Aromatic monoterpenoid glycosides from rattan stems of Schisandra chinensis and their neuroprotective activities

Article abstract of DOI:10.1016/j.fitote.2019.02.012

Six new monoterpenoid glycosides, including thymoquinol 5-O-α-L-arabinopyranosyl-(1 → 6)-β-D-glucopyranoside (1), cuminic acid 7-O-α-D-arabinofuranosyl-(1 → 6)-β-D-glucopyranosyl ester (2), p-cymene 7-O-α-D-arabinofuranosyl-(1 → 6)-β-D-glucopyranoside (3)

Full text of DOI:10.1016/j.fitote.2019.02.012

Fitoterapia134(2019)108–112
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Aromatic monoterpenoid glycosides from rattan stems of Schisandra
chinensis and their neuroprotective activities
Yan Liua,1, Jiang-Tao Guob,1, Zhi-Bin Wanga, Zu-Yi Lia, Gui-Xue Zhenga, Yong-Gang Xiaa,
⁎
⁎
Bing-You Yanga, , Hai-Xue Kuanga,
a Key Laboratory of Chinese Materia Medica, Ministry of Education of Heilongjiang University of Chinese Medicine, Harbin 150040, China
b Guizhou University of traditional Chinese Medicine, Guiyang 550000, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Six new monoterpenoid glycosides, including thymoquinol 5-O-α-L-arabinopyranosyl-(1 → 6)-β-D-glucopyr-
anoside (1), cuminic acid 7-O-α-D-arabinofuranosyl-(1 → 6)-β-D-glucopyranosyl ester (2), p-cymene 7-O-α-D-
arabinofuranosyl-(1 → 6)-β-D-glucopyranoside (3), p-methylhyd- ratropic acid 9-O-α-D-arabinofuranosyl-(1 →
6)-β-D-glucopyranosyl ester (4), (R)-p-cymene 9-O-α- D-arabinofuranosyl-(1 → 6)-β-D-glucopyranoside (5), and
(R)-p-cymene 9-O-β-D-apiofuranosyl-(1 → 6)-β-D-glucopyranoside (6), together with three known compounds,
such as (R)-p-cymene 9-O-β-D-glucopyranoside (7), thymoquinol 5-O-β-D-glucopyranoside (8), and thymoquinol
2-O-β-D-glucopyranoside (9) were isolated from the 70%-EtOH extract of Schisandra chinensis rattan stems. The
structures of the new compounds were elucidated by detailed spectroscopic analyses. Meanwhile, the neuro-
protective activities of compounds 1–9 were evaluated on the two co-culture models of microglia and neurons
cells and astrocytoma and neurons cells
Schisandra chinensis
Magnoliaceae
Monoterpenoid glycoside
Co-culture cells model
Neuroprotective
1. Introduction
glycosides, and their neuroprotective activities were evaluated on the
two co-culture cells models.
Schisandra chinensis (Turcz.) Baill., belonging to the Magnoliaceae
family, is widely distributed in northeastern China, Korea, Japan, and
Russsia [1]. The rattan stems of S. chinensis are 25.9 times as heavy as
the fruits in the whole dry plant, as the non-medicinal parts. Simulta-
neously, the rattan stems of S. chinensis are used as a folk herbal
medicine for the treatment of influenza, frostbite, asthma, and gastro-
intestinal dysfunction in northeastern China [2]. It is reported that
triterpenoids, lignans, and polysaccharides have been isolated from the
rattan stems of S. chinensis [3–7]. Several studies have shown that the
rattan stems of S. chinensis possess tonic, anti-aging, anti-hepatocarci-
noma cells, anti-hepatotoxic, and antioxidant effects [8–10]. In our
previous researches, it is interestingly found that the rattan stems could
improve the cognitive impairment and protect neuronal cells as well
[11,12]. Motivated by the discovery of new and bioactive natural
products from S. chinensis rattan stems, our group has ongoing phyto-
chemically studied. As a result, six new monoterpenoid glycosides and
three known monoterpenoid glycosides were isolated and identified
from this plant (Fig. 1). This present paper reported the isolation and
structural elucidation of the six new aromatic monoterpenoid
2. Experimental
2.1. General experimental procedures
Column chromatography (CC), silica gel (200–300 mesh, Qingdao
Marine Chemical Industry, China), ODS (50 μm, YMC, Japan), and
Sephadex LH-20 (GE Healthcare, Sweden) were used; precoated silica gel
GF254 plates (Merck, Gemany) were used for thin layer chromatography
(TLC). The chromatographic conditions were as follows: Preparative
HPLC, Waters 2535 quaternary gradient module; Waters 2414 refractive
index detectors; Sunfire C18 OBD column (19 mm × 250 mm, 10 mm,
Waters, USA). Optical rotations were measured in a Jasco P-2000 po-
larimeter (Japan). UV spectra were recorded with a Shimadzu UV-1601
spectrophotometer (Japan). IR spectra (KBr) were measured using a
Shimadzu FTIR-8400S infrared spectrometer, in cm−1. NMR spectra
were recorded with a Bruker DPX-400 spectrometer; J in Hz. HR-ESI-MS
spectra were obtained on Waters Xero Q-TOF MS spectrometer in m/z.
