Fitoterapia 96 (2014) 48–55
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Isolation and screened neuroprotective active constituents from the roots and rhizomes of Valeriana amurensis Changfu Wang a, Yang Xiao a, Bingyou Yang a, Zhibin Wang a, Lihua Wu a, Xiaolin Su a, Adelheid Brantner b, Haixue Kuang a,⁎, Qiuhong Wang a,⁎ a b
Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University of Chinese Medicine, No. 24 HePing Road, XiangFang District, Harbin 150040, China Institute of Pharmacognosy, University of Graz, Universitaetsplatz 4/I, A-8010 Graz, Austria
a r t i c l e
i n f o
Article history: Received 20 February 2014 Accepted in revised form 3 April 2014 Available online 15 April 2014 Chemical compounds studied in this article: Loganin (PubChem CID: 87691) Patriscabroside I (PubChem CID: 71453062) Keywords: Valeriana amurensis Alzheimer’s disease Iridoids Lignans PC12 cell
a b s t r a c t In previous study, we have screened the effective fraction against Alzheimer’s disease (AD-EF) from the extracts of roots and rhizomes of Valeriana amurensis, based on which neuroprotective active constituents from AD-EF were investigated. Six new compounds 1–6, including four iridoids (xiecaoside A–C and xiecaoline A), one pinane-type monoterpeneglucoside (xiecaoside D), and one phenylpropanoid glycoside (xiecaoside E) were isolated together with 11 known compounds 7–17. The structures of 1–6 were elucidated by their spectroscopic data. The protective effects of compounds 1–17 on PC12 cells with neurotoxicity induced by amyloid-beta 1–42 (Aβ1–42) was also investigated, respectively. Consequently, compound 6 and lignans 11–17 were responsible for protecting against Aβ-induced toxicity in PC12 cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Valeriana is a perennial herb and belongs to the family of Valerianaceae. There are about 30 species in genus Valeriana in China [1]. As one species of Valeriana, Valeriana amurensis (V. amurensis) is widely distributed in Russian Far East, northeast China, and North Korea [2]. The roots and rhizomes of V. amurensis, pungent and sweet in flavor and warm in
Abbreviations: AD-EF, effective fraction against Alzheimer’s disease; AD, Alzheimer’s disease; Aβ1–42, amyloid-beta 1–42; CC, column chromatography; TMS, tetramethylsilane; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. ⁎ Corresponding authors. Tel.: +86 45182193001. E-mail addresses: [email protected] (C. Wang), [email protected] (Y. Xiao), [email protected] (B. Yang), [email protected] (Z. Wang), [email protected] (L. Wu), [email protected] (X. Su), [email protected] (A. Brantner), [email protected] (H. Kuang), [email protected] (Q. Wang).
http://dx.doi.org/10.1016/j.fitote.2014.04.007 0367-326X/© 2014 Elsevier B.V. All rights reserved.
nature, possess the effects of tranquilization, regulating qi, and analgesia and have been used to treat neurological system diseases, such as neurasthenia, insomnia, hysteria, and epilepsy for thousands of years in traditional Chinese medicine [3]. To be worth mentioning, however, we reported its potential therapeutic effect on Alzheimer’s disease (AD) in previous study for the first time, meanwhile, the effective fraction of V. amurensis against AD (50% EtOH fraction from AB-8 macroporous resin column, AD-EF) has been determined [4,5]. Previously, only several compounds of germacrane-type sesquiterpenoids and lignans separated from AD-EF showed protective effect on the neurotoxicity of PC12 cells induced by amyloid-beta (Aβ25–35) [6]. To further reveal the chemical basis for the therapeutic effect of V. amurensis on AD, we continue to carry out phytochemical study on V. amurensis but a little different, and six new (four iridoid glycosides, one monoterpeneglucoside, and one phenylpropanoids glycoside) together with 11 known compounds were obtained. In this paper, the isolation and
C. Wang et al. / Fitoterapia 96 (2014) 48–55
structural elucidation of the new compounds 1–6 was described. The structures of known compounds 7–17 were identified by physico-chemical constants, spectroscopic analysis, and comparing their spectroscopic data with reference values, meanwhile, MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] assay method was used to investigate the neuroprotective effects of compounds 1–17. 2. Experimental 2.1. General Bruker DPX 400 instrument (Bruker SpectroSpin, Karlsruhe, Germany) was used to measure the NMR spectra. Tetramethylsilane (TMS) as the internal standard, chemical shifts and coupling constants were given as δ values and in Hz, respectively. The HRESIMS analyses were carried out on Xero Q Tof MS spectrometer (Waters, Milford, MA, USA). Kofler micromelting point apparatus was used to determine the melting point (m.p.). Preparative HPLC was conducted on a Waters 2535 instrument equipped with a UV-2998 and RI-2414 detector, and using the Waters Sunfire prep C18 OBD™ 10 μm (19 × 250 mmi.d.) column for preparing compounds. IR Spectra was recorded on Shimadzu FTIR-8400S (Kyoto, Japan). Macroporous resin (AB-8 Crosslinked Polystyrene, Nan Kai, Tianjin, China), silica gel (200–300 mesh, Haiyang Chemical Group Co. Ltd, Qingdao, China), and ODS-A (120A, 50 mm; YMC, Kyoto, Japan) was used for column chromatography (CC). PC12 cells (Institute of biochemistry and cell biology, Shanghai, China) were cultured in DMEM (Hyclone, NRH0020) with 5% fetal bovine serum and 1% antibiotic mixture of penicillin-streptomycin at 37 °C with atmosphere of 5% CO2. PC12 cell injury was induced by Aβ1–42 (Bioss, Beijing, China). PC12 cell viability was determined by Microplate reader (VICTOR™ × 3, PerkinElmer, Inc., Massachusetts, United States). 2.2. Plant materials We collected the roots and rhizomes of V. amurensis from the Great Xing’an Mountains area (Heilongjiang province, China) and identified by Xiaowei Du of Heilongjiang University of Chinese Medicine. The voucher specimen (No. 20100806) is deposited at the Herbarium of Heilongjiang University of Chinese Medicine, China. 2.3. Extraction and isolation The dried roots and rhizomes of V. amurensis (9.0 kg) were extracted three times (2 h for each) with 75% EtOH (72 L) under reflux and then removed the solvent. The obtained residue (1642.4 g) was suspended in water and partitioned with petroleum ether (5 × 12 L). After removal of solvent, the obtained water extract (1228.9 g) was subjected to AB-8 macroporous resin column (10 × 60 cm) and eluted with H2O, 50% and 95% EtOH successively. The obtained 50% EtOH fraction (260.6 g) is the AD-EF as previous study [4,5]. In this study, 180.0 g AD-EF was subjected to silica gel (200–300 mesh) column chromatography (CC), eluting with CH2Cl2–MeOH (from 20:1 to 1:1, v/v) to obtain fractions I–VI. Fraction I (24.8 g) was subjected to CC over silica gel, eluted with petroleum ether–EtOAc (from 25:1 to 1:1, v/v), to
49
give fractions I1–I6. Compound 4 (99.5%, 35 mg) was crystallized from fraction I4 directly in CH2Cl2. Fraction III (31.3 g) was chromatographed over silica gel, eluted with CH2Cl2–MeOH (from 15:1 to 3:1, v/v), to afford fractions III1–III5. Fraction III1 (8.5 g) was separated by ODS CC, eluted with 10–50% gradient MeOH–H2O to afford four fractions. The four fractions were separated and purified with preparative HPLC (MeOH–H2O) and compounds 6 (98.2%, 26 mg, tR = 42.0 min, 20% MeOH), 14 (97.4%, 32 mg, tR = 39.0 min, 30% MeOH), and 16 (98.8%, 48 mg, tR = 44 min, 35% MeOH) were obtained. Fraction III3 (7.2 g) was separated by ODS CC, eluted with 10–50% gradient MeOH–H2O to afford two fractions. The two fractions were separated and purified with preparative HPLC (MeOH–H2O) and compounds 10 (99.4%, 22 mg, tR = 36 min, 25% MeOH) and 13 (96.4%, 31 mg, tR = 11.0 min, 32% MeOH) were obtained. Fraction III5 (5.2 g) was separated by ODS CC, eluted with 10–30% gradient MeOH–H2O to afford two fractions. The two fractions were separated and purified with preparative HPLC (MeOH–H2O, 20%) and compounds 3 (97.4%, 27 mg, tR = 43.2 min), 7 (95.8%, 44 mg, tR = 46.1 min), and 8 (97.8%, 36 mg, tR = 48.4 min) were obtained. Fraction V (51.9 g) was subject to ODS CC, eluted with 10–50% gradient MeOH–H2O to afford compound 11 (98.5%, 47 mg, 10% MeOH) and eight fractions V1–V8. Fractions V2 (11.4 g), V4 (5.9 g), and V6 (14.5 g) were separated and purified with preparative HPLC (MeOH–H2O) to obtain compounds 2 (99.7%, 21 mg, tR = 8.1 min, 35% MeOH), 5 (95.1%, 30 mg, tR = 50.4 min, 38% MeOH), and 9 (97.2%, 33 mg, tR = 6.0 min, 35% MeOH) from fraction V2, compound 1 (96.8%, 23 mg, tR = 13.2 min, 38.4% MeOH) from fraction V4, and compounds 12 (98.2%, 44 mg, tR = 39 min, 31% MeOH), 15 (99.1%, 38 mg, tR = 51 min, 20% MeOH), and 17 (99.4%, 46 mg, tR = 14 min, 35% MeOH) from fraction V6. 2.3.1. Xiecaoside A (1) White amorphous powder, [α]22 D + 24.2° (c 0.12, MeOH); UV (MeOH) λmax (log ε) 202 (3.59) (nm); IR (KBr) νmax 3421,
Table 1 1 H NMR data of compounds 1–3 (400 MHz for 1 and 2, 500 MHz for 3, δ in ppm, J in Hz). Position
1a
1
3.95, 3.75, 4.36, 4.18, 3.24, 2.73, 2.25, 5.77,
3 5 6 7 8 9 10 11 1′ 2′ 3′ 4′ 5′ 6′ a
2a d (10.4) d (10.4) d (12.6) d (12.6) m m brd (16.3) brs
4.10, brd (10.0) 4.91, dd (6.3, 2.0) 4.28,d (7.8) 3.14, t (8.4) 3.31, m 3.23, m 3.23, m 3.81, d (11.6) 3.62, dd (11.8, 4.8)
3a
5.11, 4.44, 3.33, 2.33, 2.18, 3.86,
d (11.1) d (11.7) m dd (8.4, 13.4) m m
3.07, 1.58, 5.07, 4.40, 3.20, 3.30, 3.32, 3.38, 3.88, 3.71,
d (10.8) s 5.10, s d (7.8) t (8.2) m m m m dd (12.0, 4.9)
Measured in methanol–d4 at 30 °C.
