Hyperelatosides A-E, biphenyl ether glycosides from Hypericum elatoides, with neurotrophic activity

Article abstract of DOI:10.1039/c8ra05322g

Five new biphenyl ether glycosides, hyperelatosides A-E (1-5), one new benzoate glycoside, hyperelatoside F (6), along with nine known phenolic compounds (7-15), were isolated from the aerial parts of Hypericum elatoides. Their structures were elucidated by 1D and 2D NMR spectroscopy and HRESIMS, as well as chemical derivatization. This is the first report of the identification of biphenyl ether glycosides as plant metabolites and their possible biosynthetic pathway is proposed. Except for 3, the new phenolic metabolites exhibited significant neurotrophic activities to enhance nerve growth factor-induced neurite outgrowth in PC12 cells. In addition, the anti-neuroinflammatory and antioxidant activities of compounds 1-15 were preliminarily evaluated in vitro.

Full text of DOI:10.1039/c8ra05322g

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Hyperelatosides A–E, biphenyl ether glycosides
from Hypericum elatoides, with neurotrophic
activity†
Cite this: RSC Adv., 2018, 8, 26646
a
a
a
a
a
Xi-Tao Yan,
Zhen An, Dan Tang, Guang-Rui Peng, Chen-Yu Cao,
Yuan-Zhen Xu, Chun-Huan Li, Pei-Liang Liu, Zai-Min Jiang and Jin-Ming Gao *a
a
a
b
c
Five new biphenyl ether glycosides, hyperelatosides A–E (1–5), one new benzoate glycoside, hyperelatoside
F (6), along with nine known phenolic compounds (7–15), were isolated from the aerial parts of Hypericum
elatoides. Their structures were elucidated by 1D and 2D NMR spectroscopy and HRESIMS, as well as
chemical derivatization. This is the first report of the identification of biphenyl ether glycosides as plant
metabolites and their possible biosynthetic pathway is proposed. Except for 3, the new phenolic
metabolites exhibited significant neurotrophic activities to enhance nerve growth factor-induced neurite
outgrowth in PC12 cells. In addition, the anti-neuroinflammatory and antioxidant activities of compounds
1–15 were preliminarily evaluated in vitro.
Received 21st June 2018
Accepted 5th July 2018
DOI: 10.1039/c8ra05322g
rsc.li/rsc-advances
our ongoing search for natural neurotrophic compounds with
novel structures for potential neurodegenerative disease thera-
Plants belonging to the genus Hypericum (Hypericaceae) are well pies, 15 phenolic compounds including ve new uncommon
Introduction
15
1
known for their therapeutic uses in neurological disorders. In biphenyl ether glycosides named hyperelatosides A–E (1–5), one
particular, the extract of H. perforatum (St. John's wort), the new benzoate glycoside named hyperelatoside F (6), and nine
most known of the Hypericum species, has long been used for known, structurally related phenolic derivatives (7–15) were
2
the treatment of mild to moderate depression. Due to the isolated from the MeOH extract of the aerial parts of H. elatoides
continuous discovery of structurally novel natural products and (Fig. 1). Herein, we described the isolation and structural
their signicant biological activities, plants of the genus elucidation of these new phenolic metabolites, as well as the
Hypericum have attracted much attention from researchers in biological evaluation of their promoting effects on nerve growth
both chemistry and pharmacology. Previous chemical investi- factor (NGF)-induced neurite outgrowth in PC12 cells, inhibi-
gations on this genus have resulted in the isolation of phlor- tory effects on lipopolysaccharide (LPS)-induced nitric oxide
oglucinol
derivatives,
xanthones,
benzophenones, (NO) production in BV-2 cells, and 1,1-diphenyl-2-picrylhydrazyl
naphthodianthrones, avonoids, and coumarins as major (DPPH) free radical-scavenging capacities.
3
–6
constituents. These secondary metabolites possess extensive
biological activities, such as antidepressant, neuroprotective,
anti-neurodegenerative, antioxidant, memory-enhancing, anti-
Results and discussion
inammatory, anticancer, anti-HIV, and antimicrobial The MeOH extract of H. elatoides was subjected to liquid–liquid
7–13
activities.
fractionation to afford a hexane-soluble fraction, an EtOAc-
Hypericum elatoides R. Keller is an herbaceous plant soluble fraction, and a BuOH-soluble fraction. The active
distributed in northwest China, especially in the western region EtOAc fraction was separated by semipreparative reversed phase
14
of Shaanxi province. To date, there have been no phyto- (RP) HPLC and column chromatography (CC) using silica gel,
chemical and biological studies on this plant. In the course of RP-C , and Sephadex LH-20 to yield six new compounds (1–6)
1
8
and nine known phenolic metabolites (7–15).
Compound 1 was isolated as a brown amorphous solid. Its
a
Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of molecular formula was determined to be C22
H
O
13 by HRE-
26NaO13, 521.1271).
The IR spectrum showed absorption bands at 3418 and
26
Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, China. E-mail:
+
SIMS (m/z 521.1247 [M + Na] , calcd for C22
H
jinminggao@nwsuaf.edu.cn
b
Key Laboratory of Resource Biology and Biotechnology in Western China, College of
ꢀ
1
1
701 cm for hydroxyl and ester carbonyl groups, respectively.
Life Sciences, Northwest University, Xi'an 710069, China
1
c
The low-eld region of the H NMR spectrum (measured in
CD OD) exhibited two para-positional aromatic protons [d 7.44
College of Life Sciences, Northwest A&F University, Yangling 712100, China
†
Electronic supplementary information (ESI) available: 1D NMR, 2D NMR, and
HRESIMS of 1–6; chemical structures of 7–15. See DOI: 10.1039/c8ra05322g
3
H
(1H, s, H-3) and 6.38 (1H, s, H-6)] and two meta-coupled
26646 | RSC Adv., 2018, 8, 26646–26655
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Fig. 1 Chemical structures of compounds 1–6.
0
aromatic protons [d 6.67 (1H, d, J ¼ 2.9 Hz, H-4 ) and 6.10 (1H, aromatic carbons (dC 100.5–154.3), including eight quaternary
H
0
d, J ¼ 2.9 Hz, H-6 )] (Table 1), indicating the presence of carbons and four methine carbons, corroborating the presence
1
a 1,2,4,5-tetrasubstituted (A-ring) and a 1,2,3,5-tetrasubstituted of two tetrasubstituted aromatic rings detected in the H NMR
1
(
B-ring) benzene rings, respectively. Further, the aliphatic spectrum. Furthermore, the H NMR spectrum of 1, measured
1
region of the H NMR spectrum showed signals of three in DMSO-d
methoxy groups [d 3.87 (3H, s), 3.78 (3H, s), and 3.65 (3H, s)] groups at d
6
, displayed the downeld signals of two hydroxyl
H
H
10.03 and 8.03 (Table 1), which were attached to C-
1
3
0
and a set of sugar protons (dH 3.41–4.84). The C NMR spec- 5 position of A-ring and C-2 position of B-ring based on the
trum (Table 2) supported by DEPT experiments displayed HMBC correlations from the hydroxyl proton at dH 10.03 to C-4,
0
signals of three methoxy carbons (dC 57.1, 56.3, and 52.5), six C-5, and C-6 and from the hydroxyl proton at d 8.03 to C-1 , C-
H
0
0
characteristic glucopyranosyl carbons (d
C
104.2, 78.6, 77.7, 75.0, 2 , and C-3 . In the HMBC spectrum, the correlations from the
167.9), and 12 methoxy protons at d 3.78 to the carbonyl carbon at d 167.9 (C-
71.5, and 62.6), an ester carbonyl carbon (d
C
H
C
1
Table 1 H NMR spectroscopic data (500 MHz, d in ppm, J in Hz) for compounds 1–5
a
b
a
a
a
b
a
No.
