Journal of Medicinal Plants Research Vol. 6(11), pp.

2200-2205, 23 March, 2012

Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1776
ISSN 1996-0875 2012 Academic Journals

Full Length Research Paper

The isolation and structure identification of four

Lignans stereoisomers from Uncaria sinensis (Oliv. )
Havil.
Guangli SUN1.2, Xiaopo ZHANG1, Xudong XU1*, Junshan YANG1, Lixun LV2 andMingliang
ZHONG1,2
1
Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education,
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College,
100193, Beijing, China.
2
Hebei United University, 063000, Hebei Tangshan, China.
Accepted 3 February, 2012

The study of the chemical constituents from Uncaria sinensis (oliv.) Havil. has resulted in the isolation
and identification of four stereosturcture of lignans, which were identified as: (2R,3R,4S)-lyoniresinol-
3-O--D-glucopyranoside (1); (2S,3S,4R)-lyoniresinol-3-O--D-glucopyranoside (2); (2S,3R,4S)-
lyoniresinol-3-O--D-glucopyranoside (3); (2R,3S,4R)-lyoniresinol-3-O--D-glucopyranoside (4). All
these four compounds were obtained from the Uncaria genus for the first time. The compounds were
individually identified by one-dimensional (1D), two-dimensional (2D) nuclear magnetic resonance
(NMR) and mass spectrometry (MS) spectral analyses and comparisons with reported data, and their
absolute stereosturcture were identified by the helicity rule of exciton coupled circular dichroism for
the first time. The study also constitutes a method of the identification of absolute stereosturcture by
the helicity rule of exciton coupled circular dichroism for the first time.

Key words: Uncaria sinensis (oliv.) Havil., Rubiaceae, natural products, lignans, the helicity rule.

INTRODUCTION

The Uncaria genus in Rubiaceae comprises more than
30 species widely distributed in tropical regions, including
Asia, Africa, South America (Risdale, 1978). 12 species
of them are distributed in China, which have been found
widespread use in traditional medicine. Uncaria sinensis
(Oliv.) Havil. has been used for the treatment of
hypertension, headache and fever in Chinese
Pharmacopoeia, and found growing in Sichuan, Guangxi,
Yunnan, Hubei, Guizhou, Hunan, Shanxi, Gansu
Province, etc., in China (Luo, 1999; State Pharmacopeia
Committee of China, 2010). The isolation of alkaloids was
brought into focus in previous study of the Uncaria genus;
the previous study also led to isolation of some flavonoids
and triterpenies (Heitzman et al., 2005; Laus, 2004).
However, few information is available on lignans of U.
sinensis, and no information about the identification of
absolute configuration with the method of the helicity rule
of exciton coupled circular dichroism. In this paper, four
stereoisomers of lignans (1-4) were isolated and
identified.
EXPERIMENTAL
Collection and preparation of plant material
The medicinal material was collected at Province Guangxi in 2007,
and identified by Dr. Jing Quan Yuan at the Institute of Medicinal
Plant Development, Chinese Academy of Medical Sciences and
Peking Union Medical College. A voucher specimen has been
deposited there (Voucher specimen #20070026).
Extraction and isolation

The dried and powdered bard and branch with curve

*Corresponding author. E-mail: xdxu@implad.ac.cn. hooks of the plant (8.5 kg) of U. sinensis were

