J Nat Med (2010) 64:114116

DOI 10.1007/s11418-009-0376-y

NOTE

New naphthoquinone from the root of Lygodium japonicum

(Thunb.) Sw.
Lijuan Chen Guogang Zhang Jie He Jin Guan
Chunyuan Pan Wenzhen Mi Qing Wang

Received: 3 August 2009 / Accepted: 23 October 2009 / Published online: 4 December 2009
The Japanese Society of Pharmacognosy and Springer 2009

Abstract A new 1,4-naphthoquinone and three known

compounds were isolated from the roots of Lygodium japonicum (Thunb.) Sw. The structure of the new compound
was elucidated by two-dimensional nuclear magnetic resonance and other spectral examinations.

known compounds have been isolated for the first time from
the Lygodiaceae. Here, we report the structural characterization of these compounds by spectroscopic analysis.

Results and discussion

Keywords 1,4-Naphthoquinone
Roots of Lygodium
japonicum (Thunb.) Sw.
Spectral examinations

Introduction
Lygodium japonicum (Thunb.) Sw., belonging to the
Lygodiaceae family and that only has Lygodium [1], is
endemic to Southern China and the southwestern area of
China and commonly called zuo zhuan hui teng. This plant
species is a perennial plant, and roots of several species of
this genus are used in Chinese traditional medicine to treat
hepatitis and dysentery [2]. Chemical studies on the genus
have been reported and have revealed the presence of flavones, phenolic acids and steroidal glycosides [3, 4]. As our
chemical studies on the medicinal plants in China aimed at
searching biologically active compounds, we researched the
roots of Lygodium japonicum (Thunb.) Sw., which resulted
in the isolation of a new naphthoquinone, 6-hydroxy-2-isopropyl-7-methyl-1,4-naphthoquinone. Moreover, three
L. Chen
G. Zhang (&)
J. He
C. Pan
W. Mi
Q. Wang
School of Traditional Chinese Materia Medica,
Shenyang Pharmaceutical University,
110016 Shenyang, China
e-mail: zhangguogang824@hotmail.com
J. Guan
College of Applied Chemistry, Shenyang Institute of Chemical
Technology, 110142 Shenyang, China

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The 70% ethanolic extract of roots of Lygodium japonicum

(Thunb.) Sw. suspended in H2O was partitioned successively with CHCl3, AcOEt, n-BuOH and the n-BuOHsoluble fraction was repeatedly subjected to silica gel,
Sephadex LH-20, and ODS chromatography with various
solvent systems and to preparative HPLC to yield a new
1,4-naphthoquinone (9 mg) and three known compounds
24. The structures of the known compounds were determined by 1H-NMR, 13C-NMR and 2D NMR, by comparing
their spectroscopic data with those reported in the
references.
Compound 1 was obtained as a yellow powder, mp.
193194C. The molecular formula was determined
as C14H14O3 by HRESITOFMS (m/z 231.1004
[M ? H]?, calcd. 231.1016), along with 1H-NMR and
13
C-NMR data. The UV spectrum displayed absorption
bands at 207, 267 and 347 nm, closely resembling that
of 1,4-naphthoquinones [5]. The 13C-NMR spectrum
revealed 14 carbon resonances; in the low field area of it,
two were assigned as carbonyl carbons, eight were
assigned as aromatic carbons. However, in the high field
area of 13C-NMR spectrum, there were four carbon resonances all that assigned as sp3 carbons. By observing
these data of 13C-NMR spectrum, nucleus of naphthoquinone was revealed safely. All protonated carbons were
assigned by analysis of the HSQC spectrum (Table 1).
The 1H-NMR spectrum of 1 showed signals of two aromatic protons at d 7.30 (1H, s, H-5), 7.77 (1H, s, H-8)

J Nat Med (2010) 64:114116

115

Table 1 1H and 13C NMR data for compound 1 (300 and 75 MHz, in
DMSO-d6)
Position

HSQC

HMBC
dH, mult,
J in Hz

dC
1

183.5

156.6

131.6

185.2

110.3

160.9

131.8

129.5

124.1

10
CH3-7

6.68 (1H)

C2/C11

7.30 (1H)

C4/C9/C6

7.77 (1H)

C10/C7(CH3)/
C1/C6

131.7
16.2

2.24 (3H, s)

C6/C8

11

26.6

3.09 (1H, m)

C12 and
C13/C2/C1/C3

12 and 13

21.4

1.12 (6H, d,
J = 6.8)

C11/C2

Compound 2, was isolated as a white amorphous powder

and its elemental composition was determined to be
C27H44O7 on ESIMS: m/z 503.3 [M ? Na]?, along with
1
H and 13C-NMR (300 and 75 MHz, in CD3OD) data.
Compared the NMR data of 2 with those of a known one
[6, 7], the similar chemical shift pattern was observed.
Compound 2 was identified with b-ecdysone by comparing
spectral data with those reported [6, 7].
Compound 3, white amorphous powder, mp. 159
160C, had a molecular formula C12H20O8 determined by
HRESITOFMS (m/z 293.1257 [M ? H]?, calcd.
293.1231). The IR spectrum exhibited absorptions at 3433,
2962, 2922, 2859, 1780, 899 cm-1. Compound 3 was
safely identified with R-(-)-pantoyllactone-b-D-glucopranoside by comparing spectral data with those reported [8].
Compound 4, yellow amorphous powder, was identified
as apigenin 7-O-b-D-glucopyranoside by comparing the
physical and spectral data with those reported [9].

