Furofuran lignan glucosides from the leaves of Vitex negundo var. cannabifolia

Article abstract of DOI:10.1080/14786419.2016.1255887

Two new furofuran lignan glucosides, cannabilignin (1) and isocannabilignin (2), together with four known compounds (3–6), were isolated from the leaves of Vitex negundo var. cannabifolia. The structures of the isolates were elucidated by analysis of spec

Full text of DOI:10.1080/14786419.2016.1255887

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Furofuran lignan glucosides from the leaves of
Vitex negundo var. cannabifolia
Yue-Ting Li, Man-Man Li, Jing Sun, Zhi-Xiang Zhu, Yue-Lin Song, Dao-Ran
Pang, Jiao Zheng, Yun-Fang Zhao, Peng-Fei Tu & Jun Li
To cite this article: Yue-Ting Li, Man-Man Li, Jing Sun, Zhi-Xiang Zhu, Yue-Lin Song, Dao-Ran
Pang, Jiao Zheng, Yun-Fang Zhao, Peng-Fei Tu & Jun Li (2016): Furofuran lignan glucosides
from the leaves of Vitex negundo var. cannabifolia, Natural Product Research, DOI:
10.1080/14786419.2016.1255887
To link to this article: http://dx.doi.org/10.1080/14786419.2016.1255887
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Date: 21 November 2016, At: 08:22

Natural Product research, 2016
http://dx.doi.org/10.1080/14786419.2016.1255887
Furofuran lignan glucosides from the leaves of Vitex negundo
var. cannabifolia
Yue-Ting Lia,b, Man-Man Lia,b, Jing Suna,b, Zhi-Xiang Zhua, Yue-Lin Songa,
Dao-Ran Panga,b, Jiao Zhenga, Yun-Fang Zhaoa, Peng-Fei Tua and Jun Lia
aModern research center for traditional chinese Medicine, Beijing university of chinese Medicine, Beijing,
People’s republic of china; bschool of chinese Materia Medica, Beijing university of chinese Medicine, Beijing,
People’s republic of china
ABSTRACT
ARTICLE HISTORY
received 5 July 2016
accepted 18 october 2016
Two new furofuran lignan glucosides, cannabilignin (1) and
isocannabilignin (2), together with four known compounds (3–6),
were isolated from the leaves of Vitex negundo var. cannabifolia. The
structures of the isolates were elucidated by analysis of spectroscopic
data. Compound 3 exhibited weak inhibition of nitric oxide production
in lipopolysaccharide-stimulated BV-2 microglial cells with IC50 value
of 69.1 5.8 μM.
KEYWORDS
Verbenaceae; Vitex negundo
var. cannabifolia; lignan
glucoside
4
O
6
O
1'''
4''' OH
O
3
O
9'
1
7'''
O
8 R2
R1
6''
8'
O
1'
9
O
3'
HO
HO
1''
7'
O
OH
HO
6'
4'
1
2
R1 = OH, R2 = H
1 = H, R2 = OH
R
1. Introduction
Vitex negundo L. var. cannabifolia, is a shrub or small tree, mainly growing in Yangzi River
basin of China (Pei & Chen 1982). Different parts of this plant are used in traditional Chinese
medicine for the treatment of various diseases. Its fruits have been used to treat cough,
asthma, analgesia, diarrhoea, and beriberi (The Editorial Committee of the Administration
Bureau of Traditional Chinese Medicine 1999); the roots are utilised for the treatment of
colds, headache, toothache, malaria, and rheumatic arthralgia, while the leaves are used to
treat cough, asthma, and chronic bronchitis (The State Pharmacopoeia Commission of
People’s Republic of China’s 2010). Phytochemical investigations indicated the presence of
diterpenoids, irioid glycosides, lignans, phenolic glycosides, flavonoids, and triterpenoids in
this plant (Taguchi 1976;Yamasaki et al. 2008; Ling et al. 2010; Chen et al. 2012; Li et al. 2014,
CONTACT Jun li
drlj666@163.com
supplemental data for this article can be accessed at http://dx.doi.org/10.1080/14786419.2016.1255887.
© 2016 Informa uK limited, trading as taylor & Francis Group

2
Y.-T. LI ET AL.
