Phenylpropanoid glycosides from the fruit of Lycium barbarum L. and their bioactivity

Article abstract of DOI:10.1016/j.phytochem.2019.04.017

Fifteen phenylpropanoid glycosides, including six undescribed compounds were isolated from the fruit of Lycium barbarum L. (Solanaceae)(goji or wolfberry). Their structures were identified by detailed spectroscopic analyses. Seven known compounds were firstly isolated from the genus Lycium, in which the 1D and 2D NMR data of one compound were reported for the first time. Notably, two undescribed compounds were a pair of rare tautomeric glycoside anomers characterized by the presence of free anomeric hydroxy. Antioxidant and hypoglycemic activities of all these compounds were assessed using DPPH radical scavenging, oxygen radical absorbance capacity (ORAC), and α-glucosidase inhibitory assays, respectively. These compounds showed different levels of oxygen radical absorbance capacity, and some isolates exhibited potent antioxidant activity with greater ORAC values than the positive control (EGCG).

Full text of DOI:10.1016/j.phytochem.2019.04.017

Phytochemistry164(2019)60–66
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Phenylpropanoid glycosides from the fruit of Lycium barbarum L. and their
bioactivity
Qing-Wen Lia,1, Rui Zhangb,1, Zheng-Qun Zhoua,c,∗∗, Wan-Yang Suna, Hong-Xia Fana,c
,
Ying Wangd, Jia Xiaob, Kwok-Fai Soe, Xin-Sheng Yaoa, Hao Gaoa,∗
a Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy / Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM
and New Drugs Research, Jinan University, Guangzhou 510632, People's Republic of China
b Clinical Medicine Research Institute, The First Affiliated Hospital of Jinan University, Guangzhou 510632, People's Republic of China
c Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou 510632, People's Republic of China
d Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, People's Republic of China
e Guangdong Medical Key Laboratory of Brain Function and Diseases, GMH Institute of Central Nervous System Regeneration, Jinan University, Guangzhou, 510632,
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:
Lycium barbarum L. (Solanaceae)
Goji
Fifteen phenylpropanoid glycosides, including six undescribed compounds were isolated from the fruit of Lycium
barbarum L. (Solanaceae) (goji or wolfberry). Their structures were identified by detailed spectroscopic analyses.
Seven known compounds were firstly isolated from the genus Lycium, in which the 1D and 2D NMR data of one
compound were reported for the first time. Notably, two undescribed compounds were a pair of rare tautomeric
glycoside anomers characterized by the presence of free anomeric hydroxy. Antioxidant and hypoglycemic ac-
tivities of all these compounds were assessed using DPPH radical scavenging, oxygen radical absorbance ca-
pacity (ORAC), and α-glucosidase inhibitory assays, respectively. These compounds showed different levels of
oxygen radical absorbance capacity, and some isolates exhibited potent antioxidant activity with greater ORAC
values than the positive control (EGCG).
Wolfberry
Phenylpropanoid glycosides
Antioxidant activity
Hypoglycemic activity
1. Introduction
moieties are added onto the aglycones at the post-modification stage via
stepwise glycosylation with various glycosyltransferases, which cata-
Lycium barbarum L. (Solanaceae) is a deciduous woody perennial
plant that grows in northwestern China and other parts of Asia, and its
ripe fruits (which are named goji or wolfberry) are 1–2 cm-long bright
red ellipsoid berries (Amagase and Farnsworth, 2011). Goji has been
used widely as a functional food and traditional Chinese medicine
(TCM) for replenishing vital essence to improve eyesight and nourish
the liver and kidneys (Qian et al., 2017). Modern pharmacological
studies suggested that goji exhibited various activities, such as anti-
oxidant, antitumor, hypoglycemic, and anti-Alzheimer's disease prop-
erties (Wojdyło et al., 2018; Zhou et al., 2016b). Glycosides are the
most abundant natural products in goji, including phenylpropanoid,
coumarin, lignan, flavonoid, and dicaffeoylspermidine glycosides (Zhou
et al., 2016a,b; Zhou et al., 2017; Gao and Yao, 2019). Glycosides occur
widely in plants, microorganisms, and animals. Usually, the sugar
lyze glycosidic bond formation between a sugar and acceptor (Yang
et al., 2015; Kulkarni et al., 2018).
In our previous chemical study on goji, some phenylpropanoid
glycosides were isolated (Zhou et al., 2017). This present phytochem-
ical restudy with a larger scale led to the discovery of fifteen phenyl-
propanoid glycosides (1–15) (Fig. 1), including six undescribed com-
pounds (1–6) and seven known compounds firstly reported from the
genus Lycium. Compounds 1 and 2 were obtained as a pair of insepar-
able anomers due to the tautomerism of the hemiacetal at C-1′ in so-
lution. Considering the biological activities of goji and the relationship
between hyperglycemia and oxidative tissue damage, antioxidant and
α-glucosidase inhibitory activities of compounds 1–15 were evaluated
(Amagase and Farnsworth, 2011; Zhou et al., 2016b, 2017; Cardullo
et al., 2019). Details of isolation, structural identification, and
∗ Corresponding author.