1
1
2
3
4
4
5
3
4
6
7.44, s
7.32, s
7.35, s
7.34, s
6.85, d (8.8)
6.84, d (8.8)
6.58, dd (8.8, 3.0)
6.45, d (3.0)
6.19, d (2.9)
5.74, d (2.9)
4.60, d (7.5)
3.25, m
3.27, m
3.17, m
3.34, m
3.45, dd
6.84, d (8.8)
6.56, dd (8.8, 2.9)
6.39, d (2.9)
6.67, d (2.9)
6.17, d (2.9)
4.80, d (7.6)
3.52, m
3.49, m
3.41, m
3.44, m
3.71, dd
6.59, dd (8.8, 2.9)
6.46, d (2.9)
6.24, d (2.9)
5.91, d (2.9)
4.73, d (7.6)
3.46, m
3.41, m
3.42, m
3.25, m
3.69, dd
6.38, s
6.26, s
6.40, s
6.72, s
0
4
6
6.67, d (2.9)
6.10, d (2.9)
4.84, d (7.4)
3.52, m
3.50, m
3.41, m
6.64, d (2.9)
6.14, d (2.9)
4.72, d (7.4)
3.31, m
3.30, m
3.16, m
6.68, d (2.8)
6.33, d (2.8)
4.83, d (7.4)
3.49, m
3.48, m
3.39, m
6.72, d (2.9)
6.54, d (2.9)
4.94, d (7.7)
3.52, m
3.47, m
3.38, m
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
3.45, m
3.71, dd (12.1, 5.8)
3.37, m
3.47, dd
3.44, m
3.45, m
3.70, dd
(12.0, 5.9)
3.91, dd
3.67, dd
(12.1, 5.9)
3.86, dd
(12.1, 2.2)
3.87, s
(
11.8, 5.9)
(12.0, 4.7)
3.77, dd
(12.0, 2.4)
(11.7, 5.8)
3.62, m
(12.1, 5.7)
3.91, dd
(12.1, 2.0)
3.92, dd (12.1, 2.1)
3.75, m
(
12.0, 2.2)
4
5
7
5
2
5
2
3
2
3
4
6
-OMe
-OMe
-OMe
3.87, s
3.76, s
3.85, s
3.68, s
3.65, s
3.62, s
3.66, s
3.66, s
3.78, s
3.65, s
3.73, s
3.62, s
0
-OMe
3.70, s
3.77, s
3.59, s
8.74, s
-OH
-OH
10.03, s
8.03, s
0
0
0
0
0
0
-OH
-OH
-OH
-OH
-OH
-OH
9.08, br s
6.05, br s
5.12, d (4.4)
4.99, d (4.6)
4.31, t (5.7)
0
0
0
0
5.44, d (2.8)
5.08, br s
5.05, d (5.3)
4.64, t (5.5)
a
b
3 6
Date were recorded in CD OD. Date were recorded in DMSO-d .
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 26646–26655 | 26647

View Article Online
RSC Advances
Paper
1
3
00
0
Table 2
C NMR spectroscopic data (125 MHz, d in ppm) for proton at d
4.84 (1H, d, J ¼ 7.4 Hz, H-1 ) to C-3 (d
C
148.4),
H
compounds 1–5
respectively. These two aromatic rings were bonded via an
a
b
a
a
a
b
a
oxygen atom based on analysis of the chemical shis of C-1 (dC
No.
1
1
2
3
4
4
5
0
1
53.9) and C-1 (d 147.0) together with the molecular formula.
C
1
2
3
4
5
6
7
153.9
112.8
115.3
145.0
153.7
108.0
167.9
147.0
133.7
148.4
100.5
154.3
101.1
104.2
75.0
152.5
110.0
114.4
142.8
151.9
105.2
165.0
143.9
132.5
147.0
99.1
151.8
100.2
102.2
73.3
75.8
70.0
152.9
117.6
115.0
144.9
151.8
107.2
172.8
146.8
134.7
148.6
101.2
154.0
102.7
103.8
75.0
158.0
111.2
115.6
147.1
155.8
108.4
166.4
154.0
130.0
150.8
101.8
159.2
101.0
103.1
74.9
146.0
143.3
118.4
110.6
155.0
107.4
143.9
142.2
117.2
109.5
152.4
106.7
146.4 Acid hydrolysis followed by HPLC analysis aer
143.1 arylthiocarbamoyl-thiazolidine derivatization conrmed the
118.2
characterization of a b-D-glucopyranosyl unit. Thus, the struc-
ture of 1 was elucidated as 2-(3-O-b-D-glucopyranosyl-2-hydroxy-
110.1
155.0
106.6
5-methoxyphenoxy)-4-hydroxy-5-methoxy-methylbenzoate and
was named hyperelatoside A.
0
1
152.1
131.4
153.1
98.2
158.8
97.3
107.4
75.5
77.9
150.9
129.7
151.4
96.3
156.2
95.4
105.8
73.8
76.2
146.8
133.7
148.5
100.4
154.4
Compound 2 was obtained as a brown amorphous solid. Its
molecular formula was deduced to be C H O by HRESIMS
0
2
2
1
24 13
0
3
ꢀ
ion peak at m/z 483.1150 [M ꢀ H] (calcd for C21
23 13
H O ,
0
4
1
13
0
483.1139). The H and C NMR data of 2 (Tables 1 and 2)
5
0
6
101.2 showed similarity to those of 1, except for the lack of one
0
0
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
4
5
7
5
104.3 methoxy signals as well as the chemical shi of C-7 (d
172.8 in
C
75.0
2
C
and d 167.9 in 1). These observations indicated a carboxyl
77.7
71.5
78.6
62.6
77.7
71.6
78.5
62.6
77.8
71.5
78.6
62.6
77.8
71.6
78.6
62.6
group in 2 instead of the methoxycarbonyl group in 1 at C-2
position, which was supported by the molecular formula. In
addition, the HMBC correlations of 2 (Fig. 2) conrmed the
same connectivity as in 1. Therefore, the structure of 2 was
71.0
78.5
62.2
69.6
77.1
60.8
77.3
60.8
56.1
-OMe
-OMe
-OMe
57.1
57.0
57.0
56.4
56.1
55.2
55.4
56.4
56.3
determined
as
2-(3-O-b-D-glucopyranosyl-2-hydroxy-5-
52.5
56.3
51.5
55.3
methoxyphenoxy)-4-hydroxy-5-methoxy benzoic acid and was
given the name hyperelatoside B.
Compound 3 was obtained as a brown amorphous solid. Its
molecular formula was established as C H O by HRESIMS
0
-OMe
56.4
56.5
a
b
Date were recorded in CD
3
OD. Date were recorded in DMSO-d
6
.
2
1
22 12
+
(
m/z 489.0998 [M + Na] , calcd for C H NaO , 489.1009),
21 22 12
7
1
), from H-3 (d
H
7.44) to C-1 (d
C
153.9), C-5 (d
C
153.7), and C-7 (d
112.8) and C-4 (d 145.0),
C
and from the methoxy protons at d 3.87 to C-4 (d 145.0) (Fig. 2)
H C
suggested the presence of a methoxycarbonyl group and
a methoxy group located at the C-2 and C-4 positions of A-ring,
respectively. In addition, the locations of a methoxy group at C-
and a sugar moiety at C-3 of B-ring were established
according to the HMBC correlations observed from the methoxy
C
accounting for 11 degrees of unsaturation. The UV, IR, and
NMR data of 3 were similar to those of 1 and 2 (Table 1), indi-
cating that the structure of 3 is a biphenyl ether glycoside. The
67.9), from H-6 (d
H
6.38) to C-2 (d
C
1
H NMR spectrum of 3 revealed the presence of a 1,2,4,5-tetra-
substituted benzene ring, a 1,2,3,5-tetrasubstituted benzene
ring, two methoxy groups, as well as a sugar moiety, which were
consistent with the constituent units of 2. Similar HMBC
correlations (Fig. 2) established that a sugar moiety, a carbonyl
group, and two methoxy groups were located at the same
0
0
5
0
protons at d
H
3.65 to C-5 (d
C
154.3) and from the anomeric
Fig. 2 Key HMBC (blue arrows) correlations of compounds 1–6.