Figure 1. Compounds 1-4 isolated from U. sinensis and significant NOESY correlations ().
exhaustively extracted with 70% ethanol. The extract was
filtered and concentrated on reduced pressure until only
H2O remained. The remaining solution was sequentially
partitioned with petroleum ether, CHCl3, EtOAc and n-
BuOH. The n-BuOH partion was repeatedly subjected to
column chromatography on silica gel, MCI gel, and
reversed phase C18 gel, to yield Compounds 1 to 4
(Figure 1).
RESULTS AND DISCUSSION
Planar structure elucidation
These four compounds (1 to 4) were stereoisomers of
lignans. Their sturcture were identified as (2R, 3R, 4S)-
lyoniresinol-3-O--D-glucopyranoside (1), (2S, 3S, 4R)-
lyoniresinol-3-O--D-glucopyranoside (2), (2S, 3R, 4S)-
lyoniresinol-3-O--D-glucopyranoside (3), (2R, 3S, 4R)-
lyoniresinol-3-O--D-glucopyranoside (4). Their planar
structure were identified by one-dimensional (1D), two-
Sun et al. 2201
dimensional (2D) nuclear magnetic resonance (NMR)
and mass spectrometry (MS) spectral data compared
with the data reported (Lee, 2005). And the absolute
stereosturcture were identified by nuclear overhauser
effect spectroscopy (NOESY) and the helicity rule of
exciton coupled circular dichroism (Nakanishi et al.,
1997).
Compoud 1: A white amorphous solid, ESI-MS (m/z):
+ 1
583[M+H] . Formula, C28H38O13. HNMR (600 MHz,
MeOH) : 1.71 (1H, m, H-2), 2.09 (1H, m, H-3), 2.62 (1H,
m, Ha-1), 2.72 (1H, dd, J=4.2, 15.0 Hz, Hb-1), 3.35 (3H, s,
5-OCH3), 3.54 (1H, m, Ha-6''), 3.65 (1H, m, Hb-6''), 3.75
(6H, s, 3'-OCH3, 5'-OCH3), 3.86 (3H, s, 7-OCH3), 4.28
(1H, d, J=7.8Hz, H-1''), 4.42 (1H, d, J=6.0Hz, H-4), 6.43
13
(2H, s, H-2',6'), 6.58 (1H, s, H-8). CNMR (150 MHz,
MeOH) : 34.0 (C-1), 41.4 (C-2), 46.8 (C-3), 43.4 (C-4),
147.7 (C-5), 139.1 (C-6), 148.9 (C-7), 108.0 (C-8), 126.4
(C-9), 130.4 (C-10), 66.4 (C-2), 72.2 (C-3), 134.8 (C-
1'), 107.3 (C-2'), 149.2 (C-3'), 139.6 (C-4'), 149.2 (C-5'),
107.3 (C-6'), 104.1 (C-1''), 75.3 (C-2''), 78.2 (C-3''), 71.7
(C-4''), 78.4 (C-5''), 62.9 (C-6''), 60.3 (5-OCH3), 56.8 (7-
2202 J. Med. Plants Res.
Figure 2. Planar structure of compounds 1-4
OCH3), 57.1 (3- OCH3), 57.1 (5- OCH3). The
aforementioned data are in agreement with the reported
data (Lee, 2005) of lyoniresinol-3-O--D-
glucopyranoside (Figure 2). Compound 2: A white
+
amorphous solid, ESI-MS (m/z): 583[M+H] . Formula,
1
C28H38O13. HNMR (600 MHz, MeOH) : 1.71 (1H, m, H-
2), 2.09 (1H, m, H-3), 2.62 (1H, m, Ha-1), 2.72 (1H, dd,
J=4.2, 15.0 Hz, Hb-1), 3.35 (3H, s, 5-OCH3), 3.54 (1H, m,
Ha-6''), 3.65 (1H, m, Hb-6''), 3.75 (6H, s, 3'-OCH3, 5'-
OCH3), 3.86 (3H, s, 7-OCH3), 4.28 (1H, d, J=7.8 Hz, H-
1''), 4.42 (1H, d, J=6.0 Hz, H-4), 6.43 (2H, s, H-2',6'), 6.58
13
(1H, s, H-8). CNMR (150 MHz, MeOH) : 34.0 (C-1),
41.4 (C-2), 46.8 (C-3), 43.4 (C-4), 147.7 (C-5), 139.1 (C-
6), 148.9 (C-7), 108.0 (C-8), 126.4 (C-9), 130.4 (C-10),
66.4 (C-2), 72.2 (C-3), 134.8 (C-1'), 107.3 (C-2'), 149.2
(C-3'), 139.6 (C-4'), 149.2 (C-5'), 107.3 (C-6'), 104.4 (C-
1''), 75.3 (C-2''), 78.2 (C-3''), 71.7 (C-4''), 78.4 (C-5''), 62.9
(C-6''), 60.3 (5-OCH3), 56.8 (7- OCH3), 57.1 (3-OCH3),
57.1 (5-OCH3). The aforementioned data are in
agreement with the reported data (Lee, 2005) of
lyoniresinol-3-O--D- glucopyranoside (Figure 2).
Compoud 3: A white amorphous solid, ESI-MS (m/z):
+ 1
583[M+H] . Formula, C28H38O13. HNMR (600 MHz,
MeOH) : 1.71 (1H, m, H-2), 2.08 (1H, m, H-3), 2.62 (1H,
m, Ha-1), 2.70 (1H, dd, J=4.2, 15.0 Hz, Hb-1), 3.35 (3H, s,
5-OCH3), 3.54 (1H, m, Ha-6''), 3.65 (1H, m, Hb-6''), 3.74
