Article Type: Full Paper

Accepted Article
New Thymoquinol Glycosides and Neuroprotective

Dibenzocyclooctane Lignans from the Rattan Stems of Schisandra

chinensis

by Bing-You Yang, Jiang-Tao Guo, Zu-Yi Li, Chang-Fu Wang, Zhi-Bin Wang, Qiu-

Hong Wang, and Hai-Xue Kuang*

Key Laboratory of Chinese Materia Medica (Ministry of Education), Heilongjiang University

of Chinese Medicine, 24 HePing Road, Harbin 150040, P. R. China

(phone: +86-451-82193001; fax: +86-451-82110803; e-mail: hxkuang@yahoo.com)

Three new lignans (1-3), together with four new thymoquinol glycosides (4-7), were

isolated from 70%-EtOH extract of the rattan stems of Schisandra chinensis. The structures

of 1-7 were elucidated by detailed spectroscopic analyses, and these new compounds were

identified as pinobatol-9-O-β-D-glucopyranoside (1), 1,2,13,14-tetramethoxy-

dibenzocyclooctadiene 3,12-O-β-D-diglucopyranoside (2), 3,7-dihydroxy-1,2,13,14-

tetramethoxy-dibenzocyclooctadiene 12-O-β-D-glucopyranoside (3), thymoquinol 2-O-β-D-

apiofuranosyl-(1→6)-β-D-glucopyranoside (4), thymoquinol 2-O-α-D-arabinofuranosyl-

(1→6)-β-D-glucopyranoside (5), thymoquinol 5-O-β-D-apiofuranosyl-(1→6)-β-D-

glucopyranoside (6), and thymoquinol 5-O-α-D-arabinofuranosyl-(1→6)-β-D-

glucopyranoside (7). The neuroprotective activity of 1-7 was evaluated on PC12 cells with
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 neurotoxicity induced by amyloid-beta 1-42(Aβ1-42). 2 and 3 showed protecting activity

against Aβ-induced toxicity in PC12 cells.

Accepted Article
Keywords:

Rattan stems

Schisandra chinensis

Magnoliaceae

Neuroprotective activity

Lignans

PC12 cell

Thymoquinol glycosides

Introduction

Schisandra chinensis (TURCZ.) BAILL is a perennial herb and belongs to the family of

Magnoliaceae. S. chinensis is mainly distributed in northeastern China, Russia, Korea, and

Japan [1, 2]. Biologically active compounds, including lignans, triterpenoids, flavonoids, and

polysaccharides have been isolated from S. chinensis. In pharmacological studies,

dibenzocyclooctadiene lignans, which are the major and characteristic constituents of S.

chinensis have shown many pharmacological effects, such as anti-HIV, anti-HBV [3-6],

antitumor [7], cytotoxic, antioxidant, antihepatotoxic [8-10], anti-inflammatory [11], and

neuroprotective activities [12].

The rattan stems of S. chinensis possess tonic, anti-aging [13], anti-hepatocarcinoma

cells [14], antihepatotoxic [15] and antioxidant [16] effects, and have been used to treat

influenza, frostbite, asthma, and gastrointestinal dysfunction in China [13]. Our group had

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 found that the 70%-EtOH extract of the rattan stems of S. chinensis showed anti-

neurodegenerative activity in a Alzheimer’s disease model in rats. To further investigate the

Accepted Article
bioactive compounds from the rattan stems of S. chinensis, a phytochemical study was

performed. As a result, seven new compounds were isolated, including three lignans and four

thymoquinol glycosides. The isolation, structure elucidation, and neuroprotective activities of

all compounds are reported in this paper. All compounds were evaluated for protecting

against Aβ-induced toxicity in PC12 cells, and the results are described herein.

Results and Discussion

Structure Elucidation

Compound 1 was obtained as a white amorphous powder. The molecular formula was

assigned as C36H46O15 by HR-ESI-MS at m/z 719.2911 [M + H]+ (calc. 719.2915). The acid

hydrolysis of 1 gave only D-glucose, which was identified by GC analysis. The 1H-NMR

spectrum (Table 1) of 1 revealed the presence of one anomeric H-atom at δ(H) 4.03 (d, J =

7.8), three ABX-coupling systems at δ(H) 7.13 (d, J = 1.8), 6.79 (d, J = 8.1), 7.00 (dd, J = 1.8,

8.1), 6.30 (d, J = 2.4), 6.18 (d, J = 9.8), 7.08 (dd, J = 2.4, 9.8), 6.70 (d, J = 1.8), 6.75 (d, J =

8.2), and 6.61 (dd, J = 1.8, 8.2), a typical pattern for a 3-hydroxypropyl group at 1.78-1.79

(m), 2.57 (br. t, 7.6) and 3.53 (t, J = 6.5), four O–bearing CH2 groups at 3.67 (dd, J = 3.7,

10.7), 3.33 (m), 3.81-3.82 (m), 3.86-3.87 (m), 3.53(t, J = 6.5) 3.60 (dd, J = 5.2, 12.0) and

