New coumarins and monoterpene galloylglycoside from the stem bark of Sapium baccatum

Article abstract of DOI:10.1016/j.fitote.2019.03.011

Sapium baccatum has been traditionally used as therapeutic remedies. To support its medicinal benefits, our current phytochemical investigation attempted to further discover novel bioactive compounds from S. baccatum. Eight new phenolic compounds, namely, seven coumarins (1–7) and one monoterpene galloylglycoside (8), together with 23 (9–31) known compounds were isolated. Their structures were determined by extensive spectroscopic methods and comparison with literatures. The three pairs of enantiomers of 1, 2 and 7 were confirmed on the basis of HPLC chiral analysis, electronic circular dichroism data and optical rotations. Two coumarins (1–2) were proven to be artifacts through HPLC analysis. The inhibitory effects on TNF-α secretion were examined biologically in LPS-induced BV2 microglia cells and all of the tested compounds exhibited significant inhibitory activity, especially new compound 1 possessed stronger inhibitory effects compared to the positive control quercetin. In addition, compounds 14 and 15 showed weak antifungal activity against Candida albicans SC5314 with MIC values both at 64 μg/mL. The results laid a solid foundation for additional research on S.baccatum related to its anti-inflammatory and antifungal medicinal value.

Full text of DOI:10.1016/j.fitote.2019.03.011

Fitoterapia134(2019)435–442
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
New coumarins and monoterpene galloylglycoside from the stem bark of
Sapium baccatum
Ting Li, Shanshan Wang, Peihong Fan⁎, Hongxiang Lou
Department of Natural Product Chemistry, Key Laboratory of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan
250012, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Sapium baccatum
Coumarins
Monoterpene galloylglycoside
Anti-inflammatory
Antifungal activity
Sapium baccatum has been traditionally used as therapeutic remedies. To support its medicinal benefits, our
current phytochemical investigation attempted to further discover novel bioactive compounds from S. baccatum.
Eight new phenolic compounds, namely, seven coumarins (1–7) and one monoterpene galloylglycoside (8),
together with 23 (9–31) known compounds were isolated. Their structures were determined by extensive
spectroscopic methods and comparison with literatures. The three pairs of enantiomers of 1, 2 and 7 were
confirmed on the basis of HPLC chiral analysis, electronic circular dichroism data and optical rotations. Two
coumarins (1–2) were proven to be artifacts through HPLC analysis. The inhibitory effects on TNF-α secretion
were examined biologically in LPS-induced BV2 microglia cells and all of the tested compounds exhibited sig-
nificant inhibitory activity, especially new compound 1 possessed stronger inhibitory effects compared to the
positive control quercetin. In addition, compounds 14 and 15 showed weak antifungal activity against Candida
albicans SC5314 with MIC values both at 64 μg/mL. The results laid a solid foundation for additional research on
S.baccatum related to its anti-inflammatory and antifungal medicinal value.
1. Introduction
2. Experimental section
Sapium baccatum (Euphorbiaceae) is a tall arboreal plant with edible
sweet fruits that is distributed in China (Yunnan and Tibet) as well as
most countries of south and southeast Asia, such as Vietnam, India,
Malaysia, Sumatra, and Borneo (Kalimantan) [1–3]. This herb has been
extensively used as ingredients in Chinese prescriptions for the treat-
ment of scapulohumeral periarthritis, cerebral infarction, gouty ar-
thritis, and other conditions. It has been also commonly used for
treating abscess in Malaysia [4]. S. baccatum extracts have been proven
to possess excellent antibacterial activity and toxic effects [5–7]. Pre-
vious phytochemical studies on the bark and leaves of S. baccatum have
led to the isolation of various bioactive compounds, including eight
triterpenoids, seven tannins, three aliphatic alcohol or derivatives, one
alkaloid, and one flavonoid glycoside [2–12]. In continuing work to
discover diverse compounds and biological effects, this study aimed to
identify novel bioactive molecules from an ethanol extract of the stem
bark of S. baccatum. Therefore, seven coumarins (1–7) and one mono-
terpene galloylglycosides (8) (Fig. 1) were isolated along with 23
known compounds (9–31). In addition, anti-inflammatory and anti-
fungal activities of these compounds were evaluated.
2.1. General experimental procedures
Optical rotations were obtained on an MCP 200 (Anton Paar,
Shanghai, China). UV spectra were detected by a UV-2450 spectro-
photometer (Shimadzu, Kyoto, Japan). IR spectra were taken from KBr
disk using a Nicolet Nexus 470 FT-IR spectrophotometer (Thermo
Scientific, Waltham, MA, USA). HR-ESI-MS was implemented on a LTQ-
Orbitrap XL (ThermoFinnigan, Bremen, Germany). ESI-MS data were
obtained using an API 5500 Q-trap (AB SCIX, Concord, Ontario,
Canada). ECD spectral analysis was performed on a Chirascan spec-
tropolarimeter (Applied Photophysics, Leatherhead, UK). NMR spectra
were measured on a Bruker Avance DRX-600 spectrometer (Bruker
BioSpin Group, Billerica, MA, USA) at 600 (1H) and 150 (13C) MHz,
using TMS as internal standard. CD3OD and DMSO‑d6 were used as
solvents (Sigma-Aldrich, Shanghai, China). HPLC was performed on an
Agilent 1200 series system (Agilent Technologies) with an Eclispse
XDB-C18 (250 mm × 4.6 mm, 5 μm; Agilent, Santa Clara, CA, USA).