4
3
O
O
6
O
1'''
4'''
R3
O
O
OH
R2
R1
9'
1
7'''
O
8 R2
R1
6''
8'
O
1'
9
O
3'
4'
O
HO
HO
1''
7'
C
OH
HO
HO
HO
6'
O
HO
R1 R2
O
1
2
OH
H
H
OH
OH
OH
HO
HO
O
O
O
O
HO
R1 R2 R3
4
5
6
H
H
H
Me
H
H
OH Me
O
O
Me OH
H
3
Figure 1. structures of compounds 1–6.
2015; Lou et al. 2014; Fang et al. 2016). Our group recently reported the isolation of six new
and eight known polyoxygenated triterpenoids, which showed anti-inflammatory activity
(Li et al. 2014). In our ongoing investigation of anti-inflammatory agents from V. negundo
var. cannabifolia, two new furofuran lignan glucosides, cannabilignin (1) and isocannabilignin
(2), along with four known compounds (3–6) (Figure 1) were isolated from the 95% EtOH
extract of the leaves of V. negundo var. cannabifolia. Herein, the isolation and structural
elucidation of the isolates as well as their inhibitory effects on LPS-stimulated nitric oxide
(NO) production in BV-2 microglial cells are described.
2. Results and discussion
Cannabilignin (1) was obtained as a colourless gum, [ꢀ]2D1 −34 (c 0.1, MeOH). Its molecular
formula was determined as C32H32O14 by the 13C NMR data and a deprotonated ion at m/z
639.1744 [M − H]− in the negative-ion HR-ESI-MS, indicating 17 indices of hydrogen deficiency.
The IR spectrum indicated the presence of hydroxy (3427 cm−1) and ester carbonyl (1695 cm−1)
functionalities. The 1H NMR spectrum of 1 exhibited proton signals of two sets of ABX coupling
systems [δH 6.92 (1H, d, J = 2.0 Hz, H-2), 6.87 (1H, dd, J = 8.0, 2.0 Hz, H-6), 6.78 (1H, d, J = 8.0 Hz,
H-5); 7.09 (1H, d, J = 1.5 Hz, H-2′), 7.01 (1H, dd, J = 8.0, 1.5 Hz, H-6′), 6.83 (1H, d, J = 8.0 Hz, H-5′)],
one methylenedioxy group [δH 5.93 (2H, s, –OCH2O–)], two oxygenated methylenes [δH 4.35
(1H, dd, J = 8.5, 8.5 Hz, H-9α), 3.66 (1H, m, H-9β); 3.87 (1H, d, J = 9.5 Hz, H-9′β), 3.79 (1H, d,
J = 9.5 Hz, H-9′α)], and two oxygenated methines [δH 4.67 (1H, d, J = 6.0 Hz, H-7), 4.44 (1H, br
s, H-7′)]. These data together with the characteristic chemical shifts of 18 carbon signals at δC
149.4, 148.7, 148.3, 146.2, 136.3, 129.5, 124.3, 120.9, 118.2, 116.8, 109.0, 107.8, 92.8, 88.6, 87.6,
76.6, 71.9, 62.7 in the 13C NMR spectrum indicated that 1 was an 8′-hydroxyfurofuran lignan
derivative (Gu et al. 2004). In addition, the resonances attributable to a glucosyl moiety

NATURAL PRODUCT RESEARCH
3
[δC 104.3 (C-1″), 77.4 (C-3″), 76.0 (C-5″), 74.8 (C-2″), 71.7 (C-4″), 64.9 (C-6″); δH 4.81 (1H, d, J = 7.5 Hz,
H-1″), 4.75 (1H, dd, J = 12.0, 2.0 Hz, H-6″a), 4.39 (1H, dd, J = 12.0, 7.0 Hz, H-6″b), 3.74 (1H, m,
H-5″), 3.53 (1H, m, H-2″), 3.51 (1H, m, H-3″), 3.46 (1H, m, H-4″)] as well as a p-hydroxybenzoyl
moiety [δC 167.9 (C-7″′), 163.6 (C-4″′), 133.0 (C-2″′, 6″′), 122.2 (C-1″′), 116.4 (C-3″′, 5″′); δH 7.91
(2H, d, J = 9.0 Hz, H-2″′, 6″′), 6.85 (2H, d, J = 9.0 Hz, H-3″′, 5″′)] were observed in the NMR spectra.