3.76 (dd, J = 3.7, 12.0), three MeO groups at 3.67 (s), 3.72 (s), and 3.89 (s). The 13C-NMR

spectrum of 1 exhibited signals of a C=O group at δ(C) 183.8, two groups of C=C bond at

δ(C) 115.6, 152.9, 129.5, and 153.6, and three MeO groups at δ(C) 55.3, 56.0, and 56.5. In

the HMBC spectrum, the correlation of H-C(1''')/C(9) was observed, which confirmed that

the glucosyl group moiety should be attached to C(9). The NMR data (Table 1) of compound

1 were similar to those of pinobatol [17] except for the β-D-glucopyranoside moiety in 1. The

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 NOESY correlations of H–C(6')/H–C(7')/H–C(8) demonstrated that H–C(6'), H–C(8) and H–

C(7') are on the same side of the tetrahydrofuran ring and the dienone ring is oriented
Accepted Article
perpendicularly to the tetrahydrofuran ring. Correlations of H–C(2')/Hα–C(9)/H–C(8')/H–

C(7) indicated that H–C(2'), Hα–C(9), H–C(8') and H–C(7) are on the opposite side of the

tetrahydrofuran ring. The large coupling constant J [H-C(7), H-C(8)] = 9.7 Hz showed that

H-C(7) is trans to H-C(8). The relative configurations of C(7), C(8), C(1'), and C(7') can be

determined from the abovementioned NOE correlations. However, the relative configuration

of C(8') remains undetermined because of the single bond between C(7') and C(8'), which

allows free rotation and therefore inhibits a reliable interpretation of the NOE results. The

stereochemical view of 1 is presented in Fig. 1. The relative configuration can thus be

deduced to be (7S*,8S*,1'S*,7'R*), based on the NOE correlations and the structure reported

in [17]. Therefore, the structure of 1 was determined to be pinobatol-9-O-β-D-

glucopyranoside (Fig. 1).

Compound 2 was obtained as a white amorphous powder and its molecular formula

was determined as C34H48O16 by HR-ESI-MS at m/z 713.3018 [M + H]+ (calc. 713.3021). The

acid hydrolysis of 2 gave D-glucose. The 1H-NMR spectrum (Table 2) of 2 revealed the

presence of two aromatic H-atoms at δ(H) 6.91 (s) and 6.89 (s), two anomeric H-atoms at

4.97 (d, J = 7.4) and 5.00 (d, J = 7.4), two Me groups at 0.74 (d, J = 7.0) and 1.00 (d, J =

7.0), and four MeO groups at 3.48 (s), 3.49 (s), 3.87 (s) and 3.89 (s). The 13C-NMR spectrum

of 2 showed signals for 34 C-atoms, corresponding to two aromatic rings with four CH2

groups at δ(C) 36.1, 39.8, 62.4 and 62.6, two CH groups at 35.3 and 42.3, two Me groups at

12.8 and 22.2, and four MeO groups at 61.0, 61.0, 61.9, 61.9, suggesting the presence of a

biphenyl moiety. The 1H,1H-COSY correlations (Fig. 2) of H–C(6)/H–C(7)/H–C(8)/H–C(9),

H–C(7)/Me(17), and H–C(8)/Me(18) together with the HMBCs (Fig. 2) of H–C(11)/C(9),

C(10), C(15), and H–C(4)/C(5), C(6), C(16), implied that 2 is a dibenzocyclooctadiene lignan

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 possessing four MeO groups and two β-D-glucosyl moieties. The correlations of H–

C(1')/C(12), and H–C(1'')/C(3) suggested that two β-D-glucosyl moieties were located at
Accepted Article
C(12) and C(3), respectively. The four MeO groups of 2 were located at C(1), C(2), C(13),

and C(14) as confirmed by analysis of its HMBC spectrum (Fig. 2). The CD spectrum of 2

(negative Cotton effect at 210 nm and a positive Cotton effect at 240 nm) indicated that 2 has

a (S)-biphenyl configuration [18]. The NOESY correlations (Fig. 3) of H–C(11)/Hβ–C(9), H–

C(11)/H–C(7), H–C(4)/Me(17), H–C(4)/Hα–C(6), and H–C(8)/Hβ–C(6) in 2 indicated that

Me(17) and Me(18) are α-oriented, and 2 has a twist-boat-chair (TBC) conformation for the

cyclooctadiene ring [18]. Consequently, the structure of 2 was identified as 1,2,13,14-

tetramethoxy-dibenzocyclooctadiene 3,12-O-β-D-diglucopyranoside (Fig. 1).

Compound 3 was obtained as a white amorphous powder and its molecular formula

was determined as C28H38O12 by its positive HR-ESI-MS at m/z 589.2259 [M + Na]+ (calc.

