adenocarcinoma (A 549) and gastric carcinoma cells (SGC 7901) by using the MTT method. Unfortunately, all the isolates displayed no dramatic cytotoxicity effect. The compounds 8 and 9 exhibited weak cytotoxicity against A549 with IC50 values of 97.6 mM and 120.4 mM, while others were inactive. The study results together with the above argument led one to assume that cytotoxicity of iridoids was potentially related to molecular constitution. Understanding these relationships may give insights into chemical and pharmacological approaches for enhancing the chemopreventive effects of the iridoids and for developing novel anticancer agents. Therefore, the structure-activity relationship of the iridoids is also worth of our concern in the further research. During our search for new types of natural products possessing neuroprotective activities, we investigated the chemical constituents of the roots of V. jatamansi. By bioactivity-guided isolation, we isolated successfully isopatrinioside with neuroprotective effect from the roots of V. jatamansi (Tan et al., 2016). The previous study discovered iridoids may be bioactive component in V. jatamansi, which has been used as neurological disorder drug for thousands of

159

years in East Asia. The neuroprotective effects of the compounds (1e11) against CoCl2-induced neuronal cell death in PC12 cells were investigated by an established CCK-8 assay (Tan et al., 2016). At the concentrations of 0.05 mM, 0.5 mM, 5.0 mM, compound 6 exhibited a remarkable effect with protection rate from 18.1% to 65.0%, 71.4%. For compound 10, the moderate neuroprotective activities were performed with protection rate from 35.2% to 53.5%, 54.6%, whereas the other iridoids were inactive even at the high concentrations of 50 mM. 3. Conclusions Five iridoids (1e5) and six known analogues (6e11) were isolated from the roots of Valeriana jatamansi, most of which exhibited no cytotoxicity against A549 and SGC 7901 except compounds 8 and 9 (IC50 > 50 mM). Compound 6 and 10 showed potent neuroprotective effects. In particular, the compound 6 played an important role in the discovery and development of new chemical entities as neuroprotective drug-lead compounds. 4. Experimental 4.1. General experimental procedures NMR measurements were carried out on a Bruker-AVII-400 spectrometer (Bruker, Bremerhaven, Germany) with TMS as the internal standard. HRESIMS were obtained using Waters Synapt G2HDMS (Waters, Millford, MA, USA). UV spectra were recorded on a Shimadzu UV-260 spectrophotometer (Shimadzu, Kyoto, Japan). ECD spectra were measured on a JASCO J-1500 spectropolarimeter. IR (KBr) spectra was obtained using Perkin-Elmer one FT-IR spectrometer (PerkinElmer, Waltham, USA). Optical rotations were measured with Anton Paar MCP 200 (AntonPaar, Graz, Austria) at room temperature. MPLC was performed on a MPLC-52 system (Saipuruisi, Beijing, China). Semipreparative HPLC was performed on Shimadazu LC-10AT instrument with an SPD-10AVP detector and a YMC-Pack ODS-A column (250 mm  10 mm, 5 mm) at 210 nm. Silica gel (200e300 mesh) for column chromatography (CC) and preparative TLC was obtained from Qindao Marine Chemical Factory, Qingdao, China. Fractions were monitored by TLC, and spots were visualized under UV light (254 nm) or by spraying with 10% H2SO4 in EtOH followed by heating. Sephadex LH-20 was purchased from Pharmacia, Sweden. MCI gel (75e150 mm, Mitsubishi Chemical Corporation, Japan). All the solvent used were of analytical grade. 4.2. Plant material The roots of Valeriana jatamansi Jones were collected in June 2014 from the medicinal herb market in Chengdu, Sichuan Province, China and identified by Prof. Min Li. A rebarium specimen (ZZX-1407) was deposited in School of Pharmacy, Chengdu University of TCM, Chengdu, China. 4.3. Extraction and isolation The air-dried roots of Valeriana jatamansi Jones (5 kg), was powdered and exhaustively extracted using by percolating with 75% EtOH (3  30 L). The organic solvent was evaporated under vacuum to afford a crude extract (850 g). The residue was suspended in water (2 L) and then successively partitioned with Petroleum Ether (3  2 L), EtOAc (3  2 L) and n-BuOH (3  2 L). The EtOAc layer (170 g) was subjected to silica gel column chromatography, then eluted with a gradient of CHCl3-MeOH to afford 9 fractions (Fr.1-Fr.9). Fraction 5 (4 g) was separated by MPLC (MeOH/

