Flavonol glycosides and phenylpropanoid glycosides with inhibitory effects on microglial nitric oxide production from Neoshirakia japonica

Article abstract of DOI:10.1016/j.fitote.2021.104877

Five new flavonol glycosides (1–5), one new phenylpropanoid glycoside (6), and nine known glycosides (7–15) were isolated from the stems and leaves of Neoshirakia japonica. The structures of the new compounds were determined by detailed analysis of spectroscopic data (HRESIMS, 1D and 2D NMR) and acid hydrolysis experiment. The antineuroinflammatory effects of all the isolates were evaluated by inhibiting NO production against LPS-induced BV-2 microglial cells. Compounds 1, 8, and 9 showed more potent inhibitory activities with IC₅₀ values of 2.7, 5.5, and 4.1 μM, respectively, than that of the positive control minocycline (IC₅₀ = 15.6 μM), while compounds 7 (IC₅₀ = 17.0 μM) and 10 (IC₅₀ = 24.3 μM) also displayed inhibitory activities to a certain degree.

Full text of DOI:10.1016/j.fitote.2021.104877

Fitoterapia 151 (2021) 104877
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Flavonol glycosides and phenylpropanoid glycosides with inhibitory effects
on microglial nitric oxide production from Neoshirakia japonica
a
,
b
a
a
a
a
a
Hai-Yan Zhao , Ya-Qi Wang , Yan-Cai Li , Qian Lan , Hai-Bing Liao , Heng-Shan Wang ,
Dong Liang
a,*
a
State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Collaborative Innovation Center for Guangxi Ethnic Medicine, School of
Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004, People’s Republic of China
b
College of Food and Biochemical Engineering, Guangxi Science & Technology Normal University, Laibin 546199, 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:
Five new flavonol glycosides (1–5), one new phenylpropanoid glycoside (6), and nine known glycosides (7–15)
were isolated from the stems and leaves of Neoshirakia japonica. The structures of the new compounds were
determined by detailed analysis of spectroscopic data (HRESIMS, 1D and 2D NMR) and acid hydrolysis exper-
iment. The antineuroinflammatory effects of all the isolates were evaluated by inhibiting NO production against
LPS-induced BV-2 microglial cells. Compounds 1, 8, and 9 showed more potent inhibitory activities with IC50
Neoshirakia japonica
Flavonol glycoside
Phenylpropanoid glycoside
Antineuroinflammatory
values of 2.7, 5.5, and 4.1
μ
M, respectively, than that of the positive control minocycline (IC50 = 15.6
μM), while
compounds 7 (IC50 = 17.0
μ
M) and 10 (IC50 = 24.3 M) also displayed inhibitory activities to a certain degree.
μ
1
. Introduction
Sapium japonicum (revised as Neoshirakia japonica), as one species of
lipopolysaccharide (LPS)-induced BV-2 microglial cells were reported.
2. Material and methods
the genus Sapium in the family Euphorbiaceae, is a shrub growing in
moist places of forest or near streams and widely distributed in China,
Japan, and North Korea [1]. Many species in the genus Sapium have been
used as traditional medicines to treat eczema, scabies, detoxification,
and detumescenece [1,2]. Meanwhile, previous phytochemical and
pharmacological researches on this genus have revealed a wide array of
diterpenoids, triterpenoids, flavonoids, lignanoids, and coumarins with
significant biological activities [3].
2.1. General experimental procedures
HRESIMS data were performed on Agilent 6545 Q-TOF LC-MS
spectrometer. NMR spectra were carried out on Bruker AV-400 or AV-
600 NMR spectrometer and chemical shifts were expressed in δ (ppm)
and referenced to the solvent residual peak. Optical rotations were
recorded by a JASCO P-2000 polarimeter. Silica gel (200–300 mesh,
Qingdao Marine Chem. Co., Ltd., China), C18 reversedphase silica gel
Our previous study on chemical constituents and biological activity
of the aerial parts of S. discolor (revised as Triadica cochinchinensis) and S.
rotundifolium (revised as Triadica rotundifolia) acquired several anti-
neuroinflammatory coumarinolignoids, diterpenoids, triterpenoids, and
lignanoids [4–6], so in order to discover novel natural inhibitors of
neuroinflammation, a chemical investigation on the stems and leaves of
N. japonica was carried out. At last, six new (1–6) including five new
flavonol glycosides (1–5) as well as one new phenylpropanoid glycoside
(50
μ
m, YMC, Japan), MCI gel (CHP20, 75–150
μ
m, MCC, Japan), and
Sephadex LH-20 gel (Pharmacia Biotech, Sweden) were used for column
chromatography (CC). The other testing instruments for the purified
compounds and the materials used in the isolation were the same as
previous [7,8].
2
.2. Plant material
(
6) and nine known (7–15) compounds (Fig. 1) were isolated. Com-
′
′
pounds 2–5 are rare 2 -O-galloyl-type kaempferol glycosides. Herein,
the isolation and structural elucidation of these isolates, and their po-
tential inhibitory effects on nitric oxide (NO) production in
The stems and leaves of N. japonica were collected from Baise located
in Guangxi Zhuang Autonomous Region of China (April 2019), and were
authenticated by Prof. Shao-Qing Tang (College of Life Science, Guangxi
*
Corresponding author.
