Isolation and characterization of neolignan derivatives with hepatoprotective and neuroprotective activities from the fruits of Citrus medica L. var. Sarcodactylis Swingle

Article abstract of DOI:10.1016/j.bioorg.2020.104622

The fruit of Citrus medica L. var. sarcodactylis Swingle is a functional food with rich nutrients and medicinal values because of its content of bioactive compounds. A bioactivity-guided chemical investigation from the fruits of C. medica L. var. sarcodactylis Swingle afforded three new benzodioxane neolignans (1–3), three new phenanthrofuran neolignan glycosides (4–6), two new biphenyl-ketone neolignans (7–8), two new 1′,7′-bilignan neolignans (9–10), as well as fourteen known neolignan derivatives (11–24), which were isolated and characterized from the fruits of C. medica L. var. sarcodactylis Swingle for the first time. These neolignan derivatives were determined by extensive and comprehensive analyzing NMR, HR-ESI-MS, UV, IR spectral data and compared with the data described in the literature. Among them, compounds 1–3 and 12–13 exhibited moderate hepatoprotective activities to improve the survival rates of HepG2 cells from 46.26 ± 1.90% (APAP, 10 mM) to 67.23 ± 4.25%, 62.87 ± 4.43%, 60.06 ± 6.34%, 56.75 ± 2.30%, 58.35 ± 6.14%, respectively. Additionally, compounds 7–8 and 21–22 displayed moderate neuroprotective activities to raise the survival rates of PC12 cells from 55.30 ± 2.25% to 66.94 ± 3.37%, 70.98 ± 5.05%, 64.64 ± 1.93%, and 62.81 ± 4.11% at 10 μM, respectively. The plausible biogenetic pathway and preliminary structure–activity relationship of the selected compounds were scientifically summarized and discussed in this paper.

Full text of DOI:10.1016/j.bioorg.2020.104622

Bioorganic Chemistry 107 (2021) 104622
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Isolation and characterization of neolignan derivatives with
hepatoprotective and neuroprotective activities from the fruits of Citrus
medica L. var. Sarcodactylis Swingle
,
,
Qin-Ge Maa *, Rong-Rui Weia *, Ming Yanga, Xiao-Ying Huanga, Fang Wanga,
Jiang-Hong Dongb, Zhi-Pei Sangc
a Key Laboratory of Modern Preparation of Traditional Chinese Medicine of Ministry of Education＆ Research Center of Natural Resources of Chinese Medicinal
Materials and Ethnic Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, PR China
b College of Chemistry and Pharmaceutical Engineering, Huanghuai University, Zhumadian 463000, PR China
c College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang 473061, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
The fruit of Citrus medica L. var. sarcodactylis Swingle is a functional food with rich nutrients and medicinal
values because of its content of bioactive compounds. A bioactivity-guided chemical investigation from the fruits
of C. medica L. var. sarcodactylis Swingle afforded three new benzodioxane neolignans (1–3), three new phe-
nanthrofuran neolignan glycosides (4–6), two new biphenyl-ketone neolignans (7–8), two new 1′,7′-bilignan
neolignans (9–10), as well as fourteen known neolignan derivatives (11–24), which were isolated and charac-
terized from the fruits of C. medica L. var. sarcodactylis Swingle for the first time. These neolignan derivatives
were determined by extensive and comprehensive analyzing NMR, HR-ESI-MS, UV, IR spectral data and
compared with the data described in the literature. Among them, compounds 1–3 and 12–13 exhibited moderate
hepatoprotective activities to improve the survival rates of HepG2 cells from 46.26 ± 1.90% (APAP, 10 mM) to
67.23 ± 4.25%, 62.87 ± 4.43%, 60.06 ± 6.34%, 56.75 ± 2.30%, 58.35 ± 6.14%, respectively. Additionally,
compounds 7–8 and 21–22 displayed moderate neuroprotective activities to raise the survival rates of PC12 cells
Citrus medica L. var. sarcodactylis Swingle
Neolignan
Hepatoprotective
Neuroprotective
from 55.30 ± 2.25% to 66.94 ± 3.37%, 70.98 ± 5.05%, 64.64 ± 1.93%, and 62.81 ± 4.11% at 10
μM,
respectively. The plausible biogenetic pathway and preliminary structure–activity relationship of the selected
compounds were scientifically summarized and discussed in this paper.
1. Introduction
[9], and polyphenols [10]. Modern pharmacological investigations of
the fruits of C. medica L. var. sarcodactylis Swingle revealed a wide va-
The fruit of Citrus medica L. var. sarcodactylis Swingle, widely well
known as the bergamot or bergamot orange, belongs to the Rutaceae
family [1]. It is also named as “Foshou” in Chinese because of the shape
of its fruit like a finger. The fruit of C. medica L. var. sarcodactylis Swingle
is used as a traditional medicinal food with the functions of relieving
depressed liver, harmonizing stomach, and expelling phlegm in Chinese
folk [2]. Moreover, it was used as a traditional Chinese medicine for
treating hypertension, tracheitis, respiratory tract infections, angio-
cardiopathy, and asthma [3]. The fruits of C. medica L. var. sarcodactylis
Swingle had been chemically investigated, leading to the isolation of
some compounds including polysaccharides [1], flavonoids [4],
cumarins [5], phenolics [6], terpenes [7], carotenoids [8], amino acids
riety of biological activities, including antibiofifilm [6], antibacterial
[10], immunoregulatory [11], antioxidant [12], anticancer [13], anti-
hyperglycemic [14], cardioprotective [15], and antidepressant activities
[16].
Up to now, it is found that there are few reports on hepatoprotective
and neuroprotective activities from the fruits of C. medica L. var. sar-
codactylis Swingle. However, one article related to the neuroprotective
activity of the titled species revealed that the peels of Citrus grandis
exhibited the protection of neurons against Aβ-mediated neurotoxicity
[17]. In continuation of our ongoing study more new compounds with
biological activities from the fruits of C. medica L. var. sarcodactylis
Swingle. A bioassay-guided investigation was carried out to find further
* Corresponding authors.
E-mail addresses: maqinge2006@163.com (Q.-G. Ma), weirongrui2011@163.com (R.-R. Wei).
https://doi.org/10.1016/j.bioorg.2020.104622
Received 11 November 2020; Received in revised form 29 December 2020; Accepted 30 December 2020
Available online 8 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

Q.-G. Ma et al.
Bioorganic Chemistry 107 (2021) 104622
hepatoprotective and neuroprotective isolates from the fruits of
C. medica L. var. sarcodactylis Swingle. As a result, three new benzo-
dioxane neolignans (1–3), three new phenanthrofuran neolignan gly-
cosides (4–6), two new biphenyl-ketone neolignans (7–8), two new
1′,7′-bilignan neolignans (9–10), and fourteen known neolignan de-
rivatives (11–24) (Fig. 1) were isolated from this plant for the first time.
In this study, we report the isolation, characterization, bioactivity,
plausible biogenetic pathway, and preliminary structure–activity
Fig. 1. Structures of compounds (1–24).
2