∗∗ Corresponding author. Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy / Guangdong Province Key Laboratory of
Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou, 510632, People's Republic of China.
E-mail addresses: tzhouzq@jnu.edu.cn (Z.-Q. Zhou), tghao@jnu.edu.cn (H. Gao).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.phytochem.2019.04.017
Received 23 February 2019; Received in revised form 5 April 2019; Accepted 25 April 2019
0031-9422/©2019ElsevierLtd.Allrightsreserved.

Q.-W. Li, et al.
Phytochemistry164(2019)60–66
Fig. 1. Chemical structures of compounds 1–15.
biological effects of 1−15 are reported in this paper.
the glucopyranosyls were determined by HPLC analysis of products
obtained from acid hydrolysis and derivatization reactions by L-cy-
steine methyl ester hydrochloride and o-tolyl isothiocyanate (Tanaka
et al., 2007). Analytical HPLC was performed on the Phenomenex Ge-
mini C18 column with isocratic elution of CH3CN–H2O–HCOOH
(25:75:0.1, v/v/v) for 40 min at a flow rate of 0.8 mL/min, and the
peaks of the standard monosaccharide and sample derivatives were
recorded at tR 20.4 (D-Glc), 18.7 (L-Glc), and 20.4 (1/2) min, respec-
tively. This evidence revealed that the two glucopyranosyls of 1/2 were
D-Glc. Therefore, compounds 1 and 2 were established as 2-O-trans-p-
coumaroyl-3-O-β-D-glucopyranosyl-β-D-glucopyranoside (named lyci-
barbarphenylpropanoid J) and 2-O-trans-p-coumaroyl-3-O-β-D-gluco-
pyranosyl-α-D-glucopyranoside (named lycibarbarphenylpropanoid K),
respectively.
Due to the tautomerism of the hemiacetal at C-1′, compounds 1 and
2 coexisted as a pair of inseparable anomers in solution (Fig. 3) (Sun
et al., 2016; Chen et al., 2013). As shown in Fig. 3, the peaks of 1 and 2
were observed at tR 9.0 and 10.2 min (Phenomenex Gemini C18 column,
4.6 × 250 mm2, 5 μm; CH3CN–H2O–HCOOH 10:90:0.1, v/v/v, flow rat
1.0 mL/min; 280 nm), respectively, and their relative peak area ratio
(27.6% for 1 and 72.4% for 2) in the HPLC chromatogram was ap-
proximately 1:3, which was corresponded to those of protons re-
sonances of 1 and 2.
Compound 3 was obtained as a yellowish amorphous powder. The
molecular formula of 3 was determined as C22H30O14 by HR-ESI-MS
(m/z 541.1533 [M + Na]+; calcd. for C22H30O14Na, 541.1533). The
detailed analyses of 1D/2D NMR data of 3 (Table S3, Supporting In-
formation) determined a planar structure consisting of trans-feruloyl
and two pyranohexose units. A precise comparison of 13C NMR data of
the sugar unit with those of glycosides recorded in the literature (Bock
and Pedersen, 1983; Usui et al., 2017) and the coupling values (J1'-
2' = 7.0 Hz, J1″-2'' = 7.8 Hz) indicated that the sugar chain of 3 was
glucopyranosyl-(1 → 6)-glucopyranosyl, and the relative configurations
of the two glucopyranosyls were β. Acid hydrolysis (Tanaka et al.,
2007) indicated that both glucopyranosyls were D-Glc. Therefore,
compound 3 was identified as 1-O-trans-feruloyl-6-O-β-D-glucopyr-
anosyl-β-D-glucopyranoside, and named lycibarbarphenylpropanoid L.
Compound 4 was obtained as a yellowish amorphous powder. The
molecular formula of 4 was determined as C21H28O13 by HR-ESI-MS
(m/z 511.1425 [M + Na]+; calcd. for C21H28O13Na, 511.1428). The
detailed analyses of 1D/2D NMR data of 4 (Table S4, Supporting In-
formation) determined a planar structure consisting of trans-p-cou-
maroyl and two pyranohexose units. A precise comparison of 13C NMR
data of the sugar unit with those of glycosides recorded in the literature
(Bock and Pedersen, 1983; Usui et al., 2017) and the coupling values
(J1'-2' = 7.6 Hz, J1″-2'' = 7.8 Hz) indicated that the sugar chain of 4 was
glucopyranosyl-(1 → 6)-glucopyranosyl, and the relative configurations
2. Results and discussion
Compounds 1 and 2 were obtained as yellowish amorphous pow-
ders and are tautomeric and coexist in solution. The quasi-molecular
ion at m/z 511.1426 [M + Na]+ by HR-ESI-MS showed that the mo-
lecular formulas of 1 and 2 were C21H28O13 (eight indices of hydrogen
deficiency). The NMR spectra of the mixture of 1 and 2 showed two sets
of signals, and the corresponding integral area ratios of 1H NMR signals
of 1 and 2 were approximately 1:3 (Figure S1, Supporting Information).