E-mail address: liangdonggxnu@163.com (D. Liang).
https://doi.org/10.1016/j.fitote.2021.104877
Received 22 January 2021; Received in revised form 24 February 2021; Accepted 28 February 2021
Available online 3 March 2021
0
367-326X/© 2021 Elsevier B.V. All rights reserved.

H.-Y. Zhao et al.
Fitoterapia 151 (2021) 104877
Normal University). A voucher specimen (No. NJ-201904) has been
deposited at the Collaborative Innovation Center for Guangxi Ethnic
Medicine, Guangxi Normal University.
3 2 R
0.5 mL/min). Compound 15 (CH CN/H O, 30:70, 6 mL/min, 4.2 mg, t
48.3 min) was obtained via semi-preparative RP-HPLC from subfraction
D5c2 (13.3 mg). Subfraction D6b (20.6 mg) were performed by semi-
preparative RP-HPLC (CH
12.7 mg, t 50.2 min).
Fraction D6c (319.0 mg) was chromatographed on a Sephadex LH-20
3 2
CN/H O, 25:75, 6 mL/min) to obtain 10
(
R
2
.3. Extraction and isolation
column (2.6 cm × 120 cm, MeOH, 0.5 mL/min) to afford five sub-
fractions (D6c1ꢀ D6c5). Furthermore, subfraction D6c4 (33.1 mg) was
The air-dried stems and leaves of N. japonica (20.5 kg) were extracted
◦
four times at the temperature of 70 C (each for 3 h) by refluxing under
3 2
purified via semi-preparative RP-HPLC (CH CN/H O, 25:75, 6 mL/
the conditions of 90% EtOH-H
evaporated under reduced pressure to obtain the crude extract (2.5 kg),
which was suspended in H O and sequentially partitioned with petro-
leum ether, EtOAc, and n-butyl alcohol. The EtOAc-soluble fraction
1100 g) was subjected to macroporous resin (D101) column chroma-
tography (CC), eluting with EtOH/H O (30:70, 50:50, 80:20, and 95:5,
v/v). Then, the EtOH/H O-80:20 fraction (121.0 g) was fractionated
using MCI gel CC with MeOH/H O (30:70 to 100:0) to obtain seven
fractions (A ꢀ G). Fractions C (14.6 g) and D (14.1 g) were separated by
a silica gel (200–300 mesh) CC respectively, eluting with CH Cl /MeOH
80:1 to 1:1) of increasing polarity to obtain the following fractions:
2
O (4 × 75 L). The above filtrate was
min), leading to the isolation of 1 (2.8 mg, t
68.7 min). Subfraction D6c5 (15.8 mg) was performed by semi-
preparative HPLC (CH CN/H O, 25:75, 6 mL/min) to provide 7 (3.5
mg, t 72.5 min) and 11 (4.5 mg, t 80.9 min).
Subfractions D7c (42.0 mg) and D7d (39.2 mg) were purified using
semi-preparative RP-HPLC to yield 3 (CH CN/H O/TFA, 27:73:0.01, 6
mL/min, 30.7 mg, t 62.5 min) and 2 (CH CN/H O, 27:73, 6 mL/min,
.8 mg, t 69.5 min), respectively. Furthermore, semi-preparative RP-
HPLC (CH CN/H O/TFA, 28.5:71.5:0.01, 6 mL/min) purification of
subfraction D7e (87.0 mg) resulted in the isolation of 13 (4.6 mg, t 51.3
min), 4 (2.3 mg, t 54.7 min), and 5 (10.1 mg, t 58.4 min).
R
64.1 min) and 12 (11.2 mg,
t
R
2
3
2
R
R
(
2
3
2
2
R
3
2
2
3
R
3
2
2
2
R
(
R
R
C1ꢀ C8 and D1ꢀ D8.
Fractions C3 (2100.0 mg) and C4 (800.0 mg) were subjected to
Sephadex LH-20 columns (4 cm × 120 cm, MeOH, 0.8 mL/min) to yield
the subfractions of C3aꢀ C3d and C4aꢀ C4d. Semi-preparative RP-HPLC
′
′
2
.3.1. Kaempferol 3-O-(6 -O-feruloyl)-β-D-galactopyranoside (1)
20
Yellow amorphous powder; [
max (log ) 237 (4.47), 263 (4.46), 280.0 (4.44), 328 (4.47) nm; IR (KBr)
max 3445, 2938, 2869, 1655, 1609, 1507, 1449, 1385, 1181, 812 cm
α
]
D
ꢀ 13.8 (c 0.07, MeOH); UV (MeOH)
λ
ε
purification of C3c (42.2 mg) with CH
min) afforded 14 (11.6 mg, t
Subfraction C4d (31.7 mg) was purified using semi-preparative RP-
HPLC with CH CN/H O/TFA (27:73:0.01, 6 mL/min) to yield 8 (5.7 mg,
36.6 min) and 9 (4.1 mg, t 49.2 min).