Q.-G. Ma et al.
Bioorganic Chemistry 107 (2021) 104622
relationship of these isolates.
g) was performed on silica gel (200–300 mesh), Sephadex LH-20
(MeOH/CH2Cl2 = 1:1), Toyopearl HW-40C (95% MeOH), and semi-
preparative HPLC (λ = 210–380 nm, 3 mL/min, 15–45% MeOH) col-
umns, successively, yielded 4 (7.95 mg, 25% MeOH, tR = 28 min), 5
(9.78 mg, 30% MeOH, tR = 37 min), 6 (8.04 mg, 28% MeOH, tR = 34
min), 7 (11.86 mg, 30% MeOH, tR = 35 min), 8 (8.96 mg, 32% MeOH, tR
= 44 min), 9 (12.06 mg, 30% MeOH, tR = 34 min), 10 (9.44 mg, 33%
MeOH, tR = 46 min), 14 (12.53 mg, 32% MeOH, tR = 40 min), 15 (12.66
mg, 34% MeOH, tR = 51 min), 16 (7.88 mg, 30% MeOH, tR = 47 min),
2. Materials and methods
2.1. General experimental procedures
A Perkin-Elmer 241 digital polarimeter at 20 ℃ was utilized to
measure optical rotations. An XT5B microscopic melting point apparatus
was used to measure melting points which uncorrected. An Australia
GBC UV-916 spectrometer was used to measure UV spectral data. A
Nicolet 5700 FT-IR spectrometer with KBr pellets was utilized to record
IR spectral data. A Bruker-400 spectrometer with TMS as internal
reference was applied to obtain NMR spectral data. A Q-Trap LC/MS/MS
spectrometer and an Agilent 1100 series LC/MSD Trap SL mass spec-
trometer were utilized to measure ESI-MS and HR-ESI-MS spectral data.
17 (13.38 mg, 33% MeOH, tR = 45 min), 18 (16.50 mg, 32% MeOH, tR
=
41 min), 21 (9.45 mg, 26% MeOH, tR = 31 min), and 22 (13.22 mg, 30%
MeOH, tR = 39 min).
(7E)-1-allyl alcohol-5,6-(11-isopropyl)-furanyl-3′,5′-dimethoxy-4′-
glycerol-9′-isovalerate-3,4,7′,8′-benzodioxane neolignan (1): colorless
20
powder; [
α]
D + 4.57 (c 0.85, MeOH); mp: 204.2–204.5 ◦C; HR-ESI-MS:
A CXTH LC3050N system with a YMC-pack ODS-A column (5
μ
m, 10 ×
m/z 635.2466 [M + Na]+ (calcd. for C33H40O11Na, 635.2468); UV
250 mm) was used to perform on semi-preparative HPLC. Column
chromatographic separations were performed on silica gel (100–200 or
200–300 mesh), Toyopearl HW-40C, and Sephadex LH-20 columns. TLC
analyses were carried out on silica gel plates (GF-254) and visualized
under UV (254 nm) light and by spraying with a H2SO4/EtOH (1:9, v/v)
solution followed by heating.
(MeOH) λmax (logε): 208 (2.06), 235 (3.29), and 270 (2.57) nm; IR (KBr)
νmax: 3325, 2952, 1736, 1605, 1511, and 1498 cmꢀ 1
;
1H NMR
(DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100 MHz) data see
Table 1.
(7E,10′E,11E)-1-(9-methoxyl)-propenyl-5-hydroxy-6-prenyl-8′-
methylol-11′,16′-dihydroxy-15′,17′-dimethoxy-10′-phenylallyl alcohol-
20
3,4,7′,8′-benzodioxane neolignan (2): colorless powder; [
α]
D + 3.68 (c
0.70, MeOH); mp: 206.4–206.6 ◦C. HR-ESI-MS: m/z 641.2366 [M +
2.2. Plant material
Na]+ (calcd. for C35H38O10Na, 641.2363); UV (MeOH) λmax (log
ε): 208
(2.11), 235 (3.30), 270 (2.52), and 305 (4.17) nm; IR (KBr) νmax: 3328,
The fruits of C. medica L. var. sarcodactylis Swingle were bought from
Guangzhou, Guangdong, P. R. China, in May 2019. The plant was
authenticated by Dr. Su Zhang of Wuyang Weisen Biological Medicine
Co., Ltd,. The voucher specimen (No.FS-201905) deposited in Jiangxi
University of TCM, Nanchang 330004, China.
1611, and 1509 cmꢀ 1
;
1H NMR (DMSO‑d6, 400 MHz) and 13C NMR
(DMSO‑d6, 100 MHz) data see Table 1.
(7E,11E)-1-(9-methoxyl)-propenyl-5-hydroxy-6-geranyl-16′-hy-
droxy-15′,17′-dimethoxyphenyl-8′,11′-dimethylol-benzofuranyl-
20
3,4,7′,8′-benzodioxane neolignan (3): colorless powder; [
α]
D + 7.26 (c
0.90, MeOH); mp: 210.5–210.8 ◦C. HR-ESI-MS: m/z 725.2937 [M +
2.3. Extraction and isolation
Na]+ (calcd. for C40H46O11Na, 725.2938); UV (MeOH) λmax (log
ε): 208
(2.09), 235 (3.34), 305 (4.09), and 343 (4.25) nm; IR (KBr) νmax: 3330,
The fruits of C. medica L. var. sarcodactylis Swingle (9.5 kg) were air-
2938, 1608, and 1509 cmꢀ 1
;
1H NMR (DMSO‑d6, 400 MHz) and 13C
dried, smashed (60 mesh), and extracted with 95% EtOH (10 L) heating
◦
NMR (DMSO‑d6, 100 MHz) data see Table 1.
under reflux at 110 C for 4 h with electric heating jacket and round
1-(18,19-dimethyl)-propanol-4-hydroxyl-5,6-(13-hydroxyl-12-
methoxyl)-phenylethyl-7′-(4′-hydroxyl-5′-methoxy)-phenyl-9′-O-β-D-
glucopyranosyl-phenanthrofuran neolignan (4): colorless powder;
bottom flask. The same extraction procedure was performed by the
above method for 3 times. The extracting solution was combined and
concentrated under reduced pressure to acquire a crude extract (1.7 kg),
which followed a method described previously [18–20]. The crude
extract was suspended in water (2.5 L) and partitioned with solvents of
petroleum ether (7.0 L), EtOAc (7.0 L), and n-BuOH (7.0 L), consecu-
tively, affording petroleum ether extraction part (143.4 g), EtOAc
extraction part (300.8 g), and n-BuOH extraction part (512.5 g),
respectively. The EtOAc extraction part exhibited potential hep-
atoprotective [21] with improving HepG2 cell survival rate from
46.26% ± 1.90% (APAP, 10 mM) to 61.42% ± 3.41% and potential
neuroprotective [22] with raising the survival rates of PC12 cells from
20
◦
[
α]
+ 2.07 (c 0.45, MeOH); mp: 208.0–208.4 C. HR-ESI-MS: m/z
D
705.2520 [M + Na]+ (calcd. for C36H42O13Na, 705.2523); UV (MeOH)
λmax (logε): 205 (2.01), 272 (3.28), 320 (3.71), and 350 (4.52) nm; IR
(KBr) νmax: 3421, 1610, 1518, and 1467 cmꢀ 1; 1H NMR (DMSO‑d6, 400
MHz) and 13C NMR (DMSO‑d6, 100 MHz) data see Table 2.
1-(17-furanyl)-ethyl-4-hydroxyl-5,6-(13-hydroxyl-12-methoxyl)-
phenylethyl-7′-(3′,4′,5′-trimethoxy)-phenyl-9′-O-β-D-glucopyranosyl-
20
phenanthrofuran neolignan (5): colorless powder; [
α
]
D + 2.26 (c 0.30,
MeOH); mp: 212.1–212.3 ◦C. HR-ESI-MS: m/z 757.2475 [M + Na]+
(calcd. for C39H42O14Na, 757.2472); UV (MeOH) λmax (logε): 203 (2.04),
55.30 ± 2.25% to 67.04 ± 3.51% at 10 μM according to the biological
272 (3.22), 320 (3.27), and 346 (4.37) nm; IR (KBr) νmax: 3425, 1612,
and 1608 cmꢀ 1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6,
100 MHz) data see Table 2.
screening results.
The EtOAc extraction part was applied to column chromatography
with silica gel (100–200 mesh) and eluted by petroleum ether/EtOAc
(30:1, 15:1, 8:1, 4:1, and 1:1, v/v) to afford five fractions (A1-A5). The A3
(57.6 g, petroleum ether/EtOAc = 8:1, v/v) was carried out on a silica
gel (200–300 mesh) column with a gradient elution (petroleum ether/
EtOAc = 10:1, 8:1, and 6:1, v/v) to afford three sub-fractions (A3a-A3c).
The A3b (18.7 g, petroleum ether/EtOAc = 8:1, v/v) was performed on
silica gel (200–300 mesh), Toyopearl HW-40C (95% MeOH), Sephadex
LH-20 (MeOH/CH2Cl2 = 1:1), and semi-preparative HPLC (λ = 205–360
nm, 3 mL/min, 20–50% MeOH) columns, repeatedly, yielded 1 (7.47
mg, 30% MeOH, tR = 31 min), 2 (8.92 mg, 35% MeOH, tR = 39 min), 3
(9.45 mg, 33% MeOH, tR = 35 min), 11 (12.15 mg, 35% MeOH, tR = 45
min), 12 (14.20 mg, 32% MeOH, tR = 36 min), 13 (13.31 mg, 35%
MeOH, tR = 45 min), 19 (11.52 mg, 35% MeOH, tR = 39 min), 20 (15.54
mg, 32% MeOH, tR = 37 min), 23 (8.56 mg, 30% MeOH, tR = 42 min),
and 24 (14.24 mg, 33% MeOH, tR = 35 min). Meanwhile, the A3c (21.2
1-(17Z)-methyl-butanol-4-hydroxyl-5,6-(13-hydroxyl-12-methoxyl)-
phenylethyl-7′-(4′-hydroxy-3′,5′-dimethoxy)-phenyl-9′-O-β-D-glucopyr-
20
anosyl-phenanthrofuran neolignan (6): colorless powder; [
α]
D + 3.38
(c 0.58, MeOH); mp: 211.7–211.9 ◦C. HR-ESI-MS: m/z 733.2474 [M +
Na]+ (calcd. for C37H42O14Na, 733.2472); UV (MeOH) λmax (log
ε): 205
(2.03), 272 (3.42), 323 (3.35), and 345 (4.24) nm; IR (KBr) νmax: 3423
and 1611 cmꢀ 1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6,
100 MHz) data see Table 2.
(9′’E)-4,5-(11,12-dimethyl)-pyranyl-7′-(4′-hydroxy)-phenyl-4′’-pro-
penyl-8′-methylol-furanyl-6′’-acetyl-1′’,6-biphenyl-7-ketone neolignan
20
(7): colorless powder;
[α
]
+ 8.20 (c 0.85, MeOH); mp:
D
198.3–198.7 ◦C. HR-ESI-MS: m/z 531.1781 [M + Na]+ (calcd. for
C
32H28O6Na, 531.1784); UV (MeOH) λmax (logε): 202 (1.89), 280 (3.61),
315 (2.43), and 330 (3.62) nm; IR (KBr) νmax: 3411, 1719, 1617, and
3

Q.-G. Ma et al.
Bioorganic Chemistry 107 (2021) 104622
Table 1
1H NMR (400 MHz, DMSO‑d6) and 13C NMR (100 MHz, DMSO‑d6) of compounds (1–3).
No.
1
2
3
δH δC
δH δC
δH δC
1
-
124.6
112.3
143.0
129.6
145.6
122.4
130.4
128.9
63.5
108.6
161.2
35.8
23.1
23.1
–
131.8
111.4
143.6
130.2
146.9
118.6
130.1
125.9
73.1
-
132.0
110.8
143.3
131.5
146.7
119.1
130.2
126.1
74.0
2
6.45(s)
6.02(s)
6.04(s)
3
–
–
–
4
–
–
–
5
–
–
–
6
–
–
–
7
6.61(d,16.0)
6.52(d,16.0)
6.53(d,16.0)
8
6.29(dd,16.0,5.8)
6.21(dd,16.0,5.8)
6.20(dd,16.0,5.8)
9
4.20(d,5.8)
4.11(d,5.8)
4.13(d,5.8)
10
6.26(s)
3.48(d,7.5)
20.1
3.23(d,9.2)
21.9
11
–
5.25(t,7.5,1.5)
122.9
134.8
18.4
5.22(m)
123.9
135.8
41.1
12
3.38(m)
–
–
13
1.31(s)
1.70(s)
1.97(m)
14
1.31(s)
1.82(s)
24.3
1.46(m)
24.1
15
–
–
–
–
–
1.35(m)
44.2
1′
130.2
106.3
150.1
131.2
150.1
106.3
126.4
125.6
65.3
173.1
43.9
25.1
22.1
22.1
84.3
61.9
61.9
–
129.7
127.6
118.3
154.2
118.3
127.6
125.8
124.3
62.4
–
130.1
121.3
116.8
126.2
153.0
108.8
125.1
124.5
62.2
2′
6.58(s)
7.28(d,8.0)
7.56(dd,8.0,2.0)
3′
–
6.65(d,8.0)
7.83(d,8.0)
4′
–
–
–
–
5′
–
6.65(d,8.0)
6′
6.58(s)
7.28(d,8.0)
7.72(d,2.0)
7′
–
–
–
–
–
–
8′
9′
4.86(s)
4.18(s)
4.20(s)
10′
–
–
151.6
63.8
–
–
155.5
115.8
55.2
11′
2.30(d,6.6)
4.18(s)
12′
2.16(m)
6.21(s)
101.4
124.1
107.8
155.0
140.1
155.0
107.8
–
4.66(s)
–
13′
1.00(s)
–
122.1
106.2
149.2
137.7
149.2
106.2
–
14′
1.00(s)
6.27(s)
7.18(s)
–
15′
3.98(t,4.8)
–
16′
3.76(dd,4.8,1.0)
–
–
–
17′
3.76(dd,4.8,1.0)
–
18′
–
6.27(s)
7.18(s)
–
OCH3-3′/5′
OCH3-15′/17′
OCH3-9
CH3-12
CH3-17/18
3.71(s)
56.5
–
–
–
–
–
–
3.86(s)
56.9
3.88(s)
3.33(s)
1.88(s)
1.16(s)
57.1
–
3.34(s)
55.3
55.0
–
–
–
–
–
16.3
–
29.8
1572 cmꢀ 1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100
MHz) data see Table 3.
2.4. Hepatoprotective activity assay
(9E,9′’E)-5-isopentenyl-7′-(4′-hydroxy-5′-methoxy)-phenyl-4′’-pro-
Compounds (1–24) were evaluated for hepatoprotective activities by
penylketone-8′-methylol-furanyl-6′’-acetyl-1′’,6-biphenyl-7-ketone
the MTT method, which followed a method described previously [21].
20
neolignan (8): colorless powder; [
α
]
+ 7.54 (c 0.70, MeOH); mp:
HepG2 cells were fostered in 96-well plates (1 × 104 cells/well, 200
μL/
D
200.4–200.8 ◦C. HR-ESI-MS: m/z 577.1840 [M + Na]+ (calcd. for
well) in minimum essential media with 10% FBS and fostered for 24 h at
C
33H30H8Na, 577.1838); UV (MeOH) λmax (log
ε
): 202 (1.86), 280 (3.57),
37 ◦C [23]. The compounds (1–24) and acetaminophen were added in
318 (2.56), 332 (3.75), and 351 (4.08) nm; IR (KBr) νmax: 3410, 1720,
the culture medium to give final concentrations of 10 μM and 10 mM,
1677, 1617, and 1573 cmꢀ 1
;
1H NMR (DMSO‑d6, 400 MHz) and 13C
respectively, and incubated for 48 h at 37 ◦C. Additionally, the experi-
ment selected bicyclol as the positive group and selected DMSO as the
NMR (DMSO‑d6, 100 MHz) data see Table 3.
(7E,10E)-4,5′-dihydroxy-5-isopentenol-6-(7,8-trans allyl)-alcohol-
negative control group, respectively [24]. The MTT (0.5 mg/mL, 100 μL)
7′’-(4′’-hydroxy-3′’,5′’-dimethoxyl)-phenyl-9′,9′’-dimethylol-1′,7′-
was added to each well and incubated for 4 h at 37 ◦C. The absorbance of
20
bineolignan (9): colorless powder; [
α]
+ 6.21 (c 0.64, MeOH); mp:
selected compounds at 570 nm was measured after adding 150 μL of
206.1–206.3 ◦C. HR-ESI-MS: m/z 641.D1995 [M + Na]+ (calcd. for
DMSO. The survival rate of HepG2 cells = the mean OD of the medicated
C
34H34O11Na 641.1999); UV (MeOH) λ
(log
ε
): 202 (1.81), 230
group/the mean OD of the solvent control group [25].
(2.80), and 270 (3.06) nm; IR (KBr) νmax:m3a4x09, 1611, and 1508 cmꢀ 1
;
1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100 MHz) data
2.5. Neuroprotective activity assay
see Table 4.
(7E)-5′-hydroxy-4,5-(13,14-dimethyl)-pyranyl-6-allyl
alcohol-7′’-
Compounds (1–24) were assayed for neuroprotective activities of by
the MTT assay with desipramine as a positive control [22]. Compounds
(1–24) were dissolved in DMSO with the final concentration of DMSO in
the culture medium was 0.1% (v/v). PC12 cells were cultured in DMEM
medium supplemented with 10% fetal bovine serum, 100 U/mL pen-
(4′’-hydroxy-3′’,5′’-dimethoxyl)-phenyl-9′,9′’-dimethylol-1′,7′-bineoli-
20
gnan (10): colorless powder; [
α]
+ 6.18 (c 0.51, MeOH); mp:
203.0–203.3 ◦C. HR-ESI-MS: m/z 6D23.1895 [M + Na]+ (calcd. for
C
34H32O10Na, 623.1893); UV (MeOH) λ
(logε): 202 (1.90), 230
(2.96), 270 (3.12), and 315 (3.57) nm; IRm(aKxBr) νmax: 3410, 1612, and
1507 cmꢀ 1; 1H NMR (DMSO‑d6, 400 MHz) and 13C NMR (DMSO‑d6, 100
MHz) data see Table 4.
icilin, and 100 μg/mL streptomycin in a humidified atmosphere of 5%
CO2 at 37 ◦C [22]. PC12 cells were cultured in 96-well plates (1 × 105
cells/well) [26]. The cell viability was measured by the MTT assay for a
48 h treatment. The absorbance of compounds (1–24) was recorded at
492 nm by a microplate detector. The cell viability was indicated by a
4