The 13C NMR spectrum of 1/2 displayed 21 carbons. Based on the
DEPT-135 data, these carbons could be categorized as one carbonyl,
eight aromatic or olefinic carbons (including six sp2 methine carbons),
10 oxygenated sp3 methine carbons, and two oxygenated sp3 methylene
carbons. The 1H NMR spectrum of 1/2 exhibited six aromatic or olefinic
protons and a set of glycosyl protons. The proton signals were asso-
ciated with the directly attached carbon atoms in the HSQC experiment.
The analysis of the 1H–1H COSY experiment and the coupling values of
the protons exhibited the presence of five subunits [C-2−C-3, C-5−C-6,
C-7−C-8, C-1'−C-2'−C-3'−C-4'−C-5'−C-6′, and C-1″−C-2″−C-
3″−C-4″−C-5″−C-6″]. Based on these deduced subunits, molecular
formulas, and degrees of unsaturation, the key HMBC correlations
shown in Fig. 2 determined the planar structures of 1/2. The assign-
ments of all proton and carbon resonances are provided in Table 1.
The planar structures of 1 and 2 exhibited two pyranohexose units.
Based on the coupling values (1: J1'-2' = 8.0 Hz, J2'-3' = 9.5 Hz, J3'-4'/J4'-
5' = 9.6/8.8 Hz; 2: J1'-2' = 3.6 Hz, J2'-3'/J3'-4'/J4'-5' = 9.0/9.8 Hz), the
inside pyranohexose units of 1 and 2 were established as glucopyr-
anosyls, and their relative configurations were identified as β (1) and α
(2), respectively. A precise comparison of 13C NMR data of the sugar
unit with those of glycosides recorded in the literature (Zhou et al.,
2017; Bock and Pedersen, 1983) and the coupling values (1: J1″-
2'' = 8.0 Hz; 2: J1″-2'' = 8.0 Hz) indicated that the outside pyranohexose
units of 1 and 2 were also glucopyranosyls, and both the relative con-
figurations of glucopyranosyls were β. The absolute configurations of
Fig. 2. Key 2D NMR correlations of compounds 1/2.
61

Q.-W. Li, et al.
Phytochemistry164(2019)60–66
Table 1
NMR data of 1−4 (δ in ppm, J in Hz).
No.
1b
2b
3c
4c
a
a
a
a
δC
δH
δC
δH
δC
δH
δC
δH
1
2
3
4
5
6
7
8
127.3
131.2
116.8
161.2
116.8
131.2
146.9
115.3
168.7
96.4
127.3
131.3
116.8
161.2
116.8
131.3
147.0
115.1
168.8
91.3
127.5
111.9
149.4
150.9
116.5
124.4
148.4
114.7
167.7
95.8
127.0
131.4
116.9
161.6
116.9
131.4
148.1
114.4
167.7
95.8
7.46, d (8.6)
6.80, d (8.6)
7.47, d (8.6)
6.80, d (8.6)
7.19, br s
7.48, d (8.7)
6.81, d (8.7)
6.80, d (8.6)
7.46, d (8.6)
7.64, d (15.9)
6.36, d (15.9)
6.80, d (8.6)
7.47, d (8.6)
7.66, d (15.9)
6.38, d (15.9)
6.81, d (8.1)
6.81, d (8.7)
7.48, d (8.7)
7.73, d (15.9)
6.36, d (15.9)
7.09, br d (8.1)
7.72, d (16.0)
6.40, d (16.0)
9
1′
2′
3′
4′
5′
6′
4.73, d (8.0)
4.93, dd (9.5, 8.0)
3.82
3.51, dd (9.6, 8.8)
3.40
5.37, d (3.6)
4.83
4.11, dd (9.8, 9.0)
3.54, dd (9.8, 9.0)
3.88
5.55, d (7.0)
3.45
3.45
3.45
5.55, d (7.6)
3.41
3.45
3.45
75.4
85.1
74.7
81.3
70.1
72.8
74.0
74.0
77.7*
71.0
77.8*
71.0
70.2
77.7*
62.6
77.9*
69.5
3.59
77.8*
69.5
3.58
3.82, Ha
62.5
3.88, Ha
4.17, dd (11.2, 1.4), Ha
4.16, dd (11.3, 1.6), Ha
3.71, dd (12.0, 5.6), Hb
4.38, d (8.0)
3.17, dd (9.2, 8.0)
3.27
3.27
3.30
3.75, dd (11.9, 5.0), Hb
4.51, d (8.0)
3.21, dd (9.2, 8.0)
3.35
3.30
3.35
3.77, dd (11.2, 5.2), Hb
4.33, d (7.8)
3.21
3.34
3.26
3.26
3.84, dd (12.0, 2.0), Ha
3.65, dd (12.0, 5.3), Hb
3.89, s
3.77, dd (11.3, 5.2), Hb
4.33, d (7.8)
3.20
3.34
3.26
3.26
1″
2″
3″
4″
5″
6″
104.8
74.7
77.6*
71.3
78.1
62.6
104.9
74.8
77.7
71.3
78.0
62.5
104.5
75.1
104.5
75.1
77.8*
71.5
78.0*
71.5
77.9*
62.7
78.0*
62.7
3.82, Ha
3.63, dd (11.9, 6.1), Hb
3.88, Ha
3.66, dd (11.9, 5.8), Hb
3.83, dd (12.0, 2.0), Ha
3.65, dd (12.0, 5.3), Hb
3-OCH3
56.5
∗
Assignment may be interchanged.