589.2261). The acid hydrolysis of 3 yielded D-glucose. The 1H-NMR spectrum (Table 2) of 3

revealed the presence of two aromatic H-atoms at δ(H) 6.54 (s) and 6.91 (s), an anomeric H-

atom at 5.10 (d, J = 7.3), three CH2 groups at 2.77 (dd, J = 2.1, 14.2), 2.27 (dd, J = 8.3,

14.2), 2.57 (d, J = 13.5), 2.38 (d, J = 13.5), 3.70 (dd, J = 5.9, 12.1) and 3.91 (dd, J = 2.1,

12.1), two Me groups at 0.81 (d, J = 7.1) and 1.19 (s), and four MeO groups at 3.44 (s), 3.46

(s), 3.83 (s), and 3.89 (s). The 13C-NMR spectrum of 3 exhibited signals of two aromatic ring

C-atoms, three CH2 groups δ(C) at 35.8, 42.6 and 62.7, one CH group δ(C) at 42.2, one O-

bearing quaternary C-atom at 73.7, two Me groups at 16.3 and 29.7, and four MeO groups at

60.8, 61.0, 61.2, 61.9. The HMBCs (Fig. 2) of H–C(11)/C(9), C(10), C(15), and H–

C(4)/C(5), C(6), C(16), together with 1H,1H-COSY correlations (Fig. 2) of H–C(7)/H–

C(6)/Me(17), implied that 3 is a dibenzocyclooctadiene lignan possessing one phenolic

hydroxy group, four MeO groups, and a β-D-glucosyl moiety. The correlation of H–

C(1')/C(12) indicated that the β-D-glucosyl moiety was located at C(12). The four MeO

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 groups of 3 were located at C(1), C(2), C(13), and C(14) which was confirmed by analysis of

its HMBC spectrum (Fig. 2). The CD spectrum of 3 gave a negative Cotton effect at 218 nm
Accepted Article
and positive Cotton effect at 258 nm, indicating that 3 has a (S)-biphenyl configuration [18].

The NOESY correlations (Fig. 3) of H–C(11)/Hβ–C(9), H–C(11)/H–C(8), H–C(4)/Me(17) in

3 suggested a twist-boat-chair (TBC) conformation for the cyclooctadiene ring [18].

Correlations (Fig. 3) of H–C(4)/Hα–C(6), H–C(4)/Me(17) and Me(18)/Hα–C(6) were

observed, which determined that Me(17) and Me(18) were α-oriented. Thus, 3 was identified

as 3,7-dihydroxy-1,2,13,14-tetramethoxy-dibenzocyclooctadiene 12-O-β-D-glucopyranoside

(Fig. 1).

Compound 4 was obtained as a white amorphous powder, and showed a sodiated

molecular ion at m/z 483.1839 in the HR-ESI-MS ([M + Na]+, calc. 483.1842) corresponding

to the molecular formula established as C21H32O11. The acid hydrolysis of 4 yielded D-

glucose and D-apiose. The 1H-NMR spectrum (Table 3) of 4 revealed the presence of two

anomeric H-atoms at δ(H) 4.61(d, J = 7.5) and 4.94 (d, J = 2.3), two aromatic H-atoms at

6.93 (s) and 6.51 (s), and three Me groups at 2.18 (s), 1.17 (d, J = 6.9), and 1.19 (d, J = 6.9).

The 13C-NMR spectrum of 4 (Table 3) showed 21 C-atom signals, corresponding to a 1,2,4,5-

substituted aromatic ring, a CH group at δ(C) 28.0, three Me groups at δ(C) 16.3, 23.3, and

23.3, a β-D-glucosyl moiety, and a terminal β-D-apiosyl moiety [19-21]. In the HMBC

spectrum (Fig. 2), the key long-range correlations from Me(7)/C(1), C(2) and C(6), H–

C(8)/C(3), C(4), C(5), C(9) and C(10), H–C(9)/C(4), C(8) and C(10), H–C(10)/C(4), C(8),

and C(9), together with 1H,1H-COSY correlations of H–C(9)/H–C(8), H–C(10)/H–C(8)

showed that the aglycone of 4 was thymoquinol [22]. The key cross-peaks H–C(1'')/C(6'),

and H–C(1'')/C(2) were observed in the HMBC spectrum, which confirmed that the β-D-

apiose moiety was attached to C(6') and the β-D-glucosyl moiety was attached to C(2). Thus,

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 the structure of 4 was established as thymoquinol 2-O-β-D-apiofuranosyl-(1→6)-β-D-

glucopyranoside (Fig. 1).

Accepted Article
Compound 5, obtained as a white amorphous powder, has the molecular formula of

C21H32O11, as established on the basis of positive HR-ESI-MS data (m/z 483.1843, [M + Na]+,

calc. 483.1842). The acid hydrolysis of 5 gave D-glucose and D-arabinose. The NMR data

(Tables 3 and 4) of 5 were similar to those of 4, except for the β-D-apiosyl moiety in 4 being

replaced by α-D-arabinosyl moiety in 5. The 1H- and 13C-NMR signals of 5 were assigned by

the use of HSQC, 1H,1H-COSY, and HMBC (Fig. 2) spectra. Thus, the structure of 5 was

identified as thymoquinol 2-O-α-D-arabinofuranosyl-(1→6)-β-D-glucopyranoside (Fig. 1).