Q.-G. Ma et al.
Bioorganic Chemistry 107 (2021) 104622
Table 2
1H NMR (400 MHz, DMSO‑d6) and 13C NMR (100 MHz, DMSO‑d6) of compounds (4–6).
No.
4
5
6
δH δC
δH δC
δH δC
1
-
124.6
123.1
138.4
137.2
118.0
136.8
32.6
-
125.9
123.5
138.3
137.2
118.2
136.1
32.5
-
123.7
122.7
138.1
137.1
118.1
136.3
32.6
2
–
–
–
–
–
3
–
–
4
–
–
5
–
–
6
–
–
–
7
2.48(m)
2.47(m)
2.47(m)
8
2.42(m)
29.4
2.42(m)
29.3
2.42(m)
29.4
9
–
–
133.0
128.9
115.7
148.6
141.5
117.6
18.1
–
–
133.0
129.7
115.6
148.5
141.7
117.8
29.4
–
–
133.0
129.6
115.6
148.5
141.6
117.8
31.5
10
11
6.68(s)
6.68(s)
6.68(s)
12
–
–
–
–
–
–
13
14
6.43(s)
6.42(s)
2.25(m)
2.12(m)
–
6.42(s)
15
2.78(m)
3.28(d,6.9)
16
1.58(m)
43.2
24.5
6.03(m)
126.7
137.0
62.9
17
–
70.3
116.1
138.4
141.3
109.4
131.0
106.2
149.7
140.7
149.7
106.2
150.1
119.1
53.3
–
18
1.19(s)
29.2
7.22(s)
7.31(d,2.6)
6.33(d,2.6)
–
4.31(m)
19
1.19(s)
29.2
1.74(s)
17.5
20
–
–
–
–
–
–
1′
131.2
121.4
116.8
142.3
149.5
115.4
150.2
118.9
53.2
131.1
104.5
149.4
141.7
149.4
104.5
150.1
119.0
53.3
2′
6.98(dd,8.0,2.0)
6.39(s)
–
6.42(s)
3′
6.73(d,8.0)
–
4′
–
–
–
–
–
5′
–
6′
6.85(d,2.0)
–
6.39(s)
–
6.42(s)
7′
–
–
8′
–
–
9′
4.61(s)
4.62(s)
5.02(d,7.6)
3.71(m)
3.78(m)
3.40(m)
3.66(m)
3.58(dd,11.2,6.4)
3.90(dd,11.2,3.2)
3.81(s)
3.78(s)3.78(s)
3.78(s)
4.62(s)
1′’
5.01(d,7.6)
3.72(m)
3.80(m)
3.38(m)
3.68(m)
3.57(dd,11.2,6.4)
3.91(dd,11.2,3.2)
3.81(s)
101.3
74.2
101.2
74.0
5.02(d,7.6)
3.72(m)
3.79(m)
3.41(m)
3.66(m)
3.58(dd,11.2,6.4)
3.91(dd,11.2,3.2)
3.81(s)
101.3
74.3
2′’
3′’
77.7
77.6
77.5
4′’
71.2
71.5
71.4
5′’
76.4
76.2
76.3
6′’a
63.8
64.0
64.1
6′’b
63.8
64.0
64.1
OCH3-12
OCH3-5′OCH3-4′
OCH3-3′
57.2
57.2
57.2
3.76(s)-
–
56.4-
–
56.556.5
56.5
3.84(s)-
3.84(s)
56.7-
56.7
Table 3
1H NMR (400 MHz, DMSO‑d6) and 13C NMR (100 MHz, DMSO‑d6) of compounds (7–8).
No.
7 No. 7 No.
8 No.
8
δH δC δH δC
δH δC
δH δC
1
7.25(s)
116.2 5′ 6.64(d,8.0) 118.5 1
125.7 6′ 7.39(d,8.0) 129.4 2
144.1 7′ ꢀ 152.3 3
7.23(s)
116.4 5′
-
149.3
116.5
151.8
118.1
52.1
2
–
–
124.3 6′
6.68(d,2.0)
3
–
–
144.4 7′
–
–
4
–
140.3 8′ ꢀ 118.3 4
–
141.3 8′
5
–
119.8 9′ 4.76(s) 51.6 5
–
122.2 9′
4.78(s)
6
–
132.3 1′’ ꢀ 138.3 6
–
134.6 1′’
189.7 2′’
33.2 3′’
–
138.1
124.1
129.2
136.4
126.1
138.0
198.2
26.5
7
–
189.8 2′’ 6.72(d,8.0) 123.6 7
116.1 3′’ 7.43(dd,8.0,2.0) 129.4 8
129.0 4′’ ꢀ 136.7 9
–
6.88(dd,8.0,2.0)
8
6.64(d,10.0)
3.33(d,7.5)
7.50(d,8.0)
9
5.70(d,10.0)
5.36(d,7.5)
124.4 4′’
132.6 5′’
25.6 6′’
–
10
–
79.5 5′’ 7.85(d,2.0) 125.6 10
28.7 6′’ ꢀ 137.4 CH3-11
28.7 7′’ ꢀ 198.1 CH3-12
131.0 CH3-8′’ 2.55(s) 26.5 1′
129.4 9′’ 6.79(d,15.6) 125.8 2′
118.5 10′’ 6.60(dd,15.6,6.6) 128.7 3′
158.6 CH3-11′’ 1.95(d,6.6) 19.3 4′
–
7.90(d,2.0)
CH3-11
1.47(s)
1.47(s)
–
1.80(s)
–
–
CH3-12
1.68(s)
19.2 7′’
1′
2′
3′
4′
–
130.8 CH3-8′’
121.7 9′’
118.9 10′’
143.1 11′’
2.56(s)
7.39(d,8.0)
6.64(d,8.0)
–
6.91(dd,8.0,2.0)
8.60(d,15.8)
7.61(dd,15.8,7.8)
9.60(s)
154.7
126.0
194.1
6.70(d,8.0)
–
percentage of the non-treated control group [27].
3. Results and discussion
spectrum of compound 1 revealed a molecular formula C33H40O11 in
the light of sodium addition peak, which was determined as 635.2466
([M + Na]+ calcd. for 635.2468 for C33H40O11Na), corresponding to an
index of hydrogen deficiency of 14. The UV spectrum of compound 1
exhibited characteristic absorption bonds at λmax 208, 235, and 270 nm,
indicating the dioxane neolignan skeleton [28]. The IR spectrum of
compound 1 revealed the presence of hydroxy (3325 cmꢀ 1), carbonyl
3.1. Structure elucidation of new compounds
Compound 1 was purified as a colorless powder. The HR-ESI-MS
5