a
The indiscernible signals due to overlap or having the complex multiplicity are reported without designating multiplicity.
b
c
Measured in CD3OD (1H NMR for 600 MHz,13C NMR for 150 MHz).
Measured in CD3OD (1H NMR for 400 MHz,13C NMR for 100 MHz).
of the two glucopyranosyls were β. Acid hydrolysis (Tanaka et al.,
2007) indicated that both glucopyranosyls were D-Glc. Therefore,
compound 4 was identified as 1-O-trans-p-coumaroyl-6-O-β-D-gluco-
pyranosyl-β-D-glucopyranoside, and named lycibarbarphenylpropanoid
M.
glucopyranosyls were β. Acid hydrolysis (Tanaka et al., 2007) indicated
that these glucopyranosyls were D-Glc. Therefore, compounds 5 and 6
were identified as 4-O-[β-D-glucopyranosyl-(1 → 4)-β-D-glucopyr-
anosyl]-trans-p-coumaric acid (named lycibarbarphenylpropanoid N)
and 4-O-[β-D-glucopyranosyl-(1 → 3)-β-D-glucopyranosyl]-trans-p-cou-
maric acid (named lycibarbarphenylpropanoid O), respectively.
The known compounds, 4-O-[β-D-glucopyranosyl-(1 → 4)-β-D-glu-
copyranosyl]-trans-ferulic acid (7, CAS: 919803-07-9) (Table S7, Sup-
porting Information), lycibarbarphenylpropanoid B (8, CAS: 1988786-
72-6) (Zhou et al., 2017), lycibarbarphenylpropanoid A (9, CAS:
1988786-71-5) (Zhou et al., 2017), isosyringinoside (10, CAS 152686-
85-6) (Sugiyama et al., 1993), 4-O-β-D-glucopyranosyl-trans-caffeic
acid (11, CAS: 147511-61-3) (Nyandat et al., 1993), 3-O-β-D-gluco-
pyranosyl-trans-caffeic acid (12, CAS: 143729-78-6) (Table S8, Sup-
porting Information), 4-O-β-D-glucopyranosyl-trans-p-coumaric acid
(13, CAS: 117405-49-9) (Struijs et al., 2007), 4-O-β-D-glucopyranosyl-
cis-ferulic acid (14, CAS: 94942-20-8) (Table S9, Supporting Informa-
tion), and 4-O-β-D-glucopyranosyl-cis-p-coumaric acid (15, CAS:
Compounds 5 and 6 were obtained as a yellowish amorphous
powder. The molecular formulas of 5 and 6 were determined as
C
21H28O13 by HR-ESI-MS (5, m/z 511.1427 [M + Na]+; 6, m/z
511.1428 [M + Na]+; calcd. for C21H28O13Na, 511.1428). The detailed
analyses of 1D/2D NMR data of 5 and 6 (Tables S5 and S6, Supporting
Information) determined their planar structures consisting of trans-p-
coumaric acid and two pyranohexose units. A precise comparison of 13
C
NMR data of the sugar unit with those of glycosides recorded in the
literature (Bock and Pedersen, 1983; Veitch et al., 1998; Zi et al., 2008)
and the coupling values (5, J1'-2' = 7.7 Hz, J1″-2'' = 7.8 Hz; 6, J1'-
2' = 7.4 Hz, J1″-2'' = 7.7 Hz) indicated that the sugar chains of 5 and 6
were glucopyranosyl-(1 → 4)-glucopyranosyl and glucopyranosyl-(1 →
3)-glucopyranosyl, respectively, and the relative configurations of these
Fig. 3. The HPLC analytical chromatogram of compounds 1/2 and the tautomerism mechanism due to the hemiacetal at C-1'.
