A new alkaloid glycoside from the rhizomes of Aristolochia fordiana

Article abstract of DOI:10.1080/14786419.2014.905934

A new alkaloid glycoside named fordianoside (1), together with three known compounds arabinothalictoside (2), 6-O-p-coumaroyl-β-fructofuranosyl-(2 → 1)-α-d-glucopyranoside (3) and 4-[formyl-5-(hydroxymethyl)-1H- pyrrol-1-yl] butanoic acid (4), was isolated from the rhizomes of Aristolochia fordiana. The structure of 1 was established as (1S)-1,2,3,4-tetrahydro-7- hydroxy-1-[(4-hydroxybenzyl) methyl]-2,2-dimethyl-8-O-isoquinolinyl β-d-glucopyranoside by using chemical and spectroscopic methods including HR-ESI-MS, 1D and 2D NMR.

Full text of DOI:10.1080/14786419.2014.905934

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A new alkaloid glycoside from the
rhizomes of Aristolochia fordiana
Zhongbo Zhouab, Jianguang Luoa, Ke Pana & Lingyi Konga
a State Key Laboratory of Natural Medicines, Department of
Natural Medicinal Chemistry, China Pharmaceutical University, 24
Tong Jia Xiang, Nanjing 210009, People's Republic of China
b Xinjiang Production & Construction Corps Key Laboratory of
Protection and Utilization of Biological Resources in Tarim Basin,
Tarim University, Alaer 843300, People's Republic of China
Published online: 09 Apr 2014.
To cite this article: Zhongbo Zhou, Jianguang Luo, Ke Pan & Lingyi Kong (2014) A new alkaloid
glycoside from the rhizomes of Aristolochia fordiana, Natural Product Research: Formerly Natural
Product Letters, 28:14, 1065-1069, DOI: 10.1080/14786419.2014.905934
To link to this article: http://dx.doi.org/10.1080/14786419.2014.905934
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Natural Product Research, 2014
Vol. 28, No. 14, 1065–1069, http://dx.doi.org/10.1080/14786419.2014.905934
A new alkaloid glycoside from the rhizomes of Aristolochia fordiana
Zhongbo Zhouab, Jianguang Luoa, Ke Pana and Lingyi Konga*
aState Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China; Xinjiang
b
Production & Construction Corps Key Laboratory of Protection and Utilization of Biological Resources in
Tarim Basin, Tarim University, Alaer 843300, People’s Republic of China
(Received 29 November 2013; final version received 14 March 2014)
A new alkaloid glycoside named fordianoside (1), together with three known
compounds arabinothalictoside (2), 6-O-p-coumaroyl-b-fructofuranosyl-(2 ! 1)-a-D-
glucopyranoside (3) and 4-[formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanoic acid
(4), was isolated from the rhizomes of Aristolochia fordiana. The structure of 1 was
established as (1S)-1,2,3,4-tetrahydro-7-hydroxy-1-[(4-hydroxybenzyl) methyl]-2,2-
dimethyl-8-O-isoquinolinyl b-D-glucopyranoside by using chemical and spectroscopic
methods including HR-ESI-MS, 1D and 2D NMR.
Keywords: Aristolochiaceae; Aristolochia fordiana; alkaloid glycoside
1. Introduction
The genus Aristolochia is distributed in wide areas from the tropics to temperate zones of the
world, various species of which have been commonly used as folk medicines (Wu et al. 2005;
Kuo et al. 2012). Previous studies carried out on Aristolochia species have revealed the presence
of several classes of bioactive natural products such as aristolochic acids and their esters (Chen
et al. 2010), aristolactams (Shi et al. 2004), alkaloids (Capasso et al. 2006), amides (Chung et al.
2011), flavonoids (Machado & Lopes 2005), lignans (Zhai et al. 2004) and steroids (Navarro-
´
Garcıa et al. 2011). Aristolochia fordiana Hemsl. is widely found in southwestern China, and its
rhizome is used for the treatment of stomach ache, abdominal pain, rheumatism and skin
diseases in the Mainland China (Jiangsu New Medicine College 1977; Lopes et al. 1990; Yu
´
´
et al. 2007; Leon-Dıaz et al. 2010). Previous phytochemical and biological investigations of this
plant have led to the isolation of some neolignans (Zhou et al. 2013). In a continuation of our
phytochemical survey on the rhizomes from A. fordiana, a new alkaloid glycoside named
fordianoside (1), together with three known compounds, was isolated. In this article, we describe
the isolation and structural elucidation of the new compound.
2. Results and discussion
25
Fordianoside (1) was obtained as yellow amorphous solid with ½aꢀD þ 34:9 (c 0.06, CH3OH).
The positive HR-ESI-MS revealed a molecular ion peak at m/z 462.2119 [M]þ (calcd for
462.2122), consistent with the molecular formula of C24H32NO8. The IR spectrum exhibited the
absorptions of hydroxyl group (3552 and 3478 cm21) and aromatic rings (1638 and 1617 cm21).
The 1H NMR spectrum revealed signals of two mutually coupled protons (dH 7.02 and 6.99; both
d, J ¼ 9.0 Hz) due to a 1,2,3,4-tetrasubstituted benzene ring, two doublets (dH 7.10 and 6.73;
*Corresponding author. Email: cpu_lykong@126.com
q 2014 Taylor & Francis