Q.-G. Ma et al.
Bioorganic Chemistry 107 (2021) 104622
Table 4
1H NMR (400 MHz, DMSO‑d6) and 13C NMR (100 MHz, DMSO‑d6) of compounds (9–10).
No.
9 No. 9 No.
10 No.
10
δH δC δH δC
δH δC
δH δC
1
6.97(s)
112.7 4′ ꢀ 140.2 1
6.96(s)
112.6 4′
-
140.1
142.4
110.9
152.1
118.0
51.5
2
–
125.3 5′ ꢀ 142.4 2
–
125.2 5′
–
3
–
139.2 6′ 6.85(s) 110.8 3
138.0 7′ ꢀ 152.2 4
–
139.4 6′
6.87(s)
4
–
–
138.2 7′
–
–
5
–
116.2 8′ ꢀ 118.1 5
–
116.4 8′
6
–
129.6 9′ 4.68(s) 51.4 6
130.5 1′’ ꢀ 132.7 7
–
129.8 9′
4.68(s)
7
6.62(d,16.0)
6.63(d,16.0)
130.7 1′’
129.0 2′’
63.8 3′’
–
132.7
107.8
151.4
129.9
151.4
107.8
152.1
118.0
51.5
8
6.30(dt,16.0,5.8)
128.9 2′’ 6.36(s) 107.6 8
63.6 3′’ ꢀ 151.2 9
6.32(dt,16.0,5.8)
6.35(s)
9
4.21(d,5.8)
4.22(d,5.8)
–
10
6.86(d,16.7)
120.4 4′’ ꢀ 129.8 10
137.6 5′’ ꢀ 151.2 11
82.6 6′’ 6.36(s) 107.6 12
24.6 7′’ ꢀ 152.2 CH3-13/14
133.1 8′’ ꢀ 118.1 1′
6.65(d,10.0)
116.3 4′’
129.1 5′’
79.5 6′’
–
11
6.78(d,16.7)
5.71(d,10.0)
–
12
–
–
6.35(s)
–
CH3-13/14
1.50(s)
1.46(s)
28.3 7′’
1′
2′
3′
–
–
133.0 8′’
113.6 9′’
127.0 OCH3-3′’/5′’
–
7.21(s)
113.7 9′’ 4.68(s) 51.4 2′
126.9 OCH3-3′’/5′’ 3.79(s) 56.7 3′
7.20(s)
4.68(s)
3.80(s)
–
–
56.7
(1736 cmꢀ 1), and benzene ring (1605, 1511, and 1498 cmꢀ 1) func-
tionalities. The 1H NMR spectrum of compound 1 displayed a 7,8-trans
allyl alcohol group [28] at δH 6.61 (1H, d, J = 16.0 Hz, H-7), 6.29 (1H,
dt, J = 16.0, 5.8 Hz, H-8), and 4.20 (2H, d, J = 5.8 Hz, H-9) with 13C
NMR data δ 130.4 (C-7), 128.9 (C-8), and 63.5 (C-9) (Table 1), which
was locatedCat C-1 by the key HMBC correlations of H-2 to C-7 and H-8 to
Hz, H-11), 1.70 (3H, s, CH3-13), and 1.82 (3H, s, CH3-14) (Table 1) was
assigned to C-6 by the key HMBC correlation of H-11 to C-6 (Fig. 2).
Meanwhile, a typical AA’BB’ system [32] at δH 7.28 (2H, d, J = 8.0 Hz,
H-2′/6′), 6.65 (2H, d, J = 8.0 Hz, H-3′/5′) (Table 1) was connected to C-
7′ by the key HMBC correlations of H-2′/6′ to C-7′ (Fig. 2). Furthermore,
a 11′,16′-dihydroxy-15′,17′-dimethoxy-10′-phenylallyl moiety at δH
4.18 (2H, s, H-11′), 6.21 (1H, s, H-12′), and 3.86 (6H, s, OCH3-15′/17′)
was connected to C-4′ by the key HMBC correlation of H-2′/6′ to C-4′
(Fig. 2). The other fragments of compound 2 was established on the key
HMBC correlations of H-7 to C-9, H-10 to C-12, H-3′/5′ to C-1′, H-9′ to C-
7′, H-12′ to C-11′, H-14′/18′ to C-12′/16′ (Fig. 2), the 1H–1H COSY
correlations of H-7 to H-8, H-8 to H-9, H-10 to H-11, H-2′ to H-3′, H-5′ to
H-6′ (Fig. 2), and the key NOESY correlations of H-2 to H-9, H-7 to H-9,
H-9 to OCH3-9, H-10 to CH3-14, H-11 to CH3-13, CH3-13 to CH3-14,
OCH3-15′ to H-11′, OCH3-15′ to H-14′ (Fig. 3). Thus, compound 2 was
elucidated as (7E,10′E,11E)-1-(9-methoxyl)-propenyl-5-hydroxy-6-
prenyl-8′-methylol-11′,16′-dihydroxy-15′,17′-dimethoxy-10′-phenyl-
allyl alcohol-3,4,7′,8′-benzodioxane neolignan.
1
C-1 (Fig. 2). In the H NMR spectrum of compound 1, a 11-isopropyl-
furan ring moiety was found at δH 6.26 (1H, s, H-10), 3.38 (1H, m, H-
12), and 1.31 (6H, s, CH3-13/14) with 13C NMR data δ 108.6 (C-10),
161.2 (C-11), 35.8 (C-12), and 23.1 (C-13/14) (TableC1), which was
located at C-5/6 by the key HMBC correlations of H-10 to C-5 and H-10
to C-12 (Fig. 2). Moreover, the 1H NMR spectrum of compound 1
revealed a 1′,3′,4′,5′-tetrasubstituted benzene ring at δH 6.58 (2H, s, H-
2′/6′), 3.85 (6H, s, OCH3-3′/5′), which was attached to C-7′ by the key
HMBC correlations of H-2′/6′ to C-7 (Fig. 2). A glycerol group [29] at δH
3.98 (1H, t, J = 4.8 Hz, H-15′) and 3.76 (4H, dd, J = 4.8, 1.0 Hz, H-16′/
17′) with δC 84.3 (C-15′) and 61.9 (C-16′/17′) (Table 1) was connected
to C-4′ by the key HMBC correlation of H-15′ to C-4′ (Fig. 2). Meanwhile,
an isovalerate moiety [30] at δH 4.86 (1H, s, H-9′), 2.30 (2H, d, J = 6.6
Compound 3, a colorless powder with the molecular formula of
Hz, H-11′), 2.16 (1H, m, H-12′), and 1.00 (6H, s, CH3-13′/14′) with δ
C40H46O11, was demonstrated by its HR-ESI-MS at m/z 725.2937 [M +
C
65.3 (C-9′), 173.1 (C-10′), 43.9 (C-11′), 25.1 (C-12′), and 22.1 (C-13′/
14′) (Table 1) was assigned to C-9′ by the key HMBC correlations of H2-
9′ to C-7′/10′, H-2 to C-4 (Fig. 2). The other fragments of compound 1
were connected by the key correlations of H-7 to C-9, H-2′/6′ to C-4′, H-
16′ to C-17′, and H2-9′/12′ to C-10′ in the HMBC spectrum (Fig. 2), the
key correlations of H-7 to H-8, H-8 to H2-9, H-15′ to H2-16′, H-15′ to H2-
17′, and H2-11′ to H-12′ in the 1H–1H COSY spectrum (Fig. 2), and the
key correlations of H-2 to H-7, H-7 to H-9, H-6′ to H-9′, H-10 to CH3-13,
CH3-12 to CH3-13, CH3-13′ to CH3-14′, CH3-13′ to H-11′ in the NOESY
spectrum (Fig. 3). Consequently, compound 1 was determined as (7E)-1-
allyl alcohol-5,6-(11-isopropyl)-furanyl-3′,5′-dimethoxy-4′-glycerol-9′-
isovalerate-3,4,7′,8′-benzodioxane neolignan.
Na]+ (calcd. for C40H46O11Na, 725.2938) with 18 degrees of unsatura-
tion. Its UV spectrum showed absorption bonds at λmax 208, 235, 305,
and 343 nm, and its IR spectrum exhibited characteristic absorption
bonds at 3330, 2938, 1608, and 1509 cmꢀ 1, which revealed the char-
acteristic absorption peaks of the dioxane neolignan skeleton [28]. It
was found that UV and IR spectral data of compound 3 were similar to
compound 2, which concluded compound 3 was an analogue of com-
pound 2. The 1H and 13C NMR spectral data of compound 3 were similar
to compound 2. However, there were two different replacements be-
tween compounds 3 and 2. The H-6 was replaced by a geranyl moiety
[31] at δH 3.23 (2H, d, J = 9.2 Hz, H-10), 5.22 (1H, m, H-11), 1.97 (2H,
m, H-13), 1.46 (2H, m, H-14), 1.35 (2H, m, H-15), and 1.16 (6H, s, CH3-
17/18) (Table 1), which was verified by the key correlation of H-11 to C-
6 in the HMBC spectrum (Fig. 2); and H-4′/5′ were replaced by a 16′-
Compound 2 was obtained as a colorless powder. Its molecular for-
mula was assigned as C35H38O10 by HR-ESI-MS (m/z 641.2366 [M +
Na]+; calcd. for C35H38O10Na, 641.2363), requiring 17 degrees of
unsaturation. Its UV spectrum displayed absorptions at λmax 208, 235,
270, and 305 nm and its IR spectrum showed absorption bonds for hy-
droxy (3328 cmꢀ 1) and aromatic ring (1611 and 1509 cmꢀ 1) groups.
These characteristic absorption peaks indicated that the dioxane neo-
hydroxy-15′,17′-dimethoxyphenyl-11′-methylol-benzofuranyl
group
[33] at δH 7.18 (2H, s, H-14′/18′), 3.88 (6H, s, OCH3-15′/17′), and 4.66
(2H, s, H-12′) (Table 1), which was determined by the key HMBC cor-
relation of H-3′ to C-11′ (Fig. 2). Moreover, a typical ABX system at δ
H
7.56 (1H, dd, J = 8.0, 2.0 Hz, H-2′), 7.83 (1H, d, J = 8.0 Hz, H-3′), and
7.72 (1H, d, J = 2.0 Hz, H-6′) (Table 1) was assigned to C-7′ by the key
HMBC correlations of H-2′/6′ to C-7′ (Fig. 2). The other fragments of
compound 3 was established on the key HMBC correlations of H-7 to C-
9, H-10 to C-12, H-11 to C-13, H-14 to C-12/16, H-15 to C-13, H-2′ to C-
4′, H-3′ to C-1′, H-9′ to C-7′, H-12′/14′/18′ to C-10′, H-14′/18′ to C-16′
(Fig. 2), the 1H–1H COSY correlations of H-7 to H-8, H-8 to H-9, H-10 to
H-11, H-13 to H-14, H-14 to H-15, H-2′ to H-3′ (Fig. 2), and the key
1
lignan skeleton was in compound 2 [28]. In the H NMR spectrum of
compound 2, a 9-methoxyl-7,8 -trans propenyl group [28] at δH 6.52
(1H, d, J = 16.0 Hz, H-7), 6.21 (1H, dd, J = 16.0, 5.8 Hz, H-8), and 4.11
(1H, d, J = 5.8 Hz, H-9) with 13C NMR data δ 130.1 (C-7), 125.9 (C-8),
C
and 73.1 (C-9) (Table 1) was connected to C-1 by the key HMBC cor-
relations of H-2 to C-7 and H-8 to C-1 (Fig. 2). Moreover, a typical prenyl
moiety [31] at δ 3.48 (2H, d, J = 7.5 Hz, H-10), 5.25 (1H, t, J = 7.5, 1.5
H
6

Q.-G. Ma et al.
Bioorganic Chemistry 107 (2021) 104622
Fig. 2. Key HMBC and 1H–1H COSY correlations of new compounds (1–10).
NOESY correlations of H-2 to H-7, H-7 to H-9, H-9 to OCH3-9, H-10 to
CH3-12, H-13 to H-15, H-11 to H-14, H-15 to CH3-17, CH3-17 to CH3-18
(Fig. 3). Consequently, compound 3 was determined as (7E,11E)-1-(9-
methoxyl)-propenyl-5-hydroxy-6-geranyl-16′-hydroxy-15′,17′-
dimethoxyphenyl-8′,11′-dimethylol-benzofuranyl-3,4,7′,8′-benzodiox-
ane neolignan.
Compound 4 was obtained as a colorless powder. It was assigned the
molecular formula of C36H42O13 by the HR-ESI-MS data m/z 705.2520
7