62

Q.-W. Li, et al.
Phytochemistry164(2019)60–66
Fig. 4. The antioxidant capacity of compounds 1–15 in vitro evaluated by the ORAC method. Each value is expressed as means
SE, n = 4.
117405-48-8) (Table S10, Supporting Information) were identified by
detailed spectroscopic analyses or comparison of their spectroscopic
data recorded in the literature. Among them, the 1D and 2D NMR data
of compound 7 were firstly reported, and compounds 7 and 10–15 were
firstly isolated from the genus Lycium.
(Thermo Fisher Scientific Inc., Sunnyvale, USA), and an Alltech (Grace)
2000 ES ELSD (Alltech Co. Ltd, Portland, USA) using a Phenomenex
Gemini C18 column (4.6 × 250 mm2, 5 μm) (Phenomenex Inc., Los
Angeles, USA) and a Cosmosil Packed C18 column (4.6 × 250 mm2,
5 μm) (Nacalai Tesque Inc., Kyoto, Japan). Semipreparative HPLC was
performed on a Dionex HPLC system, which was equipped with an
Ultimate 3000 pump and an Ultimate 3000 RS variable wavelength
detector using a Phenomenex Gemini C18 column (10.0 × 250 mm2,
5 μm). Preparative HPLC was performed on a Shimadzu LC-6-AD liquid
chromatography system (Shimadzu Inc., Kyoto, Japan) with an SPD-
20A detector using a Cosmosil Packed C18 column (20.0 × 250 mm2,
5 μm). The medium pressure liquid chromatography (MPLC) system
was equipped with a dual pump gradient system, an UV preparative
detector, and a Dr Flash II fraction collector system (Lisui E-Tech Co.
Ltd, Shanghai, China). Column chromatography (CC) was performed on
HP-20 macroporous resin (Mitsubishi Chemical Corporation, Tokyo,
Japan), silica gel (200–300 mesh, Haiyang Chemical Co. Ltd, Qingdao,
China), and ODS (50 μm, YMC Co. Ltd, Tokyo, Japan).
The antioxidant activities of compounds 1–15 were evaluated using
the DPPH radical scavenging assay with vitamin C as the positive
control. Only compounds 3 and 12 exhibited moderate DPPH radical
scavenging capacity, and the DPPH radical scavenging rate of other
compounds were less than 10% (Table S11, Supporting Information).
The antioxidant activities of compounds 1–15 were also evaluated
using an ORAC assay. All exhibited different levels of oxygen radical
absorbance capacity, and most of the tested compounds exhibited
stronger oxygen radical absorbance capacity than EGCG (Fig. 4).
The hypoglycemic activities of compounds 1–15 were evaluated by
the α-glucosidase inhibitory assay with acarbose as the positive control.
None exhibited potent α-glucosidase inhibitory activity. (Table S13,
Supporting Information).
Most carbohydrates found in nature exist as polysaccharides, gly-
coconjugates, or glycosides, in which sugar units are attached to one
another or to aglycones through glycosidic bonds at the anomeric po-
sitions (Zhu and Schmidt, 2009; Ati et al., 2017). However, there are
special cases, and some rare phenylpropanoid glycosides with free
anomeric hydroxy were reported, including 6-O-trans-sinapoyl-α/β-D-
glucopyranoside, 3,6-O-dicaffeoyl-α/β-glucopyranoside, and 2,3,4,6-O-
tetragalloyl-β-D-glucopyranoside (Lou et al., 1993; Kashiwada et al.,
1992; Hussein et al., 2003). During this restudy of phytochemical
constituents on goji, two unusual glycosides (compounds 1 and 2)
characterized by the presence of free anomeric hydroxy were dis-
covered. They were determined to be a pair of inseparable glycosidic
anomers in solution, and this phenomenon is rooted in the tautomerism
of the hemiacetal at the glucopyranosyls of 1 and 2.
3.2. Plant material
The fruit of Lycium barbarum L. (Solanaceae) was both collected
(Zhongning County, Ningxia Hui Autonomous Region, China) and
identified by one of the authors (Ying Wang) in 2016. A voucher spe-
cimen (LYBA-2016-NX-ZN) was deposited in the Institute of Traditional
Chinese Medicine and Natural Products, College of Pharmacy, Jinan
University, Guangzhou, China.
3.3. Extraction and isolation
The dried goji (45.0 kg) was cold-soak extracted four times with
100 L of CHCl3 for 24 h each time. After filtration and evaporation of
the CHCl3, the residue was heated to reflux five times with 120 L of 60%
EtOH–H2O for 2 h each time. After filtration and evaporation of the
EtOH in vacuo, the concentrated solution was passed through a HP-20
macroporous resin column (20.0 × 150.0 cm2) using successive elu-
tions of EtOH–H2O (0:100, 30:70, 50:50, 95:5, v/v), yielding fractions
F1–F4.