4.02, d (12.3) 3.44, d (12.3) 1.90, 1.42, m 2.21, 2.21, 2.91, 1.19, 1.26, 4.31, 3.19, 3.36, 3.28, 3.26, 3.81, 3.62,
1.74, m m d (4.5) d (6.8) s d (7.6) t (8.4) t (8.6) t (9.1) m d (11.6) dd (4.9,12.0)
50
C. Wang et al. / Fitoterapia 96 (2014) 48–55
2873, 1671, 1632, 1445, 1381, 1326, 1169, 1078, 886, 597 cm−1; ESIMS m/z 345 (100) [M + H]+; HRESIMS [M + H]+m/z 345.1542, calcd 345.1549 for C16H24O8H; 1H and 13C NMR data, see Tables 1 and 3.
2.3.2. Xiecaoside B (2) White amorphous powder, [α] D22 − 29.3° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 215 (4.27), 223 (4.27), 281 (4.06), 324 (3.36) (nm); IR (KBr) νmax 3400, 2920, 1762, 1631 cm−1; ESIMS m/z 361 (100) [M + H]+; HRESIMS [M + H]+m/z 361.1493calcd 361.1499 for C16H24O9H; 1H and 13C NMR data, see Tables 1 and 3.
2.3.3. Xiecaoside C (3) White amorphous powder, [α]22 D − 15.8° (c 0.12, MeOH); UV (MeOH) λmax (log ε) 201 (3.13) (nm); IR (KBr) νmax 3421, 3392, 2952, 2873, 1728, 1634, 1615, 1597, 1457, 1381, 1332, 1235, 1161, 1075, 721, 637 cm−1; ESIMS m/z 363 (100) [M + H]+; HRESIMS [M + H]+ m/z 363.1661 calcd 363.1655 for C16H26O9H; 1H and 13C NMR data, see Tables 1 and 3.
2.3.4. Xiecaoline A (4) colorless needle crystal (CH2Cl2), m.p. 115–117 °C; [α]22 D − 20.7° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 240 (2.73) (nm); IR (KBr) νmax 3434, 2962, 2878, 1726, 1631, 1458, 1381, 1355, 1248, 1173, 1117, 1036, 935 cm−1; ESIMS m/z 201 (100) [M + H]+; HRESIMS [M + H]+ m/z 201.1122 calcd 201.1127 for C10H16O4H; 1H and 13C NMR data, see Tables 2 and 3.
Table 2 1 H NMR data of compounds 4–6 (400 MHz, δ in ppm, J in Hz). Position 2 3
11 3-OCH3 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ b
5a
3.76, d (12.0) 3.37, d (12.0)
4 5 6 7 8 9 10
a
4b
1.66, 1.83, 2.11, 2.65, 1.12,
m 1.47, m m dd (1.4, 4.6) d (6.8)
6a
1.77, 1.87, 1.44, 1.58, 1.72, 2.26,
d (3.7) dt (13.6, 3.3) brd (13.6) t (4.2) 1.39, m 1.53, m
1.03, 1.09, 3.71, 3.64,
s s d (13.0) d (13.0)
7.01, brs
7.02, 6.92, 6.50, 6.24, 4.16,
d (8.4) dd (8.4,1.7) d (15.9) dt (15.9, 5.6) brd (5.6)
3.93, d (9.6) 3.54, s
Measured in methanol–d4 at 30 °C. Measured in DMSO–d6 at 30 °C.
2.3.6. Xiecaoside E (6) White amorphous powder, [α]22 D − 28.3°(c 0.11, MeOH); UV (MeOH) λmax (log ε) 257 (4.78) (nm); νmax 3410, 1621, 1455, 1201, 1123, 1097, 1035, 965, 814 cm−1; ESIMS m/z 489 (100) [M + H]+; HRESIMS [M + H]+ m/z 489.1976 calcd 489.1972 for C22H32O12H; 1H and 13C NMR data, see Tables 2 and 3. 2.4. Acid hydrolysis of 1–3, 5, and 6 Acid hydrolyses of 1–3, 5, and 6 were performed according to the method reported previously [7]. Briefly, compounds (2.5 mg) were hydrolyzed with 2 mol/L H2SO4 (2.0 mL) and sugar residues were treated with trimethylchlorosilane, respectively. The obtained sugar derivatives were analyzed by GC [8]. The monosaccharide of compounds 1–3 was determined to be D-glucose (tR = 7.25 min). The monosaccharides of compound 5 were determined to be D-glucose (tR = 7.25 min) and D-apiose (tR = 8.43 min). The monosaccharides of compound 6 were determined to be D-glucose (tR = 7.25 min) and L-rhamnose (tR = 4.32 min). 2.5. Determination of cell viability The MTT assay method was used to measure the cell viability as reported in reference [6] but a little different. Briefly, the PC12 cells (8 × 103 cells/well) were cultured for 24 h in 96-well plates, and then incubated without or with compounds 1–17 (at the concentrations of 5, 12.5, 25 μM), and the cells neurotoxicity was induced by 1.5 μM aggregated Aβ1–42 [9] for 4 h. Vitamin E (VE) was used as a positive drug [10]. 20 μL MTT solutions (5 mg/mL) were added into each well and cells were incubated at 37 °C for another 4 h. Aspirated off the supernatants and then dissolved formazan crystals with DMSO. The microplate reader was used to measure the optical density of each well at 490 nm and the results were expressed as the percentages. 3. Results and discussion
1.15, s 4.44, d (7.8) 3.17, t (9.20) 3.37, m 3.20, t (9.3) 3.41, m 3.96,dd (10.6, 2.0) 3.73,brd (10.6) 4.90, d (2.7) 3.86, d (2.6)
2.3.5. Xiecaoside D (5) White amorphous powder, [α]22 D − 46.4° (c 0.13, MeOH); IR (KBr) νmax 3464, 2925, 2885, 1451, 1380, 1173, 1119, 1070, 722 cm−1; ESIMS m/z 465 (100) [M + H]+; HRESIMS [M + H]+ m/z 465.2329 calcd 465.2336 for C21H36O11H; 1H and 13C NMR data, see Tables 2 and 3.
3.82, 4.79, 3.44, 3.47, 3.31, 3.41, 3.95, 3.54, 4.64, 3.77, 3.63, 3.31, 3.59, 1.15,
s d (6.4) m m m m d (9.7) m d (1.1) m m m m d (6.2)
Compound 1 was obtained as a white amorphous powder. The molecular formula of 1 was determined as C16H24O8 by HRESIMS ([M + H]+ m/z 345.1542, calcd 345.1549). The presence of the hydroxyl groups can be confirmed by IR (3421 cm−1). In the 1H-NMR of 1, signals of a nonoxygenated methylene at δ 2.73 (1H, m, H-6a) and 2.25 (1H, brd, J = 16.3 Hz, H-6b), a nonoxygenatedmethine at δ 3.24 (1H, m, H-5), two oxygenated methylenes at δ 3.95 (1H, d, J = 10.4 Hz, H-1a), 3.75 (1H, d, J = 10.4 Hz, H-1b), 4.36 (1H, d, J = 12.6 Hz, H-3a), and 4.18 (1H, d, J = 12.6 Hz, H-3b), and three olefinic protons at δ 5.77 (1H, brs, H-7), 4.91 (1H, dd, J = 6.3, 2.0 Hz, H-11a), and 4.10 (1H, brd, J = 10.0 Hz, H-11b) were observed. Besides, the β-configured glucose unit was confirmed by coupling constant of the anomeric proton at
C. Wang et al. / Fitoterapia 96 (2014) 48–55
51
Table 3 13 C NMR data of compounds 1–6 (100 MHz for 1–2 and 4–6, 125 MHz for 3, δ in ppm). Position
1a
2a
3a
4b
5a
6a
1 2 3 4 5 6 7 8 9 10 11 3-OCH3 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″
72.6, CH2
175.0, C
180.5, C
177.7, C
72.0, CH2 156.0, C 49.6, CH 39.0, CH2 131.2, CH 144.2, C 99.6, C 59.1, CH2 105.2, CH2
71.3, CH2 144.3, C 41.0, CH 39.8, CH2 89.9, CH 85.9, C 54.0, CH 22.3, CH3 113.8, CH2
67.6, 89.8, 95.3, 34.5, 34.0, 40.2, 60.6, 21.6, 17.4,
CH2 C C CH2 CH2 CH CH CH3 CH3
65.1, 86.5, 86.6, 36.8, 33.1, 38.0, 58.6, 21.8, 16.3,
89.0, C 52.6, CH 38.6, CH2 45.5, CH 28.3, CH2 22.3, CH2 49.2, C 24.1, CH3 24.5, CH3 69.6, CH2
133.9, C 111.6, CH 147.7, C 151.0, C 118.2, CH 121.0, CH 131.5, CH 129.1, CH 63.9, CH2
104.6, CH 75.2, CH 78.1, CH 71.7, CH 78.2, CH 62.9, CH2
105.2, CH 75.5, CH 78.0, CH 71.8, CH 78.2, CH 62.9, CH2
99.4, 75.1, 77.8, 71.5, 78.1, 62.6,
CH CH CH CH CH CH2
a b
CH2 C C CH2 CH2 CH CH CH3 CH3
99.8, CH 75.3, CH 78.6, CH 72.2, CH 78.1, CH 68.2, CH2 111.4, CH 76.7, CH 80.7, C 75.2, CH2 65.8, CH2
56.9, CH3 102.9, CH 75.1, CH 77.1, CH 71.7, CH 78.0, CH 68.0, CH2 102.3, CH 72.3, CH 72.6, CH 74.2, CH 70.0, CH 18.1, CH3
Recorded in methanol–d4 at 30 °C. Recorded in DMSO–d6 at 30 °C.
