Polycyclic polyprenylated acylphloroglucinol and phenolic metabolites from the aerial parts of Hypericum elatoides and their neuroprotective and anti-neuroinflammatory activities

Article abstract of DOI:10.1016/j.phytochem.2018.12.011

A phytochemical study on the aerial parts of Hypericum elatoides led to the isolation of a previously undescribed polycyclic polyprenylated acylphloroglucinol derivative, hyperelatone A, seven previously undescribed phenolic metabolites, hyperelatones B–H, along with ten known analogues. The structures of hyperelatones A–H were elucidated by 1D and 2D NMR spectroscopy, HRESIMS experiment, single-crystal X-ray diffraction and comparison of experimental and calculated ECD spectra, as well as chemical derivatization. All compounds were evaluated for their neuroprotective activity against hydrogen peroxide (H₂O₂)-induced cell injury in rat pheochromocytoma PC-12 cells and inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV-2 microglial cells. Hyperelatones B–D and H, cinchonain Ib, and tenuiside A showed noticeable neuroprotection at concentrations of 1.0–100.0 μM. Hyperelatones D, G, and H, (?)-epicatechin, tenuiside A, and (Z)-3-hexenyl-β-D-glucopyranoside exhibited significant anti-neuroinflammatory activity with IC₅₀ values ranging from 0.75 ± 0.02 to 5.83 ± 0.23 μM.

Full text of DOI:10.1016/j.phytochem.2018.12.011

Phytochemistry159(2019)65–74
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Polycyclic polyprenylated acylphloroglucinol and phenolic metabolites from
the aerial parts of Hypericum elatoides and their neuroprotective and anti-
neuroinflammatory activities
Xi-Tao Yana, Zhen Ana, Yucui Huangfua, Yuan-Teng Zhanga, Chun-Huan Lia, Xin Chena,
Pei-Liang Liub, Jin-Ming Gaoa,∗
a Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, People's Republic of
China
b Key Laboratory of Resource Biology and Biotechnology in Western China, College of Life Sciences, Northwest University, Xi'an 710069, People's Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Hypericum elatoides R. Keller
Hypericaceae
Polycyclic polyprenylated acylphloroglucinol
Flavanone-resveratrol adduct
Phenylpropanoid-substituted flavan-3-ol
Xanthone glycoside
A phytochemical study on the aerial parts of Hypericum elatoides led to the isolation of a previously undescribed
polycyclic polyprenylated acylphloroglucinol derivative, hyperelatone A, seven previously undescribed phenolic
metabolites, hyperelatones B–H, along with ten known analogues. The structures of hyperelatones A–H were
elucidated by 1D and 2D NMR spectroscopy, HRESIMS experiment, single-crystal X-ray diffraction and com-
parison of experimental and calculated ECD spectra, as well as chemical derivatization. All compounds were
evaluated for their neuroprotective activity against hydrogen peroxide (H2O2)-induced cell injury in rat pheo-
chromocytoma PC-12 cells and inhibitory effects on lipopolysaccharide (LPS)-induced nitric oxide (NO) pro-
duction in BV-2 microglial cells. Hyperelatones B–D and H, cinchonain Ib, and tenuiside A showed noticeable
neuroprotection at concentrations of 1.0–100.0 μM. Hyperelatones D, G, and H, (−)-epicatechin, tenuiside A,
and (Z)-3-hexenyl-β-D-glucopyranoside exhibited significant anti-neuroinflammatory activity with IC50 values
Neuroprotective activity
Anti-neuroinflammatory activity
ranging from 0.75
0.02 to 5.83
0.23 μM.
1. Introduction
grown as a narrow endemic species in Northwest China. Most recently,
a series of uncommon biphenyl ether glycosides with remarkable neu-
rotrophic activity have been isolated from H. elatoides (Yan et al.,
2018). As part of our ongoing works to study the chemical diversity of
Hypericum species and their beneficial biological activities for potential
treatments of neurological disorders, the methanol extract of the aerial
parts of H. elatoides were investigated. As a result, eight previously
undescribed compounds, including a PPAP derivative (1), two flava-
none-resveratrol adducts (2–3), a phenylpropanoid-substituted flavan-
3-ol (4), and four xanthone glycosides (9–12), were isolated from H.
elatoides, along with ten known compounds (5–8 and 13–18). We re-
ported herein the isolation and structural elucidation of these pre-
viously undescribed compounds, as well as biological assessment of
their neuroprotective effects against hydrogen peroxide (H2O2)-induced
lesions of PC-12 cells and inhibitory effects on lipopolysaccharide
(LPS)-induced nitric oxide (NO) production in BV-2 microglial cells.
The genus Hypericum belongs to the Hypericaceae family and
comprises approximately 500 species of herbs, shrubs, and small trees,
most of which are placed in temperate regions of the world (Crockett
and Robson, 2011; Nürk et al., 2013). Previous phytochemical studies
on Hypericum plants revealed the presence of polycyclic polyprenylated
acylphloroglucinols (PPAPs) (Ciochina and Grossman, 2006; Yang
et al., 2018), simple phloroglucinols (Zhao et al., 2015), xanthones
(Tanaka et al., 2009; Xu et al., 2016), flavonoids (Porzel et al., 2014),
naphthodianthrones (Farag and Wessjohann, 2012), and benzophe-
nones (Zhao et al., 2015) as major components. Many of them showed
valuable biological activities, such as antidepressant (Grundmann et al.,
2010; Jat, 2013), neuroprotective (Gómez del Rio et al., 2013; Zhou
et al., 2016), memory-enhancing (Ben-Eliezer and Yechiam, 2016),
cytotoxic (Zhao et al., 2015), anti-inflammatory (Huang et al., 2011),
antimicrobial (Bridi et al., 2018), and antioxidant (Zheleva-Dimitrova
et al., 2013) properties.
Hypericum elatoides R. Keller (Hypericaceae) is a perennial herb
∗ Corresponding author.
E-mail address: jinminggao@nwsuaf.edu.cn (J.-M. Gao).
https://doi.org/10.1016/j.phytochem.2018.12.011
Received 30 October 2018; Received in revised form 30 November 2018; Accepted 16 December 2018
0031-9422/©2018ElsevierLtd.Allrightsreserved.

X.-T. Yan et al.
Phytochemistry159(2019)65–74
4' OH
OH
3'
OH
O
33
29
32
OH
8
OH
OH
1'
HO
9
O
O
2
OH
5''
4'''
OH
8
1'
2
13
10
14
HO
HO
O
4
5'
O
OH
3
27
6
1'''
34 28
10
3
23
3''
O
1''
1''
6
OH
OH
2''
8
26
7
6
20
1
6''
RO
O
O
OH
MeO
OH
OH
9
1'''
4''
15
O
18
OH
OH
16
3
5
22
O
4
5
OH
2
R = H
R =
HO
O
OH1''''
OH
3
1
HO
OH
OH
HO
OH
HO
O
O
OH
1
OH
10
O
OH
8
9
8a
4b
9a
4a
OH
HO
OH
O
7
HO
HO
HO
O
OH
OH
2
11
OH
OH
6
O
3
HO
O
5
4
OH
HO
O
O
OH
OH
OH1'
HO
HO
O
OH
O
OH
6
8
7
9
R1
O
O
O
O
OH
OH
O
O
HO
O
H3CO
OH
HO
O
OH
HO
HO
OH
HO
HO
O
O
O
HO
R2
HO
HO
OCH3
O
HO
O
OH
OH
HO
OH
HO
HO
13
14
10
11
12
R1 = OH R2 = OH
R1 = H R2 = H
OH
O
O
HO
O
HO
HO
HO
O
O
O
O
O
O
O
O
HO
HO
HO
HO
HO
HO
HO
HO
OH
OH
OH
OH
15
16
18
17
Fig. 1. Chemical structures of compounds 1–18.
2. Results and discussion
respectively. Moreover, the attachments of two methyl groups at C-5
and C-8 were evidenced by the HMBC cross-peaks from Me-22 to C-4, C-
5, C-6, and C-9 and from Me-34 to C-1, C-7, C-8, and C-28 (δC 35.9).
