Two new sesquiterpene glycosides isolated from the fresh needles of Pinus massoniana Lamb.

Article abstract of DOI:10.1080/14786419.2016.1239089

Two new sesquiterpene glycosides, namely massonside A (1) and massonside B (2), were isolated from the n-Bu extract of the fresh needles of Pinus massoniana Lamb. Their structures were established by 1D, 2D nuclear magnetic resonance and high-resolution mass spectrometry. Their biological activities were profiled by the anti-HBV and anti-HCV assays.

Full text of DOI:10.1080/14786419.2016.1239089

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
Two new sesquiterpene glycosides isolated from
the fresh needles of Pinus massoniana Lamb.
Yun-Chuan Xiao, Liang Ye, Man-Xi Zhao, Cui-Qi Yan, Wei Wang, Quan-Shu
Huang, Kai Liang, Bao-Hua Meng & Xiao Ke
To cite this article: Yun-Chuan Xiao, Liang Ye, Man-Xi Zhao, Cui-Qi Yan, Wei Wang, Quan-
Shu Huang, Kai Liang, Bao-Hua Meng & Xiao Ke (2016): Two new sesquiterpene glycosides
isolated from the fresh needles of Pinus massoniana Lamb., Natural Product Research, DOI:
10.1080/14786419.2016.1239089
To link to this article: http://dx.doi.org/10.1080/14786419.2016.1239089
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Date: 11 November 2016, At: 05:57

Natural Product research, 2016
http://dx.doi.org/10.1080/14786419.2016.1239089
OPEN ACCESS
Two new sesquiterpene glycosides isolated from the fresh
needles of Pinus massoniana Lamb.
Yun-Chuan Xiao, Liang Ye, Man-Xi Zhao, Cui-Qi Yan, Wei Wang, Quan-Shu Huang,
Kai Liang, Bao-Hua Meng and Xiao Ke
the laboratory of Innovative drug development and translational Medicine research of traditional chinese
Medicine, chengdu Kanghong Pharmaceutical co. ltd, chengdu, P.r. china
ABSTRACT
ARTICLE HISTORY
received 22 February 2016
accepted 4 september 2016
Two new sesquiterpene glycosides, namely massonside A (1) and
massonside B (2), were isolated from the n-Bu extract of the fresh
needles of Pinus massoniana Lamb. Their structures were established
by 1D, 2D nuclear magnetic resonance and high-resolution mass
spectrometry. Their biological activities were profiled by the anti-HBV
and anti-HCV assays.
KEYWORDS
Pinus massoniana lamb.;
sesquiterpene glycosides;
fresh needles; massonside a;
massonside B; hBV; hcV
1. Introduction
Pinus massoniana Lamb. (P. massoniana), is distributed throughout southern China. The
extract of fresh needles of P. massoniana is the main component of Songling Xuemaikang
capsule. The leaves of P. massoniana are recorded in the Dictionary of Chinese Materia
Medica, and have been used as a folk herbal medicine for centuries. Recent pharmacological
studies revealed that the needles of P. massoniana possess antioxidant (Chen et al. 2014),
antibacterial (Feng et al. 2010), cardiovascular protection (Wang et al. 2008), lipid regulation
(Zheng et al. 2010), and antitumour (Zheng et al. 2009) properties. Up to now, volatile oil
(Yatagai & Hong 1997), lignans (Lundgren et al. 1985), flavonoids (Shen & Theander 1985;
Patel et al. 2016), shikimate (Ma et al. 2008), proanthocyanidins (Gao et al. 2011; Shen et al.
2010) and their derivatives were isolated from P. massoniana. In our phytochemical
CONTACT Xiao Ke
txzz@cnkh.com
supplemental data for this article can be accessed here at http://dx.doi.org/10.1080/14786419.2016.1239089.
© 2016 chengdu Kanghong Pharmaceutical co. ltd.
this is an open access article distributed under the terms of the creative commons attribution-Noncommercial-Noderivatives license
(http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium,
provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