1066
Z. Zhou et al.
both d, J ¼ 8.5 Hz) because of a p-substituted benzene ring, and a singlet of six protons at dH
3.15 assigned to two N-methyl groups, which were characteristic chemical shifts and
multiplicities for a benzylisoquinoline alkaloid previously synthesised by Dwuma-Badu et al.
(1983). Other signals were attributed to the partial structures ZCH2ZCH2Z and lCHZCH2Z at
dH 5.74 (H, dd, J ¼ 7.0, 3.0 Hz), 3.83 (1H, m), 3.45 (1H, m), 3.06 (1H, dd, J ¼ 18.0, 6.0 Hz),
3.40 (1H, m) and 3.23 (2H, dd, J ¼ 18.0, 3.0 Hz). Moreover, an anomeric proton at dH 4.70 (1H,
d, J ¼ 8.0 Hz) together with six oxygenated protons at dH 3.38 (1H, t, J ¼ 8.0 Hz), 3.47 (1H, t,
J ¼ 8.0 Hz), 3.53 (1H, d, J ¼ 8.0 Hz), 3.37 (1H, overlap), 3.98 (1H, dd, J ¼ 12.0, 1.5 Hz) and
3.74 (1H, dd, J ¼ 12.0, 6.5 Hz) suggested that 1 was a glycosidic compound. The presence of D-
glucose was revealed by hydrolysis of 1 with 1 M HCl, which yielded a D-glucose as determined
by reversed-phase HPLC of its aldose thiocarbamate derivatives. In the 13C NMR spectrum, 24
carbon signals were observed, including those ascribable to a glucopyranosyl moiety (dC 107.7,
79.2, 78.0, 75.6, 71.9, 63.2), two aromatic rings, four aliphatic carbons and two N-methyl
groups. The analysis of its 2D NMR data, including HSQC and HMBC spectra, allowed for an
unambiguous assignment of all proton and carbon signals. The b-configuration of the anomeric
proton of the glucopyranosyl was assigned based on its JH1ZH2 value (J ¼ 8.0 Hz). The ROESY
correlation between d 7.02 (H-6) and 3.06 (H-4) indicated that C5ZC6 cannot be
interchangeable with C7ZC8. The HMBC cross-peaks (Figure 1) from H-1 (dH 5.74) and H-
13 (dH 7.10) to C-11 (dC 38.5) revealed the connectivity of the 1,4-disubstituted benzene ring to
C-1; cross-peaks from H-10 (dH 4.70) and H-1 (dH 5.74) to C-8 (dC 143.4) confirmed the
attachment of the glucosyl group on C-8; cross-peaks from H-3 (dH 3.81 and 3.45) and H-1 (dH
5.74) to C-Me (dC 54.5 and 52.1) indicated the presence of the two N-methyl groups. Thus, the
planar structure for 1 is depicted in Figure 1. The absolute stereochemistry at C-1 was
determined using the electronic circular dichroism experiment. The observed maxima for a
positive Cotton effect at 242 (þ1.62) nm and a negative Cotton effect at 227 (27.61) nm (Craig
& Roy 1965; Ringdahl et al. 1981; Yan et al. 2013) indicated that compound 1 had an S-
configuration at C-1. Therefore, the structure of 1 was established as (1S)-1,2,3,4-tetrahydro-7-
hydroxy-1-[(4-hydroxybenzyl) methyl]-2,2-dimethyl-8-O-isoquinolinyl b-D-glucopyranoside
and named fordianoside (Figure 1).
The structures of known compounds were identified as arabinothalictoside (2) (Yoshikawa
et al. 1993), 6-O-p-coumaroyl-b-fructofuranosyl-(2 ! 1)-a-D-glucopyranoside (3) (Georgo-
poulou et al. 2005) and 4-[formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanoic acid (4) (Chin
et al. 2003), by comparing their spectroscopic data with previously reported values (Figure 2).
All isolated compounds were tested for their cytotoxic activities on three human cancer cell
lines (human breast adenocarinoma cell line MCF-7, human hepatoma cell lines HepG2 and
human osteosarcoma cell line MG-63), and the inhibitory activities of acetylcholinesterase and
lipopolysaccharide-induced NO production in RAW 264.7 macrophages. Unfortunately, the
isolated compounds exhibited no activity in all biological evaluations.
Figure 1. Structure and selected HMBC correlations of compound 1.