(6H, s, 3'-OCH3, 5'-OCH3), 3.85 (3H, s, 7-OCH3), 4.28
(1H, d, J=7.8 Hz, H-1''), 4.42 (1H, d, J=6.0Hz, H-4), 6.43
13
(2H, s, H-2',6'), 6.58 (1H, s, H-8). CNMR (150 MHz,
MeOH) : 33.0 (C-1), 40.8 (C-2), 46.9 (C-3), 43.0 (C-4),
147.8 (C-5), 139.1 (C-6), 148.8 (C-7), 108.0 (C-8), 126.6
(C-9), 130.4 (C-10), 66.4 (C-2OCH3), 71.7 (C-3OCH3),
134.7 (C-1'), 107.1 (C-2'), 149.2 (C-3'), 139.5 (C-4'),
149.2 (C-5'), 107.1 (C-6'), 105.0 (C-1''), 75.4 (C-2''), 78.1
(C-3''), 71.8 (C-4''), 78.4 (C-5''), 63.0 (C-6''), 60.4 (5-
OCH3), 56.8 (7-OCH3), 57.1 (3-OCH3), 57.1(5-OCH3).
The aforementioned data are in agreement with the
reported data (Lee, 2005) of lyoniresinol-3-O--D-
glucopyranoside (Figure 2).
Compoud 4: A white amorphous solid, ESI-MS (m/z):
+ 1
583[M+H] . Formula, C28H38O13. HNMR (600 MHz,
MeOH) : 1.68 (1H, m, H-2), 2.13 (1H, m, H-3), 2.67 (1H,
m, Ha-1), 2.68 (1H, m, Hb-1), 3.35 (3H, s, 5-OCH3), 3.54
(1H, m, Ha-6''), 3.65 (1H, m, Hb-6''), 3.75 (6H, s, 3'-OCH3,
5'-OCH3), 3.85 (3H, s, 7-OCH3), 4.13 (1H, d, J=7.8 Hz, H-
1''), 4.22 (1H, d, J=6.0Hz, H-4), 6.41 (2H, s, H-2',6'), 6.58
13
(1H, s, H-8). CNMR (150 MHz, MeOH) : 34.0 (C-1),
41.4 (C-2), 46.8 (C-3), 43.4 (C-4), 147.7 (C-5), 139.1 (C-
6), 148.9 (C-7), 108.0 (C-8), 126.4 (C-9), 130.4 (C-10),
66.4 (C-2), 72.2 (C-3), 134.8 (C-1'), 107.3 (C-2'), 149.2
(C-3'), 139.6 (C-4'), 149.2 (C-5'), 107.3 (C-6'), 104.4 (C-
1''), 75.3 (C-2''), 78.2 (C-3''), 71.7 (C-4''), 78.4 (C-5''), 62.9
(C-6''), 60.3 (5-OCH3), 56.8 (7-OCH3), 57.1 (3-OCH3),
57.1 (5-OCH3). The aforementioned data are consistent
with the reported data (Lee, 2005) of lyoniresinol-3-O--
D- glucopyranoside (Figure 2).
Stereostructure elucidation
In the NOESY experiments, the correlations between H-2
and H-4, between H-2, 6 and H-3 (Figure 1) on
Compouds 1 and 2, between H-2 and H-3, between H-2,
6 and H-3 (Figure 1) on compouds 3 and 4, indicated
that the relative configuration of the aglycone of
Compound 1 was the same as the aglycone of
Compound 2, the relative configuration of the aglycone of
Compound 3 was the same as the aglycone of
Compound 4.
The absolute configurations were determined by
examinations of the circular cichroism (CD) spectra of
Compounds 1 to 4. The CD curves of the four
compounds should be spiltted into two parts, for the two
chromophores (two benzene rings) of -* transition of
each of the four compounds. The CD curve at 244 nm
(+11.5473) and 224 nm (-2.45488) of Compound 1
indicated the CD spectrum was a positive chiral spectrum
(Figure 3) (Nakanishi et al., 1997). According to the
helicity rule of exciton coupled circular dichroism, the
exciton coupling of the two identical chromophores with
positive chirality when Compound 1 was accepted as the
stereochemistry of C4-S (Nakanishi et al., 1997).
Consequently, Compound 1 was determined to be (2R,
3R, 4S)-lyoniresinol-3-O--D-glucopyranoside. By the
same rule, Compound 2 was determined to be (2S, 3S,
4R)-lyoniresinol-3-O--D-glucopyranoside, for the CD
curve of Compound 2 was a negative chiral spectrum at
224 nm (-5.52452) and 217 nm (+7.61588) (Figure 4).
Compound 3 was determined to be (2S, 3S, 4R)-
lyoniresinol-3-O--D-glucopyranoside, for the CD curve
of Compound 3 was a positive chiral spectrum at 244 nm
(+0.689788) and 220 nm (-0.895488) (Figure 5).
Compound 4 was determined to be (2S, 3S, 4R)-
lyoniresinol-3-O--D-glucopyranoside, for the CD curve
of Compound 4 was a negative chiral spectrum at 243.5
nm (-1.11085) and 222 nm (+0.437642) (Figure 6).
 Sun et al. 2203