The relative configuration of 1 was elucidated via the NOESY
spectrum, the key correlations of Me-22 (δH 1.41)/H-10b (δH 2.55), Me-
22/H-6b (δH 1.99), H-6b/H-7 (δH 1.76), H-6b/H-28a (δH 1.18), and H-
10b/H-28b (δH 1.38) indicated that H-7, CH2-10, Me-22, and CH2-28
were all α-oriented. The β-orientations for CH2-23 and Me-34 were
deduced from the NOESY correlations of Me-34(δH 1.16)/H-23a (δH
1.96) and Me-34/H-10a (δH 2.68). In addition, a single-crystal X-ray
diffraction pattern (CCDC 1875447) was acquired using Mo Kα radia-
tion. The crystallography data of 1 further confirmed the planar
structure and permitted unambiguous assignment of the absolute con-
figuration as (1S,5S,7S,8R) with a Flack parameter of 0(2) (Fig. 2).
Thus, the structure of 1, hyperelatone A, was elucidated as shown in
Fig. 1.
Hyperelatone B (2) was obtained as a yellow amorphous powder. Its
molecular formula was established as C29H22O8 by HRESIMS (m/z
521.1221 [M + Na]+, calcd for C29H22NaO8, 521.1212). The IR
spectrum showed absorption bands at 3405 and 1635 cm−1 for hydroxy
and carbonyl groups, respectively. The 1H NMR spectrum of 2 (Table 2)
revealed the presence of two 1,4-disubstituted aromatic rings with
AA'BB′ coupling system [δH 7.19 (2H, d, J = 8.4 Hz, H-2‴ and H-6‴),
7.00 (2H, d, J = 8.4 Hz, H-2′ and H-6′), 6.74 (2H, d, J = 8.4 Hz, H-3‴
and H-5‴), and 6.56 (2H, d, J = 8.4 Hz, H-3′ and H-5′)], a trans-olefinic
moiety [δH 6.51 (1H, d, J = 15.8 Hz, H-α) and 6.34 (1H, d, J = 15.8 Hz,
H-β], and two 1,2,3,5-tetrasubstituted aromatic rings with the protons
located at the meta-positions [δH 6.27 (1H, br s, H-6″), 6.24 (1H, br s, H-
4″), and 5.92 (2H, overlap, H-6 and H-8)], which was supported by the
HMBC correlations from the aromatic protons at δH 6.27 to C-2′′ (δC
113.1) and C-4′′ (δC 103.1), from δH 6.24 to C-2″ and C-6′′ (δC 105.9),
and from δH 5.92 to C-6 (δC 97.3), C-8 (δC 96.3), and C-10 (δC 103.4).
The remaining proton signals were attributable to an oxymethine at δH
5.75 (1H, d, J = 11.7 Hz, H-2) and a methine with vicinal coupling at
δH 4.35 (1H, d, J = 11.7 Hz, H-3), which was confirmed by the 1H–1H
COSY correlation of H-2/H-3. The large coupling constant
(J = 11.7 Hz) between H-2 and H-3 indicated their trans-
The crude MeOH extract of the aerial parts of H. elatoides was sus-
pended in H2O and partitioned successively with n-hexane, EtOAc, and
n-BuOH. The obtained extracts were subjected to repeated column
chromatography (CC) on silica gel, RP-C18, and Sephadex LH-20 to
afford eight previously undescribed compounds, hyperelatones A–H
(1–4 and 9–12), and ten known analogues (5–8 and 13–18) (Fig. 1).
Hyperelatone A (1) was obtained as a colorless oil. Its molecular
formula was determined to be C34H44O4 by HRESIMS at m/z 517.3316
[M + H]+ (calcd for C34H45O4, 517.3318), indicating 13 degrees of
unsaturation. The IR spectrum showed absorption bands of hydroxy
(3434 cm-1) and carbonyl (1640 cm−1) groups. The 1H NMR spectrum
(Table 1) exhibited resonances assignable to a monosubstituted ben-
zene ring at δH 7.36–7.52 (5H, H-17–H-21), three trisubstituted olefins
at δH 5.10 (1H, dd, J = 9.7, 1.4 Hz, H-11), 5.02 (1H, m, H-30), and 4.87
(1H, m, H-24), and eight methyl groups at δH 1.79, 1.69, 1.66, 1.66,
1.57, 1.50, 1.41, and 1.16 (each 3H, s, Me-14, 27, 13, 33, 32, 26, 22,
and 34). Analysis of the 13C and DEPT NMR spectra implied that the
structure of 1 possessed a bicyclo[3.3.1]nonane-2,4,9-trione core,
based on the presence of an enolic β-diketone system at δC 194.1 (C-2),
115.5 (C-3), and 198.0 (C-4), a nonconjugated ketone at δC 208.8 (C-9),
a methylene at δC 40.7 (C-6), a methine at δC 39.8 (C-7), and three
quaternary carbons at δC 69.8 (C-1), 55.3 (C-5), and 51.2 (C-8) (Hamed
et al., 2006; Acuña et al., 2010; Yang et al., 2015). The remaining
carbon signals were assigned to a benzoyl group (including a carbonyl
and six aromatic carbons), three prenyl groups (including six olefinic
carbons, three methylenes, and six methyls), a methylene (C-28), and
two methyl groups (Me-22 and Me-34). These observations suggested 1
to be a type-B bicyclic polyprenylated acylphloroglucinol derivative
with a benzoyl group located at C-3 position (Yang et al., 2018). Its
planar structure was established by the 2D NMR analysis (Fig. 2).
1H–1H COSY correlations of H-7 (δH 1.76)/H-23 (δH 2.05 and 1.96) and
H-28 (δH 1.38 and 1.18)/H-29 (δH 1.86 and 1.79) as well as HMBC
correlations from H-10 (δH 2.68 and 2.55) to C-1, C-2, C-8, and C-9 and
from H-28 to C-1, C-7, and C-8 confirmed the connectivities of a 4-
methyl-3-pentenyl to C-8 and two prenyl groups to C-1 and C-7,
66

X.-T. Yan et al.
Phytochemistry159(2019)65–74
of a 4′,5,7-trihydroxyflavanone (naringenin) moiety. The linkage be-
tween the naringenin unit and the resveratrol unit was determined to be
C-3–C-2″ by analysis of the HMBC correlations from H-3 to C-1″, C-2″,
and C-3′′ (δC 157.4).
Table 1
NMR spectroscopic data for compound 1 at 500 MHz (1H) and 125 MHz (13C) in
CDCl3.
Position
δC, type
δH, mult (J in Hz)
HMBC (H→C)
The absolute configurations at the chiral centers C-2 and C-3 were
established by comparing the experimental and computed electronic
circular dichroism (ECD) spectra. The ECD spectrum of 2 exhibited a
strong positive Cotton effect near 291 nm (π → π* transitions), in an
opposite manner to struthiolanone (2S,3R) (Ayers et al., 2008), which
was the first and only report of a flavanone-stilbene adduct, suggesting
the absolute configuration of 2 as (2R,3S) (Jamila et al., 2014; Vu et al.,
2016). This assignment was confirmed by computational calculations of
the ECD spectra. The calculated ECD spectrum of the (2R,3S) isomer of
2 well matched the experimental one (Fig. 4). Based on these data, the
structure of 2 was established as (2R,3S)-naringenin-(3α,2)-trans-3,5,4′-
trihydroxystilbene.
