A new flavonoid glycoside from the rhizomes and roots of Smilax scobinicaulis

Article abstract of DOI:10.1080/14786419.2014.880913

A new flavonoid glycoside, hesperetin-7-O-[β-d-glucopyranosyl-(1 → 3)]-β-d-glucopyranosyl (1), was isolated from the n-BuOH extract of Smilax scobinicaulis, together with four known flavonoid glycosides, clematine (2), ononin (3), daidzin (4) and puerarin (5). All of the five compounds were reported from this material for the first time. Their structures were determined on the basis of spectroscopic and spectrometric methods.

Full text of DOI:10.1080/14786419.2014.880913

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A new flavonoid glycoside from
the rhizomes and roots of Smilax
scobinicaulis
Jing Xua, Shixiu Fengb, Qi Wanga, Min Zhanga & Cunli Zhanga
a College of Life Science, Northwest A&F University, No. 22 Xinong
Road, Yangling, Shaanxi 712100, P.R. China
b Key Laboratory of Southern Subtropical Plant Diversity,
Shenzhen Fairy Lake Botanical Garden, Chinese Academy of
Sciences, 160 Xianhu Road, Liantang, Shenzhen 518004, P.R. China
Published online: 31 Jan 2014.
To cite this article: Jing Xu, Shixiu Feng, Qi Wang, Min Zhang & Cunli Zhang (2014) A new flavonoid
glycoside from the rhizomes and roots of Smilax scobinicaulis, Natural Product Research: Formerly
Natural Product Letters, 28:8, 517-521, DOI: 10.1080/14786419.2014.880913
To link to this article: http://dx.doi.org/10.1080/14786419.2014.880913
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Natural Product Research, 2014
Vol. 28, No. 8, 517–521, http://dx.doi.org/10.1080/14786419.2014.880913
A new flavonoid glycoside from the rhizomes and roots of Smilax
scobinicaulis
Jing Xua1, Shixiu Fengb1, Qi Wanga, Min Zhanga and Cunli Zhanga*
aCollege of Life Science, Northwest A&F University, No. 22 Xinong Road, Yangling, Shaanxi 712100, P.R.
China; bKey Laboratory of Southern Subtropical Plant Diversity, Shenzhen Fairy Lake Botanical Garden,
Chinese Academy of Sciences, 160 Xianhu Road, Liantang, Shenzhen 518004, P.R. China
(Received 28 October 2013; final version received 27 December 2013)
A new flavonoid glycoside, hesperetin-7-O-[b-D-glucopyranosyl-(1 ! 3)]-b-D-gluco-
pyranosyl (1), was isolated from the n-BuOH extract of Smilax scobinicaulis, together
with four known flavonoid glycosides, clematine (2), ononin (3), daidzin (4) and
puerarin (5). All of the five compounds were reported from this material for the first
time. Their structures were determined on the basis of spectroscopic and spectrometric
methods.
Keywords: Smilax scobinicaulis; flavonoid glycosides; hesperetin-7-O-[b-D-gluco-
pyranosyl-(1 ! 3)]-b-D-glucopyranosyl
1. Introduction
Smilax scobinicaulis (C.H.) Wright (Liliaceae), commonly known as Hei Ci Ba Qia in Chinese,
is distributed in Shaanxi, Gansu, Sichuan, Yunnan and other regions of China (Liu et al. 2002).
Its rhizomes and roots have long been used in folk medicine for the treatment of rheumatic
arthritis, lumbago, gout, tumour and inflammatory diseases (Zhang et al. 2012). Previous
phytochemical investigations of this plant led to the isolation of steroidal saponins (Zhang et al.
2013), flavonoids (Xi et al. 2011) and phenylpropanoids (Wang et al. 2013). In a continuing
research for new compounds from this plant, a series of flavonoid glycosides were obtained,
including one new flavonoid glycoside namely hesperetin-7-O-[b-D-glucopyranosyl-(1 ! 3)]-
b-D-glucopyranosyl (1) and four known flavonoid glycosides clematine (2) (Chen et al. 1993),
ononin (3) (Ma & Lou 2006), daidzin (4) and puerarin (5) (Hirakura et al. 1997) (Figure 1),
which were reported here as S. scobinicaulis compounds for the first time. In this paper, we
report the isolation of these compounds and structural elucidation of the new compound.
2. Results and discussion
The rhizomes and roots of S. scobinicaulis were extracted with EtOH. The extract was
suspended in H2O and partitioned with petroleum ether, EtOAc and n-BuOH, successively. The
n-BuOH soluble fraction was subjected to silica gel column chromatography (CC), Sephadex
LH-20, reversed phase silica gel column and semi-preparative HPLC to afford one new
flavonoid glycoside, hesperetin-7-O-[b-D-glucopyranosyl-(1 ! 3)]-b-D-glucopyranosyl (1),
along with four known flavonoid glycosides 2–5.
*Corresponding author. Email: cunli_zhang@nwsuaf.edu.cn
q 2014 Taylor & Francis