Semi-preparative HPLC separations were performed using a YMC-Pack
ODS-A (250 mm × 10 mm, S–5 μm, 12 nm; YMC, Tokyo, Japan). HPLC
chiral analysis was conducted on a Waters Delta 600 equipped with a
⁎ Corresponding author.
E-mail address: fanpeihong@sdu.edu.cn (P. Fan).
https://doi.org/10.1016/j.fitote.2019.03.011
Received 6 February 2019; Received in revised form 16 March 2019; Accepted 16 March 2019
Availableonline18March2019
0367-326X/©2019ElsevierB.V.Allrightsreserved.

T. Li, et al.
Fitoterapia134(2019)435–442
Fig. 1. The structures of the isolated compounds 1–8 from the stem bark of S. baccatum.
Waters 600 Controller and Waters 996 PDA detector (Waters, Milford,
MA, USA) and a CHIRALPAK AD-H (250 mm × 4.6 mm, 5 μm; Daicel,
Tokyo, Japan). High-speed countercurrent chromatography (HSCCC)
was performed to isolate the compounds (TBE-30A, TBEe300B, Tauto
Biotech, Shanghai, China). Medium-pressure liquid chromatography
(MPLC) was used in purifying compounds on a DUAL Pump with EYELA
UV 9000 detector (Servo Co., Ltd., Japan). Reversed phase C18 (ODS-A-
HG, 12 nm, S-50 μm; YMC, Tokyo, Japan), silica gel (200–300 mesh;
Qingdao Marine Chemical Inc., Qingdao, China), MCI gel (75–150 μm;
Mitsubishi Chemical Corp., Tokyo Japan), toyopearl HW40 (45 μm;
Tosoh Bioscience, Japan), Sephadex LH-20 (40–70 μm, Amersham
Pharmacia Biotech AB, Uppsala, Sweden), and preparative TLC (HSGF
254, 0.4–0.5 mm; Yantai, China) were used in the course of isolation
and purification.
(4.0 g) was separated on a silica gel column repeatedly to yield com-
pounds 20 (435.6 mg) and 30 (2.9 mg). PEFr2 (4.0 g) was also chro-
matographed using a similar procedure (silica gel) to yield five sub-
fractions. Compounds 23 (138.8 mg) and 31 (6.0 mg) were obtained
from the fourth subfractions by silica gel chromatography, and then
compound 29 (10.5 mg) by Sephadex LH-20 and compound 26 (4.0 mg)
by preparative TLC (petroleum ether-EtOAC 10:1). PEFr3 (3.5 g) was
also separated into five subfractions (PEFr3.1–PEFr3.5) and constantly
CC by silica gel chromatography to yield compounds 21 (6.0 mg) from
PEFr3.1, 22 (20.0 mg) and 24 (209.8 mg) from PEFr3.4.
The EA extract (90.0 g) was fractionated into 13 subfractions
(EAFr1–EAFr13) by silica gel chromatography. Compound 10
(21.3 mg) was precipitated as a yellowish solid in course of the isola-
tion. Compound 9 (3.4 mg) was obtained from EAFr4 (140.1 mg) using
toyopearl HW40 column and then reversed-phase HPLC (1.5 mL/min,
60%MeOH-0.01TFA, tR = 20.0 min). EAFr5 (315.0 mg) was separated
on a silica gel column and further purified by preparative TLC (petro-
leum ether-EtOAC 3.5:1) and preparative TLC (petroleum ether:acetone
5:1) to yield compound 25 (2.4 mg) and 28 (17.0 mg), respectively.
EAFr9 (11.1 g) was subjected to a MCI chromatography and then a
medium-pressure liquid chromatography (MPLC) eluted with gradient
MeOH-H2O to afford 17 fractions (EAFr9.1-EAFr9.17). EAFr9.2 was
further separated into 14 fractions (EAFr9.2.1- EAFr9.2.14) using
Sephadex LH-20. Further similar procedures on Sephadex LH-20,
compound 12 (5.0 mg) and 13 (3.7 mg) were acquired from EAFr9.2.2.