δ 4.28 (1H, d, J = 7.8 Hz, H-1′). Acid hydrolysis experiment further revealed a β-D-glucopyranosyl moiety existed in 1. 13 C-NMR and DEPT spectra of 1 (Tables 1 and 3) showed a hydroxymethyl at δC 59.1 (C-10), a nonoxygenatedmethine at δC 49.6 (C-5), a nonoxygenated methylene at δC 39.0 (C-6), two oxygenated methylenes at δC 72.6 (C-1) and 72.0 (C-3), an oxygenated quaternary carbon at δC 99.6 (C-9), two olefinic bonds at δC 156.0 (C-4), 105.2 (C-11), 131.2 (C-7), and 144.2 (C-8), and the remaining six signals corresponded to a β-Dglucopyranosyl moiety. The NMR data of 1 were similar to those of patriridoside G [7] and the main different lies in presence of signals at δC 59.1 and δH 4.10(2H, brd, J = 10.0 Hz) in 1 but absence of signals at δC 12.1 and 1.73 (3H, brs), which indicated that CH3-10 of patriridoside G was substituted by a hydroxyl group. The 1H-1H COSY and HSQC spectra (Fig. 2) gave two coupling sequences of C-3/C-4/C-11 and C-11/C-4/C-5/C-6/ C-7/C-8/C-10 for 1. HMBC correlations (Fig. 2) of H-10 with C-7, 8, and 9, H-1 with C-5 and 9, H-11 with C-3, 4, and 5, and H-3 with C-9 established the structure of 1. The glucosyl moiety was located at C-1 by the HMBC correlation from anomeric proton to C-1. Correlation (Fig. 2) between H-5β and H2-1 in NOESY spectra suggested a β-orientation for C-1. As a result, the structure of 1 was identified as [(5R,9S)8-hydroxymethyl-4-methylene-4,5,6,9-tetrahydro-3H-cyclopenta[b]furan-9-yl]methanol 1-O-β-D-glucopyranoside (Fig. 1) and named xiecaoside A. Compound 2 was isolated as a white amorphous powder. The molecular formula was assigned as C16H24O9 from its HRESIMS ([M + H]+ m/z 361.1493, calcd 361.1499). The IR absorptions in 3400 and 1762 cm−1 originated from the hydroxyl and lactone carbonyl groups, respectively. The 1H NMR spectrum (Table 1) gave signals for a methyl group at δ
1.58 (3H, s, H-10), three methylene groups at δ 5.11 (1H, d, J = 11.1 Hz, H-3a), 4.44 (1H, d, J = 11.7 Hz, H-3b), 2.33 (1H, dd, J = 8.4, 13.4 Hz, H-6a), 2.18 (1H, m, H-6b), 5.07 (s, H-11a), 5.10 (s, H-11b), and three methenyl groups at δ 3.33 (1H, m, H-5), 3.86 (1H, m, H-7), 3.07 (1H, d, J = 10.8 Hz, H-9). Acid hydrolysis experiment of 2 confirmed the presence of D-glucopyranosyl moiety and coupling constant of the anomeric proton (δH 4.40, 1H, d, J = 7.8 Hz, H-1′) concluded a β-configured glucose unit existed in 2. 16 carbon signals could be observed in 13C NMR and DEPT spectra (Table 3), including a methyl at δC 22.3 (C-10), a lactone carbonyl at δC 175.0 (C-1), a terminal double bond at δC 144.3 (C-4) and 113.8 (C-11), as well as a group of glucosyl moiety signals. Besides, other carbon signals such as two methylenes at δC 71.3 (C-3) and 39.8 (C-6), three methenyl groups at δC 41.0 (C-5), 89.9 (C-7), and 54.0 (C-9), a quaternary carbon at δC 85.9 (C-8) were also observed. Except for the extra glucosyl moiety signals, compound 2 had similar NMR data with jatamanin A [11]. The main difference in 13C-NMR lies in the δ values of C-7 in 2 was shifted downfield by 8.4 compared to that of jatamanin A, which indicated that the β-D-glucose unit was located at C-7. The 1H-1H COSY and HSQC spectra showed the coupling sequence for the C (9)–C (5)–C (6)–C (7) fragments. Analysis of HMBC observed correlations (Fig. 2) of H-3 with C-1, 4, and 5, H-11 with C-3, 4, and 5, H-10 with C-7, 8, and 9, and H-9 with C-1. The glucosyl moiety was located at C-7 by the correlation from the anomeric proton H-1′ to C-7. Given to the biogenetic of iridoid prescribed that H-5 and H-9 were β-oriented [7]. NOE correlations (Fig. 2) between H-9 and H-10, H-5 and H-10, but no correlations between H-9 and H-7, H-5 and H-7, suggested that H-5, H-9, and the methyl group were β-oriented and the glucosyl moiety was
52
C. Wang et al. / Fitoterapia 96 (2014) 48–55
Fig. 1. Structures of compounds 1–17.
corresponding β-oriented. Therefore, the structure of 2 was founded to be (5S,7S,8S,9S)-8-hydroxy-8-methyl-4-methylenehexahydrocyclopenta[c]pyran-1(3H)-one 7-O-β-D-glucopyranoside (Fig. 1) and named xiecaoside B. Compound 3 was obtained as a white amorphous powder and its molecular formula C16H26O9 was concluded by HRESIMS ([M + H]+, m/z 363.1661, calcd 363.1655). The IR spectrum showed absorptions for hydroxyl groups (3421, 3392 cm− 1) and lactone carbonyl group (1728 cm− 1). In the 1H NMR spectrum (Table 1) of 3, signals for two methyl groups at δ 1.19 (3H, d, J = 6.8 Hz, H-10) and 1.26 (3H, s, H-11), three methylene groups at δ 4.02 (1H, d, J = 12.3 Hz, H-3a), 3.44 (1H, d, J = 12.3 Hz, H-3b), 1.90 (1H, m, H-6a), 1.42 (1H, m, H-6b), 2.21 (1H, m, H-7a), and 1.74 (1H, m, H-7b), and two methenyl groups at δ 2.21 (1H, m, H-8) and 2.91 (1H, d, J = 4.5 Hz, H-9) could be observed. 16 carbon signals were exhibited in the 13C NMR and DEPT spectra of 3 (Table 3), including two methyl carbons at δC 21.6 (C-10) and 17.4 (C-11), three methylene carbons at δC 67.6 (C-3),
34.5 (C-6), and 34.0 (C-7), two methenyl carbons at δC 40.2 (C-8) and 60.6 (C-9), two oxygenated quaternary carbon at δC 89.8 (C-4) and 95.3 (C-5), and a lactone carbonyl carbon at δC 180.5 (C-1), as well as the six carbon signals of a glucosyl moiety. The 1H-1H COSY and HSQC spectra showed coupling sequences for the C (6)–C (7)–C (8)–C (9) and C (8)–C (10) fragments (Fig. 2). The correlations of H-3 and H-9 with C-1, C-5 and C-4 in the HMBC spectrum allowed the establishment of a iridoid structure for 3 (Fig. 2). The HMBC correlations of H-11 with C-4 and Glc H-1′ with C-5 suggested that the Me-11 and the glucosyl moieties were located at C-4 and C-5, respectively. The coupling constant of the anomeric proton (δ 4.27, 1H, d, J = 7.6 Hz, H-1′) suggested a β-configured glucose unit existed in 3 and acid hydrolysis experiment of 3 confirmed the presence of the β-D-glucopyranosyl moiety. NOESY spectrum was used to determine the relative configuration of 3. The NOESY correlations of H-10 and Glc H-1′ with H-9β, but no correlations of H-9β and Glc H-1′
C. Wang et al. / Fitoterapia 96 (2014) 48–55
53
Fig. 2. Key 1H-1H COSY, HMBC, and correlations of compounds 1–3 and 5–6, NOE correlations of compounds 1–3 and 5.
with H-11 indicated that Me-10 and 5-OGlc were β-oriented and Me-11 was α-oriented (Fig. 2). Therefore, the structure of 3 was identified as (4R,5S,8R,9S)-hexahydro-4-hydroxy4,8-dimethyl-cyclopenta[c]pyran-1(3H)-one 5-O-β-D-glucopyranoside (Fig. 1) and named xiecaoside C. Compound 4 was isolated as colorless needles (CH2Cl2) and deduced its molecular formula C10H16O4 from HRESIMS ([M + H]+ m/z 201.1122, calcd 201.1127). Compound 4 had similar NMR data (Tables 2 and 3) with 3 but no signals of the glucosyl moiety. The main difference in 13C-NMR lies in the δ values of C-5 in 4 was shifted upfield by 8.7 compared to that of 3 result from the absence of a β-D-glucose unit at C-5, which was confirmed by analyzing the 1H-1H COSY, HSQC, HMBC, and NOESY spectra of 4. Therefore, as the aglycone of 3, the structure of 4 was determined to be (4R,5S,8R,9S)hexahydro-4-hydroxy-4,8-dimethyl-cyclopenta[c]pyran-1(3H)one (Fig. 1) and named xiecaoline A. Compound 5 was obtained as white amorphous powder, and its molecular formula was determined to be C21H36O11 from the molecular ion peak ([M + H]+ m/z 465.2329, calcd 465.2336) in the HRESIMS. The 1H-NMR spectrum of 5 displayed signals from two anomeric protons at δ 4.44 (1H, d, J = 7.8 Hz, H-1′) and 4.90 (1H, d, J = 2.7 Hz, H-1″), two methyl groups at δ 1.03 (3H, s, H-8) and 1.09 (3H, s, H-9), and a hydroxymethyl group at δ 3.71 (1H, d, J = 13.0 Hz, H-10a) and 3.64 (1H, d, J = 13.0 Hz, H-10b). The 13C-NMR spectrum of 5 gave 21 carbon signals, including a C-6′-O-substituted β-glucopyranosyl moiety, an oxygenated quaternary carbon at δC 89.0 (C-1), an oxygenated methylene group at δC 69.6 (C-10), three methylene groups at δC 38.6 (C-3), 28.3 (C-5),
and 22.3 (C-6), two methyl groups at δC 24.1 (C-8) and 24.5 (C-9), two methenyl carbons at δC 52.6 (C-2) and 45.4 (C-4) and one quaternary carbon at δC 49.2 (C-7), and the remaining five signals corresponded to a terminal β-D-apiofuranosyl moiety [12–14]. Acid hydrolysis of 5 confirmed the presence of D-glucose and D-apiose. In all above, indicating 5 was a monoterpene glycoside. Analysis the 1H-1H COSY and HSQC spectra of 5 found the coupling sequence of -CH-CH2-CH-CH2-CH2- (Fig. 2). In the HMBC spectrum of 5, correlations from the anomeric proton of apiosyl H-1″ to the glucosyl C-6′ (δC 68.1), from the anomeric proton of glucosyl H-1′ to C-1, and from H-8 and H-9 to C-7 were observed, indicating that 5 was a 1, 10-dihydroxypinane glycoside (Fig. 2). The relative configuration of the aglycone in 5 was determined as shown in Fig. 2 based on NOE experiments of 5 that the correlations of H-9 with H-10, H-6β and H-2, and H-8 with H-2 and H-3β [δ 1.87 (dt, J = 13.6, 3.3 Hz)]. Consequently, the structure of 5 was determined as (2S,4R,9S)-10-hydroxypinane-1-O-β-Dapiofuranosyl-(1 → 6)-β-D-glucopyranoside (Fig. 1) and named xiecaoside D. Compound 6 was obtained as a white amorphous powder and the ion at m/z 489.1976 [M + H]+ (calcd 489.1972) in HRESIMS spectrum provided the molecular formula C22H32O12. The IR spectrum showed absorptions for hydroxyl (3410 cm−1) and aromatic rings (1621 cm−1). The 1H-NMR spectrum of 6 (Table 2) showed signals belong to a hydroxymethyl group at δ 4.16 (2H, brd, J = 5.6 Hz, H-9), a methoxy group at δ 3.82 (3H, s), two olefinic protons with trans-configuration at δ 6.50 (1H, d, J = 15.9 Hz, H-7) and 6.24 (1H, dt, J = 15.9, 5.6 Hz,
54
C. Wang et al. / Fitoterapia 96 (2014) 48–55
Fig. 3. The nueroprotective activity of the tested compounds. PC12 cells induced by Aβ1–42 were treated with the tested compounds at concentrations of 5, 12.5, 25 μM, respectively. Established MTT method was used to assay the cell viability. Data from three independent experiments are expressed as mean ± SD (n = 8), which based on the untreated control cells as 100% values. **Significant difference compared 25 and 12.5 μM of compounds 10–15 and 17 with model (**p b 0.01), respectively. *Significant difference compared 25 μM of compounds 6 and 16 with model (* p b 0.01).