Q.-G. Ma et al.
Bioorganic Chemistry 107 (2021) 104622
Fig. 3. Key NOESY correlations (blue) of new compounds (1–10). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
[M + Na]+ (calcd. for C36H42O13Na, 705.2523), requiring 16 degrees of
unsaturation. Its UV spectrum showed absorption bonds at 205, 272,
320, and 350 nm, which revealed the phenanthrofuran-type neolignan
skeleton [34]. Its IR spectrum displayed absorption peaks for hydroxy
(3421 cmꢀ 1) and aromatic ring (1610, 1518 and 1467 cmꢀ 1) function-
alities. In the 1H NMR spectrum of compound 4, a typical 18,19-dime-
77.7 (C-3′’), 71.2 (C-4′’), 76.4 (C-5′’), and 63.8 (C-6′’) (Table 2) was
connected to C-9′ by the key correlations of H-1′’ to C-9′, H2-9′ to C-7′/2
in the HMBC spectrum (Fig. 2). Moreover, a typical ABX system at δ
H
6.98 (1H, dd, J = 8.0 , 2.0 Hz, H-2′), 6.73 (1H, d, J = 8.0 Hz, H-3′), 6.85
(1H, d, J = 2.0 Hz, H-6′) (Table 2) and two methylene groups at δ 2.48
H
(2H, m, H-7), 2.42 (2H, m, H-8) were found in the 1H NMR spectrum.
The remaining fragments of compound 4 was determined by the key
correlations of H2-15 to C-17, H2-7 to C-1/5, H2-8 to C-10, H-11 to C-5,
H-14 to C-8/12, H-2′ to C-4′/7′, H-3′ to C-1′, H-6′ to C-7′, H-3′’ to C-5′’,
and H-6′’ to C-4′’ in the HMBC spectrum (Fig. 2), the key correlations of
H2-7 to H2-8, H2-15 to H2-16, and H-2′ to H-3′ in the 1H–1H COSY
spectrum (Fig. 2), and the key correlations of H-8 to H-14, H-6′ to OCH3-
5′, CH3-18 to CH3-19 in the NOESY spectrum (Fig. 3). Moreover, the D/L
isomerism of glucose moiety was confirmed by applying HPLC analyses
thylpropanol group [35] at δ 2.78 (2H, m, H-15), 1.58 (2H, m, H-
H
16), and 1.19 (6H, s, CH3-18/19) with δC 18.1 (C-15), 43.2 (C-16), 70.3
(C-17), and 29.2 (C-18/19) (Table 2) was assigned to C-1 by the key
correlations of H2-15 to C-2 and H2-16 to C-1 in the HMBC spectrum
(Fig. 2). Meanwhile, a typical β-glucopyranosyl moiety at δ 5.01 (1H, d,
H
J = 7.6 Hz, H-1′’), 3.72 (1H, m, H-2′’), 3.80 (1H, m, H-3′’), 3.38 (1H, m,
H-4′’), 3.68 (1H, m, H-5′’), 3.57 (1H, dd, J = 11.2, 6.4 Hz, H-6′’a), and
3.91 (1H, dd, J = 11.2, 3.2 Hz, H-6′’b) with δ 101.3 (C-1′’), 74.2 (C-2′’),
C
8

Q.-G. Ma et al.
Bioorganic Chemistry 107 (2021) 104622
after arylthiocarbamoyl-thiazolidine derivation [36]. The tR at 17.49
min of glucose was coincided with derivatives of D-glucose, which
compared with the retention times of the arythiocarbamoyl-thiazolidine
[36]. Therefore, the structure of compound 4 was assigned as 1-(18,19-
dimethyl)-propanol-4-hydroxyl-5,6-(13-hydroxyl-12-methoxyl)-phenyl-
ethyl-7′-(4′-hydroxyl-5′-methoxy)-phenyl-9′-O-β-D-glucopyranosyl-phe-
nanthrofuran neolignan.
hydroxyl-5,6-(13-hydroxyl-12-methoxyl)-phenylethyl-7′-(4′-hydroxy-
3′,5′-dimethoxy)-phenyl-9′-O-β-D-glucopyranosyl-phenanthrofuran
neolignan.
Compound 7 was isolated as a colorless powder, and its molecular
formula was established as
C32H28O6 through HR-ESI-MS (m/z
531.1781, [M + Na]+; calcd. for 531.1784) and NMR spectroscopic
data, suggesting 19 indices of hydrogen deficiency. Its UV spectrum
showed the absorption bonds at λmax 202, 280, 315, and 330 nm and IR
spectrum showed characteristic absorption bonds at hydroxy (3411
Compound 5 was obtained as a colorless powder, it showed an ion
peak at m/z 757.2475 [M + Na]+ in the HR-ESI-MS (calcd. for
C
39H42O14Na, 757.2472), corresponding to the molecular formula
cmꢀ 1), carbonyl (1719 cmꢀ 1), and aromatic ring (1617 and 1572 cmꢀ 1
)
C39H42O14, indicated 19 indices of hydrogen deficiency. The UV spec-
trum showed characteristic absorption bonds at 203, 272, 320, 346 nm
and its IR spectrum showed absorption bands for hydroxy (3425 cmꢀ 1),
aromatic ring (1612 and 1608 cmꢀ 1) functionalities, which indicated
the phenanthrofuran-type neolignan skeleton [34]. A closer comparison
of the 1H and 13C NMR spectral data (Table 2) with compound 4
revealed that compound 5 was an analogue of compound 4 except for
the 17-ethylfuryl group [37] at δH 2.25 (2H, m, H-15), 2.12 (2H, m, H-
16), 7.22 (1H, s, H-18), 7.31 (1H, d, J = 2.6 Hz, H-19), and 6.33 (1H, d, J
functional groups, which revealed compound 7 contained the biphenyl
ketone-type neolignan skeleton [39]. The 1H and 13C NMR spectra
showed the 11,12-dimethylpyranyl moiety [40] at δH 6.64 (1H, d, J =
10.0 Hz, H-8), 5.70 (1H, d, J = 10.0 Hz, H-9), and 1.47 (6H, s, CH3-11/
12) with δC 116.1 (C-8), 129.0 (C-9), 79.5 (C-10), and 28.7 (C-11/12)
(Table 3) was assigned to C-4/5 by the key HMBC correlations of H-9 to
C-5 and H-8 to C-6 (Fig. 2). Additionally, analysis of its NMR data
indicated the presence of a 9′’,10′’-trans propenyl unit [39] at δH 6.79
(1H, d, J = 15.6 Hz, H-9′’), 6.60 (1H, dd, J = 15.6, 6.6 Hz, H-10′’), and
= 2.6 Hz, H-20) with δ 29.4 (C-15), 24.5 (C-16), 116.1 (C-17), 109.4 (C-
1.95 (3H, d, J = 6.6 Hz, CH3-11′’) with δ 125.8 (C-9′’), 128.7 (C-10′’),
C
C
20), 141.3 (C-19), and 138.4 (C-18) (Table 2), which was connected to
C-1 by the key HMBC correlations of H2-15 to C-2 and H2-16 to C-1
(Fig. 2). A fragment of 3′,4′,5′-trimethoxyphenyl group at δH 6.39 (2H, s,
H-2′/6′) and 3.78 (9H, s, OCH3-3′/4′/5′) (Table 2) was assigned to C-7′
by the key HMBC correlations of H-2′/6′ to C-7′ (Fig. 2). The other
groups of compound 5 was connected by the key HMBC correlations of
H-15 to C-17, H-18/20 to C-16, H-19 to C-17, H-7 to C-1/5, H-8 to C-10,
H-11 to C-5, H-14 to C-8/12, H-2′/6′ to C-4′, H-9′ to C-7′, H-3′’ to C-5′’,
H-6′’ to C-4′’ (Fig. 2), the 1H–1H COSY correlations of H-7 to H-8, H-15
to H-16, H-19 to H-20 (Fig. 2), and the key NOESY correlations of H-8 to
H-14, H-2′ to OCH3-3′, H-2′/H-9′ (Fig. 3). Moreover, the D/L isomerism
of glucose moiety was confirmed by applying HPLC analyses after
arylthiocarbamoyl-thiazolidine derivation [36]. The tR at 17.48 min of
glucose was coincided with derivatives of D-glucose, which compared
with the retention times of the arythiocarbamoyl-thiazolidine [36].
Consequently, compound 5 was elucidated as 1-(17-furanyl)-ethyl-4-
hydroxyl-5,6-(13-hydroxyl-12-methoxyl)-phenylethyl-7′-(3′,4′,5′-trime-
thoxy)-phenyl-9′-O-β-D-glucopyranosyl-phenanthrofuran neolignan.
Compound 6 was isolated as a colorless powder. The molecular
formula of compound 6 was deduced as C37H42O14 in view of its HR-ESI-
MS (m/z 733.2474, [M + Na]+; calcd. for 733.2472), indicating 17
indices of hydrogen deficiency. The UV spectrum of compound 6
exhibited the characteristic absorption bonds of the phenanthrofuran-
type neolignan skeleton at 205, 272, 323, and 345 nm [34]. Its IR
spectrum exhibited the characteristic adsorptions at 3423 and 1611
cmꢀ 1 suggesting hydroxy group and benzene ring group, respectively.
The 1H and 13C NMR spectral data indicated that the chemical structure
of compound 6 resembled that of compound 5, except that the (17Z)-
and 19.3 (C-11′’) (Table 3) was connected to C-4′’ by the key HMBC
correlations of H-3′’ to C-9′’ and H-10′’ to C-4′’ (Fig. 2). Meanwhile, an
acetyl group at δH 2.55 (3H, s, CH3-8′’) was attached to C-6′’ by the
HMBC correlation of H-5′’ to C-7′’ (Fig. 2) and a 4′-hydroxy phenyl
group at δH 7.39 (2H, d, J = 8.0 Hz, H-2′/6′) and 6.64 (2H, d, J = 8.0 Hz,
H-3′/5′) (Table 3) was assigned to C-7′ by the HMBC correlations of H-
2′/6′ to C-7′ (Fig. 2). The remaining groups of compound 7 was eluci-
dated by the key HMBC correlations of H-1 to C-7/8′, H-8 to C-6/10, H-9
to C-5, H-3′/5′ to C-1′, H-9′ to C-7′, H-2′’ to C-7/6′’, and H-5′’ to C-7′’
(Fig. 2), the key 1H–1H COSY correlations of H-8 to H-9, H-2′ to H-3′, H-
5′ to H-6′, H-2′’ to H-3′’, and H-9′’ to H-10′’ (Fig. 2), and the key cor-
relations of H-2′/H-9′, H-2′/H-3′, H-5′/H-6′, CH3-12/H-9 in the NOESY
spectrum (Fig. 3). Consequently, compound 7 was elucidated as (9′’E)-
4,5-(11,12-dimethyl)-pyranyl-7′-(4′-hydroxy)-phenyl-4′’-propenyl-8′-
methylol-furanyl-6′’-acetyl-1′’,6-biphenyl-7-ketone neolignan.
Compound 8 was isolated as a colorless powder. Its molecular for-
mula was determined to be C33H30H8, based on HR-ESI-MS data at m/z
577.1840 [M + Na]+ (calcd. for C33H30H8Na, 577.1838), corresponding
to 19 degrees of unsaturation. It was concluded that compound 8 was an
analogue of compound 7 with the biphenyl ketone-type neolignan
skeleton [39] by comparing with the UV (λmax 202, 280, 318, 332, and
351 nm) and IR (3410, 1720, 1677, 1617, and 1573 cmꢀ 1). Detailed
1
analysis of the H and 13C NMR spectral data (Table 3) revealed that
compound 8 was similar to those of compound 7 except for an iso-
pentenyl [21] group at δ 3.33 (2H, d, J = 7.5 Hz, H-8), 5.36 (1H, d, J =
H
7.5 Hz, H-9), 1.80 (3H, s, CH3-11), and 1.68 (3H, s, CH3-12) (Table 3),
and above moiety was connected to C-5 by the key HMBC correlations of
H-9 to C-5 and H2-8 to C-6 (Fig. 2). Meanwhile, a group of characteristic
peaks of a propenylketone group [41] at δ 8.60 (1H, d, J = 15.8 Hz, H-
9′’), 7.61 (1H, dd, J = 15.8, 7.8 Hz, H-10H′’), and 9.60 (1H, brs, H-11′’)
methyl-butanol group [38] at δ 3.28 (2H, d, J = 6.9 Hz, H-15), 6.03
H
(1H, m, H-16), 4.31 (2H, m, H-18), and 1.74 (3H, s, CH3-19) with δC 31.5
(C-15), 126.7 (C-16), 137.0 (C-17), 62.9 (C-18), and 17.5 (C-19)
(Table 2) was located to C-1 by the key HMBC correlations of H2-15 to C-
2 and H2-16 to C-1 (Fig. 2), and a 4′-hydroxy-3′,5′-dimethoxyphenyl
group at δH 6.42 (2H, s, H-2′/6′) and 3.84 (6H, s, OCH3-3′/5′) (Table 2)
was assigned to C-7′ by the key HMBC correlations of H-2′/6′ to C-7′
(Fig. 2). The other groups of compound 6 was connected by the key
HMBC correlations of H-15 to C-17, H-16 to C-18, H-7 to C-1/5, H-8 to C-
10, H-11 to C-5, H-14 to C-8/12, H-2′/6′ to C-4′, H-9′ to C-7′, H-3′’ to C-
5′’, H-6′’ to C-4′’ (Fig. 2), the 1H–1H COSY correlations of H-7 to H-8, H-
15 to H-16 (Fig. 2), and the key NOESY correlations of H-8 to H-14, H-2′
to OCH3-3′, H-16/CH3-19 (Fig. 3). Moreover, the D/L isomerism of
glucose moiety was confirmed by applying HPLC analyses after
arylthiocarbamoyl-thiazolidine derivation [36]. The tR at 17.50 min of
glucose was coincided with derivatives of D-glucose, which compared
with the retention times of the arythiocarbamoyl-thiazolidine [36].
Therefore, compound 6 was determined as 1-(17Z)-methyl-butanol-4-
with δ 154.7 (C-9′’), 126.0 (C-10′’), and 194.1 (C-11′’) (Table 3) was
C
assigned to C-4′’ by the key HMBC correlations of H-3′’/5′’ to C-9′’ and
H-10′’ to C-4′’ (Fig. 2). Moreover, two typical ABX systems at δH 6.91
(1H, dd, J = 8.0, 2.0 Hz, H-2′), 6.70 (1H, dd, J = 8.0 Hz, H-3′), 6.86 (1H,
d, J = 2.0 Hz, H-5′) and 6.88 (1H, dd, J = 8.0, 2.0 Hz, H-2′’), 7.50 (1H, d,
J = 8.0 Hz, H-3′’), 7.90 (1H, d, J = 2.0 Hz, H-5′’) were found in the 1H
NMR spectrum of compound 8. The other groups of compound 8 was
determined by the key HMBC correlations of H-1 to C-7/8′, H-8 to C-6/
10, H-9 to C-5, H-3′/5′ to C-1′, H-9′ to C-7′, H-2′’ to C-6′’/7, H-3′’ to C-
9′’, H-5′’ to C-7′’/9′’, H-9′’ to C-11′’, H-10′’ to C-4′’ (Fig. 2), the 1H–1H
COSY correlations of H-8 to H-9, H-2′ to H-3′, H- 2′’ to 3′’, H-9′’ to H-
10′’, H-10′’ to H-11′’ (Fig. 2), and the key NOESY correlations of H-8 to
CH3-11, H-9 to CH3-12, CH3-11 to CH3-12, H-2′ to H-3′, H-6′ to OCH3-5′
(Fig. 3). Thus, compound 8 was elucidated as (9E,9′’E)-5-isopentenyl-7′-
(4′-hydroxy-5′-methoxy)-phenyl-4′’-propenylketone-8′-methylol-fur-
anyl-6′’-acetyl-1′’,6-biphenyl-7-ketone neolignan.
9