518
J. Xu et al.
OH
3'
OCH3
4'
B
O
O
O
2
3
7
A
O
1''
OH
HO
HO
OH
O
O
1'''
HO
HO
OH
HO
Figure 1. Chemical structure of compound 1.
Compound 1 was obtained as a yellow amorphous powder, the molecular formula
C28H34O16 was established from a molecular ion (m/z 649.1740 [M þ Na]þ, calcd for
C28H34NaO16) in the HR-ESI-MS and supported by the ESI-MS (m/z 649.13 [M þ Na]þ and m/z
625.52 [M 2 H]2). The UV (MeOH) spectrum exhibited absorption maxima at 256 and 285 nm,
1
suggesting the existence of a flavonoid nucleus. The H and 13C NMR assignments of 1 were
based on the DEPT and 2D NMR (COSY, HSQC and HMBC) experiments, which suggested
1
that 1 was a flavonoid glycoside with two glucose moieties. To be specific, the H NMR
spectrum displayed signals for three aromatic protons at dH 7.25 (d, J ¼ 2.0 Hz, H-20), 7.23 (d,
J ¼ 8.2 Hz, H-50) and 7.11 (dd, J ¼ 8.2, 2.0 Hz, H-60), corresponding to C-atom signals at dC
111.0, 116.2 and 120.1, respectively, in the HSQC spectrum, indicating the presence of a 1,3,4-
trisubstituted benzene ring. The remaining aromatic protons at dH 6.74 (d, J ¼ 2.4 Hz, H-6) and
6.67 (d, J ¼ 2.4 Hz, H-8) corresponding to C-atom signals at dC 97.7 and 96.6, respectively,
showed the presence of a 1,2,3,5-tetrasubstituted benzene ring. The methine proton at dH 5.35
(1H, dd, J ¼ 13.2 and 2.8 Hz, H-2) and methylene protons at dH 3.28 (1H, dd, J ¼ 17.2 and
13.2 Hz, H-3a) and 2.88 (1H, dd, J ¼ 17.2 and 2.8 Hz, H-3b), corresponding to C-atom signals at
dC 79.6 (C-2) and 43.0 (C-3), respectively, were the characteristic signals for a dihydroflavone of
1. Moreover, a methoxyl group at dH 3.76 (s) and two hydroxyl groups at dH 12.51 (s) and 11.40
(s) were also presented. In the HMBC spectrum, the correlations from H-20 to C-2 and C-60, H-50
to C-10 and C-30, H-60 and 40-OCH3 to C-40 suggested that the B ring had 30-hydroxyl-40-
methoxy-substitution. As to the A ring, correlations from H-6 to C-8 and C-10, and H-8 to C-6
and C-9 indicated that the A ring of the flavone should be 5,7-dihydroxyl substitution (Figure
S1). Therefore, the flavone moiety of 1 was deduced as hesperetin (Asztemborska et al. 2003;
Han et al. 2011).
Furthermore, in the NMR spectra, two anomeric protons observed at dH 5.70 (1H, d,
J ¼ 7.6 Hz, H-100) and 5.36 (1H, d, J ¼ 7.6 Hz, H-1000) corresponded to the C-atom signals at dC
99.7 and 106.8, respectively, indicating the presence of two sugar moieties. A combination of the
COSY, HSQC and HMBC data permitted the identification of the sugar moieties as
glucopyranoses. The J values (7.6 Hz) of the two anomeric protons indicated b-orientations of
the glycosidic linkages. The absolute configurations of the sugars were determined to be
D-glucose by acid hydrolysis of 1 and comparison with an authentic sample on thin-layer
chromatography (TLC) and measurement of its optical rotation. For the connection of the two
glucosides, the HMBC spectrum supported the 1 ! 3 linkage by the correlation from H-1000 to
C-300. Finally, the location of the glucopyranose was found to be C-7 of the aglycon on the basis
of a glycosylation shift of C-7 (dC 166.2) and the HMBC correlation from H-100 to C-7.