Compounds 6 and 7 were obtained as white amorphous powder and showed

molecular ions at m/z 483.1841 and 483.1843 in the HR-ESI-MS (both calc. 483.1842)

respectively, corresponding to the same molecular formula, C21H32O11, for both compounds.

Acid hydrolysis of 6 afforded D-glucose and D-apiose, while 7 gave D-glucose and D-

arabinose. Analysis of the 1H-, 13C- (Tables 3 and 4), 1H,1H-COSY (Fig. 2), and HMBC (Fig.

2) NMR spectra showed that aglycones of 6 and 7 were thymoquinol. In the HMBC spectrum

of 6, the cross-peaks H-C(1'')/C(6'), and H-C(1')/C(5) were observed, which confirmed that

the apiose moiety was attached to C(6') and the glucosyl moiety was attached to C(5). The

correlations of H-C(1'')/C(6'), and H-C(1')/C(5) was observed in the HMBC spectrum of 7.

Thus, 6 and 7 were identified as thymoquinol 5-O-β-D-apiofuranosyl-(1→6)-β-D-

glucopyranoside and thymoquinol 5-O-α-D-arabinofuranosyl- (1→6)-β-D-glucopyranoside.

Biological Assay. The protective effects of compounds 1-7 on Aβ1–42 induced PC12

cells neurotoxicity were investigated by using MTT assay. Aggregated β-amyloid (Aβ) has

been recognized as 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 [23]. The

PC12 cell line displays phenotypic characteristics of sympathetic neurons. PC12 cells grown

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 in the presence of Aβ1-42, have been used as a model to screen potential anti-AD candidate

drugs in vitro [24]. In this study, Aβ1-42 induced cytotoxicity (52.0 ± 3.2 % viability) in the
Accepted Article
cells when it was added at a concentration of 1.5 μM for 24 h. When PC12 cells were pre-

incubated with vitamin E (VE) or compounds 1-7, the toxicity of Aβ1–42 on PC12 cells was

significantly attenuated by VE, compounds 2 and 3 in a dose-dependent manner (Table 5),

while other compounds did not show significant protective effects on the cells at the tested

concentration (Table 5).

Conclusions

This is the first scientific report on isolation and identification of neuroprotective

compounds from the rattan stems of S. chinensis. Seven new compounds were identified,

which include three new lignans (1-3) and four new thymoquinol glycosides (4-7).

Neuroprotective activity was screened by the MTT assay. As a result, dibenzocyclooctadiene

lignans 2 and 3 showed protective effects on the PC12 cells in a dose-dependent manner

(Table 5) at the tested concentration. Thus, dibenzocyclooctadiene lignans appear to be the

anti-neurodegenerative constituents of the rattan stems of S. chinensis.

This research was supported by the program of National Natural Science Foundation

(No. 81473325) of China.

Experimental Part

General. Thin layer chromatography (TLC): precoated silica gel F254 plates (SiO2;

Merck, Gemany). Column chromatography (CC): SiO2 (200-300 mesh; Qingdao Marine

Chemical Industry, P. R. China); ODS (50 μm; YMC, Japan); and Sephadex LH-20 (GE

Healthcare, Sweden). Prep. HPLC: Waters 2535 quaternary gradient module; Waters 2414

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 refractive index detector; SunFire C18 OBD column (19 × 250 mm, 10 μm, Waters, US).

Optical rotations: Jʌsco P-2000 polarimeter (Japan). UV Spectra: Shimadzu UV-1601

Accepted Article
spectrophotometer (Japan); λmax (log ε) in nm. CD Spectra: Chirascan spectropolarimeter

(France). IR Spectra: Shimadzu FTIR-8400S infrared spectrometer. NMR spectra: Bruker

DPX-400 spectrometer; at r.t.; δ in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-

MS: Waters Xero Q TOF MS spectrometer; in m/z. GC: Fuli-9790 hydrogen flame detector;

DM-5 column (30 m × 0.25 mm, 0.25 μm; Dikma, P. R. China). Microplate reader:

PerkinElmer VICTOR® X3 (Massachusetts, United States of America).

Plant Material. The rattan stems of Schisandra chinensis (TURCZ.) BAILL. were

collected from Raohe, Heilongjiang province, P. R. China in August 2010. The botanical

identification was made by Prof. Lianjie Su, School of Pharmacy, Heilongjiang University of

Chinese Medicine. A voucher specimen (Herbarium NO. 20100899) was deposited in the

School of Pharmacy, Heilongjiang University of Chinese Medicine, P. R. China.