1
2
3
4
5
6
69.8, C
194.1, C
115.5, C
198.0, C
55.3, C
40.7, CH2
2.34, d (14.4)
1.99, dd (14.4, 7.0)
1.76, m
4, 5, 7, 8, 9, 22, 23
4, 5, 7, 9, 22, 23
1, 5, 23, 24
7
8
9
10
39.8, CH
51.2, C
208.8, C
26.2, CH2
2.68, dd (13.5, 9.7)
2.55, d (12.9)
5.10, dd (9.7, 1.4)
1, 2, 8, 9, 11, 12
1, 2, 9, 11, 12
13, 14
11
12
13
14
15
16
17
18
19
20
21
22
23
120.4, CH
135.1, C
18.4, CH3
26.3, CH3
197.0, C
Hyperelatone C (3) was obtained as a yellow amorphous powder
with a molecular formula of C35H32O13, which was determined by
1.66, s
1.79, s
11, 12, 14
11, 12, 13
HRESIMS at m/z 683.1760 [M + Na]+ (calcd for C35H32NaO13
,
683.1741). The 1H and 13C NMR spectra of 3 showed similar resonance
patterns to those found for 2 (Table 2), with an additional sugar moiety
at δH 5.04 (1H, d, J = 7.7 Hz, H-1′′′′), 3.93 (1H, dd, J = 11.9, 1.6 Hz, H-
6a′′′′), 3.72 (1H, dd, J = 11.9, 5.7 Hz, H-6b′′′′), 3.44 (2H, m, H-3′′′′ and
H-5′′′′), 3.33 (1H, m, H-4′′′′), and 3.24 (1H, m, H-2′′′′); δC 101.8 (C-1′′′′),
78.5 (C-5′′′′), 78.5 (C-3′′′′), 75.4 (C-2′′′′), 71.4 (C-4′′′′), and 62.8 (C-
6′′′′). Acid hydrolysis followed by HPLC analysis after ar-
ylthiocarbamoyl-thiazolidine derivatization confirmed the character-
ization of a D-glucose and the coupling constant (J = 7.7 Hz) confirmed
the β-anomer for the glucose moiety. The location of the β-D-gluco-
pyranosyl unit was determined to be at C-3″ by analysis of the HMBC
spectrum of 3, which showed a correlation from the anomeric proton
(H-1′′′′) to C-3′′ (δC 157.2) (Fig. 3). The trans-configuration of H-2 and
H-3 was established by the large coupling constant (J = 11.8 Hz) be-
tween H-2 and H-3. The absolute stereochemistry of C-2 and C-3 was
determined to be (2R,3S)-configuration based on the observed positive
Cotton effect at 291 nm in the ECD spectrum of 3 (Fig. 4), in agreement
with the ECD data of 2. Thus, the structure of 3 was elucidated as
(2R,3S)-naringenin-(3α,2)-trans-5,4′-dihydroxystilbene-3-O-β-D-gluco-
pyranoside.
137.1, C
129.0, CH
128.0, CH
132.7, CH
128.0, CH
129.0, CH
17.6, CH3
28.6, CH2
7.52, d (8.0)
7.36, t (8.0)
7.51, t, (7.8)
7.36, t, (8.0)
7.52, d (8.0)
1.41, s
1.96, m
2.05, m
4.87, m
15, 19, 21
16, 20
17, 21
16, 18
15, 17, 19
4, 5, 6, 9
24, 25
24, 25
26, 27
24
25
26
27
28
124.4, CH
133.2, C
18.0, CH3
26.0, CH3
35.9, CH2
1.50, s
1.69, s
24, 25, 27
24, 25, 26
1, 7, 8, 29, 30, 34
1, 7, 8, 29, 30, 34
28, 30, 31
1.18, m
1.38, m
1.79, m
1.86, m
5.02, m
29
22.7, CH2
28, 30, 31
28, 29, 32, 33
30
31
32
33
34
124.0, CH
132.2, C
17.8, CH3
25.9, CH3
18.8, CH3
1.57, s
1.66, s
1.16, s
30, 31, 33
30, 31, 32
1, 7, 8, 28
stereochemistry. The 13C NMR and HSQC spectra of 2 showed signals
for two methines at δC 83.3 (C-2) and 54.2 (C-3), a ketone at δC 200.2
(C-4), a double bond at δC 125.2 (C-α) and 132.5 (C-β), and 20 aromatic
carbons at δC 96.3–167.9, four of which were double [δC 129.4 (C-2′
and C-6′), 129.0 (C-2‴ and C-6‴), 116.5 (C-3‴ and C-5‴), and 115.6 (C-
3′ and C-5′)], corresponding to two 1,4-disubstituted aromatic rings
shown from 1H NMR spectrum. The HMBC spectrum of 2 (Fig. 3) ex-
hibited key correlations from H-α to C-1‴ (δC 130.8), C-2′′ (δC 113.1),
and C-6′′ (δC 105.9) and from H-β to C-1′′ (δC 143.1), C-1‴, C-2‴, and C-
6‴, indicating the presence of a 3,5,4′-trihydroxystilbene (resveratrol)
moiety. In addition, the HMBC correlations from H-2 to C-1′ (δC 131.5),
C-2′ and C-6′ and from H-3 to C-1′, C-2, and C-4 indicated the presence
Hyperelatone D (4) was obtained as a brown amorphous powder
with a molecular formula of C25H24O11, as inferred from the positive
HRESIMS at m/z 482.1209 [M
– ,
H2O]+ (calcd for C25H22O10
482.1213). The IR absorptions indicated the presence of hydroxy
(3436 cm-1) and ester carbonyl (1642 cm−1) groups. The 1H NMR
spectrum (Table 2) revealed the occurrence of a set of ABX coupled
aromatic protons at δH 7.00 (1H, d, J = 1.9 Hz, H-2′), 6.87 (1H, dd,
J = 8.2, 1.9 Hz, H-6′), and 6.80 (1H, d, J = 8.2 Hz, H-5′), a singlet
aromatic proton at δH 6.13 (1H, s, H-8), two oxymethine at δH 4.93 (1H,
br s, H-2) and 4.26 (1H, m, H-3), and a methylene at δH 2.93 (1H, dd,
J = 17.1, 4.5 Hz, H-4a) and 2.84 (1H, dd, J = 17.1, 2.3 Hz, H-4b),
suggesting the presence of an epicatechin skeleton in 4 (Davis et al.,
O
O
O
OH
COSY
HMBC
Fig. 2. ORTEP drawing and key 1H–1H COSY, HMBC, and NOESY correlations of compound 1.
67

X.-T. Yan et al.
Phytochemistry159(2019)65–74
Table 2
1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data for compounds 2–4 in CD3OD.
Position
2
3
4
δC, tpye
δH, mult (J in Hz)
δC, tpye
δH, mult (J in Hz)
δC, tpye
δH, mult (J in Hz)
2
3
4
83.3, CH
54.2, CH
200.2, C
5.75, d (11.7)
4.35, d (11.7)
83.3, CH
54.2, CH
199.7, C
5.84, d (11.8)
4.46, d (11.8)
80.3, CH
67.0, CH
29.6, CH2
4.93, br s
4.26, m
2.84, dd (17.1, 2.3)
2.93, dd (17.1, 4.5)
5
6
7
8
165.8, C
97.3, CH
167.9, C
96.3, CH
165.0, C
103.4, C
131.5, C
129.4, CH
115.6, CH
158.6, C
115.6, CH
129.4, CH
143.1, C
113.1, C
157.4, C
103.1, CH
158.4, C
105.9, CH
125.2, CH
165.8, C
97.5, CH
168.1, C
96.3, CH
165.0, C
103.4, C
131.3, C
129.6, CH
115.6, CH
158.5, C
115.6, CH
129.6, CH
143.5, C
114.7, C
157.2, C
102.5, CH
158.4, C
108.4, CH
124.7, CH
153.0, C
104.7, C
153.6, C
96.0, CH
156.7, C
103.3, C
132.3, C
115.4, CH
146.0, C
146.3, C
116.2, CH
119.4, CH
116.1, C
146.7, C
104.5, CH
142.3, C
146.1, C
115.3, CH
44.9, CH2
5.92, overlap
5.92, overlap
5.92, br s
5.92, br s
6.13, s
9
10
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
6″
α
7.00, d (8.4)
6.56, d (8.4)
7.05, d (8.4)
6.54, d (8.4)
7.00, d (1.9)
6.56, d (8.4)
7.00, d (8.4)
6.54, d (8.4)
7.05, d (8.4)
6.80, d (8.2)
6.87, dd (8.2, 1.9)
6.47, s
6.24, br s
6.61, d (1.9)
6.27, br s
6.51, d (15.8)
6.42, d (1.9)
6.57, d (15.9)
6.63, s
2.51, dd (14.6, 8.9)
2.98, dd (14.6, 3.6)
4.49, dd (8.9, 3.6)
β
132.5, CH
130.8, C
129.0, CH
116.5, CH
158.5, C
6.34, d (15.8)
133.3, CH
130.6, C
129.2, CH
116.5, CH
158.9, C
6.33, d (15.9)
31.4, CH
174.8, C
52.2, CH3
1‴
2‴
3‴
4‴
7.19, d (8.4)
6.74, d (8.4)
7.21, d (8.5)
6.75, d (8.5)
3.60, s
5‴
6‴
116.5, CH
129.0, CH
6.74, d (8.4)
7.19, d (8.4)
116.5, CH
129.2, CH
101.8, CH
75.4, CH
78.5, CH
71.4, CH
78.5, CH
62.8, CH2
6.75, d (8.5)
7.21, d (8.5)
5.04, d (7.7)
3.24, m
3.44, m
3.33, m
3.44, m
3.72, dd (11.9, 5.7)
3.93, dd (11.9, 1.6)
1′′′′
2′′′′
3′′′′
4′′′′
5′′′′
6′′′′
OH
OH
OH
OH
OH
OH
HO
O
HO
O
OH
HO
OH
HO
O
OH
OH
O
O
OH
O
HO
HO
HO
OH
O
MeO
HO
OH
OH
OH
O
3
2
4
Fig. 3. Key 1H–1H COSY (bold lines) and HMBC (→) correlations of compounds 2–4.