GC spectra were obtained on Fuli-9790 hydrogen flame detector; DM-5
⁎ Corresponding authors.
E-mail addresses: ybywater@163.com (B.-Y. Yang), hxkuang@yahoo.com (H.-X. Kuang).
1 Yan Liu and Jiang-Tao Guo have contributed equally to this work.
https://doi.org/10.1016/j.fitote.2019.02.012
Received 22 January 2019; Received in revised form 13 February 2019; Accepted 18 February 2019
Availableonline20February2019
0367-326X/©2019PublishedbyElsevierB.V.

Y. Liu, et al.
Fitoterapia134(2019)108–112
Fig. 1. The structures of compounds 1–9.
column (30 m × 0.25 mm, 0.25 mm; Dikma, China). The multi-detection
microplate reader was PerkinElmer (VICTOR X3, Waltham, MA, USA).
C21H30NaO11+; Calcd. 481.1681). 1H NMR (CD3OD, 400 MHz) and 13
NMR (CD3OD, 100 MHz) data (Tables 1 and 2).
C
2.2. Plant material
2.3.3. p-Cymene 7-O-α-D-arabinofuranosyl-(1 → 6)-β-D-glucopyranoside
(3)
20
The rattan stems of S. chinensis (Turcz.) Baill. were collected from
Raohe, Heilongjiang province of China, in August 2010, and authenti-
cated by Prof. SU Lian-Jie, School of Pharmacy, Heilongjiang University
of Chinese Medicine, China. A voucher specimen (Herbarium No.
20100899) had been deposited in the School of Pharmacy, Heilongjiang
University of Chinese Medicine.
White amorphous powder. [α]D
–30.5 (c 0.03, MeOH). UV
(MeOH) λmax: 213 nm. IR (KBr) vmax: 3450, 2941, 1616, 1460, 1210,
850 cm−1
.
Positive HR-ESI-MS m/z 467.1893 ([M + Na]+
,
C
21H32NaO10+; Calcd. 467.1891). 1H NMR (CD3OD, 400 MHz) and 13
C
NMR (CD3OD, 100 MHz) data (Tables 1 and 2).
2.3.4. p-Methylhydratropic acid 9-O-α-D-arabinofuranosyl-(1 → 6)-β-D-
2.3. Extration and isolation
glucopyranosyl ester (4)
20
White amorphous powder. [α]D
–28.6 (c 0.05, MeOH). UV
The air-dried and powdered rattan stems of S. chinensis (12.0 kg) were
extracted with 70% alcohol (80 L × 3, 2 h each) under reflux. The extract
were filtered and evaporated under reduced pressure. The residues
(1.72 kg) were suspended in water (25 L) and partitioned with CHCl3,
EtOAc, and n-BuOH to obtain 110.5, 360.8 and 326.3 g of extracts, re-
spectively. The crude n-BuOH extract (300 g) was subjected to column
chromatography over silica gel and eluted with CH2Cl2 − MeOH (10: 1 to
0: 1) to give Frs. I − VII. Fr. IV (50.3 g) was chromatographed on silica gel
and eluted with CH2Cl2 − MeOH (15: 1 to 3: 1) to yield Fr. IV1 − IV5. Fr.
