Lignans and triterpenoids from Vitex negundo var. heterophylla and their biological evaluation

Article abstract of DOI:10.1016/j.fitote.2016.04.020

Three new phenylnaphthalene-type lignans, vitexnegheteroins E-G (1-3), and a new polyoxygenated ursane-type triterpene, vitexnegheteroin H (9), were isolated from the seeds of Vitex negundo var. heterophylla, together with ten known compounds. Their structures were established on the basis of extensive 1D and 2D NMR experiments, as well as their mass spectroscopic data. The absolute configurations of compounds 1 and 2 were determined by comparing their experimental ECD spectra with that calculated by the time-dependent density functional theory (TDDFT) method. The isolates were evaluated for their cytotoxicities against three human cancer cell lines, inhibitory activities on lipopolysaccharide-induced NO production in murine microglial BV-2 cells, and antioxidant activities for ABTS radical scavenging.

Full text of DOI:10.1016/j.fitote.2016.04.020

Lignans and triterpenoids from Vitex negundo var. heterophylla and their
biological evaluation
Ping Hu, Da-Hong Li, Xu Hu, Sheng-Ge Li, Chun-Mei Sai, Xiao-Cong
Sun, Tong Su, Jiao Bai, Zi-Hou Wang, Zhan-Lin Li, Hui-Ming Hua
PII:
DOI:
Reference:
S0367-326X(16)30094-6
doi: 10.1016/j.fitote.2016.04.020
FITOTE 3401
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
31 March 2016
20 April 2016
22 April 2016
Please cite this article as: Ping Hu, Da-Hong Li, Xu Hu, Sheng-Ge Li, Chun-Mei Sai,
Xiao-Cong Sun, Tong Su, Jiao Bai, Zi-Hou Wang, Zhan-Lin Li, Hui-Ming Hua, Lignans
and triterpenoids from Vitex negundo var. heterophylla and their biological evaluation,
Fitoterapia (2016), doi: 10.1016/j.fitote.2016.04.020
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ACCEPTED MANUSCRIPT
Lignans and triterpenoids from Vitex negundo var. heterophylla
and their biological evaluation
a,1
a,1
a
a
a
Ping Hu , Da-Hong Li , Xu Hu , Sheng-Ge Li , Chun-Mei Sai ,
b
c
a
a
a,∗
Xiao-Cong Sun , Tong Su , Jiao Bai , Zi-Hou Wang , Zhan-Lin Li ,
a,∗
Hui-Ming Hua
a
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of
Education, and School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, Shenyang 110016, People’s Republic of China
b
College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and
Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin
300071, People’s Republic of China
c
College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of
China
*
Corresponding authors. Tel.: +86-024-23986465; fax: +86-024-23986465.
E-mail addresses: lzl1030@hotmail.com (Z. Li), and huimhua@163.com (H. Hua).
1
These authors contributed equally to this work.
1

ACCEPTED MANUSCRIPT
ABSTRACT
Three new phenylnaphthalene-type lignans, vitexnegheteroins E-G (1−3), and a new
polyoxygenated ursane-type triterpene, vitexnegheteroin H (9), were isolated from
the seeds of Vitex negundo var. heterophylla, together with ten known compounds.
Their structures were established on the basis of extensive 1D and 2D NMR
experiments, as well as their mass spectroscopic data. The absolute configurations of
compounds 1 and 2 were determined by comparing their experimental ECD spectra
with that calculated by the time-dependent density functional theory (TDDFT)
method. The isolates were evaluated for their cytotoxicities against three human
cancer cell lines, inhibitory activities on lipopolysaccharide-induced NO production
in murine microglial BV-2 cells, and antioxidant activities for ABTS radical
scavenging.
Keywords: Vitex negundo var. heterophylla; Lignan; Triterpene; Cytotoxicity;
Anti-inflammation; Antioxidation
2

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1. Introduction
The genus Vitex (Verbenaceae) consists of small trees and shrubs, with about 270
species distributed mainly in tropical and subtropical regions. About 14 species,
seven varieties, and three forms are found in China [1]. Phytochemical and related
biological investigations for the plants of Vitex genus have been reported in the past
decades [2,3]. Phytochemical investigations indicated the presence of flavonoids,
phenylpropanoids, diterpenoids, triterpenoids, iridoid glycosides, phytoecdysteroids,
phenolic glycosides, and essential oils in Vitex plants, which exhibit a wide range of
bioactivities including antibacterial, antimalarial, antiviral, antifeedant,
antiproliferative, hepatoprotective, antioxidant, antiaging, anti-inflammatory,
antipyretic, analgesic, and potential effects on menopausal symptoms [2−5]. Vitex
negundo L. var. heterophylla (Franch.) Rehd., an aromatic shrub, is widely
distributed in the hilly areas of the north, northeast, northwest, and central regions of
China. All parts of this plant have been commonly used as folk medicine. Its leaves
have been used to treat cold, fever, stroke, and urinary bleeding. Its roots and
branches are used for the therapy of bronchitis, malaria, and hepatitis. The essential
oils from the seeds and leaves are utilized for the treatment of chronic bronchitis
[
6,7].
Previous investigations on V. negundo var. heterophylla focused on the
components of the essential oil analyzed by gas chromatography-mass spectrometry
[
[
8−10], and the herbicidal and anti-inflammatory activities of the ethanol extracts
11,12]. To the best of our knowledge, though increasing interests have been given to
the genus Vitex, especially V. negundo, phytochemical and pharmacological studies
of V. negundo var. heterophylla were limited. In this study, the phytochemical
3