Compounds 1 (5.0 mg), 2 (4.0 mg), 3 (8.2 mg), and 27 (4.0 mg) were
obtained from the EAFr9.2.5 by repetitive Sephadex LH-20 and pre-
parative TLC (CH2Cl2-MeOH 9:1). EAFr9.3 was separated by CC on
toyopearl HW40 eluting in a stepwise manner with MeOH-H2O to yield
11 fractions (EAFr9.3.1-EAFr9.3.11). EAFr9.3.3 was chromatographed
by Sephadex LH-20 using an isocratic elution of 50% MeOH-H2O to
obtain seven fractions (EAFr9.3.3.1-EAFr9.3.3.7). Compound 7 was
obtained by RP-HPLC (47% MeOH-0.01% TFA, 1.5 mL/min,
tR = 23.0 min, 3.4 mg) from EAFr9.3.3.1. EAFr9.3.3.4 was purified on a
silica gel and eluted with CH2Cl2-MeOH to yield compound 11 (8.1 mg)
and EAFr9.3.3.7 through RP-HPLC with 38% MeOH (1.5 mL/min,
tR = 12.0 min) to provide compound 16 (12.3 mg). EAFr9.3.3.6 was
separated by CC on MPLC and eluted with a gradient of MeOH-H2O and
then purified via RP-HPLC with 27% MeCN-0.01% TFA to yield com-
pounds 4 (1.5 mL/min, tR = 14.0 min, 10.5 mg) and 6 (1.5 mL/min,
tR = 16.0 min, 12.7 mg). EAFr9.7 was separated by Sephadex LH-20
continuously to yield compound 5 (3.0 mg). After using a similar pro-
cess of separation on Sephadex LH-20, EAFr10 and EAFr12 were further
purified by RP-HPLC to yield compounds 8 (60%MeOH, tR = 13.0 min,
2.2. Strains, cell culture, and chemicals
C. albicans SC5314 was obtained from School of Pharmaceutical
Sciences, Shandong University (Jinan, China). BV2 microglia cells were
obtained from the China Infrastructure of Cell Line Resources (Beijing,
China). D-(+)-Glucose (Tokyo Chemical Industry Co., Ltd., Tokyo,
Japan) was used as reference substance.
2.3. Plant material
The stem bark of S. baccatum was collected from Mengla County,
Yunnan Province, PRC in November 2016 and identified by Chinese
herbal medicine of Kunming Liezhi Sales Co., Ltd. A voucher specimen
(20161118) was deposited in Dr. Fan's laboratory at the Department of
Natural Products Chemistry, School of Pharmaceutical Sciences,
Shandong University.
2.4. Extraction and isolation
The dried and powdered stem barks of S. baccatum (4.5 kg) were
extracted thrice with 95% ethanol for 2 h at reflux. After evaporation of
the filtrating solvents under reduced pressure, a part of the crude ex-
tract (500.0 g) was suspended in 90% CH3OH (1 L) and fractionated
with petroleum ether (1 × 3 L) to yield the PE extract (17.8 g). The
evaporated CH3OH residue was suspended in water (2 L) and then
partitioned in EtOAC (2 × 5 L) to generate the EA extract (105.2 g) and
H2O extract (205.3 g). The PE extract (15.0 g) was subjected to column
chromatography on silica gel and successively eluted with petroleum
and EtOAC to yield eight fractions, designated as PEFr1–PEFr8. PEFr1
436

T. Li, et al.
Fitoterapia134(2019)435–442
(MeOH) λmax (log ε) 219 (3.18), 281 (3.01), 347 (3.50) nm; IR (KBr)
νmax: 3236, 2958, 1737, 1708, 1589, 1512 cm−1；1H (600 MHz) and
13C NMR (150 MHz) data (CD3OD), see Tables 1 and 2; ESI-MS: m/z
Table 1
1H NMR spectral data of compounds 1–7.
Position
1a
2b
3b
4a
5a
6a
7a
337.4 [M + H]+
.
6
9
7.50, s
3.67, d
(17.0)
3.59, d
(17.0)
7.54, s
3.69, d
(17.0)
3.58, d
(16.9)
7.52, s
3.71, m 3.58, d
(17.1)
7.51, s
7.48, s
3.58, s
7.48, s
3.49, s
7.43, s
2.08, s
2.4.4. Baccatune D (4)
White, amorphous powder; [α]20 D-10.5 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 219 (3.28), 281 (3.10), 346 (3.57) nm; IR (KBr)
νmax: 3298, 1688, 1583, 1511 cm−1；1H (600 MHz) and 13C NMR
(150 MHz) data (DMSO‑d6), see Tables 1 and 2; ESI-MS: m/z 321.3 [M-
3.54, d
(17.0)
10
11
6.50, s
6.55, s
3.94, s
3.62, s
3.53, s
6.40, s
4.08, s
6.63, s
4.06, s
3.71, m
6.69, s
3.96, s
5.70, s
3.96, s
5.68, s
3.95, s
8-OCH3
10-OCH3
11-OCH3
7-OH
11-OH
10-COOH
3.94, s
3.59, s
H]−
.
3.66, s
2.4.5. Baccatune E (5)
11.09, s
8.57, s
12.57, s
11.08, s 11.05, s
Pale yellow, amorphous powder; UV (MeOH) λmax (log ε) 217
(3.78), 279 (3.35), 340 (3.50) nm; IR (KBr) νmax: 3298, 2958,
2845,1691, 1583, 1510 cm−1；1H (600 MHz) and 13C NMR (150 MHz)
a
1H NMR data were measured in DMSO‑d6 at 600 MHz;
b
data (DMSO‑d6), see Tables 1 and 2; ESI-MS: m/z 319.3 [M-H]−
.
1H NMR data were measured in CD3OD at 600 MHz.
2.4.6. Baccatune F (6)
14.0 mg), 14 (22%MeCN, tR = 14.0 min, 4.2 mg), 15 (22%MeCN,
tR = 22.0 min, 8.9 mg), and 17 (35% CH3OH, tR = 13.0 min, 10.8 mg).
Another part of the crude extract (500.0 g) was isolated by HSCCC
(EtOAC-nBuOH-MeOH-H2O 0.5: 2: 0.5: 4) to yield four fractions
(Fr1–Fr4). Fr3 was purified by RP-HPLC to yield compounds 18 (55%
MeOH-0.01%TFA, tR = 23.5 min, 8.2 mg) and 19 (55% MeOH-
0.01%TFA, tR = 27.0 min, 3.7 mg).