The 1H and 13C NMR data of 1 were closely related to those of (1R,2S,5R,6R)-1-hydroxy-2-(3,4-
dihydroxyphenyl)-6-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0] octane (Luo et al.
2014), except for the presence of an additional 6-O-(p-hydroxybenzoyl)-glucosyl unit at C-3′ in
1. This deduction was supported by the HMBC correlations from the anomeric proton (δH 4.81)
to C-3′ (δC 146.2) and from H2-6″ (δH 4.75, 4.39) to the carbonyl carbon (δC 167.9). Other HMBC
correlations permitted further confirmation of the planar molecular structure of 1 (Figure S19,
supplementary material). In the NOESY spectrum, NOE correlations of H-7′/H-9′β, H-7′/H-9β,
H-7/H-9′β, and H-7/H-9β indicated that H-7 and H-7′ are β-orientated. In turn, NOEs of H-8/H-
9α, H-8/H-2, and H-8/H-6 suggested the α-orientation of H-8. Compound 1 was hydrolysed
with 2.0 M trifluoroacetic acid (CF3COOH) to produce d-glucose, which was identified by HPLC
analysis using an optical rotation detector (Yoshikawa et al. 2006). Moreover, the β-configura-
tion was prompted by the large coupling constant of the anomeric proton (δH 4.81, d, J = 7.5 Hz).
The ECD spectrum of 1 showed negative Cotton effects at 223 and 273 nm, which indicated
that the absolute configuration of 1 was 7S,7′R,8R,8′S (Gu et al. 2004). Therefore, the structure
of cannabilignin (1) was established as shown.
Compound 2 (isocannabilignin) was obtained as a colourless gum,[ꢀ]2D1−18 (c 0.1, MeOH).
Its IR spectrum exhibited absorption bands for hydroxy (3431 cm−1) and ester carbonyl
(1699 cm−1) groups. The molecular formula C32H32O14 of 2 was determined to be the same
as that of 1 from the 13C NMR and negative-ion HR-ESI-MS data (m/z 639.1725 [M − H]−).
Analysis of the 1H and 13C NMR spectroscopic data of 2 showed a close structural resemblance
to 1. The major difference was that a hydroxy group was located at C-8 in 2 rather than C-8′
in 1. This assignment was supported by the HMBC correlations from H-8’ to C-1′ and C-8;
from H-2 to C-3, C-4, C-6, and C-7; from H-6 to C-2, C-4, C-5, and C-7; from methylenedioxy
protons to C-3 and C-4; from H-2′ to C-3′, C-4′, C-6′, and C-7′; from H-6′ to C-2′, C-4′, C-5′, and
C-7′; from H-1″ to C-3′; from H2-6″ to C-7″′; and from H-2″′ and H-6″′ to C-7″′. In the NOESY
spectrum, NOE correlations of H-7′/H-9′β, H-7′/H-9β, H-7/H-9′β, H-7/H-9β, and H-8′/H-9′α,
H-8′/H-2′, and H-8′/H-6′ suggested the β-orientations of H-7 and H-7′, as well as α-orientation
of H-8′. In the ECD spectrum, negative Cotton effects at 232 and 279 nm indicated that the
absolute configuration of 2 was 7R,7′S,8S,8′R (Gu et al. 2004).
The remaining four known compounds were identified as 9R-hydroxy-d-sesamin (3) (Ye
et al. 2002), vulgarsaponin A (4) (Xiao et al. 2016), 2α,3α,19α,24-tetrahydroxyolea-12-en-28-
oic acid β-d-glucopyranosyl ester (5) (Ono et al. 2009), and 2α,3α,19α,24 -tetrahydroxy-
urs-12-en-28-oic acid β-d-glucopyranosyl ester (6) (Ono et al. 2009), by comparing their
spectroscopic and physical data with those reported in literature.
Compounds 1–6 were evaluated for their inhibitory effects on the NO production in LPS-
activated BV-2 microglial cells using the Griess assay (Li et al. 2012; Huang et al. 2015).