Fractions D5 (1140.0 mg), D6 (725.0 mg), and D7 (960.0 mg) were
fractioned over RP-C18 CC (MeOH/H O, 30:70 to 100:0) of decreasing
3
CN/H
2
O/TFA (27:73:0.01, 6 mL/
ꢀ 1
ν
;
R
38.9 min) and 6 (3.1 mg, t
R
44.7 min).
1
13
H and C NMR data, see Table 1; (+) HRESIMS m/z 647.1373 [M +
+
Na] , calcd for C31
H
28
O
14Na, 647.1371.
3
2
t
R
R
′
′
′′
2
.3.2. Kaempferol 3-O-(2 -O-galloyl-6 -O-feruloyl)-β-D-galactopyranoside
(
2)
2
2
0
Yellow amorphous powder; [
α]
D
ꢀ 35.0 (c 0.09, MeOH); UV (MeOH)
polarity to obtain the subfractions of D5aꢀ D5e, D6aꢀ D6e, and
D7aꢀ D7f, respectively.
λ
ν
1
max (log
ε
) 209 (4.51), 267 (4.20), 292 (4.16), 327 (4.15) nm; IR (KBr)
max 3435, 2929, 2860, 1661, 1607, 1516, 1451, 1385, 1203, 1178,
039, 703 cm 1; 1H and C NMR data, see Table 1; (+) HRESIMS m/z
Fraction D5c (212.5 mg) was separated into five subfractions
ꢀ
13
(
D5c1ꢀ D5c5) via a Sephadex LH-20 column (2.6 cm × 120 cm, MeOH,
Fig. 1. Chemical structures of compounds 1–15.
2

H.-Y. Zhao et al.
Fitoterapia 151 (2021) 104877
Table 1
1
13
H and C NMR data of compounds 1–5.
a
a
b
a
b
NO.
1
2
3
4
5
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
δ
H
(J in Hz)
δ
C
H
δ (J in Hz)
2
3
4
5
6
7
8
9
159.2
135.5
179.6
162.9
99.9
158.2
134.6
179.4
163.0
99.8
158.3
134.6
179.4
163.0
99.9
158.8
134.3
179.1
163.0
99.8
158.8
134.3
179.1
163.0
99.8
6.10, br s
6.29, br s
6.02, d (1.8)
6.21, d (1.8)
6.07, br s
6.25, br s
6.07, d (1.8)
6.27, d (1.8)
6.03, d (1.8)
6.23, d (1.8)
165.9
94.7
165.6
94.5
165.6
94.6
165.6
94.6
165.5
94.6
158.4
105.5
122.6
132.4
116.1
161.6
116.1
132.4
105.0
72.9
158.2
105.7
122.7
132.1
116.3
161.4
116.3
132.1
100.8
74.2
158.3
105.7
122.7
132.1
116.3
161.4
116.3
132.1
100.8
74.2
158.3
105.7
122.8
132.1
116.1
161.4
116.1
132.1
100.2
75.8
158.3
105.7
122.8
132.1
116.1
161.3
116.1
132.1
100.2
75.8
1
1
2
3
4
5
6
1
2
3
4
5
6
0
′
′
′
′
′
′
′
′
′
′
′
′
8.05, d (9.0)
6.85, d (9.0)
7.92, d (9.0)
6.81, d (9.0)
7.92, d (8.8)
6.81, d (8.8)
7.93, d (8.4)
6.84, d (8.4)
7.92, d (8.8)
6.83, d (8.8)
6.85, d (9.0)
8.05, d (9.0)
5.12, d (7.8)
3.80, m
6.81, d (9.0)
6.81, d (8.8)
7.92, d (8.8)
5.61, d (8.0)
5.45, dd (10.0, 8.0)
3.84, m
6.84, d (8.4)
6.83, d (8.8)
7.92, d (9.0)
7.92, d (8.4)
7.92, d (8.8)
′
′
′
′
′
′
5.61, d (8.1)
5.71, d (8.1)
5.72, d (8.0)
5.45, dd (9.6, 8.1)
3.85, dd (9.6, 3.0)
3.90, m
5.14, dd (9.6, 8.1)
3.70, dd (9.6, 9.0)
3.43, dd (9.6, 9.0)
3.55, m
5.14, dd (9.6, 8.0)
3.70, dd (9.6, 8.8)
3.44, dd (9.6, 8.8)
3.57, m
74.9
3.57, dd (9.6, 3.0)
3.82, m
73.2
73.2
76.2
76.3
70.3
70.7
70.7
3.89, m
72.1
72.1
74.8
3.76, dd (8.4, 3.6)
4.39, dd (11.4, 8.4)
75.1
3.86, m
75.0
3.85, m
76.0
76.0
64.4
64.2
4.47, dd (11.4, 8.4)
4.16, dd (11.4, 4.2)
64.2
4.41, dd (11.2, 8.4)
4.19, dd (11.2, 4.0)
64.0
4.36, dd (11.4, 1.8)
4.23, dd (11.4, 7.2)
64.0
4.35, dd (11.6, 2.0)