1,2,4,5-tetrasubstituted aromatic ring with two protons located at the
para-position [δH 6.63 (1H, s, H-6″) and 6.47 (1H, s, H-3″)], a benzylic
methine [δH 4.49 (1H, dd, J = 8.9, 3.6 Hz, H-β)], a methylene [δH 2.98
(1H, dd, J = 14.6, 3.6 Hz, H-αa) and 2.51 (1H, dd, J = 14.6, 8.9 Hz, H-
αb)], and a methoxy group [δH 3.60 (3H, s, H-2‴)]. The 13C NMR
spectrum of 4 exhibited ten additional carbon signals comprising one
ester carbonyl (δC 174.8), six aromatic carbons (δC 146.7, 146.1, 142.3,
116.1, 115.3, and 104.5), one methoxy carbon (δC 52.2), and two ali-
phatic carbons (δC 44.9 and 31.4). These data indicated the presence of
a β-substituted phenylpropanoate unit (Foo, 1987; Lee et al., 2001).
Complete assignment of the 1H and 13C NMR signals of 4 was accom-
plished by analysis of 2D NMR spectra (Fig. 3) and by comparison with
those published NMR spectroscopic data of several phenylpropanoid-
substituted catechins (Tang et al., 2007; Huang et al., 2007; Zhang
et al., 2013). Most importantly, the key HMBC correlations from H-β to
Fig. 4. Experimental ECD spectra of compounds 2–4 and their calculated ECD
spectra at the B3LYP/6-31++G(d,p) level in MeOH.
1996; Abd El-Razek, 2007). Moreover, the characteristic epicatechin
carbons at δC 80.3 (C-2), 67.0 (C-3), and 29.6 (C-4) were also present in
the 13C NMR spectrum. The remaining proton signals were assigned to a
68

X.-T. Yan et al.
Phytochemistry159(2019)65–74
O
O
C-5 (δC 153.0), C-6 (δC 104.7), and C-7 (δC 153.6), from H-α to C-6,
from H-4 to C-5, C-9 (δC 156.7), and C-10 (δC 103.3), and from H-8 to C-
9 indicated that the phenylpropanoate unit was connected to the epi-
catechin skeleton via the linkage of C-β–C-6.
OH
OH
OH
HO
HO
HO
HO
O
O
O
O
O
O
HO
HO
HO
HO
HO
HO
The cis-configuration of H-2 and H-3 was deduced by the evidence
of a broad singlet signal due to H-2, suggesting a negligible coupling
constant between H-2 and H-3. The absolute configuration of the C-β
stereogenic center was determined via comparison of the experimental
ECD spectrum of 4 with the calculated ECD spectra of the (βS,2R,3R),
(βR,2S,3S), (βS,2S,3S), and (βR,2R,3R) enantiomers (Fig. 4). The ex-
perimental ECD spectrum of 4 showed a strong negative Cotton effect at
218 nm, consistent with the calculated curves of (βS) enantiomers
[(βS,2R,3R)-4 and (βS,2S,3S)-4], allowing assignment of the (βS) ab-
solute configuration. Moreover, the (βS,2R,3R)-4 showed the best
match. Considering the isolation of a large amount of (−)-epicatechin
(2R,3R) (7, 1341.4 mg) and its derivatives (5, 6, and 8) from H. ela-
toides, they are likely to be products of the same biosynthetic pathway,
and therefore the absolute stereochemistry of C-2 and C-3 can be as-
sumed to be (2R,3R), which was also supported by the chemical shifts
of C-ring (Abd El-Razek, 2007). Accordingly, the absolute configuration
of 4 was deduced to be (βS,2R,3R). The structure of 4 was proposed as
6-[(1S)-(2,4,5-trihydroxyphenyl)-3-methoxy-3-oxopropyl]-epicatechin.
Hyperelatone E (9) was obtained as a yellow amorphous powder
OH
OH
9
10
O
O
OH
HO
HO
HO
HO
O
O
O
O
OH
OH
O
O
OH
11
12
Fig. 5. Key 1H–1H COSY (bold lines) and HMBC (→) correlations of compounds
9–12.
4.60 (1H, t, J = 5.5 Hz, 11-OH) and two methylene signals at δH 2.87
and 2.80 (each 1H, m, H-10) and 3.51 (2H, m, H-11) (Table 3). The 13
C
NMR spectrum displayed 21 carbon signals, including a carbonyl (δC
180.3), 12 aromatic carbons (δC 93.0–162.6), a characteristic gluco-
pyranosyl moiety (δC 100.4, 77.2, 76.4, 73.3, 69.7, and 60.7), and a
hydroxyethyl group (δC 59.6 and 25.8). All these data suggested that 9
was a xanthone glycoside with a trisubstituted A-ring and a mono-
substituted B-ring (Fernandes et al., 1998). The large coupling constant
(J = 6.9 Hz) of the anomeric proton and acid hydrolysis result con-
firmed the sugar to be a β-D-glucopyranose. In the HMBC experiment of
9 (Fig. 5), the correlations from the hydrogen-bonded hydroxy proton
at δH 13.10 to C-1 (δC 159.5), C-2 (δC 109.1), and C-9a (δC 103.1), from
the methylene protons at δH 2.87 and 2.80 to C-1, C-2, and C-3 (δC
162.6), and from the anomeric proton at δH 5.05 to C-3 indicated the
locations of the hydroxy, 2-hydroxyethyl, and glucopyranosyl group at
C-1, C-2, and C-3 of A-ring, respectively. Additionally, the HMBC cor-
relations from H-8 to C-4b (δC 149.2), C-6 (δC 124.9), and C-9 (δC
that analyzed for C21H22O11 by HRESIMS (m/z 451.1233 [M + H]+
,
calcd for C21H23O11, 451.1240), accounting for 11 degrees of un-
saturation. The IR spectrum displayed absorption bands due to hydroxy
(3414 cm-1) and conjugated carbonyl (1706 cm−1) groups. The 1H NMR
data of 9 showed the presence of two hydroxy groups at δH 13.10 (1H,
s, 1-OH) and 10.06 (1H, s, 7-OH), a group of ABX coupled aromatic
protons at δH 7.53 (1H, d, J = 9.0 Hz, H-5), 7.44 (1H, d, J = 2.7 Hz, H-
8), and 7.32 (1H, dd, J = 9.0, 2.7 Hz, H-6), a singlet aromatic proton at
δ
H 6.77 (1H, s, H-4), an anomeric proton at δH 5.05 (1H, d, J = 6.9 Hz,
H-1′), and a hydroxyethyl moiety contributed by a hydroxy signal at δH
Table 3
1H NMR (500 MHz) and 13C NMR (125 MHz) spectroscopic data for compounds 9–12 in DMSO‑d6.