2
Y.-C. XIAO ET AL.
investigation, two new sesquiterpene glycosides, compounds 1 and 2, were isolated from
the title plant. In their structures, a bullatantriol and a trihydroxy endesmane skeleton each
connected a glucose via a C–O bond. This paper described the isolation and structure
elucidation of the new compounds 1 and 2, along with their anti-HBV and anti-HCV
activities.
2. Results and discussion
2.1. New compound structure elucidation
Purification of the EtOH-H2O (50:50, v/v) extract of the fresh needles of P. massoniana, using
combination of silica gel, ODS, SBC MCI gel and Sephadex LH-20 column chromatography,
gave two new sesquiterpene glycosides 1 and 2 (Figure 1).
The fresh needles of P. massoniana were extracted with EtOH-H2O (50:50, v/v) two times.
The combined extracts were concentrated by rotary evaporator and suspended in water,
then partitioned with petroleum ether (PE), AcOEt and n-Bu successively. The n-Bu part was
subjected to repeated macroporous resin D101, silica-gel, Sephadex G25, Sephadex LH-20,
ODS and SBC MCI gel column chromatography to afford the compounds 1 and 2 (Figure 1).
The structures were established by various spectroscopic analyses and chemical studies.
Compound 1 was obtained as a white amorphous powder. The IR spectrum indicated the
presence of OH (3411 cm−1). The molecular formula was determined as C21H38O8 based on
the HR-ESI-MS data (m/z 441.2451 ([M + Na]+)), besides the HR-ESI-MS gave fragment ions
at m/z 383.2408 [M + H-2H2O]+ and 221.1899 ([M + H-2H2O-C6H10O5]+), indicating the poten-
tial presence of one hexose unit.
The 13C NMR (Table S1) data combined with analysis of the heteronuclear multiple quan-
tum coherence (HMQC) of 1 revealed the remaining 21 carbon signals due to four methyls,
six methylenes, eight methines and three quaternary carbons, of which 15 were assigned
to the aglycone, and the remaining six were ascribed to a glucopyranosyl unit at δC 101.65–
61.77 ppm. Further assignments of all hydrogen and carbon signals were achieved by its
1
HMQC, H–1H COSY and HMBC spectra (Figure S1(a)). The hexose was suggested to be a
d-glucose by the comparison of data with those reported in the literature (Zhao et al. 2008).
The coupling constant of H-1′ (δH 4.15, 1H, d, J = 7.8 Hz) indicated that the d-glucose was a
β-linkage. The aglycone spectral features were closely related to those of bullatantriol (Xie
et al. 2012), except that the chemical shift of C-1 shifted downfield for 7.9 ppm, and the
correlation of δH 4.15 (H-1′) with C-1 (δC 86.13) and δH 3.30 (H-1) with C-1′ (δC 101.65) was
observed, which suggested that C-1 of 1 should be glycosylated.
Figure 1. structures of constituents from the fresh needles of Pinus massoniana lamb.