Q.-G. Ma et al.
Bioorganic Chemistry 107 (2021) 104622
Compound 9 was isolated as a colorless powder. Its molecular for-
3.2. Acid hydrolysis of compounds 4–6 and 21–22
mula was confirmed as C34H34O11 based on the HR-ESI-MS data (m/z
641.1995 [M + Na]+, calcd. for C34H34O11Na 641.1999), indicating 18
indices of hydrogen deficiency. Its UV spectrum showed absorption
bonds at 202, 230, and 270 nm, along with IR absorption of hydroxy
(3409 cmꢀ 1) and aromatic ring (1611 and 1508 cmꢀ 1) functional
groups, which concluded the 1′,7′-bineolignan skeleton [42]. In the 1H
Compounds 4–6 and 21–22 (5 mg each) were treated in 5% HCl (0.5
mL) and heated at 90 ◦C for 2 h, respectively [44]. After cooling, each
reaction mixture was extracted with EtOAc, and the aqueous layer was
neutralised with 0.1 M NaOH. As a result, the glucoses were obtained
from compounds 4–6 and 21–22, which were detected by thin layer
chromatography (TLC) with authentic sugars. The type of glucose was
identified by TLC method with authentic sugar [45]. Meanwhile, we had
confirmed the D/L isomerism of glucose moiety by applying reversed-
phase HPLC analyses after arylthiocarbamoyl-thiazolidine derivation
[36]. The glucoses were obtained from compounds 4–6 and 21–22, and
they were dried in vacuo. The residue was dissolved in pyridine (0.1 mL)
containing L-cysteine methyl ester hydrochloride (0.5 mg) and heated at
NMR spectrum of compound 9, an isopentenol moiety at δ 6.86 (1H, d,
H
J = 16.7 Hz, H-10), 6.78, 1H, d, J = 16.7 Hz, H-11), and 1.50 (6H, s,
CH3-13/14) with δ 120.4 (C-10), 137.6 (C-11), 82.6 (C-12), and 24.6
C
(C-13/14) (Table 4) was assigned to C-5 by the key HMBC correlations of
H-11 to C-5 and H-10 to C-4 (Fig. 2). In addition, a 7,8-trans allyl alcohol
group [28] at δH 6.62 (1H, d, J = 16.0 Hz, H-7), 6.30 (1H, dt, J = 16.0,
5.8 Hz, H-8), and 4.21 (2H, d, J = 5.8 Hz, H-9) and 13C NMR data δ
C
◦
130.5 (C-7), 128.9 (C-8), and 63.6 (C-9) (Table 4) was assigned to C-6 by
the key HMBC correlations of H-1 to C-7 and H-8 to C-6 (Fig. 2).
Moreover, a moiety of 4′’-hydroxy-3′’,5′’-dimethoxyl phenyl at δH 6.36
(2H, s, H-2′’/6′’) and 3.79 (6H, s, OCH3-3′’/5′’) (Table 4) was connected
to C-7′’ by the key correlations of H-2′’/6′’ to C-7′’ in the HMBC spec-
trum (Fig. 2). The structure of compound 9 was further confirmed by
analyses of the key HMBC correlations of H-1 to C-8′, H-7 to C-9, H-10 to
C-12, H-2′ to C-7′/8′’, H-6′ to C-7′, H2-9′ to C-7′, H-2′’/6′’ to C-4′’, and
H2-9′’ to C-7′’ (Fig. 2), the key 1H–1H COSY correlation of H-7 to H-8, H-
8 to H2-9, and H-10 to H-11 (Fig. 2), and the key correlations of H-1 to H-
8, 4-OH to H-10, H-2′’ to H-9′’, H-6′’ to 5′’–OCH3 in the NOESY spec-
trum (Fig. 3). Therefore, compound 9 was determined as (7E,10E)-4,5′-
60 C for 1 h. A 0.1 mL solution of O-torylisothiocyanate (0.5 mg) in
pyridine was added to the mixture, which was heated at 60 ◦C for 1 h.
The reaction mixture was directly analyzed by reversed-phase HPLC.
The tR values of peaks in the range of 17.48–17.50 min were coincided
with derivatives of D-glucose, which compared with the retention times
of the arythiocarbamoyl-thiazolidine [36].
3.3. Plausible biogenetic pathways of compounds (1–10)
It’s very important that the structures of compounds (1–10) were
determined as neolignan derivatives, which may be closely related to
biogenetic pathways, were isolated from C. medica L. var. sarcodactylis
Swingle at the same time (Scheme 1). Interestingly, the plausible
biogenetic pathway of compounds (1–3) could be traced back to ben-
zodioxane neolignan core just like many neolignans. Compound 1 went
through hydrolysis of ester bond to obtain compound 2, and compound
2 went through cyclization to afford compound 1. Compound 2 went
through cyclization and substitution to obtain compound 3. Meanwhile,
the plausible biogenetic pathway for compounds (4–6) could be traced
back to phenanthrofuran neolignan skeleton, which mainly went
through a series of potential substitution to obtain compounds (4–6).
Thus, compounds 4 and 6 went through cyclization to obtain compound
5, and compound 5 went through substitution to obtain compounds 4
and 6. In the same way, the plausible biogenetic pathway of compound 7
could be readily transformed to compound 8 went through the way of
ring opening and compound 8 transformed to compound 7 went through
cyclization. Moreover, the plausible biogenetic pathway of compound 9
could be readily changed to compound 10 by the way of cyclization and
compound 10 transformed to compound 9 went through the way of ring
opening. The further and reasonable biogenetic pathway of compounds
(1–10) of C. medica L. var. sarcodactylis Swingle will be researched in the
future.
dihydroxy-5-isopentenol-6-(7,8-trans
allyl)-alcohol-7′’-(4′’-hydroxy-
3′’,5′’-dimethoxyl)-phenyl-9′,9′’-dimethylol-1′,7′-bineolignan.
Compound 10 was obtained as a colorless powder. Its molecular
formula was established as C34H32O10 by HR-ESI-MS (m/z 623.1895 [M
+ Na]+, calcd. for C34H32O10Na, 623.1893), corresponding to 19 de-
grees of unsaturation. Its UV spectrum exhibited absorptions at λmax 202,
230, 270, and 315 nm and IR spectrum showed hydroxy (3410 cmꢀ 1
)
and aromatic ring (1612 and 1507 cmꢀ 1) functional groups, it was
concluded that compound 10 was an analogue of compound 9. A closer
comparison of the NMR spectra (Table 4) with compound 9 revealed
that compound 10 contained the 1′,7′-bineolignan skeleton [42]. The
only difference between compounds 10 and 9 was the appearance of the
13,14-dimethyl-pyran moiety [40] at δ 6.65 (1H, d, J = 10.0 Hz, H-10),
5.71 (1H, d, J = 10.0 Hz, H-11), and H1.46 (6H, s, CH3-13/14) with δC
116.3 (C-10), 129.1 (C-11), 79.5 (C-12), and 28.3 (C-13/14) (Table 4),
which was assigned to C-4/5 by the key HMBC correlations of H-11 to C-
5 and H-10 to C-6 (Fig. 2). The other fragments of compound 10 were
connected by the key HMBC correlations of H-1 to C-7/8′’, H-7 to C-9, H-
8 to C-6,H-10 to C-12, H-14 to C-12, H-2′/6′ to C-7′, H-2′ to C-8′, H-9′ to
C-7′, H-2′’/6′’ to C-4′’/7′’, H-9′’ to C-7′’ (Fig. 2), the 1H–1H COSY cor-
relation of H-7 to H-8, H-8 to H-9, H-10 to H-11 (Fig. 2), and the key
NOESY correlations of H-1 to H-8, H-7 to H-9, H-7 to H-10, 5′-OH to H-
6′, H-2′’ to H-9′’, H-6′’ to OCH3-5′’ (Fig. 3). Thus, compound 10 was
determined as (7E)-5′-hydroxy-4,5-(13,14-dimethyl)-pyranyl-6-allyl
alcohol-7′’-(4′’-hydroxy-3′’,5′’-dimethoxyl)-phenyl-9′,9′’-dimethylol-
1′,7′-bineolignan.
3.4. Statistical analysis of hepatoprotective and neuroprotective activities
In this study, compounds (1–24) were evaluated for their hep-
atoprotective activities in HepG2 cells by the acetaminophen (APAP)-
induced damage model at 10.0 μM with bicyclol as a positive group (cell
Meanwhile, fourteen known neolignan derivatives (11–24) were
isolated and characterized from the fruits of C. medica L. var. sarco-
dactylis Swingle for the first time. These isolates were elucidated to be
(7S,8R)-9′,3-dimethoxyl isoamericanol a (11) [28], (7S,8R,7′’S,8′’R)-
7,8–7′’,8′’-trans-7′,8′-Z-sesquiverniciasin A (12) [28], (7S,8R,7′’S, 8′’R)-
7,8–7′’,8′’-trans-7′,8′-E-sesquiverniciasin A (13) [27], selamoellenin B
(14) [33], dendronbibisline A (15) [34], dendronbibisline B (16) [34],
dendronbibisline C (17) [34], dendronbibisline D (18) [34], herpeto-
siols B (19) [42], herpetosiols C (20) [42], silychristin A (21) [39],
silychristin B (22) [39], (7S,8R)-threo-1′-[3′-hydroxy-7-(4-hydroxy-3-
methoxyphenyl)-8-hydroxymethyl-7,8-dihydrobenzofuran]acryl-
aldehyde (23) [41], and (-)-(7R,8S,7′E)-4-hydroxy-3,5,5′,9′-tetrame-
thoxy-4′,7-epoxy-8,3′-neolign-7′-en-9-ol (24) [43] by comparison of
their spectroscopic data and references.
viability of 75.34 ± 3.60%). As a result, compounds 1–3 and 12–13
exhibited moderate hepatoprotective activities to improve the HepG2
cell survival rates from 46.26 ± 1.90% (APAP, 10 mM) to 67.23 ±
4.25%, 62.87 ± 4.43%, 60.06 ± 6.34%, 56.75 ± 2.30%, and 58.35 ±
6.14%, respectively (Fig. 4). Among them, compounds (1–3) showed
slightly better cell viabilities than compounds (12–13). However, the
other compounds displayed no hepatoprotective activities. Meanwhile,
compounds (1–24) were also assayed for their neuroprotective activities
on PC12 cells with desipramine as a positive control (cell viability of
79.84 ± 1.55%). As shown in Fig. 5, compounds 7, 8, 21, and 22 showed
certain neuroprotective activities to raise the survival rates of PC12 cells
from 55.30 ± 2.25% to 66.94 ± 3.37%, 70.98 ± 5.05%, 64.64 ± 1.93%,
and 62.81 ± 4.11% at 10
μM, respectively. Among them, compounds 7
and 8 showed slightly better neuroprotective activities with cell
10