Position
9
10
11
12
δC, tpye
δH, mult (J in Hz)
δC, tpye
δH, mult (J in Hz)
δC, tpye
δH, mult (J in Hz)
δC, tpye
δH, mult (J in Hz)
1
2
3
4
5
6
7
8
159.5, C
109.1, C
162.6, C
93.0, CH
119.0, CH
124.9, CH
154.0, C
107.9, CH
180.3, C
155.5, C
149.2, C
120.4, C
103.1, C
25.8, CH2
160.9, C
109.7, CH
136.6, CH
107.0, CH
103.3, CH
153.0, C
144.6, C
108.2, CH
180.6, C
155.9, C
150.8, C
114.0, C
107.8, C
150.1, C
139.6, C
125.4, CH
106.2, CH
146.4, C
121.0, CH
124.1, CH
114.6, CH
182.5, C
150.0, C
145.5, C
120.4, C
108.5, C
110.8, CH
153.8
7.75, d (3.0)
6.78, d (8.3)
7.67, t (8.3)
7.03, d (8.3)
7.31, s
7.66, d (9.2)
7.06, d (9.2)
125.5, CH
119.6, CH
118.2, CH
135.5, CH
124.3, CH
125.9, CH
175.8, C
150.9, C
155.6, C
120.5, C
121.6, C
7.59, dd (9.2, 3.0)
7.67, d (9.2)
7.67, dd (8.7, 0.9)
7.88, ddd (8.7, 8.0, 1.7)
7.49, ddd (8.0, 8.0, 0.9)
8.20, dd (8.0, 1.7)
6.77, s
7.53, d (9.0)
7.32, dd (9.0, 2.7)
7.36, dd (7.9, 1.2)
7.28, t (7.9)
7.60, dd (7.9, 1.2)
7.44, d (2.7)
7.47, s
9
4a
4b
8a
9a
10
2.80, m
2.87, m
11
1′
2′
3′
4′
5′
6′
59.6, CH2
100.4, CH
73.3, CH
76.4, CH
69.7, CH
77.2, CH
60.7, CH2
3.51, m
5.05, d (6.9)
3.34, m
3.31, m
3.20, m
3.48, m
3.47, m
3.73, dd (10.2, 5.3)
13.10, s
10.06, s
5.34, d (4.2)
5.12, d (3.9)
5.08, d (5.2)
4.65, t (5.8)
4.60, t (5.5)
100.4, CH
73.1, CH
76.0, CH
69.7, CH
77.3, CH
60.7, CH2
5.14, d (7.1)
3.37, m
3.33, m
3.20, m
3.51, m
3.49, m
3.74, dd (11.0, 5.7)
12.80, s
101.1, CH
73.3, CH
76.7, CH
69.7, CH
77.1, CH
60.7, CH2
4.94, d (6.9)
3.29, m
3.31, m
3.19, m
3.36, m
101.3, CH
73.3, CH
76.4, CH
69.6, CH
77.1, CH
60.6, CH2
4.97, d (7.4)
3.29, m
3.32, m
3.21, m
3.38, m
3.47, dd (12.0, 5.9)
3.69, d (11.3)
3.51, dd (11.9, 5.9)
3.71, dd (11.9, 5.4)
1-OH
7-OH
2′-OH
3′-OH
4′-OH
6′-OH
11-OH
9.55, s
5.39, d (3.9)
5.14, d (4.7)
5.10, d (5.3)
4.66, t (5.7)
5.32, br s
5.09, br s
5.03, br s
4.57, br s
5.39, d (4.6)
5.10, d (3.7)
5.03, d (5.2)
4.59, t (5.4)
69

X.-T. Yan et al.
Phytochemistry159(2019)65–74
180.3) and from H-6 to C-4b suggested that the non-chelated hydroxy
group was attached to C-7 of B-ring. Therefore, the structure of 9 was
assigned to be 1,7-dihydroxy-2-(2-hydroxyethyl)-3-O-β-D-glucopyr-
anosylxanthone.
Hyperelatone H (12) was only appeared as a synthetic product in 1929
(Robertson and Waters, 1929). Furthermore, there has been no NMR
spectroscopic data reported for this compound by now. This is the first
report of the occurrence of 12 as a natural product and its full NMR
assignments based on the extensive 1D and 2D NMR experiments.
The remaining compounds were identified as cinchonain Ib (5)
(Beltrame et al., 2006), cinchonain Ic (6) (Nonaka and Nishioka, 1982),
(−)-epicatechin (7) (Davis et al., 1996), procyanidin B-2 (8) (Khan
et al., 1997), tenuiside A (13) (Shi et al., 2013), 2,6-dimethoxy-p-hy-
droquinone 1-O-β-D-glucopyranoside (14) (Otsuka et al., 1989), eu-
odionoside G (15) (Yamamoto et al., 2008), icariside B2 (16) (Miyase
et al., 1987), 2-phenylethyl-β-D-glucopyranoside (17) (Yoneda et al.,
2008), and (Z)-3-hexenyl-β-D-glucopyranoside (18) (Mizutani et al.,
1988) by comparison of their NMR data with the reported data in the
literature. Among them, compounds 5, 6, 13, and 15–18 were isolated
from the genus Hypericum for the first time.
Moreover, the chemical profile of H. elatoides could provide che-
motaxonomical information for verifying the systematic position of H.
elatoides in the genus Hypericum. Based on morphological character
analyses, H. elatoides was classified as either section Roscyna (Spach) R.
Keller (Robson, 1977) or section Ascyreia Choisy (Robson, 2001). Mo-
lecular phylogenetic analyses showed that H. elatoides was closely re-
lated to H. ascyron L. (a member of section Roscyna), while section
Roscyna nested with the large section Ascyreia (Meseguer et al., 2013;
Nürk et al., 2013). Our results demonstrated that H. elatoides contained
large amounts of flavonoids (Yan et al., 2018), flavanols derivatives
(4–8), xanthones (9–13), and phloroglucinol derivative (1), whereas
several characteristic secondary metabolites such as hypericin, pseu-
dohypericin, mangiferin, amentoflavone, and I3, II8-biapigenin, were
not found in H. elatoides, which is most similar to the phytochemical
profiles of the species from the sections Ascyreia and Roscyna (Kitanov
and Nedialkov, 1998; Kitanov, 2001; Crockett and Robson, 2011;
Camas et al., 2014; Cirak et al., 2016). The present study supports H.
elatoides belonging to section Roscyna or Ascyreia, and possesses a po-
tential taxonomic value for the infrageneric classification of the genus
Hypericum.
Plants belonging to the genus Hypericum are well known for their
therapeutic uses on neurological disorders (Rieli Mendes et al., 2002;
Viana et al., 2005; Sarris, 2007; Grundmann et al., 2010). The best
known species is H. perforatum L. (St. John's wort), which is widely used
for the treatment of mild to moderate depression (Sarris, 2007; Kasper
et al., 2010). Considering the fact that many previously isolated com-
pounds from the genus Hypericum showed neuroprotective activities for
antidepression potential (Oliveira et al., 2016; Xu et al., 2016; Zhou
et al., 2016), compounds 1–18 were evaluated for their protective ef-
fects against H2O2-induced PC-12 cell injury, using captopril as a po-
sitive control. As shown in Table 4, compounds 2–7 and 11–13 ex-
hibited neuroprotective activity, which significantly improved the
survival rate of PC-12 cells in a dose-dependent manner. Among them,
compounds 2–5, 12, and 13 were the most active, which may be due to
the presence of hydroxy groups and their intrinsic molecular structures.
Recent studies support that neuroinflammation plays a crucial role
in the development of depressive-like behaviors (Miller et al., 2009;
Moylan et al., 2014). Inhibition of activated microglia-induced neu-
roinflammation might be an effective therapeutic target for depression
(Catena-Dell'Osso et al., 2011; Réus et al., 2015; Dong et al., 2018).
Therefore, all the isolated compounds were evaluated for their in-
hibitory effects on LPS-stimulated NO production in BV-2 microglial
cells using Griess assay (Huang et al., 2015; Tang et al., 2018; Yan et al.,
2018). Compounds 4, 7, 11–13, and 18 showed significant inhibitory
Hyperelatone F (10) was obtained as a yellow amorphous powder.