White, amorphous powder; UV (MeOH) λmax (log ε) 218 (3.59), 281
(3.17), 347 (3.32) nm; IR (KBr) νmax: 2938, 1707, 1649, 1586,
1514 cm−1；1H (600 MHz) and 13C NMR (150 MHz) data (DMSO‑d6),
see Tables 1 and 2; ESI-MS: m/z 305.4 [M-H]−
.
2.4.7. Baccatune G (7)
Pale yellow, amorphous powder; UV (MeOH) λmax (log ε) 220
(3.74), 283 (3.62), 346 (4.00) nm; IR (KBr) νmax: 3288, 2950, 2846,
1736, 1701, 1597, 1511, 1490, 1439, 1372 cm−1；1H (600 MHz) and
13C NMR (150 MHz) data (DMSO‑d6), see Tables 1 and 2; ESI-MS: m/z
2.4.1. Baccatune A (1)
Pale yellow, amorphous powder; [α]20 D-9.6 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 217 (3.00), 279 (2.48), 347 (2.60) nm; IR (KBr)
νmax: 3285, 2954, 2911, 2846, 1721, 1688, 1585, 1512, 1489 cm−1; 1H
(600 MHz) and 13C NMR (150 MHz) data (DMSO‑d6), see Tables 1 and
2; HR-ESI-MS: m/z 373.0532 [M + Na]+ (Calcd. for C16H14O9Na,
373.0530).
293.2 [M + H]+
.
2.4.8. 3,7-Dimethyl-1-octen-3,6,7-triol-7-O-β-D-2,6-digalloylglucopyran-
oside (8)
Brown yellow, amorphous powder; [α]20 D-24.6 (c 0.10, MeOH),
UV (MeOH) λmax (log ε) 218 (3.16), 276 (3.59) nm; IR (KBr) νmax: 3271,
2974, 1693, 1611, 1534, 1448, 1319 cm−1; 1H (600 MHz) and 13C NMR
(150 MHz) data (CD3OD and DMSO‑d6), see Table 3; HR-ESI-MS: m/z
659.1938 [M-H2O + Na]+, (Calcd for C30H36O15Na, 659.1946).
2.4.2. Baccatune B (2)
Pale yellow, amorphous powder; [α]20 D-11.2 (c 0.10, MeOH); UV
(MeOH) λmax (log ε) 220 (3.00), 281 (2.48), 347 (2.60) nm; IR (KBr)
νmax
:
3340, 3232, 2959, 2924, 2854, 1711, 1600, 1509,
1484 cm−1；1H (600 MHz) and 13C NMR (150 MHz) data (CD3OD), see
2.5. Chiral analysis of compounds 1, 2 and 7
Tables 1 and 2; HR-ESI-MS: m/z 337.0556 [M + H]+, (Calcd. for
C
15H13O8，337.0554).
Briefly, compounds 1, 2 and 7 were analyzed via HPLC under the
following conditions: column, Chiralpak AD-H (250 × 4.6 mm, 5 μm);
column temperature, 25 °C; flow rate: 1 mL/min; detector, Waters 996
PDA. Compound 1: mobile phase: n-hexane:isopropanol (90/10, v/v)
with 0.1% TFA; retention time, 28.24 and 31.15 min; Compound 2:
mobile phase: n-hexane/ethanol (90/10, v/v) with 0.1% TFA; retention
time, 218.05 and 226.47 min; Compound 7: mobile phase: n-hex-
ane:isopropanol (90/10, v/v); retention time, 17. 19 and 20.25 min.
2.4.3. Baccatune C (3)
Pale yellow, amorphous powder; [α]20 D-12.5 (c 0.10, MeOH); UV
Table 2
13C NMR spectral data of compounds 1–7.
Position
1a
2b
3b
4a
5a
6a
7a
2.6. Acid hydrolysis of compound 8 and sugar analysis
2
3
4
4a
5
6
7
8
8a
9
10
11
160.4
117.0
139.2
109.0,
115.1
114.4
156.0
139.5
144.8
31.5
169.7
98.5
160.7
60.5
52.0
56.0
162.3
120.2
140.6
111.6
116.2
115.4
155.4
140.8
146.2
33.0
162.7
116.7
143.4
110.7
116.2
116.0
156.9
140.9
146.5
32.5
160.4
117.6
141.0
109.6
115.1
113.7
153.7
138.9
144.7
31.8
159.8
112.6
142.1
110.6
115.7
113.7
154.0
138.7
144.4
31.3
159.9
113.6
141.6
110.7
115.6
113.4
153.8
138.7
144.4
31.6
160.8
119.8
137.0
109.1
113.8
114.2
154.9
139.4
144.4
12.0
Compound 8 (5 mg) was added to a mixture of 1.5 mL of con-
centrated HCl, 3 mL of H2O, and 5 mL of dioxane and reacted under
100 °C for 2 h. H2O was added to the reaction mixture and then ex-
tracted with CH2Cl2 (3 × 10 mL) after completion of the reaction. The
aqueous layer was neutralized with Na2CO3 and concentrated under
reduced pressure and then purified on Sephadex LH-20 (MeOH) column
to obtain the sugar fraction. A solvent system (ethyl acetate:pyr-
idine:ethanol:water 8:2:2:1) was used for TLC identification by com-
parison of Rf and optical rotation with an authentic sample: D-Glucose,
Rf = 0.2 and optical rotation: [α]20 D + 45.0 (c 0.1, H2O).
