Three new naphthalenyl glycosides from the root bark of Juglans cathayensis

Article abstract of DOI:10.1248/cpb.60.785

Phytochemical investigations of the root bark of Juglans cathayensis DODE. led to the isolation of three new naphthalenyl glycosides, Jugnaphthalenoside A-C (1-3). Their structures were elucidated on the basis of extensive analysis of spectroscopic data. The cytotoxicities of the three new compounds were also evaluated.

Full text of DOI:10.1248/cpb.60.785

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Note
Chem. Pharm. Bull. 60(6) 785–789 (2012)
785
Three New Naphthalenyl Glycosides from the Root Bark of Juglans
cathayensis
Jia-Xiang Sun,a Xiao-Ya Zhao,b Xiao-Fang Fu,b Heng-Yi Yu,a Xue Li,a Shu-Ming Li,c and
,a
Han-Li Ruan*
a Faculty of Pharmacy, Tongji Medical College of Huazhong University of Science and Technology; Hangkong
b
Road 13, Wuhan 430030, P.R. China: Hubei Entry-Exit Inspection and Quarantine Bureau; WuHan 430050,
c
P.R. China: and Institute of Pharmaceutical Biology and Biotechnology, Philipps University of Marburg;
Deutschhausstrasse 17A, D-35037, Germany.
Received November 21, 2011; accepted March 22, 2012; published online April 2, 2012
Phytochemical investigations of the root bark of Juglans cathayensis DODE. led to the isolation of three
new naphthalenyl glycosides, Jugnaphthalenoside A–C (1–3). Their structures were elucidated on the basis of
extensive analysis of spectroscopic data. The cytotoxicities of the three new compounds were also evaluated.
Key words Juglans cathayensis; naphthalenyl glycoside; cytotoxicity
Juglans cathayensis DODE. is a perennial deciduous arbor to C-13 and C-15 (δC 140.1), and from H-11 (δH 9.06) to C-12
wildly distributed in mainland China and Taiwan province. (δC 149.9), C-13 and C-15.10) Substructure B was assembled by
The roots of this plant have been used in the folk medicine the HMBC correlations from H-7 (δH 7.41) to C-6 (δC 160.4),
for the treatment of hepatoma, lung carcinoma and esophageal C-8 (δC 146.8), C-9 (δC 140.6) and C-17 (δC 115.2). In addi-
carcinoma.1) Several naphthoquinones, naphthalenyl glucosides tion, the NMR data of the sugar part (substructure C) were
and tetralones have been isolated from Juglans species.2–15) In consistent with those of D-glucose (Table 1).17) Connection of
our previous anti-tumor activity screening study, the EtOAc substructures A, B and C with each other were suggested on
and n-BuOH-soluble fractions from the EtOH extract of the the basis of the HMBC correlations from H-11 to C-17, and
root bark of J. cathayensis showed significant anti-tumor from H-1′ (δH 5.03) to C-12 (δC 149.9). In addition, the con-
activities and two new naphthoquinones, together with six figuration of the glycosidic linkage was determined to be β
known ones, were isolated and identified.16) Further investi- on the basis of the coupling constant (7.6Hz) of the anomeric
gation on the chemical constituents of the n-BuOH fraction proton (δH 5.03).17) Considering the degree of unsaturation, the
resulted in the isolation of three new naphthalenyl glycosides, lactone carbonyl atom should be attached to C-15 through an
Jugnaphthalenoside A–C (1–3). In this paper, we describe the O-bridge, forming a six-membered lactone between substruc-