Its HRESIMS showed a [M + Na]+ peak at m/z 429.0787 (calcd for
C
19H18NaO10, 429.0798), indicating a molecular formula of C19H18O10
and 11 degrees of unsaturation. The 1H NMR data of 10 (Table 3)
showed the character of a xanthone glycoside that exhibited two hy-
droxy groups [δH 12.80 (1H, s, 1-OH) and 9.55 (1H, s, 7-OH)], an ABM
coupling system [δH 7.67 (1H, t, J = 8.3 Hz, H-3), 7.03 (1H, d,
J = 8.3 Hz, H-4), and 6.78 (1H, d, J = 8.3 Hz, H-2)], two singlet aro-
matic protons [δH 7.47 (1H, s, H-8) and 7.31 (1H, s, H-5)], and an
anomeric proton [δH 5.14 (1H, d, J = 7.1 Hz, H-1′)]. The locations of
two hydroxy groups and the sugar moiety were confirmed by 2D NMR
experiments (Fig. 5). The HMBC correlations from the hydrogen-
bonded hydroxy proton at δH 12.80 to C-1 (δC 160.9), C-2 (δC 109.7),
and C-9a (δC 107.8) indicated that the hydroxy group was attached to
C-1, which was supported by the 1H–1H COSY correlations of H-2/H-3/
H-4. The other hydroxy proton at δH 9.55 exhibited HMBC cross-peaks
with C-6 (δC 153.0), C-7 (δC 144.6), and C-8 (δC 108.2), suggesting that
the non-chelated hydroxy group was located at C-7. In addition, the
HMBC correlation from the anomeric proton at δH 5.14 to C-6 indicated
that the sugar moiety was linked at C-6. Analysis of the coupling con-
stant of the anomeric proton and acid hydrolysis result confirmed the
sugar unit to be a β-D-glucopyranose. The structure of 10 was thus
determined to be 1,7-dihydroxy-6-O-β-D-glucopyranosylxanthone.
Hyperelatone G (11) has the same molecular formula of C19H18O10
as 10 by analysis of HRESIMS (m/z 407.0978 [M + H]+, calcd for
C
19H19O10, 407.0978). IR and 1D NMR spectra revealed that these two
compounds had the same functional groups. The 1H and 13C NMR data
of 11 (Table 3) are similar to those of the known compounds, 1,2,5-
trihydroxyxanthone (Minami et al., 1994; Wu et al., 1998) and 1,5-
dihydroxy-2-methoxyxanthone (Rath et al., 1996; Tala et al., 2015),
except that
a hydroxy group in 1,2,5-trihydroxyxanthone or the
methoxy group in 1,5-dihydroxy-2-methoxyxanthone was replaced by a
glucosyl moiety. This observation suggested that 11 was probably a
1,2,5-trisubstituted xanthone, which was further confirmed by the
1H–1H COSY correlations of H-3 (δH 7.66)/H-4 (δH 7.06) and H-6 (δH
7.36)/H-7 (δH 7.28)/H-8 (δH 7.60) and the HMBC data (Fig. 5).
Moreover, the HMBC correlation from the anomeric proton at δH 4.94
(H-1′) to C-2 (δC 139.6) indicated that the glucosyl moiety was linked at
C-2. Hence, the structure of 11 was determined to be 1,5-dihydroxy-2-
O-β-D-glucopyranosylxanthone.
Hyperelatone H (12) has a molecular formula of C19H18O8 deduced
from HRESIMS peak at m/z 397.0899 [M
+
Na]+ (calcd for
C19H18NaO8, 397.0899). Comprehensive analysis of the 1D NMR
spectroscopic data (Table 3) strongly implied the presence of a mono-
substituted xanthone and a sugar unit in 12. The 1H NMR spectrum
displayed seven aromatic proton resonances at δH 8.20 (1H, dd,
J = 8.0, 1.7 Hz, H-8), 7.88 (1H, ddd, J = 8.7, 8.0, 1.7 Hz, H-6), 7.67
(1H, dd, J = 8.7, 0.9 Hz, H-5), 7.49 (1H, ddd, J = 8.0, 8.0, 0.9 Hz, H-7),
7.75 (1H, d, J = 3.0 Hz, H-1), 7.67 (1H, d, J = 9.2 Hz, H-4), and 7.59
(1H, dd, J = 9.2, 3.0 Hz, H-3) and one anomeric proton at δH 4.97 (1H,
d, J = 7.4 Hz, H-1′). The 13C NMR spectrum presented 19 carbon sig-
nals, including a carbonyl carbon (δC 175.8), 12 aromatic carbons (δC
110.8–155.6), and a sugar moiety (δC 101.3, 77.1, 76.4, 73.3, 69.6, and
60.6). The sugar moiety was characterized as a β-D-glucopyranose by
the same method as described for 3. The glycosidation position was
located at C-2 (δC 153.8) based on a HMBC correlation from the
anomeric proton (H-1′) to C-2 and 1H–1H COSY correlations of H-3/H-4
and H-5/H-6/H-7/H-8 (Fig. 5). Additionally, this assignment was con-
firmed by the downfield shift of C-2 and the upfield shifts of C-1, C-3,
and C-4a in comparison with the chemical shifts for unsubstituted ring
in xanthones (Westerman et al., 1977; Fernandes et al., 1998). Thus, the
structure of 12 was elucidated as 2-O-β-D-glucopyranosylxanthone.
effects with IC50 values of 5.83
0.23, 3.37
0.13, 3.84
0.15,
0.75 0.02, 1.39 0.03, and 2.95
0.07 μM, respectively, some of
which exhibited stronger activity than the positive controls (Table 5).
All the tested compounds did not show any significant cytotoxicity in
BV-2 cells. Among the xanthone glucosides (9–13), compounds 11–13
showed much stronger activity than 9 and 10, suggesting that the
70

X.-T. Yan et al.
Phytochemistry159(2019)65–74
Table 4
Neuroprotective effects of compounds 2–7 and 11–13 against H2O2-induced cell injury in PC-12 cells.
Compounda
Cell viability (% of control)b, concentration (μM)
0.3
1.0
3.0
10.0
30.0
100.0
Control
100.00
46.52
64.92
64.38
51.51
48.43
49.70
47.87
47.61
50.72
63.17
52.09
2.63
0.56
Modelc
Captoprild
2
3
4
5
6
7
11
12
13
0.15∗∗∗
3.99∗∗∗
1.49∗∗
0.63
72.58
73.22
60.98
59.80
61.19
52.50
48.83
49.63
69.62
65.72
0.15∗∗∗
2.52∗∗∗
0.73∗∗∗
0.67∗∗
0.73∗∗∗
5.13
77.21
78.09
70.78
70.72
71.30
54.70
53.60
55.53
76.95
68.45
0.71∗∗∗
2.39∗∗∗
0.02∗∗∗
8.52∗∗
0.95∗∗∗
2.24∗
85.14
85.66
78.31
73.31
73.60
66.00
65.50
64.63
82.71
75.43
0.41∗∗∗
0.24∗∗∗
0.11∗∗∗
0.24∗∗∗
0.28∗∗∗
2.78∗∗∗
1.06∗∗∗
1.85∗∗∗
0.39∗∗∗
1.60∗∗∗
87.45
89.68
85.78
82.00
81.65
77.47
76.72
75.00
87.27
82.55
0.11∗∗∗
0.19∗∗∗
0.80∗∗∗
1.49∗∗∗
0.30∗∗∗
0.26∗∗∗
0.37∗∗∗
0.20∗∗∗
0.32∗∗∗
0.24∗∗∗
89.34
91.98
90.96
84.74
84.10
83.86
83.26
80.20
90.08
85.12
0.15∗∗∗
0.26∗∗∗
0.41∗∗∗
2.22∗∗∗
0.28∗∗∗
1.36∗∗∗
0.09∗∗∗
0.11∗∗∗
0.88∗∗∗
0.30∗∗∗
0.13∗∗
0.04
0.45
0.80∗
0.26∗∗∗
0.32∗∗∗
0.04∗∗∗
0.04∗∗∗
0.37∗
1.47∗∗∗
0.28∗∗
0.13
0.54∗∗∗
0.84∗∗∗
*P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared with model group.
a
Compounds 1, 8–10, and 14–18 showed weak activity at test concentrations.
b
c
Each value is expressed as mean
H2O2-only treatment group.
Positive control.
SD from three independent experiments.
d
4. Experimental
Table 5
Inhibitory effects of compounds 1–18 on LPS-induced NO production in BV-
2 cells.