A portion (900.0 g) of F2 (1.9 kg) was subjected to open silica gel CC
(14.0 × 100.0 cm2) using successive elutions of CHCl3–MeOH–H2O
(80:20:2, 70:30:3, 60:40:4, 50:50:5, 40:60:6, 0:100:0, v/v/v) to yield
fractions 2.1–2.5. Fraction 2.3 (100.0 g) was subjected to ODS-MPLC
(5.0 × 80.0 cm2) using successive elutions of MeOH–H2O (5:95, 10:90,
15:85, 20:80, 100:0, v/v) to yield fractions 2.3.1–2.3.6. A portion
(20.0 g) of fraction 2.3.1 (53.0 g) was subjected to open silica gel CC
(5.0 × 60.0 cm2) using successive elutions of CH2Cl2–MeOH–H2O
(85:15:1.5, 80:20:2, 70:30:3, 60:40:4, 50:50:5, 0:100:0, v/v/v) to yield
fractions 2.3.1.1–2.3.1.7.
3. Experimental
3.1. General experimental procedures
Optical rotations were recorded on an Anton Paar MCP 200 high
precision intelligent polarimeter (Anton Paar Co. Ltd, Graz, Austria).
UV data were measured on a JASCO V-550 UV/Vis spectrometer (Jasco
International Co. Ltd, Tokyo, Japan). IR data were recorded using a
JASCO FT/IR-4600 plus spectrometer. The ESI-MS spectra were re-
corded on a Bruker amazon SL mass spectrometer (Bruker Daltonics
Int., Boston, USA). The HR-ESI-MS spectra were obtained on a Waters
Synapt G2 TOF mass spectrometer (Waters Corporation, Milford, USA).
The NMR spectra were acquired with Bruker AV 400 and 600 spec-
trometers (Bruker BioSpin Group, Faellanden, Switzerland) using the
solvent signals (DMSO-d6: δH 2.50/δC 39.5; CD3OD: δH 3.30/δC 49.0) as
internal standards. Analytical HPLC was performed on a Dionex HPLC
system equipped with an Ultimate 3000 pump, an Ultimate 3000 DAD,
an Ultimate 3000 column compartment, an Ultimate 3000 autosampler
Fraction 2.3.1.3 (683.2 mg) was subjected to MPLC on ODS CC
(2.7 × 12.7 cm2) using successive elutions of MeOH–H2O–HCOOH
(5:95:0.2, 10:90:0.2, 15:85:0.2, 20:80:0.2, 25:75:0.2, 100:0:0, v/v/v) to
63

Q.-W. Li, et al.
Phytochemistry164(2019)60–66
yield fractions 2.3.1.3.1–2.3.1.3.6. Fraction 2.3.1.3.3 (145.2 mg) was
isolated using semipreparative HPLC [12% MeOH–H2O (containing
0.2% HCOOH), 3 mL/min] to yield 13 (tR: 36.5 min, 43.6 mg) and 11
(tR: 40.2 min, 9.5 mg). Fraction 2.3.1.3.4 (180.1 mg) was isolated using
semipreparative HPLC [15% MeOH–H2O (containing 0.2% HCOOH),
3 mL/min] to yield 15 (tR: 35.6 min, 112.0 mg) and 12 (tR: 38.8 min,
13.4 mg). Fraction 2.3.1.3.6 (32.4 mg) was isolated using semi-
preparative HPLC [20% MeOH–H2O (containing 0.2% HCOOH), 3 mL/
min] to yield 14 (tR: 30.5 min, 13.4 mg). Fraction 2.3.1.5 (7.5 g) was
subjected to MPLC on ODS CC (2.7 × 25.4 cm2) using successive elu-
tions of MeOH–H2O–HCOOH (5:95:0.2, 10:90:0.2, 15:85:0.2,
Table 2
NMR data of 5−7 (δ in ppm, J in Hz).
No.
5b
6b
7b
a
a
a
δC
δH
δC
δH
δC
δH
1
2
3
4
5
6
128.6
128.4
128.3, C
129.4 7.58, d (8.6) 129.7 7.60, d (8.5) 111.2, CH 7.33, d (1.0)
116.4 7.04, d (8.6) 116.5 7.05, d (8.5) 149.1, C
158.4
158.6
148.2, C
116.4 7.04, d (8.6) 116.5 7.05, d (8.5) 114.9, CH 7.10, d (8.5)
129.4 7.58, d (8.6) 129.7 7.60, d (8.5) 122.1, CH 7.18, dd
(8.5, 1.0)
20:80:0.2,
25:75:0.2,
100:0:0,
v/v/v)
to yield fractions
7
8
141.6 7.44, d
(15.8)
119.8 6.39, d
(15.8)
142.7 7.48, d
(15.9)
118.5 6.39, d
(15.9)
143.8, CH 7.52, d
(15.9)
117.4, CH 6.46, d
(15.9)
2.3.1.5.1–2.3.1.5.7. Fraction 2.3.1.5.3 (615.8 mg) was isolated using
preparative HPLC [10% CH3CN–H2O (containing 0.1% CF3COOH),
8 mL/min] to yield 1/2 (1, tR: 26.1 min; 2, tR: 29.6 min; 41.5 mg).