Q.-G. Ma et al.
Bioorganic Chemistry 107 (2021) 104622
Scheme 1. Plausible biogenetic pathways of new compounds (1–10).
viabilities of 66.94 ± 3.37% and 70.98 ± 5.05% than compounds 21 and
22 with cell viabilities of 64.64 ± 1.93% and 62.81 ± 4.11% at 10 μM.
However, the other compounds exhibited no neuroprotective activities
in this experiment. This study will enrich the chemical constituents of
C. medica L. var. sarcodactylis Swingle and facilitate the development of
more hepatoprotective and neuroprotective agents in the future.
3.5. Preliminary structure activity relationship of active compounds
A preliminary structure–activity relationship of neolignan de-
rivatives (1–24) was summarized according to their chemical structures
and hepatoprotective and neuroprotective activities. Compounds 1–3
and 12–13 displayed certain hepatoprotective activities which
possessed the same benzodioxane skeleton. Among them, compounds
(1–3) exhibited the most obvious cell survival rates of 67.23 ± 4.25%,
62.87 ± 4.43%, and 60.06 ± 6.34%, respectively, which had the same
substitutions of 5-hydroxy-6-prenyl moiety which possibly dominated
their hepatoprotective activities. Meanwhile, compounds 7–8 and
21–22 exhibited certain neuroprotective activities which possessed the
same biphenyl-ketone skeleton. Among them, compounds (7–8) showed
the most important neuroprotective activities with increasing the cell
Fig. 4. Hepatoprotective activities of selected compounds (10
μM). n = 3,
mean ± SD. ###P < 0.001, compared with normal; *P < 0.05, ***P < 0.001,
compared with model.
survival rate at 66.94 ± 3.37% and 70.98 ± 5.05% at 10
μM,
11

Q.-G. Ma et al.
Bioorganic Chemistry 107 (2021) 104622
Technology Project of Jiangxi Health Commission (20195648,
20195650), the Science and Technology Project of Jiangxi Provincial
Department of Education (GJJ180662, GJJ180688), the Doctoral
Research Initiation Fund Project of Jiangxi University of TCM
(2018BSZR007, 2018BSZR010), and the Scientific Research Project of
First-class Discipline of Chinese Materia Medica of Jiangxi University of
TCM (JXSYLXK-ZHYAO027, JXSYLXK-ZHYAO032) .
References
[1] Y. Gao, B. Peng, Y.F. Xu, J.N. Yang, L.Y. Song, S.X. Bi, Y.Y. Chen, J.H. Zhu, Y. Wen,
R.M. Yu, Structural characterization and immunoregulatory activity of a new
polysaccharide from Citrus medica L. var Sarcodactylis, RSC Adv. 9 (2019)
6603–6612.
[2] S.D. Zhang, H.Y. Yang, J. Zeng, M. Li, Research progress of Citrus medica L. var
Sarcodactylis, China J. Trad. Chin. Med. Pharm. 33 (2018) 3510–3514.
[3] Z.C. He, F.G. Liang, Y.Y. Zhang, Y.J. Pan, Water-soluble polysaccharides from
finger citron fruits (Citrus medica L. var. sarcodactylis), Carbohydr. Res. 388 (2014)
100–104.
[4] X. Deng, X.M. Huang, D. Wu, Process optimization of total flavonoids from chuan
bergamot by supercritical CO2 extraction, J. Chongqing Univ. Arts Sci. 33 (2014)
94–98.
Fig. 5. Neuroprotective activities of selected compounds against
[5] G.L. Cui, L.Y. Li, J. Tan, Y. Zhang, Analysis and evaluation of eight active
ingredients in Citrus medica L. var. sarcodactylis Swingle from different regions, Nat.
Prod. Res. Dev. 31 (2019) 250–260.
corticosterone-induced neuronal injury in PC12 cells (10 μM). n = 3, mean ±
SD. ###P < 0.001, compared with normal; *P < 0.05, **P < 0.01, ***P < 0.001,
[6] E.W. Wang, Y.Q. Li, B.L. Maguy, Z.X. Lou, H.X. Wang, W.Q. Zhao, X.H. Chen,
Separation and enrichment of phenolics improved the antibiofifilm and
antibacterial activity of the fractions from Citrus medica L. var. sarcodactylis in vitro
and in tofu, Food Chem. 294 (2019) 533–538.
compared with model.
respectively. It is the reason that C-2/3 was replaced by the benzofuran
moiety, which possibly played an important role in mediating neuro-
protective activities. The structure–activity relationship of hep-
atoprotective and neuroprotective compounds from C. medica L. var.
sarcodactylis Swingle need further research in the future.
´
[7] A. Verzera, A. Trozzi, M. Zappala, C. Condurso, A. Cotroneo, Essential oil
composition of Citrus meyerii Y. Tan. and Citrus medica L. cv. Diamante and their
lemon hybrids, J. Agric. Food Chem. 53 (2005) 4890–4894.
[8] A.L. Fanciullino, C. Dhuique-Mayer, F. Luro, J. Casanova, R. Morillon, P. Ollitrault,
Carotenoid diversity in cultivated citrus is highly influenced by genetic factors,
J. Agric. Food Chem. 54 (2006) 4397–4406.
[9] Q.F. Zeng, Determination of amino acids and trace elements in Zhejiang Finger
Citron, J. Jiaying Univ. 28 (2010) 75–77.
4. Conclusion
[10] X. Deng, X.M. Huang, D. Wu, Study on antioxidant activity and process
optimization of extraction of polyphenols from Chuan Bergamot, Guangzhou
Chem. Ind. 42 (2014) 50–53.
In this work, three new benzodioxane neolignans (1–3), three new
phenanthrofuran neolignan glycosides (4–6), two new biphenyl-ketone
neolignans (7–8), two new 1′,7′-bilignan neolignans (9–10), along with
fourteen known neolignan derivatives (11–24) were isolated from the
fruits of C. medica L. var. sarcodactylis Swingle for the first time. Among
them, compounds 1–3 and 12–13 exhibited moderate hepatoprotective
activities to improve the survival rates of HepG2 cells from 46.26 ±
1.90% (APAP, 10 mM) to 67.23 ± 4.25%, 62.87 ± 4.43%, 60.06 ±
6.34%, 56.75 ± 2.30%, 58.35 ± 6.14%, respectively. Compounds (1–3)
showed slightly better cell viabilities than compounds (12–13). Addi-
tionally, compounds 7–8 and 21–22 displayed moderate neuro-
protective activities to raise the survival rates of PC12 cells from 55.30
± 2.25% to 66.94 ± 3.37%, 70.98 ± 5.05%, 64.64 ± 1.93%, and 62.81
[11] B. Peng, J.N. Yang, W.J. Huang, D. Peng, S.X. Bi, L.Y. Song, Y. Wen, J.H. Zhu, Y.
Y. Chen, R.M. Yu, Structural characterization and immunoregulatory activity of a
novel heteropolysaccharide from bergamot (Citrus medica L. var. sarcodactylis) by
alkali extraction, Ind. Crop. Prod. 140 (2019) 11617.
[12] M. Shojaemehr, M. Alamholo, J. Soltani, Investigation of antibacterial and
antioxidant activity of Citrus medica L extract on human pathogenic bacteria,
Avicenna J. Clin. Microbiol. Infect. 7 (2020) 8–14.
[13] N. Chhikara, R. Kour, S. Jaglan, P. Gupta, Y. Gat, A. Panghal, Citrus medica:
nutritional, phytochemical composition and health benefifits - a review, Food
Funct. 9 (2018) 1978–1992.
[14] M.H. Hetta, T.S. El-Alfy, N.Z. Yassin, R.F. Abdel-Rahman, E.M. Kadry,
Phytochemical and antihyperglycemic studies on Citrus medica L. leaves (Etrog)
growing in Egypt, Int. J. Pharmacogn. Phytochem. Res. 5 (2013) 271–277.
[15] M.A. Al-Yahya, R.A. Mothana, M.S. Al-Said, K.E. El-Tahir, M. Al-Sohaibani,
S. Rafatullah, Citrus medica “Otroj”: attenuates oxidative stress and cardiac
dysrhythmia in isoproterenol-induced cardiomyopathy in rats, Nutrients 5 (2013)
4269–4283.
± 4.11% at 10 μM, respectively. Compounds 7 and 8 showed slightly
[16] H.Y. Gao, Q. Tian, Antidepressant effect essential oils of C. medica L. var.
sarcodactylis Swingle in rats, Chin. J. Exp. Trad. Med. Formula. 18 (2012) 231–234.
[17] P.C. Kuo, Y.R. Liao, H.Y. Hung, C.W. Chuang, T.L. Hwang, S.C. Huang, Y.J. Shiao,
D.H. Kuo, T.S. Wu, Anti-inflammatory and neuroprotective constituents from the
peels of Citrus grandis, Molecules 22 (2017) 967.
better neuroprotective activities than compounds 21 and 22. These
findings shed much light on a better understanding of the hep-
atoprotective and neuroprotective activities of these neolignan de-
rivatives and provided new insights into developing better
hepatoprotective and neuroprotective drugs in the future.
[18] Q.G. Ma, R.R. Wei, B. Zhou, Z.P. Sang, W.M. Liu, Z.L. Cao, Antiangiogenic
phenylpropanoid glycosides from Gynura cusimbua, Nat. Prod. Res. 33 (2019)
457–463.
[19] Q.G. Ma, R.R. Wei, D.L. Shang, Z.P. Sang, W.M. Liu, Z.L. Cao, Hepatoprotective and
neuroprotective flavanes from the fruits of Ulmus pumila L. (Ulmaceae), Pak. J.
Pharm. Sci. 32 (2019) 2059–2064.
Declaration of Competing Interest
[20] Q.G. Ma, R.R. Wei, Z.P. Sang, Structural characterization and hepatoprotective
activity of naphthoquinone from Cucumis bisexualis, Nat. Prod. Commun. 15 (2020)
1–6.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[21] R.R. Wei, Q.G. Ma, G.Y. Zhong, J.W. He, Z.P. Sang, Isolation and characterization
of flavonoid derivatives with anti-prostate cancer and hepatoprotective activities
from the flowers of Hosta plantaginea (Lam.) Aschers, J. Ethnopharmacol. 253
(2020), 112685.
Acknowledgements
[22] Q.G. Ma, R.R. Wei, D.L. Shang, Z.P. Sang, J.H. Dong, Structurally diverse
flavonolignans with immunosuppressive and neuroprotective activities from the
fruits of Hippophae rhamnoides L, J. Agric. Food Chem. 68 (2020) 6564–6575.
[23] L. Zhen, J.Y. Zhao, S.F. Sun, Y. Li, J. Qu, H.T. Liu, Y.B. Liu, Sesquiterpenes from an
endophytic Aspergillus flavus, J. Nat. Prod. 82 (2019) 1063–1071.
[24] Q.G. Ma, R.R. Wei, X.D. Zhang, Z.P. Sang, J.H. Dong, Q.X. Lu, H.F. Huang, D.
M. Guo, L. Jiang, Tropolone derivatives with hepatoprotective and
This work was supported by Jiangxi Provincial Natural Science
Foundation (20202BAB206078, 20202BAB216036), the Scientific
Research Project of Jiangxi Administration of Traditional Chinese
Medicine (2019A002, 2019A018), the Key Scientific Research Project of
Colleges and Universities in Henan Province (19A350006), the National
Natural Science Foundation of China (81803843), the Science and
12