NATURAL PRODUCT RESEARCH
3
The NOESY (Figure S1(b)) correlations of H-5/H-1, H5/H-15 and H-6/H-14 suggested that
the H-1, H-5 and Me-15 were of α-orientation, H-6 and Me-14 were of β-orientation. Thus,
the structure of compound 1 was established as bullatantriol-1-O-β-d-glucopyranoside.
Compound 2 was obtained as a white amorphous powder. The IR spectrum suggested
the presence of OH (3427 cm−1). The molecular formula was determined to be C21H38O8,
based on the HR-ESI-MS data (m/z 441.2446 ([M + Na]+)), besides the HR-ESI-MS gave frag-
ment ions at m/z 383.2423 [M + H-2H2O]+ and 221.1904 ([M + H-2H2O-C6H10O5]+), indicating
the potential presence of one hexose unit.
1
The H, 13C NMR, HMQC and HMBC data of 2 (Table S1 and Figure S2 (a)) showed the
presence of four methyls, six methylenes, eight methines and three quaternary carbons.
Identical to compound 1, compound 2 also contained one β-d-glucopyranosyl by compre-
hensive analysis of NMR spectrum evidence.
The 13C NMR (Table S1) data combined with analysis of the HMQC of 2 revealed the
remaining 21 carbon signals due to four methyls, six methylenes, seven methines and four
oxygenated carbons, of which 15 were assigned to the aglycone, and the remaining six were
ascribed to a glucopyranosyl unit at δC 100.57–60.85 ppm. Identical to compound 1, com-
pound 2 also contained one β- -glucopyranosyl by comprehensive analysis of NMR spectrum
d
evidence. Further assignments of all hydrogen and carbon signals were achieved by its
1
HMQC, H–1H COSY and HMBC spectra (Table S1 and Figure S2(a)). The aglycone spectral
features were closely related to those of 1β, 4β, 7α-trihydroxyeudesmane (De Menezes et
al. 2004), except that the chemical shift of C-1 shifted downfield for 6.6 ppm and the chemical
shift of C-2 shifted upfield for 7.1 ppm, and the correlations of δH 4.42 (H-1′) with C-1 (δC
86.96) and δH 3.37 (H-1) with C-1′ (δC 100.57) were observed, which suggested that C-1 of 2
should be glycosylated.
The NOESY (Figure S2(b)) correlations of H-5/H-1and H5/H-15 suggested that the H-1, H-5
and Me-15 were of α-orientation, OH-7 and Me-14 were of β-orientation. Thus, the structure of
compound 2 was established as 1β, 4β, 7α-trihydroxyeudesmane-1-O-β-d-glucopyranoside.
2.2. Anti-HBV and anti-HCV activities
The two compounds identified in the present study were examined for their anti-HBV and
anti-HCV activities. Entecavir was used as a standard in the anti-HBV assay on HepG 2.2.15
cell line in vitro and both of compounds 1 and 2 exhibited no activity. Sofosbuvir was used
as a standard in the study of anti-HCV 1b replicon cell in vitro, and compound 2 showed
weak activity with EC50 at 145.2 μM, while compound 1 exhibited no activity.
3. Experimental
3.1. General experimental procedures
NMR Spectra: Bruker AV-400 spectrometer (MA, USA), at 100 (13C) MHz (probe (5 mm PABBO
BB-), pulse width (17.42 μsec), acquisition time (1.36 s), spectral width (24038.5 Hz), decou-
pling mode (completely decoupling), digital resolution (0.74 Hz)), Bruker AV-600 spectrom-
eter (MA, USA), at 600 (1H) MHz (probe (5 mm PABBO BB-), pulse width (12.88 μsec), acquisition
time (2.67 s), spectral width (12536.6 Hz), digital resolution (0.37 Hz)), resp.; (D6) DMSO and
D2O solns; δ in ppm, J in Hz. HR-ESI-MS: Bruker micrOTOF-Q mass spectrometers (MA, USA)