173.5
100.7
162.5
62.0
172.0
95.2
163.2
61.8
170.6
93.2
161.0
60.9
169.7
67.5
160.9
60.9
170.6
67.5
161.0
60.8
98.9
160.9
60.6
12
8-OCH3
10-OCH3
11-OCH3
56.3
52.8
52.1
57.1
2.7. Bioactivity assays
a
13C NMR data were measured in DMSO‑d6 at 150 MHz;
b
13C NMR data were measured in CD3OD at 150 MHz.
Compounds (1, 5, 6, 8, 12, 13, 14, 15, 18 and 22) were tested for
437

T. Li, et al.
Fitoterapia134(2019)435–442
Table 3
(hydroxyl), 2954 cm−1 (methyl), 2911, 2846 cm−1 (methylene), 1721,
1688 cm−1 (carbonyl groups), 1585, 1512, and 1489 cm−1 (phenyl
group). 1H NMR (Table 1) revealed three methoxy groups at δH 3.94
(3H, s, 8-OCH3), 3.62 (3H, s, 10-OCH3), and 3.53 (3H, s, 11-OCH3) and
a methylene group at δH 3.67 (1H, d, J = 17.0 Hz, H-9a) and 3.59 (1H,
d, J = 17.0 Hz, H-9b). The combined elucidation of the 13C NMR
(Table 2) and HSQC data suggested 16 carbon signals that were at-
tributed to three oxygenated methyls (δC 52.0, 56.0, 60.5; C-10, C-11,
8-OCH3 respectively), one sp3 methylene (δC 31.5, C-9), one sp3 me-
thine (δC 98.5, C-11), one sp2 methine (δC 114.4, C-6), seven sp2 qua-
ternary carbons (δC 117.0, 139.2, 109.0, 115.1, 156.0, 139.5, 144.8; C-
3, C-4, C-4a, C-5, C-7, C-8, C-8a, respectively), and three carbonyl
groups (δC 160.4, 169.7, 160.7； C-2, C-10, C-12, respectively). Nine
carbons, namely, C-2 (δC 160.4), C-3 (δC 117.0), C-4 (δC 139.2), C-4a
(δC 109.0), C-5 (δC 115.1), C-6 (δC 114.4), C-7 (δC 156.0), C-8 (δC
139.5), and C-8a (δC144. 8) could be attributed to a coumarin skeleton.
The HMBC correlations (Fig. 3) from H-6 (δH 7.50) to C-4a, C-5, C-7,
and C-8 verified the coumarin skeleton. The methine proton at δH 6.55
(1H, s, H-11) was located at position C-11 (δC 98.5) based on HSQC
correlation. Based on the HMBC correlation of the methine proton H-11
(δH 6.55) to C-3, C-4, C-4a, and C-12, the lactonic ring unit was con-
firmed to be substituted at C-4 and C-5. The HMBC correlation from H-
9a and H-9b to C-2, C-3, C-4, and C-10 suggested the position of a
carboxymethyl group at C-3. The locations of three methoxy groups
were determined by HMBC correlations of δH 3.94, 3.62, 3.53 with C-8,
C-10, and C-11, respectively. A literature search revealed that the
chemical structure of 1 is similar to phyllanthusiin E from the leaves of
Phyllanthus flexuosus, except for the presence of three methoxy groups
[16]. Thus, the planar structure of 1 was established. To elucidate the
configuration of the only one chiral center at C-11, chiral analysis was
performed by HPLC, and a pair of peaks with the area ration of ap-
proximately 1:1 was observed. 1a and 1b were obtained through HPLC
semipreparation, and their ECD spectra were measured as obvious po-
sitive and negative Cotton effect at 220 nm (Fig. 4). The configuration
of 1a and 1b were proposed to be S and R configuration respectively on
the basis of optical rotations 1a: [α]20D + 24.4 (c 0.05, MeOH) and 1b:
[α]20 D-26.0 (c 0.05, MeOH), in comparison with S configuration of
dehydropicrorhiza acid methyl diester ([α]14D + 17.7 (c 0.2, MeOH),
which have similar structure pattern [17]. Thus, 1 existed as a pair of
enantiomers (S and R configuration, Fig. 4). As 1 possesses a basic
skeleton similar to 2–7, for convenience, we have assigned the trivial
name Baccatune A.
Compound 2 was obtained as a pale yellow solid, whose molecular
formula was confirmed to be C15H12O9 determined by HR-ESI-MS at m/
z 337.0556 ([M + H]+, C15H13O9 calcd. 337.0554), indicating 10 de-
grees of unsaturation. The IR spectrum revealed the existence of
3232 cm−1 (hydroxyl), 2959 cm−1 (methyl), 2924, 2854 cm−1 (me-
thylene), 1711 cm−1 (carbonyl group), 1600, 1509, and 1484 cm−1
(phenyl group), in addition to the absorption bands at 3340 cm−1
(carboxyl group). Comparison of NMR data with 1 suggested a structure
with the same coumarin skeleton. However, the number of methoxy
groups differed from 1 based on the presence of a terminal carboxylic
acid moiety and the absence of correlated protons signal of 10-OCH3 to
C-10 (δC 173.5) in HMBC correlations. The remaining correlations in
the 2D spectra were comparable to 1. Chiral analysis of 2 indicated a
pair of peaks with an area of approximately 1:1, indicating that 2 is a
pair of enantiomers. Therefore, the structure of 2 was determined as
Baccatune B.