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investigation on the seeds of V. negundo var. heterophylla led to the isolation of four
new, vitexnegheteroins E−H (1−3, and 9) and ten known compounds. Their
structures were elucidated on the basis of spectroscopic analyses (IR, UV, HRESIMS,
and 1D and 2D NMR) and the TDDFT ECD calculations. The cytotoxicity,
inhibitory effects on LPS-induced NO production in murine microglial BV-2 cells,
and antioxidant activities of these isolates were also assessed. Herein, this
contribution describes the isolation, structure elucidation, and biological assessment
of the isolates.
2. Experimental
2.1. General
Optical rotations were measured on a JASCO P-2000 polarimeter. IR spectra were
recorded on a Bruker IFS-55 spectrometer (using a KBr disk method). UV spectra
were recorded using a Shimadzu UV-2201 spectrometer. HRESIMS spectra were
obtained using Bruker micrOTOF-Q mass spectrometer. CD spectra were obtained
using MOS-450 detector from BioLogic. GC was carried out on an Agilent GC
series system and performed with an HP-5 column (30 m × 0.25 mm × 0.25 µm,
Agilent, Santa Clara, CA, USA). NMR spectra were recorded on Bruker ARX-400
and AV-600 NMR spectrometers with tetramethylsilane (TMS) as an internal
standard. Semi-preparative HPLC was conducted on a YMC ODS-A column
equipped with a LC-6AD pump and a SPD-20A UV-Vis detector (Shimadzu Co.,
Ltd., Kyoto, Japan). Column chromatography (CC) was performed with silica gel
(100-200 and 200-300 mesh, Qingdao Haiyang Chemical Co., Ltd., China), ODS (50
µm, YMC Co., Ltd., Kyoto, Japan) and Sephadex LH-20 (GE Healthcare, Uppsala,
Sweden). The absorbance in bioassays was measured and recorded on Varioskan
4

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Flash Multimode Reader (Thermo Scientific, Waltham, MA, USA).
2,2′-Azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was purchased from
Sigma-Aldrich (St. Louis, MO, USA). The murine microglial BV-2 cell line was
from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.
2.2. Plant material
The seeds of V. negundo var. heterophylla were collected in July 2010 from
Huludao, Liaoning Province, China, and were identified by Prof. Jin-Cai Lu (School
of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
Shenyang, China). The voucher sample (JTZ-20100712) has been deposited in the
Department of Natural Products Chemistry, Shenyang Pharmaceutical University,
Shenyang, China.
2.3. Extraction and isolation
The air-dried seeds of V. negundo var. heterophylla (10.0 kg) were powdered and
extracted with 95% EtOH (3 × 50 L) under reflux. After removal of the solvent
under reduced pressure, the residue (540 g) was suspended in H O (1.0 L) and
2
partitioned with petroleum ether (3 × 1.2 L), CH Cl (3 × 1.2 L), EtOAc (3 × 1.2 L),
2
2
and n-BuOH (3 × 1.2 L), successively. The CH
2
Cl
2
extract (260 g) was fractionated
by silica gel column chromatography (CC) using a gradient system of
P.E.(60-90 °C)-acetone (from 100:0 to 1:1, v/v) as eluent to afford fractions a-g.
Fraction d was separated into six subfractions d1-d6 by silica gel CC with a system
of P.E.-acetone (from 100:0 to 1:1). Subfraction d5 was fractionated on a Sephadex
LH-20 column eluting with MeOH followed by preparative TLC (PTLC) to obtain
1
2 (6.2 mg) and 13 (6.5 mg). Fraction e was separated into five subfractions e1-e5
by silica gel CC with a system of CH
Cl
2 2
-MeOH (from 100:0 to 0:100). Subfraction
5