Indomethacin (IC50 = 15.4 1.3 μM) was used as a positive control. Only compound 3 showed
weak inhibition against NO production with IC50 value of 69.1 5.8 μM.

4
Y.-T. LI ET AL.
3. Experimental
3.1. General experimental procedures
Optical rotations were obtained on a Rudolph Autopol IV automatic polarimeter. IR spectra
were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrophotometer with KBr pellets.
UV spectra were measured on a UV-2401PC spectrometer. ECD spectrum was acquired on
a Jasco J-810 CD spectrophotometer. The HR-ESI-MS data were recorded on an LCMS-IT-TOF
system, fitted with a Prominence UFLC system and an ESI interface (Shimadzu, Kyoto, Japan).
The NMR spectra were recorded on a Varian INOVA-500 spectrometer operating at 500 MHz
for 1H and 125 MHz for 13C. Silica gel (200–300 mesh, Qingdao Marine Chemical Inc, Qingdao,
China), LiChroprep RP-C18 gel (40–63 μm, Merck, Germany), and Sephadex LH-20 (Pharmacia)
were used for open column chromatography. Semi-preparative HPLC was performed on a
Waters 2535 pump system (Waters Corporation, Milford, MA, USA), equipped with a 2998
photodiode array detector monitoring at 198 and 254 nm. A semi-preparative RP-HPLC col-
umn (Grace Prevail C18, 250 × 10 mm, 5 μm) was employed for the isolation. TLC was per-
formed using GF254 plates, sprayed with vanillin reagent, prepared by mixing 1 g of vanillin
with 100 mL of concentrated H2SO4, and heated until optimal colour development. All puri-
fied compounds submitted for bioassay were at least 95% pure as judged by HPLC and
supported by 1H NMR analysis.
3.2. Plant material
The leaves of V. negundo var. cannabifolia were collected in Xinyang, Henan Province, People’s
Republic of China, in September 2012, and plant authentication was performed by Prof.
Peng-Fei Tu. A voucher specimen (JLI-VNC-201209) is deposited in the Modern Research
Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine.
3.3. Extraction and isolation
The leaves of V. negundo var. cannabifolia (10 kg) were refluxed with 95% EtOH three times
(each 100 L, 2 h). After removal of the solvent under reduced pressure, the residue (3.6 kg)
was suspended in H2O (5 L) and partitioned with petroleum ether (4 × 5 L), EtOAc (4 × 5 L),
and n-BuOH (4 × 5 L), successively. The EtOAc extract (290 g) was subjected to silica gel
column chromatography (CC, 10 × 80 cm) eluting with a stepwise gradient of CH2Cl2–MeOH
(from 20:1 to 1:1) to afford fractions A–L. Fraction B (4.7 g) was subjected to silica gel CC
(3 × 47 cm) using petroleum ether–EtOAc (from 7:1 to 1:1) as the mobile phase to give 12
subfractions, B1–B12. Subfraction B2 (675 mg) was purified on a Sephadex LH-20 CC
(2 × 110 cm) eluting with CH2Cl2–MeOH (1:1) to afford 3 [68.4 mg; TLC, Rf = 0.57 (petroleum
ether–EtOAc, 2:1)]. Fraction H (4.3 g) was subjected to silica gel CC (3 × 45 cm) to give nine
subfractions, H1–H9. Fraction H6 (900 mg) was chromatographed on Sephadex LH-20 CC
(2.5 × 110 cm) eluting with CH2Cl2–MeOH (1:1) to yield 12 portions (H6a-H6l). H6f (50 mg)
was purified by semi-preparative RP-C18 HPLC (25% CH3CN, 3.0 mL/min) to produce 2 (5.2 mg,
tR = 24.0 min) and 1 (5.5 mg, tR = 26.5 min). Fraction J (3.9 g) was chromatographed over
silica gel CC (3 × 43 cm) with gradient elution of CH2Cl2–MeOH (from 20:1 to 0:1) to produce
subfractions J1–J9. Fraction J7 (579 mg) was subjected to Sephadex LH-20 CC (2 × 110 cm)
eluting with MeOH to yield J7a–J7h. Compound 4 [5.5 mg; TLC, Rf = 0.67 (CH2Cl2–MeOH,

NATURAL PRODUCT RESEARCH
5
4:1)] was obtained from subfraction J7a via ODS CC (1.5 × 19 cm) with gradient elution of
MeOH–H2O (from 55 to 100%). Subfraction J7a was purified by semi-preparative RP-C18 HPLC
(24% CH3CN, 3.0 mL/min) to give 5 (5.5 mg, tR = 13.8 min) and 6 (6.3 mg, tR = 14.5 min).