4.28, dd (11.6, 6.4)
4
.12, dd (11.4, 3.6)
′
′
′
′
′
′
′
′
′
′
′
′
′
′
′
′
′
′′
′′
′′
′′
′′
′′
′′
′′
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
3
127.6
111.5
149.2
150.5
116.4
124.1
146.8
114.9
168.7
127.6
111.5
149.3
150.6
116.4
124.2
146.9
114.8
168.6
121.6
110.6
146.4
139.9
146.4
110.6
168.0
56.4
127.0
131.2
116.8
161.2
116.8
131.2
146.6
114.6
168.7
121.6
110.6
146.4
139.9
146.4
110.6
168.0
127.0
131.2
116.8
161.2
116.8
131.2
146.6
114.6
168.7
121.5
110.6
146.4
139.9
146.4
110.6
167.7
127.6
111.5
149.3
150.5
116.4
124.3
146.8
114.9
168.7
121.6
110.6
146.4
139.9
146.4
110.6
167.8
56.4
7.02, d (1.2)
7.04, d (1.2)
7.28, d (8.4)
6.79, d (8.4)
7.31, d (8.4)
6.81, d (8.4)
7.07, d (1.6)
6.78, d (8.4)
6.80, d (8.4)
6.79, d (8.4)
7.28, d (8.4)
7.40, d (15.6)
6.05, d (15.6)
6.81, d (8.4)
7.31, d (8.4)
7.40, d (15.6)
6.06, d (15.6)
6.81, d (8.0)
6.89, d (8.4, 1.2)
7.37, d (16.2)
6.08, d (16.2)
6.89, dd (8.4, 1.2)
7.39, d (16.2)
6.10, d (16.2)
6.91, dd (8.0, 1.6)
7.39, d (15.6)
6.10, d (15.6)
′′
′′′
′′′
′′′
′′′
′′′
′′′
′′′
′′
7.16, s
7.16, s
7.16, s
7.18, s
7.18, s
7.18, s
7.16, s
3.89, s
7.18, s
3.91, s
-OMe
56.4
1
3.87, s
a
b
13
In MeOH-d
In MeOH-d
4
,
H NMR at 600 MHz, C NMR at 150 MHz.
1
13
4
,
H NMR at 400 MHz, C NMR at 100 MHz.
+
7
77.1657 [M + H] , calcd for C38
H
33
O
18, 777.1661.
2.3.6. 1-O-p-coumaroyl-6-O-feruloyl-β-D-glucopyranoside (6)
20
White amorphous power; [
α]
D
+13.8 (c 0.04, MeOH); UV (MeOH)
max 3447,
′
′
′′
2
.3.3. Kaempferol 3-O-(2 -O-galloyl-6 -O-p-coumaroyl)-β-D-
galactopyranoside (3)
λ
max (log ) 210 (4.68), 231 (4.69), 316 (4.82) nm; IR (KBr)
ε
ν
1 1
ꢀ
13
1696, 1633, 1606, 1515, 1444, 1260, 1173, 1070, 830 cm ; H and
C
2
D
0
+
Yellow amorphous powder; [
α
]
ꢀ 46.8 (c 0.07, MeOH); UV (MeOH)
NMR data, see Table 2; (+) HRESIMS m/z 525.1369 [M + Na] , calcd
λ
max (log
ε
) 211 (4.44), 267 (4.18), 279 (4.15), 314 (4.18) nm; IR (KBr)
26
for C25H O11Na, 525.1367.
ꢀ 1
ν
max 3378, 2957, 1694, 1656, 1606, 1513, 1449, 1360, 1179, 832 cm
;
1
13
H and C NMR data, see Table 1; (+) HRESIMS m/z 747.1556 [M +
+
H] , calcd for C37
H
31
O
17, 747.1556.
Table 2
1
13
H and C NMR data of compound 6.
′
′
′′
2
.3.4. Kaempferol 3-O-(2 -O-galloyl-6 -O-p-coumaroyl)-β-D-
NO.
6
NO.
6
glucopyranoside (4)
Yellow amorphous powder; [
max (log
2
0
δ
C
δ
H
(J in Hz)
δ
C
H
δ (J in Hz)
α
]
D
ꢀ 99.5 (c 0.07, MeOH); UV (MeOH)
′
1
2
3
4
5
6
95.7
74.0
77.9
71.3
76.3
64.4
5.60, d (7.8)
8
114.3
167.6
127.7
111.6
149.4
150.7
6.37, d (15.6)
λ
ε
) 209 (4.46), 270 (4.16), 293 (4.16) nm; IR (KBr) max 3454,
ν
′
ꢀ
1
1
13
3.46, dd (9.0, 7.8)
3.49, t (9.0)
9
2
935, 2869, 1644, 1513, 1448, 1384, 1186, 1061, 847 cm ; H and
C
′
′
1
+
NMR data, see Table 1; (+) HRESIMS m/z 747.1556 [M + H] , calcd for
′′
3.44, t (9.0)
2
7.20, d (1.8)
C
37
H
31
O17, 747.1556.