isolation, structural elucidation and cytotoxicities of these new tures A and B. Further analysis of the 13C-NMR data and the
compounds.
molecular formula of 1, the unusual low-field shifted carbons
C-1 (δC 153.5), C-8 (δC 146.8), C-9 (δC 140.6) and C-10 (δC
143.8) should be substituted by hydroxyl groups. HPLC analy-
Results and Discussion
Compound 1 was obtained as a dark brown amorphous pow- sis of chiral derivatives of the acid hydrolysate of 1 confirmed
der. The molecular formula of 1 was established as C23H20O12 the presence of D-glucose. The structure of 1 was thus deter-
by the [M+H]+ ion peak at m/z 489.1019 (Calcd 489.1033) in mined to be 1,8,9,10-tetrahydroxy-6H-naphtho[1,2-b]benzo[d]-
the high resolution-electrospray ionization (HR-ESI)-MS, re- pyran-6-one-12-O-β-D-glucopyranoside, and given the trivial
1
quiring fourteen degrees of unsaturation. Inspection of the H- name Jugnaphthalenoside A.
NMR spectrum of 1 revealed the presence of an AMX system
Compound 2 was obtained as a light yellow amorphous
of 1,2,3-trisubstituted benzene at δH 6.93 (1H, d, J=8.0Hz), powder with a molecular formula of C29H30O16 as deduced
7.51 (1H, t, J=8.0Hz) and 7.83 (1H, d, J=8.0Hz); Two singlets from HR-ESI-MS data (m/z 657.1424 [M+Na]+, Calcd for
at δH 7.41 (1H) and 9.06 (1H), and seven sugar protons in the 657.1432), requiring fifteen degrees of unsaturation. Acid
range of δH 5.03 to 3.37 were also observed (Table 1). The 13C- hydrolysis of 2 afforded D-glucose and L-rhamnose, which
NMR and distortionless enhancement by polarization transfer was identified on HPLC by comparison of the chiral deriva-
(DEPT) spectra of 1 showed 23 carbon signals, consisting of tives of the acid hydrolysate of 2 with the authentic sample
one lactone carbonyl, five olefinic methines, eleven olefinic derivatives. Comprehensive analysis of the 1D- and 2D-NMR
quaternary carbons, one anomeric carbon, four oxygenated spectra of 2 suggested that it was nearly identical to 1 with
aliphatic methines and one oxygenated aliphatic methylene. the exception of an extra sugar moiety rhamnose. The posi-
In addition to nine degrees of unsaturation required for one tion of the extra sugar moiety was assigned to be at C-6′ (δC
lactone carbonyl and sixteen olefinic carbons, a pentacyclic 65.4) by the HMBC correlations from H-6′ (δH 4.01, 3.72) to
structure was proposed for 1 to fulfill the needed unsatura- C1″ (δC 100.6) and from H1″ (δH 4.62) to C6′. Further more,
tion. Detailed inspection of the 1D- and 2D-NMR spectra of 1 the configuration of the glycosidic linkage of the L-rhamnose
revealed the presence of signals for three substructures A, B, was determined to be α on the basis of the coupling constant
and C (Fig. 1). Substructure A was established by the hetero- of the anomeric proton (δH 4.62, brs).18) Thus the structure of
nuclear multiple bond connectivity (HMBC) cross-peaks of 1 2 was determined as 1,8,9,10-tetrahydroxy-6H-naphtho[1,2-
from H-2 (δH 6.93) to C-1 (δC 153.5) and C-13 (δC 114.1), from b]benzo[d]pyran-6-one-12-O-α-L-rhamnopyranosyl-(1
H-3 (δH 7.51) to C-1 and C-14 (δC 125.5), from H-4 (δH 7.83) glucopyranoside, and termed Jugnaphthalenoside B.
→6)-β-D-
*To whom correspondence should be addressed. e-mail: ruanhl@mails.tjmu.edu.cn
© 2012 The Pharmaceutical Society of Japan