Fraction 2.3.1.5.4 (1.09 g) was isolated using semipreparative HPLC
[15% MeOH–H2O (containing 0.2% HCOOH), 3 mL/min] to yield 5 (tR:
25.4 min, 20.1 mg) and 4 (tR: 33.8 min, 5.0 mg). Fraction 2.3.1.5.5
(602.3 mg) was isolated using preparative HPLC [8% CH3CN–H2O
(containing 0.1% CF3COOH), 10 mL/min] to yield 10 (tR: 14.5 min,
1.7 mg), 6 (tR: 33.2 min, 4.2 mg), 3 (tR: 39.0 min, 30.3 mg), and 8 (tR:
53.5 min, 87.1 mg). Fraction 2.3.1.5.6 (1.31 g) was isolated using pre-
parative HPLC [9% CH3CN–H2O (containing 0.1% CF3COOH), 8 mL/
min] to yield fractions 2.3.1.5.6.1–2.3.1.5.6.7. Fraction 2.3.1.5.6.3
(240.6 mg) was isolated using semipreparative HPLC [15% MeOH–H2O
(containing 0.2% HCOOH), 3 mL/min] to yield 7 (tR: 52.5 min, 6.5 mg).
Fraction 2.3.1.5.6.7 (117.6 mg) was isolated using semipreparative
HPLC [22% MeOH–H2O (containing 0.2% HCOOH), 3 mL/min] to yield
9 (tR: 29.7 min, 4.1 mg).
9
168.9
168.4
167.9, C
1′
2′
3′
4′
5′
6′
99.6
72.9
74.8
79.8
75.0
60.0
5.02, d (7.7) 99.3
5.07, d (7.4) 99.1, CH
3.47 72.8, CH
75.1*, CH 3.44
5.06, d (7.8)
3.31, t (8.0)
3.46
3.44
72.0
87.6
68.1
76.5
60.5
3.32
3.51
3.32, t (8.6) 79.8, CH
3.44
3.56
3.45
3.70, Ha
3.49, Hb
75.0*, CH 3.54
3.75, br d
(12.2), Ha
3.64, dd
(12.0, 4.3),
Hb
60.0
3.72, br d
(11.6), Ha
3.64, Hb
1″
2″
3″
4″
5″
6″
103.1 4.30, d (7.8) 104.0 4.36, d (7.7) 103.1, CH 4.29, d (7.8)
73.3
76.5
70.0
76.8
61.0
3.02, t (8.4)
3.19
3.07, t (9.2)
3.19
3.71, br d
(12.5), Ha
3.42, Hb
73.9
76.1
70.2
77.0
61.1
3.09
3.20
3.05
3.20
73.3, CH
76.5, CH
70.0, CH
76.8, CH
61.0
3.02
3.20
3.07
3.20
3.72, br d
(11.6), Ha
3.42, Hb
3.70, Ha
3.40, dd
(11.7, 7.0),
Hb
3.4. Structural characterization of undescribed compounds
3-OCH3
2′-OH
3′-OH
56.5, CH3 3.81, s
5.44, d (5.8)
3.4.1. Lycibarbarphenylpropanoids J/K (1/2)
4.80, br s
4.62
5.24, d (3.0)
5.01, br s
5.01, br s
4.62
Yellowish amorphous powders; [α]2D5 +28.0 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 212 (4.22), 229 (4.17), 292 (4.35), 314 (4.45) nm;
IR (KBr) vmax 3338, 2908, 1700, 1596, 1513, 1036 cm−1; ESI-MS (po-
sitive) m/z 511.2 [M + Na]+, 999.2 [2M + Na]+; HR-ESI-MS (posi-
tive) m/z 511.1426 [M + Na]+ (calcd. for C21H28O13Na, 511.1428); 1H
and 13C NMR data see Table 1.
6′-OH
2″″-OH
3″″-OH
4″″-OH
6″″-OH
∗
Assignment may be interchanged.
a
The indiscernible signals due to overlap or having the complex multiplicity
3.4.2. Lycibarbarphenylpropanoid L (3)
are reported without designating multiplicity.