3.68, m
3′′
′
′
4.51, dd (12.0, 1.8)
4.33, dd (12.0, 5.4)
4
′
′
′′
2
.3.5. Kaempferol 3-O-(2 -O-galloyl-6 -O-feruloyl)-β-D-glucopyranoside
′
′′
′′
′′
′′
′′
′′
1
127.0
131.4
116.9
161.7
116.9
131.4
148.1
5
6
7
8
9
116.4
124.3
147.2
115.1
169.1
56.4
6.80, d (8.4)
(
5)
′
2
7.47, d (8.4)
6.80, d (8.4)
7.07, dd (8.4, 1.8)
7.63, d (15.6)
6.40, d (15.6)
2
0
Yellow amorphous powder; [
MeOH) λmax (log ) 210 (4.61), 267 (4.26), 292 (4.22), 328 (4.23) nm;
IR (KBr) max 3174, 2941, 2864, 1697, 1655, 1606, 1513, 1449, 1359,
179, 1084, 842 cm ; H and C NMR data, see Table 1; (ꢀ ) HRESIMS
α
]
D
ꢀ 100.8 (c 0.09, MeOH); UV
′
3
′
(
ε
4
′
5
6.80, d (8.4)
7.47, d (8.4)
7.72, d (15.6)
ν
′
ꢀ 1
1
13
6
3 -OMe
3.88, s
1
′
7
+
m/z 777.1666 [M + H] , calcd for C38
33
H O18, 777.1661.
1
13
In MeOH-d
4
,
H NMR at 600 MHz, C NMR at 150 MHz.
3

H.-Y. Zhao et al.
Fitoterapia 151 (2021) 104877
′′′
′′′
′′′
′′′
′′′
′′′
2
.4. Acid hydrolysis of compounds 1–6
7 (δ 7.37)/H-8 (δ 6.08) to the carbonyl C-9 (δ 168.7), from H-7
H H C
′
′′
′′′
to C-2 (δ
C
111.5)/C-6 (δ
C
124.1), and from 3 -OMe (δ
H
3.87)/H-2
′
′′
′′′
Each of 1–6 (each 1.0 mg) was added to 1.0 mL of 8% hydrochloric
(δ 7.02)/H-5 (δ
H
H
6.78) to C-3 (δ
C
149.2) indicated that a feruloyl
◦
acid. The reaction mixture was refluxed at 80 C for 6 h and extracted
moiety was esterified with 6-OH of the sugar. The other 14 aromatic
carbon signals and one carbonyl were assigned to a kaempferol skeleton
including a 5,7-dihydroxy A-ring and a 1,4-disustituted B-ring, based on
with EtOAc (3 × 2 mL) to remove the aglycone. The aqueous layer was
2
subjected to silica gel CC (EtOAc/EtOH/H O, 7:4:1) to obtain the sugar
fraction. The optical rotation of sugar fraction was afforded by JASCO P-
000 polarimeter. The positive optical rotation of the sugar were
the significant HMBC correlations from H-6 (δ
H
6.10) to C-5/C-7/C-8/C-
′
′
2
10, from H-8 (δ 6.29) to C-6/C-7/C-9/C-10, as well as from H-2 /H-6
H
1
1
′
′
compared with those of literature data, confirming the sugar to be D-
configuration [7].
(δ
H
8.05) to C-2, the H- H COSY correlations (Fig. 2) of H-2 /6 and H-
′
′
3 /5 (δ
H
6.85) together with its molecular. Meanwhile, the obvious
HMBC correlation of the anomeric proton [δ
H
5.12 (1H, d, J = 7.8 Hz)]
and C-3 implied that the sugar was connected at C-3 of the kaempferol
unit. To established the absolute configuration of the galactopyranosyl
unit, acid hydrolysis was carried out and the sugar moiety was deduced
to be D-galactose based on the positive optical rotation [11]. Therefore,
2
.5. NO production measurements and cell viability assays
The antineuroinflammatory activities of all isolates were evaluated
by Griess reaction and MTT assays including the tests of NO production
and cell viability in lipopolysaccharide (LPS) induced BV-2 microglial
cells, as described in our previous study [6,9].
′
′
1
was
established
as
kaempferol
3-O-(6 -O-feruloyl)-β-D-
galactopyranoside.
The HRESIMS data of 2 showed a hydrogen adduct ion at m/z
+
7
77.1657 [M + H] (calcd for 777.1661), consistent with the molecular
3
. Results and discussion
1
13
32
formula of C38H O18. Its H and C NMR data (Table 1) showed a high
similarity to those of 1, except for the presence of a galloyl moiety [δ
68.0, 146.4 (2C), 139.9, 121.6, and 110.6 (2C); δ
HMBC spectrum (Fig. 3), the obvious cross peaks from H-2 (δ
C
3
.1. Structural identification
1
H
7.16 (2H, s)]. In the
′
′
H
5.45)
Compound 1 was isolated as a yellow, amorphous powder with a
′
′′′
′′′′
′′′′
+
and H-2 /6 (δ
H
C
7.16) to C-7 (δ 168.0) indicated that the galloyl
sodium adduct ion at m/z 647.1373 [M + Na] (calcd for 647.1371)
from the positive HRESIMS data, which together with the 13C NMR data
unit was esterified with 2-OH of the sugar. The chemical shifts of the
′
′
′′
′′
sugar group, the coupling constants of H-1 /H-2 (8.1 Hz) and H-3 /H-
indicated that it has a molecular formula of C31
H
28
ꢀ 1
O
14. The presence of
′
′
ꢀ 1
4
(3.0 Hz), together with acid hydrolysis and optical rotation experi-
hydroxy (3445 cm ), ester carbonyl (1655 cm ), and aromatic ring
ꢀ 1
ments supported the presence of β-D-galactopyranosyl unit. Finally, the
(
1609, 1507 cm ) functionalities was evident from its IR absorption
′
′
′′
1
structure of 2 was determined to be kaempferol 3-O-(2 -O-galloyl-6 -O-
feruloyl)-β-D-galactopyranoside.