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1
Fig. 1. Key H–1H COSY and Selected HMBC Correlations of 1–3
Compound 3 was obtained as a light brown amorphous against Hep G2 and HeLa cell lines with 5-fluorouracil (5-FU)
powder with a molecular formula of C30H24O16 as deduced as a positive control and all compounds showed very weak
from HR-ESI-MS data (m/z 641.1135 [M+H]+, Calcd for cytotoxicity in comparison with 5-FU. The inhibition rates of
641.1143), requiring nineteen degrees of unsaturation. The 1–3 at 100µg/mL against each cell line were 12%, 14%, 39%
NMR data of 3 were similar to those of 1, with additional sig- and 28%, 8%, 2% respectively, while the inhibition rate of
nals for an extra galloyl moiety. Acid hydrolysis of 3 followed 5-FU was 60% and 61% with the concentration of 100µg/mL.
by HPLC analysis confirmed this conclusion. Furthermore,
the configuration of the glycosidic linkage of the D-glucose
was also determined to be β according to the coupling con-
Experimental
General Procedure Petroleum ether (PE, 60–90 C), EtO-
°
stant (7.7Hz) of the anomeric proton (δH 5.11).17) The galloyl Ac, n-BuOH, CH2Cl2, Me2CO, MeOH, and CH3CN were of
group was deduced from two chemically equivalent aromatic analytical grade and produced by Jiangsu Qiangsheng Chemi-
carbons each at δC 108.6 (C-2″, 6″) and 145.5 (C-3″, 5″), two cal Co., Ltd., China. Column chromatography (CC) was per-
substituted aromatic carbons at δC 119.5 (C-1″) and 138.5 formed on silica gel (200–300 or 300–400 mesh; Qingdao Ma-
(C-4″), and one carbonyl carbon at δC 165.9 (C-7″) in the 13C- rine Chemical Inc., Qingdao, P.R. China), MCI gel (CHP20P,
NMR spectrum, coupled with a singlet at δH 7.00 (2H, H-2″, 75–150µm, Mitsubishi Chemical Industries Ltd., Japan), and
1
6″) in the H-NMR spectrum.19) The position of the galloyl Sephadex LH-20 (GE Healthcare, U.S.A.). Fractions were
moiety was deduced from the HMBC correlations from H-6′ monitored with TLC, and spots were visualized by spraying
(δH 4.58) to C-7″ (δC 165.9) and from H-2″ and 6″ (δH 7.00) to with 5% H2SO4 in EtOH, followed by heating. Optical rota-
C-7″ (δC 165.9). Finally, the structure of 3 was identified as tion data were recorded on a Perkin 341 Polarimeter. UV data
1,8,9,10-tetrahydroxy-6H-naphtho[1,2-b]benzo[d]pyran-6-one- were obtained using a Cary 50 UV-Visible spectrophotometer.
12-O-(6-galloyl-)glucopyranoside, named Jugnaphthalenoside IR spectra were obtained on a Bruker-VERTEX-70 spectro-
C.
photometer with KBr pellets. HR-ESI-MS were recorded on a
The aglycons of compounds 1–3 were proposed to be Thermo Scientific LTQ-Orbitrap XL mass spectrometer. NMR
derived from 1,4,8-trihydroxynaphthalenen and gallic acid spectra were recorded on a Bruker-AM-400 instrument with
through the intermediates A–D by a series of biochemical re- tetramethylsilane (TMS) as an internal standard.
actions such as oxidation, hydrolysis, lactonization and glyco-
sidation.20,21) The plausible biosynthetic pathways for 1–3 were collected in Shengnongjia mountainous area of Hubei prov-
postulated and shown in Fig. 2. ince of China in September 2008, and identified by Mr. Shigui
Compounds 1–3 were tested for their inhibitory effects Shi from Shennongjia Institute for Drug Control. A voucher
Plant Material The root bark of Juglans cathayensis was