4
Y.-C. XIAO ET AL.
(The Q-TOF (ESI+) mass spectrometer was running at 4.5 kV, at a desolvation temperature
of 180 °C. The Q-TOF MS instrument was calibrated in the m/z range from 100 to 1000. All
data were processed via Agilent MassHunter Qualitative Analysis software version B.04.00.);
in m/z. ELSD-LT-II (Shimadzu Corporation, Kyoto, Japan). All solvents used were of anal. grade
(Kelong Chemical, Chengdu, P.R. China). Column chromatography (CC): Macroporous resin
D101 (Kelong Chemical, Chengdu, P.R. China), Silica gel (SiO2, 200–300 mesh; Qingdao Ocean
Chemical Industry Co., P.R. China), SBC MCI gel (Chengdu Sci-Bio-Chem Co.Ltd., Chengdu,
P.R. China), Sephadex G25 medium (GE Healthcare Life Sciences, MA, USA), ODS (GP-C18,
50 μm, Sepax Technologies, Inc, Newark, USA) and Sephadex LH-20 (GE Healthcare Life
Sciences, MA, USA). TLC Spots were visualised under UV light (254 nm) and by spraying
Vanillin-10% H2SO4 in alcohol followed by heating. Optical rotation: Perkin-Elmer-241 pola-
rimeter (MA, USA). IR Spectra: Vector 22-FTIR spectrometer (MA, USA) with KBr pellets; in
cm−1.
3.2. Plant material
The fresh needles of P. massoniana were collected from Sichuan province, P.R. China, in March
2014, and identified by Prof. Zhu-Yun Yan (College of Pharmacy, Chengdu University of
Chinese Medicine, Chengdu, P.R. China). A voucher specimen (no. 20140401) was deposited
in the Chengdu Kanghong Pharmaceutical Co. Ltd, P.R. China.
3.3. Extraction and isolation
The fresh needles of P. massoniana (24 kg) were extracted with EtOH–H2O (50:50, v/v). The
crude extract was mixed with H2O (1 L) to form a suspension, then partitioned successively
with PE, AcOEt and n-BuOH. The n-BuOH part (300 g) was subjected to CC (Macroporous
resin D101; EtOH/H2O, 0:1 → 1:0, then SBC MCI; EtOH/H2O, 0:1 → 1:9 → 2:8 → 3:7 → 1:0) to
afford five fractions. Fr.3 (EtOH/H2O, 2:8) was purified by repeated CC over Sephadex G25
(H2O), ODS (EtOH/H2O, 1:9), Sephadex LH-20 (EtOH/H2O 2:8) and SiO2 (AcOEt/EtOH 5:1, PE/
Acetone 1:2), to afford 1 (50 mg), 2 (25 mg).
Massonside A ((1R, 4S, 5R, 6R, 10R)-octahydro-4-hydroxy-6-(2-hydroxy-2-methyl-propyl)-4,
10-dimethyl-1-indenyl-β-d-glucopyranoside, 1). White amorphous powder. [ꢀ]2D0 = +3.5
(c = 0.21, MeOH). IR: 3411, 2964, 2920, 2871, 1635, 1460, 1380, 1269, 1188, 1160, 1188, 1076,
1
1022. H NMR (600 MHz, DMSO-d6) δ: 4.15 (1H, d, J = 7.8, H-1′), 3.65 (1H, dd, J = 12.0, 6.0,
H-6′a), 3.46 (1H, m, H-6′b), 3.30 (1H, dd, J = 11.4, 4.2, H-1), 3.12 (1H, dt, J = 8.4, 4.8, H-3′), 3.05
(2H, m, H-4′, 5′), 2.90 (1H, dt, J = 8.4, 4.8, H-2′), 2.17 (1H, dq, J = 10.2, 2.4, H-6), 1.93 (1H, m,
H-8a), 1.92 (1H, d, J = 13.2, H-7a), 1.68 (1H, dq, J = 12.6, 4.2, H-2a), 1.60 (1H, m, H-2b), 1.50
(2H, m, H-3a, 9a), 1.30 (2H, m, H-3b, 8b), 1.21 (1H, dd, J = 13.2, 10.2, H-7b), 1.18 (1H, m, H-9b),
1.17 (3H, s, H-15), 1.12 (3H, s, H-12), 1.10 (3H, s, H-13), 0.96 (3H, s, H-14), 0.80 (1H, d, J = 10.2,
H-5). 13C NMR (100 MHz, DMSO-d6) δ: 101.65 (C-1′), 86.13 (C-1), 77.45 (C-5′), 77.09 (C-3′), 74.03
(C-2′), 70.71 (C-4′), 70.54 (C-4), 70.30 (C-11), 61.77 (C-6′), 59.36 (C-5), 51.76 (C-7), 46.39 (C-10),
41.30 (C-3), 39.29 (C-9), 32.45 (C-8), 32.02 (C-15), 31.75 (C-6), 30.56 (C-12), 30.53 (C-13), 24.78
+
(C-2), 15.90 (C-14). HR-ESI-MS (pos.): 441.2451 ([M + Na]+, C21H38NaO8 ; Calcd 441.2459),
859.5011, 519.1871, 457.2200, 441.2451, 383.2408, 221.1899, 203.1789, 163.1479, 147.1166.
Massonside B ((1R, 4S, 5R, 7S, 10R)-decahydro-4, 7-dihydroxy-7-(1-methylethyl)-4, 10-dime-
thyl-1-naphthalenyl-β-d-glucopyranoside, 2). White amorphous powder. [ꢀ]2D0 = −57.6