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e2 was fractionated on a Sephadex LH-20 column eluting with MeOH to yield 8 (8.7
mg) and 14 (9.6 mg). Fraction f was subjected to silica gel CC using CH
2
2
Cl -MeOH
(from 100:0 to 0:100) as eluent to give six subfractions f1-f6. Subfraction f2 was
2
subjected to ODS CC eluted with MeOH-H O (20:80, 30:70, 40:60, 50:50, 60:30,
70:30 and 100:0, v/v) to afford 10 (10.7 mg). The EtOAc extract (37 g) was
subjected to silica gel CC eluting with a stepwise gradient of CH Cl -MeOH (from
2
2
100:0 to 0:100, v/v) to afford fractions A-J. Fraction C was applied to Sephadex
LH-20 CC to give subfractions C1 and C2. Subfraction C1 was further purified by
semi-preparative RP-HPLC eluted with MeOH-H O (67:33, v/v, 2.0 mL/min) to
2
afford 5 (9.2 mg, t
purified by PTLC to obtain 3 (6.7 mg). Fraction F was subjected to ODS CC using
MeOH-H O (15:85, 25:75, 35:65, 45:55, 55:45, 65:35 and 100:0, v/v) as eluent to
give seven subfractions F1-F7. Subfraction F1 was applied to Sephadex LH-20 CC
followed by semi-preparative RP-HPLC with the mobile phase of MeOH-H
58:42, v/v, 2.0 mL/min) to afford 7 (10.2 mg, t 44.0 min). Fraction G was
R R
35.0 min) and 6 (11.7 mg, t 42.0 min). Subfraction C2 was
2
2
O
(
R
subjected to ODS CC with MeOH-H O as eluent to give seven subfractions G1-G7.
2
Subfraction G1 was applied to Sephadex LH-20 CC followed by PTLC to obtain 2
(5.2 mg). Subfraction G3 was repeatedly recrystallized in methanol to afford 9 (9.9
mg). Fraction I was separated by ODS CC to yield five subfractions I1-I5.
Subfraction I2 was subjected to Sephadex LH-20 CC followed by semi-preparative
RP-HPLC eluted with MeOH-H
5.5 min). Subfraction I3 was applied to Sephadex LH-20 CC followed by PTLC to
obtain 11 (6.2 mg). The n-BuOH portion (57 g) was separated on D101 macroporous
2 R
O (47:53, v/v, 5.0 mL/min) to yield 4 (4.5 mg, t
3
resin CC and eluted with EtOH-H
2
O (30:70, 50:50, 70:30) to yield three fractions
6

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(Fr.1-Fr.3). Fraction 1 was separated into six subfractions 1a-1f by silica gel CC with
a system of CH
ODS CC followed by Sephadex LH-20 CC eluting with MeOH to obtain 1 (9.8 mg).
.3.1. Vitexnegheteroin E (1)
2 2
Cl -MeOH (from 100:0 to 0:100). Subfraction 1e was applied to
2
20
Yellowish oil; [α]
D
−99.8 (c 1.10, MeOH); CD (MeOH): 213 (∆ε +12.86), 242
(∆ε −7.81), 260 (∆ε +9.98), 316 (∆ε +9.36), 348 (∆ε −11.81) nm; UV (MeOH) λmax
−
1 1
2
59 nm; IR (KBr) νmax 3417, 2936, 1649, 1514, 1384, 1277, 1024, and 617 cm ; H
13
NMR (600 MHz, DMSO-d
6
) and C NMR (100 MHz, DMSO-d
6
) data, see Table 1;
+
+
ESIMS m/z 527 [M + Na] ; HRESIMS m/z 527.1533 [M + Na] (calcd. for
C
25
H
28NaO11, 527.1524).
2.3.2. Vitexnegheteroin F (2)
20
Yellowish oil; [α]
D
−126.3 (c 0.10, MeOH); CD (MeOH): 221 (∆ε +10.25), 248
(∆ε −2.74), 316 (∆ε +3.97), 350 (∆ε −7.49) nm; UV (MeOH) λmax 255 nm; IR (KBr)
−1 1
ν
max 3357, 2934, 1657, 1564, 1513, 1394, 1152, 1026, and 952 cm ; H NMR (600
1
3
MHz, CD
3
3
OD) and C NMR (150 MHz, CD OD) data, see Table 1; HRESIMS m/z
−
5
47.1821 [M − H] (calcd. for C27
H
31
O12, 547.1821).
2
.3.3. Vitexnegheteroin G (3)
Yellow amorphous powder; UV (MeOH) λmax 255 nm; IR (KBr) νmax 3394, 2926,
−1 1
1
640, 1513, 1384, 1274, 1150, and 1031 cm ; H NMR (400 MHz, DMSO-d ) and
6
1
3
−
C NMR (100 MHz, DMSO-d
6
) data, see Table 1; ESIMS m/z 355 [M − H] ;
−
HRESIMS m/z 355.0813 [M−H] (calcd. for C19
15 7
H O , 355.0823).
2.3.4. Vitexnegheteroin H (9)
2
0
Colorless needles; [α]
D
−75.7 (c 0.07, MeOH); UV (MeOH) λmax 258 nm; IR
−
1 1
(
KBr) νmax 3442, 2925, 1713, 1636, 1457, 1383, 1109, and 619 cm ; H NMR (400
7