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June 2012
787
specimen (P20080910) was deposited in the herbarium of the to get compound 2 (5mg).
Faculty of Pharmaceutical Sciences, Tongji Medical College
of Huazhong University of Science and Technology, Wuhan, [α]D20 −25.0 (c=0.40, pyridine); UV λmax (MeOH) nm (logε):
China.
267 (2.23), 362 (0.72), 376 (0.72); IR (KBr) υmax cm−1: 3403,
Jugnaphthalenoside A (1): Dark brown amorphous powder;
Extraction and Isolation The root bark of Juglans ca- 2958, 2927, 2856, 1731, 1688, 1614, 1396, 1355, 1222, 1073,
thayensis (20kg) was air-dried at room temperature and pul- 1030, 989; 1H- and 13C-NMR data: see Table 1; HR-ESI-
verized, then extracted with 95% aqueous ethanol (3×50L) MS (positive-ion mode) m/z: 489.1019 [M+H]+ (Calcd for
at room temperature to yield an EtOH extract. The EtOH C23H21O12, 489.1033).
extracts were suspended in water and then partitioned with
Jugnaphthalenoside B (2): Light yellow amorphous powder;
petroleum ether, EtOAc and n-BuOH to yield 190, 360 and [α]D20 −62.5 (c=0.80, pyridine); UV λmax (MeOH) nm (logε):
480g of dried extracts, respectively. The n-BuOH fraction 269 (2.14), 361 (0.65), 376 (0.65); IR (KBr) υmax cm–1: 3396,
(450g) was chromatographed on silica gel (200–300 mesh, 2926, 2856, 1686, 1614, 1577, 1394, 1354, 1226, 1061, 986;
3kg), eluted with a gradient system of CH2Cl2/MeOH (1:0, 1H- and 13C-NMR data, see Table 1; HR-ESI-MS (positive-
9:1, 8:2, 7:3, 1:1, 3:7, 1:9 and 0:1) and MeOH/H2O (9:1, ion mode) m/z: 657.1424 [M+Na]+ (Calcd for C29H30O16Na,
1:1 and 0:1) and yield fractions A–J. Fractions F (140g) was 657.1432).
chromatographed on MCI gel (75–150µm, 200g) and eluted
Jugnaphthalenoside C (3): Light brown amorphous powder;
with MeOH/H2O (gradient from H2O to MeOH) and Me2CO [α]D20 −22.7 (c=4.40, pyridine); UV λmax (MeOH) nm (log ε):
to yield 7 subfractions (F1 to F7). Subfractions F3 (15g) and 267 (2.15), 360 (0.67), 376 (0.67); IR (KBr) υmax cm–1: 3379,
F4 (5g) were further purified with repeated silica gel (300– 2924, 2856, 1692, 1613, 1577, 1394, 1352, 1226, 1078, 1015,
400 mesh, CH2Cl2/MeOH) and Sephadex LH-20 (MeOH) to 988; H- and 13C-NMR data, see Table 1; HR-ESI-MS (pos-
1
give compounds 3 (25mg) and 1 (4mg), respectively. Frac- itive-ion mode) m/z: 641.1135 [M+H]+ (Calcd for C30H25O16,
tions G (137g) was subjected to MCI gel (75–150µm, 200g) 641.1143).
to yield 7 subfractions (G1 to G7), and its subfraction G3 (8g)
Acid Hydrolysis and Sugar Analysis of 1–3 The abso-
was further purified with Sephadex LH-20 (MeOH) repeatedly lute configuration of sugar was determined according to the
Table 1. 1H- and 13C-NMR Data of 1–3 (δ in ppm, J in Hz, Recorded in DMSO-d6)
1a)
2b)
3b)
Position
δC
δH
δC
δH
δC
δH
1
2
153.5 s
112.0 d
128.3 d
112.1 d
160.4 s
107.2 d
146.8 s
140.6 s
143.8 s
108.1 d
149.9 s
114.1 s
125.5 s
140.1 s
114.2 s
115.2 s
112.4 s
103.5 d
77.3 d
153.5 s
111.9 d
128.2 d
112.1 d
160.3 s
107.1 d
146.8 s
140.6 s
143.9 s
107.7 d
149.9 s
114.0 s
125.5 s
140.0 s
114.3 s
115.2 s
112.4 s
103.6 d
76.5 d
153.4 s
112.0 d
128.3 d
112.2 d
160.3 s
107.1 d
146.8 s
140.7 s
143.9 s
107.8 d
150.0 s
114.0 s
125.5 s
140.1 s
114.3 s
115.1 s
112.3 s
103.8 d
76.2 d
6.93 (d, 8.0)
7.51 (t, 8.0)
7.83 (d, 8.0)
6.93 (d, 7.9)
7.50 (t, 7.9)
7.83 (d, 7.9)
6.94 (d, 7.1)
7.52 (t, 7.1)
7.84 (d, 7.1)
3
4
6
7
7.41 (s)
7.40 (s)
7.42 (s)
8
9
10
11
12
13
14
15
16
17
18
1′
2′
3′
4′
5′
6′
9.06 (s)
9.03 (s)
9.12 (s)
5.03 (d, 7.6)
5.02 (d, 7.1)
3.37 (m)
5.11 (d, 7.7)
3.45 (m)
3.52 (m)
3.62 (m)
3.76 (m)
4.58 (m)
c)
3.41 (m)
c)
73.4 d
3.41 (m)
73.3 d
3.42 (m)
73.4 d
c
69.1 d
3.42 (m)
3.37 (m)
68.8 d
3.45 ) (m)
68.8 d
76.6 d
75.7 d
3.52 (m)
74.3 d
60.1 t
3.90 (d, 11.1), 3.68
(dd, 12.0, 2.8)
65.4 t
4.01 (brd, 10.7), 3.72
(brd, 10.9)
62.7 t
1″
2″
3″
4″
5″
6″
7″
100.6 d
70.4 d
70.7 d
72.0 d
68.2 d
17.7 q
4.62 (brs)
3.64 (m)
119.5 s
108.6 d
145.5 s
138.5 s
145.5 s
108.6 d
165.9 s
7.00 (s)
7.00 (s)
c)
3.44 (m)
3.13 (m)
3.47 (m)
1.03 (d, 5.0)
a) 400MHz for 1H and 100MHz for 13C. b) 600MHz for 1H and 150MHz for 13C. c) Signals were overlapped.

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Vol. 60, No. 6
Fig. 2. The Plausible Biosynthetic Pathway for 1–3
literature.22) Compounds 1 (0.5mg), 2 (0.5mg), and 3 (1.0mg)
Acknowledgments Financial support from the Natural
°
were hydrolyzed with 2M HCl (MeOH/H2O, 0.5mL) at 90 C Science Foundation of China (No. 30873361) and Natural
for 2h. After that, the MeOH in the reaction mixtures was re- Science Foundation of Hubei province (No. 2006ABA125) is
moved by reduction vaporization. Then the reaction mixtures gratefully acknowledged.
were extracted with EtOAc (0.5mL). The aqueous phase was
neutralized by passing through an Amberlite IRA-67 column
and was evaporated repeatedly under reduced pressure to give
sugar fractions. The sugar fractions were each dissolved in
anhydrous pyridine (0.1mL), and then 1.0mg of L-cysteine
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