H-8), and three protons of an aromatic ring at δ 7.01 (1H, brs, H-2), 7.02 (1H, d, J = 8.4 Hz, H-5), and 6.92 (1H, dd, J = 8.4, 1.8 Hz, H-6). Two anomeric protons at δ 4.79 (1H, d, J = 6.4 Hz, H-1′) and 4.64 (1H, d, J = 1.1 Hz, H-1″) were the characteristics of β-glucose and a-rhamnose, respectively [15]. The 13C-NMR and DEPT spectra (Table 3) displayed 22 carbon signals, including signals arising from a terminal α-L-rhamnopyranosyl moiety, a C-6′-O-substituted β-Dglucopyranosyl moiety, an aromatic aglycone moiety, a hydroxymethyl group at δC 63.9 (C-9), a methoxy group at δC 56.9, and two olefinic carbons at δC 131.5 (C-7) and 129.1 (C-8). Acid hydrolysis experiment of 6 determined the presence of D-glucose and L-rhamnose. The coniferin showed similar NMR data with 6 but less a group of signals belonged to a rhamnose unit as 6 [16] and the signal of C-6′ at δC 68.0 suggested that L-rhamnose was linked to C-6′. Analysis the 1H-1H COSY and HSQC spectra of 6 showed coupling sequences for C (5)–C (6) and C (7)–C (8)–C (9) fragments (Fig. 2). The HMBC correlations of H-OCH3 with C-3, Glc H-1′ with C-4, and H-1″ with C-6′ suggested that the methoxy group, glucopyranosyl moiety, rhamnopyranosyl moiety were located at C-3, C-4 and C-6′, respectively. In addition, correlations H-7 and H-8 with C-1 indicated that the fragment of C (7)–C (8)–C (9) was located at C-1 (Fig. 2). Thus, 6 was determined to be (7E)-4-O-[α-L-rhamnopyranosyl(1 → 6)-β-D-glucopyranosyl]-ferulylalcohol (Fig. 1) and named xiecaoside E. The known compounds were identified as scabroside B (7) [8], loganin (8) [17], patriscabroside I (9) [18], olivil-4'-Oβ-D-glucopyranoside (10) [19], lariciresinol-4,4'-di-O-β-Dglucopyranoside (11) [20], olivil-4-O-β-D-glucopyranoside (12) [19], 8-hydroxylariciresinol-4'-O-β-D-glucopyranoside (13) [19], lariciresinol-4-O-β-D-glucopyranoside (14) [20], neoarctin A (15) [21], lariciresinol-4'-O-β-D-glucopyranoside (16) [22], (−)-massoniresinol 3a-O-β-D-glucopyranoside (17) [23] by comparing their NMR spectroscopic and physical data with the literature values. The protective effects of compounds 1–17 on Aβ1–42 induced PC12 cells neurotoxicity were investigated by using MTT assay. As we all known, aggregated beta-amyloid (Aβ) has been one of critical factors in the pathogenesis of AD. Aβ fragments are 39–43 amino acid peptides, among which Aβ1–42 exhibited the most neurotoxic property [24]. The PC12 cell line displays phenotypic characteristics of sympathetic
neurons and induced by Aβ for imitating the conditions of Alzheimer’s disease (AD) in vitro to screen some anti-AD candidate drugs [25]. In this study, Aβ1–42 induced cytotoxicity (51.45 ± 3.03% viability) in the cells when it was added at a concentration of 1.5 μM for 24 h. When PC12 cells were pre-incubated with VE or compounds 1–17, the toxicity of Aβ1–42 on PC12 cells was significantly attenuated by VE, compounds 6 and 10–17 in a dose-dependent manner (Fig. 3), while other compounds did not show any protective effects on the cell at the tested concentration (data not shown in Figure). Conflict of interest The authors have declared that there is no conflict of interest. Acknowledgment This research was supported by the Program of International S&T Cooperation of China (No. 2010DFA32440) and Key Laboratory of Cardiovascular Medicine Research (Harbin Medical University), Ministry of Education. References [1] Huang BK, Zheng HC, Qin LP, Zheng QM, Xin HL. Investigation on resource of genus Valeriana in China. J Chin Med Mater 2004;27:632–4. [2] Du XW, Liang CF, Song N. Study on quality standard of Valeriana amurensis. Chin J Inf TCM 2011;18:46–7. [3] You CZ. Northeast medicinal plants. first ed. Harbin: Heilongjiang Science and Technology; 1989. [4] Zuo YM, Zhang ZL, Wang QH, Xie N, Kuang HX. Effects of Valeriana amurensis on the expressions of β-APP, Aβ1–40 and caspase-3 in Alzheimer’s disease model rat’s brain. J Chin Med Mater 2010;33:233–6. [5] Zhang ZL, Zuo YM, Wang QH, Xiao HB, Kuang HX. Effects of Valeriana amurensis on the expressions of iNOS, COX-2 and IκB-α in Alzheimer’s disease model rat’s brain. J Chin Med Mater 2010;33:581–3. [6] Wang QH, Wang CF, Zuo YM, Wang ZB, Yang BY, Kuang HX. Compounds from the roots and rhizomes of Valeriana amurensis protect against neurotoxicity in PC12 cells. Molecules 2012;17:15013–21. [7] Li N, Di L, Gao WC, Wang KJ, Zu LB. Cytotoxic iridoids from the roots of Patrinia scabra. J Nat Prod 2012;75:1723–8. [8] Di L, Li N, Zu LB, Wang KJ, Zhao YX, Wang Z. Three new iridoid glucosides from the roots of Patrinia scabra. Bull Korean Chem Soc 2011;32:3251–4. [9] Bouter Y, Dietrich K, Wittnam JL, Rezaei-Ghaleh N, Pillot T, Papot-Couturier S, Lefebvre T, Sprenger F, Wirths O, Zweckstetter M, Bayer TA. N-truncated amyloid β (Aβ) 1-42 forms stable aggregates and induces acute and longlasting behavioral deficits. Acta Neuropathol 2013;126:189–205.
C. Wang et al. / Fitoterapia 96 (2014) 48–55 [10] Peng QL, Buz’Zard AR, Lau BH. Pycnogenol protects neurons from amyloidbeta peptide-induced apoptosis. Brain Res Mol Brain Res 2002;104:55–65. [11] Lin S, Chen T, Liu XH, Shen YH, Li HL, Shan L, Liu RH, Xu XK, Zhang WD, Wang H. Iridoids and lignans from Valeriana jatamansi. J Nat Prod 2010;73(4):632–8. [12] Yahara S, Shigeyama C, Ura T, Wakamatsu K, Yasuhara T, Nohara T. Cyclic peptides, acyclic diterpene glycosides and other compounds from Lycium chinense Mill. Chem Pharm Bull (Tokyo) 1993;41:703–9. [13] Kristina JS, Nadin K, Karsten S, Sven J, Gerd W, Matthias EM. Chemical composition and biological activity of Paris quadrifolia L. Z Naturforsch C 2012;67:565–70. [14] Jiang ZY, Li SY, Li WJ, Guo JM, Tian K, Hu QF, Huang XZ. Phenolic glycosides from Ficus tikoua and their cytotoxic activities. Carbohydr Res 2013;382C:19–24. [15] Guo R, Luo M, Long CL, Li ML, Ouyang ZQ, Zhou YP, Wang YH, Li XY, Shi YN. Two new lignans from Dipteronia dyeriana. Chin Chem Lett 2008;19:1215–7. [16] Sugiyama M, Nagayama E, Kikuchi M. Lignan and phenylpropanoid glycosides from Osmanthus asiaticus. Phytochemistry 1993;33(5):1215–9. [17] Nakamoto K, Otsuka H, Yamasaki K. 7-O-Acetyl loganic acid from Alangium platanifolium var. Trilobum. Phytochemistry 1988;27:1856–8. [18] Kouno I, Yasuda I, Mizoshiri H, Tanaka T, Marubayashi N, Yang DM. Two new iridolactones and their glycosides from the roots of Patrinia scabra. Phytochemistry 1994;37:467–72.
55
[19] Schumacher B, Scholle S, Hölzl J, Khudeir N, Hess S, Müller CE. Lignans isolated from Valerian: identification and characterization of a new olivil derivative with partial agonistic activity at A1 adenosine receptors. J Nat Prod 2002;65:1479–85. [20] Deyama T, Ikawa T, Nishibe S. The constituents of Eucommia ulmoides Oliv. II. Isolation and structures of three new lignan glycosides. Chem Pharm Bull 1985;33:3651–7. [21] Yong M, Kun G, Qiu MH. A new lignan from the seeds of Arctium lappa. J Asian Nat Prod Res 2007;9:541–4. [22] Shoeb M, Jaspars M, MacManus SM, Majinda RRT, Sarker SD. Epoxylignans from the seeds of Centaurea cyanus (Asteraceae). Biochem Syst Ecol 2004;32:1201–4. [23] Feng WS, Hao ZY, Zheng XH. Chemical constituents from leaves of Celastrus gemmatus Loes. Acta Pharm Sin 2007;42:625–30. [24] Lambert MP, Barlow AK, Chromy BA. Diffusible, nonfibrillar ligands derived from Abeta 1–42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A 1998;95:6448–53. [25] Isaac G, Lea RT, Erez K, Hanna R. The herbal preparation Padma® 28 protects against neurotoxicity in PC12 cells. Phytother Res 2011;25:740–3.