26648 | RSC Adv., 2018, 8, 26646–26655
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
positions as 2. Compared with the chemical shis of C-7 of 1 compound 4. Moreover, the UV and IR spectra of 5 showed
and 2 (Table 2), the appearance of an ester carbonyl carbon at dC similar absorption bands to those of 4, indicating their similar
1
13
1
66.4 in 3 suggested that an ester group presented between structures. The H and C NMR data (Tables 1 and 2) of A-ring of
0
these two aromatic rings via the linkage of C-7–O–C-2 , consis- 5 were almost superimposable to those of 4, but they differed in
tent with the 11 degrees of unsaturation required by the their B-ring, suggesting that the locations of the substituent
molecular formula. Acid hydrolysis result conrmed the sugar groups on B-ring were different between 4 and 5. The glycosyla-
0
to be a b-D-glucopyranose. Hence, the structure of 3 was char- tion site at C-3 position of B-ring of 5 was inferred from an
0
0
0
0
acterized
as
2 ,5-dihydroxy-4,5 -dimethoxy-3 -O-b-D- upeld shi of d
C
4.6 ppm for C-3 (ipso-C) and downeld shis of
0
0
0
glucopyranosyl-2-carboxy-diphenyl ether 2,2 -lactone and was
named hyperelatoside C.
d
C
2.3 ppm for C-2 (ortho-C), 2.2 ppm for C-4 (ortho-C), and
0
3.9 ppm for C-6 (para-C) (Table 2). This was further supported by
Compound 4 was obtained as a brown amorphous solid with a HMBC cross-peak between the anomeric proton at d 4.80 (H-
H
0
0
0
a molecular formula of C H O based on HRESIMS (m/z 1 ) and the corresponding aglycone carbon at d 148.5 (C-3 )
4
2
0
24 11
C
+
1
63.1205 [M + Na] , calcd for C20
NMR data of 4 (measured in CD OD, Table 1) exhibited a set of same method as described for 1. Therefore, the structure of 5 was
characteristic ABX aromatic protons at d
H24NaO11, 463.1216). The H (Fig. 2). The b-D-glucopyranosyl moiety was determined by the
3
0
0
0
H
6.85 (1H, d, J ¼ dened as 2,2 -dihydroxy-5,5 -dimethoxy-3 -O-b-D-glucopyranosyl-
8
.8 Hz, H-3), 6.59 (1H, dd, J ¼ 8.8, 2.9 Hz, H-4), and 6.46 (1H, d, J diphenyl ether and was named hyperelatoside E.
¼
2.9 Hz, H-6) and two meta-coupled aromatic protons at d
H
Compound 6 was puried as white needles with a melting
0
0
ꢁ
6
.24 (1H, d, J ¼ 2.9 Hz, H-4 ) and 5.91 (1H, d, J ¼ 2.9 Hz, H-6 ), point of 220–222 C. The molecular formula, C H O , was
1
5
20 10
+
indicating the presence of a 1,2,4-trisubstituted (A-ring) and deduced from HRESIMS (m/z 383.0946 [M + Na] , calcd for
a 1,2,3,5-tetrasubstituted (B-ring) benzene rings, respectively. It C H NaO , 383.0954). The IR spectrum showed absorption
1
5
20
10
ꢀ
1
also showed aliphatic signals for two methoxy groups [d
H
3.68 bands corresponding to a hydroxyl group (3439 cm ) and
ꢀ
1
1
(
3H, s) and 3.65 (3H, s)] and a set of sugar protons (d
H
3.25– a conjugated carbonyl group (1666 cm ). The H NMR spec-
4.73 (1H, d, J ¼ trum exhibited signals attributable to one hydroxyl group [d
.6 Hz, H-1 ). The C NMR spectrum displayed signals for 12 10.46 (1H, s, 2-OH)], one 1,2,4,5-tetrasubstituted benzene ring
4
7
.73), including an anomeric proton at d
H
H
00
13
aromatic carbons (d
C
97.3–158.8), two methoxy carbons (d
C
56.4 [d
H
7.22 (1H, s, H-6) and 6.71 (1H, s, H-3)], one anomeric proton
0
and 56.1), and a characteristic glucopyranosyl moiety (d 107.4, at dH 5.04 (1H, d, J ¼ 7.2 Hz, H-1 ), and two methoxy groups [d
C
H
1
3
7
8.5, 77.9, 75.5, 71.0, and 62.2) (Table 2). Acid hydrolysis result 3.88 (3H, s, H-8) and 3.73 (3H, s, H-9)] (Table 3). The C NMR
of 4 conrmed the sugar to be a b-D-glucopyranose. The spectrum displayed 15 carbon resonances, which were classi-
assignment of signals corresponding to A-ring was based on the ed as an ester carbonyl carbon (d 169.3), six aromatic carbons
HMBC correlations from H-3 (d 6.85) to C-1 (d 146.0) and C-5 (d 103.5–156.6), a characteristic glucopyranosyl moiety (d
155.0), from H-4 (d 6.59) to C-2 (d 143.3) and C-6 (d 107.4), 99.4, 77.1, 76.7, 73.0, 69.5, and 60.6), and two methoxy carbons
and from H-6 (d 6.46) to C-2 (d 143.3) and C-4 (d 110.6) (d 56.1 and 52.3) (Table 3). The b-D-glucopyranosyl unit was
Fig. 2). The methoxy protons at d
C
H
C
C
C
(d
C
H
C
C
C
H
C
C
(
H
3.68 exhibited a HMBC veried by the same method as described for 1. In the HMBC
correlation to C-5 (d 155.0), which indicated the connection of spectrum of 6, the methoxy protons at dH 3.88 (H-8) exhibited
C
1
a methoxy group at C-5 position of A-ring. Furthermore, the H a correlation with the carbonyl carbon at d 169.3 (C-7), which
C
NMR spectrum of 4, measured in DMSO-d6, displayed suggested that 6 is a derivative of methyl benzoate. The key
a hydroxyl proton signal at d
HMBC correlations to C-1, C-2, and C-3, supporting that 156.6), C-4 (d
a hydroxyl group was attached to C-2 position of A-ring. hydroxyl proton at d
H
8.74 (1H, s, 2-OH), which showed HMBC correlations from H-6 (d
153.3), C-5 (d 142.1), and C-7 (d
10.46 to C-1 (d 104.1), C-2 (d
103.5), and from the methoxy protons at d
142.1) (Fig. 2), indicated the locations of a hydroxyl
H
7.22) to C-1 (d
C
104.1), C-2 (d
169.3), from the
156.6), and
3.73 (H-9) to
C
C
C
C
H
C
C
1
13
Complete assignment of the H and C NMR data of B-ring C-3 (d
C
H
0
was achieved by the HMBC correlations from H-4 (d
H
0
6.24) to C-5 (d
C
0
0
0
C-2 (d 131.4), C-3 (d 153.1), C-5 (d 158.8), and C-6 (d 97.3) group and a methoxy group at C-2 and C-5, respectively. In
C
C
C
C
0
0
0
0
0
and from H-6 (d 5.91) to C-1 (d 152.1), C-2 (d 131.4), C-4 (d
C
addition, the anomeric proton at dH 5.04 (H-1 ) exhibited
H
C
C
0
9
8.2), and C-5 (d 158.8) (Fig. 2). The HMBC correlations from a HMBC correlation with C-4 (dC 153.3), indicating that the
the methoxy protons at d
4.73 to C-2 suggested the locations of a methoxy compound 6 was assigned as 4-O-b-D-glucopyranosyl-2-hydroxy-
group at C-5 and a b-D-glucopyranosyl moiety at C-2 , respec- 5-methoxy-methylbenzoate and was named hyperelatoside F.