Q.-G. Ma et al.
Bioorganic Chemistry 107 (2021) 104622
antiproliferative activities from the aerial parts of Chenopodium album Linn,
xanthine oxidase inhibitory activity from the leaves of Cyclocarya paliurus, Bioorg.
Chem. 101 (2020), 104018.
Fitoterapia 146 (2020), 104733.
[25] X.R. Peng, J.Q. Liu, C.F. Wang, X.Y. Li, Y. Shu, L. Zhou, M.H. Qiu, Hepatoprotective
effects of triterpenoids from Ganoderma cochlear, J. Nat. Prod. 77 (2014) 737–743.
[26] L. Yang, Z.M. Wang, Y. Wang, R.S. Li, F. Wang, K. Wang, Phenolic constituents
with neuroprotective activities from Hypericum wightianum, Phytochemistry 65
(2019), 112049.
[36] T. Tanaka, T. Nakashima, T. Ueda, K. Tomii, I. Kouno, Facile discrimination of
aldose enantiomers by reversed-phase HPLC, Chem. Pharm. Bull. 55 (2007)
899–901.
[37] L. Gonzalez-Cofrade, S. Oramas-Royo, I. Cuadrado, A. Amesty, S. Hortelano,
A. Estevez-Braun, B. Heras, Dehydrohispanolone derivatives attenuate the
inflammatory response through the modulation of inflammasome activation,
J. Nat. Prod. 83 (2020) 2155–2164.
[27] B.P. Jiang, Y.M. Liu, L. Le, Z.Y. Li, J.Y. Si, X.M. Liu, Q. Chang, R.L. Pan,
Cajaninstilbene acid prevents corticosterone-induced apoptosis in PC12 cells by
inhibiting the mitochondrial apoptotic pathway, Cell. Physiol. Biochem. 34 (2014)
1015–1026.
[38] Y.P. Liu, X.M. Yu, W. Zhang, T. Wang, B. Jiang, H.X. Tang, Q.T. Su, Y.H. Fu,
Prenylated chromones and flavonoids from Artocarpus heterophyllus with their
potential antiproliferative and anti-inflammatory activities, Bioorg. Chem. 101
(2020), 104030.
[28] D. Zhou, Y. Li, G. Chen, Y.Q. Yang, Y. Mi, B. Lin, W. Li, Y. Hou, N. Li, Structural
elucidation and anti-neuroinflammatory activities of lignans from the testas of
Vernicia montana, Bioorg. Chem. 97 (2020), 103690.
[39] D.D. Huang, X. Wang, L. Sun, W.S. Chen, L.N. Sun, Two new phenylpropanoids
from Penthorum chinense Pursh, Phytochem. Lett. 28 (2018) 84–87.
[40] Q.G. Ma, R.R. Wei, M. Yang, X.Y. Huang, F. Wang, Z.P. Sang, W.M. Liu, Q. Yu,
Molecular characterization and bioactivity of coumarin derivatives from the fruits
of Cucumis bisexualis, J. Agric. Food Chem. 66 (2018) 5540–5548.
[41] S.J. Zhang, Y.Y. Huang, Y. Li, Y.H. Wang, X.J. He, Anti-neuroinflammatory and
antioxidant phenylpropanoids from Chinese olive, Food Chem. 286 (2019)
421–427.
[29] F.H. Li, Z.Y. Cao, H.Q. Wang, C.K. Li, J. Fu, J. Xie, B.M. Li, R.Y. Chen, J. Kang,
Inhibition of IL-6 expression by lignans and other constituents isolated from
Schefflera rubriflora C. J. Tseng & G. Hoo, Fitoterapia 140 (2020) 104417.
[30] H.M. Wang, Z.T. Song, H.H. Xing, Z.Y. Shi, P. Wu, J. Zhang, M. Tuerhong, J. Xu, Y.
Q. Guo, Nitric oxide inhibitory iridoids as potential anti-inflammatory agents from
Valeriana jatamansi, Bioorg. Chem. 101 (2020), 103974.
[31] C.M. Xiao, X.H. Jia, H.F. Du, H.X. Zhao, C.L. Du, W.Z. Tang, X.J. Wang, Three new
C-geranylated flavonoids from Paulownia catalpifolia T. Gong ex D.Y. Hong seeds
with their inhibitory effects on xanthine oxidase, Phytochem. Lett. 36 (2020)
162–165.
[42] Y.X. Ma, H. Wang, R. Wang, F.C. Meng, Z.Y. Dong, G.W. Wang, X.Z. Lan, H. Quan,
Z.H. Liao, M. Chen, Cytotoxic lignans from the stems of Herpetospermum
pedunculosum, Phytochemistry 164 (2019) 102–110.
[32] Q.G. Ma, R.R. Wei, Z.P. Sang, J.H. Dong, Structural characterization,
neuroprotective and hepatoprotective activities of flavonoids from the bulbs of
Heleocharis dulcis, Bioorg. Chem. 96 (2020), 103630.
[43] L. Xiao, Y.Y. Huang, Y.H. Wang, J.W. Xu, X.G. He, Anti-neuroinflammatory
benzofurans and lignans from Praxelis clematidea, Fitoterapia 140 (2020), 104440.
[44] Q.G. Ma, Y.G. Wang, W.M. Liu, R.R. Wei, J.B. Yang, A.G. Wang, T.F. Ji, J. Tian, Y.
L. Su, Hepatoprotective sesquiterpenes and rutinosides from Murraya koenigii (L.)
Spreng, J. Agric. Food Chem. 62 (2014) 4145–4151.
[33] Y. Zhu, R.Z. Huang, C.G. Wang, X.L. Ouyang, X.T. Jing, D. Liang, H.S. Wang, New
inhibitors of matrix metalloproteinases 9 (MMP-9): Lignans from Selaginella
moellendorffii, Fitoterapia 130 (2018) 281–289.
[45] Q.G. Ma, R.R. Wei, M. Yang, X.Y. Huang, G.Y. Zhong, Z.P. Sang, J.H. Dong, J.
C. Shu, J.Q. Liu, R. Zhang, J.B. Yang, A.G. Wang, T.F. Ji, Y.L. Su, Structures and
biological evaluation of phenylpropanoid derivatives from Murraya koenigii,
Bioorg. Chem. 86 (2019) 159–165.
[34] L. Cheng, D.L. Guo, M.S. Zhang, L.H. Lang, S.B. Fu, Y. Deng, Y.Q. He, S.J. Xiao,
Dihydrophenanthrofurans and bisbibenzyl derivatives from the stems of
Dendrobium nobile, Fitoterapia 143 (2020), 104586.
[35] Z.J. Ye, X.A. He, J.P. Wu, J. Li, X.W. Chang, J. Tan, W.Y. Lv, H. Zhu, H.H. Sun, W.
X. Wang, Z.H. Chen, G.Z. Zhu, K.P. Xu, New prenylflavonol glycosides with
13