IV2 (9.6 g) was chromatographed on ODS, and purified with HPLC
(MeCN−H2O, 15: 85) to obtain compounds 2 (9.7 mg, tR = 17.8), 4
(15.3 mg, tR = 23.5), and 5 (7.6 mg, tR = 21.1). Fr. IV4 (14.0 g) was
chromatographed on ODS, and purified with HPLC (MeCN−H2O, 20: 80)
to give compounds 1 (12.9 mg, tR = 27.4), 3 (13.1 mg, tR = 19.7), and 6
(11.5 mg, tR = 16.2). Fr. IV5 (15.6 g) was subjected to ODS column
chromatography, and purified on Sephadex LH-20 (MeOH) to obtain
compounds 7 (10.3 mg), 8 (9.6 mg), and 9 (11.3 mg).
(MeOH) λmax: 194 nm. IR (KBr) vmax: 3442, 2940, 1625, 1461, 1235,
855 cm−1
.
Positive HR-ESI-MS m/z 481.1686 ([M + Na]+
,
C
21H30NaO11+; Calcd. 481.1683). 1H NMR (CD3OD, 400 MHz) and 13
C
NMR (CD3OD, 100 MHz) data (Tables 1 and 2).
2.3.5. (R)-p-cymene 9-O-α-D-arabinofuranosyl-(1 → 6)-β-D-glucopyranoside
(5)
20
White amorphous powder. [α]D
–29.1 (c 0.04, MeOH). UV
(MeOH) λmax: 212 nm. IR (KBr) vmax: 3443, 2924, 1618, 1455, 1215,
847 cm−1
.
Positive HR-ESI-MS m/z 467.1893 ([M + Na]+
,
C21H32NaO10+; Calcd. 467.1895). 1H NMR (CD3OD, 400 MHz) and 13
NMR (CD3OD, 100 MHz) data (Tables 1 and 2).
C
2.3.6. (R)-p-Cymene 9-O-β-D-apiofuranosyl-(1 → 6)-β-D-glucopyranoside
(6)
20
White amorphous powder. [α]D
–27.3 (c 0.03, MeOH). UV
(MeOH) λmax: 213 nm. IR (KBr) vmax: 3450, 2922, 1615, 1455, 1213,
845 cm−1
.
Positive HR-ESI-MS m/z 467.1890 ([M + Na]+
,
2.3.1. Thymoquinol 5-O-α-L-arabinopyranosyl-(1 → 6)-β-D-glucopyranoside
C
21H32NaO10+; Calcd. 467.1895). 1H NMR (CD3OD, 400 MHz) and 13
C
(1)
NMR (CD3OD, 100 MHz) data (Tables 1 and 2).
20
White amorphous powder. [α]D +19.6 (c 0.05, MeOH). UV
(MeOH) λmax
:
226 nm. IR (KBr) vmax
:
3465, 1615, 1450, 1512,
2.4. Acid hydrolysis of compounds 1–6
1270 cm−1
.
Positive HR-ESI-MS m/z 483.1843 ([M + Na]+
,
C21H32NaO11+; Calcd. 483.1840). 1H NMR (CD3OD, 400 MHz) and 13
NMR (CD3OD, 100 MHz) data (Tables 1 and 2).
C
Each compound (2 mg) was hydrolyzed in 1 mol·L−1 of HCl (1.0 mL)
for 2 h at 85 °C. The reaction mixture was cooled and partitioned between
CHCl3 (2.0 mL) and H2O (2.0 mL). The aqueous layer was washed with
CHCl3 (3.0 mL × 3), neutralized with Ba(OH)2, filtered, and evaporated
under reduced pressure. The residue was dissolved in pyridine (1.0 mL)
and 0.1 mol·L−1 of L-cysteine methyl ester hydrochloride in pyridine
(2.0 mL) was added. The mixture was heated at 60 °C for 1 h. An equal
volume of Ac2O was added with heating and incubation was continued for
2.3.2. Cuminic acid 7-O-α-D-arabinofuranosyl-(1 → 6)-β-D-glucopyranosyl
ester (2)
20
White amorphous powder. [α]D
–29.9 (c 0.05, MeOH). UV
(MeOH) λmax: 272 nm. IR (KBr) vmax: 3446, 2965, 1610, 1465, 1230,
851 cm−1
.
Positive HR-ESI-MS m/z 481.1686 ([M + Na]+
,
109

Y. Liu, et al.
Fitoterapia134(2019)108–112
Table 1
1H NMR Data of Compounds 1–6 (CD3OD).
No.