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Consequently, the structure of 1 was concluded as hesperetin-7-O-[b-D-glucopyranosyl-
(1 ! 3)]-b-D-glucopyranosyl.
3. Experimental
3.1. General experimental procedures
Optical rotations were measured with a Rudolph Research Analytical Autopol III automatic
polarimeter with MeOH as solvent (Rudolph Research Analytical, Hackettstown, NJ, USA). The
IR spectra were recorded in KBr on a Bruker Tensor 27 spectrophotometer (Bruker, Billerica,
MA, USA). The UV spectra were recorded on a Thermo Scientific Evolution 300 UV–visible
1
spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The H (400 MHz), 13C
(100 MHz) and 2D NMR spectra were recorded on a Bruker DRX-400 instrument using TMS as
internal standard (Bruker). ESI-MS were collected on a Thermo Fisher LCQ Fleet mass
spectrometer instrument (Thermo Fisher Scientific). HR-ESI-MS were carried out on a Bruker
Bio-TOF-IIIQ mass spectrometer (Bruker).
Semi-preparative HPLC was run with a Waters 1525 pump and a Waters 2489 ultraviolet
visible detector using an Xterraw prep MS C18 column (10 mm, 7.8 £ 150 mm) (Waters,
Milford, MA, USA). For CC, silica gel (200–300 mesh; Branch of Qingdao Haiyang Chemical
Co. Ltd, Qingdao, China), RP-C18 (ODS-A, 50 mm, YMC, Yantai, China) and Sephadex LH-20
(GE Healthcare Bio-Science AB, Fairfield, CT, USA) were used. TLC (5 £ 10 cm plates) was
performed on GF254 (Branch of Qingdao Haiyang Chemical Co. Ltd) plates.
3.2. Plant material
The rhizomes and roots of S. scobinicaulis C.H. were collected from Taibai Mountains of
Shaanxi Province, People’s Republic of China, in October 2010. The plant was identified by
Zhenghai Wu of Northwest A&F University. An authenticated voucher specimen (XB 00045)
has been deposited at the Herbarium of Northwest A&F University.
3.3. Extraction and isolation
The air-dried powdered rhizomes and roots of S. scobinicaulis C.H. (62 kg) were extracted with
70% EtOH at room temperature to obtain a concentrated extract, which was suspended in H2O
and partitioned successively with petroleum ether, EtOAc and n-BuOH. The n-BuOH soluble
extract (1792 g) was subjected to D101 macroporous adsorption resin and successively eluted
with 30% EtOH, 50% EtOH and 70% EtOH to afford three fractions Frs 1–3. Fr. 2 (600 g) was
subjected to silica gel CC using a step-wise gradient of EtOAc–MeOH–H2O (from 15:1:0.5 to
3:1:0.5) to afford 10 sub-fractions Frs 2a–2j. Fr. 2e (28.7 g) was chromatographed on silica gel
CC eluting with EtOAc–CH3COCH3 (from 20:1 to 1:1) in gradient to yield six sub-fractions Frs
2e1–2e6 based on TLC. Fr. 2e5 was applied to Sephadex LH-20 CC with the eluent MeOH to
yield three sub-fractions Frs 2e5a–2e5c. Fr. 2e5a was purified by reversed-phase (RP-C18) CC
and eluted with 70% MeOH 2 H2O to give compound 1 (48.6 mg). Fr. 2d (9.8 g) was
chromatographed on silica gel CC eluting with EtOAc–MeOH (from 17:1 to 3:1) in gradient to
yield four sub-fractions Frs 2d1–2d4 based on TLC. Fr. 2d1 was applied to Sephadex LH-20 CC
with the eluent MeOH to yield three sub-fractions Frs 2d1a–2d1c. Fr. 2d1b was subjected to
silica gel CC using a step-wise gradient of CHCl3–MeOH (from 30:1 to 3:1) to afford four sub-
fractions Frs 2d1b1–2b1b4. Fr. 2d1b4 was purified by semi-preparative RP-C18 HPLC with the
eluent 30% MeOH–H2O (254 nm, 2 mL/min) to give compound 3 (15.6 mg, tR ¼ 13.8 min). Fr.
2d2 was applied to Sephadex LH-20 CC with the eluent MeOH to yield four sub-fractions Frs
2d2a–2d2d. Fr. 2d2d was subjected to RP-C18 CC and eluted with 30–70% MeOH–H2O to give