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Isolation and screened neuroprotective active constituents from the roots and rhizomes of Valeriana amurensis Changfu Wang a, Yang Xiao a, Bingyou Yang a, Zhibin Wang a, Lihua Wu a, Xiaolin Su a, Adelheid Brantner b, Haixue Kuang a,⁎, Qiuhong Wang a,⁎ a b
Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University of Chinese Medicine, No. 24 HePing Road, XiangFang District, Harbin 150040, China Institute of Pharmacognosy, University of Graz, Universitaetsplatz 4/I, A-8010 Graz, Austria
a r t i c l e
i n f o
Article history: Received 20 February 2014 Accepted in revised form 3 April 2014 Available online 15 April 2014 Chemical compounds studied in this article: Loganin (PubChem CID: 87691) Patriscabroside I (PubChem CID: 71453062) Keywords: Valeriana amurensis Alzheimer’s disease Iridoids Lignans PC12 cell
a b s t r a c t In previous study, we have screened the effective fraction against Alzheimer’s disease (AD-EF) from the extracts of roots and rhizomes of Valeriana amurensis, based on which neuroprotective active constituents from AD-EF were investigated. Six new compounds 1–6, including four iridoids (xiecaoside A–C and xiecaoline A), one pinane-type monoterpeneglucoside (xiecaoside D), and one phenylpropanoid glycoside (xiecaoside E) were isolated together with 11 known compounds 7–17. The structures of 1–6 were elucidated by their spectroscopic data. The protective effects of compounds 1–17 on PC12 cells with neurotoxicity induced by amyloid-beta 1–42 (Aβ1–42) was also investigated, respectively. Consequently, compound 6 and lignans 11–17 were responsible for protecting against Aβ-induced toxicity in PC12 cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Valeriana is a perennial herb and belongs to the family of Valerianaceae. There are about 30 species in genus Valeriana in China [1]. As one species of Valeriana, Valeriana amurensis (V. amurensis) is widely distributed in Russian Far East, northeast China, and North Korea [2]. The roots and rhizomes of V. amurensis, pungent and sweet in flavor and warm in
Abbreviations: AD-EF, effective fraction against Alzheimer’s disease; AD, Alzheimer’s disease; Aβ1–42, amyloid-beta 1–42; CC, column chromatography; TMS, tetramethylsilane; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. ⁎ Corresponding authors. Tel.: +86 45182193001. E-mail addresses: [email protected] (C. Wang), [email protected] (Y. Xiao), [email protected] (B. Yang), [email protected] (Z. Wang), [email protected] (L. Wu), [email protected] (X. Su), [email protected] (A. Brantner), [email protected] (H. Kuang), [email protected] (Q. Wang).
http://dx.doi.org/10.1016/j.fitote.2014.04.007 0367-326X/© 2014 Elsevier B.V. All rights reserved.
nature, possess the effects of tranquilization, regulating qi, and analgesia and have been used to treat neurological system diseases, such as neurasthenia, insomnia, hysteria, and epilepsy for thousands of years in traditional Chinese medicine [3]. To be worth mentioning, however, we reported its potential therapeutic effect on Alzheimer’s disease (AD) in previous study for the first time, meanwhile, the effective fraction of V. amurensis against AD (50% EtOH fraction from AB-8 macroporous resin column, AD-EF) has been determined [4,5]. Previously, only several compounds of germacrane-type sesquiterpenoids and lignans separated from AD-EF showed protective effect on the neurotoxicity of PC12 cells induced by amyloid-beta (Aβ25–35) [6]. To further reveal the chemical basis for the therapeutic effect of V. amurensis on AD, we continue to carry out phytochemical study on V. amurensis but a little different, and six new (four iridoid glycosides, one monoterpeneglucoside, and one phenylpropanoids glycoside) together with 11 known compounds were obtained. In this paper, the isolation and
C. Wang et al. / Fitoterapia 96 (2014) 48–55
structural elucidation of the new compounds 1–6 was described. The structures of known compounds 7–17 were identified by physico-chemical constants, spectroscopic analysis, and comparing their spectroscopic data with reference values, meanwhile, MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] assay method was used to investigate the neuroprotective effects of compounds 1–17. 2. Experimental 2.1. General Bruker DPX 400 instrument (Bruker SpectroSpin, Karlsruhe, Germany) was used to measure the NMR spectra. Tetramethylsilane (TMS) as the internal standard, chemical shifts and coupling constants were given as δ values and in Hz, respectively. The HRESIMS analyses were carried out on Xero Q Tof MS spectrometer (Waters, Milford, MA, USA). Kofler micromelting point apparatus was used to determine the melting point (m.p.). Preparative HPLC was conducted on a Waters 2535 instrument equipped with a UV-2998 and RI-2414 detector, and using the Waters Sunfire prep C18 OBD™ 10 μm (19 × 250 mmi.d.) column for preparing compounds. IR Spectra was recorded on Shimadzu FTIR-8400S (Kyoto, Japan). Macroporous resin (AB-8 Crosslinked Polystyrene, Nan Kai, Tianjin, China), silica gel (200–300 mesh, Haiyang Chemical Group Co. Ltd, Qingdao, China), and ODS-A (120A, 50 mm; YMC, Kyoto, Japan) was used for column chromatography (CC). PC12 cells (Institute of biochemistry and cell biology, Shanghai, China) were cultured in DMEM (Hyclone, NRH0020) with 5% fetal bovine serum and 1% antibiotic mixture of penicillin-streptomycin at 37 °C with atmosphere of 5% CO2. PC12 cell injury was induced by Aβ1–42 (Bioss, Beijing, China). PC12 cell viability was determined by Microplate reader (VICTOR™ × 3, PerkinElmer, Inc., Massachusetts, United States). 2.2. Plant materials We collected the roots and rhizomes of V. amurensis from the Great Xing’an Mountains area (Heilongjiang province, China) and identified by Xiaowei Du of Heilongjiang University of Chinese Medicine. The voucher specimen (No. 20100806) is deposited at the Herbarium of Heilongjiang University of Chinese Medicine, China. 2.3. Extraction and isolation The dried roots and rhizomes of V. amurensis (9.0 kg) were extracted three times (2 h for each) with 75% EtOH (72 L) under reflux and then removed the solvent. The obtained residue (1642.4 g) was suspended in water and partitioned with petroleum ether (5 × 12 L). After removal of solvent, the obtained water extract (1228.9 g) was subjected to AB-8 macroporous resin column (10 × 60 cm) and eluted with H2O, 50% and 95% EtOH successively. The obtained 50% EtOH fraction (260.6 g) is the AD-EF as previous study [4,5]. In this study, 180.0 g AD-EF was subjected to silica gel (200–300 mesh) column chromatography (CC), eluting with CH2Cl2–MeOH (from 20:1 to 1:1, v/v) to obtain fractions I–VI. Fraction I (24.8 g) was subjected to CC over silica gel, eluted with petroleum ether–EtOAc (from 25:1 to 1:1, v/v), to
49
give fractions I1–I6. Compound 4 (99.5%, 35 mg) was crystallized from fraction I4 directly in CH2Cl2. Fraction III (31.3 g) was chromatographed over silica gel, eluted with CH2Cl2–MeOH (from 15:1 to 3:1, v/v), to afford fractions III1–III5. Fraction III1 (8.5 g) was separated by ODS CC, eluted with 10–50% gradient MeOH–H2O to afford four fractions. The four fractions were separated and purified with preparative HPLC (MeOH–H2O) and compounds 6 (98.2%, 26 mg, tR = 42.0 min, 20% MeOH), 14 (97.4%, 32 mg, tR = 39.0 min, 30% MeOH), and 16 (98.8%, 48 mg, tR = 44 min, 35% MeOH) were obtained. Fraction III3 (7.2 g) was separated by ODS CC, eluted with 10–50% gradient MeOH–H2O to afford two fractions. The two fractions were separated and purified with preparative HPLC (MeOH–H2O) and compounds 10 (99.4%, 22 mg, tR = 36 min, 25% MeOH) and 13 (96.4%, 31 mg, tR = 11.0 min, 32% MeOH) were obtained. Fraction III5 (5.2 g) was separated by ODS CC, eluted with 10–30% gradient MeOH–H2O to afford two fractions. The two fractions were separated and purified with preparative HPLC (MeOH–H2O, 20%) and compounds 3 (97.4%, 27 mg, tR = 43.2 min), 7 (95.8%, 44 mg, tR = 46.1 min), and 8 (97.8%, 36 mg, tR = 48.4 min) were obtained. Fraction V (51.9 g) was subject to ODS CC, eluted with 10–50% gradient MeOH–H2O to afford compound 11 (98.5%, 47 mg, 10% MeOH) and eight fractions V1–V8. Fractions V2 (11.4 g), V4 (5.9 g), and V6 (14.5 g) were separated and purified with preparative HPLC (MeOH–H2O) to obtain compounds 2 (99.7%, 21 mg, tR = 8.1 min, 35% MeOH), 5 (95.1%, 30 mg, tR = 50.4 min, 38% MeOH), and 9 (97.2%, 33 mg, tR = 6.0 min, 35% MeOH) from fraction V2, compound 1 (96.8%, 23 mg, tR = 13.2 min, 38.4% MeOH) from fraction V4, and compounds 12 (98.2%, 44 mg, tR = 39 min, 31% MeOH), 15 (99.1%, 38 mg, tR = 51 min, 20% MeOH), and 17 (99.4%, 46 mg, tR = 14 min, 35% MeOH) from fraction V6. 2.3.1. Xiecaoside A (1) White amorphous powder, [α]22 D + 24.2° (c 0.12, MeOH); UV (MeOH) λmax (log ε) 202 (3.59) (nm); IR (KBr) νmax 3421,
Table 1 1 H NMR data of compounds 1–3 (400 MHz for 1 and 2, 500 MHz for 3, δ in ppm, J in Hz). Position
1a
1
3.95, 3.75, 4.36, 4.18, 3.24, 2.73, 2.25, 5.77,
3 5 6 7 8 9 10 11 1′ 2′ 3′ 4′ 5′ 6′ a
2a d (10.4) d (10.4) d (12.6) d (12.6) m m brd (16.3) brs
4.10, brd (10.0) 4.91, dd (6.3, 2.0) 4.28,d (7.8) 3.14, t (8.4) 3.31, m 3.23, m 3.23, m 3.81, d (11.6) 3.62, dd (11.8, 4.8)
3a
5.11, 4.44, 3.33, 2.33, 2.18, 3.86,
d (11.1) d (11.7) m dd (8.4, 13.4) m m
3.07, 1.58, 5.07, 4.40, 3.20, 3.30, 3.32, 3.38, 3.88, 3.71,
d (10.8) s 5.10, s d (7.8) t (8.2) m m m m dd (12.0, 4.9)
Measured in methanol–d4 at 30 °C.