1
2
3
4
5
6
2
3
5
6
7
8.01 (d, 8.3)
7.37 (d, 8.3)
7.37 (d, 8.3)
8.01 (d, 8.3)
7.33 (d, 8.0)
7.20 (d, 8.0)
7.20 (d, 8.0)
7.33 (d, 8.0)
4.61 (d, 11.6)
4.85 (d, 11.6)
2.85–2.92 (m)
1.23 (d, 6.9)
7.11 (d, 8.0)
7.19 (d, 8.0)
7.19 (d, 8.0)
7.11 (d, 8.0)
2.29 (s)
7.08 (d, 8.0)
7.13 (d, 8.0)
7.13 (d, 8.0)
7.08 (d, 8.0)
2.28 (s)
7.08 (d, 8.0)
7.13 (d, 8.0)
7.13 (d, 8.0)
7.08 (d, 8.0)
2.28 (s)
6.60 (s)
6.89 (s)
2.14 (s)
8
9
3.41–3.43 (m)
1.15 (d, 6.8)
2.95–3.02 (m)
1.27 (d, 6.9)
3.77–3.79 (m)
3.02–3.05 (m)
3.00–3.05 (m)
3.65 (dd, 6.1, 9.5)
3.84 (dd, 8.2, 9.5)
1.27 (d, 7.0)
3.65 (dd, 6.1, 9.5)
3.84 (dd, 8.2, 9.5)
1.26 (d, 7.0)
10
1’
2’
3’
4’
5’
6’
1.15 (d, 6.8)
1.27 (d, 6.9)
1.23 (d, 6.9)
1.47 (d, 7.2)
4.72 (d, 7.5)
5.69 (d, 7.9)
4.32 (d, 7.7)
5.44 (d, 8.1)
4.24 (d, 7.8)
4.22 (d, 7.7)
3.41–3.42 (m)
3.39–3.41 (m)
3.42–3.43 (m)
3.50–3.52 (m)
3.75 (dd, 5.7, 11.4)
4.07(dd, 2.1, 11.4)
4.24 (d, 6.7)
3.49 (dd, 7.9, 8.7)
3.46–3.49 (m)
3.37–3.41 (m)
3.62–3.65 (m)
3.60 (dd, 5.4, 12.4)
4.04(dd, 2.2,12.4)
4.91 (d, 1.0)
3.22 (dd, 7.7, 8.9)
3.32–3.34 (m)
3.26 (dd, 8.1, 8.8)
3.33–3.36 (m)
3.15 (dd, 7.8, 8.9)
3.30–3.33 (m)
3.15 (dd, 7.7, 8.8)
3.30–3.31(m)
3.30–3.31 (m)
3.30–3.32 (m)
3.23–3.27 (m)
3.25–3.28 (m)
3.36–3.39 (m)
3.61 (dd, 6.3, 11.3)
4.01 (dd, 1.9, 11.3)
5.02 (d, 2.4)
3.40–3.44 (m)
3.52–3.55 (m)
3.38–3.43 (m)
3.62 (dd, 5.6, 11.2)
4.04 (dd, 2.2, 11.2)
4.99 (d, 1.2)
3.60 (dd, 5.4, 11.0)
3.99 (dd, 2.2, 11.0)
4.91 (d, 1.2)
3.61 (dd, 6.3, 11.3)
4.01 (dd, 2.3, 11.3)
4.97 (d, 1.2)
1”
2”
3”
4”
3.58 (dd, 6.7, 8.5)
3.46 (dd, 3.4, 8.5)
3.72–3.75 (m)
3.98 (dd, 1.0, 3.4)
3.83 (dd, 3.4, 6.0)
3.93–3.95 (m)
3.98 (dd, 1.2, 3.4)
3.83 (dd, 3.4, 6.0)
3.97–3.99 (m)
3.96 (dd, 1.2, 3.2)
3.81 (dd, 3.2, 6.0)
3.93–3.96 (m)
3.99 (dd, 1.2, 3.3)
3.82 (dd, 3.3, 6.0)
3.94–3.97 (m)
3.90 (d, 2.4)
3.97 (d, 9.6)
3.75 (d, 9.6)
3.56 (2H, s)
5”
3.41(dd, 6.3, 12.3)
3.80(dd, 3.4, 12.3)
3.60 (dd, 5.2, 11.9)
3.72 (dd, 3.2, 11.9)
3.65 (dd, 5.4, 11.8)
3.75 (dd, 3.4, 11.8)
3.63 (dd, 5.2, 11.8)
3.73 (dd, 3.2, 11.8)
3.62 (dd, 6.3, 11.8)
3.73 (dd, 3.3, 11.8)
2.6. MTT assay
Table 2
13C NMR Data of Compounds 1–6 (CD3OD).