C
0
H
3.65 to C-5 and from the anomeric glucopyranosyl moiety was attached to C-4 position. Thus,
0
proton at d
H
0
0
0
tively. An ether linkage between C-1 (d
C
146.0) of A-ring and C-1
The remaining compounds were elucidated on the basis of
(d
C
152.1) of B-ring was then established due to their downeld NMR and ESIMS data analysis as well as by comparison with
chemical shis and the molecular formula. Accordingly, literature data. They were identied as 3,5-dihydroxy-4-
0
0
0
16
compound 4 was identied as 2,3 -dihydroxy-5,5 -dimethoxy-2 - methoxyxanthone (7),
1,5,6-trihydroxy-7-methoxyxanthone
17
O-b-D-glucopyranosyl-diphenyl ether and was named hyper- (8), 1,3,7-trihydroxy-2-(2-hydroxy-3-methyl-3-butenyl)-xanthone
18
elatoside D.
Compound 5 was obtained as a brown amorphous solid. Its none (10), garcimangosone
molecular formula was determined to be C20 11 based on phloroglucinol-glucopyranoside (12), quercetin 3-O-a-L-rham-
a deprotonated ion peak at m/z 439.1247 [M ꢀ H] (calcd for nopyranoside (13), quercetin 3-O-b-D-galactopyranoside (14),
(9), 2,4-dihydroxy-3-methyl-6-O-b-D-glucopyranosyl benzophe-
19
20
D
(11), 1-(2-methylbutyryl)-
21
24
H O
ꢀ
22
23
24
20 23
C H O11, 439.1240) in HRESIMS, identical to that found for and kaempferol 3-O-a-L-rhamnopyranoside (15) (Fig. S54, ESI†).
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 26646–26655 | 26649

View Article Online
RSC Advances
Paper
1
13
Table 3 H NMR (500 MHz) and C NMR (125 MHz) spectroscopic Xanthones and benzophenones are two major constituents of
data for compound 6 (DMSO-d
6
)
1e
the plants of the genus Hypericum. The biosynthesis of
xanthone products has been studied in H. androsaemum, H.
No.
d
H
(J in Hz)
d
C
29
0
calycinum,
and
H.
perforatum.
2,3 ,4,6-Tetrahydrox-
1
2
3
4
5
6
7
8
9
104.1 ybenzophenone, which is formed from benzoic acid by the
156.6 condensation of one benzoyl CoA and three malonyl CoA units
6.71, s
7.22, s
103.5
catalyzed by benzophenone synthase, is cyclized regioselectively
to yield either 1,3,7- or 1,3,5-trihydroxyxanthone, which is then
153.3
142.1
regioselectively hydroxylated to 1,3,6,7- or 1,3,5,8-tetrahydrox-
111.5
169.3 yxanthone. Subsequent xanthone ring cleavage reaction gives
30
3.88, s
3.73, s
52.3
56.1
the biphenyl ether skeleton, which may generate the structures
of 1–5 via enzymatic reactions of decarboxylation, O-methyla-
tion using S-adenosylmethionine (SAM), and glycosylation.
The neurotrophic activities of new compounds 1–6 were
investigated in terms of their ability to enhance NGF-induced
neurite outgrowth using rat pheochromocytoma (PC12) cells
as a model system of neuronal differentiation. Fig. 3 showed
that, relative to the NGF control (100%), compounds 1, 2, and 4–
6 exerted a signicant increase in neurite-bearing cells at 1 mM.
In particular, the percentages of neurite-bearing cells for cells
treated with hyperelatosides B (2) and D (4) in the presence of
4
4
8
9
1
2
3
4
5
6
a
b
a
a
0
5.04, d (7.2)
3.26, m
3.28, m
3.16, m
3.39, m
99.4
73.0
76.7
69.5
77.1
60.6
0
0
0
0
0
3.45, dd (11.7, 5.2)
3.66, dd (11.7, 6.0)
1
2
7
2
3
4
6
-OH
-OH
-OH
ꢀ1
NGF (20 ng mL ) reached up to 192.78 ꢂ 17.71% and 228.70 ꢂ
10.46, s
12.51% relative to the NGF control, respectively. Interestingly,
0
among the biphenyl ether glycosides (1–5), only 3 had no effect
on NGF-medicated neurite outgrowth in PC12 cells at 1 mM,
suggesting that the presence of a carboxyl or hydroxyl group at
C-2 position of A-ring may be responsible for the neurotrophic
activity. Compound 2 showed much stronger activity than 1,
indicating that attachment of an active hydrogen atom to the O-
-OH
-OH
-OH
-OH
5.34, d (5.1)
5.13, d (4.6)
5.06, d (5.3)
4.58, t (5.6)
0
0
0
Among them, compounds 7, 10, and 12 were isolated from the
genus Hypericum for the rst time.
7
position of the carboxyl group could signicantly improve this
activity than attachment of a methyl group to O-7 position.
Neuroinammation has been typically involved in the
pathology of neurodegenerative diseases such as Alzheimer's
disease and Parkinson's disease. Therefore, the inhibitory
effects of compounds 1–15 on LPS-stimulated NO production in
BV-2 microglial cells were investigated to establish their anti-
neuroinammatory activities. As shown in Fig. 4, compounds
Naturally occurring biphenyl ethers have been reported to be
2
5
in some fungi in recent years, especially Aspergillus species,
26
but are rarely found in plants. In fungi, these kinds of
compounds are formed biosynthetically from the anthraqui-
none emodin, via sulochrin, and the grisandienes such as
27
geodin was previously reported. However, these intermediates
of desmethylsulochrin, sulochrin, dihydrogeodin, and geodin
are rarely as the plant metabolites. Given the isolation of ben-
zoic acid derivatives (6 and 12), benzophenones (10 and 11), and
xanthones (7–9) from H. elatoides, the possible biosynthetic
pathway of hyperelatosides A–E (1–5) is proposed in Scheme 1.
The key step of this pathway is the xanthone ring cleavage
reaction to form biphenyl ether skeleton, the immediate
precursors of which may be 1,3,6,7- and 1,3,5,8-tetrahydroxyx-
anthones. Although the formation of biphenyl ethers from
xanthones catalyzed by an enzyme system involving an oxy-
genase, has not yet detected in plants, the similar reaction has
been observed in the biosynthetic studies of biphenyl ethers
from anthraquinones in fungi. An anthraquinone ring cleavage
enzyme, named questin oxygenase, was found in the cell-free
extract of Aspergillus terreus and the reaction mechanism
underlying the ring cleavage of anthraquinone is likely to be
a chemical Baeyer–Villiger oxidation and hydrolysis of the
5, 8, and 14 exhibited inhibitory effects against NO production
in BV-2 cells at 25 mM. In order to investigate whether the
inhibitory activities of the active compounds were due to their
cytotoxicity towards BV-2 cells, the effects of compounds 5, 8,
and 14 on cell proliferation/viability were measured by 3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
method. Of these, compounds 5 and 8 showed no cytotoxicity
with LPS treatment for 24 h.
In addition, severe oxidative stress has been also implicated
in the pathogenesis of neurodegenerative diseases. The anti-
oxidant activities of compounds 1–15 were preliminarily evalu-
ated for their DPPH free radical scavenging properties at 1, 5,
10, 25, 50, and 100 mM using ascorbic acid as the positive
control (IC50 ¼ 12.45 ꢂ 0.51 mM). The result showed that
compound 14 exhibited the most signicant DPPH radical
scavenging activity with IC50 value of 11.22 ꢂ 0.38 mM (Table 4),
which showed stronger antioxidant activity than the positive
control. Its potent activity may be due to the presence of two
hydroxyl groups at C-3 and C-4 positions of the aromatic ring,
27,28
formed lactone intermediate.