4.02, d (12.3) 3.44, d (12.3) 1.90, 1.42, m 2.21, 2.21, 2.91, 1.19, 1.26, 4.31, 3.19, 3.36, 3.28, 3.26, 3.81, 3.62,
1.74, m m d (4.5) d (6.8) s d (7.6) t (8.4) t (8.6) t (9.1) m d (11.6) dd (4.9,12.0)
50
C. Wang et al. / Fitoterapia 96 (2014) 48–55
2873, 1671, 1632, 1445, 1381, 1326, 1169, 1078, 886, 597 cm−1; ESIMS m/z 345 (100) [M + H]+; HRESIMS [M + H]+m/z 345.1542, calcd 345.1549 for C16H24O8H; 1H and 13C NMR data, see Tables 1 and 3.
2.3.2. Xiecaoside B (2) White amorphous powder, [α] D22 − 29.3° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 215 (4.27), 223 (4.27), 281 (4.06), 324 (3.36) (nm); IR (KBr) νmax 3400, 2920, 1762, 1631 cm−1; ESIMS m/z 361 (100) [M + H]+; HRESIMS [M + H]+m/z 361.1493calcd 361.1499 for C16H24O9H; 1H and 13C NMR data, see Tables 1 and 3.
2.3.3. Xiecaoside C (3) White amorphous powder, [α]22 D − 15.8° (c 0.12, MeOH); UV (MeOH) λmax (log ε) 201 (3.13) (nm); IR (KBr) νmax 3421, 3392, 2952, 2873, 1728, 1634, 1615, 1597, 1457, 1381, 1332, 1235, 1161, 1075, 721, 637 cm−1; ESIMS m/z 363 (100) [M + H]+; HRESIMS [M + H]+ m/z 363.1661 calcd 363.1655 for C16H26O9H; 1H and 13C NMR data, see Tables 1 and 3.
2.3.4. Xiecaoline A (4) colorless needle crystal (CH2Cl2), m.p. 115–117 °C; [α]22 D − 20.7° (c 0.10, MeOH); UV (MeOH) λmax (log ε) 240 (2.73) (nm); IR (KBr) νmax 3434, 2962, 2878, 1726, 1631, 1458, 1381, 1355, 1248, 1173, 1117, 1036, 935 cm−1; ESIMS m/z 201 (100) [M + H]+; HRESIMS [M + H]+ m/z 201.1122 calcd 201.1127 for C10H16O4H; 1H and 13C NMR data, see Tables 2 and 3.
Table 2 1 H NMR data of compounds 4–6 (400 MHz, δ in ppm, J in Hz). Position 2 3
11 3-OCH3 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ b
5a
3.76, d (12.0) 3.37, d (12.0)
4 5 6 7 8 9 10
a
4b
1.66, 1.83, 2.11, 2.65, 1.12,
m 1.47, m m dd (1.4, 4.6) d (6.8)
6a
1.77, 1.87, 1.44, 1.58, 1.72, 2.26,
d (3.7) dt (13.6, 3.3) brd (13.6) t (4.2) 1.39, m 1.53, m
1.03, 1.09, 3.71, 3.64,
s s d (13.0) d (13.0)
7.01, brs
7.02, 6.92, 6.50, 6.24, 4.16,
d (8.4) dd (8.4,1.7) d (15.9) dt (15.9, 5.6) brd (5.6)
3.93, d (9.6) 3.54, s
Measured in methanol–d4 at 30 °C. Measured in DMSO–d6 at 30 °C.
2.3.6. Xiecaoside E (6) White amorphous powder, [α]22 D − 28.3°(c 0.11, MeOH); UV (MeOH) λmax (log ε) 257 (4.78) (nm); νmax 3410, 1621, 1455, 1201, 1123, 1097, 1035, 965, 814 cm−1; ESIMS m/z 489 (100) [M + H]+; HRESIMS [M + H]+ m/z 489.1976 calcd 489.1972 for C22H32O12H; 1H and 13C NMR data, see Tables 2 and 3. 2.4. Acid hydrolysis of 1–3, 5, and 6 Acid hydrolyses of 1–3, 5, and 6 were performed according to the method reported previously [7]. Briefly, compounds (2.5 mg) were hydrolyzed with 2 mol/L H2SO4 (2.0 mL) and sugar residues were treated with trimethylchlorosilane, respectively. The obtained sugar derivatives were analyzed by GC [8]. The monosaccharide of compounds 1–3 was determined to be D-glucose (tR = 7.25 min). The monosaccharides of compound 5 were determined to be D-glucose (tR = 7.25 min) and D-apiose (tR = 8.43 min). The monosaccharides of compound 6 were determined to be D-glucose (tR = 7.25 min) and L-rhamnose (tR = 4.32 min). 2.5. Determination of cell viability The MTT assay method was used to measure the cell viability as reported in reference [6] but a little different. Briefly, the PC12 cells (8 × 103 cells/well) were cultured for 24 h in 96-well plates, and then incubated without or with compounds 1–17 (at the concentrations of 5, 12.5, 25 μM), and the cells neurotoxicity was induced by 1.5 μM aggregated Aβ1–42 [9] for 4 h. Vitamin E (VE) was used as a positive drug [10]. 20 μL MTT solutions (5 mg/mL) were added into each well and cells were incubated at 37 °C for another 4 h. Aspirated off the supernatants and then dissolved formazan crystals with DMSO. The microplate reader was used to measure the optical density of each well at 490 nm and the results were expressed as the percentages. 3. Results and discussion
1.15, s 4.44, d (7.8) 3.17, t (9.20) 3.37, m 3.20, t (9.3) 3.41, m 3.96,dd (10.6, 2.0) 3.73,brd (10.6) 4.90, d (2.7) 3.86, d (2.6)
2.3.5. Xiecaoside D (5) White amorphous powder, [α]22 D − 46.4° (c 0.13, MeOH); IR (KBr) νmax 3464, 2925, 2885, 1451, 1380, 1173, 1119, 1070, 722 cm−1; ESIMS m/z 465 (100) [M + H]+; HRESIMS [M + H]+ m/z 465.2329 calcd 465.2336 for C21H36O11H; 1H and 13C NMR data, see Tables 2 and 3.
3.82, 4.79, 3.44, 3.47, 3.31, 3.41, 3.95, 3.54, 4.64, 3.77, 3.63, 3.31, 3.59, 1.15,
s d (6.4) m m m m d (9.7) m d (1.1) m m m m d (6.2)
Compound 1 was obtained as a white amorphous powder. The molecular formula of 1 was determined as C16H24O8 by HRESIMS ([M + H]+ m/z 345.1542, calcd 345.1549). The presence of the hydroxyl groups can be confirmed by IR (3421 cm−1). In the 1H-NMR of 1, signals of a nonoxygenated methylene at δ 2.73 (1H, m, H-6a) and 2.25 (1H, brd, J = 16.3 Hz, H-6b), a nonoxygenatedmethine at δ 3.24 (1H, m, H-5), two oxygenated methylenes at δ 3.95 (1H, d, J = 10.4 Hz, H-1a), 3.75 (1H, d, J = 10.4 Hz, H-1b), 4.36 (1H, d, J = 12.6 Hz, H-3a), and 4.18 (1H, d, J = 12.6 Hz, H-3b), and three olefinic protons at δ 5.77 (1H, brs, H-7), 4.91 (1H, dd, J = 6.3, 2.0 Hz, H-11a), and 4.10 (1H, brd, J = 10.0 Hz, H-11b) were observed. Besides, the β-configured glucose unit was confirmed by coupling constant of the anomeric proton at
C. Wang et al. / Fitoterapia 96 (2014) 48–55
51
Table 3 13 C NMR data of compounds 1–6 (100 MHz for 1–2 and 4–6, 125 MHz for 3, δ in ppm). Position
1a
2a
3a
4b
5a
6a
1 2 3 4 5 6 7 8 9 10 11 3-OCH3 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″
72.6, CH2
175.0, C
180.5, C
177.7, C
72.0, CH2 156.0, C 49.6, CH 39.0, CH2 131.2, CH 144.2, C 99.6, C 59.1, CH2 105.2, CH2
71.3, CH2 144.3, C 41.0, CH 39.8, CH2 89.9, CH 85.9, C 54.0, CH 22.3, CH3 113.8, CH2
67.6, 89.8, 95.3, 34.5, 34.0, 40.2, 60.6, 21.6, 17.4,
CH2 C C CH2 CH2 CH CH CH3 CH3
65.1, 86.5, 86.6, 36.8, 33.1, 38.0, 58.6, 21.8, 16.3,
89.0, C 52.6, CH 38.6, CH2 45.5, CH 28.3, CH2 22.3, CH2 49.2, C 24.1, CH3 24.5, CH3 69.6, CH2
133.9, C 111.6, CH 147.7, C 151.0, C 118.2, CH 121.0, CH 131.5, CH 129.1, CH 63.9, CH2
104.6, CH 75.2, CH 78.1, CH 71.7, CH 78.2, CH 62.9, CH2
105.2, CH 75.5, CH 78.0, CH 71.8, CH 78.2, CH 62.9, CH2
99.4, 75.1, 77.8, 71.5, 78.1, 62.6,
CH CH CH CH CH CH2
a b
CH2 C C CH2 CH2 CH CH CH3 CH3
99.8, CH 75.3, CH 78.6, CH 72.2, CH 78.1, CH 68.2, CH2 111.4, CH 76.7, CH 80.7, C 75.2, CH2 65.8, CH2
56.9, CH3 102.9, CH 75.1, CH 77.1, CH 71.7, CH 78.0, CH 68.0, CH2 102.3, CH 72.3, CH 72.6, CH 74.2, CH 70.0, CH 18.1, CH3
Recorded in methanol–d4 at 30 °C. Recorded in DMSO–d6 at 30 °C.