The article evaluated the influence of compounds 1–9 in co-culture
model of microglia and neurons cells, as well as co-culture model of
astrocytoma and neurons cells. Briefly, the cells were identified as pur-
ified neuronal and cells by immunofluorescence with Hoechst 33258,
respectively. Then, the primary neuronal cells were seeded and attached
in 96-well plates at a density of 1 × 105 cells/well for 24 h. The transwell
chamber (0.4 μm × 6.5 mm) was put in and added the microglia/astro-
cytoma cells (1 × 105). After incubation with compounds 1–9 (5, 20 and
50 μM) and A1–42 (20 μM) for 24 h, the cells were added with 10 mL MTT
(5 mg/mL) and incubated for 4 h at 37C. The absorbance was measured
at 490 nm with a multi-detection microplate reader [13].
NO.
1
2
3
4
5
6
1
123.4
152.0
113.1
138.4
149.0
120.3
16.2
128.3
131.2
127.7
156.6
127.7
131.2
166.7
35.6
136.2
129.6
127.3
149.7
127.3
129.6
71.7
137.9
130.2
128.6
138.5
128.6
130.2
21.1
136.9
130.0
128.3
142.5
128.3
130.0
21.1
136.9
130.0
128.4
142.5
128.4
130.0
21.1
2
3
4
5
6
7
8
27.0
35.2
46.2
40.8
40.8
9
23.6
24.1
24.5
175.2
19.2
76.5
76.5
10
1’
2’
3’
4’
5’
6’
1”
2”
3”
4”
5”
23.7
24.1
24.5
19.0
19.1
104.2
75.2
96.1
103.1
75.1
95.9
104.4
75.1
104.4
75.1
74.0
73.9
78.1
78.0
78.0
78.0
78.0
78.0
3. Results and discussion
71.6
71.6
72.0
71.5
72.0
71.7
77.1
77.7
76.8
77.6
76.7
76.5
69.4
67.9
68.1
67.8
68.1
68.7
Compound 1 was obtained as white amorphous powder. The mo-
lecular formula was determined as C21H32O11 from the [M + Na]+
signal at m/z 483.1843 (Calcd. 483.1840) by HR-ESI-MS. In acid hy-
drolysis assay, 1 yielded D-glucose and L-arabinose. The 1H NMR
spectrum (Table 1) of 1 revealed two anomeric protons [δH 4.24 (1H, d,
J = 6.7 Hz, H-1″) and 4.72 (1H, d, J = 7.5 Hz, H-1′)], two aromatic
protons [δH 6.60 (1H, s, H-3) and 6.89 (1H, s, H-6)], and three methyl
groups [δH 2.14 (3H, s, H-7), and 1.15 (6H, d, J = 6.8 Hz, H-9, 10)].
The 13C NMR spectrum (Table 2) of 1 showed 21 carbon signals, cor-
responding to a 1,2,4,5-substituted aromatic [δC 123.4 (C-1), 152.0 (C-
2), 113.1 (C-3), 138.4 (C-4), 149.0 (C-5) and 120.3 (C-6)], three methyl
groups [δC 16.2 (C-7), 23.6 (C-9) and 23.7 (C-10)], a β-D-glucopyranose
moiety [δC 104.2 (C-1′), 75.2 (C-2′), 78.1 (C-3′), 71.6 (C-4′), 77.1 (C-5′)
and 69.4 (C-6′)], an α-L-arabinopyranose moiety [δC 104.7 (C-1″), 72.4
(C-2″), 74.1 (C-3″), 69.4 (C-4″) and 66.5 (C-5″)] [14]. In the HMBC
spectrum (Fig. 2), the key correlations Me-7/C-1, C-2 and C-6, H-8/C-3,
C-4, C-5, C-9 and C-10, H-9, 10/C-4 and C-8, together with 1He1H
COSY correlations (Fig. 2) H-9/H-8, and H-10/H-8 confirmed that the
aglycone of 1 was thymoquinol [14]. In addition, the HMBC correla-
tions H-1”/C-6′, and H-1’/C-5 were observed, which confirmed that the
α-L-arabinopyranose was linked at C-6′ of the β-D-glucopyranose, and
the β-D-glucopyranose was attached to C-5. Thus, 1 was established as
thymoquinol 5-O-α-L-arabinopyranosyl-(1 → 6)-β-D-glucopyranoside
(Fig. 1).