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13
MHz, pyridine-d
5
) and C NMR (100 MHz, pyridine-d
5
) data, see Table 2; ESIMS
+
+
m/z 541 [M + Na] ; HRESIMS m/z 541.3146 [M + Na] (calcd. for C30
H
46NaO
7
,
541.3136).
2.4. Acid hydrolysis of compounds 1 and 2 and determination of the absolute
configuration of the monosaccharides
Each compound (2.0 mg) was hydrolysed with 2 M HCl (2 mL) at 95 °C for 3 h.
The reaction mixture was extracted with EtOAc. The aqueous layer was adjusted to
pH 7 and then concentrated to dryness to give a residue, which was dissolved in
anhydrous pyridine (2 mL) and mixed with L-cysteine methyl ester hydrochloride (2
mg). The mixture was heated at 60 °C for 3 h. After drying the solution,
trimethylsilylimidazole (0.2 mL) was added and heated at 60 °C for 2 h, then the
2
solution was partitioned between H O and cyclohexane. The cyclohexane layer was
concentrated and then dissolved in 200 µL of cyclohexane and analysed by GC using
an HP-5 column. Temperatures of the injector and detector were 270 °C and 280 °C,
respectively. A temperature gradient system was used for the oven, starting at 160 °C
for 1 min and increasing up to 200 °C at a rate of 4 °C/min then keeping it for 5 min,
and increasing up to 240 °C at a rate of 10 °C/min and keeping it for 10 min. The
peaks of authentic samples of
D-glucose and L-glucose after treatment in the same
manner were detected at 26.38 min and 27.03 min, respectively.
2.5. Computational methods
The Spartan 14.0 searches based on molecular mechanics with MMFF94S force
fields were performed for 7. Selected meaningful conformers were further optimized
by the time-dependent density functional theory (TDDFT) method at the
B3LYP/6-31G(d) level in Gaussian 09 program package [13], which were further
8

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checked by frequency calculation and resulted in no imaginary frequencies.
Theoretical ECD curves were calculated at the B3LYP/6-31++G(d,p) level with the
CPCM model in methanol solution and simulated using SpecDis 1.51 [14] according
to Boltzmann distributions.
2.6. Cytotoxic assay
Cytotoxic assay against human cancer cell lines in vitro was performed by a
modified 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) method
15]. Compounds 1–14 were evaluated for their cytotoxicity against human liver
[
carcinoma (HepG2), colon carcinoma (HCT116), and ovarian carcinoma (A2780) cell
lines. Doxorubicin was used as a positive control.
2.7. Anti-inflammatory assay
Murine microglial BV-2 cells were cultured at 37 °C in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% (v/v) inactivated fetal bovine
serum and 100 units/mL penicillin/streptomycin under a water-saturated atmosphere
2
of 95% air and 5% CO . The cells were seeded in 96-well culture plates and allowed
to adhere for 24 h at 37 °C. The cells were incubated for 20 h with or without 0.15
µg/mL LPS in the absence or presence of the tested compounds.
2-Methyl-2-thiopseudourea sulfate (SMT) was used as a positive control. As a
parameter of NO synthesis, the nitrite concentration was measured by the Griess
reaction using the supernatant of the BV-2 cells. Briefly, 50 µL of the cell culture
supernatant was reacted with 50 µL of the Griess reagent [1:1 mixture of 0.1%
N-(1-naphtyl)ethylenediamine in H
in a 96-well plate, and the absorbance was read on a microplate reader at 550 nm
16].
2
O and 1% sulfanilamide in 5% phosphoric acid]
[
9

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2.8. ABTS radical scavenging activity assay
The radical scavenging activities of the compounds (1–8) were carried out using
an improved ABTS decolorization assay with some modifications [17]. ABTS
+
radical cation (ABTS• ) was produced by reacting 7 mM stock solution of ABTS
with 2.45 mM potassium persulfate and allowing the mixture to stand in the dark at
+
room temperature for 12-16 h before use. The ABTS• solution was diluted with
ethanol, to an absorbance of 0.7 ± 0.02 at 734 nm. An ethanolic solution (100 µL) of
the samples at various concentrations (0.1-50 µM) was mixed with 150 µL diluted
+
ABTS• solution. After reaction at room temperature for 20 min, the absorbance at
+
7
34 nm was measured. The capability to scavenge the ABTS• was calculated using
+
the formula given below: ABTS• scavenging activity (%) = [1 − (S − SB) / (C −
CB)] × 100% where S, SB, C and CB are the absorbance of the sample, the blank
sample, the control, and the blank control, respectively.
3. Results and discussion
Fourteen compounds (Fig. 1) were isolated from the seeds of V. negundo var.
heterophylla by the systematical phytochemical investigation, among which four
1–3, and 9) were new, and compounds 4–8 and 10–14 were obtained from this
species for the first time. Ten known compounds were identified as vitecannaside B
4) [18],
-hydroxy-4-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxy-3,4-dihydr
o-2-naphthaldehyde (5) [19], vitedoin A (6) [20], vitexdoin A (7) [19],
-hydroxysesamin (8) [21], 2 -hydroxyursolic acid (10) [22], arjunglucoside (11)
23], negundoin G (12) [24], vitexilactone C (13) [25], and vitexilactone (14) [26],
respectively, by comparison of their spectroscopic data with those reported in
(
(
6
9
α
[
10