δ 4.28 (1H, d, J = 7.8 Hz, H-1′). Acid hydrolysis experiment further revealed a β-D-glucopyranosyl moiety existed in 1. 13 C-NMR and DEPT spectra of 1 (Tables 1 and 3) showed a hydroxymethyl at δC 59.1 (C-10), a nonoxygenatedmethine at δC 49.6 (C-5), a nonoxygenated methylene at δC 39.0 (C-6), two oxygenated methylenes at δC 72.6 (C-1) and 72.0 (C-3), an oxygenated quaternary carbon at δC 99.6 (C-9), two olefinic bonds at δC 156.0 (C-4), 105.2 (C-11), 131.2 (C-7), and 144.2 (C-8), and the remaining six signals corresponded to a β-Dglucopyranosyl moiety. The NMR data of 1 were similar to those of patriridoside G [7] and the main different lies in presence of signals at δC 59.1 and δH 4.10(2H, brd, J = 10.0 Hz) in 1 but absence of signals at δC 12.1 and 1.73 (3H, brs), which indicated that CH3-10 of patriridoside G was substituted by a hydroxyl group. The 1H-1H COSY and HSQC spectra (Fig. 2) gave two coupling sequences of C-3/C-4/C-11 and C-11/C-4/C-5/C-6/ C-7/C-8/C-10 for 1. HMBC correlations (Fig. 2) of H-10 with C-7, 8, and 9, H-1 with C-5 and 9, H-11 with C-3, 4, and 5, and H-3 with C-9 established the structure of 1. The glucosyl moiety was located at C-1 by the HMBC correlation from anomeric proton to C-1. Correlation (Fig. 2) between H-5β and H2-1 in NOESY spectra suggested a β-orientation for C-1. As a result, the structure of 1 was identified as [(5R,9S)8-hydroxymethyl-4-methylene-4,5,6,9-tetrahydro-3H-cyclopenta[b]furan-9-yl]methanol 1-O-β-D-glucopyranoside (Fig. 1) and named xiecaoside A. Compound 2 was isolated as a white amorphous powder. The molecular formula was assigned as C16H24O9 from its HRESIMS ([M + H]+ m/z 361.1493, calcd 361.1499). The IR absorptions in 3400 and 1762 cm−1 originated from the hydroxyl and lactone carbonyl groups, respectively. The 1H NMR spectrum (Table 1) gave signals for a methyl group at δ
1.58 (3H, s, H-10), three methylene groups at δ 5.11 (1H, d, J = 11.1 Hz, H-3a), 4.44 (1H, d, J = 11.7 Hz, H-3b), 2.33 (1H, dd, J = 8.4, 13.4 Hz, H-6a), 2.18 (1H, m, H-6b), 5.07 (s, H-11a), 5.10 (s, H-11b), and three methenyl groups at δ 3.33 (1H, m, H-5), 3.86 (1H, m, H-7), 3.07 (1H, d, J = 10.8 Hz, H-9). Acid hydrolysis experiment of 2 confirmed the presence of D-glucopyranosyl moiety and coupling constant of the anomeric proton (δH 4.40, 1H, d, J = 7.8 Hz, H-1′) concluded a β-configured glucose unit existed in 2. 16 carbon signals could be observed in 13C NMR and DEPT spectra (Table 3), including a methyl at δC 22.3 (C-10), a lactone carbonyl at δC 175.0 (C-1), a terminal double bond at δC 144.3 (C-4) and 113.8 (C-11), as well as a group of glucosyl moiety signals. Besides, other carbon signals such as two methylenes at δC 71.3 (C-3) and 39.8 (C-6), three methenyl groups at δC 41.0 (C-5), 89.9 (C-7), and 54.0 (C-9), a quaternary carbon at δC 85.9 (C-8) were also observed. Except for the extra glucosyl moiety signals, compound 2 had similar NMR data with jatamanin A [11]. The main difference in 13C-NMR lies in the δ values of C-7 in 2 was shifted downfield by 8.4 compared to that of jatamanin A, which indicated that the β-D-glucose unit was located at C-7. The 1H-1H COSY and HSQC spectra showed the coupling sequence for the C (9)–C (5)–C (6)–C (7) fragments. Analysis of HMBC observed correlations (Fig. 2) of H-3 with C-1, 4, and 5, H-11 with C-3, 4, and 5, H-10 with C-7, 8, and 9, and H-9 with C-1. The glucosyl moiety was located at C-7 by the correlation from the anomeric proton H-1′ to C-7. Given to the biogenetic of iridoid prescribed that H-5 and H-9 were β-oriented [7]. NOE correlations (Fig. 2) between H-9 and H-10, H-5 and H-10, but no correlations between H-9 and H-7, H-5 and H-7, suggested that H-5, H-9, and the methyl group were β-oriented and the glucosyl moiety was
52
C. Wang et al. / Fitoterapia 96 (2014) 48–55
Fig. 1. Structures of compounds 1–17.
corresponding β-oriented. Therefore, the structure of 2 was founded to be (5S,7S,8S,9S)-8-hydroxy-8-methyl-4-methylenehexahydrocyclopenta[c]pyran-1(3H)-one 7-O-β-D-glucopyranoside (Fig. 1) and named xiecaoside B. Compound 3 was obtained as a white amorphous powder and its molecular formula C16H26O9 was concluded by HRESIMS ([M + H]+, m/z 363.1661, calcd 363.1655). The IR spectrum showed absorptions for hydroxyl groups (3421, 3392 cm− 1) and lactone carbonyl group (1728 cm− 1). In the 1H NMR spectrum (Table 1) of 3, signals for two methyl groups at δ 1.19 (3H, d, J = 6.8 Hz, H-10) and 1.26 (3H, s, H-11), three methylene groups at δ 4.02 (1H, d, J = 12.3 Hz, H-3a), 3.44 (1H, d, J = 12.3 Hz, H-3b), 1.90 (1H, m, H-6a), 1.42 (1H, m, H-6b), 2.21 (1H, m, H-7a), and 1.74 (1H, m, H-7b), and two methenyl groups at δ 2.21 (1H, m, H-8) and 2.91 (1H, d, J = 4.5 Hz, H-9) could be observed. 16 carbon signals were exhibited in the 13C NMR and DEPT spectra of 3 (Table 3), including two methyl carbons at δC 21.6 (C-10) and 17.4 (C-11), three methylene carbons at δC 67.6 (C-3),
34.5 (C-6), and 34.0 (C-7), two methenyl carbons at δC 40.2 (C-8) and 60.6 (C-9), two oxygenated quaternary carbon at δC 89.8 (C-4) and 95.3 (C-5), and a lactone carbonyl carbon at δC 180.5 (C-1), as well as the six carbon signals of a glucosyl moiety. The 1H-1H COSY and HSQC spectra showed coupling sequences for the C (6)–C (7)–C (8)–C (9) and C (8)–C (10) fragments (Fig. 2). The correlations of H-3 and H-9 with C-1, C-5 and C-4 in the HMBC spectrum allowed the establishment of a iridoid structure for 3 (Fig. 2). The HMBC correlations of H-11 with C-4 and Glc H-1′ with C-5 suggested that the Me-11 and the glucosyl moieties were located at C-4 and C-5, respectively. The coupling constant of the anomeric proton (δ 4.27, 1H, d, J = 7.6 Hz, H-1′) suggested a β-configured glucose unit existed in 3 and acid hydrolysis experiment of 3 confirmed the presence of the β-D-glucopyranosyl moiety. NOESY spectrum was used to determine the relative configuration of 3. The NOESY correlations of H-10 and Glc H-1′ with H-9β, but no correlations of H-9β and Glc H-1′
C. Wang et al. / Fitoterapia 96 (2014) 48–55
53
Fig. 2. Key 1H-1H COSY, HMBC, and correlations of compounds 1–3 and 5–6, NOE correlations of compounds 1–3 and 5.
with H-11 indicated that Me-10 and 5-OGlc were β-oriented and Me-11 was α-oriented (Fig. 2). Therefore, the structure of 3 was identified as (4R,5S,8R,9S)-hexahydro-4-hydroxy4,8-dimethyl-cyclopenta[c]pyran-1(3H)-one 5-O-β-D-glucopyranoside (Fig. 1) and named xiecaoside C. Compound 4 was isolated as colorless needles (CH2Cl2) and deduced its molecular formula C10H16O4 from HRESIMS ([M + H]+ m/z 201.1122, calcd 201.1127). Compound 4 had similar NMR data (Tables 2 and 3) with 3 but no signals of the glucosyl moiety. The main difference in 13C-NMR lies in the δ values of C-5 in 4 was shifted upfield by 8.7 compared to that of 3 result from the absence of a β-D-glucose unit at C-5, which was confirmed by analyzing the 1H-1H COSY, HSQC, HMBC, and NOESY spectra of 4. Therefore, as the aglycone of 3, the structure of 4 was determined to be (4R,5S,8R,9S)hexahydro-4-hydroxy-4,8-dimethyl-cyclopenta[c]pyran-1(3H)one (Fig. 1) and named xiecaoline A. Compound 5 was obtained as white amorphous powder, and its molecular formula was determined to be C21H36O11 from the molecular ion peak ([M + H]+ m/z 465.2329, calcd 465.2336) in the HRESIMS. The 1H-NMR spectrum of 5 displayed signals from two anomeric protons at δ 4.44 (1H, d, J = 7.8 Hz, H-1′) and 4.90 (1H, d, J = 2.7 Hz, H-1″), two methyl groups at δ 1.03 (3H, s, H-8) and 1.09 (3H, s, H-9), and a hydroxymethyl group at δ 3.71 (1H, d, J = 13.0 Hz, H-10a) and 3.64 (1H, d, J = 13.0 Hz, H-10b). The 13C-NMR spectrum of 5 gave 21 carbon signals, including a C-6′-O-substituted β-glucopyranosyl moiety, an oxygenated quaternary carbon at δC 89.0 (C-1), an oxygenated methylene group at δC 69.6 (C-10), three methylene groups at δC 38.6 (C-3), 28.3 (C-5),
and 22.3 (C-6), two methyl groups at δC 24.1 (C-8) and 24.5 (C-9), two methenyl carbons at δC 52.6 (C-2) and 45.4 (C-4) and one quaternary carbon at δC 49.2 (C-7), and the remaining five signals corresponded to a terminal β-D-apiofuranosyl moiety [12–14]. Acid hydrolysis of 5 confirmed the presence of D-glucose and D-apiose. In all above, indicating 5 was a monoterpene glycoside. Analysis the 1H-1H COSY and HSQC spectra of 5 found the coupling sequence of -CH-CH2-CH-CH2-CH2- (Fig. 2). In the HMBC spectrum of 5, correlations from the anomeric proton of apiosyl H-1″ to the glucosyl C-6′ (δC 68.1), from the anomeric proton of glucosyl H-1′ to C-1, and from H-8 and H-9 to C-7 were observed, indicating that 5 was a 1, 10-dihydroxypinane glycoside (Fig. 2). The relative configuration of the aglycone in 5 was determined as shown in Fig. 2 based on NOE experiments of 5 that the correlations of H-9 with H-10, H-6β and H-2, and H-8 with H-2 and H-3β [δ 1.87 (dt, J = 13.6, 3.3 Hz)]. Consequently, the structure of 5 was determined as (2S,4R,9S)-10-hydroxypinane-1-O-β-Dapiofuranosyl-(1 → 6)-β-D-glucopyranoside (Fig. 1) and named xiecaoside D. Compound 6 was obtained as a white amorphous powder and the ion at m/z 489.1976 [M + H]+ (calcd 489.1972) in HRESIMS spectrum provided the molecular formula C22H32O12. The IR spectrum showed absorptions for hydroxyl (3410 cm−1) and aromatic rings (1621 cm−1). The 1H-NMR spectrum of 6 (Table 2) showed signals belong to a hydroxymethyl group at δ 4.16 (2H, brd, J = 5.6 Hz, H-9), a methoxy group at δ 3.82 (3H, s), two olefinic protons with trans-configuration at δ 6.50 (1H, d, J = 15.9 Hz, H-7) and 6.24 (1H, dt, J = 15.9, 5.6 Hz,
54
C. Wang et al. / Fitoterapia 96 (2014) 48–55
Fig. 3. The nueroprotective activity of the tested compounds. PC12 cells induced by Aβ1–42 were treated with the tested compounds at concentrations of 5, 12.5, 25 μM, respectively. Established MTT method was used to assay the cell viability. Data from three independent experiments are expressed as mean ± SD (n = 8), which based on the untreated control cells as 100% values. **Significant difference compared 25 and 12.5 μM of compounds 10–15 and 17 with model (**p b 0.01), respectively. *Significant difference compared 25 μM of compounds 6 and 16 with model (* p b 0.01).