104.7
72.4
110.0
83.3
110.0
83.3
109.9
83.2
110.0
83.2
111.0
78.0
74.1
78.8
78.9
78.8
78.9
80.6
69.4
85.7
85.9
85.8
85.9
75.0
66.5
63.0
63.1
63.0
63.1
65.7
1 h. The acetylated thiazolidine derivatives were analyzed by GC using a
DM-5 column (30 m × 0.25 mm, 0.25 μm). Temperatures of both injector
and detector were 280 °C. A temperatures gradient system was used for the
oven, starting at 160 °C and increasing up to 195 °C at a rate of 5 °C·min−1
.
As a result, the sugar derivatives from 1 were determined to be D-glucose
(tR = 16.5 min) and L-arabinose (tR = 10.6 min). The sugar derivatives
from 2 to 5 were determined to be D-glucose (tR = 16.5 min) and D-ara-
binose (tR = 9.8 min). The sugar derivatives from 6 were determined to be
D-glucose (tR = 16.5 min) and D-apiose (tR = 10.1 min).
2.5. Enzymatic hydrolysis of 7
A mixture of 7 (8 mg) and naringinase (sigma chemical co., 3 mg) in
0.1 mol·L−1 acetate buffer (pH 4.0, 1 mL) was shaken in a water bath at
40 °C for 12 h. The reaction mixture was passed through a silica gel
[CHCl3 − MeOH (9: 1)] to afford an aglycone 7a [2.2 mg], showing a
20
plus optical rotation ([α]D +10.2, c 0.08, CHCl3).
110

Y. Liu, et al.
Fitoterapia134(2019)108–112
Fig. 2. Key HMBC and 1He1H COSY correlations of compounds 1–6.
Compound 2 was obtained as white amorphous powder, and
D-glucose and D-arabinose. The 1H NMR spectrum of 4 (Table 1) re-
vealed the presence of two anomeric protons [δH 5.44 (1H, d,
J = 8.1 Hz, H-1′) and 4.91 (1H, d, J = 1.2 Hz, H-1″)], an AA'BB’-cou-
pling system [δH 7.11 (2H, d, J = 8.0 Hz, H-2, 6) and 7.19 (2H, d,
J = 8.0 Hz, H-3, 5)], two methyl groups [δH 1.47 (3H, d, J = 7.2 Hz, H-
10), 2.29 (3H, s, H-7)]. The 13C NMR spectrum of 4 (Table 2) showed
21 carbon signals, corresponding to a 1,4-substituted aromatic ring [δC
137.9 (C-1), 130.2 (C-2, 6), 128.6 (C-3, 5), and 138.5 (C-4)], a C]O
group [δC 175.2 (C-9)], two methyl groups [δC 19.2 (C-10) and 21.1 (C-
7)], a β-D-glucopyranose moiety [δC 95.9 (C-1′), 73.9 (C-2′), 78.0 (C-3′),
71.5 (C-4′), 77.6 (C-5′) and 67.8 (C-6′)], and a terminal α-D-arabino-
furanose moiety [δC (109.9 (C-1″), 83.2 (C-2″), 78.8 (C-3″), 85.8 (C-4″)
and 63.0 (C-5″)) [16]. The key HMBC correlations of H-7/C-1 and C-2,
6, H-8/C-3, 5 and C-4, and Me-10/C-4 and C-9, together with 1He1H
COSY correlations of Me-10/H-8, showed that the aglycone of 4 was p-
methylhydratropic acid [2-(p-tolyl)propanoic acid] [18]. The HMBC
correlations (Fig. 2) of H-1”/C-6′ and H-1’/C-9 indicated that the α-D-
arabinofuranose were attached to C-6′ of the β-D-glucopyranose and the
β-D-glucopyranose moiety was attached to C-9. Thus, 4 was established
as p-Methylhydratropic acid 9-O-α-D-arabinofuranosyl-(1 → 6)-β-D-
glucopyranosyl ester (Fig. 1).
showed molecular ions at m/z 481.1686 in the HR-ESI- MS ([M + Na]+
Calcd. 481.1681) corresponding to the molecular formula established,
C
21H30O11. The acid hydrolysis of 2 gave D-glucose and D-arabinose.