Cannabilignin (1): colourless gum; [ꢀ]2D1 −34 (c 0.1, MeOH); HR-ESI-MS: m/z 639.1744
MeOH
[M − H]− (Calcd for C32H31O14, 639.1719). UV
ꢀ
(log ε) 207 (2.50), 230 (2.09), 259 (2.10) nm;
max
ECD (c 0.781 mM, MeOH), λmax (Δε) 223 (−0.46), 273 (−0.77) nm; IR (KBr) vmax: 3427, 1695,
1656, 1634, 1609, 1515, 1444, 1384, 1278, 1167, 1065 cm−1. 1H NMR (500 MHz, methanol-d4):
δ 7.91 (2H, d, J = 9.0 Hz, H-2″′, H-6″′), 7.09 (1H, d, J = 1.5 Hz, H-2′), 7.01 (1H, dd, J = 8.0, 1.5 Hz,
H-6′), 6.92 (1H, d, J = 2.0 Hz, H-2), 6.87 (1H, dd J = 8.0, 2.0 Hz, H-6), 6.85 (1H, d, J = 9.0 Hz, H-3″′,
H-5″′), 6.83 (1H, d, J = 8.0 Hz, H-5′), 6.78 (1H, d, J = 8.0 Hz, H-5), 5.93 (2H, s, –OCH2O–), 4.81
(1H, d, J = 7.5 Hz, H-1″), 4.75 (1H, dd, J = 12.0, 2.0 Hz, H-6″a), 4.67 (1H, d, J = 6.0 Hz, H-7), 4.44
(1H, br s, H-7′), 4.39 (1H, dd, J = 12.0, 7.0 Hz, H-6″b), 4.35 (1H, dd, J = 8.5, 8.5 Hz, H-9α), 3.87
(1H, d, J = 9.5 Hz, H-9′β), 3.79 (1H, d, J = 9.5 Hz, H-9′α), 3.74 (1H, m, H-5″), 3.66 (1H, m, H-9β),
3.53 (1H, m, H-2″), 3.51 (1H, m, H-3″), 3.46 (1H, m, H-4″), 2.90 (1H, m, H-8); 13C NMR (125 MHz,
methanol-d4): δ 167.9 (C-7″′), 163.6 (C-4″′), 149.4 (C-4), 148.7 (C-3), 148.3 (C-4′), 146.2 (C-3′),
136.3 (C-1), 133.0 (C-2″′, C-6″′), 129.5 (C-1′), 124.3 (C-6′), 122.2 (C-1″′), 120.9 (C-6), 118.2 (C-2′),
116.8 (C-5′), 116.4 (C-3″′, C-5″′), 109.0 (C-5), 107.8 (C-2), 104.3 (C-1″), 102.4 (–OCH2O–), 92.8
(C-8′), 88.6 (C-7′), 87.6 (C-7), 77.4 (C-3″), 76.6 (C-9′), 76.0 (C-5″), 74.8 (C-2″), 71.9 (C-9), 71.7
(C-4″), 64.9 (C-6″), 62.7 (C-8).