peaks. The H NMR spectrum of 1 (Table 1) exhibited the presence of an
AX spin system [δ 6.29 (1H, br s, H-8) and 6.10 (1H, br s, H-6)], an
AA BB spin system [δ
H
′
′
′
′
Compound 3, a yellow amorphous powder, has a molecular formula
H
8.05 (2H, d, J = 9.0 Hz, H-2 , 6 ) and 6.85 (2H, d,
′
′
of C37
30
H O17, which was deduced by the positive-ion HERSIMS (m/z
J = 9.0 Hz, H-3 , 5 )], an ABX spin system [δ 7.02 (1H, d, J = 1.2 Hz, H-
H
+
13
′′′
′′′
747.1556 [M + H] , calcd for 747.1556) and C NMR data. The data of
2
), 6.89 (1H, dd, J = 8.4, 1.2 Hz, H-6 ), and 6.78 (1H, dd, J = 8.4 Hz,
1
13
′′′
′′′
its H and C NMR spectra together with the HSQC spectrum indicated
the existent of characteristic moieties including a galactose group, a
flavonol core with 5,7-disubstituted A-ring and 1,4-disustituted B-ring, a
galloyl group, an olefinic bond, and an aromatic ring. The obvious dif-
ference was the disappearance of the methoxy and 1,3,4-tirsubstituted
aromatic ring as well as the presence of another 1,4-disubstituted aro-
matic ring, compared with 2, speculating that a coumaroyl unit in 3
replaced the feruloyl unit in 2. The above deduction and the location of
these moieties were further verified by the significant HMBC correla-
H-5 )], a trans-double bond [δ
H
7.37 (1H, d, J = 16.2 Hz, H-7 ) and 6.08
′
′′
(
1H, d, J = 16.2 Hz, H-8 )], a sugar unit (δ 3.57–5.12), and a methoxy
H
3.87 (3H, s)]. Its 1 C NMR spectrum displayed 25 resonances
105.0, 74.9, 74.8, 72.9, 70.3, and
114.9 and 146.8), a
94.7–179.6) (Table 1). These
3
group [δ
aside from those of a sugar unit (δ
H
C
6
4.4), characteristic for a trans-olefinic bond (δ
C
2
methoxy (δ
C
56.4), and 22 sp carbons (δ
C
spectral properties showed many similarities with those of 8 [10],
implying 1 also being a flavonol glycoside.
Subsequently, its 2D NMR spectra strongly supported the above
inference. The structure of the sugar moiety could be fully confirmed by
the H–1H COSY correlations of H-1 (δ
3
′
′
tions, especially the correlations from H-1 (δ
H
5.61) to C-3 (δ
C
134.6),
′
′
′′′
′′′
′′′
1
′′
′′
′′
from H
2
-6 (δ
H
4.41 and 4.19)/H-7 (δ
H
7.40)/H-8 (δ
H
6.05) to C-9
H
5.12)/H-2 (δ
H
3.80)/H-3 (δ
H
′
′′
′′′
′′
′
′
′′
′′
(δ
(δ
C
168.7), from H-2 (δ
5.45)/H-2 (δ
H
7.28) to C-7 (δ
C
146.6), and from H-2
.57)/H-4 (δ
H
3.82)/H-5 (δ
H
3.76)/H
2
-6 (δ
H
4.39 and 4.12) in Fig. 2
′
′′′
′′′′
3
H
H
7.16) to C-7 (δ
C
168.0). The result of acid
together with its HSQC spectrum. Additionally, the large
J
1
′′, 2′′ value
3
hydrolysis defined the existence of D-galactose. The structure of 3 was
(
7.8 Hz) and the small J
′′, 4′′ value (3.0 Hz) (Table 1) proven that it was
3
′
′
′′
assigned as kaempferol 3-O-(2 -O-galloyl-6 -O-p-coumaroyl)-β-D-
galactopyranoside.
a β-configured galactose. The prominent HMBC cross peaks (Fig. 2) from
′′
2
the nonequivalent methylene protons H -6 and two olefinic protons H-
Compound 4 was obtained as a yellow amorphous power. Its mo-
lecular formula was the same as 3, as confirmed through its HRESIMS
+
13
ion at m/z 747.1556 [M + H] (calcd for 747.1556) and C NMR
spectroscopic data. From the 1D and 2D NMR data, it was observed that
the only difference of 3 and 4 in structure was the geometry of the sugar
′
′
′′
moiety, particularly a series of large coupling constants of H-1 /H-2
′′ ′′ ′′ ′′ ′′ ′′
(
8.1 Hz), H-2 /H-3 (9.6 Hz), H-3 /H-4 (9.0 Hz), and H-4 /H-5 (9.6
′
′
′′
Hz) in 4 whereas the relatively small coupling constant of H-3 /4 in 3,
supporting that 4 possessed a β-glucopyranosyl unit. The D-configura-
tion was determined by acid hydrolysis experiment including TLC and
the positive optical rotation results. Thus, the structure of 4 was
′
′
′′
concluded to be kaempferol 3-O-(2 -O-galloyl-6 -O-p-coumaroyl)-β-D-
glucopyranoside.