ACCEPTED MANUSCRIPT
literature.
Compound 1 was obtained as a yellowish oil and had a molecular formula of
+
C
C
25
25
H
H
28
O
11, as determined by HRESIMS at m/z 527.1533 [M + Na] (calcd. for
1
28NaO11, 527.1524). The H NMR spectrum showed signals due to one
aldehyde proton at δ 9.43 (s, H-2α), three aromatic or olefinic protons at δ 7.49 (s,
H-1), 6.91 (s, H-8), and 6.61 (s, H-5), characteristic protons for 1,3,4-trisubstituted
benzene moiety at δ 6.56 (d, J = 1.6 Hz, H-2′), 6.52 (d, J = 8.2 Hz, H-5′), and 6.21
(dd, J = 8.2, 1.6 Hz, H-6′), one methoxyl signal at δ 3.64 (3H, s), and the anomeric
13
H
proton of one β-glucopyranose moiety resonated at δ 4.01 (d, J = 7.7 Hz). The C
NMR spectrum of 1 gave signals due to one aldehyde carbonyl at δ 192.3 (C-2α),
2
fourteen sp carbons, one oxymethylene carbon at δ 68.5 (C-3
α
), one methoxyl
carbon at δ 55.6, two methine carbons at δ 41.6 (C-4) and 39.4(C-3), and six
1
13
glucopyranosyl carbons. These H and C NMR signals (Table 1) were assigned
1
1
with the aid of HSQC, HMBC, and H– H COSY (Fig. 2) experiments, indicating 1
to be a monoglucoside of vitexdoin A (7). The sugar moiety was attached at C-3 , as
deduced by HMBC correlation between the anomeric proton and C-3 . The
-configuration of the glucopyranosyl moiety was determined by acid hydrolysis
α
α
D
followed by derivatization and GC analysis. The relative configuration at C-3 and
C-4 could be assigned in trans form same as that of 7 and deduced by the signal of
H-4 (δ
dihedral angle of ca.90° between H-4 and H-3 [27]. This could also be supported by
the NOESY experiment (Fig. 3), which showed a correlation between H-4 and H-3
2.91, t-like, J = 10.4 Hz), but no correlation between H-4 and H-3. To determine
the absolute configuration of C-3 and C-4 of 1, ECD calculation of 7 was performed
H
4.37, s), which had a small coupling close to zero with H-3, revealing a
α
(δ
H
11

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by the TDDFT method [13,14] at the B3LYP/6-31++G(d,p) level with the CPCM
model in methanol solution as the relative configuration at C-3 and C-4 of 7 was the
same as that of 1. Comparison of the theoretically calculated and experimental ECD
curves of 7 (Fig. 4) led to determination of the absolute configuration of 7 as 3R,4S.
Moreover, the Cotton effects of 1 were similar with those of 7 (Fig. 5), indicating
that the absolute configuration of C-3 and C-4 in 1 was also 3R,4S. Therefore, the
structure
3R,4S)-6,7-dihydroxy-4-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-3,4-dihyd
ro-2-naphthaldehyde 3 -O- -glucopyranoside, named vitexnegheteroin E (Fig. 1).
of
compound
1
was
elucidated
as
(
α
β-D
32 12
Compound 2 was isolated as a yellowish oil. The molecular formula, C27H O ,
−
was assigned by the quasi-molecular ion peak at m/z 547.1821 [M − H] (calcd. for
1
13
C H O , 547.1821) in HRESIMS. The H and C NMR (Table 1) spectra of 2
27
31 12
were similar to those of vitecannaside B (4) except for the occurrence of one more
methoxyl group and the disappearance of one aromatic proton at δ 6.63 (s, H-5) in 4.
From these data, 2 was considered to be a 5-methoxy substituted derivative of 4,
which was confirmed by the HSQC and HMBC experiments (Fig. 2). In the HMBC
spectrum, key correlations were observed between H-1 and C-8, H-8 and C-1, H-4
and C-5, C-1′, C-2′, C-3, and C-3
connected to C-3 , as deduced by HMBC correlation between the anomeric proton
and C-3 . The coupling constant (J = 8.0 Hz) of anomeric proton indicated a
β-glycosidic linkage. The sugar moiety was assigned as -glucose by the methods
described above. The relative configuration at C-3 and C-4 could be assigned in
analogy with that of 1 and 7 by the characteristic signal of H-4 (δ 4.37, s). The
experimental ECD curves of 2 were in agreement with that of 7 setting C-3 and C-4
α, as well as H-3α and C-4. The sugar moiety was
α
α
D
H
12