Isocannabilignin (2): colourless gum; [ꢀ]2D1 −18 (c 0.1, MeOH); HR-ESI-MS: m/z 639.1725
MeOH
[M − H]− (Calcd for C32H31O14, 639.1719). UV
ꢀ
(log ε) 206 (2.50), 232 (2.00), 259 (2.03) nm;
max
ECD (c 1.563 mM, MeOH), λmax (Δε) 232 (−1.12), 279 (−1.55) nm; IR (KBr) vmax: 3431, 1699,
1653, 1634, 1609, 1515, 1446, 1385, 1280, 1168, 1064 cm−1. 1H NMR (500 MHz, methanol-d4):
δ 7.91 (2H, d, J = 8.5 Hz, H-2″′, H-6″′), 7.17 (1H, d, J = 2.0 Hz, H-2′), 7.01 (1H, dd, J = 8.5, 2.0 Hz,
H-6′), 6.93 (1H, d, J = 1.5 Hz, H-2), 6.85 (1H, overlapped, H-5′), 6.85 (1H, d, J = 8.5 Hz, H-3″′,
H-5″′), 6.84 (1H, overlapped, H-6), 6.78 (1H, d, J = 7.5 Hz, H-5), 5.92 (2H, s, –OCH2O–), 4.81 (1H,
overlapped, H-1″), 4.74 (1H, dd, J = 12.0, 1.5 Hz, H-6″a), 4.59 (1H, d, J = 5.0 Hz, H-7′), 4.55 (1H,
s, H-7), 4.39 (1H, dd, J = 12.0, 7.0 Hz, H-6″b), 4.33 (1H, dd, J = 9.0, 9.0 Hz, H-9′α), 3.91 (1H, d,
J = 9.5 Hz, H-9β), 3.77 (1H, d, J = 9.5 Hz, H-9α), 3.75 (1H, m, H-5″), 3.58 (1H, m, H-9′β), 3.52 (1H,
m, H-2″), 3.49 (1H, m, H-3″), 3.47 (1H, m, H-4″), 2.93 (1H, m, H-8′); 13C NMR (125 MHz, metha-
nol-d4): δ 168.0 (C-7″′), 163.6 (C-4″′), 148.8 (C-3), 148.7 (C-4), 148.1 (C-4′), 146.5 (C-3′), 134.1
(C-1′), 133.0 (C-2″′, C-6″′), 131.7 (C-1), 123.0 (C-6′), 122.2 (C-6), 122.0 (C-1″′), 117.3 (C-5′), 117.2
(C-2′), 116.3 (C-3″′, C-5″′), 109.4 (C-2), 108.6 (C-5), 104.2 (C-1″), 102.2 (–OCH2O–), 92.8 (C-8),
89.1 (C-7), 87.3 (C-7′), 77.4 (C-3″), 76.0 (C-5″), 75.9 (C-9), 74.8 (C-2″), 72.1 (C-9′), 71.8 (C-4″),
64.9 (C-6″), 62.4 (C-8′).
3.4. Hydrolysis of compounds 1 and 2
Each compound (2.0 mg) was dissolved in 2.0 M trifluoroacetic acid (2.0 mL) and heated at
90 °C for 2 h in a sealed tube. After cooling, the reaction mixture was extracted with EtOAc.
After repeated evaporation to dryness of the aqueous layer with MeOH until neutral, the residue
was dissolved in water (0.5 mL) and subjected to HPLC analysis under the following conditions,
respectively: HPLC column, Hypersil-APS2 (250 × 4.6 mm, 5 μm); detection, CHIRALYSER–MP
optical rotation detector; mobile phase, CH3CN–H2O (85:15, v/v); flow rate 1.0 mL/min.
Identification of d-glucose was carried out by comparison of its retention time and optical
rotation with those of authentic d-glucose (tR = 13.4 min, positive optical rotation).

6
Y.-T. LI ET AL.
3.5. Cell culture and measurement of NO production
BV-2 microglial cells were purchased from Peking Union Medical College (PUMC) Cell Bank
(Beijing, People’s Republic of China). Cell maintenance, experimental procedures, and data
determination for the inhibition of NO production are the same as previously described (Li
et al. 2012; Huang et al. 2015). Indomethacin (IC50 = 15.4 1.3 μM) was used as a positive
control.
4. Conclusion
Two new furofuran lignan glucosides, cannabilignin (1) and isocannabilignin (2), together
with four known compounds were obtained from the leaves of V. negundo var. cannabifolia.
Compound 4 was obtained from this plant for the first time, whereas compounds 5 and 6
were firstly isolated from the Vitex genus. Compound 3 showed weak inhibition against NO
production in LPS-stimulated BV-2 microglial cells with IC50 value of 69.1 5.8 μM.
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1–S20.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
The work was supported by the Program for New Century Excellent Talents in University [grant number
NCET-13-0693]; the National Natural Science Foundation of China [grant number 81573572].
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