520
J. Xu et al.
three sub-fractions Frs 2d2d1–2d2d3. Fr. 2d2d1 was purified by semi-preparative RP-C18 HPLC
with the eluent 30% MeOH–H2O (254 nm, 2 mL/min) to give compound 5 (65.7 mg,
tR ¼ 9.1 min). Fr. 2d2d3 was purified by semi-preparative RP-C18 HPLC with the eluent 30%
MeOH–H2O (254 nm, 2.5 mL/min) to give compound 4 (5.3 mg, tR ¼ 10.7 min). Fr. 2d3 was
purified by Sephadex LH-20 CC with the eluent MeOH to give compound 2 (51.1 mg).
Acid hydrolysis and sugar analysis: Compound 1 (30 mg) in 50 mL of 1 M HCl (MeOH–
H2O, 1:1) was heated under reflux for 8 h. After removal of the solvent, the residue was
partitioned between CHCl3 and H2O. The water layer was neutralised with 5% NaOH and
desalted (Sephadex LH-20, MeOH) to afford D-glucose (7 mg) [Branch of Qingdao Haiyang
Chemical Co. Ltd GF254, EtOAc–MeOH–H2O–AcOH (6.5:2.0:1.5:1.5), Rf ¼ 0.40] and by
20
measurement of its optical rotation (½aꢀD þ 608, c ¼ 0.50, H2O).
3.4. Hesperetin-7-O-[b-D-glucopyranosyl-(1 ! 3)]-b-D-glucopyranosyl (1)
26
A yellow amorphous powder, m.p. 258–2598C; ½aꢀD 2 29.308 (c ¼ 0.169, pyridine); UV lmax
(MeOH) nm (log 1): 256 (3.85) and 285 (2.96); IR ymax (KBr) (cm21): 3350, 2924, 1647, 1579
and 1450; ESI-MS: m/z 649.13 [M þ Na]þ, m/z 625.52 [M 2 H]2, 1250.9 [2M 2 H]2; HR-
ESI-MS: m/z 649.1740 (Calcd m/z 649.1739 for C28H34NaO16).
1H NMR (Pyridine-d5, 400 MHz) d: 5.35 (1H, dd, J ¼ 13.2 and 2.8 Hz, H-2), 3.28 (1H, dd,
J ¼ 17.2 and 13.2 Hz, H-3a), 2.88 (1H, dd, J ¼ 17.2 and 2.8 Hz, H-3b), 12.51 (1H, brs, OH-5),
6.74 (1H, d, J ¼ 2.2 Hz, H-6), 6.67 (1H, d, J ¼ 2.2 Hz, H-8), 7.25 (1H, d, J ¼ 2.0 Hz, H-20),
11.40 (1H, brs, OH-30), 7.23 (1H, d, J ¼ 8.2 Hz, H-50), 7.11 (1H, dd, J ¼ 8.2 and 2.0 Hz, H-60),
3.76 (3H, s, OCH3), 5.70 (1H, d, J ¼ 7.6 Hz, glu-H-100), 4.42 (1H, m, glu-H-200), 4.32 (1H, m,
glu-H-300), 4.30 (1H, m, glu-H-400), 3.99 (1H, m, glu-H-500), 4.32 (1H, m, glu-H-6a00), 4.41 (1H, m,
glu-H-6b00), 5.36 (1H, d, J ¼ 7.6 Hz, glu-H-1000), 4.12 (1H, m, glu-H-2000), 4.27 (1H, t, J ¼ 8.8 Hz,
glu-H-3000), 4.29 (1H, m, glu-H-4000), 4.04 (1H, m, glu-H-5000), 4.53 (1H, dd, J ¼ 12.0 and 2.4 Hz,
glu-H-6a000), 4.40 (1H, m, glu-H-6b000); 13C NMR (Pyridine-d5, 100 MHz) d: 79.6 (C-2), 43.0 (C-
3), 197.0 (C-4), 164.2 (C-5), 97.7 (C-6), 166.2 (C-7), 96.6 (C-8), 163.2 (C-9), 104.1 (C-10),
129.7 (C-10), 111.0 (C-20), 148.6 (C-30), 148.7 (C-40), 116.2 (C-50), 120.1 (C-60), 55.7 (40-OCH3),
99.7 (glu-C-100), 77.5 (glu-C-200), 84.0 (glu-C-300), 70.3 (glu-C-400), 78.6 (glu-C-500), 61.6 (glu-C-
600), 106.8 (glu-C-1000), 76.4 (glu-C-2000), 78.1 (glu-C-3000), 71.1 (glu-C-4000), 78.6 (glu-C-5000), 62.2
(glu-C-6000).
4. Conclusion
Five flavonoid glycosides are described for the first time from this plant and one of them is a new
compound. The result suggested that flavonoid glycoside is one of the secondary metabolites of
the plant. This work is a contribution to the knowledge of the chemistry of S. scobinicaulis.
Supplementary material
Supplementary material relating to this article is available online, alongside Figures S1–S27.
Acknowledgements
This research was supported by the National Natural Science Foundation of China (31070540) and the
Foundation of Shaanxi Agricultural Research (2011K02-03), Shenzhen Urban Management Bureau Grant
(201316), as well as the programme from Science and Technology Bureau of Xi’an (NC1121).

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Note
1. These authors contributed equally to this work.
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