Yellowish amorphous powder; [α]2D5 −8.0 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 204 (4.26), 237 (3.91), 295 (3.94), 329 (4.16) nm;
IR (KBr) vmax 3294, 2923, 1714, 1596, 1516, 1065 cm−1; ESI-MS (po-
sitive) m/z 519.2 [M + H]+; HR-ESI-MS (positive) m/z 541.1533 [M +
Na]+ (calcd. for C22H30O14Na, 541.1533); 1H and 13C NMR data see
Table 1.
b
Measured in DMSO-d6 (1H NMR for 400 MHz,13C NMR for 100 MHz).
(MeOH) λmax (log ε) 207 (4.03), 220 (3.95), 285 (4.13), 306 (4.00) nm;
IR (KBr) vmax 3300, 2917, 1685, 1604, 1513, 1077 cm−1; ESI-MS (po-
sitive) m/z 489.2 [M + H]+, 977.1 [2M + H]+; HR-ESI-MS (positive)
m/z 511.1428 [M + Na]+ (calcd. for C21H28O13Na, 511.1428); 1H and
13C NMR data see Table 2.
3.4.3. Lycibarbarphenylpropanoid M (4)
Yellowish amorphous powder; [α]2D5 −52.0 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 211 (4.08), 230 (4.09), 316 (4.48) nm; IR (KBr)
vmax 3300, 2917, 1709, 1604, 1513, 1062 cm−1; ESI-MS (positive) m/z
511.1 [M + Na]+, 999.2 [2M + Na]+; HR-ESI-MS (positive) m/z
511.1425 [M + Na]+ (calcd. for C21H28O13Na, 511.1428); 1H and 13C
NMR data see Table 1.
3.5. Acid hydrolysis
The acid hydrolysis was performed using a previously described
method with slight modifications (Tanaka et al., 2007). The compound
(1.0 mg) was hydrolyzed with 2 M of HCl for 1 h at 90 °C. After ex-
traction with EtOAc two times, the H2O layer was evaporated in vacuo
using an Eyela N-1001 rotary evaporator (Tokyo Rikakikai Co. Ltd,
Tokyo, Japan) to furnish a monosaccharide residue. The residue was
dissolved in pyridine (1.0 mL) containing L-cysteine methyl ester hy-
drochloride (1.0 mg) and heated at 60 °C. After 1 h, 20 μL of o-tolyl
isothiocyanate was added to the reaction mixture and further reacted at
60 °C for 3 h. Then the reaction mixture was directly analyzed by the
Dionex HPLC system and detected by a UV detector (at 250 nm). The
standard monosaccharides of D-Glc and L-Glc were subjected to the
same method.
3.4.4. Lycibarbarphenylpropanoid N (5)
Yellowish amorphous powder; [α]2D5 −48.0 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 208 (4.29), 221 (4.15), 285 (4.29), 306 (4.14) nm;
IR (KBr) vmax 3184, 2884, 1697, 1604, 1513, 1080 cm−1; ESI-MS (po-
sitive) m/z 489.2 [M + H]+, 977.3 [2M + H]+; HR-ESI-MS (positive)
m/z 511.1427 [M + Na]+ (calcd. for C21H28O13Na, 511.1428); 1H and
13C NMR data see Table 2.
3.4.5. Lycibarbarphenylpropanoid O (6)
Yellowish amorphous powder; [α]2D5 −14.0 (c 0.10, MeOH); UV
64

Q.-W. Li, et al.
Phytochemistry164(2019)60–66
3.6. DPPH radical scavenging assay
(2017BT01Y036), and K. C. Wong Education Foundation (Hao Gao,
2016). Assistance with Scientific English was provided by Dr L.J.
Sparvero of the University of Pittsburgh.
The DPPH radical scavenging assay was performed based on the
method with slight modifications (Cardullo et al., 2019; Xie et al., 2015;
Hidayat et al., 2017; Lu et al., 2017). In a 96-well plate, 100 μL of
0.2 mM DPPH radical solution in ethanol was added to 100 μL of the
0.2 mM sample solutions in ethanol. The mixture was wobbled for
30 min in the dark. Vitamin C was used as a positive control. The ab-
sorbance values were measured at 517 nm using a Synergy HT micro-
plate reader (Bio-Tek Instruments Inc., Burleigh, USA). All of the
samples were performed in triplicate. The DPPH radical scavenging rate
(S%) was calculated as follows: S% = [(A0 – A1)/ A0] × 100 (A1 and A0
are the absorbance of the incubation DPPH radical solution with and
without the tested sample, respectively).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2019.04.017.
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Acknowledgements
This work was financially supported by grants from the National
Natural Science Foundation of China (3171101305, 81703372, and
81703696), Chang Jiang Scholars Program (Hao Gao, 2017) from the
Ministry of Education of China, Guangdong Special Support Program
(2016TX03R280), Guangdong Province Universities and Colleges Pearl
River Scholar Funded Scheme (Hao Gao, 2014), Local Innovative and
Research Teams Project of Guangdong Pearl River Talents Program
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66