Compound 3 was obtained as a pale yellow solid, and its molecular
formula was determined at m/z 337.4 by positive ESI-MS, which to-
gether with 13C NMR spectroscopy (15 carbons) suggested a molecular
formula of C15H12O9. The IR spectrum indicated the presence of peaks
at 3236 cm−1 (hydroxyl), 2958 cm−1 (methyl), 1737, 1708 cm−1
(carbonyl groups), 1589 and 1512 cm−1 (phenyl group). Compared to
1, a methoxy carbon (11-OCH3) was not present in the 13C NMR
spectrum of 3. This can be verified by absence of CH2 with 14 m/z units
1H NMR and 13C NMR spectral data of compound 8.
Position
8a
8b
δH
δC
δH
δC
1
2
3
4
5
6
4.88, m
4.90, m
3.68, m
3.50, m
97.0
75.6
76.4
72.2
75.2
65.0
4.78, d (8.1)
4.68, dd (9.4, 8.2)
3.51, t (9.1)
3.31, m
3.58, m
4.45, dd (11.6, 1.6)
4.23, dd (11.8, 6.6)
5.08, dd (17.4, 1.7)
4.84, dd (10.8, 1.7)
5.84, dd (17.4, 10.8)
95.0
73.8
74.3
70.5
73.5
63.6
3.66, m
4.55, dd (11.7, 2.0)
4.41, dd (11.8, 6.5)
5.11, dd (17.4, 1.5)
4.86 m
1′
111.9
111.1
2′
3′
3′-CH3
4′
5.88, dd (17.4, 10.8)
145.4
84.4
25.7
38.4
144.8
82.2
25.1
37.0
1.15, s
1.09, s
1.54, m
1.70, m
1.72, m
3.75, t (6.7)
1.48, m
1.58, m
1.63, m
3.62, t (6.7)
5′
6′
7′
7′-CH3
7′-CH3
1″
2″
3″
4″
5″
6″
7″
1‴
2‴
3‴
4‴
27.6
86.1
80.32
23.7
23.2
25.8
83.7
78.2
23.3
1.13, s
1.12, s
1.03, s
1.03, s
22.1
121.4
110.3
146.5
139.8
146.5
110.3
167.5
121.7
110.2
146.4
139.7
146.4
110.2
168.3
119.4
108.7
145.4
138.4
145.4
108.7
164.8
120.0
108.6
144.5
138.1
144.5
108.6
165.7
7.10, s
6.97, s
7.10, s
7.11, s
6.97, s
6.96, s
5‴
6‴
7‴
7.11, s
6.96, s
3′-OH
6′-OH
Galloyl-OH
5.4, s
5.3, s
9.2, s
8.6, s
a
1H NMR data were measured in CD3OD at 600 MHz, 13C NMR at 150 MHz;
b
1H NMR data were measured in DMSO‑d6 at 600 MHz, 13C NMR at
150 MHz.
anti-inflammatory activity on LPS-induced BV2 microglia cells. The
secretion of TNF-α in the culture supernatants of the LPS-induced BV2
microglia cells was assessed using the same method as described in the
Ref [13], with minor modifications in that quercetin was used as po-
sitive control.
The MIC values of the compounds (1, 5, 10, 11, 13, 14, 15, 16, 26,
27, and 28) against C. albicans SC5314 were measured using the broth
microdilution based on the Clinical and Laboratory Standards Institute
(CLSI) guidelines (M27-A3), in accord with Ref [14].
3. Results and discussion
Traces of compounds 1–7 showed intense yellow fluorescence sig-
nals on TLC under UV 365 nm. During isolation and purification of
these compounds, compounds 1 and 2 were observed to emerge from
the methanol solution of pure compounds 3 and 4, respectively. The
methanol solutions of 3 and 4 that were deposited at room temperature
were measured using HPLC analysis at three time intervals (0, 4, and
10 days) [15]. The results showed that compounds 1 and 2 were arti-
facts due to the occurrence of additional new peaks that emerged in a
time-dependent manner (Fig. 2).
Compound 1 was obtained as a pale yellow solid, with a molecular
formula of C16H14O9 as determined by HR-ESI-MS at m/z 373.0532
([M + Na]+, C16H14O9Na calcd. 373.0530), indicating 10 degrees of
unsaturation. The IR spectra displayed absorption bands at 3285 cm−1
438

T. Li, et al.
Fitoterapia134(2019)435–442
Fig. 2. Time-dependent emergence of artifacts 1–2 in a methanol solution of 3–4.
(A) the emergence of 1 from 3; (B) the emergence of 2 from 4.
when compared to 1. HMBC correlations in the methine proton δH 6.63
(1H, s, H-11) still occurred at C-3 (δC 116.7), C-4a (δC 110.7), and C-12
(δC 163.2), indicating a hydroxy group substituted at C-11. A methylene
was located via HMBC correlations of its protons at δH 3.71 (over-
lapping) with C-2 (δC 162.7), C-3 (δC 116.7), C-4 (δC 143.4), and C-10
(δC 172.0). The observed 1D and 2D NMR of the remaining part of the
molecule are similar to 1. Therefore, the structure of 3 was determined
to be Baccatune C. The chiral center at C-11 of 3 may exist as S and R
configuration based on their similar ECD spectrum and optical rotation
to 1 and 2.