This suggested that the same
type of enzymes as questin oxygenase may be involved in the
formation of biphenyl ethers from xanthones in H. elatoides.
31
which agrees with previous studies. Moreover, compounds 2,
26650 | RSC Adv., 2018, 8, 26646–26655
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Scheme 1 Proposed biosynthetic pathway for biphenyl ether glycosides identified from H. elatoides (3HBL, 3-hydroxybenzoate:CoA ligase; BPS,
0
0
benzophenone synthase; B3 H, benzophenone 3 -hydroxylase; XS, xanthone synthase; X6H, xanthone 6-hydroxylase; SAM, S-adenosylme-
thionine; UDPGlc, uridine diphosphate glucose; Glc, glucopyranosyl unit).
4, 5, 8, and 13 exhibited moderate antioxidant activities. The particle size, YMC Co., Ltd., Kyoto, Japan), and Sephadex LH-20
remaining compounds were proved to have weak or no DPPH (Pharmacia Fine Chemicals, Uppsala, Sweden). Thin-layer
radical scavenging activities at concentrations of 1–100 mM.
chromatography (TLC) was performed on silica gel 60 F254
and RP-18 F254S plates (Merck KGaA, Darmstadt, Germany).
Fractions were monitored by TLC, and spots were visualized by
Experimental section
General experimental procedures
UV light (254 and 365 nm) and spraying with 10% H
2 4
SO in
ethanol, followed by heating. The purity of obtained
1
Optical rotations were measured with an Auton Paar MCP300 compounds were determined by analysis of H NMR spectra
automatic polarimeter (Anton Paar, Graz, Austria). The UV and HPLC. Horse serum (HS), fetal bovine serum (FBS), and
spectra were obtained on a Thermo Scientic Evolution-300 UV- Dulbecco's modied Eagle medium (DMEM) were purchased
visible spectrophotometer (Thermo Fisher Scientic, Waltham, from Gibco (Thermo Fisher Scientic, Bremen, Germany). NGF,
MA, USA). The IR spectra were recorded on a Bruker Tensor 27 authentic sugars, ascorbic acid, quercetin, dimethylsulfoxide
FT-IR spectrometer with KBr pellets (Bruker, Billerica, MA, (DMSO), DPPH, the growth substrate poly-L-lysine, and nutrient
USA). The NMR spectra were obtained on a Bruker Avance III mixture F-12 (Ham) were purchased from Sigma-Aldrich (Merck
500 spectrometer (Bruker, Billerica, MA, USA). HRESIMS were KGaA, Darmstadt, Germany).
recorded on an AB Triple TOF® 4600 mass spectrometer (AB
SCIEX, Redwood City, CA, USA). Semipreparative HPLC was
performed with a system consisting of LC-20AP pumps (Shi-
Plant material
madzu, Kyoto, Japan) and a SPD-20A UV/vis detector (Shimadzu, The aerial parts of H. elatoides were collected in Dongjue
Kyoto, Japan) equipped with YMC Packed C18 columns (5 mm, Mountain, Qishan County, Shaanxi Province, China, in
2
50 ꢃ 10.0 mm and 150 ꢃ 4.6 mm, YMC Co., Ltd., Kyoto, September 2016. The plant was identied by Professor Zai-Min
Japan). Column chromatography was performed on silica gel Jiang, College of Life Sciences, Northwest A&F University. A
100–200 and 200–300 mesh, Qingdao Marine Chemical Ltd., voucher specimen (Jiang 1043) was deposited in the herbarium
Qingdao, China), RP-C18 resins (YMC Gel ODS-A-HG, 50 mm of Northwest A&F University (WUK), Yangling, China.
(
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 26646–26655 | 26651

View Article Online
RSC Advances
Paper
Fig. 3 Effects of compounds 1–6 on neurite outgrowth in PC12 cells. (A) Morphological changes of PC12 cells treated with 0.1% DMSO, NGF (20
ꢀ1
ꢀ1
ng mL ), and NGF (20 ng mL ) + 4 (1.0 mM) were visualized under light microphotography. The cells treated with 4 served as a representative
example to demonstrate the enhancement of neurite outgrowth in the presence of NGF. (B) Neurite-bearing cells were quantitatively analyzed as
described in measurement of neurite outgrowth. Data are expressed as percentages of the value of NGF-treated cells (means ꢂ standard
ꢀ1
###
deviations, n ¼ 3) (D, 0.1% DMSO, control; N, 20 ng mL NGF, positive control;
p < 0.001 vs. control; *p < 0.05, **p < 0.01, ***p < 0.001 vs.
NGF control).
suspended in distilled water (3 L) and partitioned successively
with n-hexane (3 L ꢃ 3), EtOAc (3 L ꢃ 3), and n-BuOH (3 L ꢃ 3)
to yield a hexane-soluble fraction (55.6 g), an EtOAc-soluble
fraction (49.2 g), and a BuOH-soluble fraction (71.0 g), respec-
tively. The EtOAc fraction was subjected to silica gel CC and
eluted with a gradient of CH Cl /MeOH (100 : 1 to 2 : 1) to yield
2
2

een fractions (E1–E15). Fr. E3 was further subjected to RP-C18
CC (acetone–H O, 1 : 6) to give ten subfractions (E3.1–E3.10).
Fr. E3.5 was subjected to silica gel CC (CHCl –MeOH, 100 : 1) to
afford compound 7 (63.1 mg). Fr. E6 was subjected to silica gel
CC (CH Cl –MeOH, 30 : 1 to 20 : 1) to yield eight subfractions
2
3
2
2
Fig. 4 Effects of compounds 1–15 on LPS-induced NO production in (E6.1–E6.8). Fr. E6.3 was puried on a RP-C18 column (MeOH–
BV-2 microglial cells. Data are expressed as percentages of the value
H O, 3 : 1) to yield compound 8 (26.5 mg). Fr. E6.4 was puried
on a RP-C18 column (acetone–MeOH, 1 : 5) to yield compound 9
2
obtained with LPS-treated cells. (C, untreated control, 0.1% DMSO; L,
###
LPS-treated control; Q, quercetin, 5 mM, positive control;
p < 0.001
(
24.6 mg). Fr. E9 was subjected to silica gel CC and eluted with
a gradient of CHCl –MeOH–H O (15 : 1 : 0.05 to 4 : 1 : 0.1) to
yield eight fractions (E9.1–E9.8). Fr. E9.4 was further separated
by RP-C18 CC (MeOH–H O, 1 : 2 to 1 : 1) to yield seven sub-
compared with control group, **p < 0.01, ***p < 0.001 compared with
LPS group).
3
2
2
Extraction and isolation
fractions (E9.4.1–E9.4.7). Compound 6 (5.3 mg) was obtained by
the separation of Fr. E9.4.3 on a Sephadex LH-20 column
The aerial parts (1.3 kg) of H. elatoides were dried in shade, cut
into small pieces, and extracted three times (each for 8 h) with
(
MeOH–H O, 1 : 2). Compounds 1 (34.2 mg), 2 (21.5 mg), 3 (19.4
2
ꢁ
1
2 L of MeOH under reux conditions (55 C). Aer ltration
mg), and 4 (10.0 mg) were obtained by the separation of Fr.
and evaporation in vacuo, the obtained extract (334.8 g) was
26652 | RSC Adv., 2018, 8, 26646–26655
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
2
0
Table 4 Antioxidant activities of the isolated compounds by DPPH
radical scavenging assay
Hyperelatoside D (4). Brown amorphous solid; [a]
D
ꢀ44.3 (c
0
.1, MeOH); UV (MeOH) lmax (log 3) 290 (2.3) nm; IR (KBr) nmax
3402, 2950, 2837, 1650, 1455, 1410, 1111, 1022, 671 cm ; H
and C NMR data (CD OD and DMSO-d ), see Tables 1 and 2;
3 6
ꢀ
1 1
a
Compound
IC50 (mM)
1
3
+
2
4
5
8
1
1
85.50 ꢂ 3.08 HRESIMS (positive) m/z 463.1205 [M + Na] (calcd for
87.21 ꢂ 1.64
49.28 ꢂ 1.09
64.61 ꢂ 2.99
16.44 ꢂ 0.16
11.22 ꢂ 0.38
C H NaO , 463.1216).