The 1H NMR spectrum of 2 (Table 1) revealed the presence of two
anomeric protons [δH 5.69 (1H, d, J = 7.9 Hz, H-1′) and 4.91 (1H, d,
J = 1.0 Hz, H-1″)], an AA'BB’-coupling system [δH 8.01 (2H, d,
J = 8.3 Hz, H-2, 6) and 7.37 (2H, d, J = 8.3 Hz, H-3, 5)], two methyl
groups [δH 1.27 (6H, d, J = 6.9 Hz, H-9, 10)] [15]. The 13C NMR
spectrum of 2 (Table 2) showed 21 carbon signals, corresponding to a
1,4-substituted aromatic ring [δC 128.3 (C-1), 131.2 (C-2, 6), 127.7 (C-
3, 5), and 156.6 (C-4)], a C]O group [δC 166.7 (C-7)], two methyl
groups [δC 24.1 (C-9, 10)], a β-D-glucopyranose moiety [δC 96.1 (C-1′),
74.0 (C-2′), 78.0 (C-3′), 71.6 (C-4′), 77.7 (C-5′) and 67.9 (C-6′)], a
terminal α-D-arabinofuranose moiety [δC 110.0 (C-1″), 83.3 (C-2″),
78.8 (C-3″), 85.7 (C-4″) and 63.0 (C-5″)] [16]. The HMBC (Fig. 2)
correlations H-2, 6/C-7 indicated that the C]O group was located at C-
7. The HMBC correlations H-1”/C-6′, and H-1’/C-7 were observed and
indicated that the α-D-arabinofuranose was attached to C-6′ of the β-D-
glucopyranose, and the β-D-glucopyranose was attached to C-7. Thus, 2
was established as cuminic acid 7-O-α-D-arabinofuranosy-(1 → 6)-β-D-
glucopyranosyl ester (Fig. 1).
Compounds 5 and 6 were obtained as white amorphous powder.
The [M + Na]+ ion peaks at m/z 467.1893 and 467.1890 (both Calcd.
467.1895) in their HR-ESI-MS, suggested a molecular formula of
Compound 3 was obtained as white amorphous powder. The mo-
lecular formula was assigned as C21H32O10 by HR-ESI-MS at m/z
467.1893 [M + Na]+ (Calcd. 467.1891). On acid hydrolysis, 3 yielded
D-glucose and D-arabinose. The 1H NMR spectrum of 3 (Table 1) re-
vealed the presence of two anomeric protons [δH 4.32 (1H, d,
J = 7.7 Hz, H-1′) and 4.99 (1H, d, J = 1.2 Hz, H-1″)], an AA'BB’-cou-
pling system [δH 7.33 (2H, d, J = 8.0 Hz, H-2, 6) and 7.20 (2H, d,
J = 8.0 Hz, H-3, 5)], two methyl groups [δH 1.23 (6H, d, J = 6.9 Hz, H-
9, 10)]. The 13C NMR spectrum of 3 (Table 2) showed 21 carbon sig-
nals, corresponding to a 1,4-substituted aromatic ring [δC 136.2 (C-1),
129.6 (C-2, 6), 127.3 (C-3, 5), 149.7 (C-4)], two methyl groups [δC 24.5
(C-9, 10)], a β-D-glucopyranose moiety [δC 103.1 (C-1′), 75.1 (C-2′),
78.0 (C-3′), 72.0 (C-4′), 76.8 (C-5′) and 68.1 (C-6′)], and a terminal α-D-
arabinofuranose moiety [δC 110.0 (C-1″), 83.3 (C-2″), 78.9 (C-3″), 85.9
(C-4″) and 63.1 (C-5″)] [16]. The NMR data were similar to those of p-
cymene 7-O-β-D-glucopyranoside [17], except for the presence of an
additional α-D-arabinofuranose moiety in 3. In the HMBC (Fig. 2)
spectrum, the key cross-peaks H-1”/C-6′, and H-1’/C-7 were observed,
which confirmed that the α-D-arabinofuranose was linked at C-6′ of the
β-D-glucopyranose, and the β-D-glucopyranose was attached to C-7.
Thus, 3 was established as p-cymene 7-O-α-D-arabinofuranosyl-(1 → 6)-
β-D-glucopyranoside (Fig. 1).