4.1. General experimental procedures
Compound
IC50 (μM)a
Compound
IC50 (μM)a
Optical rotations were measured with an Auton Paar MCP300 au-
tomatic polarimeter (Anton Paar, Graz, Austria). ECD spectra were re-
corded with Applied Photophysics chirascan spectrometer (Applied
Photophysics, Surrey, United Kingdom). The UV spectra were obtained
on a Thermo Scientific Evolution-300 UV–visible spectrophotometer
(Thermo Fisher Scientific, Waltham, MA, USA). The IR spectra were
recorded on a Bruker Tensor 27 FT-IR spectrometer with KBr pellets
(Bruker, Billerica, MA, USA). The NMR spectra were obtained on a
Bruker Avance III 500 spectrometer (Bruker, Germany). HRESIMS were
recorded on an AB Triple TOF® 4600 mass spectrometer (AB SCIEX,
Redwood City, CA, USA). The X-ray crystallographic data was collected
using a Bruker D8 Venture diffractometer with Mo Kα radiation at
153 K (Bruker, Germany). Semi-preparative HPLC was performed on an
Agilent 1100 series instrument equipped with a quaternary pump, a
vacuum degasser, an autosampler, a diode array detector, and YMC
Packed C18 columns (5 μm, 250 × 10.0 mm and 150 × 4.6 mm, YMC
Co., Ltd., Kyoto, Japan). Column chromatography was performed on
silica gel (100–200 and 200–300 mesh, Qingdao Marine Chemical Ltd.,
Qingdao, China), reversed phase (RP)-C18 resins (YMC Gel ODS-A-HG,
50 μm particle size, YMC Co., Ltd., Kyoto, Japan), and Sephadex LH-20
(Pharmacia Fine Chemicals, Uppsala, Sweden). Thin-layer chromato-
graphy was performed on silica gel 60 F254 and RP-18 F254S plates
(Merck KGaA, Darmstadt, Germany).
1
2
3
4
5
6
7
8
9
> 30
> 30
> 30
11
12
13
14
15
16
17
18
3.84
0.75
1.39
26.96
> 30
> 30
> 30
2.95
1.07
2.60
0.15
0.02
0.03
5.83
0.23
1.07
> 30
> 30
3.37
0.13
> 30
> 30
> 30
0.07
0.04
0.06
Quercetinb
Fluoxetineb
10
a
Data are expressed as means
SD based on three independent experi-
ments.
b
Positive control.
presence of an O-glucosyl unit at C-2 or C-4 position of xantone, which
might be related to a specific molecular geometry, could improve the
inhibitory effect. Our results demonstrate that these novel phenolic
metabolites from H. elatoides have neuroprotective or/and anti-neu-
roinflammatory activities and may be useful in the prevention and
treatment of depression.
3. Conclusion
A previously undescribed PPAP, hyperelatone A (1), seven pre-
viously undescribed phenolic derivatives, hyperelatones B–H (2–4 and
9–12), together with ten known compounds (5–8 and 13–18) were
isolated from the aerial parts of H. elatoides. The structures of these
previously undescribed compounds were elucidated by spectroscopic
methods, and their absolute configurations were determined using
single-crystal X-ray diffraction crystallographic data, calculated and
measured ECD data, and chemical evidence. The flavanone-resveratrol
adducts (2 and 3) and phenylpropanoid-substituted flavan-3-ols (4–6)
were isolated from Hypericum species for the first time. In addition,
compounds 2–5, 12, and 13 showed potent neuroprotective activity
against H2O2-induced cell injury in PC-12 cells at concentrations of
1.0–100.0 μM and 4, 7, 11–13, and 18 exhibited significant inhibitory
effects on LPS-induced NO production in BV-2 cells. The present study
will facilitate the development of new neuroprotective or/and anti-
neuroinflammatory agents.
4.2. Plant material
The aerial parts of Hypericum elatoides R. Keller (Hypericaceae) were
collected in Dongjue Mountain, Qishan County, Shaanxi Province,
China (latitude 34°34′–34°35′ North and longitude 107°45′–107°46′
East), in September 2016. The plant was identified by Professor Zai-Min
Jiang, College of Life Sciences, Northwest A&F University. A voucher
specimen (Jiang 1043) was deposited at the herbarium of Northwest A
&F University (WUK), Yangling, China.
4.3. Extraction and isolation
The dried aerial parts (1.3 kg) of H. elatoides were cut into small
pieces and extracted with MeOH (3 × 12 L) under reflux conditions
(55 °C) for 8 h. After filtration and evaporation under reduced pressure,
the obtained extract (334.8 g) was suspended in distilled water (3 L)
71

X.-T. Yan et al.
Phytochemistry159(2019)65–74
and partitioned successively with n-hexane (3 × 3 L), EtOAc (3 × 3 L),
and n-BuOH (3 × 3 L). The n-hexane fraction (55.6 g) was fractionated
by silica gel CC using a stepwise gradient of petroleum ether-Me2CO
(150:1 to 2:1) as eluents to afford eighteen fractions (H1–H18). Fr. H6
was subjected to silica gel CC and eluted with a gradient of n-hexane-
Me2CO (200:1 to 5:1) to give seven subfractions (H6.1–H6.7). Fr. H6.2
was further subjected to RP-C18 CC (MeOH-Me2CO, 2:1) followed by
silica gel CC (n-hexane-Me2CO, 300:1) to yield compound 1 (430.5 mg).
The EtOAc fraction (49.2 g) was subjected to silica gel CC and eluted
with a gradient of CH2Cl2-MeOH (100:1 to 2:1) to yield fifteen fractions
(E1–E15). Fr. E9 was subjected to silica gel CC and eluted with a gra-
dient of CHCl3-MeOH-H2O (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-H2O,
1:2 to 1:1) to yield seven subfractions (E9.4.1–E9.4.7). Compound 17
(1.8 mg) was obtained from Fr. E9.4.3 by Sephadex LH-20 CC (MeOH-
H2O, 1:2). Compound 2 (23.6 mg) was obtained from Fr. E9.4.7 by RP-
C18 CC eluted first with MeOH-H2O (1:1) and then with Me2CO-H2O
(4:7). Fr. E9.5 was separated by RP-C18 CC (Me2CO-H2O, 1:6 to 1:4) to
give seven subfractions (E9.5.1–E9.5.7). Fr. E9.5.6 was subjected to RP-
C18 CC (MeOH-H2O, 2:3) followed by RP-C18 CC (Me2CO-H2O, 1:2) to
provide compounds 5 (7.7 mg) and 6 (6.0 mg). Fr. E10 was separated
by silica gel CC (CHCl3-MeOH-H2O, 15:1:0.05 to 3:1:0.1) to afford
seven subfractions (E10.1–E10.7). Fr. E10.3 was further subjected to
RP-C18 CC (Me2CO-H2O, 1:5) to yield four fractions (E10.3.1–E10.3.4).
Fr. E10.3.1 was further subjected to RP-C18 CC (Me2CO-H2O, 1:6) to
yield compound 7 (1341.4 mg). Fr. E10.3.2 was subjected to RP-C18 CC
(MeOH-H2O, 2:5 to 2:3) to afford eight fractions (E10.3.2.1–E10.3.2.8).
Compounds 4 (4.5 mg), 13 (7.0 mg), and 12 (9.1 mg) were obtained by
the separations of Frs. E10.3.2.1, E10.3.2.3, and E10.3.2.7 on repeated
RP-C18 columns (MeOH-H2O, 1:2), respectively. Fr. E10.3.4 was sub-
jected to RP-C18 CC (MeOH-H2O, 2:3) followed by RP-C18 CC (Me2CO-
H2O, 1:2) to provide compound 10 (15.0 mg). Fr. E11 was subjected to
silica gel CC and eluted with a gradient of CHCl3-MeOH-H2O (6:1:0.1 to
2:1:0.1) to afford six subfractions (E11.1–E11.6). Fr. E11.4 was further
(calcd for C29H22NaO8, 521.1212).