2
0
24
11
20
Hyperelatoside E (5). Brown amorphous solid; [a]D ꢀ28.4 (c
.02, MeOH); UV (MeOH) lmax (log 3) 290 (3.0) nm; IR (KBr) n
0
3
max
1 1
3
4
ꢀ
392, 2950, 2836, 1651, 1457, 1411, 1111, 1023, 671 cm ; H
1
3
Ascorbic acid
12.45 ꢂ 0.51 and C NMR data (CD
3
OD), see Tables 1 and 2; HRESIMS
(negative) m/z 439.1247 [M ꢀ H] (calcd for C20
ꢀ
a
23 11
H O ,
Compounds 1, 3, 6, 7, 9–12, and 15 showed weak or no activities in the
DPPH antioxidant assay and ascorbic acid was used as a positive 439.1240).
control.
ꢁ
Hyperelatoside F (6). White needles; mp 220–222 C;
2
0
[
2
1
a] ꢀ114.3 (c 0.05, MeOH); UV (MeOH) l
(log 3) 222 (3.3),
max
D
56 (2.9), 319 (3.2) nm; IR (KBr) n
3439, 2954, 1666, 1626,
max
ꢀ1
1
13
509, 1445, 1366, 1267, 1226, 1062, 694, 652 cm ; H and
), see Table 3; HRESIMS (positive) m/z
83.0946 [M + Na] (calcd for C15 20NaO10, 383.0954).
C
E9.4.5 on a RP-C18 column (acetone–H
E9.4.6 was subjected to RP-C18 CC (acetone–H
compound 10 (35.9 mg). Fr. E9.5 was separated by RP-C18 CC
acetone–H O, 1 : 6 to 1 : 4) to give seven subfractions (E9.5.1–
2
O, 1 : 4 to 1 : 2.5). Fr.
NMR data (DMSO-d
6
2
O, 1 : 2.5) to yield
+
3
H
(
2
Acid hydrolysis
E9.5.7). Fr. E9.5.2 was subjected to RP-C18 CC (MeOH–H O,
2
1
: 3) to yield compound 11 (3.4 mg). Fr. E9.5.6 was subjected to The absolute congurations of the sugar moieties of 1–6 were
32
2
RP-C18 CC (MeOH–H O, 1 : 1.4) followed by RP-C18 CC determined by the acid hydrolysis method. Each compound
(
acetone–H
2
O, 1 : 2) to provide compound 12 (4.2 mg). Fr. (approximately 1.0 mg) was separately dissolved in 1 N HCl (0.5
ꢁ
E9.5.7 was puried on a RP-C18 column (MeOH–H
2
O, 1 : 1.5) to mL), and then heated at 90 C in a water bath for 2 h. Aer
yield compound 15 (7.6 mg). Fr. E10 was separated by silica gel extraction with EtOAc two times, the H O-soluble fraction was
2
3 2
CC (CHCl –MeOH–H O, 15 : 1 : 0.05 to 3 : 1 : 0.1) to afford evaporated to dryness under reduced pressure. The residue was
seven subfractions (E10.1–E10.7). Fr. E10.3 was subjected to RP- dissolved in pyridine (0.1 mL) containing L-cysteine methyl ester
ꢁ
C18 CC (acetone–H O, 1 : 5) to yield four fractions (E10.3.1– hydrochloride (0.5 mg) and heated at 60 C for 1 h. A 100 mL
2
E10.3.4). Fr. E10.3.2 was subjected to RP-C18 CC (MeOH–H
2
O, solution of o-tolylisothiocyanate (0.5 mg) in pyridine was added
ꢁ
1
: 2.5 to 1 : 1.5) to afford eight fractions (E10.3.2.1–E10.3.2.8). and the reaction mixture was heated at 60 C for 1 h. Then each
Compound 5 (7.6 mg) was obtained by the separation of Fr. reaction mixture was analyzed by the Waters 1525 HPLC system
E10.3.2.2 on a RP-C18 column (MeOH–H O, 1 : 2). Fr. E10.6 was using a Waters 2489 UV/vis detector (at 250 nm). Analytical
separated on a silica gel column (CHCl –MeOH–H O, 4 : 1 : 0.1) HPLC was performed on the YMC Packed C18 column (5 mm,
to yield compound 13 (960.0 mg). Fr. E11 was subjected to silica 150 ꢃ 4.6 mm) with a linear gradient elution (CH CN–H O,
2
3
2
3
2
gel CC and eluted with a gradient of CHCl –MeOH–H O 20 : 80 to 40 : 60) for 30 min. The derivative of D-glucose was
3
2
(6 : 1 : 0.1 to 2 : 1 : 0.1) to afford six subfractions (E11.1–E11.6). identied in 1–6 by a comparison of the retention time with
Fr. E11.4 was further subjected to RP-C18 CC (acetone–H
to 1 : 2) to give compound 14 (161.3 mg).
2
O, 1 : 3 authentic D-glucose (tR 16.3 min), which was subjected to the
same derivatization procedure.
2
0
Hyperelatoside A (1). Brown amorphous solid; [a]
.1, MeOH); UV (MeOH) lmax (log 3) 226 (3.9), 260 (2.4), 292
2.0) nm; IR (KBr) nmax 3418, 2952, 1701, 1618, 1515, 1440, 1379,
D
ꢀ38.2 (c
0
(
Measurement of neurite outgrowth
PC12 cells were purchased from the Cell Bank of Shanghai
Institute of Biochemistry and Cell Biology, Chinese Academy of
Sciences (Shanghai, China). PC12 cells were maintained in
nutrient mixture F-12 medium supplemented with 10% inacti-
ꢀ1 1
13
3
1273, 1209, 1076, 1028, 630 cm ; H and C NMR data (CD OD
and DMSO-d ), see Tables 1 and 2; HRESIMS (positive) m/z
6
+
5
21.1247 [M + Na] (calcd for C H NaO , 521.1271).
2
2
26
13
2
0
Hyperelatoside B (2). Brown amorphous solid; [a]
D
ꢀ25.5 (c
ꢀ
1
vated HS, 5% inactivated FBS, penicillin G (100 U mL ),
0
.1, MeOH); UV (MeOH) lmax (log 3) 225 (5.4), 260 (1.0) nm; IR
ꢀ
1
ꢀ1
streptomycin (100 mg mL ), and sodium bicarbonate (2.5 g L
)
(
6
KBr) nmax 3415, 1624, 1514, 1444, 1274, 1211, 1170, 1027,
ꢁ
at 37 C in humidied atmosphere of 5% CO
2
. Morphological
ꢀ1
1
13
30 cm ; H and C NMR data (CD
3
OD), see Tables 1 and 2;
analysis and quantication of neurite-bearing cells were per-
ꢀ
HRESIMS (negative) m/z 483.1150 [M ꢀ H] (calcd for
formed using a phase-contrast microscope as previously
21 23
C H O13, 483.1139).