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Figure 2. Structures of compounds 2–4.
3. Experimental
3.1. General experimental procedures
Optical rotation was measured using a JASCO P-1020 digital polarimeter (Jasco, Tokyo, Japan).
CD spectrum was determined on a JASCO 810 spectropolarimeter (Jasco). UV spectrum was
measured with a UV-2450 UV–visible spectrophotometer (Shimadzu, Tokyo, Japan). A Bruker
Tensor 27 spectrometer (KBr discs, Bruker, Karlsruhe, Germany) was used to acquire the IR
spectrum. NMR spectra were performed in MeOD at 303 K on a Bruker AV-500 NMR
spectrometer (1H: 500 MHz; 13C: 125 MHz; Bruker) with TMS as internal standard. ESI-MS and
HR-ESI-MS data were obtained on an Agilent 1100 series (Agilent Technologies, Santa Clara,
CA, USA) LC/MSD Trap mass spectrometer (ESI-MS) (Agilent Technologies, Santa Clara, CA,
USA) and a Mariner ESI TOF spectrometer (HR-ESI-MS) (Agilent Technologies, Santa Clara,
CA, USA), respectively. Silica gel (Qingdao Marine Chemical Co., Ltd, Qindao, China),
Sephadex LH-20 (Pharmacia, Uppsala, Sweden), MCI gel CHP-20 (Mitsubishi, Tokyo, Japan)
and ODS (40–63 mm, Fuji, Tokyo, Japan) were used for open column chromatography (CC).
Preparative HPLC was performed on an Agilent 1100 Series (Agilent Technologies) coupled
with a Shim-pack RP-C18 column (200 mm £ 20 mm i.d., Shimadzu) and an Agilent 1100 Series
multiple wavelength detector (Agilent Technologies).
3.2. Plant material
The rhizomes of A. fordiana Hemsl. were collected at the Guangxi Zhuangzu Autonomous
Region, People’s Republic of China, in November 2011, and identified by Prof. Mian Zhang,
Department of Pharmacognosy, China Pharmaceutical University. A voucher specimen (No.
AF-201111) has been deposited at the Department of Natural Medicinal Chemistry, China
Pharmaceutical University.
3.3. Extraction and isolation
The dried and powdered rhizomes (3.9 kg) of A. fordiana were extracted with MeOH (3 £ 16 L)
for 2 h under reflux. After concentration under reduced pressure, the combined MeOH extract
(395.0 g) was subjected to a silica gel CC eluted with petroleum ether, EtOAc, acetone and MeOH
to obtain four subfractions (SFI–SFIV). Eighty-one grams of SFIV (MeOH) was chromatographed
on MCI gel eluted with a MeOH–H2O solvent system in gradient (0%, 10%, 30%, 50%, 70% and
100% MeOH) to obtain six subfractions (SFIV-1–SFIV-6). SFIV-3 (2.4 g, 30% MeOH) was
subjected to ODS CC eluted with MeOH–H2O (25% and 100% MeOH) to yield four subfractions
(SFIV-3A–SFIV-3D). SFIV-3A (0.63 g) was further isolated by passage over a Sephadex LH-20
column using MeOH as eluent to obtain six subfractions (SFIV-3A1–SFIV-3A6). SFIV-3A3 (47 mg)
was further purified by preparative HPLC (MeOH–H2O–formic acid 25:75:0.1) to afford 1
(22 mg). SFIV-3A4 (26 mg) was further purified by preparative HPLC (MeOH–H2O 20:80) to yield
2 (6 mg). SFIV-3B (1.23 g) was subjected to ODS CC eluted with MeOH–H2O (25% and 100%