Extraction and Isolation. The air-dried rattan stems of S. chinensis (12.0 kg) were

extracted with 70%-EtOH (3 × 80 l) under reflux for 2 h. After solvent removal, the crude

extract (1.72 kg) was suspended in H2O (25 l) and further extracted with CHCl3 (1:1), AcOEt

(1:1), and BuOH (1:1), successively, to obtain corresponding extracts after removal of the

solvent. The BuOH extract (326.3g) was subjected to CC (SiO2; CH2Cl2/MeOH 10:1 → 0:1,

gradient) to furnish seven fractions, Frs. I-VII. Fr. II (44.3 g) was separated by CC (SiO2:

CH2Cl2/MeOH 15:1 → 3:1) to yield Frs. II1-II5. Fr. II2 (11.4 g) was subjected to CC (ODS;

MeOH/H2O 30:70), to obtain compound 2 (48.3 mg). Fr. III (54.0 g) was subjected to CC

(SiO2; CH2Cl2/MeOH 10:1 → 2:1) to yield Frs. III1 - III3. Fr. III1 (14.6 g) was separated to

CC (ODS; MeOH/H2O 20:80) and purified with HPLC (MeCN/H2O 15:85) to give

compounds 4 (30.2 mg) and 3 (69.3 mg). Fr. III3 (17.0 g) was subjected to CC (ODS;

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 MeOH/H2O 20:80) and purified with HPLC (MeCN/H2O 15:85) to give 1 (19.5 mg), 6 (8.1

mg), 5 (12.6 mg), and 7 (8.3 mg).

Accepted Article
Pinobatol-9-O-β -D-glucopyranoside (= [(1S,3S,4R,5S)-1-{(1S)-2-Hydroxy-1-[4-(3-

hydroxypropyl)-2-methoxyphenoxy]ethyl}-3-(4-hydroxy-3-methoxyphenyl)-7-methoxy-

8-oxo-2-oxaspiro[4.5]deca-6,9-dien-4-yl]methyl β-D-Glucopyranoside, 1). White

amorphous powder. [α]D20 = +6.0 (c = 0.3, MeOH). UV (MeOH): 258 (3.50). IR: 3434, 1679,

1588, 1124, 1043, 786. 1H- and 13C-NMR: see Table 1. HR-ESI-MS (pos.): 719.2911 ([M +

H]+, C36H47O15+; calc. 719.2909).

1,2,13,14-Tetramethoxydibenzocyclooctadiene 3,12-O-β -D-Diglucopyranoside (=

(6R,7S)-10-(β -D-Glucopyranosyloxy)-1,2,11,12-tetramethoxy-6,7-dimethyl-5,6,7,8-

tetrahydrodibenzo[a,c][8]annulen-3-yl β -D-Glucopyranoside; 2). White amorphous

powder. [α]D20 = +9.6 (c = 0.5, MeOH). UV (MeOH): 222 (3.65), 258 (sh). CD (c = 0.02,

MeOH): 248 (–25.63), 240 (–20.36), 232 (–21.51), 210 (+32.13). IR: 3436, 2896, 1614,

1455, 1522; 1H- and 13C-NMR: see Table 2. HR-ESI-MS (pos.): 713.3018 ([M + H]+,

C34H49O16+; calc. 713.3015).

3,7-Dihydroxy-1,2,13,14-tetramethoxydibenzocyclooctadiene 12-O-β -D-

glucopyranoside (= (6R,7R)-3-(β -D-Glucopyranosyloxy)-5,6,7,8-tetrahydro-10-hydroxy-

1,2,11,12-tetramethoxy-6,7-dimethyldibenzo[a,c][8]annulen-6-yl β-D-Glucopyranoside;

3). White amorphous powder. [α]D20 = +6.0 (c = 0.4, MeOH). UV (MeOH): 218 (3.65), 258

(sh). CD (c 0.03, MeOH), nm (△ε): 258 (+38.37), 236 (+20.33), 218 (–33.45). IR: 3418,

2924, 1618, 1518, 1455. 1H- and 13C-NMR: see Table 2. HR-ESI-MS (pos.): 589.2259 ([M +

Na]+, C28H38NaO12+; calc. 589.2261).

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 Thymoquinol 2-O-β-D-Apiofuranosyl-(1→6)-β-D-glucopyranoside (= 4-Hydroxy-

2-methyl-5-(propan-2-yl)phenyl 6-O-[(2R,3R,4R)-Tetrahydro-3,4-dihydroxy-4-
Accepted Article
(hydroxymethyl)furan-2-yl]-β -D-glucopyranoside; 4). White amorphous powder. [α]D20 =

–7.5 (c = 0.3, MeOH). UV (MeOH): 223 (2.22), 284 (3.10). IR: 3442, 2941, 1461, 1155, 878.
1
H- and 13C-NMR: see Tables 3 and 4. HR-ESI-MS (pos.): 483.1839 ([M + Na]+,

C21H32NaO11+; calc. 483.1842).