H-8), and three protons of an aromatic ring at δ 7.01 (1H, brs, H-2), 7.02 (1H, d, J = 8.4 Hz, H-5), and 6.92 (1H, dd, J = 8.4, 1.8 Hz, H-6). Two anomeric protons at δ 4.79 (1H, d, J = 6.4 Hz, H-1′) and 4.64 (1H, d, J = 1.1 Hz, H-1″) were the characteristics of β-glucose and a-rhamnose, respectively [15]. The 13C-NMR and DEPT spectra (Table 3) displayed 22 carbon signals, including signals arising from a terminal α-L-rhamnopyranosyl moiety, a C-6′-O-substituted β-Dglucopyranosyl moiety, an aromatic aglycone moiety, a hydroxymethyl group at δC 63.9 (C-9), a methoxy group at δC 56.9, and two olefinic carbons at δC 131.5 (C-7) and 129.1 (C-8). Acid hydrolysis experiment of 6 determined the presence of D-glucose and L-rhamnose. The coniferin showed similar NMR data with 6 but less a group of signals belonged to a rhamnose unit as 6 [16] and the signal of C-6′ at δC 68.0 suggested that L-rhamnose was linked to C-6′. Analysis the 1H-1H COSY and HSQC spectra of 6 showed coupling sequences for C (5)–C (6) and C (7)–C (8)–C (9) fragments (Fig. 2). The HMBC correlations of H-OCH3 with C-3, Glc H-1′ with C-4, and H-1″ with C-6′ suggested that the methoxy group, glucopyranosyl moiety, rhamnopyranosyl moiety were located at C-3, C-4 and C-6′, respectively. In addition, correlations H-7 and H-8 with C-1 indicated that the fragment of C (7)–C (8)–C (9) was located at C-1 (Fig. 2). Thus, 6 was determined to be (7E)-4-O-[α-L-rhamnopyranosyl(1 → 6)-β-D-glucopyranosyl]-ferulylalcohol (Fig. 1) and named xiecaoside E. The known compounds were identified as scabroside B (7) [8], loganin (8) [17], patriscabroside I (9) [18], olivil-4'-Oβ-D-glucopyranoside (10) [19], lariciresinol-4,4'-di-O-β-Dglucopyranoside (11) [20], olivil-4-O-β-D-glucopyranoside (12) [19], 8-hydroxylariciresinol-4'-O-β-D-glucopyranoside (13) [19], lariciresinol-4-O-β-D-glucopyranoside (14) [20], neoarctin A (15) [21], lariciresinol-4'-O-β-D-glucopyranoside (16) [22], (−)-massoniresinol 3a-O-β-D-glucopyranoside (17) [23] by comparing their NMR spectroscopic and physical data with the literature values. The protective effects of compounds 1–17 on Aβ1–42 induced PC12 cells neurotoxicity were investigated by using MTT assay. As we all known, aggregated beta-amyloid (Aβ) has been one of critical factors in the pathogenesis of AD. Aβ fragments are 39–43 amino acid peptides, among which Aβ1–42 exhibited the most neurotoxic property [24]. The PC12 cell line displays phenotypic characteristics of sympathetic
neurons and induced by Aβ for imitating the conditions of Alzheimer’s disease (AD) in vitro to screen some anti-AD candidate drugs [25]. In this study, Aβ1–42 induced cytotoxicity (51.45 ± 3.03% viability) in the cells when it was added at a concentration of 1.5 μM for 24 h. When PC12 cells were pre-incubated with VE or compounds 1–17, the toxicity of Aβ1–42 on PC12 cells was significantly attenuated by VE, compounds 6 and 10–17 in a dose-dependent manner (Fig. 3), while other compounds did not show any protective effects on the cell at the tested concentration (data not shown in Figure). Conflict of interest The authors have declared that there is no conflict of interest. Acknowledgment This research was supported by the Program of International S&T Cooperation of China (No. 2010DFA32440) and Key Laboratory of Cardiovascular Medicine Research (Harbin Medical University), Ministry of Education. References [1] Huang BK, Zheng HC, Qin LP, Zheng QM, Xin HL. Investigation on resource of genus Valeriana in China. J Chin Med Mater 2004;27:632–4. [2] Du XW, Liang CF, Song N. Study on quality standard of Valeriana amurensis. Chin J Inf TCM 2011;18:46–7. [3] You CZ. Northeast medicinal plants. first ed. Harbin: Heilongjiang Science and Technology; 1989. [4] Zuo YM, Zhang ZL, Wang QH, Xie N, Kuang HX. Effects of Valeriana amurensis on the expressions of β-APP, Aβ1–40 and caspase-3 in Alzheimer’s disease model rat’s brain. J Chin Med Mater 2010;33:233–6. [5] Zhang ZL, Zuo YM, Wang QH, Xiao HB, Kuang HX. Effects of Valeriana amurensis on the expressions of iNOS, COX-2 and IκB-α in Alzheimer’s disease model rat’s brain. J Chin Med Mater 2010;33:581–3. [6] Wang QH, Wang CF, Zuo YM, Wang ZB, Yang BY, Kuang HX. Compounds from the roots and rhizomes of Valeriana amurensis protect against neurotoxicity in PC12 cells. Molecules 2012;17:15013–21. [7] Li N, Di L, Gao WC, Wang KJ, Zu LB. Cytotoxic iridoids from the roots of Patrinia scabra. J Nat Prod 2012;75:1723–8. [8] Di L, Li N, Zu LB, Wang KJ, Zhao YX, Wang Z. Three new iridoid glucosides from the roots of Patrinia scabra. Bull Korean Chem Soc 2011;32:3251–4. [9] Bouter Y, Dietrich K, Wittnam JL, Rezaei-Ghaleh N, Pillot T, Papot-Couturier S, Lefebvre T, Sprenger F, Wirths O, Zweckstetter M, Bayer TA. N-truncated amyloid β (Aβ) 1-42 forms stable aggregates and induces acute and longlasting behavioral deficits. Acta Neuropathol 2013;126:189–205.
C. Wang et al. / Fitoterapia 96 (2014) 48–55 [10] Peng QL, Buz’Zard AR, Lau BH. Pycnogenol protects neurons from amyloidbeta peptide-induced apoptosis. Brain Res Mol Brain Res 2002;104:55–65. [11] Lin S, Chen T, Liu XH, Shen YH, Li HL, Shan L, Liu RH, Xu XK, Zhang WD, Wang H. Iridoids and lignans from Valeriana jatamansi. J Nat Prod 2010;73(4):632–8. [12] Yahara S, Shigeyama C, Ura T, Wakamatsu K, Yasuhara T, Nohara T. Cyclic peptides, acyclic diterpene glycosides and other compounds from Lycium chinense Mill. Chem Pharm Bull (Tokyo) 1993;41:703–9. [13] Kristina JS, Nadin K, Karsten S, Sven J, Gerd W, Matthias EM. Chemical composition and biological activity of Paris quadrifolia L. Z Naturforsch C 2012;67:565–70. [14] Jiang ZY, Li SY, Li WJ, Guo JM, Tian K, Hu QF, Huang XZ. Phenolic glycosides from Ficus tikoua and their cytotoxic activities. Carbohydr Res 2013;382C:19–24. [15] Guo R, Luo M, Long CL, Li ML, Ouyang ZQ, Zhou YP, Wang YH, Li XY, Shi YN. Two new lignans from Dipteronia dyeriana. Chin Chem Lett 2008;19:1215–7. [16] Sugiyama M, Nagayama E, Kikuchi M. Lignan and phenylpropanoid glycosides from Osmanthus asiaticus. Phytochemistry 1993;33(5):1215–9. [17] Nakamoto K, Otsuka H, Yamasaki K. 7-O-Acetyl loganic acid from Alangium platanifolium var. Trilobum. Phytochemistry 1988;27:1856–8. [18] Kouno I, Yasuda I, Mizoshiri H, Tanaka T, Marubayashi N, Yang DM. Two new iridolactones and their glycosides from the roots of Patrinia scabra. Phytochemistry 1994;37:467–72.
55
[19] Schumacher B, Scholle S, Hölzl J, Khudeir N, Hess S, Müller CE. Lignans isolated from Valerian: identification and characterization of a new olivil derivative with partial agonistic activity at A1 adenosine receptors. J Nat Prod 2002;65:1479–85. [20] Deyama T, Ikawa T, Nishibe S. The constituents of Eucommia ulmoides Oliv. II. Isolation and structures of three new lignan glycosides. Chem Pharm Bull 1985;33:3651–7. [21] Yong M, Kun G, Qiu MH. A new lignan from the seeds of Arctium lappa. J Asian Nat Prod Res 2007;9:541–4. [22] Shoeb M, Jaspars M, MacManus SM, Majinda RRT, Sarker SD. Epoxylignans from the seeds of Centaurea cyanus (Asteraceae). Biochem Syst Ecol 2004;32:1201–4. [23] Feng WS, Hao ZY, Zheng XH. Chemical constituents from leaves of Celastrus gemmatus Loes. Acta Pharm Sin 2007;42:625–30. [24] Lambert MP, Barlow AK, Chromy BA. Diffusible, nonfibrillar ligands derived from Abeta 1–42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A 1998;95:6448–53. [25] Isaac G, Lea RT, Erez K, Hanna R. The herbal preparation Padma® 28 protects against neurotoxicity in PC12 cells. Phytother Res 2011;25:740–3.