Compound 5 had the identical molecular formula of C38
H
32
O
18 with
1
3
2
, as was evident from its C NMR and positive-ion HRESIMS data (m/z
+
1
13
7
77.1666 [M + H] , calcd for 777.1661). Inspection of its H and
C
1
1
Fig. 2. Key Hꢀ H COSY and HMBC correlations of 1.
NMR data (Table 1) and 2D NMR spectra suggested that its structure
4

H.-Y. Zhao et al.
Fitoterapia 151 (2021) 104877
1
1
Fig. 3. Key Hꢀ H COSY and HMBC correlations of 2 and 6.
shared close similarities with 2 except for the resonances of sugar region.
3.2. Anti-inflammatory effects for intervention of NO production in LPS-
induced BV-2 cells
However, the oxymethines from the sugar group had relatively large
′
′′
′
′′
′′
′′
′′
coupling constants of H-1 /H-2 (8.0 Hz), H-2 /H-3 (9.6 Hz), H-3 /H-
′′
′′
4
(8.8 Hz), and H-4 /H-5 (9.6 Hz) and almost identical chemical shifts
The cytotoxicities of all the isolated compounds against BV-2
microglial cells were tested using MTT method, and none showed
with those of 4, which implied the existence of a β-glucopyranosyl unit
with D-configuration proven by the experiment of acid hydrolysis.
obvious cytotoxicity at a dosage of 50 M. Then, their inhibitory effects
μ
′′
′′
Consequently, 5 was deduced as kaempferol 3-O-(2 -O-galloyl-6 -O-
feruloyl)-β-D-glucopyranoside.
on nitric oxide (NO) release in LPS-induced BV-2 cells were measured by
the Griess reaction [6,9]. As shown in Table 3, compounds 1, 8, and 9
exhibited exceptionally potent antineuroinflammatory activities ac-
cording to their inhibitory effects on NO production with IC50 values of
The molecular formula of 6 was ascertained as C25
H
26
O
11 based on
its 13C NMR data and positive HRESIMS ion at m/z 525.1369 [M + Na]
+
ꢀ 1
(
calcd for 525.1367). The absorption bands of hydroxy (3447 cm ),
2.7 ± 0.6, 5.5 ± 0.2, and 4.1 ± 0.2
of a known antineuroinflammatory inhibitor, minocylcine (IC50 = 15.6
± 1.8 M). Compounds 7 and 10 also showed weak inhibitory activities
with IC50 values of 17.0 ± 0.7 and 24.3 ± 1.7 M, while the other
compounds were inactive (IC50 > 50 M). A preliminary structure-
μ
M, respectively, comparable to that
ꢀ 1
ꢀ 1
carbonyl (1696 cm ), and aromatic ring (1606, 1515 cm ) were
observed in its IR spectrum. Meanwhile, the UV spectrum showed
μ
1
maximum absorption at 316 nm. In its H NMR spectrum, the signals of
μ
seven aromatic protons assignable to a 1,4-disubstituted aromatic ring
μ
[
δ
H
7.47 (2H, d, J = 8.4 Hz) and 6.80 (2H, d, J = 8.4 Hz)] and a 1,3,4-
activity relationship analysis implied that a galactopyranose group
contributed more to the influence on NO inhibitory activity than a
glucopyranose moiety (e.g., 1 vs 12 and 7 vs 11). Meanwhile, the ac-
tivity results of compounds 8–10 existing a galactopyranose unit sup-
ported the above deduction.
trisubstituted aromatic ring [δ 7.20 (1H, d, J = 1.8 Hz), 7.07 (1H, dd, J
H
=
8.4, 1.8 Hz), and 6.80 (1H, d, J = 8.4 Hz)], two pairs of trans-olefinic
protons [δ
H
7.72 (1H, d, J = 15.6 Hz), 7.63 (1H, d, J = 15.6 Hz), 6.40
(
1H, d, J = 15.6 Hz), and 6.37 (1H, d, J = 15.6 Hz)], one set of sugar
protons (δ
H
3.44–5.60 Hz), and a methoxy [δ
H
3.88 (3H, s)] exhibited
1
3
obviously. Its C NMR data and HSQC spectrum suggested that 6
possessed two carbonyls (δ 169.1 and 167.6), a six‑carbon sugar unit
95.7, 77.9, 76.3, 74.0, 71.3, and 64.4), a methoxy (δ 56.4), and the
4. Conclusion
C
(
δ
C
C
In summary, the research on the chemical components of N. japonica
2
′′
other 16 sp carbons arising from two aromatic rings and two double
obtained 15 compounds including four rare 2 -O-galloyl-type kaemp-
′
′
bonds.