CH(CH3)2

H
HO

O
5

10

H
3
2

H3C

8
H

H
CH 3

1
O

CH3

Fig. 1 The key HMBC correlations of compound 1

and one aromatic methyl proton at d 2.24 (3H, s, 7-CH3)

that were assigned by analyzing HMBC spectrum
(Table 1; Fig. 1). Additionally, d 1.12 (6H, d, J = 6.8 Hz,
H-12, H-13) and 6.68 (1H, s, H-3) correlated, respectively, with d 26.6 (C-11), 156.6 (C-2) in the HMBC
spectrum and d 3.09 (1H, m, H-11) correlated with d 21.4
(C-12 and 13), 156.6 (C-2), 131.6 (C-3), 183.4 (C-1) all
that revealed the presence of isopropyl and it connected
C-2 of quinone ring. Other detailed correlations in the
HMBC spectrum see Table 1. All these spectroscopic data
discussed above elucidated safely compound 1 as 6hydroxy-2-isopropyl-7-methyl-1,4- naphthoquinone.

Experimental
General
Melting points were determined on an X4-A micro-melting
point apparatus and were uncorrected. ESIMS spectra
were measured on an Agilent 1100 LC-MSD-Trap-SL, and
HRESIMS spectra were measured on an Bruker Daltonics MicroTOFQ. NMR spectra were measured on a
Bruker ARX-600 and 300 NMR spectrometer with tetramethylsilane (TMS) as the internal reference and chemical
shifts are expressed in d (ppm). UV spectra were recorded
on a Shimadzu UV-2201 spectrometer. IR spectra were
recorded on a Bruker IFS-55 spectrophotometer. TLC was
performed on silica gel GF254 (1040 lm; Qingdao,
China). Separation were performed by Semiprep-HPLC
named Shimadzu SPD-10A apparatus equipped with UV
detector under ODS column (i.d. 10 mm 9 200 mm).
Plant material
Lygodium japonicum (Thunb.) Sw. (Lygodiaceae), roots
collected in Anhui province of China, were identified by
asso-Prof. Jincai Lu. A voucher was deposited in the Traditional Chinese medica of Shenyang Pharmaceutical
University.
Extraction and isolation
Air-dried roots of Lygodium japonicum (Thunb.) Sw.
(4 kg) were crushed and extracted twice under reflux with
70% EtOH. Evaporation of the solvent under reduced
pressure delivered the 70% EtOH extract (around 280 g).

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The extract was partitioned successively with CHCl3,

