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 ja-

ponicum (Thunb.) Sw. The structure of the new compound

was elucidated by two-dimensional nuclear magnetic res-

onance and other spectral examinations.

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 flav-

ones, 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-iso-

propyl-7-methyl-1,4-naphthoquinone. Moreover, three

known compounds have been isolated for the first time from

the Lygodiaceae. Here, we report the structural character-

ization of these compounds by spectroscopic analysis.

Results and discussion

The 70% ethanolic extract of roots of Lygodium japonicum

(Thunb.) Sw. suspended in H2O was partitioned succes-

sively with CHCl3, AcOEt, n-BuOH and the n-BuOH-

soluble 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

2–4. The structures of the known compounds were deter-

mined 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.

193–194�C. The molecular formula was determined

as C14H14O3 by HR–ESI–TOF–MS (m/z 231.1004

[M ? H]?, calcd. 231.1016), along with 1H-NMR and13C-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 res-

onances all that assigned as sp3 carbons. By observing

these data of 13C-NMR spectrum, nucleus of naphtho-

quinone 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 aro-

matic protons at d 7.30 (1H, s, H-5), 7.77 (1H, s, H-8)

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

123

J Nat Med (2010) 64:114–116

DOI 10.1007/s11418-009-0376-y

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, respec-

tively, 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 6-

hydroxy-2-isopropyl-7-methyl-1,4- naphthoquinone.

Compound 2, was isolated as a white amorphous powder

and its elemental composition was determined to be

C27H44O7 on ESI–MS: m/z 503.3 [M ? Na]?, along with1H 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–

160�C, had a molecular formula C12H20O8 determined by

HR–ESI–TOF–MS (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-glucopra-

noside 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].

Experimental

General

Melting points were determined on an X4-A micro-melting

point apparatus and were uncorrected. ESI–MS spectra

were measured on an Agilent 1100 LC-MSD-Trap-SL, and

HR–ESI–MS spectra were measured on an Bruker Dal-

tonics MicroTOFQ. NMR spectra were measured on a

Bruker ARX-600 and 300 NMR spectrometer with tetra-

methylsilane (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 (10–40 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 Tra-

ditional 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).

Table 1 1H and 13C NMR data for compound 1 (300 and 75 MHz, in

DMSO-d6)

Position HSQC HMBC

dC dH, mult,

J in Hz

1 183.5

2 156.6

3 131.6 6.68 (1H) C2/C11

4 185.2

5 110.3 7.30 (1H) C4/C9/C6

6 160.9

7 131.8

8 129.5 7.77 (1H) C10/C7(CH3)/

C1/C6

9 124.1

10 131.7

CH3-7 16.2 2.24 (3H, s) C6/C8

CH(CH3)2

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

O

O CH3

CH3

HO

H3C

H

H

H

H

1

2

3

456

78 9

10

Fig. 1 The key HMBC correlations of compound 1

J Nat Med (2010) 64:114–116 115

123

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 CHCl3–MeOH (100:1–1:1) to

give ten fractions. Fraction 2 (2.3 g) was subjected to silica

gel column chromatography eluted with petroleum ether

(PE)–EtOAc (20:1–1:1) 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-hydroxy-

2-isopropyl-7-methyl-1,4-naphthoquinone: 9 mg). Fraction

7 (3.2 g) was repeatedly applied on silica gel column

chromatography eluted with CHCl3–MeOH (50:1–1:1) 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 MeOH–H2O (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 (0:1–1:0) 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 repeat-

edly 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 CHCl3–MeOH to

afford 2 (b-ecdysone: 35 mg). Fraction 5 (1.9 g) was

applied on silica gel column chromatography eluted with

CHCl3–MeOH (50:1–1:1) 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 7-

O-b-D-glucopyranoside: 7 mg).

6-hydroxy-2-isopropyl-7-methyl-1,4-naphthoquinone 1:

yellow powder (MeOH), mp. 193–194�C. 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 HR–ESI–MS: m/z

231.1004 [M ? H]? (calculated for C14H15O3: 231.1016).1H-NMR (300 MHz, in DMSO-d6) and 13C-NMR

(75 MHz, in DMSO-d6) see Table 1.

Compound 2: white amorphous powder, ESI–MS: 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

(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–

160�C, 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-60a), 3.48 (1H, dd, J = 18,

5.5 Hz, H-60b). 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, ESI–MS and HR–ESI–MS spectra.

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