Thymoquinol 2-O-α-D-Arabinofuranosyl-(1→6)-β -D-glucopyranoside (= 4-

Hydroxy-2-methyl-5-(propan-2-yl)phenyl 6-O-α-D-Arabinofuranosyl-β-D-

glucopyranoside; 5). White amorphous powder. [α]D20 = – 8.7(c = 0.3, MeOH). UV

(MeOH): 223 (2.22), 284 (3.10). IR: 3442, 2941, 1461, 1155, 878. 1H- and 13C-NMR: see

Tables 3 and 4. HR-ESI-MS (pos.): 483.1843 ([M + Na]+, C21H32NaO11+; calc. 483.1842).

Thymoquinol 5-O-β-D-Apiofuranosyl-(1→6)-β-D-glucopyranoside (= 4-Hydroxy-

5-methyl-2-(propan-2-yl)phenyl 6-O-[(2R,3R,4R)-Tetrahydro-3,4-dihydroxy-4-

(hydroxymethyl)furan-2-yl]-β -D-glucopyranoside; 6). White amorphous powder. [α]D20 =

–7.3 (c = 0.3, MeOH). UV (MeOH): 226 (2.22), 284 (3.10). IR: 3442, 2942, 1475, 1155, 877.
1
H- and 13C-NMR: see Tables 3 and 4. HR-ESI-MS (pos.): 483.1841 ([M + Na]+,

C21H32NaO11+; calc. 483.1842).

Thymoquinol 5-O-α-D-arabinofuranosyl-(1→6)-β-D-glucopyranoside (= 4-

Hydroxy-5-methyl-2-(propan-2-yl)phenyl 6-O-α-D-Arabinofuranosyl-β-D-

glucopyranoside; 7). White amorphous powder. [α]D20 = –8.9 (c = 0.5, MeOH). UV

(MeOH): 226 (2.22), 284 (3.10). IR: 3442, 2942, 1475, 1155, 877. 1H- and 13C-NMR: see

Tables 3 and 4. HR-ESI-MS (pos.): 483.1843 ([M + Na]+, C21H32NaO11+; calc. 483.1842).

Acid Hydrolysis and Determination of Sugar Compounds. Acid hydrolysis was carried

out as reported in [25]. Briefly, the sugar residues were obtained by hydrolysis of compounds

1-7 (2.0 mg) with H2SO4 (2.0 ml, 2 mol/l). The sugar residues were dissolved in pyridine (1.0

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 ml) and L-cysteine methyl ester hydrochloride in pyridine (2.0 ml, 0.1 mol/l) was added. The

mixtures were heated at 60° for 1 h. Then treated with trimethylchlorosilane, and stirred at 60
Accepted Article
°C for 5 min, resp. After drying the soln. with a stream of N2, the residue was extracted with

Et2O (1 ml). The sugar derivatives were analyzed by GC [26-27]. Retention times (tR) of

standards: D-apiose 12.1 min, D-arabinose 11.6 min, L-arabinose 12.4 min, D-glucose 14.9

min. As a result, the sugar derivatives from compounds 4 and 6 were determined to be D-

glucose (tR = 14.9 min) and D-apiose (tR = 12.1 min). The sugar derivatives from compounds

5 and 7 were determined to be D-glucose (tR = 14.9 min) and D-arabinose (tR = 11.6 min). The

sugar derivatives from compounds 1-3 were determined to be D-glucose (tR =14.9 min).

Determination of Cell Viability. The MTT assay method was used to measure the cell

viability as reported in reference [28]. The PC12 cells were cultured for 24 h at 37 °C in 96-

well plates (7.5 × 103 cells/well), and then incubated without or with compounds 1-7 (at the

concentrations of 5, 12.5, 25 μM), and the cells neurotoxicity was induced by 1.5 μM Aβ1–42

for 24 h [29]. Vitamin E (VE, 97.5%, Xinchang Pharmaceuticals Factory, P. R. China) at the

concentrations of 5, 12.5 and 25 μM was used as a positive control [30]. 20 μl MTT solns. (5

mg/ml) were added into each well and cells were incubated at 37 °C for another 4 h. The

supernatant was aspirated and the formazan crystals dissolved with DMSO (150 μl). A

microplate reader was used to measure the optical density (OD) of each well at 490 nm and

the results were expressed as the percentages of cell viability (ODs/OD0 × 100%, OD0 and

ODs are the optical density at 490 nm for the sample without and with test compound), resp.