11). An 27.7 ppm upfield shift of C-11 indicated the absence of hydroxy
group at C-11, which was supported by the mass difference of the 16 m/
z unit in contrast to 3. HMBC correlations of the 7-OH proton (δH 11.08)
to C-8 (δC 138.7), C-7 (δC 154.0), C-6 (δC 113.7) and 8-OCH3 (δH 3.96)
to C-8 (δC 138.7) indicated that substituents of OH and OCH3 were
assigned at adjacent carbons at C-7 (δC 154.0) and C-8 (δC 138.7), re-
spectively. The 1D NMR data of 5 was similar to that of phyllanthusiin E
methyl ester, in which a hydroxy group rather than a methoxy group
was substituted at C-8 [18]. Thus, compound 5 was assigned as Bac-
catune E.
Compound 4 was obtained as a white solid, and its molecular for-
mula was measured at m/z 321.3 ([M-H]−) based on the negative ESI-
MS, which together with 13C NMR spectroscopy (14 carbons) suggested
a molecular formula of C14H10O9. The IR spectrum indicated the pre-
sence of peaks at 3298 cm−1 (hydroxyl), 1688 cm−1 (carbonyl groups),
1583, and 1511 cm−1 (phenyl group). Furthermore, the 1H NMR and
HSQC data of 4 exhibited three hydroxy group signals at δH 11.09 (1H,
s, 7-OH), δH 8.57 (1H, s, 11-OH), and δH 12.57 (1H, s, 10-COOH),
suggesting that C-10 (δC 170.6) and C-11 (δC 93.2) were substituted by
OH instead of OCH3, which is in contrast to 1. This was also confirmed
by the ESI-MS difference of the 48 m/z unit. Except for the HMBC
correlation at δH 6.69 (1H, s, H-11) to C-3 (δC 117.6), C-4a (δC 109.6),
and C-12 (161.0), HMBC correlations of H-6 and H-9 were coincided
with 1. Thus, compound 4 was assigned as Baccatune D.Compound 4
may be mixtures of a pair of enantiomers as well owning to its similar
ECD spectrum and optical rotation to 1 and 2.
Compound 5 was obtained as a pale yellow solid, and its molecular
formula was determined at m/z 319.3 ([M-H]−) based on the negative
ESI-MS, which together with 13C NMR spectroscopy (15 carbons),
suggested a molecular formula of C15H12O8, corresponding to one
oxygen less than 3. The IR spectrum indicated the presence of peaks at
3298 cm−1 (hydroxyl), 2958, 2845 cm−1 (methyl), 1691 cm−1 (car-
bonyl groups), 1583, and 1510 cm−1 (phenyl group). The HSQC spec-
trum revealed that a methylene at δH 5.70 (2H, s, H-11) and δC 67.5 (C-
11) of 5 differed from that of 3 at δH 6.63 (1H, s, H-11) and δC 95.2 (C-
Similarly, the NMR spectra of 6 indicated a similar coumarin ske-
leton as 5, with the presence of two methylene protons at δH 5.68 (2H,
s, H-11) and 3.49 (2H, s, H-9), an aromatic proton at δH 7.48 (1H, s, H-
6), and one oxygenated methyl signal at δH 3.95 (3H, s, 8-OCH3).
However, no methyl ester was substituted on C-10 compared to 5, and
the mass spectrometric data of decreasing CH2 units were in complete
agreement with the measured molecular constitution of 6 (m/z 305.2
-
[M-H] ). Thus, compound 6 was assigned as Baccatune F.
Compound 7 was obtained as a pale yellow solid, and its molecular
formula was determined at m/z 293.2 ([M + H]+) based on the posi-
tive ESI-MS. The IR spectrum indicated the presence of peaks at
3288 cm−1 (hydroxyl), 2950, 2846, 1439, 1372 cm−1 (methyl), 1736,
1701 cm−1 (carbonyl groups), 1597, 1511 and 1490 cm−1 (phenyl
group). A detailed analysis of its 1H NMR spectrum exhibited the pre-
sence of three methyls signals at δH 3.94 (3H, s, 8-OCH3), 3.59 (3H, s,
10-OCH3), and 2.08 (3H, s, H-9); one oxygenated methine proton at δH
4.65 (1H, s, H-10); and an aromatic proton at δH 7.43 (1H, s, H-6).