ACCEPTED MANUSCRIPT
of 2 as R,S respectively (Fig. 5). Thus, the structure of compound 2 was
characterized as shown and named vitexnegheteroin F.
Compound 3 was obtained as yellow amorphous powder and its molecular
formula was determined to be C19
H
16
O
7
from its quasi-molecular ion peak at m/z
−
1
3
55.0813 [M − H] (calcd. for C H O , 355.0823) in the HRESIMS. The H NMR
1
9
15
7
spectrum showed signals due to one carboxylic proton at δ 15.82 (s), three aromatic
protons at δ 8.11 (s, H-1), 7.14 (s, H-8), and 6.68 (s, H-5), characteristic protons for
1,3,4-trisubstituted benzene moiety at δ 6.85 (d, J = 8.0 Hz, H-5′), 6.76 (d, J = 1.5
Hz, H-2′), and 6.63 (dd, J = 8.0, 1.5 Hz, H-6′), and two methoxyl protons at δ 3.80
13
(
3H, s) and 3.72 (3H, s). The C NMR spectrum of 3 revealed the presence of
2
nineteen carbon resonances, comprising one carbonyl at δ 172.0 (C-2α), sixteen sp
carbons, and two methoxyl carbons at δ 55.6 and 55.4. The NMR (Table 1) spectra
of 3 were similar to those of vitrofolal F [19] apart from the signal of one carboxylic
proton at δ
vitrofolal F, as well as a carbonyl at δ
vitrofolal F, indicating the functionality at C-2
confirmed by HSQC and HMBC experiments (Fig. 2). In the HMBC spectrum of 3,
key correlations were observed from H-1 to C-2 and C-3, H-2′ to C-4, and H-6′ to
H
15.82 (1H, s) in 3 instead of an aldehyde proton at δ
172.0 instead of the one at δ
of 3 was a carboxyl. This was
H
10.08 (1H, s) in
C
C
196.8 in
α
α
C-4. Thus, the structure of compound 3 was determined as shown and given the
trivial name vitexnegheteroin G.
Compound 9 was isolated as colorless needles with a molecular formula of
+
C
C
30
30
H
H
46
O
7
, as determined by HRESIMS (m/z 541.3146 [M + Na] , calcd. for
46NaO
7
, 541.3136). The molecular formula required eight unsaturation degrees
1
for 9. In the H NMR spectrum (Table 2), one olefinic proton at δ 6.17 (br.s, H-12),
13

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and two oxymethine protons at δ 4.29 (m, H-2) and 4.24 (m, H-3) were observed, as
well as four oxymethylene protons at δ 4.34 (2H, m, H-30), 4.25 (1H, m, H-23), and
3.75 (1H, d, J = 10.5 Hz, H-23), and five methyl groups at δ 1.62, 1.14, 1.11, 1.10,
13
and 0.84 (3H each, s), respectively. The C NMR (Table 2) spectrum revealed 30
carbon resonances including one carbonyl carbon at δ 178.8 (C-28), a pair of olefinic
carbons at δ 135.6 (C-13) and 125.8 (C-12), two oxygenated quaternary carbons at δ
87.4 (C-20) and 73.1 (C-19), two oxymethine carbons at δ 78.0 (C-3) and 68.8 (C-2),
two oxymethylene carbons at δ 66.2 (C-23) and 63.1 (C-30), and five methyl carbons
at δ 25.8, 23.4, 17.4, 16.1, and 14.2. These NMR spectral data closely resembled
those of glutinosalactone A (3
-lactone) [28], which was reported as a polyoxygenated ursane-type triterpene with
a pentahydroxy-urs-12-en-oic acid skeleton bearing a -lactone ring between C-28
β,19α,20β,24,30-pentahydroxyurs-12-en-28 oic acid
δ
δ
and C-20. In comparison of the NMR data of 9 to those of glutinosalactone A, the
main difference was an additional hydroxyl group at C-2 in 9. This was verified by
the expected carbon signals at δ 68.8 (C-2), 78.0 (C-3), and 47.3(C-1) [29]. The
1
1
deduction above was further confirmed by HSQC, HMBC, and H− H COSY
experiments (Fig. 2). The correlations of H-1/H-2/H-3, H-5/H-6/H-7, H-9/H-11/H-12,
1
1
H-15/H-16, and H-21/H-22 were observed in H− H COSY spectrum. The HMBC
correlations from H-1 to C-2 and C-3, from H-2 to C-3, from H-3 to C-2, C-4, and
C-23, from H-23 to C-3, C-4, and C-24, and from H-24 to C-3, C-4, C-5, and C-23
confirmed the substitution patterns of ring A. The relative configuration of 9 was
established by the NOESY experiment (Fig. 3), in which the correlations were
observed between H-5 and H-23, H-5 and H-3
α
, H-3 and H
α
and H-23, H-2
β
3
-25,
indicating 2-OH and 4-CH
2
OH (C-23) as
α
-orientation and 3-OH as β-orientation.
14