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Received October 2, 2015

Accepted February 24, 2016

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 Table 1. 1H- and 13C-NMR Data (400 and 100 MHz, resp.; in CD3OD) of 1. δ in ppm, J in

Hz.
Accepted Article
Position δ(H) δ(C) Position δ(H) δ(C)

1 134.7 1'' 137.4

2 7.13 (d, J = 1.8) 111.1 2'' 6.70 (d, J = 1.8) 113.6

3 149.0 3'' 151.0

4 147.2 4'' 145.9

5 6.79 (d, J = 8.1) 116.8 5'' 6.75 (d, J = 8.2) 116.2

6 7.00 (dd, J = 1.8, 8.1) 119.8 6'' 6.61 (dd, J = 1.8, 8.2) 121.4

7 5.35 (d, J = 9.7) 82.9 7'' 2.57 (br. t, J = 7.6) 32.6

8 2.82-2.83(m) 60.0 8'' 1.78-1.79 (m) 35.5

9 3.67 (dd, J = 3.7, 10.7) 65.8 9'' 3.53 (t, J = 6.5) 62.1

3.33 (m) MeO-3 3.89 (s) 56.5

1' 56.7 MeO-3' 3.67 (s) 55.3

2' 6.30 (d, J = 2.4) 115.6 MeO-3'' 3.72 (s) 56.0

3' 152.9 Glc 1''' 4.03 (d, J = 7.8) 104.4

4' 183.8 2''' 3.05-3.06 (m) 75.1

5' 6.18 (d, J = 9.8) 129.5 3''' 3.08-3.09 (m) 77.8

6' 7.08 (dd, J = 2.4, 9.8) 153.6 4''' 3.24-3.26(m) 71.3

7' 4.81 (d, J = 8.8) 85.4 5''' 3.24-3.26(m) 78.1

8' 4.36-4.37(m) 80.9 6''' 3.60 (dd, J = 5.2, 12.0) 62.4

9' 3.81-3.82 (m) 62.3 3.76 (dd, J = 3.7, 12.0)

3.86-3.87 (m)

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 Table 2. 1H- and 13C-NMR Data (400 and 100 MHz, resp.; in CD3OD) of Compounds 2 and

3. δ in ppm, J in Hz.
Accepted Article
Position 2 3

δ(H) δ(C) δ(H) δ(C)

1 152.5 152.5

2 141.6 140.2

3 152.3 150.6

4 6.91 (s) 113.2 6.54 (s) 115.5

5 141.2 135.3

6 2.21 (dd, J = 9.3, 13.0) 36.1 2.77 (dd, J = 2.1, 14.2) 35.8

2.07 (br.d, J = 13.0) 2.27 (dd, J = 8.3, 14.2)

7 1.81-1.82 (m) 42.3 1.77-1.78 (m) 42.2

8 1.89-1.91 (m) 35.3 73.7

9 2.66 (dd, J = 7.4, 12.5) 39.8 2.57 (d, J = 13.5) 42.6

2.42 (br. d, J = 12.5) 2.38 (d, J = 13.5)

10 135.8 136.2

11 6.89 (s) 116.2 6.91 (s) 116.1

12 150.8 150.8

13 142.0 142.0

14 152.6 152.7

15 126.2 126.7

16 125.0 122.8

17 1.00 (d, J = 7.0) 22.2 0.81 (d, J = 7.1) 16.3

18 0.74 (d, J = 7.0) 12.8 1.19 (s) 29.7

MeO-1 3.49 (s) 61.0 3.46 (s) 61.0

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 MeO-2 3.87 (s) 61.9 3.83 (s) 61.2

MeO-13 3.89 (s) 61.9 3.89 (s) 61.9

Accepted Article
MeO-14 3.48 (s) 61.0 3.44 (s) 60.8

1' 4.97 (d, J = 7.4) 102.2 5.10 (d, J = 7.3) 102.2

2' 3.51-3.52 (m) 75.0 3.51-3.52 (m) 75.1

3' 3.46-3.47 (m) 78.2 3.49-3.51 (m) 78.1

4' 3.39-3.41 (m) 71.6 3.37-3.39 (m) 71.5

5' 3.46-3.47 (m) 78.3 3.49-3.51 (m) 78.1

6' 3.70 (dd, J = 6.2, 12.1) 62.6 3.70 (dd, J = 5.9, 12.1) 62.7

3.92 (dd, J = 2.1, 12.1) 3.91 (dd, J = 2.1, 12.1)

1'' 5.00 (d, J = 7.4) 102.4

2'' 3.51-3.52 (m) 74.9

3'' 3.51-3.52 (m) 78.0

4'' 3.46-3.47 (m) 71.2

5'' 3.50-3.51 (m) 78.1

6'' 3.70 (dd, J = 6.2, 12.3) 62.4

3.90 (dd, J = 2.5, 12.3)

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 Table 3. 1H-NMR Data (400 MHz; in CD3OD) of Compounds 4-7. δ in ppm, J in Hz.