4.3.3. Hyperelatone C (3)
Yellow amorphous powder;
[α]20 +78.8 (c 0.1, MeOH); UV (MeOH)
D
λmax (log ε) 214 (4.5), 291 (4.2) nm; IR (KBr) νmax 3413, 2949, 2837,
1640, 1461, 1370, 1171, 1021, 668 cm−1
; ECD (c = 0.1 mg/mL,
MeOH) λmax (Δε) 220 (−26.1), 291 (+31.2), 316 (+9.9) nm; 1H NMR
(CD3OD, 500 MHz) and 13C NMR data (CD3OD, 125 MHz), see Table 2;
HRESIMS (positive) m/z 683.1760 [M + Na]+ (calcd for C35H32NaO13
,
683.1741).
4.3.4. Hyperelatone D (4)
Brown amorphous powder; [α]2D0 −30.6 (c 0.008, MeOH); UV
(MeOH) λmax (log ε) 201 (4.6), 281 (3.7) nm; IR (KBr) νmax 3436, 2955,
2844, 1642, 1464, 1018, 664 cm−1; ECD (c = 0.05 mg/mL, MeOH)
λmax (Δε) 203 (15.8), 218 (−14.5), 254 (−6.0), 290 (−2.0) nm; 1H
NMR (CD3OD, 500 MHz) and 13C NMR data (CD3OD, 125 MHz), see
Table 2; HRESIMS (positive) m/z 482.1209 [M – H2O]+ (calcd for
C
25H22O10, 482.1213).
4.3.5. Hyperelatone E (9)
Yellow amorphous powder; [α]2D0 −15.2 (c 0.05, MeOH); UV
(MeOH) λmax (log ε) 238 (3.7), 261 (3.8) nm; IR (KBr) νmax 3414, 2927,
1706, 1520, 1384, 1222, 872 cm−1 1H NMR (DMSO‑d6, 500 MHz) and
;
13C NMR data (DMSO‑d6, 125 MHz), see Table 3; HRESIMS (positive)
m/z 451.1233 [M + H]+ (calcd for C21H23O11, 451.1240).
4.3.6. Hyperelatone F (10)
Yellow amorphous powder; [α]2D0 −9.6 (c 0.1, MeOH); UV (MeOH)
λ
max (log ε) 252 (4.0) nm; IR (KBr) νmax 3431, 2942, 1639, 1475, 1280,
1224, 1027, 673 cm−1 1H NMR (DMSO‑d6, 500 MHz) and 13C NMR
;
data (DMSO‑d6, 125 MHz), see Table 3; HRESIMS (positive) m/z
429.0787 [M + Na]+ (calcd for C19H18NaO10, 429.0798).
subjected to RP-C18 CC (Me2CO-H2O, 1:2) to give compound
3
4.3.7. Hyperelatone G (11)
Yellow amorphous powder; [α]2D0 −58.3 (c 0.1, MeOH); UV (MeOH)
λmax (log ε) 240 (4.1), 261 (4.2) nm; IR (KBr) νmax 3437, 1643, 1387,
(233.3 mg). Fr. E11.5 was purified on a RP-C18 column (MeOH-H2O,
1:3) followed by a RP-C18 column (Me2CO-H2O, 1:4) to yield compound
8 (33.7 mg). The n-BuOH fraction (71.0 g) was chromatographed over
silica gel and eluted with a gradient of CH2Cl2-MeOH (20:1 to 1:1) to
afford ten fractions (B1–B10). Fr. B1 was subjected to RP-C18 CC
(MeOH-H2O, 1:6 to 1:1) to yield six fractions (B1.1–B1.6). Fr. B1.4 was
further subjected to RP-C18 CC (MeOH-H2O, 1:5) to yield compound 16
(17.0 mg). Fr. B1.5 was purified on a RP-C18 column (MeOH-H2O, 2:5)
to give compound 18 (20.1 mg). Fr. B2 was separated using RP-C18 CC
(MeOH-H2O, 1:4) followed by RP-C18 CC (Me2CO-H2O, 1:6) to yield
compound 14 (18.4 mg). Fr. B3 was subjected to RP-C18 CC and eluted
with a gradient of MeOH-H2O (1:4 to 1:1) to afford fourteen fractions
(B3.1–B3.14). Fr. B3.6 was subjected to silica gel CC (CHCl3-MeOH-
H2O, 5:1:0.1) followed by RP-C18 CC (Me2CO-H2O, 1:4) to yield com-
pound 15 (88.7 mg). Fr. B3.11 was separated by Sephadex LH-20 CC
(MeOH-H2O, 2:3) to yield compounds 9 (3.3 mg) and 11 (6.0 mg).
1026, 771 cm−1 1H NMR (DMSO‑d6, 500 MHz) and 13C NMR data
;
(DMSO‑d6, 125 MHz), see Table 3; HRESIMS (positive) m/z 407.0978
[M + H]+ (calcd for C19H19O10, 407.0978).
4.3.8. Hyperelatone H (12)
Yellow amorphous powder; [α]2D0 −40.0 (c 0.01, MeOH); UV
(MeOH) λmax (log ε) 236 (4.1), 245 (4.1) nm; IR (KBr) νmax 3455, 2962,
1640, 1017, 668 cm−1 1H NMR (DMSO‑d6, 500 MHz) and 13C NMR
;
data (DMSO‑d6, 125 MHz), see Table 3; HRESIMS (positive) m/z
397.0899 [M + Na]+ (calcd for C19H18NaO8, 397.0899).
4.4. Acid hydrolysis
The absolute configurations of the sugar moieties of 3 and 9–12
were determined by the acid hydrolysis method (Tanaka et al., 2007).
Each compound (approximately 1.0 mg) was separately dissolved in 1 N
HCl (0.5 mL), and then heated to 90 °C in a water bath for 2 h. After
extraction with EtOAc two times, the H2O-soluble fraction was evapo-
rated to dryness under reduced pressure. The residue was dissolved in
pyridine (0.1 mL) containing L-cysteine methyl ester hydrochloride
(0.5 mg) and heated at 60 °C for 1 h. A 100 μL solution of o-tolyli-
sothiocyanate (0.5 mg) in pyridine was added and the reaction mixture
was heated at 60 °C for 1 h. Then each reaction mixture was analyzed by
the Wasters 1525 HPLC system using a Waters 2489 UV/vis detector (at
250 nm). Analytical HPLC was performed on the YMC Packed C18
column (5 μm, 150 × 4.6 mm) with a linear gradient elution (CH3CN-
H2O, 20:80 to 40:60) for 30 min. The derivative of D-glucose was
identified in 3 and 9–12 by a comparison of the retention time with
authentic D-glucose, which was subjected to the same derivatization
4.3.1. Hyperelatone A (1)
Colorless oil; [α]D20 +189.3 (c 0.1, CHCl3); UV (MeOH) λmax (log ε)
226 (3.9), 260 (2.4), 292 (2.0) nm; IR (KBr) νmax 3434, 1640 cm−1 1H
;
NMR (CDCl3, 500 MHz) and 13C NMR data (CDCl3, 125 MHz), see
Table 1; HRESIMS (positive) m/z 517.3316 [M + H]+ (calcd for
C
34H45O4, 517.3318).
4.3.2. Hyperelatone B (2)
Yellow amorphous powder; [α]2D0 +56.8 (c 0.05, MeOH); UV
(MeOH) λmax (log ε) 215 (4.6), 291 (4.3) nm; IR (KBr) νmax 3405, 2931,
2842, 1635, 1463, 1362, 1268, 1167, 1024, 670 cm−1
; ECD
(c = 0.1 mg/mL, MeOH) λmax (Δε) 228 (−11.4), 291 (+14.6), 316
(+5.2) nm; 1H NMR (CD3OD, 500 MHz) and 13C NMR data (CD3OD,
125 MHz), see Table 2; HRESIMS (positive) m/z 521.1221 [M + Na]+
72

X.-T. Yan et al.
Phytochemistry159(2019)65–74
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The data are expressed as means
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Conflicts of interest
There are no conflicts to declare.
Acknowledgments
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Pharmaceut. Sci. 5, 9–13.
This study was supported by the National Natural Science
Foundation of China (No. 31800291), Natural Science Basic Research
plan in Shaanxi Province (No. 2018JQ3032), the Fundamental
Research Funds for the Central Universities (No. 2452017173), and
Scientific Startup Foundation for Doctors of Northwest A&F University
(No. 2452015356).
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Appendix A. Supplementary data
Supplementary data related to this article can be found at https://
doi.org/10.1016/j.phytochem.2018.12.011.
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