C
21H32O10 for both compounds. Acid hydrolysis of 5 gave D-glucose and
D-arabinose, and 6 gave D-glucose and D-apiose. Analysis of the 1H-, 13
C
NMR (Tables 1 and 2), 1He1H COSY (Fig. 2), and HMBC (Fig. 2) NMR
spectra showed that aglycones of 5 and 6 were p-cymene-9-ol. Com-
parison of the NMR data of 5 and 6 with that of the known compound p-
cymene 9-O β-D-glucopyranoside [19] displayed that most of them
were similar, expect for the α-D-arabinofuranose moiety in 5, and the α-
D-apiofuranose moiety in 6. The key HMBC cross-peaks of H-1”/C-6′,
and H-1’/C-9 were observed in 5 and 6. The stereochemistry at C-8 of 5
and 6 were determined by compare study with literature [19]. The
literature determined that the chemical shift and coupling constant of
H-9 in (R)-p-cymene 9-O-β-D-glucopyranoside was δH 3.64 (1H, dd,
J = 6.0, 9.8 Hz) and 3.90 (1H, dd, J = 8.2, 9.8 Hz), while that in (S)-p-
cymene 9-O-β-D-glucopyranoside was δH 3.52 (1H, dd, J = 8.6, 9.8 Hz)
and 4.02 (1H, dd, J = 6.0, 9.8 Hz). The chemical shift and coupling
constant of H-9 were at δH 3.65 (1H, dd, J = 6.1, 9.5 Hz) and 3.84 (1H,
dd, J = 8.2, 9.5 Hz) in 5 and 6, 3.64 (1H, dd, J = 6.0, 9.8 Hz) and 3.90
(1H, dd, J = 8.2, 9.8 Hz) in 7. According to chemical shift and coupling
constant of H-9 in 5–7, the stereochemistry of C-8 in 5–7 were deduced
as (R)-configuration. Furthermore, a enzymatic hydrolysis of 7 by
20
Compound 4 was obtained as white amorphous powder. The mo-
lecular formula was assigned as C21H30O11 by HR-ESI-MS at m/z
481.1686 [M + Na]+ (Calcd. 481.1683). On acid hydrolysis, 4 yielded
naringinase gave the aglycon 7a (2 mg, [α]D +10.2, c 0.08, CHCl3),
20
which showed a plus [α]D value. Since (R)-p-cymen-9-ol showed a
plus optical rotation ([α]D20 +15.7), (S)-p-cymen- 9-ol showed a minus
111

Y. Liu, et al.
Fitoterapia134(2019)108–112
Table 3
Neuroprotective against Aβ1–42 induced two co-culture cells models of compounds 1–9.
Compounds
Co-culture microglia & neurons
Co-culture astrocytoma & neuronsa
Compounds
Co-culture microglia & neurons
IC50 (μM)
Co-culture astrocytoma & neurons
IC50 (μM)
IC50 (μM)
IC50 (μM)
> 50
Donepezilb
5.7
1.4
8.1
2.3
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
38.3
> 50
40.7
38.6
> 50
3.6
Compound 1
Compound 2
Compound 3
Compound 4
38.1
40.2
> 50
28.5
2.9
> 50
34.9
> 50
33.8
37.9
> 50
> 50
27.9
6.5
5.0
4.7
5.6
5.1
4.3
1.9
2.8
a
b
IC50 was defined as the concentration that resulted in a 50% decrease in cell number. The IC50 greater than 50 μM was deemed inactive.
Positive control.
optical rotation ([α]D20 −14.8), the configuration at C-8 of 7 should be
References
R. Compounds 5–7 have same aglycone by their NMR data analysis.
And the configurations at C-8 of 5 and 6 should be R. Thus, the struc-
ture of 5 and 6 were established as (R)-p-cymene 9-O-α-D-arabinofur-
anosyl-(1 → 6)-β- D-glucopyranoside and (R)-p-cymene 9-O-β-D-apio-
furanosyl-(1 → 6)-β-D-glucopyranoside (Fig. 1).
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This work was financially supported by National Natural Science
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Creative Talents in Heilongjiang Province (UNPYSCT-2017221),
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Appendix A. Supplementary data
The following are available online: Figure S1-S34, Table S1: The
13C-NMR data of Compounds 7-9. Supplementary data to this article
can be found online at https://doi.org/10.1016/j.fitote.2019.02.012.
112