Position4 5 6 7
Accepted Article
3 6.93 (s) 6.93 (s) 6.60 (s) 6.60 (s)

6 6.51 (s) 6.51 (s) 6.88 (s) 6.88 (s)

7 2.18 (s) 2.17 (s) 2.13 (s) 2.13 (s)

8 3.19-3.20 (m) 3.19-3.20 (m) 3.45-3.46 (m) 3.45-3.46 (m)

9 1.17 (d, J = 6.9) 1.18 (d, J = 6.9) 1.15 (d, J = 6.9) 1.15 (d, J = 6.9)

10 1.19 (d, J = 6.9) 1.19 (d, J = 6.9) 1.14 (d, J = 6.9) 1.14 (d, J = 6.9)

1' 4.61 (d, J = 7.5) 4.64 (d, J = 7.5) 4.64 (d, J = 7.6) 4.64 (d, J = 7.5)

2' 3.40-3.41 (m) 3.40-3.41 (m) 3.40-3.41 (m) 3.40-3.41 (m)

3' 3.40-3.41 (m) 3.40-3.41 (m) 3.40-3.41 (m) 3.40-3.41 (m)

4' 3.37-3.38 (m) 3.39-3.40 (m) 3.38-3.39 (m) 3.39-3.40 (m)

5' 3.43-3.44 (m) 3.46-3.47 (m) 3.43-3.44 (m) 3.45-3.46 (m)

6' 3.60 (dd, J = 5.3, 10.9) 3.60 (dd, J = 5.9, 11.8) 3.57 (dd, J = 6.2, 10.9) 3.60 (dd, J = 5.9, 11.8)

3.96 (dd, J = 1.4, 10.9) 4.03 (dd, J = 2.1, 11.8) 3.98 (dd, J = 1.7, 10.9) 4.03 (dd, J = 2.2, 11.8)

1'' 4.94 (d, J = 2.3) 4.90 (d, J = 1.3) 4.94 (d, J = 2.2) 4.91 (d, J = 1.3)

2'' 3.89 (d, J = 2.7) 4.00 (dd, J = 1.3, 3.2) 3.89 (d, J = 1.4) 4.00 (dd, J = 1.3, 3.2)

3'' 3.82 (dd, J = 3.1, 5.8) 3.82 (dd, J = 3.1, 5.8)

4'' 3.73 (d, J = 9.6) 3.93-3.94 (m) 3.73 (d, J = 9.6) 3.93-3.94 (m)

3.90 (d, J = 9.6) 3.90 (d, J = 9.6)

5'' 3.56 (s) 3.60 (dd, J = 5.1, 11.9) 3.56 (s) 3.60 (dd, J = 5.1, 11.8)

3.70 (dd, J = 3.2, 11.9) 3.70 (dd, J = 3.3, 11.8)

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 Table 4. 13C-NMR Data (100 MHz; in CD3OD) of Compounds 4-7. δ in ppm

Position 4 5 6 7 Position 4 5 6 7
Accepted Article
1 127.6 127.5 123.3 123.3 1' 104.9 104.8 104.7 104.8

2 150.6 150.6 152.0 152.0 2' 75.1 75.1 75.2 75.2

3 116.7 116.5 113.0 113.0 3' 78.1 78.1 78.2 78.2

4 134.2 134.2 138.5 138.5 4' 71.5 71.8 71.7 71.9

5 150.8 150.7 149.2 149.2 5' 76.7 76.5 76.7 76.6

6 117.9 117.9 120.8 120.8 6' 68.5 68.0 68.7 68.2

7 16.3 16.3 16.2 16.2 1'' 110.9 109.9 110.9 109.9

8 28.0 28.1 27.0 27.0 2'' 78.0 83.1 78.1 83.3

9 23.3 23.3 23.7 23.7 3'' 80.6 79.0 80.6 79.0

10 23.3 23.3 23.8 23.8 4'' 75.1 85.9 75.0 85.9

5'' 65.9 62.9 65.8 63.0

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 Table 5. Neuroprotective Effects of Compounds 1-7 against Aβ1-42 Induced PC12 Cells Death

(mean ± SD, n = 8)
Accepted Article
Cell viability [%]
Compound
5 μM 12.5 μM 25 μM

1 53.3 ± 4.1 57.3 ± 4.2 64.1 ± 4.8

2 54.5 ± 3.2 69.8 ± 5.2* 84.1 ± 5.4*

3 54.7 ± 3.4 62.8 ± 3.8* 82.1 ± 4.3*

4 55.4 ± 3.1 53.1 ± 3.1 55.2 ± 4.2

5 54.7 ± 4.4 57.4 ± 4.9 55.4 ± 5.2

6 55.8 ± 3.5 52.7 ± 3.6 53.8 ± 4.4

7 54.3 ± 3.3 52.0 ± 3.3 52.9 ± 4.6

Vitamin Ea) 61.8 ± 4.8 75.7 ± 4.8 79.8 ± 5.3

a
) Vitamin E is positive control; * p < 0.01 vs. model (52.0 ± 3.2), ʻmodelʼ

group is the PC12 cells which was induced by 1.5 μM Aβ1–42 for 24 h. This

experiment was repeated three times with similar results.

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 Captions
Accepted Article
Fig. 1. Structures of compounds 1-7.

Fig. 2. Key HMBC and 1H,1H-COSY correlations of 1-7.

Figure 3. Key NOESY correlations of 1-3.

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Accepted Article

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