AcOEt and n-BuOH. The n-BuOH-soluble fraction
(50.0 g) was isolated by column chromatography on silica
gel and gradient elution with CHCl3MeOH (100:11:1) to
give ten fractions. Fraction 2 (2.3 g) was subjected to silica
gel column chromatography eluted with petroleum ether
(PE)EtOAc (20:11:1) to afford a further five fractions
(frs. 2-1 to 2-5). Fraction 2-2 was purified twice by
Sephadex LH-20 eluted with MeOH to give 1 (6-hydroxy2-isopropyl-7-methyl-1,4-naphthoquinone: 9 mg). Fraction
7 (3.2 g) was repeatedly applied on silica gel column
chromatography eluted with CHCl3MeOH (50:11:1) to
obtain six fractions (frs. 7-1 to 7-6). Fraction 7-2
(420.5 mg) was applied on Sephadex LH-20 eluted with
MeOH to give further three fractions (frs. 7-2-1 to 7-2-3).
Fraction 7-2-3 (66.3 mg) was purified by semi-preparative
ODS column using MeOHH2O (40:60) to give 3 (R-(-)pantoyllactone-b-D-glucopranoside: 7 mg). Fraction 6
(4.1 g) was isolated by ODS column chromatography
eluted with MeOH-H2O (0:11:0) to afford six fractions
(frs. 6-1 to 6-6). Fraction 6-3 (205 mg) was purified by
Sephadex LH-20 eluted with MeOH to give two fractions
(frs. 6-3-1 to 6-3-2). Fraction 6-3-2 (152 mg) was repeatedly purified by Sephadex LH-20 eluted with CHCl3
MeOH (1:1) to give two fractions (frs. 6-3-2-1 to 6-3-2-2).
Fraction 6-3-2-2 (102 mg) recrysted by CHCl3MeOH to
afford 2 (b-ecdysone: 35 mg). Fraction 5 (1.9 g) was
applied on silica gel column chromatography eluted with
CHCl3MeOH (50:11:1) to give three fractions (frs. 5-1 to
5-3). Fraction 5-3 (50.2 mg) was purified twice by
Sephadex LH-20 eluted with MeOH to give 4 (apigenin 7O-b-D-glucopyranoside: 7 mg).
6-hydroxy-2-isopropyl-7-methyl-1,4-naphthoquinone 1:
yellow powder (MeOH), mp. 193194C. UV kmax
(MeOH): 207, 267 and 347 nm. IR mmax (film on KBr) 3378
(hydroxyl), 2964 (methyl), 1660 (carboxyl), 1616, 1577,
1508 (benzene ring), 1463, 1384, 1336, 1268, 1162, 1034,
884, 727, 599 cm-1. Positive ion HRESIMS: m/z
231.1004 [M ? H]? (calculated for C14H15O3: 231.1016).
1
H-NMR (300 MHz, in DMSO-d6) and 13C-NMR
(75 MHz, in DMSO-d6) see Table 1.
Compound 2: white amorphous powder, ESIMS: m/z
503.3 [M ? Na]?, 1H-NMR (300 MHz, in CD3OD) d :
0.88 (3H, s, H-18), 0.96 (3H, s, H-19), 1.18 (3H, s, H-21),
1.19 (3H, s, H-26), 1.20 (3H, s, H-27), 3.33 (1H, m, H-22),
2.37 (1H, m, H-17), 3.14 (1H, brt, H-9), 5.80 (1H, brs,
H-7), 2.39 (1H, m, H-5), 3.85 (1H, m, H-2), 3.95 (1H, m,
H-3). 13C-NMR (75 MHz, in CD3OD) d : 37.3 (C-1), 68.5
(C-2), 68.7 (C-3), 32.9 (C-4), 51.8 (C-5), 206.5 (C-6),
122.1 (C-7), 167.9 (C-8), 35.1 (C-9), 39.2 (C-10), 21.0
(C-11), 32.5 (C-12), 48.0 (C-13), 85.2 (C-14), 31.8 (C-15),
21.5 (C-16), 50.5 (C-17), 18.1 (C-18), 24.4 (C-19), 77.9

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J Nat Med (2010) 64:114116

(C-20), 21.4 (C-21), 78.4 (C-22), 27.3 (C-25), 42.4 (C-24),

71.3 (C-25), 28.9 (C-26), 29.7 (C-27).
Compound 3: white amorphous powder, mp. 159
160C, IR mmax (film on KBr) cm-1: 3433, 2962, 2922,
2859, 1780, 899. 1H-NMR (600 MHz, in DMSO-d6) d:
4.60 (1H, s, H-3), 3.94 (2H, s, H-5), 1.02 (3H, s, 4a-CH3),
1.18 (3H, s, 4b-CH3), 4.33 (1H, d, J = 7.8 Hz, H-10 ), 3.68
(1H, dd, J = 18, 2.3 Hz, H-60 a), 3.48 (1H, dd, J = 18,
5.5 Hz, H-60 b). 13C-NMR (150 MHz, in DMSO-d6) d:
174.9 (C-2), 79.0 (C-3), 75.3 (C-5), 19.6 (4a-CH3), 23.0
(4b-CH3), 102.6 (C-10 ), 73.7 (C-20 ), 76.9 (C-30 ), 70.5
(C-40 ), 77.6 (C-50 ), 61.5 (C-60 ).
Compound 4: yellow amorphous powder, 1H-NMR
(300 MHz, in DMSO-d6), d: 12.96 (1H, s, 5-OH), 10.39
(1H, s, 40 -OH), 7.96 (2H, d, J = 8.5 Hz, H-20 ,60 ), 6.94 (2H,
d, J = 8.5 Hz, H-30 ,50 ), 6.88 (1H, s, H-3), 6.83 (1H, d,
J = 2.0 Hz, H-8), 6.45 (1H, d, J = 2.0 Hz, H-6), 5.07 (1H,
d, J = 7.5 Hz, anomeric proton). 13C-NMR (75 MHz, in
DMSO-d6), d 163.0 (C-2), 103.2 (C-3), 182.4 (C-4), 161.5
(C-5), 99.6 (C-6), 164.3 (C-7), 94.9 (C-8), 151.0 (C-9),
105.4 (C-10), 121.1 (C-10 ), 128.7 (C-20 ), 116.1 (C-30 ),
161.2 (C-40 ), 116.1 (C-50 ), 128.7 (C-60 ), 99.9 (C-100 ), 73.2
(C-200 ), 76.5 (C-300 ), 69.6 (C-400 ), 77.3 (C-500 ), 60.7 (C-600 ).
Acknowledgments We are grateful to the analytical detective
center, Shenyang Pharmaceutical University, for recording NMR,
UV, IR, ESIMS and HRESIMS spectra.

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