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H2O gradient, 40:60e90:10) to afford about 7 subfractions respectively (Fr.5.1-Fr.5.7). Fr.5.3 (86 mg) was purified by semipreparative HPLC (MeOH/H2O, 70:30) and Sephadex LH-20 (MeOH) to yield 6 (7.5 mg) and 8 (8.5 mg). Fr.5.4 (118 mg) was chromatographed over Sephadex LH-20 (MeOH) to give four subfractions (Fr.5.4.1-Fr.5.4.4). Fr.5.4.3 (50 mg) was purified by preparative TLC (petroleum ether/EtOAc,10:1) to yield 7 (8.5 mg) and 9 (6.0 mg). Fr.5.4.4 (35 mg) was purified by semipreparative HPLC (MeOH/H2O, 70:30), then followed by Sephadex LH-20 (MeOH) to yield compounds 2 (5.5 mg) and 3 (5.8 mg). Fr.5.5 (1.1 g) was further separated by MPLC (MeOH/H2O gradient,40:60e90:10), followed by a silica gel (petroleumether/acetone, 20:1e10:1) and then passed through Sephadex LH-20 column (CHCl3/MeOH, 1:1) to yield compound 1 (14.0 mg), 4 (12.0 mg) and 5(25.0 mg). Fraction 6 (5 g) was separated over silica gel CC (petroleum ether/acetone, 20:1e1:1), to afford 5 fractions (Fr.6.1eFr.6.5). Fr.6.4 (1.2 g) was successively separated by MPLC (MeOH/H2O gradient, 40:60e90:10) and repeated silica gel CC (petroleum ether/EtOAc, 5:1e1:1), purified by Sephadex LH-20 (MeOH) to yield compounds 10 (15.8 mg) and 11(12.0 mg). 4.3.1. Chlorovaltrate P (1) Colorless oil; ½a25 D : þ32:27 (c 0.04, MeOH); UV(MeOH) lmax (log ε) 289 (1.22) nm; ECD (CH3CN) 212 (Dε 12.51) nm; IR (KBr)max 3747, 3310, 2926, 2855, 1724, 1554, 1384, 1088, 1018, and 967 cm1; For 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 269.0499 [M (35Cl)þNa]þ (calcd for C11H15ClO4Na, 269.0557). 4.3.2. Chlorovaltrate Q (2) Colorless oil; ½a25 D : þ55:60 (c 0.12, MeOH); UV(MeOH) lmax (log ε) 289 (1.75) nm; ECD (CH3CN) 217 (Dε 0.11) nm; IR (KBr)max 3750, 3310, 2939, 1307, 1290, 1204, 1115, 1085, 1056, 1028, 967, 916, and 819 cm1; For 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 285.0490 [M (35Cl) þ Na]þ (calcd for C11H15ClO5Na, 285.0506). 4.3.3. Chlorovaltrate R (3) Colorless oil; ½a25 D : þ77:00 (c 0.04, CH2Cl2); UV (CH2Cl2) lmax (log ε) 297 (2.54) nm; ECD (CH3CN) 208 (Dε þ5.79), 226 (Dε 5.73) nm; IR (KBr)max 3488, 2959, 2920, 2850, 1464, 1384, 1262, 1126, 1073, 1017, 949 and 803 cm1; For 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 269.0486 [MþNa]þ (calcd for C11H15ClO4Na, 269.0557). 4.3.4. Chlorovaltrate S (4) Colorless oil; ½a25 D : þ6:89 (c 0.05, MeOH); UV(MeOH) lmax (log ε) 294 (2.15) nm; ECD (CH3CN) 212 (Dε 10.52) nm; IR (Kmax 3749, 2961, 2928, 1734, 1384, 1243, 1202, 1120, 1070, 1031, 995 and 950 cm1; For 1H NMR and 13C NMR data, see Table 2; HRESIMS m/z 493.2050 [MþNa]þ (calcd for C23H34O10Na, 493.2050). 4.3.5. Chlorovaltrate T (5) Colorless oil; ½a25 D : þ13:62, (c 0.13, CH2Cl2); UV(CH2Cl2) lmax (log ε): 296 (2.44) nm; ECD (CH3CN) 207(Dε 12.72) nm; IR (KBr)max 3357, 2958,2920, 2850, 1262, 1119, 1073, 1024 and 957 cm1; For 1H NMR and 13C NMR data, see Table 2; HRESIMS m/z 281.1002 [MþNa]þ (calcd for C12H18O6Na, 281.1001). 4.4. ECD calculation Conformational analyses for compounds (1e5) were performed via Spartan '14 software using the MMFF94 molecular mechanics force field calculation. Conformers within a 10 kcaL/mol energy window were generated and optimized using DFT calculations at

the B3LYP/6-31G(d) level. Conformers with a Bolzmann distribution over 1% were chosen for ECD calculations in MeCN at the B3LYP/6-311 þ G(2d,p) level. The IEF-PCM solvent model for MeCN was used. The calculated ECD spectra were obtained by density functional theory (DFT) and time-dependent DFT(TD-DFT) using Gaussian 09 and analyzed using GUIs GaussView (version 5.0). 4.5. Cytotoxicity assay The cytotoxic activity was measured by MTT assay. Two cancer cells, namely lung adenocarcinoma (A 549) and gastric carcinoma cells (SGC 7901), were seeded in 96-well plates and treated 24 h later with various concentrations of compounds 1e11. After 24 h of incubation, MTT was added to all wells. Plates were incubated for another 24 h, and cell viability was measured by observing absorbance at 492 nm on a varioskan flash-3001. 4.6. Neuroprotective effect assay Tan et al., 2016. Acknowledgments This research was financially supported by National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China fund (grant No. J13100340-11), the key fund project for Education Department of Sichuan (grant No.15ZA0093), the youth science and technology innovative research team fund project of Sichuan (grant No. 2016TD0006) and the project for administration of Traditional Chinese Medicine (grant No. 2016Q049). We gratefully acknowledge Prof. Cheng Yang of Si Chuan University for measuring ECD spectra. We acknowledge Dr. Y.N. Liu in Sun Yat-sen University for ECD calculation. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.05.010. References Becker, H., Chavadej, S., Thies, P.W., Finner, E., 1984. Die Struktur neuer valepotriate aus colchicinbehandelten zellkulturen von Valeriana wallichii. Planta Med. 44, 245e248. Bounthanh, C., Bergmann, C., Beck, J.P., Haag-Berrurier, M., Anton, R., 1981. Valepotriates, a new class of cytotoxic and antitumor agents. Planta Med. 41, 21e28. Fern
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