15
described. Briey, PC12 cells were seeded in poly-L-lysine-
2
0
Hyperelatoside C (3). Brown amorphous solid; [a]D ¼ ꢀ21.2
4
coated 24-well plates at a density of 2 ꢃ 10 cells per mL with
(c 0.05, MeOH); UV (MeOH) lmax (log 3) 225 (5.4), 260 (2.4), 292
normal serum medium for 24 h. The F-12 medium containing
low serum (2% HS and 1% FBS) was replaced prior to exposure
to vehicle (0.1% DMSO) or indicated reagents. The cells were
treated with tested compounds in the presence of NGF (20 ng
(
6
1.0) nm; IR (KBr) nmax 3434, 2954, 1640, 1514, 1454, 1019,
ꢀ1
1
13
3
61 cm ; H and C NMR data (CD OD), see Tables 1 and 2;
+
HRESIMS (positive) m/z 489.0998 [M + Na] (calcd for
22NaO12, 489.1009).
21
C H
ꢀ1
mL ). Cells without treatment served as a negative control.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 26646–26655 | 26653

View Article Online
RSC Advances
Paper
ꢀ
1
Cells treated with 20 ng mL of NGF served as a positive
control. One concentration experiment was repeated in three
Conflicts of interest
wells. Aer an additional 48 h of incubation, neurite outgrowth There are no conicts to declare.
of PC12 cells was observed under an inverted microscope using
phase-contrast objectives and photographed by the digital
camera. Eight images were selected randomly under a micro-
scope for each well. At least 100 cells in each of eight randomly
separated elds were scored. The cells with neurites greater
than or equal to the length of one cell body were positive for
neurite outgrowth and expressed as a percentage of the total cell
number in eight elds. Experiments were repeated at least three
times, and data are expressed as means ꢂ standard deviations.
Acknowledgements
This work was nancially supported by the Scientic Startup
Foundation for Doctors of Northwest A&F University (No.
2452015356) and the Fundamental Research Funds for the
Central Universities (No. 2452017173) and the Natural Science
Foundation of China (No. 21572182).
Determination of NO production
Notes and references
BV-2 microglial cells were purchased from Peking Union
Medical College Cell Bank (Beijing, China) and cultured in
DMEM supplemented with 10% heat-inactivated fetal bovine
1
(a) C. C. S ´a nchez-Mateo, B. Prado and R. M. Rabanal, J.
Ethnopharmacol., 2002, 79, 119; (b) M. F. Rieli, R. Mattei
and C. E. L. de Araujo, Fitoterapia, 2002, 73, 462; (c)
A. Viana, J. C. do Rego, G. von Poser, A. Ferraz,
A. P. Heckler, J. Costentin and S. M. K. Rates,
Neuropharmacology, 2005, 49, 1042; (d) O. Grundmann,
Y. Lv, O. Kelber and V. Butterweck, Neuropharmacology,
ꢀ
1
serum, penicillin G (100 U mL ), and streptomycin (100 mg
ꢀ
1
ꢁ
2
mL ) at 37 C in a humidied incubator with 5% CO . The NO
33
concentration was detected by the Griess reagent. Quercetin
was used as a positive control. BV-2 cells were seeded at the
density of 1.5 ꢃ 10 cells per mL in 96-well culture plate and
5
2010, 58, 767; (e) J. Zhao, W. Liu and J. C. Wang, Chem.
ꢀ
1
treated with each compound and LPS (1.0 mg mL ) for 24 h.
Aer that, 50 mL of cell-free supernatant was allowed to react
with an equal volume of Griess reagent (Beyotime Institute of
Biotechnology, Shanghai, China) for 15 min at room tempera-
ture in the dark. Then, the absorbance was measured at 540 nm
using a microplate reader. The cell viability of the cultured cells
was detected by MTT-based colorimetric method.
Biodiversity, 2015, 12, 309.
2
3
(a) S. S. Chatterjee, S. K. Bhattacharya, M. Wonnemann,
A. Singer and W. E. M u€ ller, Life Sci., 1998, 63, 499; (b)
J. Sarris, Phytother. Res., 2007, 21, 703; (c) S. Kasper,
F. Caraci, B. Forti, F. Drago and E. Aguglia, Eur.
Neuropsychopharmacol., 2010, 20, 747.
(a) R. Ciochina and R. B. Grossman, Chem. Rev., 2006, 106,
3
2
963; (b) I. P. Singh and S. B. Bharate, Nat. Prod. Rep.,
006, 23, 558; (c) J. J. Zhang, J. Yang, Y. Liao, X. W. Yang,
Antioxidant activity
The DPPH radical scavenging assay was performed according to
J. Z. Ma, Q. L. Xiao, L. X. Yang and G. Xu, Org. Lett., 2014,
16, 4912; (d) X. W. Yang, R. B. Grossman and G. Xu, Chem.
Rev., 2018, 118, 3508.
4 (a) Z. Q. Yin, Y. Wang, W. C. Ye and S. X. Zhao, Biochem. Syst.
Ecol., 2004, 32, 521; (b) N. Tanaka, Y. Kashiwada, S. Y. Kim,
M. Sekiya, Y. Ikeshiro and Y. Takaishi, Phytochemistry,
34
a previously described method. Ascorbic acid was used as
a positive control. The absorbance of the resulting solution of
each compound was measured at 517 nm with a spectropho-
tometer. Results were expressed as IC50 values.
2
009, 70, 1456; (c) W. J. Xu, R. J. Li, O. Quasie, M. H. Yang,
Conclusions
L. Y. Kong and J. Luo, J. Nat. Prod., 2016, 79, 1971.
5 (a) A. Hecka, B. Maunit, F. Aubriet and J. F. Muller, Rapid
Commun. Mass Spectrom., 2009, 23, 885; (b) M. A. Farag and
L. A. Wessjohann, Planta Med., 2012, 78, 488; (c) A. Porzel,
M. A. Farag, J. M u€ lbradt and L. A. Wessjohann,
Metabolomics, 2014, 10, 574.
6 (a) C. A. Ang'edu, J. Schmidt, A. Porzel, P. Gitu, J. O. Midiwo
and G. Adam, Pharmazie, 1999, 54, 235; (b)
S. A. F. Tanemossu, K. Franke, N. Arnold, J. Schmidt,
H. K. Wabo, P. Tane and L. A. Wessjohann, Phytochemistry,
2014, 105, 171.
7 (a) L. R. Jat, Int. J. Pharm. Pharm. Sci., 2013, 5, 9; (b)
Z. B. Zhou, Z. R. Li, X. B. Wang, J. G. Luo and L. Y. Kong, J.
Nat. Prod., 2016, 79, 1231.
8 (a) M. A. G. del Rio, M. I. Sanchez-Reus, I. Iglesias,
M. A. Pozo, M. Garcia-Arencibia, J. Fernandez-Ruiz,
L. Garcia-Garcia, M. Delgado and J. Benedi, CNS Neurol.
Disord.: Drug Targets, 2013, 12, 665; (b) D. Zheleva-
In summary, this study is the rst report to describe the
chemical constituents and their biological activities of H.
elatoides. Chemical investigation of the aerial parts of H. ela-
toides led to the isolation of six new phenolic compounds
including ve unusual biphenyl ether glycosides, hyperelato-
sides A–E (1–5), and one benzoate glycoside, hyperelatoside F
(
6). To the best of our knowledge, this represents the rst
report of the isolation of biphenyl ether glycosides from plants
and their putative biosynthetic pathway is proposed. Further-
more, except for 3, all the new isolates possessed potent ability
to potentiate the activity of NGF to stimulate neurite
outgrowth in PC12 cells. Compounds 5 and 8 exhibited
moderate anti-neuroinammatory activities and 14 showed
signicant antioxidant activity in vitro. Our ndings show that
the aerial parts of H. elatoides may be an excellent source of
neurotrophic phytochemicals for the prevention and treat-
ment of neurodegenerative diseases.
26654 | RSC Adv., 2018, 8, 26646–26655
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Dimitrova, P. Ncdialkov and G. Momekov, Pharmacogn. Mag.,
013, 9, 1.