Figure 3. CD spectrum of Compound 1.

Figure 4. CD spectrum of Compound 2.

2204 J. Med. Plants Res.

Figure 5. CD spectrum of Compound 3.

Figure 6. CD spectrum of Compound 4.

 Sun et al. 2205

DISCUSSION REFERENCES

Heitzman ME, Neto CC, Winiarz E, Vaisberg AJ, Hammond GB (2005).

We highlight that this is the first report on determination
of the absolute configuration of the lignans by applying
the helicity rule of exciton coupled circular dichroism. And
the method has the qualities of wide application,
convenience. The four lignans were isolated from both
Uncaria genus and U. sinensis for the first time. And the
lignans also have the widely activities, such as anti-
tumor, antivirus, protect the liver, inhibit platelet activation
factor (PAF) (Lee, 2005). The studies done in this article
played an important role of the foundation of constituents
having some connection with the application of U.
sinenses.
Ethnobotany, phytochemistry and pharmacology of Uncaria
(Rubiaceae). Phytochemistry, 66: 5-29.
Laus G (2004). Advances in chemistry and bioactivity of the genus
Uncaria. Phytother. Res., 18: 259274.
Lee DG, Jung HJ, Woo ER (2005). Antimicrobial Property of (+)-
Lyoniresinol-3-O--D-glucopyranoside isolated from the root bark of
Lycium Chinense miller against human pathogenic microorganisms.
Arch. Pharm. Res., 28(9): 1031-1036.
Luo XR (1999). Flora of China. Science Press, Beijing, 71(1): 248-258.
Nakanishi K, Berova N, Woody RW (1997). Circular Dichroism
principles and applications. Wiley-VCH, New York, pp. 362-367.
Risdale CE (1978). A revision of Mitragyna and Uncaria (Rubiaceae).
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ACKNOWLEDGEMENTS

Financial support from Chinese Traditoinal Medicine

Researches of Special Projects (Grant No. 200707007);
the technological large platform for comprehensive
research and development of newdrugs in the Twelfth
Five-Year Significant New Drugs Created Science and
Technology Major Projects (No. 2012ZX09301-002-001-
026)the chemical composition of the digital library of
traditional Chinese medicine for drug discovery in the
Twelfth Five-Year Significant New Drugs Created (No.
2011ZX09307-002 -01) are gratefully acknowledged. We
are also grateful to Dr. Jing Quan Yuan (the Institute of
Medicinal Plant Development, Chinese Academy of
Medical Sciences and Peking Union Medical College) for
kindly providing help.