1068
Z. Zhou et al.
MeOH) to yield three subfractions (SFIV-3B1–SFIV-3B3). SFIV-3B1 (0.85 g) was further isolated by
passage over a Sephadex LH-20 column and preparative HPLC (MeOH–H2O 20:80) to yield 3
(2 mg). SFIV-2 (1.71 g) was subjected to ODS CC eluted with MeOH–H2O (10%, 30% and 100%
MeOH) to yield five subfractions (SFIV-2A–SFIV-2E). SFIV-2C (744 mg) was further purified by
preparative HPLC (MeOH–H2O 20:80) to afford 4 (245 mg).
3.4. Fordianoside (1)
24
25
Yellow amorphous solid; ½aꢀD þ 34:9 (c 0.06, CH3OH); CD D1 (nm, c ¼ 3.0 £ 10
,
MeOH): þ 1.25 (322), 22.11 (271), þ 1.62 (242), 27.61 (227), þ 30.76 (208); UV (MeOH)
l
max nm (log 1) 280 (3.55), 227 (4.12), 204 (4.48); IR (KBr) nmax (cm21): 3552, 3478, 3415, 3235,
1638, 1617, 1400, 1077, 621, 478; 1H NMR (500 MHz, CD3OD): d 5.74 (1H, dd, J ¼ 7.0, 3.0 Hz,
H-1), 3.83 (1H, m, H-3) and 3.45 (1H, m, H-3), 3.06 (1H, dd, J ¼ 18.0, 6.0 Hz, H-4), 6.99 (1H, d,
J ¼ 9.0 Hz, H-5), 7.02 (1H, d, J ¼ 9.0 Hz, H-6), 3.40 (1H, m, H-11), 3.23 (2H, dd, J ¼ 18.0,
3.0 Hz, H-11, H-4), 7.10 (2H, d, J ¼ 8.5 Hz, H-13, 17), 6.73 (2H, d, J ¼ 8.5 Hz, H-14, 16), 3.15
(6H, s, 2 £ NZCH3), 4.70 (1H, d, J ¼ 8.0 Hz, H-1 £ ZGlc), 3.38 (1H, t, J ¼ 8.0 Hz, H-2
£ ZGlc), 3.47 (1H, t, J ¼ 8.0 Hz, H-3 £ ZGlc), 3.53 (1H, d, J ¼ 8.0 Hz, H-4 £ ZGlc), 3.37 (1H,
overlap, H-5 £ ZGlc), 3.98 (1H, dd, J ¼ 12.0, 1.5 Hz, H-6 £ ZGlc) and 3.74 (1H, dd, J ¼ 12.0,
6.5 Hz, H-6 £ ZGlc). 13C NMR (500 MHz, CD3OD): d 70.5 (C-1), 55.3 (C-3), 24.0 (C-4), 119.1
(C-5), 127.6 (C-6), 150.2 (C-7), 143.4 (C-8), 128.3 (C-9), 121.0 (C-10), 38.5 (C-11), 128.7 (C-12),
131.6 (C-13, 17), 116.7 (C-14, 16), 158.0 (C-15), 54.5 (CZNZCH3), 52.1 (CZNZCH3), 107.7
(C-1 £ ), 79.2 (C-2 £ ), 78.0 (C-3 £ ), 75.6 (C-4 £ ), 71.9 (C-5 £ ), 63.2 (C-6 £ ); ESI-MS: m/z
462 [M]þ, 300 [M 2 Glc]þ; HR-ESI-MS: m/z 462.2119 [M]þ (Calcd for C24H32NO8, 462.2122).
3.4.1. Acid hydrolysis of 1
Compound 1 (2.0 mg) was treated in 1 M HCl (2 mL) at 1008C for 2 h. The aqueous solution was
concentrated under reduced pressure to yield a residue, which was dissolved in pyridine (2 mL)
containing L-cysteine methyl ester hydrochloride (2 mg) and heated at 608C for 1 h. And then
10 mL of o-tolylisothiocyanate was added to the mixture, which was heated at 608C for another
1 h. The reaction mixture was dissolved in MeOH after drying in vacuo and directly analysed by
reversed-phase HPLC (acetonitrile–H2O–formic acid 25:75:0.1, 0.8 mL/min). The peak at
19.11 min coincided with the derivative of D-glucose.
4. Conclusion
In this article, a new alkaloid glycoside named fordianoside, together with three known
compounds, was isolated from the rhizomes of A. fordiana. The structure of fordianoside was
identified as (1S)-1,2,3,4-tetrahydro-7-hydroxy-1-[(4-hydroxybenzyl) methyl]-2,2-dimethyl-8-
O-isoquinolinyl b-D-glucopyranoside by 1D, 2D NMR and MS spectra analyses.
Supplementary material
Supplementary material relating to this article is available online, including 1D NMR, selected
2D NMR, HR-ESI-MS, CD spectrum and HPLC analysis of monosaccharide derivative of 1
(Figures S1–S8).
Acknowledgements
This research work was financially supported by Program for Changjiang Scholars and Innovative Research
Team in University (IRT1193) and A Project Funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions (PAPD).

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