D. Ben-Eliezer and E. Yechiam, Sci. Rep., 2016, 6, 35700.
0 N. Huang, L. Rizshsky, C. Hauck, B. J. Nikolau, P. A. Murphy
and D. F. Birt, Phytochemistry, 2011, 72, 2015.
1 L. C. Brito, A. L. R. Berenger and M. R. Figueiredo, Food 26 N. Li, H. S. Lee, N. Zhang, Y. N. Sun, J. L. Li, S. S. Xing,
Chem. Toxicol., 2017, 109, 847. J. G. Chen and L. Cui, Phytochem. Lett., 2015, 13, 286.
2 H. Zhu, C. Chen, J. Yang, X. N. Li, J. Liu, B. Sun, S. X. Huang, 27 (a) R. F. Curtis, C. H. Hassall, D. W. Jones and
T. Han and L. P. Qin, Zhongguo Tianran Yaowu, 2013, 11,
673; (e) L. L. Xu, C. C. Zhang, X. Y. Zhu, F. Cao and
H. J. Zhu, Nat. Prod. Res., 2017, 31, 1875; (f)
P. Phainuphong, V. Rukachaisirikul, S. Phongpaichit,
S. Preedanon and J. Sakayaroj, Tetrahedron, 2017, 73, 5920.
2
9
1
1
1
D. Li, G. Yao, Z. Luo, Y. Li, J. Zhang, Y. Xue and Y. Zhang,
Org. Lett., 2014, 16, 6322.
3 H. Jayasuriya, A. M. Clark and J. D. McChesney, J. Nat. Prod.,
T. W. Williams, J. Chem. Soc., 1960, 4838; (b) Z. G. Chen,
I. Fujii, Y. Ebizuka and U. Sankawa, Phytochemistry, 1995,
38, 299; (c) K. Huang, I. Fujii, Y. Ebizuka, K. Gomi and
U. Sankawa, J. Biol. Chem., 1995, 270, 21495; (d)
1
1
991, 54, 1314.
4 (a) P. L. Liu, C. Du, Z. M. Jiang and J. Cai, Xibei Zhiwu Xuebao,
011, 31, 1268; (b) Q. Sun, P. L. Liu, Q. Zhang, L. X. Cao and
Z. M. Jiang, Xibei Zhiwu Xuebao, 2012, 32, 276.
1
J.
Hargreaves,
J.
Park,
E.
L.
Ghisalberti,
2
K. Sivasithamparam, B. W. Skelton and A. H. White, J. Nat.
Prod., 2002, 65, 7.
1
5 (a) X. W. Shi, L. Liu, J. M. Gao and A. L. Zhang, Eur. J. Med. 28 I. Fujii, Y. Ebizuka and U. Sankawa, J. Biochem., 1988, 103,
Chem., 2011, 46, 3112; (b) C. C. Zhang, X. Yin, C. Y. Cao, 878.
J. Wei, Q. Zhang and J. M. Gao, Bioorg. Med. Chem. Lett., 29 (a) W. Schmidt and L. Beerhues, FEBS Lett., 1997, 420, 143;
2
015, 25, 5078; (c) R. Bai, C. C. Zhang, X. Yin, J. Wei and
(b) M. M. Gaid, D. Sircar, A. M u¨ ller, T. Beuerle, B. Liu,
L. Ernst, R. H ¨a nsch and L. Beerhues, Plant Physiol., 2012,
160, 1267; (c) I. El-Awaad, M. Bocola, T. Beuerle, B. Liu and
L. Beerhues, Nat. Commun., 2016, 7, 11472.
J. M. Gao, J. Nat. Prod., 2015, 78, 783; (d) C. C. Zhang,
C. Y. Cao, M. Kubo, K. Harada, X. T. Yan, Y. Fukuyama
and J. M. Gao, Int. J. Mol. Sci., 2017, 18, 1659.
16 Z. Zhang, H. N. ElSohly, M. R. Jacob, D. S. Pasco, L. A. Walker 30 (a) W. Schmidt, S. Peters and L. Beerhues, Phytochemistry,
and A. M. Clark, Planta Med., 2002, 68, 49.
7 N. Tanaka and Y. Takaishi, Phytochemistry, 2006, 67, 2146.
8 M. Iinuma, H. Tosa, T. Ito, T. Tanaka and M. Aqil,
Phytochemistry, 1995, 40, 267.
2000, 53, 427; (b) H. R. El-Seedi, M. A. El-Barbary,
D. M. H. El-Ghorab, L. Bohlin, A. K. Borg-Karlson,
U. G ¨o ransson and R. Verpoorte, Curr. Med. Chem., 2010,
17, 854.
1
1
1
2
2
2
9 S. E. Ukwueze, P. O. Osadebe and F. B. C. Okoye, Nat. Prod. 31 (a) P. V. Kiem, N. X. Cuong, N. X. Nhiem, V. K. Thu,
Res., 2015, 29, 1728.
N. K. Ban, C. V. Minh, B. H. Tai, T. N. Hai, S. H. Lee,
H. D. Jang and Y. H. Kim, Bioorg. Med. Chem. Lett., 2011,
21, 633; (b) X. T. Yan, S. H. Lee, W. Li, Y. N. Sun,
S. Y. Yang, H. D. Jang and Y. H. Kim, Food Chem., 2014,
156, 408.
0 Y. L. Huang, C. C. Chen, Y. J. Chen, R. L. Huang and
B. J. Shieh, J. Nat. Prod., 2001, 64, 903.
1 G. Bohr, C. Gerhaeuser, J. Knau, J. Zapp and H. Becker, J.
Nat. Prod., 2005, 68, 1545.
2 T. Fukunaga, K. Nishiya, I. Kajikawa, Y. Watanabe, 32 T. Tanaka, T. Nakashima, T. Ueda, K. Tomii and I. Kouno,
N. Suzuki, K. Takeya and H. Itokawa, Chem. Pharm. Bull.,
988, 36, 1180.
3 K. Yasukawa and M. Takido, Phytochemistry, 1987, 26, 1224.
4 C. Y. Chen, S. L. Hsieh, M. M. Hsieh, S. F. Hsieh and
T. J. Hsieh, Chin. Pharm. J., 2004, 56, 141.
5 (a) R. Liu, W. Zhu, Y. Zhang, T. Zhu, H. Liu, Y. Fang and
Q. Gu, J. Antibiot., 2006, 59, 362; (b) T. Bunyapaiboonsri,
S. Yoiprommarat, K. Intereya and K. Kocharin, Chem.
Chem. Pharm. Bull., 2007, 55, 899.
1
33 (a) Z. Huang, Z. X. Zhu, Y. T. Li, D. R. Pang, J. Zheng,
Q. Zhang, Y. F. Zhao, D. Ferreira, J. K. Zjawiony, P. F. Tu
and J. Li, J. Nat. Prod., 2015, 78, 2276; (b) J. Li, N. Li, X. Li,
G. Chen, C. Wang, B. Lin and Y. Hou, J. Nat. Prod., 2017,
80, 3081; (c) J. Wei, W. H. Guo, C. Y. Cao, R. W. Kou,
Y. Z. Xu, M. G ´o recki, L. D. Bari, G. Pescitelli and J. M. Gao,
Sci. Rep., 2018, 8, 2175.
2
2
2
Pharm. Bull., 2007, 55, 304; (c) M. J. Fang, H. Fang, W. J. Li, 34 (a) O. P. Sharma and T. K. Bhat, Food Chem., 2009, 113, 1202;
D. M. Huang, Z. Wu and Y. F. Zhao, Nat. Prod. Res., 2012,
6, 1224; (d) W. Peng, F. You, X. L. Li, M. Jia, C. J. Zheng,
(b) C. D. Zheng, G. Li, H. Q. Li, X. J. Xu, J. M. Gao and
A. L. Zhang, Nat. Prod. Commun., 2010, 5, 1759.
2
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 26646–26655 | 26655