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3 3
The NOESY correlations between H -29 and H-18, H -29 and H-12, H-12 and H-18,
H-12 and H -26, indicated a -orientation for H -29. The structure of 9 was therefore
β
3
3
characterized as a rare hexaoxygenated ursane-type triterpene with a
between C-28 and C-20 and named vitexnegheteroin H.
δ-lactone ring
The cytotoxic activities of all the isolates were evaluated against HepG2, HCT116,
and A2780 cell lines, using doxorubicin as the positive control (Table 3). Compound
6
exhibited moderate cytotoxic activity against HCT116, while cytotoxic activity of
compound 5 against HepG2 was comparable to that of the positive control.
Compound 10 showed moderate cytotoxic activity against all the selected cancer cell
lines, while compound 12 showed strong cytotoxic activity against all the selected
cancer cell lines which was comparable to the positive control DOX.
The isolated compounds (1, 3−7, 9−14) were further tested for their inhibitory
activities on LPS-induced NO production in murine microglial BV-2 cell line by the
Griess reaction [16]. SMT was used as a positive control (IC50 = 1.83 ± 0.06 µM).
All the evaluated isolates exhibited inhibitory effects on LPS-stimulated NO
production as shown in Table 4. Among them, compound 12 showed significant
inhibitory effect on NO production with an IC50 value of 0.71 ± 0.16 µM, which was
comparable to the positive control SMT. Compounds 1, 4, 9, and 13 also showed
strong inhibitory effects with IC values of 17.27 ± 6.10, 19.01 ± 2.49, 17.23 ± 2.30,
50
and 10.79 ± 1.16 µM, respectively. The cytotoxicity of the tested compounds to
BV-2 cells was measured by an MTT assay. The results indicated that compounds 1,
4
, 9, 12, and 13 exerted no cytotoxic effect at their effective concentration, while the
other compounds tested showed different levels of cytotoxicity to the BV-2 cells.
The antioxidant effects of the isolated lignans (1−8) were assessed using the
15

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ABTS radical scavenging assay. In the ABTS assay, most of the isolates showed
significant antioxidant activity, with compounds 5 (IC50 = 1.71 ± 0.22 µM), 6 (IC50
=
1
.63 ± 0.08 µM), and 7 (IC50 = 1.43 ± 0.03 µM) being the most potent. Compounds
−4 also showed strong ABTS radical scavenging activities, comparable with that of
1
the standard compound VE with an IC50 value of 3.20 ± 0.14 µM, while compound 8
exhibited dramatically weak antioxidant activities (IC50 = 99.76 ± 5.45 µM),
suggesting that the antioxidant activity of the lignans was probably related to the
structure type and the number of free phenolic hydroxyl groups [17,30].
In conclusion, phytochemical investigation led to the isolation of four new
compounds (1−3, and 9), and the first report of compounds 4−8 and 10−14 from V.
negundo var. heterophylla. Their structures were elucidated by comprehensive
spectroscopic analyses and the TDDFT ECD calculations. The in vitro evaluation of
bioactivities showed that compound 12 exhibited the most potent cytotoxic activities
against all the selected cancer cell lines and significant inhibitory effect of NO
production with an IC50 value of 0.71 ± 0.16 µM, indicating that it is as effective as,
or even more effective than the positive control SMT. Moreover, most of the lignans
showed strong ABTS radical scavenging activities. The results of bioactivities
indicated the potency of application of the seeds of V. negundo var. heterophylla as
new antioxidant and anti-inflammatory agents.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China
(No. 81172958) and key laboratory basic research projects of the Department of
16

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Education in Liaoning Province, China (NO. LZ2014044).
Appendix A. Supplementary data
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Fig. 1. Structures of compounds 1−14 from V. negundo var. heterophylla.
22

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1
1
Fig. 2. Selected HMBC and H− H COSY correlations of compounds 1−3 and 9.
23

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Fig. 3. Key NOESY correlations of 1 and 9.
24

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Fig. 4. Calculated ECD spectra of (3R,4S)-7 and (3S,4R)-7 and experimental ECD
spectrum of 7.
25