Similarly, the 1D spectra established the presence of a coumarin ske-
leton, as shown in Tables 1 and 2. The HMBC correlations of 7 were
comparable to 1, expect HMBC correlations from H-9 (δH 2.08) to C-2
(δC 160.8), C-3 (δC 119.8), and C-4 (δC 137.0), which suggested that a
methyl group was connected at C-3. Thus, the planar structure of 7 was
established. The absolute configuration at C-10 of 7 was also elucidated
by HPLC chiral analysis and optical rotations. 7a and 7b were obtained
through HPLC semipreparation and their configurations were
439

T. Li, et al.
Fitoterapia134(2019)435–442
Fig. 3. Key HMBC and HeH COSY correlations of 1–8.
confirmed be S and R configuration respectively on the basis of optical
rotations 7a: [α]20 D + 28.0 (c 0.05, MeOH) and 7b:
6.5 Hz, H-6b) with C-1‴ suggested the presence of a 2,6-digalloylglu-
copyranosyl moiety. In the remaining proton signals, ABX spin system
signals at δH 5.88 (1H, dd, J = 17.4, 10.8 Hz), δH 5.11 (1H, dd,
J = 17.4, 1.5 Hz), δH 4.86 (overlapping) were observed, which corre-
sponded to a terminal vinyl group (δC 145.4, C-2′), (δC 111.9, C-1′).
Proton signals at δH 1.15 (3H, s, 3′-Me), δH 1.13 (3H, s, 7′-Me), and δH
1.12 (3H, s, 7′-Me), together with carbon signals at δC 25.7(3′-Me),
23.7, and 23.2 (two 7′-Me) suggested the presence of three methyl
groups. The terminal vinyl group and three methyl groups, together
with the remaining five carbon signals (two quaternary carbons, two
methylene carbons, and one oxygenated methine carbon), were con-
firmed to be a monoterpenoid moiety that is closely similar to 6,7-di-
hydroxy-6,7-dihydrolinalool [21]. The position of the glycosidic
linkage was determined using NOESY spectroscopy. A NOE correlation
was observed at δH 1.13, 1.12 (3H, each, s, 7′-Me,) with δH 4.88 (1H,
overlapping, H-1), establishing the attachment of monoterpenoid
aglycone on anomeric carbon. Consequently, the structure of compound
[α]20 D-30.0 (c 0.05, MeOH). Thus, compound 7 existed as a pair of
enantiomers and was assigned as Baccatune G.
Compound 8 was obtained as a brown solid. HR-ESI-MS of 8 af-
forded a [M-H2O + Na]+ peak at m/z 659.1938 (C30H36O15Na calcd.
659.1946), suggesting a molecular formula C30H38O16 for 8. The IR
absorptions revealed the presence of peaks at 3271 cm−1 (hydroxy
groups), 2974 cm−1 (methyls), 1693 cm−1 (ester carbonyls), and 1611
and 1534 cm−1 (phenyl groups). The presence of two galloyl moieties
in 8 was proven by proton signals at δH 7.10 (2H, s, H-2″, H-6″), δH 7.11
(2H, s, H-2‴ and H-6‴) and the corresponding carbon signals at δC
168.3, 167.5, 146.5 (×2), 146.4 (×2), 139.8, 139.7, 121.7, 121.4,
110.3 (×2), and 110.2 (×2). The 1D NMR spectra also revealed the
presence of one β-glucopyranosyl unit with its anomeric proton at δH
4.78 in DMSO‑d6 (1H, d, J = 8.1 Hz, H-1, overlapped by H2O signal in
CD3OD). D-glucopyranosyl was confirmed based on TLC analysis of
products obtained from acid hydrolysis followed by Sephadex LH-20
separation and optical rotation measurement [19,20]. The long-range
HMBC correlations of δH 4.90 (overlapping, H-2) with C-1″ and those of
δH 4.55 (1H, dd, J = 11.7, 2.0 Hz, H-6a), δH 4.41 (1H, dd, J = 11.8,
8
was assigned as 3,7-dimethyl-1-octen-3,6,7-triol-7-O-β-D-2,6-di-
galloylglucopyranoside.
Known compounds were identified by comparison with the experi-
mental and reported spectroscopic data: ellagic acid (9) [22], 3,3-di-O-
440

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Fitoterapia134(2019)435–442
An antifungal investigation demonstrated that the MIC values of 14
and 15 against C. albicans SC5314 were both 64 μg/mL, indicating weak
antifungal activity. No obvious antifungal effects (MIC > 128 μg/mL)
were observed in the other tested compounds in comparsion with Ref
[14].
In summary, eight new compounds, including Baccatune A − G
(1–7), 3,7-dimethyl-1-octen-3,6,7-triol-7-O-β-D-2,6-digalloylglucopyr-
anoside (8) and 23 known compounds (9–31), were isolated from the
stem bark of S. baccatum. Compounds 1 and 2 were confirmed as arti-
facts and assigned as two pairs of enantiomers via HPLC chiral analysis,
ECD experiments and optical rotations. Compounds 3 and 4 may be
mixtures of enantiomers as well owning to their similar ECD spectra
and optical rotations to 1 and 2, however, they were not successfully
separated in present attempt. Compound 7 was confirmed as a pair of
enantiomers via HPLC chiral analysis and optical rotations.
The bioassay results of the inhibitory effects of the tested com-
pounds on LPS-induced TNF-α production revealed significant anti-in-
flammatory activity, explaining traditional use of S. baccatum from the
other aspect in comparison with Ref [35]. The observed weak anti-
fungal activity of the isolated tannins against C. albicans also justified
the pharmacological effects of S. baccatum [6]. Based on traditional
medicinal values of S. baccatum, additional studies to identify novel
bioactive compounds are warranted.
Fig. 4. ECD spectra and absolute configurations of compounds 1a and 1b.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (Grant No. 81473323). Authors thank Dr. Chang of
the same Lab for the performance of antifungal tests.
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