NATURAL PRODUCT RESEARCH
5
(c = 0.23, MeOH). IR: 3427, 2960, 2920, 2932, 2877, 1637, 1459, 1369, 1304, 1265, 1198, 1163,
1
1077, 1021. H NMR (600 MHz, D2O) δ: 4.42 (1H, d, J = 7.8, H-1′), 3.82 (1H, dd, J = 12.0, 1.8,
H-6′a), 3.61 (1H, dd, J = 12.0, 6.0, H-6′b), 3.41 (1H, m, H-3′), 3.37 (1H, d, J = 8.8, H-1), 3.33
(1H, ddd, J = 10.2, 6.0, 1.8, H-5′), 3.28 (1H, t, J = 9.0, H-4′), 3.17 (1H, t, J = 9.0, H-2′), 1.73 (1H,
dt, J = 12.0, 1.8, H-2a), 1.68 (2H, m, H-2b, 3a), 1.63 (1H, dt, J = 13.2, 3.0, H-9a), 1.56 (1H, m,
H-11), 1.52 (3H, m, H-6a, 8), 1.46 (1H, m, H-3b), 1.36 (2H, m, H-5, 6a), 1.26 (1H, dt, J = 13.2, 4.2,
H-9b), 1.03 (3H, s, H-15), 0.88 (3H, s, H-14), 0.84 (6H, d, J = 6.8, H-12, 13). 13C NMR (100 MHz,
D2O) δ: 100.57 (C-1′), 86.96 (C-1), 75.93 (C-5′), 75.93 (C-3′), 74.69 (C-7), 73.07 (C-2′), 71.49 (C-4),
69.90 (C-4′), 60.85 (C-6′), 44.92 (C-5), 38.57 (C-11), 38.33 (C-3), 37.79 (C-10), 34.17 (C-9), 28.51
(C-15), 28.35 (C-8), 27.72 (C-6), 22.65 (C-2), 16.47 (C-12), 16.38 (C-13), 11.90 (C-14). HR-ESI-MS
+
(pos.): 441.2446 ([M + Na]+, C21H38NaO8 ; Calcd 441.2459), 859.5011, 457.2203, 441.2446,
426.2894, 383.2423, 221.1904, 203.1790.
3.4. Acidic hydrolysis and HPLC-ELSD analysis
Each solution of compound 1 and 2 (5 mg) in 1 M HCl (2.0 mL) was heated at reflux for 1 h
and then the reaction mixture was neutralised with an equal volume of 1 M NaOH and
extracted with CH2Cl2 (6 mL). The sugar moiety of both 1 and 2 was identified as d-glucose
by HPLC-ELSD analysis (column: Sepax Hp-Amino (250 × 4.6 mm, 5 μm); carrier: 80% ACN in
H2O (1.0 mL/min); column temperature: 35 °C; drift tube temperature: 40 °C; N2 pressure:
350 kPa; retention time: 8.146 min) of the aqueous solution in comparison with an authentic
d-glucose.
3.5. Anti-HBV and anti-HCV activity assays
40,000 cells/well of HepG 2.2.15 cells (Wuhan Institute of Virology, Chinese Academy of
Sciences, Wuhan, P.R. China) were seeded in 96-well plates, and were incubated at 37 °C in
a humidified incubator containing 5% CO2 and were allowed to attach overnight. After incu-
bation, culture medium was replaced with fresh medium supplemented with either 0.5%
DMSO alone (control) or varying concentrations of test compounds dissolved in DMSO; this
was repeated on the fifth day. And on the eighth day, the cell supernatants were collected
to extract total HBV DNA. The qPCR assay was used to detect the HBV DNA. Entecavir (Accela
ChemBio Co., Ltd., Shanghai, P.R. China) was used as a positive control.
0.5% DMSO alone (control) and varying concentrations of test compounds dissolved in
DMSO were added in 96-well plates first, then 8000 cells/well of HCV 1b replicon cells (WuXi
AppTec, Shanghai, P.R. China) were seeded in 96-well plates, and were incubated at 37 °C in
a humidified incubator containing 5% CO2 for three days. And inhibition of HCV replication
was measured as for the stable replicon cells using Bright-Glo (Promega Corporation, MA,
USA). Sofosbuvir (Shanghai Haoyuan Medchemexpress Co., Ltd., Shanghai, P.R. China) was
used as a positive control with EC50 at 0.12 μM.
4. Conclusion
In conclusion, two new sesquiterpene glucopryanosides (1 and 2) were isolated and char-
acterised by spectrometric analysis (1 and 2D NMR, HR-MS). Compound 2 exhibited weak
anti-HCV activity with EC50 at 145.2 μM. Therefore, we believe that this plant is an important

6
Y.-C. XIAO ET AL.
source for the diverse structure of sesquiterpene glycosides and should be further investi-
gated for other biological activities.
Acknowledgements
The authors thank the Analytical and Testing Center of Sichuan University and China measurement
technology research institute for the spectral measurements. The authors thank WuXi AppTec for the
anti-HBV and anti-HCV activity measurements.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the National Standardization Project of Traditional Chinese Medicine.
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