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Fig. 5. Experimental ECD spectra of 1, 2, and 7.
26

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1
13
Table 1 H and C NMR data for compounds 1−3 (δ in ppm and J in Hz).
a
b
c
1
2
3
Position
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
1
2
7.49 s
147.8
133.4
192.3
39.4
7.47 s
149.9
130.2
192.3
41.4
8.11 s
128.6
119.7
172.0
154.9
2
3
α
α
9.43 s
9.34 s
15.82 s
3.12 m
3.57 m
3.37 m
3
68.5
3.71 dd (10.0, 4.7)
3.21 m
70.8
2
.91 t-like (10.4)
4
4.37 s
6.61 s
41.6
4.79 s
38.4
119.2
5
6
117.4
149.0
148.0
147.9
6.68 s
7.14 s
106.5
148.1
7
8
9
1
144.4
116.5
122.6
131.4
149.6
109.9
123.7
127.4
146.2
108.1
120.9
131.8
6.91 s
6.88 s
0
′
1
2
3
4
5
135.8
111.8
147.2
144.8
115.1
119.5
136.8
112.6
148.7
145.9
115.8
121.2
61.0
128.4
115.3
147.2
145.2
115.3
123.5
′
6.56 d (1.6)
6.67 d (1.9)
6.76 d (1.5)
6.85 d (8.0)
′
′
′
6.52 d (8.2)
6.56 d (8.2)
6.40 dd (8.2, 1.9)
3.54 s
6
5
7
′
6.21 dd (8.2, 1.6)
6.63 dd (8.0, 1.5)
-OCH3
-OCH
′-OCH
3
3.89 s
56.8
3.80 s
3.72 s
55.4
55.6
3
3
3.64 s
55.6
104.2
73.6
76.6
70.0
76.9
60.9
3.72 s
56.4
Glc-1
4.01 d (7.7)
3.04 m
4.25 d (7.7)
3.34−3.23 m
105.4
75.2
2
3
4
5
6
3.12 m
77.9
3.06 m
71.6
3.01 m
78.0
3.60 d (10.7)
3.83 dd (11.9, 2.3)
3.66 dd (11.9, 5.7)
62.8
3
.41 dd (11.8, 5.6)
a
b
c
Recorded in DMSO-d
Recorded in CD OD, 600 MHz for δ
Recorded in DMSO-d , 400 MHz for δ , 100 MHz for δ .
6
, 600 MHz for δ
H C
, 100 MHz for δ .
3
H
, 150 MHz for δ
C
.
6
H
C
27

ACCEPTED MANUSCRIPT
1
13
Table 2 H and C NMR data for compound 9 (δ in ppm and J in Hz).
a
9
Position
1
δ
H
δ
C
Position
16
δ
H
δ
C
2.24 m
.36 m
47.3
2.63 m
1.55 m
26.3
1
2
3
4
5
6
4.29 m
4.24 m
68.8
78.0
43.6
48.0
18.6
17
18
19
20
21
40.9
50.4
73.1
87.4
25.8
2.55 br.s
1.85 d (10.0)
1.78 m
2.71 m
1
.49 m
1.49 m
.36 m
2.24 m
2.62 m
7
32.9
22
23
25.9
66.2
1
1.47 m
8
9
40.2
47.8
38.5
4.25 m
1.94 m
3.75 d (10.5)
1.11 s
1
0
24
25
26
27
28
29
30
14.2
17.4
16.1
23.4
178.8
25.8
63.1
11
2.01 m
24.4
1.10 s
0.84 s
1.14 s
2
.00 m
1
1
1
1
2
3
4
5
6.17 br.s
125.8
135.6
42.0
1.62 s
1.78 m
25.3
4.34 m
4.34 m
1
.10 m
a
Recorded in pyridine-d
5 H C
, 400 MHz for δ , 100 MHz for δ .
a
Table 3 Cytotoxicities of isolates against three cancer cell lines (IC50, µM).
Compound
HepG2
55.48
8.24
HCT116
>100
57.52
10.18
25.00
8.17
A2780
>100
77.85
>100
13.67
7.58
2
5
6
45.11
29.41
12.59
6.49
1
0
2
1
b
DOX
3.53
3.45
a
The other nine compounds were inactive for all cell lines (IC50 > 100 µM).
b
Positive control.
28

ACCEPTED MANUSCRIPT
Table 4 IC50 values of isolates for NO inhibitory activity.
a
Compound
IC50 (µM)
17.27 ± 6.10
>30
1
3
4
5
6
7
9
19.01 ± 2.49
>1
>3
>30
17.23 ± 2.30
>3
10
11
>30
12
13
14
0.71 ± 0.16
10.79 ± 1.16
>10
b
SMT
1.83 ± 0.06
a
Values are mean ± SD of triplicate determinations.
Positive control.
b
29