ferol glycosides, a new 6 -O-feruloyl-type kaempferol glycoside, a new
phenylpropanoid glycoside, and nine known compounds. The structures
of the new compounds were elucidated by a series of NMR spectroscopic
data. The absolute configurations of sugar in 1–6 were established via
acid hydrolysis experiment and the comparison to literature data. The
preliminary anti-inflammatory tests (Fig. 4) indicated that compounds 1
and 7–10 shared obviously inhibitory activities on NO production in
The 1
H
–1H COSY correlations of oxymethylene and oxymethine
protons in Fig. 3 and their coupling constants as shown in Table 2
revealed the presence of a β-D-glucopyranosyl moiety, as also verified
through acid hydrolysis method. Based on the HMBC spectrum of 6, the
′
′
significant cross peaks from H-1 (δ
H
5.60)/H-7 (δ
H
′′
′
7.72)/H-8 (δ
H
6.37)
′
′′
to C-9 (δ
C
167.6), from H
2
-6 (δ
H
4.51, 4.33)/H-7 (δ
H
7.63)/H-8 (δ
7.47) to C-7 (δ
H
′
′
′
′
6
.40) to C-9 (δ
48.1), and from H-2 (δ
C
169.1), from H-2 (δ
H
7.47)/H-6 (δ
H
C
LPS-induced BV-2 cells with IC50 values ranging from 2.7 to 24.3
μ
M,
′
′
′′
′′
1
H
7.20)/H-6 (δ
H
7.07) to C-7 (δ
C
147.2) as
comparing with the positive control minocycline (IC50 = 15.6 ± 1.8 μ
M).
shown in Fig. 3 indicated that a p-coumaroyl group and a feruloyl group
were connected with 1-OH and 6-OH of the sugar, respectively. Thus,
the structure of 6 was illustrated as 1-O-p-coumaroyl-6-O-feruloyl-β-D-
glucopyranoside.
These isolates enriched the structure types of flavonoid glycoside from
N. japonica, in the meantime, above activity results demonstrated that
Table 3
The isolated known compounds were identified as kaempferol 3-O-
′
′
Effects of compounds 1–15 on NO production in LPS-induced BV-2 microglial
(
(
6 -O-p-coumaroyl)-β-D-galactopyranoside (7) [12], kaempferol 3-O-
′′
′′
cells.
6 -O-caffeoyl)-β-D-galactopyranoside (8) [10], quercetin 3-O-(6 -O-p-
′
′
coumaroyl)-β-D-galactopyranoside (9) [13], quercetin 3-O-(6 -O-fer-
uloyl)-β-D-galactopyranoside (10) [14], kaempferol 3-O-(6 -O-p-cou-
maroyl)-β-D-glucopyranoside (11) [15], kaempferol 3-O-(6 -O-
feruloyl)-β-D-glucopyranoside (12) [16], kaempferol 3-O-(2 -O-p-cou-
maroyl)-β-D-glucopyranoside (13) [11], 1,6-di-O-p-coumaroyl-β-D-glu-
copyranoside (14) [17], and 1,6-di-O-feruloyl-β-D-glucopyranoside (15)
Compounds
IC50
(μM)
Compounds
IC50 (μM)
′′
1
2
3
4
5
6
7
8
2.7 ± 0.6
>50
9
4.1 ± 0.2
24.3 ± 1.7
>50
′
′
10
′′
>50
11
>50
12
>50
>50
13
>50
>50
14
>50
[
18] by comparison of their MS and NMR data with literature results.
17.0 ± 0.7
5.5 ± 0.2
15
>50
a
minocycline
15.6 ± 1.8
a
Positive control.
5

H.-Y. Zhao et al.
Fitoterapia 151 (2021) 104877
Fig. 4. The effects of compounds 1 and 7–10 on NO release in LPS-induced BV-2 cells. (Data were expressed as means ± SEM (n = 3); Significance: *P < 0.001
#
compared to LPS group, P < 0.001 compared to control group; MINO = minocycline.)
kaempferol or quercetin galactopyranosides deserved to be further
investigated as anti-inflammatory agents.
[6] Y.L. Zhang, Q.M. Pan, H.B. Liao, J.K. Qin, N. Li, D. Liang, G.J. Zhang,
Coumarinolignoids and lignanoids from the stems and leaves of Sapium discolor,
Fitoterapia 133 (2019) 17–22.
[
7] H. Jiang, G.J. Zhang, Y.F. Liu, H.S. Wang, D. Liang, Clerodane diterpenoid
glucosides from the stems of Tinospora sinensis, J. Nat. Prod. 80 (2017) 975–982.
8] Q.M. Pan, Y.H. Li, J. Hua, F.P. Huang, H.S. Wang, D. Liang, Antiviral matrine-type
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Declaration of Competing Interest
The authors declare that no competing financial interests exist.
Acknowledgements
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This work was supported partially by the National Natural Science
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